Greenfield's Neuropathology [Volume 1, 9 ed.] 9781498729055

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Greenfield’s

Neuropathology

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Greenfield’s

Ninth Edition

Neuropathology Volume 1 Edited by Seth Love MBBCh PhD FRCP FRCPath Professor of Neuropathology Institute of Clinical Neurosciences School of Clinical Sciences University of Bristol Southmead Hospital Bristol, UK

Herbert Budka MD Professor of Neuropathology Consultant, Institute of Neuropathology University Hospital Zurich Zurich, Switzerland

James W Ironside CBE BMSc FRCPath FRCPEdin FMedSci FRSE Professor of Clinical Neuropathology and Honorary Consultant in Neuropathology National Creutzfeldt–Jakob Disease Surveillance Unit University of Edinburgh Western General Hospital Edinburgh, UK

Arie Perry MD Professor of Pathology and Neurological Surgery Director of Neuropathology Director of Neuropathology Fellowship Training Program University of California, San Francisco San Francisco, CA, USA

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CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2015 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Version Date: 20160121 International Standard Book Number-13: 978-1-4987-2905-5 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. While all reasonable efforts have been made to publish reliable data and information, neither the author[s] nor the publisher can accept any legal responsibility or liability for any errors or omissions that may be made. The publishers wish to make clear that any views or opinions expressed in this book by individual editors, authors or contributors are personal to them and do not necessarily reflect the views/opinions of the publishers. The information or guidance contained in this book is intended for use by medical, scientific or health-care professionals and is provided strictly as a supplement to the medical or other professional’s own judgement, their knowledge of the patient’s medical history, relevant manufacturer’s instructions and the appropriate best practice guidelines. Because of the rapid advances in medical science, any information or advice on dosages, procedures or diagnoses should be independently verified. The reader is strongly urged to consult the relevant national drug formulary and the drug companies’ and device or material manufacturers’ printed instructions, and their websites, before administering or utilizing any of the drugs, devices or materials mentioned in this book. This book does not indicate whether a particular treatment is appropriate or suitable for a particular individual. Ultimately it is the sole responsibility of the medical professional to make his or her own professional judgements, so as to advise and treat patients appropriately. The authors and publishers have also attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Contents Preface................................................................................vii Contributors........................................................................ ix Abbreviations......................................................................xiv

Volume 1 Chapter 1: General pathology of the central nervous system..................................................................................1 Harry V Vinters and BK Kleinschmidt-DeMasters Chapter 2: Vascular disease, hypoxia and related ­conditions........................................................................... 59 Raj Kalaria, Isidro Ferrer and Seth Love Chapter 3: Disorders of the perinatal period.................... 210 Rebecca D Folkerth and Marc R Del Bigio Chapter 4: Malformations................................................. 270 Brian N Harding and Jeffrey A Golden Chapter 5: Metabolic and neurodegenerative diseases of childhood..................................................................... 399 Thomas S Jacques and Brian N Harding Chapter 6: Lysosomal diseases....................................... 439 Steven U Walkley, Kinuko Suzuki and Kunihiko Suzuki

Chapter 15: Ageing of the brain........................................ 849 James Lowe Chapter 16: Dementia....................................................... 858 James Lowe and Raj Kalaria

Volume 2 Chapter 17: Psychiatric diseases..................................... 975 Margaret M Esiri, Steven A Chance, Jean Debarros and Tim J Crow Chapter 18: Prion diseases............................................. 1016 Mark W Head, James W Ironside, Bernardino Ghetti, ­ Martin Jeffrey, Pedro Piccardo and Robert G Will Chapter 19: Viral infections............................................. 1087 Seth Love, Clayton A Wiley and Sebastian Lucas Chapter 20: Bacterial infections..................................... 1192 Martina Deckert Chapter 21: Parasitic infections...................................... 1230 Sebastian Lucas Chapter 22: Fungal infections......................................... 1281 Sebastian Lucas

Chapter 7: Mitochondrial disorders.................................. 523 Patrick F Chinnery, Nichola Z Lax, Evelyn Jaros, Robert W Taylor, Douglas M Turnbull and Salvatore DiMauro

Chapter 23: Demyelinating diseases.............................. 1297 G R Wayne Moore and Christine Stadelmann-Nessler

Chapter 8: Peroxisomal disorders.................................... 562 Phyllis L Faust and James M Powers

Chapter 24: Diseases of peripheral nerves..................... 1413 Robert E Schmidt and Juan M Bilbao

Chapter 9: Nutritional and toxic diseases........................ 589 Jillian Kril, Leila Chimelli, Christopher M Morris and John B Harris Chapter 10: Trauma.......................................................... 637 Colin Smith, Susan S Margulies and Ann-Christine Duhaime Chapter 11: Epilepsy........................................................ 683 Maria Thom and Sanjay Sisodiya

Chapter 25: Diseases of skeletal muscle........................ 1515 Caroline A Sewry, Susan C Brown, Rahul Phadke and ­Francesco  Muntoni Chapter 26: Introduction to tumours.............................. 1623 Arie Perry and David N Louis Chapter 27: Astrocytic tumours...................................... 1638 Daniel J Brat Chapter 28: Oligodendroglial tumours............................ 1673 Guido Reifenberger

Chapter 12: Extrapyramidal diseases of movement......................................................................... 740 Tamas Revesz, H Brent Clark, Janice L Holton, Henry H Houlden, Paul G Ince and Glenda M Halliday

Chapter 29: Ependymal tumours.................................... 1693 Guido Reifenberger

Chapter 13: Degenerative ataxic disorders...................... 799 H Brent Clark

Chapter 30: Choroid plexus tumours............................. 1709 Hope T Richard, Jason F Harrison and Christine E Fuller

Chapter 14: Motor neuron disorders................................ 817 Paul G Ince, J Robin Highley and Stephen B Wharton

Chapter 31: Other glial neoplasms................................. 1717 Daniel J Brat V

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VI  Contents Chapter 32: Neuronal and mixed neuronal-glial tumours.......................................................................... 1726 Daniel J Brat

Chapter 40: Lymphomas and haemopoietic neoplasms...................................................................... 1850 Martina Deckert

Chapter 33: Neuroepithelial tumours of the pineal region..................................................................... 1756 Alexandre Vasiljevic, Anne Jouvet and Michelle Fevre Montange

Chapter 41: Pituitary and suprasellar tumours.......................................................................... 1870 Sylvia L Asa

Chapter 34: Embryonal tumours..................................... 1765 Charles Eberhart

Chapter 42: Cysts and tumour-like conditions....................................................................... 1908 Arie Perry

Chapter 35: Tumours of the peripheral nerves............... 1788 Arie Perry and Robin Reid

Chapter 43: Metastatic disease...................................... 1919 Matthew D Cykowski and Gregory N Fuller

Chapter 36: Tumours of the meninges........................... 1803 Arie Perry

Chapter 44: Hereditary tumour syndromes...................................................................... 1926 Arie Perry

Chapter 37: Mesenchymal non-meningothelial tumours............................................................................ 1828 Christine E Fuller and Knarik Arkun Chapter 38: Germ cell tumours...................................... 1834 Marc K Rosenblum Chapter 39: Melanocytic tumours and haemangioblastoma....................................................... 1844 Arie Perry

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Chapter 45: Paraneoplastic syndromes......................... 1945 Marc K Rosenblum Chapter 46: CNS reactions to anti-neoplastic therapies......................................................................... 1954 Arie Perry Index...................................................................................I-1

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Preface Greenfield’s Neuropathology holds a special place in the heart of most neuropathologists. It has long been a standard-bearer of our specialty. In 1921, Joseph Godwin Greenfield and Edward Farquhar Buzzard published Pathology of the Nervous System, which had a key role in defining neuropathology as a distinct specialty. The authors set out to ‘describe clearly the anatomical changes which are associated with disorders of nervous function, to discuss briefly questions of pathogenesis, and to indicate in a few words, where it is possible, the relationship between structural alterations and clinical signs and symptoms.’ In 1958, a book entitled simply Neuropathology, by Greenfield, William Blackwood, William McMenemy, Alfred Meyer and Ronald Norman, updated and greatly expanded on most of the content of Pathology of the Nervous System. Unlike Pathology of the Nervous System, however, Neuropathology did not cover neoplastic diseases (dealt with instead in a companion book, Russell and Rubinstein’s Pathology of Tumours of the Nervous System). However, tumours of the nervous system have been included in Greenfield’s Neuropathology since the seventh edition in 1997. Readers of a succession of editions over many decades have dipped into this venerable reference book seeking definitive advice and instruction on all matters neuropathological. Producing a new edition of Greenfield’s Neuropathology has therefore been both a huge privilege and a massive responsibility. It has also been a balancing act, in which we have had to reconcile the tension between the physical constraints of a two-volume book and the everexpanding amount of information encompassed within our field. Indeed, this may be the last edition of Greenfield’s Neuropathology that can be produced in hardcover printed format. Accommodating the additional information has largely involved a combination of reorganisation and restraint, together with considerably increased use of photographs and diagrams. The reorganisation has involved the merging of vascular disease, hypoxia and related conditions into a single chapter; the subdivision of movement disorders into separate chapters on extrapyramidal disorders, ataxias and motor neuron diseases; the inclusion of separate chapters on ageing and dementia, the latter encompassing an expanded section on vascular dementia; and the further subdivision of the tumour section from two chapters in the previous edition to twenty-one in the present one, which we hope will make this part of the book easier to navigate. The total number of chapters in the book has increased from twenty-four to forty-six. Restraint has been applied in relation to the inclusion of references and of some very

detailed molecular genetic and phenotypic information that is readily accessible through online resources such as OMIM, the database of Genotypes and Phenotypes (dbGaP), AlzGene and PDGene. We expect readers to look to Greenfield’s Neuropathology for guidance and perspective rather than as a substitute for bibliographic databases and search engines. The changes have involved a great deal of work on the part of our authors, who have shown unfailing courtesy and forbearance in responding to requests to condense prose, reorganise chapters and be selective in the inclusion of references. We are in their debt. Throughout, our objectives, much like those of Greenfield and Buzzard, have been to describe clearly the neuropathological changes that underlie neurological diseases, to discuss briefly their pathogenesis, and to try to relate molecular genetic, structural and biochemical alterations to clinical and neuroradiological manifestations. Once a full account has been taken of the clinical and neuroradiological manifestations of neurological disease in a particular patient, a detailed visual examination of the diseased tissue is the starting point for almost all neuropathological investigations. Much of the excitement of neuropathology comes from discovering visual clues to disease, macroscopic or microscopic, whether in a section stained simply with haematoxylin and eosin, a series of confocal laser scanning images or a transmission electron micrograph. Neuropathology remains a highly visual specialty and most of us neuropathologists obtain immense aesthetic gratification from our work. Not surprisingly, therefore, we have placed a strong emphasis on visual aspects of this reference book, which includes over one thousand completely new photographs and drawings. It also incorporates new design elements such as the alternate colour coding of chapters that is intended to allow their easier navigation. To this same end, both volumes now include full indexes to the whole book. There are also improved search, annotation and bookmarking facilities in the bundled bonus e-book version of this edition. The e-book frees users from most of the physical limitations (not least of which are the size and weight) of the printed version and can be downloaded to a wide range of mobile and electronic devices, so that it is not necessary to be online to have full access to Greenfield’s Neuropathology. Publication of this ninth edition of Greenfield’s Neuropathology would not have been possible without the support of many people, initially at Hodder Arnold and subsequently at Taylor and Francis. At Hodder Arnold, Joanna Koster, Editorial Director; Caroline

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VIII  Preface

Makepeace, Head of Postgraduate and Professional Publishing; Mischa Barrett, Project Editor; and Miriam Trent, Editorial Assistant, were closely involved in the early stages. At Taylor and Francis, Barbara Norwitz, Executive Editor; Amy Blalock, Supervisor, Editorial Project Development; Rachael Russell, Senior Editorial Assistant; and Linda Van Pelt, Senior Project Manager, Medical, all worked on different stages of the title, and one person who merits special thanks is Sue Hodgson for her invaluable help as Executive Editor. Glenys Norquay provided freelance support and Jayne Jones designed the cover and interior pages.

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We are pleased to present the ninth edition of Greenfield’s Neuropathology. We hope you obtain as much satisfaction from reading this book as we have from editing it.

S Love H Budka J W Ironside A Perry November 2014

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Contributors Knarik Arkun, md Director Neuropathology and Autopsy Service Assistant Professor Department of Pathology Tufts Medical Center Boston, MA, USA Sylvia L Asa, md, phd Medical Director Laboratory Medicine Program University Health Network Lakeridge Health & Women’s College Hospital Senior Scientist Ontario Cancer Institute Professor Department of Laboratory Medicine and Pathobiology University of Toronto Toronto, ON, Canada Juan M Bilbao, frcp (Canada) Professor Emeritus of Neuropathology St Michael’s and Sunnybrook Hospitals University of Toronto Toronto, ON, Canada Daniel J Brat, md, phd Professor and Vice Chair Translational Programs Department of Pathology and Laboratory Medicine Emory University School of Medicine Georgia Research Alliance Distinguished Cancer Scientist Atlanta, GA, USA Susan C Brown, phd Reader in Translational Medicine Comparative Biomedical Sciences Royal Veterinary College London, UK Herbert Budka, md Professor of Neuropathology Consultant, Institute of Neuropathology University Hospital Zurich Zurich, Switzerland Steven A Chance, dphil Associate Professor in Clinical Neurosciences Department of Neuropathology University of Oxford Oxford, UK Leila Chimelli, md, phd Professor of Pathology Federal University of Rio de Janeiro Rio de Janeiro, Brazil

Patrick F Chinnery, bmedsci, mbbs, phd, frcp, frcpath, fmedsci

Professor of Neurogenetics Newcastle University Newcastle upon Tyne, UK H Brent Clark, md, phd Director of Neuropathology Professor of Laboratory Medicine and Pathology, Neurology, and Neurosurgery University of Minnesota Medical School Minneapolis, MN, USA Tim J Crow, mbbs, phd, frcp, frcpsych, fmedsci SANE POWIC University Department of Psychiatry Warneford Hospital Oxford, UK Matthew D Cykowski, md Neuropathology Fellowship Program Houston Methodist Hospital/MD Anderson Cancer Center Houston, TX, USA Jean Debarros, phd, MBPsS Research Clinical Psychologist Counselling Service University of Oxford Oxford, UK Martina Deckert, md Professor Department of Neuropathology University Hospital of Cologne Cologne, Germany Marc R Del Bigio, md, phd, frcpC Canada Research Chair in Developmental Neuropathology Professor Department of Pathology (Neuropathology) University of Manitoba Winnipeg, MB, Canada Salvatore DiMauro, md Lucy G Moses Professor Department of Neurology Columbia University Medical Center New York, NY, USA Ann-Christine Duhaime, md Director Pediatric Neurosurgery Massachusetts General Hospital Nicholas T Zervas Professor of Neurosurgery Harvard Medical School Boston, MA, USA IX

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X  Contributors

Charles Eberhart, md, phd Professor of Pathology, Ophthalmology and Oncology Director of Neuropathology and Ophthalmic Pathology Johns Hopkins University School of Medicine Baltimore, MD, USA Margaret M Esiri, DM, FRCPath Neuropathology Department John Radcliffe Hospital Emeritus Professor of Neuropathology Nuffield Department of Clinical Neurosciences University of Oxford Oxford, UK Phyllis L Faust, md, phd Associate Professor of Clinical Pathology and Cell Biology Department of Pathology and Cell Biology Columbia University New York, NY, USA Isidro Ferrer, md, phd Professor Institute of Neuropathology Bellvitge University Hospital and University of Barcelona Hospitalet de Llobregat Barcelona, Spain Rebecca D Folkerth, md Director of Neuropathology Department of Pathology Brigham and Women’s Hospital Consultant in Neuropathology Boston Children’s Hospital Associate Professor of Pathology Harvard Medical School Boston, MA, USA Christine E Fuller, md Professor Pathology and Neurology Director Neuropathology and Autopsy Pathology Department of Pathology Virginia Commonwealth University Richmond, VA, USA Gregory N Fuller, md, phd Professor and Chief Section of Neuropathology The University of Texas MD Anderson Cancer Center Department of Pathology Houston, TX, USA Bernardino Ghetti, MD, FANA, FAAAS Distinguished Professor Indiana University Chancellor’s Professor Indiana University-Purdue University Indianapolis Department of Pathology and Laboratory Medicine Division of Neuropathology Indiana University School of Medicine Indianapolis, IN, USA

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Jeffrey A Golden, md Chair Department of Pathology Brigham and Women’s Hospital Ramzi S Cotran Professor of Pathology Harvard Medical School Boston, MA, USA Glenda M Halliday, phd Professor of Neuroscience and NHMRC Senior Principal Research Fellow School of Medical Sciences and Neuroscience Research Australia University of New South Wales Sydney, NSW, Australia Brian N Harding, MA, DPhil, BM, BCh, FRCPath Department of Pathology and Laboratory Medicine Children’s Hospital of Philadelphia Perelman School of Medicine University of Pennsylvania Philadelphia, PA, USA John B Harris, phd, BPharm, FSocBiol, MRPharmSoc Emeritus Professor of Experimental Neurology Medical Toxicology Centre Newcastle University Newcastle upon Tyne, UK Jason F Harrison, md, phd Neurosurgery Resident Department of Neurosurgery Virginia Commonwealth University Richmond, VA, USA Mark W Head, BSc, phd Reader University of Edinburgh Deputy Director National CJD Research & Surveillance Unit Edinburgh, Scotland J Robin Highley, DPhil, FRCPath Senior Clinical Lecturer in Neuropathology Department of Neuroscience Sheffield Institute of Translational Neuroscience University of Sheffield Sheffield, UK Janice L Holton, BSc, MBChB, phd, FRCPath Professor of Neuropathology Department of Molecular Neuroscience University College London Institute of Neurology London, UK Henry H Houlden, md, phd Professor of Neurology and Neurogenetics Department of Molecular Neuroscience University College London Institute of Neurology London, UK

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Contributors  XI

Paul G Ince, MBBS, md, FRCPath Professor of Neuropathology Head of Department of Neuroscience University of Sheffield Sheffield, UK James W Ironside, CBE, BMSc, FRCPath, FRCPEdin, FMedSci, FRSE

Professor of Clinical Neuropathology and Honorary Consultant in Neuropathology National Creutzfeldt–Jakob Disease Surveillance Unit University of Edinburgh Western General Hospital Edinburgh, UK Thomas S Jacques, phd, MRCP, FRCPath Higher Education Funding Council for England Clinical Senior Lecturer Honorary Consultant Paediatric Neuropathologist University College London Institute of Child Health and Great Ormond Street Hospital Department of Histopathology Great Ormond Street Hospital for Children NHS Foundation Trust London, UK  Evelyn Jaros, phd Clinical Scientist in Neuropathology Neuropathology/Cellular Pathology Newcastle upon Tyne Hospitals NHS Foundation Trust Honorary Senior Research Associate Institute of Neuroscience and Institute for Ageing Newcastle University Campus for Ageing and Vitality Newcastle upon Tyne, UK Martin Jeffrey, BVMS, DVM, Dip ECVP, MRCVS, FRCPath

Consultant Pathologist Pathology Department Animal Health and Veterinary Laboratories Agency (AHVLA-Lasswade) Penicuik, UK Anne Jouvet, md, phd Associate Professor of Pathology Centre de Pathologie et Neuropathologie Est Centre de Biologie et Pathologie Est Groupement Hospitalier Est Hospices Civils de Lyon Lyon, France Raj Kalaria, phd, FRCPath Professor of Cerebrovascular Pathology (Neuropathology) Institute of Neuroscience Newcastle University National Institute for Health Research Biomedical Research Building Campus for Ageing and Vitality Newcastle upon Tyne, UK

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B K Kleinschmidt-DeMasters, md Professor of Pathology, Neurology, and Neurosurgery Department of Pathology University of Colorado School of Medicine Aurora, CO, USA Jillian Kril, phd, FFSc (RCPA) Professor of Neuropathology Sydney Medical School The University of Sydney Sydney, NSW, Australia Nichola Z Lax, phd Research Associate Wellcome Trust Centre for Mitochondrial Research Institute of Neuroscience Newcastle University Newcastle upon Tyne, UK David N Louis, md Pathologist-in-Chief Massachusetts General Hospital Benjamin Castleman Professor of Pathology Harvard Medical School James Homer Wright Pathology Laboratories Massachusetts General Hospital Boston, MA, USA Seth Love, MBBCh, phd, FRCP, FRCPath Professor of Neuropathology Institute of Clinical Neurosciences School of Clinical Sciences University of Bristol Southmead Hospital Bristol, UK James Lowe, DM, FRCPath Professor of Neuropathology University of Nottingham Honorary Consultant in Neuropathology to the Nottingham University Hospitals NHS Trust School of Medicine Faculty of Medicine and Health Sciences University of Nottingham Nottingham, UK Sebastian Lucas, FRCP, FRCPath Emeritus Professor of Histopathology Department of Histopathology St Thomas’ Hospital London, UK Susan S Margulies, phd George H Stephenson Professor Department of Bioengineering University of Pennsylvania Philadelphia, PA, USA

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XII  Contributors

Michelle Fevre Montange, phd Centre de Recherche en Neuroscience de Lyon INSERM U1028 CNRS UMR 5292 Equipe Neuro-oncologie et Neuro-inflammation Université de Lyon Lyon, France G R Wayne Moore, BSc, md, CM, FRCPC, FRCPath Clinical Professor Department of Pathology and Laboratory Medicine International Collaboration on Repair Discoveries (ICORD) University of British Columbia Vancouver General Hospital Vancouver, BC, Canada Christopher M Morris, phd Senior Lecturer Medical Toxicology Centre National Institutes of Health Research Health Protection Research Unit in Chemical and Radiation Threats and Hazards Institute of Neuroscience Newcastle University Newcastle upon Tyne, UK Francesco Muntoni, FRCPCH, FMedSci Director Dubowitz Neuromuscular Centre MRC Centre for Neuromuscular Diseases University College London Institute of Child Health and Great Ormond Street Hospital for Children (GOSH) London, UK Arie Perry, md Professor of Pathology and Neurological Surgery Director of Neuropathology Director of Neuropathology Fellowship Training Program University of California, San Francisco San Francisco, CA, USA Rahul Phadke, MBBS, md, FRCPath Consultant Neuropathologist University College London Institute of Neurology National Hospital for Neurology and Neurosurgery and Dubowitz Neuromuscular Centre Great Ormond Street Hospital for Children London, UK Pedro Piccardo, md Senior Investigator Chief Transmissible Spongiform Encephalopathy Pathogenesis Section Laboratory of Bacterial and TSE Agents Office of Blood Research and Review Center for Biologics Evaluation and Research U.S. Food and Drug Administration Silver Spring, MD, USA Professor Neurobiology Division The Roslin Institute University of Edinburgh Easter Bush, UK

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James M Powers, md Professor Emeritus Department of Pathology University of Rochester School of Medicine and Dentistry Rochester, NY, USA Robin Reid, BSc, MBChB, FRCPath Formerly Consultant Pathologist Western Infirmary Glasgow, UK Guido Reifenberger, md Professor Department of Neuropathology Heinrich Heine University Düsseldorf, Germany Tamas Revesz, md, FRCPath Professor Emeritus in Neuropathology UCL Institute of Neurology University College London London, UK Hope T Richard, md, phd Neuropathology Fellow Department of Pathology Virginia Commonwealth University Richmond, VA, USA Marc K Rosenblum, md Founder’s Chair and Chief Neuropathology and Autopsy Service Memorial Sloan-Kettering Cancer Center Professor of Pathology and Laboratory Medicine Weill Medical College of Cornell University New York, NY, USA Robert E Schmidt, md, phd Professor of Pathology and Immunology Director Division of Neuropathology Medical Director Electron Microscope Facility Washington University School of Medicine St Louis, MO, USA Caroline A Sewry, phd, FRCPath Professor of Muscle Pathology Dubowitz Neuromuscular Centre Institute of Child Health and Great Ormond Street Hospital London Wolfson Centre for Inherited Neuromuscular Diseases Robert Jones and Agnes Hunt Orthopaedic Hospital Oswestry, UK

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Contributors  XIII



Sanjay Sisodiya, MA, phd, FRCP, FRCPEdin Professor of Neurology Department of Clinical and Experimental Epilepsy UCL Institute of Neurology London Consultant Neurologist Epilepsy Society National Hospital for Neurology and Neurosurgery Chalfton St Peter, UK Colin Smith, md, FRCPath Reader in Pathology University of Edinburgh Edinburgh, UK Christine Stadelmann-Nessler, md Professor Department of Neuropathology University Medical Center Göttingen Göttingen, Germany Kinuko Suzuki, md Emeritus Professor of Pathology and Laboratory Medicine University of North Carolina at Chapel Hill Chapel Hill, NC, USA Neuropathology Tokyo Metropolitan Institute of Gerontology Tokyo, Japan Kunihiko Suzuki, md Director Emeritus Neuroscience Center University of North Carolina Chapel Hill, NC, USA Robert W Taylor, phd, DSc, FRCPath Professor of Mitochondrial Pathology Wellcome Trust Centre for Mitochondrial Research Institute of Neuroscience Newcastle University Newcastle upon Tyne, UK Maria Thom, BSc, MBBS, MRCPath Senior Lecturer Institute of Neurology University College London London, UK Douglas M Turnbull, md, phd, FRCP, FMedSc Professor of Neurology Director Wellcome Trust Centre for Mitochondrial Research Director LLHW Centre for Ageing and Vitality Newcastle University Newcastle upon Tyne, UK

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Alexandre Vasiljevic, md Associate Professor of Pathology Centre de Pathologie et Neuropathologie Est Centre de Biologie et Pathologie Est Groupement Hospitalier Est Hospices Civils de Lyon Lyon, France Harry V Vinters, md Professor of Pathology and Laboratory Medicine, Neurology Chief of Neuropathology Division of Neuropathology Member of Brain Research Institute ACCESS Program Department of Cellular and Molecular Pathology University of California, Los Angeles Los Angeles, CA, USA Steven U Walkley, DVM, phd Director Rose F Kennedy Intellectual and Developmental Disabilities Research Center Head Sidney Weisner Laboratory of Genetic Neurological Disease Departments of Neuroscience, Pathology and Neurology Albert Einstein College of Medicine Bronx, NY, USA Stephen B Wharton, BSc, MBBS, phd, FRCPath Professor and Honorary Consultant in Neuropathology Department of Neuroscience Sheffield Institute of Translational Neuroscience University of Sheffield Sheffield, UK Clayton A Wiley, md, phd Professor of Pathology Director of Neuropathology PERF Endowed Chair Univeristy of Pittsburgh Medical Center Presbyterian Hospital Pittsburgh, PA, USA Robert G Will, MA, md, FRCP Professor of Clinical Neurology National Creutzfeldt–Jakob Disease Surveillance Unit University of Edinburgh Western General Hospital Edinburgh, UK

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XIV  Abbreviations

Abbreviations AA AACD AAMI ABC ABCA1 ABRA ACA ACC ACCIS

anaplastic astrocytoma age-associated cognitive decline age-associated memory impairment ATP-binding cassette ATP-binding cassette transporter 1 Aβ-related angiitis anterior cerebral artery adrenocortical carcinoma Automated Childhood Cancer Information System ACh acetylcholine AChR acetylcholine receptor ACTH adrenocorticotropin AD Alzheimer disease ADAMTS13 a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 ADC apparent diffusion coefficient ADCA autosomal dominant cerebellar ataxia ADEM acute disseminated encephalomyelitis ADK adenosine kinase ADNFLE autosomal dominant nocturnal frontal lobe epilepsy ADP adenosine diphosphate AFP alpha-fetoprotein AGA aspartylglucosaminidase AGE advanced glycosylation end product AGPS alkylglycerone phosphate synthase AGS Aicardi-Goutières syndrome AGU aspartylglucosaminuria AHLE acute haemorrhagic leukoencephalitis AHT abusive head trauma AIDP acute inflammatory demyelinating polyneuropathy AIDS acquired immunodeficiency syndrome AIP aryl hydrocarbon receptor-interacting protein AIS axon initial segment AISS axonal index sector score AL amyloidosis ALCL anaplastic large cell lymphoma ALD adrenoleukodystrophy ALK anaplastic lymphoma kinase ALL acute lymphoblastic leukemia ALS amyotrophic lateral sclerosis ALT alternative lengthening of telomeres AMAN acute motor axonal neuropathy AMN adrenomyeloneuropathy AMPA α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid

AMSAN ANA ANCA ANCL Ang-1 Ang-2 ANI AOA1 APGBD APLA ApoE APP APrP APUD AQP4 AR ARBD ARFGEF2 ASA ASDH ASE ASL AT ATP ATRT ATTR AVM BA BACE BAC BAV BBB BDNF BDV BEAN bFGF BGC BHC BMAA BMD BMP BOLD bp BPAU

acute motor sensory axonal neuropathy antinuclear antibody antineutrophil cytoplasmic autoantibody adult neuronal ceroid lipofuscinosis angiopoietin-1 angiopoietin-2 asymptomatic neurocognitive impairment early-onset ataxia with oculomotor apraxia, type 1 adult polyglucosan body disease antiphospholipid antibody apolipoprotein E amyloid precursor protein amyloid prion protein amine precursor uptake and decarboxylation aquaporin-4 androgen receptor alcohol-related brain damage adenosine diphosphate (ADP)ribosylation factor guanine exchange factor 2 arylsulfatase A acute subdural haematoma acute schistosomal encephalopathy arterial spin labelling ataxia telangiectasia adenosine triphosphate atypical teratoid/rhabdoid tumour amyloid transthyretin arteriovenous malformation Brodmann area β-site APP-cleaving enzyme bacterial artificial chromosome Banna virus blood-brain barrier brain-derived neurotrophic factor Borna disease virus brain expressed protein associated with NEDD4 basic fibroblast growth factor basal ganglia calcification benign hereditary chorea β-N-methylamino-l-alanine Becker muscular dystrophy bone morphogenetic protein blood oxygenation dependent base pair bromophenylacetylurea

XIV

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Abbreviations  XV



BRC BRRS BSE CAA CADASIL

brain reserve capacity Bannayan-Riley-Ruvalcaba syndrome bovine spongiform encephalopathy cerebral amyloid angiopathy cerebral autosomal dominant ­arteriopathy with subcortical infarcts and leukoencephalopathy CAE childhood absence epilepsy CAHS chronic acquired hepatocerebral syndrome CAMTA1 calmodulin-binding transcription ­activator 1 c-ANCA cytoplasmic antineutrophil cytoplasmic antibody CANOMAD chronic ataxic neuropathy, ­ophthalmoplegia, M-protein ­agglutination, disialosyl antibodies CAR coxsackievirus and adenovirus receptor CARASIL cerebral autosomal recessive ­arteriopathy with subcortical infarcts and leukoencephalopathy cART combined antiretroviral therapy CASK calcium-dependent serine protein kinase CBD corticobasal degeneration CBF cerebral blood flow CBS corticobasal syndrome CBTRUS Central Brain Tumor Registry of the United States CCM cerebral cavernous malformation CCSVI chronic cerebrospinal venous insufficiency CD Cowden disease CDE common data elements CDI conformation dependent immunoassay CDK5 cyclin-dependent kinase 5 CDKI cyclin-dependent kinase inhibitor CDKN2C cyclin-dependent kinase inhibitor 2C CDV canine distemper virus CEA carcinoembryonic antigen CESD cholesteryl ester storage disease CGH comparative genomic hybridization cGMP cyclic guanosine monophosphate CGRP calcitonin gene-related peptide CHD5 chromodomain helicase DNA binding domain 5 CHN congenital hypomyelinating neuropathy CHS classical hippocampal sclerosis CIM critical illness myopathy CIP critical illness polyneuropathy CIPD chronic inflammatory demyelinating polyneuropathy CIS clinically isolated syndrome CISP chronic immune sensory polyradiculopathy CK cytokeratin; creatine kinase CLA2 X-linked cerebellar ataxia

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CLL CM CMD CMRgl CMRO2 CMROGl CMT CMV CN CNC CNP CNS CNS PNET CNTF CO COL4A1 COX COX-2 CP CPCS CPM CPP CPT CR CR3 CRABP CRBP CREB CRH CRIMYNE CRMP-5 CRV CSDH CSF CSPα CT CTD CTE CTF CTL CUP CUTE CuZnSOD CVD CVS CVST CVT

chronic lymphatic leukaemia cerebral malaria congenital muscular dystrophy cerebral metabolic rate for glucose cerebral metabolic rate for oxygen cerebral metabolic rates of oxygen and glucose Charcot–Marie–Tooth cytomegalovirus cystic nephroma Carney’s complex 2ʹ,3ʹ-cyclic nucleotide 3ʹ-phosphodiesterase central nervous system central nervous system primitive ­neuroectodermal tumour ciliary neurotrophic factor carbon monoxide collagen, type IV, alpha 1 cytochrome c oxidase cyclooxygenase-2 choroid plexus chronic post-concussion syndrome central pontine myelinolysis cerebral perfusion pressure; central ­precocious puberty carnitine palmitoyltransferase cognitive reserve complement receptor type 3 cellular retinoic acid binding protein cytoplasmic retinol binding protein cyclic adenine dinucleotide phosphate response element binding protein corticotropin-releasing hormone critical illness myopathy and neuropathy collapsing response mediator protein 5 cerebroretinal vasculopathy chronic subdural haematoma cerebrospinal fluid cysteine string protein α computed tomography connective tissue disease chronic traumatic encephalopathy Colorado tick fever cytotoxic lymphocyte cancer of unknown primary corticotropin upstream ­transcriptionbinding element copper- and zinc-containing superoxide dismutase cardiovascular disease chorionic villus sampling cerebral venous sinus thrombosis cerebral venous thrombosis

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XVI  Abbreviations

CWD CX32 DAB DAG DAI DAPAT DASE DAWM DCX DEHSI DFFB DHA DHAP DHPR Dil DILS DIR DLB DLBCL DLK DM DMD DMNV DMPK DNER DNL DNMT DNT DPR DPX DRD DRPLA DSD DSPN DTI DTICH DWI DXC EA EAAT EAN EBP EBV EC ECGF1 ECM ECMO

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chronic wasting disease connexin 32 diaminobenzidine dystrophin-associated glycoprotein diffuse axonal injury dihydroxyacetonephosphate acyltransferase developmentally arrested structural elements diffusely abnormal white matter doublecortin diffuse excessive high-signal intensity DNA fragmentation factor subunit beta docosahexaenoic acid dihydroxyacetone phosphate dihydropyridine receptor dioctadecyl-tetramethylindocarbacyanine perchlorate diffuse infiltrative lymphocytosis syndrome double inversion recovery dementia with Lewy bodies diffuse large B cell lymphoma dual leucine kinase dermatomyositis Duchenne muscular dystrophy dorsal motor nucleus of the vagus dermatomyositis protein kinase delta/notch-like epidermal growth ­factorrelated receptor disseminated necrotizing leukoencephalopathy DNA methyltransferase dysembryoplastic neuroepithelial tumour dipeptide repeat di-n-butylphthalate-polystyrene-xylene dopa-responsive dystonia dentatorubropallidoluysian atrophy Dejerine-Sottas disease diffuse sensory polyneuropathy diffusion tensor imaging delayed traumatic intracerebral haemorrhage diffusion weighted imaging doublecortin episodic ataxia excitatory amino acid transporter experimental allergic neuritis elastin-binding protein Epstein-Barr virus endothelial cell; entorhinal cortex endothelial cell growth factor 1 ­(platelet-derived) extracellular matrix extracorporeal membrane oxygenation

EDH EEE EEG EET EF HS EGA EGB EGFR EGL EGR2 EIEE EL ELBW ELISA EM EMA EME EMG

extradural haematoma eastern equine encephalitis electroencephalogram epoxyeicosatrienoic acid end folium hippocampal sclerosis estimated gestational age eosinophilic granular body epidermal growth factor receptor external granule cell layer early growth response 2 gene early infantile epileptic encephalopathy encephalitis lethargica extreme low birth weight enzyme-linked immunosorbent assay electron microscopy epithelial membrane antigen early myoclonic encephalopathy electromyography

EMT eNSC ENU EPC EPMR ER ERG ERK ERM ESAM

epithelial-mesenchymal transition embryonic neural stem cell ethylnitrosourea endothelial progenitor cell epilepsy with mental retardation endoplasmic reticulum electroretinogram extracellular signal-regulated kinase ezrin, radixin and moesin endothelial cell-selective adhesion molecule erythrocyte sedimentation rate embryonal tumour with abundant ­neuropil and true rosettes embryonal tumor with multilayered rosettes ethylene-vinyl alcohol copolymer Friedreich’s ataxia fluorescence-activated cell sorting familial Alzheimer’s disease familial amyloidosis of the Finnish type focal adhesion kinase familial amyotrophic lateral sclerosis familial amyloid polyneuropathy; familial polyposis familial British dementia F-box only protein 7 focal cortical dysplasia; follicular ­dendritic cell fibrocartilaginous embolism familial Creutzfeldt-Jakob disease familial Danish dementia fibroblast growth factor 2 fatal familial insomnia formalin-fixed paraffin-embedded tissue

ESR ETANTR ETMR EVOH FA FACS FAD FAF FAK FALS FAP FBD FBXO7 FCD FCE fCJD FDD FDF-2 FFI FFPE

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Abbreviations  XVII

FG FGF FHL1 FILIP FIPA FISH FKRP FLAIR FLNA FMD fMRI FOG FPS FR FS FSH FSHD FTBSI FTD FTL FTLD FUPB1 FUS FXTAS G-CIMP GABA GAD GAG GALT GAP-43 GAT1 Gb Ose3 Cer GBE GBM GBS GC GCA GCD GCI GCL GCS GDAP1 GDNF GEMM GFAP GFP GH GHR GI

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fast-twitch glycolytic fibroblast growth factor four and a half LIM domains protein 1 filamin-A-interacting protein familial isolated pituitary adenoma fluorescence in situ hybridization fukutin-related protein fluid-associated inversion recovery filamin A fibromuscular dysplasia; Fukuyama ­muscular dystrophy functional magnetic resonance imaging fast-twitch oxidative glycolytic fasciitis-panniculitis syndrome fatigue resistant febrile seizure follicle stimulating hormone facioscapulohumeral muscular dystrophy focal traumatic brain stem injury frontotemporal dementia ferritin light frontotemporal lobar degeneration far-upstream element binding protein 1 fused-in-sarcoma protein fragile X tremor/ataxia syndrome glioma CpG island methylator phenotype gamma-aminobutyric acid gracile axonal dystrophy; glutamic acid decarboxylase glycosaminoglycan gut-associated lymphoid tissue growth-associated protein 43 glutaric aciduria type 1 globotriaosylceramide

GIST GLAST GLB1 GLD GLM GM GOM GP GPI GROD GSC GSD GSN Gsp GSS

glycogen branching enzyme glioblastoma Guillain–Barré syndrome granule cell giant cell (or temporal) arteritis granule cell dispersion global cerebral ischaemia; glial ­cytoplasmic inclusion granule cell layer Glasgow Coma Scale ganglioside-induced differentiation-­ associated protein 1 glial cell-derived neurotrophic factor genetically engineered mouse model glial fibrillary acidic protein green fluorescent protein growth hormone GH receptor gastrointestinal

HCHWA-I

gTSE GU GWAS HAART HACE HAD HAM HAN HANAC HAS HAT HB-EGF HCG HCHWA-D HCHWA-F

HCMV HD HDL HDL1 HDL2 HDL3 HE H&E HERNS HERV HES HES-1 hGH HH HHV-8 HIF

gastrointestinal stromal tumour glutamate/aspartate transporter galactosidase, beta 1 globoid cell leukodystrophy glial limiting membrane gliomesodermal tissue granular osmiophilic material globus pallidus glycosylphosphatidylinositol granular osmiophilic deposit glioma stem cell glycogen storage disease gelsolin G-protein oncogene Gerstmann-Sträussler-Scheinker disease genetic transmissible spongiform encephalopathy genitourinary genome wide association studies highly active antiretroviral therapy high altitude cerebral oedema HIV-associated dementia HTLV-1-associated myelopathy hereditary neuralgic amyotrophy hereditary angiopathy with nephropathy, aneurysms and muscle cramps high-altitude stupid human African trypanosomiasis heparin-binding epidermal growth factor human chorionic gonadotropin hereditary cerebral haemorrhage with amyloid angiopathy of the Dutch hereditary cerebral haemorrhage with amyloid angiopathy of the Flemish hereditary cerebral haemorrhage with amyloid angiopathy of the Icelandic human cytomegalovirus Huntington’s disease high density lipoprotein Huntington disease-like type 1 Huntington disease-like type 2 Huntington disease-like type 3 hepatic encephalopathy haematoxylin and eosin hereditary endotheliopathy with ­retinopathy, nephropathy and stroke human endogenous retrovirus hairy/enhancer of split hairy/enhancer of split 1 human growth hormone hypothalamic hamartoma human herpesvirus 8 hypoxia inducible factor

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XVIII  Abbreviations

HIHRATL HIMAL HIV HLA HLH HMEG HMERF HMG HMGCR H-MRS HMSN HNE HNPCC HNPP H2O2 HPC HPE HPF HPS HPV HRE HRP HS HSA HSAN HSP HSV 5-HT hTERT HTLV-I HVR IBM ICA ICAM-1 ICD ICE ICH iCJD ICP IDH IENF IFS IGF IgM IHC IHI

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hereditary infantile ­hemiparesis, retinal arteriolar tortuosity and leukoencephalopathy hippocampal malrotation human immunodeficiency virus human leukocyte antigen helix-loop-helix hemimegalencephaly hereditary myopathy with early ­respiratory failure high mobility group 3-hydroxy-3-methylglutaryl-coenzyme A reductase proton magnetic resonance spectroscopy hereditary motor and sensory neuropathy hydroxy-2-nonenal hereditary nonpolyposis colorectal cancer hereditary neuropathy with liability to pressure palsy hydrogen peroxide haemangiopericytoma holoprosencephaly high-power field haematoxylin-phloxine-safranin human papillomavirus hypoxia response elements horseradish peroxidase hippocampal sclerosis hereditary systemic angiopathy hereditary sensory and autonomic neuropathy heat-shock protein; hereditary spastic paraplegia herpes simplex virus 5-hydroxytryptamine human telomerase reverse transcriptase human T-cell lymphotropic virus I hereditary vascular retinopathy inclusion body myositis internal carotid artery; internal cerebral artery intercellular adhesion molecule-1 I-cell disease; intracellular domain interleukin-converting enzyme intracerebral haematoma iatrogenic Creutzfeldt-Jakob disease intracranial pressure intradural haemorrhage intra-epidermal nerve fibre isolated familial somatotropinoma insulin-related growth factor immunoglobulin M immunohistochemistry incomplete hippocampal inversion

IIM IL-1β ILAE ILOCA ILS IMAM IMD IML IMNM IMT INAD INCL iNOS ION IPI iPSC IPSP IRD IRES IRIS IRS ISF ISPD ISSD ITPR-1 IUGR IVH JAK/STAT JAM JME JNCL JXG kb KO KPS KRS KS KSS LA LB LCH LCMV LDD LDL LEAT LFB LFB-CV LGI1 LGMD LGN

idiopathic inflammatory myopathy interleukin-1 beta International League Against Epilepsy idiopathic late-onset cerebellar ataxia isolated lissencephaly sequence inflammatory myopathy with abundant macrophages inherited myoclonus-dystonia inner molecular layer immune-mediated necrotizing myopathy inflammatory myofibroblastic tumour infantile neuroaxonal dystrophy infantile neuronal ceroid lipofuscinosis inducible nitric oxide synthase inferior olivary nucleus initial precipitating injury induced pluripotent stem cell inhibitory postsynaptic potential infantile Refsum’s disease internal ribosomal entry site immune reconstitution inflammatory syndrome insulin receptor substrate interstitial fluid isoprenoid synthase domain-containing infantile sialic acid storage disease inositol triphosphate receptor type 1 intrauterine growth restriction intraventricular haemorrhage Janus kinase and downstream signal transducer and activator of transcription junctional adhesion molecule juvenile myoclonic epilepsy juvenile neuronal ceroid lipofuscinosis juvenile xanthogranuloma kilobase knockout Karnofsky performance status Kufor‒Rakeb syndrome Korsakoff’s syndrome Kearns-Sayre syndrome lupus anticoagulant Lewy body Langerhans cell histiocytosis lymphocytic choriomeningitis virus Lhermitte-Duclos disease low-density lipoprotein long-term epilepsy-associated tumour Luxol fast blue Luxol fast blue-cresyl violet leucine-rich glioma-inactivated 1 limb-girdle muscular dystrophy lateral geniculate nucleus

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Abbreviations  XIX

LH LIF LINCL LMNA LNMP LOH LPH LRPN LSA L-SS LTD LTP MAG MAGE-A MAP MAPK MATPase MBD MBEN MBP MCA MCB MCD MCI MCM2 MCP-1 MDC1A MELAS MEN MEN2 MERRF MFN2 MFS MGUS MHC MHC-I MHV MIBE MJD ML MLC MLD MLI MM MMN MMP MMR MNCV

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luteinizing hormone leukaemia inhibitory factor late infantile neuronal ceroid lipofuscinosis lamin A/C last normal menstrual period loss of heterozygosity lipotropin lumbosacral radioplexus neuropathy lenticulostriate artery Lewis-Sumner syndrome long-term depression long-term potentiation myelin-associated glycoprotein melanoma-associated cancer-testis antigen microtubule-associated protein mitogen-activated protein kinase myofibrillar adenosine triphosphatase Marchiafava-Bignami disease medulloblastoma with extensive nodularity myelin basic protein middle cerebral artery membranous cytoplasmic body malformation of cortical development mild cognitive impairment minichromosome maintenance 2 monocyte chemoattractant protein 1 merosin-deficient CMD mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes multiple endocrine neoplasia multiple endocrine neoplasia type 2 myoclonic epilepsy with ragged-red fibres mitofusin 2 Miller Fisher syndrome; mossy fibre spouting monoclonal gammopathy of unknown significance myosin heavy chain major histocompatibility complex class I mouse hepatitis virus measles inclusion body encephalitis Machado-Joseph disease mucolipidosis myosin light chain metachromatic leukodystrophy mucolipidosis I methionine homozygosity multifocal motor neuropathy matrix metalloproteinase mismatch repair; measles-mumps-rubella motor nerve conduction velocity

MND MNGC MNGIE MnSOD MNU MOG MPNST MPO MPS MPT MPTP mPTS MPZ MR MRC MRI mRNA MRS MRT MS MSA MSA-C MSB MSD MSH MSI mtDNA MTI MTLE MTMR2 mTOR MTR MuSK MV MVE NAA NAD+ NADH-TR NAHI NALD NAM NARP NAT NAWM

motor neuron degeneration; mild ­ eurocognitive disorder; motor neuron n disease multinucleated giant cell mitochondrial neuro-gastrointestinal encephalomyopathy manganese-containing superoxide dismutase methylnitrosourea myelin-oligodendrocyte protein malignant peripheral nerve sheath tumour myeloperoxidase mucopolysaccharidosis mitochondrial permeability transition N-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine membrane peroxisomal targeting sequence myelin protein zero magnetic resonance Medical Research Council magnetic resonance imaging messenger ribonucleic acid magnetic resonance spectroscopy malignant rhabdoid tumour multiple sclerosis multiple system atrophy; myositis-specific autoantibody cerebellar form of multiple system atrophy Martius scarlet blue multiple sulphatase deficiency melanotropin microsatellite instability mitochondrial DNA magnetization transfer imaging mesial temporal lobe epilepsy myotubularin-related protein 2 mammalian target of rapamycin magnetization transfer ratio muscle-specific kinase valine heterozygous Murray Valley encephalitis N-acetylaspartate nicotinamide adenine dinucleotide nicotinamide adenine dinucleotidetetrazolium reductase non-accidental head injury neonatal adreno-leukodystrophy necrotizing autoimmune myopathy neuropathy, ataxia and retinitis pigmentosa non-accidental trauma normal-appearing white matter

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XX  Abbreviations

NBCCS NBIA NBIA1 NBIA2 NCAM NCI NCIPC NCL NCM NECD NF NF1 NF2 NFL NFP NFT NGF NHNN NIFID NIID NINDS NINDS-PSP NIRS NK NMDA NMDAR NMO nNOS NO NOS NOTCH3 NPC NPH NPY NSAID NSC NSE NTD NTE NTS OCT OEF O-FucT-1 OH OMIM

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naevoid basal cell carcinoma syndrome neurodegeneration with brain iron accumulation neurodegeneration with brain iron accumulation, type 1 neurodegeneration with brain iron accumulation, type 2 neural cell adhesion molecule neuronal cytoplasmic inclusion National Center for Injury Prevention and Control neuronal ceroid lipofuscinosis neurocutaneous melanosis notch extracellular domain neurofilament protein neurofibromatosis type 1 neurofibromatosis type 2 National Football League neurofilament protein neurofibrillary tangle nerve growth factor National Hospital for Neurology and Neurosurgery neuronal intermediate filament inclusion disease neuronal intranuclear inclusion disease National Institute of Neurological Disorders and Stroke National Institute of Neurological Disorders and Stroke and the Society for Progressive Supranuclear Palsy near-infrared spectroscopy natural killer N-methyl-D-aspartate N-methyl-D-aspartate receptor neuromyelitis optica neuronal nitric oxide synthase nitric oxide not otherwise specified notch homolog 3 Niemann-Pick disease type C normal pressure hydrocephalus neuropeptide Y non-steroidal anti-inflammatory drug neural stem cell neuron specific enolase neural tube defect neuropathy target esterase nucleus of the solitary tract optimal cutting temperature; optical coherence tomography oxygen extraction fraction O-fucosetransferase 1 hydroxyl radical Online Mendelian Inheritance in Man

OML OPC OPCA OPIDPN ORF PACNS PAFAH PAMP PAN p-ANCA PARK1 PAS PB PBD PBH PBP PC PCD PCNA PCNSL PCP PCR PCV PD PDC PDCD PDD PDGF PDGFB PDH PECAM PEM PEO PEP PERM PES PET PGNT PGP PHF PHP PhyH PI PiB PICA PKAN

outer molecular layer oligdendrocyte precursor cell olivopontocerebellar atrophy organophosphate-induced delayed polyneuropathy open reading frame primary angiitis of the central nervous system platelet activating factor acetyl hydrolase pathogen-associated molecular pattern polyarteritis nodosa; perchloric acid naphthoquinone perinuclear ANCA Parkinson’s disease and alpha-synuclein periodic acid‒Schiff pineoblastoma peroxisome biogenesis disorder parenchymal brain haemorrhage progressive bulbar palsy pineocytoma Purkinje cell degeneration proliferating cell nuclear antigen primary central nervous system lymphoma planar cell polarity polymerase chain reaction packed cell volume Parkinson’s disease; pars distalis parkinsonism/dementia complex programmed cell death Parkinson’s disease dementia platelet-derived growth factor platelet-derived growth factor beta pyruvate dehydrogenase platelet-endothelial cell adhesion molecule protein-energy malnutrition progressive external ophthalmoplegia postencephalitic parkinsonism progressive encephalomyelitis with rigidity and myoclonus pseudotumoural encephalic schistosomiasis paraffin-embedded tissue; positron emission tomography papillary glioneuronal tumour protein gene product paired helical filament pseudo-Hurler polydystrophy phytanoyl-CoA hydroxylase pars intermedia Pittsburgh compound B postero-inferior cerebellar artery pantothenate kinase-associated neurodegeneration

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Abbreviations  XXI

PKC PLA2G6 PLAN PLP PLS PMA PMCA PMD PME PML PMNS PMP PMP2 PMS PN PNDC PNET PNMA PNS pO2 POEMS POLG POMC PPA PPB PPCA ppm pPNET PPS PPT PPTID PR PRBC PRES PRL PRNP PROMM PROP-1 ProtCa ProtS PrP PrP-CAA PRR PSAP PSD PSIR

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protein kinase C phospholipase A2, group VI PLA2G6-associated neurodegeneration proteolipid protein primary lateral sclerosis pilomyxoid astrocytoma; progressive muscular atrophy protein misfolding cyclic amplification Pelizaeus-Merzbacher disease progressive myoclonic epilepsy progressive multifocal leukoencephalopathy post-malaria neurological syndrome peroxisomal membrane protein peripheral myelin protein 2 psammomatous melanotic schwannoma pars nervosa progressive neuronal degeneration of childhood with liver disease primitive neuroectodermal tumour paraneoplastic Ma antigen peripheral nervous system partial pressure of oxygen polyneuropathy, organomegaly, endocrinopathy, M-protein, skin changes polymerase γ proopiomelanocortin primary progressive aphasia familial pleuropulmonary blastoma protective protein with cathepsin A-like activity parts per million peripheral primitive neuroectodermal tumour pentosan polysulphate; post-polio syndrome pineal parenchymal tumour pineal parenchymal tumour of intermediate differentiation progesterone receptor parasitized red blood cell posterior reversible encephalopathy syndrome prolactin PrP gene proximal myotonic myopathy prophet of Pit-1 activated protein C protein S prion protein PrP-cerebral amyloid angiopathy pattern recognition receptor prosapson post-stroke dementia phase-sensitive inversion recovery

PSP PSP-CA PSP-CST PSP-P PSP-PAGF pSS PTAH PTC PTD ptd-FGFR4 PTLD PTPR PTRF PTS Ptx2 PVH/IVH PVL PWI PXA QuIC RALDH RANO RAR RARE RC2 RCA-1 rCBF rCBV RCC RCDP RDD RDP RE REM rhNGF RIG RIM RING RIP1 RIS RNI ROS RPLS RPS Rpx RRF

progressive supranuclear palsy progressive supranuclear palsy with ­cerebellar ataxia atypical progressive supranuclear palsy with corticospinal tract degeneration progressive supranuclear palsy with parkinsonism pure akinesia with gait freezing with subsequent development of typical signs of progressive supranuclear palsy primary Sjögren’s syndrome phosphotungstic acid haematoxylin periodic triphasic complex primary (idiopathic) torsion dystonia pituitary tumour-derived FGFR4 post-transplant lymphoproliferative disorder papillary tumour of the pineal region polymerase I and transcript release factor peroxisomal targeting signal pituitary homeobox factor 2 periventricular/intraventricular haemorrhage periventricular leukomalacia perfusion weighted imaging pleomorphic xanthoastrocytoma quaking induced conversion retinaldehyde dehydrogenase response assessment in neuro-oncology retinoic acid receptor retinoic acid response element reaction centre type 2 Ricinus communis agglutinin 1 regional cerebral blood flow regional cerebral blood volume; relative cerebral blood volume renal cell carcinoma rhizomelic chondrodysplasia punctata Rosai-Dorfman disease rapid onset dystonia-parkinsonism Rasmussen encephalitis rapid eye movement recombinant human nerve growth factor radiation-induced glioma radiation-induced meningioma Really Interesting New Gene receptor-interacting protein 1 radiologically isolated syndrome reactive nitrogen intermediate reactive oxygen species reversible posterior leukoencephalopathy syndrome rhabdoid predisposition syndrome Rathke’s pouch homeobox ragged-red fibre

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XXII  Abbreviations

RRMS RSMD1 RSV RTA RTK RVCL RXR SAH SANDO SAP Sap-A Sap-B Sap-C SAR SBF2 SBMA SBP SBS SCA SCAR1 SCD SCI sCJD SCLC SCMAS SCO SCS SDF-1 SDH SDS SE SEER SEGA SF-1 sFI SFT SFV Shh SIADH SIS SKL SLD SLE Sm SMA SMARD

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relapsing-remitting form of multiple sclerosis rigid spine muscular dystrophy type 1 Rous sarcoma virus road traffic accident receptor tyrosine kinase retinal vasculopathy with cerebral leukodystrophy retinoid X receptor subarachnoid haemorrhage sensory ataxic neuropathy, dysarthria and ophthalmoparesis serum amyloid P sapsosin-A sapsosin-B sapsosin-C specific absorption rate set binding factor 2 spinal and bulbar muscular atrophy systemic blood pressure shaken baby syndrome spinocerebellar ataxia spinocerebellar ataxia recessive type 1 subacute combined degeneration spinal cord injury sporadic Creutzfeldt-Jakob disease small cell lung cancer subunit c of mitochondrial ATP synthase subcommissural organ spinal cord schistosomiasis stromal cell-derived factor 1 subdural haematoma; succinate dehydrogenase Shy-Drager syndrome spin echo; status epilepticus Surveillance, Epidemiology and End Results subependymal giant cell astrocytoma steroidogenic factor-1 sporadic fatal insomnia solitary fibrous tumour Semliki forest virus Sonic hedgehog syndrome of inappropriate antidiuretic hormone secretion second impact syndrome serine-lysine-leucine sudanophilic (orthochromatic) leukodystrophy systemic lupus erythematosus; St. Louis encephalitis Smith spinal muscular atrophy spinal muscular atrophy with respiratory distress

SMC SMN SMNA SMTM SN SNAP SNARE SND SNP SNPC SNPR SO SOD SPECT SPLTLC1 SPS SRP SSPE SUDEP SVD SVZ SWI SYN TACE TAI TBI TBP TCGA TCI TCR TEF TGA TGF TGM6 THCA TIA TLE TLR TME TMEV TNF TOCP Topo II alpha TPNH TPP TS tSAH TSC TSE TSH

smooth muscle cell survival motor neuron sensorimotor neuropathy with ataxia sulcus medianus telecephali medii substantia nigra sensory nerve action potential soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex striatonigral degeneration single nucleotide polymorphism substantia nigra pars compacta substantia nigra pars reticulata slow-twitch oxidative superoxide dismutase single photon emission computed tomography serine-palmitoyltransferase 1 stiff-person syndrome signal recognition protein subacute sclerosing pan-encephalitis sudden unexpected death in epilepsy small vessel disease subventricular zone susceptibility-weighted imaging synaptophysin TNFα converting enzyme traumatic axonal injury traumatic brain injury TATA box-binding protein The Cancer Genome Atlas total contusion index T-cell receptor thyrotroph embryonic factor transposition of the great arteries transforming growth factor transglutaminase 6 trihydroxycholestanoic acid transient ischaemic attack temporal lobe epilepsy Toll-like receptor transmissible mink encephalopathy Theiler’s murine encephalomyelitis virus tumour necrosis factor triorthocresylphosphate topoisomerase II alpha triphosphopyridine nucleotide thiamine pyrophosphate Tourette’s syndrome; Turcot syndrome traumatic subarachnoid haemorrhage tuberous sclerosis complex transmissible spongiform encephalopathy thyrotrophin

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Abbreviations  XXIII

TSP TTF-1 TTP TTR UBO UCH-L1 uPA UPDRS UPR UPS UV VaD VCAM-1 VCI vCJD VCP VEE VEGF VEP VGKC VHL VLBW VLCFA VLDL

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tropical spastic paraparesis thyroid transcription factor 1 thrombotic thrombocytopenic purpura transthyretin unidentified bright object ubiquitin carboxy-terminal hydrolase urokinase plasminogen activator Unified Parkinson’s Disease Rating Scale unfolded protein response ubiquitin-proteasome system ultraviolet vascular dementia vascular cell adhesion molecule 1 vascular cognitive impairment variant Creutzfeldt-Jakob disease vasolin-containing protein Venezuelan equine encephalomyelitis vascular endothelial growth factor visual evoked potential voltage-gated potassium channel Von Hippel-Lindau very low birth weight very-long-chain fatty acid very low density lipoprotein

VLM VM VMB VPF VPSPr VSMC VV vWF VZ WBC WE WEE WHO WKS Wlds WM WNV WSM XMEA YAC ZASP ZPT ZS

visceral larva migrans vacuolar myelopathy vascular malformation of the brain vascular permeability factor variably protease sensitive prionopathy vascular smooth muscle cell valine homozygous von Willebrand factor ventricular zone white blood cell Wernicke’s encephalopathy western equine encephalitis World Health Organization Wernicke-Korsakoff syndrome wallerian degeneration slow white matter West Nile virus widely spaced myelin X-linked myopathy with excessive autophagy yeast artificial chromosome Z-line alternatively spliced PDZ protein zinc pyridinethione Zellweger syndrome

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1

Chapter

1

General Pathology of the Central Nervous System Harry V Vinters and B K Kleinschmidt-DeMasters

Neurons..........................................................................................1 Astrocytes.......................................................................................8 Oligodendrocytes..........................................................................21 Ependyma....................................................................................25 Choroid Plexus..............................................................................29 Microglia and Macrophages..........................................................29 Distinguishing Pathological Abnormalities from Artefacts and ­Incidentals....................................................................................33 Stem Cells in the Central Nervous System – Promise, Potential and Reality...................................................................................39

Neurons The neuron is the excitable cell type responsible for the reception of stimuli and information, and conduction of electro-chemical impulses in the brain, spinal cord and ganglia. Neurons are 10–50 times less numerous than their supporting cells, the neuroglial astrocytes, oligodendrocytes and ependymal cells,27 and are estimated to constitute only 5 per cent of the cells within the cerebral grey matter.56 Yet they are responsible for the most critical and complex (arguably defining) cellular functions of the organ. They also undergo the greatest number of microscopic changes in response to acute and chronic cell injury and are the principal site of damage for several of the diseases associated with the highest morbidity and mortality in our society, i.e. cerebrovascular and neurodegenerative diseases. The complex functions of the neuron are responsible for its high metabolic demand for glucose and oxygen/ blood supply and are also reflected in its specialized morphological features. Neurons possess a nucleus, nucleolus, cytoplasm and many of the same cytoplasmic organelles found in other cells in the body. However, their extreme protein synthetic and energy requirements, the extraordinary length of their cell processes, and the need for a complex cytoskeletal architecture to support these long cell processes mandate the need for some of these subcellular structures to be better developed than in cells elsewhere in the body, or even in their neighbours, the neuroglial cells. Under normal, non-injury conditions, usually only the nuclei and cell bodies of neurons are visible to the pathologist on routine histochemical stains used in daily practice, such as haematoxylin and eosin (H&E) or Luxol

Concept of the Blood–Brain Barrier and the Neurovascular Unit....44 Determinants of Intracranial Pressure and Pressure/Volume Relationships, and Causes and Consequences of Raised Intracranial Pressure.....................................................................46 Hydrocephalus – Pathophysiology, Causes and Consequences for the CNS...................................................................................51 Acknowledgements......................................................................54 References...................................................................................54

fast blue–H&E. Immunohistochemical stains commonly employed in routine neuropathology practice to identify proximal portions of neurons (the dendrites and/or soma) include primary antibodies to synaptophysin (a presynaptic vesicle protein), NeuN (a neuronal nuclear protein), microtubule-associated protein 2 (MAP-2), and some of the three polypeptide subunits of neurofilament, which constitutes the major cytoskeletal intermediate filament type for neurons. Low (NF-L, 68 kDa), medium (NF-M, 160kDa) and heavy (NF-H, 200 kDa) subunits exist within the neuron and selective antibodies have been developed over the past 20 years against each. Early work with antibodies directed against these various NF subunits showed no staining of neuronal perikarya and dendrites with antibodies directed against the heavy 200 kDa component.47 It was subsequently recognized that the antibody directed against NF-L recognized a component in the central core of neurofilaments, and the NF-H antibody a component of the interneurofilamentous cross-bridges; because neurofilaments in mammalian axons were extensively cross-linked, it was not surprising that axons immunostained best with the antibody directed against NF-H.97 Later work further showed that a lower ratio of NH-L to NH-M and NH-H was found in dendrites and that this proportion was essential for the shaping and growth of complex dendritic trees in motor neurons.127 Antibodies were also raised to phosphorylated (SM131, NE14) and non-phosphorylated (SM132) NF subtypes. Phosphorylation of NFs was correlated with abundance of NFs and bundling and cross-linking between NF core filaments.86 Anti-phosphorylated NF antibodies showed strongest immunostaining in axons where NFs are abundant and show this cross-linking, but not in dendrites 1

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2  Chapter 1  General Pathology of the Central Nervous System

and perikarya where NFs are sparse and are present singly.86Although there are variations among cell types and the distribution of NFs changes in disease states, a general principle is that antibodies directed against non-phosphorylated subunits of NF best stain the dendrites and perikarya of neurons, whereas those against phosphorylated NFs are used to highlight axons. Neuron specific enolase (NSE), despite its name, is unfortunately not specific for neurons but does also highlight the neuronal cell body.77 Among the many definitely non-neuronal entities that NSE stains, myeloma and lymphomas can be the most problematic for the diagnostic surgical neuropathologist.168 Specific subsets of neurons can be further identified by immunostaining for calretinin, galanin or any of the various specific neurotransmitters and neuromodulators that they produce ( -aminobutyric acid (GABA), glutamine, dopamine, acetylcholine, neuropeptide Y, etc.), but these techniques are almost exclusively employed in research rather than routine daily practice. Antibodies to markers of neuronal lineage have application both in the study of normal central nervous system (CNS) and peripheral nervous system (PNS) neurons and in assessing brain tumours of possible neuronal lineage/differentiation. Antibodies have been raised to -synuclein, a presynaptic nerve terminal protein found in normal neurons, and immunostaining for this has found widest application in the study of inclusion bodies in neurodegenerative disorders. However, -synuclein immunostaining has also been found in human brain tumours manifesting neuronal differentiation, such as ganglioglioma, medulloblastoma, neuroblastoma, primitive neuroectodermal tumours and central neurocytoma.117 The proportion of tumours immunopositive for -synuclein was reported to be lower than that labelled with more commonly used neuronal antibodies, including those to synaptophysin, MAP-2, NSE and tau, but higher than the proportion positive for neurofilament or chromogranin A.117 Other neuronal markers such as TrkA, TrkB, TrkC, the 1 subunit of the GABA receptor, N-methyl-D-aspartate receptor subunit 1, glutamate decarboxylase and embryonal neural cell adhesion molecule have also occasionally been utilized to detect putative neuronal lineage in human brain tumours.270 The full extent of the cell processes of neurons, termed neurites, cannot be discerned on H&E staining and is only fully appreciable with special stains. The neurites responsible for receiving synaptic information from other neurons and for afferent conduction of electrochemical impulses towards the cell body (soma) are termed dendrites. The full arborization pattern of dendrites is best visualized using Golgi staining techniques (a time-consuming process not usually available in non-research settings). The single elongate process responsible for efferent conduction of impulses away from the cell soma is the axon. Axons can be visualized using staining techniques widely available in most diagnostic laboratories, including the modified Bielschowsky and Bodian silver histochemical stains or immunohistochemical methods that target phosphorylated neurofilaments. The number, length and position on the neuronal cell body of the branching dendrites determine the shape and morphological classification of the neuron. Unipolar neurons possess a single cell process that divides a short distance from the cell body; an example is the dorsal root ganglion cell. Bipolar neurons have an elongate cell body

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with two cell processes emerging at either end of the cell soma; examples include retinal bipolar cells and cells of the sensory cochlear and vestibular ganglia. The vast majority of neurons are, however, multipolar, with large numbers of dendrites arranged in a radiating pattern around the entire cell body (motor neurons of the spinal cord), at the apex of a triangular cell body (pyramidal cell of cerebral cortex) or near the top of a flask-shaped cell (Purkinje cell neuron of cerebellum). Multipolar neurons can be further subdivided based on the length of their efferent axonal process. Golgi type II neurons, with a short axon that terminates near the cell body, greatly outnumber Golgi type I neurons. Golgi type I neurons possess a long axon that may be up to several feet in length in the case of some motor neurons, or less lengthy in the case of pyramidal cells of the cerebral cortex or Purkinje cells of the cerebellar cortex.224 The cross-sectional diameter of the neuronal cell body, by contrast, is largely determined by the length of the axon. The size of neuronal cell bodies varies greatly, from the small, 5 μm-diameter, granule cell neurons of the cerebellum to the large, 135 μm-diameter, anterior horn cells of the spinal cord.224 The volume of the neuronal soma parallels the length of the axon for which it is responsible: the longer the axon, the larger the cell body must be – specifically, the larger the cytoplasmic volume and organelle machinery must be to sustain that axon. Hence, Golgi type I neurons have larger amounts of cell cytoplasm that are readily visible even on H&E preparations, whereas Golgi type II neurons have scant cytoplasm that may give the neuron a ‘naked nucleus’ appearance on routine stains. Examples of the latter include the ‘lymphocyte-like’ granule cell neurons of the cerebellar cortex, which have a densely basophilic nucleus but in routine preparations appear to possess no cytoplasm, or the small interneurons of the cerebral cortex, which because of the paucity of their cytoplasm may be difficult to distinguish from neuroglial cells in H&E-stained sections. The neuronal nucleus is the repository for the chromosomes and in resting, non-mitotic conditions the chromatin is generally fairly evenly dispersed throughout the nucleus. The prominent large nucleolus seen especially in Golgi type I neurons is a reflection of the need for a high rate of protein synthesis to maintain the numerous proteins within the large cytoplasmic volume, determined largely by the length of axon. The nuclear membrane is well defined on routine H&E staining, but the double-layering of the membrane and the presence of fine nuclear pores, through which substances can diffuse into and out of the nucleus, is appreciable only on electron microscopy (EM). The nuclear pores are a conduit through which newly synthesized ribosomal subunits can pass from the nucleus into the cytoplasm. The cytoplasm contains both granular and non-granular endoplasmic reticulum. The granular, RNA-containing, endoplasmic reticulum extends throughout the cell body into the proximal parts of the dendrites; it is absent from the area of cytoplasm immediately adjacent to the axon, known as the axon hillock, and from the axon itself. Subcellular organelles of the neuron can be variably appreciated on H&E staining. Components that contain appreciable amounts of DNA (nucleus) or RNA (nucleolus and abundant cytoplasmic rough endoplasmic reticulum arranged in parallel arrays known as Nissl substance) in the cell have affinity for the haematoxylin dye used in

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routine histochemical staining. Therefore, the nucleus, nucleolus and Nissl substance of large neurons manifest a distinct blue-purple colouration and are readily visible on staining with H&E. The DNA- and RNA-containing structures within the neuron can be further highlighted by other histochemical staining techniques such as the modified Nissl method, which originally used aniline but has been modified to use toluidine blue, cresyl violet or others. The monochromatic Nissl staining method is often employed by investigators interested in morphometric analyses of neuronal populations in normal or diseased states. The Nissl stain is often used to highlight neuronal loss in chronic neurodegenerative disorders. The remaining, non-DNA or RNA containing organelles in the neuron, such as the mitochondria, Golgi complex, lysosomes, neurofilaments, microtubules and microfilaments, are individually unresolvable by H&E at the light microscopic level under normal conditions. These neuronal organelles blend together within the eosinophilic, pink cytoplasm in H&E-stained normal cells and can be appreciated only on EM. The complexity of the synapse is also appreciable only by EM. A number of antibodies directed to synaptic vesicle proteins have, however, been developed that can highlight the synapse and give an indication as to its function or dysfunction. The more common of these include synaptophysin, synaptobrevin (vesicle associated membrane protein, VAMP), synaptotagmin I and synaptic vesicle protein 2 (SV2). In order to function normally, neurons require complex membrane pumps to exclude toxic calcium ions and to maintain the correct balance between internal (intracellular) and external (microenvironmental) electrolyte concentrations of sodium and potassium in order to transmit electrical signals.56 With microenvironmental damage to neuronal membranes or with energy deprivation, these pumps fail, calcium ions flood the neuron and irreparable cell damage – known as necrosis – occurs. Local oxygen deprivation, such as that seen in stroke, may result in transient (recoverable) or permanent (irreparable, necrotic) injury to the neuron (see Chapter 2, Vascular Disease, Hypoxia and Related Conditions). This oxygen deprivation can affect cell energy requirements, membrane integrity, and/or the immediate surrounding microenvironment.56 Thus calciumchannel blocking ‘neuroprotectant’ agents used in the treatment of stroke may work not just at the level of the neuron alone but also on the microvessels and supportive glial cells around them, the so-called ‘neurovascular unit’.56 At the light microscopic level, acute sustained deprivation of energy (oxygen/blood supply or glucose), however, is best appreciated in the neuron itself. The irreparable cell damage can be visualized as the brightly eosinophilic ‘red (dead) neuron’ (Figure 1.1a). This change, seen most often with ischaemia, is manifested by cell shrinkage, nuclear pyknosis, loss of nucleolar detail and loss of basophilic cytoplasmic staining as a result of dissolution of granular endoplasmic reticulum. These result in a smaller, triangular cell, condensed nuclear chromatin, loss of the nucleolus and eosinophilia of the cytoplasm (Figure 1.1a). It should be emphasized that neurons may succumb within several minutes at normal body temperature to severe deprivation of oxygen. However, when body temperature is lowered, metabolism is slowed and considerably longer time periods

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without oxygen may be endured by the human brain, with relatively lesser amounts of irretrievable neuronal loss. This explains the remarkable recovery of some people immersed for an hour or more at the bottom of a cold lake who, when retrieved and resuscitated, are able to survive in a relatively cognitively intact state! It should also be emphasized that at normal body temperature, neurons are actually irreparably damaged within minutes when subjected to complete lack of oxygen, but to fully appreciate the ‘red cell change’ in these same cells under the microscope, at least 8 hours and optimally 18–24 hours must elapse after the injury event before these changes can be confidently diagnosed. The corollary to this is that if a patient dies soon after a cardiac arrest and the family and treating physician of the deceased want to know exactly how widespread the ischaemic neuronal injury was in the patient’s brain at autopsy, the pathologist reviewing the case will be unable to answer this question by using routine autopsy techniques. A spectrum of morphological changes (‘necrophanerosis’) evolves over variable time intervals prior to final (‘definite’) necrosis; these changes depend upon a variety of factors such as the rate and extent of blood (re-) perfusion, body temperature and others. Animal studies on ischaemic cell injury in neurons often avoid this problem by using rapid perfusion-fixation and EM to detect early, subtle organelle injury. During the acute phase, brain tissue surrounding a focus of ischaemic injury has an eosinophilic neuropil and exhibits significant vacuolation due to oedema (Figure 1.1a). This should not be mistaken for the spongiform change seen in transmissible spongiform encephalopathies. These changes are considered in detail in Chapter 2, Vascular Disease, Hypoxia and Related Conditions). When neurons undergo cell death and necrosis, no effective neuronal mitosis or replenishment of neurons from stem cells is present within the adult human brain: neuron(s) and their function(s) are lost to the host. Irreversibly damaged neurons are removed over the next few days by phagocytosing microglial cells and macrophages. Astrocytes begin to proliferate in response to injury and may leave a distinctive, tell-tale indication of where the now-removed neurons formerly resided. The classic example of this is Bergmann astrocytosis in the layer of cerebellar cortex where the Purkinje cell neurons formerly resided (Figure 1.1b). Occasionally, morphological evidence of sublethal cell injury in neurons can be detected, best typified by peripheral (Figure 1.1c, bottom) and central (Figure 1.1c, top) chromatolysis. Chromatolysis refers to the response to injury usually seen in Golgi I motor neurons in the anterior horn of the spinal cord when their long axonal process is transected or severely injured. Chromatolysis can be thought of as reorganization by the cell soma and redistribution of Nissl substance in an attempt to reconstitute the axon; central and peripheral chromatolysis may be different phases of this process. If the axonal injury is too severe or the axonal transaction too proximate to the cell body, the efforts of the cell body and its chromatolytic response will be insufficient to produce a new healthy axon and the neuron will itself eventually disappear. Sublethal injury to neurons may manifest not as eosinophilic change but by cell shrinkage and atrophy. This can occur in a variety of neurodegenerative disorders but is typified by neurons affected by trans-synaptic neuronal degeneration.

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4  Chapter 1  General Pathology of the Central Nervous System (a)

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1.1 Neuronal abnormalities in diseases of the CNS. (a) Red, dead neurons with loss of nucleoli and Nissl substance after cerebral ischaemia. Note the vacuolated, oedematous background neuropil. (b) Absence of Purkinje cell neurons and gliosis, but good preservation of granule cell neurons as a result of chronic ischaemic cerebellar injury. (c) Central (bottom) and peripheral (top) chromatolysis. Nissl stain. (d) and (e) Trans-synaptic degeneration in lateral geniculate nuclei; see text for detailed description.

Trans-synaptic degeneration occurs when a neuron loses the major source of its axonal input from connecting (incoming) fibres, usually as a result of the loss of ‘upstream’ neurons that give rise to these axons. A good example of this process is seen following enucleation of one eye. Axons from retinal ganglion cells synapse on neurons in the lateral geniculate nuclei. The example of trans-synaptic degeneration illustrated in Figure 1.1d is from an autopsy performed on a female who underwent right eye removal for retinal melanoma 6 years prior to death, with subsequent wallerian degeneration of the ipsilateral optic nerve and trans-synaptic degeneration in the lateral geniculate nuclei. Because of the differing patterns of projection of axons from the ipsilateral and contralateral eye, the left lateral geniculate ganglion showed atrophy of neurons in layers 1, 4 and 6, whereas the right showed atrophy of neurons in layers 2, 3 and 5. Note the bands of preserved large cells alternating with the bands containing severely

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shrunken neurons, making them nearly invisible at low magnification (Figure 1.1d). The atrophic neurons (lower left) are readily seen at higher magnification (Figure 1.1e) and contrast with the adjacent normal neurons from preserved layers (upper left). A special variant of trans-synaptic neuronal degeneration occurs when axons emanating from the inferior olivary nucleus (ION) are disrupted and their synaptic connections lost, or input to the ION is interrupted. Lesions in the ipsilateral central tegmental tract or the contralateral dentate nucleus result in unilateral olivary hypertrophy. In these instances, the neurons individually enlarge to the degree that they collectively produce hypertrophy of the entire nucleus, visible grossly or at low magnification (Figure 1.1f), and even on high resolution neuroimaging studies carried out while a patient is alive. The illustrated example originates from a man with multiple small cavitary

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1.1 (Continued ) Neuronal abnormalities in diseases of the CNS. (f) Bilateral olivary hypertrophy; this change on any given side is due to disruption of the ipsilateral t­egmental tract or contralateral dentate nucleus. Note the area of remote infarction (arrow) in the medulla. Nissl stain. (g) Vacuolation/­fenestration of neurons in inferior olivary nucleus in the example seen in panel (f). (h) Neuronal storage diseases cause accumulation of abnormal cytoplasmic material, evidenced by cytoplasmic bloating. Tay–Sach’s ­ isorders manifest as fine vacuolation in the cytoplasm. Hunter’s disease disease illustrated. (i) Neuronal alterations in some storage d ­illustrated. (j) Neuronal enlargement and calcification of blood vessels may occur after cranial irradiation; the latter change is much more common than the former. (k) Rare pituitary adenomas (lower part of photomicrograph) manifest neuronal metaplasia (upper part), the so-called mixed pituitary adenoma-gangliocytoma.

remote infarcts that were present in the medulla (arrow) and elsewhere in the brain stem and cerebellum and that disrupted these tracts on both sides. Note the bilateral inferior olivary nuclear enlargement (Figure 1.1f). Microscopically, neurons showed characteristic vacuolation (‘fenestration’) and enlargement, accompanied by considerable astrocytosis (Figure 1.1g). The reason for this special microscopic response to trans-synaptic degeneration in the inferior olivary nucleus is unknown but involves fragmentation within the Golgi apparatus and trans-Golgi network234 and redistribution of presynaptic vesicles, as manifested by an altered pattern of synaptophysin immunoreactivity.116 Less common reactions of neurons to injury include the accumulation of abnormal cytoplasmic storage material, such

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as in inherited, autosomal recessive storage disease disorders seen in childhood (see Chapter 6, Lysosomal Diseases). Two illustrated examples depict the neuronal changes seen in Tay– Sachs disease (Figure 1.1h) and Hunter’s disease (Figure 1.1i). Neuronal enlargement and gigantism may occur in the brain tissue adjacent to a tumour after cranial radiation therapy for a nearby neoplasm, and may be accompanied by other manifestations of tissue injury such as calcification (Figure 1.1j). Unlike many epithelial cell types, neurons rarely undergo metaplasia. In rare pituitary adenomas, most often of growth hormonesecreting type, adenoma cells (Figure 1.1k, lower portion) transform focally into neurons (Figure 1.1k, top);81 these cells, phenotypically identical to other neurons, may also express small amounts of pituitary hormones.

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6  Chapter 1  General Pathology of the Central Nervous System (a)

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1.2 Inclusion bodies and abnormal deposits I. (a) Bunina body in anterior horn cell in a patient with amyotrophic lateral sclerosis (motor neuron disease). The significance of these structures is discussed in detail elsewhere. (b) Buscaino bodies (mucocytes, metachromatic bodies) in white matter can occur secondary to poor tissue fixation and post-mortem degeneration of myelin. On H&E staining, these are barely visible as pale blue bodies or almost clear vacuoles; the periodic acid–Schiff stain, used here, demonstrates these bodies strikingly. (c) Colloid bodies (hyaline inclusions) are pale eosinophilic areas within the cytoplasm of neurons and correspond on electron microscopy to dilated cisternae of endoplasmic reticulum. Although usually seen in the hypoglossal nucleus (large picture), they may also be found in the anterior horn cells of the spinal cord (top inset) and very rarely in other neurons, such as the nuclei of Clarke’s column (bottom inset, lowest left). They are of no known pathological significance and should not be mistaken for pathological accumulations of proteins or chromatolytic change. (d) Cowdry A inclusion bodies are seen in herpetic viral infections of the nervous system (herpes simplex type I and II, cytomegalovirus infection, and varicella-zoster virus infection but not infections with Epstein–Barr virus). On electron microscopy, it can be appreciated that they are due to accumulations of virions within the nucleus of the host cell. Note the clearing of the host cell nuclear chromatin centrally, with margination of chromatin at the edge of the nuclear membrane and the ‘owl’s eye’ appearance of the viral inclusion. In this case of cytomegalovirus infection, the cell cytoplasm is also enlarged (cytomegaly) and distended by viral particles. (e) Eosinophilic granular bodies (EGBs) are dot-like, refractile, proteinaceous deposits most commonly encountered in the background neuropil in or adjacent to certain types of low grade brain tumours, as here in a pleomorphic xanthoastrocytoma. They can be further highlighted by periodic acid–Schiff staining. (f) Eosinophilic crystalline inclusions can occasionally be seen in the cytoplasm of neurons of the inferior ­olivary nucleus, especially in aged individuals and are of no known pathological significance.

Although necrosis is the type of neuronal cell death that predominates in acute energy-deprivation states, neuronal apoptosis plays a critical role during embryonic development. Apoptosis or programmed cell death refers to a controlled, coordinated biochemical process leading to the death

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of affected cells and is a physiological part of normal development. In a wide variety of disparate organisms, apoptosis involves the triggering of a series of biochemical events in which caspases (cysteine aspartases) play a key role.170 Although the morphological manifestations of apoptosis

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1.2 (Continued ) Inclusion bodies and abnormal deposits I. (g) Gamna–Gandy bodies are foci containing linear, bamboo-like fibrous tissue and collagen fibres encrusted with iron pigments and calcium salts. They were originally described in the spleen in patients with congestive splenomegaly but can be seen around cavernous angiomas, cholesterol granulomas of temporal bone, pituitary adenomas, and a variety of other highly vascular primary and metastatic neoplasms and cysts in the nervous system that are subject to recurrent bouts of haemorrhage. However, when first described in the 1920s, the authors had to go to great lengths to exclude a fungal causation for these structures, which are illustrated in a colour drawing from a 1922 article. (h) Gamna–Gandy bodies, illustrated in black and white drawings of from a 1929 article by Hu et al.;102 these authors showed that there was no morphological identity between the wavy encrusted fibres (left) or waxy septate, bamboo-like fibres (right) and true fungal mycelia. (i) Gamna–Gandy bodies in tissue from a region of recurrent brain haemorrhage. (j) Granulovacuolar degeneration (of Simchowicz, granulovacuolar bodies, GVBs) appear as tiny dots within clear vacuoles that can be seen particularly in the cytoplasm of pyramidal neurons of the hippocampal gyrus in normal ageing and, to a greater extent, in patients with Alzheimer’s disease. These structures contain abnormal accumulations of several proteins including tubulin, neurofilament proteins and tau. (k) Granular mitoses (top) are clusters of chromatin often encountered in cells in highly mitotically active tissues. Although usually found, and illustrated, in the context of acute demyelinating lesions, this example comes from a case of cytomegalovirus ventriculitis and should not be mistaken for a micro-organism. Herring bodies (bottom) are spherical or ovoid eosinophilic structures with an apparent surrounding membrane that are normal findings in the posterior pituitary gland (neurohypophysis). They represent normal storage sites within axons for oxytocin and vasopressin. (l) Hirano bodies are elongate (when longitudinally sectioned) to oval (in cross-section), brightly eosinophilic neuronal inclusions that are encountered in pyramidal neurons of the hippocampal gyrus in normal ageing, and, to a greater extent, in patients with neurodegenerative diseases such as Alzheimer’s disease. Although they often seem to be extraneuronal, by electron microscopy Hirano bodies can be seen to lie within the neuronal soma or cell processes. They are composed of actin and -actinin. (b) Reproduced with permission from Graeber MB, Blakemore WF, Kreutzberg GW. Cellular pathology of the central nervous system. In: Graham DI, Lantos PL (eds). Greenfield’s Neuropathology, 7th edn. London: Arnold, 2002, pp. 123–192. (h–j) From Kleinschmidt-Demasters, BK. Gamna–Gandy bodies in surgical neuropathology specimens: observations and a historical note. Journal of Neuropathology and Experimental Neurology 2004;63:106–12. Reproduced with permission from the Journal of Neuropathology and Experimental Neurology.

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are classically described as ‘cell shrinkage, membrane blebbing and nuclear DNA condensation and fragmentation’,146 these may not be seen in non-vertebrate systems.216 Neuronal apoptosis also occurs in pathological disease states and involves similar ‘execution systems’ and proteins.170 At least 14 different mammalian caspases have been identified thus far, but these may have both death-related and death-unrelated functions in the cell.170 Neuronal necrosis and apoptosis are not always mutually exclusive processes and the co-existence of both has been emphasized in some pathological conditions.136 For instance, a shift from apoptotic to necrotic types of neuronal death may occur when energy levels are rapidly compromised.136 The practical aspect of identifying a role for neuronal apoptosis in a disease process lies in the fact that small peptide caspase inhibitors have been developed and may have therapeutic utility. Caspase inhibitors may be useful in preserving sublethally injured neurons at the perimeter (penumbra) of an acute infarct that might be less severely affected by excitotoxic-­ ischaemic injury than is the necrotic core of the infarct.170 They may also act to protect against the deleterious effects of oxygen radicals, cytokines and lipid peroxidation products that are generated in the necrotic core of the infarct and seep out to the penumbra.139,146 Among human diseases, especially prominent neuronal apoptosis is seen in the (rare) perinatal disorder, pontosubicular necrosis. In neurodegenerative diseases, apoptosis may play differing roles at different time points during the disorder, explaining why caspase inhibitors may not be universally effective therapies. In addition, apoptosis can occur without involvement of the caspase system.24 A further consideration is whether or not the preservation of neurons that would otherwise undergo apoptosis is desirable in neurodegenerative disorders such as Huntington’s disease or Alzheimer’s disease, especially if the preserved cells have aberrant function.170 A role for neuronal apoptosis has been implicated in numerous disorders other than ischaemia and neurodegenerative disease; these include spinal cord trauma, head injury194 and viral nervous system infections.51 This complex topic has been the subject of several excellent reviews (e.g. Schulz and Nicotera,215 Nicotera et al.,170 Robertson et al.,194 Paulson,180 Mattson146). Although individual cellular organelles in neurons are not distinguishable under normal, resting conditions on light microscopy using routine stains, in ageing or in disease processes, massive accumulations of some organelles can be discerned. These processes result in the development of ‘inclusion bodies’.27,76,77 Some of these have limited pathogenic implications (colloid bodies, Marinesco bodies), although others are almost exclusively seen in specific disease conditions (Lafora bodies). Yet more are seen in small numbers in ‘normal’ ageing but in significantly greater numbers in specific neurodegenerative disorders (neurofibrillary tangles, granulovacuolar degeneration/bodies, Pick bodies). Still other ‘bodies’ occur in the background tissues but are discussed here with neuronal inclusion bodies because their exact intracellular (Hirano bodies, Figure 1.2l) or extracellular (Gamna–Gandy bodies, Figure 1.2g,h) location may not be apparent in H&E-stained sections. A

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pictorial, alphabetically arranged chronology of these ‘bodies’ – most of which develop in neurons – is depicted in Figures 1.2 and 1.3. Most of these are fully identifiable on H&E staining, including Bunina bodies, colloid bodies (Figure 1.2c), granulovacuolar bodies (Figure 1.2j), Lewy bodies (Figure 1.3c and d), neuroaxonal swellings, neurofibrillary tangles, Pick bodies (Figure 1.3j, insert) and Lafora bodies (Figure 1.3a). Special silver histochemical and immunohistochemical staining, however, can further delineate these normal and abnormal accumulations. Modified Bielschowsky or Bodian histochemical silver stains generically identify neurofilamentcontaining inclusions or structures in various diseases, such as globose or flame-shaped neurofibrillary tangles (Figure 1.3h,j), Pick bodies (Figure 1.3j) and neuroaxonal swellings, also known as ‘spheroids’ (Figure 1.3g). Identification of inclusions specific for certain neurodegenerative disorders can be achieved with immunohistochemical methods that identify tau (including its isoforms), ubiquitin, huntingtin or -synuclein. It is an unresolved issue as to whether neuronal inclusions play a role in direct neuronal injury or represent a mechanism by which neurons protect themselves by sequestering abnormal proteins (reviewed by Paulson180). Neurons are post-mitotic, fully differentiated cells that have little or no capacity to regenerate effectively and reconstitute functions lost when the cell is lost. Neuronal plasticity plays an important role in development and early childhood in overcoming major areas of brain tissue damage but this ability is lost in the adult brain, in which neurons cannot be innately regrown or replaced, even by the small numbers of neural stem cells that are known to be present (discussed later).

Astrocytes Astrocytes are, together with oligodendrocytes/oligodendroglia, the two cell types in the nervous system often described as macroglia, to distinguish them from microglia (see later). These specialized glial cells outnumber neurons by over five-fold.226 Generally considered to be, in part, the CNS counterpart of fibroblasts, with a significant role in producing scar tissue (described as ‘astrocytic gliosis’, ‘astrogliosis’ or simply ‘gliosis’) within the brain or spinal cord, astrocytes are now known to have myriad physiological and biochemical functions in both brain development and maintenance of homeostasis (especially with respect to the make-up of the interstitial fluid of the brain) and may even contribute to regeneration and repair after brain/spinal cord injury.255 Many of these properties will be described in detail later. Based upon recent discoveries in molecular neurobiology, the function(s) of astrocytes within normal brain and their relationship to neurons are being so radically redefined that even the nomenclature defining these cells (in relation to neurons) has been called into question. Changes from astroglial to neuronal phenotype (in select cell populations) are now well documented, although brain parenchyma in some lesions (e.g. malformations of cortical development associated with epilepsy) contains cells that have features of both a neuronal and astrocytic phenotype.254 As one expert in the field

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1.3 Inclusion bodies and abnormal deposits II. (a,b) Lafora bodies are basophilic inclusions that are composed of polyglucosans and occur in Lafora’s disease, a neurodegenerative storage disease of children. These inclusions occur in many different types of cell and tissue, including neurons, choroid plexus, sweat glands, peripheral nerves, cardiac and striated muscle, and liver and skin. They closely resemble corpora amylacea and, like corpora amylacea, stain intensely with periodic acid–Schiff, but are usually surrounded by a corona of radiating filaments or spicules and are not restricted to the sites of predilection for corpora amylacea. In addition, corpora amylacea are infrequent in children. These figures illustrate Lafora bodies in the cerebellum. (c) Lewy bodies (brain stem type) are intracyoplasmic inclusions that represent abnormal proteinaceous accumulations consisting predominantly of -synuclein. Like many proteinaceous deposits, they are readily visualized in H&E-stained sections. They are easiest to identify in pigmented neurons, such as this one from the substantia nigra compacta, where they displace the normal intracytoplasmic, brown neuromelanin pigment. Note the targetoid appearance; however, most are not so eye-catching. Lewy bodies can be encountered in the substantia nigra compacta and especially in the locus coeruleus in normal ageing, but even in this instance may represent preclinical disease. They are more numerous and more widely distributed in patients with idiopathic Parkinson’s disease and related disorders. (d) Lewy bodies (intracortical type), when located in small neurons of the cerebral cortex, are far less well-defined in H&E-stained sections but can be highlighted by immunostaining for -synuclein or ubiquitin. Cortical Lewy bodies are usually associated with disease, not normal ageing. (e) Marinesco bodies, sometimes referred to as ‘maraschino cherry bodies’ by residents trying to remember the names of all of the various bodies for board examinations, are intranuclear eosinophilic bodies (arrow), about the same size as the nucleolus. They are largely confined to the pigmented, neuromelanin-containing neurons of the substantia nigra compacta and are usually found in aged individuals. They are proteinaceous inclusions of no known pathological significance, but are very similar to the intranuclear bodies seen in large numbers of neurons in patients with the childhood degenerative disorder, neuronal intranuclear inclusion disease. (f) Negri bodies are a pathognomonic finding in rabies viral infection (rabies viral encephalitis) of the central nervous system. These well-circumscribed intracytoplasmic, red cell-like bodies are easily overlooked, particularly when the virus fails to elicit an inflammatory host reaction. Continued

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10  Chapter 1  General Pathology of the Central Nervous System (g)

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1.3 (Continued ) Inclusion bodies and abnormal deposits II. (g) Neuroaxonal swellings (spheroids) are round or ovoid structures formed when transportation of intra-axonal neurofilaments is disrupted by axonal injury or transection. Although also discernible in sections stained with H&E they are better highlighted with silver stains, as here. They are illustrated here in the anterior horn of a patient with short-duration amyotrophic lateral sclerosis. (h) Neurofibrillary tangles (flame-shaped) are easily recognized by even novice pathologists by their flame-shaped profiles, demonstrated best with silver stains. The classical shape usually illustrated in textbooks is the one seen here in a pyramidal neuron of the cerebral cortex, and the intracytoplasmic location of the tangle, which loops around the (unstained) nucleus, is easily appreciated. Scattered tangles may be encountered in pyramidal neurons of the hippocampal gyrus in normal ageing, but they are seen in greater numbers and in a wider neocortical and brain stem distribution in patients with neurodegenerative diseases such as Alzheimer’s disease. (i) Neurofibrillary tangles (globose) contain skein-like tangles of abnormal, hyperphosphorylated tau protein and may be seen in brain stem neurons in Alzheimer’s disease or in progressive supranuclear palsy; the latter disease is illustrated here. The shape of the tangle is predicated on the shape of the neuronal cell body in which it resides. The coarse internal structure of the globose tangle distinguishes it from argentophilic Pick bodies seen in (j). Pick body-like structure associated with neurodegenerative disease. Bodian silver stain. (j) Pick bodies are intracytoplasmic bodies found in the pyramidal neurons of the hippocampal gyrus, the granule cell neurons of the dentate gyrus, smaller cortical neurons especially in layer 2, and in brain stem neurons of patients with Pick’s disease, a neurodegenerative disease associated with lobar atrophy of the frontal and temporal lobes. They have a relatively homogeneous appearance on both H&E (inset) and silver staining, in contrast to globose neurofibrillary tangles, but sometimes a degree of overlap exists. Unlike neurofibrillary tangles, Hirano bodies or granulovacuolar bodies (degeneration), Pick bodies are almost never encountered in normal aged individuals. (k) Polyglucosan bodies are histologically indistinguishable from the corpora amylacea but occur in very large numbers in individuals affected by adult polyglucosan body disease,23 illustrated here in a section of white matter from a middle-aged patient with this disorder. The variably blue-grey bodies may have a concentric, targetoid appearance (inset). Corpora amylacea are a normal finding in aged individuals, but not in so great a number, and are usually more concentrated in (but not confined to) subpial, subependymal and perivascular locations and the spinal cord. In polyglucosan body disease, heart, skeletal muscle, liver, and dermal sweat glands in addition to peripheral nerves and brain may contain these bodies. They are composed largely of sulphated polysaccharides (polyglucosans) and stain deeply with haematoxylin, periodic acid–Schiff and methyl violet. By electron microscopy, polyglucosan bodies in the nervous system are seen to consist of densely packed 6–7 nm filaments that are not bounded by a unit membrane and lie within astrocytic processes, within axons and few within the neuropil, but not within the neuronal soma.

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  Astrocytes  11

has boldly and bluntly stated, ‘… virtually every aspect of brain development and function involves a neuron–glial partnership. It is no longer tenable to consider glia as passive support cells’.17 By morphological criteria, astrocytes have been subclassified as protoplasmic (found mainly within the grey matter) or fibrous/fibrillary (located predominantly within the subcortical white matter).226 The phenotype of astrocytes is defined by the location within their cytoplasm of the intermediate filament protein, glial fibrillary acidic protein (GFAP).60,64,255 Though not all astrocytes express GFAP that is immunohistochemically detectable within the cytoplasm by light microscopy (and some non-CNS

cells do), the presence of this protein essentially remains, in daily diagnostic work, a defining feature of the cell type. GFAP is especially abundant within the cytoplasm of reactive or hypertrophic (and often neoplastic) astrocytes, though unfortunately the extent and robustness of GFAP immunoreactivity do not correlate well with the specific type or duration of CNS insult to which the astrocytes have reacted (Figures 1.4 and 1.5). GFAP immunohistochemistry has become the standard way to assess astrocytic gliosis (both qualitatively and quantitatively) in both animal studies and human CNS disease tissue examined at biopsy or autopsy. It has superseded older classic cytochemical stains such as the Holzer and phosphotungstic acid haematoxylin

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1.4 Astrocytes, reactive and neoplastic. (a) Section shows relatively evenly distributed reactive astrocytes, some (arrows) with glassy eosinophilic cytoplasm. (b) Reactive astrocytes in a region of prominent cortical vacuolation from a patient with Creutzfeldt–Jakob disease (spongiform encephalopathy). (c) Relatively hypocellular region of an infiltrating astrocytoma. Arrow indicates a plump gemistocytic astrocyte. (d,e) Photomicrographs from two different examples of gemistocytic astrocytoma, both of high grade. Note that the majority of cells in each specimen have the appearance of gemistocytes.

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12  Chapter 1  General Pathology of the Central Nervous System (a)

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1.5 Reactive astrocytic gliosis, highlighted in each specimen by GFAP (immunoperoxidase) immunohistochemistry. (a,b) Diffuse, moderately intense astrocytic gliosis, with evenly distributed strongly GFAP-immunoreactive stellate cells. (c) Section of cingulate cortex (at top) and corpus callosum (at bottom). Note prominent astrocytic gliosis in both regions, though astrocytes in the corpus callosum have less prominently stellate cytoplasm. (d) Transgenic mouse model of A amyloid deposition. Stellate astrocytes (arrow) show focal aggregation, often around amyloid deposits within cortex. (e) Slightly dysmorphic, and occasionally binucleate (arrow), astrocytes in a cortical dysplasia specimen (corticectomy for epilepsy in a child).

(PTAH) techniques, although the latter stains retain value in some settings. Vimentin and S100 are also prominent components of the astrocytic cytoplasm, though vimentin immunoreactivity in astroglial cells lacks specificity, as this epitope is expressed in many non-glial cell types. By electron microscopy, astrocytes contain abundant intermediate filaments, cytoplasmic dense bodies, gap junctions and multiple cellular processes.64,255 Astrocytes may also express a variety of growth factor receptors, including those for epidermal growth factor and basic fibroblast growth factor.60,93

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Prominent cytoplasmic GFAP immunoreactivity also characterizes neoplastic astrocytes within astrocytomas, especially gemistocytic astrocytomas, and other types of tumour-related astrocytes, e.g. the mini-gemistocytes commonly found in oligodendrogliomas (Figure 1.6) (see Chapter 26, Introduction to Tumours). The term ‘gemistocyte/gemistocytic’ used to describe an astrocyte does not, however, – ­ gemistocytes classify it as being malignant or reactive  are also common in brain tissue surrounding infarcts, vascular malformations, traumatic lesions, cerebritis/encephalitis and metastatic neoplasms, as well as in numerous other

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  Astrocytes  13 (a)

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1.6 (a–c) A predominantly oligodendroglial neoplasm (oligodendroglioma; micrographs are at various magnifications) contains n ­ umerous GFAP-immunoreactive astrocytic cells, including mini-gemistocytes. Note the different morphology of tumour cells (round, regular nuclei ­ idespread with clear cytoplasm) and the more characteristically stellate appearance of the astrocytic element. By contrast, note the w GFAP-immunoreactivity of tumour cells and their processes (d) in a predominantly astrocytic tumour (astrocytoma).

reactive settings. GFAP immunoreactive cells may even be encountered within the interstices of a metastatic neoplasm (Figure 1.7), leading to diagnostic difficulty in distinguishing an anaplastic primary glioma from a poorly differentiated metastasis. The recent discovery of mutations in the active site of isocitrate dehydrogenase (IDH1) gene in >70 per cent of intermediate-grade diffuse gliomas, i.e. diffuse astrocytomas grade II, oligodendrogliomas grade II, mixed oligoastrocytomas grade II and anaplastic variants grade III,15  and the finding that a high-fidelity antibody correlates well with a specific mutational (R132H) status33,34 have provided the pathologist with a truly tumour-specific glial marker that can be used in daily diagnostic practice. Capper et al. demonstrated that the antibody can be used to distinguish diffuse glioma from non-neoplastic reactive gliosis associated with metastases, vascular malformations, abscesses, progressive multifocal leukoencephalopathy, and ischaemic or haemorrhagic lesions (Figure 1.8).35 In addition, they showed that the IDH1 antibody was superior to p53 or Wilms Tumor 1 (WT1) antibodies in identifying neoplastic glial cells.35

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Astrogliosis is sometimes subclassified (on purely morphologic grounds) as being isomorphic (when astrocytes arrange themselves along an anatomical structure such as a tract, e.g. the corticospinal tract, in association with wallerian degeneration) or anisomorphic (cells arranged more haphazardly, as at the edges of an infarct or cerebritis/ abscess; see Figure 1.9).64 Brisk reactive astrocytic gliosis can also be associated with the proliferation of Rosenthal fibres, which represent protein aggregates in astrocytic processes that also contain ubiquitin, B-crystallin and heat shock protein HSP27 (Figure 1.10). Dominant missense mutations in the human GFAP gene are associated with a leukodystrophy (Alexander’s disease) characterized by overwhelming proliferation of Rosenthal fibres within the diseased white matter.137,159,245 The GFAP gene on chromosome 17 includes four -helical segments within the central rod domain, joined by non-helical linkers. Of interest, GFAP-null mice show relatively subtle neuropathological abnormalities, although animals that overexpress GFAP 10–15-fold manifest a fatal encephalopathy associated with prominent astrocytic swelling.159

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14  Chapter 1  General Pathology of the Central Nervous System (a)

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1.7 Both panels show GFAP-immunoreactive reactive astrocytes in an atypical teratoid rhabdoid tumour (AT/RT). Note ramified processes of the reactive astrocytes throughout the tumour.

Role of Astrocytes in CNS Development and Regeneration Experimental evidence now suggests remarkable plasticity and regenerative potential for at least some populations of astrocytes and astrocyte precursors, a view that would have been somewhat heretical as recently as 20–30 years ago.58 Since the late 1800s, radial glia have been recognized as key players in brain development. Their elongated fibres span the full width of the developing cerebral wall in most mammals. In the cerebellum, radial glia extend from the pia to the Purkinje cell layer, and are quite regularly and evenly spaced in the molecular layer (Figure 1.11). These cells, at least in the cerebrum, retain the capacity to divide. An increasingly complex understanding of their role in CNS development has coincided with more sophisticated ways to study this unique cell type.187 In the late stages of cortical development, radial glia appear to divide asymmetrically in the ventricular zone to generate (more) radial glia and intermediate progenitor (IP) cells. IP cells then divide symmetrically in the subventricular zone to give rise to multiple neurons.144 During development of the brain, radial glia (which provide the ‘guidewires’ by which neuroblasts in the germinal matrix find their way to the cerebral cortex) are thought to give rise to astrocytes.211,257 Adult astrocytes may revert to their radial glial phenotype (in tissue culture) when exposed to embryonic brain extracts.103 However, it is now clear that they can themselves also function as neural progenitor cells.84 The molecular developmental and neurobiological events in this process are extraordinarily complex, and are well reviewed elsewhere.8,89,129,158,162,226,241 Astrocytes, in addition to giving rise to new neurons in the adult hippocampus,218 are now recognized as a major component of ‘neurogenic niches’, which have the potential to generate neuroepithelial cells from the subventricular zone during early brain development and possibly also at later time point.8 Increased generation of neuronal progenitor cells after ischaemic stroke has even been demonstrated in human autopsy brain specimens originating from quite elderly individuals.143 Astrocytes secrete molecules that may support neurogenesis (fibroblast growth factor/FGF,

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insulin-like growth factor-1, glutamate, etc.) or inhibit it (astrocyte-derived bone morphogenetic protein). In the developing mammalian brain, the subventricular zone (SVZ), a germinal region of the brain, contains abundant astrocytes and astrocyte precursors, together with migrating neuroblasts, undifferentiated immature precursors and ependymal elements. In experimental animals, it has been shown that SVZ astrocytes can divide to generate neuroblasts and immature neuronal precursors, and that such astrocytes placed into tissue culture can grow into multipotent neurospheres.58 Many astrocytes may have a latent neurogenic potential that is suppressed by various inhibitory signals, or expressed only in certain well-defined anatomic locations, e.g. the subventricular zone surrounding the lateral ventricles. Experiments utilizing transgenic targeted cell fate mapping strategies have also shown that morphologically distinct GFAP-positive progenitor cells may represent the major source of cells that are key to constitutive adult neurogenesis in the adult mouse forebrain; in experimental systems, astrocytes appear to have important neuroprotective functions.80,225 Whereas astrocytic gliosis has historically been thought to inhibit axonal regeneration, experiments in rats have shown that reactive astrocytes may in fact act as a permissive substrate for axon outgrowth from neurons sensitive to (implanted) nerve growth factor (NGF) within the brain.115 Sometimes contradictory experimental data continue to fuel the debate as to whether astrocytic scarring or some cellular components overexpressed during that process of scar formation inhibits CNS regeneration. Axonal sprouting in rats is increased in lesioned septohippocampal circuits in parallel with accentuated GFAP immunoreactivity, suggesting that astrocytes may produce trophic factors (e.g. nerve growth factor and related molecules) that facilitate this reparative response.78 Yet in knockout mice that are rendered deficient in both GFAP and vimentin, improved anatomical regeneration, axonal plasticity and functional recovery have clearly been observed in lesioned spinal cords.153 Organotypic slice culture experiments have demonstrated that astroglial-associated fibronectin may play a significant role in axonal regeneration within the white matter.240

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  Astrocytes  15 (a)

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1.8 IDH1 immunohistochemistry is negative in the numerous non-neoplastic reactive astrocytes (arrows) intimately admixed with the neoplastic lymphocytes in this primary CNS lymphoma, as seen on H&E (a,b, high power), GFAP (c), and IDH1 immunohistochemistry (d). In contrast, IDH1 immunoreactivity distinguishes individually-infiltrating tumour cells (e) from reactive astrocytes (arrows) on the edge of a glial neoplasm, as well as large numbers of tumour cells in the centre of this anaplastic oligodendroglioma (f).

Trophic Effects and Influence of ­Astrocytes on Vascular Structure, Integrity and ­Physiology The physical proximity of astrocytes and their processes to CNS microvasculature (Figure 1.12), together with the obvious neuroanatomical observation that cerebral blood vessels are ‘swimming in’ a sea of astrocytes, intuitively suggests that astrocytes and molecules released by them

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influence microvascular structure and function. Astrocytes may be instrumental in subdividing brain segments into microdomains, thus defining the functional architecture of the CNS through ‘gliovascular units’.167 The observation of this intimate neuroanatomical association between glia and blood vessels was first noted by Golgi over 120 years ago.222 A modular organization has even been proposed to define the association of cerebral microvessels, neurons and astrocytes, which are now described as forming

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16  Chapter 1  General Pathology of the Central Nervous System (a)

1.9 An example of anisomorphous/anisomorphic gliosis is seen in the lining of a cystic cavity that occupies most of this gyrus, sampled at necropsy from an infant with severe perinatal brain damage. The centre of the cavity contained irregular clumps of glial fibres admixed with numerous foamy macrophages.

functional ‘neurovascular units’ (NVUs).3 Other elements of the NVU include pericytes (in the case of capillaries) and medial vascular smooth muscle cells (SMCs) in the case of arterioles. Astrocytes are a key link in these units, because they communicate with both synapses and blood vessels, as well as with other astrocytes (via gap junctions and through the release of ATP).126 They appear to act as crucial intermediaries in intercellular signalling in this putative neurovascular unit. The role of astrocytes in mediating many physiological and biochemical functions of the cerebral capillary endothelium, site of the blood–brain barrier (BBB, see later), has been established by elegant tissue grafting and transplantation, as well as cell culture experiments (for reviews, see Pardridge;174 Nag;163 Ballabh et al.13). Co-culture studies (first carried out in the mid1980s, when cerebral capillary isolation techniques became routine) have been performed in which brain-derived capillary endothelial cells252 are seeded on one side of a porous mesh separating two fluid-filled chambers, another cell type (astrocytes, pericytes, etc.) on its other side. Such protocols were used extensively to demonstrate the inductive effect of astrocytes on both structural and physiologic properties of the BBB, e.g. its well-known ‘polarity’ for transport of certain molecules.19,163,174 Though the morphological site of the BBB is widely accepted as being cerebral capillary endothelium (see later), its physiologic functions and integrity are affected by both adjacent pericytes and astrocytes in the NVU.3,13,163 The tight junction proteins that mediate many BBB functions (see also discussion later) are expressed very early in human CNS development within the germinal matrix, cerebral cortex and subcortical white matter.14 Proteins known to be crucial for calcium signalling between cells (purinergic receptors and gap junctions Cx43) are expressed mainly by perivascular astrocyte end-feet that are an invariable finding on the abluminal aspects of CNS blood vessels, both capillaries and larger arteries. Brain slice experiments show that electrical field stimulations cause an increase in astrocytic calcium, which is transmitted to perivascular end-feet, resulting in arteriolar smooth muscle cell oscillations and

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1.10 Rosenthal fibres and astroglia. This is an unusual corticectomy specimen from a child with intractable epilepsy. (a) Note numerous hyaline rod-like Rosenthal fibres aggregated at the pial surface (arrows) and in the underlying cortex. (b) Two gemistocyte-like cells (arrows), including a binucleated astrocyte (at right) are seen amid numerous Rosenthal fibres.

dilatation of these vessels.126 Molecules that may mediate communications between astrocytic end-feet and vascular smooth muscle cells include prostaglandins, epoxyeicosatrienoic acids (EETs), potassium ions and arachidonic acid. Astrocyte-mediated control of cerebral blood flow also occurs through the action of calcium transients in astrocytic end-feet.161,235 Increased blood flow that is coupled to neuronal activity (and is thus used as an indirect measure of brain activity by techniques such as functional magnetic resonance imaging [fMRI]) is modulated in part by cyclooxygenase-2 metabolites, EETs, adenosine and NO derived from neurons. Neuronal activation that results in increased astrocytic calcium is partly mediated by activation of metabotropic glutamate receptors (mGluRs). In tissue culture systems, calcium signalling may be influenced by adenosine and EETs that are produced by astrocytes. Astrocytes are even able to transmit signals to brain surface (pial) arterioles to ensure their continuous adequate supply of blood to parenchymal arterioles.

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  Astrocytes  17 (a)

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1.11 Radial glia in the cerebellum (all panels represent micrographs from GFAP-immunostained sections). (a,b) Rat cerebellum. Note GFAP-immunoreactive processes that extend from the Purkinje cell layer to the pial surface throughout the specimen, best seen at higher magnification in (b). (c) Fragment of human cerebellum adjacent to a surgically resected lesion shows similar radial glia, though in a slightly more disorganized arrangement.

Astrocytes are also a key element in the regulation of water movement into and out of the brain through the BBB (also see later). An important molecule in this physiological regulation is aquaporin-4 (AQP4), the major water channel expressed within CNS perivascular astrocytic foot processes.169 In normal circumstances, AQP4 activity is associated with osmotically induced water efflux, probably through functional linkage to ion/solute channels. In experimental animals with reduced AQP4, reduction of osmotic water efflux causes astrocytic foot processes to become swollen. In the setting of water influx to the CNS (with induced brain oedema), astrocytic foot processes swell more in wild-type animals than in AQP4-knockout (KO) mice.

Physiology, Metabolism and ­Neurochemistry of Astrocytes Astrocytes play several roles in maintaining neurochemical homeostasis within the CNS. One important way by which this occurs is through the regulation of glutamate levels in the extracellular fluid.6 Astrocytes have been described as a ‘ready source (for)

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glutamate on demand’.147 Glutamate functions as the major CNS excitatory neurotransmitter and can also act as a potent neurotoxin – so much so that glutamate toxicity has been implicated (with varying degrees of supporting evidence) as a key pathogenetic factor in diseases as different as ischaemic stroke, amyotrophic lateral sclerosis (motor neuron disease) and epilepsy. Brain extracellular glutamate is normally present at a concentration of approximately 2 μM, whereas cytosolic concentrations are in the much higher range of 1–10 mM, depending upon the cell type. Glutamate can be transported by a variety of CNS cell types, including neurons, astrocytes and even endothelia, but uptake of this neurotransmitter by astrocytes is considered quantitatively the most important. Glutamate uptake into astrocytes is mediated by both Na+-dependent and Na+-independent systems, the latter characteristically chloride-dependent glutamate/ cystine antiporters, sensitive to quisqualate inhibition. The Na+-dependent glutamate transporters are termed EAAT1 and EAAT2. Because of the huge concentration gradient against which glutamate moves to gain access to the cytosol, significant brain energy is expended in moving glutamate from extracellular fluid into cells – this is estimated to be

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18  Chapter 1  General Pathology of the Central Nervous System

1.12 A close anatomic association frequently exists between astrocytes and capillaries. Arrows indicate a cerebral capillary that contains many astrocytic foot processes on its abluminal aspect. GFAP-immunostained section.

greater than 1 ATP per molecule of glutamate transported.6 There is debate as to whether this ATP is generated by astrocytic glycolysis – tissue culture experiments suggest that this is not the case. When glutamate transporters analogous to EAAT1 and EAAT2 (GLAST and GLT-1) are ‘knocked-out’ in vivo (in rats) by use of antisense oligonucleotides, severe neurological abnormalities (e.g. paralysis) ensues in affected animals, probably the result of neurodegeneration related to excitotoxicity caused by glutamate.198 Intracellular (astrocytic) glutamate ‘handling’ may occur in several ways, though glutamine formation and its release into the extracellular space (where it may be taken up by neurons) or entry into the tricarboxylic acid cycle appear to be the most important of these. Glutamate uptake can be modulated by alterations in transporter activity and/or expression, and the transporter activity is in turn governed by both thermodynamic and kinetic factors, a detailed consideration of which is beyond the scope of this chapter. Glutamate uptake kinetics are influenced by signalling molecules (including other neurotransmitters).82 Even amyloid precursor protein (APP), a molecule that is central to the pathogenesis of Alzheimer’s disease16 and often used as a marker of axonal injury,10 can affect astrocytic glutamate levels. In tissue culture systems, APP increases glutamate uptake by a process that is sensitive to both protein kinase A and C inhibitors. Astrocytes may even participate in the determination of synaptic structure and function – synapses throughout the CNS show varying degrees of ensheathment by astrocytic processes. Astrocytic processes are more prominently distributed near synapses with a greater likelihood of

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showing ‘glutamate escape’. Glutamate released by synapses follows one (or more) of three subsequent pathways: it can (1) diffuse further between synapses, (2) be cleared by neuronal glutamate receptors, or (3) be cleared by transporters on astrocytes or their processes. The importance of each of these mechanisms varies in different regions of the CNS, determined to some extent by the degree of synaptic ensheathment by astrocytes. It appears that astrocytes are much more important synaptic glutamate sinks than are neurons.6 Of course, ‘what comes into cells … may also go out’! Glutamate release from astrocytes occurs through the process of ‘transport reversal’, through anion channels activated by cell swelling or through gap junction hemichannels.271 In the adverse circumstance of ATP ­depletion (e.g. during irreversible, severe cerebral ischaemia), the key membrane gradients keeping glutamate within astrocytes disappear, causing ‘flooding’ of the extracellular (including synaptic) spaces by this potentially neurotoxic molecule. Glutamate efflux may also occur in less dire circumstances, e.g. in response to certain signalling mediators and processes. Calcium-dependent glutamate release can occur in response to bradykinin, some prostaglandins and even extracellular ATP.6 Glutamate release may also take place as a consequence of cytoplasmic swelling; the astrocyte responds to this by the ‘expulsion’ of chloride and glutamate (among other molecules) through volume-sensitive organic osmolyte-anion channels (VSOACs). The delicate balance of astrocytic modulation of glutamate levels has practical implications in our understanding of, for example, the neurobiology of traumatic brain injury (TBI). Glutamate neurotoxicity can greatly exacerbate the secondary CNS damage that occurs after TBI. Unfortunately, one of the consequences of TBI may be downregulation of the very receptors (EAAT1, EAAT2) that can potentially ameliorate glutamate-mediated injury. Clinical intervention (to minimize secondary injury after TBI) using glutamate ­ receptor antagonists has also been largely unsuccessful as a ­preventative strategy.272 Tissue co-culture experiments utilizing retinal ganglion cells and neuroglia (including astrocytes) suggest that developing neurons in vitro form inefficient, largely inactive synapses that only become fully and vigorously functional when exposed to glial signals.183 Glia may even control the number of mature synapses.243 Neuronal stimulation may trigger electrophysiological and/or calcium responses in cultured astrocytes or experimental brain slices. In addition to glutamatergic pathways already described, NO-mediated signalling may also occur. Neuron-to-astrocyte signalling can activate subcellular compartments, the entire cell, or it can activate a multicellular astrocytic response in the form of a ‘calcium wave’ – a phenomenon that may also occur, somewhat surprisingly, in pure cultures of astrocytes.20,210 Water and ion homeostasis by astrocytes is achieved partly through hormonal mechanisms, viz. the varying effects of vasopressin (AVP), atrial natriuretic peptide (atriopeptin), angiotensinogen (AGT) and angiotensin (Ang) II on astroglial water and chloride uptake, which in turn is linked to their intrinsic osmoregulation.221 Astrocytic swelling of a pronounced degree appears to be a key element in the cerebral oedema seen in patients who experience fulminant hepatic failure. This ‘cellular

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  Astrocytes  19

oedema’ has numerous negative consequences for the CNS, including a failure of astrocytes to take up neurotransmitters, reduction in size of the extracellular space leading to abnormally elevated extracellular ion concentrations, and even vascular compromise through compression of the microvasculature. A neuropathologic correlate of hepatic encephalopathy is the presence of characteristic Alzheimer type II astrocytes, identified predominantly within cortical grey matter and deep central grey structures (Figure 1.13).64,171 This condition may be potentially severe enough to cause fatal internal herniation of brain tissue and results in severe metabolic encephalopathy.172 Its proximate cause appears to be hyperammonaemia. Various lines of evidence suggest that the pathogenesis of ammonia-induced astrocytic swelling involves oxidative stress, induction of the mitochondrial permeability transition (MPT, associated with a sudden increase in permeability of the inner mitochondrial membrane to small molecules), and intracellular accumulation of glutamine, which then act as an intracellular osmolyte. It has been hypothesized that glutamine induces both oxidative stress and the MPT. Further metabolic linkage between neurons and astrocytes may result from their utilization of specific energy substrates. Astrocytes have a prominent capacity for aerobic glycolysis and production of lactate even in the presence of normal oxygen levels. Glucose is the major energy substrate within adult CNS, but lactate and ketone bodies may serve as alternative energy substrates in prolonged starvation, diabetes mellitus or during hypoglycaemia.181 In the course of normal brain function, approximately 90–95 per cent of brain energy consumption is attributed to neurons, only 5–10 per cent to glia (especially astrocytes). Recent data suggest that glial cells may function as ‘nursing partners’ for neurons, releasing a metabolic intermediate from glucose that can be taken up and oxidized by neurons. It has also been claimed that astrocytes ‘sense’ synaptic activity at glutamatergic synapses (see earlier) and metabolize glucose into lactate that can be passed to neurons. Energy transfer from astrocytes to axons may also occur (during aglycaemic conditions) through the degradation of astrocytic glycogen to lactate, the latter molecule then being

1.13 Alzheimer type II astrocytes. These are easily appreciated in H&E-stained sections, as in this section through the basal ganglia. Arrows indicate two such cells, characterized by enlarged clear nuclei, often with tiny eccentric nucleoli.

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used by neurons as a supplementary energy source.28 Highresolution imaging methods (e.g. two-photon fluorescence imaging of nicotinamide adenine dinucleotide) can now be used to study metabolic interactions between neurons and astrocytes at the single cell level.112 For a thorough discussion of the physiological and biophysical aspects of neuronal and astrocytic metabolism in the course of afferent and efferent neural activity in the brain, see Gjedde et al.82

1

Pathological Reactions and Role in ­Neurologic Disease Neurons and astrocytes (the latter vastly outnumbering the former),226 once thought to act independently in CNS development and response to injury, are now known to be tightly linked in both processes, with well-defined cell-to-cell contacts between the two cellular elements in many regions of the CNS. Until the early 1990s, there was a widespread tendency to view reactive astroglia as generic cells with uniform biological properties regardless of their location within the CNS. This approach has been largely superseded by an appreciation of their significant functional and regional heterogeneity throughout the brain and spinal cord.93 A second assumption was that, in the face of CNS injury, supportive astroglial cells become transformed into elements that actively inhibit axonal regrowth – the classic ‘glial scar’ that poses a major barrier to CNS regeneration after brain or spinal cord injury. A third major assumption about astrocytes was that brain or spinal cord injury led directly to glial proliferation (‘astrocytic gliosis’) and associated scarring. All of these assumptions have recently been questioned or re-evaluated. In experimental models, CNS injury of only certain types (e.g. that caused by physical tearing or laceration, as often occurs in traumatic brain and spinal cord injury) reliably induces gliosis. As well, the gliosis may be regionally accentuated; furthermore, there may be an increase in the GFAP content of individual astrocytes (astroglial hypertrophy) without an actual increase in their number (hyperplasia). Neurons may substantially regulate astroglial proliferation and differentiation. Experiments using cultured cerebellar granule cells (admittedly a highly specialized type of neuron but one fairly easy to maintain in vitro) and astroglia further highlight potential neuronal–glial interactions in the response to injury. When postnatal astroglia were grown in the absence of neurons, they expressed low levels of GFAP and grew rapidly. When granule cell neurons were added to the cultures (especially at a ratio of at least 4:1), DNA synthesis (in the astrocytes) decreased substantially and GFAP protein expression increased.93 GFAP appears to be necessary but not sufficient for the formation of astroglial processes in the presence of neurons. As indicated earlier, astrocytes may support axonal growth; their degree of differentiation (rather than chronological age) may be a key determinant of glial support of axonal growth. Astroglia are also capable of expressing extracellular matrix molecules (such as laminin, heparan sulphate) and cell adhesion receptor systems – these and other molecules may play a role in axonal guidance following injury. The function (and malfunction) of both microglia (see later) and astrocytes is inextricably linked to an understanding of cytokines, low molecular weight (MW) glycoproteins that may be secreted or function as membrane-bound

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20  Chapter 1  General Pathology of the Central Nervous System

complexes.110,171 These molecules exert their actions in a paracrine or autocrine fashion. They interact with specific cell-surface receptors, most cytokines having the capability of acting as ligands for several different receptors. The biological properties of a given cytokine are determined by the receptor that is activated, more than the cytokine itself. Astrocytes both produce cytokines and are, under a variety of physiological and pathophysiological circumstances, highly responsive to them. Properties of cytokines and their receptors have been elucidated through the use of elegant (usually transgenic) animal models and tissue culture experiments. In these studies, genes specific for cytokines and/or their receptors are deleted or overexpressed, often in specific cell types or anatomically defined populations, allowing for a detailed dissection of their myriad effects on the CNS (for a detailed review, see John et al.110). Although many of these experimental studies looking at the effects of cytokines on neural cells are illuminating from the perspectives of cellular neurobiology, their relevance for understanding complex neurologic diseases is not always clear. Reactive gliosis, a non-specific but highly characteristic response to almost any type of CNS injury, can be thought of as resulting from astrocytic proliferation (hyperplasia) and enlargement (hypertrophy), both associated with distinctive patterns of gene expression. As indicated earlier, reactive gliosis can be either a positive process that results in neuroglial survival or a negative one causing diminution in neuroglial growth, migration or both processes. Targeted ablation of reactive astrocytes can cause both increased neuronal degeneration and, simultaneously, an increase in neuritic outgrowth, together with accentuated chronic inflammation and a delay in post-injury re-establishment of BBB integrity.32 Interleukin-1 (IL-1 ), tumour necrosis factor- (TNF ), IFN and transforming growth factor- 1 (TGF 1) have all been implicated as players in the initiation or modulation of reactive gliosis. Furthermore, astrocytes possess receptors for all four of these cytokines, each apparently having a different role in the astrocytic response to injury, and each response in turn is mediated by specific gene expression patterns. IL-1 , especially, appears to have a pro-inflammatory role in CNS disease, but may also be involved in CNS regeneration,110 some of these effects being modulated through ciliary neurotrophic factor (CNTF) or nerve growth factor (NGF). In vitro experiments suggest that IL-1 induces genes that are key elements in the acute or subacute immune response and include other cytokines, chemokines and several adhesion molecules. By contrast with IL-1 , IFN (produced in abundance by activated lymphocytes) appears to potentiate (rather than initiate) astrocytic gliosis, possibly through the induction of MHC class I and II molecules and chemokines, and the potentiation of IL-1 -induced expression of TNF and nitric oxide synthase (NOS) (inducible NOS type II can be expressed by astrocytes). Complex interactions among infiltrating lymphocytes, microglia and astrocytes determine the microenvironment in which the CNS functions or malfunctions. NOS II may be downregulated in astrocytes via a complex cascade that involves both microglia and IL-4 (produced by TH2 lymphocytes) acting upon TH1 lymphocytes to modify their synthesis of IFN . Both IL-1 and IFN may thus be of importance in, for example, the pathogenesis and progression of multiple sclerosis (and its experimental animal

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model, experimental allergic encephalomyelitis, EAE), in which lymphocytic infiltration into spinal cord or brain is an integral part of the neuropathologic picture.64,255 TGF 1, expressed in both astrocytes and microglia in the context of brain trauma, infarction or inflammation, is itself an apparent stimulant of astrocytic gliosis. Inhibiting TGF 1 activity prevents formation of a glial membrane at the site of CNS injury and downregulates production of extracellular matrix molecules, e.g. fibronectin and laminin. Other effects of TGF 1 include inhibition of astrocytic expression of MHC class II molecules, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and TNF , as well as the induction of numerous molecules important in CNS wound healing (fibronectin, tenascin, collagen, laminin, actin and actin depolymerizing factor among many others). TNF itself is synthesized by astrocytes, may stimulate astrocytic gliosis but may also have a part to play in CNS repair after injury. It can also be cytotoxic to both oligodendroglia and neurons. These somewhat paradoxical effects are probably affected via the distinctive signalling pathways mediated by two TNF receptors, TNFRI/p55 and TNFRII/p75; the former appears to be linked to cell death, the latter to cell viability and growth. IL-6 activates many signalling cascades, and houses the signalling receptor gp130, which is activated through the Jak/Stat pathway. In the CNS, IL-6 promotes neuronal survival and neurite outgrowth, may impact cell-fate decisions (e.g. progression of stem cells to neurogenesis versus gliogenesis), and has an immunomodulatory function in the highly complex glial cytokine network – ­abnormalities of which may result (under some circumstances) in CNS inflammation and neurodegeneration. Complex and potentially confusing as all of these interactions may appear to be, they appear to become even more so with every passing week! Summaries of the interactions among microglial and astrocyte-secreted factors are to be found in recent reviews of these cell types.226,268 Immune responses mediated through cytokines, chemokines and lymphokines, especially involving cell surface receptors, are especially important in understanding the evolution of viral infections of the CNS, especially those that impact on neurons.39 Astrocytic gliosis plays a part in virtually all neurological diseases and neuroanatomical lesions – whether they be degenerative, traumatic, metabolic, neoplastic, inflammatory or of any other aetiology. Astrocytes are commonly found in microscopic lesions as disparate as the neuritic plaques of Alzheimer disease,205 the demyelinating plaques of multiple sclerosis, and foci of viral encephalitis that have little to do with plaques! Recently, it has been suggested that astrocytes play a major role in the generation of epileptic seizures through their modulation of glutamate and calcium signalling.238 The potential roles of astrocytes in specific entities are further considered in the chapters dealing with these diseases. The molecular and cellular basis of astrogliosis itself has been the topic of substantial debate.60 Reactive gliosis appears to vary quantitatively and qualitatively – the nature of the glial response being determined by both the nature of the lesion/injury and the microenvironment in which it occurs.193 Astrogliosis is recognized by the apparent proliferation and hypertrophy of GFAP-expressing astrocytes. However, by definition this excludes a consideration (within a given lesion) of any reactive astrocytes that fail to express

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  Oligodendrocytes  21

GFAP. Furthermore, because of their apparently minimal proliferative and migratory potential, it has been suggested that reactive astrocytes (in regions of acute/subacute neural injury) may simply represent a change in the phenotype of locally residing astrocytes. The transition of resting astrocytes to activated cells is associated with the expression of new molecules not normally expressed (in the resting state), as well as the upregulation of molecules that are expressed at low levels in the resting state (for a catalogue of these molecules, see Eddleston and Mucke;60 Ridet et al.193). One of the myths to be banished in recent years is that astrocytic processes (in glial scars) are a major impediment to axonal regeneration, e.g. after traumatic spinal cord injury or stroke.93 Axons appear to grow quite happily on astrocytic scars; it has therefore been postulated that, because scars are in fact a complex admixture of various cell types, extracellular matrix components and other elements, some combination of non-astrocytic components may actually impede axonal growth after a spinal cord injury or cerebral infarct. Despite this, strategies aimed at re-establishing spinal cord function are frequently aimed at bypassing the scarred and gliotic site of cord injury, which is still perceived by many investigators as a significant barrier to axonal regeneration.30,220 Nevertheless, one strategy in the treatment of experimental spinal cord contusional or transection injury is to acutely transplant glial-restricted precursor cells (which have the potential to differentiate into oligodendroglia and astrocytes) into the lesion.50,96 When reactive astrocytes were selectively ablated from a region of spinal cord injury in a novel transgenic mouse model, the absence of astrocytes caused failure of BBB repair, leukocyte infiltration, severe demyelination with local tissue disruption, and oligodendroglial/neuronal death, resulting in pronounced neurologic deficits in experimental animals.67,225 One obvious conclusion from this work was that reactive astrocytes have important neuroprotective functions that could be harnessed in post-injury repair of neural tissue. Given the central role of astrocytes in brain energetics, water and ion homeostasis, vascular regulation, and genesis of the neurovascular unit, discussed elsewhere in this chapter, astrocytes would appear to be an attractive cell population to target in new therapeutic approaches in neuroprotection (for review, see Nedergaard and Dirnagl166). As one example, sustained astrocytic expression of the glycoprotein clusterin significantly improved brain remodelling after ischaemia in mice.106 Genetically modified astrocytes may be a way to deliver therapeutic factors into lesioned (whether artificially or by nature) portions of brain or spinal cord.182 A comprehensive review of the complex biological and pathological features of astrocytes has recently been published.226

Oligodendrocytes Oligodendrocytes are neuroglial cells with small cell bodies, few (Greek: oligos, little, few) short cell processes, and no cytoplasmic filaments. They are found in grey matter, where they cluster around neuronal cell bodies (Figure 1.14a, arrow) and are seen in the pencil fibres of white matter that course through the putamen (Figure 1.14a, asterisks). In compact regions of white matter they are often arranged in rows between myelinated fibres (Figure 1.14b), and in

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cortex often lie adjacent to neurons. The functions of oligodendrocytes are likely to be different in the grey and white matter. In the grey matter, the standard explanation for the role of oligodendrocytes that encircle neurons is that they play sustentative roles for neurons, analogous to the role of satellite or capsular Schwann cells in dorsal or peripheral sensory ganglia, or that they represent progenitor cells.27 In central nervous system white matter, oligodendrocytes are the cell type responsible for myelin formation and are thus analogous to Schwann cells in the peripheral nervous system. Oligodendrocytes must undergo a series of complex series of steps, from proliferation, migration, differentiation, to myelination before they finally are capable of producing an ensheathment of axons.26 Oligodendrocytes are among the most vulnerable cells to injury in the CNS.26 A large recent review addresses the development of oligodendrocytes, particularly in rodents26 and several of these discoveries in rodents are likely analogous in human oligodendroglial development as well. New insights into oligodendroglial development include the fact that: (1) there appears to be a common progenitor cell origin for neurons and oligodendrocytes; (2) a ventral-to-dorsal progression occurs in oligodendroglial development; (3) there are multiple origins for oligodendrocytes and (4) there is an interrelationship between axonal signalling and myelination (reviewed in Bradl and Lassmann26). Both CNS and PNS myelin can be histochemically stained with a number of different stains, the most commonly used of which is the Luxol fast blue (LFB) stain with which CNS myelin appears in paraffin sections as a slightly vacuolated robin’s egg-blue substance surrounding the axon (Figure 1.14c). In autopsy tissues, LFB may be suboptimal or yield patchy, variably intense staining; therefore, immunohistochemistry incorporating primary antibodies to, for example, myelin basic protein may better demonstrate myelinated fibres. The tinctorial properties differ slightly for PNS myelin, which appears darker blue than CNS myelin on being stained with Luxol fast blue-periodic acid Schiff (LFB-PAS). This can be easily seen at a transition zone between oligodendrocyte-mediated CNS myelin and Schwann cell-mediated PNS myelin where cranial nerves or spinal nerves exit the CNS (Figure 1.14d, 5th c­ranial nerve illustrated). Additional differences exist between ­ oligodendrocytes and Schwann cells. Schwann cells are ­ surrounded by basement membrane and have a single cell process responsible for ensheathing only a single myelin segment lying between two nodes of Ranvier, whereas the oligodendrocyte is devoid of basement membrane and its several cell processes can each form several internodal segments of myelin. Schwann cells have a more limited responsibility, with one cell responsible for one internodal segment on one axon, which they spirally enwrap to produce myelin. Oligodendrocytes, in contrast, may form as many as 60 internodal segments.224 In both the CNS and PNS, myelin serves similar functions, allowing increased speed of conduction of impulses, which propagate by saltation from node to node along myelinated axons; on loss of myelin, conduction slows or ceases. Myelin integrity requires both its formation and subsequent maintenance. Myelin formation begins at about the 16th week of intrauterine life224 and continues throughout childhood. Although myelin formation is most rapid during

1

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22  Chapter 1  General Pathology of the Central Nervous System (a)

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1.14 (a) Normal oligodendrocytes in the putamen, where they cluster around neuronal cell bodies (arrow) and are also seen in the white matter pencil fibres (asterisks). (b) In white matter, oligodendrocytes are arranged in rows between myelinated fibres. (c) Luxol fast blue-periodic acid Schiff (LFB-PAS) stain for myelin in normal white matter. (d) As demonstrated using the same histochemical LFB-PAS stain, the tinctoral properties differ for peripheral nervous system (PNS) myelin. PNS myelin appears darker blue, as seen in the illustrated transition zone between oligodendrocyte-mediated CNS myelin and Schwann cell-mediated PNS myelin, in the 5th cranial nerve root entry zone. (e) Edge of an active demyelinating plaque in multiple sclerosis (MS) shows oligodendrocyte proliferation, evidenced as a band of hypercellularity between the severely demyelinated zone (upper left) and less demyelinated edge of plaque (lower right). LFB-PAS. (f) Shadow plaque (upper left) represents an area of partial remyelination. LFB-PAS.

the first 2 years of life, diffusion tensor imaging studies suggest that myelination occurs well into the second decade.123 Maintenance of myelin demands integrity of the oligodendrocyte cell body, high energy expenditure by the cell and, should the myelin be lost, effective remyelination by oligodendrocyte precursors. Loss of, or damage to, the cell body of the oligodendrocyte almost inevitably leads to loss of the myelin produced and supported by that cell’s processes. Damage to axons in myelinated fibres leads to breakdown of the surrounding myelin sheath. Conversely, the oligodendrocyte is also important for axonal support and maintenance.61 In H&E-stained sections, the oligodendrocyte is seen as a round nucleus with evenly dispersed, dark chromatin, no

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nucleolus, and no visible cytoplasm (Figure 1.14a and b). In poorly-fixed tissues, most oligodendrocytes manifest an artefactual perinuclear clearing around the nucleus, the so-called ‘perinuclear halo’, leading to a ‘fried-egg’ appearance (Figure 1.14b). These perinuclear haloes are often not apparent in well-fixed small surgical biopsy specimens; hence identification of an oligodendrocyte – ­normal or neoplastic – cannot rest solely on the presence or absence of the ‘halo’. The processes and cytoplasm of the oligodendrocyte cannot be discerned without special histochemical stains or EM. A number of antibodies have been used immunohistochemically to identify oligodendrocytes, such as Leu-7 (CD57), anti-myelin associated glycoprotein (MAG),

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  Oligodendrocytes  23 (g)

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1.14 (Continued ) (g) Centre of old, inactive MS plaque is hypocellular and contains almost no myelin or oligodendrocytes. (h) Photomicrograph illustrates normal myelin and axon content for comparison with panel (g). (i) Oligodendrocyte nuclei with characteristic glassy, violaceous inclusion bodies in progressive multifocal leukoencephalopathy. (j) Oligodendrocyte nuclei bearing viral inclusion bodies, in subacute sclerosing panencephalitis. (k) Oligodendrocytes in multiple system atrophy contain cytoplasmic, linear, pointed, densely eosinophilic, glial inclusions, in affected regions of brain. (l) The glial cytoplasmic inclusions in multiple system atrophy are better highlighted by modified Bielschowsky silver impregnation (note that this also stains the axons).

anti-myelin oligodendrocyte protein (MOG), anti-CD44 (a cell surface glycoprotein)25 and anti-OLIG1 transcription factor.11 Several of these markers have now been recognized to be present in oligodendrocytes in differing stages of development, as reviewed by Bradl and Lassmann.26 On paraformaldehyde-fixed brain tissue, differentiated oligodendrocytes express carbonic anhydrase II, 2 :3 -cyclic nucleotide 3 -phosphodiesterase (CNP), galactosylceramide (GalC), Kir4.1 (inwardly rectifying K+ channel subunit), myelin basic protein (MBP), MAG, MOG and proteolipid protein (PLP), although myelinating oligodendrocytes express RIP and TPPP/p25 and oligodendrocyte precursor cells (OPC) express CNP, OLIG2, NG2 and O4.26 Although OLIG2 especially is being used in daily practice, it is not specific for gliomas of oligodendroglial lineage, and other markers are not part of

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the standard armamentarium of surgical neuropathologists. Hence the question, ‘What is an oligodendrocyte?’ or the corollary, ‘What is an oligodendroglioma?’31 remains a vexing one when we do not have a universally accepted immunohistochemical marker for these cells. Compounding the problem have been in vivo studies that suggest neuron-like physiological properties in cells cultured from human oligodendroglial tumours,178 as well as immunohistochemical studies demonstrating staining of oligodendroglial tumour cells for markers traditionally associated only with neurons, such as NF-H,52 N-methyl-D-aspartate receptor subunit 1 or embryonal neural cell adhesion molecule.270 The repertoire of responses to injury available to the oligodendrocyte is limited. Proliferation of oligodendrocyte precursor cells has been documented in a number of

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24  Chapter 1  General Pathology of the Central Nervous System

different disease processes including radiation injury,10 vanishing white matter disease246 and multiple sclerosis (MS) but, at least in the first two conditions, may be counterbalanced by neuronal apoptosis. Oligodendrocyte proliferation from precursor cells is most confidently identified by the pathologist at the edge of an active demyelinating plaque in multiple sclerosis (MS), as a band of hypercellularity (Figure 1.14e). This precursor cell proliferation may produce a region of partially effective remyelination, known as the ‘shadow plaque’. This appears as an area of partial, hazy myelin staining (reflecting inadequately thin sheaths) that is intermediate in intensity between normal-appearing white matter with its intense robin’s-egg blue colour on Luxol fast blue stain for myelin, and the unstained, totally demyelinated parts of a plaque (Figure 1.14f). Unfortunately, remyelination seems to be transient, because it disappears in older lesions.74 Although remyelination initially occurs as a result of recruitment of oligodendrocyte precursor cells, these may progressively become depleted,145 may become quiescent or may respond to axonal inhibitory signals and cease the myelination process.142 Whether or not oligodendrocyte apoptosis occurs in MS is still debated.16,74 In any case, the centre of an old, inactive MS plaque does not evince effective remyelination, is quite hypocellular and contains naked axons and chronic gliosis but virtually no myelin or oligodendrocyte nuclei (Figure 1.14g). The extent of this hypocellularity and oligodendrocyte loss can be best appreciated when the centre of the plaque is compared side by side with the oligodendrocyte content of normal subgyral white matter (Figure 1.14h). In many other diseases in which oligodendrocytes and myelin are lost in the CNS, no phase of oligodendrocyte proliferation has been confidently identified. In these situations, oligodendrocytes are simply injured and destroyed. A number of different viruses can infect oligodendrocytes and cause lytic infections, with loss of cell bodies and their dependent myelin sheaths. Prior to cell lysis, if sufficiently large clusters of viral particles accumulate, they may be seen in the cell nucleus as viral inclusion bodies, even on routine H&E staining. The associated margination of the normal nuclear chromatin underlines the fact that the nuclear machinery has been ‘commandeered’ by the virus for its own purposes. The characteristic glassy, violaceous inclusion bodies in progressive multifocal leukoencephalopathy (Figure 1.14i) are composed of myriad virions that fill the entire nucleus; much less frequently these form a condensed, ‘owl’s eye’ viral inclusion. Subacute sclerosing panencephalitis similarly produces relatively homogeneous viral inclusions that fill the nucleus and are associated with a margination of nuclear chromatin (Figure 1.14j). Less dramatic oligodendrocyte changes accompany almost all other disorders that damage myelin. Indeed, by the time these disorders, termed ‘leukoencephalopathies’, are encountered by the pathologist, hypocellularity, and a reduction in the number of oligodendrocyte nuclei and amount of myelin with a corresponding increase in the white matter water-to-myelin ratio, are the non-specific findings. Leukoencephalopathy is the term usually applied to non-inherited white matter damage (usually maximal in cerebral hemispheric white matter) that is a result of toxic, metabolic or ischaemic processes, with the alternate term

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‘leukoaraiosis’ also applied to chronic ischaemic white matter injury. Examples of toxic substances that cause oligodendrocyte and myelin loss include: chronic substance usage (ethyl alcohol, ‘ecstasy’, and toluene, i.e. ‘glue sniffing’), ciclosporin (US spelling cyclosporine), therapeutic radiation, and chemotherapeutic agents (carmustine and methotrexate) (Figure 1.15).70 The latter two categories of agents also affect blood vessels and produce white matter damage via ischaemic mechanisms. Oligodendrocytes are the second most vulnerable cell, after neurons, to anoxic-ischaemic central nervous system injury. In any acute deprivation of regional blood supply to grey and white matter, i.e. stroke, oligodendrocytes are lost, along with the neurons and virtually any other tissue components in the epicentre of the lesion. Significant, widespread, acute or chronic damage specifically to white matter may also occur, because of the vulnerability of oligodendrocytes to ischaemic injury. Deprivation of oxygen/ blood supply to the white matter is especially likely to affect the cerebral hemispheres. Acute hypoxic-ischaemic injury to white matter may be accompanied by a haemorrhagic component and is known as hypoxic-ischaemic leukoencephalopathy. Chronic hypoxic-ischaemic injury to white matter is often maximal in boundary zones (watershed territories) of arterial distribution in the cerebral white matter between the middle and posterior cerebral arteries and the middle and anterior cerebral arteries. It tends also to be maximal in the regions of white matter midway between the ventricular and pial surfaces, not in periventricular regions. Chronic hypoxic-ischaemic injury to white matter may result from either severe arteriolosclerosis of deep white matter blood vessels and hypoperfusion (leukoaraiosis, causing Binswanger’s disease in the most severe form) or an inherited, autosomal dominant disorder, CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). This ischaemic injury to oligodendrocytes has usually been considered to be predominantly necrotic. However, recent studies of mild hypoxic/ischaemic insults occurring in the perinatal time period have suggested that O4+ oligodendrocyte precursor cells are particularly vulnerable to injury and die by apoptotic rather than necrotic mechanisms.199 Ischaemic injury to the white matter is considered in more detail elsewhere in this text. Inherited, usually autosomal recessive, storage diseases may cause oligodendrocyte injury and loss as a result of the accumulation of abnormal storage material within the cell cytoplasm. This type of white matter injury is designated a leukodystrophy (to reflect the intrinsic dysfunction in oligodendrocyte biology in these disorders, as opposed to the acquired injury to previously normal oligodendrocytes in leukoencephalopathies), and includes a number of rare disorders, the most well known of which are metachromatic leukodystrophy, Krabbe’s disease, and adrenoleukodystrophy. Finally, oligodendrocytes contain an extensive microtubular network and express tau, which is a microtubuleassociated protein.192 In neurodegenerative disorders, tau-positive inclusion bodies may form within oligodendrocytes, usually as ‘coiled’ bodies. These inclusion bodies can also be immunostained with antibodies against ubiquitin and heat shock proteins such as B-crystallin.192

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  Ependyma  25 Ciclosporin

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Toluene

Oligodendrocyte

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1.15 Examples of toxic substances that cause oligodendrocyte and myelin loss include: chronic substance usage (ethyl alcohol, ‘ecstasy’, and toluene, i.e. ‘glue-sniffing’), ciclosporin (US spelling cyclosporine), therapeutic radiation, and chemotherapeutic agents (carmustine and methotrexate). The latter two categories of agents also affect blood vessels and produce white matter ­damage by ischaemic mechanisms.

Other inclusions are illustrated here in a case of multiple system atrophy, in which the cytoplasmic inclusions appear as linear, pointed, densely eosinophilic structures immediately adjacent to oligodendrocyte nuclei in select anatomic areas of the brain (Figure 1.14k). Although visible on careful inspection of H&E sections, they are better highlighted with silver stains as ‘Papp–Lantos bodies’ (Figure 1.14l) or by immunostaining for ubiquitin, alpha-synuclein or B-crystallin. Ubiquitination is common to many very different types of neuronal and glial inclusions and makes antibodies to ubiquitin and p62 valuable and effective generic immunostains to have available in laboratories that study neurodegenerative disorders. Reactions of the oligodendrocyte at the level of the myelin sheath include intramyelinic oedema, resulting in vacuolation of the neuropil, and myelin degeneration, resulting in myelin digestion by macrophages. Wallerian degeneration is the process that best illustrates the co-dependency of myelin sheaths and their axons. When axons are transected or otherwise severely injured, the axon distal to the transection will start to undergo dissolution. Following this, the myelin sheaths surrounding the axon start to break down into a string of myelin ovoids, separated by the oligodendrocytes that formed the myelin. These myelin debris-containing ovoids are quickly surrounded by astrocytic cell processes

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and are then phagocytosed by microglia and macrophages. Entire tracts may undergo wallerian degeneration, such as the descending corticospinal tracts in the spinal cord when a severe injury has occurred at a more proximal point in the tract (e.g. the internal capsule). Extensive wallerian degeneration is appreciable on both pre-mortem neuroimaging studies and, should the patient succumb, on gross brain examination. In the PNS, one of the more striking responses at the level of the myelin sheath of Schwann cells to injury follows chronic, repetitive myelin loss caused by various types of peripheral neuropathies. This results in exuberant proliferation of Schwann cells, usually seen as multiple concentric layers of cells around a thinly myelinated axon, evidence that the process is not fully successful. Although an analogous reaction does not affect oligodendrocytes in the CNS, in severe proximal peripheral nerve injury, onion bulb formation by Schwann cells may extend into spinal cord parenchyma.186,214

Ependyma Ependymal cells constitute the lining of the ventricular system, including the aqueduct of Sylvius and foramina that connect the different ventricles. Highly specialized

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26  Chapter 1  General Pathology of the Central Nervous System

ependymal cells play a key role in fluid homeostasis between brain parenchyma and the cerebrospinal fluid, and ependymal cells are rich in the membrane water channel protein aquaporin-4.173 This role in fluid homeostasis is relevant to both normal physiological conditions and disease states, especially ones that affect the ventricular lining (intraventricular haemorrhage, hydrocephalus, CNS infections resulting in ependymitis or ventriculitis). Ependymal cells have electrophysiological properties similar to those of astroglia.203,204 They are also seen in the spinal cord ‘central canal’, even when the canal becomes vestigial and is identified only as a somewhat disorganized collection of cells that retain their phenotype. Ependymal cells resemble the relatively monotonous cuboidal and columnar epithelia that line the gastrointestinal and respiratory tracts (Figure 1.16), but do not have a well-defined basement membrane and are immunopositive for GFAP (see later). They show prominent cilia. Interspersed among the ependymal cells are tanycytes, which have radially directed basal processes that extend into the periventricular neuropil and enwrap blood vessels, or terminate on neurons, glia or the external glia limitans.94 The innate immune response of ependymal cells may involve signalling through several pattern recognition receptors (PRRs) (see later under Identifying Microglia and Quantifying Microgliosis). Ependymal cells arise from epithelium of the neural plate and the neural tube, which develops from the neural plate. Regions in the CNS destined to show an ependymal lining later in life, demonstrate (at 6 weeks of intrauterine development) only densely cellular pseudostratified columnar epithelium that is quite mitotically active. This mitotic activity essentially ends when the ventricular lining is fully developed, never to resume. However, the pseudostratified appearance of ependyma may occasionally be found in the ventricular lining of older patients, even adults (Figure 1.16). Immunohistochemical studies of human fetal ependyma show variably strong vimentin immunopositivity as early as 8 weeks into development in most ependymal regions (including spinal cord, the lining of all ventricles and cerebral aqueduct), though this diminishes (but does not disappear entirely) by 40 weeks of gestation.203 Cytokeratin (CK-904) immunoreactivity is maximal in most regions, though surprisingly absent from the lining of the third ventricle, from 8 to 14 weeks of gestation but disappears thereafter. GFAP and S-100 antibodies show patterns of ependymal immunoreactivity that appear to be highly dependent on the precise locus within a given neuroanatomic structure being examined: as one example, GFAP immunoreactivity is prominent in the roof plate of the developing spinal cord but absent from its floor plate, although the opposite pattern of immunoreactivity is noted in the fourth ventricle and cerebral aqueduct.203 By full term, fairly consistent and robust GFAP and S-100 immunoreactivity are noted only in the ependymal lining of the lateral and third ventricles and parts of the fourth ventricle. Ependymal cells are connected to each other by gap junctions. Proteins known as connexins (Cx) are present at these junctions and contribute to ‘intercellular communication, ion homeostasis, volume control and adherent connections between neighbouring cells’.55 Connexin proteins Cx26, Cx30, Cx43, and Cx45 are mainly expressed at the apices of ependymal cells.55 Aquaporins (AQPs) are well known to be expressed in the end feet of astrocytes, but the

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AQP4 channel that controls water movement in brain is also expressed in basal-lateral areas of ependymal cells, as are other members of the AQP family.55 Ependymal cells have apical cilia that beat in a coordinated fashion and this organized beating may, in part, be due to the gap junctions. Del Bigio notes that ependymal cilia ‘may help to create concentration gradients of guidance molecules in cerebrospinal fluid that serve to direct neuroblast migration from the lateral ventricle wall into the olfactory bulb’.55 Damage to ependymal cilia has, in rare human instances, been shown to produce hydrocephalus (or may indeed result from severe and/or prolonged hydrocephalus), although there are numerous examples of mice with mutations in ciliary proteins that have been proven to develop hydrocephalus, with or without occlusion of the cerebral aqueduct.55 Ependymal cells have a relatively circumscribed repertoire of responses to injury, and only limited regenerative capacity at all ages. However, subependymal zone (SEZ) cells in a thin layer surrounding the lateral ventricle have been found to show properties of neural stem cells.206 In experimental models, injury to the cerebral cortex modestly increases metabolic activity in the SEZ (as measured by cytochrome oxidase activity), as well as its proliferative capacity. Cells in the SEZ may have the potential to repopulate lost neurons in the olfactory bulb and even regions of the cerebral cortex. Although spared in most degenerative and genetic diseases that afflict the nervous system, the ependyma is highly vulnerable in many other conditions, by virtue of its unique ‘barrier’ position and vulnerability to increases in ventricular size. It can undergo injury when stretched during the evolution of ventriculomegaly associated with hydrocephalus, a hematoma or infarct that involves the ventricular wall (e.g. germinal matrix haemorrhages that commonly extend into the ventricular cavities in distressed premature infants), and in infections or inflammatory processes that extend directly from the brain parenchyma or subarachnoid space.204 Hydrocephalus in humans is often accompanied by neuroimaging abnormalities that include a subventricular band of ‘transependymal oedema’, which may be transient and has poorly characterized neuropathological correlates. In experimental models of hydrocephalus, discontinuities and gaps in the ependymal lining are filled by the processes of subependymal astrocytes, but residual ependymal cells do not become proliferative. Neither do subependymal cells undergo metaplasia to repopulate the ependymal ventricular lining. The range of ependymal reactions to injury has been well summarized by Sarnat.204 Atrophic ependymal cells, usually a response to ventriculomegaly, are characterized by flattening and loss of their cytoplasm. Ventricular enlargement, especially when rapidly evolving and progressive, can cause stretching and tearing of the ventricular lining. Sites of rupture are more likely to occur over the smooth ventricular surface than at the ventricular angles. Subventricular astrogliosis is characterized by glial cells that proliferate, often extending into the ventricular cavity. This phenomenon, thought to occur within 1–2 weeks subsequent to the ependymal injury, is often described (somewhat inaccurately, because true inflammation is almost never a histopathological feature) as ‘granular ependymitis’, and the protrusions of glial tissue as ‘ependymal granulations’. The process is very patchy and multifocal throughout the ventricular

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  Ependyma  27 (a)

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1.16 Histological features of normal ependyma. (a) Lowmagnification view of the junction of the cerebral aqueduct (of Sylvius) with the fourth ventricle. Note small ependymalined ‘outpouchings’ (arrow), which are very common in any ependyma-lined structure. (b,c) Magnified views of the ependyma show a ‘picket fence’-like structure with a slight tendency to stratification of ependymal cells in a few foci (arrow, [c]). (d) Relatively uniform ependymal nuclei, absence of a basement membrane deep to the ependyma, and wispy cilia on the ependymal surface (arrows). (e) Occasionally, as in this image from the wall of the third ventricle, the ependyma is arrayed in ‘udderlike’ folds – a finding of no pathological significance.

lining. These subventricular glial nodules may also represent the sequelae of a fairly indolent viral infection of the CNS (see later) and are commonly seen in the CNS of patients with acquired immunodeficiency syndrome (AIDS),251 but are also commonly encountered as an incidental necropsy finding in individuals with no history of neurological disease.204 When the ependyma has been injured by intraventricular haemorrhage (e.g. extending from a large germinal matrix bleed in a premature infant), macrophages, including

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siderophages or hemosiderin-laden macrophages, may be seen at or near the locus of injury. Ependymal rosettes, characterized by tiny ‘tubules’ of ependymal cells in the periventricular region (and sometimes forming hemi-rosettes rather than complete rosettes) may represent abortive attempts at recapitulating the formation of the embryonic neural tube; they may also be the result of ependymal residua within brain parenchyma, in which luminal/ periventricular astrocytic overgrowth has occurred. Similar rosettes are seen,

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28  Chapter 1  General Pathology of the Central Nervous System

of course, in a variety of CNS tumours, most prominently ependymomas. True ependymitis (to be distinguished from granular ependymitis, see earlier) may be suppurative/purulent, e.g. when encountered in association with a bacterial or fungal meningitis or brain abscess, polymorphonuclear leukocytes will then be present in abundance within and adjacent to the ependymal lining. Such an ependymitis may evolve into a ventriculitis; in its extreme form (for instance, when untreated) this can lead to filling of the ventricular cavity by pus and ventricular abscess formation.204 Ependymitis may become so severe that it leads to fragments of ependyma being shed into the ventricular cavity and cerebrospinal fluid (CSF) pathways, rarely such cells are identified in samples obtained by lumbar puncture for evaluation of CSF cytology. Several viruses have a propensity to colonize ependyma and periventricular tissues. In the era of AIDS, this is most dramatically manifest with cytomegalovirus (CMV) infection, which can cause such a severe ependymitis/ventriculitis that a thick icing-like layer of exudative material is noted in cut sections through the fixed brain251,253 (Figure 1.17c). This ependymal infection by CMV then commonly spreads in a ‘ventriculofugal’ direction, into the brain parenchyma. Much less commonly, adenovirus has been identified as causing (a)

a more subtle ependymitis/ventriculitis (Figure 1.17 b).5 Mumps ependymitis, often with minimal inflammation, can lead to aqueductal stenosis, an important (though rare) cause of acquired hydrocephalus.204 Ventriculomegaly becomes apparent weeks to months after a clinically apparent mumps virus infection, e.g. manifest as parotitis. Aqueductal stenosis has also been described after influenza and parainfluenza 2 infections. The only ‘footprint’ of many of these viral infections is the presence of microglial nodules in close proximity to the ependyma, and immunocytochemical evidence of viral infection within ependymal cells. In experimental animals, human respiratory syncytial virus (RSV) infection can also lead to viral antigen within ependymal cells and, eventually, aqueductal stenosis with hydrocephalus. Primary CNS neoplasms may extend to the ependymal lining (Figure 1.17a) and sometimes breach this barrier to gain access to the ventricular cavity, facilitating spread of such a tumour through the CSF pathways. It is hardly surprising that the ependyma is vulnerable to infection by numerous pathogens, especially viruses, given its anatomically critical locus at the interface between CSF and brain parenchyma. Viruses may use specific receptors (e.g. CAR, JAM, CD46 and CD55) to target the ependymal and subependymal microenvironment. Choroid plexus (b)

(c)

1.17 Ventricular/ependymal lining involved by neuropathologic lesions. (a) Widely infiltrating malignant primary brain tumour extends to the ependymal lining of the lateral ventricle. In one area, tumour appears (asterisk) to extend into the ventricular cavity. Other regions of the ependyma (arrows) show disruption. (b) Adenovirus encephalitis and ependymitis in a child with AIDS. Ventricular cavity is indicated by the ‘V’ (at left). Note almost complete loss of the ependyma, with spongy change and oedema in the periventricular region. (c) Cytomegalovirus ependymitis/ventriculitis affecting the fourth ventricle in a human immunodeficiency (HIV)-infected patient with AIDS. Note patchy denudation of the ependymal lining, with scattered cytomegalic cells (arrows) in or immediately adjacent to the ependyma. Sparse inflammatory cells are present in the periventricular neuropil.

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  Microglia and Macrophages  29

epithelium, with anatomic similarities to ependyma, contains some cells that express pattern recognition receptors (PRRs) that in turn bind to pathogens opsonized with C3.94 Thus both ependyma and choroid plexus epithelium, functionally linked by having crucial functions in maintaining ionic and fluid homeostasis in the brain, also play a direct role in preventing its colonization by micro-organisms, and maintaining the CSF in a sterile condition.

Choroid Plexus Choroid plexus is a villous, frond-like, convoluted structure located within ventricles and composed of epithelial cells, fenestrated blood vessels, and stroma; it is involved in the production of cerebrospinal fluid.269 Four separate areas of brain contain choroid plexus: each lateral ventricle, the third ventricle, and the fourth ventricle. Choroid plexus cells are epithelial cells derived from neuroectoderm and thus constitute a subtype of macroglia. In early development, the epithelial cells are tall and pseudostratified, at intermediate stages of development they contain cytoplasmic glycogen, but by later stages of development they become cuboidal and lose glycogen.269 Unlike ependymal cells that carry cilia, choroid plexus epithelial cells have frequent microvilli and cilia are rarely found. At their basal aspect, choroid plexus epithelial cells lie on a basal lamina and adjacent to highly fenestrated blood vessels that allow the choroid plexus (CP) to produce CSF from the blood. Wholesale diffusion of blood-borne substances, however, does not occur between the blood and CSF because of the presence of tight junctions between the CP epithelial cells. Tight junctions in CP cells contain the proteins occludin, claudin-3, claudin-5, and endothelial selective adhesion molecule (ESAM).269 A comprehensive review of the functions of choroid plexus in health and different disease states has recently been published.269 Abnormalities in CP cells are few in number, although progressive calcification of the stroma of the choroid plexus occurs with ageing. A amyloid and Biondi ring tangles accumulate in CP epithelial cells in Alzheimer’s disease, with the latter also seen to accumulate as part of normal ageing.269 The amyloid source for A amyloid accumulation in CP cells has been suggested to be uptake from CSF. Biondi rings are biochemically and ultrastructurally different from either neurofibrillary tangles or A amyloid, and the exact nature of these structures is still being explored. Interestingly, some workers have suggested that Biondi ring tangles are among the earliest manifestations of Alzheimer disease.155

Microglia and Macrophages Microglial cells have historically been considered the cells within the CNS that respond to invasion of the parenchyma by viral agents, have important phagocytic functions and constitute the neural component of the reticuloendothelial system. A variety of observations, many of them originating in neuropathological specimens from patients, have led to a reformulation of the putative role of microglia in various diseases and responses of brain and spinal cord to injury.121,247 It has become frustratingly clear that some ailments in which

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microglial proliferation is a key element, e.g. Rasmussen encephalitis associated with intractable partial epilepsy and cerebral hemiatrophy in children, do not have an obvious viral aetiology and may in fact be autoimmune disorders.1,18,66 Microglia may also play a significant role in the progression of lesions seen in diseases of the CNS that are not primarily inflammatory, e.g. Alzheimer’s disease, and under some circumstances may even have trophic/nutritive functions.121 Thus, our understanding of microglial function and potential has greatly expanded in recent years. Much of this has also come about as a consequence of the AIDS pandemic. It became manifestly clear early in the worldwide epidemic of infection by the human immunodeficiency virus (HIV) that understanding the CNS consequences of direct HIV infection of the brain required a sophisticated understanding of the role of its microglial cells, the major cell type that harbours this retrovirus and is productively infected by it.49,185 For these and other reasons, the ‘rediscovery’ of the importance of microglia has been reflected (as pointed out in a recent mini-symposium on this cell type) by a massive increase in the literature pertinent to their biology – between 2001 and 2005, almost 3600 articles had been published on microglia, more than in the prior 15 years!57

1

Identifying Microglia and Quantifying Microgliosis For decades, the origin of microglia/macrophages in the CNS has been controversial – the question has been formulated, somewhat simplistically, as ‘Do they originate in the brain or the bone marrow or in both sites?’ The current view is that blood-derived monocytes move into the brain during early embryonic development, then differentiate into microglia that share many surface markers or antigens with their blood-borne and visceral counterparts, monocytes and macrophages.121 Under normal circumstances, microglia are inconspicuous bystanders in the scaffolding of the brain and spinal cord. Unlike neurons or ependymal cells, they are not identifiable by their striking and distinctive morphological characteristics or anatomical location. They are estimated to comprise as many as 15 per cent of cells in some parts of the CNS.94 Though cells with characteristic microglial morphology had been recognized previously – probably even by Nissl in the late 1800s – their discovery and confirmation as a distinctive cell type is widely attributed to the work of del Rio Hortega and Penfield in 1927. They and other investigators seeking to study microglial biology in the early to mid1900s utilized the silver carbonate method to demonstrate their presence in histological specimens. These methods have been largely supplanted by immunohistochemical stains of microglia/macrophages with primary antibodies directed against macrophage/microglial epitopes and surface antigens or receptors involved with immune system activation, including integrins and the ligands for ICAM-1. Frequently used markers are CD45 (relatively non-specific), CD68 (Figure 1.18), HAM (human alveolar macrophage)-56, CD11b (Mac1), CD11c (LeuM5), CD64 (an immunoglobulin receptor), MHC Class I antigen, MHC Class II antigen (HLA-DR), Ricinus communis agglutinin I lectin (RCA),121 and a newer marker of great utility, Iba1.268 Some microglia express both MHC class I and II antigens and may interact biologically with both T-helper (T4) and T-cytotoxic

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30  Chapter 1  General Pathology of the Central Nervous System (a)

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1.18 Microglia and macrophages; light microscopic and immunohistochemical features in and adjacent to brain infarcts. (a,b) A subacute infarct is highlighted using the antiCD68 antibody, which demonstrates clusters of cells with well-defined cell membranes and coarse, foamy or granular cytoplasm that is strongly CD68-immunoreactive. In the older literature, these are sometimes described as compound granular corpuscles or gitter cells, terms that are now rarely used. (c) H&E-stained section from the centre of an old infarct shows numerous foamy macrophages (arrows), some containing haemosiderin. (d,e) Widely and evenly distributed (activated) CD68immunoreactive microglia at the edge of a recent cerebral infarct, shown at low magnification in (d), at higher ­magnification in (e). Note the rod-like morphology of many of these cells.

(T8) lymphocytes at the same time. Microglia are also demonstrated histochemically (in the mouse) by staining for nucleotide diphosphatase (NDPase).267 Quantitative estimates of microglial number in the mouse fascia dentata have been provided through the use of unbiased stereological cell counting techniques; estimated numbers of microglia in this structure were on the order of 12 000.266

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Microglial Types Microglia were subclassified using the older silver carbonate technique as being amoeboid, ramified or of intermediate form.121 They are now more commonly described as resting, activated or amoeboid phagocytic microglia, but are known to modify their structure and repertoire of expressed

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  Microglia and Macrophages  31

cell surface antigens in response to their ambient microenvironment. Thus there are almost certainly many subsets of microglia, defined by the molecules they secrete and their immunophenotype. Some may be more harmful to the CNS when activated, although others may have significant protective functions.217 Activated microglia usually take on a ‘rod shape’ within neural parenchyma. However, their definitive identification relies upon immunohistochemistry (see p. 29 and Figure 1.18). Perivascular cells surrounding brain capillary endothelium share phenotypic properties of both microglia and smooth muscle cells. Perivascular macrophages and macrophages in the subarachnoid space express higher levels of CD45, MHC class II antigen, and ‘pattern recognition receptors’ (e.g. CD14) than do their parenchymal counterparts, and turn over more quickly, with replenishment by bone marrow-derived elements.110 When studied by electron microscopy, microglia have few microtubules and intermediate filaments (unlike astrocytes) but numerous cytoplasmic dense bodies that presumably contain molecules that facilitate their phagocytic potential. Their cell surface extends into pseudopodia and filopodia. Given the ability of microglia to sequester themselves unobtrusively within the microanatomy of the CNS, it is somewhat surprising that they can be coaxed from this setting and isolated in relatively pure tissue culture preparations – this has even been achieved from human autopsy brain tissue, obtained with a very short post-mortem autolysis time.258In vitro, they can be easily induced to reveal their phagocytic properties, e.g. by co-culturing the cells with latex beads, which they avidly take up into their cytoplasm. Primary cultures of microglia are difficult to obtain in pure form, but a number of different permanent/established cell lines have a microglial (immunohistochemical) phenotype.121 Microglia in vitro can be stimulated to proliferate by molecules that originate from microglia themselves, e.g. some cytokines (e.g. IL-1 , IL-4, IFN ) and appear to be tightly regulated by colony-stimulating factors, such as macrophage colonystimulating factor (M-CSF, constitutively produced by astrocytes) and granulocyte/macrophage colony-stimulating factor (GM-CSF, expressed primarily during development). M-CSF is crucial to maintaining the normal population of ramified microglia within the brain, although GM-CSF promotes proinflammatory activity.

Functions of Microglia As for astrocytes (see earlier), whether proliferation and activation of microglia within the CNS have predominantly deleterious consequences for the brain and spinal cord or have beneficial effects has been the subject of intense investigation and reconsideration. Microglia have numerous functions.87,94,110,121,268 They are, in addition to being immunological sensors within the CNS and antigen presenting cells (APCs), certainly ‘factories’ for the production of a variety of cytokines and chemokines. The former are low molecular weight proteins including interleukins (ILs), tumour necrosis factors (TNFs), interferons (IFNs), transforming growth factors (TGFs), and colony stimulating factors (CSFs). Cytokines are important elements in not only the modulation of inflammation and immune responses but also the physiological processes vital to CNS growth and development. Chemokines, by

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contrast, are small (8–10 kDa) inducible, secreted proinflammatory molecules crucial to various immune and inflammatory responses; they activate specific types of leukocytes and act as chemoattractants. Using reverse transcription polymerase chain reaction methodology, human microglia have also been shown to express transcripts for numerous cytokine receptors, as well as gp130 (an IL-6 receptor component), ciliary neurotrophic factor, and chemokine receptor CXCR4, an important co-receptor for HIV entry into (human) microglia. It remains controversial whether microglia are major producers of nitric oxide (NO). Microglia can also synthesize various neurotrophins and other molecules vital to neuronal survival, including nerve growth factor (NGF), neurotrophin 3 (NT3), brainderived neurotrophic factor (BDNF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF). This may explain the apparent neuroprotective function of microglia in some settings.217

1

Pathological Reactions of Microglia Under normal circumstances, microglial cells represent uncommitted (undifferentiated, immature, resting) myeloid progenitors within the brain, capable of differentiating toward a dendritic morphology (under the influence of granulocyte/macrophage colony-stimulating factor [GM-CSF]) or a histiocytic morphology (in response to macrophage colony-stimulating factor (M-CSF) and/ or cytokines).110 Maintenance of the immature state of microglia is attributed, at least in part, to TGF and IL-10. The relatively ‘immune privileged’ status of the CNS is ascribed to the relative absence of cells that express MHC class I, as well as the constitutive microglial expression of macrophage migration inhibition factor (MIF, also an inhibitor of natural killer [NK] cells) and interleukin-1 receptor antagonist (IL-1Ra), which exerts its inhibitory effect by binding to the IL-1 receptor but not transmitting a signal as a result of this union. TGF is a growth factor with numerous effects on processes as diverse as immune homeostasis, angiogenesis, extracellular matrix remodelling, apoptosis and migration. TGF 1 knockout mice develop a fatal multisystem autoimmune disease. IL-10 inhibits the production of numerous proinflammatory cytokines (e.g., IL-1, IL-6, IL-12, TNF ). Microglial activation occurs through either stimulation/activation of pattern recognition receptors (a major component of the innate immune response, see later) or the adaptive immune response itself. The innate immune response is the initial line of defense in response to a variety of pathogens, one that does not require prior exposure to foreign antigens in order to be ‘triggered’.120 Cellular elements of this innate immune response include macrophages, neutrophils, dendritic cells and natural killer cells, in addition to CNS microglia. Component cells of the innate immune system must be able to recognize antigens by virtue of a predetermined set of conserved receptors, ones expressed on many micro-organisms; these conserved structural motifs are known as pathogen-associated molecular patterns (PAMPs), although the cellular receptors that recognize them are described as pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are a family of PRRs expressed on cells of the innate immune system. Microglia

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32  Chapter 1  General Pathology of the Central Nervous System

are known to express several TLRs, including TLR2, 3, 4 and 9.120 Activated microglia secrete, in addition to proinflammatory chemokines and cytokines (e.g. TNF ), several toxic molecules that may damage structural CNS components, e.g. reactive nitrogen intermediates (RNIs), reactive oxygen species (ROS) and derivatives of arachidonic acid, and upregulate MHC class I and II expression.

Microglia in Disease/Injury States The role of microglia in the pathogenesis and progression of various diseases and forms of brain/spinal cord injury and infections is considered in detail in other chapters of this book, but will briefly be introduced here. A common experimental paradigm has been to consider microglial responses to ischaemic, traumatic or seizure-related brain injury in experimental animals, often focusing on the hippocampus.72 In elegant experiments using green fluorescent protein (GFP)-expressing (GFP+) bone marrow-derived cells and highly specific phenotypic markers, investigators using a modest entorhinal cortex lesion (that results in local axonal degeneration and microgliosis) have been able to demonstrate the time course of the microglial reaction, as well as the relative contributions of ‘resident’ and ‘immigrant’ microglia to the tissue response.267 Whereas the immigrant (GFP+) microglial response reached a maximum at 7 days post-injury, the resident microglial reaction appeared to peak at about 3 days. Resident microglia, when undergoing early activation, express the stem cell antigen CD34.132 Microglia (including resting and perivascular cells) play both beneficial and potentially harmful roles in the evolution of focal brain infarcts and global ischaemia.48 Microglia – at least ones that are not rendered necrotic by a significant ischaemic insult – have been shown to become activated and increase in number within minutes to hours of ischaemic brain injury. Taking on various morphologies (amoeboid, round), these cells probably represent a combination of microglia that have undergone local proliferation and those that have migrated from the penumbra of the ischaemic lesion. Microglial/macrophage responses have been studied in the spinal cords of patients who had experienced significant cerebral infarcts at time intervals ranging from 4 days to 4 months prior to death. An increase in the number of microglia/macrophages (labelled with antibodies to HLA-DR and CD68) was observed in the contralateral anterior horn of the spinal cord when the cerebral infarct had occurred less than 2 weeks prior to death, but was much more pronounced in the ipsilateral corticospinal tracts (probably a function of ongoing wallerian degeneration) weeks or months after the stroke.212 Localized spinal cord injury may also be induced (in rats) by injection of zymosan, which causes axonal injury and focally pronounced demyelination in the adjacent spinal cord tissue, injury that appears to be mediated by activation of microglia and macrophages).184 Microglial activation/proliferation need not be caused by a direct injury to the neural parenchyma. In rats, transection of the facial nerve generates an increased number of microglia in the facial nucleus (from which the nerve obviously originates) – this response is at a maximum 3 days after nerve transection.165 Microglia subsequently isolated from this axotomized facial nucleus could be cultured and were shown to proliferate in vitro in an autocrine fashion.

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The possible role of microglia (and other aspects of neuroinflammation) in the exacerbation of developmental brain injuries has been reviewed.38 Subtle alterations in microglial structure (including increases in heterogeneous cytoplasmic inclusions) and immunophenotype (increased cell numbers expressing the ED1 macrophage marker, complement receptor 3/CR3) have been shown to occur with age in experimental animals.41 Microglial changes (in both size and number) as a function of age are difficult to quantify in the human brain. Several factors are responsible for this. The brain undergoes physiological atrophy beginning in the sixth decade of life, meaning that assessments of microglial number (in autopsy brain specimens) must be undertaken using rigorous unbiased stereological techniques in order to provide meaningful data. Subtle morphological agerelated changes are difficult to quantify in this extremely polymorphous cell population. However, human brain appears to show an increased number and density of MHC Class II cells as a function of age. Common neurodegenerative diseases (e.g. Alzheimer’s disease [AD] and Parkinson’s disease [PD]) are associated with activation and proliferation of microglia either focally or diffusely within the CNS. Although few would claim that activation of microglia is the key aetiological factor underlying these diseases, there is growing, compelling evidence for their probable role in the progression of neurodegeneration.140,149 The implication of this scenario is that interference with microglial (and, for that matter, astroglial, see earlier) activation might stabilize or even somewhat ameliorate the disease while not curing it.160 There is evidence from tissue culture systems, for instance, that the presence of microglia exacerbates the neurotoxicity of A (1-42) peptide, which accumulates in the brains of patients with AD. Certainly activated microglia are easily demonstrated around A plaques in the cerebral cortex in AD. In AD, CD68-immunoreactive microglia are more abundant within the subcortical white matter than in the overlying cortex.256 In an inflammation-mediated rat model of PD, nigral dopaminergic neuronal degeneration clearly postdated a period of microglial activation, suggesting that the latter may have contributed to the former. Microglial activation and increase in number occurs in both the spinal cord and motor cortex of patients with motor neuron disease/amyotrophic lateral sclerosis (MND/ALS) but this could be argued to be as much a result of the neurodegeneration in these regions as a cause of it.150 Nevertheless, evidence continues to accumulate that microglia and their secreted products are of some importance in this disorder. One study of MND/ALS spinal cord tissue from both sporadic and familial cases found immunohistochemical and molecular evidence (mRNA expression profiles reflecting, e.g. increases in the chemokine MCP-1) suggesting the involvement of immune and inflammatory – including microglial  –  responses in exacerbating neuronal degeneration in spinal cords of affected patients.95 Microglial activation has even been suggested as one mechanism of progression in the trinucleotide repeat disease, Huntington disease.202 Although generally a helpful process in terms of fending off or at least containing potentially harmful pathogens (e.g. viruses that have gained entry to the CNS),

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  Distinguishing Pathological Abnormalities from Artefacts and Incidentals  33

microglial activation may also be harmful. As one example of the latter, radiation-induced microglial activation has been shown to interfere with hippocampal (granule cell) neurogenesis – this may be associated with clinical cognitive decline commonly seen in cancer patients undergoing CNS radiotherapy.118 The putative molecular pathogenesis of this phenomenon is by no means clear; however, brain inflammation associated with radiation-induced microglial activation may lead to overproduction of IL-6, which in turn interferes with neuronal production (and facilitates the generation of astroglia) from pluripotent neuronal progenitor cells that reside in the hippocampus. This observation reflects a newly emerging role for microglia in brain repair and perhaps even regeneration.87,268 Brain microglia and macrophages have been shown to express neurotrophins (neurotrophin-3, nerve growth factor) that can then selectively regulate microglial proliferation and even their function.63 Microglia may also be involved in directing the migration and differentiation of neural precursor cells, even playing a part in influencing the differentiation of adult and embryonic neural precursor cells toward a well-defined neuronal phenotype.2 Microglia and macrophages may also be seen in substantial numbers in resected glioneuronal brain tumours that presented with intractable seizures. (Indeed, the more microglial/macrophage immunohistochemical markers are applied to brain tumour surgical material, the more obvious it has become that these cells are frequently a major component of both low- and high-grade glial tumours of all types!) In epilepsy-related neoplasms, the density of microglia was reported to correlate with preoperative epilepsy duration and seizure frequency.9 This suggests either a role for microglia in causing the seizures or an inflammatory response to the neoplasm or the seizures themselves. As regards the relationship between infiltrating microglia within primary brain tumours, it is possible that diminution of the immune effector functions of these microglia enhances tumour growth, although microglia-derived cytokines and growth factors may facilitate tumour cell proliferation and invasion into normal brain parenchyma.260 Microglia and other inflammatory cells are particularly abundant in brain tissues of patients with viral encephalitis. In this context, small collections of such cells are often described as inflammatory or microglial nodules (Figure 1.19). The former term is preferred, because microglia are often only one cellular component of a collection of inflammatory cells. Often, in viral encephalitis microglia accumulate around an infected neuron, as part of the process of neuronophagia (Figure 1.19b). Microglial activation is a prominent feature of Rasmussen encephalitis (RE) – a paediatric inflammatory brain disorder causing partial epilepsy and hemiatrophy, probably as the result of the actions of (autoimmune) cytotoxic T-lymphocytes or an (as yet) unidentified viral pathogen and is probably an important contributor to neurodegeneration in this uncommon disorder.1,66 In RE, microglia are abundant among other inflammatory and reactive cells that infiltrate the CNS, including T-lymphocytes and astrocytes (Figure 1.20). Microglial cells and macrophages are the main types of cell that harbour HIV-1 in patients with HIV encephalitis/HIV-associated dementia; a marker for HIV-1 in the

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1.19 (a) Microglial nodule in the white matter. (b) A cluster of microglia around a ‘dying’ neuron (neuronophagia).

brain is the presence of multinucleated microglia/macrophages (Figure 1.21). A recent review on microglial cells has been published as part of a symposium on the biology and pathology of glial cells and particularly focuses on the role of microglia in neurodegenerative disorders, CNS trauma, and pain hypersensitivity syndromes following nerve injury (neuropathic pain due to aberrant excitability of dorsal horn neurons and mediated by microglial activation due to chemokine CCL2).87 An extensive review of microglial function has also been published by Wirenfeldt et al.268

Distinguishing Pathological Abnormalities from Artefacts and Incidentals One of the greatest challenges for diagnostic autopsy or surgical pathologists, or researchers using human tissue in their studies, is to determine whether a new feature they are viewing grossly or microscopically in the tissue they are studying is real or relevant to their study. Central and peripheral nervous system anatomy is challenging enough in the first place, and the difficulties in tissue interpretation can be further compounded by the fact that these tissues are subject to a plethora of artefacts, incidental findings and variations of normal. Tangential cuts through complex anatomical areas can erroneously give the impression of an abnormality that

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34  Chapter 1  General Pathology of the Central Nervous System (a)

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1.20 Rasmussen encephalitis. The panels illustrate fields from skip-serial sections through a corticectomy specimen; the surgery was performed for intractable seizures. (a) CD68-immunoreactive cells are found in clusters, and often closely apposed to neurons (arrows). (b) Other cell types present in this inflammatory infiltrate include astrocytes, (c) and lymphocytes immunoreactive for both ICAM-3, and (d) CD3 (T-lymphocytes).

1.21 A case of HIV encephalitis. Multinucleated cells containing minimal cytoplasm (arrows) are characteristic of HIV-1 infection of the brain.

does not exist. Normal tissues can be compressed, distorted, or can predominate in what is otherwise thought to be a tissue block containing solely neoplasm. Oedema in the tissue,

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and increased water content of CNS tissue in general, often lead to poor tissue fixation or preservation. Encountering patient tissues from extremes of the age spectrum may lead to misinterpretation of what is simply a normal, age-related change. Many ‘bodies’, cellular inclusions, pigments, crystals, clefts, unusual reactions to tissue injury, and surgical or treatment-related inert substances may be encompassed within human tissues of pathological interest.27,76,77 These structures can be diagnostically challenging for autopsy and surgical pathologists, as well as for researchers using the tissues in their studies. Most of the inclusions occur within neurons and represent either accumulations of virions in infected cells or abnormal proteins or cytoskeletal components in neurodegenerative or storage diseases. We have tried to illustrate some of the more common of these changes, mostly as seen in sections stained by the routine haematoxylin and eosin (H&E) method most often used by diagnostic and research pathologists. Some of these inclusion bodies and abnormal deposits are described earlier, and alphabetically listed and illustrated in Figures 1.2 and 1.3. Further CNS and PNS artefacts, incidentals, pigments and miscellaneous bodies are illustrated in Figures 1.22 to 1.26.

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  Distinguishing Pathological Abnormalities from Artefacts and Incidentals  35

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1.22 Artefacts and incidentals found at autopsy. (a) Displacement of cerebellar tissue from herniated cerebellar tonsils may occur when the necrotic, softened cerebellar tissue (right) is displaced from the posterior fossa and travels within the subarachnoid space to surround the spinal cord (left), where it is found at autopsy; this is a characteristic finding in patients who have suffered nonperfused (‘respirator’) brain change. (b) Artefactual displacement of individual neurons (arrow) into the leptomeninges can sometimes be encountered at autopsy, especially in the cerebellum. (c) Cerebellar granular cell layer autolysis (‘état glacé’) is a frequent finding at autopsy and is characterized histologically by the dissolution and smudgy appearance of this layer of neurons. The cerebellar granular cell layer presumably undergoes enzymatic alterations soon after death, especially if an elevated body temperature is not lowered by prompt refrigeration of the body in the morgue. This layer of cells is the ‘pancreas of the brain’ when it comes to autolytic change! Unlike anoxic injury to the cerebellum, the nearby Purkinje cells (which are considerably more vulnerable than the granule cells to hypoxia and ischaemia) are intact with normal basophilic cytoplasm. Contrast this autolytic change with the normal intact granule cell neurons seen in Figure 1.2a. (d) Ferrugination of axons secondary to dystrophic mineralization in a patient with a remote infarction of the brain stem, as illustrated here, can be easily mistaken for organisms, especially if the mineralization is not noticed before special stains are applied. Ferrugination of dead neurons (‘tombstoning’) can also occur in old infarcts and is more often illustrated in textbooks but less easily misinterpreted. (e) Calcification of the basal ganglia is commonly encountered at autopsy in older patients and may be especially prominent in individuals with chronic hypertension, but it is seldom as striking, or grossly apparent, as seen here. Note the yellow-white discolouration in both the globus pallidus and the nearby white matter. Microscopic calcification is also common in the endplate of the dentate gyrus and in the hilum of the dentate nucleus of the cerebellum in aged individuals. However, the degree of calcification in this 62-year-old male, who had an ‘undefined progressive neurological disorder’, is extreme, probably disease producing, and at the pathological end of the spectrum, so-called Fahr’s disease. Coronal brain section at the level of the amygdala. Less extreme calcification is incidental finding. (f) Calcification of the basal ganglia provides an excellent opportunity to capitalize on your friendship with your local radiologist and obtain a post-mortem radiographic image, as was done here for the brain illustrated in panel (e). Continued

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36  Chapter 1  General Pathology of the Central Nervous System (g)

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1.22 (Continued ) Artefacts and incidentals found at autopsy. (g) Lipomas are one of the more common incidental mass lesions encountered at autopsy and can be located in a variety of usually midline sites, including the collicular plate of the midbrain, as illustrated here. The patient was asymptomatic from this lesion. (h) Meningiomas are one of the other frequent incidental mass lesions found at autopsy, especially in elderly patients. This tiny, flat, plaque-like, subdural-based lesion, seen on a whole mount section, was found in an older woman with multiple sclerosis and was too small to be responsible for any of her symptomatology. (i) Rosenthal fibres are brightly eosinophilic, sausage-like inclusions in astrocytic processes that are associated with long-standing gliosis. Although they are most commonly illustrated in the context of a childhood disorder, Alexander’s disease, or in neoplasms such as pilocytic astrocytomas, pleomorphic xanthoastrocytomas or ganglion cell tumours, this example represents an incidental, massive accumulation of Rosenthal fibres in reaction to a macrophage-filled infarct in the cerebellum. This represented the only site of Rosenthal fibre deposition in the brain of this elderly woman. (j) Swiss cheese artefact develops when gas-forming bacteria, usually Clostridia sp. derived from the gastrointestinal tract, grow post-mortem within brain tissue. Note the concentration of these massive vacuoles in deeper brain areas where the formalin fixative used in autopsy preservation of tissues has not penetrated. The brain was immersed in formalin at the time of autopsy and the formalin penetrated a few centimetres into the tissues and killed the post-mortem overgrowth of micro-organisms in the more superficial areas. On microscopic inspection, no host inflammatory reaction is present around these vacuoles, but numerous bacteria can often be found. (k) Toothpaste artefact is seen when the spinal cord is removed in a less-than-gentle fashion at the time of autopsy and portions of the cord become ‘intussuscepted’ or squeezed into cord at lower or higher levels. This is illustrated here with Luxol fast blue–PAS staining, which highlights the pencil-like cores of displaced cord. This artefactual occurrence is particularly likely if the cord is already fragile and damaged by infarction or tumour.

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  Distinguishing Pathological Abnormalities from Artefacts and Incidentals  37 (a)

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1.23 Artefacts, incidentals, interpretation problems and inert substances found in surgical pathology specimens. (a) Adipose tissue in dura mater (top) is more often observed on neuroimaging studies as a normal finding but when it is included in a surgical pathology specimen, such as in this patient who had falcine meningioma removed with the adjacent attached dura, can be misinterpreted. Also note the small amount of surgically induced haemorrhage within the specimen. (b) Pacinian corpuscles (top) are eyecatching, concentric, onion skin-like normal structures that occasionally show up in peripheral nerve (bottom) biopsy specimens. (c) Lipid vacuoles in choroid plexus cells can be seen in either surgical or autopsy pathology material and occur with normal ageing; the change is of no known pathological significance. Choroid plexus can also undergo a number of other age-related changes including cystic degeneration, xanthogranuloma of choroid plexus, calcification of the collagenous stroma, psammoma body calcification of the meningothelial cells entrapped in the normal choroid plexus, and formation of Biondi rings within the cytoplasm of choroid plexus cells, seen best with PAS (inset) or silver stains. The latter are also of unknown significance and are not associated with any specific disease. (d) Macrophages cause no problem in interpretation when they occur in cohesive sheets or lie within cavities of tissue damage in response to injury. However, in acute demyelinating lesions that prompt biopsy (as illustrated here) they diffusely permeate a neuropil that is largely intact except for the loss of myelin. Their admixture with reactive astrocytes further adds to the diagnostic difficulty; some examples are misdiagnosed as mixed oligoastrocytomas. (e) Chronic subdural hematomas can be resident to large numbers of eosinophils and even foci of extramedullary haematopoiesis, both of which are seen here. Although metastatic neoplasms, including hematopoietic neoplasms, often involve the dura, this granulation tissue response should not be interpreted as neoplastic, or even abnormal. Continued

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38  Chapter 1  General Pathology of the Central Nervous System (f)

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1.23 (Continued ) Artefacts, incidentals, interpretation problems and inert substances found in surgical pathology specimens. (f) Artefactually compressed choroid plexus (top) within a surgical pathology specimen from a patient with colloid cyst of the third ventricle (arrows) can mimic a vascular malformation or even a choroid plexus papilloma unless the collapsed lumen and inconspicuous lining of the colloid cyst are recognized (arrows). (g,h) Entrapped normal meningothelial cell nests within a slowly growing, low-grade brain tumour (arrow) are a rare occurrence. The identity of the entrapped cells can be proven by immunostaining for epithelial membrane antigen (h). More commonly, corpora amylacea can also become encompassed by an infiltrating glioma; this usually occurs in an older adult whose brain contains numerous corpora amylacea as part of normal ageing. (i) Textilomas (gossypibomas)191 can occur in response to the surgical haemostatic packing material (arrow) that is employed intraoperatively, eliciting a brain mass that mimics recurrent brain tumour on neuroimaging.191 The foreign body reaction may occur in response to gelatin sponge, oxidized cellulose and microfibrillar collagen (resorbable agents) or cotton or rayon-based hemostats (non-resorbable). Mass lesions usually follow abdominal surgery, not brain surgery, but CNS examples are well documented. The appearance of the inert substance differs, depending on the agent used. (j) Bioglue is an inert, bioadhesive surgical substance composed of bovine serum albumin and glutaraldehyde that is usually employed in cardiac surgical repair procedures; in this patient it was used in a neurosurgical operation. This 23-year-old woman had undergone a posterior cervical spinal cord untethering procedure several months previously and had had Bioglue placed over her dural suture as a sealant for a cerebrospinal fluid leak. She then developed a fluid collection in the posterior cervical tissue and aseptic meningitis, prompting wound revision and removal of the soft tissue and Bioglue. This specimen shows the granulomatous foreign-body type response and the macrophages containing tiny droplets of eosinophilic Bioglue (arrow); the large central inert blob of Bioglue was homogeneously densely eosinophilic, and visually uninteresting. (k) Embolization of arteriovenous malformations134 with polyvinyl alcohol (PVA) prior to operative removal of the lesion can yield striking intravascular spicules of inert substance occluding many, but often not all, vascular channels of the malformation.134 Note the patent channel in extreme upper left part of the panel. This 30-year-old woman presented with a spontaneous haemorrhage from her cerebellar arteriovenous malformation (AVM) and required three embolization procedures before the lesion could be removed. The material can cause vessel wall necrosis, neutrophilic infiltrates that simulate an acute infection, and a foreign body giant cell reaction.

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  Stem Cells in the Central ­N ervous System – Promise, Potential and Reality  39 (a)

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1.24 Colours, clefts and crystals in the nervous system. Bilirubin staining in the damaged central nervous system of immature infants (kernicterus) occurs when the CNS is injured and there is damage to the blood–brain barrier, allowing access of high levels of unconjugated bilirubin from the blood into the brain. Both the anoxia, sepsis or acidosis that was part of the original injury and the bilirubin itself may injure the brain. Free (unconjugated to albumin) bilirubin is reported to bind to phospholipids and gangliosides in cell membranes and to interfere with neuronal oxygen consumption and oxidative phosphorylation.227 The morphological result is bright yellow discolouration of the subthalamic nucleus, hippocampus, thalamus, globus pallidus (all seen in [a]) and cranial nerve nuclei (3rd cranial nerve nucleus illustrated in [b]). The cerebellar vermis and dentate nuclei can also be affected. The pigmentation may be lost after formalin fixation of the autopsy brain.

Stem Cells in the Central ­Nervous System – Promise, Potential and Reality The field of stem cell research is advancing so rapidly that any information on the subject that appears in a textbook is outof-date before the book is in print.233 Nevertheless, a comprehensive symposium on stem cells in the CNS appeared in 2006 in Brain Pathology42,207,208,228,273 and served as an up-to-date reference for neuropathologists to that time. More recently, the potential role of embryonic (neural) stem cells (eNSCs) and induced pluripotent stem cells (iPSCs) in treating various neurologic disorders (especially traumatic brain and spinal cord injury) has been summarized in several reviews.128,130 eNSC grafts may partially restore cord function after transection in a rat model.100 Stroke therapy may eventually be revolutionized through the use of iPSCs, NSCs, and even mesenchymal stem cells, though several barriers to their effective use must be overcome (e.g. cell homing

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to appropriate tracts, survival and tracking, not to mention safety issues).91 Multiple sclerosis (MS) patients may benefit from SC therapy, with an emphasis on cells that differentiate towards an oligodendroglial phenotype – the important caveat being that axonal injury and loss are now accepted as being mediators of long-term MS disability (see Chapter 23, Demyelinating Diseases).105 Stem cells are defined as cells with the potential for selfrenewal and multilineage differentiation. Neural stem cells from the adult mammalian central nervous system were first confidently isolated in 1992.190 It is now appreciated that neural stem cells, as well as glial progenitors, exist in multiple adult brain sites, including the subventricular zone, the lining of the lateral ventricles, the dentate gyrus, the hippocampus, and the subcortical white matter (reviewed by Sanai et al.201). In humans, the largest of these germinal zones is the subventricular zone. As noted earlier, although the presence of these stem cells is now undisputed, their innate ability effectively to replenish injured or dead neurons is highly limited. Nevertheless, overwhelming interest has been

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40  Chapter 1  General Pathology of the Central Nervous System (a)

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1.25 Pigments and crystals. (a) Bilirubin staining of damaged central nervous system tissue can occur when the blood–brain barrier is acutely or subacutely broken down and the patient has an elevated bilirubin level due to liver disease. This is an example of active central pontine myelinolysis, characterized by its typical triangular midline location in the basis pontis at the level of the 5th cranial nerve, occurring in a typical patient with severe concomitant liver failure and bilirubin elevation. Note how the greenish discolouration extends beyond the immediate nidus of the tissue injury. (b) Biliverdin staining can be seen around lesions with massive, recurrent haemorrhages and will occasionally elicit a multinucleated giant cell reaction by the host. This non-iron-containing pigment forms when erythrocytes are disrupted within the ingesting macrophage; the iron in haemoglobin is then oxidized to the trivalent state, forming methaemoglobin. The haem and globin then dissociate and the iron is liberated from hemin by the microsomal enzyme, haem oxygenase, yielding iron and biliverdin.131 Biliverdin does not stain with the Perls’ iron reaction. Note the more abundant robin’s egg blue iron-containing material within macrophages also seen in the lesion. (c) Biliverdin pigment manifests a yellowish gold colour in contrast to the granular golden brown, refractile appearance of the haemosiderin within macrophages. (d) Cholesterol clefts also occur in areas of recurrent haemorrhage, including in the incidental xanthogranulomas often encountered at autopsy in choroid plexus located at the trigone of the lateral ventricle. Xanthogranulomas of the choroid plexus are incidental findings usually in aged individuals, but the cholesterol clefts illustrated here are seen in a pathological lesion, a cholesterol granuloma of temporal bone. (e) Crystals due to mucus can occur within the inspissated contents of colloid cysts or Rathke cleft cysts, manifest a spike-like faint yellow or non-stained colouration as seen here, and can be mistaken for the ‘sulphur’ granules of Actinomyces sp. infection. (f) Crystals due to mucus also stain with PAS. Same case as panel (e).

generated in the possibility of therapeutically manipulating/ expanding exogenous or endogenous stem cell populations and transplanting them into the CNS for the treatment of neurodegenerative disorders, spinal cord injury and multiple sclerosis. Any successful use of stem cells as therapy requires

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an understanding of how neurons differentiate and commit to lineage during normal embryology.21,83,88,219 Identifying the factors responsible for proliferation, symmetrical versus asymmetrical cell division, and differentiation is critical. For example, proliferation and expansion of neural stem cells

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  Stem Cells in the Central ­N ervous System – Promise, Potential and Reality  41 (g)

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1.25 (Continued ) Pigments and crystals. (g) Epidermoid cysts, as a gross specimen (top) and a touch preparation made at the time of intraoperative consultation (bottom) for this cerebellopontine angle mass (microscopic, H&E), can have a pearly white cyst content that can grossly mimic true crystals and can contain anucleate clusters of angular squamous cells. Pseudo-crystal. (h) Haemosiderin pigment (intracellular) is much finer, less refractile, and more likely to mimic melanin pigment when it is within tumour cells rather than macrophages (compare with Figure 1.4c) and may require a Perls’ stain to confidently exclude melanin (inset). An anaplastic meningioma with intracellular iron pigment is illustrated. (i) Haematoidin pigment is an artefactual black, dot-like pigment that occurs in areas of fresh haemorrhage and represents a chemical reaction between haemoglobin in red blood cells and formalin fixative, not a true breakdown product of haemoglobin. This pigment appears birefringent when examined under polarized light. (j) Melanosis of the dentate nucleus (melanosis cerebelli) is a rare finding at autopsy and manifests with large, rounded globules of extracellular, homogeneous, non-refractile pigment that can be compared in size to the adjacent lipofuscin-containing neurons of the dentate nucleus. This material is Fontana-positive, bleaches with potassium permanganate and is thought to be composed of sulphur. It is probably artefactual, may represent melanization of lipofuscin and is almost never associated with cerebellar symptomatology. (k) Ochronosis of the dura mater occurs only rarely, in patients who have the autosomal recessive disorder alkaptonuria, characterized by increased urinary excretion of homogentistic acid. The black pigment is usually found in joints, the cardiovascular system, kidney and skin, but in this reportable example it was found in the dura mater. Gross photograph of lateral surface of formalin-fixed brain with attached, reflected dura mater. (k) Reproduced with permission from Liu W, Prayson RA. Dura mater involvement in ochronosis (alkaptonuria). Arch Pathol Lab Med 2001;125:961–3.141

isolated from developing brain depend on the presence of basic fibroblastic growth factor or epidermal growth factor, and withdrawal of these factors will result in differentiation of the stem cells into neurons, astrocytes or oligodendrocytes

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(reviewed by Brüstle29). The localized microenvironment into which neural stem cells are transplanted has a strong influence on the stem cells and provides the signals that influence the cells to form the correct synapses and chemical phenotype

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42  Chapter 1  General Pathology of the Central Nervous System (a)

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1.26 The many guises of melanin, lipofuscin and amyloid in the central nervous system (CNS) and peripheral nervous system (PNS). (a) Meningeal naevi represent both an uncommon incidental finding and a deposition of a colour/pigment in the brain. Although melanocytic melanin pigment is commonly found at the base of the brain at autopsy, particularly around the ventral medulla or hypothalamus, and especially in dark-skinned individuals, visible melanocytic accumulation elsewhere is rare. This example from the temporal lobe of a young Hispanic girl was mistaken by the referring coroner for an area of remote haemorrhage. (b) Meningeal naevi are much less exciting microscopically, where they appear as linear melanocytes within the leptomeninges. The elongate shape of the cells containing pigment, coupled with the fine black brown cytoplasmic content, distinguishes these cells from haemosiderin-containing macrophages (compare with Figure 1.4b). (c) Lipofuscin is seen in several locations within the neurons of older individuals but not normally in children. Although the coeliac ganglion is illustrated here, more commonly observed sites include neurons of the dorsal root ganglia, the dentate nucleus of the cerebellum and the inferior olivary nucleus. Note the satellite Schwann cells surrounding the neurons and the fact that the Nissl substance is arranged at the periphery in this type of neuron. (d) Lipofuscin also accumulates with age in large neurons, particularly the Golgi type I neurons of the anterior spinal cord, cerebral cortex and brain stem. Note the displacement of the basophilic Nissl substance in this neuron from the anterior horn cell region, as well as the artefactual perineuronal space. (e) Neuromelanin accumulates with age in neurons and represents a by-product of the metabolism of neurotransmitters in dopaminergic (substantia nigra) and noradrenergic (locus coeruleus) neurons. Young children do not start to manifest either gross or significant microscopic accumulation of neuromelanin pigment in these neurons until about the age of 7 years. (f) Melanin (melanocytic melanin) can occur in a variety of different types of central and peripheral nervous system ­neoplasms; a melanotic medulloblastoma is illustrated here.

appropriate for that anatomical area, but gene transfer or other techniques may aid in manipulating neuronal cell differentiation and commitment.73 Reviews of numerous aspects of neural stem cell biology have been published (see also previous paragraph).29,73,152,233,248

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Neural stem cells share many properties with the component cells in gliomas, such as high motility and proliferative potential, association with blood vessels and white-matter tracts, and immature antigenic profiles that reflect activation of developmental signalling pathways.201

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  Stem Cells in the Central ­N ervous System – Promise, Potential and Reality  43 (g)

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1.26 (Continued ) The many guises of melanin, lipofuscin and amyloid in the central nervous system (CNS) and peripheral nervous system (PNS). (g) Amyloid (cerebral amyloid angiopathy) is most commonly encountered in the central nervous system in conjunction with Alzheimer’s disease but can also occur as a primary vasculopathy, cerebral amyloid angiopathy. The amyloid in blood vessels of the cortical grey matter and leptomeninges is quite obvious on Congo red staining. The amyloid core in the centre of neuritic plaques can also be visualized with Congo red stain (arrow) but is better highlighted with thioflavin-S staining (inset). (h) Amyloid (primary cerebral amyloidoma) can also occur in large, tumour-like deposits in the cerebrum that cause mass effect and require neurosurgical intervention. These are not associated with cerebral amyloid angiopathy, Alzheimer’s disease, inherited forms of cerebral amyloid angiopathy or systemic amyloidosis. The amyloid appears to be formed focally and locally for unknown reasons, but has the staining characteristics of all amyloids. (i) Pseudoamyloid deposits can also occur in brain. These appear to be large conglomerates of proteinaceous material and lack the affinity for Congo red dye or the thioflavin-S immunofluorescence of true amyloid. (j) Amyloid deposits due to systemic amyloidosis do not affect the central nervous system, because of the presence of the blood–brain barrier, but massive deposits are often found in the peripheral nervous system, as seen in this nerve from a patient with widespread systemic amyloidosis. (k) Amyloid deposits due to systemic amyloidosis show affinity for Congo red stain. (f) Reproduced with permission from USCAP Case 2006-5 Illustrated. (h) Reproduced with permission from USCAP Case 2006-3 Illustrated. (i) Reproduced with permission from Case 1991-3, AANP Diagnostic Slide Session Illustrated.

Both neural stem cells and glioma tumour cells demonstrate hedgehog and Wnt pathway activity and expression of nestin, epidermal growth factor-receptor, telomerase and PTEN.201 Nestin (named for ‘neuroepithelial stem cell’) is an intermediate filament strongly expressed in neuroepithelial cells but not in differentiated cells.29 Self-renewing, multipotential cells in human tumours

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have been recognized by their expression of CD133 cell-surface markers.79,223 Neural stem cells are thought to be particularly vulnerable to oncogenic transformation. In animal models, highly proliferative stem cells within the subventricular zone show the highest degree of susceptibility to chemical or viral oncogenesis. In a tumour model in which avian sarcoma

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44  Chapter 1  General Pathology of the Central Nervous System

virus was injected into neonatal dog brains, gliomas initially developed in the periventricular regions, but as the tumours increased in size, their relationship with the subventricular zone diminished until they were found at day 10 deep within the white matter, unconnected to the subventricular zone.249 This model may explain how spontaneous adult human gliomas can still originate in periventricular neural stem cells, yet be located in lobar areas by the time they are discovered clinically. Further evidence in support of the concept that human gliomas may arise from neural stem cells is the fact that neural stem cells share many properties with gliomas (see earlier). The strongest evidence implicating neural stem cells in brain tumour initiation, however, comes from the studies that showed that injection of as few as 100 CD133+ stem cells isolated from human brain tumours into NOD-SCID (non-obese diabetic, severe combined immunodeficient) mice produced a neoplasm, as many as 105 CD133- cells did not.223 The best-documented example of a human brain tumour that originates from progenitor cells is the medulloblastoma, a highly malignant posterior fossa brain tumour in children that is thought to arise from neural stem cells present in the external granule layer of the cerebellum.231 The mechanisms of tumourigenesis are still incompletely understood, but studies demonstrate that abnormal coexpression of REST/NRSF (a transcriptional repressor of neuronal differentiation genes) and c-Myc oncogene in neural stem cells ‘causes cerebellum-specific tumours by blocking neuronal differentiation and thus maintaining the “stemness” of these cells’.231 This highly malignant tumour arises through mutations in the sonic hedgehog developmental signalling pathway that normally controls self-renewal in the cerebellar cells of the external granule cell layer. Various developmentally regulated genes that are important in normal brain development and the evolution of neoplasia, e.g. in medulloblastoma (SHH, PTCH, WNT, Notch, etc.) have even suggested molecular subclassifications of this tumour type that may have relevance for prognosis and appropriate treatment.214 In addition to their potential therapeutic roles in transplantation and reconstitution of neurons and axons irreparably lost in neurodegenerative disorders or trauma, neural stem cells have been suggested as possible delivery vectors for therapeutic genes.73

Concept of the Blood–Brain ­Barrier and the Neurovascular Unit The BBB is one of three well-defined barrier sites that separate blood from neural parenchyma and the fluid that bathes it, within the CNS; the others are the arachnoid epithelium, which forms the layer of the leptomeninges immediately ‘deep’ to the dura mater, and the choroid plexus epithelium, site of ongoing synthesis of the cerebrospinal fluid, thus site of the blood–CSF barrier. Barrier properties of these structures are determined by intercellular tight junctions that reduce intercellular (paracellular) permeability pathways for large molecules (for reviews, see Abbott et al.,3 Ballabh et al.,13 Najjar et al.,164 Bicker et al.22).

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BBB permeability changes have been implicated in disorders as diverse as Alzheimer’s disease and neuropsychiatric diseases; successful delivery of drugs through the blood to the CNS is obviously dependent upon their making their way through the BBB (and surrounding cells, e.g. pericytes and astrocytes). Abnormalities of the NVU have been suggested to be of aetiologic importance in the pathogenesis of Alzheimer’s disease and mixed dementias resulting from a combination of cerebrovascular disease and AD changes within the brain.104,274 Virtually all CNS neurons are located within 8–20 μm of a brain capillary. The anatomical ‘site’ of the BBB is the cerebral capillary endothelium. A more encompassing (and recently popular) term, the ‘neurovascular unit’, is now used to describe the neuroanatomical cohesiveness and combined activity (especially important in cerebral blood flow and the brain’s response to stroke) of brain microvessels together with their surrounding neurons and glia.36 Current knowledge of the regulation of cerebrovascular tone by perivascular nerves is well summarized by Hamel.90 Definition of the concept of the BBB, and identification of its anatomic site as being cerebral capillary endothelium, was achieved in the second half of the last century using elegant ultrastructural tracing techniques, e.g. injection of high molecular weight horseradish peroxidase (HRP) into animals prior to their sacrifice. Extensive characterization of BBB physiology continued through the 1980s and 1990s, when various BBB carrier/transport systems were identified, sometimes by examination of isolated cerebral capillaries and even cells cultured from them.252 Recent comprehensive textbooks have summarized our current knowledge of BBB physiology, biochemistry and molecular pathology, including the novel and innovative techniques that have been used to examine the regulation of its unique biological properties.163,174 Understanding BBB physiology has immense implications for the therapy of neurological diseases, because the vast majority of large molecules that might be of benefit in the treatment of such ailments do not readily cross the BBB. Novel strategies are being developed to overcome this problem, especially through the use of fusion proteins that can utilize specific BBB transporters to gain access to the CNS parenchyma.176,177 The BBB has to ensure the supply of the CNS with nutrients, the efflux of waste molecules from the brain, restriction of ionic and fluid movements between blood and brain or spinal cord, and protection of the CNS from significant fluctuations in blood ionic composition that might result from intense exercise or ingestion of a large meal. It has a high electrical resistance (up to 2000  /cm2) and effectively regulates the brain/spinal cord microenvironment (i.e. its interstitial fluid) in part by separating neuroactive agents that act both centrally and peripherally.3 Brain capillary endothelium is characterized by tight intercellular junctions, abundant cytoplasmic mitochondria, and a comparatively low pinocytotic rate, this last feature correlating with a lower rate of transcytosis/endocytosis than is observed in peripheral endothelia. The structure of interendothelial junctions that define the BBB is itself highly complex. Two of the most important components of tight junctions are a group of proteins with four transmembrane domains and two extracellular loops, known as occludin and the claudins.3,163 Adherens junctions, which are intertwined with tight junctions, contain

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  Concept of the Blood–Brain ­B arrier and the Neurovascular Unit  45

vascular endothelial cadherin (VE-cadherin), although platelet-endothelial cell adhesion molecule (PECAM) functions as a mediator of homophilic adhesion. Catenins serve to provide linkage between adherens junctions and the endothelial cytoskeleton. Other junctional elements include members of the immunoglobulin superfamily, junctional adhesion molecules (JAMs) and endothelial cell-selective adhesion molecule (ESAM). The brain capillary endothelial cytoplasm contains a number of adaptor and regulatory/ signalling proteins, whose function is to bind to the membranous proteins and modulate their interactions with the actin/vinculin-based cytoskeleton. Among these are zonula occludens 1, 2 and 3 (ZO-1,2,3), calcium-dependent serine protein kinase (CASK), cingulin and junction-associated coiled-coil protein (JACOP). Other proteins vital to signalling and regulation at the BBB include multi-PDZ-protein 1 (MUPP1), partitioning defective proteins (PAR3 and PAR6), membrane-associated guanylate kinase, ZO-1-associated nucleic acid-binding protein (ZONAB), afadin (AF6) and regulator of G-protein signaling 5 (RGS5). It is important to remember that certain periventricular structures within the CNS lack a BBB or contain a porous or ‘leaky’ one, allowing for rapid entry of blood-borne molecules into the local CNS parenchyma. These structures, known as the circumventricular organs (CVOs) and including the median eminence, neurohypophysis, subfornical organ and the area postrema in the floor above the obex of the fourth ventricle, are populated by neurons highly specialized for chemosensitivity and/or neurosecretion; the location of these neurons within the CVOs allows them to respond rapidly to bloodand CSF-borne substances, including potentially injurious ones, and initiate homeostatic neural and neurohumoral responses. Although the neuroanatomic site of the BBB is cerebral capillary endothelium, this distinctive endothelium shows dynamic interactions with numerous other cell types. CNS capillary endothelial cells are usually surrounded by pericytes and/or are ensheathed by astrocytic foot processes; indeed astrocytes are often thought of as the cells that link the BBB to neural function (see also earlier section on interactions between astrocytes and the microvasculature). Bidirectional interactions between CNS capillary endothelial cells and their neighbouring cellular elements have been demonstrated using novel co-culture techniques.19 Molecules hypothesized or proven to be of importance in the maintenance of BBB integrity include TGF , basic fibroblast growth factor (bFGF), glial cell derived neurotrophic factor (GDNF) and angiopoietin-1 (ANG1). Some properties of the BBB are influenced by the glycosaminoglycan-rich basement membrane (especially its components heparin sulphate proteoglycan and agrin) that envelops the basal (abluminal) aspect of cerebral capillary endothelial cells and separates them from adjacent pericytes or the foot processes/end feet of perivascular astrocytes. Several studies have shown that anatomical proximity and interaction between the perivascular astrocytes and the capillary endothelium plays a critical role in determining the properties of the BBB. Astrocytic processes or end feet that are in close proximity to cerebral capillary walls show distinctive anatomic and molecular features, including a high density of orthogonal arrays of particles, which contain aquaporin 4 and the Kir4.1 potassium channel. Axonal terminals

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synapse on the smooth muscle cells of arterioles, whose endothelium may also have BBB functions.3,163,174 Various agents (e.g. ATP, histamine) can affect endothelial physiology and functions by ligand–receptor interactions, which in turn may be coupled to intracellular calcium release. Such ligand–receptor interactions can take place on either the luminal or abluminal aspect of CNS capillary endothelium, with resultant release of molecules into either the blood or the intercellular space of the CNS. There is indeed not only a blood–brain barrier, but also a brain–blood barrier. How do molecules (including toxins) gain entry to the CNS across the BBB at sites where it is intact, i.e. most of the CNS? This depends very much on the size and biological properties of the molecules involved. Water-soluble molecules may pass through interendothelial junctions. Lipid-soluble substances (e.g. barbiturates, ethanol) and small gases such as oxygen and carbon dioxide cross the endothelial cell membranes (both luminal and abluminal) with relative ease. Specific transport proteins exist for several families of molecules, e.g. glucose transporter 1 (GLUT1) for glucose, LAT1 for large neutral amino acids, P-glycoprotein, excitatory amino acid transporters EAAT13, carriers for ciclosporin A, zidovudine, vinca, etc., a list that contains several commonly administered therapeutic agents. Some BBB transport systems are polarized, i.e. present or active on only the luminal or abluminal endothelial membrane, a property that may be induced, at least in part, by astrocytes.19 Genes selectively expressed within cerebral capillary endothelium include those for carrier-mediated transporters, efflux transporters and receptor-mediated transporters.175 Receptor-mediated transcytosis is responsible for the BBB transport of some large proteins, e.g. insulin and transferrin, although others (e.g. plasma proteins, including albumin) cross this barrier by adsorptive transcytosis. The BBB is a highly dynamic structure, vulnerable to modulation by many factors and molecules. Impairment of the normal BBB function may well be one of the most common pathways by which neurological symptoms occur, in entities as aetiologically diverse as CNS neoplasms (vasogenic oedema), HIV and other viral infections, Alzheimer’s disease and a variety of intoxications. Increasing BBB permeability through injury of the brain capillary endothelium (which may also cause severe derangement of normal CNS metabolism) occurs through the action of numerous agents representing various families of molecules, including bradykinin, histamine and glutamate; purine nucleotides; adenosine; phospholipase A2, arachidonic acid, prostaglandins and leukotrienes; interleukins; TNF and macrophage-inhibitory proteins; free radicals and NO.3,163 With CNS inflammation, especially purulent meningoencephalitis, interendothelial junctions may open, leading to severe (sometimes fatal) cerebral oedema with a marked rise in intracranial pressure. Starvation and hypoxia may lead to GLUT1 transporter upregulation at the BBB. Factors that have the ability to cause the opposite effect, i.e. improved BBB function secondary to its ‘tightening’, include steroids, noradrenergic agents and increased intracellular cAMP. Structural or functional alterations of the BBB in disease states are often inferred from neuroimaging studies but less often directly demonstrated by morphoanatomical investigations. Evidence of BBB ‘leakiness’ is usually

1

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46  Chapter 1  General Pathology of the Central Nervous System

surmised, in autopsy brain specimens, by looking for evidence of seepage of serum proteins (e.g. fibrinogen) into perivascular (especially pericapillary) brain parenchyma.174 Such immunohistochemical investigations can be problematic because of post-mortem artefacts, and should therefore be carefully controlled by use of selected comparable ‘normal’ tissue specimens. Even so, when controls are chosen to match age and post-mortem autolysis interval of the ‘disease’ specimen, agonal factors that may influence BBB permeability cannot always be accurately assessed. The other approach to evaluating anatomical integrity of cerebral capillary endothelium – quantitative ultrastructural analysis of brain microvessels – relies on the availability of scarce brain biopsy material that has been appropriately harvested and processed, to ensure preservation of morphological details, which can be studied by electron microscopy.229 Despite these limitations, many studies have clearly demonstrated BBB abnormalities in human disease states. Other mechanisms of disease-related BBB disruption have been inferred from experimental (including transgenic) animal models.163 In acute brain injury, as with CNS trauma and ischaemic stroke, microvascular leakage may lead to fatal cerebral swelling secondary to vasogenic oedema (see later). One mechanism for this in animal studies appears to be hypoxiainduced expression of vascular endothelial growth factor (VEGF).213 BBB abnormalities have been suggested to be a major cause of neurological morbidity in AIDS patients;174 the molecular pathogenesis of this microvascular injury may have to do with direct effects of HIV proteins on cerebral capillary endothelium. HIV-1 Tat protein, for example, has been shown to alter tight junction protein expression and distribution in brain endothelial cells in vitro and can furthermore induce oxidative and inflammatory pathways in the endothelium.7,239 The BBB may be transiently opened in epileptogenic foci that cause intractable seizures.3 BBB abnormalities have also been found in neurodegenerative diseases, e.g. Alzheimer’s disease230 and Parkinson’s disease. In the latter, brain capillary dysfunction may result from reduced efficacy of P-glycoprotein.3 Direct morphometric evaluation of brain biopsies from a small number of AD patients has shown several features suggestive of barrier ‘leakiness’, including diminished mitochondrial density within endothelial cytoplasm (perhaps a correlate of decreased BBB ‘work capability’), an increased number of capillary profiles containing pericytes, and abnormalities of inter-endothelial tight junctions.229,230 The specific role of BBB abnormalities in the progression of such chronic conditions remains to be ascertained; the abnormalities may be relatively non-specific, which does not exclude their playing a part in disease pathogenesis. Brain neoplasms are perhaps the situation in which subacute or chronic cerebral oedema secondary to BBB abnormalities is the greatest cause of direct morbidity and mortality. The molecular basis for this has been suggested to be underexpression of the tight junction proteins occludin, and claudins-1 and -5, as well as malfunction of aquaporin-4 (AQP4).173 AQP4 expression is prominently upregulated around malignant brain tumours. Further discussion of brain tumour-related brain oedema appears elsewhere in this text, as well as the role of microvascular lesions in the pathogenesis of inflammatory/demyelinating conditions (e.g. multiple sclerosis).

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Determinants of Intracranial Pressure and Pressure/­Volume Relationships, and Causes and Consequences of Raised ­Intracranial Pressure The Pathology of Intracranial ­Expanding Lesions An expanding mass lesion within a rigid cranial cavity will trigger a number of closely interrelated events, the first of which is distortion of the adjacent brain.259 The major factor responsible for spatial compensation is a reduction in the volume of intracranial CSF. This is achieved by a reduction in the volume of the cerebral ventricles, subarachnoid space and extracerebral CSF cisterns. Compression of the major intracranial venous sinuses may also contribute to spatial compensation within the cranial cavity. The basic sequence of events can therefore be summarized as local deformity, reduced volume of CSF, shift and distortion of the brain and eventually (in the intact skull) the appearance of internal hernias, i.e. displacement of brain tissue from one intracranial component into another, or into the spinal canal. These displacements result from the development of pressure gradients between intracranial compartments and lead to secondary vascular complications such as haemorrhage and ischaemia.157,261 When the skull surrounding an expanding intracranial mass is not rigid, e.g. the unfused skull in infants or a displaced flap of bone resulting from skull fracture or surgery, displacement of the brain may occur through the bony defect as an external cerebral hernia.

Supratentorial Expanding Lesions Expansion of an intrinsic lesion within a cerebral hemisphere – irrespective of the etiology of the ‘mass’ – results in compression of adjacent brain structures and overall expansion of the hemisphere (Figure 1.27). Sulci on the surface of the brain become narrowed and overlying gyri are flattened against the dura mater, obliterating the subarachnoid space. Reduced cerebral perfusion pressure in a patient with a high intracranial pressure is the major factor causing perisulcal infarcts.107 As the mass continues to expand, the lateral ventricles on the side of the lesion and the third ventricle become reduced in size and there is contralateral displacement of the midline structures: the pericallosal arteries, interventricular septum, thalamus, hypothalamus, third ventricle and midbrain. Clinical and neuroradiological studies have suggested that acute lateral displacement of the midbrain and hypothalamus may be fatal in the absence of established cerebral herniation.156,195 Obliteration of the contralateral foramen of Monro may lead to enlargement of the contralateral lateral ventricle and a further increase in intracranial pressure. The sylvian fissure becomes narrowed and the lesser wing of the sphenoid bone may produce a groove on the inferior surface of the frontal lobe. The floor of the third ventricle is displaced towards the basal cisterns and the mammillary bodies become wedged into a narrowed interpeduncular fossa.

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Determinants of Intracranial Pressure and Pressure/­Volume Relationships 47

(a)

(b)

1

(d)

(c)

(e)

1.27 Cerebral oedema and herniation. (a,b) Coronal slices through the brain from a patient with a recent (24–36 hours) right middle cerebral artery territory infarct. Note the enlargement of the right cerebral hemisphere, with dusky discolouration and effacement of normal anatomical landmarks at the cortex–white matter junction. There is partial effacement of the right lateral ventricle. Arrows indicate bilateral uncal grooving from transtentorial herniation, which is wider on the right (a). Only right uncal grooving is seen in (b). (c,d) Cerebral oedema related to high-grade gliomas. (c) A large left cerebral hemispheric (predominantly frontal) expansile lesion (arrows) after partial surgical resection. There is diffuse left hemispheric oedema. (d) Coronal view of a large infiltrative necrotic and haemorrhagic mass in the left cingulate gyrus (patient died within a few days of biopsy), extending into the corpus callosum (arrowheads). Note subfalcine herniation of the cingulate gyrus, which also shows blurring of its cortex–white matter junction. Arrows indicate one of the biopsy needle tracks, at some distance from the neoplasm. (e) Large right frontal mass lesion (chloroma, indicated by arrows) with pronounced oedema of the right cerebral hemisphere and marked right-to-left shift of midline structures.

Although this sequence of events may occur with any expanding lesion within a cerebral hemisphere, certain displacements are selectively affected by the site of the lesion.

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An expanding lesion in the frontal lobe will produce displacement of the free margin of the anterior part of the falx cerebri; the posterior part of the falx is rarely displaced laterally

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48  Chapter 1  General Pathology of the Central Nervous System

because it is firmly tethered at this level. A lesion in the temporal lobe will produce disproportionately severe shift of the third ventricle and will displace upwards the sylvian fissure and the adjacent branches of the middle cerebral artery. As the lesion continues to expand, the next stage is the development of internal cranial hernias. The major sites of intracranial herniation are at the falx cerebri, tentorium cerebelli and foramen magnum.45,135,154,209

Supracallosal Subfalcine (or ­Cingulate) Hernia Expansion of a mass in the frontal or parietal lobe will eventually result in herniation of the ipsilateral cingulate gyrus under the free edge of the falx to produce selective displacement of the pericallosal arteries away from this lesion and from the midline (Figure 1.27d). This may compromise circulation through the pericallosal arteries and result in infarction of the parietal parasagittal cortex, manifesting clinically as a weakness or sensory loss in one or both legs. A wedge of pressure necrosis may occur along the groove where the cingulate gyrus makes contact with the falx.197 If the brain returns to its normal shape as a result of emergency treatment, this wedge of necrosis can be taken as a reliable marker of previous herniation at this site.

Tentorial (Uncal or Lateral ­Transtentorial) Hernia Any supratentorial expanding hemispheric mass may produce herniation of the ipsilateral uncus and medial part of the parahippocampal gyrus medially and downward through the tentorial incisura; this occurs most frequently when the mass is located in the temporal lobe (Figure 1.28). The width of this hernia is influenced by variations in the capacity of the tentorial incisura,43 as well as the size and location of the mass lesion. As the parahippocampal gyrus herniates, the midbrain is narrowed in its transverse axis and the cerebral aqueduct becomes compressed. The contralateral cerebral peduncle is pushed against the opposite free tentorial edge,69 and the ipsilateral oculomotor nerve becomes compressed between the petroclinoid ligament or the free edge of the tentorium and the posterior cerebral artery. The oculomotor nerve is at first only flattened where it is compressed and angulated over the posterior cerebral artery, but later there is often haemorrhage into the nerve. The resulting paralysis of oculomotor nerve produces ptosis and dilatation of the pupil ipsilateral to the lesion, with loss of the direct response to light shone in the affected eye and of the consensual response to light shone in the opposite eye. There is loss of upward and medial movement of the eye and in its resting position it deviates laterally, because of unopposed action of the VIth cranial nerve. Dilatation of the pupil is the earliest consistent sign of tentorial herniation and may occur before there is any impairment of consciousness.71,108,109,154,189,232,250 As the tentorial hernia enlarges, a wedge of haemorrhagic necrosis appears along the lines of the groove in the parahippocampal gyrus. Compression of the contralateral cerebral peduncle against the free edge of the tentorium may lead to infarction, with or without haemorrhage in the dorsal part of the peduncle and adjacent tegmentum.119 This

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1.28 Wide left uncal grooving (arrows) in autopsy brain specimen from a patient with a large, inoperable brain tumour in the left temporal and parietal lobes.

lesion (Kernohan’s notch) may produce weakness followed by extensor rigidity in the limbs ipsilateral to the expanding lesion. This phenomenon has been studied by magnetic resonance imaging (MRI), and is seen most often in older patients with brain atrophy and a pronounced degree of midbrain shift as a complication of chronic subdural hematoma.40 It is much more common for tentorial herniation to be associated with contralateral limb weakness and eventual extensor rigidity, owing to compression of the cerebral peduncle on the side of the mass lesion by direct pressure from the herniating brain. Any increase in the pressure gradient is commonly associated with abrupt worsening of the patient’s neurological status, such as onset of decerebrate rigidity and loss of consciousness. Expansion of a supratentorial mass lesion may therefore be responsible for initiating tentorial herniation and establishing the beginnings of a transtentorial pressure gradient. Subsequently, any process that would normally induce a diffuse increase in intracranial pressure will increase the transtentorial pressure gradient and accentuate the process of herniation; major degrees of lateral midline shift may cause blockage of the foramen of Monro and narrowing of the cerebral aqueduct, resulting in hydrocephalus.

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Determinants of Intracranial Pressure and Pressure/­Volume Relationships 49

Other sequelae of raised intracranial pressure and tentorial herniation include the compression of arteries; occlusion of the anterior choroidal artery may lead to infarction in the medial part of the globus pallidus, in the internal capsule and in the optic tract. Compression of a posterior cerebral artery, the blood vessel most commonly affected, may lead to infarction in the thalamus, in the temporal lobe including the hippocampus, and of the medial and inferior cortex and subcortical white matter in the occipital lobe. Compression of a superior cerebellar artery may lead to cerebellar infarction. Infarction of the occipital cortex and cerebellum under these circumstances is often intensely haemorrhagic. These vascular effects usually occur on the same side as the tentorial hernia, but may also be bilateral and very occasionally contralateral.236 Clinical and neuroradiological studies of survivors of transtentorial herniation have revealed a spectrum of complications, which range from a transient ‘locked-in’ syndrome263 to more profound neurological deficits, the severity of which is generally related to the degree of herniation as assessed neuroradiologically.244

Central Transtentorial Herniation This form of herniation occurs particularly in response to frontal and parietal lesions or to bilateral expanding lesions such as chronic subdural hematomas. It results from caudal displacement of the diencephalon and the rostral brain stem and may be preceded by a lateral transtentorial hernia. If intracranial pressure (ICP) increases rapidly in association with lesions of this type, both parahippocampal gyri may herniate through the tentorial incisure, leading to the formation of a circular or ring hernia that is most evident posteriorly and may compress the tectal plate. The clinical manifestations are bilateral ptosis and failure of upward gaze, followed by loss of the pupillary light reflex. Although major degrees of ‘diencephalic downthrust’65 are readily identifiable on neuropathological examination, minor degrees of caudal displacement of the brain stem are less easy to identify, even in a properly fixed brain. The evidence for downward axial displacement of the brain stem in the herniation process has emerged from both experimental237 and human post-mortem studies.92 One study196 obtained MR images of a patient showing the clinical manifestations of central transtentorial herniation and failed to establish downward axial displacement of the brain stem. Autopsy studies in patients in whom central herniation has been clinically established show backwards and downwards displacement of the mammillary bodies, compression of the pituitary stalk and caudal displacement of the posterior part of the floor of the third ventricle, which comes to lie below the level of the tentorial incisure. Focal infarction may occur in the mammillary bodies and in the anterior lobe of the pituitary gland, owing to impaired blood flow through the long hypothalamohypophysial portal vessels. The thalamus becomes distorted with elongation of individual neurons, and the oculomotor nerves become elongated and angulated. Infarction in territories supplied by the anterior choroidal, posterior cerebral and superior cerebellar arteries is also a frequent occurrence. The clinical correlates of this state are loss of consciousness, decerebrate rigidity and bilateral dilatation of the pupils with loss of light response. The systemic blood pressure becomes elevated as a result of increased sympathetic

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activity and the heart rate slows. Important areas in the brain stem associated with arterial hypertension appear to be the floor of the fourth ventricle and the nucleus of the tractus solitarius, especially on the left side.68,98 Alterations in respiration are also common.101,237

1

Haemorrhage and Infarction of the ­Midbrain and Pons This is a common and often terminal event in patients with supratentorial expanding lesions, high ICP and tentorial herniation. Emphasis is usually placed on the occurrence of haemorrhage because this is obvious macroscopically, but microscopic examination shows infarction to be at least as frequent as haemorrhage. Both types of lesion occur adjacent to the midline in the tegmentum of the midbrain (Figure 1.29) and in the tegmental and basal parts of the pons. First described by Duret,255 there has always been considerable debate about the pathogenesis of the haemorrhage and ischaemia. The most important factors are likely to be caudal displacement and anterior–posterior elongation of the rostral brain stem caused by side-to-side compression by the tentorial hernia, coupled with relative immobility of the basilar artery. With progressive displacement, the central perforating branches of the basilar artery that supply the rostral brain stem become stretched and narrowed,92 leading to spasm, infarction or haemorrhage (Figure 1.30).111 According to Klintworth,124,125 brain stem haemorrhage is more likely when high ICP and axial brain stem shift have suddenly been reduced by surgical decompression, resulting in an increase in blood flow in the previously ischaemic brain stem.

Tonsillar Hernia (Foraminal Impaction, ­Cerebellar Cone) Downward displacement of the cerebellar tonsil through the foramen magnum occurs as an early complication of

1.29 Transtentorial hernia: brain stem haemorrhage. A large centrally located haemorrhage is present in the midbrain as a consequence of transtentorial herniation. The cerebral peduncle on the right is compressed and distorted. Reproduced with permission from Ironside JW, Pickard JD. Raised intracranial pressure, oedema and hydrocephalus In: Graham DI, Lantos PL (eds). Greenfield’s Neuropathology, 7th edn. London: Arnold, 2002, pp. 193–233.

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50  Chapter 1  General Pathology of the Central Nervous System

remain symmetrical and there is no lateral shift of midline structures.4 Nevertheless, bilateral tentorial hernias may occur, their size depending on the rate and severity of brain swelling and the dimensions of the tentorial incisura. Caudal displacement of the diencephalon and brain stem, and central transtentorial herniation are the major contributors to the neurological dysfunction and vegetative disturbance that may result in a fatal outcome in such patients.

Infratentorial Expanding Lesions

1.30 Transtentorial hernia: brain stem haemorrhage. Histological examination of the midbrain from the case illustrated in Figure 1.29 shows distortion of the white matter and multiple perivascular haemorrhages extending into the parenchyma as a consequence of transtentorial herniation. Reproduced with permission from Ironside JW, Pickard JD. Raised intracranial pressure, oedema and hydrocephalus In: Graham DI, Lantos PL (eds). Greenfield’s Neuropathology, 7th edn. London: Arnold, 2002, pp. 193–233.

expanding masses in the posterior cranial fossa, but may also occur in association with supratentorial space-occupying lesions.113 The pathognomonic indication of this form of brain herniation is haemorrhagic necrosis at the tips of the cerebellar tonsils and a groove on the ventral surface of the medulla, where it is compressed against the anterior border of the foramen magnum. The accompanying distortion of the spinomedullary junction results in apnoea, which may occur at a stage when consciousness is still preserved. However, tonsillar herniation is usually the last in a sequence of intracranial events, at least one of which will already have been responsible for loss of consciousness. Most patients at this stage will also exhibit other abnormal neurological signs, such as decerebrate rigidity and impairment of brain stem reflexes. This latter situation is more likely if the source of raised ICP is a supratentorial expanding lesion; isolated apnoea is usually a sequel to an expanding lesion within the posterior cranial fossa.

Diffuse Brain Swelling When intracranial pressure has become elevated as a result of diffuse brain swelling, the ventricles become small but

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Hydrocephalus, with enlargement of both lateral ventricles and the third ventricle, is the most common abnormality associated with expanding lesions in the posterior cranial fossa, whether they be in the fourth ventricle, within or outside the cerebellum. When the lesion is not in the midline, the aqueduct and fourth ventricle are both compressed and displaced contralaterally. Tonsillar herniation occurs most rapidly with a supratentorial expanding lesion. Occasionally, the posterior inferior cerebellar arteries may be compressed, resulting in infarction in the inferior part of one or both cerebellar hemispheres. Herniation of the superior surface of the cerebellum may occur in an upward direction through the tentorial incisure, and is termed ‘reversed tentorial hernia’. If the posterior cranial fossa lesion has been expanding very slowly, upward herniation of the superior vermis of the cerebellum can produce considerable distortion of the temporal lobes. The clinical manifestations of upward tentorial herniation (Figure 1.31) are sudden appearance of bilateral extensor rigidity and loss of the pupillary light reflex. This is most likely to occur when sudden decompression by CSF drainage of enlarged lateral ventricles is carried out in the presence of an undecompressed expanding lesion in the posterior fossa.

External Cerebral Hernias These occur as rare complications of rapidly expanding supratentorial masses when there is a displaceable defect in the skull, usually surgical or traumatic. This may amount to small protrusions of cortex through cranial burr holes, but if a larger cerebral decompression has been undertaken, major portions of the cerebral hemisphere may herniate through the calvarial defects. Haemorrhagic pressure necrosis occurs

1.31 Upward transtentorial herniation, secondary to a cerebellar mass lesion. Note the notch, indicated by an arrow, most pronounced on the superior aspect of the left cerebellar hemisphere. A small Duret (midbrain) haemorrhage is also noted.

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  Hydrocephalus – ­P athophysiology, Causes and Consequences for the CNS  51

at the edge of the hernia, with swelling of brain tissue within the defect, because of venous obstruction and vasogenic oedema. Continuing herniation will result in extensive ischaemic or haemorrhagic necrosis of the involved cortex and white matter. A recent study of acute intraoperative brain herniation during elective surgery found that most cases were due to extra-axial haemorrhage (subarachnoid or intraventricular), rather than the intraparenchymal haemorrhages and acute brain oedema occurring in patients with severe head injury who undergo emergency neurosurgery.262 Accordingly, the outcome for such elective surgical patients is better than for those with severe head injury.

Hydrocephalus – ­Pathophysiology, Causes and Consequences for the CNS The term ‘hydrocephalus’ defines a state or condition of the brain in which circulation of the cerebrospinal fluid is altered such that there is resultant expansion of fluidfilled intracranial compartments (Figure 1.32).53,54 It can affect individuals at any age, and since the 1950s has been treatable, largely by the use of shunts that redirect excess CSF from the brain or intracranial cavity to another site, usually the peritoneal cavity; these shunts are often encountered at necropsy in the course of brain removal (Figure 1.32). Most of the CSF is produced by the choroid plexus (at a rate estimated to be 500 mL/day;46 because CSF volume ranges between 90 and 150 mL, this is replaced or renewed 4–5 times per day), although brain extracellular fluid is responsible for a significant, though lesser, (a)

amount. Hydrocephalus is a result of decreased clearance of CSF from the lateral and third ventricles through the aqueduct of Sylvius and thence the exit foramina of the fourth ventricle, decreased absorption of CSF into the venous system at the parasagittal arachnoid granulations over the cerebral convexities, or excessive production of CSF from a choroid plexus papilloma or (rarely) choroid plexus hyperplasia, the latter being much the least common aetiology.75 A diagnosis of hydrocephalus is not itself an aetiological diagnosis. Neoplastic, malformative, haemorrhagic, degenerative and other causes of hydrocephalus are considered in their respective chapters. Here we shall deal with cellular/molecular mechanisms germane to the consequences of hydrocephalus for the brain and consider some aetiologies, as well as selected animal models that bear on the pathogenesis. An excellent monograph on the pathology of hydrocephalus, with unsurpassed descriptions of its causes and consequences, was published by Russell almost 60 years ago but remains topical in the twenty-first century.200 Hydrocephalus is not a benign condition and results in deleterious consequences for the brain. These are dependent upon the duration and magnitude of the ventricular enlargement (ventriculomegaly) as well as the age at onset and rate of progression.53,54 Hydrocephalus may cause clinical symptoms that include motor, cognitive and endocrine disturbances. As ventriculomegaly progresses, the ependymal lining of the ventricles is compromised (ependymal cells are largely non-proliferative, see earlier), in particular over the periventricular white matter. Subependymal gliosis may result from movement of CSF into the interstitial space of the brain, with resultant oedema. Periventricular white matter may become rarefied, devoid of oligodendroglia and gliotic.

1

(b)

1.32 Hydrocephalus in a young child. (a) Note massive enlargement of the ventricular system (lateral ventricles are illustrated), with thinning leading almost to obliteration of the subcortical white matter. Despite this, the overlying neocortex appears relatively normal. (b) A young child who had hydrocephalus caused by a posterior fossa mass. The apparent loss of deep grey matter in the right cerebral hemisphere is an artefact of a slightly asymmetrical cut. Note large catheter tip in the right lateral ventricle; shunting was only partly successful in treating the hydrocephalus.

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52  Chapter 1  General Pathology of the Central Nervous System

However, in the authors’ experience, evidence of direct axonal injury in the form of neuroaxonal spheroids is observed only rarely, even in relatively severe hydrocephalus, when the process has progressed slowly over many years. In acute hydrocephalus, there may be petechial haemorrhages and evidence of injured axons, a change that can be highlighted using amyloid precursor protein (APP) immunohistochemistry.59,188 Atrophy of the corpus callosum is commonly observed, as a consequence of both axonal stretching and pressure from above caused by an unyielding falx cerebri. Compression atrophy of the adjacent fimbria/fornix may effectively disconnect the hippocampal formation from the mammillary bodies (and produce trans-synaptic degeneration in the latter), causing memory storage and retrieval deficits. Animal studies suggest that myelin injury, possibly caused by oligodendroglial damage resulting from ventricular enlargement, may antedate axonal disruption.53,54 Hypothalamic nuclei may be distorted or injured by the hydrocephalus (especially in children), possibly explaining the frequent neuroendocrine abnormalities observed in affected individuals. The brain stem and cerebellum are relatively spared in hydrocephalus. The cerebral cortex is comparatively spared of injury except in cases of severe hydrocephalus, in the course of which cortical thinning can occur. This may be accompanied by polygyria, not to be confused with polymicrogyria. Atrophy of the basal ganglia and hypothalamic nuclei may occur. Neurodegenerative changes, when present (identifiable in the form of cytoplasmic shrinkage and vacuolation), are relatively non-specific and thought to most likely reflect retrograde change secondary to axonal injury. There may be loss of dendritic spines and a reduction of synaptic vesicle proteins. Neurofibrillary tangle formation in affected neuronal cell bodies has been attributed to longstanding hydrocephalus in adults, but may simply represent coincidental Alzheimer-type change. Effects of hydrocephalus on the choroid plexus itself are variable and include (subtle) epithelial atrophy, cytological changes, and ‘stromal sclerosis’, possibly reflecting diminished secretory activity.53,54 Findings of unclear significance include alterations of various receptors and glycoproteins in the circumventricular zones of hydrocephalic brains. From a neuroanatomical perspective, it seems logical that a disease process that causes stretching and distortion of axons (coincident with ventriculomegaly) leads to axonal and myelin abnormalities, whereas neuronal alterations may be secondary. Nevertheless, the mechanisms by which these alterations occur are incompletely understood. Molecular phenomena of likely importance in producing these changes include those of aetiologic significance in traumatic brain injury (TBI), another situation in which long axons are extremely vulnerable to stretching and tearing.148 Damage to periventricular axons results in part from calcium-mediated activation of proteolytic calpains, molecules that injure cytoskeletal proteins.53,54 Cerebral blood flow and oxidative metabolism may be reduced, especially in the deep white matter, in infants, children and adults with hydrocephalus; in adults, this process may be aggravated by concomitant hypertension and atherosclerotic cerebrovascular disease. The BBB (see earlier) may serve as an alternative route for regulation of brain water in hydrocephalics and manifest increased pinocytotic activity (which is quite low in normally functioning

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cerebral capillary endothelium). Elevated nitric oxide synthase activity and NO production may play protective roles in hydrocephalus, though the details are poorly understood. Some aetiologies of hydrocephalus will be briefly considered. Acute intraventricular haemorrhage (IVH) in premature neonates is a major clinical problem, especially as increasingly young preterm infants are salvaged.37 Even though the incidence of IVH appears to be declining, it occurs in one in five infants with a birth weight of less than 1000 g. Many are destined to subsequently develop cerebral palsy. The abnormalities in CSF flow in surviving infants that result in hydrocephalus are the direct consequence of meningeal fibrosis, arachnoiditis and subependymal astrogliosis, all of which lead to impairment of its normal flow and absorption. In addition to compressive and ischaemic mechanisms contributing to hydrocephalus in these infants, there appears to be increased parenchymal and perivascular deposition of extracellular matrix proteins, probably as the result of upregulation of TGF . It is of note that astrocytic overexpression of TGF 1 in transgenic mice leads to perivascular astrocytosis, increased perivascular deposition of extracellular matrix proteins such as laminin and fibronectin and striking ventriculomegaly.44 Free radical mediated brain parenchymal injury may also contribute to morbidity after ventricular haemorrhage. Abnormalities of the cerebral aqueduct (Figure 1.33), including agenesis, atresia, gliosis, forking and membranous occlusion, are further discussed elsewhere in this text. The terminology used to describe these aqueductal anomalies is inconsistent and often confusing.12,138,151,242 It has been suggested that aqueductal stenosis can be the

1.33 Aqueductal agenesis – one (comparatively) rare cause of congenital hydrocephalus. This condition may be X-linked. Arrows indicate region of the midbrain where the aqueduct would be expected.

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  Hydrocephalus – ­P athophysiology, Causes and Consequences for the CNS  53

result of hydrocephalus, rather than its cause.264 Rare X-linked forms of aqueductal stenosis have been recognized for almost 60 years.99,133 Hydrocephalus is often discovered in adults, a population in whom it is almost certainly underestimated. The incidence of adult-onset chronic hydrocephalus has been estimated at 2.6 per 100 000;62 in other words, adult-onset hydrocephalus may account for approximately half of the 80 000 cases of hydrocephalus diagnosed in the United States annually. It may represent decompensation of a compensated congenital hydrocephalus (e.g. secondary to aqueductal stenosis) that becomes symptomatic for the first time only in adulthood. Acquired neurological diseases that lead to meningeal fibrosis may result in hydrocephalus; the most common are subarachnoid haemorrhage and meningitis, especially suboptimally treated purulent or granulomatous meningitis. Normal-pressure hydrocephalus (NPH), a type of communicating hydrocephalus, classically presents with the triad of gait disturbance, urinary incontinence and cognitive impairment, though it may manifest only one or two of these symptoms (most often gait disturbance) and can be entirely asymptomatic. The neuropsychological phenomena observed in affected patients include those common in subcortical frontal lobe disorders (inattention, forgetfulness, diminished intellectual agility), as well as apathy, emotional lability and disinhibition. First described in 1965 and sometimes known as Hakim–Adams syndrome, NPH is treated by ventricular shunting.62 Though it is said to account for 5–10 per cent of dementia, NPH is encountered only infrequently in dementia autopsy series, including ours – perhaps because the patients respond well to shunting. At autopsy, NPH manifests as enlargement of the lateral and third ventricles, out of proportion to the degree of cerebral cortical atrophy (Figure 1.34) – a dissociation that is generally not found in AD brain specimens, notwithstanding the fact

1.34 Normal pressure hydrocephalus (NPH). This condition, one potentially treatable cause of cognitive impairment, has caused ventriculomegaly (affecting the third and lateral ventricles) with macroscopic preservation of the cortical ribbon. Note that, unlike in patients with Alzheimer disease, the hippocampi are relatively normal in size, with no evidence of atrophy.

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that not all AD brains are strikingly atrophic. However, a note of caution is in order: when brain biopsies from individuals undergoing shunt placement for idiopathic NPH are carefully examined, they often show evidence of AD-related lesions, in the form of A plaques and, less commonly, amyloid angiopathy, neuropil threads and neurofibrillary tangles;85 the severity of AD-related abnormalities in these study patients correlated with the severity of their dementia.

1

Treatment of Hydrocephalus, and Its Effects on CNS Structure/Function The treatment of hydrocephalus usually includes therapy of the underlying causal condition, when this is possible. For example, treatment of ventriculomegaly secondary to a posterior fossa mass is surgical resection of this mass. Other modalities used to treat hydrocephalus over the decades have included attempts at ablation of the choroid plexus, third ventriculostomy and (most commonly) a shunt that redirects CSF from the brain to the peritoneal cavity or the cardiac chambers.53,54 The introduction of the flexible, biocompatible shunt tubing now used in these procedures has dramatically altered the outlook for people with hydrocephalus, leading to pronounced symptomatic improvement in treated patients. However, although shunting is effective at relieving symptoms caused by hydrocephalus, it does not totally reverse resultant pathological abnormalities. The ventricular size in shunted patients does not always return to normal after shunting. Successful shunting leads to less severe atrophy of the corpus callosum and fornix than would be expected in untreated hydrocephalus, but there is residual periventricular gliosis. This may serve as an impediment to reconstitution of normal white matter in the periventricular region. As indicated earlier, the ependyma has very limited regenerative potential, thus regions of ependymal loss that occur in the evolution of hydrocephalus are not repaired. This may lead to persistent abnormalities of interstitial fluid content within the underlying brain parenchyma. Clinically, shunt placement to treat hydrocephalus is one of the most commonly performed operations by paediatric neurosurgeons and the procedure has completely changed the natural history of hydrocephalus. Before shunting was available, hydrocephalus led to progressive neurological deterioration and early death, often before the third decade of life.179 The procedure, however, is associated with significant complications, including shunt malfunction and shunt infection.179 In a recently published, 20-year follow-up of patients who were younger than age 15 years at the time of their first shunt placement, 2.9 per cent died of shunt failure, 81 per cent required at least one shunt revision, and among those with revision, the mean number of revisions was 4.2.179 Thus, a large National Institutes of Health workshop on hydrocephalus has called for additional research regarding better treatment options and further investigation of ‘pathophysiological and recovery mechanisms of neuronal function’.265 Pharmacological therapy of hydrocephalus has included administration of carbonic anhydrase inhibitors (e.g. acetazolamide, which can reduce CSF production by

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54  Chapter 1  General Pathology of the Central Nervous System

over 60 per cent) and furosemide. Attempts to prevent hydrocephalus in infants following intraventricular haemorrhage (see earlier) by infusion of urokinase, streptokinase and tissue plasminogen activator in affected patients have met with limited success.53,54 Given some of the neurobiological similarities between axonal injury associated with hydrocephalus and brain injury after trauma/stroke, neuroprotective approaches are also being tried.

Acknowledgements This chapter would not have been possible without the photographic assistance of Ms Lisa Litzenberger and Carol Appleton and the manuscript preparation work of Ms Susan Peth.

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2

Chapter

2

Vascular Disease, Hypoxia and Related ­Conditions Raj Kalaria, Isidro Ferrer and Seth Love

Introduction..................................................................................59 Terminology..................................................................................59 Development of the CNS Vascular System.....................................61 Anatomy of the CNS Vasculature...................................................65 Physiology of the Cerebral Circulation...........................................73 Pathophysiology of Cell Death in Ischaemia and Hypoxia...............76 Consequences of Hypoxic Insults..................................................81 Disorders of CNS Hypoxia.............................................................86 Diseases Affecting the Blood Vessels............................................91 Haematological Disorders.......................................................... 125

Introduction The efficient functioning of the central nervous system (CNS) relies on an uninterrupted supply of oxygenated blood and nutrients, particularly glucose. The transportation of these fuels requires sufficient blood flow through a healthy cerebral vasculature with the capacity to respond appropriately to metabolic demands. If the oxygen or glucose content or the flow of blood falls below the level needed to maintain nervous tissue viability, this precipitates a series of acute and longer term changes within the brain parenchyma. The removal of metabolic wastes such as lactate by venous drainage also plays an important role in cerebral function. This chapter describes the regulatory mechanisms that protect brain perfusion and oxygenation, the diseases that affect cerebral blood vessels and the damage to brain tissue that results from disturbances in brain oxygenation and cerebral blood flow (CBF).

Terminology Several purely clinical and radiological terms have come into general use even in pathology to describe specific ­syndromes and lesions. Table 2.1 provides the currently used definitions of key terms and concepts. Hypoxia describes a reduction in oxygen supply or content but is also sometimes applied to conditions in which the metabolic ­utilization of oxygen is impaired (histotoxic hypoxia). Ischaemia means a lack of blood supply but is

Consequences of Cerebrovascular Disorders and Impact on Brain Tissues..............................................................128 Venous Thrombosis and Infarction..............................................162 Small Vessel Diseases of the Brain..............................................163 Haemorrhagic Stroke and Consequences....................................174 Vascular Diseases of the Spinal Cord..........................................183 Vascular Diseases of the Pituitary Gland.....................................187 Acknowledgements....................................................................188 References.................................................................................188

usually used to denote a reduction in blood supply below the level needed to maintain tissue function. The term hypoxia is often combined with ischaemia (hypoxia–­ ischaemia) although, as indicated later, the general or casual use of this expression is not recommended. Tissue hypoxia is a consequence of ischaemia; however, hypoxia is itself anti-ischaemic, as it stimulates an increase in CBF.3,149 In most forms of hypoxia, CBF is increased, so that supply of nutrients and removal of potentially damaging end products continue unabated. During ischaemia, in addition to the decreased blood supply, the removal of damaging metabolites is also impaired. Brain ischaemia may accompany systemic hypoxia in specific circumstances, such as strangulation and cardiorespiratory arrest, in which cases the term describing the particular clinical event should be used unless the CBF or partial pressure of oxygen (pO2) is known. The pO2 is the pressure that the oxygen would exert in a liquid or gas if it alone occupied the total volume, regardless of other molecules that may be present and ­irrespective of the total pressure. Hypoxia and ischaemia are different pathophysiological states. Ischaemia is always pathological but not so hypoxia. Hypoxia is graded as one ascends in altitude; it blends smoothly with physiology but not directly with pathology. Ischaemia interfaces more directly with tissue pathology. In itself, low oxygen tension in the blood is incapable of causing cerebral necrosis, but ischaemia of even 2 minutes957 can result in necrosis within selectively vulnerable brain regions of the brain, e.g. the hippocampus. The term hypoxia is often qualified to indicate whether it refers to the means of delivery or utilization of oxygen. 59

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60  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions Table 2.1  Terminology: definitions of key terms and concepts Term

Description

Type of damage in brain tissue

Comment

Anoxia

No oxygen

Not a specific or useful term by itself

Carries no specific meaning in the intact organism

Anoxaemia

No oxygen in blood

Impossible to assess in intact animal

An impossibility without cardiac bypass and removal of all blood O2

Anaemic hypoxia

Low blood haemoglobin

No brain-damaging potential

Actually protective for stroke because of favourable rheology

Asphyxia

Inability to breathe

Can cause brain necrosis if ­ischaemia results

Includes suffocation, strangulation and some chemicals (cyanide, sulphide, azide) which paralyze breathing centres in ­medulla oblongata

Carbon monoxide (CO) toxicity

CO in blood, displacing O2 from haemoglobin sites

Necrosis in pallido-reticularis, plus typical ischaemic distribution

Complex triad effected: anaemia ­(haemoglobin occupation by CO), histotoxic hypoxia (by binding to iron-rich globus pallidus), and global ischaemia due to heart failure

Haematoma

Localized bleeding (e.g. ­intracerebral, sub-arachnoid or sub-dural) from ruptured ­vessels or aneurysms

Haemorrhagic strokes result in tissue injury by causing compression of tissue from expanding bleeds

Not to be confused with hemiangioma

Hypoxia

Low oxygen, not further specified (tissue, blood, atmosphere)

See specific entities

Not a useful term without further ­qualification

Hypoxia/ischaemia

Combination of hypoxia and ischaemia

Hypoxia and ischaemia cause even greater necrosis

Occurs in strangulation and hanging; widely used incorrectly to describe pure ischaemia; cardiac arrest encephalopathy and global ischaemia are better terms, if that is what is meant

Hypoxaemia

Low oxygen in blood

Reversible synaptic alterations without neuronal necrosis

Seen in respiratory tract disease (larynx, trachea, bronchi, bronchioles), not in pure cardiovascular disease; tends to occur in younger patients; causes tissue hypoxia that is not necrotizing

Hypobaric hypoxia

Hypoxaemia accompanying decrease in ambient pO2

Reversible synaptic alterations (at very high altitudes), but without neuronal necrosis

Temporary synaptic alterations produce ‘high-altitude stupid’ (HAS) syndrome; capillary leakage produces high altitude cerebral oedema (HACE), which is potentially lethal; both reverse on descent or on increasing inspired O2

Histotoxic hypoxia

Tissue utilization of oxygen impaired

No brain-damaging potential without accompanying hypotension

Examples: poisoning by cyanide, sulphide and azide

Ischaemia

Cessation of blood flow to tissue; no perfusion

Variable cellular damage, neurons most vulnerable

Often also used (albeit imprecisely) to describe reduced blood flow — oligaemia

Oligaemia

Low blood flow, hypoperfusion

Selective vulnerability

Close to normal but still insufficient

Tissue hypoxia (global ischaemia)

Low tissue pO2 due to global ischaemia

Necrosis (both pan-necrosis and selective neuronal ­necrosis) in brain regions of selective ­vulnerability

Decreased tissue pO2 due to imbalance between delivery and utilization everywhere in brain

Tissue hypoxia ­(focal ischaemia)

Low tissue pO2 due to focal ischaemia

Necrosis is usually pan-necrosis and does not spare glia

Decreased tissue pO2 due to imbalance between delivery and utilization in focal arterial distribution

Watershed infarction

Localized to the border zones between territories of two major arteries (e.g. anterior cerebral artery [ACA] and middle cerebral artery [MCA] or MCA and posterior cerebral artery [PCA])

Ischaemic injury

Analogous to a lawn watered by multiple sprinklers: occlusion of the hose leads to a dry lawn in the territory centred on a sprinkler (or artery), but low pressure (hypotension) leads to a dry lawn between the sprinklers.

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  Development of the CNS Vascular System  61

Hypoxic, anaemic and histotoxic hypoxia describe states in which, respectively, oxygen supply, blood oxygen transport and tissue utilization of oxygen are impaired (Table 2.1). Hypoxaemia is low blood oxygen content. Anoxaemia is zero blood oxygen (a physiological impossibility). Anoxia is a term often used, although there cannot be total absence of oxygen in the body. Ambient oxygen, however, can be zero, as for example on inhalation of pure nitrogen,449 in drowning939 and in an unscheduled space walk. In clinical brain ischaemia, systemic hypoxaemia is usually absent. Conversely, hypoxic states are usually not accompanied by ischaemia. Pure hypoxaemia of the brain can result in a prolonged coma of 2 weeks, from which a complete and remarkable recovery is possible,360,890 whereas prolonged coma after cardiac arrest or global ischaemia carries a very poor prognosis. Because hypoxia tends to occur in younger patients, recognition of a pure hypoxic insult, without accompanying ischaemia, is important in determining clinical prognosis. Hypoxia, thus, needs to be distinguished from ischaemia, while taking note that at tissue level, ischaemia always causes low tissue oxygenation (tissue hypoxia). In ischaemic injury, the perfusion range between the threshold (in millilitres of blood per 100 grams of brain per minute, or mL/g/min) below which there is impairment of electrophysiological responses or function and the threshold below which irreversible damage occurs (typically around 15–18mL/100g/min) is termed the penumbra. The restitution of flow above the functional threshold can reverse the deficits without permanent damage. However, attempts to define precise ischaemic thresholds below which damage consistently takes place encounter difficulty because this depends on interacting factors including age, temperature, blood glucose concentration, and duration of ischaemia. Magnetic resonance (MR) imaging and MR spectroscopy

are useful for assessing the effects of hypoxia and ischaemia on brain chemistry, structure and function. The term stroke describes an acute disturbance or loss of brain function resulting from brain ischaemia or haemorrhage. The types of stroke and their pathological manifestations are described in detail later in this chapter.

Development of the CNS Vascular System Normal and Abnormal Vasculogenesis and Angiogenesis The formation of the brain vascular system is a tightly regulated developmental process. It involves an intricate interplay between the mesodermally derived vascular cells and the neuroectodermally derived CNS (Figure 2.1). Vasculogenesis is the differentiation of mesodermal precursors into endothelial cells whereas angiogenesis is the formation of new vessels from preexisting vessels or plexuses. Embryonic blood vessels consist of endothelial cells and pericytes that organize and expand into highly branched conduits. This process is controlled by signalling systems involving a large number of specific receptors and their ligands, in addition to mediators of mitogenic, chemotactic, proteolytic and adhesive activities.98,641,778 The regulation of embryonic or pathological angiogenesis when hypoxia- or tumour-induced is predominantly controlled by the same molecules (Table 2.1). There has been much therapeutic interest in tumour-induced angiogenesis, but the understanding of pro- and anti-angiogenic mechanisms has ­implications for cerebral ischaemia, vascular malformations, neurodegenerative disorders, CNS trauma, multiple sclerosis and diabetic retinopathy.98,804,853 The development

Vasculogenesis FGF2

Angiogenesis

VEGF

VEGFR2

FGFR

2

VEGFR1

Ang1

VEGF

Ephrin

Tie2

VEGFR1,2

EphR

PDGF

TGF

PFGFR TGF R

PDGF Mural cell Pericyte Mesoderm cells Haemangioblast Haemangioblastic EC proliferation cord and tube formation recruitment

Pruning Connection, maturation and remodelling /remodelling

Haematopoietic cells

2.1 Processes of vasculogenesis and angiogenesis in the brain. Growth factors regulate differentiation of mesodermal cells into haemoangioblasts, which give rise to endothelial cells that proliferate to form cords and capillary tubes. Pericytes are recruited as support cells, with concomitant basal lamina production. Multiple growth factors activate specific receptors to model and prune branching vessels. Ang1, angiopoietin; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; TGFβ, transforming-growth factor β; TGFβR, TGFβ receptor; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor. Adapted from Augustin et al.76 and Patel–Hett and D’Amore.778 Diagram courtesy of Y Yamamoto, Yamaguchi University Graduate School of Medicine, Japan.

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62  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

of the vascular system (Box 2.1) is initiated around the third week of gestation, when splanchnopleuric mesodermal precursor cells at the periphery of blood islands in paraxial mesoderm differentiate into haemangioblasts. This occurs under the influence of mesoderm-inducing factors of the fibroblast growth factor family, which interact with vascular endothelial growth factor receptor 2 (VEGFR-2) on mesodermal precursor cells. The haemangioblasts differentiate into vessel-forming angioblasts and haematopoietic stem cells. Angioblasts cluster and acquire lumina, to form interconnecting tubes that constitute the primitive vascular plexus. Angioblasts from the splanchnopleuric region migrate into the head region to form a perineural vascular plexus around the developing brain (extracerebral vascularization). Later, this plexus develops into meningeal arteries and veins. After development of the primitive perineural vascular plexus, brain blood vessels are formed (intracerebral ­vascularization) by capillary sprouts from the pre-existing vessels in this plexus. New blood vessels in adult organs are formed by similar angiogenesis. The capillary sprouts penetrate the developing CNS and extend into the subventricular zone, forming a new plexus there. Next, another capillary plexus is formed in the intermediate zone between the subventricular precursor cell zone and the cortical plate. Finally, the cortical plate is vascularized from the deep ­layers outwards. Hypoxia is considered to be the major regulator of angiogenesis (Box 2.1). In the presence of oxygen, the α-subunit of hypoxia-inducible (transcription) factor 1 (HIF-1α) is hydroxylated by HIF-prolylhydroxylases, binds to a multimolecular complex that includes von Hippel–Lindau (VHL) tumour suppressor protein, and is rapidly degraded in proteasomes. Hypoxia inhibits the hydroxylation of HIF-1α and, as a result, constitutively expressed HIF-1β can bind to HIF-1α. Heterodimeric HIF-1 thus accumulates and is translocated to the nucleus where it binds to hypoxiaresponsive elements to activate transcription of numerous hypoxia-inducible genes (estimated at 2–3 per cent of all

genes), including genes of the pivotal vascular endothelial growth factor (VEGF) family (Figure 2.2).174 Perineural angioblasts differentiate into endothelial cells, which are attracted by different chemotactic factors. A most important chemotactic stimulus is VEGF, produced by neural progenitor cells in the periventricular matrix zone, towards which the VEGFR-2-expressing cells migrate.188 VEGFR-2 is essential for the proliferation of endothelial cells, their migration and survival.935 Endothelial and blood cells are not formed in mice lacking VEGFR-2. VEGFR-1 is expressed later than VEGFR-2; the former acts as a negative regulator of VEGF and seems necessary for the assembly of angioblasts into functional blood vessels. The primary endothelial structures need to be stabilized by the formation of pericytes and smooth muscle cells, in which process angiopoietin-1 (Ang-1) ligand binding to TIE-2 receptors on endothelial cells is essential.76,946 This stabilization through Ang-1/TIE-2 signalling also regulates

�� �

� ��

O2 2-oxoglutarate Prolyl hydroxylase

HIF-1

Fe 2

Hypoxia

CO2 succinate P HIF-1 P

OH

HIF-1

VHL cofactors E3 ubiquitin ligase complex

HIF-1 Ub Ub Ub Ub

P HIF-1 P

HIF-1

Box 2.1.  Key events in the development of the cerebral vasculature ●●

●●

●●

●●

●●

●●

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Mesodermal precursor cells in paraxial mesoderm differentiate into haemangioblasts, induced by FGFs signalling via VEGFR-2 on precursor cells. Angioblasts form the perineural vascular plexus around the developing brain (vasculogenesis, extracerebral vascularization: leptomeningeal vascularization). Capillary sprouts emerge from the primitive plexus (angiogenesis) and penetrate into the brain, beginning from deeper layers upwards (intracerebral vascularization). Angiogenesis is downregulated postnatally and follows the metabolic needs of the growing CNS. In adult humans, fewer than 1 per cent of the endothelial cells proliferate. Angiogenesis may be re-upregulated, for example in ischaemia (the most important cause of the reactivation), upon metabolic demand and in neoplasia. Bone marrow derived endothelial progenitor cells (EPCs) also promote postnatal vasculogenesis and neovascularisation in ischaemic tissues. Their contribution is confirmed by the presence of various cytokines and other secreting proangiogenic factors in EPCs, such as VEGF and Ang-1.

p300/CBP

P HIF-1 P

co-activator

5´-[A/G]CGTG-3´ HIF-1 degradation

VEGFA, EPO SLC2A1, PKM2 RORyt

2.2 HIF-1 stabilization and activity under normoxic and hypoxic conditions. Under normoxia, hydroxylation at specific proline residues leads to binding of HIF-1α to VHL followed by HIF-1α destruction via the ubiquitin/proteasome pathway. During hypoxia, HIF-1α subunit is stabilized and dimerizes with the ubiquitously expressed HIF-1β subunit. Nuclear translocation is initiated, followed by binding of the HIF-1 heterodimer to hypoxia response elements (HREs) of enhancers and promoters of specific target genes. OH, hydroxyl group; VHL, von Hippel– Lindau tumour suppressor protein; P, phosphorylated subunit. Diagram adapted from Trollman and Gassmann1021 and redrawn courtesy of Y Yamamoto, Yamaguchi University Graduate School of Medicine, Japan.

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  Development of the CNS Vascular System  63

the permeability of developing vessels. Sprouting and induction of further angiogenesis in mature vessels require destabilization of endothelium and pericyte ­ contacts by angiopoetin-2 (Ang-2), an antagonist of Ang-1. Another set of ligands and receptors, ephrins, play a decisive role in determining the arterial or venous identity of the vessels (Figure 2.1; Table 2.2). Other signalling pathways with precise functions in endothelial-mural cell interactions during vascular development and maturation involve platelet-derived growth factor-B (PDGFB) and transforming growth factor-β (TGF-β) and their respective receptors.323 During sprouting, the extracellular matrix is degraded by proteolysis, enabling migration of the proliferating endothelial cells. The blood vessels penetrating the neuroectoderm form intracerebral branches of various sizes. This, together with the regression of supernumerary vessels, creates the vascular tree. The blood vessels mature,

through recruitment of pericytes and, in larger blood vessels, also smooth-muscle cells and fibroblasts and the formation of contacts with astrocytic processes to form a tight blood–brain barrier (BBB) (see Chapter 1). The active phase of angiogenesis ceases soon after birth, after which the cerebral vasculature is expanded only to meet the needs of the growing brain, mainly by elongation of the pre-existing blood vessels. Experimental evidence suggests complete new ‘loops’ are formed from existing vascular networks.406 In the adult brain, angiogenesis is normally minimal (fewer than 1 per cent of endothelial cells incorporate thymidine) but may be reactivated in pathological conditions,395 e.g. hypoxia, ischaemia, trauma and neoplasia. The newly activated angiogenesis is mainly regulated by the same signalling molecules as during development (Figure 2.1; Table 2.2). Because VEGF and its receptors are hypoxia-inducible, their expression is upregulated in glial

2

Table 2.2  Key mediators in neovascularisation including vasculogenesis and angiogenesis Parent molecule

Isoforms/ Receptors

Gene(s)

Function

Hypoxia-inducible (transcription) factor 1 (HIF-1)

HIF-1α; HIF-1β

HIF1A; HIF1B

In the presence of oxygen HIF-1 is degraded, but it persists under conditions of hypoxia. During hypoxic episodes, HIF-1 is translocated into the nucleus and binds to hypoxia-responsive elements (HREs) of several genes, including vascular endothelial growth factor (VEGF) family, to activate their transcription (Figure 2.2) and regulate angiogenesis.

Fibroblast growth factor 2 (FGF2)

Basic FGF

FGF2

FGF family members bind heparin and possess broad mitogenic/ angiogenic activities. FGFs activate receptors (FGFRs) on endothelial cells or indirectly stimulate angiogenesis by inducing the release of angiogenic factors from other cell types.

Vascular endothelial growth factor A (VEGFA)

VEGFR-1 (FLT1); VEGFR-2 (KDR, Flk-1)

VEGFA FLT1

VEGFA is expressed in neuroectodermal cells and is the ligand for VEGFR-2 on endothelial cells. VEGF is an important chemotactic stimulus for endothelial cells (ECs). VEGFR-2 is essential for the proliferation, migration and survival of ECs.

Angiopoietin-1 (ANG1)

TIE-1; TIE-2

ANGPT1 TIE1 TEK

The ANG and TIE family binary switch mechanism allows vessels to maintain quiescence, while remaining able to respond to angiogenic stimuli. ANG-1 is the ligand binding to TIE-2 receptors on perivascular cells to induce formation of pericytes and smooth muscle cells around the vessels.

Ephrin

EPH receptor

EPHRIN EPHB4

EPH receptors and their ligands, the ephrins, regulate cell-contactdependent patterning and can generate bidirectional signals. Ephrin-B2 and its receptor EPHB4 regulate vessel morphogenesis by several mechanisms.

Delta-like ligand (DLL4)

NOTCH

DLL4 NOTCH

NOTCH signalling is involved in vessel-branching; tip cells migrate and stalk cells proliferate. EGFR-2 upregulates DLL4 expression in tip cells and in neighbouring stalk cells, DLL4 activates NOTCH, which modulates VEGFR-2 and VEGFR-1.

Platelet-derived growth factor (PDGF)

PDGFR-β

PDGFB PDGFRA PDGFRB

The ligand/receptor pair PDGFB/PDGFR-β are involved in pericyte recruitment.

Transforming growth ­factor β (TGFβ)

TGFβR1, TGFβR2

TGFB1 TGFB2 TGFBR1 TGFBR2

TGFβ regulates proliferation, differentiation and survival of many cells. TGFβR binding activates multiple intracellular pathways resulting in phosphorylation of receptor-regulated SMAD (small ‘mothers against’ decapentaplegic) proteins. Both TGFβ1 and 2 stimulate expression of VEGF, plasminogen activator inhibitor and certain metalloproteinases involved in vascular remodelling, angiogenesis and degradation of the extracellular matrix.

Chemokine (C-X-C motif) ligand 12 (CXCL12)

Chemokine (C-X-C motif) receptor 4 (CXCR4)

CXCL12 CXCR4

CXC ligand 12 is a haemostatic chemokine, whose major function is to regulate haemopoietic-cell trafficking.

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64  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

cells or endothelial cells at the periphery of infarcts.98 VEGF expression in glioblastomas, especially in the perinecrotic palisading cells, is enhanced up to 50-fold and accompanied by a parallel increase in VEGFRs on the endothelium of the neoplastic blood vessels. Analogous upregulation occurs in the angiopoietin/TIE systems.834 Studies in mice challenge the previous notion of CNS angiogenesis as a passive process driven primarily by demands for oxygen and other nutrients by the growing neuronal populations. Angiogenesis in the mouse telencephalon progresses in an orderly, ventral-to-dorsal gradient regulated in a cellautonomous manner by compartment-specific homeobox transcription factors.1042 These same transcription factors, Nkx2.1, Dlx1, Dlx2 and Pax6, confer compartmental identities on telencephalic neurons and progenitor populations and therefore regulate the development of telencephalic neuronal networks and vascular networks, underscoring shared mechanisms in CNS vascular and neuronal development.1042 The identification of endothelial progenitor cells (EPCs) in peripheral blood as haematopoietic cells with the ability to differentiate into endothelial cells has also changed the dogma that vasculogenesis is only an embryogenic process.59 CXCL12, a chemokine involved in both embryonic and tumour angiogenesis, may mediate signalling to promote the formation of new vessels by recruiting circulating EPCs or directly enhancing migration or growth of endothelial cells.601,992

Hypoxia-independent tumour angiogenesis occurs in haemangioblastomas in patients with VHL, because the loss of VHL tumour suppressor gene function hinders degradation of HIFs, which thus accumulate and upregulate hypoxiaregulated genes in the absence of hypoxia (Figure 2.2). It has also been suggested that some tumours may create vascular channels lined by tumour cells instead of endothelium, a phenomenon called vascular mimicry, which was first described in melanomas305 and has been claimed to occur even in astrocytomas.1116 The orderly formation of the vasculature is vulnerable to disruption at several stages, resulting in vascular malformations of the brain (VMBs). The identification of gene mutations and risk factors associated with cerebral cavernous malformations (CCMs), sporadic brain arteriovenous malformations (AVMs), and the arteriovenous malformations of hereditary haemorrhagic telangiectasia has provided new insights and enabled the development of genetic testing and animal models for these diseases.590,1065 Genes associated with angiogenesis and vascular remodelling are involved in VMBs (see Table 2.3). Studies suggest the angiogenic process most severely disrupted by VMB gene mutations is that of vascular stabilization subsequent to the ­formation of vessel tubes and recruitment of vascular smooth muscle cells.590 Mutations in VMB genes also render vessels vulnerable to rupture when challenged by other genetic or environmental factors.

Table 2.3  Genes and protein products involved in vascular malformations and aneurysms Classification

Gene

Protein product

Putative Function

CCM1

KRIT1

KRIT1, ankyrin repeatcontaining protein

Binds β-catenin, stabilizes interendothelial junctions associated with actin stress fibres. Three CCM genes (CCM1, CCM2, and CCM3) have been identified to date. All 3 corresponding proteins are expressed in vascular endothelium and associated with cytoskeletal and interendothelial junction proteins and components of certain signal transduction pathways.

CCM2

CCM2

Malcavernin

Cellular responses to osmotic stress; modulates mitogenactivated protein (MAP) kinase and RhoA GTPase signalling and is part of a complex with MAP2K3, MAP3K3 and RAC1.

CCM3

PDCD10

Programmed cell death protein 10, TF-1 cell apoptosis-related protein 15

Cell proliferation and transformation (cancer cell lines); modulates extracellular signal-regulated kinase (ERK).

HHT1

ENG

Endoglin, CD105

TGFβ superfamily co-receptor; modulates signalling by TGFβ type II receptor, ALK-1 and ALK-5. Mutations in ENG or ALK1(ACVRL1) cause HHTs.

HHT2

ACVRL1

ALK-1, Activin A receptor type II-like 1, serine/threonine-protein kinase receptor R3

Type I receptor for TGF-β family ligands BMP9/GDF2 and BMP10. It is an important regulator of normal blood vessel development. On ligand binding, forms a receptor complex consisting of two type II and two type I transmembrane serine/threonine kinases.

HHT (a juvenile form)

SMAD4

Mothers against decapentaplegic homologue 4, deletion target in pancreatic carcinoma 4

Common downstream mediator of multiple TGFβ superfamily signalling pathways.

CCM, cerebral cavernous malformation; ECs, endothelial cells; HHT, hereditary hemorrhagic telangiectasia.

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  Anatomy of the CNS Vasculature  65

Anatomy of the CNS Vasculature

(a)

2

Arterial Blood Supply General aspects The metabolism of the brain is almost solely aerobic. The brain lacks significant energy reserves and requires a continuous supply of well-oxygenated blood. The exceptionally high demand for circulating blood and oxygen is reflected in the disproportionately high rate of CBF compared with flow to other parts of the body: 20 per cent of the cardiac output and 15 per cent of oxygen consumption in an adult at rest, although the brain makes up only 2 per cent of the body weight.

Large Arteries of the Brain The blood to the brain is supplied by two pairs of large arteries. The anterior flow (approximately 70 per cent of CBF) enters the cranial cavity through the internal carotid arteries, and the posterior flow (approximately 30 per cent of CBF) through the vertebral arteries, which fuse to form the basilar artery. These two systems anastomose via the anterior and posterior communicating arteries at the base of the brain to form the circle of Willis (Figure 2.3).299,402 There can be considerable variation in the relative size of the vertebral arteries and those forming the circle of Willis.239 In the most common variants of the circle, there is hypoplasia of the precommunicating segment of the anterior cerebral artery (A1) or the posterior cerebral artery (P1).275 Variations in the circle of Willis may affect the relative flow rates in proximal arteries with the circle.984 Another important anastomotic pathway is between the external and internal carotid arteries via the ophthalmic arteries. When this anastomotic network is normal, then the occlusion of even one of the four main arteries will not necessarily lead to insufficient regional CBF. For example, collateral circulation is usually sufficient for slow occlusion of the distal segment of an internal carotid artery (ICA) by atherosclerosis or surgical clamping for treatment of a fusiform aneurysm, not to cause infarction.389 Patients surviving with only one patent vertebral artery demonstrate the efficiency of the collateral circulation. The perfusion territories of the main arteries in the brain and brain stem are illustrated in Figures 2.4 to 2.7.

(b)

ACA

MCA AcoA ICA PcoA

PCA SCA

BA AICA

VA

Leptomeningeal and Pial Arteries The leptomeningeal anastomoses are in the subarachnoid space over the surface of the brain, where the territories of the distal branches of the anterior, middle and posterior cerebral arteries overlap in the border or watershed zones (Figure 2.4a–c). Corresponding border zones are also formed between the superior and inferior cerebellar arteries (Figure 2.4d). The leptomeningeal anastomoses are located at the periphery of the arterial trees and these zones tend to be the first to be deprived of sufficient blood flow in the event of arterial hypotension or a reduction in perfusion due to raised intracranial pressure.

Intraparenchymal Arteries The branches of the arteries running in the subarachnoid space penetrate the brain parenchyma (Figure 2.8). These branches include both deep and superficial perforating

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PICA ASA

2.3 Main arteries of the circle of Willis. (a) Structure of the main arteries that anastomose to form a complete circle, in this case dissected from the base of the brain of a 70-year-old man. Some atherosclerosis can be seen. Note the small calibre of the posterior communicating arteries, which are often narrower in older people. (b) Schematic diagram of the circle of Willis showing the most common sites of atheroma (circles). ACA, anterior cerebral artery; AcoA, anterior communicating artery; AICA, anterior inferior cerebellar artery; ASA, anterior spinal artery; BA, basilar artery; ICA, internal carotid artery; MCA, middle cerebral artery; PCA, posterior cerebral artery; PCoA, posterior communicating artery; PICA, posterior inferior cerebellar artery; SCA, superior cerebellar artery; VA, vertebral artery. Images kindly provided by T Polvikoski and A Oakley, Newcastle University, UK.

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66  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions (a)

4c

(b)

4 4b

5d

1

5e

5d

5f

5

5g

7

5h

6

5b

3b

5l

3

5c

3a

5k

2

5j

2a

5i

2d

(c)

(d) 4f 4e 4g 4d 4 4c 6g 6f 6 6e

6c 6b 6a 6 3b 3

3a 2a 2

2.4 Perfusion territories of the cerebral and cerebellar arteries (see also Figure 2.6). (a) Basal view; (b) lateral view; (c) midline view; (d) cerebellum and brain stem. The border zones (watershed areas) between the territories are indicated by shading. 1, internal carotid artery; 1a, anterior choroidal artery; 2, vertebral artery; 2a, posterior interior cerebellar artery; 2b, paramedian branch; 2c, lateral bulbar branch; 2d, anterior spinal artery; 3, basilar artery; 3a, anterior inferior cerebellar artery; 3b, superior cerebellar artery; 3c, paramedian branch; 3d, short circumferential branch; 4, anterior cerebral artery; 4a, recurrent artery of Heubner; 4b, anterior communicating artery; 4c, medial orbitofrontal artery; 4d, frontopolar artery; 4e, callosomarginal artery; 4f, internal frontal branches; 4g, pericallosal artery; 5, middle cerebral artery; 5a, lenticulostriate artery; 5b, upper division; 5c, lower division; 5d, lateral orbitofrontal artery; 5e, ascending frontal (candelabra) branch; 5f, central (Rolandic) artery; 5g, anterior parietal artery; 5h, posterior parietal artery; 5i, temporal polar artery; 5j, anterior temporal artery; 5k, posterior temporal branches; 5l, angular artery; 6, posterior cerebral artery: 6a, quadrigeminal artery; 6b, posterior choroidal artery; 6c, thalamogeniculate artery; 6d, thalamo-perforating artery; 6e, anterior temporal artery; 6f, posterior temporal artery; 6g, calcarine artery; 6h, paramedian branch; 6i, short circumferential branch; 6j, long circumferential branch; 7, posterior communicating artery; 7a, hypothalamic artery. Adapted from Romanul.858  With permission from Wolters Kluwer.

a­ rteries. The deep perforators branch off the main ­cerebral arteries at the base of the brain and consist of (i) the lenticulostriate arteries (LSAs), which emerge from the first segments of the anterior and middle cerebral artery (MCA) to supply the basal ganglia, and (ii) perforant branches, which leave the posterior cerebral and posterior communicating arteries and supply the thalamic nuclei. In the striatum, the

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distribution of microvascular territories from the lateral LSA, medial LSA and the recurrent artery of Heubner coregister with each of the functional corticostriatal zones, the sensorimotor, associative and limbic zones, with greater density of both large penetrating vessels and small pre-­capillary arterioles and capillaries within the matrix compared to the striosomes.282 The superficial perforators originate from the

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  Anatomy of the CNS Vasculature  67

2

2.5 Arterial supply territories depicted in an adult brain sliced in the coronal plane. The extent of the three main arterial territories in the cerebrum along the rostro-caudal plane are shown: anterior cerebral (magenta), middle cerebral (blue) and posterior cerebral (yellow). The vascular supply to the striatum (delineated in green) includes the lateral lenticulostriate arteries, medial lenticulostriate arteries and the recurrent artery of Heubner (most medial), all of which branch off from the middle cerebral artery.282 The hippocampal formation is perfused predominantly by the posterior cerebral artery with a minor contribution from the anterior choroidal artery. The slices include several small infarcts (white circles) in the right frontal lobe, in the territory of the middle cerebral artery. Original material from the Newcastle Cognitive Function After Stroke study and kindly provided by T Polvikoski, Newcastle University, UK.

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68  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

4

4 5 5a 4a

4

5

5a 4a 7a 1a

5

6

4

4

4

4

5

5

5

5a

6b

7a 1a

6b 6d 7a

5a 1a 6

6

5a 6c 1a

6b 6d 7a 6

6b

4 4 5

5

6b 6c

1a

6

6 3b 6j

1a 6i

3b

6j

1a

6h

6h 3a 3b

3c

3c

6h 3c

3a 3a

2a

3a

3d

3d

3d

3b 3d

6i

2c 3c

2c 2b

2b

2.6 Arterial supply territories in coronal planes of the cerebrum and brain stem in greater detail. The key to the numbers is in the legend to Figure 2.4. Adapted from Romanul.858 With permission from Wolters Kluwer.

pial branches of the anterior, middle and posterior cerebral arteries over the surface of the brain and are of variable length: short ones supply the cortex and longer ones (the medullary arteries) the deep white matter. Short penetrators also exist in the brain stem as paramedian branches of the basilar artery. The perforators are end arteries, i.e. they have very limited collateral connections with neighbouring blood vessels until they branch into capillaries. The capillaries do interconnect but their collateral flow is so local and restricted that the occlusion of a perforator usually results in a small region of ischaemic damage, described as a lacunar infarct (see later). The deep and superficial perforators do not

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anastomose deep in the brain but meet in junctional zones (Figure 2.8a), where subcortical infarction may occur.121 There is increasing interest in the magnetic resonance imaging of structural changes with high tensile magnets (e.g. 3 and 7 Tesla) that allow identification of small lacunar lesions in vivo (Figure 2.9).

Arteries of the Spinal Cord The main arterial blood supply of the spinal cord is provided by one (occasionally two) anterior and two posterior spinal arteries (Figure 2.10). Branches from vertebral arteries join in the midline to form the (usually) single anterior

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  Anatomy of the CNS Vasculature  69

2

2a 3 3a 3b 6 1a 4 5 5a

2.7 Maps of arterial supply territories in horizontal planes through the cerebrum, cerebellum and brain stem. The key to the numbers is in the legend to Figure 2.4. Adapted from Sacco888 and Savoiado.903 With kind permission from Springer Science and Business Media.

spinal artery, responsible for the greater part of the blood supply to the spinal cord. The anterior spinal artery may be of variable size or even discontinuous at different levels, depending on the pattern of replenishing tributary arteries along its passage downwards. Posterior spinal arteries are even more irregular, deriving from vertebral or posterior inferior cerebellar arteries. Along the spinal canal, branches from larger arteries coursing along the spine enter the spinal canal through the intervertebral foramina and give off (i) a dural branch, which supplies the dural sleeve around the root, then (ii) a branch to serve the nerve roots and finally (iii) a medullary (tributary) branch to replenish blood to the anterior and posterior spinal arteries (Figure 2.10b). The term ‘radicular artery’ is commonly used to describe the combination of these three branches, not solely the branch supplying the nerve roots. The number of the tributary radicular arteries varies from 4 to 10. In the superior (cervicothoracic, C1 to

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T1–2) territory, the tributary arteries emerge from vertebral arteries and costocervical or thyrocervical trunks (Figure 2.10a). In the intermediate (midthoracic T1–2 to T8) and inferior (thoracolumbar, T9 to conus medullaris) territories, the tributary arteries emerge from the intercostal or lumbar arteries as their dorsal rami (Figure 2.10b). The most important and largest tributary artery is arteria radicularis magna (of Adamkiewicz), which enters the spinal canal at a variable level, between T8 and L2, below which spinal artery blood flow is mainly downward. A border zone is created, usually at a lower thoracic level than the traditionally stated T4.193,975 The anterior spinal artery gives off sulcal (also called sulco-commissural or central) arteries. These supply the anterior central part of the cord, in an alternating pattern on the left and the right side (Figure 2.10c). This alternate pattern of distribution explains why occlusion of a sulcal artery can manifest as spinal hemisection, typically the

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70  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions (a)

(b)

1 2 3

4

2.8 (a) General pattern of cerebral penetrating arteries. (b) The cortical penetrators reach three different depths (1–3). The longest penetrators (4) continue into the white matter. Adapted from Romanul.858

(a)

(b)

(c)

2.9 Sub-cortical infarction. (a) CT scan in an 82-year-old female with longstanding arterial hypertension, who had a small, clinically silent lacuna in the right basal ganglia (arrow). She died acutely of a large fresh atherothrombotic infarct in the territory of the left middle cerebral artery, which appears hypodense in this scan. (b) Several lacunar infarcts in lenticulostriate territories seen bilaterally on T2-weighted MRI in the axial plane of an elderly subject. (c) The lacunar change seen by CT scan was confirmed as a lacunar infarct (extreme right arrow) elongated in the direction of the lenticulostriate perforating arteries. There are also perivascular cavities (left arrows), best seen in the left caudate nucleus.

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  Anatomy of the CNS Vasculature  71

2

Basilar artery Vertebral artery Vertebra

Anterior spinal artery

Anterior spinal artery

Spinal branch of costocervical trunk C1–7

C4 spinal branch of vertebral artery Costocervical trunk

Radial veins Medullary artery (anterior branch)

Thyreocervical trunk Subclavian artery Spinal branch of thyreocervical trunk

Coronal venous plexus

Anterior spinal artery T4 (T1–T5) intercostal artery (spinal branch)

Medullary vein

Radicular artery from dorsal ramus intercostal artery

T7 (T6–T10) intercostal artery (spinal branch)

T1–12

Medullary artery (posterior branch)

Dural artery

Left posterior spinal artery Anterior branch Medullary artery

T12 (T8–L2) intercostal artery (spinal branch) (Adamkiewicz artery) Dural branch

Anterior spinal artery Renal artery

Artery to nerve roots Posterior branch

(b)

L1–5 Common iliac artery (a)

Posterior spinal artery

(c)

Anterior spinal artery

Sulcal artery

2.10 (a) Blood supply to the spinal cord. The main anterior (usually single) and posterior (usually paired) spinal arteries arise from the vertebral arteries, and receive tributaries from the intraosseous vertebral, intercostal, lumbar and other arteries that enter the spinal canal through the intervertebral formina at multiple levels. The levels at which the different tributaries enter the spinal canal vary considerably. (b) Blood supply of the spinal cord at a segmental level. (c) Blood supply within the spinal cord. The anterior spinal artery gives rise to sulcal arteries. In the depths of the anterior median fissure, alternate sulcal arteries deviate either left or right to supply the corresponding side of the cord. (b) Adapted with permission from Rosenblum et al.868 (c) Reproduced with permission from DeGirolami and Kim.238 © 2005 Wiley-Blackwell.

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72  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

Brown–Séquard syndrome. The branches from the posterior spinal arteries supply the posterior horns and columns. The circumferential vascular plexus receiving blood from the radicular and spinal arteries supplies the superficial parts of the cord (Figure 2.10b,c).

Venous Sinus Systems Venous Drainage of the Brain The venous circulation of the brain (Figure 2.11) differs from the common, consistent antiparallel (i.e. running in parallel but in opposite directions) orientation of arteries and veins in most other organs. In addition, the cerebral venous drainage employs the dural sinuses as the final intracranial collecting blood vessels.39,858 The blood from most of the white matter and cortex of the cerebral hemispheres is drained by veins of various lengths, antiparallel to the pial penetrating arteries. In general, there are fewer veins than perforating arteries and the long veins also drain the cerebral cortex while coursing through it. After the veins of the superficial or cortical network exit the parenchyma and enter the subarachnoid space, they turn towards the dural sinuses. In the suprasylvian and paramedian regions, the frontal, parietal and occipital superior cerebral veins run upward to drain into the superior sagittal sinus. In the parasylvian region, the middle cerebral veins drain via the superficial sylvian vein

1 10 13

11 11 10

9 13 16 1

18

15 12

1

4

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3

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2a

7 2b 5

2.11 Anatomy of venous drainage pathways of the brain. In the case of paired blood vessels, only the right is shown in dark colour. The veins in black are on the surface of the brain; those depicted with dashed lines are within the parenchyma. 1, superior sagittal sinus; 2a, transverse portion of lateral sinus; 2b, sigmoid portion of lateral sinus; 3, confluence of sinuses; 4, straight sinus; 5, internal jugular vein; 6, superior petrosal vein; 7, inferior petrosal vein; 8, cavernous sinus; 9, inferior sagittal sinus; 10, frontal veins; 11, parietal vein; 12, occipital vein; 13, vein of Trolard; 14, vein of Labbé; 15, great vein of Galen; 16, internal cerebral vein; 17, basal vein; 18, superficial sylvian vein. Adapted from Ameri and Bousser.39 With permission from Elsevier.

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to the cavernous sinus. On the posterior lateral and inferior surfaces of the temporal lobe, and on the lateral and inferior surfaces of the occipital lobe, the veins drain into the lateral sinuses. The middle cerebral veins connect superiorly, through the vein of Trolard, with the superior sagittal sinus, and inferiorly, through the vein of Labbé, with the lateral sinus. The number and location of the cortical veins vary considerably, which makes angiographic verification of their patency difficult. The superficial veins have thin walls, no tunica muscularis and no valves, permitting dilation and flow of venous blood in various directions. These features, together with numerous anastomoses, help to achieve efficient collateral flow in the case of venous thrombosis. Within the parenchyma of the hemispheres, the veins of the superficial system anastomose extensively with the internal cerebral and basal veins of the deep network. The deep veins collect blood from the deep grey matter at the base of the brain and the choroid plexus of the lateral ventricles and drain into the centrally located great cerebral vein of Galen. The latter joins the straight sinus at the apex of the cerebellar tentorium. The inferior sagittal sinus, running along the lower edge of the falx, also joins the straight sinus. The straight sinus then merges with the superior sagittal and occipital sinuses at the confluence of sinuses (torcula herophili). The bulk of the venous blood flows bilaterally via the transverse and sigmoid sinuses (which together form the lateral sinus), through the jugular foramen into the jugular vein. The right lateral sinus is commonly larger than the left. In 14 per cent of cases, the transverse portion of the left sinus is not visualized on angiography, an anomaly that may be relevant when investigating venous thrombosis. Dural sinuses also receive blood from the diploë of the skull bones and are connected with the extracranial veins via the emissary veins, which traverse the cranium. The posterior fossa veins drain the cerebellum and brain stem. At the surface, the veins form a subarachnoid plexus, from where blood drains in three directions: from the superior part it drains into the great cerebral vein of Galen, from the anterior part into the petrosal sinuses, and from the posterior and lateral parts into the adjacent straight, occipital and lateral sinuses.

Venous Drainage of the Cord The venous drainage of the cord in general corresponds to the vascular architecture of the arterial supply of the cord (see earlier), but the number of veins within and around the cord, as well as exiting the spinal canal through the intervertebral foramina, is greater than that of arteries.350 Within the cord, there are three main venous networks, anastomosing more freely than the arteries: the anterior part of the cord is drained by the anterior spinal vein, and the posterior part by a single posterior midline vein as opposed to paired posterior spinal arteries. From the periphery, radially oriented veins drain into the superficial plexus of veins around the cord. Radicular veins convey the blood into paravertebral and intervertebral plexuses, which drain into the azygous and pelvic veins. Because these veins do not have valves, there is a high potential for infections from the abdominal cavity to spread into the spinal cord.

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  Physiology of the Cerebral Circulation  73

Histology of Cerebral Vessels and Barrier of the Brain The microscopic structure of the extracranial parts of the carotid and vertebral arteries is similar to that of other large arteries, whereas intracranial cerebral blood vessels have several distinct structural features for the specific functions and protection of the CNS (Figure 2.12). The endothelial cells of the intracranial blood vessels are joined by tight junctions and have no fenestrations. The muscle layer of the intracranial arteries is thinner than in extracranial arteries of a similar size, the external elastic lamina lacking and the adventitia leaner. To complement these features the brain is endowed with structurally unique protective systems. The intracellular organelles within endothelial cells have specific features related to BBB and transcellular transport functions.40b The cerebrospinal fluid (CSF)–blood barrier is controlled by specialized epithelial cells of the choroid plexus.832 The exchange between the ependymal cells and ventricle walls is also a regulated surface. In addition to the perivascular drainage routes,1082 the brain also has a lymphatic-like pathway, recently described as the glymphatic system.457 This is thought to be an anatomically distinct drainage system or paravascular pathway for CSF and interstitial fluid (ISF) exchange that facilitates efficient clearance of solutes and waste from the brain.1

Physiology of the Cerebral Circulation

2

The physiology and regulation of the CBF and the exchange of metabolites between blood and parenchyma across the BBB are described in Chapter 1. Autoregulation of CBF is the first order mechanism that ensures flow and the supply of oxygen, glucose and nutrients through the vascular beds. Cerebral resistance arteries dilate or constrict during changes in arterial pressure. Functional hyperaemia, or coupling of CBF to neural activity, is another vital mechanism whereby the neurovascular unit maintains the homeostasis of the cerebral microenvironment. Several different signals, including vasoactive peptides and nitric oxide, mediate the regional increases in CBF in tandem with waves of neural activity in different brain regions. Besides the BBB (see Chapter 1), the choroid plexus (CP)–CSF barrier system ensures not only CSF secretion but also constant scrutiny of nutrients and harmful substances to maintain homeostasis.276 However, none of these protective mechanisms is immune to ischaemic insults or the effects of ageing and disease.

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���������������� CSF

AE

AE

EC

EC

BM

VSMC

P

IN

IN

FB

��������������

������� ����

CSF

Dura Arach

SAS

PIA

2.12 Schematic illustration of structural components of the perfused surfaces of the brain. AE, astrocytic endfeet; BM, basement membrane; EC, endothelial cell; IN, innervation; P, pericyte; VSMC, vascular smooth muscle cell; FB, fibroblast; CSF, cerebrospinal fluid; Arach, arachnoid; SAS, subarachnoid space. Diagram kind courtesy of Y Yamamoto, Yamaguchi University Graduate School of Medicine, Japan.

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74  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

Microcirculation and Neuronal Metabolism As arterial perforators penetrate the brain parenchyma, they branch repeatedly, reducing in size until they end in capillaries that allow blood cells to pass only in single file. The capillaries vary in density throughout the brain, being more abundant in regions with high metabolic rates. Capillaries have long been known to be distributed more densely in grey than white matter (Figure 2.13).221 Cerebral blood flow averages 50 mL/100 g of brain/min. This value for human CBF is an integrated average value for grey matter, where flow rates are greater than 80 mL/100 g/min, and white matter, where rates can be as low as 20–25 mL/100 g/min. Within grey or white matter, capillary density is richer where metabolic and, consequently, oxygen-delivery requirements are higher.147 Within the white matter, capillary density is twice as high in the pyramidal tract as the fasciculus cuneatus.221 Within the grey matter of the inferior colliculus, capillary density correlates with both the metabolic rate for glucose and the CBF.375 The rate of consumption of glucose and oxygen, per gram of brain tissue, is higher in species with small brains1015 and in infants.109 Small brains have a greater density of

neurons (Figure 2.14), not glia, and this relationship has remained constant during phylogenesis. The metabolic correlate of this is that glycolytic rate remains constant across species,1016 whereas the rate of oxidative phosphorylation (aerobic metabolism) increases in smaller brains1015 because of increased neuronal density (Figure 2.14). The decrease in density of cortical neurons from the mouse (~142 500 neurons/mm3) to rat (~105 000 neurons/mm3), cat (~30 800 neurons/mm3), human (~10 500 neurons/mm3) and whale (~6800 neurons/mm3) is accompanied by a corresponding reduction in the rate of oxidative phosphorylation per unit volume of brain tissue. These phylogenetic and ontogenetic variations may explain the differential effects of hypoxia and ischaemia in these species (see later). Cerebral blood flow is modulated by cerebral metabolism and, like the metabolic rate, is higher per gram of tissue in smaller brains, e.g. of rats or gerbils, than in humans. Glucose use per gram of a rat brain is roughly twice that of a human.116 This is reflected anatomically in a higher capillary density (and reduced diffusion distance for oxygen) in species with small brains and in the developing brain. Capillary density also increases on adaptation to high altitude or reduced oxygen intake (see Hypoxic Neovascularization). (a)

2.13 Cerebral blood vessels in cortex and white matter. Just to the left of centre, an arteriole penetrates about two-thirds of the cortex before ramifying. The vascular density is much lower in the white matter (bottom part of the photomicrograph) than in the cerebral cortex, reflecting the neuronal and synaptic energy requirements. Across other species as well, neuronal density determines blood vessel density. Capillary density also increases in adaptation to altitude (see text). Factor VIII immunocytochemistry.

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(b)

2.14 Neuronal density in rat (a) versus human (b), in homologous areas of cortex. The neuronal density is higher in the rat. Neuronal density determines synaptic density, metabolic rate, capillary density, susceptibility to ischaemia and threshold of necrosis due to lactic acid production. Measured in moles of glucose used per gram of brain per minute, metabolic rate in the rat is twice that in man. Capillaries and venules are also more numerous per unit volume in rats (a) than in humans (b).116

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  Physiology of the Cerebral Circulation  75

Cerebral Blood Flow Regulation Mechanisms controlling CBF have been investigated intensively and are local rather than global: hypoxic increases in brain blood flow occur independent of sensorineural input from peripheral hypoxia sensors.557,1019 Rather, local brain activity determines blood flow in any particular brain region, depending on oxygen and glucose consumption. In the intact brain, CBF, cerebral metabolic rate for oxygen (CMRO2) and cerebral metabolic rate for glucose (CMRgl) are normally coupled, rising and falling in synchrony. This is termed local autoregulation or functional hyperaemia. From the ~50 mL of blood delivered to the brain every minute, 25 μmol of glucose is extracted, stoichiometrically requiring the simultaneous extraction of 150 μmol (3 mL) of O2 for its oxidation: C6H12O6 → 6CO2 + 6H2O. The normal coupling mechanism matching CBF to cerebral metabolism is still unknown. Candidates include pH (H+), adenosine, nitric oxide and O2-derived free radicals. Whatever the mechanism, coupling normally occurs Protein synthesis

�

Selective gene exp.

�

CMRG, lactate

�

Selective neuronal loss

Glutamate release

pH PCr

mL/100g/min

� Infarct

ATP �

K+, Ca2+

2.15 Thresholds for cerebral blood flow and critical events at which metabolic disturbances occur. PCr, phosphocreatine. Adapted from Hossman et al.439

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between primary, activity-driven increases or decreases in brain glucose metabolism, and both oxygen utilization and blood flow. In contrast to most of the pathophysiological states, isolated hypoxaemia impairs neither CBF-metabolism coupling nor cerebral circulatory responses.260,697 The average human CBF value of 50 mL blood/100 g brain/min is not only an average for grey and white matter but also an average of regional brain blood flow coupled to local activity. In addition to the fine-tuning of local blood flow, the brain maintains or autoregulates constant overall CBF within the upper and lower limits of the autoregulatory range (usually cited as 50–160 mmHg) during fluctuations in systemic arterial pressure (Figure 2.16). If blood pressure falls to too low a level, perfusion drops. In man, this occurs in the range of 70–93 mmHg for different individuals. Human studies of the upper limit of auto­regulation are ethically difficult because vascular rupture and haemorrhage can result, not only excessive perfusion, if blood pressure is too high. The upper limit of auto­regulation is probably just over 160 mmHg for most individuals. Mathematical modelling suggests that the brain can maintain constant blood flow over a blood pressure range of approximately 69–153 mmHg.326 Certain conditions or age-related alterations in the systemic circulation, and degenerative changes in the extracerebral resistance arteries, can shift the lower and upper limits of the autoregulatory plateau to cause hypertensive encephalopathy or cerebral hypoperfusion, which may not necessarily cause ischaemic injury as obvious as in stroke, but an oligaemia that leads to disruption of the microcirculation, damage to the cerebral endothelium, breach of the BBB and oedema. Neurogenic responses via the brain stem autonomic nuclei may also disrupt CBF autoregulation. In addition, pathological changes of vascular smooth muscle and altered release of metabolic factors (e.g. vasoactive substances, cytokines) can influence autoregulatory responses. Although the usual focus of attention in hypoxia and ischaemia is on the use and supply of brain oxygen and glucose, also important is the removal of metabolic wastes, such as lactate. There is good evidence that accumulation of H+ contributes to cerebral necrosis.558,720

Cerebral blood flow (ml/100g/min)

When CBF drops, the critical points at which ischaemic injury occurs vary according to the level of metabolism. Ischaemic thresholds for infarction are lower in species with large brains, at approximately 12 mL/100 g/min in the larger non-human primate brain64 compared to 45 mL/100 g/min in the small rodent brain.472 The ratio of the normal blood flow to the threshold for infarction remains roughly constant across species, however, at 3:1 (in humans, flow drops from ~50 mL/100 g/min to 15–18 mL/100 g/min before infarction occurs) (Figure 2.15). Animals with small brains and greater neuronal densities have higher rates of basal blood flow to satisfy the higher rates of metabolism and, therefore, have higher absolute CBF thresholds for infarction. Thresholds for ischaemic damage are related to metabolic activity.472 Neuropil necrosis can result not only from vascular occlusion but also from hypermetabolism and acidosis. This is exemplified by Wernicke’s encephalopathy (see Chapter 9), in which acidosis-induced neuropil necrosis (sparing neurons) can be precipitated or exacerbated by a glucose load.

2

75 Autoregulatory plateau

50

Breakdown points Breakthrough points

25

0

50 100 150 Mean arterial blood pressure (mmHg)

2.16 Autoregulatory thresholds for maintenance of cerebral blood flow (CBF). Constant CBF is maintained in the 50–150 mmHg range of mean arterial blood pressure. Below and above this range, the CBF varies with the arterial blood pressure. In hypertensive patients, the curve shifts to the right (dashed line).

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76  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

Interstitial fluid drainage pathways The total volume of interstitial fluid (ISF) in the human adult brain is approximately 200–250 mL, 15–18 per cent of brain weight.832 It is distributed in the narrow extracellular space between the parenchymal cells and their processes. Its precise origin has not been established. Some of the ISF may be derived from passage across the endothelium of CNS capillaries, some from CSF entering from the ventricles and subarachnoid space, and some from neuronal and glial metabolism. The pial–glial layer on the surface of the brain and the ependymal lining of the ventricles allow free exchange between ISF and CSF. About 10 per cent of ISF is formed by the metabolic activity of parenchymal cells.1 ISF has an important role in the homeostasis of the extracellular environment. This homeostatic function necessitates that ISF is not static but flows within the extracellular space and is continuously renewed, as drainage via specific pathways172,456,1082 provides space for fresh ISF.1,1081 The motive force for ISF flow is assumed to be derived from systolic vascular pulsations. In animal experiments, ISF has been shown to drain from the white matter into ventricular CSF, whereas from the cortex the flow of ISF occurs in the reverse direction to the blood flow in the penetrating arteries, i.e. towards the surface.172 There are two pathways of ISF drainage: the bulk flow of particulate tracers such as Indian ink or fluospheres has been shown to occur in the expanded perivascular spaces between arterial tunica media and surrounding astrocytic end feet, whereas soluble and particulate tracers but not cells follow the basal lamina of capillaries and arteries.172,1081 In humans, the flow of ISF cannot be traced directly but the drainage pattern of ISF has been inferred from the distribution of amyloid β-peptide (Aβ) and development of cerebral amyloid angiopathy (CAA).1082 Soluble Aβ is formed in the parenchyma, carried in the ISF and drained along the perivascular basement membrane of capillaries

(a)

and arterioles. As cerebral blood vessels become more rigid with age, the drainage may slow down, increasing the poly­ merization and deposition of Aβ in the walls of the vessels along which it drains. This may further impair flow along the perivascular drainage pathways. Impaired perivascular drainage of ISF may contribute to other forms of CAA and dementias characterized by the deposition of aggregated proteins, such as amyloid familial British dementia (ABri), amyloid familial Danish dementia (ADan), cystatin C and transthyretin.1082 In experimental animals, the perivascular drainage of antigens to cervical lymph nodes is important in the development of an immune response,1083 and in humans these drainage routes may facilitate the spread of immune cells and inflammation from the primary site of tissue damage.1 ISF pathways have also been implicated in the spread of malignant cells, in the pharmacokinetics of drugs within the CNS and in potential therapeutic approaches using stem cells.2

Pathophysiology of Cell Death in Ischaemia and Hypoxia Neuronal Cell Death Mechanisms Besides necrosis, several ‘non-accidental’ mechanisms of neuronal death have been described. They include delayed neuronal death, apoptosis, autophagy and necroptosis.201,1041 These ‘programmed’ mechanisms involving specific signalling pathways have molecular signatures but cannot readily be distinguished on routine histopathological examination. The extent of necrotic tissue damage is conventionally classified into two categories, selective neuronal necrosis, which affects neurons but spares glia, and pan-necrosis, in which all tissue elements die — neurons as well as glia and blood vessels — in time progressing to cavitation. If the aetiology

(b)

2.17 Selective neuronal necrosis versus pan-necrosis. (a) Selective neuronal necrosis in the hippocampus of a 78-year-old woman with septic shock. The acutely necrotic neurons toward the upper left part of the figure are abnormally acidophilic (‘red’ neurons). The selective neuronal death spares the glia and neuropil. (b) Pan-necrosis: left middle cerebral artery embolic infarct in a 51-yearold male. The necrosis (upper left part of the figure) involves all cellular elements and neuropil, producing a bubbly appearance surrounding acidophilic neurons. Inset: Sharp demarcation of the necrotic tissue, which at low power has a 'geographical' contour. The sharp, undulating border cuts across cell processes and crosses over grey and white matter boundaries, implying an abnormal microenvironment (likely to be acidosis) rather than a cellular mechanism. Bars = 50 μm; inset, bar = 500 μm.

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  Pathophysiology of Cell Death in Ischaemia and Hypoxia  77

of pan-necrosis is ischaemia, the term infarction is applied. Selective neuronal necrosis and pan-necrosis (Figure 2.17) can both affect virtually any brain region. Selective neuronal necrosis should not be confused with selective vulnerability, which refers to the phenomenon whereby global brain insults cause focal lesions that predominate in certain brain regions (see later), the specific location depending on the type of insult. In ischaemic states, selectively vulnerable areas can show either pan-necrosis or selective neuronal necrosis.

Selective Neuronal Necrosis Comparison of the patterns of neuronal death in ischaemia, hypoglycaemia and epilepsy71 reveals that all three insults can cause selective neuronal necrosis (Table 2.4). This is because neurons are more vulnerable than astrocytes to death by overstimulation, not by virtue of their higher metabolic rates or larger size705 but as a result of excessive release of excitatory amino acids.24,198 In selective neuronal necrosis, recovery is abetted by the surviving neuropil and, despite the drop out of neurons, the surrounding cellular elements appear healthy.701

Pathophysiology: Excitotoxicity It has long been known755,756 that neurons can be damaged by overstimulation by glutamate or other excitatory amino acids such as aspartate. The neuroexcitatory activity of acidic amino acids coincides with their neurotoxic potential.757

Glutamate is a ubiquitous neurotransmitter,1074 and acts via four glutamate receptors:712 (i) N-methyl-d-aspartate (NMDA) receptors, (ii) kainate receptors, at which kainate is a favoured agonist (although the marine toxin domoic acid, associated with outbreaks of shellfish poisoning, appears to have even higher affinity), (iii) α-amino-3-hydroxy5-methyl-4-isoxazole propionic acid (AMPA) receptors and (iv) metabotropic receptors, so named because they lead to activation of intracellular regulatory transduction events without necessarily generating transmembrane ion fluxes. The first three glutamate receptor subtypes cause ion fluxes accompanied by water movement across the dendritic cell membrane. They play a role in the morphological expression of excitotoxicity by causing dendritic swelling, but axons are spared. When subjected to different patterns of stimulation, the NMDA receptors have the capability of causing longterm changes in synaptic efficiency. These may persist for hours to days or even longer and are likely to be involved in memory. The prolonged facilitation of synaptic transmission is known as long-term potentiation (LTP), and the prolonged reduction of synaptic transmission as long-term depression (LTD).117 LTP is associated with postsynaptic alterations in dendrites and spines.630 NMDA receptors are highly concentrated in the CA1 pyramidal neurons of the hippocampus. This may explain the selective vulnerability of Sommer’s sector to the three insults of ischaemia, hypoglycaemia and epilepsy. Cell death in these conditions can be attributed to several common mechanisms.842

2

Table 2.4  Comparison of some clinical and metabolic aspects of ischaemia, hypoxia, hypoglycaemia and epilepsy Feature

Global ischaemia

Hypoxaemia

Hypoglycaemia

Epilepsy

Patient age (usual)

Old

Young

Young

Young–middle aged

Spreading depolarisation

+

+

+

++

↓ ATP

+++

+

+

++

↓ Phosphocreatine

+++

+

+

+

↑ Lactate

+++

+

+

+

Fatty acid catabolism

+++

0

0

0

Protein catabolism

+++

0

0

+

Steady state attained

No

Yes

Yes

No

Gene activation

All genes studied ­affected

Some genes activated

Some genes activated

Many genes activated

Tissue pathology

Selective neuronal ­ ecrosis and infarction n

Synaptic alterations only; no cell death

Selective cell death

Selective cell death

Local perfusion

Decreased

Decreased

Decreased/ increased

Increased

Acute-phase reactants in serum

All619

Erythropoietin only

Clinical context

Cardiac arrhythmia or ­arrest, profound ­hypotension (older age group)

Anaphylaxis, asthma, bronchitis, bronchiolitis, epiglottitis, short anaesthetic accidents (younger age group)

Hypoglycaemic encephalopathy, coma

Myoclonus, seizures

Clinical decerebration

+++

+

++

+

Clinical recovery

±

+++

+++

++

ATP, adenosine triphosphate.

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78  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

Pan-necrosis and Infarction Pan-necrosis occurs in ischaemia but also in MELAS (mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes), to some extent in Wernicke’s encephalopathy, and experimentally in the pars reticulata of the substantia nigra in epilepsy, non-ischaemic conditions all characterized by extreme local tissue acidosis and unimpaired or increased blood flow. Large regions of infarction leave only a fluid-filled cyst, within which and recovery of any nature at the tissue level is impossible. Multiple adjacent small infarct cavities can eventually close to form a glial scar.701

Pathophysiology: acidosis and pan-necrosis Acidosis is a key alteration in pan-necrosis, damaging both neurons and glia88,560,720 but sometimes sparing endothelial cells63,425 perhaps because of metabolic acid washout by the circulation. In MELAS, the distribution of necrosis does not conform to arterial territories and is unaccompanied by ischaemia, although microvascular changes may develop.588 Pan-necrosis in MELAS and some other mitochondrial diseases probably relates to local tissue acidosis (see later). Neuropil acidosis possibly accounts also for the neuronal soma-sparing necrosis seen in Wernicke’s encephalopathy and, occasionally, ischaemia. Pan-necrosis occurs when all cell types are overwhelmed by a drop in tissue pH.721,942 Lactic acid is toxic and has a pKa of 3.83. It tends to lower the tissue pH but the drop in pH is not due simply to equimolar H+ and lactate production. It also results from protons released by the hydrolysis of ATP under anaerobic conditions.777 Conditions altering lactate production can leave pHi unaltered,710 and low tissue pH does not always accompany high lactate levels.444,777 Intracellular buffering and compartmentation of lactate in glia558 allow independent modulation of lactate levels and acidosis.559 The dissociation of acid from lactate production has led to the terms lactosis and acidosis to describe accumulation of lactate and protons.777 There is both hypermetabolism and excitation in pan-necrotic states. Wernicke’s encephalopathy is a good example in which there is dendritic swelling1072 and tissue acidosis.388 Similarly, in ischaemic or epileptic pan-­necrosis of the substantia nigra there is dendritic swelling,463,464 which also accompanies a profound tissue acidosis.465 Acidosis of only a mild degree can protect against excitotoxic selective neuronal death,1008 but severe acidosis overwhelms this minor protective action. Acidotic pannecrosis can manifest with axonal swelling, sparing dendrites (Figure 2.18). This finding may be accounted for by the protective effect of mild acidosis on excitotoxicity.1008 A clear demarcation within the tissue (Figure 2.17) between necrotic and non-necrotic brain suggests a threshold effect. With time the intervening neuropil is removed and pannecrosis appears as a fluid-filled cyst surrounded by neuropil containing fibre-forming astrocyte.

Acidosis and acidophilic ‘pink’ neurons The swelling of dendrites is an early morphological feature in neuronal necrosis and is evident in ischaemia,1102 hypoglycaemia74 and epilepsy.466 The dendritic location of excitatory receptors accounts for the dendritic swelling

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2.18 Selective swelling of dendrites in early selective neuronal necrosis. Synapses are marked S on the dendritic side of the synaptic membrane densities. Dendrites are subject to ion fluxes caused by excitatory activity, leading to transmembrane water fluxes and swelling. The dendritic mitochondria are also swollen, to several micrometres in diameter. The axon terminals that synapse with the swollen dendritic spines are not swollen and contain dark mitochondria. This axon-sparing dendritic lesion is a hallmark of the excitotoxic neuronal death, seen in ischaemia, hypoglycaemia and epilepsy. Bar = 1 μm.

(Figure 2.18). An early subsequent change is an increased affinity for acid dyes, i.e. acidophilia, that occurs whenever a cell or tissue dies. In nerve cells acidophilia is not due merely to the loss of the basophilic ribonucleic acid that constitutes Nissl substance but the nucleus and other cell structures show acidophilia. Acidophilic neurons in pan-necrosis and selective neuronal necrosis appear similar (Figures 2.19 and 2.20). They will take up any acid dye of whatever colour, ­including safranin, which is yellow. In some centres haematoxylin–phloxine–safranin (HPS) staining is routinely used. The commonly used terms ‘eosinophilic neuron’, ‘pink neuron’ and ‘red neuron’ reflect the widespread histological use of eosin dye. Acidophilic neurons are important to distinguish from dark neurons (biopsy artefact), as the latter are not injured lethally but represent perturbed neurons at the time of fixation (see Experimental neuropathology, later in chapter).

Specificity of acidophilic neurons Acidophilic neurons are seen in several acute, non-­ischaemic causes of neuronal death, such as viral encephalitis or experimental seizure activity. The term ‘ischaemic neuron’ should thus be avoided as a morphological descriptor of neuronal acidophilia, as it is too specific with respect to cause. Necrotic neurons develop an affinity for acid dyes regardless of the cause of neuronal death.

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  Pathophysiology of Cell Death in Ischaemia and Hypoxia  79

Delayed Neuronal Death � � �

�

�

�

�

2.19 Selective swelling of axons in early pan-necrosis. Axons (Ax) are subject to transmembrane water fluxes and swelling. Dendrites (De) are not swollen and contain dark mitochondria. This selective axon lesion is almost a mirror image of the excitotoxic lesion in selective neuronal death and suggests an entirely different mechanism for pan-necrosis, not an exaggeration of the mechanism of selective neuronal necrosis. Pan-necrosis, often sparing of the nerve cell body, is seen in ischaemia, ­ epilepsy with focal acidosis, and other acidotic, neuropil-­cavitating conditions such as Wernicke’s disease and mitochondrial encephalopathies. Neuropil cavitation, or pannecrosis, does not result from pure hypoglycaemia, as acidosis cannot occur. Note that axonal mitochondria are swollen to several micrometres in diameter. Bar = 1 μm.

Electron microscopy of the acidophilic neuron The ultrastructural features of acidophilic neurons are those of cellular necrosis, not apoptosis. Acidophilic neurons have mitochondrial flocculent densities and large, confluent breaks in the nuclear and cell membrane (Figure 2.20). Cell membrane rupture precedes nuclear membrane abnormalities, the reverse order of apoptosis. Concomitant with the appearance of cytoplasmic acidophilia is the ultrastructural appearance of mitochondrial flocculent densities (Figure 2.19) related to irreversible protein alterations in the cell. Mitochondrial flocculent densities have been shown to be proteinaceous by their disappearance when the tissue is injected with trypsin or proteinase.1022

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‘Delayed neuronal death’ or ‘maturation phenomenon’, was first applied to observations in the hippocampus of rodents subjected to global ischaemia469,539 but may also occur after brief, focal ischaemia.713 The fundamental observation is that neurons do not die immediately after the 5- to 10-minute period of global ischaemia but rather in the hours to days afterwards. In both experimental conditions and humans,438 delayed neuronal death refers to the slow neuronal degeneration occurring even remotely days after an episode of complete ischaemia of short duration rather than that following chronic, partial incomplete ischaemia.236,730

2

Cell Death: Apoptosis versus Necrosis and Necroptosis The death of cells in the CNS following injury may occur by combinations of apoptosis, necrosis, and hybrid forms along an apoptosis–necrosis continuum (‘necroptosis’). Apoptosis is the best characterized form of programmed cell death in vascular diseases and the principal type of delayed neuronal death in ischaemia. Mild ischaemic injury preferentially induces cell death by an apoptotic-like mechanism rather than necrosis. Although some controversy prevails,842 there is good evidence from experimental studies that apoptosis predominates in the hypoperfused regions of the brain, e.g. the penumbra. The triggers of apoptosis include oxygen free radicals, ionic imbalance, DNA damage, protease activation and death receptor ligand binding. There are two main types of apoptotic signalling cascade after cerebral ischaemia. Apoptosis can be initiated by internal events (the intrinsic pathway) involving disruption of mitochondria and the release of cytochrome C. The other principal pathway involves the binding of ligands to cell surface death receptors such as the Fas and tumour necrosis factor (TNF) receptors (extrinsic pathway).143 Both pathways result in the activation of a series of downstream caspases, leading to DNA cleavage and cell death.617

Necroptosis Until recently necrosis was considered an uncontrolled mode of cell death. It is now known that necrosis can be regulated by several molecular mediators.201 ATP depletion is considered a key factor in initiating ischaemia-induced necrotic cell death. Serine/threonine kinase receptor-interacting protein 1 (RIP1) plays a crucial role in the initiation of necrosis by ligand-receptor interactions. Programmed necrosis (necroptosis) during ischaemic injury may be activated upon stimulation of death receptors by several ligands (TNFα, FasL and Trail) that can also activate apoptosis.

Autophagy Autophagy (Greek ‘self eating’) is a regulated cell process that is activated for bulk removal of cellular proteins and organelles. Depending on the circumstances, the role of autophagy in cell death can be cytoprotective or cytotoxic. In autophagic degradation, proteins are targeted and transported to membrane-enclosed vesicles, which then fuse with lysosomes allowing their contents to be degraded by

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80  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

2.20 Electron-microscopic features of acidophilic neurons. Colour inset: Acidophilic neurons are seen in the dentate gyrus of the hippocampus. Electron microscopy of such a field shows three neurons (N) with the typical coarse tigroid nuclear pattern of necrosis, with ruptured nuclear and cytoplasmic membranes, and amorphous cytoplasm. An astrocyte (A) contains glial cytoplasmic fibrils. Two macrophages (M) are at the lower left. Greyscale inset: early features of necrosis are rupture of the cell membrane (black arrow) whereas the nuclear membrane (white arrow) is still intact. Two mitochondrial flocculent densities, also characteristic of necrosis, are in white circles. Bar = 1 μm; colour inset, bar = 100 μm.

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  Consequences of Hypoxic Insults  81

lysosomal enzymes such as cathepsins B and D. Besides soluble cytosolic proteins, effete organelles such as mitochondria and peroxisomes or regions of the Golgi and endoplasmic reticulum are removed by autophagy. Autophagy can be activated after ischaemic injury,819,825 in both neurons and glia.609 There are three basic types of autophagic processes: macroautophagy, microautophagy and chaperonemediated autophagy. The autophagy pathway is mediated by up to 50 autophagy-related genes and their encoded proteins such as ATG5, ATG12 and microtubule-associated protein light chain 3 (LC3).825

Selective Vulnerability What is Selective Vulnerability? Many of the insults that may be delivered to the entire brain produce only restricted regions of brain damage. Almost all diseases in neuropathology show selective vulnerability but little is known of the mechanisms. Because a pattern of selective vulnerability constitutes a type of ‘signature’ of a disease or a class of disease processes, it is diagnostically important. It may also provide insights into the pathogenesis of brain injury caused by a specific deficiency, genetic defect or toxin.

Selective vulnerability in hypoxic and ischaemic states Several factors contribute to selective vulnerability. Much progress has been made in understanding some forms of selective vulnerability. Experimental studies have shown that transient global cerebral ischaemia (GCI) causes accumulation of glutamate in the extracellular fluid of the brain,106 leading to ‘excitotoxic neuronal death’: over-stimulation of NMDA receptors, excessive neuronal entry of Ca2+, stimulation of a range of enzymes such as neuronal nitric oxide synthase, phospholipases and calpain, oxidative and enzymatic damage to macromolecules, and cell death. The NMDA receptor is abundant in the dendritic fields of vulnerable neurons693 in the CA1 field of the hippocampus.615,965 The synaptic connections that facilitate LTP make these hippocampal neurons particularly vulnerable. Such vulnerability is probably an exaggeration of the normal physiological mechanism that allows long-term changes in excitability. The selective vulnerability of CA1 over CA3 neurons may relate to higher concentrations of NMDA receptors on CA1 neurons, and their less robust and smaller perikarya, with less Nissl substance. Other pathogenetic mechanisms may also be involved. For example, other excitatory receptors, such as AMPA receptors, may play a role in neuronal death after transient GCI308,348 Neurotoxicity may be enhanced by zinc, known to be present in synaptic vesicles and co-released with transmitter:309,951 zinc released into the synaptic cleft has been estimated to reach potentially neurotoxic concentrations.63,441 Zinc in the hippocampus, however, is present primarily in en passant axonal dilatations of mossy fibres ending on CA3 neurons,321 which are relatively resistant. There are numerous enzymatic differences between the vulnerable CA1 zone and the resistant CA3 zone.315 The dentate granule cells of the hippocampus are rich in NMDA receptors and yet are relatively resistant to

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ischaemia. In hypoglycaemic brain damage, where massive quantities of aspartate and also glutamate are released into the brain extracellular fluid,899 the dentate gyrus is clearly vulnerable, in a pattern showing a relationship to CSF spaces.74,75 It thus seems that access of an excitatory neurotransmitter to vulnerable neurons and their receptive fields must be considered, in addition to the intrinsic properties of the neuron, to give a comprehensive explanation of selective vulnerability. In most brain regions, GCI causes selective loss of GABAergic neurons. Selective loss of GABAergic neurons occurs in the cerebral cortex,950 thalamus (lateral reticular nucleus314,872,957) and striatum.307 In contrast, in the hippocampus the CA1 zone shows preservation of GABAergic interneurons,487 in spite of the stronger excitatory input that CA1 GABAergic neurons receive.1110 The hilus of the hippocampus shows loss of somatostatinergic innervation of GABAergic neurons, rather than loss of GABAergic neurons.488 Selective GABAergic neuron loss is one plausible explanation for epilepsy after ischaemic states.950,958 The fundamental cause of selective destruction of neurons containing GABA is unclear but may be related to activation of excitatory receptors other than those of the NMDA subtype.872 Other examples of selective vulnerability include that of the cerebellar Purkinje cells to transient GCI. This was initially difficult to reconcile with the calcium hypothesis of neuronal cell death, because calcium was thought to enter neurons mainly by NMDA-gated channels or by voltagesensitive Ca21 channels activated by depolarization. The use of cerebellar cultures enriched in Purkinje cells allowed the discovery of an AMPA subtype of glutamate receptor with direct Ca2+ permeability,627 perhaps explaining the vulnerability of cerebellar Purkinje cells to degeneration in excitotoxic situations such as GCI. Sometimes, selective vulnerability of a particular brain region is striking and easily correlated with a known pathogenetic factor. For example, the globus pallidus and pars reticularis of the substantia nigra are the two brain regions richest in iron and vulnerable to necrosis in CO toxicity (see Carbon Monoxide Toxicity, later in chapter). The high affinity of the CO molecule for the cytochrome haem iron in these regions far exceeds that of oxygen itself. This is a good example of histotoxic hypoxia. Similarly, necrosis of the putamen implicates methanol. Large neuronal size does not adequately explain selective vulnerability. For example, the largest neurons in the brain, the Betz cells of the motor cortex, are relatively resistant to a transient global ischaemic insult.705

2

Consequences of Hypoxic Insults As in the case of ischaemia, cerebral hypoxia results if the process or cascade of oxygen (O2) delivery is interrupted from through the route from ambient air to the brain. The pO2 decreases in steps from the ambient air, through the lungs and blood and finally to the brain. O2 molecules always flow from an area of higher partial pressure to an area of lower partial pressure. Each step in this cascade allows O2 molecules to ‘flow down’ a portion of the gradient, culminating in the delivery of O2 to the tissue. At sea level, these steps give rise to an overall drop in pO2 from

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82  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

50

Mo unt Eve res t

% Saturation

60 40 30 20 10 0

70 60 50 40 30 20

0 10 20 30 40 50 60 70 80 90 100 pO2

160

Mount Everest Sea level

140

80

10 0

(b)

90

20

120 100 80

pO2

60

10

40

Pressure (kPa)

gh Hi

70

de itu alt

100

20 0

0 ai n ch on dr ia

80

level Sea

Br

90

ito

100

Pressure (mmHg)

(a)

M

Several mechanisms act together over acute and chronic adaptation to hypoxia (Box 2.2) to uphold CBF,149 cerebral oxygenation, the cerebral metabolic rate and the cerebral rate of oxygen consumption (CMRO2). Experimental work

Formation of new vessels is stimulated in the brain by hypoxia,120 mediated by a sequence of gene activation (Figure 2.2). HIF-1α activates VEGF, erythropoietin and glycolytic enzymes927 and genes in many other classes.829 HIF-1α is the key upstream mediator of hypoxia.217,926 The primary oxygen-sensing mechanism that turns on HIF-1α is a reduced O2-dependent degradation (see earlier). HIF-1 is a heterodimer of HIF-1α and HIF-1α, subunits and the increased HIF-1α allows heterodimerization of these subunits. This induces a conformational change that allows the complex to bind to hypoxia response elements. Induction of VEGF is the critical event in the growth of new capillaries, preceding brain vascularization itself.650,1067 Similar upregulation and neovascularisation also occur in experimental models of intermittent hypoxaemia that simulate sleep apnoea.89 These changes all occur along a smooth spectrum of physiology, not pathophysiology, because HIF-1α levels vary over the physiological range of O2 tension.485 However, damage can occur when newly formed vessels leak, especially under the high CBF conditions of hypoxia. This leakage is the basis for brain water accumulation and high-altitude cerebral oedema, which can be fatal384 or leave permanent neuropsychiatric impairment because of damage to the globus pallidus.1032

Al ve ol i Ar te rie s

Brain Compensation in Hypoxaemia

Hypoxic Neovascularization

ta ir

Hypoxaemia (Table 2.1) may result from restrictive or obstructive pulmonary parenchymal disease, upper airway obstruction or a low inspired oxygen concentration, such as during high-altitude mountaineering (Figure 2.21). At normal pO2 values, over 98 per cent of the oxygen-carrying sites on the haemoglobin molecule are occupied by oxygen in arterial blood, with most molecules having all four sites occupied by O2 and only a few having three O2 molecules. In the superior sagittal sinus, roughly 70 per cent of the oxygen-carrying sites on haemoglobin molecules are occupied, i.e. three sites carry O2 on most haemoglobin molecules and two sites carry O2 on some. The oxygen extraction fraction (OEF) is thus 28 per cent (98 minus 70), implying that 28 per cent of the oxygen carried into the brain is extracted by passing through the brain. The OEF can increase in states of hypoxia, as part of the adaptation of the brain to hypoxia. In addition to O2 carried on haemoglobin, a small amount of O2 is dissolved in the blood (roughly 0.3 mL/dL), none of which is returned from the tissue to venous blood.415 In hyperbaric oxygen treatment of profound anaemia, the dissolved oxygen alone is sufficient to supply tissue needs.1003

en

Hypoxaemia

shows a slight decline in phosphocreatine and ATP and an early increase in tissue lactate, followed by the achievement of a new, hypoxic steady state. Subsequent to these metabolic adjustments, brain pH and tissue pCO2 remain stable during hypoxaemia,432 indicating that tissue washout and removal of wastes are unimpaired.

Am bi

21 kPa (158 mmHg) in ambient air, to 4 kPa (30 mmHg) as the normal value for tissue pO2. Values for focal partial pressures of O2 in mitochondria are even lower.

2.21 Oxygen availability to brain. (a) Oxygen–haemoglobin dissociation curve, showing sigmoid shape of saturation as a function of pO2; 25 per cent, 50 per cent, 75 per cent and 100 per cent saturation correspond to, on average, one, two, three and four O2 molecules per molecule of haemoglobin. At sea level, most oxygen delivered in arterial blood consists of the fourth O2 molecule and often the third, leaving two or three O2 molecules in venous blood haemoglobin molecules and an average venous saturation of 70 per cent. At 4 kPa (30 mmHg) on Mount Everest, however, haemoglobin is just over 50 per cent saturated; under such circumstances of extreme altitude, even arterial blood reaching the brain carries only two or three O2 molecules per haemoglobin (as does venous blood at sea level). At the summit of Mount Everest, venous blood departs the brain with only one or two O2 molecules per haemoglobin molecule, utilizing a different part of the oxygen–haemoglobin dissociation curve to effect oxygen delivery at altitude. (b) Stepwise drop in pO2 at each successive step of oxygen delivery. Although the ambient pO2 on top of Mount Everest is equal to the brain tissue pO2 at sea level, a staircase of declining pO2 at every step along the anatomical pathway of O2 delivery nevertheless exists at all altitudes. The staircase is considerably flattened on Mount Everest and in the most hypoxic patients. The lowest recorded arterial pO2 in a living human was 1 kPa (7.5 mmHg),360 with a corresponding venous pO2 of 0.27 kPa (2 mmHg).

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  Consequences of Hypoxic Insults  83 Box 2.2.  Hypoxic response mechanisms ●●

●●

●●

●●

●●

●●

Hyperventilation: occurring almost immediately and ­accompanied by a respiratory alkalosis. Hypoxic hyperventilation is due to an increase in tidal volume. Oxygen extraction from blood: oxyhaemoglobin dissociation curve shifts to the right, decreasing affinity for O2 with the ultimate consequence of decreasing brain tissue pO2. Glycolysis: net effect is a relative preservation of ATP at the expense of phosphocreatine. CBF: hypoxic hyperaemia compensates for the decreased arterial blood O2 by increasing flow through the tissue ­microcirculation even when brain tissue pO2 is low. Erythropoietin: production increases circulating ­haemoglobin. Capillary density: hypoxic brain neovascularization is important for adaptation over time.

All above mechanisms act together during acute and chronic hypoxia to uphold cerebral oxygenation, the cerebral metabolic rate and even the cerebral rate of oxygen consumption (CMRO2). ATP, adenosine triphosphate; CBF, cerebral blood flow; pO2, partial oxygen ­tension.

Tissue pO2 Measurement in Hypoxia There is a direct proportional relationship between brain tissue pO2 and the FiO2.857 Measurement of brain tissue pO2 shows that hypoxaemia leads to a further decrease in the tissue pO2 from the normal. Tissue pO2 levels can fall from 2.7–5.4 kPa (20–40 mmHg) to < 1 kPa (< 5 mmHg) during severe hypoxaemia780 and can reach < 0.25 kPa (0–2 mmHg) when breathing 2–3.5 per cent O2.912 The effect is a flattening of the steps in the staircase of oxygen delivery (Figure 2.21b). In the non-injured brain subjected to iatrogenic ischaemia, e.g. during neurosurgical aneurysm clipping, a tissue pO2 of 1.3 kPa (10 mmHg) can be tolerated before tissue hypoxia becomes critical and tissue pH decreases.273,430 Brain tissue pO2 monitoring appears to offer prognostic value in head injury, correlating with CBF better than either brain pCO2 or pH.261 Poor outcome in head injury was found to correlate with tissue pO2 of 300 000) supervene, when cerebral haemorrhage (not ‘anaemic hypoxia’) can result.312 As in hypoxia, therefore, there is no evidence that uncomplicated anaemia causes brain damage.

Hypoxia versus Ischaemia The mechanisms of tissue damage in hypoxia and ischaemia are quite different. In hypoxaemia, only delivery of O2 is impaired, not removal of products of metabolism. Because CBF is maintained or increased in hypoxia,3,207,343,796 other molecules continue to be delivered to the brain. Waste products such as CO2 and H+ also continue to be removed unabated, accounting for normal tissue pCO2 and pH in pure hypoxaemia.431 Hypoxia is thus a much simpler and less dire primary insult than ischaemia.636 When hypoxia is added to ischaemia, necrosis occurs, and hypoxia then modulates the degree of damage.688

Neurotransmission Failure and Energy Failure In the hypoxic brain, neurotransmission ceases before energy failure.21,64,664 For example, there is no evidence of energy failure in the medulla at an appropriately early time that would account for hypoxic apnoea.267 Lactate accumulation occurs without energy failure,895 and so energy

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failure cannot be invoked to explain hypoxic lactate accumulation. Lactate begins to increase when the oxygen content of inspired air drops to 12 per cent, corresponding to a saturation of O2 (SaO2) of 50 per cent.381 When inspired oxygen is 7 per cent, corresponding to an SaO2 of 23–35 per cent, there is some hydrolysis of phosphocreatine but ATP levels are maintained.381 Still lower inspired oxygen concentrations of 2–3 per cent cause electroencephalogram (EEG) changes and the phosphocreatine/creatine ratio decreases.912 Events in hypoxia do not progress beyond this reversible pathophysiological state of electrical failure and early energy failure. A steady state is reached,381 unlike in ischaemia. Unlike in ischaemia the release of glutamate106 is absent in pure hypoxaemia.780 As a consequence, the stage of tissue necrosis is never reached in the brain during hypoxaemia.

Gene Expression in Hypoxia and Ischaemia Analysis of gene activation in hypoxia and ischaemia shows further differences between these two kinds of insults (Table 2.4). A new steady state is achieved in hypoxia, with upregulation of mainly regulatory genes whereas in ­ischaemia there is mass activation of genes, in association with tissue destruction (see earlier). Many classes of genes are differentially induced and regulated by hypoxia and ischaemia. These include genes for transcriptional regulators (c-fos, c-jun, junB, TIS8 (5zif268), krox-20), stress proteins (e.g. heat-shock proteins), glucose transporters, haem oxygenase, growth factors, ­interleukin-converting enzyme (ICE)-like proteases, bcl family (e.g. ICE, Nedd2, Yama/CPP32, bcl-2, bcl-x) and ­caspases involved in apoptosis. Some genes have adaptive value when stimulated,233,525,1107 whereas others may be harmful.60,226,277 Thresholds for activation of gene ­transcription vary. Even physiological stimuli and ­ neurotransmitter ­(acetylcholine) release can activate genes such as the transcriptional regulator c-fos.363,448,854,880 Stronger ­stimuli turn on heat-shock proteins, and still stronger stimuli switch on the genes for apoptosis. In ­ hypoxaemia, one of the first genes to be upregulated is that for e­rythropoeitin.659 Some genes stimulated are ­pro-apoptotic, such as BNIP3.150 Other genes, such as that for haem oxygenase, are turned on by ischaemia but not hypoxia alone.108 The genes encoding heat-shock ­proteins such as hsp72,538,742 tumour suppressor genes199 and transcriptional regulators, such as c-fos, c-jun, junA and junB,210,760 are all stimulated by ischaemia (Table 2.4).

Clinical Differences between Hypoxia and Ischaemia A major reason to distinguish hypoxic and ischaemic insults688,943 clinically is to predict the outcome of global insults that result in coma. Hypoxic coma, although rare, is reversible and tends to occur in young people. If a grave prognosis is spuriously ascribed to a coma accompanying a pure hypoxic insult, then treatment could be withdrawn from a patient who has not suffered widespread brain necrosis. In medicolegal review, it is important to determine whether cardiorespiratory arrest or only respiratory arrest

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  Consequences of Hypoxic Insults  85

has occurred. The prognosis is very different if cardiac arrest has not occurred and blood pressure has been maintained. Total cerebral ischaemia of only 2 minutes can cause neuronal necrosis957 whereas even profound arterial hypoxia, without cardiac arrest or hypotension, does not.360,847,890 Causes of pure hypoxia include allergic reactions and tracheobronchial infections (see Table 2.4). Mostly young people demonstrate this syndrome of respiratory failure without heart arrest. Hypoxic coma persists for up to 2 weeks, usually followed by a complete recovery.360,847,890 The underlying brain-repair process is synaptic, accounting for this time course and eventual recovery. If only hypoxia has occurred, no neuropathological abnormalities will be detectable on conventional examination,360,847,890 whereas GCI causes either extensive brain necrosis209 or the changes of a non-perfused brain if capillary closure has occurred. The neuropathologist is often called upon to confirm a diagnosis of non-perfused (‘respirator’) brain. This pathophysiological and pathological distinction between hypoxia and ischaemia is thus important for the pathologist as well as the clinician.

Hyperoxic Brain Damage Hyperoxia alone at normobaric pressures does not seem to damage the adult brain, but at hyperbaric pressures, such as those encountered during diving,100,257,258 100 per cent O2 is toxic and causes brain necrosis.86 Early changes are seen in cell processes and especially in mitochondria.87 In the neonate, the situation is different, as even normobaric hyperoxia can cause widespread neuronal necrosis.19 The combination of hyperoxia and unilateral carotid ligation has the converse effect to that of hypoxia and carotid ligation. In hyperoxia alone, i.e. in the hemisphere contralateral to the ligation, necrosis is seen, but in the ipsilateral, carotid-ligated hemisphere, there is no necrosis.86 Thus, the reduced flow protects the ligated ipsilateral hemisphere from hyperoxic damage. Toxicity of hyperbaric gases is not discussed here. See review 237 on the narcotic and toxic properties of CO2, O2 and N2 (nitrogen narcosis) at normal and high pressures.

Asphyxia Hypoxic conditions arise from asphyxia, a term that denotes an inability to breathe. Forensic analysis distinguishes three causes: (i) suffocation, (ii) strangulation and (iii) chemical asphyxia.746 Chemical asphyxia paralyzes breathing by causing hyperpolarization of the respiratory control neurons in the medulla oblongata. Although suffocation and strangulation are discussed here, more detailed analysis is available in textbooks of forensic neuropathology.746 Suffocation includes environmental suffocation e.g. lack of breathable O2 in the ambient environment, smothering and gagging (blockade of external air passages), choking (bolus or aspiration, manual compression of trachea), drowning (H2O contacting larynx, causing laryngospasm) and mechanical suffocation as in a snake encircling and progressively tightening around the victim’s thorax. Strangulation implies neck constriction in addition to ventilatory obstruction and includes band strangulation (a band is tightened around the neck), garrotting (a rope or a finer,

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neck-cutting ligature is tightened slowly, from behind), manual strangulation (occlusion of neck by fingers and hands) and mugging (forearm around the neck applied from the rear). In the special case of hanging, the weight of the individual is used to tighten a constricting band or rope around the neck. ‘Birth asphyxia’ refers to the mechanical constriction of the thorax in the birth canal during delivery and is physiological, ending with the first breath.

2

‘Birth Asphyxia’: Neonatal Hypoxia and Cerebral Palsy Hypoxia is physiological in utero. The fetus has different, ‘stickier’ haemoglobin, facilitating O2 transfer from mother to fetus. The temporary inability to breathe due to chest wall compression during a vertex delivery is normal if the umbilical cord is supplying oxygenated blood, as are physiological decelerations in fetal heart rate. Breech delivery, with the umbilical cord pressed against the pelvic rim until delivery of the aftercoming head, initially seems a plausible risk factor for cerebral ischaemia, cerebral necrosis and cerebral palsy. Large studies of breech deliveries, however, have shown no risk associated with vaginal delivery versus caesarean section for the development of subsequent neurological deficits.225,767 Converging evidence suggests that brain necrosis and cerebral palsy are not causally related to the birth process except in rare instances. The loose attribution of cerebral palsy to ‘birth asphyxia’ should be avoided. The generally litigious climate attributing cerebral palsy to obstetric or neonatal paediatric malpractice is based on spurious claims. Cerebral palsy has an incidence of roughly 1.5–3 per 1000 live births, generally unchanged over time.795 Cerebral palsy is now known not to be linked to hypoxia of birth and rates have remained unchanged over the time period in which increased oxygen has been loaded successfully into newborn infants.724 Hypoxia even in adult animals688,780 and adult humans360,847,890 causes no necrosis unless accompanied by ischaemia. The neonatal brain has a lower metabolic rate and is even less sensitive than in the adult, and so the neonatal hypoxic brain does not even activate some genes such as HSP32 that are turned on by ischaemia.108 Epidemiological evidence over decades indicates a general success of the medical establishment in increasing newborn blood oxygen levels. The major effect has been not a decline in cerebral palsy but rather a decreased incidence in the diagnosis of ‘birth asphyxia’.1099 Neither the birth process itself nor isolated transient hypoxia has causal association with cerebral palsy, but several other disorders have been discovered to be associated with a risk. These include low birth weight, disorders of coagulation and intrauterine exposure to infection or inflammation, all of which show a positive association with cerebral palsy.528,724

Histotoxic Hypoxia This describes the interference by toxins in the inability of the cell to use oxygen as an electron acceptor in the mitochondrial electron transport chain, preventing the oxidation of glucose to CO2 and H2O. Sulphide (S2) cyanide (CN2) and azide (NaN3) can all inhibit mitochondrial cytochrome oxidases, sulphide being slightly more potent than cyanide

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86  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

and azide being relatively weak.956 Sulphide,8 cyanide137 and azide670 can also cause immediate apnoea and death, a phenomenon too rapid to be explained by inhibition of metabolism. This is due to their immediate effect on respiratory neurons of the medulla.

Sulphide, Cyanide and Azide Exposure to sulphide is seen clinically in a number of circumstances. Hydrogen sulphide is formed in sewers and, together with occupational exposure to H2S-containing gas (‘sour gas’) in the oil and gas industry,156 accounts for most cases of histotoxic hypoxia the neuropathologist is likely to encounter. Cyanide exposure occurs industrially, and in suicide and homicide attempts, because the chemical is easily available. Granular crystallized prussic acid, Zyklon B, was used in Hitler’s genocide in the Second World War in the gas chambers. The admixture with acid produced free cyanide gas, lethal at 300 parts per million (ppm). Although azide is an important industrial chemical, used also in rocket fuels and as a herbicide, insecticide and molluscicide, it is no longer used as a fumigating agent in buildings. Historically, there was widespread use of both cyanide and azide to rid ships, buildings, rooms and apparatuses of both infection and infestation by insects. Sometimes, the fumigator perished because of the action of these agents, in a manner similar to workers exposed to natural gas or sewers that contains H2S. Gaseous sulphide smells of rotten eggs, and cyanide smells of apricot seeds or bitter almonds. Exposure to any of these three agents causes brain damage, but heart failure always supervenes in sulphide-,8 cyanide-405 and azide-related670 injury. Hence, the brain damage is attributable to the associated hypotension. Inhaled H2S passes through the lungs into the blood and dissociates in an aqueous equilibrium: H2S ⇌ H+ + HS− ⇌ 2H+ + S2−. Exposure to low to moderate ambient concentrations of H2S at 20–50 ppm causes eye and lung irritation.614 Very low concentrations are sought by people for ‘cures’ at sulphur springs, where the associated air has the faint odour of rotten eggs, believed to be healthful. Higher concentrations of H2S paralyse the olfactory nerves and sense of smell, making it impossible to recognize the signal rotten-egg odour. The mechanism of immediate death is too fast to be accounted for by necrosis of cells due to cytochrome binding. Inhalation of 500 ppm sulphide causes immediate apnoea, related to hyperpolarization of neurons in the medulla oblongata that control breathing.551,833 Together, the anosmia and apnoea obviate the possibility of life-saving self-removal in H2S exposure. The immediate death often leads to a scenario in which a missing person exposed to H2S is sought after by rescue workers, who are themselves then overcome by the gas. No neuropathological changes are usually evident in such cases. If exposure is survived, however, brain necrosis can be evident. Whether necrosis is due to direct histotoxicity, cardiac hypotension or standstill is not clear in these physiologically uncontrolled human observations. Cardiac function and blood pressure remain unknown at the time of exposure, and GCI to the brain can thus never be ruled out. Experimental work suggests that the cerebral necrosis relates to the potent and immediate depression of blood pressure by cyanide or sulphide. Exposure to even very high (supra-lethal

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without ventilation) concentrations of these agents is incapable of producing cerebral necrosis unless hypotension supervenes.84,137 Cyanide causes severe perturbations in cerebral energy metabolism, including reduction in cytochrome oxidase activity, depletion of ATP and production of lactate but not brain necrosis.637 The situation is similar with sulphide exposure, in which necrosis does not occur even after several hours of EEG isoelectricity (flat EEG). In one series, a single ventilated animal that received a very high dose of sulphide (a supra-lethal dose in the unventilated animal) showed cerebral necrosis;84 physiological monitoring of this animal had revealed persistent hypotension to 200 g/L) and haematocrit (>60 per cent). In patients with polycythaemia vera the risk of ischaemic stroke is increased up to five times, with a lesser increase in those with secondary polycythaemia.195,400 Even an abnormally high haematocrit irrespective of high red blood cell count carries an increased risk of stroke, which may occur secondary to dehydration.731 In the Framingham study, if the haematocrit was over 42–45 per cent the risk of stroke increased two-fold.514 Similarly, the risk of cerebral infarction increased significantly when haematocrit values were above 45 per cent, especially if severe atherosclerosis was evident.1005 In patients with polycythaemia, stroke occurs because of hyperviscosity, which decreases CBF and gives rise to multifocal small infarcts.291 Whether the sluggish flow results in the formation of thrombi or the stagnation of the blood itself causes ischaemia has not been established. Increased numbers of white blood cells can also lead to infarction, most likely because of stasis. An excessive concentration of plasma proteins, e.g. in plasma cell dyscrasias, can lead to hyperviscosity, too. A hyperviscosity syndrome quite often complicates Waldenström’s macroglobulinaemia, a neoplastic disease of B-lymphocytes in which there are high levels of plasma immunoglobulin M (IgM), but other paraproteinaemias can also give rise to this complication.766

Anaemias The low blood haemoglobin of anaemia causes decreased oxygen-carrying capacity. Compensatory mechanisms during anaemia usually ensure adequate transport of oxygen to the brain. Low haemoglobin does not itself cause strokes.514 The risk of strokes increases if additional factors affect blood flow.

Sickle Cell Disease Sickle cell disease is one of the best known monogenic disorders. It belongs to a group of haemoglobinopathies in which the abnormal β-chains of the sickle haemoglobin S aggregate and polymerize to form rigid filamentous structures, ‘tactoids’, which deform erythrocytes into sickle cells.125 Although the rheological properties of sickled erythrocytes suggest that microvascular occlusion might result primarily from intravascular aggregation, an additional major contributor is likely to be the extensive cranial vasculopathy that affects many patients. This is characterized by stenosis of the extracranial and intracranial segments of the internal carotid artery, and the anterior, middle and posterior cerebral arteries. The vasculopathy results from abnormal proliferation of fibroblasts and vascular smooth muscle cells in the vessel wall; contributory factors probably include increased blood flow as a result of anaemia, abnormal adherence of erythrocytes to the endothelium, haemolysis, endothelial activation, leukocyte adhesion, elevated production of endothelin-1 and scavenging of nitric oxide by cell-free haemoglobin dimers.976 Intracranial haemorrhages and fat embolism may also occur.884 Of the

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anaemias, sickle cell disease is the most commonly associated with silent infarction and stroke. Strokes occur most often in children under the age of 15 years. About 15 per cent of children with sickle cell disease experience cerebrovascular disorders. Cerebral infarcts occur in about 75 per cent and intracerebral haemorrhages in some 20 per cent, and these changes often occur bilaterally. In sickle cell disease, children with seem to have a greater risk of ischaemic stroke, and adults, intracranial haemorrhage.749 Recent, pathway analyses based on genome-wide association studies have shed light on the importance of the TGF-β superfamily and oxidative stress in the pathogenesis of complex traits in sickle cell disease.290

2

Beta-Thalassaemia Major The thalassaemias are another group of hereditary anaemias associated with strokes.125,677,1096 In these, either the α- or the β-chain of the haemoglobin A molecule carries a genetic defect. Beta-thalassaemia major is the most clinically important of the thalassaemias. Patients may carry one of over 200 homozygous β-chain mutations, resulting in reduced or no β-globin synthesis and excessive α-globin, which precipitates within the red blood cells. The main feature of β-thalassaemia major is hypochromic, microcytic anaemia due to impaired production and haemolysis of erythrocytes. The disease usually manifests soon after birth. Patients have an increased risk of thrombotic stroke, to which the post-splenectomy thrombocytosis contributes. Cerebral haemorrhage has also been reported as an occasional complication of blood transfusion in β-thalassaemia.

Platelet Abnormalities Both thrombocytosis and thrombocytopenia have been associated with TIAs, ischaemic stroke and intracerebral haemorrhage. The risk of micro-occlusion is increased if the platelet count is above 400 000 or if the platelets are abnormally adhesive. Neurological complications are most common in thrombotic thrombocytopenic purpura (TTP), which is also known as thrombotic micro-angiopathy or Moschowitz disease.766 It is a rare disorder that primarily affects women aged 20–50 years. In TTP, platelets form thrombi, which occlude mainly cerebral and renal micro-vessels. In parallel, platelets are consumed to such an extent that thrombocytopenia and petechial and purpuric haemorrhages occur.809 TTP is caused by deficient activity of the metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13),889,1023 which cleaves von Willebrand factor (vWF), a glycoprotein that is produced by endothelial cells and plays an important role in the formation of blood clots. In the absence of normal ADAMTS13 activity, large multimers of vWF accumulate, bind platelets and cause thrombosis, leading to the depletion of vWF multimers from the circulation. In idiopathic TTP, the impairment of ADAMTS13 activity is caused by inhibitory antibodies in the absence of underlying disease. In secondary TTP, deficient ADAMTS13 activity occurs in the context of other conditions, including neoplasia, pregnancy and HIV infection, or as an adverse reaction to several medications, such as ticlopidine and immunosuppressive drugs.

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128  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

Rare familial forms of TTP are caused by mutations in the ADAMTS13 gene.548,889 Several single nucleotide polymorphisms may also affect the expression and function of ADAMTS13.1024 Neurological symptoms in TTP,988 are often dramatic and include seizures, stupor, coma and stroke. The changes on neuroimaging526 and histopathological examination may be minimal, even in lethal cases.7 The lumina of microvessels, predominantly in the grey matter, are occluded by hyaline, eosinophilic platelet thrombi (Figure 2.63). These can be highlighted by immunohistochemistry with antibody to CD61. In addition, the thrombi may contain fibrin and factor VIII. Endothelial hyperplasia may be prominent, and sometimes the blood vessel wall is necrotic, whereas the surrounding parenchyma may seem nearly normal. In severe cases, multiple small cerebral infarcts are present in the territory of the occluded microvessel. Rarely large vessel occlusion occurs. Alpha2β1-Integrin (glycoprotein Ia–IIa) is one of the major collagen receptors on platelets via which platelets adhere to collagen exposed in damaged vessel walls and become activated. The density of this receptor molecule is regulated by two linked, silent polymorphisms (C807T and G873A) in the α2 gene coding sequence. Compared to individuals homozygous for C807, those homozygous or heterozygous for the T807 allele have higher α2β1-integrin density, enhancing adhesion to subendothelial collagen and promoting thrombus formation. The genotype T807 was shown to be an independent risk factor for stroke in young patients (85

1 in 33

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  Consequences of Cerebrovascular Disorders and Impact on Brain Tissues  129 Table 2.6  Range of crude stroke incidence rates (per 100 000 person years) and pathological subtypes among all ages in different populations in high and low-to middle-income countries High-income countries (range low to high) Year

Low- to middle-income countries (range low to high)

Total

CI

ICH

SAH

Total

CI

ICH

SAH

1970–1979

125–460

n.r.

n.r.

n.r.

15–50

n.r.

n.r.

n.r.

1980–1989

156–466

n.r.

n.r.

n.r.

202–217

n.r.

n.r.

n.r.

1990–1999

131–451

137–264

24–48

4–9

167–281

n.r.

n.r.

n.r.

2000–2008

112–223

101–174

10–23

2–10

73–165

47–92

17–44

4–16

2

CI, cerebral infarction; ICH, intracerebral haemorrhage; SAH, subarachnoid haemorrhage; n.r., not reported. Data from Feigin et al.284 and Lovelock et al.625 The 18 high-income countries included Australia, Barbados, Denmark, Estonia, Finland, France, Germany, Greece, Italy, Ireland, Japan, Norway, New Zealand, Portugal, Sweden, Netherlands, UK and USA. The 10 low to middle income countries were Brazil, Chile, French West Indies, Georgia, India, Nigeria, Mongolia, Sri Lanka, Russia and Ukraine.

significantly in Japan, whereas that of SAH has remained fairly constant.993

Stroke incidence Increasing age is the strongest risk factor for stroke (Table 2.5). The risk for a child under 15 years of age is 1 in 100 000, whereas it is 1 in 33 for people aged 85 years and over. The incidence of stroke varies greatly according to the age distribution of the population under study. It is higher in western countries and Japan because of the relatively high proportion of elderly people in these countries. Corresponding trends in stroke incidence (see later) are observed in younger (60

‘

‘

‘

‘

Down syndrome

>20

‘

‘

‘

‘

HCHWA-Dutch type

50

APP

p.Glu693Gln

Unknown

Aβ

HCHWA-Flemish type

45

‘

p.Ala692Gly

‘

‘

HCHWA-Italian type

50

‘

p.Glu693Lys

‘

‘

50-66

‘

p.Asp694Asn

‘

‘

60

‘

p.Glu693Gly; p.Glu693Δ

‘

‘

50-70

‘

p.Leu705Val

‘

‘

20-30

CYST C

Cystatin C

Protease inhibitor

ACys

GEL

Gelsolin

Actin binding

AGel

Hereditary Aβ-CAA

HCHWA-Iowa type HCHWA-Arctic type HCHWA-Piedmont type HCHWAIcelandic type (I) FAF FBD

45-50

BRI2

ABri precursor protein

Unknown

ABri

FDD

30

BRI2

ADan precursor protein

Unknown

ADan

FAP/MVA

35

TTR

Transthyretin

Transport protein

ATTR

PrP-CAA

38

PRNP

Prion protein

Infectious agent

APrP

AD, Alzheimer disease; APP, Amyloid precursor protein; CAA, cerebral amyloid angiopathy; FAF, familial amyloidosis, Finnish type; FAP/MVA, familial amyloidotic polyneuropathy/meningovascular amyloidosis; FBD, familial British dementia; FDD, familial Danish dementia; HCHWA, hereditary cerebral haemorrhage with amyloid angiopathy; PrP-CAA, prion protein cerebral amyloid angiopathy. Data from Revesz et al.839

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  Consequences of Cerebrovascular Disorders and Impact on Brain Tissues  161

in ischaemia, where an acidosis develops. This lack of acidosis in hypoglycaemia likely accounts for the impossibility of producing hypoglycaemic pan-necrosis. Pan-necrosis is commonly seen in more severe tissue ischaemia, in which acidosis is probably a critical part of the pathogenesis of brain infarction. The lack of infarction in hypoglycaemic brain damage thus probably has a simple biochemical explanation. Even in the most severe form of pure hypoglycaemic brain damage, infarction does not occur; damage is limited to selective neuronal necrosis. Neuronal necrosis in hypoglycaemia usually involves the cerebral cortex, hippocampus75,507 and caudate nucleus.507 The spinal cord1007 is rarely involved but the cerebellum635 is never affected. The granule cells of the dentate gyrus are usually conspicuously normal in ischaemic brain damage, being the last neuronal type within the hippocampus to be affected by ischaemia. In hypoglycaemia, possibly because of the extracellular overflow of large quantities of aspartate into the CSF,899 necrosis of dentate granule cells, which contain excitatory receptors on the superficial molecular layer close to the ventricular fluid, occurs in experimental animals74,1077 and man.75,635 (Figure 2.107). Another potentially distinguishing feature is the presence of Purkinje cell necrosis in ischaemia but not in hypoglycaemia. This may relate to the glucose transporter of the cerebellum, which is more efficient than elsewhere in the brain.574 Reversible changes, such as mitochondrial swelling seen by electron microscopy in experimental material, do occur in the cerebellum in severe hypoglycaemia.11 The distribution and degree of neuronal necrosis in the cerebral cortex also tend to differ between hypoglycaemia and ischaemia. A superficial distribution of neuronal necrosis has been described in hypoglycaemic brain damage in humans587 and animals.294,362 This contrasts with an intracortical distribution of neuronal necrosis to the middle cortical laminae in global ischaemic insults. Hypoglycaemic brain damage also gives a paucity of intracortical necrosis, with a widespread even distribution of necrotic neurons over the hemisphere,75,507 accounting for the high CBF in persistent coma.17 The hemispheric distribution of neuronal necrosis does not show the predilection for arterial boundary zones seen in ischaemic brain damage (see Table 2.3). Ultrastructurally, axon-sparing lesions imply that excitatory compounds are present in the extracellular space, binding selectively to dendrites and causing selective dendritic swelling due to receptor activation, followed by ion and water fluxes across the dendritic membrane. Dendritic swelling is a salient feature of hypoglycaemic neuronal death (Figure 2.15), unlike in ischaemia, in which glutamate106 rather than aspartate899 excitatory action predominates.

Neonatal Hypoglycaemia and Cerebral Palsy Low glucose levels in the neonatal period are physiological and there is no good evidence for the concept of hypoglycaemic cerebral palsy. The few articles alleging neuronal death in neonatal hypoglycaemia have other causes discernible by history alone, most frequently ischaemia.43 In a medicolegal case (personal communication with Ronald N Auer), a blood glucose level of almost zero (1 mg/dL)

�����������

(a)

2

(b)

2.107 Hypoglycaemic brain damage. Asymmetrical damage is common in hypoglycaemic brain damage. This case shows a normal thick band of nuclei in the dentate gyrus of the right hippocampus (a) but near-total loss of the dentate granule cell band on the left side (b). Such loss of dentate granule cells in ischaemia would be accompanied by loss of selective vulnerability and total necrosis of all neuronal elements, but here the dentate and CA1 are first affected. Reproduced with permission from Auer.69 © 2005 Wiley-Blackwell.

was found in an awake and normally crying infant, alleged at that time to have been undergoing hypoglycaemic brain damage. The likely cause of abnormal neurological function or structural brain abnormalities that are associated with neonatal hypoglycaemia is related to the overt or covert gestational diabetes in the mothers. The fetus produces insulin in response to maternal hyperglycaemia, as glucose crosses the placenta. The reactive hyperinsulinaemia often, if prolonged, leads to proliferation of the β-cells of the pancreas and other cells producing insulin in ectopic locations. The high insulin is secreted in a physiological attempt to counteract the fetal hyperglycaemia but causes macrosomia, as insulin is the growth hormone of the fetus (after birth, growth hormone, from the pituitary, takes over from insulin in determining body size and morphology). The fetal high insulin levels affect the growth and development

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162  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

of the fetal brain to result in abnormalities, often even ­macroscopic, with high morbidity.240

Cerebral venous thrombosis (CVT) was long considered a rare, usually fatal disease, with a characteristic clinical picture of headache, seizures, focal signs, increased intracranial pressure, papilloedema and coma. The diagnosis was often established only at autopsy. Use of angiographic methods has changed our understanding of CVT.563 Three-dimensional MR flow imaging33 has shown that CVT is more common and often less serious than previously thought. The symptoms may be non-specific and mild. Headache and a clinical picture of benign intracranial hypertension should prompt appropriate imaging investigations (Figure 2.108).799 The incidence of CVT is not known.39 The risk is considered to be approximately equal for both genders. CVT can occur in all age groups, from neonates to the elderly. Among women there seems to be a peak in younger age groups, which probably reflects the association of CVT with pregnancy and the use of oral contraceptives.39,168,259 The detection of milder forms of CVT and recognition that most patients, whose disease would not previously have been recognized, recover after recanalization of the thrombosed blood vessel, has contributed to the decrease in mortality to 5.5–13 per cent.39,168,1001 Neuropathological experience is largely confined to infrequent fatal cases.

to effective antimicrobial and anticoagulant therapy and improved diagnosis of non-infective CVT. The most common site of septic CVT is the cavernous sinus and the most common infective agent Staphylococcus aureus, spreading from an infection in the middle third of the face, sphenoid or ethmoid air sinus, or a dental abscess. Otitis media and mastoiditis may induce septic thrombosis in the lateral sinus, and infections of the scalp may extend via the diploic and emissary veins through the skull bone to the sagittal sinus. In immunosuppressed and chronically debilitated patients, various opportunistic microorganisms, including fungi and cytomegalovirus, can cause CVT.254,675 The altered hormonal status in young women during pregnancy, the puerperium or associated with the intake of oral contraceptives is an important risk factor for CVT.39,168,259 Similarly, an altered state of coagulability associated with any surgery or condition affecting the patient’s general health, such as malignancy and malnutrition, predisposes to CVT, as do systemic connective tissue and inflammatory diseases.32 Behçet’s disease was responsible for 10 in a series of 40 cases from Saudi Arabia.229 Thrombosis may be promoted simply by stagnation of blood flow, e.g. in congenital or congestive heart disease or dehydration. Certain haematological disorders, including thrombocythaemia and sickle cell disease, increase the risk of CVT. Disorders leading to thrombophilia, e.g. Factor V Leiden; deficiencies of antithrombin III, protein C, protein S or plasminogen;224,902,918,989 or the presence of acquired factors inducing hypercoagulability, e.g. antiphospholipid antibodies,595,815 are rare causes but should be considered, particularly when no other cause is obvious.

Aetiology of Venous Thromboses

Pathogenesis and Pathology

CVT can be caused by a multitude of different conditions. In the past, infections were by far the most common cause564 but in a 1992 series of 110 cases only 8.2 per cent had an infective aetiology.39 The decrease is attributable

The cerebral venous network includes many well-developed collaterals (see earlier). Thrombosis has to be extensive, i.e. to involve a sinus or a major part of such, before venous return and capillary blood flow are sufficiently restricted to cause ischaemic damage. Vasogenic oedema develops and is aggravated by the engorgement of arterial blood vessels proximal to the occlusion. The combination of ischaemic injury and arterial engorgement results in haemorrhagic infarction. Thrombolytic therapy in CVT does not carry the same risk of expansion of haemorrhage as in arterial thrombosis. This is because recanalization leads to lowered intravascular pressure in the region of haemorrhagic infarction. The most common sites of CVT are the superior sagittal sinus (72 per cent; Figures 2.108 and 2.109), lateral sinuses (70 per cent combined) and straight sinus (13 per cent), but thrombosis commonly extends to several sinuses or veins.39 The anatomical variation of the left lateral sinus can cause diagnostic confusion; thrombosis of the sinus can cause infarction of the ipsilateral basal ganglia but CVT may be misdiagnosed if, as sometimes happens, the sinus lacks connection with the confluence of sinuses and its transverse portion is hypoplastic. Localized thrombosis of cerebral veins, especially cortical ones, rarely results in tissue damage. However, CVT of the deep internal veins and the great vein of Galen is likely to cause severe damage to the basal ganglia and brain stem.824

Venous thrombosis and infarction Clinical Picture and Incidence

2.108 Superior sagittal sinus thrombosis. T1-weighted gadolinium-enhanced magnetic resonance image of a patient with thrombosis of the anterior part of the sagittal sinus (arrowheads). Scan courtesy of O Salonen, Helsinki University Central Hospital, Helsinki, Finland.

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  Small Vessel Diseases of the Brain  163 (a)

acquire a β-sheet secondary structure and, through an intermediate stage of protofibrils, form highly insoluble cross-βsheet quaternary fibrils. These bind dyes such as Congo red (in which case, they show apple-green birefringence under polarized light) and thioflavin S and T (which fluoresce under ultraviolet light) (Figure 2.110). Because of the characteristic binding of Congo red to amyloid-laden vessels, the condition was originally described as congophilic angiopathy. The term CAA is more appropriate and widely used, although the angiopathy can involve blood vessels in the cerebellum and brain stem as well as the cerebrum. Among the different amyloidogenic proteins, seven are associated with CAA (Table 2.9). In sporadic CAA, the amyloid fibrils are composed of Aβ, whereas in the rare hereditary CAAs all seven proteins are implicated.

2

Amyloid β Angiopathies (b)

Sporadic Amyloid Angiopathies General Aspects The most common types of CAA are those associated with deposition of Aβ,65,623 a cleavage product of amyloid-β precursor protein (APP), encoded on chromosome 21. Deposition of Aβ in the walls of cerebral blood vessels is seen in sporadic CAA, both associated with and independent of sporadic Alzheimer disease (AD), and in Down

(a)

2.109 (a) Thrombosis of the superior sagittal sinus has caused haemorrhagic necrosis and intraparenchymal haemorrhage in the parasagittal brain parenchyma. (b) The occluding thrombus is seen within the sinus.

The most familiar pathological findings in CVT are those caused by superior sagittal sinus thrombosis: haemorrhagic infarction, parasagittal haemorrhages extending to the white matter, and marked oedema (Figure 2.109). The appearances on microscopy are largely the same as in any haemorrhagic infarct but there may be more profuse leukocytic invasion, because the patent arteries allow ready inflow of reactive inflammatory cells.

Small Vessel Diseases of the Brain

(b)

(c)

Sporadic and Familial Cerebral Amyloid Angiopathy Cerebral amyloid angiopathy (CAA) comprises a group of protein-misfolding disorders characterized by the extracellular deposition of fibrillar proteins with amyloid properties in the walls of blood vessels of the brain and meninges.837,838 It is an important cause of brain hemorrhage in the elderly.1052 CAA may also lead to circulatory disturbances and cognitive impairment. To date, over 25 unrelated proteins are known to generate different types of amyloidosis.876 Despite their biochemical heterogeneity, all amyloid fibrils share certain unique physicochemical properties. Amyloid proteins

�����������

2.110 Cerebral amyloid angiopathy (CAA). A penetrating artery from a non-demented patient who died of an intracerebral haematoma. The arterial walls are thickened by amorphous substance, which is coloured red with alkaline Congo stain (a) and gives typical apple-green birefringence (b). (c) Leptomeningeal blood vessels with abundant amyloid in their walls. Amyloid plaques are present in the adjacent cerebral cortex. Thioflavin S fluorescence.

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164  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions Table 2.10  Molecular genetics of small vessel diseases of the brain Disorder

Onset Age (y)†

Gene

Chromosome locus

Protein

Protein type/ function

Predicted dysfunction(s)

CADASIL (most common); autosomal dominant)

20–60

NOTCH3

19p13.2-p13.1

NOTCH3

Transmembrane cell signalling receptor

Aberrant cell-cell signalling, activates unfolded protein response and impaired gene transcription (NICD)

CARASIL (Maeda syndrome); autosomal recessive

20–30

HTRA1

10q25.3-q26.2

HTRA1

Serine protease

Promotes serine-proteasemediated cell death, suppresses TGFβ expression

RVCL disorders: HERNS (Chinese descent); CRV (cerebroretinal vasculopathy); HVR (hereditary vascular retinopathy)

30–50

TREX1

3p21.3-p21.2

TREX1

3′→5′-prime exonuclease DNase III

Disruption of cell death mechanisms, impaired DNA degradation and repair

COL4A1 and COL42related disorders (stroke syndromes); autosomal dominant

14–49

COL4A1; COL4A2

13q34

COL4A1 and A2

Collagen IV, α1 and α2 chains, constituent of BM

Weakening of vascular basement membranes

Hereditary multi-infarct dementia of the Swedish type

28–38

Not known

No linkage to

-

-

-

PADMAL (pontine autosomal dominant microangiopathy and leukoencephalopathy)/ subcorticalangiopathicencephalopathy

12–50

Not known

No linkage to

-

-

-

Hereditary small vessel disease of the brain (SVDB)*

36–52

Not known

Linkage to Chromosome 20

-

-

-

NOTCH3

NOTCH3

*Several other disorders prominently characterized by leukoencephalopathy and cognitive impairment have been described in isolated families. †Age of onset signifies when first cerebrovascular event or gait disturbance due to spasticity was recorded. BM, basement membrane; CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; CARASIL, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy; RVCL, autosomal dominant retinal vasculopathy with cerebral leukodystrophy; NICD, NOTCH intracellular domain.

syndrome. Cerebrovascular Aβ accumulation has also been reported in patients with dementia pugilistica1006 and in both cerebral and spinal vascular malformations.399

Epidemiology Sporadic Aβ-CAA rarely affects people under 60 years of age. In those over 60, the prevalence is a little over 30 per cent,279 increasing with age from just under 15 per cent in the seventh decade to 45 per cent after 80 years.622,987 The high prevalence of CAA in AD indicates that these two disorders are interrelated: in AD, the proportion of patients with CAA increases with advancing age, reaching 80–90 per cent.185,279,812 Furthermore, genetic polymorphisms or mutations that increase the risk of AD are also risk factors for CAA.733 APOE ε4 allele in AD favours vascular over parenchymal deposition of Aβ in a dose-dependent manner.185,827 The odds ratio for CAA increased approximately 3-fold for ε4 heterozygotes and 14–17 fold for ε4 homozygotes,366,812 relative to individuals without the ε4 allele. There is also more severe CAA in ε4 homozygotes but not ε4 heterozygotes, than in people lacking an ε4 allele.1063 The extent to which ε4 confers an increased risk of Aβ-CAA in non-AD patients is unclear; Love et al.622 found no

�����������

association between ε4 and the prevalence of Aβ-CAA in the absence of AD or cerebral haemorrhage. Although protective against AD (see Chapter 16), APOE ε2 is a risk factor for CAA.624,996 Sporadic Aβ-CAA is responsible for about 5–12 per cent of primary non-traumatic PBHs and is the most common cause of lobar haemorrhage.345 However, in a large retrospective neuropathological study of brains from elderly individuals (mean age 78 years), a lower prevalence of CAA in those with than without PBH did not support the concept that CAA is the most important risk factor for PBH in the aged, and suggested that other risk factors including hypertension play a larger role even in this group.480 The association of APOE genotype with Aβ-CAA-related haemorrhage (CAAH) has also been somewhat controversial. In an unselected population, APOE genotype did not associate with lobar PBH.924 A meta-analysis found only marginally significantly increased likelihood of PBH among people with APOE ε2 and a non-significant trend toward increased PBH among those with ε4.973

Clinical diagnosis Sporadic CAA is predominantly a silent disease without overt clinical symptoms. Despite the successes in the

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  Small Vessel Diseases of the Brain  165

development of positron emission tomography (PET) methods to detect CNS amyloid with tracers such as Pittsburgh compound B (PiB) there are no established methods to detect Aβ-CAA.367,489 However, PiB binding was moderately increased in most patients with probable CAA-related intracerebral hemorrhage.632 Non-AD associated CAA manifests most often when it becomes severe enough to cause rupture of a diseased small artery, with resulting PBH. In the intra vitam search for the cause of PBH, gradient echo (T2*weighted) MRI has become an important non-invasive tool, because it detects micro-haemorrhages1051 and the presence of these is used in the Boston criteria for estimating the clinical likelihood that PBH has resulted from CAA.364,544 Microhaemorrhages or microbleeds detected by gradient echo MR imaging occur preferentially in regions of high amyloid load as detected by PiB PET.249 Definite diagnosis necessitates pathological verification. Probable CAAH can be diagnosed if two or more acute or chronic lobar macro-haemorrhages or micro-haemorrhages are identified without any other definite cause of intracerebral haemorrhage, such as excessive warfarin treatment, antecedent head injury, stroke, neoplasm, vascular malformation, vasculitis or blood dyscrasia/ coagulopathy. CAAH is designated as possible when a single lobar haemorrhage is encountered in a patient older than 55 years with no other obvious cause of cerebral haemorrhage. Clinicopathological correlation in a series of 39 cases of PBH, confirmed CAAH in all 13 patients fulfilling ‘probable’ diagnostic criteria but in only 16 (62 per cent) of those who had been diagnosed as having ‘possible’ CAAH.544 Rarely, Aβ-CAA may present as a mass lesion.132 Severe Aβ-CAA is an occasional cause of rapidly progressive dementia, associated with ischaemic damage to the white matter, and petechial cortical haemorrhages or infarcts (see also Aβ-related angiitis).361,612

Pathology The appearance of brains with CAA depends largely on the consequences of the vascular disease. Major CAAHs tend to occur in the frontal or frontoparietal regions and are usually of lobar type. Cerebellar haemorrhages occur but less often.1048 In addition to lobar haemorrhages, CAA may cause cortical petechial haemorrhages or small infarcts, and focal or diffuse white matter ischaemic changes. There may be accompanying AD pathology.365,618 The CAA preferentially affects small arteries and arterioles (Figure 2.110). Most often, CAA has a patchy distribution and, in contrast to the distribution of CAAHs, tends to be most severe in the occipital and parietal meninges and cortex.1049 The hippocampus is usually spared and the white matter and basal ganglia are involved only rarely. The arterial walls are thickened by the deposition of amyloid. This appears amorphous, intensely eosinophilic, periodic acidSchiff (PAS)-positive, Congo red-positive and birefringent, and fluorescent under ultraviolet light in sections stained with thioflavin S or T. In mildly affected blood vessels the amyloid is deposited in a reticular pattern in the basement membrane surrounding smooth muscle cells in the tunica media.1082 More severe disease is associated with confluent accumulation of amyloid in the tunica media accompanied by destruction of the smooth muscle cells, and deposition of Aβ amyloid in the adventitia as well. Concentric

�����������

separation of the amyloid-laden media and adventitia often causes severely affected vessels to have a ‘lumen within a lumen’ appearance. Aβ can also accumulate in the walls of capillaries, albeit less often, predominantly in association with APOE ε4624,996 and mainly in the deeper laminae in the parahippocampal and occipital cortex. In the meninges, larger blood vessels, particularly veins, sometimes show accumulation of amyloid in the outer adventitia only. The presence of abnormally round, thick-walled blood vessels in the meninges or cortical parenchyma should always signal possibility of CAA. The presence of Aβ can be demonstrated immunocytochemically. The predominant form of Aβ in arterioles and arteries is Aβ40, that in capillaries mainly Aβ42.765 Electron microscopy reveals extracellular deposits of randomly oriented, straight, unbranched filaments of indefinite length with a diameter of approximately 6–9 nm (Figure 2.111). CAA tends to be associated with accentuated perivascular neurofibrillary pathology, particularly if the CAA is severe. Systematic morphometric analysis of sections of frontal, temporal and parietal cortex from 51 AD brains revealed that phospho-tau labelling of neurites around Aβ-laden arteries and arterioles significantly exceeded that around non-Aβ-laden blood vessels, which was, in turn, greater than the immunolabelling of cortex away from blood vessels.1093 Aβ-CAA may also be associated with an inflammatory reaction, of which two main patterns have been distinguished. The first consists of the accumulation around Aβ-laden vessels of lymphocytes, macrophages and microglia, and occasionally multinucleated giant cells, but without actual vasculitis. Eng et al.278 described the clinical and pathological findings in 7 such patients (from a consecutive series of 42 cases of CAA). The patients all presented with subacute cognitive decline or seizures, most had white matter abnormalities on neuroimaging and 5 were homozygous for APOE ε4. Most improved clinically and radiologically after immunosuppressive treatment. Other patients may develop a granulomatous vasculitis similar to that in primary angiitis of the

(a)

2

(b)

2.111 CAA. (a) Electron micrograph of approximately 10-nmthick amyloid filaments in the wall of a cerebral artery from a patient with Aβ-cerebral amyloid angiopathy (Aβ-CAA). (b) The walls of intracerebral arteries from a patient with familial British dementia are strongly immunopositive with an antibody to amyloid BRI. (b) Courtesy of Dr T Revesz, Institute of Neurology, London, UK.

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166  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

CNS (PACNS).41 The fairly stereotyped clinical, neuroradiological and neuropathological features in these patients, and other evidence of an immune response to Aβ, prompted Scolding et al. to define a clinical entity of Aβ-related angiitis (ABRA).922 This is described in more detail earlier (see Inflammatory Diseases of the Cerebral Vasculature). ABRA manifests at a later age (60–70 years) than PACNS but may show a similar, if sometimes short-lived, beneficial response to immunosuppressive therapy.922 Aβ is usually abundant in affected blood vessels but scant in the cerebral cortex, where activated microglia containing cytoplasmic Aβ may be present, occasionally in a plaque-like distribution. White matter oedema and rarefaction are common.

Pathogenesis The pathogenesis of CAA is still an intensive topic of research. Three possible sources of Aβ accumulation in vessel walls may be considered. (i) Systemic: Aβ is derived from cells throughout the body and is carried in plasma and transported bidirectionally to and from the brain parenchyma by specific receptors in the vessel walls.232 (ii) Vascular: Aβ is produced locally by vascular smooth muscle cells, endothelium and pericytes,506 all of which express APP.717 (iii) Drainage: Aβ accumulates because of impairment of its clearance from the CNS. Aβ formed by neurons within the CNS is cleared from the interstitial fluid by several processes, including enzymatic degradation within the brain parenchyma and the walls of blood vessels,683,714 transcytosis across the BBB, with endothelial cell uptake mediated by specific receptors including lipoprotein receptor-related protein 1,933 and drainage, together with other constituents of the interstitial fluid, along the perivascular extracellular matrix, to meningeal arteries and probably cervical lymph nodes.1084 This drainage may be impaired in older people, as vascular disease reduces arterial pulsations (thought to supply the motive force for perivascular drainage), with resulting accumulation of Aβ in the arterial wall, further impeding vascular pulsatility.1084 Precipitation within the perivascular extracellular matrix of amyloidogenic solutes such as Aβ in the course of their removal from the brain is probably the cause of most types of CAA.1082 Reduced Aβ-degrading enzyme activity within the vessel wall may be a contributory factor: neprilysin activity was lowest in meningeal blood vessels from patients with most severe CAA, even after adjusting for smooth muscle content.684 Raising or lowering neprilysin activity respectively decreased or increased the death of cultured human cerebrovascular smooth muscle cells on exposure to Aβ. Exclusively neuronal production of Aβ is sufficient to cause CAA.419,420 Affected blood vessels in Aβ-CAA may show segmental dilation or fibrinoid necrosis.643 Serial sectioning and computer-assisted three-dimensional image analysis ­suggest that the following sequential steps lead to blood vessel rupture and haemorrhage:639 (i) accumulation of amyloid in the arterial wall, (ii) death of smooth muscle cells, (iii) dilation (formation of micro-aneurysms) of the artery and (iv) breakdown of the BBB, (v) deposition of plasma proteins in the vessel wall (fibrinoid necrosis), and finally (vi) rupture and haemorrhage. Fibrinoid change was more marked than deposition of amyloid at sites of dilation and rupture.639 Fibrinoid change was also significantly associated with possession of APOE

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ε2,663 which carries an increased risk of haemorrhage in people with Aβ-CAA. The death of vascular smooth muscle cells in CAA seems to depend at least partly on their uptake of Aβ and this, in turn, is influenced by APOE genotype.886 In transgenic mice with the Swedish double mutation in APP, degradation of extracellular matrix proteins by MMP-9 is pivotal in the rupture of Aβ-laden arteries.592

Hereditary Amyloid β-Peptide Cerebral Amyloid Angiopathy Aβ-CAA is familial in the syndromes of hereditary cerebral haemorrhage with amyloid angiopathy of the Dutch631 (HCHWA-D) and Flemish413 (HCHWA-F) types and in the different forms of familial Alzheimer disease (FAD). Mutations in APP that cause haemorrhages are mostly located within the Aβ domain and involve substitutions of amino acids 21–23 of Aβ, whereas those responsible for FAD are most often located in APP next to but outside of Aβ. In HCHWA-D the mutation is E693Q,599 and in HCHWA-F it is A692G. Autosomal dominant HCHWA-D causes recurrent brain haemorrhage in middle-aged normotensive individuals.126 The CAAH is fatal in about two-thirds of patients; survivors develop a vascular type of dementia that is independent of AD plaques and tangles.718 CAA is also prominent in those with the Arctic E693G, Italian E693K and Iowa D694N APP mutations, considered to be primarily FADs. In the latter two, CAAH is a common feature, along with dementia. In those FADs associated with presenilin-1 mutations, CAA has been reported to be more common if the presenilin-1 mutation is located beyond codon 200.645

Other Cerebral Amyloid Angiopathies Icelandic Hchwa Hereditary cerebral haemorrhage with amyloid angiopathy of the Icelandic type (HCHWA-I, hereditary cystatin C amyloid angiopathy) is a rare autosomal dominant CAA associated with fatal brain haemorrhages in young and middleaged normotensive adults.5,484 In addition to cerebral and meningeal arteries, extracerebral tissues, including the skin, show deposition of ACys-amyloid.102 The amyloidogenic protein is a 120-amino-acid cysteine proteinase inhibitor, cystatin C, with a glutamine-to-leucine substitution in codon 68 due to a CTG to CAG point mutation in exon 2.340,598 The low extracellular concentration of mutated inhibitor of cysteine proteinases in the brain may contribute to the destruction of the amyloidotic blood vessels.1000

BRI2 Gene-Related Cerebral Amyloid Angiopathies An autosomal dominant CAA with non-neuritic plaque and neurofibrillary tangle formation, clinically characterized by dementia, spastic tetraparesis and cerebellar ataxia with onset around the sixth decade was originally described by Worster–Drought et al.665,803 The disease was called familial British dementia (FBD) when the defective gene BRI2 was discovered.1046 A mutation at another site on the same BRI2 gene causes the disorder that was first described in a Danish family as heredopathia ophthalmo-oto-encephalica,837 but

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  Small Vessel Diseases of the Brain  167

later renamed familial Danish dementia (FDD). It is characterized by cataracts and ocular haemorrhages in the third decade, followed by hearing problems and, in the fourth to fifth decades, cerebellar ataxia and dementia. Although CAA in both FBD (with deposition of ABri) and FDD (with deposition of ADan) is severe, with concentric splitting and occlusion of affected vessels (Figure 2.111b), haemorrhages are rare. CAA also affects small arteries and arterioles in white matter as well as in systemic organs. In FDD, Aβ is sometimes co-deposited with ADan in both vessels and parenchyma. In both disorders, amyloid plaques, hyperphosphorylated tau-positive neurofibrillary tangles and neurites are also present in the brain parenchyma. FBD and FDD are both caused by a novel amyloidogenic mechanism.876 The BRI2 gene is located on chromosome 13 and encodes a putative type II single-spanning transmembrane precursor protein of 266 amino acids with a molecular weight of ~30 kDa. In both FBD and FDD, the precursor molecule BRI-PP is elongated by 11 amino acids. In FBD, a single base substitution in the stop codon of BRI2 gene results in a larger, 277-residue precursor; release of its 34 C-terminal amino acids generates the highly insoluble ABri amyloid subunit with a relative molecular weight of 4 kDa. In FDD, a decamer duplication insertion before the normal stop codon 267 also results in a 277-residue precursor molecule with a 34-amino-acid amyloidogenic peptide, ADan.1047

Amyloid Transthyretin-Related Cerebral Amyloid Angiopathy The transthyretin (TTR) gene is located on chromosome 18 and encodes a transporter protein for thyroid hormone and retinol-binding protein. Amyloid transthyretin (ATTR) derives from a partially folded monomer of mutated TTR that is prone to form amyloid fibrils. Currently more than 100 different mutations have been identified in TTR. In addition to systemic ATTR deposition, common consequences of which include peripheral neuropathy and cardiomyopathy, nine mutations are associated with oculoleptomeningeal amyloidosis with prominent meningeal CAA.115,640 This is especially marked in patients with the Hungarian (D18G) and Ohio (V30G) mutations and produces a rusty brown colour to the pia mater.334,791 These patients suffer from dementia, cerebellar ataxia, motor dysfunction, and decreased vision and hearing.640

Gelsolin-related cerebral amyloid angiopathy In gelsolin-related familial amyloidosis of the Finnish type,390 the amyloid AGel is deposited systemically, particularly in the skin, peripheral nerves and cornea. In the CNS, deposition of AGel is widespread in spinal, cerebral, and meningeal blood vessels, and extensive extravascular deposits are present in the dura, spinal nerve roots and sensory ganglia.542 Clinical manifestations include atrophic bulbar palsy, ataxia, minor cognitive impairment, facial palsy, peripheral neuropathy and corneal lattice dystrophy.

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Gelsolin is an 80-kDa (cytoplasmic) to 83-kDa (plasma) protein involved in the gel–sol transformation of actin. It is encoded by the GEL gene on chromosome 9. The two mutations in AGel amyloidoses are both in codon 654, either G:A or G:T, resulting in substitutions D187N or D187Y. The cleaved product AGel is composed of amino acids 173–243.

2

Prion protein-related cerebral amyloid angiopathy CAA in patients with prion disease usually results from vascular deposition of Aβ. CAA due to prion protein (PrP) deposition is rare and associated with stop mutations in PRNP. In one family with Gerstmann–Sträussler–Scheinker syndrome, PrP amyloid (APrP) was extensively deposited in parenchymal and leptomeningeal vessels and in the surrounding neuropil.339 A TAT to TAG point mutation in codon 145 (Y145STOP) resulted in an N- and C-terminally truncated amyloidogenic 7.5-kDA PrP fragment composed of 70 amino acids. Y163STOP, Y226STOP and Q227STOP PRNP mutations were also reported to cause APrP-CAA.477,839

Hereditary small vessel diseases Molecular genetic studies have identified several monogenic conditions that involve small vessels and predispose to ischaemic and haemorrhagic strokes and diffuse white matter disease (Table 2.10). Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) remains the most common hereditary SVD. Other hereditary SVDs include cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), retinal vasculopathy with cerebral leukodystrophy (RVCL), and the collagen type IV, α1 (COL4A1)-related disorders.246,1104 The hereditary SVDs, albeit with variations in phenotype, demonstrate convergent effects of microangiopathy on cerebral grey and white matter, leading to cognitive impairment.

CADASIL CADASIL as we know it today was probably first described in 1955 in a family that was reported to have Binswanger’s disease.1035 Subsequently, several families with multiple Table 2.11  Causes of non-traumatic parenchymal brain haemorrhage Cause

Percent

Hypertension

50

Cerebral amyloid angiopathy

12

Anticoagulants

10

Tumours

8

Illicit and licit drugs

6

Arteriovenous malformations and aneurysms

5

Miscellaneous

9

Adapted from Feldman.286

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168  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

strokes and dementia were reported under various designations. In 1993, the disease was linked to a locus in chromosome 19 in two French families and given the acronym CADASIL.1013 The defective gene on chromosome 19 and its protein product were identified in 1996.492 To date, there are likely more than 700 CADASIL families worldwide among all ethnicities, with an estimated prevalence of 4–5 per 100 000.508,715,831

Clinical Features The cardinal clinical features of CADASIL are (i) migraine with aura, often exceptionally severe, sometimes to the extent of causing hemiparesis; (ii) recurrent ischaemic strokes; (iii) psychiatric symptoms; and (iv) cognitive decline, with eventual dementia.181 CADASIL patients may have their first stroke before the age of 30 years but the peak is around 40–50 years. White matter changes are detectable on T2-weighted MRI well before clinical symptoms, temporopolar hyperintensities being the most characteristic finding (Figure 2.112). Decreased cerebral circulation can be demonstrated by PET1027 and bolus-tracking MRI.148,180 Multiple small infarcts, detectable on T1-weighted MRI, cause cognitive decline between 40 and 70 years, primarily in executive frontal lobe functions, followed by impairment of memory and other cognitive functions, leading to the development of subcortical dementia. About 80 per cent of CADASIL patients aged over 65 years are demented.247 Only symptomatic therapy is available, and death usually ensues within 15–25 years of the first strokes.

(a)

Pathology The characteristic neuropathological feature is non-arteriosclerotic, non-amyloid arteriopathy,511,881 mainly affecting penetrating small and medium-sized arteries of the white matter but also present in leptomeningeal blood vessels. The symptoms are almost exclusively neurological although the arteriopathy is generalized and also affects, albeit less severely, blood vessels in systemic organs, including skin, skeletal muscle and peripheral nerve.881 The vascular smooth muscle cells degenerate with accompanying accumulation of basophilic, PAS-positive granules in the tunica media. The vessel walls become markedly thickened and fibrotic (Figure 2.113), particularly in the white matter, leading to narrowing of the lumina, impaired circulation and multiple infarcts (Figure 2.114).676 Electron microscopy shows destruction of vascular smooth muscle cells and deposition of pathognomonic clumps of granular osmiophilic material (GOM) between the degenerating vascular smooth muscle cells and in plasmalemmal indentations of these cells, particularly on the abluminal aspect (Figure 2.115). It is not exactly known how early GOM appears but it has been detected in skin biopsies of asymptomatic patients before the age of 20 years and was abundant in the cerebral arterioles of a 32-year-old patient.676 Vessels in the cerebral white matter are most severely affected and where most infarcts occur. Cortical grey matter and the retina are relatively spared but infarcts in the basal ganglia are common (Figure 2.114) and infarcts also occur in the brain stem.

(b)

2.112 (a) T2-weighted fluid attenuation inversion recovery (FLAIR) image of a 62-year-old female patient with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) who had several subcortical ischaemic events. The temporopolar hyperintensities are pathognomonic for CADASIL. (b) In a moderately demented homozygous CADASIL patient with a history of several strokes, the MRI shows extensive confluent hyperintensities. Scan (b) courtesy of T Kurki, Turku University Hospital, Turku, Finland.

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  Small Vessel Diseases of the Brain  169

2

(d)

2.113 Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The media and the adventitia of a penetrating artery from the parietal white matter of the patient in Figure 2.112b are markedly thickened, with accumulation of basophilic (a), periodic acid-Schiff (PAS)-positive (b), granular non-amyloid material in the media, and fibrosis of the adventitia. Immunolabelling for smooth muscle actin (c) shows irregular degeneration of the smooth muscle cells. (d) Accumulation of material that is immunopositive for Notch-3 extracellular domain (N3ECD) in association with degenerating vascular smooth muscle cells.

2.114 Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The brain of a 63-year-old female patient, who had presented with adult-onset migrainous headache with visual aura and who had her first stroke at the age of 52 years. Thereafter, strokes recurred several times per year and she became demented, with other stroke-related deficits. There are multiple subcortical infarcts, most obvious in the deep grey matter and left parasagittal frontal white matter.

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In accord with other SVDs (see later), CADASIL is associated with alterations in retinal microvessel: arteriolar narrowing can be revealed by fundoscopy and fluorescence angiography.181 Autopsy shows thickened vessel walls with fibrosis, accumulation of granular material, pericyte degeneration and loss of vascular smooth muscle cells in the central retinal artery and branches.397 Demonstration of GOM by electron microscopy in dermal biopsies offers a means of intra vitam diagnosis (Figure 2.115)511,881 and was reported to be completely congruent with genetic screening.1004 Immunohistochemical demonstration of Notch3 extracellular domains (N3ECD; Figure 2.115c) in skin arteries is also helpful in establishing diagnosis495 provided clinical symptoms are consistent with the disease. These methods are practical when diagnosing new families with suspected disease, because genetic screening can be labour intensive and costly.

Genetics The aberrant gene in CADASIL is NOTCH3, located at chromosome 19p13. In humans, NOTCH3 spans 33 exons and encodes a single transmembrane receptor protein, NOTCH3, of 2321 amino acids (Figure 2.116). NOTCH3

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170  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions (b)

(a)

(c)

� �

�

2.115 Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). (a) Lowpower electron micrograph of a small dermal artery in a skin biopsy. There are numerous deposits of granular osmiophilic material (GOM; arrows) in plasmalemmal indentations of smooth muscle cells (smc) some of which appear to be degenerating. The basal lamina of the cells is irregularly thickened. (b) Higher magnification of a biopsy from a 19-year-old patient demonstrates the typical granular appearance of GOM (arrows), and the pinocytotic vacuoles that are often seen in the adjacent smooth muscle cell cytoplasm (visible here beneath the upper GOM deposit). (c) Immunostaining with specific antibody demonstrates the accumulation of N3ECD in the walls of small arteries deep in the dermis. (c) Reproduced with permission from Kalimo and Kalaria.508 © 2005 Wiley-Blackwell.

has pivotal functions in stem cell renewal, proliferation, differentiation, and apoptosis during organogenesis, including vasculogenesis. NOTCH family genes are highly conserved within all species: orthologous genes have been identified in organisms from nematodes to humans.57,583 In adult human tissues, NOTCH3 appears to be expressed predominantly in vascular smooth muscle cells.494 Upon ligand (transmembrane Delta and Jagged molecules) binding, NOTCH33 is cleaved at sites S2 and S3, and the released intracellular domain enters the nucleus to regulate transcription (Figure 2.116). The ligands and the functional responses to NOTCH signalling in adult human tissues are not fully known. More than 200 different mutations in NOTCH3 segregate with CADASIL. The vast majority (~70 per cent) are missense point mutations. Almost all of the mutations result in either substitution of a wild-type cysteine by another amino acid or vice versa, in one of the 34 epidermal growth factor-like (EGF) repeats in N3ECD (Figure 2.116). In addition, small deletions have been described, which cause loss of one or three cysteine residues:248,493 instead of the normal six cysteine residues, the mutant EGF repeats contain three, five or seven cysteine residues. Two de novo mutations as well as two non-cysteine mutations have been reported.1104

Pathogenesis The pathogenesis of CADASIL remains elusive. The uneven number of cysteine residues affects the formation of disulphide bridges and changes the three-dimensional structure and NOTCH3 receptor.493,494 The formation of abnormal disulphide bridges could affect receptor trafficking, processing, specificity for ligand binding and/or signal transduction. In vitro studies suggested that apart

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from mutations in the ligand-binding site of the NOTCH3 receptor, mutations in NOTCH3 do not appreciably impair signal transduction activity, NOTCH3 processing, or signalling to CBF1/RBP-Jκ activation (EGF repeats 10–11; Figure 2.116) Thus, loss of signalling by mutated NOTCH3 may not always be the direct cause of the disease. Over the years, the accumulation of misfolded, nondegradable N3ECDs that accumulate within GOM1105 may affect the function of vascular smooth muscle cells and interfere with perivascular drainage.173 Proteomic studies on cultured vascular smooth muscle cells from a CADASIL patient revealed differences in the expression of proteins involved in protein degradation and folding, indicating that mutant NOTCH3 causes endoplasmic reticulum stress and activates the unfolded protein response.455,978 In HEK293 cell lines, mutant forms of NOTCH3 were prone to aggregation and retention in the endoplasmic reticulum; they reduced cell proliferation and increased sensitivity to proteasome inhibition.978

CARASIL (Maeda syndrome) Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) was first described as Maeda syndrome.55 The acronym CARASIL was applied later but neglects the skeletal pathology and could cause confusion with CADASIL. The predominantly normotensive patients, generally from families with consanguinity, have severe non-amyloid arteriopathy, leukoencephalopathy and lacunar infarcts together with characteristic spinal anomalies and alopecia. Thus far, few Japanese families with CARASIL have been described.38,737 CARASIL has also been reported in China and exists in other countries.

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  Small Vessel Diseases of the Brain  171 ������ �

����� �

(DSL)Ligand+NECD?

Maml

Notch receptor

Delta

Transcription Gene expression and/or repression e.g. HES,HERPS, Mash1

NICD translocation

Jagged Kuz and PS dependent -secretase

2

RBP-Jk

Deltex?

TACE and PS dependent -secretase? ����� Lfng

Furin-like

2.116 Schematic illustrates NOTCH receptor generation and signalling mechanism. The putative Notch signalling pathway involves a complex series of proteolytic cleavage events eventually mediating transcriptional activation of a target gene. NOTCH is glycosylated by O-fucosetransferase1 (O-FucT-1) and Fringe, in the endoplasmic reticulum (ER) and the trans-Golgi network, respectively, after translation. NOTCH first undergoes constitutive cleavage at a site designated S1, by a furin-like convertase in the trans-Golgi network, to form a functional heterodimeric receptor, which is presented on the cell surface. The leucine zipper domain in Notch extracellular domain (NECD) may regulate dimerization. NOTCH ligands such as Jagged and Delta are activated by endocytosis to interact with NOTCH, and NECD is trans-endocytosed by the ligand-presenting cell in a Mib1/Neur1-dependent process. The NECD dissociation enables second cleavage at S2 site by ADAM-10, adisintegrin and metalloprotease/TNF-α converting enzyme (TACE). The S2-cleaved NOTCH receptor is ubiquitinated (Ub) and endocytosed in a clathrin/AP2/epsin1-dependent or -independent way to be either cleaved at S3 site by γ-secretase to generate an intracellular domain (ICD), or destined for lysosomal degradation. ICD translocates to the nucleus and binds to a DNA binding protein, CSL (for CBF1/RBP-Jκ, Suppressor of Hairless and Lag-1). The following recruitment of the coactivator, Maml (mastermind-like), promotes transcriptional activity of its target genes, such as the hairy/enhancer of split (Hes). Diagram redrawn courtesy of Y Yamamoto, Yamaguchi University Graduate School of Medicine, Japan.

Clinical features Maeda syndrome begins at the age of 20–45 years and usually leads to death within about 7.5 years.318,1108 It is more common in males and manifests with recurrent lacunar strokes, predominantly involving basal ganglia and brain stem structures. Most patients develop cognitive impairment. Patients also have orthopaedic manifestations, including intervertebral disc herniations, kyphosis, o­ssification of spinal ligaments and various osseous deformities. Alopecia is a characteristic feature. T2-weighted MRI reveals diffuse high-signal abnormalities in the white matter (most marked in the deep white matter), and multiple lacunar infarcts in the basal ganglia, thalamus brain stem and cerebellum.318,1108

Pathology Small penetrating arteries and arterioles (100–400 μm in calibre) in the white matter and basal ganglia are most severely affected. The intima shows marked fibrous thickening and the internal elastica is fragmented. Vascular smooth muscle cells in the media are destroyed and adventitia is thinned. GOM deposits are not present. The lumen is usually narrowed but segmental dilation can occur. There is reduced

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myelin staining of cerebral white matter, with relative sparing of U-fibres. Multiple small foci of softening and cavitation are seen in the cerebral white matter, basal ganglia, thalamus and brain stem.55,1108 Maeda syndrome appears to be a generalized disorder but the vascular pathology in other organs is less severe. Skin biopsy is not helpful for diagnosis.

Genetics CARASIL co-segregates with the 2.4-Mb region on chromosome 10q, which contains mutations in the HTRA1 gene.393 Thus encodes the serine protease HTRA1, known to influence multiple processes including transforming growth factor β (TGF-β) signalling and perhaps the metabolism of APP, which includes several HTRA1 cleavage sites. Both nonsense and missense mutations of the HTRA1 gene have been identified in CARASIL families. The mutations result in reduced HTRA1 protease activity (21–50 per cent) or loss of the protein.393

Pathogenesis In CARASIL, the adventitial layers of vessels are profoundly thin, with reduced immunolabelling of type I, III and VI collagens. Degeneration of both the medial and adventitial

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172  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

layers is likely to impair autoregulation and functional hyperaemia. Immunohistochemical analysis of the cerebral vessels reveals increased extra domain-A region of fibronectin and versican in the thickened intima and TGF-β1 in the media of arterial walls. In CARASIL, the mutant variants of HTRA1 are unable to repress the phosphorylation of SMAD proteins, effectors of TGF-β signalling (Figure 2.117).393,1104 TGF-β signalling induces synthesis of extracellular matrix proteins including fibronectin and versican and promotes vascular fibrosis.991 TGF-β signalling is also involved in angiogenesis and vascular remodelling. The findings in CARASIL extend the spectrum of diseases associated with the dysregulation of TGF-β signalling. Defective TGF-β signalling due to mutations in the TGF-β receptors leads to hereditary haemorrhagic telangiectasia, whereas activation of TGF-β signalling contributes to Marfan’s syndrome and associated disorders.991

Dysregulation of inhibition of TGF-β signalling has been linked to alopecia and spondylosis, which are characteristic of CARASIL. A single-nucleotide polymorphism in the promoter region of HTRA1 that results in elevated levels of HTRA1 is a genetic risk factor for the neovascular form of age-related macular degeneration.

Retinal Vasculopathies with Cerebral Leukodystrophy (RVCL) Hereditary endotheliopathy with retinopathy, nephropathy and stroke (HERNS), cerebroretinal vasculopathy (CRV) and hereditary vascular retinopathy (HVR) were reported independently but linkage analysis demonstrated that they are allelic disorders or different phenotypes of same disease spectrum.761 They map to the same locus on chromosome 3p21.1-p21.3 and are now described as autosomal

���

� ��

JAGGED1

TGF-b NOTCH3

TGF-b receptor

Smad4

p38 MAPK/ HSP27

RhoA/RhoK

ALK-5

ALK-1

Smad2/3

Smad1/5/8

VSMC contraction

NICD

Migration Proliferation UbiquitinProteasome System

HDAc CoR RBP-J

HDAc MamI RBP-J

HES/HRT PDGFR-b

VSCM maturation Fibrosis ECM synthesis

HTRA-1 pro-TGF-b

TGF-b

ER

2.117 Signalling pathways in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Schematic shows putative crosstalk and mediators between NOTCH3 and TGF-β signalling pathways, which converge to cause changes in vascular smooth muscle cell (VSMC) maturation, fibrosis and extracellular matrix (ECM) synthesis. In NOTCH3 pathway, after final cleavage by γ-secretase from the receptor the NOTCH extracellular domain (NICD) translocates to the nucleus to form a complex with RBPJ to induce transcriptional activation of target genes. In the TGF-β pathway, HTRA1 protease activity inhibits TGF-β signalling via ALK5/Smad 2/3, leading to cell maturation and control of extracellular matrix production. ALK, activin receptorlike kinase or active TGF-β type I receptor; ECM, extracellular matrix; NECD, NOTCH extracellular domain; NICD, NOTCH intracellular domain; Rho, Rho protein kinases; Smad, mediators of transcriptional activation; VSMC, vascular smooth muscle cells. Diagram courtesy of Y Yamamoto, Yamaguchi University Graduate School of Medicine, Japan.

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  Small Vessel Diseases of the Brain  173

dominant retinal vasculopathy with cerebral leukodystrophy (RVCL).246,845

Clinical features The RVCLs all cause progressive central visual impairment. In CRV, symptoms begin during the fourth or fifth decade. Subjects experience migraine-like episodes, transient ischemic attacks and strokes with motor and sensory loss, headaches, personality changes, depression and anxiety. In the late stages, subjects show cognitive impairment. Death occurs within 10 years of onset. Neuroimaging shows diffuse white matter changes consistent with SVD. Renal disease occurs in HERNS and CRV, whereas HVR is associated with Raynaud phenomenon.1104 Neurological complications of CRV and HERNS lead to death before 55 years, whereas HVR patients live longer.1104 Common ophthalmological findings in these disorders include capillary dropout and vascular tortuosity, aneurysms and telangiectasia. No specific therapy is available.

Pathology Lesions occur in the pons, cerebellum and basal ganglia in addition to the frontal and parietal lobes, and consist of foci of necrosis with negligible inflammation. Blood vessels have multilayered endothelial basement membranes and thickened adventitia. Affected vessels are often occluded by fibrin thrombi, resulting in small infarcts in the surrounding white matter.761 In the retina, there are micro-aneurysms of arterioles and foci of capillary telangiectasia; in advanced stages, the damaged arteries become occluded, giving rise to retinal infarcts. In CRV, cerebral pathology is similar to that of HERNS:761 fibrosis of the walls of small and medium-sized arteries and veins within the white matter and basal ganglia lead to obliteration and foci of parenchymal necrosis. Abnormalities of the microvasculature in the liver and kidney are not as prominent as in the brain.

Genetics SVDs in the RVCL group are caused by carboxyl terminal truncations causing frameshifts in the TREX1 gene, which encodes an autonomous (non-processive) 3′–5′ DNAspecific exonuclease found in all mammalian cells. Whilst heterozygous mutations in TREX1 cause RVCL, homozygous mutations in the same gene are linked to the typical autosomal recessive form of Aicardi–Goutières syndrome (AGS).845 AGS manifests as a progressive encephalopathy of early onset, brain atrophy, demyelination, basal ganglia calcifications and chronic lymphocytic proliferation in the CSF.

Pathogenesis Mutations in the carboxyl terminus of TREX1 do not diminish the DNA-specific exonuclease activity of the protein but disrupt the predicted transmembrane domain and prevent TREX1 from localizing to the normal perinuclear site; the mutant proteins instead diffuse freely throughout the cytoplasm.524 It is unclear how this leads to the phenotype of RVCL.

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Rarer Vasculopathies and Angiopathies Several individual families with hereditary SVDs have been described that are not explained by any of the known gene defects. Hereditary systemic angiopathy (HSA) manifests with cerebral calcifications, retinopathy, progressive nephropathy, hepatopathy and microangiopathy reminiscent of a RVCL variant.1094 Patients present in their midforties with visual impairment, migraine-like headaches, skin rashes, seizures, motor paresis and cognitive decline. The retinal microvessels undergo progressive occlusion leading to ischaemic retinopathy with subsequent optic disc atrophy and formation of capillary aneurysms. Pathological changes include foci of necrosis in the white matter, with prominent perivascular inflammation, oedema and astrocytic gliosis. There is evidence of proliferation of microvessels, many with hyperplastic endothelium and severely thickened walls, and some showing fibrinoid necrosis or thrombosis.1094

2

SVD Associated with Collagen Type IV, A1 (Col4a1) and A2 (Col4a2) Mutations The recently recognized spectrum of COL4A1and 2-related disorders encompasses a number of conditions with features of SVD of varying severity.20,801,1034 The COL4A1 gene is located on chromosome 13q34 and consists of 52 exons spanning approximately 158 kb. Mutations are associated with four major phenotypes:579 (i) perinatal haemorrhage with porencephaly in survivors, (ii) hereditary infantile hemiparesis, retinal arteriolar tortuosity and leukoencephalopathy (HIHRATL),1033 (iii) SVD with Axenfeld–Rieger anomaly (anterior segment dysgenesis of the eye) and (iv) hereditary angiopathy with nephropathy, aneurysms (typically of the internal carotid artery) and muscle cramps (HANAC). Other clinical manifestations can include Raynaud’s phenomenon, kidney defects and cardiac arrhythmias. COL4A1 mutations cause variable degrees of retinal arteriolar tortuosity, and abnormalities of endothelial basement membranes in the skin and cerebral vasculature have been documented. Neurological manifestations of COL4A1 and COL4A2 mutations depend on the age of disease onset but can vary even within families. Affected individuals may present with infantile hemiparesis, seizures, visual loss, dystonia, strokes, migraine, mental retardation, or cognitive impairment and dementia. Single or recurrent intracranial haemorrhages may occur in non–hypertensive young adults: spontaneously, subsequent to trauma or as a result of anticoagulant use. Stroke is often the first presentation of the disease, with a mean age of onset of 36 years.579 Neuroimaging shows typical features of SVD including diffuse leukoencephalopathy with most severe involvement of posterior periventricular regions, subcortical infarcts, cerebral microbleeds, and dilated perivascular spaces. Cases with porencephaly have large fluid-filled cavities that appear as paraventricular cysts.579 Autosomal dominant COL4A1-related disease is described in at least 12 European caucasian families, with 100 per cent penetrance. Several other familial disorders with an SVD phenotype have been reported but the genetic basis not yet established.1104

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174  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

Haemorrhagic Stroke and Consequences

but also microbleeds: small, perivascular, clinically silent extravasations of blood.368,546,863 Micro-bleeds can be detected in about 5 per cent of older adults without clinical cerebrovascular disease.546 Micro-bleeds are demonstrable in association with 47–80 per cent of larger spontaneous PBHs and 18–78 per cent of people with ischaemic cerebrovascular disease.546 They are thought to be harbingers of future major PBH.27,546,1051 Hypertension and CAA are numerically the most important causes of PBH (Table 2.11). Hypertension is thought to be responsible for about half of PBHs but the numbers have been decreasing with effective antihypertensive treatment. The cause of PBH affects its location in the CNS (Table 2.13).286 The common, large parenchymal bleeds are often associated with micro-bleeds and subacute ischaemic lesions.370

Parenchymal brain haemorrhage The terms parenchymal brain haemorrhage (PBH) and intracerebral haemorrhage (ICH) are used interchangeably, although PBH is preferred, as the haemorrhage can involve the cerebellum and/or brain stem as well as the cerebrum.

Epidemiology and causes The relative frequency of PBH in first-time stroke varies widely between different populations. East–West differences were evident even prior to the reduction in stroke incidence over recent years (Table 2.8).300 Annual incidence estimates in studies of white populations vary between 11 and 20 (crude) or 16 and 32 (age-adjusted) per 100 000.303,735 The incidence of PBH is dependent on the ethnic background and is, for example, markedly higher in African-Americans (in Kentucky, USA, 48.9 versus 26.6 per 100 000)300 and Far Eastern populations (47 (adjusted) in Japan462 and over 60 in Taiwan).443 Estimates of primary ICH and SAH in low- to middle-income countries during the period 2000– 2008 were almost twice the rates in high-income countries (primary IAH 22 per 100 000 versus 10 per 100 000 and SAH 7 per 100 000 versus 4 per 100 000, respectively).284 In several populations, the incidence of PBH has declined over the past three decades (Figure 2.118).586,877,994 The mortality rate in PBH is higher than for ischaemic strokes: the 1-month case-fatality rate is estimated at 35–50 per cent.283 Modern imaging methods provide reliable intra vitam identification of PBH and have markedly improved the accuracy of diagnosis (Figure 2.119). T2*-weighted MRI allows the detection of not only the larger haematomas

Hypertensive haemorrhage The decline in the incidence of PBH is largely attributable to improvements in diagnosis and treatment of hypertension.140,519,822,877 Hypertension was the cause of almost 90 per cent of all PBHs in the 1940s, 70–80 per cent in the 1970s319 and only 45–60 per cent in the 1980s and 1990s (Table 2.13).919,1097 PBH constitutes a relatively high proportion of strokes in Far Eastern populations, reflecting the high prevalence of hypertension.527,554,931 PBH is often considered to be a one-time event, with recurrent bleeding being rare compared with that from saccular aneurysms, vascular malformations or CAA.519 Rebleeding has, however, become more common with increased survival of high-risk patients. Recurrence rates from 1.8 to 6 per cent are reported, recurrence being more common in people with persistence of high diastolic blood pressure.50 Hypertensive PBH most commonly occurs in the deep cerebral grey matter (putamen and thalamus, about 60

Proportional frequency (%) of stroke pathological type

90 80 70 60 50 40 30 20 10 0

1980–89

1990–99

2000–08

High-income countries IS

PICH

SAH

2000–08 Low to middle income countries

UND

2.118 Worldwide trends in relative frequency of intracerebral and subarachnoid haemorrhage relative to ischaemic stroke. The graph compares the frequencies of stroke types in high-income countries and low- to middle-income countries across different study periods (pooled estimates). IS, ischaemic stroke; PICH, posterior intracerebral haemorrhage; SAH, subarachnoid haemorrhage; UND, undefined pathological type of stroke. Graph courtesy of V Feigin, AUT University, Auckland, New Zealand.

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  Haemorrhagic Stroke and Consequences  175 (a)

(b)

(d)

(e)

(c)

2

2.119 Images in a patient with an acute haematoma in the left putamen (down arrow) and clinically silent micro-bleeds (up arrow) in the basal ganglia. The micro-bleeds are detectable only in the transverse T2*-weighted gradient-echo image (a) as foci of low signal intensity. (b,c) show, in the same plane of section as (a), a computed tomography (CT) scan (b) and T1-weighted spin echo (SE) (c), T2-weighted SE (d) and fluid-associated inversion recovery (FLAIR) (e) magnetic resonance (MR) images.

Table 2.12  Sites of non-traumatic parenchymal brain haemorrhage Haemorrhage

n

per cent

Lobar

65

31

Frontal

17

Parietal

11

Temporal

9

Occipital

9

Two lobes

42

Three lobes

12

Deep supratentorial

107

Adapted from Massaro AR et

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51

Putamen

48

Thalamus

43

Caudate

9

Deep infratentorial

37

18

Cerebellum

70

Pons

30 209

al.652

per cent

46

One lobe

Total

per cent

100

With permission from Lippincott Williams & Wilkins/Wolters Kluwer Health.

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176  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions Table 2.13  Proportional distribution of parenchymal brain haemorrhage (PBH) by location in different populations Study

Total number of PBH

Lobar (per cent)

Deep cerebral (per cent)

Brain stem (per cent)

Cerebellum (per cent)

Cincinnati, USA

1038

35

49

6

10

Izumo, Japan

350

15

69

9

7

Southern Sweden

341

52

36

4

9

Jyväskylä, Finland

158

34

49

7

11

Dijon, France

87

18

67

6

9

Perth, Australia

60

30

52

7

10

Results obtained from several sources.42,300,303,352,462,735

per cent), the hemispheres (lobar, 20 per cent), cerebellum (13 per cent) and pons (7 per cent). The diameter of the arterial vessels at these sites ranges from 50 to 200 μm. Rupture of non-aneurysmal arterioles damaged by hypertension is regarded as the main cause of hypertensive haemorrhages; the role of Charcot–Bouchard microaneurysms as a source of PBH is uncertain (see Hypertensive Angiopathy, earlier).183,1062 Generalized degenerative change in small arteries and arterioles, with perivascular accumulation of haemosiderin (microbleeds), is readily demonstrable pathologically, whereas microaneurysms are relatively rare. Identifying the source of a particular hypertensive bleed is generally not possible because the ruptured part of the affected small artery is likely to have been destroyed by the haemorrhage.

Cerebral amyloid angiopathy Sporadic CAA is responsible for up to 12 per cent of primary non-traumatic PBH. Sporadic CAA is typically associated with lobar (i.e. peripherally located) ICH (it is responsible for about 30 per cent), particularly in elderly normotensive patients (Figures 2.110, 2.112 and 2.120). Severe CAA is associated with high risk of PBH.1057 Microhaemorrhages are demonstrable in up to 80 per cent of CAA patients on T2*-weighted MRI.863 The ‘Boston criteria’ estimate the clinical likelihood of PBH being CAAH.364,544 A diagnosis of definite CAA requires pathological verification. Probable CAAH can be diagnosed if two or more acute or chronic lobar macrohaemorrhages or microhaemorrhages are identified, without any other definite cause of PBH (such as excessive warfarin treatment, antecedent head injury, stroke, neoplasm, vascular malformation, vasculitis or blood dyscrasia/coagulopathy). Possible CAAH is diagnosed when a single lobar haemorrhage occurs in a patient over 55 years with no other obvious cause of cerebral haemorrhage. In one prospective series with pathological follow-up, a diagnosis of probable CAA proved to be to 100 per cent accurate but CAA could be confirmed in only 62 per cent of cases diagnosed as possible.544 CAAH can occur at multiple sites and progress over time. CAAH tends to be superficial and is quite often associated with secondary SAH (Figure 2.120). Rupture of amyloid-laden arteries may sometimes occur directly into the subarachnoid space, causing primary SAH (see

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Subarachnoid Haemorrhage). The rigid and fragile arterial walls may make haemostasis difficult to achieve after neurosurgical evacuation of the haematoma.369,1062 Antiplatelet and anticoagulant medication and head injury probably increase the risk of CAAH.662 CAA increases the risk of PBH after fibrinolytic treatment of acute myocardial infarction.786 It is unclear whether or not hypertension increases the risk of CAAH, although antihypertensive medication tended to lower the risk of probable CAAH (defined according to the Boston criteria, see earlier) in patients who had previously experienced a stroke or TIA.54 The risk of CAAH is marginally increased in APOE ε2 carriers and shows a trend towards association with the ε4 allele.973

Haemorrhage Caused by Anticoagulants and Antithrombolytics Nearly 1 per cent of patients treated with anticoagulants are reported to experience intracranial haemorrhage during therapy. The risk of PBH is increased about 8–11-fold.401,519 During treatment of ischaemic stroke with anticoagulants, not only may the infarct not only undergo haemorrhagic transformation (see earlier), but bleeding from damaged blood vessels may also produce sizable haematomas. Anticoagulation prolongs the time for the breached vessels to be resealed, making haematomas larger and their prognosis worse. The mortality of PBH patients on anticoagulation is about twice the overall mortality of PBH.301 Thrombolytic therapy may similarly cause PBH. The incidence of PBH after thrombolysis for acute myocardial infarction is about 0.3–0.8 per cent.468 The risk of PBH is considerably higher when the thrombolytic treatment is given for acute cerebral ischaemia. The incidence of PBH (often fatal) was reported to be 3.5 times higher among 5216 patients treated with thrombolysis than in placebotreated patients. Despite this, there was a significant net reduction in the proportion of patients who died or became dependent on carers.1068 The frequency of haemorrhagic events increases with delay of treatment beyond 3 hours.1031

Tumour Haemorrhage Both primary and metastatic intracranial tumours can cause major PBH. In about half such cases, PBH is the first manifestation of the tumour.608,642 The overall frequency of

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  Haemorrhagic Stroke and Consequences  177

tumour haemorrhage but not acutely in non-neoplastic PBH. Metastases are the most common cause of tumour-related PBH. Among primary tumours associated with PBH, glioblastomas predominate, in approximate proportion to their relative frequency (it is perhaps surprising that PBH does not occur more often in these tumours in view of the profusion of abnormal blood vessels).913 Bronchogenic carcinoma, choriocarcinoma, melanoma and renal cell carcinoma are the metastatic tumours most often responsible for PBH.519

(a)

2

Drugs and Intraparenchymal Haemorrhage

(b)

The risk of PBH among drug abusers is markedly increased, especially among users of cocaine, heroin and sympathomimetics such as phenylpropanolamine, phencyclidine and amphetamines.596,949 Drug abuse was identified as a predisposing factor in 47 per cent of stroke patients under 35 years.503 In drug abusers and even in alcoholics, the haematomas are commonly lobar, in the subcortical white matter. The bleeding usually develops within minutes to a few hours after drug use. Two main pathogenetic mechanisms have been proposed. The more likely is that the haemorrhage results from an acute rise in the blood pressure. Arteritis has also been reported but usually diagnosed on the angiographic appearance rather than histology, and in some reported cases with pathological confirmation the changes may reflect medial necrosis due to vasospasm rather than inflammatory vascular damage.934 Ethanol also increases the risk, at the high end of the J-shaped relationship between haemorrhage and consumption. The relative risk for PBH was 2.4 if more than 400 g of ethanol was consumed per week.347

Haemorrhage from Arteriovenous Malformations, Angiomas and Aneurysms

2.120 Haemorrhage associated with cerebral amyloid angiopathy (CAA). (a) Typical lobar and superficial distribution of haematomas caused by rupture of Aβ-laden blood vessels. Both haematomas are largely intraparenchymal but communicate with the subarachnoid space. (b) Large lobar haematoma complicating CAA. Image (b) kindly provided by T Polvikoski, Newcastle University, UK.

spontaneous PBH in intracranial neoplasms ranges from 1.4 to 10 per cent, with a mean of 2–3 per cent. Conversely, the frequency of intracranial neoplasms in spontaneous PBH ranges from 0.8–7.4 per cent.913 Abnormal blood vessels in the tumours are thought to be responsible for the haemorrhage. The rich vascularity helps in differential diagnosis, because ring-enhancement on CT scanning is common in

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The common location of arteriovenous malformations (AVMs) at the interface of the parenchyma and the subarachnoid or intraventricular space increases the risk of haemorrhage in any or all of these three compartments. SAH from AVMs is considerably less common than intraventricular and intracerebral bleeds.47 Cavernous angiomas are less often a source of major PBH than are arteriovenous malformations, but minor, usually subclinical bleeding that results in local accumulation of haemosiderin is almost invariable. Clinically significant haemorrhage is reported in 9–25 per cent of sporadic241,809 and 9 per cent of familial cavernomas.573 The surgical specimen of an arteriovenous malformation rarely presents diagnostic difficulties. Some arteriovenous malformations may be difficult to detect within a PBH, but an otherwise unusual location of a small haematoma may indicate the diagnosis. For microscopic analysis sampling of all suspected areas within the wall of the haematoma cavity is recommended.1062 Rupture of a saccular aneurysm may cause a PBH when the fundus of the aneurysm is embedded in the parenchyma. Because the parent artery of these aneurysms lies in the subarachnoid space, the haematoma originates close to the basal surface of the brain and extends to a variable depth into the parenchyma, sometimes all the way to the ventricle (Figure 2.121). Bacterial emboli that cause an infective aneurysm commonly lodge within small intraparenchymal

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178  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

2.121 Intraparenchymal and intraventricular haemorrhage caused by ruptured saccular aneurysm. (a) The tip of the ruptured aneurysm in the intracranial part of the left internal carotid artery is embedded in the overlying parenchyma. The massive haemorrhage has extended through the basal ganglia into the lateral ventricle. (b) A close-up view shows the thinning of the wall of the aneurysm, which ruptured at its tip.

arteries. PBH caused by an infective aneurysm may look indistinguishable from a hypertensive haemorrhage (Figure 2.51b). The risk of rupture of infective aneurysms has been estimated at 3–7 per cent for patients with endocarditis. The rupture tends to occur within the first 5 weeks of endocarditis, and carries a mortality rate of the order of 80 per cent. Infective aneurysms are small in size and fragile in structure. They are often destroyed by the haemorrhage and difficult to identify post mortem. The clinical history and associated findings suggestive of infection, especially in the cardiac valves (Figure 2.51a), may help in determining the cause of the bleed. SAH may be caused by aneurysms or arteriovenous malformations (see Subarachnoid Haemorrhage).

Clinical Features and Imaging of Parenchymal Brain Haemorrhage In general, smaller PBHs manifest with focal neurological deficits. In larger supratentorial PBHs, the clinical picture is usually related to the mass effect of the haematoma. The bleeding may be monophasic and relatively brief, being stopped by quick clotting and tamponade. However,

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enlargement of the haematoma can continue for up to 20 hours.142 The expansion is ascribed to continued bleeding from the primary site and is significantly associated with elevated systolic blood pressure.53 Thus, neurological deterioration after PBH may result not only from secondary oedema and ischaemia but also from continued enlargement of the haematoma. Motor deficits occur in both deep and lobar PBHs but are more common in deep haemorrhages. Severe headaches and seizures are frequent in lobar haemorrhages,179,519 whereas visual deficits are more often associated with deep bleeds.652 Decreased levels of consciousness (55–60 per cent) and coma (20 per cent) are almost as frequent in lobar and deep PBH. Raised intracranial pressure and the risk of transtentorial herniation relate to the size of the haematoma and the surrounding oedema. Small pontine PBHs may cause focal sensory or motor impairments,516,519 whereas larger pontine PBHs usually cause coma leading rapidly to death from compression of the vital centres. Cerebellar haemorrhages are characteristically associated with vertigo and nausea. They may rapidly obstruct the circulation of CSF, with consequent acute life-threatening hydrocephalus.516,519 In routine practice, CT scans are used for acute diagnosis of PBH (Figure 2.119a). CT shows a hyperdense region for 1–2 weeks, after which it is transformed into a hypodense lesion and occasionally becomes calcified.532,565,1079 MRI, especially T2*-weighted, reveals acute haemorrhages as well as CT,532 but in an emergency situation may be less accessible. In visualizing older haemorrhages, T2*-weighted MRI is superior (Figure 2.119b).27,938 The prognosis of PBH is dependent on the size and location of the haemorrhage, the patient’s age and whether there is intraventricular extension. The 30-day case-fatality after supratentorial PBHs varies in population-based studies from 25 to 72 per cent, with a weighted mean of 48 per cent. In hospital-based series the numbers range from 27 per cent to 54 per cent, with a weighted mean of 35 per cent.303 Deep PBHs have a 4–5 per cent higher fatality rate, probably reflecting the greater likelihood of intraventricular extension. Intraventricular extension increases the fatality rate to 78 per cent.302,652 An increase in the size of the haematoma from below 20 mL to over 80 mL raises the fatality rate from 16 to 82 per cent.303 The mortality of cerebellar haemorrhages is similar to or greater than that of supratentorial PBH. Over 80 per cent of patients with large pontine haemorrhages succumb, whereas only about 5 per cent of unilateral tegmental haemorrhages are fatal.202

Anatomical Aspects of Parenchymal Brain Haemorrhages Supratentorial haemorrhage Large PBHs are usually divided into lobar haemorrhages, and deep haemorrhages in the basal ganglia and thalamus.519,822 The ratio of lobar to deep haemorrhages varies considerably between different countries and ethnic groups (Table 2.14).303,652 Hypertension is common among patients with deep PBHs, occurring in up to 80 per cent,

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  Haemorrhagic Stroke and Consequences  179

compared with a prevalence of 31–55 per cent in patients with lobar haemorrhages. The relatively higher frequency of deep PBHs in black compared to white people at younger ages (35–54 years) has been ascribed mainly to higher rates of untreated hypertension.300 As far as lobar PBHs are concerned, CAA, arteriovenous malformations (often of very small size) and leukaemia (Figure 2.122) are more common causes than hypertension.407,516,652 On average, Table 2.14     Cumulative mortality rates after aneurysmal subarachnoid haemorrhage Time after bleed

Cumulative mortality (per cent)

Before medical care Day 1

15 25–30

Week 1

40

Month 1

55

Month 6

60

Year 1

63

Year 5

65

Adapted from Broderick et al.141 and Fogelholm R et al.304

patients with lobar haemorrhages are 4–9 years older than those with deep haemorrhages (65–68 years vs 59–61.5 years),300,652 which probably reflects the increase in prevalence of CAA with age. This is consistent with a Swedish study in an aged population in which lobar PBHs were much more common than deep PBHs (Table 2.12). The association with CAA may also explain why lobar haemorrhages are more common in women, who live longer than men. Lobar PBHs are, on average, larger than deep PBHs, reflecting the greater volumes of the hemispheres than the deep nuclei. In the Stroke Data Bank the majority of the lobar PBHs exceeded 50 mL, whereas most deep PBHs were smaller than 15 mL.652 The proximity of the ventricular system to deep haemorrhages accounts for their propensity to extend into the ventricular system.652

2

Infratentorial Haemorrhage Cerebellar and brain stem haemorrhages constitute approximately 15–18 per cent of all primary PBHs in both Western and Far Eastern populations (Table 2.13). Most cerebellar haemorrhages are hemispheric. The great majority of brain stem haemorrhages are restricted to the pons. The medulla oblongata is a rare primary site.90 Pontine haemorrhages usually destroy large parts of the basal or tegmental pons. CT scanning has also allowed intra vitam detection of small, nonfatal, infratentorial haemorrhages. In the cerebellum, these small PBHs are usually located in the vicinity of the fourth ventricle. In the pons they occur unilaterally in the tegmentum.202,535 Primary pontine haemorrhages have been subclassified on the basis of their size and anatomical location.202,516

Pathology and Pathogenesis of Parenchymal Brain Haemorrhage

2.122 Intracerebral lobar haemorrhage in a patient with ­leukaemia. The presence of leukaemic cells gives the haematoma a variegated appearance.

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Pathologists encounter PBHs mostly at autopsy. Surgical removal of the haematoma is recommended only in selected patients.407,523 If an operation is performed, however, careful sampling of the haematoma cavity and detailed histopathological analysis reveal the definite cause of the PBH in a surprisingly high proportion of patients (e.g. Figures 2.37 and 2.36).1062 At post-mortem examination, PBHs are readily identified. Even small old lesions can be recognized by their orange tinge from haemosiderin. However, determination of a definite cause of the bleeding may be difficult, and even a thorough search of multiple sections may be inconclusive. The site, size and multiplicity of the PBHs should be considered, as well as the presence of any structural vascular abnormalities such as aneurysms, arteriovenous malformations or CAA.1079 Regardless of their cause, haematomas have a similar appearance. The time course of haematoma resorption and the response of the surrounding parenchyma have been analyzed systematically in experimental animals and the findings shown to correlate well with those in humans.481,980 Fresh haematomas are sharply demarcated, with only limited spread of erythrocytes into the adjacent parenchyma. A rim of surrounding neurons and glia undergoes necrosis during the first day and oedema increases. The inflammatory reaction in the surrounding tissue is similar to that in infarcts. However, polymorphonuclear leukocytes may appear only a little later, by 48 hours, possibly because blood

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180  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

flow immediately surrounding the haematoma is impeded by compression. Macrophages assume the appearance of siderophages instead of foam cells, and blood-derived pigment may also be seen within astrocytes. The time course for the formation of haemosiderin in the CNS is the same as elsewhere in the body. It can be detected by Perls’ method (Prussian blue stain) 1 day after commencement of phagocytosis. Haemosiderin may still be present several years later at the site of the haematoma within the astrocytes and macrophages in the walls of the cavity. It has been estimated that the clot is resorbed at about 0.7 mm/day. Astrocyte proliferation around the haematoma begins within 1 week, with simultaneous neovascularization, causing characteristic ring enhancement around the haematoma on CT.980 The pathogenesis of the tissue damage in PBH has been less studied than that of the damage in cerebral ischaemia. Mechanisms responsible for primary injury at or around the site of the haemorrhage include direct disruption of neurons and axons. Given the considerable size of the haematoma, the extent of this type of injury is often surprisingly limited, as evident from the remarkable abatement or even complete disappearance of PBH on CT scans.565 This has been attributed to the splitting rather than destruction of fibre tracts. Brain ischaemia, both in the vicinity of the haematoma and global, plays a key role in the development of permanent damage in PBH.481,536,667,711,722 By comparing the effects of injected autologous blood and inflated balloons of the same volume in the caudate nucleus, researchers demonstrated that the mass effect alone caused a reduction in local blood flow below the ischaemic threshold but that ischaemia was aggravated by substances derived from the blood clot even without a significant rise in the intracranial pressure. PBH with intraventricular extension raised the intracranial pressure still further and decreased cerebral perfusion pressure. After balloon inflation, the ischaemic damage progressed even when the balloon was deflated, indicating the existence of a surrounding penumbral zone, although if the deflation occurred within 10 minutes, the extent of the lesion at 24 hours was smaller than that after longer inflation times. Calcium-channel blockers and immunosuppression were reported to ameliorate the ischaemic injury, suggesting that there is a therapeutic window for PBH, although the period seems to be short.667

Subarachnoid Haemorrhage General Aspects In subarachnoid haemorrhage (SAH), the bleeding occurs in the subarachnoid space, alone or in conjunction with bleeding elsewhere in the CNS. In most populations, primary non-traumatic SAH represents about 5–9 per cent of all strokes (Table 2.14). The annual incidence of SAH from verified intracranial aneurysms is about 10–11 per 100 000 in most Western countries (range 6–17), with higher numbers reported from Japan, some parts of the USA and Finland but lower numbers from New Zealand, some parts of the USA and Scandinavia, excluding Finland.123,461,668,901 Unlike the incidence of most other types of stroke, that of SAH has not decreased over the past 30 years. The incidence of aneurysmal SAH increases almost linearly with age, from below 1 per 100 000 before the age of 20 years to about 40 per 100 000 after 65 years. The median age of

�����������

onset of a first SAH is 50–60 years. Deaths caused by ruptured aneurysms constitute 16–24 per cent of all patients dying from cerebrovascular diseases.304 Modern neuroimaging techniques allow demonstration of the cause of SAH in the great majority of cases. Rupture of a saccular aneurysm is by far the most frequent cause of non-traumatic SAH and accounts for about 85 per cent of cases (range 57–94 per cent). AVMs are responsible for about 5–10 per cent. In approximately 10–15 per cent, the source of bleeding cannot be identified by neuroimaging,304 perimesencephalic haemorrhage being the most common among these.1037 Secondary SAH may occur in connection with PBH, blood being forced through the cortex into the subarachnoid space (Figure 2.120) or with intraventricular haemorrhage, when the blood follows CSF routes into the basal cisterns. In some patients with CAAH there is associated bleeding into the subarachnoid space and in a small proportion the haemorrhage is exclusively subarachnoid.1101

Rupture of Saccular Aneurysms and Aneurysmal Subarachnoid Haemorrhage The rate of rupture of saccular aneurysms has been estimated at approximately 1–2 per cent per year. Larger aneurysms, in particular, tend to increase in size with time. In one series, over a median period of 47 months, none of 53 aneurysms under 9 mm in diameter enlarged, whereas four of the nine aneurysms with initial diameters of 9 mm or larger increased in size, as measured by MR angiography.794 Size is a major independent risk factor for rupture, and a diameter of about 9–10 mm seems to represent a critical watershed.502,970 In patients with multiple aneurysms, when ruptured ones are clipped, the size of other aneurysms that rupture later increases significantly during the follow-up period.502 The growth of the aneurysm fundus seems to occur by passive yield to blood pressure with simultaneous reactive formation of granulation tissue.970 It has been proposed that atherosclerosis contributes to the growth, and associated inflammation accelerates the rupture of aneurysms.522,555 About 10–43 per cent of major aneurysmal ruptures are preceded by clinical warning symptoms, usually within the preceding 1–3 weeks: headache, nausea, neck pain, cranial nerve palsies or visual defects.235,499,808 These may be caused by local effects of aneurysmal expansion or possibly by ‘warning leaks’. The rupture of a critically weakened aneurysm wall, most commonly at its fundus (Figures 2.48 and 2.121), frequently follows an acute rise in blood pressure during physical stress. It occurs significantly more often during waking hours, particularly in the morning because of diurnal variations in blood pressure. Much higher transient pressure peaks are evident during the waking hours.1044 The variations in blood pressure are accentu­ eople and in hypertensive subjects, owing ated in elderly p to diminished compliance of vessel walls. In a series of 250 patients with aneurysmal SAH, Vlak et al.1055 identified 8 trigger factors for rupture of saccular aneurysms, all known to cause surges in blood pressure: coffee consumption, cola consumption, anger, being startled, straining for defaecation, sexual intercourse, nose blowing and vigorous physical exercise. The rupture most often causes SAH, which may be complicated by vasospasm and infarction.5,423 Less frequently, the rupture results in PBH (Figure 2.121).1045

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  Haemorrhagic Stroke and Consequences  181

A ruptured aneurysm can usually be identified in vivo by angiography, CT or MRI (Figure 2.123). The distribution of subarachnoid blood may indicate the source of haemorrhage. Rupture of an aneurysm in the circle of Willis usually gives rise to blood in the basal cisterns. Bleeding from an aneurysm of the ACoA or an ACA often results in a haematoma between the frontal lobes, which may extend caudally above the corpus callosum. Blood from a ruptured MCA aneurysm tends to have an asymmetrical distribution, in and around the affected sylvian fissure. When the aneurysm (arising from the middle cerebral, anterior cerebral or anterior communicating artery) is embedded in the surrounding brain parenchyma, blood from the ruptured aneurysm may penetrate into brain tissue, resulting in PBH, which may even extend into the ventricular system (Figure 2.121). After SAH, blood often refluxes into the ventricular system; conversely, after PBH, intraventricular blood may spread throughout the ventricular system and into the subarachnoid space via the foramina of the fourth ventricle. Occasionally, SAH may penetrate spontaneously into the subdural space. Histologically, the blood in the subarachnoid space is contained by the arachnoid membrane and the pia mater surrounding the blood vessels. The pia mater on the surface of the brain appears to be continuous with the leptomeningeal ensheathment of the blood vessels in the subarachnoid space. Such structural organisation seems usually to

2

1

3

prevent direct passage of red blood cells from the subarachnoid space to the perivascular spaces within the brain.452 Aneurysmal SAH has a high mortality. It accounts for about 16–24 per cent of all deaths from cerebrovascular disease.901 About 40 per cent of patients die from the initial haemorrhage. Cumulative mortality rates after SAH304 show that about 40 per cent of patients die from the initial haemorrhage (Table 2.15), 30 per cent of those within the first 24 hours. Without surgical or endovascular intervention, one-third of patients who survive the initial haemorrhage die from recurrent bleeding within 6 months of the initial episode.498,871 Factors influencing survival include age, history of hypertension and, particularly, the amount of subarachnoid blood evident on early neuroimaging.424,750About 70 per cent of patients who are still alive 6 months after the haemorrhage may return to normal life, whereas 20 per cent are partially and 10 per cent severely disabled.304

2

Rebleeding About 15–20 per cent of patients experience further bleeding, particularly during the first 24 hours.460,498,1037 The cumulative risk of rebleeding after primary SAH is approximately 15 per cent by 7 days, 25 per cent by 14 days and 50 per cent by 6 months. After 6 months, the rate of rebleeding is about 3 per cent per year. After 10 years the risk approaches that of an unruptured aneurysm. Approximately two-thirds of the rebleeds are fatal, mortality increasing with successive bleeds. Platelets are responsible for primary haemostasis after the rupture of an aneurysm. Coagulation factors and collagen subsequently secure the scar. A reduced ability of platelets to aggregate after primary SAH has been invoked as an explanation for the early rebleeds.317,500 Later rebleeds have been attributed to lysis of the plugging clot by increased fibrinolytic activity in the CSF and plasma. Antifibrinolytic therapy decreases the risk of rebleeding by about 50 per cent, but the resulting reduction in mortality from rebleeding is offset by deaths from delayed cerebral ischaemia.521,864

Non-aneurysmal Subarachnoid Haemorrhages In about 10–15 per cent of patients with definite SAH, no aneurysm or AVM is detected on digital subtraction Table 2.15     Comparison of the pathological changes in global ischaemia and hypoglycaemia Global ischaemia Hypoglycaemia

2.123 Saccular aneurysm. X-ray arteriography (an oblique projection) of a patient with subarachnoid haemorrhage discloses a ruptured saccular aneurysm of the anterior communicating artery (arrow 1). The secondary vasospasm in the pericallosal artery (arrow 2) has rendered it markedly narrower than the middle cerebral artery (arrow 3). Scan courtesy of M Porras, Helsinki University Central Hospital, Helsinki, Finland.

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Cerebral cortex: gross distribution

Watershed

Uniform

Cerebral cortex: layers involved

Middle laminae

Superficial laminae

Hippocampus

CA1, CA3 (dentate only if severe)

CA1, dentate

Cerebellum

Watershed

Absent

Brain stem

Can be involved

Absent

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182  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

angiography.849 In two-thirds of these patients, (approximately 7–10 per cent of all SAHs), the accumulation of blood is predominantly anterior to the midbrain and pons without spread to the sylvian or anterior interhemispheric fissure. This entity is described as perimesencephalic haemorrhage. It is reported to be the second most common type of SAH1037 and usually affects young, male, non-hypertensive patients. The clinical course is much more benign than that of aneurysmal SAH. The headache begins more slowly, i.e. over minutes rather than in seconds. Seizures, focal neurological signs or unconsciousness are exceptional. Neither rebleeding nor delayed ischaemia occurs, and the prognosis is ­favourable with conservative treatment.1037 The precise source of the haemorrhage is difficult to identify but detailed imaging may suggest leakage from veins or capillaries around the midbrain or bleeding from a small arterial aneurysm.656,1036 In 2.5–5 per cent of patients with this perimesencephalic pattern of blood accumulation, the bleeding originates from a posterior fossa aneurysm. Other suggested sources of SAH in the absence of a detectable aneurysm or other vascular abnormality include lenticulostriate or thalamoperforating vessels,29 micro-aneurysms or micro-angiomas obliterated by the haemorrhage, saccular aneurysms undergoing spontaneous ­thrombosis after rupture, and segmental defects or necrosis of the tunica media of small arteries.29,354,849 SAH patients without a detectable aneurysm or other vascular abnormality have a better prognosis than those with aneurysmal haemorrhages, even in the absence of any specific treatment. Rebleeding or delayed cerebral ischaemia is rare.851,920,1037 AVMs seldom cause only SAH and bleeding is often associated with intraventricular and PBH. These are more common complications of AVMs than ruptured saccular aneurysms.47,860,1037 Saccular aneurysms sometimes develop on the feeding artery of an AVM (in approximately 10–20 per cent) and these may also rupture, giving rise more often to PBH than SAH.1037 The annual risk of rupture of previously unruptured AVMs has been estimated at about 2–3 per cent.357 Enlarging malformations and those with a single draining vein have a higher risk of bleeding.689,1060

Complications of Subarachnoid Haemorrhage

disabled as a result of brain infarction, with only 40 per cent of the deficits being reversible. Arterial vasospasm is usually demonstrable angiographically (Figure 2.123) but transcranial Doppler sonography is a useful method for non-invasive repeated examinations to monitor the spasm.376 Vasospasm may persist even after death and can be demonstrated by post-mortem angiography.515 Severe diffuse vasospasm with more than 50 per cent reduction in vessel calibre is usually associated with reduced global and regional CBF. Reduction in CBF correlates more closely with ischaemic symptoms than does the severity of angiographic vasospasm.1092 In spite of severe vasospasm, CBF may be maintained by compensatory mechanisms, such as an increase in blood pressure and collateral circulation. CBF may be critically reduced, however, even in the absence of angiographic vasospasm, if SAH is associated with intracerebral haematoma, oedema or hydrocephalus. A minimum global CBF of 30–33 mL/100g/minute and regional CBF of 15–20 mL/100g/minute are needed to prevent the development of infarcts and permanent neurological deficits.655 The amount of subarachnoid blood in the basal cisterns and fissures within 3 days of the initial haemorrhage is highly predictive of the risk of delayed cerebral ischaemia and brain infarction. However, the site of these complicating lesions is not always related to the location of the ­maximum subarachnoid blood.424,750 The volume of intraventricular blood is an independent predictor of ischaemia, as is the duration of the initial unconsciousness.436 Although the presence of blood in the CSF seems to be the crucial initiating event, the pathogenesis of arterial vasospasm is incompletely understood.215,250,633 A host of compounds released from the subarachnoid blood clot has been proposed to be vasoconstrictive. Vasoconstrictive substances may also come from the circulation; morphological abnormalities suggesting increased BBB permeability have been described after SAH.292,1092 These include increased endothelial pinocytosis and channel formation, opening of interendothelial tight junctions, endothelial detachment and destruction, and intraluminal platelet adhesion and aggregation on to the damaged endothelium within a few hours of SAH.292 Contrast enhancement in the basal cisterns indicates increased arterial permeability in patients with SAH.1092 Protracted contraction of the smooth muscle cells may result from the effect of vasoconstrictive agents (e.g. oxyhaemoglobin, endothelin-1, thromboxane A2, catecholamines, serotonin) and/or impairment of vasodilatation by mediators such as prostacyclin, endothelium-derived relaxing factor and NO.215,228

Arterial Vasospasm and Delayed Cerebral Ischaemia

Increased Intracranial Pressure and Hydrocephalus

Vasospasm of the cerebral arteries, and associated delayed cerebral ischaemia and infarction are important causes of morbidity and mortality in SAH, affecting about 20–30 per cent of patients.4 The risk of ischaemia and infarction may be diminished by improved therapy, including nimodipine and maintenance of cerebral perfusion pressure.4,293,1038 The clinical manifestations of delayed cerebral ischaemia do not usually begin before the fourth day after the initial SAH and reach their maximum around the seventh day. Delayed cerebral ischaemia is a serious complication. About 30 per cent of patients with delayed cerebral ischaemia die and another 30 per cent become permanently

During the first few days after SAH, the majority of patients have increased intracranial pressure, which does not necessarily correlate with the amount of subarachnoid or intraventricular blood.424 It seems to be caused largely by impaired reabsorption of CSF owing to the presence of blood in the subarachnoid space.139 Increased intracranial pressure after SAH may also be caused by the mass effect of a space-occupying haematoma or brain oedema. Ventricular dilatation occurs in about 20 per cent of patients during the acute phase and is related to the amount of intraventricular rather than subarachnoid blood.418 About 10 per cent of patients develop delayed

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  Vascular Diseases of the Spinal Cord  183

communicating hydrocephalus, often with symptoms of ‘normal-pressure hydrocephalus’, which can usually be alleviated by shunting.418 This post-haemorrhagic hydrocephalus may result from meningeal fibrosis.892

with aortic coarctation or bilateral vertebral occlusion. In these cases spinal artery flow is increased because of their recruitment into the collateral circulation that bypasses the obstruction.651

Other Complications

Vascular Malformations

Hypothalamic lesions (both perivascular haemorrhages and microinfarcts) have been described in patients dying soon after SAH. These may be associated with fluid and electrolyte imbalance. Electrocardiographic abnormalities and elevations of serum creatine kinase activity are common after SAH. These may be associated with a reduction in cardiac output, which increases the risk of cerebral ischaemia if vasospasm ensues.660 Repeated SAH may cause leptomeningeal siderosis.

There is a lack of consensus on the definitions and classification of spinal vascular malformations.581,967 The 1987 scheme of Rosenblum et al.868 is often used. A new classification was proposed by Spetzler et al.967 but has been criticized.

Vascular Diseases of the Spinal Cord In general, the types of disease that affect blood vessels in the brain and brain stem also involve the spinal cord vasculature. In this section, the differences and special aspects of spinal cord circulatory diseases are discussed. The distribution of vascular diseases of the spinal cord reflects the anatomy of its vasculature (see earlier).

Atherosclerosis of Spinal Cord Arteries Atherosclerosis does not usually affect spinal arteries severely. They may escape the brunt of this disease process because of their smaller size. However, spinal perfusion may be affected secondarily because the feeding arteries are often involved by atherosclerosis, which may reduce blood flow to the spinal cord. For example, the blood flow through the radicular arteries from a sclerotic aorta may be compromised at their aortic origin. Spinal cord infarction occasionally complicates surgical treatment of atherosclerotic aortic aneurysms (see later).

Vascular Malformations of the Spinal Cord Similar types of malformation occur in the intracranial and intraspinal compartments, but their relative frequencies vary markedly. The variation is probably due to the smaller size of the blood vessels within the spinal canal and haemodynamic differences in different parts of the CNS.

Aneurysms Isolated aneurysms not associated with AVMs are very rare in spinal arteries, although saccular and dissecting aneurysms and even giant aneurysms are reported.651,954,967 They occur in the larger spinal arteries, for example the anterior spinal artery954 or the artery of Adamkiewicz.1050 Aneurysms associated with intraspinal arteriovenous malformations are more common, however (see Spinal Dural Arteriovenous Fistulae, below).967 The high-pressure, high-volume turbulent flow in the feeding vessels to AVM is thought to be responsible.868 Haemodynamic factors also explain the occurrence of aneurysms in association

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2

Spinal dural arteriovenous fistulae Arteriovenous fistulae are low-flow malformations.868 The anatomy of these was characterized by selective angiography in the 1980s. The most common intraspinal vascular malformation (accounting for about one-third) is the spinal dural arteriovenous fistula. This has also been termed type I spinal AVM, angioma racemosum venosum, Foix–Alajouanine syndrome and angiodysgenetic necrotizing myelopathy.238 Onset is usually after the third decade, with the peak during the fifth and sixth decades. The clinical picture is commonly one of progressive, often painless paraparesis. In a dural arteriovenous fistula, the shunting of blood occurs from a normal dural artery, most commonly at the thoracic or upper lumbar level (T4 to L3), through a fistula inside the dura sleeve of the emerging spinal nerve, into a single, structurally abnormal vein of the perimedullary venous plexus (Figure 2.124a).103 On the basis of their age distribution and histopathology, arteriovenous fistulae are considered to be acquired malformations, in many cases probably related to trauma. Under increased intravascular pressure, the veins dilate and elongate, with variable thickening and fibrosis of their walls (arterialization), and form a meandering venous conglomerate, which is more prominent on the dorsal surface of the cord (Figure 2.124b). Dural arteriovenous fistulae almost never bleed. They cause ischaemic damage to the cord that can, if untreated, lead to extensive cord necrosis (Figure 2.124c). The ischaemia is ascribed mainly to spinal venous hypertension. Arteriovenous fistulae can usually be treated successfully by endovascular or surgical occlusion.

Spinal Intradural Arteriovenous Malformations Intradural AVMs are a heterogeneous group of malformations that are often congenital. Many manifest during the first decade, with the peak age of presentation in the second and third decades.868 They can affect any part of the spinal cord. The malformations have a nidus: a glomus of abnormal vessels either intramedullary or both extramedullary and intramedullary, and in the juvenile type occupying the whole spinal canal. They are generally high pressure, high flow malformations, supplied by single or multiple branches from spinal (medullary) arteries of the cord. This explains their tendency to bleed (in about onethird of symptomatic patients) and the occasional associated spinal bruit. These malformations have a tendency to

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184  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions (a)

(b)

Vertebra

Medullary artery

Dilated coronal venous plexus

Dural artery

Dorsal intradural arteriovenous fistula

(c)

2.124 Spinal dural arteriovenous fistula. (a) Schematic drawing and (b) ex vivo appearance. The increased pressure at which blood is shunted to veins causes marked dilation and elongation of the veins, which become tortuous, with their walls thickened and fibrotic (arterialized). (c) In transverse section, the dilated veins are seen posterior to the extensively necrotic cord. Luxol-fast-blue–cresyl violet. (a) Adapted with permission from Rosenblum et al.868

cause SAH of acute onset accompanied by back and root pain. They may present with progressive paraparesis and bowel and bladder dysfunction. Structurally, spinal AVMs are similar to their intracranial counterparts (Figure 2.52).

Spinal Cord Ischaemia Infarcts in the spinal cord are less common than those in the brain. Their pathogenesis differs in several respects, although our present understanding is limited by the scarcity of epidemiological studies.193 The smaller volume of the

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spinal cord is one explanation for the lower frequency of spinal infarcts. More importantly though, the smaller spinal arteries are relatively spared from atherosclerosis and thromboemboli. Most often, the infarct is caused by major vascular disease, the operative correction of a circulatory problem involving the aorta, or by vascular malformations of the spinal vasculature. Because of the variable and complicated anatomy of the vascular supply to the spinal cord and technical difficulties in collecting specimens at autopsy, the ultimate cause of spinal cord infarcts often remains undetermined.

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  Vascular Diseases of the Spinal Cord  185

Ischaemic Lesions Due to Aortic Diseases Surgery-Associated Ischaemic Myelopathies Vascular operations requiring cross-clamping of thoracic aorta carry considerable risk of spinal infarction and consequent paralysis.359,798 Repair of thoracoabdominal aneurysms is one of the main reasons for such operations.193 Until the early 1980s, these carried a risk of paralysis of up to 41 per cent in the most complex cases.975 The main reason for paralysis was the loss of tributary blood flow to the lower spinal cord through intercostal or lumbar arteries below the level of T8, particularly if there was loss of flow through the important great radicular artery of Adamkiewicz (Figure 2.10). If these were oversewn during the operation, the risk of paralysis increased to 61 per cent, whereas their preservation and reimplantation reduced the risk considerably. The development of elaborate surgical, and electrophysiological and polarographic monitoring of cord function during the operation; hypothermia; d ­ istal aortic perfusion by atriofemoral shunting; CSF drainage and postoperative hypertension have reduced the frequency of paraparesis to 3–4 per cent.975 In the late 1990s, the development of endovascular stent grafts provided a method for repair of thoracic aortic aneurysms and aortic dissections in which aortic clamping is not used. However, intercostal arteries are usually covered by the stent, which may cause spinal (a)

cord ischaemia. To prevent this stents that allow reimplantation of intercostal arteries are being developed.194 In correcting aortic coarctation associated with a patent ductus arteriosus (when the aorta is cross-clamped), the risk of paraparesis is much lower, because of the well-developed collaterals in these patients. In neonates and infants, the risk of ischaemic spinal damage is about 0.3 per cent. In older children and adults it is about 2.6 per cent.238 The ischaemic lesion commonly occurs below the midthoracic watershed region, because the flow through the upper spinal arteries is not sufficient to sustain this part of the cord without the supply through the great radicular artery (Figure 2.10). In less severe cases the ventral cord shows ischaemic damage but in severe cases most of the cord may undergo necrosis (Figure 2.125).

2

Aortic Dissection Aortic dissection usually occurs as a complication of degenerative disease of the aortic wall. This may be atherosclerosis (often also associated with aneurysmal dilation) or a connective tissue disease such as Marfan’s syndrome. It has also been reported in association with pregnancy, hypothyroidism and aortic stenosis. A tear in the intima allows blood to penetrate into the media and, often boosted by hypertension, to dissect the aortic wall. In about 2–8 per cent of

(b)

(c)

2.125 (a) Ischaemic lesion in the lower lumbar spinal cord. The lesion was caused by a dissecting aortic aneurysm. (b) Extensive necrosis of the cord above and below the (c) completely necrotic segment of the cord.

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186  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

patients, aortic dissection presents with paraplegia or paraparesis and sensory loss associated with bowel and bladder dysfunction.1120 As the dissection extends downwards, often from the aortic arch, it may shear off or occlude intercostal or lumbar arteries, cutting off blood flow to the spinal cord through the dorsal branches of these arteries. The ischaemic lesions are variable in extension and topography. They tend to occur at the midthoracic T4–T6 level, the watershed region and a level at which the intercostal arteries are often involved (Figure 2.10). If the dissection extends downward to involve the artery of Adamkiewicz, the lesion is usually maximal at the T10–L1 level. The upper part of the cord is rarely damaged because it receives its blood supply from the vertebral arteries and costocervical or thyrocervical trunk. The latter is not affected by the dissection. The damage primarily involves the grey matter of the cord.

Embolic Occlusion Emboli to the spinal vasculature are most commonly dislodged from an atherosclerotic aorta. The risk is increased when the aorta is subjected to operative procedures. The topography and extent of the ischaemic lesions depend on the anatomic pattern of spinal vasculature. As single events the symptoms may be trivial, but recurrent atheromatous embolism cause progressive myelopathy.238 A special type of embolic disease of the spinal cord is that caused by fibrocartilaginous emboli (FCE).238,272 This is uncommon and only about 30 cases have been verified histopathologically (Figure 2.126). FCE has a bimodal age distribution, one peak in young adulthood and another in late middle age, with predominance in females.272,1012 Its pre-mortem diagnosis is difficult because spinal biopsy is hardly ever performed and neither the clinical features nor neuroimaging is specific. Spinal cord infarction of unknown origin but occurring a few hours or days after minor spinal trauma should raise the suspicion of FCE. This occurs in the absence of a major vertebral bone lesion or of evidence of herniation of the nucleus pulposus into the vertebral body.

The pathogenesis of FCE has not been established. The spinal trauma may force disc material into vertebral body sinusoids. A rare, neoplastic embolic process is angiotropic lymphoma (see Chapter 40). The intravascular growth and embolic spread are because of impaired inability of the malignant lymphoid cells that lack surface adhesion molecule CD18a to extravasate.476 The neurological symptoms of IML are highly variable and non-specific but ischaemic myelopathy is relatively common.

Hypotensive Spinal Ischaemia Hypotension is often an aggravating factor in i­schaemic myelopathy complicating aortic surgery or dissec­ tion.359,741,798 Other low-flow conditions such as c­ardiac arrest and severe hypotensive shock238 and perinatal hypoxia–ischaemia947 can also result in ischaemic spinal cord injury. The anatomical pattern of the spinal a­ rteries results in an arterial border zone of ischaemic vulnerability in the lower thoracic region. Clinical studies have indicated that the mean level of the arterial watershed in global spinal ischaemia is T9 (in contrast with the classical view of a watershed at T4).193 However, the pattern of the arterial supply is so variable that the vulnerable zone may even be lumbosacral (Figure 2.125).78 In perinatal cases, lumbosacral segments were affected most severely.947 Watershed infarction is thought to be less common in the spinal cord than the brain. This suggests that its development requires additional local circulatory factors besides the systemic arterial hypotension. However, patients with cerebral watershed infarcts are often obtunded and not examined thoroughly for spinal lesions; and in the event of death, post-mortem examination of the CNS is often limited to the brain. Within the cord there is another watershed zone between the anterior and posterior spinal arteries. After severe ischaemia the cord may be almost completely necrotic (Figure 2.125). After less severe ischaemia or in less severely affected regions, the anterior grey matter bears the brunt of the damage, with better preservation of the surrounding white matter. In mildly affected cases and regions, there may be selective necrosis of the anterior grey matter only.

Venous Infarction

2.126 Fibrocartilaginous embolus in the anterior spinal artery. This embolus caused infarction of the medulla and anterior part of the cervical spinal cord. Reproduced from Kase et al.517 With permission from Lippincott Williams & Wilkins/Wolters Kluwer Health.

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The most common type of venous infarction of the spinal cord is probably that associated with AVMs, usually in association with dural arteriovenous fistulae. The shunting of blood to the venous side through the fistula raises the venous pressure in the confined space of the spinal canal to such an extent that ischaemia ensues. Venous congestion rather than venous occlusion occurs. The infarction is thus non-haemorrhagic. Non-haemorrhagic venous infarction may also occur in the absence of a malformation but the pathogenesis in these cases is unclear. Acute venous infarction can be caused by thrombosis of intramedullary or meningeal veins, e.g. due to cancerrelated thrombophilia. In such cases, the infarct is usually haemorrhagic, the onset sudden and associated with pain.

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  Vascular Diseases of the Pituitary Gland  187 (a)

(b)

2

2.127 Pituitary apoplexy. (a) A large chromophobe macro-adenoma underwent necrosis, leading to the patient’s death as a consequence of increased intracranial pressure and panhypopituitarism. (b) The adenoma cells are necrotic and polymorphonuclear leukocytes have invaded the tissue. Images courtesy of A Paetau, Helsinki University Central Hospital, Helsinki, Finland.

Progression is rapid and the prognosis poor. The central parts of the cord are affected most severely. There may be an intramedullary haematoma. A specific form of venous ischaemia occurs in association with brain death. The venous drainage in the upper cord is upwards into the cranial cavity. In brain death, the high intracranial pressure prevents this flow. If survival continues for some days the cervical and upper thoracic cord undergo venous infarction with circumferential necrosis in the cervical cord and radially oriented perivenous haemorrhages in the thoracic cord.238

Spinal Haemorrhage Haemorrhages within the spinal canal can occur in the same four compartments with respect to the meninges as in the cranial cavity, i.e. intraparenchymal, subarachnoid, subdural and extradural.238,816 Intramedullary haemorrhage (haematomyelia) is often caused by trauma (see Chapter 10). Other causes are spinal AVMs, which are often located completely or partially within the cord and thus may bleed into the parenchyma. Bleeding diatheses (including due to anticoagulant therapy), intramedullary neoplasms or a syrinx may also be a cause or source of haemorrhage. On the other hand, intramedullary haemorrhages are seldom attributable to the common causes of brain haemorrhage, such as hypertension and amyloid angiopathy. A peculiarity of the spinal cord is the occurrence of small petechial haemorrhages of unknown pathogenesis. These are regularly found in asymptomatic patients.238

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Spinal aneurysms may rupture, as do their intracranial counterparts, but are very uncommon on intraspinal arteries unless associated with AVMs. They are responsible for blood in the spinal subarachnoid space in under 1 per cent of all SAHs.967 More commonly, ruptured AVMs cause SAH of primary intraspinal origin. Often the blood in the spinal subarachnoid space is a result of downward spread from an intracranial SAH. Spinal subdural and epidural haemorrhages have the same predisposing factors as spinal haemorrhages.816,883 In addition, lumbar puncture is a fairly common precipitating factor. Although spinal epidural haemorrhages are caused by trauma less often than their intracranial counterparts, minor trauma and straining can cause rupture of the thinwalled veins of the epidural plexus in the loose connective tissue surrounding the cord, especially posteriorly.

Vascular Diseases of the Pituitary Gland Ischaemic or haemorrhagic necrosis of the pituitary gland can result from a number of insults: local, such as trauma, neoplasms and compression by adjacent aneurysms (usually arising from the anterior part of the circle of Willis), and systemic or more generalized, such as hypotension, irradiation, ICH and increased intracranial pressure. Pituitary apoplexy describes haemorrhagic necrosis of the pituitary. The term is reserved for the full-blown syndrome, evolving over hours to 1–2 days (see later). Most commonly, apoplexy occurs in the context of a pituitary adenoma,

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188  Chapter 2  Vascular Disease, Hypoxia and Related C ­ onditions

diagnosed or previously unknown, without predilection for a specific immunotype but often relatively large. In addition, apoplexy rarely complicates non-adenomatous pituitary disorders, such as abscess, metastatic tumour and lymphocytic hypophysitis.266 It is uncommon, occurring as an acute serious complication in 1.6–2.8 per cent of pituitary adenomas. Lesser foci of haemorrhage or necrosis are sometimes detected by imaging or histology in adenomas from patients with minor or no related symptoms, and may explain the reported incidence of up to 13 per cent. An outlying incidence was reported of 21 per cent.734 The clinical symptoms include severe headache, nausea and vomiting, motor ophthalmologic and visual disturbances resulting from compression of optic chiasm, and confusion or disturbed consciousness. Neutrophils infiltrating the necrotic tissue can spread into the CSF and, in previously undiagnosed cases of adenoma, may simulate purulent meningitis (Figure 2.127). Histopathological findings may be difficult to evaluate, because of extensive necrosis or haemorrhage, and infiltration by inflammatory cells. Upon immunohistochemical examination of the glandular remnants and taking into account the clinical history, it is often possible to determine the type of underlying adenoma. Sheehan’s syndrome is another disorder caused by necrosis of the pituitary gland. It is usually related to severe hypotension as a result of postpartum haemorrhage.929

With improvements in individual medical care, Sheehan’s syndrome has become an extreme rarity under normal obstetric conditions. For example, Sheehan’s syndrome was not detected among 55 patients with excessive obstetric haemorrhage.285 The clinical symptoms reflect panhypopituitarism and include amenorrhoea, failure of lactation, weight loss, weakness, loss of pubic hair, breast atrophy and psychiatric disturbances.285 Histopathological examination of the pituitary gland typically discloses extensive central necrosis with a rim of viable cells at the periphery. Sometimes lesser degrees of destruction may be evident. The neurohypophysis is usually spared.

Acknowledgements This chapter updates and combines considerable ­information from Chapters 2 and 3 in the eighth edition. The ­previous authors (R Auer, G Sutherland, H Kalimo and M Kaste) are thanked for their substantial ­contributions, ­scholarship and generosity to allow us to re-use the m ­ aterial. We also thank A E Oakley, Newcastle University, for several ­illustrations. The contribution by Sylvia Asa to the section on vascular diseases of the pituitary gland is also gratefully acknowledged.

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1093. Williams S, Chalmers K, Wilcock GK, Love S. Relationship of neurofibrillary pathology to cerebral amyloid angiopathy in Alzheimer’s disease. Neuropathol Appl Neurobiol 2005;31:414–21. 1094. Winkler DT, Lyrer P, Probst A, et al. Hereditary systemic angiopathy (HSA) with cerebral calcifications, retinopathy, progressive nephropathy, and hepatopathy. J Neurol 2008;255:77–88. 1095. Wolf HK, Anthony DC, Fuller GN. Arterial border zone necrosis of the spinal cord. Clin Neuropathol 1990;9:60–65. 1096. Wong V, Yu YL, Liang RH, et al. Cerebral thrombosis in beta thalassemia/ hemoglobin E disease. Stroke 1990;21:812–16. 1097. Woo D, Haverbusch M, Sekar P, et al. Effect of untreated hypertension on hemorrhagic stroke. Stroke 2004;35:1703–8. 1098. Woolfenden A, Albers G. Cardioembolic stroke. In: Fisher M, Bogousslavsky J eds. Current review of cerebrovascular disease. Boston, MA: Butterworth Heinemann, 1999:93–105. 1099. Wu HM, Huang SC, Hattori N, et al. Selective metabolic reduction in gray matter acutely following human traumatic brain injury. J Neurotrauma 2004;21:149–61. 1100. Wytrzes LM, Chatrian GE, Shaw CM, Wirch AL. Acute failure of forebrain with sparing of brain-stem function: electroencephalographic, multimodality evoked potential, and pathologic findings. Arch Neurol 1989;46:93–7. 1101. Yamada M, Itoh Y, Otomo E, Hayakawa M, Miyatake T. Subarachnoid haemorrhage in the elderly: a necropsy study of the association with cerebral amyloid angiopathy. J Neurol Neurosurg Psychiatry 1993;56:543–7. 1102. Yamamoto K, Hayakawa T, Mogami H, et al. Ultrastructural investigation of the CA1 region of the hippocampus after transient cerebral ischaemia in gerbils. Acta Neuropathol (Berl) 1990;80: 487–92. 1103. Yamamoto Y, Ihara M, Tham C, Low RW, Slade JY, Moss T, Oakley AE, Polvikoski T, Kalaria RN. Neuropathological correlates of temporal pole white matter hyperintensities in CADASIL. Stroke 2009;40:2004–11. 1104. Yamamoto Y, Craggs L, Baumann M, Kalimo H, Kalaria RN. Molecular genetics and pathology of hereditary small vessel diseases of the brain. Neuropathol Appl Neurobiol 2011; 37:94–113. 1105. Yamamoto Y, Craggs LJL, Watanabe A, et al. Brain Microvascular Accumulation and Distribution of the NOTCH3 ectodomain and GOM in CADASIL. J

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Chapter

Disorders of the Perinatal Period Rebecca D Folkerth and Marc R Del Bigio

Introduction................................................................................210 Approach to Evaluation of Perinatal Specimens...........................213 Brain Growth..............................................................................214 Cellular Components of the Developing Human Central Nervous ­System.......................................................................................216 Hypoxic-Ischaemic Injury to the Developing Brain.......................227 Grey Matter Lesions....................................................................232 White Matter Lesions..................................................................235 Combined Grey and White Matter Lesions...................................239

Introduction Consideration of perinatal brain disorders requires that the neuropathologist understands terminology used in obstetrics. A normal pregnancy lasts approximately 40 weeks from the last normal menstrual period (LNMP). In the ‘ideal’ 28 day menstrual cycle fertilization occurs on the 14th day of that cycle. Birth between 37 and 42 weeks after the LNMP is considered full term. Gestational age is the time from the LNMP to birth, postmenstrual age (typically used in the circumstance of premature birth) is the gestational age plus the chronological age (time elapsed from birth). Corrected age is the chronological age minus the number of weeks born before 40 weeks gestation.153 (See Table 3.1.) Conceptional age (gestational age minus 2 weeks) is not a term recommended for use in obstetrics but is a necessary concept for understanding the terminology of comparative development (e.g. the Carnegie stages).406 The embryonic period extends to the end of the 8th week after fertilization (Carnegie stage 23), and the fetal period extends thereafter until birth. The formal definition of the ‘perinatal period’ is from 20 gestational weeks to 28 postnatal days. In this chapter, however, we will consider pathological processes occurring through the first postnatal year (i.e. the end of infancy), because the neuropathology of this period is unique, given that this is a setting of rapid brain growth quite different from that in the mature brain. Very roughly, the human brain is 1/3 adult weight at full term birth, 2/3 adult weight at 1 year, and near adult weight by ~10 years, although the brain is not fully mature until the middle of the third decade of life (Figure 3.1; Box 3.1; Table 3.1).587 During the fetal and infantile period, there are rapid changes in neuronal differentiation, synaptogenesis,

Cerebral Haemorrhages..............................................................242 Birth Trauma...............................................................................247 Hypoglycaemia...........................................................................248 Kernicterus.................................................................................249 Infection.....................................................................................250 Sudden Infant Death Syndrome..................................................255 Epilogue.....................................................................................257 References.................................................................................258

dendritic arborization, axonal elongation, gliogenesis and myelination. Quantitative magnetic resonance (MR) imaging studies underscore the dramatic changes occurring over the last half of human gestation in the volume, surface area and sulcation of the cerebral cortex, as well as in the microstructural organization of the white matter (Box 3.2). All of these organizational events occur after the ground plan for the central nervous system (CNS) is laid down in the embryonic period and after neuronal proliferation and migration in the cerebral hemispheres is largely complete, i.e. by the end of 20–22 gestational weeks. In essence, the pathology from mid-gestation through infancy reflects cellular and tissue reactions brought about by a complex interplay of disease and rapidly changing developmental processes. Good evidence indicates that the most vulnerable time for cortical injury is toward the end of the gestational period, perhaps including the perinatal period, whereas the time associated with the greatest capacity for plasticity is 1 to 2 years of age.297 The developmental changes occur so rapidly that responses to the same insult may vary considerably from mid-gestation through infancy, as demonstrated by hypoxic-ischaemic injury to cerebral white matter preferentially in the preterm infant and to grey matter preferentially after the neonatal period. Moreover, different types of brain lesion may be restricted to very specific time periods within this overall period (Figure 3.2). This latter phenomenon is due in part to the rapid changes in peak periods of maturation of different cellular and molecular components of the brain, e.g. the peak occurrence of neuronal migration and differentiation in early gestation compared with the peak period of myelination in infancy. The focus upon the period from mid-gestation to

210

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Introduction  211

100

3

50

0 20 wk 30 wk Term

3 mo

6 mo

9 mo

12 mo

16 mo

20 mo

24 mo

Adult

3.1  Graph showing brain growth expressed as a percentage of adult brain weight in early life. Mid-gestation through infancy (the first postnatal year of life) is a critical period in brain growth, at the end of which the brain attains 75 per cent of adult weight. Note that adult weight is reached by 10–15 years age.

Table 3.1  Definitions of preterm and post-term terminology Terminology

Definition

Comment

Post term

>42 weeks

213

Term

>37 to 37 to G mutation, but the precise pathogenetic mechanism for vessel calcification remains to be determined.

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7.11 Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes. (a) Extensive cortical ablation/laminar necrosis over the occipital, lateral parietal and lateral inferior temporal lobes in a fixed right cerebral hemisphere. Note the depressions of the pia-arachnoid-cortex at gyral crests and the more preserved cortex next to sulci. (b) Cortical ablation at the gyral crest (between arrows) and severe loss of myelinated fibres in the underlying white matter; the arcuate myelinated fibres underlying the unaffected cortex in the sulci are relatively spared; posterior temporal lobe. Loyez myelin stain, bar = 1000 μm. (c) Prominent astrogliosis at the interface between the ablated cortex (lower right corner) and white matter, where milder astrogliosis is present (upper left corner), glial fibrillary acidic protein immunohistochemistry (IHC). Haematoxylin counterstain, bar = 200 μm. (d) Infarct-like lesion involving the molecular, Purkinje and granular cell layers of the cerebellar cortex and the adjoining white matter. Haematoxylin and eosin, bar = 200 μm. (e) Purkinje cell in the vicinity of the infarct-like lesion showing abnormally disorganized dendrites with thickening of primary, secondary and tertiary dendritic branches, COX-I IHC. Haematoxylin counterstain, bar = 50 μm. Adapted with permission from Betts et al.13

The cerebellum is often severely affected in MELAS, with features ranging from devastating ischaemic-like lesions, to microvacuolation of the cortex and white matter, atrophy of the folia and focal Purkinje cell loss.128 Abnormal Purkinje

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cell dendrites, including cactus formations, have also been reported in several patients with the 3243A>G mutation.240 These formations were first described as a characteristic finding in Menkes’ kinky hair disease.158 They are thought to be

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Mitochondrial Encephalopathies—Neuropathology  543 (b)

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7.12 Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes. (a) Temporal cortex with loss of gyral crests over the lateral and inferior surfaces of temporal gyri (arrows). Enlarged insert (double shaft arrows) illustrates surface grey matter ablation in the superior temporal gyrus with a rim of surviving grey matter at the edge. The latter is partially highlighted by photography. Cystic structures may form at the edge of the crest crater abutting the grey matter margin – ball and arrow heads. The floor of the crater-like formations consists of white matter covered with pia-arachnoid membrane. Smaller areas of surface gyral ablation are present at the lateral surface of the middle and inferior temporal gyri. These features are virtually pathognomonic of MELAS. The section is from a 52-year-old woman. (b) Frontal cortex and white matter showing central infarcts and demyelination in a 60-year-old woman. Loyez stain. Adapted with permission from Betts et al.13

caused by abnormal accumulation of mitochondria within the dendrites, similar to the abnormalities in ragged-red skeletal muscle fibres.165,267 It is unclear why cactus formations seem to occur only in the cerebellum. They may reflect a selective vulnerability of Purkinje cells to metabolic disturbances. A number of different hypotheses have been proposed to account for the pathogenesis of the ischaemic-lesions and neuronal cell death in MELAS. One of the most commonly cited hypotheses suggests that abnormalities within the vascular smooth muscle and endothelial cells of the MELAS brain constitute the pathogenic basis of the brain lesions (the primary vascular hypothesis). This hypothesis is based on the observations of aggregated, enlarged mitochondria in smooth muscle and endothelial cells of the cerebral blood vessel in MELAS patients.58,62,160,183,248 These changes are most marked in pial arterioles and small arteries, whose vasculature plays an important role in the autoregulation of cerebral blood flow. Further support to this hypothesis is provided by the observation of arterioles strongly reactive to succinate dehydrogenase (strongly succinate dehydrogenase-reactive blood vessels, SSVs) in MELAS patients.73 In addition, an immunohistochemical study of MELAS brains demonstrated that, although the expression of the nDNAencoded FeS subunit of complex III was normal, the expression of the mtDNA-encoded COX II subunit was markedly reduced in small arteries and arterioles.242 It has been suggested that these changes in the cerebral blood vessels could lead to aberrant vascular tone, resulting in local ischaemia and the stroke-like episodes.62 Recent work has confirmed widespread respiratory chain deficiency in the cerebral blood vessels of the brains from MELAS patients.13,129 COX-deficient blood vessels

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were observed in various brain regions from MELAS and MERRF patients harbouring the m.3243A>G and the m.8344A>G point mutation, respectively. In contrast, cerebral vessels were found to be COX-positive when similar regions from control brains were examined. Importantly, high levels of the m.3243A>G mutation were also found in COX-deficient vessels; a mutant load of 99 per cent was observed in large leptomeningeal arteries, compared to 95 per cent ±3 per cent in smaller cortical arterioles. The mutation load in surrounding normal cortex was lower (76 per cent).13 These findings strengthen the suggestion that vascular changes are important in the pathogenesis of the ischaemic-like infarcts in MELAS. However, because respiratory chain deficient vessels were found in all regions of the brain examined, the deficits alone cannot explain cortical selectivity of the lesions. An extension of the vascular hypothesis is the suggestion that changes in the blood–brain barrier (BBB) are important factors in the development of neuropathological changes in MELAS, and indeed other mitochondrial diseases.108,182,183,243 Again, this hypothesis is based on the observations of abnormal mitochondria and respiratory chain deficiency in the cerebral vasculature in addition to the findings of similar changes in the epithelial cells of the choroid plexus in MELAS patients.182,243 Furthermore, Tanji and colleagues showed by immunohistochemistry deficiency of the mtDNA-encoded subunit II of COX and normal expression of the nDNA-encoded FeS subunit of complex III in MELAS choroid epithelial cells. The authors suggested this implied that the m.3243A>G mutation was abundant in the epithelial cells and the mutation had reached the threshold level required to impair

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7.13 Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes. (a) Partial laminar necrosis at the gyral crest of occipital cortex proceeds from the upper to deeper cortical layers. Upper cortical layers show paler staining, prominent microvacuolation and severe neuron loss, whereas the deeper cortical layers show less prominent microvacuolation. Cresyl fast violet. (b) Enlarged portion of the deeper cortical layers boxed in (a), showing neurons at various stages of necrosis (empty arrows) and capillary hypertrophy (filled-in arrow). Cresyl fast violet. (c) Low concentration of the mtDNA-encoded cytochrome c oxidase (COX-I) mitochondrial enzyme in the microvacuolated upper cortical layers and a high concentration of COX-I in the deeper cortical layers, serial section to (a). COX-I immunohistochemistry. (d) Enlarged area portion of cortex boxed in (c) at an interface between the deeper cortical layers affected by the neuronal necrosis (high concentration of COX-I in right third of image) and a relatively intact cortex (moderate concentration of COX-I in the neuropil in left two-thirds of image); note a relatively intact pyramidal neuron (filled-in arrow) containing a high concentration of COX-I. COX-I immunohistochemistry. (e) Activated microglia (filled-in arrow) and necrotic neurons (empty arrows) in deeper cortical layers, serial section to (a) and (c). CD68 immunohistochemistry. (f) Macrophages (filledin arrows) within neuropil and in the vicinity of a cortical blood vessel (chevron) in upper cortical layers, serial section to (a) and (c). CD68 immunohistochemistry. (g) Activated astroglia (filled-in arrows) in the vicinity of the partial laminar necrosis. Glial fibrillary acidic protein immunohistochemistry. (c–g Haematoxylin counterstain. a,c: bar = 300 μm; b: bar = 20 μm; d–g: bar = 30 μm.) Adapted with permission from Betts et al.13

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7.14 Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes. (a) mtDNA-encoded COX-I mitochondrial enzyme is either absent (empty arrow) or reduced in concentration (filled-in arrow) in morphologically preserved polymorphic neurons of hippocampal CA4 segment; the fine granules in the neuropil surrounding the neurons represent synaptic labelling; the blood vessel wall (chevron) shows absence of COX-I, COX-I immunohistochemistry (IHC) with haematoxylin counterstain. (b) nDNAencoded succinate dehydrogenase (SDH) mitochondrial enzyme is present at high concentration in all the hippocampal polymorphic neurons and in the blood vessel wall (chevron); the fine granules in the neuropil surrounding the neurons represent synaptic labelling; serial section to (a). SDH IHC and haematoxylin counterstain levels. (c) Dystrophic microcalcification in vessel walls of putamen H&E. (d). Severe microvacuolation, neuron loss, apoptotic neuron (filled-in arrow) and axonal spheroids (empty arrows) in the gracile nucleus. H&E. (a,b,d: bar = 30 μm; c: bar = 50 μm.) Adapted with permission from Betts et al.13

mitochondrial protein synthesis. Based on these observations and on the presence of COX II deficiency in the microvasculature, the authors went on to ascertain if a breakdown in the BBB permeability had occurred in the cerebral cortex of MELAS patients. They found evidence of serum protein (fibrinogen) in the superficial and deep layers of both infarcted and non-infarcted cortex, indicating that a breakdown of the BBB does occur in MELAS patients.243 Indeed, a recent study has shown that high levels of the m.3243A>G and m.8344A>G point mutations are associated with COX-deficiency within the endothelium. There is also evidence in MELAS brains of a loss of endothelial cell integrity with reduced immunoreactivity for tight junctional proteins, including occludin and zona-occludins 1 and extravasation of fibrinogen into the grey matter and white matter neuropil.129 An alternative hypothesis to explain the distribution of the infarct-like lesions and necrosis within specific CNS regions blames mitochondrial energy failure not only in the

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brain vasculature but also within the neurons (the metabolic hypothesis).58 To support this hypothesis, Gilchrist and colleagues provided electron microscopic evidence of abnormal mitochondria within neuronal, smooth muscle and endothelial cells. According to the metabolic hypothesis, the distribution of the infarct-like lesions in the posterior temporal and occipital cortex may indicate a greater metabolic demand on energy-challenged cortical cells in these than in other brain regions. This is supported by MR spectroscopic data, which shows increased concentrations of lactate and impaired oxidative metabolism within the focal cortical lesions during an acute episode, with return to normal after clinical resolution.104,148 Furthermore, positron emission tomography (PET) scanning revealed that impaired glucose uptake in MELAS patients, with or without CNS symptoms, was most prominent in the occipital and temporal regions. However, it was not established whether this decrease was due to impaired cerebral perfusion or to higher metabolic rate of neurons in these regions.161

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Another explanation for the unusual topographical distribution of cortical neuropathology relates to different threshold levels for the m.3243A>G mutation of neurons in different brain regions. For example, neurons in the posterior cortex of MELAS patients may have a lower threshold for the m.3243A>G mutation and a higher vulnerability to the respiratory chain defect, thus explaining the selective distribution of the pathology. However, in one MELAS patient there was little correlation between the threshold levels in different neuronal populations and the distribution of neuropathological changes. The threshold for the m.3243A>G mutation was surprisingly low in the hippocampus (between 31 per cent and 43 per cent) whereas m.3243A>G levels greater than 70 per cent were observed in COX-positive neurons within the occipital cortex and cerebellum, indicating that the threshold level in these neurons was >70 per cent. These observations are inconsistent with the hypothesis that cell loss is determined by the threshold level for the 3243A>G mutation. Yet another hypothesis posits a neurovascular cellular mechanism and was proposed by Iizuka and colleagues, who suggest that neuronal hyperexcitability or seizure activity is the predominant trigger of ischaemic-like lesions in mtDNA disease.90,93 Evidence in support of this theory stems from clinical studies in patients with the m.3243A>G mutation, of whom 72 per cent suffer from headaches and 50 per cent are affected by epileptic seizures and hemianopia. Because EEG-recorded seizure activity often correlates with the lesion foci in the acute stages, this suggests that epileptic activity is responsible for driving the structural changes in the brain. It has been suggested that the cause of neuronal hyperexcitability is the presence of the mtDNA defect within neurons, astrocytes and microglia, which alters extracellular ion homeostasis and induces membrane instability, eventually resulting in impaired neuronal networking and manifesting as epilepsy. In conjunction with these changes, impaired mitochondrial function in the microvasculature is likely to cause impaired cerebrovascular reactivity and loss of BBB integrity, which could lead to mismatched neuronal activity and blood supply and may even perpetuate the seizure activity. The prolonged epileptic activities are considered likely to drive the progressive spread of the lesions. Neuroradiological imaging studies have provided some insight into the cellular mechanisms of the stroke-like lesions both in the acute and in the chronic stages, but many also show contradictory results. In the acute stages, measures of cerebrovascular perfusion using SPECT showed evidence of hypoperfusion hours after seizure activity,117,181 whereas others have shown hyperperfusion in the days, and hypoperfusion in the months, after the event.91 Measures using diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) to tease out the cellular and vascular contributions showed high DWI signals and either normal or decreased ADC,96,184,265 suggestive of vasogenic and cytotoxic oedema. In the chronic stages, in the areas of MRI T2-hyperintensities, magnetic resonance spectroscopy (MRS) demonstrates decreased N-acetyl-aspartate and presence of lactate peaks, indicative of irreversible changes in cortical regions.121,255 The conflicting imaging results could be due to a number of factors, including the definition of temporal events and interpretation bias.

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The formation of infarct-like lesions in patients with mtDNA disease is increasingly being recognized as part of the disease process in patients harbouring genetic defects other than the m.3243A>G, including m.8344A>G and POLG mutations, impacting on disease morbidity and mortality. For this reason, understanding the physiopathology of stroke-like lesion is an important area under intense investigation.

Myoclonic Epilepsy with Ragged-Red Fibres On external examination, the brain often appears unremarkable, although there may be brown discoloration and shrinkage of the dentate nucleus. Microscopically, severe neuronal loss and astrocytosis are observed in the dentate nucleus and may be accompanied by grumous degeneration (Figure 7.15). Neuronal loss is less severe in the red nuclei, pons and basal ganglia. Moderate loss of pallidal neurons, particularly of the outer segment, is common but is rarely associated with mineralization. The gracile and cuneate nuclei and Clarke’s column in the spinal cord may also be affected. Within the cerebellum, loss of Purkinje cells is generally moderate. Quantitative assessment of neuronal cell density has revealed 85 per cent, 67 per cent and 75 per cent reduction in neuron density in the inferior olives, Purkinje cells and dentate nucleus, respectively.127 In fact, ischaemiclike lesions have been documented in the cerebellar cortex in association with biochemical defects and pathological changes in the microvasculature.129 Studies have also demonstrated the accumulation of abnormally large mitochondria in the cerebellar cortex and dentate nuclei of patients.56,242,243 Despite PET studies showing evidence of decreased cortical metabolism with normal cortical blood flow and cerebral pH, loss of cerebral cortical neurons is rare.12 Biochemical defects in respiratory chain complexes I, III and IV are common in MERRF.229 Immunohistochemical studies revealed selective and severe deficiency of mtDNAencoded subunits of complex I and of subunits I and II of complex IV (COX-I and COX-II) in the surviving neurons of the dentate nucleus and medullary olive (Figure 7.15). However, this deficiency was also present in Purkinje cells and in scattered neurons within the medullary nuclei and cerebral cortex. A micro-dissection study of mutated mtDNA in a MERRF patient has documented that the percentage of m.8344A>G mutant DNAs was similar in neuronal somata and in adjacent neuropil and glia.271 In this study, mutant mtDNA was approximately 97 per cent in Purkinje cells compared to 80 per cent in anterior horn cells. However, the distribution of mutant mtDNA did not correlate well with the degree of cell loss, which was 45 per cent in the dentate neurons and only 7 per cent in Purkinje cells. A similar study has shown high levels of mutated mtDNA in individual neurons from the inferior olives, Purkinje cells and dentate nucleus at 86.1, 91.9 and 87.4 per cent, respectively.127 The authors concluded that additional factors probably contribute to cell death in MERRF. Indeed, a recent study has shown an increased mtDNA copy number in pathologically affected regions of the brain, including the putamen, hippocampus and caudate nucleus.20 Whether this reflects a compensatory mechanism remains to be elucidated.

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Mitochondrial Encephalopathies—Neuropathology  547

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7.15 Myoclonic epilepsy with ragged-red fibres (MERRF). (a) A T2-weighted image showing scattered cerebral infarcts, particularly in the occipital lobes. (b) Haematoxylin and eosin stain showing an infarcted region in the occipital cortex (×55). (c) Succinate dehydrogenase and cytochrome c oxidase histochemistry on sections of frozen ileum from a control and from a patient with MERRF (×2.5). Note the reduced stain for COX in all layers in the patient’s sample. Adapted from Tanji K, Gamez J, Cervera C, et al. The A8344G mutation in mitochondrial DNA associated with stroke-like episodes and gastrointestinal dysfunction. Acta Neuropathol [Burl], 2003;105:69–75. With kind permission of Springer Science and Business Media.

Leigh Syndrome In LS cases coming to autopsy, neuropathological lesions may be varied and widespread, and involve grey and white matter from the optic nerves to the spinal cord. Cystic degeneration or areas of softening are characteristically visible as symmetrical lesions occurring in a distinct topographical distribution affecting particularly substantia nigra and periaqueductal grey matter in the midbrain, and the putamen in basal ganglia. In some cases, lesions may also be visible in the thalamus or subthalamic nuclei (Figure 7.16a). Microscopic lesions are more widespread and commonly

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involve the dorsal pons, inferior olives in the medulla, roof nuclei in the cerebellum, and posterior columns and anterior horns in cervical spinal cord (Figure 7.16b). In the subcortical nuclei, the putamen may be more affected than the caudate nucleus and the subthalamic nucleus may be selectively involved. The cerebral cortex and white matter are generally spared, as are the mammillary bodies. The lack of involvement of the mammillary bodies is an important feature in juvenile or adult cases because it helps differentiate Leigh syndrome from Wernicke’s encephalopathy. The microscopic features are distinctive and in established lesions consist of ‘vasculonecrotic’ lesions characterized by

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548  Chapter 7  Mitochondrial Disorders (a) (b)

mb

(c)

7.16 Leigh syndrome. (a) Focal basal ganglia infarcts in caudate nucleus, dorsal putamen and globus pallidus (arrows) with sparing of mammillary body (mb). Similar infarcts in (b) midbrain and (c) pons involving periaqueductal tissue, red nucleus and colliculi (arrows). (Nissl and Loyez stains.)

a combination of tissue rarefaction with varying degrees of spongiform neuropil change, and capillary prominence suggesting either local capillary aggregation or capillary proliferation. This unusual capillary response is within or adjacent to affected grey or white matter. Recent lesions may show neuropil, probably cytotoxic, oedema and swollen cell

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processes. As the lesion evolves, this is followed by reactive neurone and astroglial cell changes, microglial proliferation and eventual tissue necrosis. In grey matter lesions, neuronal ischaemic-type change and neuron loss predominate, and in white matter demyelination, axonal loss, axonal spheroid formation and spongy change may be apparent.

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The type of lesion found may be dependent on the brain area affected, and cytotoxic oedematous lesions in the putamen may differ from necrosis occurring in periaqueductal grey matter. Although neuronal loss and neuronal ischaemic change may be prominent in the affected areas, it is often possible to identify apparently intact individual neurones in areas showing severe rarefaction or neuropil degeneration. Surviving neurones are an important clue to the differential diagnosis of such infarct-like lesions, because their presence helps differentiate LS lesions from the more common infarcts associated with hypoxic-ischaemic insults. Preferentially or selectively involved brain areas in LS include substantia nigra, inferior colliculi and periaqueductal grey, dorsal medulla, spinal cord (especially dorsal columns and anterior horns), cerebellar roof nuclei and adjacent white matter, pontine tegmentum, corpus striatum (especially putamen), inferior olives in medulla, subthalamic nuclei and thalami. In cerebellar folia, Purkinje cell abnormalities and loss may be associated with neurone loss in the inferior olives (Figure 7.16a-c). Beyond childhood, documented LS cases are uncommon and their aetiology is more controversial. Nagashima and colleagues report a predilection for midbrain, brain stem and thalamic lesions in this age group and a more rapid clinical course.168

Leber Hereditary Optic Neuropathy Histopathological data is only available on a few cases of LHON and none during the acute phase of visual loss.27 The most striking finding in the molecularly confirmed cases112,201–203 was the dramatic loss of the retinal ganglion cell layer and retinal nerve fibre layer, predominantly affecting the central fibres, with variable sparing of the periphery. Evidence of axoplasmic abnormalities was also observed with focal accumulation of mitochondria and cytoplasmic debris. No inflammatory cells were seen. Focal demyelination and occasional regions of re-myelination were associated with numerous glial cells and occasional macrophages filled with lipofuscin, suggestive of ongoing neurodegeneration long after the subacute visual loss. Additional features were seen in a patient with two mtDNA mutations,111 in whom residual retinal ganglion cells contained swollen mitochondria and double-membrane bodies containing calcium. This family was unusual because some members exhibited additional clinical features similar to MELAS. Additional clinical features in patients with LHON have also been documented, including peripheral neuropathy (see Mitochondrial Peripheral Neuropathies, p. 552) and a Leigh-like encephalopathy with dystonia.102 Anita Harding first described a multiple sclerosis (MS)-like illness in eight women from families with LHON due to the m.11778G>A mutation of mitochondrial DNA (mtDNA).71 Subsequent reports have described clinically definite MS in males23,187 and females71 with other LHON mtDNA mutations,82,110 usually in patients presenting with prominent optic nerve dysfunction. It is currently not clear whether these rare cases arise purely by chance, or whether mtDNA mutations are aetiologically linked to the pathogenesis of the MS-like disorder. Recent pathological examination of one case with the m.14484T>C mutation (complicated by Hashimoto’s thyroiditis) revealed a spectrum of neuropathological

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changes, including actively and inactively demyelinating plaques in the white matter and optic nerve, vacuolation and cystic necrosis with CD8-positive T-cells in the frontal lobe, axonal damage and vacuolation of white matter,120 implicating immune mediated mechanisms. In addition, spinal cord degeneration has also been described in one LHON patient.101

7

Multiple mtdna Deletions Disorders The neuropathological changes in a single patient with multiple mtDNA deletions were originally described by Cottrell and colleagues39 (Figure 7.17). At the time, the molecular defect was not known, but subsequent sequencing of nuclear DNA from postmortem samples revealed two autosomal recessive POLG mutations, p.Ala467Thr and p.X1240Gln. Macroscopically, the brain showed moderate atrophy, and the brain stem and the cerebellum were reduced in size. Neurohistopathological changes included massive neuronal depletion in the inferior olivary nuclei in the medulla and moderate to severe Purkinje cell loss with less neuronal depletion in the dentate nucleus. Moderate microgliosis was present in the red nuclei, while the pons remained unaffected. This distribution of pathology is indicative of cerebello-olivary atrophy and is likely to be responsible for the cerebellar ataxia observed in this patient and in other individuals with multiple mtDNA deletions. Massive myelin loss and associated axonal loss and astrogliosis were observed in the dorsal columns of the cervical spinal cord, and in dorsal spinal roots. Severe neuronal degeneration was seen in dorsal root ganglia and paraspinal sympathetic ganglia with evidence of respiratory chain deficiencies for complexes I and IV in remaining cells. Laser microcapture dissection of individual sensory neurons confirmed the presence of multiple mtDNA deletions, and quantification of mtDNA copy number revealed a significant reduction in mtDNA content.128 Sural nerve showed features of chronic axonal degeneration and regeneration. In the spinal cord, the ventral and lateral myelin tracts and motor neurons were intact. These changes are likely to account for the sensory neuropathy afflicting this patient. Sections of the midbrain revealed severe neuronal depletion from the substantia nigra, without specific neuronal cytopathology or Lewy body formation. This pathology is likely to explain the parkinsonism symptoms observed in patients. Cottrell and colleagues39 observed that most COXdeficient neurons were found in the reticular formation, nucleus, ambiguous, caudate nucleus, putamen, globus pallidus and pontine nuclei. The lowest levels were found in the cerebellum, hippocampus, motor cortex and spinal cord. Thus, the proportional distribution of COX-deficient neurons did not always correlate directly with the degree of neuropathological damage, as regions of high neuronal loss had relatively low proportions of COX-deficient cells. Further, other clinically affected CNS regions had higher levels of COX-deficient neurons without significant cell loss. These findings indicate that additional factors must be involved in determining neuronal susceptibility to multiple mtDNA deletions in the pathogenesis of this disease. These factors may include neuronal dependence on oxidative phosphorylation or thresholds for apoptosis, in which mitochondria play a pivotal role.

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7.17 Multiple mtDNA deletion disorder. (a) Severe myelin pallor in the dorsal columns of the mid-cervical spinal cord. (b) Medulla oblongata showing loss of myelin from the inferior olivary nucleus. (c) Irregular myelin pallor of the central and foliar cerebellar white matter. (d) Diffuse astrocytic gliosis of the occipital cortex with sparing of the lower laminae. (e) Microvacuolation of superficial layers was prominent in all neocortical regions. (f) Reactive astrocytes in the depleted pyramidal layer of hippocampal sector CA1. (g) Dorsal root ganglion cells. (h) Satellite cell clusters (nodules of Nageotte) throughout the dorsal root ganglia indicating neuronal loss and focal macrophage immunoreactivity (i). (j) Enzyme histochemistry of the spinal ventral horn showing cytochrome c oxidase (COX)-deficient/SDH positive anterior horn cells (blue) contrasting with the normal brown stain of the other large motor neurons. (k) A single COX-deficient lower motor neuron. (l) Normal COX staining in these neurons. (m–p) Varying populations of neurons (m, inferior olive; n, hippocampal dentate cells; o,p: hippocampal pyramidal cells). Stains: a,b,c: cresyl violet/Luxol fast blue; d,f: GFAP; e,g,h: haematoxylin and eosin; I: CD68; m,n,o,p: COX-SDH histochemistry. Scale bars: a,b: 3 μm; c: 5 μm; d,e: 100 μm; f,j,m,n,o: 50 μm; h,i,k,l,p: 25 μm; g: 12.5 μm. Adapted from Cottrell DA, Ince PG, Blakely EL, et al. Neuropathological and histochemical changes in a multiple mitochondrial deletion disorder. J Neuropathol Exp Neurol, 2000; 59: 621–7. Reproduced with permission from Lippincott Williams & Wilkins/Wolters Kluwers Health.

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The neuropathology of two siblings from a Swedish adPEO family has been investigated.156 Both patients harboured the mtDNA POLG p.Tyr955Cys mutation and died at similar age, 60 and 61 years. Microscopic examination revealed severe neuronal loss of pigmented neurons in the substantia nigra of both patients, without the presence of Lewy bodies, in agreement with a recent study.14 The cerebellum and white matter were without obvious changes. Interestingly, one patient also displayed pathology typical of Alzheimer’s disease (AD), including numerous diffuse neuritic plaques throughout the cerebral cortex and neurofibrillary tangles in the hippocampus, entorhinal cortex, amygdala and nucleus of Meynert. It may be purely coincidental that this patient developed AD pathology in addition to changes associated with multiple mtDNA deletions. However, the accumulation of mtDNA point mutations and deletions is thought to play an important role in ageing and degenerative disorders, such as AD. Therefore, it is possible that the higher levels of mtDNA mutations present in this patient may have contributed to the early development of Alzheimer pathology in the brain of this patient. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with multiple deletions and depletion of mtDNA in skeletal muscle.78,190 In 1976, Okamura and associates reported the first case as ‘congenital oculoskeletal myopathy with abnormal muscle and liver mitochondria’.185 Since then, more than 35 additional individual with MNGIE have been described, with several acronyms: myo-, neuro-, gastrointestinal encephalopathy (MNGIE),9 polyneuropathy, ophthalmoplegia, leukoencephalopathy and intestinal ­pseudo-obstruction ­uscular dystrophy (POLIP);224 oculogastrointestinal m (OGIMD);95 and mitochondrial encephalomyopathy with sensorimotor polyneuropathy, ophthalmoplegia and pseudoobstruction (MEPOP).198 In 1999, Hirano and colleagues identified mutations in the gene encoding thymidine phosphorylase (TYMP), located on chromosome 13.32qter, as the cause of MNGIE. This enzyme usually catabolizes thymidine to thymine and 2-deoxy-d-ribose 1-phosphate. In MNGIE patients, mutations in TYMP severely reduce the enzyme activity in leukocytes and presumably in other tissues.179 As a consequence of the TYMP defect, average plasma levels of thymidine are elevated nearly 50-fold in patients. The accumulation of thymidine is likely to alter deoxynucleoside and nucleotide pools and consequently impair mtDNA replication, repair, or both, leading to mtDNA abnormalities (depletion, multiple deletions and point mutations). The most prominent and debilitating symptom of MNGIE is gastrointestinal dysmotility, which is due to neuromuscular dysfunction. Any portion of the enteric system, from the oropharynx through the small intestine, may be affected.133 Histological abnormalities, including increased numbers of abnormal appearing mitochondria, are present in both intestinal smooth muscles and the enteric nervous system.9,76,95,138 Recent studies document mtDNA depletion combined with mitochondrial proliferation and smooth muscle cell atrophy in the external layer of the muscularis propria in the stomach and small intestine with loss of interstitial cells of Cajal, pacemaker cells responsible for stimulating gut contraction.59,60

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In contrast, the clinical neurological features of MNGIE are relatively mild. One of the most consistent features of MNGIE is a leukoencephalopathy observed as T2 hyperintensity on MRI. This striking leukoencephalopathy is not manifested neurologically or neuropsychiatrically as dementia or mental retardation. Furthermore, the MRI findings lack a pathological correlation, such as demyelination or gliosis at autopsy.238 Szigeti and colleagues hypothesised that TYMP may play a role in BBB function, and that the loss of function of this enzyme results in BBB breakdown causing subtle oedema, which correlates with the white matter changes observed on MRI in patients.238 Using albumin immunohistochemistry, they investigated the intracellular albumin immunoreactivity in astrocytes and neurons in two MNGIE cases and age-matched normal controls. They found a statistically significant difference in the number of albumin-positive cells between the MNGIE cases (26.08 ± 6.29) and the healthy control subjects (7.59 ± 3.17) (Figure 7.18c,d). Furthermore, the white matter capillaries and astroglial cells also indicated a striking loss of TP expression in MNGIE brains (Figure 7.18a,b). However, further investigation is required to elucidate the importance of TP in the maintenance of BBB, and it remains unknown whether BBB dysfunction is a primary or secondary phenomenon of MNGIE. The most severe neurological manifestation of MNGIE is the neuropathy and this is discussed later.

7

Mitochondrial DNA Depletion Syndromes Neuropathological investigations of MDS have increased in recent years. A post-mortem study of an infant with a documented mutation in the DGUOK gene (4-bp GATT duplication: nucleotides 763–766 in exon 6) revealed hepatocerebral pathology. The liver showed cirrhosis with small and middle-sized nodules and peripheral fibrosis. In the central nervous system, foci of spongy degeneration in the white matter of cerebral and cerebellar hemispheres were associated with mild astrogliosis. The cerebral cortex and subcortical grey nuclei were normal without any evidence of axonal degeneration. The cerebellar cortex showed focal Purkinje cell loss with Bergmann gliosis, while the dentate nucleus was unaffected.53 Muscle biopsies from members of two families with mutations in the TK2 gene (family one: homozygous Ala181Val substitution; family two: substitutions Cys108Trp and Leu257Pro) showed severe depletion of mtDNA and features of progressive dystrophic process, including ragged-red fibres, COX deficiency, great variability in fibre size and shape and increased content of connective and fat tissue; the central nervous system was not examined histologically.57

Alpers–Huttenlocher Syndrome Macroscopically, the cerebral cortex is variably involved in AHS, showing patchy thinning and discoloration, with a striking predilection for the striate cortex. Microscopic changes affect all areas but affect the calcarine cortex most severely and include spongiosis, neuronal loss and astrogliosis progressing down the cortical layers frequently accompanied by signs of capillary proliferation.227 In the liver,

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7.18 Mitochondrial neurogastrointestinal encephalomyopathy. Thymidine phosphorylase (TP) immunoreactivity is markedly decreased in the white matter of MNGIE patients (a) compared to controls (b). Albumin-positive reactive astrocytes are rare in healthy control subjects (d), but are prominent in MNGIE (c), reflecting increased blood-brain barrier permeability. Adapted from Szigeti K, Sule N, Adesina AM, et al. Increased blood–brain barrier permeability with thymidine phosphorylase deficiency. Ann Neurol 2004;56:881–6. With permission of John Wiley and Sons. Copyright © 2004 American Neurological Society.

the pathological changes include fatty change, hepatocyte loss, bile duct proliferation, fibrosis and often cirrhosis.70 In addition to the cortex, lesions have been found in subcortical gray nuclei and cerebellar cortex of an infant with AHS.225 Central-peripheral axonopathy affecting the deep sensation carried by the peripheral nerve fibres and the posterior tracts of the spinal cord, due to neuronal loss in the sensory ganglia, has also been reported in a juvenile patient with AHS.225

Infantile Onset Spinocerebellar Ataxia Neuroradiological imaging typically reveals cerebellar cortical, olivopontocerebellar and spinocerebellar atrophy. Measures of the acute diffusion coefficient (ADC) following onset of epilepsy reveal multiple small hypointensities involving the whole hemisphere, thalamus and caudate nucleus. These hypointensities are suggestive of cortical oedema localized to non-vascular territories. DWI shows hyperintensities within the oedematous regions similar to early ischaemic changes. Neuropathological features

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include patchy laminar cortical necrosis in the occipital cortex, basal ganglia, thalami and subthalamic nucleus.137 Immunohistochemical studies have shown a selective loss of complex I subunits in the cerebellum and frontal cortex, whereas subunits of complexes II and IV remain unchanged. Molecular genetic investigation reveals mtDNA depletion in the cerebrum and cerebellum, with residual amounts of mtDNA at levels 5–20 per cent of control tissues.67

Mitochondrial Peripheral Neuropathies Diseases of the central nervous system and muscle have taken central stage among disorders due to mitochondrial dysfunction (‘mitochondrial encephalomyopathies’) and have often overshadowed peripheral nerve involvement, which, however, is extremely frequent. This should come as no surprise because considerable energy is required for axonal transport and for the synthesis and deposition of the myelin sheath.

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Clinically, mitochondrial peripheral neuropathies (PNs) are predominantly distal and sensory, with blunted or absent deep tendon reflexes. Electrophysiology is usually consistent with axonal PN and shows decreased sensory and motor amplitudes and relatively preserved conduction velocities, although some patients have mainly demyelinating PN, with prolonged distal latencies, slowed conduction velocities and absent F waves. Sural nerve biopsy often shows loss of large and small myelinated fibres, thinly remyelinated fibres and demyelinated axons. Abnormal mitochondrial, some containing paracrystalline inclusions, can be seen in Schwann cells, axons and in endothelial or smooth muscle cells of endoneurial and perineurial arterioles. Peripheral nerves are rarely—if ever—affected in isolation by mitochondrial dysfunction, and PNs are usually part of syndromic disorders. A rational classification of the mitochondrial PNs can be based on a combination of genetic and clinical criteria: whether they are due to mutations in mitochondrial DNA (mtDNA) or in nuclear DNA (nDNA); and whether neuropathy is a major or minor clinical component.

PN Due to mtDNA Mutations This group of disorders can, in turn, be subdivided according to the type of the mutation: some mutations, such as large-scale mtDNA rearrangements (single deletions, duplications, or both together) and point mutations in tRNA or rRNA genes, impair mitochondrial protein synthesis in toto, whereas mutations in protein-coding genes affect specifically the respiratory chain complex to which the mutated protein belongs. Rearrangements of mtDNA are usually de novo events and the related disorders are sporadic; point mutations in tRNA genes are usually maternally inherited; point mutations in protein-coding genes can be maternally inherited or de novo – and often somatic – events resulting in sporadic and tissue-specific disorders.

Neuropathy as a Major Clinical Component Neuropathy dominates the clinical picture in three conditions, two (MERRF and MELAS) due to mutations in tRNA genes, the other (NARP) due to mutations in the gene encoding the ATPase 6 component of complex V.

Myoclonic Epilepsy with Ragged-Red Fibres Neuropathy is present in about 30 per cent of patients with typical MERRF and the m.8344A>G mutation in tRNALys46 but is even more frequent (80 per cent) in the MERRF variant associated with multiple symmetric lipomatosis (MSL), also known as Madelung disease.171,172 These patients have multiple non-encapsulated, often disfiguring lipomas in the neck and shoulder-girdle region and a predominantly axonal sensorimotor neuropathy. Other symptoms include cerebellar ataxia, neurosensory hearing loss, optic atrophy and mitochondrial myopathy with ragged-red fibres, which are COX-negative histochemically. Sural nerve biopsies show a predominantly axonal neuropathy with loss of large myelinated axons.33,193 Ultrastructural studies have shown abnormal mitochondria with amorphous matrix both in axons and in Schwann cells. A recent study found combined

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central and peripheral demyelination with reduced motor conduction and absent sensory action potentials in a child with the m.8344A>G mutation.

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Mitochondrial Encephalomyopathy, Lactic Acidosis, Stroke-like Episodes In this most common of mtDNA-related and maternally inherited disorders, the frequency of PN was estimated at 22 per cent,105 but a recent study of 30 patients with typical MELAS and the common m.3243A>G mutation in tRNALeu(UUR) has revealed that 77 per cent had abnormal nerve conduction measures.106 Of these, 43 per cent had sensory abnormalities only, 35 per cent had both sensory and motor abnormalities, and 22 per cent had motor abnormalities only. Electrophysiological changes indicated axonal or mixed neuropathy in 83 per cent of patients and demyelinating neuropathy in the remaining 17 per cent. Symptoms of PN were present in only half of the patients, but almost all had decreased reflexes or distal sensory abnormalities on exam, especially in the legs. Male gender and older age seemed to contribute to the genetic disposition to develop PN. The common occurrence of a subclinical PN in MELAS/m.3243A>G patients makes them vulnerable to dichloroacetate, a lactate-lowering agent often used anecdotally in mitochondrial patients. This risk became evident during a randomized, placebo controlled therapeutic trial of dichloroacetate, which had to be interrupted because of peripheral nerve toxicity.107

Neuropathy, Ataxia and Retinitis Pigmentosa Neuropathy is a defining feature in this maternally inherited disorder, also characterized by developmental delay, ataxia, retinitis pigmentosa, seizures and dementia, and typically associated with the m.8993T>G mutation in ATPase 6 gene of mtDNA.80 Patients have both proximal neurogenic and distal limb weakness, absent ankle jerks and loss of vibratory sensation. Muscle biopsies often show features of denervation but no ragged-red fibres. When the mutation load surpasses 90 per cent, the clinical presentation is in infancy and the CNS is predominantly affected (maternally inherited Leigh syndrome, MILS). Nerve conduction studies typically reveal a sensorimotor neuropathy with lengthdependent axonal deficits.

Neuropathy as a Minor Clinical Component Kearns-Sayre Syndrome Despite the multisystem nature of this disorder due to single deletions of mtDNA, PN is usually subclinical and often revealed only by electrophysiological studies, possibly because post-mortem studies are rare. One early clinicopathological study demonstrated peripheral myelin loss in spinal motor and sensory nerve roots and in cranial nerves of a KSS patient.64

Leber’s Hereditary Optic Neuropathy LHON is a maternally inherited subacute, painless loss of vision inexplicably more common in men than women and almost invariably associated with three mutations

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in genes encoding subunits of complex I, m.11778G>A, m.3460G>A and m.14484T>C. Although the optic nerve is the target organ in this disease, PN is rare.178 Sural biopsy in one patient with the clinical diagnosis of LHON (not confirmed genetically), spastic paraplegia and PN, showed signs of axonal degeneration and demyelination.189

Peripheral Neuropathies Due to nDNA Mutations From the genetic point of view, these disorders fall into multiple subgroups, including: (i) mutations in genes encoding respiratory chain subunits; (ii) mutations in genes encoding assembly proteins; (iii) defects of intergenomic signaling (mtDNA multiple deletions; mtDNA depletion; defective translation of mtDNA); and (iv) mutations in genes controlling mitochondrial motility, fusion and fission.

Neuropathy as a Major Clinical Component Neuropathy characterizes the clinical picture in some defects of intergenomic signaling with multiple mtDNA deletions and mtDNA depletion (MNGIE, SANDO), and in two forms of Charcot-Marie-Tooth type 2A due to defects of mitochondrial fusion or motility.

Mitochondrial Neurogastrointestinal Encephalomyopathy The defining clinical features of this autosomal recessive disorder due to mutations in the gene (TYMP) encoding the enzyme thymidine phosphorylase (TP) are gastrointestinal dysmotility, cachexia, ophthalmoplegia, PN and leukoencephalopathy.179,180 PN is present to a more or less severe degree in all patients with MNGIE and, in a few, it is the predominant or the presenting symptom. The neuropathy involves the legs more than the arms and is manifested by stocking-glove sensory loss and areflexia. Nerve conduction studies show features of demyelination in about 75 per cent of patients and features of mixed axonal and demyelinating (a)

neuropathy in 25 per cent. Muscle biopsy usually shows signs of denervation (fibre type grouping, group atrophy and target fibres), together with mitochondrial proliferation (ragged-red fibres, COX-negative fibres). Molecular studies of muscle show both multiple mtDNA deletions and some degree of mtDNA depletion. Absence of TP activity in the buffy coat and greatly increased blood levels of thymidine are useful diagnostic clues.234 Nerve biopsies show loss of myelinated fibres, segmental demyelination and remyelination and occasional onion bulb formation (Figure 7.19). Ultrastructural studies have shown abnormal mitochondria in Schwann cells.204 In a few patients with MNGIE, demyelination predominates and the clinical presentation may mimic chronic inflammatory demyelinating polyneuropathy (CIDP) or Charcot–Marie–Tooth disease.11,204

Sensory Ataxic Neuropathy with Dysarthria and Ophthalmoplegia This autosomal recessive syndrome is one of the protean clinical expressions of mutations in the gene (POLG) encoding polymerase , an enzyme involved in the synthesis, replication and repair of mtDNA.136 The cardinal features of SANDO are sensory ataxic neuropathy, dysarthria, and progressive external ophthalmoplegia (PEO). Depression is often part of this syndrome. The muscle biopsy shows COX-negative ragged-red fibres and multiple mtDNA deletions are detectable in muscle and other tissues. One patient with SANDO harboured a heterozygous mutation in the C10orf2 gene (previously known as Twinkle and now renamed PEO1), suggesting that autosomal dominant inheritance is sometimes possible. Patients have loss of vibration and position sense in the legs, mildly decreased pinprick and temperature sensation, sensory ataxic gait and areflexia. Nerve conduction studies show absent sensory responses in all limbs, although motor responses and conduction velocities are relatively preserved. Sural nerve biopsies show loss of large and small myelinated axons, with regenerative clusters and endoneurial fibrosis, but without (b)

7.19 Mitochondrial neurogastrointestinal encephalomyopathy, sural nerve. (a) The nerve shows loss of myelinated fibres, scattered isolated thinly-myelinated fibres and a few small regenerative clusters. Semithin plastic section, toluidine blue. (b) This electron micrograph shows a small onion bulb. No ultrastructural abnormalities of mitochondria were encountered. With thanks to Dr Hays, Columbia University Medical Center, New York.

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abnormal mitochondria.260 A recent study provides evidence that SANDO may be a sensory ganglionopathy with degeneration of sensory axons.130

CMT 2A Mitochondria move on microtubular rails propelled by motor proteins, usually GTPases called kinesins. Nowhere is mitochondrial motility more important than in the peripheral nervous system, as axonal flow is essential to carry ATP-generating mitochondria all the way from the soma of an anterior horn motor neuron to the neuromuscular junction (to use but one example). Mitochondrial motility is strictly related to mitochondrial fusion and fission. The machinery for mitochondrial fusion requires several proteins, including two outer membrane GTPases, mitofusin 1 (MFN1) and mitofusin 2 (MFN2), and a third dynamin-related GTPase, OPA-1, which is located in the inner mitochondrial membrane. Similarly, the machinery for mitochondrial fission involves several proteins acting in concert, especially a GTPase called dynamin-related protein 1 (DRP-1). Interestingly, mutations in two genes, one (KIF1B) encoding a kinesin motor protein, the other (MFN2) encoding mitofusin 2, have been associated with CMT 2A, an autosomal dominant axonal neuropathy characterized by symmetric, distal, motor and sensory neuropathy, with normal or minimally slowed nerve conduction velocities.126,273 Mutations in OPA1, encoding a fusion protein, cause autosomal dominant optic atrophy (AOA), the mendelian counterpart of LHON,2,43 whereas mutations in KIF5A, encoding a kinesin motor protein, cause a variant of autosomal dominant spastic paraplegia.51

Neuropathy as a Minor Clinical Component POLG Mutations Although PN is a defining clinical component of SANDO, it is also very common and often a dominant feature in patients with autosomal dominant or recessive PEO and other mutations in POLG. ‘Mild axonal neuropathy’, ‘stocking-glove numbness’, ‘impaired vibration’ and ‘impaired vibration, sensory ataxia, areflexia’ have been described in patients harbouring heterozygous mutations,52,143 and in two com­ iopsies in pound heterozygous patients.52,143 Sural nerve b two patients showed loss of large and small myelinated fibres, axonal degeneration and fibrosis,144 and demyelination and axonal loss.79 Electrophysiological assessment of PN in 11 patients harbouring POLG mutations reveals

a predominantly sensory neuronopathy with evidence of motor fibre involvement in later life. In agreement with this, post-mortem investigation of spinal cord and dorsal root ganglion tissues from a patients harbouring compound heterozygous autosomal recessive POLG mutations revealed striking neuronal cell loss from the dorsal root ganglia with evidence on-going degenerative changes.129,155 In addition, severe respiratory chain deficiencies of complexes I and IV were found in remaining cells. Molecular analysis showed a striking reduction of mtDNA content in remaining neurons, which is likely responsible for the high levels of mitochondrial respiratory deficiency.130

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Leigh Syndrome Leigh syndrome is a neurodegenerative disorder of infancy or early childhood defined neuropathologically by bilateral symmetrical foci of spongiform degeneration and vascular proliferation in the basal ganglia, thalamus, and brain stem. Clinically, these children show developmental regression, brain stem dysfunction (recurrent vomiting, nystagmus, abnormal respiration) and seizures. Retinitis pigmentosa characterizes the maternally inherited form (MILS), which is often seen in families with NARP (see  p.  530). LS has been associated with a great variety of biochemical mitochondrial defects. Aside from NARP/MILS, most forms of LS are due to mutations in nuclear genes encoding subunits of the pyruvate dehydrogenase complex (PDHC), subunits of respiratory chain complexes (complex I and complex II) or assembly proteins (complexes I, III, IV and V). Although PN is not a major clinical feature of LS (or is subclinical and overshadowed by the encephalopathy), ‘acute polyneuropathy’,236 ‘Guillain-Barré syndrome’36 and polyneuropathy65 were reported in three children with biochemically undefined LS. The biochemical or molecular aetiologies were also unknown in four children with neuropathologically defined LS, whose sural nerve biopsies showed primary demyelination and remyelination and loss of both myelinated and unmyelinated axons.61 Demyelinating neuropathy was described in three children with LS and cardiomyopathy, who were homozygous for a mutation (E140K) in SCO2, but documentation of the PN was limited to neurogenic atrophy in muscle biopsies and decreased nerve conduction velocities in one case.99 A sural nerve biopsy from a 5-year-old child with COX deficiency and a homozygous nonsense mutation in SURF1 showed loss of large diameter myelinated fibres and thin myelin sheaths in remaining fibres.206

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Andreu AL, Tanji K, Bruno C, et al. Exercise intolerance due to a nonsense mutation in the mtDNA ND4 gene. Ann Neurol, 1999b; 45: 820–3. Andrews RM, Griffiths PG, Johnson MA, Turnbull DM. Histochemical localisation of mitochondrial enzyme activity in human optic nerve and retina. Brit J Ophthalmol, 1999; 83: 231–5. Antonicka H, Mattman A, Carlson CG, et al. Mutations in COX15 produce

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8

Chapter

Peroxisomal Disorders Phyllis L Faust and James M Powers

Introduction................................................................................562 Peroxisomes...............................................................................563 Peroxisomal Disorders................................................................563 Leukodystrophies and Other Defects in Myelination....................568 Group I: Disorders of Peroxisome Biogenesis...............................569 Group II: Disorders with Morphologically Intact Peroxisomes and a Single Protein Deficiency..........................575

Introduction Peroxisomes are intracytoplasmic, single membrane-bound organelles that play major roles in diverse reactions of lipid metabolism as well as cellular scavenging of peroxides and reactive oxygen species. Many of their functions are highly conserved throughout evolution, although some are unique to particular species.80 Peroxisomes and mitochondria are interrelated biochemically, and defects in both organelles have been reported in the Zellweger spectrum62,158 and adreno-leukodystrophy (ALD). Both peroxisomes and mitochondria are involved in β-oxidation of fatty acids and the α-oxidation of phytanic acid.191 Long-chain fatty acids are predominantly oxidized in mitochondria, whereas oxidation of very long-chain fatty acids (VLCFAs  ≥ C22) and branched-chain fatty acids is initiated in peroxisomes and then completed in mitochondria.194 In yeast and plants, fatty acid β-oxidation is confined to peroxisomes. Peroxisomes participate in both generation and scavenging of cellular reactive oxygen species (ROS).162,192 Peroxisomes also have metabolic interactions with endoplasmic reticulum (ER), including breakdown of dicarboxylic acids generated by ω-oxidation of fatty acids in the ER, and activation of hepatic ER stress responses in peroxisomal disorders.80,178 The number, size and function of peroxisomes change dramatically with cellular type and environmental stimuli and common transcriptional regulators, such as peroxisome proliferatoractivated receptors, coordinately control enzyme levels in peroxisomes, mitochondria and ER.80,163 Peroxisomes and mitochondria also share key components of their division machinery and increasing evidence supports a functional interaction of peroxisomes with ER. Peroxisome interactions with lipid droplets and the lysosomal-endosomal system are increasingly recognized. Novel peroxisome functions, such

Group III: Others..........................................................................583 Specific Treatment of Peroxisomal Disorders...............................583 Considerations on the Cellular Pathogenesis of peroxisomal Disorders........................................................583 Dedication..................................................................................584 References.................................................................................584

as signalling platforms for antiviral innate immunity39 and tuberous sclerosis complex regulation of mammalian target of rapamycin (mTORC1) and autophagy in response to ROS,204 continue to be discovered. Peroxisomal disorders have been estimated to be responsible for approximately 10 per cent of human heritable metabolic diseases. They produce systemic, multiorgan lesions, in which the central nervous system (CNS), eyes, skeleton, liver and adrenal glands are most severely involved.135 The diagnosis of peroxisomal disorders is based initially on clinical symptomatology, followed by confirmatory laboratory studies.65,171,193 Most of the peroxisomal disorders are transmitted in an autosomal recessive pattern, except for ALD and adrenomyeloneuropathy (AMN), which are X linked. Dysmorphic facies or rhizomelia (shortening of forelimbs), particularly when accompanied by hypotonia and seizures in the neonatal period, or signs of central white matter disease (e.g. spasticity, ataxia, aphasia), myelopathy and neuropathy in male children or adults, should alert the clinician to the possibility of a peroxisomal disorder. Plasma VLCFA levels, particularly C26:0, are elevated in almost all peroxisomal disorders and are therefore an excellent, albeit technically challenging, initial laboratory-screening test. If VLCFA levels are abnormal and the patient is suspected of having a peroxisomal disorder other than ALD or AMN, then plasma levels of bile acid intermediates (dihydroxycholestanoic acid, trihydroxycholestanoic acid [THCA]), pristanic acid, phytanic acid, pipecolic acid and docosahexaenoic acid (DHA) should be measured. Diagnosis may require functional enzyme assays of de novo plasmalogen biosynthesis, fatty acid β-oxidation, phytanic acid α-oxidation and immunostaining for peroxisomal matrix/ membrane proteins in fibroblast cultures.96 Characteristic patterns of brain involvement seen in magnetic resonance

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  Peroxisomal Disorders  563

imaging (MRI) may assist in diagnosis, particularly in biochemically atypical patients.130,185 Prenatal diagnosis in at-risk families through analyses of VLCFA and moleculargenetic analyses is available for most peroxisomal disorders and is highly reliable. The identification of mutant peroxin (PEX) genes allows the identification of heterozygotes or carrier status and has become the definitive test of peroxisome biogenesis disorders (PBDs).171,202 Newborn screening methods have been developed, although not yet widely implemented, for X-ALD and other peroxisomal β-oxidation disorders.176 If the diagnosis is not made antemortem and a peroxisomal disorder is suspected, then samples of plasma, brain, liver and adrenal gland should be frozen at autopsy for biochemical analysis. Sections of brain, liver and adrenal gland should be fixed for light and electron microscopy and immunohistochemistry,36 small samples of skeletal muscle and spleen snap-frozen for molecular genetic studies, and a sample of skin removed for fibroblast culture. Even if such measures are not taken, such as in retrospective archival investigations, VLCFA and phytanic acid determinations can be made on formalin-fixed wet tissue samples of brain and adrenal gland112 because of the postmortem stability and insolubility of these metabolites in aqueous solution.

35 weeks of gestation. As in other mammals, the prominence of peroxisomes in neurons decreases with postnatal age. Catalase-positive glia are identified in deep white matter at 31–32 weeks of gestation and, throughout the remainder of gestation, they appear to shift from deep to superficial white matter.74 The peroxisome is named for its peroxide-based ­respiration, in which a variety of oxidases generates hydrogen peroxide, which is decomposed by catalase or ­peroxidatically to yield O2 and water (Figure 8.1b).32,192 The ­relevance of peroxisomes in mammalian cells was only fully appreciated after morphological and biochemical abnormalities of this organelle were noted in a few rare human diseases. Peroxisomal (and mitochondrial) abnormalities were recognized first in the cerebro-hepato-renal syndrome of Zellweger (ZS),62 which is the prototype PBD.65 These diseases display biochemical defects in most or all of the known functions of peroxisomes in humans, which are executed by over 50 matrix enyzmes, including: β-oxidation of long-chain fatty acids, VLCFA, pristanic acid, cholestanoic acids and eicosanoids; α-oxidation of phytanic acid; pipecolic acid degradation; glyoxylate detoxification; glutaryl-CoA metabolism; and the biosynthesis of etherphospholipids (plasmalogens), DHA, cholesterol and dolichol.191,192,193 Additional and more current information is available on the European PeroxisomeDB website (www.peroxisomedb.org).

8

Peroxisomes Peroxisomes, originally called ‘microbodies’ by electron microscopists, are found in all nucleated cells of plants (glyoxysomes) and mammals. Human peroxisomes are characterized ultrastructurally by a granular, moderately electron-dense matrix surrounded by a unit membrane, a shape that varies from primarily spherical, with a diameter of approximately 0.1–1.0 μm, to elongated tubular (reticulum) (Figure 8.1a). They were initially visualized by enhanced electron density after diaminobenzidine staining, as a result of peroxidatic activity of the matrix enzyme catalase.62 Immunofluorescence microscopy with antibodies to peroxisomal matrix and membrane proteins now allows more specific detection.88,157 The presence of membranebound particulate (i.e. peroxisomal) versus cytosolic (soluble) catalase activity has been the traditional biochemical marker. Much of our understanding of mammalian peroxisomes has been derived through studies of hepatic and renal tubular peroxisomes (reviewed by Depreter et al.36). The investigation of yeast and Chinese hamster ovary cell peroxisomes, highly homologous to those of mammals, yielded rapid and dramatic insights into peroxisomal biogenesis and diseases.65,103,121 The peroxisomes in the CNS are smaller (micro-peroxisomes) than those in the liver and kidney73 and are biochemically diverse, with functional differences between cell types and brain regions.3,156 In the mature mammalian CNS peroxisomes are most abundant in oligodendrocytes, whereas in the developing CNS they are also abundant at the termini of developing neurons and have been implicated in the early determination of neural polarity.18,73 In human fetuses, catalase-positive neurons are observed in the basal ganglia, thalamus and cerebellum at 27–28 weeks and in the frontal cortex at

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Peroxisomal Disorders Peroxisomal disorders have been classified into two main categories: the peroxisome biogenesis disorders (PBDs), in which the peroxisome lacks most or multiple content proteins and sometimes membrane, and single-enzyme ­ ­disorders, in which there is loss of a specific component of a peroxisomal metabolic pathway (Table 8.1). In PBDs, previously referred to as generalized peroxisomal diseases, morphologically identifiable or ­ biochemically particulate peroxisomes are absent or ­ severely deficient, owing to a fundamental defect in their assembly; this leads to loss of multiple peroxisomal enzymatic functions. Many peroxisomal matrix enzymes, such as those involved in β-oxidation, need to be imported if they are to be biologically active. Proteins that control peroxisome assembly and division are called ­ ­ peroxins (Pexp) and all are nuclear-encoded (by PEX genes). Distinct cellular machineries sort matrix and membrane proteins to peroxisomes. Matrix proteins are synthesized on free polyribosomes and are post-translationally imported into pre-existing peroxisomes.80,103 Matrix proteins are sorted by two distinct sequences, known as peroxisomal targeting signal (PTS) type 1 and type 2, which are recognized by cytosolic import receptors, Pex5p and Pex7p, ­respectively, followed by translocation across the membrane through a complex import machinery. Peroxisomes import fully folded, c­ofactor-bound and even oligomeric matrix proteins. Most m ­ ammalian matrix proteins contain a carboxy-terminal serine–lysine– leucine (SKL) sequence characteristic of PTS1, whereas thiolase, alkyl-dihydroxyacetone ­ phosphate (DHAP)

��������

564  Chapter 8  Peroxisomal Disorders (a)

m

m p

p

p

(b)

VLCFA - CoA ALD/AMN

����

����

ABCD1 PTS2

PTS1 ZS-NALD-IRD

RCDP Acyl-CoA oxidase

Polyunsaturated fatty acids

Acyl-CoA oxidase deficiency

DHA DHAP Fatty acids

a-oxidation

Bifunctional protein b-oxidation

Acyl-DHAP

BPD

Phytanoyl-CoA hydroxylase

ARD

Thiolase

Alkyl-DHAP

Thiolase deficiency Plasmalogens

����� ��� �

Phytanic acid

Pristanic acid

b-oxidation CO2, H2O

Phospholipid CO2, H2O

b-oxidation

� �����

8.1 (a) Peroxisomes (p) with limiting single membranes and a homogeneous granular matrix admixed with mitochondria (m) ­displaying internal cristae, electron-dense granules and limiting double membranes in a hepatocyte. Electron microscopy of a biopsy. (b) Metabolic pathways of neuroperoxisomal disorders. ABCD1, adenosine triphosphate-binding cassette D1; ALD, adreno-­ leukodystrophy; AMN, adrenomyeloneuropathy; ARD, adult Refsum’s disease; BPD, bifunctional protein deficiency; CoA, coenzyme A; DHA, docosahexaenoic acid; DHAP, dihydroxyacetone phosphate; IRD, infantile Refsum’s disease; NALD, neonatal adreno-leukodystrophy; PTS1, peroxisomal targeting signal 1; PTS2, peroxisomal targeting signal 2; RCDP, rhizomelic chondrodysplasia punctata; VLCFA, very long-chain fatty acid; ZS, Zellweger syndrome.

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  Peroxisomal Disorders  565 Table 8.1  Classification of peroxisomal disorders Group I: PBD and multiple peroxisomal functions Cerebro-hepato-renal Zellweger syndrome (ZS) Neonatal adreno-leukodystrophy (NALD) Infantile Refsum disease (IRD) Rhizomelic chondrodysplasia punctata (RCDP), type I, classical

Group II: D  isorders with morphologically intact peroxisomes and a single protein deficiency A. Pseudo-PBD 1. Acyl-CoA oxidase deficiency (pseudo-NALD) 2. Bifunctional protein deficiency (BPD) 3. Di- and trihydroxycholestanoic deficiencies 4. RCDP types II, III (DHAP acyltransferase, alkyl DHAP ­synthase deficiencies) 5. Zellweger-like syndrome B. ALD and AMN C. Refsum’s disease, classical, adult (ARD) and atypical Refsum’s disease D. Miscellaneous 1. Mulibrey (muscle–liver–brain–eye) nanism 2. Peroxisomal racemase deficiency 3. Glutaric aciduria, type III (glutaryl-CoA oxidase deficiency) 4. Hyperoxaluria type I (alanine glyoxylate aminotransferase deficiency) 5. Acatalasaemia

Group III: Others ALD, adreno-leukodystrophy; AMN, adrenomyeloneuropathy; CoA, coenzyme A;  DHAP, dihydroxyacetone phosphate; PBD, peroxisome biogenesis disorder

synthase and ­phytanoyl-CoA ­hydroxylase display PTS2 amino-terminal sequences, which are p ­ ­roteolytically cleaved after import into the peroxisome. The import of peroxisomal membrane proteins (PMPs) remains less understood and controversial.80,186 Some PMPs (class I) may be imported directly from cytosol to peroxisomes, with sorting via the Pex19 receptor/chaperone that recognizes internal membrane peroxisomal targeting sequences (mPTS). The Pex19p-PMP complex then interacts with Pex3p (and Pex16p) at peroxisomal membranes to mediate PMP insertion. However, recent studies demonstrate that many PMPs (class II) are initially trafficked through the ER into a specific pre-peroxisomal compartment, where interactions with Pex3p and Pex19p lead to budding of vesicles for subsequent delivery to peroxisomes. Peroxisomes can form both by growth and division (fission) of pre-existing organelles, with the ER providing membrane lipids, as well as de novo via the ER; this occurs in a multistep maturation pathway initiated by Pex11p.163 Key ­components for both peroxisomal and mitochondrial fission are the dynamin-like GTPase DLP1/Drp1 and the DLP1 membrane adaptors Fis1 and Mff. Early attempts to understand the pathogenesis of PBD were largely classical morphological studies,45,190 followed shortly thereafter by biochemical approaches. At about the same time, complementation analysis, in

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which abnormal fibroblasts from two different patients are induced to fuse into a multinucleated heterokaryon, was applied to PBDs.42,113 As with other diseases, correction of the abnormalities occurs only if one abnormal cell provides the gene product that is deficient in the other, thereby reflecting distinct genotypes. Complementation analysis has identified thirteen distinct genotypes in PBDs, with characteristic clinical phenotypes;65,113,115 all ­display an impairment of matrix protein import and three have defects in peroxisome membrane biogenesis (Table 8.2).103,130 At present, more than thirty different ­peroxins have been identified in various species; although many are conserved from lower to higher eukaryotes, there may be some functional redundancy. Complementation analysis may fail to identify other putative human PEX genes, such as a distinct peroxisomal and mitochondrial fission defect due to mutation in the DLP1 gene.196 PBD patients with milder clinical phenotypes may be diagnostically challenging, with variably defective or even normal peroxisomal functions in plasma and/or fibroblasts. The majority of peroxins are involved in matrix protein import, forming distinct subcomplexes that mediate PTS receptor-cargo docking and translocation and receptor recycling and degradation machinery. PEX 3, 16 and 19 are ­ critical for peroxisomal membrane protein import (Table 8.2). Genotype–phenotype correlations have been poor because defects in the same gene can produce different phenotypes and defects in different genes can produce the same phenotype.65,115,141 For example, the ZS phenotype is ­associated with all human PEX genes excepting PEX 7. The most common complementation group is group 1, because of a defect in PEX 1, in which the ZS, neonatal adreno-leukodystrophy (NALD) and infantile Refsum’s disease (IRD) phenotypes (referred to as the Zellweger spectrum) are seen in decreasing order of prevalence and severity.30 A defect in PEX 1 is responsible for about 60–70 per cent of ZS–NALD–IRD phenotypes; PEX6 and PEX12 (16 per cent and 9 per cent of PBD patients, respectively) comprise other more common genotypes.42,202 The phenotypic variability with the same genotype largely correlates with the nature of the gene defect and the resultant deficiency of mRNA/protein, leading to a variable import defect of matrix proteins. For example, the G843D missense mutation in PEX1 has approximately 15 per cent residual matrix protein import activity and correlates with the mildest IRD phenotype, although the 2097inST frameshift mutation has no import activity and is associated with the most severe ZS phenotype.30 A comparable situation has been found with PEX 5, 6, 7, 10 and 12.65,115 The temperature sensitivity of peroxisomal permeability may also play a role.79 Most patients with Zellweger spectrum, with an incidence of one per 25000–50000 births, have a defect in PTS1-matrix protein import. By contrast, rhizomelic chondrodysplasia punctata (RCDP) type 1, which is the only PBD associated with PEX 7, displays a selective defect in PTS2-matrix protein import. Over 90 per cent of RCDP patients have defects in PEX 7. A common mutant allele, PEX 7 L292ter, is found in almost 50 per cent of patients with RCDP; this causes reduced amounts of PEX 7 with no residual activity. On the other hand, some missense mutations result in PTS2

8

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566  Chapter 8  Peroxisomal Disorders Table 8.2  Human PEX-genes and peroxins PEX-gene

Peroxin function

Phenotype

PEX 1

AAA-ATPase; matrix protein import, PTS receptor recycling

ZS, NALD, IRD

PEX 2

RING-finger PMP; E3 ligase and PTS receptor ubiquitination

ZS, IRD

PEX 3

PMP biogenesis and Pex19 receptor; de novo formation

ZS

PEX 5

PTS1 receptor, predominantly cytoplasmic; matrix protein import

NALD, ZS, IRD

PEX 6

AAA-ATPase; matrix protein import, PTS receptor recycling

ZS, NALD

PEX 7

PTS2 receptor, predominantly cytoplasmic; matrix protein import

RCDP, ARD

PEX 10

RING-finger PMP; E3 ligase and PTS receptor ubiquitination

ZS, NALD

PEX 12

RING-finger PMP; E3 ligase and PTS receptor ubiquitination

ZS, NALD, IRD

PEX 13

PMP; matrix protein import, docking complex component

ZS, NALD

PEX 14

PMP; matrix protein import, docking complex component

ZS

PEX 16

PMP-targeting; proliferation; de novo formation

ZS

PEX 19

PMP receptor and chaperone; de novo formation

ZS

PEX 26

PMP; matrix protein import, membrane anchor for Pex6

ZS, NALD, IRD

AAA, ATPases associated with diverse cellular activities; ARD, adult Refsum’s disease; ATP, adenosine triphosphate; IRD, infantile Refsum’s disease; NALD, neonatal ­adreno-leukodystrophy; PMP, peroxisomal membrane protein; PTS1, peroxisomal targeting signal 1; PTS2, peroxisomal targeting signal 2; RCDP, rhizomelic chondrodysplasia punctata; ZS, Zellweger syndrome

receptors with some residual activity and milder RCDP phenotypes.19,191 Four cases of ‘hyperpipecolic acidaemia’ have been reported. They were initially assigned to the PBDs, but the legitimacy of this common biochemical abnormality as a separate disease entity among the PBDs has been rejected.65 One, at least, has a PEX 1 mutation.191 Hence, ‘hyperpipecolic acidaemia’ has been deleted from Table 8.1. The two reported autopsy cases, on patients who died at or before 27 months of age, are included historical continuity.25,57 Minor dysmorphic here for ­ features, hepatomegaly, developmental delay, h ­ ­ypotonia, pigmentary retinopathy and progressive neurological ­ dysfunction are reported. Micro-nodular cirrhosis and ­ hepatocytic glycolipid inclusions were seen, in addition to dilations of renal tubules. The neuropathological findings were discordant. In neither case were abnormal neuronal migrations or significant brain atrophy noted. In one case, the white matter of brain showed multiple areas of demyelination and astrocytosis, most prominently in the internal capsule, pons, medulla and cerebellum. Abnormal, often striated, material was observed in both macrophages and astrocytes. Macrophages also contained sudanophilic spherical droplets and the striated macrophages were periodic acid–Schiff (PAS)-positive and contained angulate lysosomes with spicules. Myelin was interpreted as degenerate ultrastructurally, but post-mortem autolysis may have complicated this interpretation.57 In the other case, the cerebral white matter was hypoplastic (similar to RCDP type I case of Agamanolis and Novak1 later in this chapter) and showed a moderate decrease in myelin staining without myelin breakdown. There was also a noteworthy accumulation of 1- to 1.5-mm, PAS-positive, diastase-­ resistant, alcian blue-negative, non-­ sudano­ philic, non-fluorescent granules in satellite cells and astrocytes, including perivascular foot processes. This

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material seemed to correlate ultrastructurally with irregular membrano-vesicular profiles within astrocyte cytoplasm.25 The adrenal glands in both cases were reported to be within normal ­limits, but rare PAS-positive striated macrophages were found within the adrenal cortex of the Gatfield case ­ (personal observation, slide courtesy of Dr Daria Haust).

General Pathology and Neuropathological Overview of Peroxisomal Disorders The major organ systems involved in peroxisomal dis­ orders are the CNS, peripheral nervous system (PNS), skeleton, eyes, liver, adrenal gland and kidney.135 In the PBDs and those single enzyme deficiencies that simulate them clinically (pseudo-PBDs), a range of extraneural lesions is usually seen: dysmorphic facies; stippled calcifications to shortened proximal long bones (rhizomelia); portal fibrosis to micro-nodular cirrhosis and steatosis; PAS-positive macrophages with a­ ngulate lysosomes containing trilaminar spicules (Figure 8.2); s­triated adrenocortical cells containing lamellae and lipid profiles to adrenal atrophy; and renal cortical micro-cysts to macrocysts.37,135 The eyes typically display cataracts, atypical pigmentary retinopathy, degeneration of photoreceptor cells, ganglion cell loss and optic atrophy. Angulate lysosomes with spicular inclusions in retinal macrophages and electron-opaque membranous cytoplasmic bodies in ganglion cells are observed ultrastructurally.29 Of the PBDs, ZS is the most severe clinically and biochemically and in general results in the most severe lesions. However, skeletal involvement is most impressive in RCDP and its phenocopies, hepatic macrophages in IRD and lymphoid/thymus macrophages in NALD.135 The characteristic ultrastructural inclusion in PBD is the membrane-bound angulate lysosome. This contains

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  Peroxisomal Disorders  567

rigid-appearing trilaminar spicules consisting of two electron-dense leaflets of 3–5 nm thickness, which are separated by a regular electron-lucent space of 6–12 nm. These structures are also seen in brain, adrenal, retina and rarely other macrophages of ALD and AMN.29,58,143 In spite of their prominence in ­peroxisomal disorders, especially PBDs, they are non-specific.38 For example, they have been reported in skin biopsies of other degenerative metabolic diseases, ­particularly in late infantile and juvenile ­ neuronal ceroid lipofuscinoses.41 Hence, their ­identification in macrophages of the CNS or an affected

(a)

visceral organ is of paramount diagnostic importance. They contrast ­morphologically with the most characteristic inclusions of ALD and AMN, which are linear to gently curved lamellae, and clear lipid profiles lying free in the cytoplasm of adrenocortical, Leydig and Schwann cells138,139 and brain macrophages133,159 (Figure 8.3). Lamellae are not m ­ embrane-bound and ­ fundamentally consist of two 2.5-nm, electron-dense leaflets separated by a variable electron-lucent space of 1–7 nm. They often lie within or at the edge of large clear spaces (lamellar-lipid profiles). Cells that possess spicules or lamellae/lamellarlipid profiles are birefringent and retain their ­birefringence after acetone extraction.36,85 The ­lamellae/lamellar-lipid profiles, apparently in contrast to the s­picules,36 can be extracted with xylene or n-hexane. This non-polar lipid has been identified as abnormal cholesterol esters containing saturated VLCFA.78 Additional details about morphology, composition and formation are available.36,134,142,143 In view of the known facts that the only major biochemical defect in ALD is the accumulation of saturated (except for minimal monounsaturated) VLCFAs and that VLCFAs are increased in all PBDs and pseudoPBDs, angulate lysosomes with spicules in peroxisomal disorders most probably contain biochemically modified VLCFA. Peroxisomes vary greatly in their morphology; they are almost undetectable, atrophic or hypertrophic in PBD, malformed to normal in pseudo-PBD, and normal in most other single enzymopathies. Mosaicism has also been documented.36 Neuropathological lesions in peroxisomal disorders are of three major types: (i) abnormalities in neuronal migration or positioning, which are characteristic of PBD and pseudo-PBD, particularly ZS; (ii) defects in the formation or maintenance of central white matter, the former typically seen in PBD and the latter in ALD and AMN and NALD; and (iii) post-developmental neuronal degenerations, which are most frequent in AMN, PBD and Refsum’s disease.141 Except for ZS, NALD, ALD and AMN, pathological data on most peroxisomal disorders are scant and should be considered provisional.135 The prototypes of the PBD group, ZS and, of the single

8

(b)

8.2 Inclusions in PBDs. (a) Angulate lysosomes containing trilaminar straight spicules in a central nervous system macrophage from a symptomatic adreno-leukodystrophy heterozygote. Electron microscopy of autopsy material. (b) Angulate lysosome containing trilaminar inclusions in hepatocyte of infantile Refsum’s disease. Electron microscopy of a biopsy.

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8.3 ALD. Electron micrograph of lamellae and lamellar-lipid ­profiles (arrows) among dilated smooth endoplasmic reticulum and variably sized mitochondria in a 22-week fetal zone adrenocortical cell of adreno-leukodystrophy. Autopsy.

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568  Chapter 8  Peroxisomal Disorders

protein deficient group, ALD and AMN, will be emphasized. Abnormal white matter is prominent in ZS and predominant in NALD and ALD; consequently, an ­overview of primary diseases of myelin136 can provide a perspective for the white matter lesions described later.

Leukodystrophies and Other Defects in Myelination The term ‘leukodystrophy’ is generally used to describe genetic (inherited) and progressive disorders that primarily and directly affect CNS myelin. The classic leukodystrophies have been historically classified as ‘dysmyelinative’ primary diseases of myelin. Other diseases displaying a comparable confluent loss of myelin but lacking the genetic, progressive or primary myelin involvement have been referred to as ‘leukoencephalopathies’. The genetic defect in leukodystrophies may result in synthesis of biochemically abnormal myelin or in a molecular abnormality in myelin-forming cells, usually oligodendrocytes, which adversely impacts myelin in other ways. Irrespective of the biochemical/molecular abnormality, the end result is typically a confluent destruction, or failed development, of central white matter. In the latter and much less common ‘hypomyelinative’ leukodystrophies, such as Pelizaeus– Merzbacher disease (PMD), a molecular defect in oligodendrocytes impedes the formation of myelin. There also are a number of neonatal-infantile diseases, including PBD, in which it is difficult to decide whether there is hypomyelination and, if so, whether it represents a delay in CNS myelin formation or an arrest of myelination. Both of these situations are ‘hypomyelinative’ at autopsy and, at least theoretically, could be considered as ‘hypomyelinative leukodystrophies’. However, a delay in CNS myelination is usually not progressive and, hence, it may be more appropriate to consider these as leukoencephalopathies. It takes considerable experience with numerous controls and a sound knowledge of the timing of regional myelinogenesis in the CNS21,95 to make these determinations neuropathologically. MRI, particularly on T2-weighted images, has considerably improved our sensitivity of diagnosis of white matter disorders, and the same patterns of classic gross neuropathological lesions can be appreciated more easily.130,161,184,185 Longitudinal studies using MRI, especially with modifications including magnetization transfer and diffusion anisotropy, provide powerful pathogenetic insights. Most leukodystrophies fall into the dysmyelinative category, where myelin is initially formed to a variable extent but subsequently breaks down. The myelin may break down (i) because it is biochemically abnormal, such as in metachromatic leukodystrophy (MLD) and ALD; (ii) because an oligodendroglial toxin accumulates because of the molecular defect in the oligodendrocyte, such as has been proposed in globoid cell leukodystrophy (GLD, Krabbe disease); or (iii) for unknown reasons, such as in sudanophilic (orthochromatic) leukodystrophies (SLD). When myelin is catabolized, its protein and lipid constituents, particularly galactolipids (cerebroside, sulphatide [cerebroside sulfate]) and cholesterol, are liberated and can be appreciated with traditional carbohydrate (e.g. PAS) and

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lipid (e.g. oil red O) stains. Galactolipids are PAS-positive; sulphatide, which contains anionic sulfate groups, is also metachromatic. When cells with normal catabolic enzymes degrade biochemically normal myelin, these staining reactions are ephemeral. However, if galactocerebrosidase or arylsulphatase (arylsulfatase) activity is absent or markedly diminished, these staining qualities persist. Consequently, in GLD lacking galactocerebrosidase the myelin debris that accumulates is PAS-positive, although in MLD it is PAS-positive and metachromatic. The liberated cholesterol is esterified primarily by macrophages and usually persists for much longer as cytoplasmic vacuoles (lipophages, gitter cells, compound granular corpuscles). Therefore, if the myelin is biochemically ‘normal’ and the host possesses normal catabolic enzymes, then the myelin debris consists primarily of cholesterol esters. Normal cholesterol esters, the ‘floating fraction’ of neurochemists, are stained convincingly with neutral lipid or sudanophilic dyes, hence the term ‘sudanophilic’. As cholesterol esters are not metachromatic, they are also ‘orthochromatic’.136 Histological examination reveals a dynamic process, with various cells participating at specific times. In general, the affected myelin sheaths display morphological changes of vacuolation, blebbing, fragmentation and loss of stainability with traditional myelin stains, which is accompanied by cellular reactions characteristic of each disease. Most commonly, in the dysmyelinative leukodystrophies, axons lacking myelin sheaths are admixed with numerous macrophages containing myelin debris, which is often diagnostically distinctive, and hypertrophic or reactive astrocytes. Later, the macrophages migrate to perivascular spaces around venules, either to die there by apoptosis or to exit the brain; the reactive astrocytes involute and produce a chronic astroglial scar, which may be anisomorphic or isomorphic. In the ‘hypomyelinative’ leukodystrophies, there is little need of a macrophage response but reactive astrocytosis is usually prominent. The classic dysmyelinative leukodystrophies (ALD, MLD, GLD and probably some SLD) involve defects in myelin lipids that are qualitatively similar in the CNS and PNS; hence, involvement of both CNS and PNS myelin is commonly observed. If the defect involves myelin proteins that are largely restricted to one compartment (e.g. proteolipid protein, in CNS myelin), then the myelin lesions are likewise restricted (e.g. to the CNS in PMD). All leukodystrophies mentioned earlier display similar macroscopic CNS features: reduced brain weight, optic atrophy, bilaterally symmetrical diffuse loss or lack of deep cerebral and cerebellar white matter, which is replaced by firm tan to grey astrocytic gliosis, relative sparing of subcortical arcuate (‘U’) fibres, atrophy of the corpus callosum and compensatory (ex vacuo) hydrocephalus. Light and electron microscopic examination shows that each leukodystrophy has its own characteristic and usually diagnostic lesions, in addition to the common features of reduced myelin staining, loss of oligodendrocytes, relative sparing of axons, macrophages containing myelin debris and reactive astrocytosis to fibrillary astrocytic gliosis. Axonal loss is greater and inflammatory cells, other than macrophages, are conspicuously absent in the leukodystrophies when compared with the prototypic demyelinative diseases, multiple sclerosis (MS) and acute disseminated (allergic) encephalomyelitis (ADEM). ALD is the exception, because

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  Group I: Disorders of Peroxisome Biogenesis  569

it is markedly inflammatory and mimics MS.133,136,159 ALD differs from MS, however, by the typical localization of lymphocytes just within (rather than at) the demyelinative edge, by the relative paucity of B-lymphocytes and plasma cells,148 and by the T-helper cell-cytokine pattern.109 In MS and ADEM, lymphocytic or lymphocytic/plasmacytic reactions, more axonal sparing, lesion asymmetry when bilateral, random involvement of subcortical fibres, sudanophilic myelin debris and restriction of lesions to the CNS reflect the immune destruction of biochemically normal central myelin, in which myelin proteins appear to be a major target. There is an additional group of primary diseases of myelin, sometimes referred to as ‘myelinolytic’ or ‘spongy myelinopathies’, which share the common histopathological feature of spongy or vacuolated myelin due to intramyelinic oedema. The splits in myelin usually occur at the intraperiod (extracellular face) line. They typically elicit little to no macrophage or reactive astrocytic response. Despite these common histopathological features, their aetiologies are diverse, usually toxic-metabolic and include vitamin B12 deficiency, aminoacidurias and mitochondrial disorders.136 Canavan’s disease bridges this classification scheme by exhibiting a spongiform myelinopathy (i.e. myelinolytic) and a confluent, progressive and genetically determined myelin lesion (i.e. a leukodystrophy). Peroxisomal disorders are not characterized by spongy myelin, except in one ­family with atypical Refsum’s disease, in which a mitochondrial defect was not completely excluded.181 Table 8.3 summarizes the main histopathologic, biochemical and molecular features seen in peroxisomal disorders.

Group I: Disorders of Peroxisome Biogenesis

8

‘Classic’ Zellweger Syndrome These infants usually present at birth with characteristic dysmorphic facies, seizures, severe hypotonia and profound psychomotor retardation. They usually die in the first year of life. Additional clinical and radiological findings include cataracts, pigmentary retinopathy, optic atrophy, sensorineural hearing deficits, equinovarus deformity, hepatomegaly and stippled calcifications of the patellae, femora and humeri. Systemic pathological findings may also include biliary dysgenesis, ventriculoseptal defects, islet cell hyperplasia and hypoplasia of the thymus and lung. Renal microcysts, predominantly cortical and varying from 0.1 to 0.5 cm, arise from both tubules and glomeruli.17,124,127,203 The adrenal cortex displays scattered and infrequent adrenocortical striated cells with lamellar-lipid profiles and PAS-positive macrophages with spicules.63 Many of these abnormalities have been documented in affected fetuses.144 Hepatic peroxisomes originally had been reported as absent, and mitochondrial abnormalities were observed;62 subsequently, immunofluorescent studies identified remnant peroxisomes present as ‘membrane ghosts’, representing enlarged vesicles with peroxisomal membrane proteins but lacking matrix pro­ teins157 (reviewed in Depreter et al.36). Deficiency of Pex3p, Pex16p or Pex19p impairs PMP biogenesis, and peroxisomal membranes do not form. As a result of multiple enzyme losses, elevations in VLCFA and phytanic, pristanic, pipecolic,

Table 8.3  Overview of peroxisomal disorders ZS

NALD

RCDP

BPD

ALD

AMN

Cer

Cer

CNS

Cer

Cer

SC

GM

WM

GM

WM

Histopathology

Dysgenesis

De

Microencephaly

Dysgenesis

De

Axonopathy

Misguided neurons

+++

+

0?

++

0

0

Cer/Cbl myelination

Hypo

De

Hypo

Hypo/De

De

Dys/De

Oligodendroglial loss

±

++

±

±

+++

++

Astrocytosis

++

++

±

±

+++

+

Microgliosis

±

++

±

±

+++

+++

Inflammation

0

++

0

+

+++

±

Adrenal lamellae

+

+

0

+

+++

+++

Angulate lysosomes

+

++

0

+

+

+

Biochemical defect

Multiple

Multiple

↓ Plasmalogens

↑ VLCFA ↑ Cholestanoic acids

↑ VLCFA

↑ VLCFA

Molecular defect

PEX 1*

PEX 1*

PEX 7

D-BP

ABCD1

ABCD1

Import

Import

Import

Activity

Import

Import

Major localization

*PEX 1 most common gene defect. For other PEX genes, see Table 8.2. 0, negative; ±, rare; +, mild; + +, moderate; + + +, severe. ABCD1, adenosine triphosphate-binding cassette D1; ALD, adreno-leukodystrophy; AMN, adrenomyeloneuropathy; BPD, bifunctional protein deficiency; Cbl, cerebellar; Cer, cerebral; CNS, central nervous system; D-BP, dextro isomer of bifunctional protein; De, demyelination; Dys, dysmyelination; GM, grey matter; Hypo, hypomyelination; NALD, neonatal adreno-leukodystrophy; PEX, peroxin; RCDP, rhizomelic chondrodysplasia punctata; SC, spinal cord; VLCFA, very long-chain fatty acid; WM, white matter; ZS, Zellweger syndrome.

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570  Chapter 8  Peroxisomal Disorders

di- and tri-hydroxycholestanoic acids with decreases in ­plasmalogens and DHA are evident. Phytanic acid is derived exclusively from dietary sources, whereas pristanic acid ­ comes from phytanic acid breakdown and dietary sources; hence, the presence and prevalence of both may vary with age, diet and catabolic enzyme activity.191,193

Neuropathology of Zellweger Syndrome The major abnormalities are in the CNS, where defective neuronal migrations dominate.45,190 These infants classically show a unique combination of centrosylvian or parasylvian pachygyria (medial) and polymicrogyria (lateral and extending into the lateral frontal lobe and the lateral parietotemporo-occipital region) (Figure 8.4); this localization for the major neocortical malformation is consistent but may be associated with other areas of polymicrogyria and pachygyria. There may also be an abnormal vertical tilt to the sylvian fissure. The limbic areas of the brain are typically spared. Coronal sections of the cerebrum exhibit a thickened cortex, with either excessive superficial plications or obvious subcortical heterotopias. Microscopy of the micro-polygyric cortex usually reveals fusion of the molecular layers and better preservation of the supragranular cortex (Figure 8.5). The outer cortex is typically occupied by medium to large pyramidal cells destined for deep cortex, whereas the usually superficial neuronal populations are detained in deep cortex and subcortical white matter. The pachygyric cortex has a similar but more severe alteration; the subcortical heterotopias are likewise more prominent (Figure 8.6). These cortical abnormalities differ from those of classic four-layered polymicrogyria or lissencephaly-pachygyria in that all neuronal classes seem affected, with those destined for the outer layers tending to be more impeded. The cerebellum is grossly unremarkable, but it is common to find heterotopic Purkinje cells (often polydendritic) or combinations of Purkinje cells and granule cells in the white matter, especially in the nodulus. There is dysplasia of claustra, medullary olives and often the dentate nuclei. These dysplasias are not true heterotopias but, rather, appear to reflect a problem with neuronal positioning or the terminal stages of migration.135,141 For example, the olives and dentate nuclei may lack their normal serpiginous configuration or consist of discontinuous islands of neurons; the olives may display peripheral palisading of neurons (Figure 8.7). Striated and globose PAS-positive macrophages, containing abnormal lipid cytosomes, have been identified in grey and white matter of cerebrum and cerebellum (Figure 8.8).2,33,200 These abnormal lipids are detectable with proton MR spectroscopy.68 Morphological evidence of a restricted neuronal lipidosis is seen in the form of striated neuronal perikarya and dystrophic spheroids containing lamellar and lipid profiles in Clarke’s and lateral cuneate nuclei (Figures 8.9–8.11).146 Ventricular enlargement is common, as are periventricular cysts and ependymal abnormalities.65 Brains from fetuses at risk of developing ZS show neocortical migratory defects as early as postmenstrual estimated gestational age (EGA) 14 weeks, in the form of micropolygyric ripples and subtle subcortical ­heterotopias. Thin abnormal cortical plates with more obvious s­ubcortical heterotopias occur at EGA 22–24 weeks (Figure 8.12). Astrocytes, neuroblasts, immature neurons, radial glia and

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8.4 Zellweger syndrome. Coronal section of a 4-week-old PEX 1 male with Zellweger syndrome, demonstrating bilateral parasylvian medial pachygyria with prominent subcortical heterotopia and asymmetric lateral polymicrogyria, particularly of insular cortex. Nissl.

8.5 Zellweger syndrome. Micropolygyric heterotopia in Zell­ weger syndrome, with fewer pyramidal and more granular ­neurons than its pachygyric counterpart.

8.6 Zellweger syndrome. Pachygyric heterotopia in Zellweger syndrome, consisting of many pyramidal neurons.

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8.7 Zellweger syndrome. Discontinuous and simplified inferior olive with peripheral palisading of neurons in a 4-week-old PEX 1 male with Zellweger syndrome.

8.10 Zellweger syndrome. Lamellae and lamellar-lipid profiles (arrow) between mitochondria in dorsal nucleus of Clarke perikaryon of a 12-week-old male with Zellweger syndrome. Electron microscopy of autopsy material.

8.8 Zellweger syndrome. Electron-opaque membranous ­cytoplasmic bodies, typical of Zellweger syndrome, in an astrocyte of occipital cortex in a 13-week-old male with Zellweger syndrome. Autopsy.

8.11 Zellweger syndrome. Electron micrograph of l­amellae and lamellar-lipid profiles in axonal swelling within dorsal nucleus of Clarke of the same patient as in Figure 8.10. Autopsy.

n

8.9 Zellweger syndrome. Striated neuron (n) of dorsal nucleus of Clarke, with spheroid (arrow) in the same patient as in Figure 8.8.

PAS-positive macrophages contain abnormal pleomorphic cytosomes; these include electron-opaque membranous cytoplasmic bodies, perhaps representing gangliosides ­containing saturated VLCFA. Some neurites also exhibit lamellar and lipid profiles. Dysplastic a­ lterations of the inferior olivary and dentate nuclei are present, as well as renal

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cortical microcysts, fetal zone adrenocortical s­triated cells and patellar mineralization.147 These fetal lesions support the proposal that the insult (presumably ­metabolic) causing the neocortical migration defect is ­operating throughout the entire neocortical neuronal migratory period. The CNS white matter in ZS is commonly abnormal (Figure 8.13) but does not contain inflammatory cells (lymphocytes or plasma cells). Although some authors have referred to this as a leukodystrophy,127 most would not, and the nature of the defect is still unclear.2,135 It appears to be dysmyelinative and primarily hypomyelinative, in that there is usually little sudanophilic lipid accumulation in macrophages. Reactive astrocytosis may be relatively inconspicuous in immature white matter or severe, particularly in

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8.14 Zellweger syndrome. Subcortical cerebral white matter of another patient syndrome, with reactive astrocytosis. Glial fibrillary acidic protein.

8.12 Zellweger syndrome. Abnormal cortical plate with subcortical heterotopia (arrows) in incipient pachygyric superior parietal gyrus of a 22-week-old male fetus with Zellweger syndrome.

areas with heterotopias (Figure 8.14); it can even extend throughout the white matter of the neuraxis. Many abnormal lipid cytosomes are present in astrocytes and oligodendrocytes,200 some of which might be VLCFA-gangliosides, and plasmalogen deficiency could interfere with normal myelination.135,141 Myelin pallor may reflect a decrease in the normal complement of myelinated axons, reflecting the severe pachygyria–polymicrogyria; this is supported by more severe involvement of the posterior limb of the internal capsule underlying areas of perisylvian polymicrogyria (Figure 8.13). Additionally, superimposed hypoxic– ischaemic–acidotic damage due to seizures, systemic metabolic abnormalities and chronic debilitation are probable complicating factors. It is important to emphasize that, irrespective of the precise pathogenesis of the white matter lesion in ZS, it is not inflammatory and differs morphologically from that of ALD. Many of the gross neuropathological features of ZS can be appreciated in living patients with MRI (Figure 8.15).130,184,185

Neonatal Adreno-Leukodystrophy

8.13 Zellweger syndrome. Coronal section of a 4-week-old PEX 1 male, demonstrating an asymmetric paucity of myelin most notably in the posterior limbs. Luxol fast blue–periodic acid–Schiff.

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This highly variable PBD has features in common with both ZS and ALD but is less severe.82,106,182 Clinically, infants with NALD resemble those with ZS, but they usually die around 36 months of age and survival can be highly variable, even extending into adolescence.92 Despite its name, NALD is transmitted as an autosomal recessive trait and the coexistence of NALD and ALD in the same kindred has never been reported. Hepatic lesions are common. Peroxisomes in the liver have been reported as missing, decreased in size or enlarged, with associated biochemical abnormalities in all peroxisomal functions75 (reviewed by Depreter et al.36). Mitochondrial abnormalities have also been documented.75 Adrenocortical atrophy with striated adrenocortical cells mimics the adrenal pathology of ALD. The diffuse distribution of PAS-positive macrophages with angulate lysosomes containing spicules in the liver, thymus, spleen, lymph node, lung, gastrointestinal tract and adrenal gland contrasts with their hepatic predominance in IRD. The PNS in NALD has been unremarkable or has shown evidence of demyelination and thin myelin sheaths, and lamellae and lipid profiles (see ALD) have been visualized in

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8.16 ALD. Inflammatory demyelinative lesion in occipital white matter of a 14-year-old male with neonatal adreno-leukodystrophy. Luxol fast blue–periodic acid–Schiff.

systemic PAS-positive macrophages, inflammatory demyelinative CNS lesions and increases in saturated C26:0; ZS exhibits chondrodysplasia, renal microcysts, pachygyria– polymicrogyria with dysplastic claustra, dentate nuclei and olives and the accumulation of both s­aturated and monounsaturated C26:0.48,92

8.15 Zellweger syndrome. Axial T2-weighted image from a 3-month-old PEX 1 male, showing sparse myelination, frontal pachygyria, parasylvian polymicrogyria and some ventriculo‑ megaly. Reprinted with kind permission of Springer Science and Business Media and Prof. MS van der Knaap from van der Knaap and Valk.184

Schwann cells.111 One case of NALD demonstrated severe atrophy of the auditory sensory epithelium and tectorial membrane.77 Retinopathy with a ‘leopard spot’ appearance may be a distinctive clinical feature.48 The CNS in NALD, in contrast to ZS, shows greater involvement of white matter than disrupted neuronal migration. Both ZS and NALD are associated with a slight increase in brain weight and infants are often macrocephalic. Heterotopic Purkinje cells are observed. Neuronal loss has been reported in the olives, dentate nuclei and thalami of two patients with NALD. PAS-positive macrophages are present in the cortex of the cerebrum and, particularly, the cerebellum, similar to ZS, as are rare swollen neurons in the arcuate nucleus and perhaps in the pons.182 Polymicrogyria may be diffuse, focal or multifocal and associated with subcortical heterotopias; pachygyria and dysplastic olives have not been reported. Diffuse dysmyelination/demyelination of cerebral and cerebellar white matter, often with a prominent perivascular lymphocytic reaction, is another distinguishing CNS feature and resembles ALD (Figure 8.16). PAS-positive macrophages containing both spicules and lamellae are present and variable degrees of reactive astrocytosis to chronic fibrillary astrogliosis are seen. Sparing of the arcuate fibres has been reported and sudanophilic macrophages are commonly observed within white matter lesions. In summary, NALD displays adrenal atrophy,

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Infantile Refsum’s or Phytanic Acid Storage Disease Patients with infantile Refsum’s disease (IRD) have mental retardation, dysmorphic facies of a minor degree, retinitis pigmentosa, sensorineural hearing deficits, failure to thrive and hypocholesterolaemia.131 All peroxisomal biochemical parameters are abnormal and peroxisomes are either deficient or reduced in number75 (reviewed by Depreter et al.36). The clinical course is relatively mild compared to NALD, with presentation after the neonatal period and survival at least into the early teens. Some patients who are phenotypically IRD develop clinical and radiological evidence of a progressive leukoencephalopathy.130 A distinct Zellweger spectrum phenotype is a late-onset, white matter disease with a central cerebellar predilection, clinically characterized by precipitous neurologic regression in late infancy, adolescence or adulthood.8 The most conspicuous pathological alteration in IRD is the prominence of PAS-positive macrophages containing angulate lysosomes in liver and accumulation of spicular structures in hepatocytes.166 Abnormal mitochondria have been noted.75 Hepatomegaly with micronodular cirrhosis was reported at autopsy in a 12-year-old boy.179 The adrenal glands were interpreted as hypoplastic but the appearance is more likely to have resulted from atrophy. CNS migration defects were not observed, but the cerebellar cortex was diffusely small; granule cells were preferentially reduced in number and Purkinje cells were abnormally situated in the molecular layer. MRI ­studies184 and similar neuropathology in chronic RCDP indicate that this probably represents cerebellar atrophy.149 The cerebral white matter demonstrated focal decreases in the number of myelinated axons, particularly in the periventricular region, corpus callosum, corticospinal tracts and optic nerves. MRI studies have shown

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more widespread ­ cerebral involvement, including the posterior limb and pyramidal fibres in brain stem.130,184 Inflammatory cells were not seen but there were numerous perivascular collections of non-sudanophilic and PASnegative macrophages and dense gliosis. Occasional perivascular macrophages were also noted in brain stem and cerebellar white matter. Ultrastructurally, brain macrophages contained lamellae but lacked ­angulate lysosomes. Astrocytes also displayed lamellar inclusions. Two ‘atypical’ IRD siblings, who died at 3.5 years and 8 months, have also been reported. Marked adrenal atrophy with striated cells (not illustrated) and sparse PAS-positive macrophages was associated with displaced Purkinje cells in the molecular layer, peripheral palisading of neurons in the olive and cirrhosis with PAS-positive macrophages.26 With respect to the sensorineural hearing deficit, which is also seen in ZS, NALD, ARD, RCDP, acyl-CoA oxidase deficiency and bifunctional protein deficiency ­ (BPD), one patient with IRD exhibited good preservation of ganglion cells and nerve fibres in the organ of Corti but severe ­atrophy of the sensory epithelium and stria vascularis.179

Rhizomelic Chondrodysplasia Punctata, Type I, Classic Classic RCDP presents at birth with severe shortening and stippled calcifications of the humerus and femur, vertebral defects, joint contractures, dysmorphic facies, psychomotor and growth retardation, cataracts and optic atrophy, sensorineural hearing deficits and microcephaly.169 Most also have ichthyosis. Survival in RCDP is better than once thought, with 90 per cent surviving to age 1 year and 50 per cent surviving to 6 years.199 Some milder variants may have chondrodysplasia without rhizomelia, cataracts and less severe growth and mental deficiency.130 Plasmalogen deficiency is even more severe than that seen in ZS. VLCFA are normal. Dihydroxyacetonephosphate acyltransferase (DAPAT) and alkylglycerone phosphate synthase (AGPS) enzymes, which initiate peroxisomal plasmalogen synthesis, are also deficient.191 Pex7p imports AGPS into peroxisomes and DAPAT activity requires the presence of AGPS. The phytanic acid oxidation defect, because of deficient Pex7p import of phytanoyl-CoA hydroxylase (PhyH), approximates that of adult Refsum’s disease and ZS. In some hepatocytes peroxisomes cannot be identified, whereas in others they are irregularly shaped and enlarged.36,65 Few post-mortem examinations of infantile cases have been undertaken.59,132 Although most RCDP infants are microencephalic, no satisfactory morphological correlation for their severe psychomotor retardation has been found. Microscopical examination of the CNS in patients who died at about 1 year of age was essentially unremarkable; however, in one these brains, the inferior olives showed focal discontinuities and were considered to be a mild form of the defect seen in ZS.132 The white matter of a girl aged 3 years with RCDP was diffusely reduced in size but appeared to be normally myelinated, except in the occipital lobe; diffuse cerebellar degeneration and a corresponding neuronal loss in the olives were observed.1 We confirmed the microencephaly (Figure 8.17) in two patients with chronic RCDP and focal dysplasia of the olive in one;

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8.17 RCDP. Microencephalic brain of an 11-year-old boy with rhizomelic chondrodysplasia punctata, with reduced volume of frontal white matter.

they both displayed severe cerebellar ­atrophy because of losses of Purkinje and granule cells (Figure 8.18). Neuronal loss in the olives and a variable p ­allor of myelin with ­corresponding reactive ­astrocytosis were also noted. Thus, post-developmental cerebellar atrophy occurs in RCDP patients with prolonged survival. Phytanic acid accumulation (diet dependent), perhaps in concert with reduced tissue plasmalogen levels, has been proposed to cause the apoptotic death of Purkinje and granule cells by altering calcium homeostasis.149 Neocortical migration defects are generally considered to be absent, supporting the hypothesis that the migration defects in PBD and pseudo-PBD are due, at least in part, to elevated VLCFA.147 However, one patient with short humeri and femora, widespread stippled calcifications, collecting tubule and glomerular cysts and cataracts displayed pachygyria of the posterior frontal and pararolandic region and focal microgyria of the frontal pole; designation of this case as RCDP remains uncertain as it preceded the era of biochemical or genetic confirmation.141 Another patient, clinically classified, although not genetically confirmed, as RCDP, had pachygyria and polymicrogyria of bilateral frontal lobes on neuroimaging.61 MRI in severe RCDP phenotypes reveals delayed myelination, regressive white matter changes with a parietooccipital predominance, ventricular dilatation and cerebellar atrophy.6,130

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8.18 RCDP. Loss of granule neurons and Purkinje cells, worse distally, in atrophic cerebellum of a 9-year-old girl with rhizomelic chondrodysplasia punctata.

A PEX 7 knockout mouse provides some interesting insights into this disorder. Neocortical lamination abnormalities were detected, but so were elevations in VLCFA (deficient Pex7p-mediated import of thiolase [ACAA1]) that are not seen in human RCDP. The authors speculated that the neuronal migration defect was due to a combination of elevated VLCFA and decreased plasmalogens (reviewed in da Silva31).

Group II: Disorders with Morphologically Intact Peroxisomes and a Single Protein Deficiency Pseudo-Peroxisome Biogenesis Disorders Acyl Coenzyme A Oxidase Deficiency (Pseudo-neonatal Adreno-Leukodystrophy) Over thirty cases have been reported (reviewed in Wang et al.195).100,193 Although dysmorphic features were not observed in some patients, mild anomalous facial features were noted in others. All paediatric patients displayed moderate to severe neonatal hypotonia, seizures and psychomotor retardation. Sensorineural hearing deficits and abnormal electroretinograms were reported. The only biochemical abnormality was elevated VLCFA; hepatic peroxisomes were heterogeneous in size, many enlarged and angulated, and they were increased in number (reviewed by Depreter et al.36). Macrophages with angulate lysosomes were not identified. Neuroradiological studies usually indicate an absence of migration defects, diffuse and progressive CT hypodensities in cerebral white matter, a thin corpus callosum and cerebellar ‘hypoplasia’ (probably atrophy). However, several patients have had cortication defects, such as perisylvian polymicrogyria or pachygyria. The white matter of one progressive case demonstrated abnormal contrast enhancement, suggesting an inflammatory component, such as in NALD and ALD. With MRI, the white matter abnormality was seen as high signal intensity in T2-weighted images but contrast material was not administered.100 The distribution of the lesions was mainly cerebellum, cerebral

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peduncles and brain stem in the early stages but also posterior cerebrum later. Neuropathologic data are rare. Two siblings, one of whom had haematopoietic cell transplantation, had inflammatory demyelination of cerebral white matter as severe as that in ALD with a comparable posterior predominance. Less inflammatory demyelination was observed in cerebellar white matter and basis pontis, again comparable to ALD. In keeping with the dual localization of acyl coenzyme A oxidase in glia and neurons,47 an olivopontocerebellar-like degeneration was also noted. Severe neuronal losses in the cerebellar and cerebral (particularly motor) cortex, as well as dentate, olivary and basis pontine nuclei, resulted in severe cerebellar and pontine atrophy, moderate cerebrocortical atrophy and corresponding secondary tract degeneration of cerebral and cerebellar peduncles (see Wang et al.195). A knockout mouse model has been developed but neuropathological data were not provided.5

8

D-Bifunctional Protein Deficiency (2-Enoyl Coenzyme A Hydratase/D-3 Hydroxacyl Coenzyme A Dehydrogenase) This deficiency is by far the most common of the pseudoPBD single enzymopathies.198 The deficient enzyme is dbifunctional protein (D-BP; also referred to as multifunctional protein 2, multifunctional enzyme II or D-peroxisomal bifunctional enzyme), not L-bifunctional protein, as originally reported.188,197 BPD is a more severe clinical phenotype than acyl-CoA oxidase deficiency.198 Biochemically, these patients demonstrate elevations in VLCFA, bile acid intermediates and perhaps phytanic and pristanic acids but erythrocyte plasmalogens are normal.191,193,198 Clinically, they often resemble the severe Zellweger phenotype, with neonatal hypotonia and seizures, facial dysmorphia, psychomotor retardation, neuronal migration defects (polymicrogyria) and hypomyelination (Figure 8.19). Most affected children die within the first two years of life. Milder BPD cases may have longer (≥ 7.5 years) survival and peroxisomal biochemical abnormalities may be lacking in plasma. The clinical, biochemical, imaging and (to some extent) neuropathological spectrum of a large cohort of these patients has been reported.53 Peroxisomes may be reduced in number, enlarged or undetectable.53 The first reported patient with BPD had clinical features of NALD; he died at 5.5 months of age. Autopsy findings consisted of mild portal fibrosis, glomerular microcysts and adrenal atrophy with ‘lipid-containing balloon cell’; striated cells were not reported. The CNS revealed polymicrogyria, focal heterotopias, ‘demyelination’ of cerebral white matter and periventricular cysts.197 In another case, previously classified as atypical acyl-CoA oxidase deficiency, there was dysmyelination and inflammatory demyelination of the CNS white matter (occipital lobes and cerebellum), resembling that of NALD and ALD. There were also some mild cerebral and cerebellar heterotopias, microgyria (but not pachygyria or polymicrogyria) and focal dysplasia of olivary and Clarke’s nuclei. PAS-positive macrophages with angulate lysosomes and adrenocortical atrophy with striated cortical cells were present.119 A third patient displayed centrosylvian pachygyria-polymicrogyria, reminiscent of, but milder than, ZS, diffuse hemispheric hypomyelination with subcortical heterotopic neurons, Purkinje cell heterotopias and simplified

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biosynthesis enzymes in RCDP type 2 (DAPAT deficiency) or RCDP type 3 (AGPS deficiency) produces clinical phenotypes indistinguishable from classic RCDP. Neuroradiological abnormalities of CNS myelination have been found in RCDP type 2.193 A neonatal patient with RCDP type 2 reportedly had some heterotopias, but the description ‘multiple heterotopic foci of immature neurons in the vicinity of the third ventricle’ suggests normal findings in an infant. The olive was also reported to be ‘broadened’.69

Adreno-Leukodystrophy and Adrenomyeloneuropathy

8.19 PBD. Polymicrogyric pattern in sylvian fissure and white matter abnormalities in an 8-month-old female with D-bifunctional protein deficiency.

convolutions of the dentate nucleus and inferior olive.91 In summary, focal polymicrogyria and an inflammatory leukoencephalopathy, more consistent with NALD, as well as centrosylvian pachygyria-polymicrogyria, a non-inflammatory leukoencephalopathy and olivary dysplasia, more consistent with ZS, have been reported in BPD (Figure 8.19). The single patient reported with thiolase deficiency (3-oxoacyl-CoA thiolase deficiency; pseudo-ZS) was later classified as BPD. This patient resembled classic ZS, with increased VLCFA and bile acid intermediates, dysmorphic facies, profound hypotonia and typical pathologies in liver, kidney and adrenal.64,164 However, the inflammatory demyelination and astrocytosis of cerebellar white matter, heterotopic subcortical Purkinje cells, focal polymicrogyria,135 and adrenal atrophy with striated cells were more reminiscent of NALD. Additional clinical presentations are reported in BPD. A male aged 4 years with BPD demonstrated optico-cochleodentate degeneration as well as frontoparietal and insular microgyria (normal lamination), cerebellar atrophy, cerebral and particularly cerebellar white matter loss and a severe axonopathy of sural nerve.165 Two siblings, the longest surviving females reported with a mild BPD mutation, developed ovarian dysgenesis, along with hearing loss and ataxia that were clinically defined as Perrault syndrome.129 In view of the evidence of oxidative stress in ALD and the ZS knockout mice, it was somewhat surprising that it was also found in BPD but not in the PBD.52 Phospholipid analysis of brain tissue from a BPD patient revealed declines in myelin lipids indicative of dysmyelination, as well as decreased plasmalogens in grey matter, despite intact plasmalogen synthesis enzymes in BPD (reviewed in da Silva31). The remaining diseases in the pseudo-PBD category  – Zellweger-like syndrome,173 in which multiple peroxisomal enzymes were deficient, di- and tri-hydroxycholestanoic acidaemia27 and RCDP type 334  –  are rare and lack neuropathological data. Isolated deficiency of plasmalogen

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X-linked ALD has two major phenotypes: juvenile (childhood cerebral) ALD and its adult variant AMN.13,116 Much less frequent types (accounting for fewer than 15 per cent of cases) are adolescent and adult cerebral ALD, which differ from juvenile only in the age of onset; adrenal insufficiencyonly (addisonian); asymptomatic; and rare olivopontocerebellar99,123 and spinocerebellar107 types. Approximately 65 per cent of heterozygote (female) carriers demonstrate the AMN phenotype, usually mild and later in life.13,83 The molecular genetics of ALD has been a field of intense activity in the past two decades, but genotype– phenotype correlations are lacking. The same genetic defect is associated with juvenile ALD, AMN and even addisonianonly ALD.93,141 This has generated the hypothesis that a modifier gene and/or environmental factors are responsible for the phenotypic variation (reviewed by Moser).114 CD1 gene polymorphisms (see later) do not seem to contribute to the phenotypic variation,7 but head trauma, activity of the mitochondrial enzyme manganese superoxide dismutase (SOD2), and loss of AMP-activated protein kinase α-1 (AMPKα1), may.22,155,204 It is also unclear how the genetic defect in ABCD1 (formerly ALDP) is related to the major biochemical abnormality identified in ALD – elevations of saturated VLCFA in blood and tissues. Previously, it was believed that the major determinant was decreased activity of VLCF ­acyl-CoA synthetase (lignoceroyl-CoA ligase) with a consequent elevation of VLCFA. Current evidence suggests that the major physiological function of ABCD1 is the transport of VLCFA-CoA across the peroxisomal membrane.94 VLCFAs have been identified in many myelin components, but especially in cholesterol esters,78 gangliosides,76 phosphatidylcholine172 and proteolipid protein.16 The ALD gene (ABCD1) is localized to the Xq28 region, occupies 21–26 kilobases (kb) of genomic DNA, contains 10 exons and encodes an mRNA of 3.7–4.3 kb to translate a protein of 745–750 amino acids. This ALD protein (ABCD1), instead of being the deficient synthetase, was found to be an integral membrane protein of peroxisomes with the properties of an ATP binding cassette (ABC) half-transporter.35,94,118 ABCD1 (ALDP) mRNA is expressed in all tissues but is highest in the adrenal glands, intermediate in the brain and almost undetectable in the liver. ABCD1 is strongly expressed in microglia, astrocytes and endothelial cells; oligodendrocytes have little to no ABCD1, except those in the corpus callosum and internal capsule. ABCD1 is not detectable in the fibroblasts of about two-thirds of patients with ALD. A large number of mutations have been identified: approximately 54 per cent are missense, 25 per cent frameshift, 10 per cent nonsense and 7 per cent large deletions. A mutational hotspot is

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  Group II: Disorders with Morphologically Intact Peroxisomes and a Single Protein Deficiency  577

noted in exon 5. All mutations examined, except for about a third of the missense, do not express detectable ABCD1 in fibroblasts, although all have ABCD1 mRNA.93 The juvenile or childhood cerebral form usually presents between 6 and 9 years of age with behavioural, auditory, visual and gait abnormalities or adrenocortical insufficiency (Addison’s disease). The disease is rapidly progressive, typically leading to death within 3 years.159 MRI confirms the gross neuropathological findings described previously as well as the usual progression of the lesions from parietooccipital to frontal lobes. Enhancement at the advancing edge of the demyelinative process is highly characteristic (reviewed by Moser114 and van der Knaap and Valk184), and MRI patterns can predict the progression of the cerebral disease (Figure 8.20).101 The adrenal cortex and testis are the only two non-neural organs that show significant lesions.134,135,139 Adrenocortical cells, particularly those of the inner fasciculata-reticularis, become ballooned and, of ­diagnostic import, striated as a result of accumulations of lamellae, lamellar-lipid profiles and fine lipid clefts (Figures 8.21–8.23).138 The striated material, consisting of the same abnormal cholesterol esters as in CNS striated lipophages,85 appears to lead to cell dysfunction, atrophy and apoptotic death.137,142 Histoenzymatic decreases in mitochondrial α-glycerophosphate dehydrogenase, 3-β-hydroxysteroid dehydrogenase and reduced triphosphopyridine nucleotide (TPNH) diaphorase have been reported and they show excessive peripheral cytolysis under the electron microscope. (a)

Moreover, the striated cells appear to adapt poorly to a tissue culture environment.142 Ultimately, primary atrophy of the adrenal cortex ensues. Inflammatory cells are rarely observed and are probably an epiphenomenon; anti-adrenal antibodies are not detected. The adrenal cortex in AMN displays the same qualitative changes as most adrenal glands of patients with ALD but tends to be more atrophic; this is related to the longer duration of hypoadrenalism and consequent corticosteroid replacement therapy in AMN.134 The same striated adrenocortical cells have been identified in ALD heterozygotes, both symptomatic and asymptomatic, but limited to small, multifocal clusters.145 This resembles ZS but is in striking contrast to ALD or AMN and to the fetal adrenal zone in affected fetuses, where this lesion is diffuse.143 Testicular abnormalities in prepubertal males with ALD are usually present only at the ultrastructural level and consist of lamellae and lipid profiles in the interstitial cells of Leydig and their precursors. The testis in AMN and adult cerebral ALD demonstrates the same Leydig cell alterations as noted already in childhood ALD but there is also Leydig cell loss. No inflammatory cells have been seen, except in a single unpublished case of adult ALD. Degenerative changes in the seminiferous tubules in AMN appear indistinguishable from those of adult cerebral ALD and vary from a ­maturation arrest of spermatocytes to a Sertoli cell-only phenotype, in which all germ cells are depleted. Ultrastructurally, vacuolation of the Sertoli cell’s endoplasmic reticulum f­ollowed by widened intercellular spaces appears to be the initial lesion of the seminiferous tubules.140

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(b)

8.20 Magnetic resonance images from an 8-year-old male with juvenile (cerebral) adreno-leukodystrophy. (a) T2-weighted image, showing the typical pattern of symmetrical parieto-occipital lesions (bright white). (b) Contrast-enhanced T1-weighted image, showing intense enhancement of the rim of the lesions (bright white). Reprinted with kind permission of Springer Science and Business Media and Prof. MS van der Knaap from van der Knaap and Valk.184

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578  Chapter 8  Peroxisomal Disorders

8.21 ALD. Ballooned adrenocortical cells of adreno-leukody­s­ trophy and adrenomyeloneuropathy, with diagnostic striations.

8.22 ALD. Lamellae and lamellar-lipid profiles in adrenocortical cells of juvenile adreno-leukodystrophy. Medullary cell granules on left. Biopsy.

8.23 ALD. Predominantly fine, clear lipid clefts in an adrenocortical cell of another juvenile patient with adreno-leukodystrophy. Biopsy.

The Neuropathology of Adreno-Leukodystrophy

fibres are relatively spared but the posterior cingulum, corpus callosum, fornix, hippocampal commissure, posterior limb ­ of the i­nternal capsule and optic tracts are typically involved (Figure 8.25). The cerebellar white matter usually exhibits a similar, but milder, confluent loss of myelin and sclerosis. Secondary corticospinal tract degeneration extending down through the peduncles, basis pontis, medullary pyramids and spinal cord is characteristic (Figure 8.26). The brain stem may also display primary demyelinative foci, especially in the basis pontis. The spinal cord is spared, except for the descending tract degeneration. Cerebral and cerebellar grey matter may

In addition to the gross and microscopic features common to most leukodystrophies, ALD has its own distinguishing characteristics.134,159 Mild to moderate premature atherosclerosis can be seen in adult patients. The loss of myelin is almost always most prominent in the parieto-occipital region (Figure 8.24). The advancing edges, usually frontal, are more asymmetrical and may display a white to pink ‘softening’ that blends imperceptibly into normal white matter. Cavitation and calcification of white matter may be seen in severe cases. Arcuate

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8.24 ALD. Bilaterally symmetrical, confluent demyelination of parietal white matter, particularly of the posterior limb of the internal capsule, with sparing of subcortical myelin in adrenomyeloneuropathy/adreno-leukodystrophy.

8.25 ALD. More severe loss of myelin, including most arcuate fibres, in juvenile adreno-leukodystrophy. Luxol fast blue– haematoxylin and eosin.

be intact or may be atrophic if there is severe (e.g. cavitary) damage to the centrum semiovale or cerebellar white matter. Histopathologically, there are marked losses of myelinated axons (myelin, axons) and oligodendrocytes. Apoptotic nuclear changes have not been seen in oligodendrocytes with traditional stains but some appear pyknotic. Random preservation of individual myelinated axons in foci of myelin loss is common but these myelin sheaths may be thin or irregular. The advancing edges of myelin loss are sites of intense perivascular inflammation by ­lymphocytes and ­lipophages and the demyelinated white matter shows d ­ epletion of oligodendrocytes and reactive ­astrocytosis (Figures 8.27 and 8.28). The predominant lipophage has granular to vacuolated cytoplasm, which is intensely sudanophilic and PASpositive. The second type stains less intensely and usually demonstrates striated cytoplasm because of the presence of clear clefts. The number of striations generally correlates inversely with sudanophilia and PAS positivity. Striated macrophages retain birefringence after acetone extraction, although granular macrophages generally do not.85 This non-polar lipid is cholesterol esterified with saturated

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8.26 ALD. Loss of myelinated fibres in asymmetrical medullary pyramids of the same patient as in Figure 8.23. Luxol fast blue– haematoxylin and eosin.

8.27 ALD. Subacute inflammatory demyelinative lesion of juvenile adreno-leukodystrophy.

VLCFA,78 and it does not accumulate until after the phase of myelin breakdown.85,134,175 Small numbers of perivascular lymphocytes, hypertrophic astrocytes and lipophages are noted even in sclerotic lesions. The lymphocytes display both T-cell, including CD4+ and B-cell phenotypes,66 with the T-cell predominant in two studies.81,148 Plasma cells are much less frequent. Immunoglobulins identified in pathological regions were elutable at neutral pH, implying that they are not bound to a tissue component and are probably due to a disruption of the blood–brain barrier.15 Inflammatory foci are usually most intense immediately within the advancing edge, where myelin and oligodendrocytes have already been lost, axons are relatively spared and many interstitial lipophages are present. It was primarily this finding that prompted a two-stage pathogenetic theory:134,135,139,159 first, a biochemical defect in the myelin membrane leads to its breakdown (dysmyelination); second, an inflammatory immune response directed at a CNS antigen exposed during this dysmyelination causes additional and more extensive destruction of myelin (demyelination). Dysmyelinative foci (myelin pallor and

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580  Chapter 8  Peroxisomal Disorders

8.28 Inflammatory demyelinative lesion of ALD. Perivas­ cular lymphocytes and lipophages, interstitial lipophages (arrows) and reactive astrocytes are prominent in demyelinated white matter displaying a reduction in oligodendrocytes.

a few PAS-positive macrophages), analogous to, but less common than, those in AMN, can be found in otherwise normal white matter far beyond the advancing demyelinative edge. Such abnormalities can be appreciated with proton MR spectroscopy, which also indicates axonal pathology.44 It is plausible to speculate that these abnormalities correlate with those areas of biochemically affected, but grossly ‘normal’, white matter, particularly with VLCFA-phosphatidylcholine.175 Perilesional white matter contains increased VLCFA in lyso-phosphatidylcholines, which induce microglia apoptosis and macrophage recruitment from the periphery.13 CD8-positive cytotoxic lymphocytes (CTLs) are the most prevalent lymphocytes and appear to be associated intimately with interfascicular oligodendrocytes of ‘normal’ white matter, which suggests a prominent pathogenetic role for CTLs in the early myelin lesion of ALD.81 Hypertrophy, and perhaps hyperplasia, of astrocytes and macrophage infiltration are also seen just outside the advancing edge, where some splitting of myelin sheaths and oedema is observed. These astrocytes and macrophages outside and at the edge show both tumour necrosis factor α (TNF-α) and interleukin 1 (IL-1) immunoreactivities. Intercellular adhesion molecule (ICAM) and class I and II upregulation are also noted. These data generated the additional hypotheses that cytokines, particularly TNF-α, initiate the secondary phase of inflammatory demyelination, in which macrophages and T-lymphocytes are the main effector cells.148 Subsequent studies demonstrated an upregulation of TNF receptor II mRNA. The absence of the cell-mediated immunosuppressive cytokine interleukin 4 (IL-4) and the presence of γ-interferon aligned the T-helper cell response in ALD, closer to the TH1 subtype.109 Others have provided evidence for TNF-α, interleukin 12 (IL-12) and γ-interferon.102,125 Unstimulated ALD patient-derived lymphocytes have increased proinflammatory gene expression, including IL-6 and inducible nitric oxide synthase (iNOS), compared to control- or heterozygote carrier-patient derived ­ lymphocytes.168 Oxidative stress and damage in the form of an excess of iNOS and abnormal protein nitration have been identified in the inflammatory demyelination of ALD;60,152 the

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presence of the highly toxic peroxynitrite can be inferred from these latter findings. More recently, firm evidence for a TH1 response (γ-interferon and IL-12) and the participation of 4-hydroxynonenal (HNE), a toxic byproduct of lipid peroxidation that is a self-propagating element, has been provided.152 The close association of CD8+ CTLs, macrophages, HNE and myelin debris in areas outside the active demyelinative edge also suggests an early role for oxidative damage.152 The immunolabelling of astrocytes and macrophages for CD1b, CD1c and CD1d raised the possibility of lipid antigen presentation in the pathogenesis of the inflammatory demyelination and that CD1 may be a modifier gene81 (reviewed by Heinzler et al.70). However, more recent evidence mitigates against CD1 as a modifier.7 Abnormal myelin lipids and proteins, particularly gangliosides and proteolipid protein, containing saturated VLCFA, have been proposed as the cytokine trigger/immunologic target.114,134,135,141 The death of oligodendrocytes in ALD appears to be mainly lytic (not apoptotic, in contrast to adrenocortical cells) and mediated by the granule exocytosis pathway of CD8 CTLs,81 peroxynitrite and HNE.152 The wallerian-like degeneration of the corticospinal tracts exhibits equivalent losses of axons and myelin, mild hypertrophy of astrocytes and a few lipophages. In contrast to non-ALD secondary tract degeneration, ALD tract degeneration often contains foci of perivascular lymphocytes and striated lipophages.134,159 Ultrastructural examination of old, sclerotic lesions usually reveals little more than astrocytic processes filled with dense intermediate filaments. Rarely, crystals, consistent with hydroxyapatite, are seen within myelinated axons and in the extracellular space. Active lesions contain demyelinated intact axons, demyelinated degenerate axons, thinly myelinated axons, a variety of inflammatory cells and two populations of macrophages. The predominant macrophage contains myelin debris and opaque lipid droplets (cholesterol esters), whereas the other contains lipid droplets, lamellae, lipid profiles and angulate lysosomes containing spicules (Figure 8.29).134 The latter macrophage is the type that retains birefringence after acetone extraction. Beyond this active edge of demyelination, the extracellular space is enlarged; splitting and fragmentation of myelin sheaths are observed.159,172 Convincing evidence of lamellar inclusions in oligodendrocytes is rare.174 The extent and intensity of CNS white matter lesions in ALD do not correlate with clinical or pathological involvement of the adrenal cortex. A possible role for androgens in triggering the onset of ALD and AMN has been suggested.134

Neuropathology of Adrenomyeloneuropathy Typically, patients with AMN present with stiffness or clumsiness of the legs in their third or fourth decade, which progresses slowly but inexorably over the next few decades to severe spastic paraparesis.67 Patients have been divided into ‘pure’ and ‘cerebral’, the latter term indicating the presence of brain lesions.114 In patients with AMN, as well as in symptomatic female heterozygotes, the spinal cord bears the brunt of the disease (Figure 8.30).134,141,150,160 Loss of myelinated axons and a milder loss of oligodendrocytes are observed in the long

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  Group II: Disorders with Morphologically Intact Peroxisomes and a Single Protein Deficiency  581

8

8.31 AMN. More localized loss of myelinated fibres in both the gracile tracts and the postero-lateral columns in another patient with adrenomyeloneuropathy. Modified Bielschowsky. Silver impregnation.

8.29 Electron microscopy in ALD. Central nervous system macrophage in juvenile adreno-leukodystrophy, containing predominantly fine, clear lipid clefts. Autopsy.

8.32 AMN. Loss of myelinated fibres and myelin ovoids in the gracile tract of adrenomyeloneuropathy. Semi-thin longitudinal section. Toluidine blue.

8.30 AMN. Severe loss of myelinated fibres in long tracts of adrenomyeloneuropathy spinal cord, with sparing of the propriospinal fibres. Luxol fast blue–periodic acid–Schiff.

ascending and descending tracts of spinal cord, especially in the fasciculus gracilis and lateral corticospinal tracts (Figure 8.31). The pattern of fibre loss is consistent with a distal axonopathy, in that the greatest losses are observed in the lumbar corticospinal and cervical gracile and dorsal spinocerebellar tracts. Axonal loss is usually commensurate with, or greater than, myelin loss. Gangliosides containing VLCFA, present in AMN axonal membranes, are postulated to be the major pathogenic element,134,135,141,150 ­perhaps by interfering with neurotrophic factor–receptor interactions. Sudanophilia and inflammation are minimal or absent. Astrocytic gliosis, usually isomorphic, is moderate, but the predominant reactive cells are activated microglia. Some sparing of individual myelinated fibres is noted, even in severely affected tracts. Perivascular accumulations of striated and granular P ­ AS-positive lipophages are

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present, particularly in relatively preserved tracts. Spinal grey matter is unremarkable. In s­emi-thin and thin sections, the affected tracts may show segmental demyelination, myelin corrugation, axonal ovoids, axons with thin myelin sheaths and probably axonal atrophy (Figure 8.32).150,153 Macrophages with pleomorphic cytoplasmic inclusions, mainly spicules, have been visualized. The ­severity of the myelopathy does not appear to correlate with the duration of neurological symptoms, presence or duration of endocrine abnormalities or extent of supraspinal neuropathological lesions. Atrophy of neurons in the dorsal root ganglia, ­predominantly ­involving the largest and without appreciable neuronal loss, and lipidic mitochondrial inclusions have been demonstrated.151 Thus, this slowly progressive myelopathy with a late onset and perikaryal preservation should be amenable to therapeutic intervention. The involvement of cerebral and cerebellar white ­matter in AMN is variable but it is usually minimal compared with ALD.134,141,150,160 In some cases, there may be no lesions of white matter; most, if not all, probably contain at least microscopic dysmyelinative foci, measuring millimetres in diameter, of myelin pallor with relative to total ­axonal and oligodendrocytic sparing, activation of microglia, and the recruitment of PAS-positive, striated macrophages (Figure 8.33). Reactive astrocytosis and lymphocytes are

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582  Chapter 8  Peroxisomal Disorders

8.33 AMN. Dysmyelinative lesion of the cerebral white matter displaying myelin pallor and PAS-positive macrophages in adrenomyeloneuropathy. Luxol fast blue–periodic acid–Schiff.

absent or minimal. These abnormalities probably explain some of the neuropsychological deficiencies43 and spectroscopic abnormalities40 in ‘pure’ AMN. Other patients with AMN ­demonstrate confluent losses of myelin staining in the cerebrum and, to a lesser extent, the cerebellum, ­without significant inflammation and with sparing of arcuate fibres.160 Still others display inflammatory demyelinative lesions with axonal loss, qualitatively similar to ALD but much more localized. Mixtures of these lesions may ­coexist in the same patient. Involvement of the posterior limbs can result in secondary corticospinal tract degeneration in the midbrain, pons, medulla and spinal cord, accompanied by mild reactive astrocytosis and a few ­lymphocytes. The ­ concurrent finding of primary inflammatory demyelinative foci in the basis pontis can further complicate the interpretation of pyramidal tract degeneration. In AMN, however, the pyramidal tracts more commonly seem to undergo a dying-back axonopathy and pyramidal signs appear early in AMN, even ‘pure’ AMN. Thus, the corticospinal tracts in some patients with ‘cerebral’ AMN may undergo both anterograde and retrograde axonal degeneration, whereas patients with ‘pure’ AMN appear to develop only a retrograde, dying-back axonopathy. Finally, about 25 per cent of patients with AMN experience the myeloneuropathy for several decades before they develop confluent cerebral inflammatory demyelination qualitatively and usually quantitatively similar to childhood cerebral ALD (AMN/ALD); they usually die within a few years of this transition148 (reviewed by Moser et al.116). In addition to these primary dysmyelinative and inflammatory demyelinative lesions, patients with AMN also demonstrate noninflammatory, bilateral, fairly symmetrical supraspinal lesions displaying comparable losses of axons and myelin (i.e. system degenerations). These involve the medial and lateral lemnisci, brachium conjunctivum, middle and inferior cerebellar peduncles, optic system, and particularly the geniculo-calcarine tracts.134,160

Refsum’s Disease, Classic or Adult, and Atypical Refsum’s Disease Peroxisomal phytanoyl-coenzyme A hydroxylase (phytanic acid α-hydroxylase) is the enzyme deficient in most patients

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diagnosed clinically as having ARD.84 In a small subset of patients, the defect is in PEX 7, not the hydroxylase.19,183 In this subset, defects in plasmalogen synthesis and peroxisomal thiolase are also noted. In classical ARD, phytanic acid elevation is the only biochemical abnormality, as a result of deficiency in the initial α-oxidation of phytanic acid that occurs in peroxisomes; the subsequent β-oxidation of its degradative product, pristanic acid, in humans is essentially completed in the peroxisome.191 Phytanic acid is a 20-carbon branchedchain fatty acid solely of dietary origin; hence, dietary restriction can be an effective treatment, particularly for the peripheral neuropathy. The typical patient with ARD presents before 20 years of age with decreased visual acuity because of pigmentary retinopathy, peripheral neuropathy, cerebellar ataxia, sensorineural hearing deficits, cardiac problems and dry skin to ichthyosis.170 Many of these features are typical of PBD. Chronic RCDP has comparably elevated phytanic acid levels and ichthyosis but demonstrates low plasmalogens and cerebellar atrophy,149 whereas ARD has normal plasmalogens and lacks cerebellar atrophy. In view of the fact that dietary therapy is so successful, neuropathological studies are essentially restricted to those performed decades ago.23 Neuropathological abnormalities are most prominent in peripheral nerve, where onion bulbs predominate. Osmiophilic granular, granulomembranous and crystalloid bodies are seen in Schwann cell cytoplasm.46 The relationship of any of these structures to the increased phytanic acid found in peripheral nerve in this disease is unclear. Oil red O-positive lipid droplets, presumably phytanic acid,23 in the CNS have been noted in the leptomeninges, subpial glia, ependymal cells and choroid plexus epithelium. Excessive lipid is also detected in the pallidum and around cerebral and retinal blood vessels.180 Other CNS lesions include system degenerations, with marked loss of myelin (presumably myelinated fibres) in olivary and dentate nuclei and olivocerebellar fibres. Loss of myelin (presumably myelinated fibres) and gliosis also occur in the superior and middle cerebellar peduncles, pyramidal tracts and medial lemnisci. Neuronal loss is noted in the inferior olivary, dentate, cochlear, vestibular, gracile and cuneate nuclei. Thus, the CNS lesions of ARD seem to resemble those of AMN and myoclonus epilepsy with ragged-red fibres (MERRF; see Chapter 7, Mitochondrial Diseases), in that they are mainly tract degenerations but with superimposed lipid accumulations. Cerebral cortical neurons, Purkinje cells and spinal ganglion cells are also reduced in number. Degeneration and loss of anterior horn cells and ascending tract degeneration have been interpreted as secondary to the peripheral neuropathy. MRI studies are not available. The reason why myelin is the major site of disease in the PNS but not the CNS, is probably that phytanic acid concentrations in the PNS of ARD are much greater104 and PNS myelin has a higher turnover rate. A knockout mouse model has been generated, which has exhibited signs of peripheral neuropathy and ataxia, Purkinje cell loss, astrocytosis and an upregulation of calcium-binding proteins in the CNS.54 Most of the few remaining conditions listed in Table 8.1 are rare. Patients have biochemical evidence of per­ oxisomal dysfunction but lack either neuropathological data or noteworthy neurological features. Mulibrey (muscle–liver–brain–eye) nanism (Perheentupa syndrome)

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  Considerations on the Cellular Pathogenesis of Peroxisomal Disorders  583

is the most recently recognized peroxisomal disorder and manifests as severe perinatal growth retardation, dysmorphic features, pericardial constriction and hepatomegaly. A J-shaped sella, small yellow deposits in the ocular fundi, slight muscular weakness and ventriculomegaly are also seen. The disease is most common in Finnish people. Mutations have been identified in TRIM37, which encodes a RING-B-box-coiled-coil subfamily of the zinc-finger protein that is localized to peroxisomes.86 TRIM37 functions as an ubiquitin E3 ligase;87 markers of peroxisomal dysfunction in plasma and fibroblasts (e.g. excess VLCFA) are lacking. Three patients with a sensorimotor neuropathy and elevated levels of pristanic acid and C27 bile acid intermediates were found to have a deficiency of α-methylacylCoA racemase, which is responsible for the conversion of pristanoyl-CoA and the C27 bile acyl-CoAs to their (S)-stereoisomers.51 A relapsing encephalopathy with seizures and cognitive decline has also been reported in three adults.177

in ALD. Dietary restriction and treatment with glyceryl trierucate/glyceryl trioleate (Lorenzo’s oil), in spite of the publicity and its ability to reduce plasma VLCFA, has not had a statistically significant effect on the progression of AMN or ALD187 but may have helped to delay the onset of cerebral disease in asymptomatic boys with normal MRI findings.117 Modulation of microsomal elongases (ELOVL1, ELOVL6) have the potential to reduce VLCFA levels.13,122 In view of the mitochondrial defects and presence of reactive species and oxidative damage in human ALD and mouse ABCD1 –/– tissues, antioxidant (e.g. N-acetylcysteine) and mitochondrial-enhancing therapies are under consideration.14 Riboflavin appeared to have a favourable biochemical effect on the first patient reported with glutaric aciduria, type III.11 Dietary restriction has had a profound impact on ARD. Phytanic acid restriction also had a positive clinical effect on an ataxic patient with di- and tri-hydroxycholestanaemia27 and another ataxic patient with increased plasma levels of phytanic, pristanic and C27 bile acids.28

Group III: Others

Considerations on the Cellular Pathogenesis of Peroxisomal Disorders

An archival case of orthochromatic leukodystrophy with epithelioid cells of Norman–Gullotta (case 1) had elevated VLCFA and equivocally elevated phytanic acid as well as ‘typical lamellar inclusions’.112 One ataxic patient had increased concentrations of pristanic acid, phytanic acid and C27 bile acids;28 two other patients had a peripheral neuropathy, one of whom was also ataxic, and they demonstrated panperoxisomal dysfunction.105 Autosomal recessive cerebellar ataxia is rarely associated with mild PBDs, as recently identified in patients with mild PEX2 gene mutations.110,167 Another patient with a neuromuscular disorder resembling Werdnig–Hoffmann disease also had a panperoxisomal defect.10 Finally, patients with the typical, but milder, biochemical abnormalities of ZS may present with pigmentary degeneration of the retina and sensorineural hearing loss alone.65

Specific Treatment of Peroxisomal Disorders Docosahexaenoic acid has been used in the treatment of PBD with reported success;108,120 however, a doubleblind trial did not show efficacy.126 Small molecules with chaperone-like properties have enhanced residual protein activity in cells from PBD patients with intermediate and milder phenotypes;12,205 this includes betaine, which is currently in clinical trials for patients with Pex1-G843D mutations (ClinicalTrials.gov Identifier: NCT01838941). Ether lipid precursors will rescue plasmalogen levels and organ pathology in Pex7 knockout mice and RCDP patient cells,20,201 but efficacy in RCDP patients remains to be determined. Patients with ALD and AMN who are addisonian need glucocorticoid replacement therapy. Bone marrow transplantation in patients with ALD with mild neurological symptomatology has been its most effective therapy thus far.128 Gene therapy is becoming a realistic possibility.24 Immunomodulatory and immunosuppressive drugs have failed to prevent progression of cerebral neuroinflammation

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8

Much has been learned about the morphological, biochemical, cellular and molecular intricacies of peroxisomes and their disorders. However, our insights on how these metabolic defects cause the various degenerative brain phenotypes remains limited. Based on biochemical/neuropathological correlative studies of human patients, primarily post-mortem, saturated VLCFA have been implicated as major pathogenic culprits in most peroxisomal disorders, particularly ALD and AMN. We now know the physiological role of ABCD1 and that ABCD1 deficiency leads to a failure in the import of VLCFA-CoA into the peroxisome and pathogenic elevations in saturated VLCFA. The relative insolubility of VLCFA at normal body temperature and their incorporation into membrane and myelin constituents, perhaps in particular phosphatidylcholine in ‘normal’ white matter,175 have an adverse impact on the fluidity of these membranes by increasing their viscosity.72,142 This is thought to lead to myelin instability with dysmyelination and, especially in ALD, to inflammatory (immune) demyelination.81,148 Their incorporation into axonal membranes, particularly as gangliosides, may interfere with normal receptor–neurotrophic factor interactions, resulting in perikaryal and axonal atrophy with axonal loss in AMN.150,151 Mouse models have provided considerable insight into pathogenetic mechanisms in peroxisomal disorders. Several ZS knockouts (KOs) have been generated, including of PEX2, PEX5 and PEX13; all display abnormal neocortical neuronal migrations and early neonatal lethality (reviewed in Baes4,5). ZS mice have mitochondrial abnormalities with evidence of oxidative stress.9 In addition, defective hepatic and CNS peroxisomes both contribute to neuronal migration defects,98 and systemic metabolic abnormalities, such as elevations of bile acid precursors, may impact on neuronal migration.49,50 Deletion of PEX5 in all neural cell types by Nestin-Cre causes cerebellar hypomyelination followed by diffuse demyelination, axon loss and neuroinflammation

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throughout the brain. Defective peroxisomal β-oxidation alone does not disturb neocortical migration in mice; however, D-BP KO mice do develop defects similar to NESPEX5–/–,189 but less severe and later in onset, suggesting that additional factors, possibly a lack of plasmalogens, contribute to the early phenotype of peroxisome deficiency in brain.5 Plasmalogen deficiency in DAPAT KO leads to defects in myelination, paranode organization and cerebellar Purkinje cell innervation; mice with combined β-oxidation and plasmalogen deficiencies show more severe phenotypes, further suggesting a synergistic effect of these metabolic defects.5,31,129 Pex1-G844D knock-in mouse recapitulates many classic features of mild PBD cases, providing a model for Pex1-G843D patients, with longer postnatal survival, retinopathy with cone photoreceptor degeneration, and normalization of peroxisomal β-oxidation in mutant fibroblasts with chaperone-like compounds.71 Three laboratories have generated KO mice lacking ABCD1; none of these has developed the cerebral inflammatory demyelination or the oxidative damage152 typical of human ALD. Rather, spinal axonal degeneration, more reminiscent of AMN, occurs, particularly if ABCD2 is also knocked out.154 We have demonstrated mitochondrial abnormalities in the ABCD1 KO and found that the rate of peroxisomal VLCFA β-oxidation is related directly to the rate of mitochondrial long-chain fatty acid oxidation (reviewed by Heinzer et al.70). However, it has confirmed our belief that the most fundamental CNS abnormality is axonal and probably in cell membranes,139 both of myelin sheaths and axons. Recent studies using the ABCD1 KO have strongly supported the pathogenic role of saturated VLCFA and a prominent and early role for oxidative damage in the pathogenesis of ALD and AMN (reviewed in Galea56). Perhaps most importantly, it has provided another link to a concomitant mitochondrial abnormality in ALD and AMN,55 which was first demonstrated in adrenocortical cells in the 1970s.139,142 Our data also suggest that an alternative possibility – increased microsomal elongation122 – is an additional explanation for the elevations in VLCFA. Evidence for primary oligodendrocyte dysfunction in ALD pathogenesis is provided by mice with CNPaseCre-directed, oligodendrocyte specific deletion of PEX5.89 Similar to human X-ALD, these CNP-PEX5–/– mutants develop progressive symmetric subcortical demyelination, severe axonal loss, VLCFA accumulation and neuroinflammation, including infiltrating CD8+ T-cells and cytokine production. It has been postulated that ABCD1-deficient peroxisomes accumulate secondary peroxisomal changes over time that ignite the inflammatory response in brain; for

instance, a concomitant depletion of plasmalogens is noted in human X-ALD demyelinated brain tissue.31 Peroxisomes in oligodendrocytes are found concentrated in paranodal loops, which are sites of axon–glial interaction that may provide trophic support to axons independent of myelin. CNP-PEX5−/− mice also develop peripheral neuropathy, associated with vesicle filled swellings at the paranodes.90 A similar mechanism has been proposed for phytanic acid in the PNS myelin of ARD. This branched-chain fatty acid, with a ‘thorny’ configuration, may take the place of normal straight-chain fatty acids and also cause a membrane (myelin) instability.104,170 In the cerebellar atrophy of chronic RCDP, and perhaps IRD, the tissue plasmalogen deficiency is viewed as a possible contributing factor.149 Toxicity of phytanic acid and pristanic acid is mediated by multiple mitochondrial dysfunctions, generation of reactive oxygen species and dysregulation of intracellular Ca2+ signalling pathways in glial cells.97 The importance of DHA to retinal photoreceptor cells suggests a pathogenetic role for its deficiency in the atypical pigmentary retinopathy of PBD. Thus, there is considerable evidence that abnormal fatty acids accumulating in peroxisomal disorders, particularly saturated VLCFA and phytanic acid, are incorporated into cell membranes (including myelin and axons) and perturb their micro-environments. This would lead to dysmyelination and dysfunction, atrophy and death of vulnerable cells. Deficiencies in tissue plasmalogens and DHA may contribute to their vulnerability in PBD. However, whether these peroxisomal biochemical accumulations and/or deficiencies can fully explain the various tissue pathologies is still debated, and information on how peroxisome deficiencies may affect intracellular signalling pathways, including disturbance to membrane-raft domains and other proteinlipid modifications, awaits further research. Finally, there is increasing recognition of pathogenic roles for oxidative stress/damage and concomitant mitochondrial dysfunction in both groups of peroxisomal disorders.

Dedication We dedicate this chapter to our friend and collaborator for three decades, Hugo W. Moser, MD, who passed away on 20 January 2007. His commitment to peroxisomal diseases, particularly ALD, and his establishing of the Peroxisomal Disease Center in the Kennedy Krieger Institute, enabled the study of these perplexing diseases and provided much of the pathological material displayed in this chapter.

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588  Chapter 8  Peroxisomal Disorders 158. Sarnat H, Machin G, Darwish H, Rubin S. Mitochondrial myopathy of cerebrohepato-renal (Zellweger) syndrome. Can J Neurol Sci 1983;10:170–77. 159. Schaumburg HH, Powers JM, Raine CS, et al. Adreno-leukodystrophy: a clinical and pathological study of 17 cases. Arch Neurol 1975;33:577–91. 160. Schaumburg HH, Powers JM, Raine CS, et al. Adrenomyeloneuropathy: a probable variant of adreno-leukodystrophy. II: general pathologic, neuropathologic and biochemical aspects. Neurology 1977;27:1114–19. 161. Schiffmann R, van der Knaap MS. Invited article: an MRI-based approach to the diagnosis of white matter disorders. Neurology 2009;72(8):750–9. 162. Schrader M, Fahimi HD. Peroxisomes and oxidative stress. Biochim Biophys Acta 2006;1763:1755–66. 163. Schrader M, Bonekamp NA, Islinger M. Fission and proliferation of peroxisomes. Biochim Biophys Acta 2012;1822:1343–57. 164. Schram AW, Goldfischer S, van Roermund CWT, et al. Human peroxisomal 3-oxoacyl-coenzyme A thiolase deficiency. Proc Natl Acad Sci U S A 1987;84: 2494–6. 165. Schruder JM, Hackel V, Wanders RJA, et al. Optico-cochleo-dentate degeneration associated with severe peripheral neuropathy and caused by peroxisomal d-bifunctional protein deficiency. Acta Neuropathol 2004;108:154–67. 166. Scotto JM, Hadchouel M, Odievre M, et al. Infantile phytanic acid storage disease, a possible variant of Refsum’s disease: three cases, including ultrastructural studies of the liver. J Inherit Metabol Dis 1982;5:83–90. 167. Sevin C, Ferdinandusse S, Waterham HR, Wanders RJ, Aubourg P. Autosomal recessive cerebellar ataxia caused by mutations in the PEX2 gene. Orphanet J Rare Dis 2011;6:8. 168. Singh J, Giri S. Loss of AMP-activated protein kinase in X-linked adrenoleukodystrophy patient-derived fibroblasts and lymphocytes. Biochem Biophys Res Commun 2014;445:126–31. 169. Spranger JW, Opitz JM, Bidder U. Heterogeneity of chondrodysplasia punctata. Humangenetik 1971;11:190–212. 170. Steinberg D. Refsum disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D eds. The metabolic and molecular bases of inherited disease, Vol. II. New York: McGraw–Hill, 1995:2351–69. 171. Steinberg S, Chen L, Wei L, et al. The PEX gene screen:molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum. Molec Genet Metab 2004;83:252–63. 172. Suzuki K, Grover WD. Ultrastructural and biochemical studies of Schilder’s disease: I. Ultrastructure. J Neuropathol Exp Neurol 1970;29:392–404. 173. Suzuki Y, Shimozawa N, Orii T, et al. Zellweger-like syndrome with detectable hepatic peroxisomes: a variant form of peroxisomal disorder. J Paediatr 1988;113:841–5. 174. Takeda S, Ohama E, Ikuta F. Adrenoleukodystrophy: early ultrastructural

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changes in the brain. Acta Neuropathol 1989;78:124–30. 175. Theda C, Moser AB, Powers JM, Moser HW. Phospholipids in X-linked adrenoleukodystrophy white matter-fatty acid abnormalities before the onset of demyelination. J Neurol Sci 1992;110: 195–204. 176. Theda C, Gibbons K, Defor TE, et al. Newborn screening for X-linked adrenoleukodystrophy: further evidence high throughput screening is feasible. Mol Genet Metab 2014;111:55–7. 177. Thompson SA, Calvin J, Hogg S, et al. Relapsing encephalopathy in a patient with α-methylacyl-CoA racemase deficiency. BMJ Case Rep 2009;pii: bcr08.2008.0814. 178. Thoms S, Grostrokennborg S, Gärtner J. Organelle interplay in peroxisomal disorders. Trends Mol Med 2009;15: 293–302. 179. Torvik A, Torp S, Kase BF, et al. Infantile Refsum’s disease: a generalized peroxisomal disorder–case report with postmortem examination. J Neurol Sci 1988;85:39–53. 180. Toussaint D, Danis P. An ocular pathologic study of Refsum’s syndrome. Am J Ophthalmol 1971;72:342–7. 181. Tranchant C, Aubourg P, Mohr M, et al. A new peroxisomal disease with phytanic and pipecolic acid oxidation. Neurology 1993;43:2044–8. 182. Ulrich J, Herschkowitz N, Hertz P, et al. Adreno-leukodystrophy: preliminary report of a connatal case–light and electron microscopical, immunohistochemical and biochemical findings. Acta Neuropathol 1978;43:77–83. 183. Van den Brink DM, Brites P, Haasjes J, et al. Identification of PEX 7 as the second gene involved in Refsum disease. Am J Hum Genet 2003;72:471–7. 184. Van der Knaap MS, Valk J eds. Magnetic resonance of myelination and myelin disorders, 3rd edn. Berlin: Springer– Verlag, 2005. 185. van der Knaap MS, Wassmer E, Wolf NI, et al. MRI as diagnostic tool in earlyonset peroxisomal disorders. Neurology 2012;78:1304–8. 186. van der Zand A, Braakman I, Tabak HF. Peroxisomal membrane proteins insert into the endoplasmic reticulum. Mol Biol Cell 2010;21(12):2057–65. 187. Van Geel BM, Assies J, Haverkort EB, et al. Progression of abnormalities in adrenomyeloneuropathy and neurologically asymptomatic X-linked adreno-leukodystrophy despite treatment with ‘Lorenzo’s oil’. J Neurol Neurosurg Psychiatry 1999;67:290–99. 188. Van Grunsven EG, van Berkel E, Mooijer PAW, et al. Peroxisomal bifunctional protein deficiency revisited: resolution of its true enzymatic and molecular basis. Am J Hum Genet 1999;64:99–107. 189. Verheijden S, Beckers L, De Munter S, Van Veldhoven PP, Baes M. Central nervous system pathology in MFP2 deficiency: Insights from general and conditional knockout mouse models. Biochimie 2014;98:119–26. 190. Volpe JJ, Adams RD. Cerebro-hepatorenal syndrome of Zellweger: an inherited

disorder of neuronal migration. Acta Neuropathol 1972;20:175–98. 191. Wanders RJA. Metabolic and molecular basis of peroxisomal disorders: a review. Am J Med Genet 2004;126A:355–75. 192. Wanders RJ, Waterham HR. Biochemistry of mammalian peroxisomes revisited. Annu Rev Biochem 2006;75:295–332. 193. Wanders RJA, Barth PG, Heymans HSA. Single peroxisomal enzyme deficiencies. In: Scriver CR, Beaudet AL, Sly WS, Valle D eds. The metabolic and molecular bases of inherited disease, 8th edn. New York: McGraw–Hill, 2001:3219–48. 194. Wanders RJ, Ferdinandusse S, Brites P, Kemp S. Peroxisomes, lipid metabolism and lipotoxicity. Biochim Biophys Acta 2010;1801:272–80. 195. Wang R, Monuki E, Powers J, et al. Effects of hematopoietic stem cell transplantation on acyl-CoA oxidase deficiency: a sibling comparison study. J Inherit Metab Dis 2014;[Epub ahead of print]. 196. Waterham HR, Koster J, van Roermund CW, et al. A lethal defect of mitochondrial and peroxisomal fission. N Engl J Med 2007;356:1736–41. 197. Watkins PA, Chen WW, Harris CJ, et al. Peroxisomal bifunctional enzyme deficiency. J Clin Invest 1989;83:771–7. 198. Watkins PA, McGuinness MC, Raymond GV, et al. Distinction between peroxisomal bifunctional enzyme and acyl-CoA oxidase deficiencies. Ann Neurol 1995;38:472–7. 199. White AL, Modaff P, Holland–Morris F, Pauli RM. Natural history of rhizomelic chondrodysplasia punctata. Am J Med Genet 2003;118A:332–42. 200. Wisniewski T, Powers J, Moser A, Moser H. Ultrastructural evidence for a gliopathy in cerebro-hepato-renal (Zellweger) syndrome. J Neuropathol Exp Neurol 1989;48:366. 201. Wood PL, Khan MA, Smith T, et al. In vitroin vivo plasmalogen replacement evaluations in rhizomelic chrondrodysplasia punctata and Pelizaeus– Merzbacher disease using PPI-1011, an ether lipid plasmalogen precursor. Lipids Health Dis 2011;10:182. 202. Yik WY, Steinberg SJ, Moser AB, Moser HW, et al. Identification of novel mutations and sequence variation in the Zellweger syndrome spectrum of peroxisome biogenesis disorders. Hum Mutat 2009;30: E467–80. 203. Zellweger H. The cerebro-hepato-renal (Zellweger) syndrome and other peroxisomal disorders. Dev Med Child Neurol 1987;29: 821–9. 204. Zhang J, Kim J, Alexander A, et al. A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS. Nat Cell Biol 2013;15:1186–96. 205. Zhang R, Chen L, Jiralerspong S et al. Recovery of PEX1-Gly843Asp peroxisome dysfunction by small-molecule compounds. Proc Natl Acad Sci U S A 2010;107: 5569–74.

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Nutritional and Toxic Diseases Jillian Kril, Leila Chimelli, Christopher M Morris and John B Harris

Introduction................................................................................589 Malnutrition................................................................................589 Alcohol Intoxication.....................................................................594 Disorders of the Hypothalamic-Pituitary Axis...............................597 Metabolic Disorders....................................................................602

Introduction The clinical and pathological sequelae of nutrition deficiencies depend on the type and severity of the deficiency and the developmental stage at which the deficiency is experienced. The developing central nervous system is most vulnerable during periods of rapid growth, whereas, in many instances, the adult nervous system is less vulnerable because of the body’s propensity to protect the physiological demands of the brain. Nutritional deficiencies can result from either intrinsic or extrinsic factors. Undernutrition occurs when total protein-calorie intake is inadequate. Social and environmental reasons, as well as chronic disease, can result in inadequate dietary intake or impaired absorption. In developed countries, patients with malignancy or HIV-AIDS are commonly susceptible to undernutrition, whereas in developing countries external factors are often responsible for undernutrition. Malnutrition occurs where there is an imbalance of nutrients. This may be a highly specific deficiency of a trace element, amino acid or vitamin, or a more global deficiency such as protein malnutrition in the case of kwashiorkor (see later). Malnutrition can result from lifestyle factors such as vegetarianism or from a wide variety of medical and psychiatric conditions. Patients with gastroenterological disease or who have undergone gastroenterological surgery are particularly vulnerable to malnutrition because of impaired absorption of nutrients. Malnutrition can also occur in subjects with increased utilization of nutrients and in patients with chronic liver disease because the liver acts as a store for a number of nutrients, especially vitamins.

Malnutrition Maternal nutritional status can affect the development of a number of organ systems in the infant, including the brain. There is also an expanding body of literature linking maternal

Neurotoxicology..........................................................................609 Motor Nerve Terminal.................................................................624 Skeletal Muscle..........................................................................624 Acknowledgements....................................................................625 References.................................................................................626

undernutrition and later life risk of disease, especially cardiovascular disease and diabetes. Severe maternal undernutrition, especially in the last trimester,132 results in infants with lower birth weight and smaller circumference of head and abdomen whereas mild or moderate maternal undernutrition, or differences in maternal nutrient intake, appears to have little effect on brain size.307 Cognitive performance measured at school age of low birth weight infants has been shown to be comparable with that of normal weight infants,95,144 suggesting no permanent abnormality in these children. Postnatal nutritional disorders are seen in the childhood diseases of marasmus, as a result of a severe reduction in protein and energy intake and kwashiorkor, due to insufficient protein intake accompanied by adequate energy intake. Although these disorders are considered ends of a spectrum of nutritional insufficiency, in many instances there is considerable overlap (marasmic kwashiorkor) and they are collectively referred to as protein-energy malnutrition (PEM). It is difficult to assess the nervous system consequences of PEM as such markedly abnormal nutritional states are almost invariably accompanied by deficiencies of essential amino acids, vitamins and minerals. Furthermore, the functional consequences of childhood nutritional deficiency are often not easily separated from other environmental factors such as socio-economic status, medical care and psychomotor stimulation.190 The neurological features of PEM are alterations in mentation (irritability and apathy) and, during the recovery period, a myelopathy.542 Reduced brain weight59 and neuroimaging evidence of cerebral atrophy and ventricular dilatation11,388 relative to age-matched controls have been reported in up to 75 per cent of PEM cases. The effects of PEM are most marked when they occur in the first 2 years of life because this is a period of maximum brain growth.132 Complete reversal of the atrophy is seen following nutritional rehabilitation.11 Neuropathologically, the consensus from human and experimental animal studies is that postnatal undernutrition leads to impaired myelin development. Reduced 589

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myelination in infants with PEM has been shown using magnetic resonance imaging (MRI)313 and lipid profiling.337 In addition, the ratio of myelinated to unmyelinated axons is reduced in the corpus callosum and pyramidal tracts.570 Other neuropathological changes in experimental animals following undernutrition are reviewed by Bedi.36 Undernutrition in the adult results from a variety of causes including famine, self-imposed starvation, cachexia accompanying chronic disease, anorexia nervosa and other eating disorders. Such conditions may result in one or more of the specific deficiencies described later or in a generalized brain atrophy that is largely reversible on nutritional rehabilitation.

Thiamine Deficiency Thiamine (vitamin B1) is integral to maintaining adequate cerebral energy supplies through its involvement in brain glucose metabolism. In its phosphorylated form thiamine is used as a cofactor by three major enzymes: α-ketoglutarate dehydrogenase, pyruvate dehydrogenase complex and transketolase.67 In addition, thiamine has non-cofactor roles in nerve conduction and membrane transport.230 The daily requirement for thiamine has been estimated at 1.0–1.5 mg162 and as uptake and turnover rates are comparable,67 thiamine deficiency can occur rapidly if body stores are depleted. In the human, thiamine deficiency results in beriberi or Wernicke’s encephalopathy (WE). Beriberi is most often seen in Asian populations where it has both cardiac and peripheral nervous system manifestations. Central nervous system involvement is rare.293 Clinically, beriberi manifests as a sensorimotor polyneuropathy of weakness and dysaesthesia. Pathologically, there is axonal degeneration particularly of the large myelinated fibres and secondary segmental demyelination.392 WE is commonly associated with alcoholism because chronic alcohol ingestion results in increased thiamine utilization, reduced gastrointestinal uptake and impaired phosphorylation of thiamine to the phosphorylated form.73 However, WE has also been reported in an array of conditions that affect nutrition including prolonged intravenous feeding,159 malignancies, infections, AIDS,72 starvation and hyperemesis gravidarum.98 Although WE is usually reported in adults, nine cases of WE were reported in infants following feeding with a formula devoid of thiamine.150 In recent years, the increasing frequency of bariatric procedures for the treatment of obesity has seen an increase in the n ­ umber of cases of WE. Aasheim1 reviewed 100 cases of WE identified following bariatric surgery and found that persistent vomiting, glucose loading and parenteral feeding are risk factors for developing WE in these patients. In another study of 318 patients having undergone gastric by-pass surgery, 18.6 per cent had thiamine deficiency 1 year after surgery,107 indicating impaired thiamine status is common in this population. Impaired thiamine status is also reported in patients with heart failure571 and diabetes527 suggesting risk of WE is also high in non-alcoholic populations. Clinically, WE is characterized by the ‘classical triad’ of ocular motor abnormalities, cerebellar dysfunction and altered mental state, which might manifest as disorientation, confusion or even coma.211,554 However, it is well recognized that the pathology of WE may develop without recorded

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evidence of clinical signs and that the majority of patients with WE do not have all three triad signs.211 Operational criteria with a high degree of specificity and sensitivity have been developed for the identification of WE.78 These criteria require the presence of at least two of four signs: ocular motor abnormalities, cerebellar dysfunction, altered mental state or mild memory impairment, evidence of dietary deficiency defined as impaired thiamine status on laboratory measures, a BMI 100 mg/day) can result in liver damage and skin irritation. Macroscopically, the brain in pellagra is normal. Microscopically, there is chromatolysis of neurons characterized by eccentrically placed nuclei and loss of Nissl substance (Figure 9.7). Chromatolysis is most frequently found in the pontine nuclei and dentate of the cerebellum, although it is also seen in other brain stem nuclei and posterior horn cells.221 In nutritionally derived pellagra, the cerebral cortex is rarely affected, whereas in isoniazidinduced pellagra the cerebral cortex, and in particular Betz cells, were affected in all cases in addition to the pontine nuclei.250 The polyneuropathy found in patients with niacin deficiency has been shown to be due to demyelination, but in longstanding cases axon degeneration also occurs.565

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9.6 Vitamin B12 deficiency. (a) Thoracic spinal cord, showing spongy vacuolation in the posterior and lateral white matter columns, typical of combined subacute degeneration of the cord. Loyez myelin stain. (b) Acquired immunodeficiency syndrome (AIDS) myelopathy: note the confluent vacuolation in the posterior and lateral columns and milder vacuolation in the anterior columns. The appearances are similar to those in (a). Reproduced with permission from Petito CK, Navia BA, Cho ES, et al. Vacuolar myelopathy pathologically resembling subacute combined degeneration in patients with the acquired immunodeficiency syndrome. N Engl J Med 1985;312:874–9. Copyright 1985 Massachusetts Medical Society.

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9.7 Chromatolytic neuron in the basis pontis in pellagra. The nucleus is eccentric and there is margination of Nissl substance. Scale bar, 20 μm. Slide courtesy of Dr Michael Rodriguez.

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Folic Acid Folate (vitamin B9) as its tetrahydrofolate derivatives is necessary for three important metabolic processes; the synthesis of purine nucleotides; the conversion of homocysteine to methionine in conjunction with vitamin B12; and the synthesis of deoxythmidylate monophosphate which is required for DNA synthesis. Deficiency of folate during pregnancy has been associated with an increased incidence of neural tube defects447 and supplementation has been shown to reduce the risk of birth defects.432 Some medications with folate antagonist activity, such as antiepileptics, have also been associated with neural tube defects whereas others have not,343 indicating specificity of action. In rare cases, congenitally impaired folate transport across the intestine and blood–brain barrier has been described in young children resulting in a progressive neurological disorder.440 The role of folate deficiency in CNS disease in the adult is controversial. Low serum folate levels are relatively common, especially in the elderly, alcoholics and those with gastroenterological disease. A wide variety of symptoms, including muscle weakness and depression, have been attributed to folate deficiency with little consensus. Nevertheless, Parry405 did describe 20 patients with neurological abnormalities in which folate deficiency was demonstrated in the absence of B12 deficiency and in which symptoms improved with folate supplementation. Ten showed a peripheral neuropathy, eight had SCD of the cord and two had a myelopathy. Folate-responsive changes in mental function were also described in nine patients. The author suggested that each of these neurological deficits could be accounted for by disruption of folate-dependent methionine synthase. The link between folate deficiency, elevated serum homocysteine and vascular disease, depression and dementia has prompted renewed interest in the neurological sequelae of folate deficiency.

Alcohol Intoxication Alcohol-Related Brain Damage The neurological consequences of alcohol abuse are many and varied.123 There are primary CNS effects of alcohol, such as intoxication and chronic toxicity, and secondary effects from medical and lifestyle factors. Many alcoholics are poorly nourished, have coexisting medical conditions, suffer from additional psychiatric conditions or are at greater risk of infections or trauma. Consequently, the neuropathological examination of the brain of an alcoholic patient may reveal a great deal of pathology, and distinguishing the direct effects of alcohol from these other factors may be difficult. In broad terms, the neuropathology can be divided into that which occurs in alcoholics with either nutritional deficiencies (e.g. WKS, pellagra) or liver disease (viz. hepatic encephalopathy) and that which occurs in alcoholics without other disease (alcohol-related brain damage, ARBD).292 Macroscopically, the brain of an ARBD patient shows atrophy. Harper and Blumbergs208 showed a 70-g reduction in mean brain weight between alcoholics and controls. This has been confirmed in a number of other studies209,498 and by a comparison of brain volume and intracranial cavity

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volume.210 In non-alcoholic subjects, the mean pericerebral space is 8.3 per cent, whereas in ARBD, it is 11.3 per cent and 14.7 per cent in alcoholics with WKS. Quantitative morphometry has shown the loss of brain tissue is largely due to a decrease in white matter volume,122,210,297 a finding that has been confirmed using volumetric MRI.418 The mean white matter reduction is 14 per cent,210 although this is most marked in alcoholics with WKS and less severe in ARBD where it is largely restricted to the frontal lobe.297 This may, however, represent a dose phenomenon rather than an effect of thiamine deficiency as white matter volume is negatively correlated with maximum daily alcohol consumption.297 MRI studies have also found a decrease in cortical volume in alcoholics,258,418 especially in the frontal lobes.420 The magnitude of atrophy is greater in older alcoholics than young alcoholics, despite similar drinking histories suggesting increased susceptibility of the ageing brain.418,419 Abstinence from alcohol results in, at least partial, resolution of atrophic changes,477,532 yet this capacity declines with advancing age.294 Neuronal loss in alcoholics occurs in discrete anatomical regions. In the cerebral cortex, the loss is restricted to the superior frontal cortex (BA8),297 however the magnitude of loss (mean 23 per cent) is less than that which can be reliably identified using routine neuropathological examination. Neuronal loss does not occur from the primary motor, anterior cingulate or temporal cortices.295,297 There is atrophy, but not neuronal loss in the hippocampus, which has been shown to relate to whether or not the patient was still drinking at the time of death.205 In subcortical regions, there is neuronal loss from the supraoptic and paraventricular nuclei of the hypothalamus, but not from the mammillary bodies,204 anterior principal or mediodorsal nuclei of the thalamus,204 serotonergic dorsal raphe,26 locus ceruleus,197 basal forebrain115 or cerebellum.27 Neuronal loss from the hypothalamus is related to maximum daily alcohol consumption.206 The reasons for this anatomical specificity of susceptibility to alcohol toxicity are unclear. However, regional variations in the subunit composition of amino acid neurotransmitter receptors has been hypothesized as one potential mechanism.134 The clinical manifestations of alcohol abuse are predominantly disorders of the frontal lobes reflecting the greater damage in this region.585 Executive functions such as planning ability, self-regulation, goal setting and working memory have all been shown to be impaired in alcoholics.31,172 Cerebellar ataxia and incoordination are common in cases with cerebellar atrophy.554

Central Pontine Myelinolysis and Marchiafava–Bignami Disease In rare instances, chronic alcoholics can also develop other neurological conditions. Hepatic encephalopathy (see section later), central pontine myelinolysis (CPM) and Marchiafava–Bignami disease (MBD) are examples of such conditions. CPM is due to the rapid correction of electrolyte imbalances, especially chronic hyponatraemia,336 but has been reported with other electrolyte imbalances such as hypophosphataemia149 leading to the description of the osmotic demyelination syndromes.336 CPM is usually seen

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in patients who are markedly malnourished or debilitated such as alcoholics, but has also been reported in patients with advanced liver disease, following liver transplantation, and in those with HIV-AIDS or severe burns. Limiting the rate of correction of chronic hyponatraemia to ≤8 mmol/L per day significantly reduces the occurrence of CPM. Clinical manifestations depend on the severity of the lesion. MRI studies have identified cases with early disease and complete recovery. More severe cases result in a biphasic course where initially there are encephalopathy ­ and seizures from hyponatraemia and then, following correction, deterioration occurs again several days later.336 In the second phase, there are quadriparesis, dysarthria and dysphagia when lesions are restricted to the basis pontis, and additional oculomotor abnormalities if the tegmentum is also involved. When lesions are extensive a ‘locked-in’ syndrome may ensue.336 Pathologically, CPM is characterized by demyelinating lesions in the midline of the basis pontis. These are symmetrical, triangular or ‘butterfly’-shaped lesions that appear as pallor on myelin-stained preparations (Figure 9.8a). Microscopically, there is loss of myelin with relative preservation of axons in early lesions (Figure 9.8b), but more long-standing lesions also show axonal degeneration and the presence of foamy macrophages. Extrapontine lesions were recognized many decades ago and have been described in up to 50 per cent of cases of CPM in the cerebellum, lateral geniculate body, external capsule, hippocampus, putamen and cerebral cortex.175 Microscopically, these lesions are similar to those in the pons. MBD is extremely rare and is mostly described in reports of isolated cases. In recent years, the greater availability of (a)

MRI and the utility of this technique in identifying discrete alterations in brain signal have meant a larger number of cases have been identified, but few of these have been examined pathologically. Lesions in the corpus callosum can be identified as hyperintensities on T2-weighted, FLAIR and diffusion-weighted sequences. In a study of the progression of radiological changes in MBD, areas of hyperintensity were seen to persist for 4 months, but to be largely resolved by 11 months.166 Classical descriptions of MBD describe three patterns of clinical symptoms: acute MBD, which is a severe neurological disorder of altered consciousness and seizures that rapidly progresses to death; subacute MBD, in which there are interhemispheric disconnection, abnormalities of higher cognitive function and gait disturbance; and a chronic form characterized by progressive dementia.166 However, this classification has recently been challenged. In the largest review of MBD to date, Heinrich and colleagues226 collated data from 50 cases arising from 41 publications. They found a predominance in males (M:F, 3.2:1) and a mean age of 46.6 years (range, 26–66). They also found two distinct subtypes using clinical and radiological parameters. Type A occurred in 19 cases (38 per cent) and was characterized by a major disturbance of consciousness (stupor or coma) and extensive lesions on MRI. These cases were more likely to die or to have a major disability.226 Type B occurred in 29 (58 per cent) cases, had no or minor impairment of consciousness, partial callosal lesions on MRI and minimal impairment.226 Pathologically, the brain in MBD may be normal or may exhibit thinning and discolouration or cavitation of the ­corpus callosum. Microscopically, there is a spectrum of severity from demyelination with preservation of axons to necrosis and cystic cavitation. There is a loss of oligodendrocytes, upregulation of astrocytes and foamy macrophages. Rarely, there is involvement of other white matter tracts such as the centrum semiovale, cerebral peduncles or optic chiasm. The aetiology of MBD is unknown. It was originally described in Italian red wine drinkers and attributed to an unidentified toxin in the wine, yet subsequent cases in

9

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9.8 (a) Central pontine myelinolysis, showing the symmetrical, triangular region of myelin loss. (b) Higher magnification view, showing the loss of myelin within the region of myelin loss (towards the right), but preservation of neurons. Myelin in the adjacent part of the pons (towards the left) appears normal. Luxol fast blue and cresyl violet. Slide courtesy of Dr Michael Rodriguez.

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people who drink other types of alcoholic beverage have discounted this hypothesis. Clinical improvement following treatment with vitamin supplements166 has led to the current theory that MBD is due to vitamin deficiency.

Hepatic Encephalopathy Hepatic encephalopathy (HE) is a syndrome of neurological dysfunction that occurs in patients with acute or chronic liver disease. There are acute and chronic forms of HE depending on the time course and severity of the liver disease. The clinical pattern and pathology of HE is independent of the aetiology of the liver disease. HE associated with acute liver failure (HE type A154) is a disorder of rapid onset that occurs in the setting of fulminant liver failure, such as with paracetamol overdose. In the early stages, patients exhibit an increase in muscle tone and altered mental state that progresses to stupor and coma. Death is common secondary to raised intracranial pressure from cerebral oedema. Neuropathologically, there is brain swelling with a decrease in ventricular volume, flattening of gyri and herniation. Microscopically, oedema is evident and ultrastructural studies have revealed that this is due to swelling of perivascular astrocytes.270 Traditionally, chronic HE, often seen following chronic alcoholism, was often referred to a portal-systemic encephalopathy because it commonly occurs as a result of the shunting of portal blood into the systemic circulation.69 This could happen either spontaneously as a result of portal hypertension in patients with cirrhosis or following surgery to alleviate variceal bleeding.69 However, the recognition that shunting can occur in the absence of hepatic disease has led to the suggestion that HE be classified as type B if encephalopathy is associated with portal-systemic bypass in the absence of hepatic disease and type C if the encephalopathy is associated with cirrhosis and portal hypertension or portal-systemic shunting.154

The neuropathology of chronic HE is largely restricted to the astrocyte (Figure 9.9). Morphologically, astrocytes undergo Alzheimer type II change in which they show enlarged, pale nuclei with a rim of chromatin and prominent nucleoli. Astrocyte pairs and triplets are seen, but evidence of mitosis has not been found.383 In severe cases, nuclei become lobulated and contain granules of glycogen. Astrocytes also lose their immunoreactivity for glial fibrillary acid protein and contain increased numbers of mitochondria.383 Alzheimer type II astrocytes are most ­commonly seen in grey matter regions, such as the pons, cortical grey matter and putamen. In the cerebral cortex, they are often found in the deep layers. Alzheimer type II change in astrocytes is also seen other metabolic encephalopathies including uremia and hypercapnia and, in infants, hypoxia and hypoglycaemia.383 The pathogenesis of HE has been studied extensively in both humans and animal models following portacaval anastomosis and the underlying disorder is one of astrocyte-neuron trafficking.71 Cirrhosis of the liver results in elevated blood and brain ammonia levels and it is ammonia toxicity that is the leading hypothesis for the causation of HE. Astrocytic changes similar to Alzheimer type II change can be induced by hyperammonaemia in experimental animals and tissue culture and is also seen in patients with congenital hyperammonaemia due to inherited disorders of enzymes involved in the urea cycle.68 Millimolar concentrations of ammonia in the brain impairs postsynaptic inhibitory neurotransmission. Although there is substantial ­evidence for a central role of ammonia in HE, the mechanism by which ammonia causes the cerebral dysfunction is not entirely clear.457 Neuronal abnormalities in HE are not apparent on routine neuropathological examination and recovery of neurological function in HE patients following treatment of the underlying liver disease adds weight to the viewpoint that HE is a functional rather than a structural brain disorder. Nevertheless, there is evidence of persistent neurological abnormalities in some patients suggesting neuronal damage can occur.70

Uraemic Encephalopathy

9.9 Astrocyte doublet showing Alzheimer type II change, in a case of hepatic encephalopathy. The astrocytes are larger than normal (inset) and show margination of nuclear chromatin. Scale bar, 10 μm.

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Uraemic encephalopathy can manifest as a spectrum of neurological abnormalities from mild changes in cognitive function through to delirium and even coma. It can be associated with motor (e.g. tremor, asterixis, myoclonus), visual and sleep disturbances, alterations in consciousness and seizures.58,303 Peripheral neuropathy is also frequently reported in patients with uraemia.161,303 Neurological function is usually improved with successful treatment of the renal failure. Historically, cases with dialysis dementia, a progressive, often fatal syndrome of motor disturbance, personality change, psychosis and seizures, were described but these are now rare due to improvements in dialysis. Accumulation of aluminium was found to be the basis of dialysis dementia (see this chapter). The pathogenesis of uraemic encephalopathy has not been fully elucidated. A wide variety of compounds has been implicated including the accumulation of urea and other metabolites, alterations in amino acid neurotransmitter systems and hormone imbalance, especially parathyroid

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hormone.58,303 Macroscopically, the brain is often normal. In rare cases, CPM and extrapontine myelinolysis can occur. Microscopically, Alzheimer type II astrocytes are seen and in some cases areas of perivascular neuronal degeneration and demyelination.

Disorders Of The HypothalamicPituitary Axis The hypothalamus and pituitary gland are considered together in view of their close structural and functional relationships. We have covered neither the neuropathology of vascular lesions nor the consequences of physical injury to the head nor the biological aspects of hypothalamic structure and function. For reference, we have tabulated the principal products of the hypothalamus and pituitary gland (Table 9.1). We have concentrated on the major clinical manifestations of disorders of the hypothalamic–pituitary axis and their neuropathological associations.

Precocious Puberty Puberty is characterized by maturation of the ­hypothalamic– pituitary–gonadal (HPG) axis, the appearance of secondary sexual characteristics, acceleration of growth and, ultimately, the capacity for fertility. Disorders of pubertal development may occur at any of the steps in this maturational process, leading to either precocious or delayed puberty.261 Precocious puberty, defined as the premature development of secondary sexual characteristics and a post-­ pubertal endocrine profile, is associated with elevated blood levels of gonadotrophin hormone releasing hormone (GnRH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) and sex steroids. Early activation of the HPG axis gives rise to central precocious puberty, whereas lesions that secrete gonadotrophin-like substances and androgen- or oestrogen-producing neoplasms cause precocious pseudo-puberty. Misdiagnosis is a common problem. One survey of 104 cases referred for evaluation identified

nine cases of true precocious puberty; the majority of children referred were benign normal variants of the general population.268 It is generally agreed that the age of onset of central ­precocious puberty (CPP) is under 8 years in females and 9 years in males, although it is suspected that it may be much earlier. The aetiology of organic CPP is multifactorial and is related directly to the location and type of lesion. Demonstrable hypothalamic pathology is seen in fewer than 10 per cent of cases of CPP. Despite the absence of a demonstrable lesion, idiopathic precocious puberty is presumably hypothalamic in origin. It is a prominent feature of the McCune–Albright syndrome, a disorder not associated with observable hypothalamic lesion.288 CPP can be divided into two major groups. The first includes lesions that elaborate GnRH – for example, hypothalamic hamartoma in which CPP is associated with a childhood epileptic syndrome characterized by gelastic seizures, agenesis of the corpus callosum, Dandy–Walker complex and grey matter heterotopias.193,485 The second group includes endocrine-inactive lesions that non-specifically affect hypothalamic centres engaged in the control of sexual maturation. Reported lesions include germ-cell tumours,352,381 pineal cysts,130 gliomas,454 infections, such as congenital human immunodeficiency virus (HIV) infection239 and congenital toxoplasmosis,482 suprasellar arachnoid cysts,507 hydrocephalus, Angelman’s syndrome and trauma.48 Also implicated are craniopharyngioma (the tumour associated most often with pubertal delay), Rathke’s cleft cyst,354 duplication of the hypophysis with thickening of the hypothalamus,127 sectioning of the pituitary stalk secondary to Langerhans cell histiocytosis,367 neurofibromatosis type 1556 and tuberous sclerosis complex.7 Rutland and colleagues reported the third case of hypomelanosis of Ito associated with precocious puberty in a girl aged 5 years, with extensive cerebral involvement, suggesting a central mechanism of precocious puberty,467 although two previously reported cases had abnormal gonads and responded to therapy, indicating a peripheral mechanism. A prevalence of 27.5 per cent of familial cases among 147 patients with idiopathic CPP has been reported by

9

Table 9.1  Principal hormones of the hypothalamic-pituitary axis Hypothalamic hormone

Pituitary hormone regulated

Location of hormone in pituitary

GHRH

GH

Anterior pituitary

Somatostatin

GH↓

TRH

TH

Anterior pituitary

GnRH

FSH, LH

Anterior pituitary

CRH

ACTH

Anterior pituitary

ADH

Produced in hypothalamus, neurally transferred to pituitary

Posterior pituitary

OT

Produced in hypothalamus, neurally transferred to pituitary

Posterior pituitary

ACTH, adrenocorticotrophic hormone; ADH, antidiuretic hormone (vasopressin); CRH, corticotrophin releasing hormone; FSH, follicle stimulating hormone; GH, growth hormone; GHRH, growth hormone releasing hormone; GnRH, gonadotrophin hormone releasing hormone; LH, luteinizing hormone; OT, oxytocin; TH, thyrotrophic hormone; TRH, thyrotrophic hormone releasing hormone.

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598  Chapter 9  Nutritional and Toxic Diseases

Phillip and Lazar.421 Segregation analysis of this cohort suggested an autosomal dominant transmission with incomplete sex-dependent penetrance. Grosso reported three patients presenting with CPP in whom karyotype analysis demonstrated abnormal chromosomal patterns.189 One showed XXX syndrome, which is commonly characterized by premature ovarian failure. The second patient had a chromosomal aberration involving an imprinted chromosomal region, deletion of which is commonly associated with Prader–Willi syndrome and hypogonadotrophic hypogonadism. The third patient was a boy carrying a rare duplication of chromosome 9. All had degrees of mental retardation, were treated with luteinizing hormone releasing hormone (LHRH) analogues and did not progress with precocious sexual development. Iliev and colleagues reported a girl aged 5 years with precocious puberty and high levels of testosterone as a result of a mixed gonadal dysgenesis with a testosterone-producing gonadoblastoma.249

Ectopic Production of Hypothalamic Hormones Hormones of hypothalamic origin are referred to as releasing hormones (RH) or hypothalamic hormones.192 A variety of tumours, most neuroendocrine in nature, are known to produce hormones indistinguishable from releasing hormones of hypothalamic origin in terms of their endocrine effects. Of these, the production and effects of ectopic GHRH and corticotrophin releasing hormone (CRH) have been best documented. GnRH and thyrotrophin releasing hormone (TRH) can also be produced ectopically. Vasopressin (antidiuretic hormone; ADH) is the principal posterior lobe-related hormone to be produced ectopically. Less obviously ‘ectopic’ in origin is tumour-associated hypersecretion of oxytocin, vasoactive intestinal peptide (VIP), substance P and somatostatin.

Growth Hormone Releasing Hormone GHRH was first isolated and characterized as a 44-residue peptide from a rare tumour of the pancreas, which had induced acromegaly in the absence of a pituitary tumour. The physiology of GHRH and the diagnosis and treatment of GHRH-mediated acromegaly has been reviewed.135 GHRH-producing neurons have been well characterized in the hypothalamus by immunostaining techniques. Hypothalamic tumours, including hamartomas, choristomas and gangliocytomas, may produce excessive GHRH, with subsequent GH hypersecretion and acromegaly. Immunoreactive GHRH is present in several neoplasms, including carcinoid (bronchial, thymic) tumours, pancreatic cell tumours, small cell lung carcinoma, medullary ­carcinoma of the thyroid, adrenal adenomas and phaeochromocytoma.50,396 Clinical acromegaly due to tumour GHRH production in these patients is, however, very uncommon (Figure 9.10).470

Corticotrophin Releasing Hormone CRH is expressed in the brain, pituitary and many peripheral tissues, including adrenal medulla (mostly in layers

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(b)

(a)

(c)

R

(d)

L

R

L

9.10 Ectopic hypothalamic hormone secretion by a bronchial carcinoid tumour. (a) Nests and sheets of tumour cells in a carcinoid tumour. (b) The cells are immunopositive for GHRH. (c) A contrast-enhanced computed tomography (CT) scan shows marked enlargement of the pituitary, as a result of growth hormone (GH) cell hyperplasia, which (d) remitted after resection of the tumour.

of cells adjacent to the cortex). In the brain, the presence of CRH in the cerebellum and the limbic system suggests a role distinct from that in the hypothalamic–pituitary axis.192 The evaluation of Cushing’s syndrome is extremely complex and the ectopic production of CRH must be considered in its differential diagnosis. In one child, it was due to ectopic production of CRH by a ganglioneuroblastoma.587 Because most CRH-producing tumours also secrete adrenocorticotrophic hormone (ACTH), the ectopic production may represent a paracrine phenomenon in addition to an endocrine phenomenon. Ectopic CRH may also indirectly provoke pituitary ACTH secretion. This dual mechanism of ACTH production may explain the resistance of the tumour to feedback inhibition and a CRH stimulation response indistinguishable from that observed in pituitary-dependent Cushing’s syndrome.

Gonadotrophin Releasing Hormone GnRH appears to be involved in synaptic transmission in sympathetic ganglia and may function as a neuromodulator in its multiple other locations in the nervous system. It has been shown that, during development, some GnRH neurons remain in the nasal cavity, including the olfactory and vomeronasal mucosa. GnRH receptors are also expressed by chemosensory neurons. The elucidation of GnRH neurons in this site, in addition to the

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Disorders of the Hypothalamic-Pituitary Axis  599



hypothalamus, provides a link between the anosmia and isolated gonadotrophin deficiency in patients with some forms of Kallmann’s syndrome (Figure 9.11) and related developmental abnormalities.192

Somatostatin Somatostatin, an inhibitor of GH secretion, has multiple roles. There is extensive evidence that somatostatin originates from the immune system and has significant immunomodulatory activity. Thymic epithelial and dendritic cells synthesize somatostatin and express type 2 somatostatin receptors. Somatostatin is recognized to be part of an immunoregulatory circuit that inhibits production of interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), CRH and substance P at sites of (chronic) inflammation.566 Scintigraphy with labelled analogues of somatostatin has shown the presence of several types of receptor in pathological peripheral tissues, including the retro-orbital tissue in Graves’ disease, breast cancer, malignant melanomas, small cell lung cancer and bronchial carcinoid tumours.386 In fact, all ­so-called neural crest or neuroendocrine tumours (insulinoma, glucagonoma, gastrinoma) have been shown to express somatostatin receptors and to respond, sometimes dramatically, to long-acting analogues of somatostatin. In addition, prostatic tumours contain cellular elements that are immunoreactive for a variety of peptides, such as somatostatin, bombesin, TRH, chromogranin A and serotonin.202

Box 9.1.  Syndrome of inappropriate antidiuretic hormone secretion: case reports

9

Mineta et al.347 described a patient with small cell carcinoma of the tonsil who developed the syndrome of inappropriate antidiuretic hormone secretion (SIADH) due to antidiuretic hormone (ADH) hyperproduction, as demonstrated by immunohistochemistry. Ikegami et al.248 reported two patients in whom SIADH occurred during the initial loading period of amiodarone therapy but who improved by reduction of the dose without discontinuation of the drug. Previously reported cases were related to long-term use of amiodarone. Kanda et al.265 demonstrated severe hyponatraemia in a 68-year-old man who had poor appetite and was disoriented. MRI showed an adenoma that pushed the pituitary stalk upwards. After adenomectomy, serum sodium level returned to normal without any treatment. The findings suggested that the local pituitary tumour had caused exaggerated secretion of arginine vasopressin, resulting in SIADH. Iida et al.247 described a 52-year-old man presenting with vomiting, general fatigue and hyponatraemia, consistent with SIADH. He had transient lymphocytic panhypophysitis associated with SIADH, leading to diabetes insipidus after glucocorticoid replacement. SIADH has also been described in ­association with relapsing multiple sclerosis.371 The patient, with a ­previous history of recurrent sensorimotor disturbance and visual deficit, developed bilateral motor weakness in the upper limbs and systemic malaise. SIADH was diagnosed, and it was suggested that demyelinating lesions in the ­hypothalamus might have caused the transient increased ADH secretion.

Antidiuretic Hormone/Vasopressin Ectopic ADH production is most often associated with pulmonary neoplasms and infection, particularly tuberculosis. In addition, adenocarcinoma of the prostate with ectopic ADH production has been reported.252 Ectopic expression of vasopressin V1b and V2 receptors in the adrenal glands of patients with familial ACTH-independent macronodular adrenal hyperplasia has been reported.315 Neither of these receptors is known to be normally expressed in the adrenal gland. The syndrome of inappropriate antidiuretic hormone secretion (SIADH) (see Box 9.1) is not an uncommon (a)

(b)

disorder. It is characterized by symptomatic hyponatraemia, with high urine osmolarity, and can be caused by a variety of conditions (Table 9.2). Unlike diabetes insipidus, for which the anatomical basis is usually obvious, that of SIADH is poorly understood. Whereas stimulatory osmoreceptors lie close to the ADH-producing magnocellular nuclei, inhibitory pathways are dispersed, their centres lying as far afield as the brain stem.547 As a result, lesions remote from the hypothalamus and posterior pituitary may result in ADH release. In addition, ectopic production of ADH, particularly by tumour cells (see earlier), may be responsible for SIADH. The treatment of ADH excess consists of water restriction and slow re-establishment of sodium balance; too rapid an elevation of sodium may result in central pontine myelinolysis.384

Diabetes Insipidus

9.11 Kallmann’s syndrome. (a) Normal density of gonadotrophs labelled with anti-FSH antibody. (b) Marked reduction in the density of FSH-positive gonadotrophs in Kallmann’s syndrome – the effect of diminished hypothalamic stimulation.

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Diabetes insipidus is a heterogeneous condition characterized by polyuria and polydipsia and caused by a lack of ADH secretion, its physiological suppression following excessive water intake, or the resistance of the kidney to its physiological actions.171 Physiological water balance is dependent upon functioning hypothalamic osmoreceptors, the capacity of the hypothalamus to produce ADH, the structural and functional integrity of the pituitary stalk and posterior lobe, the presence of renal vasopressin receptors, and a normal response to thirst. Diabetes insipidus may, therefore, be central (i.e. pathology is present in the brain or pituitary gland, causing a deficiency of ADH synthesis in the hypothalamus and/or of secretion from the neurohypophysis) or

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600  Chapter 9  Nutritional and Toxic Diseases Table 9.2  Disorders associated with excessive antidiuretic hormone (ADH) Neoplasia (extra-CNS)

Carcinoma of the lung, tonsil, duodenum, pancreas, ureter and prostate

Table 9.3  Diabetes insipidus: variants and causes Central diabetes insipidus

Primary

Hereditary: autosomal dominant, sex-linked recessive

Lymphoma, leukaemia, Hodgkin’s disease

Wolfram (DIDMOAD) syndrome

Mesothelioma

Secondary

Ewing’s sarcoma

CNS disease

Trauma (surgical trauma, ‘stalk section’, head injury)

Trauma, neurosurgery

Neoplasia

Infection: meningitis (tuberculous), encephalitis, brain abscess, malaria

Primary (craniopharyngioma, glioma, germ cell tumour)

Hydrocephalus Delirium tremens

Infection

Encephalitis, meningitis (bacteria, fungi)

Vascular

Hypoxic tissue injury Postpartum pituitary necrosis

Guillain–Barré syndrome

Intraventricular haemorrhage

Multiple sclerosis

CSF leak after transsphenoidal surgery

Haemorrhage: intracerebral, subarachnoid, subdural

Systemic disease

Tuberculosis Cavitary aspergillosis Pneumonia (viral, bacterial or fungal) Positive-pressure

ventilationa

Endocrine diseases

Addison’s disease, myxoedema, hypopituitarism, lymphocytic panhypophysitis

Miscellaneous

Cirrhosis with ascitesa Myocardial infarctiona Congestive heart failurea Acute intermittent porphyria Postoperative statea

Drugs

Vasopressin, oxytocin, chlorpropamide, chlorothiazide, clofibrate, carbamazepine, nicotine, phenothiazines, cyclophosphamide, morphine, barbiturates, amiodarone

CNS, central nervous system. aDisorders that may induce ‘appropriate’ ADH hypersecretion.

nephrogenic (when the kidney is unable to produce concentrated urine, because of the insensitivity of the distal nephron to ADH). Central diabetes insipidus may be idiopathic or caused by a variety of lesions (Table 9.3). A thorough investigation to rule out CNS lesions needs to be undertaken before considering a diagnosis of idiopathic central diabetes insipidus, because in many cases it is caused by a germinoma, craniopharyngioma, Langerhans’ cell histiocytosis, sarcoidosis, local inflammatory, autoimmune or vascular diseases, or trauma. Central diabetes insipidus is a common complication of transsphenoidal surgery, but it may also be caused by or

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Hypothalamic disease

Aesthesioneuroblastoma

Tumours (glioma, etc.)

Pulmonary disease

Idiopathic: autoimmune disease (?)

Sarcoidosis, Langerhans’ cell histiocytosis Extramedullary haemopoiesis

Nephrogenic diabetes insipidus

Primary

Hereditary (X-linked): vasopressin-unresponsive

Secondary

Electrolyte disturbance (hypokalaemia, hypercalcaemia) Chronic renal disease Drugs (lithium, colchicine, gentamicin)

CSF, cerebrospinal fluid; DIDMOAD, diabetes insipidus, diabetes mellitus, optic atrophy, neural deafness.

associated with an intraoperative cerebrospinal fluid (CSF) leak, a craniopharyngioma, a Rathke cleft cyst or a microadenoma. The first three conditions are risk factors for persistent diabetes insipidus,376 but diabetes insipidus is usually transient in nature. Autoimmune central diabetes insipidus is likely in young patients with a clinical history of autoimmune disease and radiological evidence of pituitary stalk thickening.425 It is probable that many cases of idiopathic central diabetes insipidus are actually autoimmune central diabetes insipidus.425 Nephrogenic diabetes insipidus, due to various causes (Table 9.3), may result in severe dehydration and electrolyte imbalances. Reversible nephrogenic diabetes insipidus results in resolution of the condition. Long-term treatment with lithium may result in irreversible nephrogenic diabetes insipidus.168 Hereditary diabetes insipidus with a variable age of onset is exceedingly rare, representing only 1 per cent of cases of central diabetes insipidus. Hereditary diabetes insipidus encompasses vasopressin-sensitive degenerative

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Disorders of the Hypothalamic-Pituitary Axis  601



disorders of magnocellular neurons in the supraoptic and paraventricular nuclei269 and may also be seen in Wolfram’s syndrome,137 a disorder characterized by atrophy of the hypothalamic nuclei, and degeneration of the optic pathway, pons and cerebellum.85 Given the variety of causes of central diabetes insipidus, the prognosis varies considerably. Resultant diabetes insipidus may be transient or permanent, depending upon the level of the lesion and its degree of tissue destruction. In most cases of hypothalamic involvement, both supraoptic and paraventricular nuclei are affected; a destructive lesion proximal in the neurosecretory system, such as the upper portion of the pituitary stalk, produces permanent diabetes insipidus due to retrograde axonal degeneration and atrophy of the magnocellular nuclei (Figure 9.12).321 A low stalk lesion or destruction of the posterior pituitary produces only temporary diabetes insipidus because a small proportion of axons originating in the supraoptic nucleus terminates high in the median eminence and is consequently spared. Furthermore, destruction of the stalk at a low level permits axonal regeneration.117 Biopsy of an enlarged pituitary stalk should be reserved for patients with a ­hypothalamic–pituitary mass and progressive thickening of the pituitary stalk, because spontaneous recovery may occur. Neuronal loss must be extensive and high, involving the infundibular region or median eminence to result in permanent deficits. Autopsy studies are few but demonstrate striking neuronal loss and gliosis.54,185 Similar changes have been described in ‘idiopathic diabetes insipidus’.185 In occasional cases, magnocellular nuclei appear morphologically normal but lack vasopressin immunoreactivity.370

abnormality of GnRH secretion; Kallman’s syndrome describes an inherited condition of GnRH abnormality in association with anosmia.77,528 The presenting features of patients with hypothalamic hypogonadism are delayed puberty and/or incomplete sexual development. In males, these include prepubertal testes, micropenis, cryptorchidism, and in females delayed breast bud development and abnormal pubic and axillary hair. The neuropathological consequences of hypothalamic hypogonadism are hypoplasia of the lateral tuberal nuclei, an increase in neurons in the subventricular nucleus and a marked increase in gonadotrophs in the anterior pituitary.287 Pulsatile GnRH therapy is usually effective. A variety of midline malformations may also be associated with GnRH deficiency. For example, hypogonadism is associated regularly with hypothalamic lesions due to sarcoidosis, Langerhans’ histiocytosis and a variety of neoplasms. Hypogonadism is a common finding in idiopathic haemochromatosis, usually due to iron deposition in gonadotrophs (Figure 9.13).39 One report however,

9

(a)

Hypothalamic Hypogonadism This condition results from the deficient or dysrhythmic release of GnRH. The amplitude and rhythmic frequency of release regulates the secretion of LH and FSH. The term ‘idiopathic hypothalamic hypogonadism’ is used broadly to describe the condition in all patients with an unexplained (b)

9.12 Familial central diabetes insipidus. Marked reduction in the size and number of secretory neurons. The patient, a 70-year-old male with lifelong diabetes insipidus and several affected family members, had a serum vasopressin level of 1.5 pg/mL, which did not rise with dehydration. Courtesy of Dr C Bergeron, University of Toronto, Toronto, Ontario, Canada.

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9.13 Pituitary haemosiderosis. (a) In the adenohypophysis of a patient with haemochromatosis, there is deposition of iron in scattered hormone-producing parenchymal cells. (b) Co-localization reveals that the majority of cells containing iron (blue) are gonadotrophs that contain β-follicle stimulating hormone (FSH) (brown).

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602  Chapter 9  Nutritional and Toxic Diseases

described a clinically hypogonadal male with haemochromatosis and laboratory evidence for a combined defect in hypothalamic and pituitary function.496 Idiopathic hypothalamic hypogonadism has been associated with polyostotic fibrous dysplasia (Albright’s ­syndrome).241 Hypogonadotrophic hypogonadism may be associated with exercise-related and psychogenic ­amenorrhoea (also known as hypothalamic amenorrhoea) and anorexia, but the precise mechanisms involved are unclear. The evaluation of hypothalamic hypogonadism involves the exclusion of other systemic disorders and broader disorders of the hypothalamus, such as panhypopituitarism.582

Hypothalamic Dwarfism In addition to pituitary disease and end-organ resistance to GH, dwarfism may result from hypothalamic dysfunction. In many instances, as a result of their proximity and shared circulation, the hypothalamus and pituitary are simultaneously involved by inflammatory, infectious, neoplastic, congenital or neonatal diseases associated with growth retardation. Underlying causes include bacterial meningitis, granulomatous disease, Langerhans’ cell histiocytosis, hypothalamic neuronal hamartoma and neoplasms, particularly craniopharyngioma and germ cell tumours. Aside from the more often idiopathic nature of hypothalamic growth retardation in early childhood, this may also be due to disorders in the pre-, peri-, and postnatal periods.111 Midline developmental defects, such as septo-optic dysplasia, may also be responsible.20,360,427 An autosomal recessive form of hypothalamic dwarfism is associated with failure to produce GHRH. In such cases, somatotrophs are structurally normal and produce GH.452 Patients with mitochondrial encephalomyopathies, especially MELAS (mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes) and MERRF (myoclonus epilepsy with ragged red fibres), have also been reported to have hypothalamic–pituitary dysfunction (see also Chapter 7).391

Diencephalic Syndrome The diencephalic syndrome is a rare but potentially lethal cause of failure to thrive in infants and young children. Clinical features are severe emaciation, normal linear growth, normal or precocious intellectual development, ophthalmological abnormalities, including late optic atrophy, and signs of hypothalamic dysfunction such as euphoria, hyperkinesis, hypertension and hypoglycaemia.66,262,408,409,429 Although extremely rare, diencephalic syndrome can occur in adults.351,483 In almost all instances, the syndrome is associated with CNS tumours in the hypothalamic–optic pathway region, such as an optic pathway glioma (pilocytic astrocytoma),157,429,557 craniopharyngioma351,483 or germ cell tumours.352 The syndrome usually improves with removal of the tumour.121 Van der Wal and colleagues reported leptomeningeal spread of a chiasmatic pilocytic astrocytoma in a child presenting with diencephalic syndrome.548 Despite treatment with chemotherapy and radiation, the tumour recurred and progressed to a high-grade astrocytoma. One rare case of pilocytic astrocytoma and diencephalic

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syndrome occurring together in a 30-year-old woman with neurofibromatosis type 1 has been described.281 MR imaging revealed a tumour in the chiasmatic–hypothalamic region, which invaded the thalamus, brain stem and cerebellum. Some reports157,429 indicate these tumours are usually larger, occur at a younger age, and behave more aggressively than similarly located tumours not presenting with diencephalic syndrome. The suggestion has been made that in a child affected by diencephalic syndrome and hypothalamic juvenile pilocytic astrocytoma, the patient should be reviewed carefully for evidence of tumour dissemination.412 Developmental cysts and inflammatory lesions are less often the cause of diencephalic syndrome. Some cases are congenital. Involvement of the anterior hypothalamus is a common feature.408 Accordingly, diencephalic syndrome should be considered in any child with emaciation despite adequate caloric intake and an inappropriately euphoric mood.121

Metabolic Disorders of the Pituitary Amyloid deposition involving the pituitary gland occurs as part of a systemic disorder and is seen occasionally in pituitary adenomas, most commonly prolactinomas.45,46,306,359,558 The amyloid is deposited in vessel walls or the interstitium, as an extracellular, amorphous, eosinophilic substance that shows apple green birefringence with Congo red under polarized light. Haemosiderosis of the pituitary glands of patients with haemochromatosis (Figure 9.13) shows preferential deposition in gonadotrophs,278,324 explaining the most frequent endocrine clinical manifestation of hypogonadism.

Metabolic Disorders Metabolic Encephalopathies Metabolic encephalopathies are a diverse group of disorders. Primary metabolic encephalopathies are those resulting from inherited metabolic abnormalities; those resulting from hypoxic-ischaemic states, systemic diseases and toxins are secondary (Table 9.4). The brain has extensive demands for oxygen and energy substrates, requiring approximately 20 per cent of total body oxygen utilization and approximately 75 per cent of hepatic glucose production. An adequate blood supply is clearly essential, and limiting either oxygen or glucose can result in metabolic dysfunction. Furthermore, as aerobic oxidation of glucose is the sole source of energy in the brain under normal conditions, the consequences of hypoglycaemia are rapid and serious. Primary metabolic disorders (inborn errors of metabolism) are normally rare, and unique abnormalities are reported on occasion.5 The number of disorders described has increased steeply in recent decades as a result of improvements in the range, accuracy and availability of diagnostic tests and expansion of our knowledge of the molecular basis of brain function. In general, these conditions are characterized by the disruption of a metabolic pathway and the resulting accumulation of precursor substrates and the depletion of immediate or subsequent products. Such disorders can result from mutations or

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Metabolic Disorders  603

Table 9.4  Causes of metabolic encephalopathy Primary

Mitochondrial disorders Urea cycle disorders Amino acid metabolism disorders Organic acid metabolism disorders Fatty acid metabolism disorders Lysosomal disorders Peroxisomal disorders Porphyria

Secondary

Hypoxia

Anaemia Pulmonary disease Asphyxia Ischaemia

Cardiac or cardiovascular disease

Hypotension Hypertension Microvascular disease

Systemic disorders

Hypovolaemia

Disorders of Amino Acid Metabolism

Hyperviscosity

These disorders are due to inborn errors of amino acid metabolism, e.g. phenylketonuria, hyperglycinaemia, maple syrup urine disease, homocystinuria, disorders of the urea cycle and others. Almost all are autosomal recessive. Most manifest early in neonates, presenting with variable combinations of symptoms and signs, particularly vomiting, feeding difficulties, irritability, weight loss, fits, abnormal movements, respiratory distress, metabolic acidosis, lethargy and coma. In view of the non-specific nature of many of these signs, the process of investigation should be, first, the exclusion of conditions such as meningitis, intracranial haemorrhage, hypoxic-ischaemic disorders and cerebral malformations. The possibility of a disorder of amino acid metabolism should then be considered, particularly if there is a similar history in the family or parental consanguinity. Screening for metabolic diseases should target glycaemia, gasometry, ionogram, ammonaemia, ketonic acidaemia and the more specific assessment of enzymatic dosages in the urine, blood and other tissues. These diseases provoke great clinical interest, because early diagnosis often allows treatment that prevents future irreparable cerebral damage. There have been relatively few recent advances in the neuropathology of disorders of amino acid metabolism since the reviews by Martin and Schlote335 and Crome and Stern.113 In most cases, the lesions are non-specific, the most common change being spongiosis and cavitation of the white matter and gliosis. Cavitation is relatively rare and its presence usually depends on the duration of the pathologic process. An exception is homocystinuria, in which the lesions are usually ischaemic in appearance. Disorders of urea cycle enzymes, which cause the accumulation of precursors of urea (ammonia and glutamine), induce widespread Alzheimer ­

Hepatic disease Renal disease Malnutrition Electrolyte imbalance Endocrine dysfunction Infection Toxins Alcohol Medications and other drugs Heavy metals Organic compounds Carbon monoxide

other DNA abnormalities, and involve abnormalities of enzymes or other proteins involved in the regulation, activation or transport of metabolites. Disorders may be inherited in an autosomal, X-linked or mitochondrial pattern (see OMIM for current information, www.ncbi.nlm. nih.gov/omim). The clinical presentation and age of onset of primary metabolic encephalopathies vary widely with the major determinant being the pathway involved. Abnormalities that affect essential pathways usually have an early age of onset and marked clinical manifestations, whereas others

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may have a later onset, more gradual course or little or no clinical deficit. The range of clinical signs is also broad, varying from focal deficits to global deficits involving all functional domains. The clinical diagnosis of a primary metabolic encephalopathy can be difficult.5 A combination of clinical history (including family history), clinical examination, biochemistry, histology and genetic analysis may be required in order to arrive at a diagnosis. In some situations, for example in storage disorders, electron microscopy of blood cells or skin may reveal morphological features that assist in diagnosis and alleviate the need for brain biopsy. Secondary or acquired metabolic encephalopathies are encountered more frequently and, in many instances, are life-threatening complications of systemic disease (Table 9.4). Most result in global cerebral impairment, which may manifest as altered conscious level, epilepsy, or psychiatric and/or motor abnormalities.299 Additional focal abnormalities and cerebral signs can also exist. Clinical signs in many of these encephalopathies may be reversed on correction of the underlying systemic disorder, and fluctuation in their clinical course may reflect variations in organ function. The course of secondary metabolic encephalopathies is also variable, with some presenting as coma or progressing rapidly to coma and others having a more indolent course.

9

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604  Chapter 9  Nutritional and Toxic Diseases

type II disease change in astrocytes. Important determinants of the ­neuropathological changes are the age of the patient (length of survival) and the efficacy of treatments, such as dietary restriction. A number of different mechanisms may play a role in causing the structural and functional deficits seen in these disorders. Some amino acids are neurotransmitters (e.g. glycine) or are required in the formation of neurotransmitters (e.g. tyrosine). Other amino acids, when present in excessive amounts, can interfere with mitochondrial function, and deficits of others may lead to abnormalities of lipid and protein metabolism. Furthermore, the ketoacids and other by-products of metabolic failure in amino acid metabolism (e.g. ammonia) may induce toxicity by interfering with various cellular functions, including neurotransmission.

Phenylketonuria Phenylketonuria (PKU) is an autosomal recessive disorder, and one form of hyperphenylalaninaemia (defined as a plasma phenylalanine level above 0.12 mM/dL) which is associated with impaired cognitive development if not detected and treated during the first few weeks of life. This inborn error of metabolism results from a deficiency of the phenylalanine hydroxylase (PAH) gene, which synthesizes tyrosine from phenylalanine. Non-PKU hyperphenylalaninaemia is a benign condition as a result of a defect in the hydroxylation of phenylalanine to tyrosine. The different clinical manifestations in the two phenotypes suggest that the degree of phenylalanine excess influences pathogenesis.560 There is great geographical and ethnic variation in the incidence of PKU, with 5–190 cases per 1 million births.459 Numerous different mutations have been identified in a variety of populations with many being found at relatively high frequencies.590 A classical description of PKU involves a normal birth and an infant that seems to develop normally for several months, before development slows and then stops, when the child drifts into irreversible mental retardation.92 Newborn screening for PKU began 35–40 years ago in most industrialized countries. Because of this initiative and the early institution of phenylalanine-restricted diets, there are now many young adults with this disease that have normal or near-normal intellectual function. Up to 10 per cent of adults with classic PKU, and possibly 50 per cent of those with milder variants, may not need treatment; after adolescence, intelligence does not appear to deteriorate, at least into early adulthood, even if diet therapy is discontinued or poorly controlled. Blood levels of phenylalanine are poor prognostic indicators, but remain the most important indicator that dietary control is needed; it has been suggested (but not rigorously tested) that brain levels may be more helpful. Despite dietary control, neuropsychological and psychosocial problems develop frequently, needing focused and intensive support by healthcare providers.125 MRI studies show white matter changes in a significant proportion of cases.524 The severity of these changes correlates with blood phenylalanine concentrations, as well as with brain phenylalanine concentrations measured by MR spectroscopy.353 Dezortova and colleagues used MRI to study the pathological changes observed in periventricular white matter. Known PKU lesions characterized by T2 enhancement in periventricular white matter were observed in all patients.129 The MR spectra from the lesioned areas

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showed a significant decrease in choline concentration and support the hypothesis that the T2 increase in a PKU lesion reflects an increase in water mobility, which might be explained by changes in the volume of the extracellular space and possible abnormalities in myelin sheaths. Neuropathological changes in untreated patients occur in regions that develop postnatally, particularly with respect to myelination of the subcortical white matter and spinal cord and the growth of axons, dendrites and synapses in the cerebral cortex.32 Because of seizures, hippocampal pyramidal and cerebellar Purkinje cell loss is present.284 In addition, a small minority of brains show evidence of progressive white matter leukodystrophy, with white matter spongiosis, gliosis and delayed myelination. Biochemical studies reveal diminished levels of cerebral lipids and proteolipids.334 The pathological changes are thought to be due to toxic effects of phenylalanine and/or its metabolites, and it is assumed that they can be prevented by dietary therapy during infancy and childhood, but direct confirmation by neuropathological studies is lacking.245 It is important to note that, despite dietary control, patients with PKU usually have moderate hyperphenylalaninaemia throughout life. The fetus is sensitive to increased maternal levels of phenylalanine; an affected fetus shows facial dysmorphism, microcephaly, intrauterine growth retardation, developmental delay, congenital heart disease, mental retardation and other malformations.319 In one UK study of the impact of a maternal phenylalanine-restricted diet in women with PKU,314 of 228 live births, metabolic control for the first 12–16 weeks of gestation had most influence on outcome.314,319 In a 15-year international study, women with PKU on a phenylalaninerestricted diet before conception showed microcephaly in 27 per cent of children, and 7 per cent with serious congenital heart disease.280 Improvements in control of phenylalanine levels have reduced these problems.426

Hyperglycinaemia Two distinct types of hyperglycinaemia are recognized, and both can present as a life-threatening illness in the newborn. In ketotic hyperglycinaemia (KHG), there is a primary metabolic block in the catabolism of some organic acids, leading to a severe acidosis and hyperglycinaemia. The second type, non-ketotic hyperglycinaemia (NKHG), also known as glycine encephalopathy, is an autosomal recessive disorder caused by a defect in the glycine cleavage system (GCS), a complex of four proteins encoded by separate genes.19,236,301,539 With hyperglycinaemia, large amounts of glycine accumulate in body fluids, including the CSF, and are classically associated with neonatal apnoea, lethargy, hypotonia, poor feeding, seizures and deep coma, followed by severe psychomotor retardation in those who survive. In a series of 65 patients, one-third of the subjects died, 8 during the neonatal period and 14 thereafter. Median age at death for boys was 2.6 years, which compares with less than 1 month for girls. Of 25 patients living more than 3 years, ten were able to walk and say or sign words; all were boys, revealing a striking gender difference in mortality and developmental progress.236 In NKHG, neuropathological changes are similar to those described in other amino acidopathies, with

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spongiosis and gliosis of the white matter. An abnormal corpus callosum and/or dilation of the ventricular system have been associated with especially poor motor and speech development. In a study of the brains of three infants, there was a dramatic reduction in the volume of the white matter of the cerebral hemispheres (Figure 9.14).492 In a study of a 17-year-old patient, the myelinopathy appeared to be static when compared with neonatal cases, suggesting that the neurological deficits could be due to neurotransmitter abnormalities rather than damage to myelin.9 Glycine is the major inhibitory neurotransmitter in the spinal cord and brain stem, but it may also play a role as a transmitter in the cerebral cortex, and the high levels that can be measured in CNS tissue in this disease may account for the clinical syndrome.42,229

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9.14 Non-ketotic hyperglycinaemia (glycine encephalopathy). Brain slice from a male infant who was normal at birth but fed poorly, became jittery and increasingly hypotonic and had recurrent seizures. Metabolic studies revealed non-ketotic hyperglycinaemia. He died at 1 month. (a) Macroscopic examination of the brain revealed partial agenesis of the corpus callosum (a common finding in this disease) and slight, ill-defined yellow discolouration of the white matter (arrows). (b) Histology showed marked ­spongiosus as a result of intramyelinic oedema (illustrated here in the cerebellar vermis), and reactive astrocytosis. Courtesy of Seth Love, University of Bristol, UK.

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Electron microscopy shows that the vacuoles in this condition lie within the myelin sheath.8 The white matter lesions are bilateral and symmetrical and located in the dorsal brain stem, cerebral peduncles and posterior limbs of the internal capsule, the expected sites of abnormality in vacuolating myelinopathy. The lesions are more conspicuous and extensive on diffusion-weighted MR images than on T2-weighted images.274,480 Using sequential MR imaging with T2-signal intensity and restricted diffusion, white matter changes have been documented.364 At 3 weeks, the abnormalities were consistent with a vacuolating myelinopathy, and these increased in extent by 3 months. At 17 months, diffusion restriction had disappeared, probably because of the coalescing of myelin vacuoles, and there was evidence of axonal loss. Using MR spectroscopy in NKHG, the existence of glycine disposal pathways producing increases in CSF lactate, and increased creatine in CSF and brain from an early age.114,555 The cerebral N-acetyl aspartate to myo-inositol-glycine ratio was identified as a prognostic indicator of the disease. Three atypical variants of NKHG have been described: neonatal, infantile and late-onset.131 Atypical variants have heterogeneous clinical presentations, in contrast to the uniform severe neurological symptoms in classical NKHG. In the neonatal variant, presentation is similar to the classical form, but the subsequent outcome is significantly better. Mental retardation and behavioural abnormalities are prevalent in both infantile and lateonset forms, although the phenotype in late-onset atypical NKHG is more heterogeneous. Hyperglycinaemia in atypical neonatal and infantile NKHG is caused by a deficient GCS. The cause of hyperglycinaemia in atypical late-onset NKHG is uncertain, although several mutations of the P-protein GLDC gene have been identified, along with the T-protein (AMT) and H-protein (GCSH). Some cases with GLDC mutation are associated with residual glycine decarboxylase activity, and early therapeutic intervention may be crucial in order to improve the outcome in patients harbouring such mutations. Identification of more mutations causing atypical NKHG and information about the mutations’ effects on enzyme activity may help to predict the course of patients with a milder phenotype, as well as those who may respond to early therapeutic intervention. Transient NKHG is rare.16 It is characterized by clinical and biochemical findings similar to those seen in classic NKHG. Abnormalities in amino acids partially or completely remit in a period ranging from days to months. Because most patients exhibit normal development, distinguishing the transient form from the classic form is important.

9

Maple Syrup Urine Disease Genetic disorders of branched-chain alpha-ketoacid (BCKA) dehydrogenase metabolism produce amino acidopathies and various forms of organic aciduria, with severe clinical consequences. A metabolic block in the oxidative decarboxylation of BCKA caused by mutations in the mitochondrial BCKA dehydrogenase complex (BCKDC) results in increased branched-chain amino acids (BCAA) and their ketoacids (BCKA), causing maple syrup urine disease (MSUD) and acute or chronic encephalopathy.510

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Classically, this disease presents as a fulminating neurological disorder with vomiting, lethargy, hypertonicity, convulsions and a peculiar odour (maple syrup) of the urine. Rapid deterioration may lead to death, and mental retardation accompanies longer survival. The primary approach to therapy is to modify the diet early in life in order to reduce BCAA and BCKA, thus preventing or minimizing brain dysfunction. There are presently five known clinical phenotypes for MSUD: classic, intermediate, intermittent, thiamine-responsive and dihydrolipoamide dehydrogenase (E3)-deficient. This classification is based on severity of disease, response to thiamine therapy, and the affected gene.103 Reduced glutamate, glutamine and γ-aminobutyric acid (GABA) concentrations in the cerebrum are considered to be the cause of MSUD encephalopathies. The long-term restriction of BCKA in the diet and orthotopic liver transplantation have been effective in controlling plasma BCKA levels and mitigating some of the neurological manifestations. To date, approximately 100 mutations have been identified in four of the six genes that encode the human BCKDC catalytic machine. Chuang and colleagues have documented a strong correlation between the presence of mutant E2 proteins and the thiamine-responsive MSUD phenotype.103 Changes in MRI in 14 juvenile and adult patients with MSUD consisted of an increased signal in white matter on T2-weighted images, which is compatible with a disturbance of water content and dysmyelination. Areas affected most commonly were the mesencephalon, brain stem, thalamus and globus pallidus; supratentorial lesions were restricted to severe cases. The severity of dysmyelination does not correlate well with acute neurotoxicity but is correlated with median plasma BCAA concentrations over the 6–36 months prior to imaging.475 The principal neuropathological findings in untreated cases are spongiosis and gliosis of the white matter, aberrant orientation of neurons, and abnormalities of dendrites and dendritic spines.263 Secondary effects are hypoxia and hypoglycaemia, which may be associated with classic crises and can complicate the pathological picture. An acute axonal neuropathy was demonstrated in a woman aged 25 years with MSUD, who developed areflexia, generalized weakness and distal sensory loss over 1 week.277 Electrodiagnostic studies indicated an acute axonal polyneuropathy, and sural nerve biopsy revealed acute wallerian degeneration without inflammation. Peripheral neuropathy associated with MSUD may become more common as chronic dietary restrictions and improved management of the disease allow survival into adulthood.362 MSUD has been identified in Poll-Hereford and PollShorthorn cattle in Australia. The disease is first seen within 2 days of birth. Affected calves are weak and develop limb rigidity and opisthotonus by day 5. Intramyelinic vacuoles, myelin oedema and axonal swellings are reproducible findings (Figure 9.15).212 Plasma and CSF contain high levels of BCAA.

Homocystinuria Homocystinuria is an autosomal recessive inborn error of the methionine metabolic pathway. In its principal form, it is due to cystathionine-beta-synthase (CBS) deficiency

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9.15 Section of cerebellum from a Poll–Hereford calf with maple syrup urine disease. (a) There is spongiosis of the white matter. The granule cell and molecular layers appear normal. Phosphotungstic acid-haematoxylin. (b) Electron microscopy from specimen in (a), showing that the vacuoles are intramyelinic. Courtesy of Dr PAW Harper.213 Copyright © 2008, John Wiley and Sons.

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and is characterized by increased plasma homocysteine.365 Homocystinuria may be more common than previously reported,448 and screening of newborns for homocystinuria through mutation detection is recommended. The clinical syndrome is more complex than with other aminoacidurias, and abnormalities can be present in the eye, skeleton, vasculature and CNS.120 Mental retardation is common in infants who are not treated from birth, but a structural explanation for this is not evident. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood.436 The predominant neuropathological lesions relate to thromboembolic disease. Involvement of cerebral vessels produces infarcts in the cerebrum, cerebellum, midbrain and thalamus.365 Thrombi in the dural sinuses have also been reported.108 Arterial walls often have fibrous intimal thickening, even in children.38,318,567 Combined methylmalonic aciduria and homocystinuria is the most common inborn error of cobalamin metabolism due to MMACHC gene mutation.317,462 This complex disorder presents within 12 months of age with severe neurological, haematological and gastrointestinal abnormalities. Clinical manifestations include hypotonia, failure to thrive and poor feeding. Ophthalmological and dermatological changes are common.116 Diffuse supratentorial white matter oedema and dysmyelination is the typical MR picture at presentation, whereas loss of white matter bulk characterizes later stages of the disease. White matter damage is probably caused by reduced methyl group availability and non-physiological fatty acid toxicity, whereas focal gliosis results from homocysteine-induced toxicity to the endothelium. Hydrocephalus, which can be a feature, may result from diffuse intracranial arterial stiffness, known as reduced arterial pulsation hydrocephalus. The disease is commonly believed to be a disease of infants, but adult-onset forms have been identified.460 Other disorders of amino acid metabolism, such as tyrosinaemia,22,187,465 and disorders of histidine, proline and hydroxyproline metabolism have been reviewed extensively elsewhere.113,335

Urea Cycle Disorders Various disorders cause hyperammonaemia during childhood, including those caused by inherited defects in urea synthesis and related metabolic pathways. These disorders can be grouped into two types: disorders of the enzymes that make up the urea cycle, and disorders of the transporters or metabolites of the amino acids related to the urea cycle.145 Ornithine transcarbamylase (OTC) deficiency is probably the most common, but there are four other enzymes where mutation can result in elevated levels of blood ammonia as a result of the failure of conversion of ammonia to urea, giving rise, respectively, to arginaemia, arginosuccinic aciduria, citrullinaemia and hyperornithinaemia. OTC shows an X-linked mode of inheritance, and most males with low residual OTC activity present with severe chronic hyperammonaemia in the neonatal period. Female carriers have a milder form of the disease. The syndrome results from a deficiency of the mitochondrial enzyme OTC, which catalyses the conversion of ornithine and carbamoyl

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phosphate to citrulline. OTC mutations can be divided into two groups: those with neonatal onset and completely abolished enzyme activity, and those with later onset and partial and variable enzyme deficiency.181 The clinical syndrome in urea cycle disorders (UCDs) is related to hyperammonaemia. Except for arginaemia, which can present as a progressive tetraplegia and mental retardation,472 the UCDs have a similar clinical presentation. In the neonatal period and after an initial period when they appear well, infants develop poor feeding, drowsiness, lethargy, hypothermia, tachypnoea and apnoea. When OTC occurs among males in the neonatal period, it is likely to be lethal, and CNS injury in OTC deficiency may have occurred in utero. The diagnosis should be considered if coma with cerebral oedema and respiratory alkalosis occur for no obvious reason. The signs and symptoms of childhood UCDs are often vague but nevertheless recurrent; fulminant presentations associated with acute illness are common. A disorder of urea cycle metabolism should be considered in children who have recurrent symptoms, especially neurological abnormalities associated with periods of decompensation. In late infancy, affected individuals may also present with vomiting and mental changes, and in many cases the precipitant is a protein load, e.g. a change of diet or infection.500 Additional disease-specific symptoms are related to the particular metabolic defect. These specific clinical manifestations are often due to an excess or lack of specific amino acids.145 Nassogne and colleagues reviewed the clinical presentation of 217 patients with UCDs, including 121 patients with neonatal onset forms and 96 patients with late-onset forms.373 The latter may present at any age and carry a 28 per cent mortality rate and a subsequent risk of disabilities. Routine laboratory tests, including measurement of plasma ammonia concentration, can indicate a potential UCD; ammonia levels above 200 μmol/L are usually caused by inherited metabolic diseases. Specific metabolic testing and, ultimately, enzymatic or genetic confirmation are necessary, however, in order to establish a diagnosis. In the case of prenatal diagnosis, this is possible with a chorionic villus sample or amniotic fluid cells.181,196 Neuropathological findings in congenital OTC deficiency are variable, ranging from a microscopically normal brain with Alzheimer type II astrocytic change to severe damage in cerebral cortex and deep grey matter structures. For example, one report describes the cerebral hemispheres of a female aged 6 years as ‘little more than bags of leptomeninges’, whereas her cousin, aged 8 years and affected by the same disorder, had no macroscopic abnormality of her brain.62 Epilepsy may well cause some of the more devastating neuropathological damage reported in these disorders. MRI imaging in urea cycle disorders shows changes in white matter connectivity.188 In Holstein–Friesian calves affected by citrullinaemia due to argininosuccinate synthetase deficiency, there is spongiosis of the grey matter in the cerebral cortex, with vacuolation of the cytoplasm of astroglial cells (Figure 9.16).214 The acute clinical encephalopathy is associated with hyperammonaemia and a relative increase in glutamate-mediated excitatory activity.133 Treatment consists of a low protein diet and alternative pathway drugs, such as sodium benzoate and

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seen at autopsy. Although a previously unreported finding in propionic acidaemia, diffuse grey matter vacuolization has been described in other fatty acid metabolic disorders.153

Porphyrias

9.16 Section of cerebrum from a Holstein–Friesian calf with citrullinaemia. There is expansion of the cytoplasm of the astroglial processes in the neuropil and the astrocytic end feet surrounding blood vessels, with a reduced density of organelles. Courtesy of Dr PAW Harper.214 With permission from Elsevier.

phenylbutyrate.61 Haemodialysis or haemofiltration should be instituted if ammonia concentrations exceed 500 μmol/L or if they do not fall promptly.316 Administration of specific amino acids and use of alternative pathways for discarding excess nitrogen have been recommended, but although combinations of these treatments are employed extensively, the prognosis in severe cases remains unsatisfactory.145

Propionic Acidaemia Propionic acidaemia is a disorder of branched-chain amino acid and odd-chain fatty acid metabolism. The clinical features typically begin shortly after birth, with rare cases presenting in young adulthood. Episodic decompensations are characterized by dehydration, lethargy, nausea and vomiting, as well as occasional neurological sequelae. Propionic acidaemia is one of the most frequent organic acidurias, but information is rather limited. Data on 49 patients with propionic acidaemia from Europe471 identified by selective metabolic screening, showed 86 per cent were of early-onset propionic acidaemia with presentation within the first 90 days of life. The mortality rate was one-third. The defect in the propionyl-coenzyme A carboxylase enzyme, causes accumulation of toxic organic acid metabolites. Neuropathological findings have not been characterized extensively but include white matter spongiosis in neonates. Widespread grey matter vacuolization may be

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The porphyrias are a heterogeneous group of disorders that result from inherited or acquired defects of metabolism characterized by overproduction and excretion of porphyrins or their precursors, with dysregulation of one of the eight enzymes in the haem biosynthetic pathway.83,160 Each of the porphyrias is characterized by a unique pattern of overproduction, accumulation and excretion of haem biosynthesis intermediates.382 They are classified as erythropoietic or hepatic, depending on the primary organ in which excess production takes place. Only the hepatic porphyries, including acute intermittent porphyria and porphyria cutanea tarda (PCT), produce neurological disease. The porphyrias rarely manifest before puberty. Factors that may precipitate acute disease include certain drugs (e.g. barbiturates, sulphonamides, griseofulvin, meprobamate, phenytoin, succinimides, steroids), infections, starvation and menstruation (some women experience attacks just before menstruation). A comprehensive review of the history, pathophysiology, classification and treatment of porphyria should be consulted for further information.385 Acute intermittent porphyria (AIP) due to mutation in hydroxymethylbilane synthase (HMBS) is an inherited metabolic disease with an autosomal dominant pattern of inheritance with reduced penetrance where only 10–20 per cent of mutation carriers will develop symptoms.416 Because the biochemical parameters of patients and their non-affected relatives overlap, the diagnosis may remain undetermined during the symptom-free phase, however, mutation detection in AIP provides high clinical benefit. The neurological manifestations of AIP commonly present as a peripheral autonomic and motor neuropathy.35,345 Neuropsychiatric symptoms such as anxiety, depression, insomnia, disorientation, hallucinations and paranoia, as well as seizures and cranial nerve neuropathies, are also reported.345 During an acute attack, which includes various neurovisceral symptoms, measurement of urinary porphobilinogen is the method of choice to confirm diagnosis. Rare cases with compound heterozygous or homozygous mutation of HMBS have been described with early severe motor and neurological manifestations, sensory nerve conduction deficits due to denervation, with delayed myelination seen on MRI and in one case porencephaly.43,232,503 Neuropathological studies of the porphyrias have revealed a range of inconsistent findings. Hierons reviewed the neuropathological literature on acute porphyria and presented five cases.233,234 Lesions are either minimal or absent, except for those that can be explained on the basis of associated hypoxia during severe attacks. Suarez and colleagues presented the morphological findings of 35 patients with AIP and reported diffuse neuronal loss, occipital cerebral ischaemia, perivascular pigmentation, diffuse perivascular demyelination, cytolysis of Betz cells or no obvious

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abnormality.511 Nuclear chromatolysis of cranial nerves has been reported, as has Purkinje cell loss. Two reports have documented neuronal loss in the supraoptic and paraventricular nuclei of the hypothalamus, which was proposed as a mechanism for the hyponatraemia observed in some porphyric patients. Central pontine myelinolysis has also been observed.513 MRI studies have shown multiple cerebral lesions during attacks of acute porphyria.10,276,300 The lesions, which may be ischaemic, are multifocal and resolve after several weeks.75 One hypothesis is that because nitric oxide synthase is a haem protein and nitric oxide is a vasodilator, unopposed cerebral vasoconstriction due to decreased nitric oxide from severe haem deficiency may occur during acute attacks.300 The peripheral autonomic, motor and sensory disturbances are due to an axonal neuropathy. Distal degeneration of posterior column fibres has been reported in some cases. Chromatolysis of neurons in peripheral ganglia and the spinal cord, particularly among the anterior horn cells, is reported frequently. Degeneration of various spinal cord tracts and dorsal root ganglia has also been reported.234,478,578 The peripheral nerves themselves may show segmental demyelination and axonal swelling and fragmentation.577

Neurotoxicology In previous editions, the late David Ray provided a ­succinct summary of the subject: ‘Neurotoxicology is the science dealing with adverse effects produced in the nervous system by synthetic and naturally occurring chemicals. It is not an exact science, in the sense that the adverse effects may be subtle, protracted and multifactorial in origin. The biological targets utilized by the toxins may result in no obvious structural pathology, despite causing significant behavioural change. In many cases, toxicity may be caused not by direct action on a neuron or its neurites, but on its metabolic state, the localized circulation, or the availability of glucose or oxygen.’ The general population is exposed to a very wide range of doses of potentially toxic agents, and little is known about the effects of long-term low-level exposure to neurotoxic agents or of the potential for reversibility of neurotoxicity, particularly in the ageing nervous system. Currently, there is little direct evidence that acute exposure to environmental toxins will lead to a decline in cognitive function or the development of psychiatric behaviour except in relatively specific cases (e.g. following exposure of the developing brain to lead or the memory loss that may follow severe domoic acid poisoning). It is however, difficult to be confident that prolonged exposure to low levels of neurotoxic agents is without subtle behavioural changes in a vulnerable subset of the population. Such possibilities present significant problems for the processes of diagnosis and prognosis, and also mean that neuropathological data are sparse. For the majority of neurotoxins and neurotoxic exposures, descriptions of neuropathological changes are likely to be confined to animal models because the nature of human poisoning is rare and cases often do not come to

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autopsy or to specialist centres where appropriate neuropathological investigation can be undertaken. The lack of access to the brain, spinal cord and the peripheral nervous systems ensures that neuropathology tends only to describe and analyse tissues obtained at autopsy. The data, therefore, reflect end-stage disease and are often difficult to place in the context of aetiology and development. Although neuropathy may be a common finding, nerve conduction studies are the only likely investigation given the reduced frequency of (sural) nerve biopsy. Many of the studies on neurotoxic exposures are now almost of historical nature because we are more aware of the potential for specific chemicals to cause toxicity and therefore controls on their use have led to reduced exposure and consequently decreased incidence of clinical cases in developed countries. There are specific problems associated with certain neurotoxins in developing countries, where exposure is less regulated, or unique geography brings localized neurological conditions. That said, we are now entering an era where the effects of low level and chronic exposure to specific toxins are likely to become more apparent, and such exposures, although showing similar effects to acute exposures, may show subtle differences in clinical symptoms and neuropathological changes. Clinical indications are therefore likely to help to pinpoint the nature of the chemical exposure, and imaging-based techniques such as MRI, positron emission tomography (PET), single photon emission computed tomography (SPECT) will provide clues to the systems involved and thereby the possible toxic exposures. It cannot be overemphasized that very detailed occupational and specific lifestyle history becomes crucial for those investigations where industrial exposure (e.g. lead or manganese) or geography (e.g. arsenic) is suspected, and is key in identifying potential neurotoxic chemicals. Neuropathological studies in animals need to be treated with some degree of caution because direct comparison can be confounded with differences in physiology and neural responses. Perhaps the exceptions here are neuropathological investigation in primates where the physiology is sufficiently similar although, as with human studies, these are understandably rare. Even when animals are used, note should be taken of the route of administration, its dose and formulation as well as the general suitability of the species itself.141 These concerns are frequently ignored in academic studies but are rather more rigidly adhered to in the protocols laid down by US and OECD guidelines for the study of potentially neurotoxic agents. It is of interest that the guidelines identify the need to ‘assess the effects of any substance on learning memory and performance’. As non-invasive structural and functional imaging becomes more available and more sensitive, it may become possible to directly visualize changes appearing as the result of exposure to neurotoxic agents, to make longitudinal studies of both patients and experimental animals, and therefore relate the neurotoxic assault to clinical tests of function and behaviour. To date, however, the application of imaging techniques to toxicology remains minimal. Notwithstanding these caveats, considerable detail on the potential mechanisms of neurotoxins and their pathological changes has been produced through small animal studies.

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Neurotoxins Affecting Mitochondrial Energy Production The high metabolic demands of the nervous system means that disturbances of energy supply either by altered oxygenation or by inhibition of cellular energy production can cause rapid changes in neural activity and over prolonged periods considerable neuropathological changes. Chemicals that can affect energy production can provide relatively selective damage to the nervous system as with certain mitochondrial toxins, or neuropathological changes in combination with peripheral effects for example with cyanide poisoning. In general, there is a relative selectivity for basal ganglia structures with necrosis of the globus pallidus and putamen being common, but cortical structures, and in particular the hippocampal formation can be equally affected. Given the high energy demands of the nervous system, high level exposures can often have rapid effects, though equally chronic lower level exposures can still cause neuropathological changes.

Carbon Monoxide One of the most commonly encountered neurotoxins is carbon monoxide (CO) and in the UK alone there are between 200 to 500 deaths per year associated with CO intoxication, and perhaps 4000 or more associated with non-fatal exposure leading to hospitalization.200 Given the numbers of individuals who may be inadvertently exposed to CO because of faulty heating systems or the use of CO as a means of self-harm, CO intoxication is often encountered in emergency situations and the consequences are well described. Individuals are often comatose or very drowsy following exposure, and breathing is shallow with tachycardia, and the individual may have seizures, ataxia, often with signs of cyanosis. Cardiac injury is common in moderate to severe intoxication (carboxyhaemoglobin levels in excess of 20 per cent) and is a considerable cause of mortality.228 Prior symptoms, particularly in cases of accidental exposure, are persistent headache, nausea, abdominal pain, fatigue, confusion and cognitive impairment, and exacerbation of other medical conditions. These symptoms are however not specific and therefore the possibility of misdiagnosis exists, particularly given the problems of measuring CO levels in blood. Chronic low level exposure to CO not resulting in acute hospitalization may have a risk of longterm effects on the nervous system though this is lacking in any critical detail. Certainly, up to half of individuals who survive acutely debilitating CO exposure will have delayed or continued neurological or cognitive problems and the use of more fuel-efficient engines in cars means that it is more difficult to cause lethal CO poisoning in cases of self-harm resulting in a change in the spectrum of illness associated with CO exposure.237,407 Hyperbaric oxygen therapy is suggested as the method of choice for recovery with improved outcome, although this is often unavailable and there may be disadvantages.237 The principal mode of action of CO is adduction to haemoglobin to form carboxyhaemoglobin (HbCO), which has approximately 200 times greater affinity for haemoglobin than oxygen. Consequently, there is decreased oxygen capacity of the blood and gradual anoxia. CO and HbCO

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levels of over 20 per cent in blood are normally seen in those individuals with acute intoxication with levels in normal individuals of below 1 per cent, although over 10 per cent blood HbCO can be seen in smokers.222 Blood levels of HbCO can rapidly drop following removal from exposure and within a few hours can return to normal making clinical investigation difficult where patients are not suspected as being exposed, because routine pulse oximetry is not able to detect HbCO.228 Other haem-containing proteins are also adducted by CO, and respiratory chain enzymes in mitochondria are a prime site of inhibition by CO with inhibition of cytochrome c oxidase (Complex IV) persisting long after CO levels return to normal.349 The neurological and neuropathological effects of CO are therefore due to a combination of acute hypoxia and more prolonged impairment of neuronal energy production. Similar biochemical changes also occur with cyanide exposure and the neuropathological changes also show parallels. CO also acts as an intracellular and local signalling molecule, being produced by cells in a manner analogous to nitric oxide and hydrogen sulphide and therefore acute local effects on cell signalling may be an additional factor in the mechanism of action of CO. One such finding is perhaps associated with the common frontal headache seen in CO intoxication, causing altered blood flow both cortically and in the basal ganglia, and which may be a contributory factor in the prominent necrosis seen in the globus pallidus.363 The greater affinity of CO for fetal haemoglobin means that its actions on the fetus are greater than on the mother, and exposure to CO has been reported in children born to exposed mothers and also cigarette-smoking mothers.285 The outcome of severe CO exposure with recovery has most often been investigated by means of imaging using MRI, CT or SPECT/PET imaging. Typical changes include bilateral necrosis of the globus pallidus (Figure 9.17), although these changes are not universally present and may depend on exposure levels.93 Haemorrhagic infarction of the globus pallidus can be seen on MRI,44 along with ischaemic change in the putamen and medial parts of the thalamus,474 as well as infarction of white matter and atrophy of the corpus callosum.428 One study showed that just over half of cases showed globus pallidus necrosis and a quarter showed putaminal involvement.389 Reduced basal ganglia volume appears to be associated with reduced verbal memory and is present in up to 88 per cent of some studies,237 suggesting that the globus pallidus lesion is not necessarily a typical feature in all cases. Pallidoreticular damage and thalamic changes are seen in many severely affected cases.93,167,272,537 This extended damage from the globus pallidus can be observed using susceptibility weighted imaging and is associated with more severe outcome including poorer performance on a wide range of neuropsychiatric tests, and parkinsonian features with concomitant reduction in dopaminergic imaging markers.93 Extrapyramidal syndromes of varying presentation involving dystonia, parkinsonism, and chorea100,101,402 are consequently common following high exposures to CO and show associated pathology. Delayed neurological impairment following recovery of consciousness after CO is frequent, involving cognitive impairment.240,402 With very severe cases of CO poisoning with recovery, there may be considerable cortical atrophy and prolonged

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9.17 Pallidal lesions in a case of carbon monoxide intoxication. After recovery from acute CO exposure, only minimal abnormality is seen within the globus pallidus on CT imaging (a), but there is a well-demarcated area of reduced signal (arrows) in the globus pallidus on T1-weighted imaging (b) corresponding with a region of necrosis and heterogeneous hyperintense signal on T2-weighted imaging (arrows in c). Images courtesy of Prof Jiun-Jie Wang and Dr Chiung-Chih Chang. Adapted from Chang et al.93 By permission of Oxford University Press on behalf of The Guarantors of Brain.

cognitive and neuropsychiatric impairment.93,449 Similarly, decreased blood flow to frontal lobes can be observed using SPECT in CO exposed individuals even in the absence of major neuropsychiatric impairment563 and appears to correlate with decreased neuropsychiatric and neurological performance.102,481,540 Early study suggested up to 90 per cent of individuals will show signs of cognitive impairment in a variety of domains including attention and processing speed along with changes in mood, with up to 70 per cent showing signs of hippocampal atrophy.227 Although MRI and also magnetic resonance spectroscopy (MRS) may indicate evidence of damage in key areas such as the basal ganglia (Figure 9.17),

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pallidoreticular system and white matter, in some instances there is neurological and neuropsychiatric impairment in the absence of changes on imaging.433 These imaging data indicate that pathological changes in cortical structures are relatively common following CO exposure and may reflect metabolic impairment due to CO in cortical neurons. Damage to white matter and fibre tracts are common following CO intoxication and using prospective MRI data shows that white matter changes are present as a major finding following CO poisoning, even in the absence of any major neurological complications.23,139 Bilateral hyperintense lesions in the white matter including centrum

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semiovale indicative of demyelination are frequently seen275 although in one study only 12 per cent of CO-exposed individuals showed white matter hyperintensities with no differences in centrum semiovale hyperintensities.404 This white matter damage may, however, be seen on FLAIR images even in the absence of changes on conventional T1 and T2 MRI images suggesting that simple structural MRI may not accurately reflect existing white matter change.368 White matter damage has been followed in CO poisoning where increased choline and decreased N-acetylaspartate were associated with areas of demyelination.369,469 The suggestion has been made that this white matter damage is an immune-mediated change involving production of myelin basic protein adducts similar to that seen in experimental autoimmune encephalomyelitis,523 although in the absence of clinical support for this, this needs to be treated with caution. Early experimental studies in the cat suggest that rather than true primary demyelination, there is a central wallerian degeneration with die-back of axons.238 Elevated S100B or myelin basic protein in the CSF or serum may be useful predictors of the extent of white matter damage in the CNS,63 although the utility of serum measurements of these proteins has been questioned.443 Most often, individuals with CO exposure coming to autopsy will be acute exposure cases due to suicide with no or very limited survival period after exposure. Acute fatal exposure to CO shows chromatolysis of neurons in the hippocampus and in cerebellar Purkinje cells indicative of anoxic change. The seminal work of Lapresle and Fardeau describing neuropathological changes following CO exposure and subsequent periods of survival before death describes several groups of neuropathological changes.311 Principal amongst these are the white matter changes involving the centrum semiovale, corpus callosum and within the cerebral peduncles where often confluent foci of demyelination and/or necrosis can be seen.311 Four groups of cases showing white matter change were described with Group 1 cases of short post-exposure survival (4–5 days) showing small multifocal areas of necrosis in the white matter and corpus callosum, often associated with changes in the hippocampus, cerebellum, putamen and thalamus. Group 2 cases often of longer survival duration showed marked necrosis within the centrum semiovale. Group 3 cases of intermediate duration of 2–3 weeks, frequently showed recovery then relapse associated with confluent areas of spongy demyelination with sparing of axons and perivascular myelin (‘Grinker’s myelinopathy’) and associated necrosis. A final Group 4 shows cases with limited changes often in the hippocampus and cortex of focal necrosis and spongy change perhaps indicating a more anoxic change. In all these groupings, however, there is a continuum of pathology with variable degrees of change. The white matter changes and axonal damage in CO intoxication show the presence of axonal bulbs and torpedoes, which can be seen using amyloid precursor protein immunostaining in the areas of demyelination.136 Astrocytic hypertrophy and microglial activation typically accompanies these areas of necrosis and demyelination. Also within these areas, necrotic changes are observed involving lipid and haemosiderinladen macrophages and in the globus pallidus and hippocampus calcium deposits within the neuropil surrounding vessels. Vascular changes involving the presence of enlarged

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perivascular spaces, regions of capillary and arterial necrosis, endothelial loss and swelling, and accompanying red cell extravasation are common, particularly in areas of necrosis within the anterior globus pallidus. Experimental reproduction of some of the features of CO poisoning suggests that decreased blood flow to the globus pallidus may be a cause of the necrosis possibly due to CO action on the vasculature504 and that the ‘Grinker’s myelinopathy’ may be due to a combination of initial hypoxia due to CO followed by secondary hypotension at a later stage corresponding with the diphasic clinical presentation seen in the patients of Group 3 of Lapresle and Fardeau.311,394 Corresponding with the areas of necrosis such as in the globus pallidus, putamen, substantia nigra and thalamus, there is associated neuronal loss and this can be seen in the hippocampus, particularly in CA1 and CA3, and also in patchy areas within the cerebral cortex and loss of cerebellar Purkinje cells.4,136,311 Neuropathologic investigation shows oedema in the basal ganglia and hippocampus along with the presence of both apoptotic and necrotic neurones.543

N-Methyl-4-Phenyl-1,2,3,6Tetrahydropyridine Considerable attention has been given to the possibility that environmental exposure to a neurotoxin is associated with the development of Parkinson’s disease. This hypothesis stems from the finding that human exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can cause acute-onset parkinsonism119,309 and from epidemiological studies of Parkinson’s disease that indicate that disease risk may be increased by increased exposure to pesticides in particular. MPTP was initially identified as a contaminant in preparations of methyl-4-phenyl-4-propionoxypiperidine (desmethylprodine) a synthetic opioid used illegally, following identification of clinically acute onset parkinsonism in the synthetic chemist who had prepared desmethylprodine.119 At autopsy 18 months later, the individual showed typical nigral degeneration with Lewy bodies, although the association with MPTP was not made at the time. Following development of acute onset l-dopa responsive parkinsonism in several drug users309,572 and also in an industrial chemist exposed to MPTP,308 MPTP was identified as the specific chemical, with reproduction of clinical symptoms in experimental animals with an estimated total dose of between 160 and 640 mg of MPTP over several weeks sufficient to cause parkinsonism in man.309 Although humans and primates are sensitive to the effects of MPTP,65,257,289,309 rodents are generally resistant with rats only showing reversible nigral pathology at near lethal systemic doses, and mice showing slightly higher sensitivity, though this is strain dependent.199 The selectivity of MPTP in causing parkinsonism is achieved by MPTP being able to cross the blood–brain barrier as a result of a relatively neutral charge, whereas in astrocytes it is converted by monoamine oxidase-B via the intermediate compound MPDP86 to the active metabolite MPP+.97,333 The generally accepted view is that MPP+ is selectively taken up by the dopamine transporter into dopaminergic neurones256,333 and actively concentrated in mitochondria441 where it acts as an inhibitor of the mitochondrial respiratory chain, selectively inhibiting complex I and causing depletion of cellular

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energy supply and cell death.378,442 Although the pathological changes seen in MPTP-induced parkinsonism (nigral degeneration and Lewy bodies) are identical to typical Parkinson’s disease,119,308,309 it is unlikely that any further cases will ever be apparent to routine pathology. The active metabolite, MPP+, is, however, the chemical cyperquat, which is used as a herbicide along with similar compounds such as paraquat (1,1′-dimethyl-4,4′-bipyridinium), which are thought to act like MPTP by causing inhibition of complex I of the mitochondrial respiratory chain. In rodents, although paraquat shows nigral degeneration,57,332,340,522 there are differences in mechanisms of action,57,451 which suggest that paraquat produces its effects by redox cycling with indirect mitochondrial damage.521 In man, paraquat toxicity, at least acutely, is not associated with direct neurological complications, although most reports show patchy intracerebral necrosis particularly in the white matter in paraquat fatalities.184,242,366 This distribution of pathology may relate to the abnormal oxygen consumption caused by paraquat poisoning,580 the severe lung damage and subsequent hypoxia, myocardial depression and depletion of cellular NADP. Of note, paraquat poisoning is difficult to treat when certain levels of paraquat have been ingested, and patients may survive for several days before death with supportive care. The neuropathology of poisoning may change with time. Similarly, diquat (1,1′-ethylene-2,2′-bipyridyldiylium) causes intracerebral haemorrhage, often of the brain stem260,463,549 but also within the globus pallidus, similar to CO poisoning, with recoverable hemiparesis,468 but may also cause persistent parkinsonism.479 The long-term neurological consequences of paraquat or diquat following recovery from exposure are unknown. Animal studies have provided considerable support for the concept of mitochondrial toxins and development of Parkinson’s disease. In primates, MPTP exposure either acutely or chronically causes a relatively selective degeneration of dopaminergic neurones in the substantia nigra with accompanying gliosis,65 but also of the paranigral (A10) group.564 This cell loss also shows accompanying necrosis, possibly due to the rapid onset and appears either acutely518,564 or chronically.203,551 Mitochondrial inclusions are seen in macaques treated with MPTP518 and in baboons this appears to involve the deposition of α-synuclein in the remaining neurons, although these do not have the appearance of typical Lewy bodies (Figure 9.18).289 Depletion of neurons in other brain areas is also present with loss of dopaminergic neurons and accompanying gliosis in the hypothalamus of marmosets,174 and in some instances locus caeruleus neurons are lost518 and serotonergic neuron changes as evidenced by decreased serotonin markers in the striatum.411 The pesticide rotenone ((2R,6aS,12aS)-1,2,6,6a,12,12ahexahydro-2-isopropenyl-8,9-dimethoxychromeno[3,4-b] furo[2,3-h]chromen-6-one) has received considerable attention recently as a toxin based model for Parkinson’s disease. Rotenone is normally used as a general purpose non-specific insecticide and in some cases for the elimination of fish and is found naturally occurring in several different genera of leguminous (pea) plants such as Derris eliptica. As a highly specific and sensitive mitochondrial complex I inhibitor, rotenone is also used in biochemical assays of mitochondrial function186 and chronic administration of rotenone has been used to model aspects of Parkinson’s disease.41,82 Pathology is reproduced in rats only near to the maximal

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9

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9.18 Sections through the substantia nigra of the common marmoset (Callithrix jacchus) after treatment with the neurotoxin MPTP. (a,b) The sections were labelled with anti-α-synuclein antibody and show diffuse and finely granular α-synuclein in the cytoplasm of remaining neurons along with proliferation of glial nuclei. Images kindly supplied by Dr Sarah Salvage, Dr Atsuko Hikima and Professor Peter Jenner of Neurodegenerative Diseases Research Group, Institute of Pharmaceutical Science, School of Biomedical and Health Sciences, Guy’s Campus, King’s College, London UK.

tolerable dose and many animals die acutely showing cerebral perivascular haemorrhage and oedema in the CNS and similar changes in peripheral organs, comparable to other

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agents causing anoxia.17 For some of the rats that survive older dosing regimens, there is selective degeneration of nigral neurons and the presence of amorphous α-synuclein positive inclusions41,487 accompanied by early microglial activation.486 A similar pathology can also be achieved by infusion of the rotenoid deguelin into rats.76 Acute ingestion of rotenone and fatality in man is rare and does not appear to cause nigral degeneration, although multiple small haemorrhagic infarcts were described in one case372 and in another.406 In this respect, rotenone, at least acutely, shows some similarities to carbon monoxide and cyanide toxicity in causing acute anoxic change. In one case of poisoning in a child who died 8 hours after intake of a mixture containing 6 per cent rotenone, the brain showed signs of an ‘acute hypoxic episode’, but no specific details were given.128

3-Nitropropionic Acid 3-Nitropropionic acid is a fungal metabolite of the genus Arthrinium and was found to be responsible for human poisonings through consumption of contaminated sugarcane. 3-Nitropropionate is a structural analogue of succinic acid and irreversibly inhibits succinate dehydrogenase, complex II of the mitochondrial respiratory chain. Local injection of malonate, a reversible inhibitor of succinic dehydrogenase, produces a very similar lesion.34 Cases, often children, develop convulsions followed by coma and death.224,348 Dystonia often occurs following recovery from acute symptoms and CT scans show low-density lesions predominantly in the putamen.224,348 No neuropathological findings on human cases have been reported in western literature. Pathology in animals has been studied owing to the similarity with Huntington’s disease pathology, with mice,182 rats198 and primates showing lesions in a number of brain areas with a clear sequence of relative vulnerability (see Table 9.5). There is selective loss of medium-sized neurons in the putamen,379 with vascular breakdown preceding neuronal loss, lesser involvement of the globus pallidus, and only occasional involvement of the caudate or claustrum,164,328 although in some human cases a more diffuse lesion occurs.224 Repeated lower-dose administration in rats574 and primates leads to selective neuronal death, confined to GABAergic/substance P-expressing medium-sized spiny neurons in the rostrolateral caudate/putamen.399,455 Lesions caused by 3-nitropropionic acid are characterized by mitochondrial swelling in neurons, first in processes and then in cell bodies. Axonal swelling and myelin splitting occur mainly in the internal capsule, resembling that seen after hypoxic/ischaemic damage. Astrocytic mitochondria were involved less prominently than those in neurons, with oligodendrocytes minimally affected. The vasculature was involved at all stages of lesion development, with a decrease in endothelial cell cytoplasmic density, thrombin accumulation in capillaries, aggregates of perivascular protein and petechial haemorrhages.450 This vascular pathology described earlier164,379,450 has not been noted by some,33 possibly because they are examining end-stage lesions where there is vascular repair. There may be a useful analogy with the primarily vascular and astrocytic lesions produced by m-dinitrobenzene456 or the primary astrocytic lesions produced by α-chlorohydrin,89 which produce a secondary neuronal loss that becomes difficult to distinguish

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Table 9.5  Topography of damage produced by metabolically acting neurotoxic agents or hypoglycaemia in the rat brain Inferior colliculia,b,c Auditory cortex Anterior cingulate cortex Superior olivesa,b,c Sensorimotor cortexf Frontal cortexf Medial geniculate nucleia Parietal cortex Lateral habenular nuclei Cochlear nucleia,b,c Visual cortex Caudate nucleib,d,e,f Vestibular nucleia,b,c,d Thalamic nucleia,d Superior colliculi Cerebellar nucleib,c,d Hippocampusb,d,f Red nucleia,b Inferior olivesa Septal nuclei Pontine nucleia Globus pallidusd Cerebellar cortexf Substantia nigrad,e Hypothalamus Amygdala Corpus callosum Cerebellar white matter Brain regions are listed in decreasing order of glucose phosphorylation rate in the normal conscious rat.375 Neurotoxic agents: a3-chloropropanediol; b6-aminonicotinamide; c1,3-dinitrobenzene; d3-nitropropionate; erotenone; fhypoglycaemia.

from primary neuronal damage at survivals of greater than 2 days.

Cyanide Toxicity Block of oxidative energy metabolism by hydrogen cyanide or cyanide salts can produce neuropathology by inhibition of mitochondrial cytochrome oxidase (Complex IV). Cyanide toxicity outside of self-harm is seen most commonly as a result of inhaling hydrogen cyanide from burning plastics,12 although cyanide salts are used in electroplating, as nitroprusside in the treatment of hypertension, and cyanogen chloride has been used as a chemical warfare agent. In

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forensic cases, cyanide toxicity can be seen in victims of house fires where carbon monoxide will be an additional neurotoxicant.12 The effects of cyanide poisoning closely resemble those of hypoxia when not acutely fatal and show similarities to carbon monoxide poisoning with parkinsonism or dystonia being a common finding.151,183,410,458,546 MRI shows symmetrical striatal, cerebral and cerebellar lesions after cyanide poisoning, often with delayed onset,437,461 with reduced dopaminergic markers in the striatum indicative of nigral pathology.458,586 One report of pathology, however, showed no dopamine neuron loss in the substantia nigra despite parkinsonism, which may indicate that reduced striatal signal is due to striatal rather than nigral pathology.544 Histopathology of cyanide survivors shows cerebral, striatal and cerebellar neuronal loss with one report showing pseudolaminar necrosis.453 In individuals dying after house fires where cyanide or carbon ­monoxide may be contributors, the presence of intranuclear ubiquitinated Marinesco-like bodies and diffuse ubiquitinated staining in substantia nigra dopaminergic neurons has been reported.435 Hydrogen sulphide also acts on cytochrome oxidase in a similar manner to cyanide, and acute poisoning with hydrogen sulphide (commonly encountered in the oil-refining industry) produces similar effects.

Methyl Bromide Methyl bromide poisoning usually results from its use as a fumigant and antifungal agent, and despite aims to reduce its use in developed countries its use is nonetheless widespread. Methyl bromide and methyl chloride intoxication show acute headache, vomiting and nausea progressing to convulsions and coma.244,488 The production of petechial haemorrhages, oedema and neuronal loss in the cerebral and cerebellar cortices, dentate and brain stem nuclei220 suggests a metabolic basis for toxicity.87,170 In some instances, mammillary body and inferior colliculus pathology is seen with a Wernicke’s-like distribution,505 although the mechanism is not understood. A distal sensorimotor polyneuropathy is also seen. Ataxia is noted at least acutely and MRI in several instances has shown signal changes in the splenium of the corpus callosum28,266 and in the dentate nucleus.515 The bioactivation of methanol to the cytochrome oxidase inhibitor formate suggests that methanol toxicity may be due to impairment of energy metabolism.377 Formate is normally present in the body, but elevated levels are associated with ocular lesions in primates497,520 and folate deficiency enhances its neurotoxicity. Although methanol initially produces an acute intoxication similar to that of ethanol, this is succeeded by nausea, vomiting, headache, loss of visual fields, and extrapyramidal signs (rigidity, akinesia, dystonia). The extrapyramidal signs are associated with selective necrosis of the putamen, similar though not identical to the effects of hypoxia.320,342,539 Methanol produces retinal oedema and axonal swelling in the anterior segment of the optic nerve along with considerable gliosis and loss of vascular markers.484,539

Organophosphorus Compounds Some of the more widely used insecticides belong to the organophosphorus (OP) group, which are widely used in agriculture and sometimes in home use, but also as

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chemical warfare agents (e.g. sarin, soman). Given their widespread use, OP compounds are readily available and often used in cases of self-harm.140 Although acute effects are well recognized and due to inhibition of acetylcholinesterase, long-term, low-grade use of some OP compounds results in organophosphate-induced delayed polyneuropathy (OPIDPN), characterized by a distal distribution and delayed onset,326 though some evidence suggests low-dose exposure may have peripheral effects in the absence of overt neuropathy506 and potentially neuropsychiatric problems.29,40 A dying-back axonopathy affects sensory, motor and autonomic neurons, although in mild cases degeneration may be confined to the extreme distal (intramuscular) processes of motor fibres. Other than an accumulation of smooth endoplasmic reticulum, there are negligible morphological changes before the onset of axonal degeneration and functional loss 10–14 days after acute exposure.52 Some large-diameter fibres are particularly sensitive, including the proprioceptive afferents from muscle spindles. Within the CNS, long tracts in the spinal cord, medulla and cerebellum are involved. Young people and animals are relatively resistant to OPIDPN. In a series of 12 high-dose intoxications, clinical improvement was observed in (peripheral) lower motor neuron function over 1–2 years, but not centrally.552 Mechanistic studies have shown that the nature of the neurotoxicity produced by an organophosphorus ester can be predicted by its relative ability to interact with either acetylcholinesterase (acute cholinergic toxicity) or neuropathy target esterase (OPIDPN).358 It is possible to classify agents in terms of producing neuropathy at doses that are not acutely toxic (tri-o-cresyl phosphate), that are acutely toxic (mipafox) but survivable, that are survivable only with acute therapy (trichlorophon, dichlorvos), or that have little or no neuropathic potential at any dose (sarin, paraoxon). The first two classes are banned from use as pesticides, but some agents from the third class have produced neuropathy in people who have attempted suicide and have required intensive care therapy. No current pesticides produce OPIDPN at dose levels that fail to produce severe acute cholinergic signs, but there have been several hundred predominantly historical cases of OPIDPN due to the lubricant tri-o-cresyl phosphate.562 The mechanism of OPIDPN is at present uncertain, because the physiological role of neuropathy target esterase (NTE) is unclear. Mutation in this gene (patatin-like phospholipase domain containing protein 6, PNPLA6) causes an autosomal recessive disorder with remarkable similarities to OPIDN with an upper and lower motor axonopathy and spinal cord lesions,439 suggesting that impairment of NTE function causes OPIDN.

9

Metals Exposure to different metals has been established as a cause of either generalized disease or specific symptoms that can often indicate the nature of toxic exposure. Given the nature of exposure to metals, toxicity often results from industrial use or, often with childhood exposures, where parental activities involving industrial exposure to metals leads to exposure of the infant. Regulation of exposure has largely reduced toxicity in developed countries, however, there are still circumstances, many accidental, which can lead to neurotoxicity.

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Lead

Lead has long been established as having both acute and chronic effects on the nervous system. While in developed countries there are strict regulations on exposure, there are still instances, often because of poor occupational health monitoring, where lead neurotoxicity occurs. Lead toxicity is to a certain extent dependent on the chemical form of lead (organic versus inorganic lead salts), although the outcome is often the same. High-level exposure to inorganic lead (greater than 100 μg/dL blood) can result in gastrointestinal problems (‘lead colic’), encephalopathy and peripheral neuropathy. Exposure can occur from old paint, soil, water standing in lead pipes, glazes in non-commercial pottery, lead glass and leaded petrol. The encephalopathy is seen more commonly in children than in adults, who require much higher exposures to develop lead encephalopathy. Characteristic effects of acute lead poisoning are vomiting, ataxia, apathy, convulsions and coma. Morphological correlates are brain oedema,430 particularly of the cerebellum, which can be observed on MRI24,401; focal neuronal necrosis with a disproportionate astrocytic reaction; swelling of endothelial cells associated with increased vascular permeability; and petechial haemorrhage with subsequent vascular proliferation observed experimentally.431 These changes are prominent in the neocortex and cerebellum. Astrocytes appear to play an important role in limiting lead exposure, and astrocytic changes are a prominent feature of lead toxicity.380 Acute lead toxicity in neonatal animals is also characterized by prominent oedema and petechial haemorrhage.106 The strong binding of lead to plasma proteins means that its actions are restricted largely to the peripheral nervous system (PNS) and the brain vasculature, except in neonates. Organic lead compounds enter the CNS and produce prominent neuronal loss with mitochondrial swelling in the hippocampus, striatum and brain stem experimentally though human toxicity is rare.79,177,559 Lead has a potent effect on the retina, retinal toxicity representing the most sensitive effect of lead in adult experimental animals with experimental evidence for apoptotic death of rods and bipolar cells.158 The peripheral neuropathy produced by lead is a primary motor and segmental demyelination with secondary axonal loss. As with lead encephalopathy, vascular damage is a prominent feature. Lead neuropathy is now seen only rarely, although significant reductions in radial nerve conduction velocity have been seen in groups of workers with exposures close to the neuropathic threshold.142 Lead remains a common developmental neurotoxicant.282 Although high-level neonatal lead exposures with marked effects on intellectual development are rare, lower exposure levels producing small but significant effects are more common and blood lead above 10 μg/dL requiring intervention.282 Maternal blood lead levels in excess of 10 μg/dL are associated with demonstrable abnormalities in the electroretinogram at 7–10 years of age.282 Damage due to lead is greatest in the prefrontal cortex, hippocampus and cerebellum.156 Animal studies have shown ­sensitivity to be greatest in the later stages of brain development, with delayed synaptogenesis and impaired learning performance though gross pathology is not apparent.374 The mechanisms of lead toxicity are not clear, but lead substitutes for calcium and interferes with NMDA receptor expression

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and function, which could perturb brain development.372 Competition between lead and calcium for intestinal absorption leads to the greater bioavailability of lead, and hence neurotoxicity, if there is a lack of calcium in the diet.

Aluminium Aluminium is the most abundant metal in the earth’s crust and excessive exposure is associated with acute and chronic encephalopathy. Early use of tap water for renal haemodialysis resulted in a predominantly reversible e­ncephalopathy in patients. This appeared as a slow onset progressive cognitive impairment with dysarthria and apraxia, myoclonus with specific EEG changes and eventual coma, which was subsequently found to be linked to aluminium in the tap water.15 Patients dying during these acute episodes showed generalized cortical spongiform pathology, although few other changes.64 Aluminium accumulates in cortical neurons via transferrin-mediated uptake with little further distribution providing the relative selectivity for cortical pathology.361 Subsequent studies also showed cases on continuous ambulatory peritoneal dialysis,499 but also the potential for further cases as a result of the use of aluminium containing phosphate binders used to control hyperphosphataemic osteomalacia in renal patients. These aluminium compounds have been replaced with calcium-, magnesium- and more recently lanthanum-containing compounds.325 Contamination of a water supply in the UK with aluminium sulphate in 1988 was associated with some neuropsychological effects in the local population, but these could not be attributed directly to aluminium.398 Chronic aluminium exposure has been proposed as a contributor to Alzheimer’s disease as a result of the finding of raised aluminium in some studies and experimental evidence for a form of neurofibrillary degeneration following intracerebral administration of aluminium salts,112 but no consistent epidemiological evidence has been found to support this.

Mercury Mercury has long established its role as a major neurotoxin although, as with many metals, the picture of toxicity varies with the state of mercury as organic mercury, metallic mercury (Hg0) or mercury salts (Hg+). A particular place also exists within neuropathology owing to the use of mercuric chloride in Golgi impregnation methods and in neurosurgery in the past use of Zenker’s fluid. Metallic mercury, being poorly absorbed through the gut, has low toxicity when ingested, and shows low acute toxicity when injected during cases of self-harm or accident, but chronic exposure to mercury vapour or potentially metallic mercury is associated with a progressive neuropsychiatric disorder of insidious onset, characterized by nervousness, emotional instability, depression, anorexia, insomnia, cognitive problems, and development of intentional tremor with micrographia, myoclonus, and in terminal cases, coma and death. Gingival disease and ‘erythism’ is also seen in typical cases.49 MRI has demonstrated ­generalized cerebral and cerebellar atrophy.568 Metallic mercury is suggested to preferentially enter large cortical motor neurons, which may explain the symptoms of intentional tremor.400 In one report of acutely fatal mercury vapour inhalation, perivascular inflammation and multiple areas of cortical and white

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matter necrosis with accompanying gliosis were seen.267 Mercury injection has also been associated chronically with myoclonus.438 Exposure to inorganic mercury salts, such as mercuric chloride (Hg2+) or mercurous salts (Hg22+), is regarded as more toxic than metallic mercury as a result of the enhanced absorption. Mercury salts have a long history involving a variety of uses as treatments for syphilis, skin whitening creams, and teething aids in children with consequent reports of toxicity. Mercuric chloride is acutely toxic causing major kidney damage and has a lowest lethal dose (LDLO) of about 30 mg/kg. One report showed white matter hyperintensities indicative of vascular damage following exposure in a child.37 Peripheral polyneuropathy and accompanying central effects have been also been observed following chronic use with demyelination and axonal degeneration being found in nerve biopsy.124 Organic mercury is generally more toxic than mercury salts and has been associated with major toxicity. Ethyl mercury toxicity has been rarely reported, though in one report following ingestion of contaminated meat, progressive gait disturbance and ataxia developed with eventual deterioration, coma and death. In non-fatal cases, eventual recovery occurred with some constriction of visual fields suggesting damage to the primary visual cortex. Neuropathology on fatal cases showed demyelination of cranial nerves and extensive loss of motor neurons at multiple levels within the spinal cord along with chromatolysis of remaining neurons. Loss of granule cells in the cerebellum was a feature with axonal torpedoes in Purkinje cells. Cortical gliosis with loss of neurons was observed with the visual cortex being affected; additional small neuron loss was seen in the caudate and putamen with gliosis.105 Dimethyl mercury is one of the most toxic compounds known with exceptionally rare fatalities and major nervous system pathology. The archetypal case, due to skin absorption of a few hundred microlitres of dimethyl mercury, resulted in progressive weight loss, nausea, diarrhoea, paraesthesia in the extremities, tinnitus, gait disturbance, progressive neuropsychiatric decline and eventual coma with death approximately 10 months later. The brain was grossly atrophic with cortical thinning, neuronal loss and accompanying gliosis, with marked cerebellar degeneration and loss of granule cells.493 Methyl mercury has been associated with several cases of mercury intoxication owing to its widespread use in industry as part of chlorine and sodium hydroxide production, plastics production, and use as a fungicide for seed storage. Large scale poisonings have been seen in Japan in 1956–60 after environmental contamination,143 Iraq in 1972 after its use as a fungicide,13 and in Canada following environmental contamination.341 In adults, pathology is almost entirely confined to the nervous system with progressive symptoms including paraesthesia of the extremities and lips, decreased concentration, fatigue, speech impairment, ‘erythism’, dysphagia, constricted visual fields and eventual blindness, ataxia, tremors, incoordination, abnormal reflexes, severe hearing loss, permanent CNS damage, coma and death with an estimated 25 mg whole body exposure required to give paraesthesia.143 The neuropathological damage can be very selective, with loss of small neurons of the anterior primary visual cortex, somatosensory cortex, and superior temporal cortex and deep cerebellar granule cell loss

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with relative sparing of Purkinje cells with the presence of stellate bodies on staining with silver staining.147,148,243 Peripheral neuropathy is seen, sensory nerves being more affected, with wallerian degeneration and degeneration of gracile tracts,148 this being persistent but mild and possibly related to damage to the somatosensory cortex.541 The exact mechanism of methyl mercury toxicity is unknown, but methyl mercury is converted slowly to thiol-bound inorganic mercury, affecting glutathione production and cysteine containing proteins.195 Methyl mercury is a potent developmental neurotoxicant, with multiple examples of fetal and infant exposure following outbreaks. Affected cases show cerebral atrophy with abnormal cortical layering as a consequence of disordered neuronal migration,99 particularly in the primary visual cortex, but also in the precentral, post-central and lateral temporal cortices, cerebellar atrophy with Purkinje cell and focal granule cell loss, degeneration of gracile tract fibres, and a sensory peripheral neuropathy.148 The pathology is least selective with earlier exposure and is always less selective than that seen in adults. Pathology is not seen in infants exposed to moderately elevated exposures to methyl mercury as a result of diet.310

9

Manganese Although manganese is an essential element, overexposure via mining and the production of alloys and batteries, and potentially use of manganese as a flux in welding are occupational causes of exposure, with over 50 000 individuals in the UK exposed through the workplace. Manganese toxicity may also be a feature in intravenous drug abusers using ‘ephedrone’ (2-methylamino-1-phenylpropan-1-one, methcathinone) manufactured using potassium permanganate.494 Hepatic disease, particularly hepatic encephalopathy (see earlier under Hepatic Encephalopathy, p. 596) when there is portal vein shunting also causes abnormal retention of manganese.217,291 Several studies have shown neuropsychiatric disturbances in individuals exposed industrially to manganese without the presence of overt neurological symptoms with workers showing increased levels of anxiety, fatigue, decreased fine motor skills, with persistence of these effects even after exposure ends.51,344 These symptoms are present in highly exposed individuals who also show variously dystonia, parkinsonism, or chorea with no or poor response to l-dopa. MRI in cases of hepatic disease or in welders exposed to high levels of manganese shows hyperintensity of the globus pallidus on T1-weighted imaging in many cases with cirrhosis259,279,331 and a characteristic selectivity of manganese neurotoxicity (Figure 9.19). Damage is largely restricted to the globus pallidus, where there is neuron loss, demyelination and gliosis, and in the substantia nigra zona reticulata with relative sparing of the dopaminergic neurons in the zona ­compacta, unlike Parkinson’s disease, although dopaminergic dysfunction is seen in non-human primates (suggesting that striatal dopamine release is compromised).191 There is also some involvement of the subthalamic nucleus and mammillary bodies in post-mortem cases.579 Once neuronal loss has developed, characteristic motor problems such as rigidity, ataxia and fine tremor persist, although chelation therapy has shown some reversal if brain manganese

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618  Chapter 9  Nutritional and Toxic Diseases (a)

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9.19 MR images from a patient with chronic manganese toxicity, showing strongly hyperintense signal in the globus pallidus and substantia nigra bilaterally. The images were obtained using a 1.5-T system in the sagittal (a,b) and axial (c,d) planes, with SE T1-weighted images 6.0 mm thickness/1.0 mm spacing. Images kindly supplied by Dr MC Valentini, Chief of the Neuroradiological Department, University of Turin, Italy.

levels can be reduced.231 Selective retention of manganese in the striatum and globus pallidus, interference with iron homeostasis and damage to mitochondria are likely mechanisms of pathogenesis.21

Arsenic A distal polyneuropathy develops 10–20 days after a single exposure to inorganic arsenic, as occurs after malicious administration of arsenic salts, occupational exposure, burning high-arsenic-content coal322 and drinking water from deep wells in arsenic-rich strata.545 Sensory signs can

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sometimes be seen in advance of motor signs, but they are usually seen together. Cranial nerve involvement has also been described. Overt arsenic poisoning causes gastrointestinal effects followed by development of a distal motor and sensory neuropathy that is characterized by a dying back axonopathy of myelinated fibres with limited regeneration.576 Functional recovery can take several years. Exposure to arsine gas (AsH3) can also cause a similar neuropathy. The toxicity of arsenic is probably mediated by the trivalent form, which inhibits formation of acetyl co-enzyme A formation by pyruvate dehydrogenase by a mechanism involving redox reactions.545

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Thallium A predominantly peripheral, but also central, dying-back axonopathy with distal paraesthesia and hyperaesthesia followed by muscle weakness, cranial neuropathy, optic neuropathy with lens opacities, autonomic involvement, and alopecia with characteristic blackening of the hair roots, is seen after occupational or malicious poisoning by thallium.88,466,516 Long, large-diameter sensory fibres are most sensitive, and central involvement is usually late and limited to long tracts. High doses produce poorly characterized neuropsychiatric features and encephalopathy,525 plus choreoathetosis, tremor and dystonia, the morphological accompaniments of which include vascular damage and neuronal loss. The effects of thallium closely resemble those of arsenic, and they may share a common mechanism via inhibition of the pyruvate dehydrogenase complex.88,525

Tin A selective excitotoxicity is produced by the fungicide trimethyl tin and also with triethyl lead. Trimethyl tin produces a selective and early increase in limbic system excitability in experimental animals.444 The pattern of neuropathology in the hippocampus, pyriform cortex, amygdaloid nucleus, neocortex and various brain stem nuclei is typical of excitotoxicity,14 with dendritic vacuolation and pyramidal neuronal necrosis. Not dissimilar changes have been seen in humans with white matter changes on MRI that resolved.246,290 It has been proposed that trimethyl tin may be concentrated specifically in vulnerable neurons because of their greater expression of a mitochondrial protein, stannin (stanniocalcin), which binds trimethyl tin.14 Disruption of the structure of central and peripheral myelin is a feature of poisoning with triethyl tin with myelin swelling found mainly in the CNS and optic nerve, with effects on peripheral nerve being restricted largely to the spinal nerve roots. Triethyltin is also directly ototoxic. Oligodendrocytes do not seem to be damaged directly, and the myelin swelling is completely reversible if secondary damage does not develop. In France in 1954, 290 people were poisoned by a preparation containing 5 per cent triethyl tin to treat boils. Of these, 110 died with central myelin swelling and raised intracranial pressure. Damage to myelin may be mediated by disruption of the charge interactions that hold myelin lamellae in tight apposition, but true demyelination is produced by chemicals damaging oligodendrocytes or Schwann cells. Both myelin disruption and demyelination impair nerve conduction and both are usually reversible, although the myelin sheath around remyelinated axons remains thin and intranodal lengths are often shorter than normal.476

Excitotoxins The production of sustained neuronal hyperexcitation can generate neuronal death by compromising ionic homeostasis. Damage is usually focal and common features are postsynaptic swelling and selective neuronal necrosis, commonly caused by direct s­timulation of ­neuronal ­glutamate receptors. The density of these largely determines vulnerability, but other factors such as inherent local excitability (e.g. high in limbic circuits) and local

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lack of a blood–brain barrier (e.g. in the area postrema) are also important. Exogenous glutamate, because of the high-activity reuptake systems in neurons and astrocytes that limit exposure, is neurotoxic only after exceptionally high doses. An excessively sustained release of endogenous glutamate as a consequence of brain injury, stroke or epilepsy, however, is a significant cause of ­secondary neurotoxic injury. The similarity of exogenous chemical-induced excitotoxicity and that of hypoxia–­ischaemia (see Chapter 2) is a reflection of the importance of excitotoxic release of endogenous glutamate seen during reperfusion. Although in human material it is often difficult to dissociate the consequences of poisoning from incidental hypoxia, this is possible in experimental animal studies, enabling primary excitotoxicity to be recognized.

9

Domoic Acid Domoic acid first came to light as a potential neurotoxin in man following an outbreak of amnesic shellfish poisoning on Prince Edward Island in Canada.413 Affected individuals had eaten mussels contaminated with a marine diatom of Pseudo-nitzschia spp. that produce domoic acid and developed abdominal cramps, vomiting, diarrhoea, nausea, severe headache and, in some instances, short-term memory loss.413 In a subgroup of exposed individuals, confusion, disorientation, seizures, myoclonus and coma were seen, with death in a small number, although comorbidities due to age may have been confounding factors.413,519 Longer-term peripheral motor or sensorimotor neuronopathy and/or axonopathy are observed in severely intoxicated people.519 Neuropathology in four individuals who showed neurological complications after exposure and died from 7 days to more than 3 years after exposure showed necrosis and loss of neurons in the hippocampus with some selectivity for the CA1, CA3 and CA4 neurons and neuron loss in the amygdala.91,519 Additional sites of pathology, predominantly consisting of neuronal loss and accompanying gliosis, were the claustrum, olfactory cortex, septum, nucleus accumbens and, in some instances, the medial thalamus, insula cortex and orbitofrontal cortex, although the brain stem motor nuclei and spinal cord were spared.519 Although CNS effects are major factors causing disability, peripheral effects on the heart and other organs by domoic acid may contribute to fatal outcomes.434 Current regulatory control of shellfish quality and domoic acid levels makes the likelihood of outbreaks of domoic acid intoxication unlikely, although there are still instances of illegal harvesting of contaminated shellfish and mild poisoning that may cause long-term neuropsychiatric impairment. Pseudo-nitzschia spp. contamination of shellfish is almost global, with increasing reports of fish contamination. Domoic acid is structurally similar to glutamate and kainic acid (found in red algae) and has its effects by acutely activating ionotropic glutamate AMPA/kainate receptors, which are most concentrated in limbic regions.414 This causes influx of calcium into neurons and depolarizationinduced glutamate release, which leads to excitotoxicity.96 Toxicity is essentially limiting because domoic acid shows poor gut absorption and blood–brain barrier penetration. Rats and mice appear relatively insensitive to oral doses of domoic acid compared to human exposures with 1–5 mg/kg domoic acid thought to give symptoms in man253,413,508

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although in macaques, intraperitoneal doses that cause symptoms appear similar to the rat or mouse.536 Pathology in primates and also rodents occurs principally in the limbic system with pyramidal neurons in the hippocampus, predominantly CA1 and CA3 neurons, affected following kindling seizure activity and shows striking similarity to human pathology.18,473,501,509,514,535 Pathology is however seen mostly in animals that show seizures, which is also accompanied by microglial reactivity and astrogliosis.18 In the macaque, mouse and rat, pathology is also seen in the hypothalamus and area postrema and other circumventricular organs.60,534 In sea lions (Zalophus californianus), which are frequently affected off the coast of California as a result of consumption of contaminated anchovies, pathology is located within the limbic system with hippocampal atrophy and loss of CA1, CA3 and CA4 neurons and also granule cell loss, which may be unique to this species because it has not been observed in human cases. Chronic low-grade toxicity may also be a problem in sea lions in causing development of chronic epilepsy.179

Organophosphates Excitotoxicity is not restricted to glutamatergic neurons of the limbic system, and there are several other examples of neuronal loss due to primary excitotoxicity. Cholinergic excitotoxicity has been described following severe poisoning with acetylcholinesterase inhibitors in animals. The lesions all involve primary neuronal necrosis. In rats, Soman produces brain lesions of a severity proportional to the signs of acute poisoning, and control of seizures markedly reduces damage.490 The lesions in primates comprise diffuse cortical damage, and more focal neuronal loss and necrosis within the amygdala, piriform cortex, hippocampus, caudate nucleus and thalamus.56 The nature of this damage, plus the association with seizures, suggests that the pathogenesis involves a combination of hypoxia secondary to breathing difficulties, direct cholinergic mediated excitotoxicity and secondary recruitment of glutamatergic excitotoxicity.502 Direct toxicity is shown by the persistence of significant damage after effective seizure control and artificial ­ventilation.490 Although there have been many clinical reports of poisoning, no morphological observations appear to have been made in survivors, though one study suggests subtle MRI changes with decreased grey matter and increased ­ventricular volume following possible lowdose sarin/cyclosarin exposure.225 In a large series of poisonings with organophosphorus pesticides with acute neurological deficits, persistent polyneuropathy, predominantly sensory, was present indicating long-term damage.255 A report of acute sarin poisoning of sufficient severity to cause seizures and dyspnoea described memory impairment and learning problems 6 months after exposure suggestive of hippocampal damage.219 Other organophosphorus poisonings have shown an asymmetrical blood flow deficit in the temporal lobe and basal ganglia, or in the primary visual cortex.80,562 These effects follow high-dose exposure, but chronic low-level exposure to organophosphorus cholinesterase inhibitors has also been associated with impaired cognition.264 A causal association has not been established and no morphological investigations have been made.

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The origin of the hyperexcitation produced by the insect repellent N,N-diethyl-m-toluamide (DEET) is unclear, but it has been the cause of acute coma, seizures and other effects in humans after exceptionally large dermal exposures, mostly in young girls.397 Pathology has not been characterized in humans, although in rats coma, EEG spiking and myelin vacuolation restricted entirely to the cerebellar motor nuclei have been reported.553 The abnormally high levels of nerve activity produced by pyrethroid insecticide poisoning in experimental animals can cause a peripheral axonal degeneration closely resembling wallerian degeneration.81 This is seen only after exposures producing marked hyperexcitability, which is due to activity on sodium channels and is probably the axonal equivalent of central excitotoxicity.53,445 Very selective excitotoxicity is produced by some other agents in the cerebellum, an area not normally affected by excitotoxicity. The fungal tremogen penitrem-A found in mouldy foodstuffs, is associated with initial Purkinje cell dendritic changes, reactive vascular and astrocytic responses and eventual cellular necrosis,90,327 related to its action on high-conductance calcium-dependent potassium channels. The industrial intermediate L-2-chloropropionic acid, damages only granule cells within the cerebellum, although it also produces neuronal loss in the medial habenular nucleus, pontine grey matter and inferior olivary nucleus, which is blocked by NMDA antagonists.138 Some images illustrating selective pathology in experimental rats are shown in Figure 9.20. Reports of toxicity in man are unknown.

Neuropathies Associated with Solvents and Other Chemicals The most important non-therapeutic chemicals causing peripheral neuropathy are the solvents n-hexane and methyl-n-butyl ketone. As with most other neurotoxins, the hazard is well recognized, and occupational poisoning is not seen in well-regulated industries, though abuse through ‘glue sniffing’ with coincident hypoxia due to rebreathing is seen. Additional hydrocarbon solvents, such as toluene, are often also involved. Prolonged exposure to n-hexane or methyl-n-butyl ketone results in a dying-back of distal sensorimotor axonopathy, with motor loss being more prominent. In man, this is characterized by axonal swellings and focal demyelination in large myelinated axons. Electrophysiological studies show that recovery is faster in sensory than motor nerves, although often delayed94,138,302 and, as with remyelination, there is often thinning of new myelin sheets. There is also electrophysiological evidence for central auditory and visual fibre tract damage with little evidence of reversibility.94 Experimental animal studies have shown that the first effect is a decrease in fast axoplasmic transport, followed by development of distal axonal swellings in central and peripheral nerves, with accumulation of neurofilaments. Axonal degeneration occurs distal to these swellings. With higher doses, the degeneration is more proximal. In myelinated fibres, the neurofilaments accumulate proximal to the nodes of Ranvier. Both nhexane and methyl-n-butyl ketone produce their effects via a common metabolite, 2,5-hexanedione with the capacity to cyclicize by reaction with lysine residues on proteins such as neurofilaments, which can then become cross-linked.588

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(a)

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9.20 Illustrations of selective and vascular pathology in experimental rats. In each case, the distribution of the lesions is determined by inherent vulnerability of the susceptible cells or brain areas rather than by the local concentration of the chemical agent. (a) Regional selectivity of 3-chloropropanediol lesions within brain stem nuclei. (b) Lesions produced by a high intrathecal dose of gadolinium–diethylenetriamine penta-acetic acid (Gd-DTPA), a nuclear magnetic resonance imaging agent. Although the agent was given via the ventricular system, pathology was in deep, rather than periventricular, structures. (c) Selective loss of cerebellar Purkinje cells produced by penitrem-A. Sections courtesy of Colin Willis and Christopher Nolan, MRC Applied Neuroscience Group, Nottingham, UK.

Because the cross-linking pyrrole–pyrrole bond requires an oxidized intermediate, the process is accelerated by oxidative stress and depletion of glutathione.589 One report

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Neurotoxicology  621

suggests the occurrence of parkinsonian symptoms following chronic solvent exposure with nigral loss, although an absence of Lewy bodies.417 A similar condition is seen with industrial exposure to high levels of carbon disulphide. This results in a distal dying-back of axonopathy and damage to cochlear hair cells leading to deafness.110 Such exposures are now rare. The mechanism is thought to involve crosslinking of axonal proteins via dithiocarbamate metabolites of carbon disulphide.495 This hypothesis is supported by the close similarity of carbon disulphide and n-hexane axonopathies in experimental animals. Toluene is one of a very few organic solvents that have been shown to produce neuropathological damage following repeated high-level exposures,155 but chronic lower level exposures also cause neuropathic changes.489 These exposures have been associated with solvent abuse with hypoxia contributing, but because the lesion is a pure central myelinopathy, primary toxicity is likely. Magnetic resonance imaging has shown white matter loss, with cerebellar, cerebral and brain stem atrophy.575 At the microscopic level, demyelination of major central tracts, especially of periventricular white matter, accompanied by astrocytosis and microglial activation, but without major axonal or neuronal loss, appears to be characteristic.283 Evoked potential studies show corresponding delays in central conduction times. The mechanism of myelin loss appears to involve initial concentration of the lipophilic toluene in myelin, with oxidative metabolism of the toluene resulting in local redox stress.339 Toluene and also xylene and styrene can cause ototoxicity. Toluene produces ototoxicity,357 and in experimental animals these effects are seen at lower exposure levels when there is also exposure to noise.329 Occupational measurements have strongly suggested that similar hearing loss may be seen in workers exposed to high, but not uncommon, levels of xylene or toluene if there is significant coexposure to noise.355,356 Experimental animal studies indicate that repeated exposure to styrene at very high concentrations produces loss of outer hair cells in the mid-frequency range.583 Mechanistic evidence suggests that cochlear hair cell toxicity may be related to local oxidative stress.561 An analogous synergy in the central auditory pathway has been described between noise exposure and chemical toxicity in experimental rats given the redox disruptor m-dinitrobenzene.446 A slowly reversible trigeminal anaesthesia follows highlevel acute and chronic exposure to trichloroethylene, a solvent and (formerly) an anaesthetic.30,152,312 Conduction studies suggest a greater effect on small-diameter fibres. Axonopathy and some neuronal loss involving the trigeminal, facial, oculomotor and auditory nuclei have been described in one case.74 In experimental animals, trigeminal nerve myelin is the primary target,30 with lipid peroxidation mediated probably by oxidative metabolism to dichloroacetylene. The reason for the special susceptibility of the cranial nerves is unclear. Trichloroethylene has recently been linked to parkinsonism in an industrial setting169 and potentially multiple system atrophy (Blain, personal communication), with an association being seen in epidemiological studies.178 Experimental studies have shown reduced dopaminergic neurons in the substantia nigra323 possibly through production of the mitochondria

9

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622  Chapter 9  Nutritional and Toxic Diseases

toxin 1-trichloromethyl-1,2,3,4-tetramthyl-beta-carboline (TaClo) via metabolism of trichloroethylene through chloral.55,273 Acrylamide (2-propenamide) is well characterized as being able to produce a central and peripheral axonopathy through occupational or accidental exposure. There have been multiple human poisoning incidents, but given the knowledge concerning acrylamide neurotoxicity very few since control of exposure was introduced.25,176 Acute exposure to significant amounts of acrylamide produces seizures and cerebellar signs, which are followed by peripheral neuropathy. Acute poisoning shows a greater reversibility than chronic poisoning.223 Despite a short half-life in the body, acrylamide shows cumulative toxic effects, with even weekly exposures producing a fully additive effect in experimental animals.165 It induces a dying-back axonopathy of the deep touch and muscle spindle afferents before motor fibres become involved,165 resulting in numbness, sweating, peeling of the skin on the hands and feet, some muscle weakness and an unsteady gait, with a marked sensory component. These effects are slowly reversible. One of the first effects seen in experimental animal studies is inhibition of fast axonal transport,346 with subsequent distal paranodal accumulation of neurofilaments. Unmyelinated fibres are also susceptible, and a sympathetic and parasympathetic axonopathy has been produced. Cerebellar Purkinje cells show an accumulation of neurofilaments at the axon hillock. No human post-mortem studies have been reported, although sural nerve biopsy has shown loss of large-diameter fibres. Most clinical studies have been carried out by monitoring nerve conduction velocity or vibration sensitivity.25 Chemotherapy with cisplatin and related agents results in a dose-limiting peripheral distal large-fibre sensory neuropathy and ototoxicity, but its potential for producing a central axonopathy is limited by poor penetration of the blood–brain barrier. Selective dorsal root ganglion cell pathology has been shown in animal models,529 although myelin breakdown and axonal loss is seen in sural nerve biopsy.526 The mechanism of toxicity is unknown, but is associated with inhibition of axonal transport and selective uptake into hair cells via the organic cation transporter.104,464 Similarly, chemotherapy with paclitaxel, ­vincristine or suramin can cause a mixed distal sensorimotor neuropathy, with loss of large myelinated fibres.84 The early neuropathic pain produced by vincristine has been shown to be associated with hyperresponsiveness and microtubular disorganization (but not loss) of C-fibres in experimental animals.531 Therapeutic use of thalidomide in multiple myeloma therapy causes neuropathy involving the dorsal root ganglia and is associated with degeneration in the dorsal columns detectable by MRI in some, but not all, patients.173,251 Therapeutic use of isoniazid causes a dose-dependent distal sensory and motor peripheral neuropathy affecting myelinated and unmyelinated nerves, and occasionally central fibres,387 particularly in individuals less able to metabolize isoniazid via acetylation.581 Acute neurotoxicity is associated with seizures. Although parenteral pyridoxine is an effective antidote, this is not always available.517 In experimental animals, distal axonal degeneration is preceded by focal accumulation of smooth endoplasmic reticulum.393 Intraneuronal oedema and loss of the blood–nerve barrier

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are seen along with regenerating fibres adjacent to degenerating fibres.254 Hexachlorophene is used widely and safely as a topical antimicrobial, but when first introduced and used at high concentrations (3–6 per cent) caused central and peripheral myelinopathy. Babies and young children were particularly susceptible. The central myelin damage was most marked in long tracts at the level of the brain stem.491 Hexachlorophene also damages retinal photoreceptors.

Developmental Neurotoxicology Several neurotoxins produce adverse effects on the developing brain after prenatal or postnatal exposure, often at lower doses than required in adults. Developmental neurotoxicity is usually irreversible with distinctive neuropathological features attributable either to the specific chemical or to the timing of the exposure relative to the stage of brain development. In the case of several agents, such as polychlorinated biphenyls, effects in animals and humans can only be seen functionally. Valproate is an effective antiepileptic drug, but early toxicity studies in mice indicated its potential to produce developmental abnormalities, which have been confirmed in children born to mothers taking valproate during the first trimester of pregnancy. The children developed spina bifida, an effect subsequently shown to be dose related.47 At lower doses, more subtle effects are seen with cognitive impairment and behavioural disturbances, an effect seen also with carbamazepine and lamotrigine.109 Although valproate can interfere with folate metabolism, valproate embryo toxicity in mice is not preventable with folate supplementation,201 and it is possible that inhibition of histone deacetylases, leading to altered gene transcription is key, an observation supported by the similar teratogenicity of the histone deacetylase inhibitor trichostatin A.194 Another antiepileptic drug, phenytoin, also produces developmental neurotoxicity, with microcephaly and (in mice) damage to the cerebellum.390 Vitamin A (retinol) overdose during early pregnancy is teratogenic in both animals and humans, resulting in multiple defects, including microcephaly and disturbances of neuronal migration.218 Similar effects are seen with isotretinoin for the treatment of acne.305 Children and fetuses show a cluster of congenital abnormalities including underdeveloped jaw, heart defects, optic nerve and retinal degeneration, external ear and neural tube abnormalities. Lower doses in animals induce behavioural defects and oxidative stress.126 By contrast, in older children and adults, headache and pseudo-tumour cerebri (increased intracranial pressure with no biochemical or morphological abnormalities) are the main dose-limiting effects when retinoic acid is used in tumour therapy.6

Bites and Stings Envenoming bites and stings are inflicted by a very wide range of animals: snakes, spiders, scorpions, bees, wasps, cone snails and fishes are just a few of those implicated regularly in incidents of envenomation. The venom is a complex mixture of small polypeptides and other molecules elaborated in a specialized venom gland and inoculated via

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Neurotoxicology  623



a hollow or grooved fang or stinging part. Most venoms serve several functions: the capture of prey, defence and the initiation of a digestive process. Components targeting the PNS and the circulation are common constituents of venom, causing neuromuscular weakness, degeneration of peripheral nerves and skeletal muscle, and abnormalities of coagulation. Neurotoxic signs are particularly common following bites by snakes of the families Elapidae, a family of short-fanged snakes that includes kraits, mambas, coral snakes and cobras, and the Hydrophidae, the sea snakes. Envenoming bites by these snakes cause the classical signs of ptosis, dysphonia, and inability to smile and generalized neuromuscular weakness. Clotting times may be prolonged, but haemorrhage is rare. Necrosis may involve the skin and skeletal muscle. The venoms of vipers of the families Viperidae and Crotalidae are rarely neurotoxic, but coagulopathies and extensive haemorrhage are usual. The coagulopathy and systemic haemorrhages seen in association with many envenomings by vipers and crotalids (pit vipers) are usually of no special interest to the neuropathologist, unless the bleed is into the CNS. Central haemorrhages are relatively uncommon, but seem to occur most often following bites by the large vipers of South America and by Russell’s viper (Daboia spp.) of South East Asia. With Russell’s viper, a central bleed into the pituitary gland is not uncommon (Figure 9.21), giving rise to a delayed and irreversible pituitary failure.286,538 Cone snail stings can be fatal. The cone snails that are of particular interest in this context are the fish-eating cones, which produce venom that is powerfully neurotoxic; death results from neuromuscular paralysis. The venoms of spiders, scorpions and sea anemones contain toxins that cause neurotoxic signs of hyper- or hypoactivity in the PNS (summarized by Goonetilleke and Harris180,215). These toxins are described later according to their principle anatomical targets. There are numerous ill-defined reports of long-lasting neurological problems resulting from envenomations, particularly following snake bites. It is probable that most problems arise when overtight ligatures have been applied to the bitten limb resulting in anoxic tissue damage. Formal studies of human neuropathology following envenoming have only rarely been reported in the clinical literature. All the data reported herein, unless explicitly stated otherwise, have been generated following work using experimental animals or isolated cells and tissues.

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9

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Post-Junctional Acetylcholine Receptor Venoms of cone snails and the elapid and hydrophid snakes all contain toxins that target the α-subunit of the postsynaptic acetylcholine (ACh) receptor at the neuromuscular junction, thus preventing the binding of ACh and causing neuromuscular paralysis. Specific antivenoms accelerate the disassociation of toxin from receptor and reverse the paralysis. Anticholinesterases may accelerate recovery. In the absence of either of these therapies, assisted ventilation will keep the patient alive until disassociation occurs naturally at 12–24 hours post-envenoming. No pathological signs have ever been documented in association with the transient blockade of the neuromuscular ACh receptor.

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9.21 Panhypopituitarism following envenoming bite by Russell’s viper in Burma. (a) Daboia siamensis, Tharawaddy, Burma. (b) Autopsy revealed haemorrhagic infarction of the anterior pituitary in a Burmese victim of an envenoming bite by D. siamensis. (c) Patient with signs of panhypopituitarism developing years after severe envenoming by D. siamensis. Copyright Professor David Warrell, University of Oxford, Oxford, UK.

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Motor nerve terminal

(also known as the psycho-organic syndrome) has been proposed as a lasting effect that is distinct from the compound-specific effects of high-level hexane exposure (axonopathy) or toluene abuse (encephalopathy). One useful feature is that there is little further progression of neuropsychiatric symptoms once occupational exposure to solvents has ceased.163 White spirit (a petroleum distillate, also known as Stoddard fluid in the United States), styrene and xylene are the solvents most commonly cited. In some countries (notably Denmark, Finland, Norway and Sweden), impaired cognitive function associated with chronic solvent exposure has been considered diagnostic of an industrial injury if other CNS diseases can be excluded.533 Although commendable from the social welfare viewpoint, this broad definition of causation has led to practical problems regarding differential diagnosis, and the causal relationship between organic solvents as a class and encephalopathy is not recognized in, for example, the USA or the UK, where regulatory limits are set solely by acute toxic potential. A number of epidemiological studies have, however, shown that neurobehavioural deficits are associated with estimates of past, but not current, solvent exposure level,271,550 although additional factors, such as ethanol consumption and employment status, are also important.163 Relatively few new cases are now being diagnosed, because exposure levels are falling.533 There is no suggestion that medical use of transitory high doses of other organic solvents, such as trichloroethylene and halothane, as anaesthetics has led to a comparable condition. Neuropathological and imaging studies have failed to differentiate organic solvent syndrome from other conditions, such as dementia, seen in non-exposed people. A single case of chronic heptanone solvent exposure ­showing multiple small white matter foci by MRI has been reported.568 Experimental animal studies of l­ong-term exposure to white spirit have indicated that there are poorly reversible neurochemical effects and an increase

Numerous toxins target the motor nerve terminal. Ω-Conotoxins in cone snail venoms block voltage-gated calcium channels, blocking transmitter release. The only pathological sign is the accumulation of synaptic vesicles in the nerve terminal. Neurotoxic phospholipases are common constituents of elapid venoms. These toxins cause the depletion of synaptic vesicles and the degeneration of both nerve terminal and axonal neurofilaments (Figures 9.22 and 9.23). Recovery of function is slow and therapeutic intervention ineffective. Assisted ventilation may be required for more than a month. The recovery of function may be associated with abnormal patterns of skeletal muscle innervation and the delayed appearance of a poorly defined peripheral neuropathy.216,298 Toxins of scorpions, spiders and sea anemones target ion channels involved in action potential regulation, causing hyperactivity and enhanced transmitter release or reduced excitability, but there are no pathological features of note.

Skeletal muscle Snake venoms from all major envenoming species may cause a severe rhabdomyolysis that results in acute and potentially fatal renal failure.235

Controversies and Uncertainties Three areas of neurotoxicology are the subject of continuing uncertainty and so are grouped here. The first relates to the poorly defined encephalopathy that has been associated with long-term, low-level exposure to organic solvents.533 Chronic solvent-related encephalopathy

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9.22 Electron micrographs of rat neuromuscular junction nerve terminals. (a) Normal terminal containing numerous synaptic vesicles. The nerve terminal is covered by Schwann cell (S) and fibroblast (F) processes. (b) Twenty four hours after exposure to the neurotoxic phospholipase A2. Note the loss of synaptic vesicles. A few vesicles remain fused to the nerve terminal membrane (arrows).

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Acknowledgements  625

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9.23 Neuromuscular junctions in soleus muscles of the rat. In the normal neuromuscular junction (a), a single axon (red) innervates the end plate, within which it forms fine terminal branches that cover the acetylcholine receptors (green). In muscle (b), 5 days after the axon had regenerated following exposure to phospholipase A2 (β-bungarotoxin), sprouts emerge from nodes of Ranvier to innervate three individual muscle fibres in close proximity to each other.

of GFAP expression (biochemical assay) in the cerebellum without overt cytopathology.304 Controversy is not restricted to observational clinical studies, where some ambiguity is to be expected, it is also a feature of some experimental studies. Thus, the pyrethroid insecticides are generally believed to be purely functional toxicants, producing hyperexcitability via reversible actions on ion channels.445 Some published animal studies, however, have indicated that repeated lowlevel exposure to permethrin has the potential to produce neuronal death indicated by scattered densely eosinophilic neurons and a reduction in neuronal counts, and also by a loss of neurofilament staining and reactive astrocytosis.2 Quantification indicated hippocampal neuronal loss of 27–32 per cent,3 and two other structurally and functionally unrelated chemicals, DEET and pyridostigmine bromide, also produced a similar pattern of neuropathology. Another pyrethroid, alpha-cypermethrin, produced a somewhat similar pattern of neuropathology in rats at a higher dose,330 with neuron loss in CA3 of the hippocampus, hypothalamus and cerebral cortex, with ‘slight’ loss of cerebellar Purkinje cells. A combination of severe neuronal loss in the hippocampus and neocortex and an

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increase in c-Fos and c-Jun immunoreactivity in the neocortex of rats given a single high dose of a third pyrethroid, deltamethrin, has been described,573 though this was potentially related to seizure-associated excitotoxicity. In contrast to most of the earlier findings, regulatory studies using less sensitive measures, but higher pyrethroid exposure levels have yielded uniformly negative neuropathological findings after both single and repeated dose administration.569 Hence, the consequences of repeated exposure to low levels of these agents remain somewhat unclear.

Acknowledgements CMM is supported by the UK Health Protection Agency, Department of Health, and the UK Medical Research Council. JBH is funded through grants from the Wellcome Trust, the Royal College of Veterinary Surgeons of the United Kingdom and the Dubai Millennium Foundation. We are indebted to the late David Ray who provided much of the section on neurotoxicology within this chapter. His wisdom and generosity will be missed.

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References 1. 2.

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5. 6.

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10 10 CHAPTER

Trauma Colin Smith, Susan Margulies and Ann-Christine Duhaime

Introduction................................................................................637 Pathology Associated with Fatal Head Injury...............................643 Intracranial Haemorrhages..........................................................646 Brain Injury Secondary to Raised Intracranial Pressure................652 Blunt Force Head Injury; Diffuse Injury........................................655 Penetrating Injuries.....................................................................665 Perinatal Head Injury...................................................................666

INTRODUCTION Epidemiology Neurotrauma is a major cause of morbidity and mortality worldwide, and yet in many cases an avoidable one, being a reflection of the availability of fast, personal transport accounting for many road traffic accidents; the easy availability of firearms in many societies; casual violence in society, often fuelled by alcohol; and the lax approach to health and safety in some countries. In western societies, road traffic accident–related neurotrauma has been in decline over the past few decades. However, in developing countries, where medical care is often already overstretched, the incidence has been increasing.364 The human brain is highly vulnerable to injury, and injury can compromise the quality of life through profound cognitive and neurobehavioral dysfunction.86 Traumatic brain injury (TBI) is an overwhelming and major global public health problem and one of the most important causes of morbidity and mortality in both industrialized and developing countries.415 Estimates by the World Health Organization (WHO) indicate 57 million people internationally have been hospitalized with one or more TBIs.326 In the United States, recent statistics from the National Center for Injury Prevention and Control (NCIPC) reveal that approximately 1.7 million cases of TBI were reported each year from 2002 to 2006 (www.cdc.gov/traumatic­braininjury/ pdf/blue_book.pdf). Of those patients, about 1.36 million (80 per cent) were treated and discharged from emergency departments, 275 000 were h ­ospitalized and 52 000 died from their injuries. The leading causes of TBI were falls (35.2 per cent), motor vehicle crashes (17.3 per cent), being struck by or against events (16.5 per cent), assaults (10 per cent) and unknown/other events (21 per cent). Children aged 0–4 years,

Abusive Head Trauma in Children................................................666 Mild Traumatic Brain Injury, Concussion and Sports-related Brain Injuries.................................................669 Long-term Sequelae of Brain Injury.............................................670 Repetitive Head Injury and Chronic Traumatic Encephalopathy..........................................................671 References.................................................................................673

adolescents aged 15–19 and adults over the age of 65 are the age groups most likely to sustain a TBI, and children aged 0–14 account for almost 500 000 emergency department visits per annum. Adults aged over 75 years have the highest rates of TBIrelated hospitalization and death. At the beginning of 2005, 3.17 million people in the United States were living with permanent disability as a consequence of TBI,478 highlighting the associated financial burden. In the United Kingdom, more than 1 million patients attend hospital each year suffering from head injury.218 Based on the Glasgow Coma Scale (GCS) (Table 10.1),442 about 90 per cent of this group have a mild head injury, 5 per cent have moderate and 5 per cent severe head injury (GCS: mild 15–13, moderate 12–9, severe 8 or less). Approximately 20 per cent are admitted to hospital for observation and 5 per cent are transferred to specialist neurological care. Most serious injuries result from road traffic accidents (RTA), but most head injuries follow a fall (40 per cent) or an assault (20 per cent). As the epidemiological data outlined earlier clearly demonstrate, traumatic brain injury remains a major cause of morbidity and mortality throughout the world, affecting young and old alike. Our understanding of the pathological consequences of head injury has been developed through observations on human autopsies and animal models developed to investigate pathophysiological mechanisms. One has to be aware of the limitations of both these approaches: autopsies mostly provide information relating to the most severe end of the clinical spectrum of TBI, fatal outcome; and animal models do not reproduce the polypathology of human brain injury, and there are likely to be significant differences in the anatomical basis of injury and cellular responses between species. 637

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638  Chapter 10  Trauma TABLE 10.1  Glasgow Coma Scale Eye opening

Best verbal response

Best motor response

Spontaneous

4

To sound

3

To pain

2

None

1

Oriented

5

Confused conversation

4

Inappropriate words

3

Incomprehensible sounds

2

None

1

Obeys commands

6

Localises to pain

5

Flexion-withdrawal

4

Flexion abnormal

3

Extension

2

None

1

The injuries sustained and the outcome of TBI are determined and modified by many factors such as age and preexisting illness, and genetic factors are also important.214,251

Classification of Traumatic Brain Injury Classification systems can be pathological, clinical or mechanistic. Currently there is no single classification of traumatic brain injury that completely encompasses all

the clinical, pathological and cellular/molecular features of this complex process. In 2007, a workshop convened by the National Institute of Neurological Disorders and Stroke (NINDS), supported by the Brain Injury Association of America, the Defense and Veterans Brain Injury Center and the National Institute of Disability and Rehabilitation Research, reviewed the current status of classification systems and arrived at recommendations for classifications to support translational and targeted therapies.377 In addition, attempts have been made to define common data elements for TBI to help standardize clinical trial reporting.111,268 Severe TBI, as assessed by GCS, can be caused by a range of pathophysiological processes (Figure 10.1), and any therapy undergoing clinical trial is unlikely to be effective in managing all of these. Although some studies suggest TBI mortality has shown a steady decline over the past few decades,262 because of increased use of a protocol-driven approach to the initial management of the head-injured patient, standardizing therapeutic management in the acute phase, more recent epidemiological studies indicate no clear decrease in TBI-related mortality or improvement in overall outcome.372 Therapies derived from animal models have, to date, had little impact on outcome. The reasons for this are likely to be multifactorial, but the heterogeneity of human TBI is clearly a major factor.267 A clear recommendation of the NINDS workshop was that a multidimensional classification system should be developed to support clinical trials in TBI, allowing targeted therapies for specific pathophysiological injuries to be tested. Pathological classifications can be anatomical, describing injuries as focal or diffuse, or pathophysiological, in which injuries are subdivided into those that are primary (immediate direct consequences of the physical force of the trauma) and those that are secondary (delayed and/or

(a)

(b)

(c)

(d)

(e)

(f )

10.1 Varied computed tomography (CT) appearance of traumatic brain injury (TBI) patients presenting with unconsciousness after head injury, all graded as having severe head injury. A range of differing pathologies is seen. (a) 5-month-old with seizures and bilateral acute subdural haematoma (ASDH). (b) 16-year-old boy with extra-axial haemorrhage and multifocal contusions after a snowmobile crash. (c) 6-week-old struck by falling object, with depressed fracture, cortical laceration, and intracerebral and intraventricular hemorrhage. (d) 14-year-old boy with gunshot wound. (e) 6-year-old boy who was in a high-speed car crash and has radiological changes of diffuse axonal injury (DAI). (f) 16-year-old boy in car crash with ASDH (coronal view).

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Introduction  639

TABLE 10.2  Mechanisms of traumatic brain injury

TABLE 10.3  Classification of traumatic brain injury

Mechanism

Main pathology

Focal

Diffuse

Impact

Vascular (haemorrhages)

Scalp lacerations

Global ischaemic injury

Traumatic axonal injury

Skull fractures

Traumatic axonal injury/diffuse vascular injury

Contusions/lacerations

Brain swelling

Inertial loading

Traumatic axonal injury

Penetrating

Local tissue necrosis

Blast

Brain swelling

indirect results of the trauma). A number of clinical classifications have been developed over the years, with the GCS being the most widely used,442 although this scale is less useful in paediatric assessment and is a poor discriminator in mild head injury. Mechanistic classifications describe impact, inertial loading, penetrating and blast injuries (Table 10.2). Impact injuries require the head to make contact with an object, with the forces potentially being transmitted to the brain. Injuries produced by inertial forces result from the differential movement of the brain relative to the cranial cavity, or of different parts of the brain relative to each other. It is worth noting that most clinical injuries have elements of both contact and inertial forces acting on the brain. Penetrating injuries produce damage when an object passes through the protective covering of the skull resulting in direct parenchymal damage; in the case of firearm injuries, there is also a significant element of tissue damage caused by the pressure cavities produced by the projectile passing through brain tissue.479 Blast injuries are the least well described pathologically and are seen in an industrial, military or terrorist situation in which the shock waves from an explosive device can result in injuries to the brain parenchyma.247 This chapter will follow the standard outline for the neuropathological description of traumatic brain injury – focal and diffuse injuries (Table 10.3) – and will then detail the neuropathological features associated with penetrating and blast injuries, before considering spinal injuries and finally the specific paediatric injuries seen with obstetric TBI and abusive head trauma. We will then consider mild TBI, particularly in relation to sport, and finally the long-term consequences of TBI.

Experimental Models Animal models of TBI have helped to advance the study of the cellular and molecular responses to TBI. Animal models allow an opportunity to control the physiological parameters of the model and to focus specifically on a single type of focal or diffuse injury. Human tissue typically shows polypathology, and many of the cellular and molecular responses are due to secondary injuries, such as ischaemia, rather than reflecting the primary tissue response, such as axonal injury. However, it is important not to overinterpret the data from animal models, and a number of clinical trials of promising pharmacological therapies have failed owing to a lack of recognition of the polypathology in human brain injury, and of differences in host factors.

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10

Intracranial haemorrhage Focal lesions secondary to raised intracranial pressure

Rodents remain the cornerstone of animal modelling of TBI, and a number of systems have been developed to model focal and diffuse pathologies.471 However, large animal models, including dogs, sheep, swine and primates, have an important role to play in TBI research103 and often show closer anatomical and physiological similarities to humans than do rodents. Indeed, it is surprising that so large a number of studies have addressed diffuse white matter injury in rodents, when rodents have so little white matter to study. Recent reports indicate that rodents show limited fidelity to human genomic and proteomic responses, injury timecourses, and grey and white brain matter distribution,110,387 which implies that there are challenges in applying what is learned about injury in the rodent brain to the human. Regardless, animal models are a valuable tool for understanding how head impacts and sudden head movements translate to brain deformations and how brain deformations result in a spectrum of brain injuries, from mild to severe. Significant advances have been made in detailing the functional anatomy of the brains of larger animals, allowing clearer association between the pathological assessment of injury and functional outcomes as assessed by a range of behavioural tests.383 Studies using larger animals can also incorporate detailed post-injury neurocritical care monitoring,133 providing valuable pathophysiological information post-injury.

Models of Focal Traumatic Brain Injury The three main experimental models of focal TBI are weight drop, fluid percussion and controlled cortical impact.316 The weight drop model124 is most often a rodent model that uses a weight falling freely under gravity to produce a focal impact. The skull is usually not opened and fractures are common unless the skull is protected with a plate or disk. The controlled cortical impact method in rodents95 uses a rigid impactor directly onto the exposed dura, causing an underlying contusion; in other variations, impact may occur directly onto the cortical surface. Midline fluid percussion injury297 requires the skull to be opened by trephination over the sagittal suture. A fluid bolus is accelerated onto the dural surface, the force being modified by variation of height from which the pendulum used to accelerate the bolus is released. The fluid bolus also can be applied laterally for a different distribution of injury (see later). These models are useful for modelling focal lesions such as contusions and haematomas. Rodents are most

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640  Chapter 10  Trauma

frequently used in these models, although they can be modified for larger animals.22,272

Diffuse Brain Injury Models Models to study diffuse traumatic axonal injury have been developed for rodents and larger animals. Much of the initial work on diffuse axonal injury was done using nonhuman primates and the inertial acceleration brain injury model;144 this method has also been used extensively in swine models.131,373 Some of the work relating to the basic science of axonal injury has been undertaken on the optic nerve stretch model; this model, developed in guinea pigs147 and modified for rodents,376 produces pure white matter injury that can be studied at different stages post-injury. In vitro models have been developed specifically to assess the pathophysiology of axonal stretch; the models allow a controlled one-directional stretch466 and hold the potential for highthroughput analysis of potential therapies, more so than with established animal models.412 Other animal models to replicate inertial injury include automated and manual shaking of species from rodents to lambs,125,414 models that aim to allow study of so-called ‘shaken baby’ syndrome and repetitive mild TBI.

Mixed Focal and Diffuse Brain Injury The most widely used model for producing a mixed brain injury is the lateral fluid percussion model.298 In this model, the craniotomy is moved from the sagittal suture (midline fluid percussion model) to a lateral position. Although the site of the impulse is unilateral, the pathology produced is bilateral.

Models of Penetrating Brain Injury Historically, a number of animal models have been used to study ballistic brain injury, including non-human primates.82 Most penetrating ballistic brain injury work currently undertaken in animals uses a rodent model.462 This model does not use a fired projectile but rather an inflatable penetrating probe that can simulate the cavity produced by a missile. The probe extends into brain parenchyma in a controlled fashion from a stereotactic frame. Newer models use modified air rifle pellets,350 and behavioural tests have been developed as part of the assessment in this model.395

Models of Blast Injury Of all the animal models used to investigate traumatic brain injury, those modelling blast injury are the least well reported and characterized. Most use pressure generators to produce a blast wave within a shock tube and this blast wave can, to a degree, be controlled and scaled.116,366 This approach has been used with smaller animals such as rats63 and pigs,91 and cognitive function has been shown to be affected in rodents even at low levels of blast injury.380

Primary Injuries Primary injuries constitute various types of tissue disruption that occur as a direct consequence of the application of

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force to the nervous system and its coverings. These i­njuries in themselves cannot be avoided by therapeutic intervention, only prevented. They include scalp lacerations, skull fractures, several types of intracranial haemorrhage, cortical contusions and diffuse traumatic axonal injury. However, as will become apparent, the division between primary and secondary injury is to some extent artificial in that many of these processes are closely linked. In diffuse traumatic axonal injury, for example, although the initial damage is primary, the degenerative process evolves over a period of hours. From the perspective of current and evolving therapies, it is useful to consider the concept of a delayed primary injury that represents tissue that although injured is not initially irreversibly injured, such that there is a window for therapeutic intervention: the primary forces associated with the TBI initiate molecular cascades that if untreated lead to irreversible damage. Most neuroprotective therapies target this delayed primary injury. The avoidance or limitation of secondary injuries, which are injuries not directly related to the initial trauma but arising as a consequence of downstream events such as an evolving mass lesion, are a major focus of neurosurgical intervention and aggressive critical care management, such as the prevention of seizures and the management of intracranial pressure. In human neurotrauma, by far the most common type of force applied to the nervous system is dynamic loading, in which the forces are applied rapidly. These may be subdivided into impulsive and impact types of dynamic loads. Impulsive loading refers to the head being accelerated or decelerated without a direct impact, whereas in impact loading the head strikes an object. The consequences of such dynamic loading are dependent on many variables and form the basis of the study of the biomechanics of head injury.

Biomechanics Biomechanical studies can provide insight into mechanisms of primary TBI, including the interrelationships among the forces experienced during impact, head and neck movements, tissue stiffness of the materials that compose the head/neck complex, deformation of structures at the macroscopic and microscopic level and ­biological responses to the various forces imposed on the head. The biological responses may be immediate or delayed, may be structural (torn vessels and axons) or functional (changes in blood flow or neurological status) and may differ with maturation. Biomechanical investigations typically include direct measurements of loading conditions and responses in humans, animals ­ and anthropomorphic surrogates (i.e. crash test dummies); ­ visualization of tissue responses to prescribed loads in order to characterize the responses of complex geometries or composite structures; mechanical property ­testing of individual components to identify changes with age; computational models to predict how tissues will deform during impact or rapid head rotations; and identification of the time-course of cell or tissue responses to specified deformations in order to define thresholds associated with various types of injuries.

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Biomechanics investigators can use human data obtained prospectively, for example via sensors,53,85,374 or retrospectively, by means of crash reconstructions, to help understand TBI scenarios. To obtain kinematic information in more controlled settings, human-like anthropomorphic surrogates (i.e. crash test dummies) and laboratory-based studies are used to re-enact film and witness accounts of sports-related events in order to estimate the forces of impact and head movements (kinematics), but surrogates cannot be used to predict or measure brain injuries or tissue distortions. Instead, results obtained using surrogates must be correlated with animal studies, autopsy reports, and patient records to infer biological responses to kinematic loading conditions or else with computational models to infer tissue deformations resulting from a head rotation or impact. Computational models are used to estimate the tissue distortions and stresses that may result from a rapid head motion or head impact. Data on brain and skull tissue stiffness that can be incorporated into these models are available for young children (infants and toddlers) and adults,74,355 but there are very limited data for older youths. Like surrogates, computational models cannot predict injury; rather, predicted tissue distortions are correlated with animal or human data. Early studies demonstrated that the brain tissue distortions and stresses in the skull that are associated, respectively, with axonal injury and skull fracture are smaller in young children than in adults.74,358 Biomechanical injury thresholds are most often used to re-enact actual or idealized scenarios to identify tissue distortions associated with acute injury, rather than longterm consequences, repeated exposures, or predisposing biological conditions. It is unknown if deformation injury thresholds for previously injured tissue, which may be hypoxic or metabolically compromised, are lower than for normally functioning tissue. The critical deformations associated with various types and severity of the specific brain injury of interest377 are age and injury specific, such that the magnitude and rate of the distortion required to rupture a blood vessel are different from those required to injure an axon. It is widely accepted that smaller deformations may be associated with brief functional changes (deficits in synaptic transmission, signalling pathways and membrane permeability)305 and that larger deformations may cause permanent structural changes.60,117 Thus, tissue distortions and the rates of tissue deformation associated with concussion (with no lingering neural or vascular structural changes visible on radiological imaging or pathology) are probably lower than those for more severe brain injuries,148 so it is inappropriate to rely on a single threshold for all head injuries. Currently, because human data and computational models are limited, researchers use alternative idealized experimental preparations, such as animals, tissues and isolated cells, to create controllable settings with similar predisposing conditions among subjects and reproducible mechanical loads. Animal models are useful for measuring physiological responses, neuropathology and neurofunctional changes at prescribed time points after injury and have been discussed in more detail earlier in this chapter. Using the tools described, researchers have determined that with or without a helmet, when the head contacts

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Introduction  641

a stationary or moving object, there is a rapid change in velocity and a possible deformation of the skull. Skull deformation may produce a local contusion or haemorrhage if the deformations of the tissues exceed their injury thresholds. When the properties of the contact surfaces are softer or allow sliding or deformation, the rate of velocity change (acceleration or deceleration, depending on whether the velocity is increasing or decreasing) is lower. Similarly, if there is no head contact but only body contact, the deceleration of the moving head is usually lower than when the head is contacted directly. After the initial rapid change in velocity caused by impact to the head or body, the subsequent motion of the head is influenced by the location of that initial point of contact and the interaction between the head, neck and body. There are three possible types of responses to head contact. First, if the contact is directed through the centre of the mass of the brain (centroid), there may be linear motion and no rotation of the head (e.g. a weight dropping down onto the top of the head or a blow to the back of the head that moves the ears and nose forward without neck flexion or extension). Animal studies have shown that these purely linear motions produce little brain motion or distortion and no concussion.182,338 However, most often the contact force is not directed through the centroid of the brain, a situation that is referred to as a non-centroidal impact. After a non-centroidal contact, the head may rotate without a linear motion (e.g. as in shaking the head to indicate ‘No’). This purely rotational motion produces a distortion of the brain’s neural and vascular structures within the skull because the brain is softer than the skull and loosely coupled to it. More commonly, though, a head impact produces a change in head velocity that is associated with both linear acceleration and rotation of the head. This combined rotational and linear motion may occur because the contact is glancing (further away from the rotation centre), the body continues to move after the head is restrained by the contact surface, or the head bounces or rebounds after contact. Internal structures of the head, such as the falx cerebri and tentorium cerebelli, influence how the brain moves within the skull and can cause local brain regions to be markedly deformed in certain directions of head rotation, so that sagittal and coronal rotations may produce more severe injuries in primates at lower accelerations and velocities.144 In addition, animal and human studies have shown a general trend that higher rotational velocities and ­accelerations – rather than linear accelerations – tend to cause larger diffuse brain deformations and worse diffuse brain injuries224 and that head injuries depend on the direction of head motion as well as on the magnitude of rotational kinematics.119,145 Animal studies have indicated that it is important to limit the duration of exposure to acceleration, because research has shown that concussions occur when the duration of acceleration is increased.339 Furthermore, animal studies have demonstrated that the location of brain deformation affects the resulting injury.60,117,451 Paediatric head injury, while sharing some similarities with adult injuries, differs with respect to the immaturity of many of the components of the developing nervous system. At birth, the bones of the cranial vault lack diploë and ossification is incomplete, with bony elements being joined by fibrous or cartilaginous tissue. Myelination of the human

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brain begins in utero and continues into early adult life. Myelination is particularly active in the first two years of life. As a result of these ongoing processes of maturation, the child’s brain may respond differently to an adult brain for a given force. The age at which the head injury is sustained was demonstrated to be important in determining the ­vulnerability to and recovery from a focal injury in a piglet model.108,109 Piglets of different ages were injured using a scaled cortical impact model and then sacrificed­ 7 days after injury. Assessment of the brains demonstrated smaller lesions in the younger animals despite c­ omparable injury inputs.108 The authors concluded that vulnerability to focal mechanical trauma increases ­progressively during maturation. Magnetic resonance imaging (MRI) assessment of similar animals over a longer time period (24 hours, 1 week and 1 month post injury) showed that in the younger animals the lesions reached maximal volumes earlier and resolved more quickly.108 Less damage was also found in younger animals with scaled subdural haematomas.114 However, different results were obtained by Raghupathi et al. for inertial injuries, in which similar strains led to greater relative susceptibility in younger subjects.358 Thus, relative vulnerability may vary with specific species, age and injury type. In summary, further research is needed to define the direction-specific and brain region–specific thresholds for linear and rotational accelerations associated with TBI across the age spectrum.

Secondary Injury It is apparent that the primary injury can underlie pathophysiological changes to the cerebral environment ­ and initiate molecular and cellular changes that can have a significant impact on the outcome of the head-injured individual. TBI is heterogeneous; a patient who is in a coma with diffuse axonal injury may have very different pathophysiological perturbations from those of a patient with multiple cortical contusions, brain swelling and reduced cerebral perfusion. Two pathophysiological mechanisms that are key elements in secondary brain injury are energy depletion and disturbed calcium homeostasis.67 In addition, neuroinflammation is a common response to TBI. The neuroinflammatory response may be beneficial initially, but damaging over time.405

Vascular and Metabolic Consequences TBI tends to result in significant changes to cerebral blood flow (CBF). One study of 125 patients with severe TBI documented three different phases in CBF after TBI: an initial phase of hypoperfusion on the day of injury, followed by a hyperaemic phase lasting 1–3 days, and, finally, a phase of vasospasm lasting 4–15 days.277 However, whether altered CBF in itself results in tissue injury is uncertain because TBI also results in a hypometabolic state,450 such that the reduced cerebral perfusion does not in itself necessarily ­represent an ischaemic environment. Techniques to assess tissue oxygen extraction fractions are required to assess the impact of ischaemia properly in different brain regions after episodes of TBI.76

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The forces associated with the primary injury lead to deformation of the neuronal cellular membrane resulting in alterations in membrane ion flows,404 although this membrane disruption appears to be potentially reversible.123 These changes at the cellular membrane can lead to potassium efflux and sodium and calcium influx, and the release of excitatory amino acids, particularly glutamate, that can activate N-methyl-d-aspartate (NMDA) receptors resulting in further calcium influx.122 This alteration of the ionic homeostasis can lead to spreading membrane depolarization, a phenomenon reported in 50–60 per cent of severely head-injured patients,183 exacerbating a non-ischaemic metabolic crisis.239 Within minutes of brain trauma and ion movement, there are attempts to restore the ionic homeostasis. This is an energy-dependent process, and ­ there is significant increase in a local glucose ­metabolism ­resulting in a localized increase in lactate production.217 In cortical contusions, a pericontusional ­ penumbra is described that also shows metabolic derangement, the derangement increasing closer to the contusion core.469 Mitochondria undergo ultrastructural changes in different regions of human ­contusions, ­possibly in response to the changing metabolic activity.25 In the paediatric setting, particularly in neonates and infants, the metabolic responses are different.24 The immature brain is more able to metabolize ketones, having a ­six-fold increase over the adult brain in the ability to metabolise β-hydroxybutyrate (β-OHB). It has been suggested that as the brain matures, the increase in local glucose metabolism reflects increased local synaptogenesis.72 In addition, the immature brain has a greater vulnerability to excitotoxicity and ischaemia.200 Studies in children have demonstrated hypoperfusion and low oxygen metabolic index in brain tissue after TBI, in keeping with mitochondrial dysfunction.357 Calcium influx associated with excessive NMDA receptor activation (excitotoxicity) after trauma can result in mitochondrial damage,252 increased free radical production and activation of calcium-dependent proteases, such as caspases, calpains and phospholipases.367 Damage to ­mitochondria can result in cellular necrosis, apoptosis or autophagy.67 Apoptosis can be initiated by extrinsic and intrinsic pathways, the extrinsic pathway being activated by Fas or tumour necrosis factor-alpha (TNF-α) ligand binding, the intrinsic pathway by cytochrome-c release from the mitochondria-activating caspase-3. An early post-TBI increase was described in apoptosis-related proteins,61,219 and in human TBI studies, morphological changes ­consistent with apoptosis were noted in contusions, peaking 24–48 hours after injury, although still detected at 10 days,413 and in the white matter up to 12 months after an episode of TBI.465

Neuroinflammation The inflammatory response develops rapidly after an episode of blunt force head injury. Neutrophils, lymphocytes and circulating monocytes infiltrate the damaged tissue, and there is local microglial and astrocytic activation. Blood–brain barrier dysfunction with cellular extravasation can be seen around contusions in the acute phase. The rodent weight-drop and cortical impact models of

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TBI have shown that neutrophils accumulate in damaged tissue within 24 hours of trauma, as measured by myeloperoxidase (MPO) activity and immunohistochemistry.73 In humans, vascular margination by neutrophils is seen in contusions by 24 hours, and by 3–5 days there is lymphocyte and monocyte infiltration, and both microglial and astrocytic activation.191 Microglial activation can clearly be demonstrated by 72 hours post injury in human studies,118 although lymphocyte numbers decline rapidly, suggesting they have a limited role in the overall inflammatory response.150 However, microglial cells and macrophages can be seen associated with contusions beyond the acute phase, and the microglial response is not limited to the local environment of the contusion.150 An imunohistochemical study of contusions from autopsy cases with survival times ranging from 24 hours, with severe also showing posturing and motor deficits.246 DAI contributes to at least 35 per cent of cases of fatal TBI142 and is the major cause of severe disability and persistent vegetative state after TBI.157,225 Approximately 65 per cent of patients with clinically mild DAI have a good outcome, whereas only 15 per cent with severe DAI have a good outcome. Because of the microscopic nature of the pathophysiological correlate of DAI, conventional imaging techniques lack the sensitivity to detect structural changes. CT has limited value in assessing DAI, and scans can appear normal in severe head injury.258 However, very small haemorrhages, sometimes referred to as micro-bleeds, often co-localize with areas of axonal damage and can act as a surrogate for white matter injury. There are MRI sequences that are particularly suited to identifying blood products, such as T2* gradient echo, which can be useful in the clinical setting to support a diagnosis of DAI (Figure 10.19). Newer techniques do appear to offer greater sensitivity in assessment of DAI, although these techniques still require detailed validation, particularly with post-mortem correlation. Diffusion tensor imaging (DTI) is an MRI technique that measures restricted diffusion of water, building images of white matter tracts, and this technique has been shown to identify more lesions than other more routine MRI techniques, such as T2* gradient echo.195 Susceptibility weighted imaging is used to demonstrate very small haemorrhages that are less well demonstrated by other imaging modalities (Figure 10.20). Because it is a relatively new technique, standardization of methodology is still evolving.403 There are many papers in which DTI has been applied to animal models of TBI,250 but to date there has been no correlation of DTI ­ isruption in signal changes with actual microscopic tissue d human brain tissue.

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10.19 (a) Axial computed tomography (CT) scan in acute traumatic brain injury (TBI) showing areas of haemorrhage. The patient was unconscious at the time of admission, and both the clinical picture and CT appearances are consistent with diffuse axonal injury (DAI). (b) A T2* gradient echo magnetic resonance image (MRI) highlights the haemorrhages.

Histopathological Identification of Traumatic Axonal Injury The macroscopic appearances of diffuse TAI are variable, ranging from an essentially macroscopically normal brain to extensive frontal petechial haemorrhages, parasagittal white matter lesions, focal corpus callosal haemorrhage, and haemorrhage in the dorsolateral midbrain and pons.

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10.20 Magnetic resonance image (MRI) of a 14-year-old girl struck by a car, with immediate unconsciousness lasting several hours, followed by gradually improving lethargy. Susceptibility weighted imaging shows small haemorrhages that are seen as black dots (arrow) in the corpus callosum.

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10.21 A more centrally placed haemorrhagic lesion in a case of diffuse traumatic axonal injury. The haemorrhagic lesion extends into the ventricular system, and there is damage in the region of the septum pellucidum and fornices. There is no evidence of subfalcine herniation, and there was no mass lesion.

Three degrees of diffuse TAI have been described: mild, moderate and severe.10 In grade 1, there are microscopic changes in the white matter of the cerebral cortex, corpus callosum, dorsolateral midbrain and pons and the cerebellar peduncles. Grade 2 is distinguished by focal lesions, usually haemorrhagic, restricted to the corpus callosum; these lesions are typically laterally placed in the corpus callosum, but can extend medially to involve the interventricular septum and fornix (Figure 10.21), and are separate from the more midline corpus callosal haemorrhagic infarction seen with subfalcine herniation. In grade 3, additional focal haemorrhagic lesions are seen in the dorsolateral quadrants of the rostral brain stem. In the acute stage, the haemorrhages may be unilateral or bilateral (Figure 10.22a). If the patient has survived for several weeks, the lesions are granular and brown (Figure 10.22b), and with time become cystic and shrunken, being best seen using glial fibrillary acidic protein (GFAP) immunohistochemistry. Of 122 cases of diffuse TAI identified from the Glasgow neurotrauma post-mortem archive, ten cases were of grade 1, 29 cases of grade 2 (in 11 of which the haemorrhagic lesions were only identified microscopically) and 83 cases of grade 3, with 34 of the cases having microscopic lesions only.10 The more severe forms of diffuse TAI are associated with parasagittal white matter lesions9 and deep basal ganglia haematomas.8 An earlier study of diffuse TAI by the Glasgow group described the clinicopathological correlation of 45 cases, and found all to be unconscious from the time of injury and to remain in coma for a prolonged period.5 However, 17 of the 122 cases reported in the subsequent study10 had a

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10.22 (a) An acute bilateral dorsolateral haemorrhagic lesion involving the dorsolateral pons of a 35-year-old male who sustained a road traffic accident, was unconscious from the time of injury and died within a few hours of the accident, never recovering consciousness. (b) Midbrain of a 23-year-old male, victim of car traffic accident. Unconscious from the time of injury, the patient was maintained on a ventilator until death several weeks after the accident. An old haemorrhagic lesion (arrow) is seen in the dorsolateral midbrain.

lucid interval, 15 having a partial lucid interval ­(confused but lucid) with grade 2 diffuse TAI, and two having a complete lucid interval with grade 1 diffuse TAI. These patients died from other pathology, mostly related to brain swelling. However, as discussed later, such data need to be interpreted with caution. When assessing a brain post mortem for diffuse TAI, extensive sampling is required;138 this should include the genu and splenium of corpus callosum, frontal parasagittal white matter, posterior limb of the internal capsule, cerebellar hemisphere, midbrain and pons, including the superior or middle cerebellar peduncle. TAI has been shown to be accentuated in the caudal part of the corpus callosum,240 and it is recommended that both parts of this structure are sampled. Several techniques have been used to identify damaged axons. As discussed later, trauma disrupts the normal axonal flow of proteins, causing proteins to accumulate at points where there is perturbation of normal axonal function, resulting in axonal swelling. These swellings occur along the length of an involved axon, giving an appearance called axonal

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10.23 Eosinophilic axonal bulbs (axonal ‘retraction balls’) can be seen in areas of damaged white matter after about 15–18 hours’ survival. Eosinophilic axonal bulbs may be seen several months after an episode of head injury, and in this setting they may not immunostain with antibody to β-APP. H&E ×40.

varicosities. If the axonal pathology progresses to actual disconnection, a single swelling, called an axonal bulb or axonal ‘retraction’ bulb, is seen. These swellings are eosinophilic in haematoxylin- and eosin-stained sections (Figure 10.23), and can also be detected by silver stains, although a survival of 15–18 hours is required before axonal bulbs can be identified using these techniques. Immunohistochemistry is the most sensitive technique, and a range of proteins that move along the axon via the fast axonal transport system were assessed in human material.396 This study found β-amyloid precursor protein (β-APP) to be the most specific and sensitive marker in human material, and immunohistochemistry for β-APP remains the most widely used method for detecting disruption to normal axonal flow. Older texts refer to a minimum survival of 2 hours before β-APP accumulation is seen, and 3 hours until axonal bulbs are identified in diffuse TAI.301 However, owing to changes in the antibody clone used and advances in antigen retrieval techniques, the time frame for detection of axonal injury has changed significantly. More recent literature has described β-APP accumulation in paediatric cases 35–45 minutes after TBI154 and 35 minutes after TBI in adults.193 Indeed, one study of cases described as dead at the scene showed β-APP accumulating in the neuronal cytoplasm within minutes, and occasional beaded axons within the white matter.318 The axonal pathology evolves over at least 24 hours and then plateaus, remaining easily identifiable for about 10–14 days after injury, after which the staining intensity lessens such that by 3–4 weeks after injury β-APP immunoreactivity is difficult to identify.164 As noted previously, β-APP accumulation is not specific to trauma and may be seen in axonal disruption of any aetiology, such as ischaemia96 and hypoglycaemia.97 When assessing a case of possible TBI, it is always important to be cognisant of other potential causes of axonal pathology, particularly ischaemia. Ischaemia in TBI cases is common, and can cause extensive β-APP accumulation throughout the white matter. However, ischaemic axonal injury is diffuse, and the accumulation of β-APP shows a geographic or

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10.24 Different patterns of immunostaining can be identified to help differentiate between ischaemic and traumatic white matter injury. (a) β-APP immunostaining highlights a solid band of axonal injury, delineating an area of ischaemic damage (β-APP, ×4). (b) The susceptibility to axonal injury is determined by the orientation of the white matter bundles, typical of traumatic axonal injury. The example presented is from the pons, and the damaged axons are in one orientation, whereas the intervening undamaged axons are in another. β-APP, ×10.

contiguous pattern, whereas diffuse TAI follows a specific anatomical distribution, often highlighting axonal damage in one white matter pathway, whereas an adjacent differently orientated pathway does not show axonal damage (Figure 10.24). Specific patterns of β-APP immunohistochemical staining have been described for TAI and ischaemic axonal injury, and it is possible to distinguish them from each other,164,363 although in some cases the ischaemic axonal injury is so extensive it is not possible to comment on the presence or absence of underlying TAI. It is important when reviewing the historical TAI literature to consider that papers describing axonal injury detected with silver stains and haematoxylin and eosin will have underrepresented the axonal pathology, and in many of the papers no consideration was given to other causes of axonal injury, particularly ischaemia. Papers describing axonal bulbs detected using silver stains in TBI cases with brain swelling and/or intracranial haemorrhage may reflect ischaemic damage rather than TAI. A review of cases with a diagnosis of diffuse TAI over three decades in the Glasgow neurotrauma post-mortem archive compared a 1968–1972 cohort (151 cases),4 a 1981–1982 cohort (112 cases)161 and a cohort from 1987–1999 (226 cases, unpublished). In the earlier cohorts diffuse TAI was diagnosed using haematoxylin and eosin and silver stains, whereas in the later cohort all cases were assessed using β-APP immunohistochemistry.

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10.26 A coronal section through the frontal lobes, short survival (hours) after a road traffic accident. Petechial haemorrhages are widespread, scattered throughout the white matter. 10.25 With longer survival (after several weeks) β-APP becomes an unreliable marker of axonal injury as the staining intensity fades. However, in these cases, CD68 immunoreactivity can be helpful in highlighting degenerating pathways (wallerian degeneration), again showing specific susceptibility related to axonal orientation. In this example from the pons, in a patient with seven months’ survival in coma after an episode of traumatic brain injury (TBI), the corticospinal pathways are highlighted. CD68 immunostaining, ×10.

Diffuse TAI was reported in 41 per cent of the later cohort, compared to 18 per cent (1968–1972) and 33 per cent (1981–1982). Grading was compared between the 1987– 1999 cohort and the published data from the 1968–1982 cohort:10 grade 1, 8 versus 17 per cent; grade 2, 24 versus 7 per cent; grade 3, 68 versus 17 per cent. Although it is possible that the epidemiology of diffuse TAI changed between these cohorts, it is likely that the extensive axonal injury seen with silver stains in some of the older cohorts was at least partly attributable to ischaemic damage. In long-term survival, the wallerian degeneration of the white matter tracts can be highlighted by immunohistochemistry for phagocytic markers, e.g. with antibody to CD68 (Figure 10.25). CD68 immunoreactivity can be seen in white matter pathways for many years after diffuse TAI.407

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Haemorrhagic Lesions Associated with Rotational Injury As described earlier, focal haemorrhagic tissue tears are seen in the corpus callosum and dorsolateral brain stem in severe rotational injury. Other focal haemorrhagic lesions are seen in severe TBI, as part of the spectrum of rotational injury. Diffuse vascular injury refers to extensive petechial haemorrhages extending through the white matter, particularly the frontal white matter, associated with immediate unconsciousness and a poor prognosis, often being seen in cases described as ‘dead at the scene’ (Figure 10.26). The pathology is thought to occur at the time of injury and to represent the shearing of many small parenchymal blood vessels.4 Histological examination demonstrates perivascular haemorrhages (Figure 10.27). It is important to undertake histological examination in TBI cases with

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10.27 (a) A coronal section through the parasagittal frontal white matter showing extensive petechial haemorrhages. (b) A microscopic section from (a) showing prominent perivascular haemorrhage. The vessel can be seen within the centre of the haemorrhage. H&E, ×20.

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a short survival period and no obvious mass lesion even if diffuse vascular injury cannot be identified macroscopically, because microscopic lesions are often present in the absence of obvious macroscopic lesions.349 Parasagittal white matter lesions are often seen in cases with macroscopic or microscopic diffuse vascular injury. In addition, subcortical haemorrhages are a frequently neglected but important additional marker of a severe rotational head injury: these lesions lie at the grey–white matter interface, particularly in the inferior frontal regions, and are not associated with overlying cortical contusions. The final haemorrhagic lesion seen in severe rotational injuries is the traumatic basal ganglia haematoma described previously. These haemorrhagic lesions are seen in severe rotational TBI cases, usually road traffic accidents or falls from a height, and predominantly occur in association with grade 3 dTAI, but also sometimes with grade 2 dTAI. As noted earlier, patients with these haemorrhagic lesions have a poor prognosis.

Clinicopathological Correlation That there exists a spectrum of clinical and pathological changes associated with rotational forces has been clearly demonstrated. Clinically, this extends from mild concussion through to DAI, with immediate prolonged coma and poor prognosis. The pathological correlates of these clinical states are still being evaluated. It is clear that the clinical and radiological entity know as DAI is associated with severe diffuse TAI, with corpus callosum and dorsolateral brain stem lesions, and with a higher incidence of diffuse vascular injury and parasagittal white matter injury. A study of 14 cases of diffuse vascular injury found that all cases were road traffic accidents and had associated diffuse TAI grade 2 or 3, and all patients died within 24 hours of the injury.349 Studies using a porcine non-impact model suggest that it is the brain stem pathology that is the anatomical correlate of immediate unconsciousness and coma42 and that the duration of coma is related to the extent of brain stem axonal pathology.410 However, at the milder end of the clinical spectrum the association is less secure. The structural basis of concussion is unknown and discussed in more detail later. Focal axonal injury, particularly involving the corpus callosum, is increasingly recognized, but the clinical consequences are unknown. It is seen not infrequently in cases with other obvious causes of death, such as assaults with fatal stab wounds and road traffic accidents with massive thoracic and abdominal injuries, and the clinical significance of such a focal lesion is currently unknown. Likewise diffuse TAI in the absence of any lesions in the corpus callosum or dorsolateral brain stem has an uncertain clinical correlation. It has been reported in mild TBI with no loss of consciousness33,34 and described in association with a lucid interval.10 A neuropathologist can be confident when faced with a TBI case of immediate coma ultimately resulting in death, with no intracranial mass lesions but diffuse TAI and lesions in both the corpus callosum and the rostral brain stem, that the cause of unconsciousness is diffuse TAI. Presented with a similar case but with no focal haemorrhagic lesions, if the post-mortem examination is essentially negative but there is diffuse TAI, it is reasonable to suggest

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diffuse TAI as the cause of unconsciousness. However, this grade 1 pathology does not necessitate a period of unconsciousness and is, in itself, a survivable injury. As such, the existing grading system while conceptually useful does not offer clear clinicopathological correlation between grades; grade 1 can be both a fatal and a survivable pathology.

Causes of Diffuse Traumatic Axonal Injury Diffuse TAI is typically associated with high-velocity rotational forces, such as road traffic accidents and falls from a height.10,33 Simple falls, i.e. a fall from one’s own height or less, typically cause a rapid deceleration and ASDH and/or contusions. Severe diffuse TAI has not been demonstrated in simple falls, but is seen in falls from a height.6,87 Diffuse TAI has been described in the setting of assaults, although it must be remembered that reliable descriptions of the incidents are often lacking in this setting. Graham et al.162 reported 15 fatal assault cases and described grade 3 diffuse TAI in 10, grade 2 in 1, and grade 1 in 4. Other single case reports exist in the literature.201 Since more sensitive clones of β-APP antibodies have been introduced, axonal injury is now being seen much more frequently in assault cases, particularly focal TAI;138 however, the relevance of this pathology to the death of those concerned is uncertain.

The Pathophysiology of Axonal Damage in Traumatic Brain Injury Initially, trauma-induced axonal injury described in human brains was thought to be the result of axons being disconnected by shearing forces at the time of the impact (primary axotomy) leading to axonal retraction and axoplasmic pooling.431 We now know that only a minority of axons undergo primary axotomy, the majority being damaged as a consequence of focal axolemmal perturbations and degenerating over a period of time after the initial insult.289 Primary axotomy, i.e. direct shearing through the axon at the time of injury, is uncommon, but has been identified in small diameter fibres by electron microscopy in the non-human primate.288 However, most of the axonal damage is secondary and delayed. Morphological studies in a cat model showed an anterograde tracer, horseradish peroxidase (HRP), accumulating at sites of axonal ­dysfunction within 60 minutes of the injury, indicating altered axolemmal permeability, with axotomy developing between 6 and 12 hours post injury.354 The time-course does appear to be species specific, being most rapid in rats and of longer duration in cats and pigs, the longest duration being described in man.353 How the rotational forces associated with TBI modify ionic regulation in focal axonal segments remains to be fully determined. The initial hypothesis developed from animal models suggested that pores were produced in the axonal membrane as a direct consequence of the forces associated with TBI, the process being referred to as mechanoporation. This was thought to occur within minutes and continue for several hours. In vitro studies have offered an alternative hypothesis. In an axonal stretch model, rapid influx of calcium occurred into the axon, but this appeared to be temporally related to a sodium influx through specific sodium channels.466 This group suggested that stretch causes activation of mechanosensitive sodium channels, resulting in sodium influx and activation of voltage-gated

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calcium channels (VGCC), in turn causing calcium influx and an increased intra-axonal calcium concentration. Further work by this group suggests a negative feedback mechanism whereby calcium influx results in proteolysis of one unit of the sodium channel, causing persistently elevated intra-axonal levels of calcium.204 Sodium channels are found at the highest density at nodes of Ranvier, and the earliest structural changes occur at this site.11 With failure of the sodium/calcium exchange system, additional calcium is released from intra-axonal stores, including the axoplasmic reticulum and mitochondria.423 Fast axonal transport is a function of microtubules, and microtubule fragmentation occurs rapidly after stretching along multiple points of an axon. Undulations develop in the axons, and there is disruption to fast axonal transport, causing axonal swelling and a risk of fragmentation.439 The varicosities associated with this process are described in human diffuse TAI,440 and microtubule and neurofilament fragmentation have been demonstrated in other models.287 Not all axons show the same level of vulnerability. Small-diameter unmyelinated axons are most susceptible to injury in both in vitro and in vivo systems.422 An increased calcium level causes the activation of calcium-dependent proteases (calpains) and caspases. This ­ results in the modification of neurofilament subunits, leading to the accumulation of dephosphorylated neurofilaments and damage to other intra-axonal proteins, resulting in local impairment of axonal transport with resultant axonal swelling. Over time, this is followed by loss of continuity of the axon and wallerian degeneration. Calpains have been shown to be important contributors to ongoing cytoskeletal degeneration in wallerian degeneration.266 Immunohistochemistry for β-APP only identifies axons in which anterograde axoplasmic flow has been disrupted as a result of microtubule fragmentation, and there is likely to be a second population of axons in which structural changes are present but in which axonal transport continues. An important consequence of the activation of calcium-dependent proteases is the fragmentation of ­ neurofilament proteins. Studies using animal models of TBI have shown that β-APP and RMO14, a marker of neurofilament compaction, highlight distinct populations of damaged axons.275,429 This has been demonstrated in both piglets and humans in our laboratory (Figure 10.28). Neurofilament protein changes were shown to evolve over time in a pig TBI model: neurofilament-light (NF-L) accumulated in axons within 6 hours, but neurofilamentmedium (NF-M) and neurofilament-heavy (NF-H) did not accumulate until 3 days after injury.65 Along with alteration of the neurofilament structure, other components of the cytoskeleton, such as spectrin and ankyrin, become damaged, eventually resulting in axonal disconnection with closing of the damaged axolemmal membrane on each side of the disconnected axon.412 These observations highlight the need for a reappraisal of how traumatic axonal injury is diagnosed in humans, and how these different molecular changes correlate with clinical outcome. In particular β-APP-immunoreactive axons are not necessarily irreversibly damaged and may only be an indicator of transient dysfunction of axonal flow, whereas neurofilament compaction is more likely to represent an end-stage process.

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10.28 Double-label immunofluorescence of traumatically injured axons in the human corpus callosum, showing intraaxonal β-APP accumulation (green) and phosphorylated neurofilament (SMI-34 antibody) immunoreactivity (red). Axons co-expressing β-APP and phosphorylated neurofilament protein appear yellow. Although there is some overlap, there are many axons showing changes consistent with neurofilament compaction that do not show β-APP accumulation.

10.29 A damaged axon within the corpus callosum in a case of diffuse traumatic axonal injury (TAI). Varicosities have formed along the length of the axon, possibly related to microtubule fragmentation. β-APP immunostaining, ×40.

In the setting of human diffuse TAI, although the detection of β-APP is the most widely used marker of axonal injury, the neuropathologist needs to be careful in its interpretation. Accumulation of β-APP within axons in the absence of varicosities is common and is most likely a reflection of increased β-APP production within the cell body. It is particularly common in cases of global ischaemia with widespread neuronal cytoplasmic staining, and may not be a direct consequence of trauma. Varicosities along the length of an axon (Figure 10.29) appear to be related to microtubule fragmentation, an early step in axonal damage. Axonal bulbs (Figure 10.30) are likely to represent disconnection of the two ends of the damaged axon. Of all these

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10.30 Swellings develop at the disconnected ends of damaged axons. β-APP immunostaining, ×40.

pathologies, it is possible that only the axonal bulb represents irreversible damage.

Brain Swelling Brain swelling is a common finding in fatal TBI. Increased ICP associated with brain swelling remains the most common cause of death in severe TBI.311 The swelling may be focal or diffuse and is mostly due to oedema, an increase in the water content of the brain tissue, and congestion, an increase in the cerebral blood volume, with oedema accounting for most brain swelling.274 Oedema can be classified as cytotoxic, in which there is abnormal water retention by injured cells; vasogenic, in which blood–brain barrier (BBB) breakdown leads to the passage of plasma proteins and water into the extracellular compartment; and hydrocephalic (or interstitial), in which, as a result of increased intraventricular pressure, CSF is forced from the ventricle into the periventricular extracellular space.328 In the setting of TBI, the swelling may be focal, as a result of contusions or ICH; diffuse within one cerebral hemisphere, typically secondary to an overlying ASDH;259 or diffuse in both cerebral hemispheres. Adjacent to contusions and ICH, there is physical disruption of the tissues, including BBB, and loss of the normal autoregulation within the local vasculature. The development of cerebral oedema appears to be principally due to severe disruption of the BBB involving endothelial cells, tight junctions, astrocytes or a combination of all components. MRI studies in rodents have shown that the BBB allows passage of fluid immediately after a closed blunt force head injury, but returns to normal function within 30 minutes.26 After injury, there is altered expression of several proteins associated with BBB function including tight junction proteins and caveolin-1, a major component of the caveolae involved in transporting fluids.329 Experimentally BBB proteins can be modulated to increase the movement of water from the brain parenchyma to the vascular system.54 Aquaporin-4 (AQP-4) is an important molecule involved in water homeostasis in the brain, found predominantly in astrocyte cell membranes.343 Interference with AQP-4 expression has been shown to reduce cerebral oedema in TBI models.135

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Oedema can develop rapidly after TBI in animal models,48,49 and rapid brain swelling within 20–30 minutes of injury has been described by CT scan.226,476 Diffuse swelling of one cerebral hemisphere is most typically associated with an adjacent ASDH. Even after this is removed surgically, the hemisphere may swell. The reasons for brain swelling in this context are not entirely understood but are likely to include a combination of a non-reactive vascular bed and local ischaemic injury. Hypermetabolism due to subclinical seizures or excitotoxic injury may also contribute.44,104,202,231,312 In the early CT era, the concept was introduced of ‘diffuse brain swelling’ as an entity seen primarily in paediatric head injury. Early studies suggested that this was due to cerebral hyperaemia with increased blood volume.43 It was posited to reflect relative hyperaemia, although other studies suggested that the degree of hyperaemia was insufficient to cause cerebral swelling.324 The increased prevalence in children is supported by post-mortem studies160 where a specific type of ‘malignant’ brain swelling may be seen in the absence of significant ischaemic injury. However, the concept of ‘malignant’ oedema as an entity peculiar to childhood has been challenged. In a CT-based study, Lang et al.235 found diffuse swelling of both cerebral hemispheres to be associated equally with paediatric and adult head injury, and to have a more aggressive course in adults. Bilateral brain swelling seems to be less common with modern TBI management, and this may relate to more care in the field to avoid hypoxia and ischaemia, more precise methods for fluid management with euvolemic dehydration, early ventriculostomy and decompression and improved imaging. Nonetheless, there are differences between normal cerebral blood flow and reactivity at different stages of maturation,70,393,434,468 and these may influence the physiological propensity to brain swelling. At autopsy, brain swelling is easily recognized by flattening of the gyri, sulcal compression, ventricular compression and midline shift if the swelling is unilateral. However, these features have not been found to be reliable for grading the severity of brain swelling,186,271 and we suggest that brain swelling should simply be recorded as either present or absent, rather than as mild, moderate or severe.

Brain Stem Injuries Brain stem lesions may be primary, as a direct consequence of the forces at the moment of injury, or secondary, as a result of the brain stem displacement associated with increased ICP. Brain stem lesions are common, being seen in 60 per cent of patients with severe TBI in one MRI study.126 A good-prognosis group was defined on MRI with ventral lesions or superficial dorsal lesions, whereas a poor-­ prognosis group had deep dorsal brain stem lesions.397 Pathologically, primary brain stem injury has been considered to be a component of diffuse TAI, representing a severe form of the injury, discussed previously.3 Al-Sarraj and colleagues17 extended this concept and differentiated two separate forms of primary traumatic brain stem injury: that associated with diffuse TAI, and that which occurs in isolation, representing direct trauma to the brain stem, termed focal traumatic brain stem injury (FTBSI). The brain stem lesions of diffuse TAI were discussed earlier (pp. 656–657).

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Blunt Force Head Injury; Diffuse Injury  663



Twelve cases of FTBSI were identified in a cohort of 319 TBI cases.17 The FTBSI was due to a complex fall from height or an accelerated fall (7/12), an assault (4/12) or severe impact on the top of the head (1/12). The longest documented survival was 2 days in this group, although most were found dead. Ten of the cases had skull fractures, eight involving the occipital bone extending into the posterior fossa, and two involving the base of the skull. The main pathological finding within the brain was haemorrhage in the brain stem. Contusions and lacerations of the brain stem may be seen as a consequence of skull fractures around the foramen magnum, sometimes seen in the setting of extreme hyperextension of the neck. In severe hyperextension of the neck, partial or complete pontomedullary or cervicomedullary avulsion can occur (Figure 10.31).244 These lesions should not be dismissed as artefactual changes induced by poor post-mortem methodology, and most neuropathologists with experience in TBI examination will see cases. They are mostly related to road traffic accidents, and are seen particularly in pedestrians rather than in those in the vehicle, or in motorcyclists involved in accidents. Most individuals die immediately, but there are case reports of occasional examples where there is a survival of several days to weeks.347 In one case series, 36 examples of pontomedullary avulsion were found in 988 TBI post-mortem cases, representing 3.6 per cent of the total.402 Eight cases had pontomedullary avulsion in

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the absence of other brain pathology, 17 had avulsion with other brain stem lacerations and pathology elsewhere in the brain and 11 had brain stem lacerations outside the pontomedullary region, possibly fracture contusions/lacerations. Brain stem injuries were overrepresented in the motorcycle injuries group of this cohort, accounting for 41.7 per cent of the total.

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Spinal Cord Injuries Spinal cord injuries (SCIs) are common, the prevalence ranging from 11.5 to 57.8 cases per million people in the population.1 The aetiology of SCI varies between countries, but road traffic accidents, falls, sports-related injuries and assaults are consistent causes between studies.1 Age at the time of injury, neurological status at the time of injury and extent of injury were identified as predictors of survival.449 In the paediatric population, SCI is uncommon, with cervical spine injuries accounting for 1.5 per cent of the National Pediatric Trauma Registry cases over a ten-year period.21 Most cases involving younger children are due to road traffic accidents, whereas those involving adolescents are mostly sports related.344 Younger children are more likely to have high spinal injuries, typically involving ligaments rather than bone, than will older children and adults. Spinal traumatic EDH is exceptionally rare.222 When spinal EDH is seen, it is usually spontaneous or related to therapeutic procedures. Cranial SDH can migrate to the spinal region,249 but primary traumatic spinal SDH is also seen. It has been described in cervical30 and thoracolumbar regions.168 As with spinal EDH, there are a range of non-traumatic causes of spinal SDH, including coagulopathy, neoplasms, vascular malformations and therapeutic procedures. Penetrating SCIs are rare outside military situations. Symptoms are related to the level of the injury, with high cervical lesions having a high mortality. Haemorrhage and tissue necrosis are seen in relation to the penetrating lesion. Closed SCIs are the most common traumatic cord lesions in clinical practice, and are associated with fracture or dislocation of the spine (Figure 10.32). Cervical region SCI was seen in 5.4 per cent of patients with moderate or severe TBI.190 SCI can be a consequence of ­hyperflexion/­hyperextension movements, particularly in the

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10.31 A severe brain stem injury caused by neck hyperextension during a motorcycle crash. There is partial separation of the brain stem at the pontomedullary junction (pontomedullary rent). Although the survival time was very short (minutes) haemorrhages could be seen in the tissue on either side of the tear.

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10.32 Fracture of the spinal column resulting in direct compression of the spinal cord.

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10.33 A cross-section of cervical spinal cord, showing central haemorrhagic infarction after a contusional spinal cord injury.

spinal region; compressive forces, such as a fall from height landing on the top of the skull; and rotational movements, causing fracture dislocations, most typically associated with thoracolumbar lesions. Neuropathological examination in acute SCI may show compressive or contusional injuries. Compressive SCI may be due to fractures or displacement of intervertebral disc material, sometimes after mild trauma,207 the pathology being a consequence of vascular compromise of the compressed tissue. In the early stages of compression, the cord shows venous congestion, progressing to necrosis and central cystic cavitation. Contusional SCI is more dramatic, typically associated with vertebral body fractures, in some cases with dislocation. The direct blunt force trauma to the spinal cord results in parenchymal haemorrhage and oedema that causes fusiform swelling of the cord. Bleeding is initially petechial, but these lesions coalesce to form more extensive haemorrhages, usually situated centrally within the spinal cord (Figure 10.33). Ischaemia develops, resulting in central infarction of the cord. The tissue damage extends for several levels above and below the site of direct injury. Axonal injury is prominent, and macrophage infiltration is seen after several days. In young children, distraction injuries can occur, typically involving the upper cervical spine or cervicomedullary junction. These are most often related to immobilization in restraints during highspeed road traffic accidents, or to crush injuries in which the head is run over by a vehicle. Immunohistochemistry for axonal injury (β-APP) and macrophage infiltration (CD68) can be useful in defining SCI. However, extensive peripheral β-APP immunoreactivity in the upper cervical region may reflect a boundary-zone infarct, particularly in the setting of a ‘respirator brain’, rather than direct spinal cord trauma, and should be interpreted with caution (Figure 10.34). Transection of the spinal cord is seen with extreme force, usually associated with dislocated fractures. In one study of 22 SCI cases, 38 per cent showed complete transection.45

Focal Vascular Injuries We have discussed vertebral artery disruption as the cause of massive basal tSAH. However, a more common trauma-associated injury of the vertebral artery is dissection. Vertebral artery dissection develops when there is

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10.34 Extensive peripheral β-APP immunoreactivity seen in the high cervical spinal cord region. Although there was history of trauma, no associated soft tissue or bony injury was found at autopsy, and in this case β-APP accumulation has resulted from ischaemic rather than traumatic axonal injury. This part of the spinal cord is a recognized border-zone (watershed) region. It is important to be aware of this pattern of immunostaining to avoid potentially considering this a primary traumatic injury. β-APP immunostaining, ×2.

disruption of the intima of the vessel, allowing thrombus to form, which can lead to infarction of part of the brain stem (particularly lateral medullary syndrome) or rarely the spinal cord.194 The site of dissection is most commonly adjacent to the first and second cervical vertebrae.256 Dissection can follow relatively minor trauma, and symptoms can develop over several days. Typical symptoms include ataxia, vertigo and nausea. An association has been suggested with neck manipulation.187 Paediatric cases are also described.62,319 Internal carotid artery injuries secondary to trauma are well described, particularly after hyperextension of the neck.23 In one study of 67 patients with blunt force carotid artery injury, 89 per cent were due to road traffic accidents, and 6 per cent to assaults.120 One patient had a fatal transection of the internal carotid artery, while all other patients had dissection, with or without thrombus, cavernous sinus fistulae or pseudo-aneurysms. These injuries are often associated with skull fracture. Extracranial common carotid or external carotid artery injury is typically secondary to blunt force trauma to the neck and results in dissection with subsequent thrombosis. Traumatic intracranial aneurysms can develop after blunt force or penetrating head injuries, and have a mortality of up to 50 per cent.101 The aneurysms can be classified as follows: true aneurysms, in which incomplete vessel wall damage leads to subsequent dilatation of the damaged section of wall; false aneurysms (pseudo-aneurysms), which damage the full thickness of the vessel wall, with a false wall being formed by surrounding soft tissue structures; and mixed aneurysms, which histologically show a mixture of both, with a dilated section of vessel wall from which there has been bleeding, forming a haematoma that acts as a false wall. A carotid cavernous fistula can develop after maxillofacial trauma, with direct communication between the

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internal carotid artery and the cavernous venous sinus. Presentation may be acute or up to several weeks following injury, and typically includes pulsatile proptosis, orbital and ocular erythema, headache and visual loss. Cerebral venous sinus thrombosis (CVST) can be associated with TBI. However, one study concluded that CT venography should only be undertaken if a fracture extends across a dural venous sinus or jugular bulb, the risk being greater for occipital than parietal bone fractures.92 Only 7 per cent of this high-risk study group developed venous infarction. At autopsy, it is important to remember that CVST and cortical vein thrombosis can be a consequence of reduced cerebral perfusion after TBI. In the most extreme situation, the so-called respirator brain,330,460 secondary CVST and cortical vein thrombosis are very common. Radiologically, no association has been demonstrated between CVST and ASDH.304

PENETRATING INJURIES Penetrating injuries are injuries in which an object enters the cranial cavity; in strict terms, a penetrating injury is one in which the missile enters the cranial cavity but does not exit, whereas a perforating injury is one where the missile also exits. The resulting pathology is very much determined by the nature of the missile. Sharp objects, such as knives, long nails or metal poles, may pierce the skull and extend into the underlying brain parenchyma causing local damage. In young children, objects may enter the cranial cavity through the orbital roof or nasopharynx, most often in association with a fall. They produce a haemorrhagic tract through the regions of parenchyma into which the object extends (Figure 10.35). High-velocity missiles, such as bullets, cause considerably more damage, the extent of the damage being related to the velocity of the missile; high-velocity military weapons produce greater tissue damage than small firearms. Ballistic penetrating brain injuries are associated with a high mortality. A low GCS on admission, an associated intracranial haematoma, age >40 years, a trajectory passing through the ventricles and/or both hemispheres, and a unilaterally fixed dilated pupil are associated with a poor outcome.18,278 A comparison of penetrating brain injuries between military and civilian groups found a significantly

10.35 The haemorrhagic tract caused by a penetrating ballistic missile. There is cystic cavitation of the direct tract, but also extensive surrounding haemorrhagic infarction.

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Penetrating Injuries  665

lower mortality in the military group (5.6 per cent military mortality, 47.9 per cent civilian mortality). There was a higher rate of intracranial pressure monitoring and neurosurgical intervention in the military group, although the difference in mortality between the two groups was probably due to a range of factors.102 In civilian gunshot wounds, a good outcome can be seen in cases with rapid neurosurgical intervention, injury to the non-eloquent brain regions, and absence of injury to the brain stem and major vessels.253 As the missile passes through the brain parenchyma, it produces localized tissue damage, and in the wake of the missile a cavity forms, its size being determined by the energy of the projectile. The localized damage is a result of crushing of tissue by the missile passing through brain parenchyma. Studies in animal models have indicated that a penetrating ballistic injury causes local tissue damage including haemorrhage and necrosis, and initiates a biphasic inflammatory response: in the acute phase, there is cytokine expression and neutrophil infiltration; the delayed response involves white matter degeneration distant from the site of direct tissue injury and develops some days after the injury.463,464 In addition, after a penetrating head injury, cerebral blood flow and cerebral metabolism are reduced.227 The tract of damaged tissue is roughly the same diameter as the projectile, unless there is a degree of yaw in the path of flight of the projectile, in which case the tract may be of greater diameter. A temporary cavity forms as the projectile passes through the brain and stretches surrounding tissue rather like the ripples spreading as a diver enters a swimming pool.189 The final size of the cavity along the trajectory of the projectile is determined by the velocity and shape of the projectile, deformation of the projectile (such as mushrooming or flattening), fragmentation of the projectile and twisting or oscillation of the projectile about its flight axis (yaw).121 At post-mortem examination of ballistic penetrating brain injuries, fragments of the bullet may remain within brain parenchyma. If available, post-mortem radiology can be useful in identifying fragments prior to brain dissection. Three different zones have been described in penetrating missile injuries of the brain:254 a central permanent cavity that contains necrotic brain tissue and blood, an intermediate zone with less tissue necrosis and parenchymal haemorrhages and a marginal zone with tissue discolouration. Both the intermediate and the marginal zones are related to the temporary cavity. Around the permanent cavity, there is axonal fragmentation and haemorrhagic extravasation. Axonal damage reduces radially, moving away from the permanent cavity.332 By analysis of cases with a survival time beyond 2 hours, the tissue reaction has been further characterized.333 CD68-immunoreactive macrophages demarcated a 1–2 mm necrotic zone around the permanent cavity, with β-APP immunoreactive damaged axons being seen in the surrounding tissues. In addition, β-APP immunoreactive axons could be seen remote from the permanent cavity.334 A diffuse distribution of damaged axons was described in one series of 14 cases of gunshot wounds to the head, with extensive involvement of the brain stem in all cases.228 Contusions may be seen at sites distant from the permanent cavity, particularly involving the lower cerebellum (owing

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to impact with the foramen magnum) and frontal and temporal lobes.

Blast Injuries Traditionally, the study of blast injuries focused on the damage caused by blast waves to air-filled viscera, such as the lungs in the thoracic cavity. However, increasingly, and particularly in relation to the recent conflicts in Iraq and Afghanistan, attention has been focused on possible injuries to solid viscera, and the brain in particular. The abrupt pressure changes associated with a blast can lead to a mild head injury, and, in particular, symptoms suggestive of concussion. Long-term sequelae in the form of impaired concentration and memory problems have been described with a greater frequency after blast than non-blast traumatic brain injuries.455 The cellular basis of this injury is to date poorly defined, although there is considerable research activity in this field. The cellular responses produced by blast injuries, including microglial and astrocytic activation, were reviewed by Leung et al.247 The presence or absence of white matter injury in the form of TAI is controversial. Diffusion tensor imaging has demonstrated diffuse disruption of white matter integrity in blast TBI.88 In a non-human primate model exposed to blast TBI, distorted apical dendrites were described in the cortex, and there was loss of hippocampal CA1 pyramidal neurons, Purkinje cell dendritic degeneration, astrocytosis and upregulation of astrocytic aquaporin-4 and oligodendrocyte apoptosis, but no significant axonal injury.263 Saljo et al.379 described redistribution of phosphorylated neurofilaments from the axon to the neuronal cell body in an animal model of blast injury. A possible explanation as to why damage to axonal transport mechanisms has not been convincingly demonstrated in blast-injury models, as demonstrated in both blunt force and penetrating head injuries, may be either that a different white matter degenerative process occurs or that the current methods used to identify axonal damage are not sensitive enough to do so in thinner non-myelinated axons.432 Proteomics has been proposed as a powerful research tool that could be used to investigate the subcellular responses to blast injury,14 although to date few data have been generated by this approach.

PERINATAL HEAD INJURY Perinatal head injuries can develop as a result of excessive moulding of the cranial bones, or excessive force applied to the skull during delivery. Hyperextension of the neck during delivery may result in craniocervical injuries. This section will concentrate on perinatal skull fractures, traumatic intracranial haemorrhages and SCI. In his comprehensive monograph, Govaert et al.155 urged caution in the assessment of incidence figures for cranial birth trauma, although he did note the clear decline in incidence from the 1950s through to the 1980s, from roughly 3 per 1000 to 0.5 per 1000 live births. Linear skull fractures were described in up to 10 per cent of births in one series.169 They are due to direct force on the bone, typically related to the use of forceps. Depressed

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(ping-pong) skull fractures are also associated with the use of forceps.113 In themselves skull fractures, if appropriately managed, are not life-threatening. However, they can be associated with significant intracranial haemorrhage. Perinatal EDH is rare, accounting for 2 per cent of perinatal traumatic haemorrhages in one post-mortem series.433 Linear skull fracture is seen in most cases, and the bleeding is from meningeal arteries or veins. Other causes include parieto-temporal bone overlapping and excessive bending of the calvarial bone, rupturing underlying vessels. Perinatal SDH is common, as discussed later in regard to abusive head trauma. However, clinically significant perinatal SDH is rare and is traumatic in aetiology. Tentorial laceration results in massive infratentorial ASDH. The tear may involve combinations of the vein of Galen, straight sinus or transverse sinus. Occipital osteodiastasis refers to the separation of the joints within the developing occipital bones, and this can result in damage to the occipital sinus and laceration of the cerebellum, causing infratentorial haemorrhage.459 Laceration of the falx is less common than laceration of the tentorium cerebelli, and usually the tear is close to the junction of the falx and tentorium cerebelli. Bleeding is usually from the inferior sagittal sinus, with the haematoma forming above the corpus callosum. Finally, ASDH may be due to bridging vein rupture over the convexity of the brain. These lesions can be bilateral.426 Perinatal SCI results from excessive traction and/or rotation during delivery. SCI is uncommon with cephalic deliveries, but when it does occur it is a devastating injury involving the upper cervical spinal cord (Figure 10.36a) and in some cases causing complete transection of the cord.399 In breech delivery, the site of injury is usually in the lower cervical/upper thoracic region.270 In the acute phase, there is parenchymal haemorrhage, in some cases secondary to dislocation of the vertebral bodies, although the neonatal spinal column is particularly elastic.452 The demonstration of a vital tissue reaction, such as macrophage infiltration or axonal swellings and axonal β-APP immunoreactivity, can be important in demonstrating that the injury was antemortem and does not represent a post-mortem artefact. If the neonate survives the acute injury, chronic degenerative changes develop, particularly cystic degeneration of the ­spinal cord parenchyma (Figure 10.36b).

ABUSIVE HEAD TRAUMA IN CHILDREN Although head injury is relatively common in the paediatric population, the vast majority of cases are mild, causing few acute clinical concerns. As with adult head injury, the outcome in children is partly determined by the force of the injury and whether the injury involves primarily contact or inertial forces, and their magnitudes and distribution. Head injury in childhood may be due to a variety of causes ­including road traffic accidents, falls, injuries sustained in recreational and competitive activities, and assault. Of particular interest in this section is abusive head trauma (AHT). The largest pathological study of fatal human paediatric traumatic brain injury looked at patients between the ages of 2 and 15 years; the results are referred to in the following discussion.160 Skull fractures were recorded in 72 per cent of the cases, with the majority being linear. It is

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Abusive Head Trauma in Children  667

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10.36 Perinatal spinal cord injury. This term infant had a complex delivery requiring forceps. Apgar scores were poor. The neonate was transferred to a neonatal intensive care unit and maintained on a ventilator for several days prior to death. At autopsy, there was obvious high-cervical spinal cord damage (a) which, on longitudinal section, showed cystic cavitation (b). Panel (a) courtesy of Dr Paul French, Glasgow, UK.

important not to confuse a linear skull fracture with sutures or Wormian bones, and the pathologist should be aware of this potential pitfall.29 Contusions were seen in 92 per cent, large intracranial haematomas in 34 per cent and intracerebral haematomas in 16 per cent, of which 8 per cent were burst lobes, including two cases of posterior fossa haematoma. From this study, the incidence of major TBI pathological substrates is similar to that in adults. However, children under the age of one year were not represented in this study, and AHT has a peak incidence in infants (by definition aged 12 months or under), with an incidence of 28.9 per 100 000 per year in infants compared to an incidence of AHT of 4.1 per 100 000 per year in five-year-olds in one study.386 Child abuse can involve neglect, emotional abuse, physical abuse and sexual abuse. Scottish government statistics from 2002 recorded 1900 children as victims of child abuse, 33 per cent of whom suffered physical abuse. Of this group, 1.8 per cent had brain injury, with brain injury accounting for 0.6 per cent of all child abuse.313 The concept of abusive brain injuries in children has been known for many years,50,176 and has had several names attached such as battered baby syndrome51 and, more commonly, shaken baby syndrome (SBS). The latter name reflects the proposed mechanism resulting in the typical triad

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10.37 Bilateral thin film subdural haematomas (SDH) typical of that in abusive head trauma (AHT). Computed tomography (CT) imaging (a) and post-mortem (b) appearances. The skull cap has been removed with the dura intact, and thin acute subdural bleeds are seen bilaterally.

described in these cases: ASDH, usually thin film and bilateral (Figure 10.37); retinal haemorrhages; and ischaemic encephalopathy. Shaking was proposed to cause an acute whiplash-type injury. However, a biomechanical study highlighted the low forces generated by shaking compared to much higher forces generated by inflicted impact, the latter being more in line with the forces predicted to be required for ASDH and diffuse TAI.105 The study suggested that shaking alone was unlikely to generate the forces required to produce the typical pathology. This was supported by post-mortem findings in this same study, which showed evidence of impact injury in all fatal cases. Biomechanical data have subsequently been conflicting as to whether shaking alone can cause a fatal injury.79,340 Non-accidental trauma (NAT) and non-­accidental head injury (NAHI) are two commonly used terms, although currently the term abusive head trauma (AHT) is recommended by the American Academy of Pediatrics:71 all three terms avoid attributing injuries to any specific mechanism, recognizing that different mechanisms can cause the variety of injuries observed in this context. There are numerous studies detailing the clinical and radiological characteristics associated with AHT. However, all such studies have problems with case selection, because often the diagnosis of AHT can be subjective and requires a critical assessment of a caregiver’s history against the infant’s injuries on presentation. In children 50–500 mg/ dL

↓ 50–500 mg/ dL

↓ 50 cells/mm3) or polymorphonuclear leukocytes in the CSF,820 all these features being exceedingly unusual in MS. In addition, an absence of cerebral lesions at clinical onset was, at that point, also felt to be a feature of NMO.820 A pivotal milestone in the understanding of the pathogenesis of NMO in 2004 was the discovery that it was associated with a circulating immunoglobulin that bound to cerebral microvessels, perivascular regions, the pia and subpial regions.423 This highly specific antibody was called ‘NMO-IgG’ and was subsequently found to recognize aquaporin 4,424 a water channel concentrated in the foot processes of astrocytes adjacent to pia and blood vessels

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23.86 Magnetic resonance (MR) imaging of neuromyelitis optica (NMO). Spinal cord swelling and hyperintensity extend over several segments in the T2-weighted scan (a). There is linear enhancement in the lesion on the T1-weighted scan with gadolinium– diethylenetriamine penta-acetic acid (Gd-DTPA) (b). Reproduced from Wingerchuk et al.820 With permission from Lippincott Williams & Wilkins/Wolters Kluwer Health.

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  Neuromyelitis Optica  1375

and in the basolateral cell membrane of ependymal cells.28 In view of this, the diagnostic criteria were subsequently modified to include the detection of aquaporin 4 antibody as a supportive criterion,821 which was also reflected in the slightly modified versions that were to follow.494,685 There is a variety of assays for NMO-IgG and no one method has been universally accepted. At present, indirect immunofluorescence is the preferred technique. The significance of patients with clinical NMO who are negative for NMOIgG is uncertain. Possible explanations include the use of a relatively insensitive assay to detect the antibody or the existence of a subset of NMO patients in whom the autoimmunity is directed toward a different as yet undiscovered antigen.341

NMO Spectrum It soon became apparent that in addition to the classic presentation of optic nerve and spinal cord involvement, the presence of circulating aquaporin 4 antibody was also associated with a variety of other presentations, some of which reflected involvement of either the optic nerve or spinal cord alone and some of which did not involve either of these sites. Thus, the concept of ‘NMO spectrum’ disorders was born, of which there are four general categories,472 as follows.

Spatially Limited NMO-Spectrum Disorders These are instances where only optic nerves or only longitudinally extensive myelitis is present with detection of NMO-IgG. Many, but not all, may go on to show signs and symptoms at the site that was not initially affected.472 Thus, NMO-spectrum (as well as MS) enters into the differential diagnosis of what clinically has been termed ‘transverse myelitis’93 or ‘transverse myelopathy’, i.e. an acute or subacute isolated non-compressive intramedullary spinal cord syndrome. Most of these patients who have longitudinally extensive involvement are NMO-IgG-positive and thus considered to have a NMO-spectrum disorder.93 However, at least 35 per cent with an extensive longitudinal myelopathy are not NMO-IgG-positive;93 these presumably, at least for now, are idiopathic and cannot be classified as part of the NMO spectrum.

Asian ‘Optico–spinal MS’ ‘Optico–spinal MS’ (OSMS) is much more common in non-Caucasian than Caucasian patients and has been extensively studied in Japan. It too, as the name indicates, presents with optic neuritis and spinal cord involvement. It is now felt that those patients with longitudinally extensive myelitis have NMO and indeed many do have the NMO antibody.93 However, there is a group of Asian OSMS patients whose spinal lesions are not extensive and who are NMO-IgG negative, and who are therefore felt to have MS with optic and spinal plaques, rather than NMO.746 A third group of Asian OSMS patients are positive for NMO-IgG and have longitudinally extensive spinal lesions characteristic of NMO/NMO spectrum,

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but also have periventricular brain lesions characteristic of MS.473 This has led to the suggestion that there may be an overlap in the MRI appearance in classic MS (also referred to as ‘Western’ MS as it is seen predominately in Caucasian populations) without NMO-IgG positivity and those with NMO with NMO-IgG positivity, and may indicate a common mechanism for the formation of periventricular plaques in both of these settings.473 In any event, it appears that the distinction between NMO and the various clinical expressions of ‘Asian’ OSMS is not as clear cut as between NMO and ‘Western’ MS. Why this is so is not clear, and is fertile ground for future studies.

23

Atypical NMO Although the initial diagnostic criteria for NMO excluded the possibility of brain lesions, it is now known that the brain MRI may show lesions in up to 60 per cent of longstanding cases that fulfil current diagnostic criteria for NMO.583 These brain lesions comprise nonspecific lesions that are often asymptomatic, in a minority of cases lesions that are typical of MS, or in some cases (particularly paediatric), cerebral, diencephalic and brain stem lesions in locations that are atypical of MS but where aquaporin 4 channels are concentrated (Figure 23.87).583 These lesions that are atypical for MS are now believed to be typical for NMO/NMO spectrum. Thus, hypothalamic lesions are thought to be responsible for the endocrinopathies sometimes seen in NMO, and have also been recognized as the basis for narcolepsy and coma secondary to reduced production of hypocretin (orexin).133 Persistent nausea and hiccoughs, which occasionally may be the presenting symptoms and frequently abate, are due to lesions in the area postrema and associated chemoreceptor trigger zone in the floor of the fourth ventricle.589 The corpus callosal lesions in NMO/NMO spectrum are large and oedematous – again different from MS plaques at that location.521 A further atypical white matter lesion associated with NMO/NMO spectrum is that of posterior reversible encephalopathy syndrome (PRES), thought to be vasogenic oedema secondary to disturbance of water handling as a result of an aquaporin 4 channel abnormality in the setting of hypertension or sudden fluid shifts.453

Association with Other Disorders and Other Antibodies Aquaporin 4 autoimmunity may coexist with other autoimmune conditions such as myasthenia gravis, Graves’ disease, Hashimoto’s thyroiditis,472 pernicious anaemia, ulcerative colitis, primary sclerosing cholangitis, idiopathic thrombocytopenic purpura,685 and coeliac disease.819 Patients with NMO may also have a high prevalence of anti-neuronal antibodies, such as anti-glutamic acid decarboxylase and anti-collapsin response mediator protein-5 (CRMP-5).472 NMO has been associated with systemic lupus erythematosus (SLE) and Sjögren’s syndrome, neither of which, however, shows NMO-IgG positivity in the absence of a myelopathy or optic neuritis.819 This has led to the notion that these two rheumatological diseases and NMO are separate entities coexisting in patients with

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1376  Chapter 23  Demyelinating Diseases

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23.87 Cerebral lesions in atypical neuromyelitis optica (NMO). NMO lesions are indicated by yellow arrows. (a) Longitudinal corticospinal tract lesions involving internal capsule (a and b) and cerebral peduncle (c) and extensive hemispheric lesions that are tumefactive (d) or spindle-like (e). (b) Periependymal lesions in the diencephalon abutting the third ventricle (a and b) and in the brain stem abutting the fourth ventricle (c-e). (c) Periependymal lesions abutting the lateral ventricles (a), which with involvement of the full thickness of the corpus callosum may produce an arch-bridge sign (b), and longitudinal medulla oblongata lesions that are contiguous with cervical spinal cord lesions (c-e).

From Huh S-Y, Min JH, Kim W, et al. The usefulness of brain MRI at onset in the differentiation of multiple sclerosis and seropositive ­neuromyelitis optica spectrum disorders. Mult Scler 2014;20:695–704. © 2014, SAGE Publications. Reprinted by permission of SAGE.

a propensity to autoimmunity. Thus, it is now thought that the myelopathy and optic nerve involvement that may be evident in SLE and Sjogren’s syndrome is not due to involvement of those structures by these connective tissue

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diseases but rather due to co-existing NMO.819 In addition to autoimmune disorders, NMO has also been described as a paraneoplastic syndrome, where the relationship is also probably an autoimmune phenomenon.311

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  Neuromyelitis Optica  1377

Clinical Features and Imaging NMO may present as a monophasic or relapsing disorder, and the level of NMO-IgG correlates with clinical outcome and reflects disease activity.341 The optic neuritis may be unilateral or bilateral and may affect the chiasm and optic tract. The visual deficits, although similar in kind to those in MS, tend to be more severe in NMO and often permanent, in contrast to the usual recovery from optic neuritis in MS. The same is true of the myelopathy, which produces symptoms and signs referable to the spinal level of its most rostral extent. Thus, the acquisition of progressive neurologic deficit in NMO is quite different from MS. In NMO, it is stepwise and due to the accumulated residua from individual relapses, whereas in MS it is gradual and generally independent of relapses.685 The spinal cord lesions tend to involve the cervical and upper thoracic cord, and they may also extend into the medulla oblongata. As such, they are frequently associated with respiratory failure, rarely seen in MS, and this can be a cause of death.820 The spinal lesions of NMO produce a complete transverse myelopathy with symmetrical neurological signs, as opposed to the asymmetrical and milder spinal involvement of the MS relapse.685 Clinical cerebral involvement is relatively unusual but certainly may occur, particularly in children, manifesting as the clinical syndromes of the atypical forms of NMO/NMO spectrum discussed earlier. As noted previously, the finding of a CSF pleocytosis, particularly if polymorphonuclear leukocytes and/or eosinophils are present, also distinguishes NMO from MS.820 The massive increase in GFAP in the CSF during relapses is no doubt due to the destruction of astrocytes in the NMO lesions (see later under Neuropathology, this page).502 The single most useful MRI feature of NMO is the longitudinally extensive myelopathy spanning three or more vertebral levels (Figure 23.86).820 These lesions often show irregular enhancement during a relapse.685 In addition to its longitudinal extent, the myelitis of NMO also differs in its cross-sectional appearance from the spinal MS plaque, being central and often associated with spinal cord swelling in contrast to the more peripheral, posterior and lateral locations of the MS plaque. The optic nerve(s) often show enhancement and the abnormality may extend to the chiasm.685 In addition to abnormalities seen in the brain by routine imaging described earlier, diffuse abnormalities in normal-appearing grey matter227 and in the normal-appearing white matter in NMO660 can be demonstrated by non-routine MRI techniques. Intravenous corticosteroids have been used in the treatment of relapses.818 Plasmapheresis or intravenous immunoglobulin has also been employed.472,818 The response to the latter treatment and to rituximab, a monoclonal antibody directed against CD20 on the surface of the B-cell,472,818 is further evidence of the importance of humoral immunity in the pathogenesis of this disorder. Non-specific immunotherapy has also been used in NMO.818 Interferon-β, frequently used in the treatment of MS, is less effective in NMO and in some cases may worsen the clinical manifestations.472

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It should also be noted that some patients presenting with optic neuritis and a myelopathy will not necessarily have NMO or an NMO-spectrum disorder, as defined earlier, and that other conditions such as MS (see earlier under Asian ‘Optico–spinal MS’, p. 1375), ADEM and some systemic diseases may present in this fashion. To encompass such conditions and in an attempt to reverse the nosological confusion that has plagued the terms ‘Dévic’s disease’ and ‘neuromyelitis optica’ in the past, it has been suggested that these be referred to as ‘Dévic syndrome’ whereas the pure form of NMO with distinct clinical and pathological features be termed ‘Dévic disease’,679 now referred to as NMO/NMO-spectrum disorders. Alternatively, it may be best to designate a patient with an optic–spinal clinical syndrome who fulfils the NMO diagnostic criteria as NMO, and for the cases in whom the underlying condition has been determined to be a condition other than NMO to simply indicate the disorder (MS, ADEM, etc.) presented as an optico–spinal syndrome. Those cases in whom the underlying condition has not been determined could be designated as an optico–spinal syndrome of undetermined classification. These distinctions are clearly important, because treatment for these groups of patients is different.

23

Neuropathology NMO lesions may show heterogeneity of histopathological features. Thus lesions may show demyelination and/or necrosis, which may progress to cystic cavitation (Figure 23.88a,b).441,463 The spinal cord lesions involve grey and white matter and are usually centrally located (Figure 23.88a). Optic nerves show demyelination, which in most studies is inactive,441,560 or necrosis with cyst formation.501 These findings probably reflect the sampling time rather than an indication of the early features of the optic nerve lesion. Necrosis is associated with axon end-bulbs. In contrast to MS, the perivascular inflammatory infiltrates contain very few T-lymphocytes but many eosinophils and neutrophils (Figure 23.88c,d); the degranulation of these leukocytes is thought to be the cause of the necrosis.441 The presence of CCR3, a receptor for the eosinophil chemotactic cytokine eotaxin, may be responsible for the recruitment of eosinophils. In addition, there is deposition of immunoglobulins M (IgM) and G (IgG) (Figure 23.88e), particularly the former, and complement C9 neoantigen around the thickened hyalinized blood vessels (Figure 23.88f).441 In these regions, there is also loss of immunoreactivity for aquaporin 4 and often GFAP (Figure 23.89).501 Moreover, in NMO lesions, C9 neoantigen is also evident on astrocytic foot processes where aquaporin 4 molecules are concentrated.501,640 Some lesions show demyelination, whereas others do not (Figure 23.90), but aquaporin 4 loss is evident in all NMO lesions, despite the degree of demyelination.501,640 Of particular note are lesions in the floor of the fourth ventricle, where there is intense inflammation and aquaporin loss, with reactive GFAP-positive astrocytes and preserved myelin, indicating that aquaporin 4 loss antedates loss of astrocytes and myelin; these

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1378  Chapter 23  Demyelinating Diseases (a)

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23.88 Neuromyelitis optica (NMO). Spinal cord lesion in NMO. There is extensive destruction of the parenchyma (a,b), with perivascular infiltrates of eosinophils (c) and polymorphonuclear leukocytes (d), and perivascular deposition of immunoglobulin (e) and C9 neoantigen (f). (a,b) Luxol fast blue (LFB)–periodic acid-Schiff (PAS); (c,d) haematoxylin and eosin; (e) immunohistochemistry for immunoglobulin; (f) immunohistochemistry for C9 neoantigen. Adapted from Lucchinetti et al.441 By permission of Oxford University Press on behalf of The Guarantors of Brain.

medullary lesions have been surmised to be reversible and the underlying cause of transient nausea and hiccoughing in NMO (Figure 23.91).589,640 The loss of aquaporin 4 in astrocytes in early NMO lesions is in contrast to the early MS plaque, where aquaporin 4 is upregulated in reactive astrocytes702 and is then expressed in a lesional stage-related manner, being absent only in chronically demyelinated MS plaques.640 Examination of lesions of different ages explains the heterogeneity of the lesions in NMO. In early lesions, myelin is still present501 and axons are intact but apoptotic oligodendrocytes and expansion of the extracellular space or vacuolation consistent with intramyelinic edema may be evident.560 These early lesions show fragmenting

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GFAP-positive astrocytes and macrophage phagocytosis of GFAP-positive debris.560 The demyelination that follows is characterized by macrophages that now no longer contain GFAP-positive debris but rather LFB-positive myelin debris and neutral lipid.560 Within these lesions, which are eventually depleted of stellate astrocytes, emerges a population of small unipolar or bipolar GFAP-positive aquaporin 4-negative cells thought to be astrocyte precursors (Figure 23.90c).560 As the lesion matures and demyelination goes to completion, astrocytes form elongated processes that are again positive for GFAP and negative for aquaporin 4. These astrocytes tend to be in bundles running between demyelinated axons and macrophages; the tendency to form bundles of astrocytic processes appears

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  Other Central Nervous System Inflammatory Demyelinating Diseases  1379

represents the end-stage of the tissue destruction mediated by the products of inflammatory cells and the immune response.441 It is of interest that, unlike in MS, cortical demyelination is not a feature of NMO.588 The histopathologic features of these aquaporin 4-depleted lesions, with localization of immunoglobulin and complement to perivascular regions where this molecule is normally concentrated in astrocytic foot processes, is consistent with the current prevailing concept that humoral autoimmunity to aquaporin 4 is the basis of this disorder that is distinct from MS. Despite this progress, however, it is presently unclear as to why the NMO lesions have a particular predilection for the optic nerves and spinal cord. Hypotheses include oedema within the non-yielding confines of the spinal cord pia mater and the optic canal and possible peculiarities of the microvasculature at these sites.441,609

(a) AD

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23

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Acute Disseminated Encephalomyelitis and Related Disorders History and Nosology CV

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23.89 Astrocytes and myelin in neuromyelitis optica (NMO). Glial fibrillary acidic protein (GFAP) (a) and aquaporin 4 (AQP4) (b) are absent in NMO lesions, whether they are actively demyelinating (AD) or chronic active (CA), but myelin basic protein (MBP) (c) is variably spared. At higher magnifications, GFAP-positive reactive astrocytes (d, arrows) are seen at the lesion edge and perilesional parenchyma. Only fragments of GFAP-positive debris (d, arrowheads) remain in the cavitating lesion (CV). No AQP4-positivity is seen in the lesion (e). (a,d) Immunohistochemistry for GFAP; (b,e) immunohistochemistry for AQP4; (c) immunohistochemistry for MBP. From Misu T, Fujihara K, Kakita A, et al. Loss of aquaporin 4 in lesions of neuromyelitis optica: distinction from multiple sclerosis. Brain 2007;130 (Pt 5):1224–34.501 With permission of Oxford University Press on behalf of The Guarantors of Brain.

to be characteristic of NMO and is a further characteristic that distinguishes it from MS. It appears that necrosis, although usually a prominent feature in NMO, occurs relatively late, as it is evident in old lesions.560 It presumably

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Acute disseminated encephalomyelitis (ADEM), an inflammatory demyelinative disorder of the CNS with a presumed autoimmune pathogenesis, has been referred to by a variety of terms in the literature that emphasize its triggering events, histopathology, pattern of lesion distribution or immunopathogenesis.748 Such terms include post-infectious encephalomyelitis, post-vaccinal encephalomyelitis, post-infectious multifocal encephalitis, perivenous encephalomyelitis, acute perivascular myelinoclasis, disseminated vasculomyelinopathy, acute demyelinating encephalomyelitis, hyperergic encephalomyelitis, postvaccinal perivenous encephalomyelitis, and post-encephalitis demyelination.748 The first reports of the disorder emerged as descriptions of post-infectious and post-vaccinal perivenous encephalomyelitis in the early 1920s.609

Epidemiology The incidence of ADEM is estimated to be approximately 0.2–0.8/100 000 per year.561 It appears to be somewhat more frequent in males and is more frequent in young children and adolescents than in adults, 80 per cent of cases occurring before 10 years of age.561,678 ADEM has been reported following a large number of viral, bacterial, spirochetal, rickettsial561 and protozoan464 infections and after vaccinations561 and the disorder tends to occur more in the winter and spring months.172,748 It has also been reported as a paraneoplastic syndrome.561,735

Clinical Features and Neuroimaging Acute disseminated encephalomyelitis usually presents with an abrupt onset of a neurological syndrome, the differential diagnosis of which is quite broad and

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23.90 Astrocytic loss and astrocytic progenitors in neuromyelitis optica (NMO). An NMO lesion shows an absence of myelin on Luxol fast blue (LFB) staining (a). However, immunohistochemistry for glial fibrillary acidic protein (GFAP) shows astrocytic loss well beyond the area of demyelination (b). High magnification of the area indicated by the upper box in B shows there are elongated GFAP-positive astrocytic progenitors in this region (c). A more recently involved area (d), indicated by the lower box in B, is GFAP-negative in the absence of macrophages and GFAP-positive debris, indicating astrocytic pathology antedates demyelination in NMO. From Parratt JD, Prineas JW. Neuromyelitis optica: a demyelinating disease characterized by acute destruction and regeneration of perivascular astrocytes. Mult Scler 2010;16:1156–1172.560 © 2010, SAGE Publications. Reprinted by permission of SAGE.

includes various infectious diseases.748 There is often a history of infection, or rarely vaccination, in the preceding few weeks. The clinical presentation, which may involve the motor, sensory, cerebellar, brain stem and spinal systems in any combination, tends to be polysymptomatic, as opposed to the monosymptomatic presentation that usually characterizes an MS relapse.172,561 Some patients present with optic neuritis, which is usually bilateral, unilateral involvement being very unusual in ADEM, and if present, should raise the possibility of MS or NMO.172 However, particularly characteristic of ADEM is the occurrence of encephalopathic symptoms and signs, including seizures, alterations in mental status and/or reduced level of consciousness.172 Fever with pleocytosis in peripheral blood and CSF are common.172 Headache, nausea, vomiting and meningismus may also be manifest.172,748 Elevated CSF protein is a common finding whereas oligoclonal bands are unusual,172

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being more frequent in older individuals.561 Rarely concomitant involvement of the peripheral nervous system has been reported, and this appears to be a more likely occurrence in adults.748 The clinical distinction of ADEM from MS is often difficult. Criteria have been developed by the International Pediatric MS Study Group (IPMSSG) for the diagnosis of ADEM and its distinction from paediatric MS on an operational basis and, no doubt, will be modified as future research unravels these two closely related entities.393 These criteria require the clinical syndrome to be polysymptomatic and encephalopathic with no other aetiologies to explain its occurrence, followed by clinical or MRI improvement, although there may be fluctuating symptoms, signs and MRI findings over a 3-month period.393 However, it has been shown that not all patients with ADEM necessarily have encephalopathy,21 and depressed level of consciousness appears

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  Other Central Nervous System Inflammatory Demyelinating Diseases  1381 (a)

23

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23.91 Area postrema lesion in neuromyelitis optica (NMO). (a) Area postrema lesion in a patient with NMO (a, arrows), who presented with an episode of nausea and intractable vomiting shows loss of aquaporin 4 (AQP4), compared to the control area postrema (b, arrows) in a patient with acute MS (b). High magnification of the region indicated by the right arrow in (a), (c) shows depletion of AQP4, with sparing of the immediate subependymal region. The AQP4 loss is particularly perivascular (d). Note the contrast to the control (e) and (f), which shows preservation of AQP4-positive astrocytic perivascular processes in a comparable area (indicated by the left arrow in (b). Immunohistochemistry for AQP4. Scale bars: (a), (b), 1 mm;

23.92 Acute disseminated encephalomyelitis (ADEM) magnetic resonance imaging (MRI). T2-weighted MRI shows bilateral hyperintense poorly-marginated lesions in central, periventricular and juxtacortical white matter (a), internal capsules and thalami (b). From Tenembaum S, Chitnis T, Ness J, Hahn JS. Acute disseminated encephalomyelitis. Neurology 2007;68(Suppl 2):S23–36.748 With permission from Lippincott Williams & Wilkins/Wolters Kluwer Health.

The disease is usually monophasic, with good recovery, both clinically and by imaging, even in the absence of treatment.748 However, death during the illness and residual neurological deficits may occur, more commonly in adults than children.363 Despite the overall good prognosis, it is becoming apparent that patients with ADEM may be left with mild cognitive impairment.363,561,748 Treatment is directed against the inflammatory/immune response and consists of corticosteroids as a first line of defence and usually IVIg therapy or plasmapheresis as alternatives, should the disease be steroid unresponsive.561,748

(c), 100 μm; (d), (f), 50 μm; (e), 200 μm. From Popescu BF, Lennon VA, Parisi JE, et al. Neuromyelitis optica unique area postrema lesions: nausea, vomiting, and pathogenic implications. Neurology 2011;76:1229–37.589 With permission from Lippincott Williams & Wilkins/Wolters Kluwer Health.

to correlate better with the characteristic neuropathology of ADEM than does encephalopathy.841 Moreover, occasional patients with MS may have encephalopathy at their first presentation.363 IPMSSG MRI criteria for the diagnosis of ADEM (Figure 23.92) include multifocal (or rarely a single) lesion(s) greater than 1 cm in size in the supratentorial and infratentorial white matter or in the spinal cord; grey matter, particularly the basal ganglia and thalamus, is frequently involved.393 Periventricular and corpus callosal lesions are less common than in MS, but may be more common in adults than children with ADEM.561,748 The incidence of gadolinium-enhancing lesions is quite variable, having been described in 30 to 100 per cent of patients in various studies.748 MRS shows reduction of NAA and elevated lactate, without elevation of choline, in the lesions; these changes reverse after normalization of the clinical status and T2-weighted abnormalities.748 Diffusion-weighted imaging and perfusion-weighted imaging show variable results.748

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Recurrent and Multiphasic ADEM and Their Relationship to MS Although most patients with ADEM experience a single episode of inflammatory demyelination, there are a minority of patients that go on to have one or more further attacks of the disease. As would be anticipated, the number of such patients varies from study to study, depending on the criteria used for the diagnosis of ADEM and for relapse and the duration of the follow up. The figures vary from 5 to 21 per cent.561,748 The IPMSSG has defined ‘recurrent ADEM’ as a recurrence of the same symptoms and signs as the initial event without any new involvement by clinical and MRI parameters and without a better explanation than disease relapse, occurring at least 3 months after the initial episode and/or 1 month after cessation of steroid treatment.393 ‘Multiphasic ADEM’ is defined as a new clinical event, with the same temporal restrictions just noted, still fulfilling the criteria for ADEM diagnosis but now with evidence of new anatomic regions of involvement by clinical and MRI assessment and partial or complete resolution of the original lesions on MRI.393 The latter scenario, because there is now evidence of dissemination in time and space, makes the distinction from MS problematic and controversial. However, current

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IPMSSG recommendations state that the initial ADEM event should not be used for the determination of space and time dissemination and the distinction from MS can be made on the basis of the fulfilment of diagnostic criteria for an ADEM event (as discussed earlier) applied to the second event;393 but as also noted earlier, these diagnostic criteria do not capture nor distinguish all cases of MS and ADEM. The issue becomes particularly troubling when more than two distinct events occur in a patient with the diagnosis of ADEM. IPMSSG suggests that such cases should be considered extremely suspicious for MS.393 The distinction between ADEM and MS is important, because the former is short-lived and is treated with anti-inflammatory agents, whereas the latter is a lifelong disease that can be treated with long-term immune suppression or immunomodulation. Although it is clear some patients with ADEM, both adults678 and children,748 subsequently fulfil the diagnostic criteria for and ‘convert to’ MS, the exact risk for this is not yet clear, variously reported as 0 to 29 per cent.561,748 Risk factors that have been reported to be operative include initial presentation with optic neuritis or brain stem syndrome, older age at presentation, family history of MS, periventricular lesions, fulfilment of 2005 McDonald criteria for MS at the first ADEM presentation, elevated CSF albumin, female gender, peripheral nervous system involvement, absence of encephalopathy, and absence of seizures, but by no means are any of these absolute.428,561,748 Thus, it is clear from the earlier discussion that the boundaries between ADEM and MS are ill-defined. Hopefully, future research will help clarify whether these are two separate entities or are varied temporal and pathogenetic manifestations of the same underlying disease process. The pathologic features of the two are distinct, but even here there are occasional cases that show histopathologic features of both.

23.93 Acute disseminated encephalomyelitis (ADEM). Fatal ADEM in a 70-year-old male with a 7-week history. Lesions were located predominantly in the brain stem and cerebellar white matter. The pons and medulla were swollen and were softened. Courtesy of Dr LS Forno, Veterans Affairs Health Care System, Palo Alto, CA, USA.

Pathology In patients who die during the acute phase of the disease, the brain may appear swollen and congested, with evidence of transtentorial and cerebellar tonsillar herniation. Swelling of the brain stem or spinal cord is seen in cases in which lesions are concentrated in these regions (Figure 23.93). In contrast to cases of MS of comparable duration in which newly forming lesions may be evident macroscopically in the freshly sliced brain, in cases of ADEM there is usually little to see, apart from swelling.609 Some petechial haemorrhages may sometimes be seen in the corona radiata, brain stem and cerebral cortex.297 In most cases, numerous microscopic perivenous lesions are present throughout the CNS, but they can be limited to a single region. Although they are most numerous in white matter (Figure 23.94), the lesions frequently involve deeper layers of the cerebral cortex and other grey matter regions. Involvement of periventricular blood vessels is also common. Narrow zones of subpial demyelination in the spinal cord and brain stem, and rarely in the cerebral and cerebellar cortex, may also

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23.94 Acute disseminated encephalomyelitis (ADEM). 34-year-old male who died on the sixth day of a neurological illness. There was no preceding febrile illness or vaccination. Necropsy revealed a swollen brain, with numerous small perivascular foci of demyelination throughout the cerebrum. Heidenhain. Courtesy of Dr CJ Bruton, formerly of Runwell Hospital, Wickford, Essex, UK.

be seen.183,468 Marginal demyelination may involve the circumference of the cord or may be especially prominent along the anterior median fissure and more laterally dorsal to the anterior horns. Mild lymphocytic

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  Other Central Nervous System Inflammatory Demyelinating Diseases  1383

meningitis is invariably present. Occasionally, inflammatory demyelination may be present in the spinal ganglia and nerve roots, peripheral nerves and cranial nerves (Figure 23.95).468 The characteristic lesions of ADEM consist of small veins that are often engorged and are surrounded by foamy macrophages (Figures 23.96 and 23.97), with or without a minor lymphocytic/mononuclear cell component.183,281 The vascular inflammatory infiltrates are sometimes associated with necrosis of the blood vessel wall and fibrinous exudates.297 The immediately adjacent parenchyma appears pale and is undergoing active demyelination and sometimes even necrosis.297 The occasional occurrence of necrosis in the vasculature and the parenchyma in this otherwise demyelinating condition highlights the overlap of ADEM and acute haemorrhagic leukoencephalitis (see later under Acute Haemorrhagic Leukoencephalitis, p. 1386). Occasionally, neutrophils are seen in the perivascular cuffs and, as do lymphocytes, sometimes accompany the macrophages in the perivascular parenchyma.609 Macrophages typically contain LFB-positive myelin fragments and neutral lipids (Figure 23.98). Mitotic figures and pyknotic nuclei are relatively common among the perivascular and infiltrating cells.10 Reactive astrocytes are usually relatively inconspicuous. Demyelination remains restricted to extended serpentiginous perivenular sleeve-like hypercellular zones (Figures 23.96 and 23.99), but the size of these zones is variable. In cases preceded by viral illnesses, occasionally microglial nodules are observed.297 In patients who die within a few days of onset, there may be minimal or no apparent changes in myelin but leptomeningeal and perivascular inflammatory infiltrates are usually present,297 whereas in patients dying later, the lesions may encompass larger volumes around the vessels with innumerable foamy macrophages and more extensive myelin loss. Although they are preserved relative to myelin loss, axons show typical features of acute injury (Figure 23.99).261 In typical ADEM, the foci appear to be of similar histological age.183 Some patients may have focal lesions with features that overlap with those in acute haemorrhagic leukoencephalitis.297 Some patients have an associated necrotic myelopathy.312 Patients dying months or years after clinical recovery have shown either no evidence of the earlier illness or variable degrees of perivascular gliosis, fibrosis of perivascular spaces, perivascular rarefaction of myelinated tissue, or more diffuse myelin loss.210,459 A recent report has documented cortical lesions in ADEM consisting of perivenous demyelination, subpial demyelination, and aggregates of activated microglia that were not necessarily associated with demyelination or were adjacent to pyramidal neurons in cortical layer III.841 Although the occurrence of non-confluent perivenous lesions are characteristic of ADEM and are distinguishable from the large confluent perivenous lesions of MS, there are occasional cases in which the clinical history or pathology overlap. Thus, there are rare cases with a protracted history thought to be MS that at autopsy have the characteristic lesions of ADEM.25,547 Whether such cases would be

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23.95 Cranial nerve involvement in acute disseminated encephalomyelitis (ADEM). Same case as shown in Figure 23.93. The third cranial nerve shows diffuse mononuclear cell infiltration, demyelination and some axonal injury. Note the axonal spheroids (a) and the loss of myelin (b). (a) Haematoxylin and eosin; (b) Luxol fast blue (LFB)–Bielschowsky.

23.96 Acute disseminated encephalomyelitis (ADEM). Same case as shown in Figure 23.94, showing a sleeve-like perivascular demyelination in central white matter. Mallory. Courtesy of Professor JW Prineas, University of Sydney, Sydney, Australia.

diagnosed as ADEM now using current clinical criteria is unclear. There are also cases in the older literature labelled as acute MS that have had an antecedent infectious exanthematous illness.166,330,771

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23.97 Acute disseminated encephalomyelitis (ADEM). Enlarged view of lesion illustrated in Figure 23.96, showing a perivascular cellular infiltrate composed largely of macrophages and other mononuclear cells. Mallory. Courtesy of Professor JW Prineas, University of Sydney, Sydney, Australia.

23.98 Acute disseminated encephalomyelitis (ADEM). Same case as shown in Figure 23.94. Higher magnification of a small perivenous demyelinating lesion, showing myelin loss associated with a perivascular macrophage infiltrate. Luxol fast blue (LFB)–Nissl.

Pathogenesis The pathogenesis of ADEM is uncertain. Attempts to recover virus from the brain and to demonstrate viral antigens and viral nucleic acid in affected CNS tissues in ADEM have, for the most part, been negative. This, together with the fact that the pathological changes are unlike those seen in known viral and other pathogen infections, argues against direct invasion of the CNS by an infectious agent as the cause of ADEM. Moreover,

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23.99 Acute disseminated encephalomyelitis (ADEM). Same case as shown in Figure 23.93. A high-power magnification demonstrates acute axonal injury as beading in a demyelinated focus. Note the absence of blue staining of myelin. Combined Luxol fast blue (LFB)–Bielschowsky.

the clinical and pathological uniformity among most cases following infection or vaccination argues in favour of a common autoimmune pathogenesis. The immune response has been postulated to be directed against inoculated CNS tissue components in the post-vaccination cases and possibly involving molecular mimicry between the pathogen and CNS tissues in post-infectious cases. Alternatively, it has also been proposed that the disorder is due to non-specific activation of a pre-existing antimyelin immune response during an inflammatory process.748 As MS, ADEM is also associated with Class II MHC alleles; in the case of ADEM, these include HLADRB1*01, HLA-DRB*03, HLA-DRB1*1501 and HLADRB5*0101.748 Also consistent with an autoimmune reaction is the observation that the usual time periods between infection or immunization and onset of the neurological illness are similar to those observed in acute EAE models (see later under Experimental Autoimmune [Allergic] Encephalomyelitis, p. 1387). Moreover, there are several studies in which MBP-reactive lymphocytes have been detected in the peripheral blood and CSF in patients with post-infectious ADEM.291a Serum antibodies to MOG have also been found in ADEM.539,687 In addition to the neuropathological similarities between ADEM and EAE lesions, in both conditions recovery from acute clinical disease appears to correlate with widespread T-cell apoptosis.62,568 Further, autoimmune demyelination after a viral infection has experimental counterparts (see later under Pathogen-induced Models, p. 1389).

Rabies Post-vaccinal Encephalomyelitis History Neuroparalytic accidents from ADEM complicating rabies vaccination were first recognized within a few years of the introduction by Louis Pasteur of an attenuated virus

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  Other Central Nervous System Inflammatory Demyelinating Diseases  1385

vaccine prepared from desiccated infected rabbit spinal cord. Vaccines of this type were replaced in the early 1900s by Semple-type vaccines prepared using formalin- or phenol-treated infected neural tissue from a variety of animal species. In the 1930s, it was shown that inoculation of neural tissue into experimental animals produced perivenous demyelination.636 Subsequent vaccines prepared using suckling mouse brain, duck embryo and human diploid cell lines have reduced the incidence of neurological complications. The incidence of ADEM associated with neural rabies vaccines is 1:7000 to 1:300, but with non-neural rabies vaccines this incidence drops to 1:15 000.399

Clinical and Pathological Features Clinically, the disorder presents as a transverse myelitis, encephalomyelitis, optic neuritis or polyradiculitis closely resembling the Guillain-Barré syndrome, the last with lesions restricted to the PNS. In other respects, the clinical, neuroimaging and pathological features resemble those of other forms of ADEM, but spinal cord clinical presentations are the most common. In addition to the known nature of the inciting stimulus and the close pathological resemblance to EAE in animals, an autoimmune pathogenesis has been proposed, particularly in patients with chronic neurological complications. Rare cases of rabies post-vaccinal encephalomyelitis have also shown changes resembling those seen in acute MS.609

In the 1950s, a monophasic form of rabies post-vaccinal encephalomyelitis with prominent cerebral symptoms, especially memory loss and personality change, was described in Japan.767 Fever, meningeal signs and impaired consciousness were present at the onset of the illness, and the patients who survived improved without recurrences.696 This disease had been relatively common in Japan, but subsequently disappeared for unknown reasons. Nine autopsied cases had lesions that closely resembled those seen in acute MS, i.e. large, sharply circumscribed, inflammatory demyelinating lesions with hypercellular borders that were often located adjacent to lateral ventricles (Figure 23.100). Unlike acute MS, however, there were also microscopic perivascular demyelinating and inflammatory lesions disseminated throughout the brain and spinal cord. All lesions appeared to be of the same histological age. These cases, therefore, exhibited clinical features of ADEM, but had neuropathological features in common with both ADEM and MS, i.e. they may also be considered to be ‘transitional cases’, as discussed earlier. Other cases have been reported of patients inoculated with neural tissue who subsequently developed MS-like plaques: one was a patient with rabies post-vaccinal

23

(a)

Transitional Cases The concomitant occurrence of the histopathologic features of non-confluent thin perivenous sheaths of inflammatory demyelination of ADEM and the larger confluent plaques of MS, although quite rare, is well established both in patients with a clinical presentation compatible with ADEM and those clinically felt to have acute MS.10,224,232,389, 547,575,684,767,770,771,841 This observation again points out the ill-defined boundary between ADEM and MS. The controversy as to whether this observation indicates ADEM and MS are related diseases9 or these are just two superimposed diseases546 is not resolved.299,841

(b)

Human Experimental Autoimmune (Allergic) Encephalomyelitis The inadvertent or vaccinational inoculation of CNS tissue in humans can produce an ‘allergic encephalomyelitis’. The most common such occurrence is evident after neural rabies vaccines (see earlier under Rabies Post-vaccinal Encephalomyelitis, p. 1384). As noted, the vast majority of cases with neuroparalytic instances after rabies vaccine show the histopathology of ADEM. Similarly, ADEM may be seen after inoculation of CNS tissue for reasons other than rabies vaccination. Among such cases are that of a woman who had been given repeated injections of xenogeneic CNS tissue as ‘fresh cell therapy’ and who developed a fatal coma with CNS lesions suggestive of ADEM.270 Therapeutic immunization trials have occasionally led to unexpected adverse effects that suggest EAE-like mechanisms.225

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23.100 Rabies post-vaccinal encephalomyelitis. Cerebral form in a 41-year-old Japanese male. The patient died 90 days after the first inoculation of Calmette vaccine. Confluent and perivascular foci of demyelination are apparent adjacent to the lateral and third ventricles. (a) Woelcke; (b) thionine. Reproduced from Uchimura and Shiraki.767 With permission from Lippincott Williams & Wilkins/Wolters Kluwer Health.

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1386  Chapter 23  Demyelinating Diseases

encephalomyelitis who later developed a progressive neurological illness and who died 6 years after vaccination with changes typical of longstanding MS present at autopsy.754 Another developed MS-like lesions acutely after receiving multiple injections of a preparation containing neural tissue as treatment for Parkinson’s disease (Figure 23.101).684 These rare cases again illustrate the unresolved relationship between ADEM and MS. The clinical and pathological features of disseminated encephalomyelitis and acute MS are compared in Table 23.10.

Acute Haemorrhagic Leukoencephalitis Clinical Features and Neuroimaging First recognized as a discrete entity by Weston Hurst in 1941326 and hence sometimes referred to as ‘Weston Hurst disease’, acute haemorrhagic leukoencephalitis (AHLE) is a usually fatal disease characterized clinically by an abrupt onset of fever, neck stiffness and neurological deficits, often progressing rapidly to seizures and coma. In most fatal cases, death occurs within 1–5 days.11,326 The CSF pressure is usually elevated, with an increase in protein content and a pleocytosis chiefly of neutrophils, often with red blood cells. An elevated erythrocyte sedimentation rate and a peripheral neutrophil leukocytosis are common.609 The presenting clinical picture is, therefore, similar to that of ADEM, but the course of AHLE is usually more fulminant and ADEM is rarely fatal. The disease has been seen in most age groups, including infant siblings.483 It appears to be rare in elderly people. In approximately 50 per cent of cases there is a prodromal febrile illness, almost always an upper respiratory tract infection, 1–13 days before onset. Usually the offending pathogen cannot be documented, but AHLE has occasionally been associated with herpes simplex, varicella zoster, human herpes virus 6, measles, mumps and (a)

(b)

23.101 Human experimental autoimmune encephalomyelitis (EAE). Case of a 51-year-old man who had received seven injections of calf neural tissue over 18 months as a treatment for Parkinson’s disease. He developed an encephalitis that resulted in death after 10 weeks. The resemblance to acute multiple sclerosis (MS) is striking. (a) Heidenhain. (b) Cresyl violet. Reproduced from Seitelberger et al.684 With kind permission of Springer Science and Business Media.

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Epstein–Barr viruses. Rare cases have also been associated with Mycoplasma pneumoniae, laparotomy and gastrointestinal disturbances.578,609 Morbidity is usually high in surviving patients, but patients with recovery following early treatment have been reported. Treatment modalities have included various combinations of corticosteroids, immunoglobulin, cyclophosphamide and plasma exchange.748 The CT and MR imaging features of ADEM and AHLE are similar, but the latter has larger lesions, more oedema and mass effect and punctate haemorrhages. The distinction between the two is difficult. However, early in the course of AHLE, there are hyperintense areas in diffusion-weighted images and low signal intensity in maps of the apparent diffusion coefficient.580 The use of 2D gradient recalled echo T2*-weighted imaging and susceptibility-weighted imaging to detect haemorrhage may also be helpful in the diagnosis of AHLE.352,580

Macroscopic Pathology At autopsy, the brain appears congested and swollen, sometimes asymmetrically in patients with unilateral signs. Cingulate, parahippocampal and tonsillar herniations are frequent. Multiple petechial haemorrhages affect the central part of the centrum ovale, internal capsule, subcortical white matter and corpus callosum and involve grey and white matter in the midbrain, pons, medulla, and cerebellar peduncles (Figure 23.102). Confluent haemorrhages or large asymmetrical foci of necrosis with cavitation may be present. The spinal cord may also be affected, and rare cases have been restricted to the brain stem, cerebellum or cord.117,580,609 Affected grey matter areas are discoloured and oedematous.

Microscopic Pathology Histologically, perivascular lesions consist of ball or ring haemorrhages surrounding necrotic venules, sometimes with fibrinoid exudates within the blood vessel wall or extending into adjacent tissue. There are cuffs of mononuclear cells and neutrophils (Figures 23.103 and 23.104), but to a lesser degree in cases in which the duration of the illness has been brief. There is also substantial axonal injury in these areas,261 and oedema is usually conspicuous. The extent of microvascular damage and consequent haemorrhage is greater in AHLE than ADEM (Figure 23.105). Neutrophils are also frequently present in the leptomeninges. Many of these features have been reported in stereotactic biopsy samples.262 Demyelination per se is not usually evident in these haemorrhagic lesions and when it is present is usually associated with necrosis.297 However, it should be noted that some perivascular demyelinative lesions of the type seen in ADEM may be also seen in AHLE, but apparently not in the first 4 days.273

Pathogenesis Most authors conclude that AHLE represents a hyperacute form of ADEM, and that the two disorders represent a spectrum of the same disease process. This is supported by the fact that in ADEM, there are instances where the haemorrhagic lesions similar to those of AHLE are also present, and vice versa. As in ADEM an allergic mechanism is suggested by the appearance of the lesions, which in the case of AHLE

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  Animal Models of Human Demyelinating Diseases  1387 Table 23.10  Clinical pathological features of acute disseminated encephalomyelitis and acute multiple sclerosis Acute disseminated encephalomyelitis

Acute multiple sclerosis

Stimulus

Antecedent infection, immunization, vaccines in most cases

No recognized preceding infection or immunization

Prodromal illness

Headache, fever

None

Neurological illness

Meningism, stupor, focal signs

Focal signs

Course usually non-progressive

Variably progressive course

Recovery rapid and often complete

Recovery variable; may be rapid and complete

Relapses rare

Relapses common

Often does not progress to MS

Progresses to chronic MS

Diffuse vascular congestion and swelling

Vascular congestion and swelling limited to lesions

Lesions usually not visible or appear as narrow rings of discoloured tissue around small blood vessels

Lesions several millimetres to several centimetres in diameter

Innumerable small perivascular lesions throughout central nervous system grey and white matter; may be restricted to a particular region

Variable number of lesions in grey and white matter of the brain, spinal cord and optic nerves

Subpial and subependymal demyelination common

Subpial and subependymal demyelination common

Narrow perivenous sleeves of demyelination that retain same shape and size throughout illness

Within days, individual lesions appear as large, irregularly shaped plaques

Margins sharp

Margins sharp; growth by radial extension or by confluence of periplaque perivascular lesions or concentric bands of demyelination

Age of lesions uniform

Lesions of different ages

Demyelination associated with appearance of macrophages

Demyelination associated with appearance of macrophages

Astrocytic reaction inconspicuous

Astrocytes numerous and enlarged, with mitoses and multinucleate and giant forms

Perivascular and parenchymal lymphocytic infiltration and appearance of neutrophils

Perivascular and parenchymal lymphocytic infiltration; blood vessel wall destruction and granulocytic infiltration usually absent

Axonal injury present

Axonal injury present

Macroscopic features

Lesion number and location

Microscopic features

resembles those seen in ‘hyperacute’ EAE where a modified adjuvant containing pertussis vaccine is administered427 with the immunogenic CNS tissue.

Animal models of human demyelinating diseases Animal models contribute indispensable insight into human CNS demyelinating diseases. They elucidate complex mechanisms of CNS immunity, tissue injury, chronic degeneration, regeneration, and the failure of spontaneous repair. They are also employed for advances in the management of patients, for example through testing therapies and neuroimaging techniques. Representative models are listed in Table 23.11.

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23

Experimental Autoimmune (Allergic) Encephalomyelitis In 1935, it was discovered that the repeated inoculation of normal rabbit brain into non-human primates over several months resulted in a neurological illness that resembled ADEM.636 It was subsequently shown that the latency to disease onset could be markedly reduced by inoculation of the neural tissue in complete Freund’s adjuvant (comprised of an emulsion of killed Mycobacterium tuberculosis in oil)347 and that the disease could be induced in small laboratory animals.427 Later chronic EAE was introduced, some with a relapsing-remitting course and with large demyelinative lesions that resembled those of MS (Figures 23.106 and

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1388  Chapter 23  Demyelinating Diseases (a)

(b)

23.102 Acute haemorrhagic leukoencephalitis. 22-year-old male who developed a rapidly fatal neurological disease 2 weeks following an upper respiratory tract infection. (a) There are multiple scattered petechial haemorrhages, oedema and focal diffuse haemorrhage in the cerebral hemispheric white matter. The patient was on a respirator, and there is additional cortical hypoxic–ischaemic damage. (b) Multiple petechial haemorrhages in the cerebellar white matter and basis pontis.

23.103 Acute haemorrhagic leukoencephalitis. Same case as shown in Figure 23.102. The subcortical white matter shows multiple foci of inflammatory demyelination, with diffuse oedema in the adjacent, more intact white matter. Klüver–Barrera.

23.107).617 The evolution of EAE models in the second half of the twentieth century paralleled the discovery and biochemical characterization of diverse myelin and other CNS tissue components and advances in understanding molecular and cellular immunology and neurobiology, particularly the mechanisms of demyelination (Figures 23.108 and 23.109). Immunization protocols progressed from the use of whole CNS tissue homogenates to purified myelin protein components for EAE

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23.104 Acute haemorrhagic leukoencephalitis. 42-year-old male with a 3-day history following pneumonia. Perivascular neutrophil infiltrates in cerebral white matter. Luxol fast blue (LFB)–Nissl. Courtesy of Dr CJ Bruton, formerly of Runwell Hospital, Wickford, Essex, UK.

induction. Following the identification of encephalitogenic epitopes, synthetic peptide immunogens and protocols involving cell transfer of antigen-specific T-cell lines and clones have been employed widely. Although T-cell-mediated autoimmunity was thought to be the basis for most forms of EAE, more recently the marmoset model with MOG as the encephalitogen appears to be antibody-mediated (Figure 23.110).

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  Animal Models of Human Demyelinating Diseases  1389

Pathogen-Induced Models Several CNS virus infections address the possible viral aetiology of MS. In these models, demyelination may be a result of oligodendrocyte infection and death, persistent infection that impairs oligodendrocyte functions, a virusspecific immune response that mediates so-called ‘bystander damage’ of myelin and oligodendrocytes (i.e. by elaboration of toxic molecules such as TNF and NO), or crossreactive immunity.609,730

23

Theiler’s Murine Encephalomyelitis Virus

23.105 Acute haemorrhagic leukoencephalitis. Acute haemorrhagic demyelinating lesion. There is marked destruction of both myelin and axons, with prominent fibrin leakage into the parenchyma. Luxol fast blue (LFB)–Bielschowsky preparation.

As in human demyelinating diseases, susceptibility to EAE, its clinical manifestations, and its pathological and immunopathological mechanisms are heterogeneous and are influenced by genetic and epigenetic factors.204, 206a,207,731,855 The range of clinical EAE phenotypes now includes hyperacute, acute monophasic, chronic progressive and relapsing-remitting forms; CNS lesion features generally correlate with the disease duration and clinical severity. In addition to inflammatory demyelination, axonal damage is also recognized as a feature of EAE (Figure 23.109),617 but the progressive neurodegenerative phase of MS has not yet been addressed to any significant extent in EAE models, but some emerging models may offer some promise in this area.402 In this regard, it would be of great interest to determine if the autoimmune process in these experimental models can induce progressive axonal degeneration. Recently, transgenic mice have been developed with transgenes for T-cell receptors that recognize specific myelin encephalitogenic epitopes or MHC alleles that are associated with autoimmune demyelination and these animals spontaneously (i.e. without inoculation of the encephalitogen) develop EAE.402 Transgenic mice with transgenes for cytokines and chemokines have also been developed.670 In several T-cell receptor transgenic mouse models, there are distinct CNS lesion localization patterns that correspond to those in human disease subtypes, such as opticospinal.402 Knockout mice have also been used in the study of autoimmune demyelination, such as the studies showing the importance of IL17 in its pathogenesis.402 Also the utilization of ‘humanized’ mice, wherein the pertinent human transgene is inserted into murine DNA, will lead to further insights into the pathogenesis and immunopathogenesis of experimental demyelination and its relationship to MS.40,612 EAE is used extensively worldwide as a demyelinating disease model, as a paradigm for organ-specific autoimmunity and for determining efficacy of potential therapies of MS and other demyelinating diseases.293

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Virulent strains of Theiler’s murine encephalomyelitis virus (TMEV), a non-enveloped, single-stranded RNA picornavirus, induce lethal encephalitis following intracerebral injection into mice. Avirulent strains first cause mild, transient encephalitis due to neuronal infection, followed by a persistent infection that results in demyelination secondary to both lytic infection of oligodendrocytes and immune-mediated mechanisms.638 Oligodendrocytes proliferate in recent lesions, and there is both remyelination and local recurrent inflammatory demyelinating activity in chronic infections (Figure 23.111).484,522,637a

Mouse Hepatitis Virus The A-59 strain of mouse hepatitis virus (MHV), an enveloped single-stranded mRNA coronavirus, produces multifocal demyelination following intracranial injection into C57Bl/6 mice. Demyelination due to lytic infection of glial cells, and motor signs appear early in the infection; when the virus is cleared, there is widespread remyelination and corresponding functional recovery. The JHM strain of MHV is also toxic to oligodendrocytes and can produce a persistent and recurrent demyelinating disease.474

Semliki Forest Virus Semliki forest virus (SFV) is an alphavirus of the Togavirus family. Following systemic infection, the avirulent A7 strain produces small perivascular foci of demyelination associated with the site of entry into the CNS. Focal demyelination is thought to result from activities of cytotoxic CD8+ T-cells on virus-infected oligodendrocytes and from production of antibodies that recognize myelin components.219,504

Canine Distemper Virus Canine distemper virus (CDV) is an enveloped paramyxovirus closely related to human measles virus that causes immunosuppression and a demyelinating neurological disease in dogs.821a In the early stage, there is disseminated encephalomyelitis with demyelination that is associated with viral replication in the white matter, restricted infection of oligodendrocytes and microglial activation. A progressive or relapsing disease course associated with chronic inflammatory demyelination may be

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1390  Chapter 23  Demyelinating Diseases Table 23.11  Selected animal models of demyelinating diseases Type

Examples

Representative reference/review

Autoimmune

Acute and chronic EAE induced by active sensitization or adoptive transfer of T-cells sensitized for myelin and other CNS antigen epitopes

Kurschus et al.402

Raine and Genain617 Viral

Genetic

Toxic

Nutritional/metabolic

Physical manipulations

Theiler’s murine encephalomyelitis virus (TMEV) infection

Rodriguez et al.638

Mouse hepatitis virus (JHM) encephalomyelitis

Matthews et al.474

Semliki forest virus (SFV) encephalitis

Mokhtarian et al.504

Canine distemper encephalomyelitis

Vandevelde and Zurbriggen778

Visna

Georgsson254

Herpes simplex virus (HSV) Type I

Kastrukoff et al.352

Herpes simplex virus (HSV) Type II

Georgsson et al.255

Naturally occurring and inbred myelin-deficient and dysmyelinating mouse mutants: Jimpy, Twitcher, Quaking, Shiverer mice

Duncan et al.191

Myelin-deficient and dysmyelinating rats

Duncan et al.191

Myelin antigen-specific T-cell receptor transgenic mice that develop spontaneous EAE

Kurschus et al.402

Transgenic mice with transgenes for cytokines and chemokines

Scheikl et al.670

‘Humanized’ mice

Attfield et al.40

Lysolecithin Kotter et al.385a Ethidium bromide

Black et al.81

Cuprizone

Kipp et al.369

Triethyl tin

Aleu et al.15a

Hexachlorophene

Lampert et al.408a

6-aminonicotinamide

Schneider and Cervos-Navarro674a

Hypocholesterolaemic agents

Suzuki and Zagoren736a

Cyanide

Brierley et al.103a

Electrolyte-induced demyelination

Rojiani et al.642

Swayback (copper deficiency)

Innes and Shearer335

Spinal cord compression

Jones et al.346,583a

Cerebrospinal fluid exchange

Bunge et al.119

Heat injury

Sasaki and Ide665a

X-irradiation in young animals

Blakemore and Patterson82a

X-irradiation in adult animals

Mastaglia et al.469a

CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis.

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  Animal Models of Human Demyelinating Diseases  1391

due to interactions between macrophages and antiviral antibodies. Non-cytolytic selective spread of the virus and restricted infection may permit viral persistence and escape from immune surveillance (Figure 23.112).778

23

Visna Visna is a naturally occurring retroviral infection of the nervous system in ruminants that can manifest demyelinative lesions,254 thought to be due to a bystander effect of the inflammation.609

Herpes Simplex Virus 23.106 Experimental autoimmune encephalomyelitis (EAE). Large demyelinating lesion in the spinal cord of a juvenile Hartley guinea pig 7 weeks after sensitization for EAE. Toluidine blue. Courtesy of Drs Y Maeda and R Maeda, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA.

Herpes simplex virus (HSV) Type I inoculation into the trigeminal ganglion of mice results in demyelinative white matter lesions.356 The virus is present in a variety of cell types in the lesions and the pathogenesis of tissue damage appears to be related to inflammation, a viral cytopathic effect and immune-mediated mechanisms.356,609 Intracerebral or genital introduction of HSV Type II in mice leads to a necrotizing encephalitis or predominately white matter disease with viral-induced oligodendrocyte death.255,609

Genetic Models Dysmyelinating animal mutants are important models for investigating the physiology and development of myelin constituents and elucidating the pathogenesis of inherited human diseases of CNS myelin. Models with counterparts in human leukodystrophies include twitcher mice and canine globoid leukodystrophy (see Chapter 6, Lysosomal Diseases). Despite having a primary genetic basis, these models provide critical insights into CNS pathophysiology that are highly relevant to human demyelinating diseases.191 Various clinically and pathologically distinct phenotypes of spontaneous mutants, such as Jimpy and Shiverer mice and myelin-deficient rats, are due to mutations of myelin genes. These animals have been particularly useful for testing cell transfer therapies.191 Recombinant technology has also produced transgenic mouse models that elucidate the roles of specific antibodies, cytokines and other molecular mediators of inflammatory demyelination (see also under Experimental Autoimmune [Allergic] Encephalomyelitis, p. 1387).402

Toxic and Other Chemical Models of Demyelination

23.107 Experimental autoimmune encephalomyelitis (EAE). Large demyelinated lesion in the spinal cord of a juvenile Hartley guinea pig 10 weeks after sensitization with guinea pig central nervous system white matter for induction of EAE. Toluidine blue. Courtesy of Drs Y Maeda and R Maeda, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA.

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Chemicals that are toxic to oligodendrocytes or myelin sheaths are often used to create experimental demyelination. In many cases, the precise mechanisms of the demyelination, i.e. whether due to oligodendrocyte dysfunction or death, myelin sheath injury or a combination of these mechanisms, are not clear. The chemical models are sometimes referred to as ‘gliotoxin models’ and are induced either by systemic delivery, such as in drinking water, or by injecting directly into the CNS white matter or another site. Because the induced injury is generally limited to either a fixed time period or a single injection, it is monophasic and the resulting demyelination is frequently followed by remyelination.

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1392  Chapter 23  Demyelinating Diseases (a)

(b)

23.108 Experimental autoimmune encephalomyelitis (EAE). Rabbit 14 days after immunization with bovine central nervous system white matter. Electron micrographs. (a) Obliquely sectioned myelinated fibre is enveloped by a macrophage. Early demyelination involves the attachment of myelin to a coated pit on the macrophage surface (arrowhead). (b) Enlarged view of the area indicated by the arrowhead in (a). Reproduced from Epstein LG, Prineas JW, Raine CS. Attachment of myelin to coated pits on macrophages in experimental allergic encephalomyelitis. J Neurol Sci 1983;61:341–8.206a With permission from Elsevier. (a)

(b)

(c)

23.109 Experimental autoimmune encephalomyelitis (EAE). Border of chronic EAE lesion (right side of fields), with intact white matter in the brain of a C57Bl/6 mouse 3 months after immunization with an encephalitogenic peptide of myelin oligodendrocyte glycoprotein. At this late stage, the lesion shows scant inflammation but there is marked demyelination (a,b). Axonal injury and depletion are evident in (c). (a) Haematoxylin and eosin; (b) Luxol fast blue (LFB)–periodic acid-Schiff (PAS); (c) Bielschowsky.

Systemically Administered Agents The most widely used systemic chemical model involves feeding mice with a diet containing cuprizone, a copper-chelating agent that causes demyelination in particular brain regions in mice. This lesion has been used widely in remyelination studies444 and as a means of

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studying demyelination and remyelination in transgenic and knock-out mice.369,728 Other, less frequently used models involve the systemic administration of triethyl tin,15a 6-aminonicotinamide,674a hexachlorophene,408a cyanide,103a and hypocholesterolaemic drugs in young animals736a (see Table 23.11).609

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  Animal Models of Human Demyelinating Diseases  1393

Focally Administered Agents

23.110 Experimental autoimmune encephalomyelitis (EAE). Evolving spinal cord lesion in a marmoset with myelin oligodendrocyte glycoprotein (MOG)-induced EAE. Intramyelinic oedema is the dominant pathological alteration in this animal examined 28 days after sensitization. Toluidine blue. Courtesy of Dr CS Raine, Albert Einstein College of Medicine, Bronx, NY, USA.

Two frequently used chemicals that induce demyelination following local administration into the CNS are lysolecithin385a and ethidium bromide.81 Lysolecithin (Figure 23.113) is a phospholipid that acts by solubilizing myelin membranes via a general detergent effect. Ethidium bromide is a DNA-intercalating agent that prevents DNA replication and disrupts RNA and thus protein synthesis, leading to cell death. A particular advantage of these models is that there is precise control over anatomical localization of the lesion, and it is thus easily sampled or manipulated by, for example, cell transplantation.81 Other chemicals that have been used to induce demyelination by direct injection are 6-aminonicotinamide, diphtheria toxin, calcium ionophores, and zymosan.590 Focal areas of demyelination can also be created by direct injection of antibodies against myelin components or antibody-producing cells.651

23

Nutritional/Metabolic Models Demyelination has been produced by an electrolyte disturbance resulting from hypertonic saline.642 Although this is a primary demyelinating disorder, its mechanism of induction is particularly pertinent to central pontine myelinolysis (see Chapter 9, Nutritional and Toxic Diseases). Copper deficiency has been associated with a demyelinating disease in animals referred to as ‘swayback’.335

Physical Injury Central nervous system myelin fibres are particularly susceptible to physical injury, a property that has been exploited in several experimental models of demyelination. A technique of historical importance, although used rarely now, is that of CSF barbotage, in which small volumes of CSF are injected forcibly on to the surface of the spinal cord. It was with this technique that the process of remyelination following superficial demyelination was first described.119 In spinal cord injury models, acute compression or concussion of the cord induces inflammation and demyelination, which are common features of human spinal cord injury.346,583a Other CNS injury models, such as chronic cord compression, X-irradiation in young and adult animals,82a,469a and thermal injury,665a also result in demyelination, whereas cold probe injury tends to induce more axonal damage (see Table 23.11).609

Limitations of Demyelinating Disease Models and Their Relevance to Human Diseases 23.111 Theiler’s murine encephalomyelitis virus (TMEV). Remyelination by oligodendrocytes (O) is seen in the spinal cord of a SJL/J mouse infected with the DA strain of TMEV and treated with serum from a mouse hyperimmunized with spinal cord homogenate. The remyelination is characterized by myelin sheaths that are inappropriately thin for the diameter of the axons they ensheath (stars). A demyelinated axon is present (arrowhead). Reproduced with permission from Rodriguez.637a Copyright © 2006, John Wiley and Sons.

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The scientific value of animal models of human demyelinating diseases cannot be overestimated and, indeed, they often provide impetus for analogous studies of human diseases. It should be emphasized, however, that although EAE models closely replicate most features of human ADEM, no naturally occurring animal CNS disease corresponds to MS. The nosological distinction between ADEM and acute MS is, therefore, particularly important in this context. Because of the strong

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1394  Chapter 23  Demyelinating Diseases

23.112  Canine distemper. 10 days after onset, nerve fibres are seen to undergo active demyelination by encircling macrophage processes (arrowheads). Distemper virus can be seen in the macrophage cytoplasm (arrow). There is a denuded axon at the upper left. Electron micrograph. L, lymphocyte; O, oligodendrocyte. Reproduced by permission from Macmillan Publishers Ltd: Laboratory Investigation from Wisniewski H, Raine CS, Kay WJ. Observations on viral demyelinating encephalomyelitis: canine distemper. Lab Invest 1972;26:589–99.821a Copyright 1972.

23.113 Lysolecithin-induced remyelination. The demyelination that follows lysolecithin is followed by remyelination, shown here 21 days after its injection. Macrophages (m) contain droplets of neutral lipid. Many axons (asterisks) have thin remyelinated myelin sheaths. Reproduced from Kotter MR et al.385a With permission of John Wiley and Sons. Copyright © 2001 Wiley-Liss, Inc.

similarities between EAE models and ADEM, a close relationship between MS and ADEM would support the widely held view that EAE is a relevant laboratory model for MS. Morphological and other evidence, based on the existence of ‘transitional’ or overlapping cases with

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features of both MS and ADEM, and of findings in cases of rabies post-vaccinal encephalomyelitis and of ‘human EAE’, provide further conceptual support for a close relationship. There are, however, numerous factors intrinsic to animal disease models that preclude their faithful recapitulation of human diseases. The models are generally induced in uniform, genetically homogeneous test groups, either by an artificial induction process (sensitization, infection) or through genetic manipulation. Therefore, they contrast dramatically with the spontaneous diseases that develop in outbred human populations. Often overlooked, furthermore, are the large disparities in lesion size between typical MS plaques and microscopic inflammatory demyelinating foci in small animal models. In particular, EAE is by definition an autoimmune disease. The view that the human demyelinating diseases are also autoimmune diseases (with the possible exception of post-infectious and post-vaccination encephalomyelitides; see earlier under Other Central Nervous System Inflammatory Demyelinating Diseases, p. 1379) continues to rest largely on circumstantial, although strongly suggestive, evidence that implicates CNS myelin, oligodendrocytes or other antigen(s) as autoimmune targets in MS. Thus, despite multiple common pathological features and pathogenetic mechanisms, the relationships of human diseases (particularly MS) to EAE (and to other currently used models) remain approximate. As a further precaution, the therapies that may be successful in treatment trials in animal models may not be beneficial and can be harmful in MS. Table 23.12 compares features of MS and major types of animal demyelinating disease models currently employed.

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  References  1395 Table 23.12  Features of multiple sclerosis and experimental demyelinating diseases MS

EAE models

Viral-induced models

Chemical/toxic models

Genetic models (spontaneous and induced)

Inflammation

Yes

Yes

Yes

Not usually

Variable

Demyelination

Yes

Yes

Yes

Yes

Yes

Acute axonal injury

Yes

Yes

Yes

Variable

Often

Chronic neurodegeneration

Yes

Yes

Yes

Usually minimal

Often

Known aetiology or pathogenetic agent

No

Yes

Yes

Yes

Yes

Autoimmune mechanisms

Presumed but unproven

Yes

Some

No

Variable

Peripheral nervous system involvement

Variable

Variable

Variable

Variable

Variable

23

EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis.

Acknowledgements The authors wish to record their gratitude to Professor John W Prineas for permission to reprint many of his illustrations

in this chapter, and to Dr Raymond A Sobel for his contributions to portions of the text that have been retained from the previous edition. Dr Moore’s research is supported by grants from the Multiple Sclerosis Society of Canada.

References 1.

2.

3.

4.

5.

6.

7. 8.

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24 24 Chapter

Diseases of Peripheral Nerves Robert E Schmidt and Juan M Bilbao

Introduction..............................................................................1413 Clinicopathological Features of Neuropathy...............................1414 Normal Structure of Peripheral Nerve........................................1416 Basic Pathological Mechanisms................................................1424 Inherited Neuropathy................................................................1435 Inflammatory and Infectious Neuropathies................................1453 Neuropathies Associated with Infectious Diseases....................1461 Vasculitic Neuropathy...............................................................1470 Amyloid Neuropathy..................................................................1474

Introduction The axons and dendrites of peripheral nerves arise from ganglion cells lying within the ventral grey matter of the spinal cord, from brain stem nuclei, and from neurons situated within the sensory ganglia of cranial and spinal nerves and the ganglia of the autonomic nervous system and enteric plexuses. They end in synaptic contact with other neurons in the dorsal horns of the spinal cord or other sensory nuclei in the central nervous system (CNS), with autonomic ganglia or with secretory or muscular effector cells; alternatively, in the case of sensory axons, they end as free terminal arborizations or specialized terminals within a variety of encapsulated sense organs.504 Nerve trunks, their branches and distal twigs are composite structures of nerve fibres, Schwann cells, layers of connective tissue and perineurial cells and vessels. This unique arrangement of tissues derived from the CNS, the neural crest and mesenchyme constitutes the peripheral nervous system (PNS). The PNS comprises the ten lower cranial nerves, nerve roots of the spinal cord, spinal ganglia, plexuses, nerve trunks and their terminations in skin and muscle, as well as the autonomic ganglia and nerves. At any level, this system may be affected by a variety of multifocal and systemic disorders. Because nerves travel through subcutaneous tissues and narrow anatomical channels, they are often involved in lacerations, blunt trauma and repetitive injuries leading to entrapment neuropathies and pressure palsies.

Neuropathy of Dysproteinaemia................................................1480 Diabetic Neuropathies...............................................................1485 Toxin-Induced Neuropathies.....................................................1492 Metabolic Neuropathies............................................................1498 Miscellaneous Neuropathies.....................................................1499 Neuropathies Related to Lymphoreticular Proliferative Disorders.1502 Acknowledgements..................................................................1503 References...............................................................................1503

The traditional pathological classification of peripheral nerve disorders emphasized the component of the PNS most likely to be affected, thus:504 ●●

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neuronopathies consist of conditions affecting loss of neurons (lower motor neurons and/or sensory and autonomic neurons) that contribute nerve fibres to ­ peripheral nerves; axonopathies occur when there is axonal degeneration, with preservation of the cell body. More often, in generalized axonopathy, the degeneration commences distally and advances proximally towards the cell body in a ‘dying back’ manner. Rarely, the axonal degenerative process begins proximally and progresses downstream. Axonopathies may also be focal; disorders that directly affect the Schwann cell or myelin lead to primary segmental demyelination. When demyelination occurs as a consequence of axonal influences, it is termed ‘secondary demyelination’; Conditions that primarily affect connective tissue and vasculature often have secondary effects on the nerve fibre and Schwann cells.

It is this biological interdependence of the ganglion cell, the axon or dendrites, the Schwann cell and the supporting connective vascular tissue that explains the stereotyped response of the nerve fibre to a variety of different pathological insults.

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1414  Chapter 24  Diseases of Peripheral Nerves

Clinicopathological Features of Neuropathy Three fundamental features characterize a neuropathy clinically: ●● ●● ●●

spatial distribution; time course; fibre type involvement.325

A fourth feature, axonal versus demyelinating, is largely an electrophysiological determination.

Spatial Distribution Polyneuropathies may occur diffusely or in a multifocal manner. The most common pattern is a length-dependent process in which the distal parts of extremities are affected first, symmetrically, with progression evolving from toes to ankles and from fingertips to wrist, and into the classic glove–stocking pattern. Many aetiologies can cause this ‘distal axonopathy’, including toxic and metabolic conditions, genetic disorders and inflammatory and paraneoplastic diseases. At the other extreme are focal and multifocal neuropathies, which in classic form are designated clinically as ‘mononeuritis multiplex’ (Table 24.1). However, the progression of multifocal peripheral nerve disease with summation of insults affecting contiguous nerves on both sides of the body, as may occur in polyarteritis nodosa (PAN), blurs the clinical distinction between multiple mononeuropathy and symmetrical polyneuropathy, thus setting the stage for misdiagnosis.

Time Course Consideration of time course is helpful in assessing a patient with peripheral neuropathy. Subacute processes evolving over days to a month are usually inflammatory, such as Guillain–Barré syndrome (GBS), hyperacute vasculitic syndrome and paraneoplastic sensory neuropathy, although some toxic neuropathies can evolve rapidly as well. Processes developing over a period of 3–5 years often have a genetic basis, even when there is no family history of a neuromuscular disorder. Paraproteinaemic neuropathy and some metabolic conditions may also have a slow, indolent course. Neuropathies that evolve over many years without causing major disability often elude diagnosis despite extensive testing (cryptogenic neuropathies) and are particularly common in elderly patients.

Fibre Type Involvement Most neuropathies affect sensory and motor function. In many cases, sensory symptoms and signs tend to dominate initially, because the longest axons are those supplying the feet and toes, whereas motor involvement necessitates progression of the disease to involvement of more proximal segments of nerve serving major muscle groups. Commonly, sensory involvement affects both large and small fibres. Dysfunction of large fibres produces deficits in vibration and joint position sensation, and impairment of deep tendon reflexes that are transmitted by 1A afferent sensory axons. This pattern is non-specific. There is a narrow range of diagnoses for polyneuropathies affecting small fibres (Table 24.2). Pure sensory neuropathies other than small-fibre neuropathy are uncommon; aetiologies include paraneoplasia, paraproteinaemia, sensory chronic inflammatory demyelinating polyneuropathy (CIDP) and genetic causes. The diagnosis of pure motor neuropathy is fraught with difficulty because of the overlapping clinical features of peripheral nerve motor dysfunction with diseases of the lower motor neuron. Known causes of pure motor neuropathy include multifocal motor neuropathy (MMN) and CIDP. When nerve conduction and electromyographic (EMG) studies suggest demyelination in the pathogenesis of a neuropathy, the differential diagnosis is restricted to GBS, CIDP, Charcot–Marie–Tooth (CMT) disease, leukodystrophies, a few toxins and, rarely, mitochondrial disease. However, there is frequent discordance between electrophysiological and histological characterization of a neuropathic process.325 Compounding the problem of diagnosing a polyneuropathy is the patient who presents with concurrent medical conditions such as cancer, and subsequent therapy, and underlying diabetes. Such conditions may result in clinical and electrophysiological changes of their own that hinder attempts to arrive at a diagnosis. Furthermore, patients with generalized peripheral neuropathy appear more susceptible to compression neuropathies. Although histological assessment of a peripheral nerve trunk may provide important clues for the diagnosis of a polyneuropathy and occasionally may unmask amyloid deposits or vasculitis as the cause of a cryptogenic polyneuropathy, it is obvious that nerve biopsy is not a screening test for neuropathy.62 The current clinical approach to a patient with peripheral neuropathy comprises: ●● ●●

●●

Table 24.1  Multifocal neuropathies Hereditary neuropathy with liability to pressure palsies Neuritic leprosy Sarcoidosis Necrotizing vasculitis Multiple entrapments Multifocal motor neuropathy Diabetic neuropathy Multifocal chronic inflammatory and demyelinating  ­polyneuropathy Multiple intraneural deposits of amyloid

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●●

history and clinical examination; examination of kindred even without a phenotype suggestive of a familial neuromuscular disorder; nerve conduction, EMG and sensory studies; autonomic testing;

Table 24.2  Small fibre neuropathy Diabetes (most common) Amyloid neuropathy Alcoholic neuropathy Fabry disease Pseudo-syringomyelic form of Tangier disease (extremely rare) Hereditary sensory and autonomic neuropathy Acute pandysautonomia Idiopathic distal sensory neuropathy (common)

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  Clinicopathological Features of Neuropathy  1415 ●●

●●

laboratory testing, including blood glucose testing, assessment for monoclonal gammopathy and, in the appropriate clinical setting, specific antibody assays, including anti-glycolipid antibody and anti-Hu antibody; examination of spinal fluid (which rarely provides a specific diagnosis); adipose tissue, salivary gland, nerve, muscle or skin biopsy.

In patients with symptoms of peripheral nerve dysfunction, clinical examination and electrophysiological studies may be normal or borderline abnormal; in such cases, it is likely that the microscopic examination of a subcutaneous nerve will not result in a specific diagnosis. Limitations of nerve biopsy relate to the small size of tissue removed, the great susceptibility of peripheral nerve to artefacts when a strict method for handling and processing is not followed and the absence of ganglion cells in the sample. Because some subcutaneous nerves contain nerve fibres that are far removed from their parent ganglion cell (the separation may exceed 1 metre in nerves to the lower limb), any estimate of disease affecting the perikaryon is a prediction made by inference of abnormalities in distal dendrites and axons. When compared with the dimension of the cell body, axons and dendrites in peripheral nerve may constitute the largest component of the total mass of the neuron; however, only small segments of nerve bundles are visualized in a nerve biopsy. Since the 1990s, the indication for nerve biopsy has decreased considerably because genetic testing can now identify the majority of neuropathies of CMT-1, CMT-X, hereditary neuropathy with liability to pressure palsies (HNPP), familial amyloidosis, spinocerebellar ataxias (SCAs) and many of the genetically determined metabolic conditions that produce peripheral neuropathy. Detailed information on biopsy site, technique of tissue removal, complications of the procedure, specimen handling, and the methods used for tissue preparation, which comprise paraffin embedding, histostains and immunostains, plastic-resin embedding and transmission electron microscopy, post-fixation maceration and ‘teasing’ and ‘snap freezing’, are found in a number of texts.34,324,325,447 The diagnostic yield of the peripheral nerve biopsy varies considerably but it remains a necessary part of the analysis of peripheral neuropathy.91,113,324,325,516 In many specimens the morphological abnormality is non-specific, without providing clues as to a definite aetiology. Furthermore, in some instances, autopsy studies have shown that histological change, however specific, proximal to the biopsy site, may be associated with ‘downstream’ (at biopsy site) nonspecific axonal degeneration. For example, in a large series of subcutaneous nerve biopsies performed over a 33-year period and comprising nearly 1000 consecutive cases at the University of Toronto, a specific diagnosis was established only in about 29 per cent of cases.34 Although in many patients with peripheral neuropathy all types of nerve fibre are involved, some neuropathic processes affect only small-diameter fibres. This syndrome of small-fibre neuropathy is a source of frustration for the clinician, because the search for a specific diagnosis with physical examination, electrodiagnostic studies and even subcutaneous nerve biopsy often results in an absence of objective findings. The implementation of skin biopsy for the

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demonstration of depletion of dermal and intra-epidermal nerve fibres (IENFs) provides the rationale for a morphological–quantitative diagnosis for a neuropathy.239 Patients with neuropathies involving small-diameter fibres (a category that includes some cases of distal, painful peripheral neuropathy) experience reduced sensitivity to pain and temperature, and dysaesthetic burning and searing pains that are more troublesome at night. The mechanisms underlying painful neuropathies are complex and may involve contribution by multiple levels of the CNS and PNS.343,564 Muscle strength, myotendinous reflexes, and motor and sensory nerve conduction velocities (which measure largefibre function) are typically normal; as a result, no objective evidence for peripheral nerve dysfunction has been documented. The differential diagnosis of predominantly smallfibre neuropathy is limited (Table 24.2). In these cases, skin biopsy has a number of advantages over conventional nerve biopsy because IENF density can be decreased significantly, despite normal sural nerve morphometry. The fine innervation of the human epidermis was originally described in 1868 by Paul Langerhans, then a medical student.263 Using gold chloride impregnation (0.5 per cent solution) of skin obtained from surgical and autopsy specimens, he observed, and illustrated with a drawing, free nerve endings in the epidermis between keratinocytes. In 1928, working with methylene blue staining and metallic (both gold and silver) techniques, Kadanoff published detailed drawings of human IENFs and their mode of termination as free endings.295 In 1959, Arthur and Shelley demonstrated IENFs using intra vital and in vitro techniques of methylene blue staining and examining sections 50 μm thick under oil immersion,15 providing the first quantification of IENFs in humans at different sites. They estimated the number of nerve endings per unit area and found a range of individual variation and a rostral to caudal gradient of innervation. However, this method was cumbersome and time-­consuming; in addition, because the results could not be photographed, there was a paucity of convincing illustrations. Arthur and Shelley’s findings were confirmed more than 30 years later by the discovery that the antibody to neuropeptide protein gene product (PGP) 9.5 can be used to visualize almost any nerve fibre. Combined with the advances provided by the confocal microscope, which allows the three-dimensional reconstruction of thick sections of skin, this stimulated research into the cutaneous innervation and the study of patients with small-fibre neuropathies.239,265 Because the small-diameter nerve fibres traverse the skin perpendicular to the epidermis, the number of epidermal neurites per millimetre can be quantitated in punch skin biopsies, and normative reference ranges for nerve fibre densities are being reported. Punch biopsy of non-glabrous skin of limbs is preferred. The fresh sample is immediately immersed in a chilled paraformaldehyde-based fixative, cryosectioned at 50 μm and immunostained for PGP 9.5. Additional immunolabelling for other neuropeptides allows assessment of the innervation of sweat glands, arterioles, papilla and hair follicles. One of the limitations of this technique is the need to have an adequate bank of control specimens from the different parts of the body to correlate with the biopsy sites used. In addition, the normal sample has to be obtained from age-matched controls that have been subjected to rigorous neurological and electrophysiological

24

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1416  Chapter 24  Diseases of Peripheral Nerves

studies to rule out subclinical disease. Furthermore, the ­distribution density of epidermal nerve fibres differs in various parts of the body: studies have shown the density of epidermal fibres to be lower in men and to decrease with age (range 4.5–26.5/mm).

Normal Structure of Peripheral Nerve The basic architecture of a peripheral nerve trunk is illustrated in Figure 24.1 and the micro-anatomy is shown in Figure 24.2 (see also Figure 24.3 and 24.5 for normal ultrastructural appearance of peripheral nerve components). Normal peripheral nerves consist of a bundle of fascicles encased in a fibrovascular stroma termed the epineurium. Individual peripheral and autonomic nerve fascicles are further ensheathed by perineurium, a specialized structure

functionally akin to the arachnoid membrane. Contained within the perineurium is the endoneurium, consisting of a collagenous matrix housing the axons, Schwann cells, fibroblasts, macrophages, mast cells and capillaries.

The Epineurium The epineurium consists of massed collagen fibrils (types 1 and 3) interspersed with occasional elastin fibres, fibroblasts, mast cells, and the small arterial and venous blood vessels that supply the capillary plexus of the underlying nerve. Superficially, the epineurium merges with the surrounding areolar connective tissue of the deep fascia, and on its deep surface with the outermost layer of the perineurium. It is distinguishable as a separate sheath only in the largest nerves and nerve trunks; its thickness diminishes as the nerves pass distally, and a distinct epineurial layer cannot usually be identified around the smaller peripheral branches in most species.

The Perineurium

Peri

The perineurium, or sheath of Henle, consists of layers of flattened cells, interspersed by thin layers of fine collagen fibrils aligned parallel to the axis of the nerve. The number of layers is highest proximally, progressively diminishing to a single layer at the finest distal cutaneous and intramuscular nerve branches. In larger nerves, septa composed of two or three layers of perineurial cells arising from the inner aspect of the sheath may subdivide individual funiculi into

Endo Epi

C

24.1 Architecture of normal peripheral nerve. Five individual fascicles are shown, with surrounding epineurium (Epi). Endo, endoneurium; Peri, perineurium.

Endo

e

BM

S Epi

F P

A

24.2 Micro-anatomy of peripheral nerve. A, axon; Endo, endoneurium; Epi, epineurium; F, fibroblast; P, perineurium; S, Schwann cell.

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24.3 Normal perineurial ultrastructural appearance. Note the thin perineurial cells, both faces of which are covered by a thick basement membrane (BM). The connective tissue between the layers shows collagen fibrils (C) and elaunin (e).

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  Normal Structure of Peripheral Nerve  1417

24

MA C

24.4 Renaut body. UA

two or more subsidiary fascicles. Blood vessels penetrating the perineurium to supply the endoneurial capillary plexus also carry sleeves of perineurial cells with them into the endoneurium for short distances. In sections, the individual perineurial cells appear as thin sheets of cytoplasm, often no more than 0.1 μm thick, that contain occasional small cisterns of endoplasmic reticulum and numerous pinocytotic vesicles that open on to both the external and the internal surfaces of the cell (see Figure 24.3). The cells are somewhat thicker in their central zones (1.0–1.5 μm), where two equally thin layers of cytoplasm enclose a thin discoid nucleus. Both faces of the perineurial cells are covered by basal lamina, which may become thickened when compared with the basal laminae of other cells, even in quite young subjects, and to which the cell membrane may be anchored by scattered hemi-desmosomes. The connective tissue between these layers of cells consists of collagen fibrils (40–80 nm) and occasional elastic fibrils. The outer layers may also contain occasional fibroblasts and mast cells. At their margins, the individual perineurial cells overlap and interdigitate and are linked by tight junctions between their apposed membranes. Renaut bodies (see Figure 24.4) are normal subperineurial structures that may be better shown in epon-embedded sections than in tissue embedded in paraffin. They are thought to be perineurial in origin, with constituent cells being epithelial membrane antigen (EMA)-positive and sometimes being mistaken for pathological structures.

The Endoneurium The interstices between the fibres of peripheral nerves are packed with collagen fibrils in a mucopolysaccharide ground substance. This inter-fibre matrix constitutes the endoneurium. The great majority of the collagen fibrils run longitudinally in parallel with the nerve fibres (see Figure 24.5) and have a relatively uniform diameter in the range 30–65 nm. In addition, each nerve fibre is surrounded immediately outside the Schwann cell basal lamina by a thin sleeve of much finer, more irregularly disposed reticulin fibres. The principal cellular constituents of the endoneurium are fibroblasts. These are angular cells lacking a basal lamina that lie free between the endoneurial collagen fibrils. The endoneurial fibroblast population

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24.5 Normal endoneurial ultrastructural appearance. Note abundant collagen fibrils (C) within the matrix running in parallel with the axons. MA, myelinated axon; UA, unmyelinated axon.

has been reported to range from 5 per cent to 25 per cent of the nuclear profiles visible in transversely sectioned peripheral nerves, and it is believed to be responsible for the production of the major part of the extracellular endoneurial connective tissue. Other cells that may be found within the endoneurium are mast cells and macrophages. Resident macrophages are a normal constituent of the endoneurium, contributing some 2–5 per cent of the population. They are situated predominantly near blood vessels or the inner aspect of the perineurium, and they tend to have a dendritic morphology, with their processes oriented along the longitudinal axis of the nerve. These CD68 immunoreactive cells are the principal intrinsic antigen-presenting cells of the PNS and express many of the antigens common to other tissue macrophages, including major histocompatibility complex (MHC) class I and class II antigens (see review181). Injury to, or disease of, peripheral nerves results in a rapid influx of a further haematogenously derived population of macrophages at the site of the lesion, the trigger for recruitment being the presence of degenerating axons. These cells play the predominant role in myelin phagocytosis, probably aided by the Schwann cells.

Function of Peripheral Nerve Sheaths A primary function of the connective tissue that contributes to the structure of the peripheral nerve sheath is a mechanical one. The massed array of collagen in parallel

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1418  Chapter 24  Diseases of Peripheral Nerves

with the nerve fibres has considerable mechanical strength and resilience, which may serve to protect the nerve fibres from the stresses imposed by movement of the bodily parts through which they run. The sheaths of peripheral nerves are also resistant to penetration by foreign materials and infective agents. A considerable body of experimental work has demonstrated that the principal barrier to the entry of materials into the peripheral nerve is the perineurial sheath, particularly its innermost layers in which the constituent cells are linked by a continuous series of tight junctional complexes. Such material that does enter the endoneurium appears to be transported actively across the perineurial cells via pinocytotic vesicles. The perineurial barrier may be disrupted by mechanical trauma or osmotic shock. The perineurial barrier may be circumvented at the sites of penetration of the perineurium by blood vessels and at the peripheral open ends of the perineurial tubes, particularly at the neuromuscular junction; the latter has been suggested to be an important portal of entry for viruses and toxins into the PNS.

Vasculature of Peripheral Nerves Peripheral nerves are supplied by branches from their local regional blood vessels that ramify and branch within the epineurium. Pre-capillary vessels penetrate the perineurium at an oblique angle and are ensheathed for a short distance within the endoneurial compartment by internal prolongations of the innermost layers of the perineurial sheath. They then divide to supply the endoneurial plexus, which appears to consist nearly exclusively of capillaries. The plexus has a predominantly longitudinal orientation and drains directly to collecting blood vessels situated outside the perineurium, with few smooth muscle containing venules being found within the endoneurium. The contacts between the edges of adjacent capillary endothelial cells of the endoneurial circulation are completely sealed by tight junctions, and, in most species, this arrangement provides a second and effective barrier to the penetration of higher-molecular-weight materials into the endoneurial compartment. This blood–nerve barrier is,

however, much less effective in the spinal nerve roots and in the dorsal root and autonomic ganglia, to which plasma proteins and exogenous tracer molecules such as horseradish peroxidase have relatively unimpeded access. The anatomical basis for this difference has been attributed to a lack of tight junctions between the capillary endothelial cells of the endoneurial compartment at these sites and the presence of ‘fenestrated’ capillaries within the dorsal root ganglia of some species.

Nerve Fibres The nerve fibres are readily distinguishable into two classes, myelinated and unmyelinated, according to the presence or absence of a densely staining sheath of myelin around the axon. All axons of the PNS are ensheathed by chains of satellite or Schwann cells along the greater part of their length; the term ‘Remak cell’ is used occasionally to distinguish Schwann cells that surround unmyelinated axons. Myelin is a proteophospholipid material produced by Schwann cells enclosing the axon. Myelinated fibres are distinguished further by their generally much larger calibre, having diameters in the range 2–20 μm in human peripheral nerves. Unmyelinated axons are always of small diameter and pass through the nerve trunk in groups of 8–15 within a common chain of Schwann cells. Nerve fibres have been classified according to their calibre into three groups: A, B and C (Table 24.3).

Transmission of the Nerve Impulse The most significant axonal function is its capacity to transmit signals over considerable distances in the form of propagated action potentials. The basis of this property lies in the polarization of the axolemma, the internal surface of which is maintained at a negative potential of around 70 mV by energy-dependent adenosine triphosphate (ATP)-utilizing, ionic-pumps that are integral components of the membrane. The principal partitioned ion is sodium, and this is pumped outwards into the extracellular space. Electrical or mechanical disturbance of the axolemma at any point along

Table 24.3  Classification of mammalian peripheral nerve fibre types Type

Subtype

Myelinated

Function

Diameter (μm)

A

α

Yes

Proprioception, somatic motor

12–20

70–120

β

Yes

Touch, pressure, motor

5–12

30–70

γ

Yes

Motor to muscle spindles

3–6

15–30

δ

Yes

Pain, cold, touch

2–5

12–30

Yes

Preganglionic autonomic

distal limb weakness Weakness usually non-progressive White matter changes on brain MRI in all cases by 6 months Intelligence normal but may be reduced in cases with structural brain changes Epilepsy common Feeding difficulties common Frequent spinal rigidity and scoliosis Respiratory complications and respiratory failure common Marked elevation of CK

Ullrich CMD: Hyperlaxity of distal joints Contractures of proximal joints Dislocation of hips Respiratory insufficiency Delayed ambulation, achieved in some but not all Round face with prominent ears Hyperkeratosis; abnormal scar formation Prominent calcanei Scoliosis CK normal or mildly elevated

25

Note: Although described separately, Bethlem myopathy and forms intermediate between Ullrich and Bethlem congenital muscular dystrophies are essentially a continuum of clinical severity. In both these latter milder allelic variants, the general features can be similar to Ullrich congenital muscular dystrophy, but the maximum function and strength of individuals with Bethlem are significantly better (with acquisition of running abilities and forced vital capacity always above 80 per cent). Intermediate patients typically lose the ability to walk towards the late teens or early 20s, and develop respiratory insufficiency in the third decade of life.

Integrin α7 deficiency (very rare): Delayed motor milestones Mild muscle weakness CK normal

CK, creatine kinase; MRI, magnetic resonance imaging.

fibres may be present, particularly in cases of Ullrich CMD (see Figure 25.70). Some of these features also occur in some congenital myopathies, and because there is pathological and clinical overlap, these should be considered in the differential diagnosis (see Congenital Myopathies and Allied Disorders, p. 1580).

General Pathological Features Muscle pathology in CMDs may appear considerably more severe than expected from the clinical picture, and the degree of pathology cannot be used as an indication of disease severity or prognosis. All variants of CMD share common pathological features, although the degree of abnormality is variable. It is not possible to identify a particular form of CMD from the histological and histochemical features alone, most of which are non-specific, and immunohistochemistry is essential for making a diagnosis and directing molecular analysis. Although the name ‘dystrophy’ implies muscle fibre necrosis and regeneration at the pathological level, these are not striking in all variants, at least in the quadriceps muscle. Other muscles, i.e. paraspinal axial muscles, as in many disorders, show dystrophic changes. In some variants, such as RSMD1, the overall pathology may resemble a myopathy rather than a dystrophic process with necrosis. All CMDs show the typical features of other muscular dystrophies (see General Histological and Histochemical Abnormalities). The amounts of fibrosis and adipose tissue are variable, but both may be marked (Figure 25.70). A prominent inflammatory infiltrate may be present.331 With oxidative enzyme stains, areas of mitochondrial depletion resembling cores, or aggregation of peripheral mitochondria resembling lobulated

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Congenital Muscular Dystrophies Associated with Sarcolemmal and Extracellular Matrix Proteins Laminin α2-Deficient Congenital Muscular Dystrophies (MerosinDeficient Congenital Muscular Dystrophy)  This form of CMD

(MDC1A) is caused by mutations in the LAMA2 gene on chromosome 6q25. Patients invariably present at birth or in the first few weeks of life. Hypotonia and muscle weakness may be associated with failure to thrive and respiratory or feeding problems, but severe respiratory failure at birth is not a feature. Contractures may be present, but severe arthrogryposis is rare. Most mutations result in the complete absence of laminin α2 protein, or only traces of detectable protein, and are always associated with a severe phenotype. Box 25.6 gives some details of the laminin chains in skeletal muscle and their nomenclature.11 Patients with MDC1A rarely acquire the ability to stand independently and almost invariably cannot walk unaided, but they are usually able to sit unsupported. Some LAMA2 mutations can result in partial protein reduction and are usually associated with a milder phenotype, which can resemble a LGMD (Figure 25.71).407 The use of two antibodies to laminin α2 is particularly useful in these cases, and a reduction in labelling is then more apparent with an antibody to the 300 kDa fragment of laminin α2. Serum CK

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1568  Chapter 25  Diseases of Skeletal Muscle (a)

(b)

(c)

(d)

25.70 Representative views from various forms of congenital muscular dystrophy showing the variable degree of fibre size variation and fibrosis. (a) Merosin-deficient MDC1A, (b) Ullrich congenital muscular dystrophy, (c) muscle–eye–brain disease (H&E) and (d) the unevenness of NADH-TR staining (minicores) in a case of muscular dystrophy with rigid spine (RSMD1).

Box 25.6.  Laminins Laminins are components of the basal lamina, and 16 variants have been identified, all of which are composed of a heterotrimer of α, β and γ chains. The previous names of merosin (M) and laminins A, B1 and B2 were replaced by a nomenclature based on the diverse α, β and γ chains. A new scheme has been proposed using three Arabic numerals, based on the α, β and γ chain numbers. For example, the laminin 211 has the chain composition α2–β1–γ1. still often referred to as merosin. The predominant trimers in muscle are 211 (laminin 2 composed of α2–β1–γ1 chains) and 221 (laminin 4, previously known as S-merosin, composed of α2–β2–γ1 chains). Thus, mutations in the gene for laminin α2 affect both variants. The laminin β2 chain has an important role at the neuromuscular junction and is also present on extrajunctional sarcolemma. Laminin α2 is important for the correct assembly and maintenance of the muscle fibre basal lamina, and ultrastructural studies reveal abnormal basal lamina surrounding the fibres in patients with ‘merosin-deficient’ congenital muscular dystrophy (MDC1A) and in laminin α2 dy mouse models. Laminin α2 undergoes spontaneous proteolytic cleavage within the C-terminal G-domain, resulting in 80-kDa and 300-kDa fragments. In cases with a partial reduction of laminin α2, the reduction may be apparent only with antibodies to the 300-kDa fragment. Western blotting can also be used to detect laminin α2 defects, but not all commercial antibodies are suitable.

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levels are always elevated. Although cognitive function is usually normal, most patients with MDC1A have increased signal intensity in the white matter on T2-weighted brain MR imaging by 6 months of age, although there are a few exceptions in cases with partial reduction of laminin α2.407 Some patients (about 5 per cent) also show structural brain changes, such as occipital agyria.337 Mental retardation and epilepsy may be present in these cases. Laminin α2 labelling of intramuscular nerves in cases with LAMA2 mutations is also absent. In one rare case, absent labelling of nerves was associated with apparently normal labelling of the sarcolemma.107 Laminin β2 is associated with laminin α2 in nerves (as a component of laminin 221), as well as being abundant at neuromuscular junctions and on the sarcolemma.378,495 Laminin α2 immunoreactivity in the skin can be used as an alternative to muscle biopsy; it is absent from the epidermal–­ dermal junction, sensory nerves and glands in patients with MDC1A (Figure 25.72). Laminin α2 is absent from all blood vessels of skin and muscle but is present on blood vessels in the brain.478 A number of proteins are secondarily affected in MDC1A. Laminin β2 and α7 integrin are reduced on the sarcolemma, but laminin α5 and α4 chains are overexpressed.402

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Inherited Muscle Disorders  1569



Table 25.14  Summary of the main clinical features of congenital muscular dystrophies associated with defects in ­proteins that affect the glycosylation of α-dystroglycan and may show a secondary reduction in laminin α2 Fukuyama congenital muscular dystrophy

Walker–Warburg syndrome

MDC1C (‘FKRP’ congenital muscular dystrophy)

Muscle–eye–brain disease

MDC1D (LARGE gene, rare, a few reported cases only)

Prevalent in Japan

Most severe form of CMD, short life expectancy

Severe muscle weakness at birth

Hypertrophy of leg muscles and tongue neonatal presentation

Severe myopia and eye involvement

Severe generalized muscle weakness at birth

Very severe muscle weakness at birth

No contractures at birth

Normal brain and intelligence in milder cases variable severity (mild to severe)

White matter changes

Standing with support achieved, rarely ambulant

Contractures at birth or soon after

Sitting delayed but usually achieved

Severe cases have structural brain changes and mental retardation

Epilepsy in about 30 per cent

Progressive contractures and scoliosis

Severe structural brain changes with type II lissencephaly

Standing with support may be temporarily achieved

Sitting and ambulation achieved in milder but not severe cases

Subtle structural brain changes

Hypertrophy of calves, quadriceps and tongue

Cerebellar hypoplasia, brain stem hypoplasia and hydrocephalus

Progressive respiratory muscle weakness

Severe cases may have eye involvement and resemble MEB or WWS muscle hypertrophy

Severe mental retardation at age 17 years

Severe brain involvement with type II lissencephaly

Severe eye abnormalities with microphthalmia, cataracts, glaucoma, cerebellar cysts, hypoplastic optic nerve

Dilated cardiomyopathy

Moderately elevated CK

Severe mental retardation

Marked elevation of CK

Joint contractures

Epilepsy in most cases by age of 3 years

Absent psychomotor development

Pronounced elevation of CK

Ocular involvement in about 50 per cent of cases

Severe brain involvement with type II lissencephaly

Dilated cardiomyopathy from second decade

Cerebellar cysts, flat brain stem

Respiratory failure in second decade

Severe mental retardation

25

Marked elevation of CK MDC1B is not included in this table because the gene has not been identified. In common with the above disorders muscle biopsies from the few cases identified show abnormal glycosylation of α-dystroglycan and a secondary reduction of laminin α2. Note. Each phenotype is associated with defects in more than one gene (see Table 25.11). Although the table describes the phenotype in the original group of patients in whom gene mutations were identified, there is considerable overlap in the genes responsible as well as several recently identified.

However, when assessing all these proteins, age and developmental regulation must be taken into account. Because laminin α2 is expressed in chorionic villi on the basal lamina beneath the trophoblast, immunohistochemical studies of chorionic villi samples can be useful for prenatal diagnosis. Absence of laminin α2 from the trophoblast is highly suggestive of MDC1A, but a combined molecular genetic approach is recommended (Figure 25.73).312 A retrospective study from five international centres confirmed the reliability of combined molecular genetic and immunohistochemical studies of chorionic villi for the prenatal diagnosis of MDC1A in cases with an absence of laminin α2.471 Immunolabelling for laminin β2 is also reduced in

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the MDC1A trophoblast. The reliability of immunohistochemical studies of chorionic villi in patients with a primary partial deficiency, and in patients with secondary deficiency, is unknown. Integrin α7 Congenital Muscular Dystrophy  This is a very rare

form of CMD, and only three Japanese cases have been reported.189 Immunohistochemical studies identified an absence of integrin α7 from the sarcolemma and mutations in the corresponding gene (ITGA7) were identified. The morphological changes in muscle were mild and necrosis was not a feature, although regenerating fibres were seen in one patient. Integrin β1D was slightly reduced and laminin

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1570  Chapter 25  Diseases of Skeletal Muscle (a)

(a)

(b)

(b)

25.71 (a) Immunolabelling of laminin α2 in control muscle showing normal labelling compared with (b) reduced labelling on several fibres in a case with mutations in the LAMA2 gene.

(a)

25.72 Immunolabelling of laminin α2 in skin from (a) a control and (b) a case of ‘merosin-deficient’ MDC1A with absence of laminin α2 at the junction of the epidermis and dermis and from the sensory nerves.

(b)

25.73 Immunolabelling of laminin α2 in a chorionic villus from (a) a control and (b) a fetus with merosin-deficient MDC1A showing an absence of laminin α2.

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α2 was normal. Analysis of integrin α7 ­ localization in CMD muscle is limited because application of ­commercial antibodies is limited and sarcolemmal levels are low in ­ infants. Some details on various integrins in muscle are given in Box 25.7.59,272 Integrin α9 Congenital Muscular Dystrophy  A locus on chromosome 3p23-21 is associated with a form of CMD with some clinical features resembling Ullrich CMD, and integrin α9 was suggested as the candidate gene.449 Mutations in the ITG9 gene have been reported at an international meeting but not yet published in a peer reviewed article. There are no published reports of integrin α9 expression in the cases linked to this locus, but pathological studies of a few muscle biopsies showed central nuclei in several muscle fibres and features resembling a centronuclear myopathy (see Congenital Myopathies and Allied Disorders, p. 1580).449 Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy 

Ullrich CMD is one of the most common forms of CMD.86 It is caused by defects in the genes encoding collagen VI, a major protein of the extracellular matrix with enhanced labelling seen at the sarcolemma. Collagen VI is composed of three α-chains, α1(VI), α2(VI) and α3(VI), encoded by the COL6A1 and COL6A2 genes on chromosome 21q25.3 and the COL6A3 gene on chromosome 2q37. Mutations in each gene can result in Ullrich CMD. No mutations have been found in the α5 and α6 chains of collagen VI in patients with an Ullrich CMD or Bethlem phenotype.375 In muscle, collagen VI plays a structural role and anchors the muscle fibre basement membrane to the extracellular matrix. Collagen VI may also be important for organization of components of the extracellular matrix, such as fibronectin, and for the regulation of intracellular events, including apoptosis.205,209 It was initially believed that recessive mutations in COL6A genes caused the Ullrich phenotype and that dominant mutations caused the milder Bethlem phenotype. However, dominant mutations have also been identified in severely affected patients with an Ullrich phenotype, and patients with a phenotype intermediate between Ullrich CMD and Bethlem myopathy identified.1,35 Thus, there is a Box 25.7.  Integrins Integrins are transmembrane heterodimers composed of α and β chains. The complex at the myotendinous junction (MTJ), neuromuscular junction (NMJ) and sarcolemma of adult muscle fibres is composed of the α7 (mainly the B-splice variant) and β1D (muscle-specific) chains. In addition, the α3 and αv subunits are also found at the NMJ and MTJ. Integrin α7β1D is a major cell surface receptor for laminin α2, and both α7 and β1D immunolabelling may be secondarily reduced in primary laminin α2 deficiency and in other forms of congenital muscular dystrophy with a secondary reduction of laminin α2. The MTJ is severely disrupted in the muscles of integrin α7-deficient transgenic mice, with loss of the characteristic digit-like extensions and retraction of the sarcomere from the muscle membrane, suggesting that impairment of force transmission across the MTJ causes the muscle weakness.

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Inherited Muscle Disorders  1571

clinical spectrum, and the effect of a particular mutation on the production and function of collagen VI, rather than the mode of inheritance, determines severity. The clinical features of patients with Ullrich CMD include contractures and hypotonia at birth, torticollis and hip dislocation, rigidity of the spine, kyphosis and follicular hyperkeratosis and keloid formation (see Table 25.12). They show marked distal laxity of the fingers and ankles, and protrusion of calcanei may also be present. There can be clinical overlap with congenital myopathies, such as those caused by mutations in the RYR1 gene, and with connective tissue disorders such as Ehlers–Danlos syndrome. Muscle weakness is usually milder in cases of Bethlem myopathy, particularly in adults, but weakness and progression of disease are variable. The typical features of Bethlem myopathy are summarized in Table 25.13, but as emphasized previously there is a spectrum of severity and some patients follow an intermediate course between the clinical extremes of Ullrich CMD and Bethlem myopathy. Weakness in Bethlem myopathy, as in Ullrich CMD, affects proximal more than distal muscles, and legs more than arms. A proportion of patients become wheelchair-bound during the course of adult life, although respiratory insufficiency, invariable in Ullrich CMD, is ­exceptional. Contractures characteristically affect the long finger flexors causing an inability to oppose the palms in a ‘prayer sign’. Elbow, knee, hip and ankle contractures also occur in most patients, in association with rigidity of the spine. Abnormal scar formation (hypertrophic or atrophic) may also be seen. There is clinical overlap of some features with those that occur in the Emery–Dreifuss muscular dystrophies and LGMD2A, and sometimes they may not be easy to distinguish clinically, although the cardiac defects seen in Emery–Dreifuss muscular dystrophies are not a feature of collagen VI disorders. Muscle imaging shows a characteristic pattern in Ullrich CMD and Bethlem myopathy that is useful for differential diagnosis.280,281 There is a characteristic pattern of muscle involvement, particularly of the vastus lateralis and rectus femoris, with the periphery more affected than the belly of the muscles. Marked, progressive replacement of muscle with fibrous and adipose tissues can also be apparent. A variable reduction in collagen VI immunolabelling can be seen in Ullrich CMD, but normal labelling does not exclude a defect, and biopsies from patients with a mild Bethlem phenotype often show no detectable alteration. Some cases show a complete absence or an unequivocal reduction, whereas in others the ­reduction is subtle and may only be apparent at the sarcolemma, with normal labelling of the endomysium (Figure 25.74).209 The absence or reduction of collagen VI may also be apparent around axons and blood vessels, but this is not a universal phenomenon. Double immunohistochemical labelling with an antibody to another protein, such as perlecan, collagen IV or collagen V, to assess the integrity of the basal lamina may be necessary to identify this subtle reduction (Figure 25.75). Laminin β1 labelling of the sarcolemma may be reduced in some cases of Bethlem myopathy compared with normal intensity of blood ­vessels. However, this has been observed in other

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1572  Chapter 25  Diseases of Skeletal Muscle (a)

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An absence of immunolabelling of collagen VI in chorionic villi samples can also be useful for the prenatal diagnosis of Ullrich CMD, but experience is limited to patients in whom muscle of the proband showed absent immunolabelling of collagen VI.42 In addition to the congenital muscular dystrophies discussed earlier, there are several other myopathies caused by defects in proteins of the extracellular network that clinically overlap with connective tissue disorders such as Ehlers–Danlos.480 The defective proteins identified in humans include various types of collagen, tenascin-C and tenascin-X, fibrillin, perlecan, and secondary alterations of biglycan and decorin expression in human dystrophic muscle have been observed.507 Patients with tenascin-X defects have mild muscle weakness, myalgia and fatigue, and muscle biopsies from some cases show mild myopathic features and reduced immunolabelling of tenascin-X.479,482 In addition, mutations affecting collagen XII have recently been identified in patients with clinical similarities to UCMD and Bethlem myopathy.

Congenital Muscular Dystrophies Associated With Hypoglycosylation of α-Dystroglycan

25.74 Immunolabelling of collagen VI in (a) a control and (b) a case of Ullrich congenital muscular dystrophy (CMD) showing absence of collagen VI from the sarcolemma, perimysium and endomysium. Note also the labelling round the nerve axons and blood vessels in the control and the retained labelling around the large blood vessels in case of Ullrich CMD.

disorders and is an age-related feature usually only seen in adults and adolescents. Immunolabelling of myosins in Ullrich CMD shows a population of fibres of varying size with fetal myosin and there may be several fibres that coexpress fast and slow myosin (Figure 25.76). Collagen VI is expressed in most connective tissues and its presence in skin can be studied immunohistochemically, and a clear reduction may be seen in some affected cases.228,286 Normal immunolabelling of collagen VI in muscle or skin, however, does not exclude a defect in a gene for collagen VI. Studies of cultured fibroblasts suggest that changes in collagen VI may also be useful for identifying abnormalities.210,225 Fibroblasts from cases of Ullrich CMD clearly show abnormalities, but fibroblasts from Bethlem myopathy patients show more variable results. There is also evidence from studies of cultured fibroblasts that the interaction with fibronectin may be abnormal in some cases.373 A secondary reduction in collagen VI from fibroblasts in vitro is seen when tenascin-X is deficient, also reflecting the importance of the complex interactions of extracellular proteins.294

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Both α- and β-dystroglycan are central components of the DAG complex at the sarcolemma and are expressed in other tissues in addition to muscle (see Figure 25.64). They are the post-translational products of a single gene. β-Dystroglycan is the transmembrane component, the C terminus of which interacts directly with dystrophin, and the N terminus is non-covalently bound to the C terminus of α-dystroglycan. The core protein of α-dystroglycan has a predicted molecular mass of 72 kDa, but following modest N-glycosylation and extensive O-glycosylation in ­different tissues, its mass is increased to 156 kDa in muscle and 120 kDa in brain. O-glycosylation of several serine–­threonine-rich sites on the mucin-like domain are believed to be crucial for its binding to a number of ligands, including laminin α2. The epitopes for two commercial antibodies to α-dystroglycan, IIH6 and VIA4–1, probably lie in this region. The structures of some of the O-linked mannose glycans on α-dystroglycan have been elucidated and its formation involves the action of several glycosyltransferases that add different monosaccharides in a stepwise manner. Defects in several genes result in hypoglycosylation of α-dystroglycan. The group of muscle disorders caused by this secondary feature are collectively known as the ‘dystroglycanopathies’. There is considerable overlap of phenotype with more than one gene associated with the various phenotypes. The central nervous system is frequently involved. Clinical severity associated with the genes is broad and some of the same genes are associated with limb-girdle forms of dystrophy. A new classification of this group of disorders has been proposed that takes into account the genes responsible, the involvement of the brain and eye, and the presence or absence of mental retardation.158 This has been adopted by OMIM, but is not yet widely used and the clinical names are still relevant (see Tables 25.11 and 25.14). Several of the defective genes encode proven or putative glycosyltransferases (POMT1, POMT2, POMGNT1, FKTN, FKRP, LARGE). Novel methods and next-generation sequencing are identifying defects in additional genes of uncertain

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Inherited Muscle Disorders  1573



25

25.75 Double immunolabelling of collagen VI and perlecan and the merged images in (a-c) a control and (d-f) a case of Ullrich congenital muscular dystrophy showing the very reduced sarcolemmal collagen VI but normal labelling of perlecan.

(a)

(b)

(c)

25.76 Serial sections immunolabelled for (a) fetal myosin, (b) slow and (c) fast myosin in a case of Ullrich congenital muscular dystrophy showing a population of fibres of varying size with fetal myosin and coexpression of more than one isoform in several fibres.

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1574  Chapter 25  Diseases of Skeletal Muscle

function that are also associated with hypoglycosylation of α-dystroglycan and its function, such as isoprenoid synthase domain-containing protein (ISPD), transmembrane protein 5 (TMEM5), β-1, 3-n-acetylglucosaminyltransferase 1 (B3GALNT2), guanine diphosphate mannose pyrophosphorylase B (GMPPB), glycosyltransferase-like containing protein (GTDC2) and protein kinase-like protein SgK196 (SGK196) (see Table 25.11).55,61,76,159,207,271,369,428,485,498 This is an expanding area, and further causative genes will undoubtedly be identified. In addition, genes thought to only be involved with N-glycosylation also result in hypoglycosylation of α-dystroglycan. These include genes encoding members of the dolichol-phosphate-mannose synthase family of proteins (DPM1, DPM2 and DPM3) and link disorders with glycosylation defects to the congenital muscular dystrophies.27,204,245,289 Hypoglycosylation of α-dystroglycan has also been shown to have a role in the progression of carcinoma cells and to correlate with prognosis.16 Mutations in the gene DOLK, which encodes the dolichol kinase responsible for the formation of dolichol phosphate, have so far only been associated with a dilated cardiomyopathy, although elevated serum CK was present in several patients.244 The hypoglycosylation of α-dystroglycan discussed earlier is a secondary phenomenon and a consequence of various gene defects. Mutations in the DAG1 gene that encodes dystroglycan cause LGMD2P (see Recessive Limb-Girdle Muscular Dystrophies, p. 1561). The main clinical features of each of the ‘dystroglycanopathies’ are listed in Table 25.14. Structural brain or eye involvement is common in patients at the severe end of the clinical spectrum of each gene defect. This suggests that the clinical features of a patient affected by a dystroglycanopathy are related to the functional effect of the mutation rather than to the gene involved. At the severe end of the spectrum, mutations in POMT1, POMT2, FKRP, POMGNT1, FKTN, ISPD, TMEM5,, B3GALNT2, GTDC2, GMPPB and SGK196 result in type II lissencephaly, ‘flat’ brain stem, a hypoplastic cerebellum and absent corpus callosum.50,207 Immunohistochemistry and immunoblotting of muscle reveal a variable reduction in glycosylation of α-dystroglycan but usually normal β-dystroglycan (in contrast to the reduction of both α- and β-dystroglycan in DMD) and is a useful control. The extent of the reduction of α-dystroglycan immunolabelling often correlates, but not always, with disease severity.50,211 Research studies of the core protein of α-dystroglycan show that is often normal, but studies are limited by the lack of commercial antibodies that are suitable for both immunohistochemistry and immunoblotting.211 A secondary reduction of laminin α2 is common when α-dystroglycan is hypoglycosylated. The extent of this reduction is variable, but complete absence of laminin is never observed, in contrast to cases with a primary laminin α2 defect. Other proteins reported to show a secondary reduction in some of these CMD variants include laminin β2, perlecan, P180, and integrin α7 and β1D chains, but the maturation-related expression of some of these proteins was not always taken into account.91,92,374,434,495 Enzyme activity of POMGnT1 can also be assessed. It has been found to be reduced in muscle and cultured fibroblasts from patients with a mutation in the corresponding gene. Kinetic abnormalities have also been identified.87,509

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Congenital Muscular Dystrophies due to Defects in Proteins of the Sarcoplasmic Reticulum: Rigid Spine Muscular Dystrophy Although some degree of spinal rigidity is associated with several neuromuscular disorders, it is an invariable complication of one form of CMD (designated RSMD1). Rigid spine muscular dystrophy type 1 is caused by recessive mutations in the gene encoding selenoprotein N1 (SEPN1) on chromosome 1p35–36. Mutations in the SEPN1 gene manifest pathologically as multiple cores in muscle fibres (multi-minicore disease), a myopathy with ‘Mallory body’like inclusions, and fibre type disproportion.83,137,139 There is considerable clinical and pathological overlap between these disorders, and it is likely that they represent a clinicopathological spectrum instead of distinct entities. Selenoproteins constitute a family of enzymes that contain a selenium atom in the form of a seleno-cysteine in the catalytic site and are involved in oxidation–reduction reactions. Selenoprotein 1 (SEPN1) is a membrane-bound glycoprotein localizing to the rough endoplasmic reticulum. It is expressed in several tissues, including skeletal muscle, heart, brain, lung and placenta.297 It is most highly expressed in fetal muscle. Mutations have been found in most of the 13 exons except exon 3, and most lead to a premature stop codon. The specific function of SEPN1 is not fully understood, but it is believed to be involved in oxidative stress and calcium homeostasis.247 Patients affected by RSMD1 usually present with axial weakness and mild proximal muscle weakness, and there is selective involvement of the diaphragm resulting in respiratory insufficiency (Table 25.15). Scoliosis typically accompanies the condition. There is clinical overlap with Emery–Dreifuss muscular dystrophy (see later), but there is no cardiac involvement in RSMD1. RSMD1 is usually stable, and most patients retain the ability to walk for life, provided that the scoliosis and respiratory problems are managed appropriately. Serum CK is usually normal. In addition to variation in fibre size, muscle pathology may show a mild increase in endomysial connective tissue and an increase in internal nuclei, although these are not usually abundant. An uneven pattern in oxidative enzyme stains, sometimes in the form of multiple core-like lesions, is often associated with mutations in the SEPN1 gene. Multiple cores may also be seen in cases with a mutation in the RYR1 gene, which may cause diagnostic confusion.214 In SEPN1 cases, however, the core-like lesions often occur in both fibre types Table 25.15  Summary of the main clinical features of the congenital muscular dystrophy with rigid spine associated with a defect in selenoprotein N1 of the endoplasmic reticulum (RSMD1) Axial hypotonia in first few years of life Normal motor milestones Ambulation usually achieved and maintained into adulthood Rigidity of spine in most cases Progressive scoliosis Nasal speech Pronounced respiratory insufficiency leading to respiratory failure Normal or mild elevation of creatine kinase Clinical, pathological and molecular overlap with multi-minicore disease and Mallory body myopathy

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and a two-fibre type pattern is usually retained, whereas in RYR1 cases marked predominance of type 1 fibres is common and the cores are often restricted to type 1 fibres. Antibodies to SEPN1 have been used in research studies to show the absence of the 70-kDa SEPN1 band in immunoblots of fibroblasts cultured from a patient with RSMD1.2 At present, changes in SEPN1 levels cannot be ascertained on sections by immunohistochemistry and there are no reported secondary protein abnormalities. In keeping with the absence of necrosis and low CK there are usually very few, or no, fibres with fetal myosin. Absence of the protein has been shown in fibroblasts from affected patients, but there are no studies on human sections reported. A reduced number of satellite cells has been noted in patients and in an animal model and it has been suggested that SEPN1 may function in regeneration of muscle.65

Congenital Muscular Dystrophies Associated with Nuclear Membrane Proteins It is now appreciated that some early onset disorders, classified as a congenital muscular dystrophy, are caused by mutations in nuclear membrane proteins and can be lethal.282,345,474 In early onset cases associated with mutations in the lamin A/C gene, respiratory insufficiency is rapidly progressive and spinal rigidity and scoliosis typically develop. Neck weakness resulting in a ‘dropped head’ may also be a feature, although not specific for lamin A/C mutations. Muscle pathology is variable and may be mild. Features include variation in fibre size, an increase in connective tissue, necrosis and regeneration and inflammation. No immunohistochemical abnormalities of sarcolemma proteins or nuclear proteins are seen. Mutations in the SYNE1 gene encoding nesprin-1 have been found to cause a rare congenital muscular dystrophy with adducted thumbs.483 The two cases from the single family identified were hypotonic and had muscle weakness, ptosis, ophthalmoplegia, mild mental retardation and mild cerebellar hypoplasia. Serum CK was moderately elevated and muscle pathology showed non-specific myopathic features.

Other Congenital Muscular Dystrophies A number of other disorders have been designated as a congenital muscular dystrophy, but not all cases have onset within the first 6 months of life or have a clear ‘dystrophic’ pathology with fibre necrosis, regeneration and fibrosis. For example, cases with defects in the gene encoding choline kinase B (CHKB) can show a spectrum of age at onset and severity.178,296,348 The distinctive pathology of this disorder is discussed with those with mitochondrial defects (see Mitochondrial Myopathies, p. 1602).

Emery–Dreifuss Muscular Dystrophies The clinical features of the X-linked and autosomal forms of Emery–Dreifuss muscular dystrophy are similar, but often more severe in the latter. Emery–Dreifuss muscular dystrophy is invariably associated with both cardiac and skeletal muscle involvement. Both disorders present with muscle weakness and early contractures of the elbow, the Achilles tendons and the spinal extensor muscles. The contractures

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Inherited Muscle Disorders  1575

are progressive, and rigidity of the spine often becomes marked. Skeletal muscle involvement typically precedes the cardiac abnormalities, which are evident before the third decade of life and are the most deleterious aspect of both disorders, being characterized by atrioventricular conduction disturbances and heart block. Dilated cardiomyopathy can also be found in autosomal dominant Emery–Dreifuss muscular dystrophy, but less commonly in the X-linked variant. Involvement of skeletal muscle is typically humeroperoneal, but can also feature scapular involvement. Striking wasting of the upper arms and lower legs is often apparent in both conditions, which are almost indistinguishable, although subtle differences in the pattern of muscle involvement can be demonstrated using muscle MR imaging.280 The dominant form is more common than the X-linked form and generally more severe, with earlier onset – even congenital in a few instances.282,285 Except in cases with onset in infancy, ambulation is usually retained for life. Serum CK levels are usually normal, or mildly or moderately elevated, but never at the levels found in DMD or BMD. The X-linked form is caused by mutations in the STA gene on chromosome Xq28, which encodes for emerin.302,504 Emerin is a 34-kDa nuclear protein with a hydrophobic C terminus anchored in the nuclear membrane and an N-terminal tail projecting into the nucleoplasm. The STA gene has six exons, and mutations have been found throughout the gene, with no ‘hotspots’. Most are nonsense or frameshift mutations or occur at splice sites. The majority of mutations result in absence of localized protein, which can be demonstrated with antibodies.267 Rare cases have been reported in which emerin expression is reduced rather than absent.62,108 Female carriers rarely manifest with muscle weakness but are at risk of cardiac involvement. The absence of emerin in a proportion of nuclei can be detected in the skin and buccal cells of carriers (Figure 25.77).268,376 If buccal cells are investigated, it is essential that only viable cells are assessed; parallel studies of nuclear lamins are a useful control, because they appear normal. Autosomal dominant Emery–Dreifuss muscular dystrophy is caused by dominant mutations in the LMNA gene on chromosome 1q11–23; de novo mutations are common. This gene is alternatively spliced to produce lamin A and lamin C, which are intermediate filament proteins that localize to the nuclear lamina.503,504 The nuclear lamina has been attributed with a role in chromatin organization, gene regulation and cell signalling. A direct interaction between lamin A and emerin has been demonstrated in vitro, which together with the similar clinical phenotype of the two forms of Emery–Dreifuss muscular dystrophy provides evidence that these two proteins form a functional link at the nuclear envelope. Immunolabelling of emerin and lamin A/C in the autosomal dominant form of Emery–Dreifuss muscular dystrophy appears normal. Some antibodies that specifically detect lamin A but not C show labelling of very few nuclei in mature human muscle (Figure 25.78); this is thought to be due to epitope masking, because it is not seen with all antibodies. Similarly, differences in the immunolabelling of lamin B1 with different antibodies are thought to be due to epitope masking.462 Antibodies to epitopes common to both lamin A and C show labelling of all nuclei

25

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1576  Chapter 25  Diseases of Skeletal Muscle (a)

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25.77 Immunolabelling of (a) emerin, (b) counterstained with DAPI in a skin biopsy from a carrier of X-linked Emery– Dreifuss muscular dystrophy showing areas of nuclei with and without emerin.

(Figure 25.78b). Some studies have suggested that there is reciprocal expression of lamin B1 and emerin, with emerin in myonuclei and lamin B1 in endothelial cell nuclei.269 Lamin B2 appears to be present in all nuclei, and internal nuclei show similar emerin and lamin labelling as peripheral nuclei (Figure 25.78c). In common with other intermediate filament proteins, lamins possess a highly conserved α-helical coiled-coil rod domain, which plays a crucial role in lamin–lamin interactions. Mutations occur throughout the gene, although many are found in the common α-helical rod domain of exons 1–10. These mutations result in no detectable alteration in lamin A/C immunolocalization. Mutations in the LMNA gene underlie the cause of several allelic conditions, including Emery–Dreifuss muscular dystrophy, LGMD1B, dilated cardiomyopathy with conduction system disease, familial partial lipodystrophy, Charcot–Marie–Tooth disease type 2B1, mandibuloacral disostosis, premature ageing disorders and restrictive dermopathy.504 These are now often collectively referred to as the ‘laminopathies’, but skeletal muscle is affected in only some of them (autosomal dominant Emery–Dreifuss muscular dystrophy, LGMD1B). Some severely affected infants with axial weakness have also been found to have de novo mutations in the gene encoding lamin A/C and it is often worth considering this gene in a severely affected infant or neonate in whom the muscle biopsy shows nonspecific myopathic changes and/or inflammation.345 These

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(c)

25.78 Immunolabelling of (a) lamin A (b) lamin A/C and (c) lamin B2 in a case of autosomal dominant Emery– Dreifuss muscular dystrophy that are all comparable to controls. Note the relatively few nuclei labelled with this antibody to lamin A (other antibodies can show labelling of more nuclei) and the similar labelling of internal and peripheral nuclei.

cases are considered to overlap with congenital muscular dystrophies (see earlier) and some have been described as having a ‘dropped head’ syndrome.71,345 The explanation for the phenotypic diversity is unclear. Research, however, suggests that tissue-specific differences in the arrangement of the lamin-associated protein complexes at the nuclear

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envelope may underlie some of the tissue specificity in these disorders. Additional genes encoding proteins of the nuclear envelope are also associated with a dominant Emery–Dreifuss muscular dystrophy (EDMD) phenotype. Identification of the genes that encode nesprin 1 and 2 (SYNE 1 and SYNE2), a protein known as LUMA (encoded by TMEM43), and identification of a mutation in the genes encoding LAP2α, LAP1B and SUN proteins (lamin A-associated protein), confirm this and emphasize the importance of the nuclear envelope and the complex of proteins that interact with emerin and lamins.22,249,275,277,508 Defects in nesprin are also responsible for a recessive disorder that presents with arthrogryposis.9 A protein in the nuclear matrix, matrin-3, is responsible for a dominant distal myopathy with vocal cord and pharyngeal weakness but this has a different phenotype to Emery–Dreifuss muscular dystrophy.397 The main histological and histochemical features of the two forms of Emery–Dreifuss muscular dystrophy are similar. In quadriceps biopsies, variation in fibre size is not markedly abnormal; occasional atrophic fibres are common, and some fibres may be hypertrophic. Internal nuclei may be few or numerous, with more than one per fibre (Figure 25.79). Necrotic fibres are generally rare, and there is usually only a mild, or little, increase in adipose or connective tissue (Figure 25.79). There are exceptions, however, such as a severely affected boy found to have a mutation in both the STA and the LMNA genes.308 This case illustrates the importance of considering the possibility of a causative mutation in more than one gene, particularly those in which mutations are common, such as LMNA. Biopsies may contain small basophilic fibres with a slightly granular appearance (Figure 25.80). These might be regenerating fibres, because they express fetal myosin and an increased amount of desmin; however, generally only a few fibres express developmental myosins, suggesting that there is little ongoing muscle fibre regeneration. It is not clear whether this reflects a lack of necrosis, which precedes regeneration, or whether there is a malfunction in the regenerative process. In support of the latter, in vitro studies suggest that myoblasts deficient in either lamin A/C or emerin display delayed differentiation kinetics and have a decreased differentiation potential. This may, therefore, partly explain the dystrophic phenotype observed in patients with Emery–Dreifuss muscular dystrophy.147 A two-fibre type pattern is usually maintained with oxidative enzyme stains and ATPase. There is a tendency for the type 1 fibres to be smaller, but not usually to the degree seen in congenital myopathies. A predominance of type 1 fibres may also occur. Non-specific structural changes such as cores can also occur. Immunolabelling of all proteins associated with the sarcolemma is normal in both forms of Emery–Dreifuss muscular dystrophy, with the exception of laminin β1. Reduced laminin β1 labelling on the sarcolemma may be apparent in some cases, but all blood vessels, including the capillary network, show normal reactivity. Labelling of laminin α2 and laminin γ1 is normal and useful as controls.117 The reduced sarcolemmal labelling of laminin β1 is not specific for Emery–Dreifuss muscular dystrophies and may be seen in Bethlem myopathy and other

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25

25.79 A case of autosomal dominant Emery–Dreifuss muscular dystrophy showing only mild variation in fibre size, mild fibrosis, an increase in internal nuclei and no necrosis. H&E.

25.80 Small basophilic fibres in an 8-year-old child with autosomal dominant Emery–Dreifuss muscular dystrophy. H&E.

myopathies, including LGMD2I. It is an age-related phenomenon, observed in adult and adolescent patients, but rarely in young children. Electron microscopy may demonstrate aggregation of chromatin and a lack of attachment of chromatin to the nuclear membrane. 49 Abnormalities of the nuclear envelope have also been reported in skeletal muscle nuclei and cultured skin fibroblasts. 140,321 The specificity of these findings to Emery–Dreifuss muscular dystrophy remains unclear.

Disorders Associated with Deletions or Expansions of Repeated Sequences Most gene mutations responsible for a neuromuscular disorder are deletions, duplications or splice site or missense changes, which disrupt the sequence of the reading frame or, more rarely, affect their promoter regions. A few conditions, however, are caused by more unusual mechanisms, which involve deletions or expansions of a nucleotide

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1578  Chapter 25  Diseases of Skeletal Muscle

repeat sequence. These are facioscapulohumeral muscular dystrophy (FSHD), the myotonic muscular dystrophies, and oculopharyngeal muscular dystrophy (OPMD). With the advent of reliable molecular testing for FSHD and the myotonic dystrophies, ­muscle biopsies are performed less often and their role has diminished. Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies, with an estimated prevalence of 1 in 20 000.103,425,472 The clinical features of FSHD are summarized in Table 25.16. FSHD is inherited dominantly, although cases at the severe end of the clinical spectrum are often the product of de novo dominant mutations. Penetrance, based on clinical presentation, is age dependent and most cases show clinical signs before the age of 20 years. Infantile cases with onset recognizable before the age of 10 years are seen in some families. Anticipation is also well recognized, although unexplained, with earlier onset in successive generations. Severity may relate to size of the deletion of the fragment associated with FSHD. The molecular defect responsible for FSHD is a deletion of copies of a 3.3-kb DNA repeat fragment in the subtelomeric region of chromosome 4q (D4Z4) in association with a 4qA161 haplotype. In normal individuals, this fragment varies in size from 50–300 kb, with 11 or more D4Z4 repeats, but patients with FSHD have 11 or fewer repeats.425,472 It is not clear how the reduction in the size of the 4q fragment causes disease, but recent studies indicate that a crucial step in the disease is the inefficient repression of the retrogene DUX4 in a microsatellite repeat array.425 Molecular diagnosis of FSHD relies on a double digest with the EcoRI and BlnI restriction enzymes, in order to distinguish the chromosome 4q fragment from the similar fragment on chromosome 10q26. The fragments from both chromosomes are detectable with the p13E–11 probe, but the chromosome 10 fragment contains a BlnI restriction site that is absent from the chromosome 4 fragment. Thus, these two enzymes completely digest the chromosome 10 fragment, leaving the chromosome 4-related fragments. Confusion can arise, however, because interchromosomal exchange of the repeat regions occurs in a few normal individuals, resulting in hybrids of chromosome 4 and 10 fragments. Germline mosaicism may also hamper molecular analysis, and false negative and false positive results can be obtained, requiring the use of additional probes. The double-digest analysis, however, identifies the majority of cases. About 95 per cent of cases with the typical phenotype show contraction of the 4q fragment (FSHD1) and the other 5 per cent do not show contraction, but have hypomethylation at the D4Z4 locus (FSHD2).103 Although muscle biopsies are now performed less often for the diagnosis of FSHD, we summarize here the pathological features that have been observed and may alert pathologists in atypical cases. The abnormalities are non-specific, and the degree of change is variable and may be influenced by the clinical involvement of the sampled muscle. Variation in the size of both fibre types is common, but some biopsies from minimally involved muscles may show only scattered very small fibres. These fibres have been described as atrophic, but the expression of developmentally regulated proteins in them raises the possibility that they represent attempts at regeneration.117 Fibre type grouping is not a feature, but clusters of small fibres, as in

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Table 25.16  Main clinical features of facioscapulohumeral muscular dystrophy Age of onset variable, congenital (rare) to adulthood. Most cases present in the first 2-3 decades of life Autosomal dominant inheritance with variable penetrance Facial weakness Shoulder girdle weakness with difficulty raising arms Peroneal muscle weakness Scapular winging Generalized muscle wasting, often asymmetrical Weakness of abdominal muscles Variable progression – minimal or slow, may lead to loss of ambulation Hearing loss Retinal vasculopathy Mild to moderate elevation of CK that is age and sex dependent Rare congenital cases with extreme repeat deletions and severe weakness, cognitive involvement and severe neurosensory hearing loss and epilepsy have been described; these cases may never acquire ambulation and need to be differentiated from congenital dystrophy.

BMD, have been put forward as evidence of denervation. These fibres, however, also show proteins associated with immaturity, and there are no electrophysiological data to support the proposal of denervation. Fibre necrosis is not usually marked in FSHD, but it can occur. Similarly, an increase in fibrous and adipose tissues may be seen. Internal nuclei may be numerous, but they are usually not increased. An inflammatory response is frequent and may vary from mild to profuse. In contrast to inflammatory myopathies and LGMD2B, overexpression of sarcolemmal MHC class I antigens is rarely observed in FSHD.363 There are no specific immunohistochemical abnormalities and, although reduced sarcolemmal laminin β1 has been observed, this is not a consistent feature and can be seen in other disorders. Myotonic dystrophies are common autosomal dominant disorders. They are characterized by myotonia in association with muscle weakness and wasting. These disorders also affect several other tissues, including cardiac muscle. Two forms of myotonic dystrophy (DM1, DM2) are recognized; they are caused by defects in two different genes. Type 2 is also known as proximal myotonic myopathy (PROMM). Both DM1 and DM2 are caused by expansion of a nucleotide repeat: DM1 by expansion of a CTG repeat in the 3ʹ untranslated region of a gene on chromosome 19q, and DM2 by a CCTG repeat expansion in the first intron of the ZNF9 gene on chromosome 3q. The chromosome 19 protein is a putative kinase (DM protein kinase [DMPK]) and that encoded by ZNF9 is a zinc finger protein. Both disorders are believed to result from ‘toxic RNA’ produced by the expansion, which interferes with a number of RNA-binding proteins, such as muscleblind and SIX5 in the nucleus.151,384,463 Type 1 myotonic dystrophy is a common disorder with an estimated prevalence of about 1 in 8000 in Caucasians, whereas DM2 is rarer but is particularly common in some populations.437 There are no reported congenital cases of DM2, but congenital presentation of DM1 is well recognized; almost invariably, a mildly affected mother transmits the mutant gene. Although clinical and electrophysiological assessments are often sufficient to make a presumptive diagnosis of DM1 or DM2, histopathological examination can

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help in less obvious cases and plays an important role in the diagnosis of congenital DM1 cases. Type 1 myotonic dystrophy is caused by an increase in the number of repeats in the DMPK gene. In normal individuals there are between 4 and 40 CTG repeats, but in patients with DM1 this is increased to 50 or more–­sometimes over 1000. In general, there is a correlation between the size of the repeat and the clinical severity and age at onset: patients with fewer than 100 repeats are affected more mildly than those with more than 1000 repeats (e.g. congenital patients). Anticipation in DM1 is common, with successive generations showing more clinical severity. There is also somatic variability and instability in the size of the repeat expansion with the number being greater and more unstable in muscle than in blood lymphocytes. Type 2 myotonic dystrophy is caused by an increase in the number of chromosome 3 ZNF9 gene CCTG repeats. In normal individuals, the number of repeats ranges from 10 to 30, but in patients with DM2 this is increased to many thousands. Anticipation and somatic variability also appear to occur in DM2, but the correlations are less clear than in DM1. There does not appear to be a congenital form of DM2. Myotonia is common to both DM1 and DM2, but the pattern of muscle weakness is different. In DM2 there is early proximal muscle involvement, in contrast to the distal pattern seen in DM1, hence the name (PROMM) commonly attributed to DM2.259,384 Facial weakness is common in DM1 but rare in DM2. Ptosis and facial/neck weakness are characteristic features of DM1. Similarly, cardiac conduction defects and CNS involvement are common in DM1 but less so in DM2. Diaphragmatic weakness leads to respiratory insufficiency and is often a cause of death in DM1. Cataracts occur in both DM1 and DM2. Other associated features of both include frontal balding and gonadal atrophy. Molecular analysis of the myotonic dystrophies to ­confirm a clinical diagnosis is highly reliable and muscle biopsies are now less often performed. The diagnosis of patients with DM2, however, may not be as clinically apparent as for DM1, and a muscle biopsy from a DM2 patient is then more likely to be taken. Histopathological information on DM2 has increased, because more molecularly confirmed cases have been studied and differences between DM1 and DM2 are apparent.117,313,391,477 Both forms show a pronounced number of multiple internal nuclei (which may be in chains) and variation in fibre size. The pathology is progressive with loss of muscle fibres and increasing amounts of fat and fibrous tissue. Sarcoplasmic masses with disorganized myofibrillar material and dilated sarcoplasmic reticulum are typical of DM1. Ring fibres are also more common in DM1 than DM2. Early changes in DM1 are atrophy of type 1 fibres and hypertrophy of type 2 fibres. In DM2, in contrast, atrophy affects type 2 fibres more than type 1 fibres. Prominent nuclear clumps are an early feature in DM2, which label with antibodies to fast and fetal myosin isoforms, but are a late feature in DM1.391,477 Congenital forms of DM1 may show many central nuclei, and the pathology is very similar to that of myotubular myopathy. The number of fibres with slow myosin, however, is less in congenital DM1 and the two disorders can be distinguished with antibodies to muscleblind.409,422 Molecular

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Inherited Muscle Disorders  1579

analysis of the DM1 locus is essential in all neonates with abundant central nuclei. Oculopharyngeal muscular dystrophy is usually transmitted as an autosomal dominant disorder, but a few recessive cases have also been reported. The molecular defect is short GCG expansions of the first exon of the polyadenylate-binding protein nuclear 1 gene (PABPN1) on chromosome 14q11.1.39 Normal individuals have six GCG repeats in the first exon of the PABPN1 gene, but in OPMD there are an additional two to seven repeats. The disorder has a worldwide distribution, but it is particularly prevalent in French Canadian and Bukhura Jewish populations.39 OPMD is a late-onset disorder characterized by early ptosis and dysphagia. The disorder progresses slowly, with increasing weakness of several muscles, including those of the face, eyes and limbs; a nasal voice also develops. There is no cardiac involvement, and CK is usually normal or occasionally mildly elevated. The pathological feature of note is the presence of rimmed vacuoles that are often more common in type 1 than type 2 fibres. The vacuoles often contain acid phosphatase. Electron microscopy shows that they are autophagic and contain osmiophilic membranous myelinlike whorls and cytoplasmic debris. Electron microscopy also reveals the other particular feature of OPMD – the presence of intranuclear tubular filaments. These can be identified in semi-thin toluidine blue-stained resin sections as pale or clear areas. These filaments are about 0.25 μm in length, with an outer diameter of 8.5 nm and an inner diameter of 3 nm. They are unbranched and their orientation is variable. They are seen only in muscle nuclei and never in the cytoplasm or nuclei of other cell types, such as satellite, endothelial and interstitial cells. Thus, the nuclear inclusions of OPMD are distinct from the 15- to 18-nm filaments seen in IBM and distal myopathies with rimmed vacuoles. The number of affected nuclei in OPMD varies between muscles (2–5 per cent) but is rarely more than 9 per cent per plane of section. Antibodies to PABPN1 localize to the nuclear inclusions, and poly(A) RNA can be detected in them with in situ hybridization.21 They are also recognized by antibodies to ubiquitin and proteosomal subunits.8 Other pathological features of OPMD, some of which may be age-related, include variation in fibre size, nuclear clumps, occasional internal nuclei, moth-eaten fibres, corelike areas and whorled fibres. Mitochondrial aggregates, as in lobulated fibres, may be seen. A few ragged-red fibres and fibres devoid of cytochrome oxidase, reflecting the presence of abnormal mitochondria, may also occur. Oculopharyngodistal myopathy (OPDM) is distinct from OPMD although they share some clinical and pathological similarities. It is not associated with the GCG expansion of the PABPN1 gene and the gene responsible is not known. Both autosomal dominant and recessive inheritance of OPDM has been described.120,293,383,473 The typical ­presentation is earlier than OPMD and includes ptosis, ophthalmoparesis, facial and bulbar weakness and distal limb weakness, in contrast to the proximal weakness of OPMD. Cardiomyopathy may also occur.451 Serum CK levels are mildly elevated. Muscle biopsies show variation in fibre size, rimmed vacuoles and occasional fibres negative

25

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1580  Chapter 25  Diseases of Skeletal Muscle

for cytochrome c oxidase. The 8 nm nuclear inclusions typical of OPMD are absent but nuclear and cytoplasmic tubular filaments variously reported to be 10–12 nm, 14–16 nm or 16–18 nm occur.

Congenital Myopathies and Allied Disorders The congenital myopathies emerged as a distinct group of disorders in the 1950s and 1960s, alongside the wider application of muscle histochemistry and electron microscopy. They are clinically, genetically and pathologically a heterogeneous group of disorders defined by, and named after, characteristic morphological features on muscle biopsy, such as nemaline myopathy, central core disease, multi-minicore disease and myotubular myopathy. It is now apparent, however, that there is considerable pathological, genetic and clinical overlap with the same pathological feature being associated with more than one gene, and defects in the same gene being associated with more than one abnormal structure (Table 25.17). Onset of congenital myopathies is usually at birth or in early childhood, with hypotonia and muscle weakness (‘floppy baby’), but some adult cases with similar histopathological features have been reported, and a rare case with hypertonia and a ‘stiff’ phenotype has been reported.354 Muscle weakness may be predominantly proximal and of limb-girdle distribution, or it may be more generalized. In some cases, weakness may show marked involvement of the axial muscles and the face, and a few may show prominent distal involvement. A long ‘myopathic’ face is a common feature, particularly in nemaline myopathy and myotubular myopathies, and extraocular involvement occurs in some disorders, such as myotubular/­ centronuclear myopathies and in some cases with mutations in the ryanodine receptor gene (RYR1). The progression of the muscle weakness may be slow or minimal, but there is often disproportionately severe weakness of the respiratory muscles. Arthrogryposis may occur in some severe cases of nemaline myopathy and RYR1-related core disease, and in association with defects in various genes encoding myofibrillar proteins.177,239,367,427,439,442 Lordosis, spinal rigidity, scoliosis and joint laxity are common, and hip dislocation is a particular feature of RYR1-related core disease. Intelligence is usually normal. Creatine kinase levels are usually normal or elevated only mildly. Differential involvement of muscles is a feature of several congenital myopathies, and muscle MR imaging reveals characteristic patterns that can help direct molecular analysis in childhood and adult cases.284,431 Most of the structural features that characterize these disorders are non-specific and occur to a variable extent in several disorders. As stated earlier, there is also considerable pathological overlap between the various congenital myopathies. Mutations in different genes can lead to the presence of the same histopathological feature, and mutations in the same gene can give rise to a variable clinical phenotype. Several of the genes responsible for the more common congenital myopathies have been identified (see Table 25.17) and there is now a wider appreciation of the clinical and pathological phenotypes associated with them.217,318,489 Inheritance of the congenital myopathies may be autosomal dominant, autosomal recessive or X-linked recessive, and there is a high incidence of de novo dominant mutations.

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The most common congenital myopathies are nemaline myopathy, core myopathies (central core, multi-minicore) and myotubular/centronuclear myopathies. Rare disorders characterized by diverse morphological features, such as hyaline bodies, dilation of the sarcoplasmic reticulum (sarcotubular myopathy), reducing bodies, fingerprint bodies, zebra bodies, cylindrical spirals and lamellar bodies, have also been identified. Some of these have a molecular basis, but it is not clear whether they are all genetic entities.160,400 Most of the congenital myopathies share some histopathological features. Hypotrophy of type 1 fibres is seen in several/most of the conditions, and there is often a marked predominance or uniformity of type 1 fibres. Antibodies to myosin isoforms confirm the slow phenotype of most fibres, but there may be some hybrid fibres with more than one isoform. Necrosis and regeneration are not typical features, but they may occur. Scattered, very small fibres containing fetal myosin (sometimes referred to as ‘pinpricks’) are often seen, but it is not clear whether these represent attempts at regeneration, because fetal myosin can be expressed for a variety of reasons. Centrally placed nuclei characterize myotubular and centronuclear myopathies but can also occur in association with RYR1- related core myopathy. Fibrosis is rare in congenital myopathies, but it can occur, especially in RYR1-related core myopathies.

Congenital Myopathies with Structural Defects Nemaline Myopathy Inheritance of nemaline myopathies is autosomal dominant or autosomal recessive, with a significant number of de novo dominant cases. Mutations in several genes have been identified (see Table 25.17), many of which encode thin filament proteins.405 There is further genetic heterogeneity, because some cases do not link to any of the published loci, and next-generation sequencing is identifying gene variants that are currently under investigation. Mutations in the genes encoding skeletal α-actin (ACTA1) and nebulin (NEB) are the most common. Most mutations in ACTA1 are dominant or de novo dominant, with a few rare recessive cases. In contrast, all reported NEB mutations are recessive.489 Most cases of the common childhood type result from NEB mutations, and there is a particular deletion in the Ashkenazi Jewish population.489 Mutations in the genes for α- and β-tropomyosin (TPM3, TPM2) are rare, and mutations in the gene for troponin T (TNNT1) seem to be restricted to the Amish population in North America.489 There is a broad clinical spectrum associated with the presence of rods.489 The severe congenital/neonatal forms show marked hypotonia and an absence of spontaneous movements and respiration at birth. Some cases may display the fetal akinesia sequence.239 The term nemaline myopathy is usually applied to cases with congenital hypotonia and weakness, and those of adult onset (sporadic late onset nemaline myopathy) are often of autoimmune origin, several of which have a gammopathy.24,67,320,489 These cases are better considered as ‘myopathies with rods’, to distinguish them from the congenital nemaline myopathies. In addition, cases described as nemaline myopathy with onset in childhood or early adulthood, characterized by slowness of movement, have dominantly inherited mutations in the KBTBD13 gene.377 These cases therefore differ from

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Inherited Muscle Disorders  1581



Table 25.17  The structural and genetic defects in congenital myopathies and allied disorders Gene

25

Gene locus

Inheritance

Protein

ACTA1

1q42

AD or AR

Skeletal α-actin

NEB

2q.2

AR

Nebulin

TPM3

1q2

AD or AR

α-Tropomyosin

TPM2

9p13

AD or AR

β-Tropomyosin

TNNT1

19q13

AR

Troponin T

CFL2

14q12

AR

Cofilin-2

KBTBD13

15q25.31

AD

KBTBD13

KLHL40

3p21

AR

KLHL40

KLHL41

2q31

AR

KLHL41

RYR1

19q13

AD

Ryanodine receptor 1

ACTA1

1q42

AD or AR

Skeletal α-actin

NEB

2q.2

AR

Nebulin

TPM2

9q13

AD

β-Tropomyosin

Nemaline rods and nemaline myopathy

Other myopathies with abundant rods Rods and cores

Rods and caps

Congenital myopathies with cores* Central core disease

RYR1

19q13

AD or AR

Ryanodine receptor 1

Multi-minicore disease

SEPN1

1p36

AR

Selenoprotein N1

Congenital myopathy +/- cardiomyopathy

TTN

2q31 14q11

AR AD

Titin Slow myosin heavy chain

MYH7

Central nuclei and centronuclear myopathies Myotubular myopathy

MTM1

Xq28

XLR

Myotubularin

Centronuclear myopathy

DNM2

19p13

AD

Dynamin 2

BIN1

2q14

AR

Amphiphysin 2

RYR1

19q13

AD

Ryanodine receptor 1

CCDC78

16p13.3

AD

Coiled-coil domaincontaining protein 78

TTN

2q31

AR

Titin

Congenital myopathy & fatal cardiomyopathy

Surplus protein congenital myopathies Actin aggregation

ACTA1

1q42

AD

Skeletal α-actin

Zebra body myopathy

ACTA1

1q42

AD

Skeletal α-actin

Cap disease

TPM2

9q13

AD

β-Tropomyosin

TPM3

1q2

AD

α-Tropomyosin

ACTA1

1q42

AD

Skeletal α-actin

ACTA1

1q42

AD

Skeletal α-actin

SEPN1

1p36

AR

Selenoprotein N1

TPM3

1q2

AD

α-Tropomyosin

TPM2

9q13

AD

β-Tropomyosin

Congenital fibre type disproportion

Continued

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1582  Chapter 25  Diseases of Skeletal Muscle Table 25.17  The structural and genetic defects in congenital myopathies and allied disorders (Continued) Gene

Gene locus

Inheritance

Protein

MYH7

14q11

AD

Slow myosin heavy chain

RYR1

19q13

AD or AR

Ryanodine receptor 1

HACD1

10p12

AR

3-hyroxyacyl-CoA dehydratase 1

Congenital myopathies characterized by distal involvement and/or distal arthrogryposis

NEB

2q2

AR

Nebulin

TPM2

9q13

AD

β-Tropomyosin

MYH3

17p13

AD

Myosin heavy chain 3

MYH8

17p13

AD

Perinatal myosin

TNNI2

11p15

AD

Troponin I

TNNT3

11p15

AD

Troponin T3

MYBPC1

12q23.2

AD

Myosin-binding protein C, slow type

ECEL1

2q37.1

AR

Endothelin-converting enzyme-like 1

AD, autosomal dominant; AR, autosomal recessive; XLR, X-linked recessive; XLD, X-linked dominant;?, inheritance currently uncertain. +/- with or without. Some disorders with early onset caused by mutations in myosin heavy chains, and disorders with reducing bodies and spheroid bodies are not included in this table (see section on Sarcomeric Proteins, p. 1589). *Core-like

lesions occur in association with defects in several genes (see text).

other forms of nemaline myopathy by their lack of neonatal hypotonia and a muscle pathology that shows hypertrophy of type 1 fibres and atrophy of type 2 fibres, in addition to cores. Defects in the genes of other members of the Kelch family, however, have recently been identified as causing severe recessive forms of nemaline myopathy (KLHL40, KLHL41) and others are under investigation (see gene table of neuromuscular disorders at www.musclegenetable.fr).355 The most common form of nemaline myopathy presents with hypotonia in early infancy or childhood, and patients have delayed motor milestones and generalized weakness predominantly affecting the facial and axial muscles. There is poor muscle bulk, and feeding and respiratory problems are common. Independent ambulation is achieved, and the disorder is non-progressive or mildly progressive. Some cases show a predominant distal weakness but not all show rods.246,488 The characteristic feature of nemaline myopathy is the presence of rod-shaped structures staining red with Gomori trichrome (Figure 25.81). They are often in clusters at the periphery of fibres near nuclei, or they may appear throughout the fibre. If fibres are very small, rods may be difficult to identify without high-power optics. In addition to rods, varying degrees of Z-line disruption and core-like areas devoid of mitochondria and showing pronounced myofibrillar disruption may be present.217 The areas of abundant rods do not stain for oxidative enzymes and should not be confused with cores with disrupted myofibrils. Accumulation of actin filaments can occur with or without the presence of rods in cases with ACTA1 mutations.163 With electron microscopy, rods are seen as electrondense structures, which may be rod-like or sometimes ovoid (Figure 25.82). They are frequently parallel to the

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longitudinal axis of the sarcomeres, and their form depends on the plane of section. Some rods may be derived from Z-lines, because they show continuity with Z-lines, have a similar lattice structure and contain similar proteins. The major constituent of both rods and Z-lines is α-actinin. They also contain tropomyosin, and proteins associated with Z-lines, such as actin and myotilin. As with Z-lines, desmin occurs at the periphery of rods, but not within them. In most cases, rods are cytoplasmic, but rare cases caused by ACTA1 mutations also demonstrate nuclear rods.200,220,492 It is rarely possible to predict the defective gene from pathology, with the exception of the accumulation of actin filaments and presence of nuclear rods, which are associated with ACTA1 mutations in most cases. Only a minority of ACTA1 cases, however, show these features. In patients with recessive NEB mutations, immunohistochemistry shows nebulin is present in all muscle fibres.400,487 In the absence of any genetic clues, molecular analysis usually begins with ACTA1, because it has only six exons, in contrast to the giant NEB gene, which has 183 exons. Rods may also occur in normal eye muscles, at myotendinous junctions, in ageing muscle and occasionally in a variety of disorders. Nuclear rods can occur in association with mutations in the genes encoding plectin and ZASP, but the phenotype in these cases is different from the congenital nemaline myopathy cases.15,325

Core Myopathies Areas devoid of oxidative enzyme stains are the characteristic pathological feature of the ‘core myopathies’. However, there is considerable variability and overlap in

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Inherited Muscle Disorders  1583

25

25.81 A case of nemaline myopathy showing numerous redstained rods in peripheral clusters and throughout some fibres. Gomori trichrome.

Box 25.8.  The ryanodine receptor of skeletal muscle The RYR1 gene consists of 106 exons with a cDNA greater than 15 kb and encodes the skeletal muscle ryanodine receptor 1 protein (RyR1). It is a large transmembrane tetrameric ligandgated calcium-release channel in the terminal cisternae of the sarcoplasmic reticulum and has a major role in the regulation of cytosolic calcium levels and excitation–contraction coupling. The predicted structure of the ryanodine receptor suggests that the calcium-release channel is located in the C-terminal domain of the protein, whereas the remaining N-terminal domains constitute the visible foot structure that interacts with the dihydropyridine receptor (DHPR) in the T-tubule. Genotype– phenotype correlations have suggested that mutations in the cytoplasmic N-terminal domain and the cytoplasmic central domain mostly result in malignant hyperthermia susceptibility, rather than in central core disease, whereas mutations affecting the C-terminal exons are a ‘hotspot’ and more commonly result in central core disease. Recessive mutations associated with core-like lesions occur in all parts of the gene. The majority of mutations in the RYR1 gene are missense mutations, although small in-frame deletions have also been detected.

the clinical phenotype and pathology that can make differential diagnosis difficult, and to refer to this group of disorders as ‘core myopathies’ can be helpful. We have, however, retained the two historical categories of central core disease and multi-minicore disease because these are familiar terms to pathologists, but emphasize the overlap between them. Central Core Disease  In 1956, Magee and Shy published

details of muscle biopsies from a family in which fibres showed amorphous central areas.260 The name ‘central core disease’ was suggested later.169 Since then, many cases with a similar pathology have been identified, the gene responsible has been identified (the ryanodine receptor 1 gene -RYR1 on chromosome 19q13) (Box 25.8), and a greater understanding of the phenotype appreciated.217,229,344 The inheritance of most RYR1 mutations is autosomal dominant; several sporadic de novo dominant cases have

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25.82 Electron micrograph of a case of nemaline myopathy showing electron-dense structures that are rod-shaped when sectioned longitudinally or ovoid in shape when sectioned transversely.

also been reported and there are also recessive cases.217,405 Cases with a severe presentation, with features of the fetal akinesia sequence, have also been reported, some of which are associated with recessive inheritance.367 These severely affected infants require ventilation at birth, and death may occur in infancy. In contrast, some of these patients may show considerable improvement, and it may be possible to wean them off ventilation; one reported child eventually became independently ambulant.367 The RYR1 gene is also responsible for malignant hyperthermia susceptibility, although additional loci are also linked to this.219 Most, if not all, patients affected by central core disease are considered to be ‘at risk’ of malignant hyperthermia susceptibility, and appropriate precautions need to be taken. It is now appreciated that the range of clinical and pathological features associated with RYR1 mutations is broad. RYR1-related core disease is one of the most common congenital myopathies. Patients typically have hypotonia at birth and developmental delay. Weakness is more pronounced in the pelvic girdle and axial muscles than in the upper limbs. Facial involvement is usually mild, and lack of complete eye closure may be the only finding. However, some cases show ophthalmoplegia and ptosis.217 Orthopaedic complications such as scoliosis, talipes and dislocation of the hips are common. Contractures, other than tendon Achilles tightness, are rare, but many affected individuals have marked ligamentous laxity, occasionally associated with patellar instability. Apart from the most severe neonatal cases and some patients with congenital dislocation of the hips, most patients achieve independent walking. The course of RYR1-related core disease is often static or slowly progressive over prolonged periods of time.217 Primary cardiac involvement is not a feature, and respiratory involvement is usually milder than with other congenital myopathies, except in the severe neonatal cases. Serum CK activity is usually normal or only mildly elevated. A striking feature of RYR1-related core disease is the differential muscle involvement, and muscle MR imaging

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1584  Chapter 25  Diseases of Skeletal Muscle

reveals a characteristic pattern that can be helpful when selecting the site for a muscle biopsy and for directing molecular genetics.431 The name ‘central core disease’ reflects the pale central zones of muscle fibres, which are devoid of mitochondria and, therefore, oxidative enzyme staining (Figure 25.83). The core usually extends a considerable way down the fibre, and associated myofibrils are often hypercontracted (structured) or, in some cases, very disrupted (unstructured). The cores may be rimmed by PAS-positive material, and desmin may accumulate peripherally or internally within them. Additional proteins such as αB-crystallin, small heat-shock proteins, myotilin and filamin C also accumulate within cores.117,400,405 Although cores may be central, they can also be peripheral, and more than one may be present in cross-section. Clearly demarcated cores are not always evident, and some cases may show only subtle unevenness of oxidative enzyme stains or multiple focal areas of disruption, which resemble minicores. In particular, muscles from very young patients may show only type 1 uniformity or predominance, suggesting there is an age-related development of the cores (see Figure 25.83).406

Fibre size variation is often mild, but fibre hypertrophy is common, particularly in adults. Some cases may show only fibre type disproportion with small type 1 fibres.84 If fibre typing is retained, the cores have a predilection for type 1 fibres; however, fibre type uniformity is common, with most fibres staining as type 1 fibres. A few fibres may coexpress fast myosin, and there may be a few very small fibres with fetal myosin scattered through the biopsy (often referred to as ‘pinpricks’; see Figure 25.53c). Some biopsies may show rods and cores, and occasionally rods may be an obvious feature.214,299 Cores are not specific for a RYR1 mutation and can also occur following tenotomy or with neurogenic atrophy, where they are target-like (see Figure 25.56), and in association with several other gene defects, including mutations in the ACTA1, MYH7, TTN, CFL2, KBTBD13 genes.60,130,215,217,221,318,400,443,489 Cores can also coexist with rods and be associated with RYR1, NEB, CFL2 or KBTBD13 mutations.300,366 Internal nuclei had not been considered a feature of central core disease in early studies, but it is now appreciated that they can be an important indicator of RYR1 mutations (Figure 25.84). In some cases internal nuclei

(a)

(b)

(c)

(d)

25.83 Images showing the variability of oxidative enzyme staining that can be seen in association with mutations in the RYR1 gene. In (a) no cores are visible but in (b) there are typical large cores; in (c) there are multiple large cores; and in (d) minicores and uneven staining. Note in all fibre type uniformity and absence of fibre type differentiation. (a, b, d), NADH-TR; (c), cytochrome c oxidase.

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Inherited Muscle Disorders  1585

25

25.84 Internal nuclei and excess connective tissue and fat in a case of RYR1 core myopathy. Note also the wide variation in fibre size. H&E.

25.85 Low power view showing the pronounced adipose tissue in the same case as in Figure 25.84. H&E.

may be numerous, and several may be in a central position, resembling centronuclear myopathy.499 Similarly, an increase in connective tissue was not considered a feature of ‘classic’ central core disease, but it can occur, and occasionally there may be extensive adipose tissue (Figure 25.85). In such cases, the separation of fascicles by adipose tissue and fibrous tissue has caused diagnostic confusion with a muscular dystrophy. Some of these samples may show only subtle unevenness of oxidative enzyme stains, whereas others show large classic cores or multiple small cores. Multicore Myopathy  This is a relatively non-progressive

congenital myopathy characterized by multifocal areas of degeneration in muscle fibres, which prompted the name ‘multicore disease’. There have been various reports of this disorder encompassing a broad range of clinical phenotypes, but with similar histopathological features. The defining histopathology consists of multiple small areas that are devoid of oxidative enzymes, lack mitochondria and show focal disruption of the sarcomeric pattern (Figure 25.86). Core-like areas, however, can be associated with defects in several genes in addition to RYR1 and SEPN1, including ACTA1, MYH7, MYH2, CFL2, TTN, CCDC78 and KBTBD13, as well as in some forms of muscular dystrophy, some myasthenic conditions and in association with denervation. Four clinical categories of patients with minicores have been identified, and their molecular defects are being defined.138 The most common phenotype shows marked axial weakness, with spinal rigidity, scoliosis, torticollis and respiratory involvement that are often disproportionate to the overall muscle weakness. This phenotype is similar to RSMD1, and the two disorders are now often considered allelic because both are caused by recessive mutations in the gene for selenoprotein N1 (SEPN1). The pathological spectrum associated with SEPN1 mutations also includes cases with Mallory bodies.137 A second clinical group with multiple minicores and similar proximal and axial weakness also shows a variable association with external ophthalmoplegia. These cases have mutations in the RYR1 gene.213,298 Minicores and

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25.86 Multiple minicores in a case with ‘minicore myopathy’ caused by mutations in the SEPN1 gene. Note also the two-fibre pattern and uneven staining in both fibre types. Cytochrome c oxidase.

opththlamoplegia are also features associated with mutations in the MYH2 gene encoding myosin IIA (see later). RYR1 mutations have also been found in a third phenotypic group with similarities to patients with central core disease.298 The fourth group comprises rare patients with antenatal onset, generalized arthrogryposis, dysmorphic features and mild to moderate reduction of respiratory function. Primary cardiac dysfunction is not a feature of any of these groups. The multicore cases associated with RYR1 mutations can be considered part of a clinicopathological spectrum of ‘core myopathies’, and the term ‘multiminicore’ disease can then be reserved for cases with SEPN1 mutations. The presence of minicores or the absence of typical central cores can make it difficult to distinguish between SEPN1 and RYR1 cases. Pathological features that can help are the preservation of a two-fibre type pattern and the presence of minicores in both fibre types in SEPN1 cases, versus the type 1 uniformity that is common in RYR1 cases.

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1586  Chapter 25  Diseases of Skeletal Muscle

Internal nuclei may be occasional, or sometimes profuse, in SEPN1 cases. Rods are not a feature of SEPN1 cases, although there have been several reported cases of minicores in association with an abundance of additional structural defects, including rods and whorled fibres. Some of these cases have been shown to result from mutations in the RYR1 gene or the ACTA1 gene, but many of the early cases are still molecularly unresolved.117,217,400

Myotubular and Centronuclear Myopathies Early descriptions of muscle biopsies with abundant central nuclei led to the introduction of the terms ‘myotubular myopathy’, because central nuclei are a feature of fetal myotubes, and ‘centronuclear myopathy’.412,423 The clinical and pathological features described in these early papers would probably be interpreted differently in the light of more recent advances. The term ‘myotubular myopathy’ is now often reserved for the X-linked disorder caused by mutations in the myotubularin gene (MTM1), and the term ‘centronuclear myopathy’ for the molecularly heterogeneous group of autosomal disorders characterized by central nuclei.365 X-Linked Myotubular Myopathy  X-linked myotubular myopa-

thy, caused by mutations in the MTM1 gene on chromosome Xq28 encoding myotubularin, is a severe condition with onset in utero. Pregnancy is complicated by polyhydramnios, and there is often a history of miscarriages and neonatal death in the maternal line. Features include marked neonatal hypotonia, a variable degree of external ophthalmoplegia, feeding difficulties and respiratory failure at birth, which is often fatal. Some affected infants may survive if the respiratory problems in the neonatal period can be managed.273 Female carriers may manifest with a variable degree of muscle weakness that ranges from mild to severe, usually depending on the pattern of X chromosome inactivation; hypotonia from birth and the inability to stand or to walk in a female carrier has also been reported.216,446

Myotubularin is expressed in most tissues. It is a dual-specificity phosphatase that dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol (3,5)-bisphosphate.242 Its exact function is unclear, but evidence suggests a role in triad formation.3,196 Preliminary studies indicated that myotubularin was localized to nuclei, but later studies showed that it is essentially cytoplasmic.196 Antibodies to myotubularin fail to detect endogenous protein in muscle sections, but alterations in its levels have been studied by immunoprecipitation.457 A large number of different mutations, distributed throughout the gene, have been identified, which may be missense, nonsense or splice site mutations, point mutations, small or large deletions, or insertions. The MTM1 gene is part of a family with several members that share sequence homology. These genes are clearly candidates for other neuromuscular disorders, and the genes for myotubularin-related protein 2 and myotubularin-related protein 13/SBF2 are mutated in forms of Charcot–Marie–Tooth neuropathy and myotubularinrelated protein 14 (hJUMPY) in rare cases of autosomal centronuclear myopathy, the latter being thought to be a gene modifier.304,456 The characteristic pathological feature of cases with MTM1 mutations is centrally placed nuclei that occupy a large volume of the fibre (Figure 25.87a), and are present in both fibre types. In longitudinal section, these are spaced regularly down the fibre, not in chains as in regenerating fibres (Figure 25.87b); thus, the plane of transverse section influences the number of observed central nuclei. The number of fibres with central nuclei can vary between muscles and may not be numerous at birth.191,382 The area around the central nuclei is often devoid of myofibrils and appears as holes with ATPase staining or myosin immunolabelling. With oxidative enzyme and PAS stains, the central area is stained darkly, reflecting aggregation of mitochondria and glycogen, and there may also be pale subsarcolemmal halos around many fibres (Figure 25.88). As in many congenital myopathies, type 1 fibres may be predominant, and most fibres are small in diameter (hypotrophic and/or atrophic), particularly type 1 fibres. (b)

25.87 Central nuclei in a case of X-linked myotubular myopathy in (a) transverse and (b) longitudinal section. Note in transverse section not all fibres show a central nucleus that probably relates to the spacing of the nuclei down the fibre as seen in (b). The basophilic central granularity relates to accumulation of mitochondria. H&E.

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Inherited Muscle Disorders  1587

(a)

(b)

(c)

(d)

25

25.88 (a) NADH-TR activity concentrated in the centre of the fibres and pale peripheral halos in a case of X-linked myotubular myopathy. Note also the lack of differentiation into fibre types. (b) Cytochrome c oxidase shows similar features but one larger fibre has a loop of higher activity, as seen in necklace fibres. (c, d) Radial strands in an autosomal case of centronuclear myopathy with a DNM2 mutation. (c), PAS; (d), NADH-TR.

An unusual pathological feature seen in some MTM1 carriers and also some neonatal cases is ‘necklace fibres’, noted by Romero and colleagues.28,190 A milder adult male case was also reported to show them. Necklace fibres have a basophilic loop internally within the fibre and near the sarcolemma. This loop is associated with internal nuclei, increased oxidative enzyme activity and PAS staining, and shows increased desmin, αB-crystallin and SERCA immunolabelling (Figure 25.88b). It is not yet clear if necklace fibres are specific to MTM1 mutations, as fibres with a similar appearance but lacking the nuclei on the loop have been observed in centronuclear cases with DNM2 mutations250 (see later), and in one case with a RYR1 mutation (personal observation). The presence of abundant desmin and vimentin has been put forward as evidence of a developmental defect,379 but it is not a feature of all fibres with central nuclei. The immunolabelling of myosin isoforms shows that fibres with central nuclei have the fast or slow isoform of mature muscle, without fetal myosin, indicating that maturation, at least with regard to myosin isoforms, does occur in myotubular myopathy.401 Expression of NCAM, utrophin

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and laminin α5 has also been reported in myotubular myopathy cases.191 A similar pathological appearance, with abundant central nuclei, is seen in cases of congenital myotonic dystrophy, and an expansion of the DM1 gene should always be considered in neonates with the pathological picture of myotubular myopathy (see Myotonic Dystrophies, p. 1578). Autosomal Centronuclear Myopathies 

Several cases of centronuclear myopathy not linked to Xp28 have been identified, some of which have treatable myasthenic-like symptoms.27,361 Some are familial whereas others are sporadic. They are molecularly and clinically heterogeneous. Inheritance may be recessive or dominant and some causative genes have been identified (see Table 25.17). Mutations in the gene encoding dynamin 2 on chromosome 19q13.2 and BIN1 encoding amphiphysin 2 are now both recognized as autosomal causes of centronuclear myopathy.119,458 Screening of the human sequence databases has also implicated the MTMR14 gene456 and it is reported to have a modifying role in autophagy.476 In

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1588  Chapter 25  Diseases of Skeletal Muscle

addition, next-generation sequencing has identified a family with a d ­ ominant mutation in the gene encoding the coiled-coil domain-containing protein 78 (CCDC78), a protein enriched in the perinuclear region and triads.261 Biopsies from this family not only showed central nuclei but also core-like lesions. Dynamin 2 is involved in membrane remodelling, endocytosis and membrane trafficking, actin assembly and centrosome cohesion and it interacts with amphiphysin 2.119,452 Onset in cases with mutations is usually in adolescence or adulthood, but neonatal onset has also been identified.29,183 The condition is characterized by slowly progressive muscle weakness. Distal involvement precedes involvement of the limb girdles, trunk and neck muscles. Loss of ambulation is rare. Bilateral ptosis is almost invariable, and involvement of extraocular muscles is common. Peripheral axonal neuropathy is present in some patients, and it is notable that DMN2 mutations are also responsible for dominant Charcot–Marie–Tooth disease type 2B.512 The mutations responsible for centronuclear myopathy are missense changes in the central domain of the gene, whereas mutations that cause Charcot–Marie–Tooth disease type 2B are restricted to the pleckstrin homology domain. Muscle imaging shows a pattern distinct from other neuromuscular disorders, which may help to direct molecular analysis. It is characterized predominantly by involvement of lower leg muscles, with mild involvement of the posterior thigh and gluteus maximus.142,438 Muscle biopsies in DMN2 cases show central nuclei surrounded by a zone devoid of organelles, type 1 fibre hypotrophy and predominance, and oxidative enzyme and PAS stains that exhibit a spoke-like pattern radiating from the centre of the fibre (Figure 25.88c,d). It is not clear whether this is a specific feature associated with DNM2 mutations. Radial strands were less c­ommon in the three rare cases identified with BIN1 mutations, and electron microscopy suggests the central nuclei form chains rather than being spaced, a feature that may also be seen in DNM2-related cases.458 Cases with BIN1 mutations have shown subsarcolemmal vacuolation, and triad morphology is abnormal in MTM1, DNM2 and BIN1 related centronuclear myopathies, suggesting a common pathogenic link.458 The presence of cores together with central nuclei suggested a relationship with RYR1 mutation cases and several cases with central nuclei and RYR1 mutations have now been identified.217,499 There are apparent founder RYR1 mutations in the South African population.499 An early onset disorder, in which internal and some central nuclei are a pronounced feature, is caused by homozygous mutations in the C-terminal kinase domain in the gene encoding titin (TTN).60 In addition to hypotonia and proximal and distal weakness, spinal rigidity, contractures and dilated cardiomyopathy develop. The pathological features seen in muscle biopsies include multiple internal nuclei, type 1 fibre uniformity, cores of varying size devoid of oxidative enzyme staining, and sometimes internal basophilia in occasional fibres. Immunohistochemistry shows an absence of titin only with an antibody specific to the M-line C-terminal domain and immunoblots show a secondary absence of calpain-3.

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Congenital Fibre Type Disproportion There has been a longstanding debate as to whether the presence of small type 1 fibres in the absence of other pathology (congenital fibre type disproportion [CFDP]) represents a distinct disease entity. Defects in ACTA1, SEPN1, TPM2, TPM3, RYR1 and MYH7 genes can all result in fibre type disproportion without any other pathological defect.82,84,85 It is not known if the typical pathological features, such as rods or cores, associated with these genes might develop at a later age in the cases reported, because repeat muscle ­biopsies at a later age have not been reported. Type 1 hypotrophy has also been reported in some patients with congenital myasthenia, but these were not molecularly confirmed and myasthenic symptoms can occur in association with defects in several genes.176 Further genetic heterogeneity associated with fibre type disproportion is likely. The HACD1 gene is also associated with CFDP in humans and with central nuclei in dogs.

Other Early Onset Myopathies with Structural Defects A number of other structural features are associated with early onset disorders resembling congenital myopathies, the molecular causes of which are now known. Cap-like structures of focal peripheral disorganized myofibrils are associated with defects in the TPM2, TPM3 and ACTA1 genes, all of which are also associated with other pathological features, such as nemaline rods. The caps lack, or show reduced, staining for NADH-TR and ATPase (in contrast to ‘hyaline bodies’ in myosin storage myopathy - see later) and reduced immunoreactivity for myosin but high reactivity for many sarcomeric proteins including actin, nebulin, tropomyosin, troponin T and desmin. Some disorders were classified as congenital myopathies before identification of the defective gene, but they have now been reclassified as part of the clinicopathological spectrum of other disorders. For example, spheroid body myopathy is caused by defects in the gene encoding ­myotilin (MYOT) and is considered to be a variant of a myofibrillar myopathy (see later). Reducing body myopathy is a particularly severe progressive disorder with onset that is not always congenital; it is discussed later with other conditions caused by defects in the FHL1 gene. ‘Hyaline bodies’ were described as a feature of a congenital myopathy and represent accumulation of slow myosin, caused by defects in the gene encoding slow myosin (MYH7). Disorders of MYH7 are discussed further later (see Myopathies Caused by Defects in Sarcomeric Proteins). A number of other cases have been reported with unusual ultrastructural features that include cylindrical spirals, fingerprint bodies, hexagonal arrays. It is not yet clear if these are all genetic entities.160

Congenital Myopathies Characterized by Distal Involvement and/or Distal Arthrogryposis Distal arthrogryposis is a symptom of several disorders and several clinical forms with varying severity have been described. Distal arthrogryposis type 1, 2A and 2B have been associated with mutations in different sarcomeric proteins including β-tropomyosin (TPM2),435,440 fast troponin T (TNNT3),436 fast troponin I (TNNI2),226 embryonic myosin heavy chain (MYH3),441,460 perinatal (fetal) myosin

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heavy chain (MYH8)459 and myosin-binding protein-C, slow type (MYBPC1).177 Other forms have recently been shown to be associated with the ECEL1 gene.110,274 Histopathological studies are limited, because the number of molecularly confirmed cases is small and the pathological spectra not known. The changes observed are non-specific and include small type 1 fibres in some cases with MYH3 or ECEL1 mutations, and small type 2 fibres cases with TNNI2 mutations. Central basophilia has been observed in the rare cases with mutations in the slow myosin-binding protein-C.177

Myopathies Caused By Defects in Sarcomeric Proteins Defects in several myofibrillar proteins of the skeletal muscle sarcomere are associated with neuromuscular disorders. Aggregation of proteins (e.g. actin, myosin, desmin) is a feature of some of these.161,162,393 Components of the thin filaments (actin, tropomyosins, nebulin) and associated proteins are involved in nemaline myopathies (see Congenital Myopathies and Allied Disorders, p. 1580). Defects in telethonin cause a form of LGMD (see Recessive Limb-Girdle Muscular Dystrophies, p. 1561). In this section we discuss myopathies caused by defects in myosins and titin (see also Congenital Myopathies and Allied Disorders, p. 1580, and Limb-Girdle Dystrophies, p. 1561) and proteins of the Z-line that are associated with a group of disorders commonly referred to as myofibrillar myopathies. FHL1 is often also included within this group as it is a myofibrillar protein and some cases have similar pathological features to myofibrillar myopathies, although there is a broad spectrum associated with FHL1 (see Table 25.18).

Myopathies Associated with Mutations in Genes Encoding Myosin Heavy Chains Myosins are a family of highly conserved proteins that exist is several isomeric forms encoded by different genes. The isoforms expressed in skeletal muscle currently associated with a myopathy are (i) slow myosin, expressed in type 1 fibres (and in the heart) encoded by the MYH7 gene; (ii) myosin IIa expressed in fast 2A skeletal muscle fibres encoded by the MYH2 gene; and (iii) embryonic and perinatal myosin expressed in developing muscle and in regenerating fibres encoded by the MYH3 and MYH8 genes, respectively.439 Defects in MYH3, MYH8 and MYBPC1 are associated with syndromes characterized by distal arthrogryposis (see Congenital Myopathies and Allied Disorders, p. 1580). The MYH7 gene encoding slow/β-cardiac myosin is associated with either a cardiomyopathy or skeletal myopathy, or sometimes both.162 Age of onset varies from childhood to middle age. Weakness may be predominantly distal, as in Laing early onset distal myopathy with a characteristic ‘hanging big toe’, or scapuloperoneal or of a limbgirdle distribution. Talipes, hip dislocation and knee flexion contractures can be present in the most severe cases. Some cases show subsarcolemmal aggregates of slow myosin in type 1 fibres. These were previously referred to as hyaline bodies (‘hyaline body myopathy’), but with the identification of slow myosin within them caused by mutations in the MYH7 gene the disorder is now referred to as ‘myosin storage myopathy’. The areas of myosin aggregation

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Inherited Muscle Disorders  1589

are pale pink with H&E and pale green with Gomori trichrome staining and are unstained (except at the periphery) with stains for oxidative enzymes and desmin but positive for ATPase, in contrast to caps (see Congenital Myopathies and Allied Disorders, p. 1580; Figure 25.89). With electron microscopy the accumulated material has a granular and slightly filamentous appearance.117 Myosin storage is not seen in other cases with MYH7 mutations or in cases with Laing myopathy. Muscle pathology in these can include small type 1 fibres (sometimes resembling fibre type disproportion), predominance of type 1 or type 2 fibres, occasional necrotic and regenerating fibres, mild fibrosis, increased internal nuclei, core-like areas, lobulated fibres, rimmed vacuoles, tubulofilamentous inclusions and ring fibres.117,439 Both dominant and recessive mutations in MYH2 can cause a myopathy. The dominant form was identified in a large Swedish family with congenital joint contractures but no hypotonia. Weakness is predominantly proximal with atrophy of the quadriceps femoris muscle, which may affect ambulation. In the recessive form, muscle weakness is mild to moderate and muscle MRI shows a distinctive pattern with predominant involvement of the medial gastrocnemius. Ophthalmoplegia is a feature of both forms. Histopathology in dominantly inherited cases shows mild changes in young cases with focal core-like areas in type 2A fibres, which are reduced in number and size. In adult cases pathological features are more pronounced and rimmed vacuoles containing p62 are present. Recessively inherited cases show an absence of 2A fibres and non-specific myopathic changes. Some cases may show fibre type uniformity and lobulated fibres. In contrast to dominant cases rimmed vacuoles or protein aggregation are absent.439

25

Disorders Associated with Defects in Titin Titin is a giant protein that stretches from the M-line to the Z-line. Defects in only some domains have been identified, but next-generation sequencing is increasing the number of mutations detected. The phenotype of some is defined as a limb-girdle dystrophy (see Limb-Girdle Muscular

25.89 Pale-stained areas (‘hyaline bodies’) corresponding to accumulation of slow myosin in a case with a mutation in the MYH7 gene. H&E.

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1590  Chapter 25  Diseases of Skeletal Muscle Table 25.18  Features of myopathies caused by defects in sarcomeric proteins Popular name

Locus

Gene symbol

Protein

Inheritance

Cardiac involvement

Peripheral neuropathy

Desminopathy

2q35

DES

Desmin

AD(rare AR)

Yes

Yes

αBcrystallinopathy

11q-25.3

CRYAB

αBcrystallin

AD

Yes

Possibly

Myotilinopathy

5q31

MYOT (TTID)

Myotilin

AD

Yes

Yes

Zaspopathy

10q25.3

LDB3

ZASP

AD

Yes

Yes

Filaminopathy

7q32.1

FLNC

Filamin C

AD

Yes

Yes

Bag3opathy

10q25

BAG3

Bag3

AD

Yes

Yes; severe axonal neuropathy

Early onset; severe progression

FHL1opathy

Xq26.3

FHL1

Fhl1

XD

Yes

Not ­reported

Broad phenotype; childhood onset cases with severe progression

17p13

MYH3

Myosin heavy chain 3

AD

–

–

Arthrogryposis

17p13

MYH8

Perinatal myosin

AD

–

–

Arthrogryposis

12q 23.2

MYBPC1

Myosinbinding protein-C, slow type

AD

–

–

Arthrogryposis

Myosin storage myopathy and Laing distal myopathy

14q11

MHY7

Slow myosin heavy chain

AD

No

Not ­reported

Occasional arrthyhmias

Inclusion body myopathy

17p13

MYHC2A

Fast myosin heavy chain IIA

AD, AR

Not ­reported

Not ­reported

Ophthalmoplegia

Distal myopathy and HMERF

2q321

TTN

Titin

AD

Not ­reported*

Not ­reported

Other features

Cataracts

AD, autosomal dominant; AR, autosomal recessive; Bag3, Bcl-2-associated athanogene 3; FHL1, four and a half LIM domains protein 1; HMERF, hereditary myopathy with early respiratory failure; XD, X-linked dominant; ZASP, Z-line alternatively spliced PDZ protein. *See Limb-Girdle Muscular Dystrophies and Congenital Myopathies and Allied Disorders for recessive conditions of titin.

Dystrophies, p. 1558), whereas others have a more distal phenotype and a severe neonatal form with cardiomyopathy has been reported (see Congenital Myopathies and Allied Disorders, p. 1580).60,466,467 Muscle pathology in titin distal myopathy shows features of a muscular dystrophy with variation in fibre size, fibrosis, adipose tissue and internal nuclei, but necrotic fibres are rare.181 Rimmed vacuoles are seen in the tibial muscles, which may show accumulation of ubiquitin, but the other proteins and Congo red seen in myofibrillar myopathies (see later) are not present. Immunohistochemistry using exon-specific antibodies to the C-terminal M-line M8/M9 region shows an absence of titin, but titin is detected with antibodies to other domains.181 Titin has a binding site for calpain-3 and immunoblots show a secondary reduction of calpain.

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Mutations in the A-band domain of titin are responsible for HMERF.322,335 Presentation is from early adulthood and respiratory complications are common. Muscle MRI shows a characteristic severe involvement of the proximal parts of the thighs and the anterior and lateral compartments of the lower limbs. Muscle biopsies in HMERF show variability in muscle fibre size with atrophic and hypertrophic fibres, many fibres with numerous internal nuclei and focal areas with frequent split fibres. Eosinophilic inclusions or deposits are a typical feature but not present in all biopsies. These inclusions are reddish or dark green with Gomori trichrome staining and some of them have the appearance of cytoplasmic bodies. The cytoplasmic body-like inclusions stain positively for filamentous actin using fluorescent-labelled phalloidin. Rimmed vacuoles are usually present, and

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staining for oxidative enzymes shows unstained, ‘rubbedout’ regions. Immunohistochemistry can show sarcolemmal NCAM and accumulation of various proteins including desmin, dystrophin, titin, myotilin and αB- crystallin.117

Z-Line Associated Proteins and Four-and-a-Half LIM Domain 1 (FHL1) Disorders with various unusual inclusions have been reported, including spheroid bodies,164 sarcoplasmic bodies,122 cytoplasmic bodies165 and granulofilamentous material.132 Molecularly they are heterogeneous, but several of them share histopathological features, in particular the accumulation of proteins such as desmin. This led to the use of the terms ‘desminopathies’ and ‘desmin-related myopathies’. Accumulation of several proteins that also occurs in IBM is seen, and the terms ‘hereditary inclusion body myopathy’ and ‘protein surplus myopathies’ have been suggested. As the molecular basis of these disorders is gradually unravelled, the term ‘myofibrillar myopathies’ has been adopted for those caused by or associated with defects in Z-line proteins.403 We also include here disorders caused by abnormalities in the FHL1 gene as some cases can have similar pathological features. Plectin is included by some authors, but these cases do not always show rimmed vacuoles, in contrast to myofibrillar myopathies. Mutations in PLEC cause a form of LGMD and muscular dystrophy with epidermolysis bullosa, some with a neuromuscular junction transmission defects (see Recessive Limb-Girdle Muscular Dystrophies, p. 1561; and Myasthenic Syndromes, p. 1596). Many cases of myofibrillar myopathy are sporadic, but inheritance in most (where it can be determined) is autosomal dominant, although rare recessive mutations in the desmin and αB-crystallin genes have also been identified. Disorders associated with FHL1 are X-linked, dominantly inherited.146,339,393,395 Defects have been found in the genes encoding, desmin, αB-crystallin, myotilin, filamin C, ZASP, Bag3 and FHL1 (Table 25.18, Box 22.9).395 There is further heterogeneity because no causative gene mutations have been detected in several patients with the clinicopathological phenotype of a myofibrillar myopathy. Proteins that interact with Z-line proteins or play a role in maintaining myofibrillar integrity are likely candidates. The age at onset in myofibrillar myopathies can be in childhood, adolescence or adulthood; many occur in adulthood.393 Although there is a spectrum of presentation many cases are of adult onset, often late (beyond the fourth decade), particularly in cases with ZASP, myotilin or filamin C defects. Cases with defects associated with the desmin gene tend to present earlier than those with ZASP, myotilin or filamin C defects. Cases with defects in FHL1, Bag3 and αB-crystallin can manifest in childhood.393,395 Muscle weakness is slowly progressive but may be rapid in early onset cases, particularly those with FHL1 or BAG3 defects who can show a severe, rapid progression. Weakness may be proximal or, more frequently, distal or both, or scapuloperoneal, and may be asymmetrical in some cases with FHL1 defects. Muscle weakness may be accompanied by muscle wasting, stiffness or aching, cramps and sensory symptoms. Facial weakness is uncommon, but dysarthria and swallowing difficulties may occur in some older patients. Wasting of hand muscles occurs in cases with mutations in the

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Inherited Muscle Disorders  1591 Box 25.9.  Defective proteins that cause a myofibrillar myopathy

25

Desmin is a highly conserved intermediate filament of skeletal, cardiac and smooth muscle. It localizes to the extramyofibrillar space around the Z-line where it forms a peripheral lattice with plectin and links the myofibrils to the nuclei, mitochondria and sarcolemma. Plectin binds to β-dystroglycan, dystrophin and utrophin, and alternative splicing of plectin transcripts gives rise to eight protein isoforms, of which four are highly expressed in muscle (1, 1b, 1d and 1f). Mutations affecting the 1f isoform have been found in a recessively inherited form of limb-girdle muscular dystrophy. Desmin filaments are 10 nm in diameter, intermediate between actin and microtubules, although originally said to be intermediate between actin and myosin. Identified mutations sometimes impair assembly of desmin filaments. αB-Crystallin is a cytoplasmic small heat-shock protein with a chaperone role in protecting the intermediate filament network from stress-induced damage. Two α-crystallin forms (A and B), encoded by different genes, have been identified; both are abundant in the lens, where they are thought to have a role in preventing the formation of cataracts. Myotilin is a Z-line-associated protein strongly expressed in skeletal muscle but more weakly in cardiac muscle. It binds to α-actinin and filamin C. It also binds actin and plays a role in myofibrillogenesis. Most identified mutations in the MYOT gene occur in the serine-rich N-terminal domain of exon 2. The antibody commonly used and marketed by Leica shows a fibre typing effect, the reason for which is not known. ZASP is a protein of the Z-line that is predominantly expressed in skeletal and cardiac muscle. There are various splice site isoforms, three of which are expressed in skeletal muscle. All isoforms have a N-terminal PDZ domain important for proteinprotein interactions, and a domain important for interaction with α-actinin. Filamin C is part of a family of proteins and its expression is restricted to skeletal and cardiac muscle. It cross-links and stabilizes actin, and binds to several Z-line proteins, and to γ- and α-sarcoglycan at the sarcolemma. Patients with mutations in the actin-binding domain have a distal phenotype but do not show the typical muscle pathology of myofibrillar myopathies. Bcl-2 associated athangene 3 (Bag3) is a co-chaperone protein and is also part of a family of proteins. It is highly expressed in skeletal and cardiac muscle but is also present at low levels in other tissues. It participates in the degradation of misfolded or aggregated proteins by complexing with heat-shock proteins. It has a role not only in muscle but also in neural tissues and tumour cells and various cellular activities. It also has a role in apoptosis, consistent with the finding of apoptotic nuclei in cases with a mutation. Four and a half LIM domains protein 1 (FHL1) is another protein that is part of a family of proteins. Its function is not fully known but it is believed to participate in cell growth, differentiation, sarcomeric assembly and to be a regulator of muscle mass. It has three isoforms and mutations affecting all isoforms result in a more severe phenotype. The severity of the phenotype and presence of reducing bodies in a biopsy correlates with the domain that the mutation affects, with mutations in the LIM domain 2 being associated with the severe phenotype and the presence of reducing bodies in most cases.

actin-binding domain of filamin C, but the cases reported do not show the typical pathology of myofibrillar myopathies, in particular they have no vacuoles.118 Peripheral neuropathy is present in a significant proportion of patients,

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1592  Chapter 25  Diseases of Skeletal Muscle

particularly in cases with BAG3 mutations, some of which show myotonic discharges. Cardiac involvement is common (arrhythmia, or dilated or hypertrophic cardiomyopathy), particularly in those cases with a mutation in the gene encoding desmin, when it may precede or coincide with skeletal muscle weakness; it is also a feature of BAG3 cases with childhood onset. Cardiac involvement occurs in cases with mutations affecting filamin C and FHL1, but is rarer in those with defects in myotilin. Respiratory failure is present in several cases of myofibrillar myopathies, especially those that present early. Cataracts are associated with mutations in the αB-crystallin gene, but may not be present in ­childhood cases.173 Scapular winging is common and other clinical features include rigid spine, scoliosis and contractures.393 The pattern of contractures, muscle weakness and cardiac involvement in some patients with mutations in the FHL1 gene has been assigned to a form of Emery–Dreifuss muscular dystrophy,173,232 but the clinical and pathological features in these cases are similar to cases of myofibrillar myopathy with scapuloperoneal weakness. These cases could therefore be considered as part of the spectrum associated with mutations in FHL1 rather than as a form of Emery–Dreifuss muscular dystrophy, which has defects in nuclear membrane proteins.385 Serum CK is usually normal or mildly elevated, but may be more elevated in Bag3 or some myotilin-related cases. Muscle MRI can also be helpful in differential diagnosis.143 Mutations in the myotilin gene were first associated with a dominant form of limb-girdle muscular dystrophy, LGMD1A, but it is now appreciated that there is a spectrum of phenotypes associated with the gene defect and that this is an allelic disorder to that now known as a myofibrillar myopathy.34 Mutations in the genes encoding myotilin and filamin C are also associated with the presence of spheroid bodies.144,256 There are two founder mutations in ZASP, and mutations have been shown to be responsible for the late-onset distal myopathy described by Markesbery et al. in 1974.171 Mutations in the actin-binding domain of filamin C cause a distal myopathy, but the pathology is not that typically observed in a myofibrillar myopathy and rimmed vacuoles are not a feature.

In addition to non-specific pathological features, such as muscle fibre atrophy and hypertrophy, fibre splitting and excess endomysial connective tissues, abundant internal nuclei and eosinophilic regions that are stained more darkly than surrounding myofibrils with the Gomori trichrome stain are characteristic (see Figure 25.41b). Some inclusions may be stained red with Gomori trichrome and are cytoplasmic bodies, spheroid bodies or reducing bodies. Reducing bodies can be distinguished from cytoplasmic bodies using the menadione NBT method without substrate, which stains reducing bodies and accompanying accumulated protein darkly (Figure 25.90). Reducing bodies are usually only seen in cases with a FHL1 mutation in the LIM domain 2 region, most of which are the severe childhood onset cases, but some myofibrillar material may also appear darkly stained with menadione NBT or show pale staining in other cases of myofibrillar myopathy (see Figure 25.90). Female carriers with FHL1 mutations in this domain may also show reducing bodies but are often clinically milder, possibly with X-inactivation having a role. Reducing bodies are not a feature of milder cases with mutations in other domains, although rare exceptions have been reported.135 The pathology in FHL1-related cases can be focal. The dark areas with the Gomori trichrome stain represent disrupted myofibrillar material and lack staining for oxidative enzymes, contributing to a ‘wiped-out’ or ‘rubbed-out’ appearance (see Figure 25.42). Some dark areas are also congophilic, but they are not metachromatic with the crystal violet stain, in contrast to amyloid deposits in inclusion body myositis. The Congo red stain is best viewed under fluorescence, using an excitation filter in the red range, as for rhodamine or Texas red. Rimmed and unrimmed vacuoles are a feature of myofibrillar myopathies but are not apparent in all cases. A few inflammatory cells may be present, but inflammation is not usually pronounced; necrosis and regeneration may occur, but are not usually extensive. Fibre type grouping and groups of atrophic fibres of both types may be present, consistent with a peripheral neuropathy, and nerves may show loss of myelin and increased fibrosis. Neurogenic features can be particularly apparent in BAG3-related cases. A predominance of type 1 fibres may occur in some cases. (b)

25.90 Menadione NBT staining without substrate in (a) a case of myofibrillar myopathy with a mutation in the MYOT gene and (b) a case with a mutation in the FHL1 gene. Note the weak staining associated with the accumulated myofibrillar material in (a) and strong staining of whole fibres and focal staining corresponding to reducing bodies in (b).

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Abnormal fibres demonstrate the accumulation of several proteins including desmin, αB-crystallin, syncoilin, ubiquitin, myotilin, filamin C, caveolin-3, dystrophin, β-amyloid precursor protein, Xin, filamentous actin, gelsolin, heatshock proteins, NCAM, phosphorylated Tau, TDP43, p62 and prion protein (see Figure 25.54). Accumulation of FHL1 may also be seen. Several of these proteins are also seen in sporadic inclusion body myositis, hereditary forms of ‘inclusion body myopathy’ caused by defects in the GNE gene (UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine kinase) and the VCP-related and other distal myopathies with rimmed vacuoles, which can cause diagnostic difficulties. For diagnosis it is rarely necessary to study a large panel of antibodies (see later). Electron microscopy reveals various degrees of myofibrillar disruption, with Z-line streaming, accumulation of Z-line material, accumulation of granulofilamentous material (Figure 25.91) and inclusions of various types. Tubulofilamentous inclusions occur in myofibrillar as well as other myopathies with rimmed vacuoles. Cytoplasmic bodies with a halo of radiating filaments are common, as are myelin-like whorls and autophagic debris.385,393,395 It is rarely possible to predict the molecular defect of a myofibrillar myopathy from its histopathological features, but some features can help in the differential diagnosis from other myopathies with rimmed vacuoles (see later).

Distal Myopathies Several disorders have predominant involvement of distal muscles and a recent paper by Udd shows a useful flow chart for diagnosis.465 Some of these disorders have already been discussed within the spectra of conditions caused by various genes, such as nebulin (see Congenital Myopathies and Allied Disorders, p. 1580), anoctamin 5 and dysferlin (see Recessive Limb-Girdle Muscular Dystrophies, p. 1561), and titin, myosin and myofibrillar myopathies genes (see Myopathies Caused by Defects in Sarcomeric Proteins, p. 1589). In this section we discuss other disorders with distal involvement, in particular Welander myopathy, myopathy caused by mutations in the gene encoding UDPN-acetylglucosamine-2 epimerase/N-acetylmannosamine kinase (GNE), myopathy caused by the gene encoding the VCP), and the rare distal myopathy with vocal cord and pharyngeal involvement (VCPDM) caused by mutations in the matrin 3 gene. Several of these are sometimes referred to as inherited inclusion body myopathies (h-IBM), because they have rimmed vacuoles and other pathological features that resembles that seen in sporadic forms of IBM (see Inflammatory Myopathies, p. 1605), but they usually lack inflammatory cells. Welander myopathy maps to chromosome 2p13 and the defective gene has recently been shown to be the cytotoxic granule-associated RNA-binding protein, TIA1.180 It is a late-onset disorder in which weakness initially affects the extensor muscles of the fingers and progresses to the lower legs and all muscles of the hand. Muscle biopsies show dystrophic-like features with variation in fibre size and fibrosis. Rimmed vacuoles and nuclear and cytoplasmic inclusions are a feature. Immunohistochemistry shows accumulation of several of the proteins seen in IBM, including p62 and TPD43, but inflammation is absent.

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Inherited Muscle Disorders  1593

Included among ‘hereditary inclusion body myopathies’ is the myopathy known as ‘quadriceps-sparing myopathy’. This is a slowly progressive disorder characterized by early weakness in the distal lower limb muscles that progresses to proximal weakness, but the quadriceps muscles remain relatively strong. Involvement of the upper limbs, and sometimes the distal arm and hand muscles, including the finger flexors (a feature of sporadic IBM), occurs in a few cases. Onset may be in late adolescence or more often in adulthood. It is the same disorder as Nonaka myopathy.223 The condition is recessively inherited and a founder homozygous mutation, originally identified in the Persian Jewish population, is also present in other countries in the Middle East. This condition has also been described in other populations.5,41 The number of autophagic rimmed vacuoles is variable, but congophilia is reported to be absent, in contrast to myofibrillar myopathies.41 Additional features include variation in fibre size, with fibre hypertrophy and scattered angular atrophic fibres, nuclear clumps, internal nuclei, necrosis and an increase in connective and adipose tissues. With electron microscopy, collections of cytoplasmic 15to 21-nm tubulofilamentous structures are found, similar to those seen in sporadic IBM. The protein product of the GNE gene is a key enzyme involved in the synthesis of sialic acid. Reduced sialylation of NCAM has been shown on immunoblots, but it is not yet clear if this is the pathogenic mechanism in the disease. Studies of α-dystroglycan in a single study suggested a reduction in glycosylation, but this is not a consistent finding.41 A multisystem dominantly inherited disorder with distal involvement and rimmed vacuoles that also presents with Paget’s disease and frontotemporal dementia is well recognized in some patients (e.g. inclusion body myopathy with early-onset Paget disease and frontotemporal dementia [IBMPFD]). It is caused by missense mutations of the gene encoding p97/VCP.493 Immunolabelling of VCP and TDP43 is useful and shows positive inclusions that can be sarcoplasmic and intranuclear in IBMPFD patients. The VCP inclusions can be present in vacuolated and non-vacuolated fibres and co-localize with ubiquitin.493

25

25.91 Electron micrograph of granulofilamentous material at the periphery of a fibre in a case with a mutation in the gene encoding desmin.

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1594  Chapter 25  Diseases of Skeletal Muscle

Rimmed vacuoles in association with distal weakness are also a feature in muscle biopsies from patients with a late-onset dominantly inherited distal myopathy with vocal cord and pharyngeal involvement (VCPDM). This was shown to be caused by defects in the gene encoding matrin 3 in the only two families described.397 In addition, rimmed vacuoles are also seen in patients with either a dominant or recessively inherited disorder known as oculopharyngodistal myopathy that is molecularly distinct from oculopharyngeal muscular dystrophy (see section on Disorders with Deletions or Expansion of Repeat Sequence, p. 1577).120,451 Differential diagnosis in hereditary disorders with rimmed vacuoles and IBM is complicated by overlapping clinical and pathological features, but some pathological aspects can be useful in differential diagnosis. In myofibrillar myopathies ‘rubbed-out’ or ‘wiped-out’ fibres are reported to be a consistent feature of cases with mutations in the genes encoding desmin and αB-crystallin, whereas cases related to ZASP and myotilin are reported to show more vacuoles.80 The large accumulations of proteins and the dark staining material seen with Gomori trichrome are features of myofibrillar myopathies not seen in IBM, and MHC-1 upregulation is less in myofibrillar myopathies compared with IBM. Inclusion body myositis and myofibrillar myopathies show congophilic amyloid deposits, but these are not a consistent feature of other myopathies with vacuoles; however, they have been observed in cases with mutations in VCP.430 Electron microscopy studies have revealed subtle differences in relation to the genotypes of myofibrillar myopathies.78 The granulofilamentous material is a feature of myofibrillar myopathies, but the tubulofilamentous inclusions can occur in several myopathies with vacuoles. As with all neuromuscular disorders, clinical correlations are essential and muscle MRI is having an increasing role.143 The most useful panel of techniques for studying these disorders is H&E, Gomori trichrome, oxidative enzymes, Congo red, desmin, myotilin, αB-crystallin, dystrophin, TDP-43, VCP supplemented by menadione NBT and FHL1 (if reducing bodies are thought to be present) and electron microscopy. In HMERF, identification of F-actin with phalloidin in the cytoplasmic-like bodies can be useful. Kelch proteins are a large family of proteins with various functions that include protein binding and transcriptional activation. A mutation in the gene encoding Kelch 9 was identified in a large family with an autosomal dominant distal myopathy. Onset was in childhood or early adulthood and muscle pathology showed atrophy and hypertrophy of fibres, increased internal nuclei, fat and connective tissue, and uniform fibre typing.77 Mutations in Kelch 13 (KBTBD13) are associated with a rare form of autosomal dominant nemaline myopathy (see section on Congenital Myopathies and Allied Disorders, p. 1580). Next-generation sequencing is identifying other members of this large family that may be responsible for a neuromuscular disorder (see Nemaline Myopathies, p. 1580).

the sarcolemma, sarcoplasmic reticulum and T-tubules. Ion channels are complex multidomain transmembrane proteins, and numerous mutations in their genes, disrupting ion movement, have been identified (Table 25.19). These mutations result in disturbed excitability, in the form of either hyperexcitability (myotonia) or inexcitability (periodic paralysis); thus, these conditions are collectively referred to as ‘ion channelopathies’. There is significant clinical overlap between the channelopathies, and defects in the same gene can give rise to different phenotypes (see Table 25.19) and fall into two main groups: those with myotonia or those with periodic paralysis.218,350 Defects in the calcium-release channel, the ryanodine receptor 1 of the sarcoplasmic reticulum, cause malignant hyperthermia and core myopathies, whereas Brody disease is caused by disturbances in a calcium pump of the sarcoplasmic reticulum. A number of channelopathies affecting specifically the heart or the CNS are also recognized, such as long QT syndrome and episodic ataxia, respectively. The diagnosis of ion channelopathies is usually based on clinical assessment and detailed electrophysiological studies. Muscle biopsy does not often contribute to the process. Pathological features include variation in fibre size, increase in internal nuclei, and vacuoles or tubular aggregates (Figure 25.92). A genetic cause of tubular aggregate myopathy has recently been shown to be mutations in the calcium sensor stromal interaction molecule 1 (STIM1).32 The vacuolation is thought to appear in a sequence that starts with focal proliferation and dilation of the sarcoplasmic reticulum and T-tubules and ends with large membrane-bound vacuoles. The dilated sarcoplasmic reticulum may contain amorphous granular material, cell debris and myelin-like whorls and stain for NADH-TR and acid phosphatase. Immunolabelling shows the presence of dystrophin and β-spectrin, but not laminin. Glycogen deposits, focal reduction of mitochondria, myofibrillar disruption and Z-line streaming have also been reported.117 Tubular aggregates are derived from the sarcoplasmic reticulum or endoplasmic reticulum exit sites and are often restricted to type 2 fibres.72,386 They stain red with Gomori trichrome, are basophilic with H&E, and show reactivity for NADH-TR, myoadenylate deaminase, non-specific esterase and phosphofructokinase, but not SDH or ATPase activity (see Figure 25.92). Immunohistochemistry shows several proteins associated with them, including SERCAs, heat-shock proteins, triadin, calsequestrin, dysferlin and emerin (in one case) but not mitochondrial proteins.72 With the electron microscope, the aggregates appear as a honeycomb of tubules, and different types have been observed.117 Tubular aggregates are not specific for ion channel disorders and they are a feature of some inherited myasthenic disorders (see later), as well as a variety of other conditions.156

Ion Channel Disorders

Malignant hyperthermia is a severe complication of general anaesthesia and is characterized by a rapid and sustained rise in temperature following the intravenous administration of anaesthetic. It is accompanied by generalized muscle rigidity, tachycardia, tachypnoea and cyanosis. Extensive

Generation of action potentials and contraction of myofibrils require the movement of ions, in particular sodium, potassium, chloride and calcium ions, through channels in

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Malignant Hyperthermia

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Inherited Muscle Disorders  1595

Table 25.19  Ion channel disorders of skeletal muscle Clinical syndrome

Type of ion channel

Gene

Locus

Inheritance

Myotonia congenita (Becker)

Chloride channel

CLCN1

7q35

Recessive

Myotonia congenita (Thomsen)

Chloride channel

CLCN1

7q35

Dominant

Potassium-aggravated myotonia

Sodium channel

SCN4A

17q23

Dominant

Paramyotonia congenita

Sodium channel

SCN4A

17q23

Dominant

Hyperkalaemic periodic paralysis

Sodium channel

SCN4A

17q23

Dominant

Normokalaemic periodic paralysis

Sodium channel

SCN4A

17q23

Dominant

Hypokalaemic periodic paralysis

Calcium channel Sodium channel

CACNA1S SCN4A

1q32 17q23

Dominant Dominant

Hyperkalaemic or hypokalaemic periodic paralysis (Andersen’s syndrome)

Potassium channel

KCNJ2

17q

Dominant

Thyrotoxic periodic paralysis

Potassium channel

KCNJ18

17p11

Unknown/ sporadic

Congenital myasthenic syndrome

Sodium channel

SCN4A

17q23

Recessive

Malignant hyperthermia

Calcium channel ­Calcium  channel

RYR1 CACNA1S

19q13 1q32

Dominant Dominant

Tubular aggregate ­myopathy

Influences calcium release-activated ­calcium  channels

STIM1

11p15

Dominant

muscle necrosis follows, with subsequent myoglobinuria and possible renal shutdown. Serum potassium is elevated and CK is grossly elevated. Many general anaesthetic agents can trigger the reaction, including those containing halogenated hydrocarbons such as halothane, and succinylcholine. Individuals ‘at risk’ can be diagnosed using the in vitro contracture test (IVCT), which measures muscle tension in vitro when the sample is exposed to halothane or caffeine.13 Malignant hyperthermia is inherited as an autosomal dominant trait. Several loci have been linked to susceptibility to malignant hyperthermia, and two genes have been identified – RYR1 and CACNA1S – that encode the calcium-release channel in terminal cisternae of the sarcoplasmic reticulum (ryanodine receptor) and the α-subunit of the voltage-gated DHPR, respectively.13 Because mutations in RYR1 are also responsible for a core myopathy, all patients with a RYR1 defect are considered at risk for malignant hyperthermia. Linkage to chromosome 17q in several malignant hyperthermia-susceptible families suggests the sodium channel gene, SCN4A, as a possible candidate gene for malignant hyperthermia. The muscle pathology associated with malignant hyperthermia is mild and non-specific. Rhabdomyolysis, with marked fibre necrosis and regeneration, is seen immediately after an episode of malignant hyperthermia, but samples taken at other times may show only minor changes, such as

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25

scattered smaller fibres and fibres with central nuclei. Thus, routine evaluation of muscle is not of value in diagnosing the disorder or in predicting susceptibility to malignant hyperthermia.

Brody Disease Calcium ions are removed from the cytosol after muscle contraction by the combined action of Ca2+ ATPases in the sarcoplasmic reticulum (SERCA), cell membrane and mitochondrion. In mammals, type 2 fibres express the SERCA1 isoform encoded by the ATP2A1 gene on chromosome 16p12, whereas type 1 muscle fibres express the SERCA2 isoform encoded by the ATP2A2 gene on chromosome 12q23. Recessive mutations in the ATP2A1 gene are responsible for Brody disease, a disorder characterized by painless cramps and impairment of muscle relaxation.481 Muscle contraction is normal, but the relaxation phase becomes increasingly slow during exercise. An absence of SERCA1 protein from fast fibres has been detected using isoform-specific antibodies in some, but not all, patients with an ATP2A1 mutation. This was associated with total loss of enzymatic activity. Other studies have demonstrated reduced ATPase activity and expression of a non-functional protein. Therefore, the presence of SERCA1-positive fibres does not exclude Brody disease.481

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1596  Chapter 25  Diseases of Skeletal Muscle (a)

(b)

25.92 Serial sections of tubular aggregates that are (a) basophilic with H&E, (b) red with Gomori trichrome, (c) intensely stained in type 2 fibres with NADH-TR but (d) negative for cytochrome c oxidase activity. There is also loss of oxidative enzyme activity from the centre of several fibres in this case.

Myasthenic Syndromes Defects of neuromuscular junction transmission are broadly categorized on the basis of their aetiology – genetic or acquired. Myasthenia gravis is an acquired autoimmune disorder in which autoantibodies against various antigens of the neuromuscular junction can be demonstrated, in particular to the AChR and to MuSK, a tyrosine kinase receptor. Both of these receptors are on the postsynaptic membrane of the neuromuscular junction.320 Recently, autoantibodies to low density lipoprotein receptor-related protein-4 (LRPP4) have also been identified in some patients with myasthenia gravis.334 Antibodies to voltage-gated calcium channels on the presynaptic membrane cause Lambert– Eaton syndrome, and neuromyotonia results from antibodies to a presynaptic voltage-gated potassium channel. In addition, antibodies to various muscle proteins can be detected, including to myosin, actin, α-actinin, titin, filamin, vinculin, tropomyosin and the ryanodine receptor.134,241 Neonates of mothers with autoantibodies may show transient myasthenia (see later). There are also reports of cases with an autoimmune disorder of caveolin-3 that also have antibodies to AChR and myasthenia gravis.390 These cases

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show a mosaic pattern of immunolabelling of muscle fibres with antibodies to caveolin-3. Inherited variants of myasthenia are generally congenital disorders that result from mutations in genes encoding various critical presynaptic or postsynaptic proteins (Table 25.20).129 Abnormal fatigability is the characteristic symptom of patients affected by most variants of myasthenia. Muscle weakness and fatigability are generalized in myasthenia gravis, with weakness of the ocular muscles and ptosis being the most common presenting symptoms. There is a high incidence of miscarriages in females with myasthenia gravis. Transient neonatal myasthenia affects about one in seven infants born to myasthenic mothers and may produce life-threatening weakness requiring urgent treatment. It results from maternal antibodies to the embryonic acetylcholine receptor.359 The infant is usually affected at birth with general hypotonia and weakness, but symptoms may sometimes be delayed. Arthrogyposis is a severe complication in some cases. The condition is self-limiting with gradual recovery of the infant, usually within 2–4 weeks. Confirmation of myasthenia is usually provided by pharmacological tests, such as the response to intravenous

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Inherited Muscle Disorders  1597

TABLE 25.20  Congenital myasthenic syndromes and known gene defects Location of defect

Protein

Gene

Locus

Inheritance

ChAT

CHAT

10q11.2

AR

25

Presynaptic Defects in ACh resynthesis Paucity in synaptic vesicles

?

Lambert–Eaton-like congenital myasthenia

?

Synaptic End plate AChE deficiency

Collagen tail of AChE

COLQ

3p24.2

AR

β2 Laminin deficiency

Laminin β2 chain

LAMB2

3p21

AR

ACh receptor α subunit

CHRNA1

2q24–q32

AR

ACh receptor δ subunit

CHRND

2q33–q34

AR

ACh receptor ε subunit

CHRNE

17p13

AR

ACh receptor α subunit

CHRNA1

2q24–q32

AD

ACh receptor β subunit

CHRNB1

17p11–p12

AD

ACh receptor δ subunit

CHRND

2q33–q34

AD

ACh receptor ε subunit

CHRNE

17p13

AD, AR

ACh receptor β subunit

CHRNB1

17p11–p12

AR

ACh receptor δ subunit

CHRND

2q33-q34

AR

ACh receptor ε subunit

CHRNE

17p13

AR

Rapsyn

RAPSYN

1p11

AR

MuSK

MUSK

9q31.3–q32

AR

Abnormalities of ­cytoskeleton

Plectin

PLEC

8q24–qter

AR

Anomaly of muscle sodium channel

Sodium channel α-subunit

SCN4A

17q23

AR

Docking protein 7

DOK7

4p16.2

AR

Glutamine-fructose 6-phosphate transaminase 1

GFPT1

2p12–p15

AR

Dolichyl-phosphate

DPAGT1

11q23.3

AR

α-1,3/1,6-mannosyltransfer­ase homologue of yeast ALG2

ALG2

9q 22.33

AR

UPD-N-acetyl glucosamine transferase subunit ALG14 homologue

ALG14

1p21.3

AR

Postsynaptic Fast channel syndromes

Slow channel syndromes

ACh deficiency

Abnormalities in clustering of ACh receptors

Defects in end plate development and maintenance

N-acetylglucosamine phosphotransferase

ACh, acetylcholine; AChE, acetylcholinesterase; AD, autosomal dominant; AR, autosomal recessive; ChAT, choline acetyltransferase.

edrophonium (an acetylcholine esterase inhibitor), or a trial of oral pyridostigmine, or by electrophysiological studies, such as the response decrement of the motor action potential to repetitive stimulation of a nerve, or more specifically by the presence of ‘jitter’ on single-fibre electromyography, or by the finding of serum antibodies

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to acetylcholine receptors or MuSK. Electrophysiology and a response to therapy, such as pyridostigmine, have revealed an associated myasthenic syndrome in some patients with mutations in genes not known to be associated with neuromuscular junction proteins, including TPM3, BIN1 and DNM2.79,251,305 In addition, patients

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1598  Chapter 25  Diseases of Skeletal Muscle

with myasthenic symptoms and muscle biopsies showing central nuclei, one of which had a mutation in the MTM1 gene and others that have not been resolved molecularly, have been reported.361 Muscle biopsy has a limited role in the diagnosis of myasthenic syndromes, and pathological features, if present, are non-specific. In our experience a myasthenic or metabolic condition should be considered if a patient is clinically severely affected and the muscle pathology is minimal. The pathological features may include variation in fibre size, collections of lymphocytes, type 2 fibre predominance and core-like areas.230 In some congenital cases (molecularly unresolved), selective atrophy of type 1 fibres resembling fibre type disproportion was reported.176 Some cases may show pronounced fibre hypertrophy.117 Detailed studies of nerve terminals and the neuromuscular junction in autoimmune myasthenia show abnormal binding of complement (C3, C9 and the membrane attack complex), immune complexes, and the autoantibodies derived from the serum of affected patients. A particular and common feature of muscle biopsies from patients with congenital myasthenia caused by defects in glutamine-fructose-6-phosphate transaminase (GFPT1) and dolichyl-phosphate N-acetylglucosamine phosphotransferase (DPAGT1) and also in some cases with ALG2 mutations is tubular aggregates.174,199,399 These genes, together with ALG14, emphasize the importance of N-glycosylation at the neuromuscular junction.96 Tubular aggregates are not usually a feature associated with defects in the other genes that cause myasthenia, for example they are absent in patients with mutations in DOK7. The involvement of the neuromuscular junction in patients with epidermolysis bullosa simplex caused by mutations in the gene encoding plectin (PLEC) has been noted over the years.15,145 A detailed study of the muscle pathology in two additional rare cases showed a variety of pathological changes.396 These included variation in fibre size, an increase in internal nuclei, large clusters of peripheral nuclei, fibrosis, necrosis and regenerating fibres, uneven distribution of oxidative enzyme stains, fibre type predominance, an increase in calcium demonstrated with alizarin red, and multiple endplates spread over a wide area. Electron microscopy revealed disruption of myofibrils, occasional nemaline rods, aggregates of mitochondria and degeneration of neuromuscular junctions. Immunolabelling of plectin with antibodies to two different domains showed a reduction in labelling from sarcoplasmic areas and a slight reduction in sarcolemmal labelling. Although both patients reported in this paper also had epidermolysis bullosa simplex with typical skin changes, not all patients with mutations in the PLEC gene show clinical features evocative of neuromuscular junction involvement, despite the high concentration of plectin at neuromuscular junctions.175

Metabolic Myopathies There are many disorders of glycogen and lipid metabolism and of mitochondrial function, but histopathological assessment of muscle is helpful in only a few.25 In general, defects of ‘substrate utilization’, whether of glycogen or fatty acids, result in two main clinical syndromes. One is associated with acute, recurrent episodes of muscle pain,

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exercise intolerance, cramps and myoglobinuria, and the second with permanent muscle weakness. The diagnosis of these conditions relies on specialized biochemical techniques and confirmatory genetic testing. This section therefore concentrates on selected conditions where pathological studies are helpful in suggesting a metabolic problem. Some lysosomal diseases of muscle, such as Danon disease and the vacuolar myopathy XMEA, may have features similar to glycogenosis and are included in this section. For a description of metabolic conditions affecting muscle and their biochemical diagnosis, the reader is referred to various reviews.112,113,248,360

Disorders of Glycogen Metabolism and Glycolysis Disorders due to enzymes involved in the synthesis and degradation of glycogen and in some of the steps of glycolysis are referred to as ‘glycogenoses’. The numerical classification of these disorders suggested by Cori has found wide acceptance.95 The inheritance of glycogenoses is autosomal recessive, except for type IX (phosphoglycerate mutase deficiency) and the hepatic form of type VII (phosphorylase b kinase deficiency), which are X-linked. The following sections highlight aspects of selected glycogenoses.

Muscle Glycogen Depletion Defects in enzymes responsible for glycogen synthesis lead to a pronounced depletion of glycogen in skeletal muscle, and the disorders described also show cardiac involvement. Defects in glycogen synthase encoded by GSY1 have been reported in a few rare cases and designated ‘muscle glycogen storage disease 0’.235 Defects in the gene encoding glycogenin (GYG1) have also been identified (type XV).303

Type II Glycogenosis (Acid Maltase Deficiency) Acid maltase (acid α-glucosidase) is a lysosomal enzyme that degrades glycogen by hydrolysing its 1,4-links. Absence of this enzyme results in glycogen accumulation in membranebound areas of lysosomal origin in several tissues, but mainly muscle. Three clinical types can be distinguished: a severe infantile form (Pompe disease), a juvenile form and a form with adult onset, although ‘Pompe’ is often now used for all forms.117,278 The severe form (Pompe disease) is usually fatal in infancy and affects both cardiac and skeletal muscles. Affected infants are usually floppy from birth or during the first few months of life, and may clinically resemble patients with infantile SMA. Pompe disease can be distinguished from SMA by diaphragmatic and cardiac involvement and a marked elevation of serum CK. Children affected by the juvenile form have no cardiomyopathy, but have predominantly axial and proximal muscle weakness, and their symptoms may resemble those of a rigid spine syndrome. The prognosis of juvenile patients, and of adult patients presenting with limb girdle weakness, is dependent on the management of the respiratory insufficiency. These patients have often been given a clinical diagnosis of LGMD.

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Inherited Muscle Disorders  1599

(a)

25.93 Pronounced vacuolation in a severe infantile case of Pompe disease. The spaces contain accumulation of glycogen. H&E.

Muscle fibres in Pompe disease have a pronounced vacuolar appearance, with large PAS-positive deposits of ­glycogen (Figure 25.93). The glycogen is digested by diastase, but some resistant material may remain. Ultrastructurally, the glycogen is characteristically located in membranebound areas and in large lakes of freely dispersed granules. Glycogen is lost easily during processing, and the excess may not always be apparent. Accumulation of glycogen in vacuoles can be demonstrated easily in lymphocytes, and this is a useful diagnostic test. A dried blot spot test for testing enzyme activity is also available. Because the enzyme is lysosomal, there is also abundant acid phosphatase activity in the vacuoles. Granular bodies positive for acid phosphatase that also reduce menadione NBT (similar to reducing bodies) may also be a feature.410 The muscle pathology in milder cases is variable, but increased glycogen and acid phosphatase are usually apparent. The vacuolation may be extensive or minimal, or have a punctate appearance, or it may be confined mainly to type 1 fibres (Figure 25.94). With immunohistochemistry, some vacuoles may be surrounded by dystrophin and spectrin, and sometimes also laminins, but this is not a universal feature and not as prominent as in Danon disease or XMEA, in which many vacuoles are associated with both plasmalemmal and basal lamina proteins (see later). There may also be abundant MHC-I labelling associated with the vacuoles and on the sarcolemma in acid maltase deficiency.117 In some cases, vacuoles can be absent, and in this situation distinction from LGMD is important.

Type V Glycogenosis (McArdle Disease) The muscle isoform of phosphorylase (myophosphorylase), which is encoded on chromosome 11, is defective in this recessive disorder.347 The defect results in absence of enzyme activity, and in most cases this is the result of an absence of protein. There are two common hotspot mutations in northern European populations (R50X and G205s). Two other isoforms of phosphorylase, encoded by

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25

(b)

25.94 (a) Vacuoles in some fibres in an adult case of acid maltase deficiency that contain (b) acid phosphatase.

different genes on chromosomes 20 and 14, are expressed predominantly in the brain and liver, respectively. Fetal muscle, regenerating fibres and myotubes in vitro express the brain isoform, and this can be detected in the immature fibres in biopsies (Figure 25.95). Similarly, intrafusal fibres in muscle spindles and the smooth muscle of blood vessels express the brain isoform. Patients with McArdle disease present with cramps on exertion. Weakness and muscle pain occur, and there may be transient myoglobinuria. There is a high incidence of chronic fatigue/pain, depression and anxiety, and a ‘second wind’ phenomenon (in which exercise can be endured better after a short pause) is usually present.347 Muscle biopsies in type V glycogenosis may show relatively few abnormalities on light microscopy. There may be some degenerating, regenerating and necrotic fibres, but the most consistent finding is the presence of subsarcolemmal ‘blebs’, which contain PAS-positive glycogen and resemble vacuoles but are not membrane bound (Figure 25.96). The excess glycogen may be more apparent at the ultrastructural level. Sometimes glycogen accumulates between the plasma membrane and the basal lamina.117

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1600  Chapter 25  Diseases of Skeletal Muscle (a)

(a)

(b)

(c)

25.96 (a) Peripheral vacuolar-like ‘blebs’ that (b) contain glycogen in a case of McArdle disease. (a), H&E; (b), PAS.

except in cases with defects in glycogen synthase (see earlier), because endogenous glycogen is required for demonstration of the enzyme in sections. The reaction fades in aqueous mountants but is retained following dehydration and mounting in synthetic mountants, such as di-n-butylphthalate-polystyrene-xylene (DPX).

Type VII Glycogenosis (Tarui Disease)

25.95 (a) Normal staining of phosphorylase in a control compared with (b) an absence from all mature fibres in a case of McArdle disease but staining of regenerating fibres that (c) express fetal myosin (arrows) because of the presence of the brain isoform encoded by a different gene.

The absence of phosphorylase can be demonstrated readily by histochemical reaction, but this must always be performed with a positive control (see Figure 25.95). The results are unequivocal, and McArdle disease is the only disorder to show a complete absence of enzyme activity,

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This disease is caused by a deficiency of phosphofructokinase, which catalyses the conversion of fructose-6-phosphate to fructose-1,6-diphosphate. Clinical features are similar to those of McArdle disease, but a haemolytic anaemia may also occur in phosphofructokinase deficiency.311,455 There is a particular prevalence in Japanese and Jewish Ashkenazi populations.349 Severe infantile and adult cases have also been described. Infants may show CNS involvement, and the distinction from CMD variants with associated brain involvement or mitochondrial diseases is important. Muscle biopsies show non-specific changes on light microscopy and excess glycogen at the ultrastructural level, although this may not be pronounced. Diastase-resistant polyglucosan deposits positive for PAS may also be present,

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Inherited Muscle Disorders  1601



which accumulate with age.262 Absence of phosphofructokinase activity can be detected histochemically but is better determined biochemically.

Glycogenoses with Polyglucosan Bodies Additional disorders caused by defects of enzymes associated with glycogen metabolism have also been reported and most of them are associated with exercise intolerance, but not always with glycogen storage.112 Mutations in the branching enzyme encoded by the GBE1 gene cause type IV glycogenosis and result in the accumulation of abnormal glycogen, commonly referred to as polyglucosan. This is in contrast to defects in type III glycogenosis caused by defects in the debranching enzyme in which glycogen appears normal.117 Polyglucosan bodies are often diastase-resistant PASpositive bodies, and are the typical pathological hallmark associated with GBE1 mutations, but some cases do not show them and not all are PAS-positive. Polyglucosan bodies are not specific for type IV glycogenosis and can also occur in phosphofructokinase deficiency, Lafora disease and an adult disease characterized by neurogenic involvement.56,97

Lysosomal Glycogen Storage with Normal Acid Maltase Danon Disease Danon disease is a vacuolar myopathy with normal acid maltase levels.433 The gene responsible has been mapped to Xq24 and encodes the lysosomal-associated membrane 2 protein (LAMP-2). Not all cases show glycogen storage, and the disorder is sometimes referred to as ‘X-linked vacuolar myopathy’.263 Onset is in childhood and is characterized by severe hypertrophic cardiomyopathy, a mild and relatively stable myopathy, and variable mental retardation. Creatine kinase levels are elevated, even in preclinical cases. Muscle weakness and atrophy affect the shoulder and neck muscles, but there may also be distal involvement. Female carriers may also manifest.433

(a)

In addition to abnormal variation in the size of both fibre types, the striking feature is the presence of numerous vacuoles containing glycogen. Invaginations of the sarcolemma are also common. The vacuoles are lined by a membrane that labels with antibodies to dystrophin, β-spectrin, laminin chains and other sarcolemmal proteins (Figure 25.97). The membrane and content of the vacuoles are labelled with some lectins, such as wheat germ agglutinin, Ulex europaeus I agglutinin (UEA-1) and Limas flavus agglutinin. This has been suggested as a possible way to distinguish the vacuoles from those seen in acid maltase deficiency, which show little or no labelling with lectins.468 With the electron microscope, a basal lamina is seen on the inner surface of some vacuoles, which contain abundant amounts of granular, osmiophilic debris. Immunohistochemistry and immunoblots show a virtual absence of LAMP-2, indicating that this is useful in the assessment of muscle biopsies.263,468

25

X-Linked Myopathy with Excess Autophagy The pathology in Danon disease is remarkably similar to that seen in XMEA, in which sarcolemmal proteins are also found on vacuoles but they are clinically distinct.264 No cardiac involvement or mental retardation has been reported in XMEA. Onset is typically in adults, although a more severe and probably allelic variant with onset in childhood has been reported. Calcium deposits can be detected in subsarcolemmal areas, and complement C5b–9 (membrane attack complex) and acetylcholinesterase can be demonstrated on the sarcolemma in XMEA and may help to distinguish it from Danon disease. Duplication of the basal lamina is abundant and debris seen between the layers.264 The genetic defect that causes XMEA is a defect in the VMA21 gene that encodes a V-ATPase involved in lysosomal function.352 Some similar pathological features to those described in XMEA can occur in other conditions associated with autophagy, such as Vici syndrome and in molecularly unresolved cases.99

(b)

25.97 A case resembling X-linked myopathy with excessive autophagic vacuoles (XMEA) with vacuoles that are immunolabelled with antibodies to (a) β-spectrin and (b) laminin γ1. Indentations of the sarcolemma can also be seen.

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1602  Chapter 25  Diseases of Skeletal Muscle

Disorders of Fatty Acid Metabolism The oxidation of fatty acids is a major energy source and occurs in mitochondria. A heterogeneous group of disorders is caused by defects in the β-oxidation metabolic pathway. These disorders fall into two broad groups: those in which muscle symptoms are the main complaint, and those in which muscle involvement is part of a systemic condition. In the former, presenting symptoms and signs may be proximal or diffuse muscle weakness, or muscle pain, particularly following prolonged exertion, which may be associated with muscle necrosis and myoglobinuria. The predominant muscle symptoms in children, in whom muscle involvement is part of a systemic illness, are hypotonia and generalized muscle weakness. There may be cardiac involvement and global delay. The two types of carnitine palmitoyltransferase M deficiency (CPTI, CPTII) are relatively common and characterized by recurrent myoglobinuria, which can be precipitated by prolonged exercise, fasting and intercurrent illnesses. Carnitine deficiency is characterized by mild weakness, hypotonia and life-threatening cardiomyopathy. The contribution of muscle pathology to the investigation of these disorders has diminished with advances in molecular analysis and biochemical assays. In patients with carnitine deficiency, excess lipid storage gives a striking vacuolar-like appearance corresponding to the non-membranebound lipid droplets, and may be associated with structural abnormalities in mitochondria. In patients with CPT deficiencies, however, the amount of lipid may not be increased, and the overall pathology is minimal or absent. Similarly, in other disorders of fatty acid metabolism, including β-oxidation defects, only mild non-specific abnormalities, or no abnormalities, may be seen, and any increase in lipid is difficult to quantify. If a muscle biopsy is taken soon after an episode of myoglobinuria in CPT deficiency, necrotic or regenerating fibres may be present.238

Mitochondrial Myopathies The mitochondrial myopathies are a heterogeneous group of multisystem disorders in which abnormalities in mitochondrial function may or may not be associated with structural abnormalities in the mitochondria. Overlapping clinical features are present in the various mitochondrial disorders, and further complexity arises because the same molecular abnormality may produce divergent clinical features. Particular neuromuscular features that suggest a mitochondrial disease are exercise intolerance, fatigue, rhabdomyolysis, ptosis, ophthalmoplegia and a raised serum lactate. Neonatal and infantile hypotonia are well-recognized presenting features.336 Involvement of tissues other than skeletal muscle is common, with clinical and radiological features of CNS involvement, cardiac involvement, retinal degeneration, hearing loss, endocrine disorders, and liver, gastrointestinal and peripheral nerve involvement. Mitochondria have a pivotal role in the final common pathway for aerobic metabolism–oxidative phosphorylation (OXPHOS), composed of five complexes. Components of these complexes are under the control of both nuclear (nDNA) and mitochondrial (mtDNA) genomes. The nuclear genome encodes most of the subunits of the enzyme complexes, assembly proteins and many of the factors necessary

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for mtDNA replication, transcription and translation, and defects show Mendelian inheritance. The mitochondrial genome, however, encodes 13 subunits of the OXPHOS system, ribosomal RNA (rRNA) and transfer RNA (tRNA) involved in the mitochondrial protein synthesis, and defects are maternally inherited and influenced by heteroplasmy, threshold effect and mitotic segregation (Box 25.10). For biochemical studies, muscle tissue must be snap frozen in liquid nitrogen within 30 minutes of performing the biopsy to avoid loss of enzyme activity. Isopentane interferes with the measurement of complexes I, II and III, but copy number can be studied in samples frozen in isopentane. Cultured skin fibroblasts are used for β-oxidation studies. Muscle pathology is variable and can range from striking abnormalities, typical of a mitochondrial disease, to non-specific or minimal. Adults with mitochondrial disease are more likely to have an abnormal muscle biopsy than children. The absence of pathology does not exclude a mitochondrial problem. In addition to routine histological stains, enzyme histochemistry for NADH-TR (complex I),

Box 25.10.  Pathobiology and genetics of mitochondrial disease Oxidative phosphorylation (OXPHOS) is carried out by the mitochondrial respiratory chain comprising five multisubunit enzyme complexes located on the inner mitochondrial membrane and the process generates adenosine triphosphate (ATP). The pathway is under dual control of both nuclear (nDNA) and mitochondrial (mtDNA) genomes. The nuclear genome encodes most of the subunits of the enzyme complexes, assembly proteins and many of the factors necessary for mtDNA replication, transcription and translation. The mitochondrial genome encodes 13 subunits of the OXPHOS system, as well as the ribosomal RNA (rRNA) and transfer RNA (tRNA) involved in the mitochondrial protein synthesis. Transmission of mitochondrial diseases is therefore governed both by Mendelian genetics and by mitochondrial genetics, the latter of which is influenced by heteroplasmy, threshold effect, mitotic segregation, and maternal inheritance. Nuclear defects are a major cause of mitochondrial disease as the majority of the mitochondrial proteins, including some respiratory chain subunits, are nuclear encoded. They can affect (i) synthesis of structural or assembly proteins of the respiratory chain; (ii) other components of the mitochondrial respiratory chain such as coenzyme Q10; (iii) intergenomic signalling impairing replication, maintenance or translation of mtDNA leading to qualitative (multiple deletions) or quantitative (depletion) alterations in mtDNA that accumulate during life; (iv) synthesis of inner mitochondrial membrane phospholipids; and (v) mitochondrial motility and fission (mitodynamics). Human mtDNA is a small16.5 kb circular double-stranded DNA molecule of which there are many thousand copies within each nucleated cell. It contains 37 genes: 13 structural genes encoding essential mitochondrial respiratory chain subunits, 2 rRNA genes and 22 tRNA genes. With rare exceptions, all surviving mtDNA is derived from the mother’s ovum at conception. mtDNA mutations are therefore inherited maternally. mtDNA mutations may affect only a proportion of mitochondria, leading to a mixture of mutant and wild-type mtDNA within a cell (heteroplasmy). The proportion of mutant mtDNA must exceed a critical threshold before a biochemical defect manifests. This threshold varies between tissues and partly explains the tissue variability in mitochondrial disease. A site-map for the mitochondrial mutation databases maintained by the Human Genome Variation Society can be accessed at www.hgvs.org/ dblist/mito.html.

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Inherited Muscle Disorders  1603



SDH (complex II), COX (complex IV), and the combined COX/SDH technique are the most useful techniques (see Figure 25.32). There are no reliable histochemical techniques to demonstrate complexes III and V. The role of immunohistochemistry is limited to demonstrating the presence of the enzymes and is of less diagnostic use than assay of enzyme activity. Pathological features include fibre atrophy, but little or no hypertrophy, an increase in internal nuclei, and necrosis and fibre regeneration in cases with myoglobinuria. Fibre type differentiation is rarely abnormal, although accumulation of abnormal mitochondria may occur more often in type 1 fibres. In conditions with associated peripheral nerve involvement, there may be fibre type grouping. Mitochondrial proliferations are an important diagnostic clue, regardless of the molecular defect. Extreme mitochondrial proliferation gives rise to ‘ragged-red fibres’ (RRF) that show enhanced red staining with the Gomori trichrome technique and have a granular basophilic appearance with H&E (see Figure 25.32). Staining is often pronounced at the periphery of fibres and also disrupted internally. RRFs have a high proportion of mutant mitochondrial genomes that is segmental. RRFs react intensely for SDH and NADH-TR and may lack COX activity (Figure 25.98), but this is not a universal feature and fibres with enhanced COX activity may be present. Neither RRFs nor COX-negative fibres are disease specific and they can occur with ageing and as a secondary feature in several inherited and acquired disorders.117 Several studies have attempted to quantify the number of RRFs and/or COX-negative fibres in mitochondrial myopathies and in association with normal ageing. Various proportions of abnormal fibres have been regarded as diagnostic of mitochondrial disease including more than 2 per cent RRFs and/or more than 2 per cent COX-negative fibres if the individual is under 50 years of age, or more than 5 per cent COX-negative fibres if the individual is over the age of 50 years. It has been suggested that identification of any RRFs in an individual less than 30 years of age should raise the suspicion of mitochondrial disease. More

recent work has suggested that an occurrence of more than 0.5 per cent COX-negative fibres in the absence of a primary muscle disease should raise the possibility of mitochondrial disease. In addition, the number of COX-negative fibres may vary in different muscles with more being observed in the deltoid than in the quadriceps.419 In children, COXnegative fibres outnumber RRFs, which are rare in children below 5 years of age with mitochondrial disease. Subsarcolemmal mitochondrial aggregates (SSMA), however, are thought to be more prevalent. The ­modified paediatric criteria proposed in 2002 included more than 2 per cent SSMAs as a minor diagnostic criterion and ­continues to be suggested for the diagnosis of ­mitochondrial respiratory chain disorders in children younger than 16 years of age. Recently, however, it has been proposed that muscle biopsies of paediatric patients with low a percentage of SSMA (≤4 per cent) are significantly more likely to have respiratory chain enzyme complex deficiency than patients with increased SSMA percentage (≥10 per cent). A mtDNA depletion syndrome and coenzyme Q10 deficiency should be considered in patients with a low SSMA score (≤4 per cent).291 Ultrastructural abnormalities of mitochondria include alteration in shape, increase in size, disruption and distortion of cristae, and paracrystalline or osmophillic inclusions (Figure 25.99). Electron microscopy may also reveal varying degrees of myofibrillar loss and disruption, and an increase in intracellular lipid, which can often be detected with oil red O or Sudan black stains. Ultrastructural changes are usually non-specific, an exception may be mitochondria with simplified cristae filled with homogenous staining material, reported to be a feature unique to mtDNA depletion.38 In the absence of changes seen with light microscopy, mitochondria, in our experience, are usually ultrastructurally normal, except in endothelial cells (unpublished data), although some changes have been reported in endothelial cells.364,380 Given the lack of specificity of the findings and the time and expense involved, the routine use of electron microscopy in the investigation of mitochondrial disease is questionable.

25

(b)

25.98 A case of a mitochondrial myopathy with mitochondrial DNA depletion caused by a TK2 mutation showing (a) granular basophilic fibres and (b) several fibres devoid of cytochrome c oxidase activity that show only blue staining for SDH that is enhanced in some fibres (ragged blue fibres). (a), H&E; (b), combined COX/SDH.

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1604  Chapter 25  Diseases of Skeletal Muscle (a)

(b)

25.99 Electron microscopy of structurally abnormal mitochondria with (a) paracrystalline inclusions and abnormal cristae and (b) circular inner membranes and inclusions as well as paracrystalline inclusions.

tRNA cause the benign reversible infantile COX-deficient myopathy that presents as a severe myopathy at birth or in the first few weeks of life, with spontaneous recovery between 5 and 20 months of age.198,469 An early onset myopathy with muscle wasting and mental retardation is linked to defective phosphatidylcholine biosynthesis due to mutations in the CHKB gene encoding choline kinase beta. The muscle pathology is distinctive, with myofibres showing central mitochondrial depletion and peripheral giant mitochondria (Figure 25.100).296,348

Other Metabolic Disorders Myoadenylate Deaminase Deficiency

25.100 A case with mutations in the CHKB gene showing large mitochondria towards the periphery of the fibres and pale-stained central areas that do not show disrupted myofibrils with electron microscopy. Note also the blue staining of some fibres due to an absence of cytochrome c oxidase. COX/ SDH.

There is no absolute correlation between genotype and the clinical, biochemical and pathological phenotype in mitochondrial disease, especially for mtDNA mutations. The presence of RRFs and COX-negative fibres in a mosaic and segmental distribution is typical of heteroplasmic mtDNA deletions seen in Kearns–Sayre syndrome (KSS) and progressive external ophthalmoplegia (PEO) or mt tRNA mutations seen in myoclonic epilepsy with raggedred fibres (MERRF) and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS).179,445 In MERRF and MELAS, mitochondrial proliferation also occurs in blood vessels that stain strongly for SDH. Both RRFs and blood vessels in MERRF are COX negative, whereas those in MELAS are typically COX positive, probably as a result of an even distribution of mutant and wildtype mtDNA in these fibres and vessels. Increased lipid in myofibres, including RRFs, is a feature seen in KSS and PEO.388 Mutations in two genes encoding mitochondrial

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Myoadenylate deaminase (MAD) is the enzyme that catalyses the deamination of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and there are several isoforms encoded by different genes. The muscle isoform is encoded by the myoadenylate deaminase deficiency (AMPD1) gene and is more abundant in type 2 fibres, giving a two-fibre type pattern on histochemistry. In addition, type 1 fibres also express the E isoform. The enzyme technique stains tubular aggregates but because they also stain without substrate present this may relate to components of the incubation medium. The histochemical reaction for MAD is negative in affected individuals, but symptoms are mild and non-­ specific (exercise intolerance, aches and cramps, normal or slightly elevated CK). Because there is a common mutation in the gene in the normal population, the significance of absent enzyme activity has been questioned.

Acquired Disorders There are various disorders affecting muscle that are acquired rather than genetic. These include inflammatory myopathies, myopathies related to endocrine abnormalities and vitamin deficiencies, myopathies induced by drugs and toxins, critical illness myopathy, infectious myopathies and myopathies associated with neoplasia and ageing.

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Inflammatory Myopathies Idiopathic Inflammatory Myopathies The classical idiopathic inflammatory myopathies (IIM) include polymyositis (PM), juvenile and adult dermatomyositis (JDM and DM) and IBM. However, the spectrum of inflammatory muscle diseases is much wider and includes immune-mediated and non-immune necrotizing myopathies, focal myositis, granulomatous myositis, graft-versus-host disease, fasciitis/myofasciitis, panniculi­ tis, connective tissue diseases including overlap syndromes, brachiocervical inflammatory myopathy and the vasculitides. Overlapping clinical and pathological features between the IIMs and other inflammatory conditions can present considerable diagnostic challenges. Serology and muscle imaging have an increasing role in the diagnostic pathway for inflammatory muscle diseases. As with other immunogenic disorders, there is evidence that implicates a role for genes, both within and outside the MHC complex, that modulate the immune response in IIM. Such associations include differing IIM phenotypes for anti-Jo-1 and anti-PM-Scl antibody cases despite a shared HLA8.1 ancestral haplotype, association of DPB1*0101 with anti-Jo-1 positivity but not anti-Scl-1, GM13 allotype being a confirmed risk factor in IIM, and potential influence of HLA8.1 ancestral haplotype on disease susceptibility and expression in IBM.74 Specific genes at polymorphic loci such as HLA DRB1*0301, TNF-α308A and GM 3 23 5,13 are risk factors for all of the major clinical groups. Other alleles are specific to particular antibody phenotypes.358 Consistent and accurate classification of IIMs is more than just semantics; accurate delineation of homogenous subgroups is helpful for predicting response to treatment, clinical outcome, the development of therapeutic trials, and for epidemiological studies. The Bohan and Peter criteria formulated in 1975 have been widely used, but are limited and do not take account of pathological and clinical developments in the field. The classification excludes IBM and immune necrotizing myopathies, now well-recognized entities, and better criteria have been established.195 Better understanding of the immunopathogenesis, histopathology, muscle imaging and specific autoantibody associations has led to refined IIM criteria through large-scale consensus efforts. (For aspects on pathogenesis see Box 25.11 and various reviews6,100). The European Neuromuscular Centre classification for IIMs, based on clinical, muscle pathology and other laboratory criteria, proposes five categories: (i) IBM; (ii) PM; (iii) DM; (iv) non-specific myositis; and (v) immune-mediated necrotizing myopathy.75,111 A review encompassing historical approaches to classification as well as more recent attempts to refine knowledge of immunopathological and autoantibody associations in inflammatory myopathies has recently been published.194 The onset of polymyositis or dermatomyositis in adults is usually associated with muscle pain, accompanied by variable muscle weakness. The weakness may be proximal and symmetrical, as in LGMD, or asymmetrical with varying distribution, but it is rarely selective. Tenderness and swelling of muscles may accompany weakness. Progression may be rapid and severe, but most cases follow an insidious subacute or chronic course. Rapid progression is associated

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Acquired Disorders  1605 Box 25.11.  Pathogenesis of polymyositis, dermatomyositis and inclusion body myopathy

25

Immunopathogenesis of polymyositis (PM) and inclusion body myositis (IBM) is based on antigen-directed cytotoxicity mediated by CD8+ T-cells invading non-necrotic, major histocompatibility class I (MHC-I) expressing muscle fibres. This CD8+/MHC-I immunological synapse is identical in PM and IBM, despite the latter being refractory to immunosuppression. The major cytotoxic effector mechanism in PM and IBM is mediated via the perforin pathway. In PM and IBM, there is evidence of variable upregulation of pro-inflammatory cytokines chemokines as well as various adhesion and extracellular matrix molecules such as VCAM, ICAM, thrombospondins and metalloproteinases on myofibres and some autoinvasive CD8+ T-cells. These molecules consolidate the immunological synapse by enhancing T-cell activation, inducing co-stimulatory molecules and facilitating cell adhesion. BB1/CD28 and ICOS/ ICOS-L interactions in PM and IBM denote participation in antigen presentation, clonal expansion and T-cell co-stimulation. In IBM, in addition to the immunopathogenic changes in common with PM, there is a prominent degenerative process characterized by fibre vacuolation and accumulation of conformationally modified proteins similar to those seen in neurodegenerative diseases such as Alzheimer disease. Abnormalities in AβPP processing such as increased BACE1 and γ-secretase contribute to Aβ overproduction. Aβ has recently been demonstrated in IBM muscle and plasma. Conformationally modified Tau protein has been identified recently in IBM muscle and is implicated in IBM pathogenesis. Dermatomyositis (DM) is considered to be a humorally mediated microangiopathy; the primary antigenic target being the vascular endothelium of the endomysial capillaries and to a lesser extent of the larger blood vessels. Putative antibodies directed against the endothelial cells activate the complement cascade leading to C5b-9, the terminal lytic component of the complement pathway, being deposited early in the disease course. Demonstration of a majority of B-cells and CD4+ T-cells in the perimysial and perivascular foci and plasmacytoid dendritic cells in the perimysial foci further points to humorally mediated mechanisms in DM. Certain immunopathogenic mechanisms are shared across the different IIM subtypes. Regenerating fibres in PM/DM express Toll-like receptors TLR3 and TLR7. Necrotic cell debris drives cytokine production in vitro in part through TLR3 signalling, and necrotic/regenerating fibres may amplify the immune response in PM/DM. Involvement of the type I interferon system in the pathophysiology of idiopathic inflammatory myopathies (IIMs) has emerged in recent years, as well as the role of dendritic cells (DCs). Myeloid DCs are present in substantial numbers in PM/IBM muscle, whereas in DM muscle, plasmacytoid DCs predominate. Besides innate and adaptive immune responses, non-immune mechanisms may participate in muscle damage in IIMs.

with gross elevation of CK. Rhabdomyolysis and myoglobinuria may also occur, but this is rare in dermatomyositis. Joint contractures affecting the ankles and other large joints are common. There is a well-established association between malignancy and dermatomyositis in adults.506 The tumour and myopathy usually present within a short time of each other, but occasionally the myopathy may significantly precede identification of the tumour. In dermatomyositis, involvement of skin vasculature manifests as a heliotrope or violaceous rash, particularly over the eyes and malar regions of the face, and as erythema around the nail beds and over the knees and elbows. In

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1606  Chapter 25  Diseases of Skeletal Muscle

severe cases, the entire skin becomes tight, shiny and reddened. In chronic juvenile cases of dermatomyositis, there may be calcinosis. Calcium is deposited in the subcutaneous tissue and in the supportive connective tissues within muscle, but not in muscle fibres themselves. Juvenile dermatomyositis differs from the adult form. It is always an idiopathic condition and is not associated with malignancy. Although muscle weakness is always present, it may be extremely variable. Children with dermatomyositis usually have systemic symptoms, such as mood swings, malaise, listlessness and lethargy, which may be the presenting feature or characterize a relapse. Inclusion body myositis is one of the most common disorders of muscle in patients aged over 50 years. It is more common in males than females, and usually sporadic. However, some hereditary disorders sharing pathological features of inclusion body myositis, but lacking an inflammatory component, have been described and are sometimes referred to as ‘hereditary inclusion body myopathies’. The underlying gene defects in some of these inherited forms have been identified, and are discussed earlier (see Z-line Associated Proteins and Four-and-a-Half LIM Domains Protein 1, p. 1591; and Distal Myopathies, p. 1593). IBM is distinguished from polymyositis and dermatomyositis by a more insidious onset, with both proximal and distal weakness, which is often asymmetrical. Dysphagia, weakness of the wrist, finger flexor muscles and of ankle dorsiflexion are common.100 IBM patients are usually nonresponsive to steroid therapy. Serum CK activity in all forms is frequently, but not invariably, elevated, and a normal level does not exclude a diagnosis of inflammatory myopathy. Other laboratory investigations that may be helpful are a raised erythrocyte sedimentation rate (ESR), serum autoantibodies, and serum and urinary compounds such as myoglobin and neopterin. The ESR is raised in only a proportion of cases, and there is no correlation between its level and the degree of muscle weakness. Myositis-specific autoantibodies (MSAs) are detected in one-third of IIM cases. They are highly disease specific, can appear months before the onset of symptoms, correlate with disease activity and disappear on disease remission. There is increasing evidence that MSAs can allow more homogenous grouping in PM/DM, and can help in assessing prognosis and developing therapies. Antiaminoacyl-tRNA-synthetase antibodies are the most prevalent. The most common of these is anti-Jo-1; patients with these antibodies show a high frequency of moderate to severe myositis (PM or adult DM) with characteristic extramuscular manifestations, including interstitial lung disease, Raynaud’s phenomenon, low grade fever, arthritis and mechanic’s hands – the antisynthetase syndrome. Antisignal recognition particle (anti-SRP) and anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibodies are typically associated with immune-mediated necrotizing myopathies. It should be noted that a degree of overlap exists between MSAs and their disease associations; for instance, antisynthetase antibodies typically associated with the antisynthetase syndrome with or without myositis (PM/DM) may also occur in immune necrotizing myopathies. New autoantibodies are continually being identified and, to date, around 60–80 per cent of patients with autoimmune myositis seem to have at least one MSA.63,74,197 The

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various clinical associations with MSAs are summarized in Table 25.21. Electromyography shows a characteristic pattern, with a combination of spontaneous fibrillation potentials, similar to those seen in denervation, and polyphasic short-duration potentials on voluntary contraction, as in myopathies. Imaging techniques are having an increasing role, and show increased signal in relation to oedema and inflammatory changes in subcutaneous fat.486 In muscle biopsies the presence of lymphocytes that may invade muscle fibres is a key feature of myositis, but the degree of inflammation is variable and may be absent. Some cases may show a fasciitis (see later). The presence of sarcolemmal MHC-I antigens, however, is universal in all forms and a useful diagnostic marker that can be present in the absence of other signs of inflammation or other pathology (Figure 25.101).100,117 MHC-I immunolabelling within muscle fibres may also be seen. In DM, MHC-I upregulation may be focal or restricted to the perifascicular regions and not be as diffuse and widespread as in PM/ IBM. Inflammation is not only a feature of inflammatory myopathies, but can also be prominent in various muscular

(a)

(b)

25.101 Immunolabelling of major histocompatibility class 1 antigens in (a) a control and (b) a case of juvenile dermatomyositis. Note the sarcolemmal labelling in the case of dermatomyosits and normal labelling of blood vessels in both.

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Acquired Disorders  1607

Table 25.21  Clinical associations of myositis-specific antibodies Antibody

Target antigen

Clinical association

25

Antibodies associated with antisynthetase syndrome Anti-amino-acyl-tRNA synthetase

Amino-acyl-tRNA synthetase

Antisynthetase syndrome (myositis, interstitial lung disease, Raynaud’s phenomenon, arthritis. mechanic’s hands, fever, DM skin rash)

-Jo-1 (most common)

-Histidyl

Myositis (PM/ adult DM), ILD

-PL7

-Threonyl

Hypomyopathic disease, prominent lung involvement

-PL12

-Alanyl

DM skin rash, ILD

-OJ

-Isoleucyl

DM skin rash, ILD

-EJ

-Glycyl

–

-KS

-Asparginyl

DM skin rash, ILD

-Ha

-Tyrosyl

Myositis, ILD

-Zo

-Phenylalanyl

Myositis, ILD

Antibodies associated with dermatomyositis Anti-Mi-2

NuRD

Classic DM, decreased risk of malignancy, more severe rash, good response to steroids, good prognosis

Anti-p155/140

TIF1 family

Children: ulceration Adults: malignancy

Anti-p140

NXP2

Children: calcinosis Adults: ILD

Anti-SAE

SAE

Skin manifestations precede myositis

Anti-CADM-140

MDA-5

CADM, ILD, cutaneous ulceration, palmar papules, rapidly progressive ILD

Anti-MJ

Nuclear matrix protein 2 (NPX2)

High frequency of calcinosis, arthritis, joint contractures and absence of truncal rash

Antibodies associated with immune-mediated necrotizing myopathies Anti-SRP

Signal recognition particles (cytoplasmic RNA complexes)

Aggressive disease, poor steroid response, cardiac involvement

Anti-HMGCR

3-hydroxy-3-methylglutaryl-coenzyme A reductase

Progressive myopathy in patients with statin use despite cessation of statin

Antibodies associated with inclusion body myositis Anti-p44

5′-nucleotidase IA

IBM

DM, dermatomyositis; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; IBM, inclusion body myositis; ILD, interstitial lung disease; PM, polymyositis.

dystrophies, particularly in association with necrotic fibres, and MHC-I is often overexpressed on the sarcolemma (see sections on muscular dystrophies). Abnormal variation in fibre size is often present, but hypertrophy is absent or less pronounced than in muscular dystrophies. Other pathological features include internal nuclei, basophilic fibres, an increase in connective ­tissue (usually less than in muscular dystrophies), moth-eaten fibres or fibres with core-like areas, and fibre splitting. There may also be loose oedematous separation of muscle fibres with interspersed fibrous tissue. Perifascicular atrophy is a particular feature of dermatomyositis, which is not

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seen in polymyositis or IBM, and when present should raise the suspicion of DM even in the absence of inflammation (see Figure 25.26). Histochemically, perifascicular fibres are of both types and often show intense and aggregated NADH-TR activity. Many of these small fibres express proteins associated with immaturity, such as fetal myosin, desmin and NCAM, and differentiating them from regenerating fibres is then difficult. Necrosis and regeneration and a characteristic ­vacuolar degeneration may be extensive, particularly in JDM (Figure 25.102). Macrophages and T-cells invade the fibre after it becomes necrotic. Necrosis may be segmental and

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1608  Chapter 25  Diseases of Skeletal Muscle (a)

25.102 Vacuolar degeneration in a case of dermatomyositis. H&E.

involve clusters of fibres, or it may involve single fibres. In dermatomyositis, areas of infarction, characterized by groups of pale-staining necrotic fibres, may be present. Necrotic fibres may show a peripheral cuff of basophilia, corresponding to regeneration, a feature rarely seen in ­muscular dystrophies. Acid phosphatase activity is associated with the presence of inflammatory cells and macrophages and is also increased in the fibres.117 T-cells, B-cells, dendritic cells and macrophages are the principal infiltrating cells. They occur in the perimysium and endomysium, are often perivascular and may partly invade blood vessel walls. However, true vasculitis is not a feature of PM/DM and if present should raise the possibility of an associated connective tissue disease. Eosinophils are not usually seen. The proportion and distribution of the various inflammatory cell types differ in polymyositis and dermatomyositis. In polymyositis, the infiltrate is predominately endomysial, with a large number of CD8+ T-lymphocytes. These surround and invade non-necrotic muscle fibres, a feature not found in dermatomyositis (Figure 25.103a). B-cells, in contrast, are predominantly perivascular and rarely located in the endomysium in polymyositis. In dermatomyositis, the cells are predominantly perivascular and perimysial, although some may be endomysial, and there is a higher proportion of B-cells and CD4+ cells than in polymyositis. A variable proportion of CD4+ cells in PM and DM are dendritic cells. Macrophages may accompany CD8+ T-cells in invading MHC-I restricted apparently ­ ­ non-necrotic fibres in PM. Occasionally in DM, the p ­ erimysium may harbour prominent ribbon-like ­configurations of macrophages with basophilic cytoplasm; this pattern has been referred to as inflammatory myopathy with abundant macrophages (IMAM), a condition that should be distinguished from macrophagic myofasciitis.101 Blood vessels in dermatomyositis often have thickened walls and capillaries may be enlarged. With electron microscopy, endothelial cells of capillaries and arterioles are seen to contain tubuloreticular inclusions. These are an early pathological feature. Another early feature of DM is deposition of the membrane attack complex C5b–9 on endomysial capillaries. Surface deposition of C5b–9 may be seen on a limited number of fibres and within necrotic

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(b)

25.103 (a) Immunolabelling of CD8+ cells in the endomysium and some that appear to be invading a fibre in a case of polymyositis. (b) Immunolabelling of p62 shows cytoplasmic inclusions in a vacuolated fibre and peripheral labelling in an adjacent non-vacuolated fibre in a case of inclusion body myositis.

fibres as a non-specific feature. In both adult and juvenile dermatomyositis, there is depletion of capillaries, and this is also an early feature seen in the absence of other pathology. Because MHC-I is present on all blood vessels, it can be used to assess the number of capillaries, but this may be visualized better with the endothelial marker CD31 (PECAM-1). Basal lamina markers such as Ulex europaeus or laminin α5, are also useful as the capillaries are highlighted against the negative (Ulex) or weak (laminin α5) labelling of the sarcolemma. In DM, the perimysium contains abnormal vessel fragments, perivascular inflammation and increased CD31. Perifascicular atrophy and capillary pathology are concentrated near the avascular perimysium.333 A scoring system has been devised for evaluating muscle biopsies in JDM assessing pathological severity in four domains (inflammatory, vascular, muscle fibre and connective tissue) and has been shown to have good interobserver reliability and correlation with clinical severity.491 The main pathological features of inclusion body myositis are fibre atrophy and hypertrophy, an increase in internal nuclei and endomysial connective tissue, necrosis, with

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Acquired Disorders  1609



invasion of fibres by phagocytes, and basophilic regenerating fibres. Cytoplasmic bodies, disruption of the myofibrillar pattern and core-like areas lacking NADH-TR activity are also seen. There is often a greater than expected number of COX-negative fibres, and electron microscopy reveals structurally abnormal mitochondria. As in PM, there is widespread MHC-I upregulation and endomysial inflammation composed predominantly of autoinvasive cytotoxic CD8+ cells and macrophages that surround and invade MHC-I restricted non-necrotic fibres. The inflammation can vary from florid to focal to sparse. Rimmed vacuoles are also a typical feature of inclusion body myositis (see Figure 25.43) but the number is variable and they may be sparse. Intranuclear inclusions may be seen. Multiple or single foci of amyloid deposits are present in vacuolated and non-vacuolated fibres, and best observed with Congo red staining viewed with fluorescence using an excitation filter suitable for rhodamine or Texas red. Some deposits are also seen with polarized light. The amyloid deposits contain Aβ42, plaque-like inclusions corresponding to 6–10 nm fibrils electron microscopically or aggregates of phosphorylated Tau, appearing with electron microscopy as 15–21 nm twisted paired helical filaments and best demonstrated as squiggly, linear or round p62/sequestrosome1 positive inclusions (Figure 25.103b).7 Many other proteins also accumulate in IBM, including Tar-DNA–binding protein-43 (TDP-43), AβPP, ubiquitin, presenilin 1, apolipoprotein E, α-synuclein, prion protein, nuclear membrane proteins and survival motor neuron protein. Many of these proteins are found in the brains of patients with Alzheimer’s disease, which has led to the hypothesis that a cascade of events leads to cell degeneration and misfolding of proteins.6,101 TDP-43 and p62 currently seem to be the most useful to study in possible cases of IBM, but neither are specific and they can be seen in a variety of other disorders, including myofibrillar myopathies and cases with GNE or VCP mutations that show rimmed vacuoles (see Distal Myopathies, p. 1593).115 Electron microscopy reveals cytomembranous debris, myelin-like whorls corresponding to the basophilic granules lining vacuoles. None of these changes is diagnostic for IBM, and absence of the characteristic paired helical filaments or amyloid fibrils does not exclude a diagnosis of IBM. Immune-mediated necrotizing myopathy (IMNM), also known as necrotizing autoimmune myopathy (NAM), is a relatively newly recognized subgroup of IIM, and encompasses a heterogeneous group of diseases that share common pathological features of predominant myonecrosis, myophagocytosis and regeneration, scattered endomysial and perimysial macrophages with little or no lymphocytic inflammation in the biopsy. Some cases are associated with various autoantibodies. Clinical onset and progression are similar to PM/DM with subacute onset of symmetrical, proximal limb-girdle weakness. CK levels are usually markedly elevated (>10 times upper limit of normal) and EMG shows an irritable, myopathic pattern. Variable ­cardiac and lung involvement is also reported. Marked fibre size variation and hypertrophy may be seen, and this, in c­ ombination with the necrosis and regeneration and the subacute proximal weakness, may lead to the misdiagnosis of a ­ dystrophy (Figure 25.104). IMNM can be a­ ssociated with specific autoantibodies such as anti-SRP, ­ anti-HMGCR, antisynthetase and anti-PMScl, connective tissue disease,

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25

(b)

(c)

25.104 Immune-mediated anti-SRP necrotizing myopathy showing (a) pronounced necrosis of muscle fibres but little inflammation, (b) diffuse upregulation of major histocompatibility class 1 antigen on most fibres and (c) sarcolemmal complement C5b-9 labelling on a fibre (arrow).

paraneoplastic syndrome and viral infections such as HIV and hepatitis C. Variable, sometimes diffuse, strong MHC-I upregulation on non-necrotic fibres and prominent complement C5b-9 deposition on capillaries is reported in ­anti-SRP, anti-HMGCR and antisynthetase, and paraneoplastic

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1610  Chapter 25  Diseases of Skeletal Muscle

IMNM (see Figure 25.104). Fragmented perimysial connective tissue with alkaline phosphatase staining is seen in antisynthetase and paraneoplastic IMNM.426 Statins (HMGCR inhibitors) can cause a toxic myopathy, which is self-limiting and may recover upon discontinuation of the drug.265 The presentation is variable including muscle pain and weakness, raised CK, and rarely rhabdomyolysis. Muscle biopsies can reveal myonecrosis, lipid accumulation or type II atrophy. The very rare necrotizing myopathy with pipestem capillaries shows distinct thickening of the capillary walls with complement deposition, and deposition of granular amorphous material in their basement membranes is seen electron microscopically. All reported patients had severe systemic disease such as neoplasia, vasculitis and interstitial lung disease. In contrast to non-IMNM due to exposure to drugs or toxins, IMNM cases show significantly greater MHC-I upregulation, capillary complement deposition, more inflammation and regeneration. In addition, in IMNM, a stronger Th1/classically activated macrophage M1 response was observed with elevated interferon-γ, TNF-α, interleukin-12 and STAT-1 levels in muscle. B-cells and high expression of CXCL13, a B-cell chemoattractant, were observed in a subset with defined antibodies. These findings may potentially lead to the development of new biomarkers and therapeutic targets in IMNM.341

Connective Tissue Disorders Patients with IIM can present with or develop a connective tissue disease (CTD). They can have typical symptoms of CTD, such as dry mouth and eyes (Sjöogren syndrome) or renal involvement (SLE), as well as IIM-related muscle symptoms – so called ‘overlap myositis’. The most common secondary autoimmune diseases in overlap myositis are systemic sclerosis and mixed CTD. Myositis-associated antibodies (MSAs) are seen in both IIM as well as CTD and overlap myositis; for example, the majority of patients with myositis-scleroderma overlap syndrome are positive for anti-PM/Scl. Muscle biopsies may show fibre necrosis and inflammation, often with a vasculitis. True vasculitis is not seen in IIM and when present should prompt a search for an underlying CTD. In SLE, blood vessel walls may be thickened and may contain the tubuloreticular inclusions found in dermatomyositis. Non-specific myopathic changes, such as type 2 fibre atrophy, are also common in connective tissue disorders.

­ iastase-resistant, PAS-positive of CD68+ macrophages with d basophilic cytoplasm, with a minor mainly CD8+ lymphocytic component often around blood vessels (Figure 25.105). Myofibres remote from the infiltrate are typically intact but the alum can reach the brain. Electron microscopy shows spicules of osmiophilic aluminium oxyhydroxide surrounded by discontinuous lysosomal membranes.155 Inflammatory myopathy with abundant macrophages is a recently defined entity. Patients present with a proximal myopathy; some are clinically diagnosed with DM based on a skin rash. The inflammation is seen typically at the periphery of the fascicles in the epimysium and perimysium comprising non-cohesive ribbons of CD68+ macrophages remote from necrotic myofibres and lacking the inclusions seen in macrophagic myofasciitis. In contrast to typical DM infiltrates, the macrophages are of the acute inflammatory MRP14+ type, CD20+ B-cells, CD3+ T-cells and CD123+ plasmacytoid dendritic cells are sparse. Capillary complement deposits are absent. Presently, it is unclear if IMAM and DM are pathogenically distinct or if they reflect different stages of the same disease.51 Fasciitis-panniculitis syndrome (FPS) comprises a group of disorders characterized by skin induration caused by chronic inflammation and fibrosis of the subcutaneous fascia and myosepta, as well as septal and lobular panniculitis. The prototype disorder is idiopathic eosinophilic fasciitis (Shulman syndrome) characterized by diffuse fasciitis comprising fibrosis and infiltrates of diffuse and perivascular, mainly CD8+ T-cells and/or eosinophils, eosinophilia and hypergammaglobulinaemia without visceral involvement or Raynaud’s phenomenon. Eosinophils may be absent in the chronic stage or after steroid treatment. The disease can be triggered by exposure to toxins, treatment or trauma, and infections such as borreliosis and Mycoplasma arginine.243 Histologically, FPS shows a subcutaneous septal-fascial-perimysial collagenous scaffold accompanied by an infiltrate of lymphocytes, plasma cells and histiocytes into the adjacent fat. Depending on the aetiology, eosinophils and mast cells can be identified. Secondary associations of FPS include cancer, graft-­ versus-host reaction, insect bites, post-traumatic or post-radiation reactions, and Sweet syndrome.

Fasciitis/Myofasciitis with Panniculitis Fasciitis and panniculitis encompass a diverse clinicopathological spectrum of diseases where the inflammatory changes are centred on the fascia and subcutaneous fat respectively. The pathology may extend to the periphery of the fascicles. STIR MR imaging reveals hyperintense signal change in the fascial planes. A small proportion of individuals who receive vaccines containing aluminium oxyhydroxide as an adjuvant present with delayed onset of myalgia, chronic fatigue and cognitive dysfunction, and exhibit very long-term persistence of alum-loaded macrophages at the site of vaccination forming a lesion called macrophagic myofasciitis. There is focal infiltration of the epimysium, perimysium and perifascicular endomysium by circumscribed collections and sheets

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25.105 A case of macrophage fasciitis induced by vaccine showing abundant macrophages in the perimysium. H&E.

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Other rare causes of fasciitis include necrotizing fasciitis due to bacterial infections, systemic non-Langerhans’ histiocytosis and echovirus infection in congenital X-linked and acquired hypogammaglobulinaemia.

Granulomatous Myositis, Focal Myositis and Brachiocervical Inflammatory Myopathy Granulomatous myositis is a rare disease characterized by discrete perimysial or endomysial epithelioid granulomas that are usually non-necrotizing, with variable interstitial inflammation and myonecrosis. Clinical presentation is usually that of proximal weakness and myalgias. Bulbar involvement may occur, and flexion contractures may be prominent in some. The association with sarcoidosis is well known and the prevalence of asymptomatic granulomatous myositis in sarcoid is reported to be as high as 50–80 per cent. Significant MHC-I upregulation may be seen in some cases mimicking IIM. Less well recognized is the association with infections (parasites, brucellosis, tuberculosis, syphilis), paraneoplastic manifestation in lymphoma, graft-versushost disease, inflammatory bowel disease (Crohn’s), CTD (rheumatoid arthritis) or as an autoimmune overlap syndrome with myasthenia gravis, myocarditis, thyroiditis and thymoma. The diagnosis of idiopathic/primary/isolated granulomatous myositis is one of exclusion. Focal myositis is a rare entity that presents clinically as a solitary, intramuscular mass lesion and histologically corresponds to an inflammatory pseudotumour, with variable myopathic, focal neurogenic and inflammatory changes, and fibrosis. The infiltrate is macrophage and T-cell rich, with a prominent B-cell and plasmacytoid dendritic cell component present when the lesion is actively inflamed. MHC-I and IgG4 is weakly present, the latter may be linked to fibrosis. Common misdiagnoses include haematological or soft tissue malignancy, primary or proliferative myositis, myositis ossificans and inflammatory myofibroblastic tumour. Careful attention to the morphology and immunophenotype and the clinical presentation is necessary to avoid these pitfalls. Spontaneous regression is seen over time in most cases. There is no clear genetic or acquired etiological link. More recently it has been suggested that focal myositis may be a neurogenic phenomenon.257 Patients with brachiocervical inflammatory myopathy (BCIM) syndrome have progressive weakness in the proximal regions of the arms and neck. Histology reveals an active myopathic process with focal perimysial and perivascular inflammation with prominent B-cells, endomysial dendritic cells and complement C5b-9 staining of the endomysial connective tissue. There is a strong association with systemic autoimmune disorders and presence of antinuclear antibodies. There is a good response to steroids. It is unclear if BCIM represents a distinct form of autoimmune inflammatory myopathy.

Vasculitis of Skeletal Muscle Vasculitis is infrequently encountered in skeletal muscle. The frequency of muscle involvement in systemic vasculitis is poorly defined. Clinicians use a two-tiered system of definite vasculitis when there is transmural inflammation with fibrinoid necrosis, mural destruction or leucocytoclasia, and probable vasculitis when there is a dense

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Acquired Disorders  1611

perivascular cellular cuff or transmural inflammation without the destructive features. Additional evidence of vascular damage including haemosiderin deposits, fibrous scarring or organization is used by many investigators. The presence of neurogenic atrophy, presumably due to concomitant peripheral nerve involvement (if present), points to a multisystem process. A degree of overlap occurs with IIM because perivascular inflammation can be prominent in DM an IMNM, leading to a potential misdiagnosis. However, a necrotizing component is unusual in IIM and if present points to a systemic vasculitic process such as polyarteritis nodosa, rheumatoid vasculitis, Wegener’s, Churg–Strauss or drug-induced anti-neutrophil cytoplasmic antibodies (ANCA) vasculitis or sarcoidosis.340 Vasculitis restricted to skeletal muscle is even rarer. Granulomatous vasculitis may be seen in sarcoidosis and Wegener’s disease. Eosinophils may be present in the infiltrates of Churg–Strauss vasculitis. A combined superficial peroneal nerve and peroneus brevis muscle biopsy has been shown to increase the diagnostic yield of vasculitis. However, no such advantage was noted combining sural nerve with vastus lateralis biopsy. The choice of biopsy may vary depending on the institutional preference, but the muscle and/or nerve biopsied should be clinically and/or electrophysiologically affected.

25

Viral and Bacterial Myositis Several infections are known to induce an acute myositis.125 These include influenza viruses, Coxsackie viruses, Epstein– Barr virus, cytomegalovirus, herpes simplex virus, hepatitis C virus, human immunodeficiency virus and human T-cell lymphotropic virus type 1. A variable degree of muscle fibre necrosis and inflammatory infiltration occur. Onset can be acute, with weakness and marked muscle tenderness that involves all major muscle groups. Myoglobinuria may be present. Symptoms often improve rapidly once the acute phase is over, even in the absence of specific therapy.

Drug and Toxic Myopathies A variety of drugs and toxins can affect muscle and nerves.117 Some of these are common, whereas others are rare. Statins are now widely prescribed and are a common cause of myalgia and raised CK. Alcohol is also a common cause of myopathy, and drugs of abuse frequently cause muscle necrosis. Several other prescribed drugs that may induce myopathy include corticosteroids, ciclosporin, D-penicillamine, chloroquine, vincristine and zidovudine. Toxic effects can be induced in muscle by various dietary supplements, such as geranium, by excess vitamin E and by snake venoms. The pathological changes induced in muscle by drugs are non-specific, are variable in degree, and may be focal or diffuse. Necrosis, which may be acute, is common, as is type 2 fibre atrophy. A well-established side effect of steroids is type 2 fibre atrophy, particularly of type 2B fibres. Steroids can also induce selective loss of myosin from A-bands in ‘critical illness myopathy’. This may affect patients receiving high doses of corticosteroids and neuromuscular blocking agents to assist mechanical ventilation and occurs when the paralysing agent is withdrawn.

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1612  Chapter 25  Diseases of Skeletal Muscle

A vacuolar myopathy can be induced by some antimalarial drugs, such as chloroquine.117 These autophagic vacuoles contain membranous myelin-like whorls, and cell debris and curvilinear bodies are a feature associated with chloroquine. Vincristine and colchicine can also induce vacuoles and the formation of spheromembranous bodies, which are thought to be derived from the sarcoplasmic reticulum (Figure 25.106). A second type of vacuolar change is associated with hypokalaemic agents, such as diuretics, laxatives and liquorice derivatives. These vacuoles are thought to originate from T-tubules. The effects of zidovudine on mitochondria are reflected by the presence of ragged-red fibres and ultrastructurally abnormal mitochondria. Geranium-based elixirs and dietary supplements can also influence mitochondrial function, and ragged-red fibres, COX-negative fibres and ultrastructural mitochondrial damage may be seen.

Endocrine and Systemic Myopathies Hormones have an important role in maintaining normal muscle function, and myopathies have been described in association with either an excess or a deficiency of several, in particular thyroid and parathyroid hormones, glucocorticoids, growth hormone and insulin.117 In most cases, muscle involvement is an incidental feature of the disorder but often reversible with correction of hormone levels. Muscle symptoms may be the presenting features of endocrine imbalance and prompt the diagnosis of an underlying disorder, such as thyrotoxicosis. There is often a predominant proximal weakness with varying degrees of wasting, and in many cases the weakness is disproportional to the degree of muscle wasting. Other symptoms may include muscle pain, cramps, stiffness, periodic paralysis (hypothyroidism) and ocular involvement (hyperthyroidism). Muscle pathology may be minimal or may show non-specific changes such as type 2 fibre atrophy, type 1 fibre hypertrophy or an increase in internal nuclei. Additional pathological features may be necrosis, an increased percentage of type 1 fibres, and inflammatory infiltrates, which can make the differential diagnosis with polymyositis difficult, especially if MHC-I is not studied. Hypothyroidism during pregnancy can also affect the expression of myosin isoforms and fibre typing in the fetus.387 Vitamin deficiencies such as vitamin B12, C, D or E result in abnormalities, some of which affect muscle. There are both acquired and hereditary forms of vitamin E deficiency. Malabsorption of vitamin E can lead to spinocerebellar ataxia, dysmetria, areflexia and loss of vibratory sensation.109 Muscle biopsies show typical electron-dense inclusions that are positive for acid phosphatase, non-specific esterase and are autofluorescent.117 Selenium deficiency, especially in association with vitamin E deficiency can result in muscle symptoms such as myalgia and weakness. Muscle biopsies can show non-specific changes such as type 2 fibre atrophy and mitochondrial changes have been reported.69,356 Malignancies can be associated with various neurological symptoms. Type 2 fibre atrophy is a common nonspecific feature associated with malignancies. Inflammatory myopathies, in particular dermatomyositis, are associated with malignancies, especially pulmonary, gastrointestinal, ovarian and nasopharyngeal carcinomas.506

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25.106 Vacuoles of varying size in a case treated with colchicine. H&E.

Amyloid is a proteinaceous material with a fibrillar structure that stains red with Congo red and is biorefringent with polarized light. It can accumulate (amyloidosis) both intracellularly and extracellularly in several tissues, and may be secondary to malignancies, chronic inflammatory conditions, genetic diseases (see Recessive Limb-Girdle Muscular Dystrophies, p. 1561) and also to ageing.416 Amyloid myopathy in the majority of patients results from the deposition of amyloid immunoglobulin light chains, chiefly the lambda type. Clinical features include proximal muscle weakness but distal muscle weakness may occur. Dysphagia, macroglossia and/or muscle pseudo-hypertrophy are frequent but not consistent findings. Congestive heart failure and cardiomyopathy can be complications. Muscle biopsies show perivascular and endomysial/perimysial deposition of amyloid, and neurogenic atrophy of muscle fibres may be present. Electron microscopy shows blood vessels and muscle fibres coated with amyloid fibrils.68,117

Muscle and Ageing Sarcopenia (loss of muscle mass) is common in ageing and there is considerable research into its pathogenesis.206 The reduction of muscle mass is due both to muscle fibre atrophy, particularly type 2 fibres, and to loss of muscle fibres, resulting in a higher proportion of type 1 fibres. Although exercise maintains the size and strength of fibres, no way to halt the loss of type 2 fibres has been found. Mitochondrial DNA shows age-related accumulation of mutations in postmitotic tissues, and an increase in the number of COXnegative fibres and ragged-red fibres is seen in the muscle.168

Acknowledgements We are grateful to Dr Cecilia Jimenez-Mallebrera for her valuable contribution to this chapter in the previous edition. The financial support of the National Specialist Commissioning Team for Rare Neuromuscular Disorders to the Dubowitz Neuromuscular Centre for Congenital Muscular Dystrophies and Congenital Myopathies is gratefully acknowledged. Also funding from the Muscular Dystrophy Association of America to SCB is gratefully acknowledged.

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  Epidemiology And Aetiology  1623

26 26 Chapter

Introduction to Tumours Arie Perry and David N Louis

Epidemiology and Aetiology......................................................1623 Animal Models of Nervous System Tumours..............................1628 Classification and Grading........................................................1632

Epidemiology and aetiology Intracranial and spinal tumours account for only 2 per cent of all malignant neoplasms, but in children they are the second most common tumour type and the most common cause of cancer death. Of all intracranial tumours, roughly 40 per cent originate from neuroepithelium, 35 per cent from meningothelial cells, 14.4 per cent from oral ectoderm (pituitary adenomas and craniopharyngiomas), and 7.5 per cent from peripheral nerve sheath elements. Lymphomas and germ cell tumours account for 2.3 and 0.5 per cent, respectively.27 The 2007 classification of the World Health Organization (WHO) for central nervous system tumours lists over 120 different types and subtypes.108 The incidence, localization, age distribution, biological behaviour and patient survival differ greatly among them. For these reasons, it is also likely that the aetiologies and biological underpinnings of many central and peripheral nervous system (CNS and PNS) tumours vary.

Incidence and Mortality Cancer registry data are greatly influenced by whether meningeal, cranial nerve and spinal cord tumours are included in addition to neoplasms of the brain.44 Often, incidence data include only malignant neoplasms, but the ICD-0 and efforts of the Central Brain Tumor Registry of the United States have made major strides to include all brain tumour data. There has been some concern over a possible increase in the incidence of brain tumours since the 1980s, but most authors agree that this apparent increase is largely due to the introduction and more frequent utilization of high-resolution neuroimaging, which has greatly improved the sensitivity of detecting brain tumours.89,121,125 Population-based s­tudies have not shown increases in brain tumour incidence in Scandinavia since the early 1980s.106 Mortality in patients with brain tumours is highly dependent on the type of tumour, with some lesions (WHO grade I) acting in a relatively benign manner and others (WHO

Acknowledgements..................................................................1633 References...............................................................................1633

­ alignancy; grades II–IV) representing different degrees of m patients with grade IV lesions typically have survival times of less than 2 years from diagnosis, and roughly 2–5 years for grade III, 5–10 years for grade II and >10 years for grade I. In addition, the mortality/incidence ratio for brain tumours reflects the effectiveness of ­diagnostic and ­therapeutic measures. Mortality is lower overall in women, given their lower incidence of gliomas and increased ­predisposition to meningiomas. Not surprisingly, mortality and morbidity are also highly associated with site of presentation, both increasing considerably when eloquent brain is involved.

Age and Sex Preferential manifestation in specific age groups is a ­hallmark of CNS tumours and often yields, together with tumour site, a limited differential diagnosis. The age ­distribution of brain tumours is bimodal, with the first peak in children (e.g. medulloblastoma, pilocytic ­ astrocytoma, ependymoma) and the second, larger peak in adults aged 45–70 years, mainly due to glioblastomas and ­meningiomas. Information for each tumour type is included under each tumour entity. In general, gliomas and embryonal tumours occur more frequently in males, whereas meningiomas preferentially affect females. Meningiomas account for 43 per cent of primary intracranial tumours in women as compared to only 22 per cent in men.27 In spinal meningiomas, the preferential occurrence in women is even stronger, with female/ male ratios in modern surgical series ranging from 3 to 9:1.109,122,152 Another striking preferential occurrence is germinoma of the pineal region, which occurs approximately 10 times more frequently in boys than in girls.108

Regional, Socioeconomic and Genetic Variation Descriptive epidemiological studies show some geographical variation in the incidence of brain tumours, which generally tends to be highest in developed, industrial countries.132 1623

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1624  Chapter 26  Introduction to Tumours

In Western Europe, North America and Australia, there are between 6 and 19 new cases of primary intracranial tumours per 100 000 inhabitants each year.27,57,132 Whereas lower frequencies were previously reported, similar incidence rates are now also seen in Japan.121 In multiracial countries, Caucasians are affected more frequently than people of African, Hispanic, or Asian descent and this difference has also been observed in children, with some studies further suggesting genetic differences in gliomas among racial groups.167,186 For example, glioblastomas and germ cell tumours are 3.5 times more frequent in the USA in Caucasians than in African Americans.45 Socioeconomic associations have been found in some studies, but not in others.8,84 Molecular epidemiological studies over the past decade have suggested possible links between inherited genetic polymorphisms and brain tumour development. These are based on the logical premise that functional variability in genes (such as those involved in DNA repair or detoxification processes) across the population could predispose certain individuals to brain tumours and could affect the response of some people to individual therapies (the latter known as ‘pharmacogenomics’). The most recent approach utilizes genome wide association studies (GWAS) with thousands of single nucleotide polymorphisms (SNPs) applied to large cohorts. For instance, one international series evaluating 75 glioma families found evidence of Mendelian inheritance and a susceptibility locus at 17q12-21.32.161 Other GWAS have discovered and confirmed a number of risk alleles (some common and others rare), with ­ increasing cumulative risk found with increasing ­ numbers of risk alleles present. Of interest, several of these risk loci involve genes already known or suspected to be c­ritical in glioma biology, including 5p15.33 (TERT), 7p11.2 (EGFR), 9p21.3 (CDKN2A/CDKN2B) and the TP53 polyadenylation signal, plus several others for which the gene functions have yet to be determined with respect to gliomagenesis, including 8q24.21(CCDC26), 11q23.3 (PHLDB1) and 20q13.33 (RTEL1).56,153,160,165,187 Most recently, ­finemapping of the 8q24.21 region revealed a variant tightly associated with risk of oligodendroglial tumours and IDH–mutant astrocytic gliomas, with odds ratios ranging from five- to six-fold risk for people carrying the risk variant in rs55705857. This result was confirmed in independent series of glioma cases and controls.88 Approximately 40 per cent of glioma cases of the above types carry this variant compared to only about 8 per cent of controls or people with other types of glioma. Some further suggest that similar germline polymorphisms involving SSBP2 (single-stranded DNA-binding protein 2) on 5q14.1189 and DNA repair enzymes, such as LIG4, BTBD2, HMGA2 and RTEL1 genes, are associated with survival times in glioblastoma patients.103 Polymorphisms in DNA repair genes have similarly been implicated outside the setting of GWAS, including ERCC1, ERCC2, XRCC1, XRCC3, MGMT, PARP1, RAD51 and XRCC7.41,102,104,115,179,193 To confirm these associations, however, larger studies are required. Various individual candidate genes have also been explored, with mixed results. For example, a number of studies have evaluated glutathione S-transferase 1 (GSTM1), because gene deletions have been associated with increased risk for e­nvironmentally induced cancers (e.g. smoking-related lung cancer). Despite

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some earlier suggestions of an association, however, a recent meta-analysis of 12 published studies concluded that there is no increased risk between polymorphisms of GSTM1 or other GST variant haplotypes and glioma risk.191 It is also likely that there are complex interactions between race/ethnicity, genetic polymorphisms, tumour genotype, patient age and gender.186 It has long been observed that TP53 mutations are more common in astrocytic tumours from younger adults and, to some extent, from females.107 In a study of TP53 mutations from a population-based study in the San Francisco Bay area, astrocytomas were much more likely to harbour TP53 mutations in non-white (African American and Asian) than white (Latino and non-Latino) patients with both TP53 mutations and EGFR amplifications being considerably less common in carriers of the MGMT variant 84Phe allele.186 Similarly, younger patients are more likely to develop low-grade gliomas and secondary glioblastomas with mutations of IDH mutations, CIC, and/or PDGFRA, as well as chromosome 1p/19q co-deletions, whereas older adults are more prone to primary glioblastomas with EGFR gene amplifications and monosomy 10.62,70,77,116,190

Aetiology With the exception of inherited neoplastic syndromes (see Chapter 44, Hereditary Tumour Syndromes) and prior irradiation, the aetiology of human brain tumours is still largely unknown. Numerous epidemiological studies have been performed, but most associations with environmental, dietary and lifestyle factors either have not been statistically significant or are inconsistent.125,129

X-Irradiation X-ray irradiation (both therapeutic and non-therapeutic) is the sole environmental factor clearly associated with an increased risk of brain tumours (also see Chapter 46, Reactions to Antineoplastic Therapies). In most reported cases, radiation was administered for treatment of the fungal disease tinea capitis or of a cranial tumour unrelated to the radiation-induced neoplasm, although epidemiological studies following atomic bomb detonations in Japan have similarly provided compelling evidence.136,192 Radiation-induced meningiomas and other tumours (schwannomas, gliomas, sarcomas and embryonal tumours) have been most frequently observed after lowdose irradiation for tinea capitis,175 and after high-dose radiation for primary brain tumours.6,33,151 Typically, they arise within the field of irradiation. Multiple and highgrade (II or III) lesions appear to be more frequent than among sporadic meningiomas, with latency being roughly inversely linked to radiation dose. Sarcomas (mostly fibrosarcomas and malignant peripheral nerve sheath tumours) of the dura, meninges, or nerves are less frequent but, given their extreme rarity sporadically, are highly indicative of iatrogenic origin if diagnosed in patients with prior therapeutic irradiation.168 In a report of seven cases, the mean latency was 8 years.28 Children receiving prophylactic CNS irradiation for acute lymphocytic leukaemia (ALL) appear at particularly high risk

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  Epidemiology and Aetiology  1625

for subsequent astrocytomas (low and high grade).55,123,125 Less frequently, tumours are classified as CNS primitive neuroectodermal tumour (PNET) or other embryonal neoplasms.33,125 In a follow-up of long-term survivors, 7 of 468 children (1.9 per cent) treated for ALL (median dose, 24 Gy) developed primary intracranial neoplasms.177a In a retrospective cohort study of 14 361 children treated for cancer, there was over seven-fold overall risk of subsequently developing a CNS tumour (odds ratio [OR], 7 for glioma; 10 for meningioma), the dose response for excess relative risk being linear, such that children treated with 30–44.9 Gy of cranial irradiation peaked at 21-fold increased risk of gliomas.123 Second primary CNS tumours have similarly been observed after irradiation of pituitary adenoma,33 craniopharyngioma,15 pineal parenchymal tumours74 and germinoma,33 scalp tumours and many other lesions. Although a causative role of radiotherapy is generally acknowledged, the possible risk of diagnostic X-rays (e.g. in dental care) has been unclear. A modern series reported a 4.9-fold increased risk (95 per cent confidence interval [CI], 1.8–13.2) of meningioma for patients getting annual panorex films before 10 years of age;35 given that less irradiation is now given for dental series than in the past, it is unclear whether similar risks will apply going forward. Long-term low-dose radiation exposure of nuclear workers has not been definitively implicated to increase risk of brain tumours.23 After the nuclear accident in Chernobyl, the incidence of childhood brain tumours in neighbouring Sweden did not change significantly, although further follow-up is likely needed.172

Occupational Exposure Analytical epidemiological studies have revealed an increased risk of brain tumour development in association with certain occupations,157,171 for example in physicians, farmers, dentists, firefighters, metal workers and workers in the rubber industry. Attempts to identify a specific exposure or causative environmental agent have generally been unsuccessful,125 although a recent study suggested an association between carbon tetrachloride exposure and glioblastoma.124 The somewhat increased incidence of CNS neoplasms in anatomists, pathologists and embalmers raised the possibility of a role for formaldehyde, but increased brain tumour risk has not been observed in workers exposed to formaldehyde in industrial settings.125 Similarly, multicentre cohort studies have not substantiated the hypothesis that occupational exposure to vinyl chloride carries enhanced risk of developing brain tumours.162 Polycyclic aromatic hydrocarbons have also been associated with increased brain tumour risk, including in children.39 Several studies have pointed to a positive association with farm work,18,171 possibly accounted for by herbicides, pesticides and their derivatives,10,11 but these associations remain questionable. Some studies show a slightly elevated risk for white-collar workers, including social science professionals,18 financial workers, managers and people of higher socioeconomic status, but these trends are also inconsistent. Parental exposure may also influence risk of paediatric gliomas. A slightly increased risk of brain tumours has been found in children of electrical or chemical workers or children of fathers involved in hobbies with toxic exposures, such as pesticides.114,146 Once again though, such associations remain controversial as other series have shown no increased risk.112

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Electrical and Magnetic Fields Although a weak association of brain tumours with electromagnetic field exposure has been previously reported, over 30 years of research has failed to establish a definite link.57,125,157 Similarly, although some studies have suggested that it is specifically residential electromagnetic field exposure (determined by the electrical wiring configuration around houses or electrical blankets) that leads to increased childhood cancer, including brain tumours,154,182 these observations have not been subsequently confirmed.65,127,137

26

Cellular Telephones There has been extensive media coverage addressing the question of whether cellular telephone use is associated with brain tumour risk, as well as legal cases claiming an association. Cellular phones utilize radio-frequency waves that fall between radiowaves and microwaves. Exposure is measured as the specific absorption rate (SAR), which is the amount of radio-frequency energy absorbed from the telephone into the local tissues. Radio-frequency wave exposure is related to the duration and frequency of cellular phone use, with increased use implying increased exposure.58 Scientific studies addressing the question of whether brain tumours are related to cellular telephone use have primarily involved two approaches: (1) exposure of cells or animals to a radio-frequency field similar to that of cellular phone usage, followed by measurement of various end points, and (2) epidemiological studies. In general, the studies using experimental systems to evaluate the effects of such radio-frequency exposures have not shown biological effects that are directly relevant to the situation encountered in human brain tumours. Thus, although biological effects may be found after exposure, these may have little to do with human brain tumourigenesis. More importantly, well-conducted epidemiological studies have failed to document clear associations with increased risk of gliomas, meningiomas or vestibular schwannomas, possibly with the exception of those at the highest exposure levels, although biases in study designs have prevented definitive interpretations.24,40,58,71,85,91 Furthermore, tumours have not occurred disproportionately on the side of head on which the telephone was used. Of note, one group from Sweden has reported an increased risk for brain tumours ipsilateral to the side of cell phone use, not for malignant tumours, but only when vestibular schwannomas are included in the analysis.67–69 However, these data have been called into methodological question; for example, although an increased number of these tumours was noted ipsilateral to phone use, the overall number of tumours was not increased, which would problematically suggest that cell phone use may have a protective effect against tumours contralateral to the side of cell phone use. No dose–response relationships have been noted in these studies and issues of recall bias may also be operative. For these reasons, the current literature does not support a role for cellular phone use in brain tumourigenesis. Nonetheless, it has been noted that caveats remain concerning long-term cellular phone usage, i.e. that further, long-term follow-up studies are needed to exclude long-term effects. The ongoing risk may decrease with time, given the overwhelming shift from analogue to

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1626  Chapter 26  Introduction to Tumours

digital methodologies, because radio-frequency wave exposure appears lower with digital cellular phones.58

Smoking No definite association exists between smoking and brain malignancies.80 Despite occasional reports suggesting a weak association with passive smoking in women and during pregnancy,38 no clear associations between parental tobacco smoking and the incidence of childhood brain tumours have been found. A prospective study of a large birth cohort in Sweden reported that children of women who smoked during pregnancy (particularly children in the 2–4-years age group) had an increased incidence of benign and malignant brain tumours (hazard ratio 1.24).17 In addition, another study found an association between childhood astrocytomas and paternal smoking history (OR 1.4).39 However, a meta-analysis of 12 published observational studies, representing a total of 6566 patients, showed no clear association between maternal smoking during pregnancy and risk of childhood brain tumour development.80 Similarly, a metaanalysis of 20 previously published series found no definite link between patient smoking history and glioma development.110 Nonetheless, limitations in study designs prevent definitive conclusions based on available data. Regardless, if an increased risk exists, then the effect appears small.

Dietary N-nitroso Compounds and Other Considerations Because N-nitroso compounds are potent neurocarcinogens in rodents, numerous epidemiological studies have evaluated their possible role in brain tumourigenesis. Nitroso compounds have been detected in nitrite-preserved food and in beer, but they can also be formed in the stomach after uptake of their chemical precursors, nitrate/nitrite and ­secondary amines. The results of several studies suggest that the risk of developing a primary brain tumour may be slightly higher in people with a high intake of meat, in particular cooked ham, processed pork and bacon.125 A meta-analysis of nine epidemiological studies showed that dietary cured meat intake of all types had a relative risk of 1.48, suggesting a 48 per cent increased risk of glioma in adults ingesting high levels of cured meat.81 On the other hand, the large population-based NIH-AARP Diet and Health Study found no convincing associations.51 Of interest, this same study found a potential protective effect of caffeine on glioma development, with a hazard ratio of roughly 0.70.52 In some studies, an inverse association was reported for high intake of fruit and vegetables and of vitamin C, which is known to block the endogenous formation of nitrosamines;125 in others, however, no association could be found.51 Similarly, no associations with aspirin or non-steroidal anti-inflammatory drug (NSAID) use or with nitrite in drinking water have been found.43,166

Trauma Anecdotal reports have documented the occurrence of gliomas119,173 and meningiomas7,156 at the site of prior head injury. A causal relationship is difficult to prove, although an association would be biologically plausible, because

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trauma induces a strong proliferative cellular response. Epidemiological studies have shown a weak, but inconsistent or non-significant, association with adult and perinatal traumatic head injury73,78,94,150,158 or combined perinatal and adult head trauma.66 A large, international case–­control study revealed an elevated risk with an odds ratio of 1.5 for meningiomas in male adults.138

Viruses SV40 was iatrogenically introduced on a large scale into human populations in North America and Europe between 1955 and 1962 through SV40-contaminated polio vaccines.22 SV40 sequences have been identified in a variety of human neoplasms, raising the question of a possible aetiological role.19 JC virus has an extensive nucleotide sequence homology with SV40, but the host range is distinctly different. Although SV40 does not infect human cells, latent JC infection is very common, with a serological prevalence of 40–60 per cent in most developed countries. In immunosuppressed patients, JC virus is reactivated and may cause progressive multifocal leukoencephalopathy (PML). Investigations of SV40 or JC virus in brain tumourigenesis have primarily evaluated whether viral sequences can be detected in primary tumours. However, one case–control study looked at the association between antibodies to JCV and SV40 from serum collected 1–22 years before diagnosis and incidence of primary malignant brain tumours.144 JCV and BK virus infection was high in the study population (77 and 85 per cent, respectively), whereas antibodies to SV40 were less prevalent (11 per cent). The odds ratio for subsequent brain tumour development was 1.46 for JCV, 0.66 for BKV and 1.00 for SV40. Furthermore, there were no significant differences between cases and controls in having antibodies to JCV, BKV or SV40, arguing against a major role for exposure in brain tumour risk. The literature on whether SV40 or JC virus is present in human brain tumours has reported highly variable results. In a series of papers evaluating brain tumours, SV40 sequences were detected in approximately 35 per cent of cases.19,22,79,111 In one study, 25–56 per cent of brain tumours of Swiss patients contained SV40 sequences, but these were not detectable in a similar series from Finland, a country where SV40-contaminated polio vaccine was not used,126 consistent with the hypothesis that SV40 in human brain tumours originates from SV40contaminated polio vaccine. However, a selective increase in the incidence of brain tumours has not been reported in populations that received SV40-contaminated polio vaccine, and incidence rates for brain tumours are similar in countries that did or did not use SV40-contaminated vaccine.126 Furthermore, careful study involving expert laboratories from two large centres evaluated the prevalence of SV40, JC and the related BK viral sequences in 225 brain tumours using polymerase chain reaction (PCR) followed by Southern hybridization, as well as real-time quantitative PCR. In the face of stringent controls, the laboratory using PCR followed by Southern hybridization found only three JCV-positive, three BKV-positive and three SV40-positive cases and the laboratory employing real-time quantitative PCR found only one positive tumour (for SV40). This group concluded that JCV, BKV and SV40 are rare in brain tumours.145 When viewing the literature on this subject, one must consider that

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  Epidemiology and Aetiology  1627

small sample sizes and differences in underlying patient populations, laboratory techniques and quality control measures could all contribute to the variability of reported results.145 The role of these viruses in brain tumourigenesis therefore remains sub judice. A somewhat more compelling case has been made for the role of human cytomegalovirus (HCMV) in gliomas, although this issue similarly remains controversial. This association was first suggested in 2002 when HCMV transcripts and proteins were detected in 22 of 22 glioblastomas, 5 of 5 lower-grade diffuse gliomas, and none of the 23 meningiomas and non-neoplastic brain samples studies.37 Whereas some groups were unable to replicate these findings, the majority similarly found evidence of HCMV in glioblastomas using immunohistochemistry (IHC), in situ hybridization, PCR, electron microscopy, PCR coupled with DNA sequencing, enzyme-linked immunosorbent assay, and flow cytometry.36,54,140 The most commonly applied method is IHC with antibodies directed against the immediate early 1 (IE1) protein. Nonetheless, methodological differences may be responsible for interlaboratory discrepancies, with ultrasensitive techniques being touted as necessary for detecting relatively low levels of infection, particularly when working with paraffin embedded tissue, which not surprisingly is less reliable than frozen tissue and generally works best if the case is relatively recent. Unlike other models of viral oncogenesis, data suggest that HCMV is not involved in neoplastic transformation, but merely facilitates malignant progression. This putative role is based on evidence that viral infection or HCMV-encoded proteins promote genomic instability, cellular proliferation, angiogenesis, evasion of growth suppressors, cellular migration, replicative immortality via telomerase activation, decreased cell death via antiapoptotic activity, and induction of tumour promoting inflammation.36,54,140 Evidence also suggests that viral infection of glioma cells requires PDGFRA expression, a common finding in diffuse gliomas. Nonetheless, this association is still poorly understood and some have postulated that PDGFRA haplotype differences may be associated with variable levels of susceptibility to infection. Based on the lack of active viral production and the detected proteins being expressed, HCMV infection in human gliomas does not correspond to the classic definitions for either lytic or latent stages of disease, prompting the hypothesis that this is a ‘persistent’ form of infection. Additional evidence supporting a potential role for this virus includes: (1) a murine CMV glioma model whereby enhanced tumourigenicity and decreased survival times are reported in CMV infected versus control mice and (2) preliminary data suggesting enhanced patient survival times in two clinical trials, one from the Karolinska Institute in Stockholm utilizing the antiviral drug valgancyclovir, and the other from Duke University using autologous CMV pp65 RNA loaded dendritic cells.36,54 One observation that is difficult to reconcile is that the viral genome only appears to involve a minority of cells within the tumour, although some data suggest a possible explanation being that the stem cell compartment is preferentially targeted.140 Although many unresolved questions remain and there is clearly much work still to be done, the following statement was published by a group of experts: ‘a consensus was reached that there is sufficient evidence to conclude that HCMV sequences and viral gene

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expression exist in most, if not all, malignant gliomas; that HCMV could modulate the malignant phenotype in glioblastomas by interacting with key signaling pathways; and that HCMV could serve as a novel target for a variety of therapeutic strategies’.54 Lastly, there are rarer tumour subsets with viral associations, including that of Epstein–Barr virus (EBV) with lymphomas (see Chapter 40, Lymphomas and Haemopoietic Neoplasms) and Smooth Muscle Tumours (see Chapter 37, Mesenchymal Non-meningothelial Tumours). EBV is a member of the herpes viral family and is one of the most common human viruses worldwide. Its potential role in lymphoproliferative disorders systemically is well known and, in the brain, it is most frequently associated with primary CNS B-cell lymphomas of immunocompromised hosts, including those with AIDS, organ transplants, or immunosuppressive regimens administered for other disorders.14,26,97 It is similarly thought to play a role in very rare examples of intracranial leiomyomas and leiomyosarcomas in immunocompromised patients.29,64,164

26

Allergy and Autoimmune Disease Epidemiologic studies demonstrating inverse relationships between gliomas and allergic diseases (including asthma and eczema) and/or borderline increases in serum IgE levels have been surprisingly consistent, with overall risks of roughly 0.3 to 0.7 for most associations.20,101,113,184 Perhaps most compelling is a recent nested case–control study using archived serum specimens from Norway of 594 case subjects and 1177 controls, where IgE levels measured at least 20 years prior to diagnosis were inversely associated with subsequent risk of developing a glioma.159 Besides asthma, the most common autoimmune disease inversely associated with gliomas is diabetes (OR, 0.63).16 Of interest, one study found that prior exposure to chickenpox was also associated with lower risks for WHO grade II and III oligodendrogliomas (OR, 0.5–0.6).113 Studies of meningiomas have been less constant; whereas a relatively small study of 197 meningioma patients failed to find an association,16 a more recent investigation of 1065 patients reported a similar inverse association with allergies (OR, 0.64) as seen in gliomas.185 Overall, the observed relationships between allergies, autoimmune disease and brain tumours suggest a role for immune regulation.

Immunosuppression Epidemiological studies previously found an increased incidence of primary CNS lymphoma, although this may have peaked in the mid-1990s, subsequently dropping dramatically in HIV patients as a result of the efficacy of HAART therapy, but still continuing to rise slightly in immunocompetent elderly patients.75,147,177 In non-treated or poorly treated HIV patients, the acquired immunodeficiency syndrome (AIDS) is associated with an increased risk of developing primary malignant CNS lymphomas, with the risk estimated up to 3600-fold higher for patients with AIDS than the general population.40a Most examples are EBVpositive diffuse large B-cell lymphomas.14,47,83 Primary involvement of the CNS has been found to occur in 22 per cent of patients with post-transplant

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1628  Chapter 26  Introduction to Tumours

non-Hodgkin’s lymphoma.135 The risk appears lower for patients with renal transplants (1–2 per cent) than for those with heart, lung or liver transplants (2–7 per cent).155a It has been estimated that approximately 2 per cent of all patients with prolonged immunosuppression develop primary CNS lymphomas and those with iatrogenic immunosuppression are similarly at risk.97,133

Familial Brain Tumour Syndromes and Clusters A number of hereditary syndromes feature nervous system tumours; these are discussed at greater length later (see Chapter 44, Hereditary Tumour Syndromes). The syndromes and their respective nervous system tumours include neurofibromatosis 1 with diffuse astrocytomas, pilocytic astrocytomas, neurofibromas and malignant peripheral nerve sheath tumours; neurofibromatosis 2 with schwannomas, meningiomas, meningioangiomatosis and ependymomas; schwannomatosis with multiple schwannomas; tuberous sclerosis with subependymal giant cell tumours; von Hippel–Lindau disease with haemangioblastomas; Li–Fraumeni syndrome with malignant gliomas and PNETs; Turcot syndrome with malignant gliomas and PNETs; Cowden syndrome with dysplastic gangliocytoma of the cerebellum (Lhermitte–Duclos); basal cell naevus (Gorlin) syndrome with medulloblastoma; and a host of less common syndromes. For nearly all of these conditions, the respective causative genes have been identified and this knowledge has provided invaluable information concerning the molecular basis of brain tumourigenesis. Non-syndromic clusters of brain tumours have been reported in families, but it is unclear whether such clusters arise because of genetic or environmental factors. A study of 154 patients from 72 families revealed that parents and children were affected in 33 families, siblings in 27 families, and spouses in 12 families. Notably, these tumours did not involve multiple generations or present at early age. In addition, the cases tended to cluster in time, with 47 per cent of the familial and 50 per cent of the husband–wife cases occurring within a 5-year span. These data suggested that environmental exposures may explain such clustering.63 Another study of 25 546 relatives of 396 patients with glioma found no statistically significant increase in gliomas or other CNS tumours in these relatives.128 Nonetheless, a report of 5088 these relatives of 639 probands with gliomas favoured a multifactorial mendelian model and rejected a model postulating a purely environmental cause.46 Another study of the Utah Population Data Base similarly suggests an increased heritable risk in first-degree relatives of astrocytoma patients.13 It is therefore likely that such clusters represent a combination of multigenic and environmental causes.

Animal models of nervous system tumours The use of animal models has played an important role in understanding key pathways in brain tumourigenesis and in providing preclinical systems for evaluation of potential therapies. Although there are clear differences between each model and the human counterparts, knowledge of

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these models provides selected but important insights into human brain tumour formation and progression, and possibly response to therapy. Spontaneously occurring nervous system tumours in animals are neither common nor stereotypical in their presentation, thus creating a substantial need for experimental tumour models. Animal models of nervous system tumours have been undertaken with a number of goals: (1) to identify environmental chemicals or viruses involved in tumour aetiology; (2) to elucidate molecular pathways operative in tumour initiation and progression; and (3) to provide preclinical models for novel therapeutic testing. Most animal modelling has shifted decidedly towards transgenic mouse models, also known as genetically engineered mouse models (GEMMs) because these can recapitulate some of the molecular and phenotypic characteristics of the human tumours. In general, such models hold greater promise for elucidating pathways and for providing as relevant preclinical models as possible. Nonetheless, xenograft models are still in common use and there is a long productive history in experimental models to evaluate chemicals and viruses that might be involved in brain tumour aetiology. The following section discusses spontaneous neoplasia, tumour xenografts, older models based on chemical or viral tumourigenesis and the transgenic approaches that are now widely used.

Spontaneous Nervous System Neoplasia in Animals Spontaneous nervous system tumours are uncommon in animals. Some strains of mouse and rat have slightly elevated rates of primary brain tumours. This has assumed importance primarily in the determination of whether particular carcinogenic agents generate tumours above the baseline rate. Given their low incidence, such tumours are not of experimental value, although cell lines can be generated from these for subsequent use. Glial and meningeal tumours are moderately common in dogs and cats, and are occasionally used for experimental purposes or for genetic comparisons with human counterparts.48,72 For instance, cell lines derived from spontaneous canine gliomas can be used to generate intracerebral xenografts in dogs rendered immunotolerant after allogeneic, subcutaneous tumour growth. It has been suggested that such a model could permit evaluations of new glioma therapies for brain tumours that would not be feasible in smaller, immunocompromised or inbred animals.9 Additionally, these tumours closely resemble the human counterpart.21 The expense of using this model and the perceived difficulties of working with domestic dogs has, however, prevented widespread use. Domestic dog species are highly inbred, as a result of a directed policy pursued by breeders over the past few hundred years. As a result, different breeds have different propensities to individual diseases. In dogs, brachycephalic (short-snouted) breeds have a predisposition to gliomas, whereas dolichocephalic (long-snouted) dogs more commonly get meningiomas. These canine tumours bear striking similarities to their human counterparts. Importantly, the characterization of the dog genome now creates the possibility of linking disease predisposition to the various

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  Animal Models of Nervous System Tumours  1629

polymorphic loci that determine breed specificity and presumably breed disease predilection.131 It therefore remains possible that critical insights into human gliomas and meningiomas will be derived from studies linking dog brain tumours to specific genetic loci.

Xenograft Models Xenograft models of brain tumours have been the core of experimental and preclinical brain tumour work. Most xenograft models employ well-established cell lines, either human or rodent, that are transplanted in either the brain (orthotopic) or heterotopic sites (mostly subcutaneous). Not surprisingly, the shift has been towards the former, ­coupled with sensitive bioluminescence techniques for monitoring tumor growth.130 Many of these have been characterized for key genetic changes.86 Xenograft models are highly reproducible in that tumours nearly always develop and follow stereotypical time courses, making them valuable for evaluating new therapies; indeed, most preclinical trials conducted over the past few decades have been based on xenografts. They are also relatively simple and inexpensive, not requiring complicated mouse transgenesis. However, xenograft models suffer from disadvantages as well. Nearly all glioma xenograft models are non-invasive; because glioma invasion is perhaps the cardinal feature impeding effective therapy in humans, this is a major modelling flaw and many agents that appear promising in xenografts do not fare well in human trials. In addition, some murine lines were originally induced via chemical mutagenesis (see under Chemical Models, this page), other lines appear more sarcomatous than glial and most widely used human lines have been passaged for decades. For these reasons, their direct relevance to human brain tumours remains an open question. Finally, xenografting itself raises immunological issues. For example, human cell lines transplanted into rodents will survive only in immunocompromised animals, creating a substantial complicating variable for translation to the human condition. Major advances have recently made xenograft models more representative of the human counterparts. For instance, although most glioblastomas with gene amplifications lose amplification when grown in vitro, serial passaging as subcutaneous flank xenografts in nude mice yields tumours with retained EGFR- and PDGFRAamplified status.60 Importantly, when transplanted from the flank via short-term culture to the brain of nude mice, these lines create tumours that infiltrate into the surrounding brain. The tumours have glial characteristics, but they show necrosis only rarely and do not display microvascular proliferation. Additionally, xenograft models have been important in formulating cell origin hypotheses and the concept of the glioma stem cell (GSC), a putative cell capable of self-renewal and recapitulation of the histopathological and molecular heterogeneity seen in the tumour of origin. Although controversial, GSCs share some properties with non-neoplastic neural stem cells (NSCs), often expressing early developmental markers, such as CD133, Nestin, SOX2, OLIG2, Bmi1, Nanog, CD44, CD15 and integrin alpha 6.31 For instance, one study demonstrated that as few as 100 human CD133-positive GSCs injected into brains

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of immunodeficient mice could generate viable xenografts, whereas as many as 100 000 CD133-negative cells could not.163 In another example, transduction of mutant N-myc into NSCs from forebrain followed by orthotopic transplantation resulted in formation of gliomas, whereas the same approach using NSCs from cerebellum or brain stem yielded medulloblastomas and PNETs, respectively; this study suggested that both the tumour location and timing of the NSC extraction during development influenced the type of tumour that developed.169

26

Chemical and Virus-Based Models The generation of brain tumour models in rodents through the use of chemical carcinogens or tumourigenic viruses has had a long and productive history in neuro-oncology. Such models suffer, however, from being less ‘faithful’ to human disease biology than more recent transgenic approaches. The following section provides a brief overview of these models and the reader is referred to earlier editions of this text,100 as well as other reviews,12,149 for further information.

Chemical Models Alkylating agents are the most effective chemicals for inducing CNS neoplasms in animals. Nitrosourea derivatives, particularly methylnitrosourea (MNU) and ethylnitrosourea (ENU), cause a high incidence of CNS neoplasms in rats after systemic administration.49 Ethyl-nitrosourea and related ethylating agents (1,2-diethylhydrazine, 1-phenyl-3,3-diethyltriazene) are particularly powerful when administered as a single dose transplacentally or shortly after birth.87 The susceptibility of the rat CNS to these agents begins at the tenth prenatal day (E10), increases gradually and reaches its maximum at birth, when a single dose is approximately 50 times more effective than in adult rats. N-nitrosomethylurea and related methylating agents (1,2-dimethylhydrazine, 1-phenyl-3,3-dimethyltriazene) are less effective transplacentally, but induce tumours after repeated administration of small weekly doses to adult rats.50,139,170 Of particular interest have been ENU-induced schwannomas of cranial and peripheral nerves, which invariably contain a T : A to A : T transversion at nucleotide 2012 (codon 664; Val to Glu) in the transmembrane domain of the neu proto-oncogene; however, no neu mutations have been found in human schwannomas.155

Oncogenic Viruses and Viral Oncogenes Earlier studies utilized oncogenic viruses to induce brain tumours, whereas more recent approaches have expressed specific viral oncogenes in a transgenic and hence more directed fashion. The use of oncogenic viruses generates brain tumours of various sorts, depending on the virus and mode of delivery, but the relevance of such models has been questioned by the lack of direct epidemiological evidence linking such viruses to human brain tumours. Nonetheless, several oncogenic viruses induce a high incidence of tumours in rats after postnatal intracerebral injection. These are discussed briefly later, but the reader is referred to earlier ­editions of this text,100 as well as other reviews,12,149 for further information. Directed expression of viral ­oncogenes has found more favour in

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1630  Chapter 26  Introduction to Tumours

that these oncogenes affect specific pathways involved in human brain tumourigenesis, but models have shifted to direct genetic manipulation, as discussed later (see under Transgenic Mouse Models, below). As discussed earlier (under Epidemiology and Aetiology), there have been intriguing suggestions that SV40 virus may have a role in brain tumourigenesis. This DNA virus and its transforming gene, large T antigen, exert oncogenic effects through binding and inactivation of two key cellular regulatory molecules: p53 and pRb (as well as the other pocket protein members of the pRb family). When inoculated intracerebrally in newborn hamsters, SV40 induces choroid plexus papillomas and ependymomas.53 Transgenic expression of SV40 large T antigen can generate PNETs, retinoblastomas or choroid plexus tumours, depending on the promoter utilized.59 Directed expression of a mutant large T antigen (T121) that specifically inactivates pRb family members, under control of the GFAP promoter, results in high-grade astrocytic tumours.188 JC virus has an extensive sequence homology with SV40. However, whereas SV40 does not infect human cells, latent JC infection is common, with a serological prevalence of 40–60 per cent in most developed countries. In immunosuppressed patients, JC virus is the cause of progressive multifocal leukoencephalopathy. JC virus causes brain tumours, mostly PNETs, after intracerebral injection in newborn hamsters.178,196 Two Colombian owl monkeys inoculated with JC virus derived from human PML cases developed brain tumours after 16–25 months that closely resembled human astrocytomas.105 Transgenic mice expressing the early region of JC virus developed medulloblastoma/PNET in the cerebellum and the surrounding brainstem at 9–13 months of age.99 The BK virus, which has extensive sequence homology to JC and SV40 virus, primarily affects other organs such as the urinary tract, but can induce choroid plexus papillomas and ependymomas after intracerebral injection in newborn Syrian golden hamsters.174 Polyoma virus can be tumourigenic when inoculated at high titre into newborn mice. A variety of tumours can be induced, including gliomas in rats.1 Intracerebral injection of various strains of Rous sarcoma virus (RSV) can produce intracranial neoplasms in a variety of animals. The resulting tumours may be malignant astrocytomas or meningeal sarcomas, depending on the site of inoculation.12 Simian adenovirus (SA7) has induced choroid plexus papillomas and medulloblastomas in the hamster.32,117 Transgenic mice expressing human papillomavirus (HPV16) E6 and E7 open reading frames under control of the human beta-actin promoter developed brain tumours at a penetrance of 71 per cent between 2.5 and 10 months of age.2a Most frequent were anaplastic neuroepithelial tumours associated with the ependyma of the third ventricle; other tumours were choroid plexus neoplasms and pituitary carcinomas.

Transgenic Mouse Models Transgenic mouse models have revolutionized experimental neurooncology. These models all employ genetic manipulation to generate mice that are highly susceptible to specific brain tumour types. There are numerous approaches to generating such mice. Some of the key issues relating to

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Box 26.1.  Considerations in evaluating mouse brain tumour models Is the genetic manipulation that results in the mouse brain tumour also found in human brain tumours? Is there a reasonable genotype–phenotype correlation between mouse and human disease? Does the model overexpress a tumour-promoting oncogene or inactivate a tumour suppressor gene? Furthermore, is overexpression to physiological or supraphysiological levels, and is inactivation complete or partial downregulation? Is the nature of the genetic manipulation homozygous or hemizygous? This applies not only for the original genetic manipulation, but also for subsequent experiments in which one line is crossed into a second transgenic line. Additionally, somatic events must be evaluated to determine if, for example, the second copy of a hemizygously inactivated tumour suppressor has been lost during the process of tumourigenesis. Is the genetic manipulation in all cells or is it confined to a particular cell type or set of cell types? In this regard, the use of cell-specific promoters can direct an oncogenic effect to one organ and cell type. Is the genetic deregulation constant or inducible? Use of a particular promoter will direct expression or inactivation to cells when that promoter is ‘turned on’. However, use of an inducible promoter, which involves addition or removal of a compound from the animal's drinking water, can turn on and/or turn off gene expression according to an experimental protocol. Another method to induce genetic deregulation at a specific time involves introduction of viruses bearing oncogenes or other genes that induce subsequent genetic consequences (e.g. Cre recombinase). Because the deregulation of some genes during development can be lethal or can produce a phenotype altered in many undesirable ways, inducible systems may allow distinctions to be made between developmental and non-developmental effects. Are there strain-specific effects on the expression of the phenotype? Some genetic manipulations produce gliomas in one genetic strain, but nerve sheath tumours in another.141 How penetrant is the model? If the model is not highly penetrant, can the phenotype be clearly separated from age-­ dependent tumour development in inbred strains?

evaluating the models are listed in Box 26.1. The optimal preclinical model would: (1) accurately reproduce the human phenotype histopathologically and molecularly, (2) deliver short latency and high penetrance, (3) be easy to generate and utilize, and (4) incorporate a built-in molecular mechanism to assess therapeutic efficacy, such as a bioluminescent reporter.82 Achieving all of these in one model has not been possible to date and one must consider that even in primary human tumours, genomic studies have revealed multiple molecular subsets for each diagnostic category, such that there are now attempts at modelling individual genetic subtypes. Therefore, each model offers its own distinct advantages and disadvantages, although testing prospective therapeutic agents on multiple genetically distinct GEMMs would likely provide the highest yield and reduce potential false negatives. Additionally, there has been an increasing recognition that not only the neoplastic cells themselves, but also their microenvironment is critical in determining tumour development and phenotype.82,120,148 For instance, data suggest that Nf1 heterozygous (one mutant and one wild-type allele) inflammatory cells, such as mast cells and

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  Animal Models of Nervous System Tumours  1631

microglia, are necessary for the formation of neurofibromas and optic gliomas in GEMMs.5,194 To date, GEMMs have been generated using many approaches. Modelling has focused most heavily on malignant gliomas, although GEMMs have been generated for a wide variety of tumour types. Some of these models have been more ‘true’ to the human condition, either in their genetic makeup or in their histological characteristics, than others. Some of the more relevant models of gliomas, medulloblastomas, nerve sheath tumours and meningiomas are covered elsewhere and the reader is referred to a number of reviews for additional information.25,31,82,90,118,120,180 Models of other nervous system tumours are discussed in subsequent chapters.

Malignant Gliomas The suggested approach to classification of GEM gliomas follows the nomenclature of the WHO ­systems.180 GEM high-grade astrocytomas have been generated using a wide variety of approaches,82 many employing GFAP promoters to encourage oncogenic events in astrocytic cells. Whereas most strategies targeting only a single tumour suppressor or oncogene have failed, highly efficient tumourigenesis has generally been achieved by combining activation of receptor tyrosine kinase (RTK) pathways with dysregulation of the cell cycle,31 with some models appearing more discrete and others more infiltrative as in the human counterpart. Further incorporation of progression associated alterations, such as Pten loss, generally results in highgrade gliomas exclusively. Most recently, GEM glioblastomas have begun to be used to understand the role of cancer stem cells and the relationship of such cells to chemotherapeutic resistance in glioblastomas.2,30 More focal malignant gliomas have been generated via combined inactivation of the Nf1 and Tp53 genes. One approach generated mice with heterozygous inactivation of Nf1 and Trp53 in a cis configuration (residing on the same chromosome). These animals developed a range of histologically typical low-grade astrocytomas to glioblastomas with a penetrance of 100 per cent at 6 months.141 Strikingly, this was strain-specific, because the same transgenic manipulation in a different strain resulted in malignant peripheral nerve sheath tumours.34 A more directed approach was achieved via inactivation of both Nf1 and Trp53, specifically in astrocytic cells or their precursors, with GFAP driving Cre expression.195 Low-grade astrocytomas developed in 100 per cent of these mice, eventually progressing to high-grade tumours with all of the classic histological findings of glioblastoma. For these reasons, such mice may prove to be a highly useful model of astrocytoma formation and progression. The most directed approach to yield high-grade astrocytomas that closely recapitulate the histological features of human glioblastoma involves the avian retrovirus RCAS system.76 In this approach, transgenic expression of an avian retroviral receptor is driven by either the nestin or GFAP promoter; viruses expressing specific oncogenes are then introduced via intracerebral ­injection. Only those cells expressing the retroviral receptor can be infected by the virus and express the oncogene. Mimicking activation of the EGFR pathway by

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introducing activated Ras and mimicking inactivation of the PTEN pathway by introducing Akt expression, both in nestin-expressing progenitor cells, produces histologically classic glioblastomas in about one-quarter of mice by approximately 2 months of age.76 Other methods have been utilized to generate astrocytoma models, including introduction of activated EGFR into cultured cortical astrocytes from Ink4a-deficient mice, recapitulating two cardinal events in glioblastoma formation (EGFR overexpression and p16 deletion).3 Overexpression of EGFR drives these astrocytes to a less differentiated state. Remarkably, these cells will then generate tumours of divergent (astrocytic, oligodendroglial and neuronal) differentiation when introduced into nude mouse brains. Oligodendrogliomas have resulted from a variety of genetic manipulations. Driving v-erbB expression (homologous to EGFR) under control of the S-100B promoter generates diffuse tumours that are histologically identical to oligodendrogliomas. Crossing these mice into an Ink4adeficient or Tp53-deficient background then produces high-grade oligodendrogliomas.181 Using the RCAS system, oligodendrogliomas have been produced by overexpressing PDGF-B in nestin-expressing neural progenitors, inducing oligodendrogliomas in about 60 per cent of mice by 3 months of age; on the other hand, PDGF transfer to GFAP-expressing astrocytes more often produced oligoastrocytomas, in about 40 per cent of mice by 3 months. Additional loss of Ink4a, as in other models, created highgrade tumours.42

26

Medulloblastomas and Embryonal Neoplasms Medulloblastomas and other embryonal neoplasms have been generated using a variety of genetic manipulations.118 One of the most relevant to human medulloblastoma has been heterozygous inactivation of the Ptc gene, which results in medulloblastoma in fewer than 20 per cent of mice.61 Penetrance jumps to over 95 per cent and is accelerated to under 12 weeks of age in mice that lack p53.183 Other groups have targeted a variety of other molecules in various combinations, including Tp53, Rb1, Shh, SmoA1, Inc4c, N-Myc and c-Myc producing models of medulloblastoma and/or CNS PNET,118 with overexpression of the latter often associated with large cell or anaplastic features.134 For atypical teratoid/rhabdoid tumour, a Smarcb1+/− GEMM has been reported, although the majority of tumours develop in the soft tissue rather than the CNS.142

Nerve Sheath Tumours Genetically engineered murine nerve sheath tumours have primarily involved genetic inactivation of Nf1, Nf2, Smarcb1 and Prkar1a, mimicking human neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), schwannomatosis and Carney complex, respectively; a full description of these models is beyond the scope of this text and the reader is referred to an excellent review.25 As mentioned, the microenvironment appears to be key in some of these models and Schwann cell conditional knockouts are needed in others. Similar to GEM gliomas, strain background similarly has an influence in GEM nerve sheath tumours.

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1632  Chapter 26  Introduction to Tumours

Classification and grading Classification and Grading Systems Tumours of the nervous system present a bewildering variety of histological appearances. Therefore, it is not ­ surprising that many attempts have been made to produce classifications that accurately reflect prognosis and response to particular therapies. The aim to produce such a classification has been frustrated not only by lack of knowledge concerning histogenesis and tumourigenesis at a biological level, but also by the variably subjective nature of histological parameters. The classification of neoplasms can be based on morphological features, biological behaviour, cells of origin, histological resemblances, expression of particular molecules and genetic abnormalities. The introduction of modern investigative methods into neuro-oncology has been instrumental in devising classifications that take more than one aspect of each tumour into consideration. Electron microscopy, tissue culture, immunohistochemistry, biology and molecular genetics have all substantially contributed to the understanding of the neoplastic process and, in doing so, have greatly improved the definitional criteria on which tumour classifications are based. It must be emphasized that tumour classification is a dynamic process that is nestled between pathological approaches to diagnosis, radiological techniques, biological advances and improvements in therapy. Classification can, and must, change in response to improved understanding and abilities in each of these areas. For example, the advent of a new effective therapy raises the possibility that particular histological or molecular features correlate with response to the new therapy. On the other hand, molecular advances now commonly outpace clinical neuro-oncology. For instance, recent high throughput genomics data suggest that there are at least four molecular subtypes each of glioblastoma and medulloblastoma.98,143,176 Nevertheless, there are not targeted therapies available for each of these subtypes. Furthermore, although classification systems must be dynamic, they must refrain from excessive flux, in order to retain stability in the treatment of individual patients. In this regard, changes in classification must be based on highquality validated data. Many classification systems have been put forth for brain tumours over the past 100 years.4,92,95,96,149,197,198 In 1979, the WHO published histological typing of tumours of the CNS.197 It was subsequently revised multiple times by teams of experts with subsequent publications in 1993, 2000 and 2007.95,96,108 The WHO classification adopted the basic principle of histological typing: tumour entities are defined primarily by morphological appearances, including constituent cell types and tissue patterns. However, the results of modern investigative techniques, particularly those of genetics and immunohistochemistry, were taken into consideration when available. The overall aim was to classify, whenever possible, neoplasms according to their biological properties. That being said, there are numerous, mostly rare entities for which no significant biological information exists, and these tumours are classified solely on the basis of their light-microscopic characteristics. In this regard, it must be recognized that histological appearances may not reflect

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cells of origin, i.e. that a tumour having cells resembling astrocytes does not necessarily result from transformation of astrocytes. Grading attempts to assign numerical values to expected ‘natural’ biological behaviours. This immensely complex problem is unlikely to be sufficiently addressed by simply assigning histological grades to intracranial neoplasms ranging from I (benign) to IV (most malignant). This numerical system, based on the similar grading of carcinomas, was first developed by Kernohan and colleagues.93 The revised classification has adapted these criteria within the framework of the internationally accepted coding system.108 However, the grading of rare or newly defined tumour entities requires continuous reassessment, because their ­biological behaviour becomes clearer with longer follow-up studies of larger cohorts. Two general points should also be emphasized. First, histological variations within the same tumour may render grading of small biopsies a treacherous exercise, a difficulty that may be overcome by multiple, image-guided stereotactic biopsies and close communication between the neuropathologist and the neurosurgeon. Second, the importance of factors independent of histology should not be overlooked in prognosis: patient age, extent of surgical removal and clinical performance status are the most important prognostic factors for high-grade malignant gliomas.

Clinical Approach to Brain Tumour Diagnosis Brain tumour diagnosis by the pathologist is based on amalgamation of a wide variety of data derived from clinical information and specialized assays. In general, knowledge of clinical history, radiological features, neurosurgical findings and histopathological evaluation is required for accurate diagnosis. In many cases, immunohistochemistry and special stains provide valuable ancillary information. Less commonly, electron microscopy is used. Increasingly, molecular diagnostic assays also provide valuable diagnostic, prognostic, or even predictive information. The basic features of each of these parameters are discussed later. From a clinical point of view, the following information must be found before evaluating a brain tumour sample: patient age and gender; the location of the tumour; neuroradiological findings; and pertinent past medical history. For example, with knowledge of patient age and tumour location, the differential diagnostic possibilities can be narrowed and prioritized to a remarkable degree. Neuroradiological features, including the exact location, nature of the margins and imaging characteristics (e.g. enhancing versus nonenhancing, pattern of enhancement, perfusion and diffusion weighted imaging data, etc.), also allow prioritization of the diagnostic possibilities. Moreover, imaging features may provide information not available from the tissue sample; for instance, the finding of a ring-enhancing parenchymal tumour on neuroimaging in the presence of a WHO grade II or III astrocytoma on biopsy argues strongly that a glioblastoma has been sampled inadequately. Histochemical stains do not play a major role in the classification of brain tumours, but may be of help to

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  References  1633

demonstrate reticulin or mucin or other structures of diagnostic importance. Immunohistochemistry, on the other hand, is of great importance in tumour classification and, to a lesser extent, tumour grading by use of proliferation markers. Most immunohistochemical assays evaluate lines of differentiation, for example GFAP in glial tumours and cytokeratins in epithelial malignancies. This application is covered in the following chapters, when each of the entities is discussed. It should be borne in mind that ‘differentiation’ in malignant tumours may be biologically distinct from normal development and that tumours may undergo divergent differentiation. In this regard, one should not conclude tumour cell of origin from studies of tumour differentiation. The need for electron microscopy has diminished in diagnostic neuropathology with the advent of improved immunohistochemical approaches and the emerging role of molecular genetic analysis, but may still be of great utility in rare settings, such as histologically atypical ependymal neoplasms or poorly differentiated tumours in general. Molecular analysis has made considerable inroads into diagnostic tumour neuropathology. The application of molecular genetics to neuro-oncology is well established in the field of lymphoma diagnosis, where monoclonality of immunoglobulin and T-cell receptor gene rearrangements may be diagnostically useful. Other molecular alterations characterize particular tumours and can be used for ­diagnosis, such as specific translocations in sarcomas or chromosome 22 loss and INI1 mutations in atypical teratoid/rhabdoid tumours (ATRT). In addition to diagnostic aid, some markers are associated with enhanced prognosis, such as the presence of MGMT promotor methylation in glioblastomas or IDH gene mutations in diffuse gliomas in general. Most widespread are assays for parameters that potentially affect treatment decisions such as the type of chemotherapy or radiation therapy; 1p and 19q testing in oligodendroglial tumours conforms to this scenario, as may EGFR analysis in non–small cell lung carcinoma or ER,

PR and HER2 studies in metastatic breast cancer. Of interest, some assays initially assessable only through molecular techniques may now be interrogated with standard immunohistochemistry. Examples include the loss of INI1 protein expression in ATRT and the detection of the R132H mutant IDH1 protein in diffuse gliomas. The following two sections closely follow the 2007 WHO classification. It should be stressed, however, that not all tumours fit well within the standard WHO entities. Not uncommonly, individual brain tumours defy exact classification. In such a situation, it makes little sense to attempt to force a lesion into one of the existing entities, because the behaviour of the tumour may not correspond with that of the existing entity. For tumours that do not conform to the WHO definitions, descriptive diagnoses suffice, as long as they convey the critical prognostic and predictive information needed by the clinician. Caveats must also be issued regarding WHO grades because these primarily reflect ‘natural history’. Grade I tumours are benign in the sense that, if they can be resected, they can be cured, but not all grade I brain tumours can be resected, particularly those lying deep within the brain. Grade IV tumours, at the other end of the spectrum, are highly malignant tumours with a generally poor prognosis, but the natural behaviour can be altered radically using available therapies (e.g. medulloblastoma). Furthermore, although all tumours can be assigned a WHO grade, most clinicians only utilize grades in the management of gliomas, particularly for the astrocytic group of neoplasms.

26

Acknowledgements The authors are grateful to Dr Margaret Wrensch, Neurological Surgery and Institute of Human Genetics, University of California San Francisco, for providing valuable input regarding the latest GWAS and allergy study data.

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180. Weiss WA, Israel M, Cobbs C, et al. Neuropathology of genetically engineered mice: consensus report and recommendations from an international forum. Oncogene 2002;21:7453–63. 181. Weiss WA, Burns MJ, Hackett C, et al. Genetic determinants of malignancy in a mouse model for oligodendroglioma. Cancer Res 2003;63:1589–95. 182. Wertheimer N, Leeper E. Electrical wiring configurations and childhood cancer. Am J Epidemiol 1979;109:273–84. 183. Wetmore C, Eberhart DE, Curran T. Loss of p53 but not ARF accelerates medulloblastoma in mice heterozygous for patched. Cancer Res 2001;61:513–16. 184. Wiemels JL, Wilson D, Patil C, et al. IgE, allergy, and risk of glioma: update from the San Francisco Bay Area Adult Glioma Study in the temozolomide era. Int J Cancer 2009;125:680–7. 185. Wiemels JL, Wrensch M, Sison JD, et al. Reduced allergy and immunoglobulin E among adults with intracranial meningioma compared to controls. Int J Cancer 2011;129:1932–9. 186. Wiencke JK, Aldape K, McMillan A, et al. Molecular features of adult glioma associated with patient race/ethnicity, age, and a polymorphism in O6methylguanine-DNA-methyltransferase. Cancer Epidemiol Biomarkers Prev 2005;14:1774–83. 187. Wrensch M, Jenkins RB, Chang JS, et al. Variants in the CDKN2B and RTEL1 regions are associated with highgrade glioma susceptibility. Nat Genet 2009;41:905–8. 188. Xiao A, Wu H, Pandolfi PP, et al. Astrocyte inactivation of the pRb pathway predisposes mice to malignant astrocytoma development that is accelerated by PTEN mutation. Cancer Cell 2002;1:157–68.

189. Xiao Y, Decker PA, Rice T, et al. SSBP2 variants are associated with survival in glioblastoma patients. Clin Cancer Res 2012;18:3154–62. 190. Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009;360:765–73. 191. Yao L, Ji G, Gu A, Zhao P, Liu N. An updated pooled analysis of glutathione S-transferase genotype polymorphisms and risk of adult gliomas. Asian Pacific J Cancer Prev 2012;13:157–63. 192. Yonehara S, Brenner AV, Kishikawa  M, et al. Clinical and epidemiologic characteristics of first primary tumors of the central nervous system and related organs among atomic bomb survivors in Hiroshima and Nagasaki, 1958-1995. Cancer 2004;101:1644–54. 193. Yosunkaya E, Kucukyuruk B, Onaran  I, et al. Glioma risk associates with polymorphisms of DNA repair genes, XRCC1 and PARP1. Br J Neurosurg 2010;24:561–5. 194. Zhu Y, Ghosh P, Charnay P, Burns DK, Parada LF. Neurofibromas in NF1: Schwann cell origin and role of tumor environment. Science 2002;296:920–2. 195. Zhu Y, Guignard F, Zhao D, et al. Early inactivation of p53 tumor suppressor gene cooperating with NF1 loss induces malignant astrocytoma. Cancer Cell 2005;8:119–30. 196. Zu Rhein GM, Varakis JN. Perinatal induction of medulloblastomas in Syrian golden hamsters by a human polyoma virus (JC). Natl Cancer Inst Monogr 1979;51:205–8. 197. Zülch K. Histological typing of tumours of the central nervous system. International histological classification of tumours. Geneva: World Health Organization, 1979. 198. Zülch K. Brain tumours. Their biology and pathology. Berlin: Springer, 1986.

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27 Chapter

Astrocytic Tumours Wayne JMoore Daniel Brat and Christine Stadelmann-Nessler

Introduction..............................................................................1638 Overview and Biology of Diffuse Astrocytic Tumours..................1638 Diffuse Astrocytoma (WHO grade II)...........................................1645 Anaplastic Astrocytoma.............................................................1648 Glioblastoma.............................................................................1650

Introduction Astrocytomas are central nervous system (CNS) tumours formed by neoplastic cells displaying astrocytic ­differentiation. They include the common diffusely infiltrative a­ strocytomas, as well as the less common, l­ow-grade ­circumscribed variants. Although there is occasional ­histological overlap, these two groups should be considered clinically and biologically distinct. The diffusely infiltrative astrocytomas are subdivided by degree of malignancy into diffuse astrocytoma, WHO grade II, anaplastic astrocytoma (AA), WHO grade III and glioblastoma, WHO grade IV.143 The circumscribed astrocytic tumours are distinctive clinicopathologic entities, yet share a more indolent clinical course; they include pilocytic astrocytoma, pleomorphic xanthoastrocytoma (PXA) and subependymal giant cell astrocytoma (SEGA).

Overview and biology of diffuse astrocytic tumours The astrocytic neoplasms, collectively referred to as ‘diffuse astrocytic tumours, (WHO grade II diffuse astrocytoma, WHO grade III anaplastic astrocytoma and WHO grade IV glioblastoma), are the most common primary tumours of the cerebral hemispheres in adults.52,143 They are related both clinically and biologically and share two cardinal characteristics: (1) diffuse infiltration of involved CNS structures and (2) an inherent tendency to progress to a more malignant phenotype. Also characteristic are preferential location in the cerebral hemispheres, more frequent presentation in adults than children, and a wide range of histopathological features, genetic alterations and biological behaviour.143

Glioblastoma Variants...............................................................1657 Pilocytic Astrocytoma................................................................1659 Pleomorphic Xanthoastrocytomas.............................................1663 Subependymal Giant-Cell Astrocytomas....................................1665 References...............................................................................1667

Incidence Diffusely infiltrating astrocytic tumours are the most frequent CNS neoplasms and account for more than 60 per cent of all intra-axial brain tumours.52 Although there may be slight regional variation, estimates suggest an annual incidence of 0.58, 0.36 and 3.19 per 100 000 for grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma) and grade IV (glioblastoma), respectively.52

Age and Gender Histological grade correlates directly with age of presentation. Entities within the grade I category, such as pilocytic astrocytoma and SEGA, tend to arise in childhood and adolescence. Diffusely infiltrative astrocytomas, WHO grade II, typically affect young adults, whereas glioblastoma shows a peak incidence in the sixth decade. Anaplastic astrocytoma occupies an intermediate position. Population-based studies show a mean age at diagnosis of 39 years for diffuse astrocytoma, WHO grade II and 61 years for glioblastoma.174,176 Children may also develop the entire range of diffuse astrocytomas, although less frequently than adults. Males are affected more frequently, with a male-female ratio ranging from 1.4 to 1.5:1.

Sites Diffuse astrocytomas preferentially arise in the cerebral hemispheres, particularly the frontotemporal region, often occupying the subcortical or deep white matter. However, they can occur throughout the neuraxis, including the spinal cord. Interestingly, diffuse astrocytomas rarely involve the cerebellum as a primary site. In children, the brain stem and thalamus are characteristic locations for diffuse

1638

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Overview and Biology of Diffuse Astrocytic Tumours  1639

astrocytomas. Circumscribed forms of astrocytomas tend to occur in stereotypic locations. Pilocytic astrocytomas arise most frequently in the cerebellum, brain stem, optic pathways and hypothalamus. SEGAs are almost always ­ localized to the lateral ventricle near the foramen of Monro. PXAs arise most frequently in the temporal lobes, but also in other cerebral hemispheric locations.

series reveal patient survivals of 6–8 years for diffuse astrocytomas, 2–4 years for anaplastic astrocytomas and 12–16 months for patients with glioblastoma.143,231 Populationbased studies that include all patients with disease rather than only those who qualify for clinical trials indicate survival times of 5.6 years for diffuse astrocytoma, 1.6 years for AA, and less than 6 months for GBM patients.170

Clinical Features

Cells of Origin

Focal neurological deficits are related to tumour location and may include weakness, sensory abnormalities or visual disturbance. Non-localizing signs and symptoms are also common, particularly headaches, seizures and altered consciousness. In the setting of lower-grade lesions, seizures may be present for years before the onset of other clinical signs and symptoms. Ultimately, patients develop increased intracranial pressure owing to mass effect.

The cells of origin for astrocytomas and other malignant gliomas remain enigmatic. Most glioma classifications have postulated that astrocytomas arise from astrocytes, and oligodendrogliomas from oligodendrocytes.142 However, the number of actively dividing glial cells in the normal brain is low, making this possibility unlikely. Work in both animal models and primary gliomas has suggested that malignant gliomas arise from progenitor cells (Figure 27.1).75,76 Neuroectodermal stem cells that reside in adult mammalian brains have a proliferative potential, are migratory and can pursue diverse paths of differentiation – all features intrinsic to glioma cells and likely characteristics for neoplastic cells of origin. Differential targeting of oncogenic alterations to either progenitor

Grading of Diffuse Astrocytomas and Survival Diffuse astrocytic tumours range from slowly growing, relatively localized lesions to high-grade malignancies that involve large expanses of brain. Significant indicators of increasing grade in gliomas include cytological atypia, cellular density, mitotic activity, microvascular proliferation and necrosis. The grading system that is most widely utilized is the WHO classification.143 As with other forms of neoplasia, grading of astrocytomas is based on the most malignant areas, assuming that this population drives the course of disease. Importantly, both astrocytic differentiation and tumour grading are determined morphologically and subject to interpretation.70 The diagnosis of an infiltrative astrocytoma (WHO grade II) is applied when individual tumour cells showing astrocytic differentiation infiltrate CNS parenchyma.47,143 Classification of infiltrating tumours as astrocytic versus oligodendroglial depends on cell shape, appearance and the character of the nuclei.70 In astrocytomas, nuclei are elongate, hyperchromatic and irregular, generally lacking prominent nucleoli and perinuclear halos. The histopathologic distinction of grade II from grade III depends on the identification of mitotic activity, with the finding of even a few mitotic figures sufficient to establish the diagnosis of anaplastic astrocytoma, WHO grade III. As compared to grade II astrocytoma, AA also has increased cellular density, as well as greater nuclear pleomorphism and atypia. For the diagnosis of glioblastoma (GBM; WHO grade IV), either microvascular hyperplasia or necrosis, often with pseudopalisading (or both), is required.26,143 In the past, necrosis within a malignant glioma was often viewed as the sole criterion for the diagnosis of GBM. However, studies have emphasized that vascular proliferation and necrosis are biologically linked, so that either feature can be used for the diagnosis of GBM.20,47 In addition to histological grade, patient survival depends on a variety of clinical features, including patient age29,228 and neurological status, as reflected in the Karnofsky performance score,228 tumour location and treatment, e.g. extent of surgical resection,2,15,165 radiotherapy2,128 and chemotherapy.231,232 Data from clinical trials and institutional

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27

Paediatric AA/GBM H3F3A, DAXX TP53, ATRX Primary GBM

EGFR, PDGFRA PTEN, TP53 CDKN2A/B, NF1 TERT promoter

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Diffuse astrocytoma RB1, CDK4 CDKN2A, MDM2, PTEN

Anaplastic astrocytoma, GBM

Pilocytic astrocytoma PXA

IDH1,2 IDH1-mutated infiltrating glioma

TP53 ATRX

BRAF

1p/19q CIC, FUBP1 TERT promoter

Oligodendroglioma

RB1, CDK4 CDKN2A

Anaplastic oligodendroglioma

27.1 Progenitor cells, genetic alterations and tumour differentiation. A glioma progenitor cell could arise either from a stem cell population or from a differentiating progenitor cell population. Astrocytomas can develop through numerous pathways of acquired gene mutations, amplifications and deletions. Those that are IDH mutant are clinically and biologically distinct from those that are IDH wild type. Among IDH mutant gliomas, the resultant phenotype of astrocytoma or oligodendroglioma depends on specific sets of genetic changes. TP53 and ATRX mutations are typical of astrocytomas, whereas co-deletion of 1p and 19q and mutations of CIC and FUBP1 are typical of oligodendroglioma.

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1640  Chapter 27  Astrocytic Tumours

cells or maturing astrocytes has demonstrated that glial progenitors are more permissive to oncogenic transformation than mature astrocytes. For instance, overexpressing oncogenic Ras and Akt in progenitors results in mouse brain tumours that are histologically similar to glioblastomas, yet targeting more mature astrocytic progenitors is less oncogenic.92 Furthermore, malignant gliomas themselves likely contain tumour stem cells, a relatively primitive population responsible for repopulating tumours as they develop and progress; such cells may represent transformed variants of normal neural progenitors.50,134,223 The existence of tumour stem cells may have major therapeutic implications as well, because therapies that do not ablate them will ultimately prove ineffective.

Neoplastic Transformation In many epithelial malignancies, genetic changes occurring in the progression from normal to hyperplastic to dysplastic to malignant have been identified. For diffuse astrocytic tumours, the first identifiable step is already a low-grade malignancy (WHO grade II astrocytoma) and knowledge of the earliest oncogenic events is limited. Mutations in isocitrate dehydrogenase 1 (IDH1) gene are frequent (70–80 per cent) in grade II and III astrocytomas, oligodendrogliomas, and oligoastrocytomas, as well as the GBMs resulting from progression of lower grade gliomas (secondary GBMs).177,264 Mutations in IDH2 have also been described, but are much less frequent. IDH mutations are infrequent in primary (de novo) GBMs. Because mutations are common to both grade II astrocytomas and oligodendrogliomas, it has been suggested that this oncogenic step is one of the first to occur in the development of diffuse gliomas, with later genetic alterations determining astrocytoma or oligodendroglioma lineages, such as TP53 mutations and 1p/19q losses, respectively (Figure 27.1). IDH mutations lead to the production of the oncometabolite 2-­ hydroxyglutarate, which ­ inhibits the function of numerous α-ketoglutarate–dependent enzymes.243 Inhibition of the family of histone demethylases and the TET family of 5-methylcytosine hydroxylases has profound epigenetic effects and leads directly to a hypermethylator phenotype that has been referred to as the CpG Island Methylator Phenotype (G-CIMP).167 Importantly, over 90 per cent of IDH1 mutations in the diffuse gliomas occur at a specific site and are characterized by a base exchange of guanine to adenine within codon 132, resulting in an amino acid change from arginine to histidine (R132H). Because of this consistent protein alteration, a monoclonal antibody has been developed to the mutant protein, allowing its detection in paraffin-embedded specimens (mIDH1R132H).37 The reported sensitivity and specificity for identifying IDH1 mutant gliomas using this antibody was 94 and 100 per cent, respectively. Furthermore, the ability of this antibody to detect only minor mIDH1R132H populations makes this method more sensitive than sequencing in specimens with minimal tumour. The p53 tumour suppressor protein encoded by the TP53 gene on chromosome 17p plays a central role in numerous cellular processes, including cell cycle arrest, response to DNA damage, apoptosis, angiogenesis and differentiation. In grade II astrocytomas that are IDH mutant,

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inactivating ­mutations are present in the vast majority.176 In glioblastoma, the p53 pathway may be deregulated by alterations of other components in the signalling pathway, including amplifications of MDM2 or MDM4 or losses of CDKN2A-ARF.95,96 Indeed, The Cancer Genome Atlas (TCGA) project has catalogued these related gene alterations, finding that this family is dysregulated in nearly 90 per cent of GBM.27,36 A small number of genes related to histone modification, chromatin remodelling and telomere maintenance are mutated in diffuse astrocytomas as an early event and are tightly coupled to TP53 mutation. ATRX   (α-thalassaemia/ mental-retardation-syndrome-X-linked) mutations are present in 33 per cent of grade II and 46 per cent of grade III gliomas and are highly correlated with astrocytic differentiation, both by morphologic and molecular assessment.139 They are also present in 80 per cent of secondary, but only 7 per cent of primary GBMs. ATRX alterations are closely coupled with mutations in IDH1/2 and TP53 across all astrocytoma grades. Mutations of ATRX lead to alternative lengthening of telomeres (ALT), a telomerase independent mechanism of maintaining telomeres in cancer.83,219 Interestingly, ATRX mutations are uncommon in lower-grade paediatric astrocytomas and in gliomas from adults appear to be mutually exclusive with 1p/19q co-deletions and gene mutations associated with oligodendrogliomas (CIC, FUBP1). Another gene related to chromatin remodeling, H3F3A, is mutated in astrocytomas and appears to be an early event, mostly in paediatric high-grade astrocytomas.219 In one series, H3F3A mutations were noted in over 40 per cent of such cases. ATRX and TP53 mutations were also present in these samples and showed a considerable degree of overlap with H3F3A mutations. There are two types of recurrent somatic mutations that occur in H3F3A, with one resulting in amino acid substitution at K27 and the other at G34. Each of these has an impact on the ability of H3.3 to regulate gene expression and leads to alterations in transcriptional profile.233 The K27 mutated tumours occur in the youngest children in midline sites (thalamus and pons), whereas the G34 mutated tumours preferentially involve teenagers and young adults with cerebral hemispheric gliomas. H3F3A mutations are mutually exclusive with IDH mutations. Mutations in DAXX (death-domain associated protein), encoding yet another chromatin remodelling subunit, have also been described in paediatric high-grade astrocytomas, but at a much lower frequency.219 The presence of mutations in these chromatin remodelling genes is also tightly coupled to the finding of ALT. ALT occurred most frequently in those astrocytomas with combined ATRX/ H3F3A/TP53 mutations. Many growth factors and their receptors are overexpressed in grade II astrocytomas, including plateletderived growth factor (PDGF), fibroblast growth factors (FGFs), vascular endothelial growth factor (VEGF) and EGFR. PDGF ligands and receptors are expressed approximately equally in all astrocytoma grades, suggesting that overexpression is an initial event.35 Introduction of PDGF into the rodent white matter is sufficient to induce neoplasia and malignant transformation, highlighting the role of this signal transduction network in gliomagenesis.5,6

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Overview and Biology of Diffuse Astrocytic Tumours  1641



Differentiation and Tumour Phenotype A wide variety of cellular patterns have been described in astrocytomas, including fibrillary, gemistocytic, small cell, protoplasmic, sarcomatous, epithelioid, granular cell, giant cell and mixed with oligodendroglioma (oligoastrocytoma). Factors that determine tumour phenotypes are unclear. Mouse modelling studies have shown variations in tumour morphology depending on the type of cell transformed, as well as the oncogenic combination.92 For example, overexpression of Ras and Akt in progenitors yields tumours with astrocytic differentiation, whereas overexpression of PDGF-B in the same cells produces tumours resembling oligodendroglioma.7,46 The occurrence of oligoastrocytomas in both humans and animal models provides insight to the origins and pathways of glial differentiation. Morphologically distinct regions in oligoastrocytomas have similar genetic alterations, indicating that these are clonal lesions, albeit with striking phenotypic diversity.120 Most human astrocytomas do not display overt neuronal differentiation. Nonetheless, as more sensitive neuronal markers are utilized, many glial neoplasms seemingly coexpress neuronal and glial antigens, either uniformly or focally. This includes rare but distinct variants, such as malignant gliomas with PNET foci and rosetted glioneuronal tumours.186,238 It is likely that most ‘gliomas’ will prove to be more heterogeneous, particularly when developing from pluripotent progenitors.

Understanding glioma phenotype must also take into account that astrocytic tumours are associated with ­specific genetic alterations, including TP53 and ATRX mutations, whereas oligodendrogliomas are strongly associated with 1p/19q losses, CIC mutations and FUBP1 mutations. In this regard, it may be possible that astrocytomas and oligodendrogliomas arise from the same cells of origin, yet genetic events drive differentiation along one or another pathway (Figure 27.1). It is interesting to note, however, that those oligodendroglial tumours with 1p and 19q loss preferentially affect particular areas of the brain, raising the possibility that specific precursor populations in different brain regions transform along distinct genetic pathways to reach common phenotypic end points, such as oligodendroglioma.268

Invasion Diffuse gliomas of all grades and morphologies display a remarkable tendency to infiltrate the brain, confounding therapeutic attempts at local control (Figure 27.2). Patterns of brain invasion by gliomas are stereotypic.266 For instance, there is preferential invasion along white matter tracts: many gliomas cross the corpus callosum to form ‘butterfly’ lesions; other gliomas remain confined to the white matter, stopping abruptly at the grey–white matter junction. Still other migratory patterns give rise to the so-called ‘secondary structures of Scherer’, including preferential growth

Proteases: MMP2, MMP9, uPA, cathepsin B

Perivascular spread Subpial growth

27

ECM: tenascin, vitronectin, fibronectin

EGFR FAK

Pial surface

Integrins Perineuronal satellitosis

Neuron cell body

Single tumour cell

Axons Spread along white matter tracts Cerebral cortex

White matter

27.2 Invasion. Malignant glioma cells show preferential invasion along white matter tracts, around neurons and blood vessels, and in the subpial region. Molecular events relating to invasion of single cells include elaboration of proteases such as MMP2, MMP9, uPA and cathepsin B; expression of integrins that interact with extracellular matrix (ECM) components, such as tenascin, vitronectin and fibronectin, that are themselves expressed by tumour cells; and activation of FAK-mediated cellular signalling pathways either via EGFR or integrin signalling. Adapted with permission from DN Louis.141

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1642  Chapter 27  Astrocytic Tumours

around neurons (‘perineuronal satellitosis’), perivascular aggregation and subpial spread. These infiltrative tendencies suggest that glioma cells have either a tropism for particular sites or a restricted ability to invade other regions. Moreover, glioma invasion is best viewed as the combined ability to migrate and to modulate the extracellular space. Unfortunately, investigations of glioma invasion have been hampered by a paucity of representative experimental models that mimic the human disease. Invasion by glioma cells reflects a dynamic interplay between cell–cell adhesion, remodelling of the extracellular matrix and cell motility.14,143 Glioma invasion of brain is biologically distinct from carcinoma invasion because of the single cell nature of the former and the distinctive extracellular matrix of the brain. In general, the extracellular matrix of the brain is ill defined and scant, consisting primarily of hyaluronic acid, except in two areas: around blood vessels and at the pial surface (glia limitans). At these sites, there is a well-defined and more traditional basal lamina that includes collagens. Notably, glioma cells preferentially involve the perivascular and subpial spaces – places where basal lamina is well defined – but they also involve perineuronal and white matter locations – places where it is not. It has been suggested that the expression of the chemokine SDF-1α by neurons, blood vessels, subpial regions and white matter tracts may guide the infiltration of glioma cells towards these structures, because tumour cells express the receptor for SDF-1α, CXCR4, with highest expression by those invading glioma cells organized around neurons and blood vessels, in subpial regions, and along white matter tracts.266 Moreover, neuronal and endothelial cells exposed to VEGF upregulate the expression of SDF-1α, thereby enhancing the chemoattraction in hypoxia. Investigations into astrocytoma invasion have highlighted the complex nature of cell–cell and cell–­extracellular matrix interactions.14,196 Proteases are elaborated by glioma cells, which appear to play a significant role, including cysteine, serine and metalloproteinases. These degrade the extracellular environment to facilitate migration, but also remodel the environment in a manner that facilitates tumour cell growth. Most studies have focused on matrix metalloproteinases MMP2 and MMP9, the serine protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR), and the cysteine protease cathepsin B.196 Their expression increases with glioma grade, and their interference in vitro results in decreased invasive and/or migratory properties. Studies of interactions between glioma cell surface molecules and extracellular matrix molecules have shown that gliomas express a variety of integrin receptors that mediate interactions with molecules in the extracellular space. The integrin heterodimers most clearly implicated have been α2β1 (interacting with tenascin), α5β1 (interacting with fibronectin), α6β1 (interacting with laminin) and αvβ3 (interacting with vitronectin).246 Activation of integrins through interactions with extracellular ligands results in alterations of the cellular cytoskeleton, promoting locomotion. Focal adhesion kinase (FAK), an intermediate signalling molecule in glioma migration, is a cytoplasmic tyrosine kinase expressed in high-grade gliomas and activated by EGFR and integrins such as αvβ3 and αvβ5.202 FAK subsequently signals through pathways that affect proliferation, survival and migration.164

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Many of the growth factors expressed in astrocytomas, such as FGF, EGF and VEGF, also stimulate migration. Significantly, those glioblastomas with EGFR gene amplification do not demonstrate uniform distribution of the amplified cells within the tumour.175 Rather, EGFRamplified cells are preferentially located at the infiltrating edges.229 Given that EGFR gene amplicons are found on double minute chromosomes, which must be maintained by positive selection during cell division, this suggests that EGFR amplification provides selective advantage at the infiltrating edge. Further support that EGFR signalling contributes to invasion is provided by gene expression profiling data. Overexpression of the vIII EGFR mutant, a constitutively active EGFR variant, results in upregulated expression of multiple genes associated with invasion, including metalloproteinases (MMP1 and MMP13) and collagens.125 Furthermore, inhibition of EGFR appears to reduce invasion.180

Tumour Progression Diffuse astrocytomas nearly always recur, typically showing histopathological evidence of tumour progression, including increased nuclear atypia, mitotic activity and, eventually, microvascular proliferation and/or necrosis. Glioblastomas developing along this pathway have been termed secondary glioblastomas,215 in contrast to primary glioblastomas, which develop clinically de novo, i.e. without an identifiable, less malignant precursor lesion.171 Nearly all de novo GBMs are IDH wild type, whereas the vast majority of secondary GBMs are IDH1 or IDH2 mutant.264 Although the acquisition of anaplastic features is an inherent property of diffuse astrocytomas, there is temporal variability in progression. Occasional WHO grade II astrocytomas do not change histological grade for over ten years.173 Others, especially those that are IDH wild type, rapidly progress to malignancy within one or two years. Molecular changes underlying malignant progression of astrocytomas parallel histological changes and clinical course.171 Deregulation of cell cycle control as well as activation of receptor tyrosine kinase (RTK) and PI3K-AKTmediated signalling pathways is strongly related to progression of grade II gliomas to higher-grade counterparts, including glioblastoma. At a simplified level, many alterations can be divided into two large groups: those related to deregulated RTK-AKT signalling and those related to deregulated p53-Rb cell cycle control/apoptosis cascade (Figure 27.3). At a histological level, WHO grade III tumours have increased cellular density, mitotic activity and proliferative indices compared to grade II tumours. At a molecular level, many genetic alterations target cell cycle regulatory genes. Most of these converge on critical cell-cycle regulatory complexes that include the CDKN2A/ p16, cyclin-dependent kinase 4 (CDK4), CDK6 and retinoblastoma (Rb) proteins. Individual components in this pathway are altered in up to 50 per cent of anaplastic astrocytomas, higher in those that are IDH wild type, and in nearly all glioblastomas. In the TCGA analysis of Rb pathway genetic alterations, the most frequent alterations included CDKN2A/p16 deletions (52 per cent), CDKN2B deletions (47 per cent), CDK4 amplification (18 per cent),

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Overview and Biology of Diffuse Astrocytic Tumours  1643

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EGFR

p16

ARF

Cell cycle control Apoptosis Growth

cdk4/ cdk6 cyclin D

MDM2/ MDM4

p53

phospho-Akt

pRb

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RB1 mutation (11 per cent), CCND2 amplification (2 per cent) and CDK6 amplification (1 per cent).27,36 Chromosome 9p loss occurs in approximately 50 per cent of anaplastic astrocytomas and glioblastomas, with 9p deletions primarily affecting the region of the CDKN2A gene, which encodes the p16 and the ARF proteins. The CDKN2A gene is inactivated either by homozygous deletion or, less commonly, by point mutations or hypermethylation. The role of p16 in progression has been confirmed in mouse models overexpressing either PDGF or EGFR, in which a p16 null background is sufficient to change the phenotype from low-grade to anaplastic.46,260 Loss of chromosome 13q occurs in onethird to one-half of high-grade astrocytomas, with the RB1 gene preferentially targeted by losses and inactivating mutations. RB1 and CDKN2A alterations rarely occur together in the same tumour, suggesting that their losses are functionally similar. Amplification of the CDK4 gene, located on chromosome 12q13-14, provides an alternative to subvert cell-cycle control and facilitate progression to glioblastoma. CDK4 amplification, CDK6 amplification and cyclin D1 or D2 overexpression appear to represent alternative events to CDKN2A deletions and RB alterations, because these genetic changes only rarely occur in the same tumours.95,245 Chromosome 10 loss occurs in 60–95 per cent of glioblastomas, but less commonly in grade II or III diffuse astrocytomas.172 The PTEN gene at 10q23.3 has been strongly implicated as a glioma-related tumour suppressor on chromosome 10q, with mutations identified in about 25–30 per cent of glioblastomas. PTEN functions as a protein tyrosine phosphatase and has 3ʹ phosphoinositol phosphatase activity; in addition, PTEN has an amino-terminal domain with homologies to tensin and auxilin. Thus, PTEN may regulate cell migration via affecting focal adhesion kinase (FAK) and may regulate cell proliferation via control of the Akt s­erine/threonine kinase. Introduction of wild-type PTEN into glioma cells with mutant PTEN leads to growth suppression.263 Allelic losses have been found at other loci in high-grade astrocytomas, particularly on 19q (up to 40 per cent of anaplastic astrocytomas and glioblastomas), suggesting a ­progression-associated glial tumour suppressor gene.

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27

(CDKN2A)

PTEN

p21

27.3 Progression from grade II to grade III and IV malignant gliomas is associated in nearly all cases with genetic changes that activate the growth receptor tyrosine kinase signalling pathways that include EGFR, PDGFR, PTEN, Akt and NF1 and that inactivate the cell cycle control pathways that feature p53 and pRb. ↑, upregulation; ↓, downregulation. Adapted with permission from DN Louis.141

EGFR is a transmembrane receptor tyrosine kinase, whose ligands include EGF and TGF-α. The EGFR gene is the most frequently amplified oncogene in astrocytic tumours, being amplified in approximately 40 per cent of all glioblastomas, but rarely in lower grade astrocytomas.171,172 Glioblastomas with EGFR gene amplification display overexpression of EGFR at both the mRNA and protein levels, suggesting that activation of this growth signal pathway is integral to malignant progression. Downstream targets include the Shc-Grb2-ras pathway and the PI3K-AKT pathway. Approximately one-third of glioblastomas with EGFR gene amplification also have specific EGFR deletions (the vIII mutant), which produce truncated cell surface receptors with constitutive tyrosine kinase activity. Such truncated EGFR receptors are capable of conferring dramatically enhanced tumorigenicity to glioblastoma cells. Although EGFR amplification is the most frequent and best studied oncogenic event involving a receptor tyrosine kinase with downstream signalling of PI3K-ATK and Ras pathways, other family members have also been implicated. Amplification of PDGFRA is noted in approximately 13 per cent of glioblastomas, based on high density SNP analysis of copy number alterations.36,248 Fluorescence in situ hybridization (FISH) – based assays of PDGFRA, which can identify lower level copy number alterations than genomic averaging techniques, suggest that PDGFRA amplifications occur in a higher frequency, one study finding PDGFRA amplifications in 21 per cent of adult GBMs and 29 per cent of paediatric GBMs.189 This study also concluded that the PDGFRA amplification was a poor prognostic feature in IDH1 mutated adult GBM, removing the prognostic advantage normally associated with IDH1 mutations. In addition to EGFR and PDGFRA amplification, less frequent alterations that enhance intracellular signalling of the RTK, PI3K and Ras pathways include c-Met amplification, activating mutations in PI3K, mutations and deletions of PTEN and mutations and deletions of NF1.27,36

Necrosis and Hypoxia Necrosis is one of the diagnostic features of glioblastoma and a key driving force behind its behaviour.197,208 Indeed, no histological feature is more powerful in predicting

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1644  Chapter 27  Astrocytic Tumours

prognosis among the diffuse gliomas.143 The accelerated tumour growth that accompanies necrosis could be due to a highly malignant state achieved by the neoplasm, a selection pressure due to necrosis that encourages outgrowth of highly malignant cells, or the effects of hypoxia and other factors that are associated with necrosis (Figure 27.4). Necrosis could potentially arise from a number of ­mechanisms. In the setting of rapidly dividing cells with high metabolic demands, regions of tumour distant from blood vessels may develop necrosis when metabolic demands exceed supply (diffusion-limited hypoxia and necrosis).208 Necrosis may also arise following vaso-occlusion associated with intravascular thrombosis, which is frequently seen in GBM, but not lower-grade gliomas (perfusion-limited hypoxia and necrosis).236 One possible molecular culprit that could promote intravascular thrombosis is tissue factor,

which is highly overexpressed by neoplastic cells and could act as a local pro-coagulant.22,209 One theory suggests hypoxia follows vaso-occlusion and thrombosis within a high-grade astrocytoma and leads to the active migration of tumour cells outward towards a more functional vascular supply, thus ‘clearing’ a central region that undergoes necrosis. The latter hypothesis is based on work showing that those glioblastoma tumour cells surrounding necrotic centres (so-called ‘pseudo-palisades’) are less proliferative and more apoptotic than adjacent cells, suggesting that they accumulate from their migration.22,23 These perinecrotic cells also express hypoxia-inducible genes, such as HIF1α and VEGF;23 and in vitro studies have demonstrated that hypoxia induces an increase in cellular migration and in gelatinase activity. Morphometric analysis of the central necrotic zones of pseudo-palisades in glioblastomas

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Tumour cells

Hypoxic region

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Migration of cells away from hypoxic centre

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Necrosis Palisade (c) ����������������

Clonal selection VEGF

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Other angiogenic factors

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27.4 Hypoxia, necrosis and angiogenesis. The development of hypoxia, necrosis and angiogenesis is biologically linked. (a) Localized hypoxia appears to upregulate migration-­associated genes, leading to migration of tumour cells away from a central hypoxic centre (b). (c) Necrosis then ensues in the central region, sometimes in association with vascular thrombosis and a ‘palisade’ of densely packed tumour cells develops. (d) Palisading cells express angiogenic factors such as VEGF, leading to adjacent angiogenesis that includes so-called ‘glomeruloid’ microvascular proliferation. Another consequence of hypoxia may be clonal selection of malignant cells that are able to survive selection pressure. Adapted with permission from DN Louis.141

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have demonstrated abnormal and sometimes ­thrombosed vessels in over half, suggesting an initial vaso-occlusive event may have initiated the hypoxia/necrosis cycle. Factors that initiate vaso-occlusion are not clear, yet endothelial cells of small vessels may be driven toward apoptosis and regression as a result of angiopoietin 2 expression.91,265 The development of hypoxia may have other downstream effects as well. For example, hypoxia may act as a selective force that leads to the emergence of highly malignant and apoptosis-resistant tumour cells bearing inactivated p53.69 Hypoxia also facilitates angiogenesis by the release of growth factors such as VEGF and IL-8.25 Expression profiling of pseudo-palisading cells of glioblastomas also supports the notion that perinecrotic regions contain highly malignant clones.53 These, in turn, imply increased metabolic demand, likely causing greater hypoxia or necrosis and feeding a vicious cycle. Lastly, the development of hypoxia occurs centrally in the neoplasm, with many of its influences leading to the radial expansion of GBM in a rapid fashion.

Angiogenesis Markedly increased vascular density is a hallmark of glioblastoma and occurs in two pathologically discernible forms: (1) increase in vessel number because of enhanced small vessel formation and (2) an abnormal form of angiogenesis most commonly known as ‘microvascular ­ proliferation’ (Figure 27.4). The diffuse increase in vascular density is not always an obvious feature on haematoxylin and eosin (H&E)-stained sections, but can be appreciated using immunohistochemistry for endothelial cells. Microvascular proliferation is a complex accumulation of proliferating endothelial and perivascular cells that lead to tufting and budding of the vasculature within neoplastic tissue. The multilayering of these often hyperplasticappearing cells is a feature that correlates with aggressive behaviour in GBM.47 The most characteristic form is the so-called ‘glomeruloid’ body (because of their resemblance to renal glomeruli), which are large and complex threedimensional structures composed of both proliferating endothelial and smooth muscle cells, representing the most exuberant form of angiogenesis.21,74,143 Microvascular proliferation is irregularly distributed in glioblastomas. Most notably, complex glomeruloid bodies form semicircular garlands that hug regions of necrosis, highlighting the interrelationship between hypoxia/necrosis and angiogenesis. Less commonly, microvascular proliferation can be found at the invading edges, far from necrosis, and presumably as a result of angiogenic factors expressed by invading glioma cells. The biological underpinnings of microvascular proliferation are complex, with many angiogenic growth factors and their receptors found in glioblastomas. VEGF and PDGF are expressed by tumour cells whereas their tyrosine kinase receptors, VEGF receptors 1 and 2 for VEGF and the PDGF-β-receptor for PDGF, are expressed on endothelial cells.21 VEGF and its receptors may also be responsible for breakdown of the blood–brain barrier and tumoural oedema in glioblastoma, by causing vascular permeability. VEGF is a downstream target of a number of the activated signalling pathways, e.g. via EGFR and Akt, and is upregulated by numerous oncogenic events. Interleukin-8 (IL-8)

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Diffuse Astrocytoma (WHO Grade II)  1645

is another hypoxia-inducible cytokine that is expressed in gliomas and has potent angiogenic activity.25 Numerous lines of investigation support the notion that angiogenesis within gliomas is supported and enhanced by bone marrow progenitor cells. Studies in animal models indicate that these progenitors localize to angiogenic vessels in high-grade gliomas and are incorporated into their walls. Whether these cells are capable of transdifferentiating into endothelial cells or pericytes or have additional roles continues to be investigated.55

27

Diffuse Astrocytoma (WHO Grade II) Diffuse astrocytoma, WHO grade II, is characterized by advanced cellular differentiation and widespread brain invasion by individual neoplastic cells. It manifests most frequently in young adults, with a peak incidence in the fourth and fifth decades. It may be located in any region of the CNS, including the spinal cord, but preferentially involves the cerebral hemispheres. Symptoms depend on location, but seizures are common.143

Neuroimaging By computed tomography (CT), diffuse astrocytomas present as ill-defined, homogeneous masses of low density without contrast enhancement. Magnetic resonance (MR) imaging demonstrates low signal intensity on T1-weighted and higher signal intensity with better defined borders on T2-weighted sequences (Figure 27.5a). Diffusion weighted imaging (DWI), perfusion weighted imaging (PWI) and MR spectroscopy (MRS) are helpful in estimating tumour grade pre-operatively, with diffuse astrocytomas typically having low relative cerebral blood volume (rCBV), high apparent diffusion coefficients and low Cho/Cr and Cho/NAA ratios as compared to higher grade astrocytomas.4,9

Macroscopic Appearances Astrocytomas expand the involved brain, have ill-defined borders and are slightly firm, yellow-white and homogeneous, with occasional cysts. Infiltration often leads to enlargement and distortion, but not destruction, of involved grey or white matter. Extensive micro-cyst formation may cause a gelatinous appearance.

Microscopy Diffuse astrocytomas infiltrate the involved brain as single cells of variable density and cytologic atypia (Figure 27.5b–h). Normal nervous system cells and structures, including neurons, their axons, glial cells and blood vessels, are all typically entrapped within the lesion. Mitotic activity is generally absent, although isolated mitoses found after long searches of large resection specimens may not connote increased malignancy in the same way as finding a single mitosis in a needle biopsy.63 Three major variants have been distinguished: fibrillary, gemistocytic and protoplasmic, with granular cell morphology representing a rare, yet intriguing variation (Figure 27.6a–c).

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1646  Chapter 27  Astrocytic Tumours (a)

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27.5 Diffuse astrocytoma, WHO grade II. (a) On MRI, diffuse astrocytomas, such as that in the right frontal lobe in this axial FLAIR image, are hyperintense on T2-weighted or FLAIR imaging, typically centred in the white matter and expand the involved brain. (b) In tissue sections, individual astrocytoma cells with enlarged, atypical nuclei are noted infiltrating the CNS parenchyma. (c) Cytologic preparations demonstrate the oblong nuclei of astrocytoma cells and thin, elongate fibrillary processes. (d) Diffuse astrocytomas will occasionally show prominent microcystic architecture. (e) Invasion of the cortex by diffuse astrocytomas is often accompanied by peri-neuronal satellitosis. (f) Infiltrating astrocytoma cells occasionally accumulate in the subpial zone of the cortex. (g) Astrocytoma cells infiltrating along white matter tracts. (h) Immunohistochemistry for neurofilament highlights the entrapped axons adjacent to infiltrating neoplastic cells..

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Diffuse Astrocytoma (WHO Grade II)  1647



Fibrillary astrocytomas are most common, being characterized by scant to indiscernible cytoplasm, creating the appearance of ‘naked nuclei’ (Figure 27.5b). Cytologic preparations are better for demonstrating the elongate, delicate eosinophilic glial process that emerge from individual tumour cells (Figure 27.5c). Nuclear atypia, i.e. enlarged, irregular hyperchromatic nuclei, often resembling ‘Idaho potatoes’ is a histological hallmark distinguishing tumour cells from normal and reactive astrocytes. Neoplastic cell processes form a loose fibrillary matrix that is most often indiscernibly intertwined with the neuropil meshwork of the normal brain parenchyma. A gradient of increasing cell density from normal brain to hypercellular tumour can often be appreciated in fibrillary astrocytomas and is helpful in establishing a diagnosis of an infiltrative form of glioma (Figure 27.6a). Micro-cysts containing mucinous fluid dominate the histological picture in a subset (Figure 27.5d). The diffusely infiltrative pattern sometimes forms so-called ‘secondary structures of Scherer’: perivascular satellitosis, subpial growth and perivascular spread (Figure 27.5e–h). Immunohistochemistry for neurofilament can be used to highlight the infiltration of white matter tracts by neoplastic astrocytes (Figure 27.5h). GFAP is usually expressed by neoplastic cells, although the GFAPpositive fibrillary matrix of the brain parenchyma may render interpretation difficult. Nuclear staining with OLIG2 may be helpful in establishing glial lineage.104,136 Tumour cells also usually show S-100 protein immunoreactivity in the nucleus and cell processes. Proliferative activity, as determined by the Ki-67/MIB-1 labelling index, is usually less than 4 per cent.

Gemistocytic astrocytomas are characterized by a conspicuous fraction of cells with abundant glassy eosinophilic cytoplasm and eccentric nuclei (Figure 27.6b). Stout, randomly oriented processes extend from cells and intermix with the normal neuropil. The cells consistently express GFAP. Perivascular lymphocyte cuffing is frequent. MIB-1 labelling is usually less than 4 per cent, with the gemistocytic cells having a lower rate of proliferation than intermixed fibrillary or small astrocytoma cells. Granular cell astrocytomas are characterized by sheets or interspersed large, round tumour cells packed with eosinophilic, PAS-positive granules (Figure 27.6c).38 They may be confused with macrophage-rich lesions in some instances, given the abundance of large, lysosome-filled cells. Transition to typical infiltrating astrocytoma is noted in the majority of cases, yet others consist almost entirely of atypical granular cells. Lymphocytic infiltrates are common. Given their highly unusual morphology, determining their glial differentiation often requires immunohistochemistry for GFAP, S-100 protein or OLIG2. Protoplasmic astrocytoma, a less common variant, has been described as containing neoplastic astrocytes with small cell bodies with few, thin processes and a low content of glial filaments. Nuclei are mostly round to oval and the cellularity is low. Mucoid degeneration and micro-cyst formation are common and the MIB-1 labelling index, is often less than 1 per cent. This lesion is not well defined and is considered by some to be a non-specific pattern, rather than a true variant.

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27

27.6 Astrocytoma variants. (a) Fibrillary astrocytomas are the most frequent morphology and typically appear as elongate hyperchromatic nuclei haphazardly arranged in a densely fibrillar matrix. Note the gradient of increasing cellular density, which is helpful in establishing an infiltrative pattern of neoplasia. (b) Gemistocytic astrocytoma with copious eosinophilic cytoplasm. (c) Granular cell astrocytomas contain neoplastic cells with abundant cytoplasm packed with lysosomes. (d) Diffuse astrocytomas sometimes contain long, thin ‘microglia-like’ neoplastic cells, and this finding is particularly characteristic of gliomatosis cerebri.

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1648  Chapter 27  Astrocytic Tumours

Gliomatosis cerebri is a clinicopathologic diagnosis that implies extensive concurrent involvement of multiple lobes (at least three) or brain compartments by an infiltrating glioma.114 Most examples appear astrocytic, yet oligodendroglial forms have also been described. Microscopically they usually resemble fibrillary astrocytomas of grade II or III. Long, thin mildly hyperchromatic ‘microglia-like’ astrocytoma nuclei are typical (Figure 27.6d). As with other infiltrating gliomas, there are often secondary structures, including subpial or subependymal condensation, perivascular aggregates, and perineuronal satellitosis. Although gliomatosis is characterized by widespread involvement by diffusely infiltrating astrocytoma, foci of dedifferentiation to GBM may also develop, either at the time of clinical presentation or later in the disease progression.

Grading Diffuse astrocytomas correspond to WHO grade II.143

Molecular Genetics Mutations in isocitrate dehydrogenase 1 (IDH1) are frequent (60–80 per cent) in grade II astrocytomas,177,264 with mutations in IDH2 much less common. In all forms of diffuse gliomas, the mutant forms of IDH1 and IDH2 cause the accumulation of the oncometabolite 2-hydroxyglutarate, which leads to the hypermethylator (G-CIMP) phenotype. Immunohistochemistry for mutant IDH1 (mIDH1R132H) is highly valuable in distinguishing grade II astrocytomas from diagnostically challenging reactive astrocytic proliferations.37 The sensitivity for identifying IDH1 mutant gliomas of any kind using this monoclonal antibody was 94 per cent and the specificity was 100 per cent. TP53 mutations are present in over 60 per cent of grade II astrocytomas,255 even higher in those that are IDH mutant, and in up to 80 per cent of gemistocytic variants.257 Nuclear p53 protein accumulation is similarly frequent but does not always reflect a mutation.255 During malignant progression of astrocytomas, the frequency of TP53 mutations does not increase significantly, indicating that this is an early genetic event and already present in the first biopsy in the vast majority of cases.222,254,255 The association of germline TP53 mutations and predilection to astrocytomas also argues for a role for p53 deregulation early in tumorigenesis.116 Genomic and gene expression studies on the diffusely infiltrative astrocytomas of grades II and III have shown three clearly distinguishable classes.68,106 One group is characterized by both IDH and TP53 mutations; the second group is characterized by IDH mutations, with wild-type TP53; and the third group is IDH wild type. The last group behaves very aggressively and is associated with much shorter survival times. ATRX   (α-thalassaemia/mental-retardation-syndromeX-linked) mutations are also common in grade II astrocytomas, but are uncommon in oligodendrogliomas.139 ATRX alterations occur nearly exclusively in astrocytomas harbouring mutations in IDH and TP53; they are associated with alternative lengthening of telomeres (ALT), a telomerase independent mechanism for maintaining long telomeres in neoplasms.

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Comparative genomic hybridization (CGH) analyses have repeatedly demonstrated that the most frequent genomic imbalances are the gain of chromosome 7q and amplification of 8q.166,218 Less common alterations include chromosome 22q loss100 and chromosome 6 deletions.153 Methylation of the gene encoding O6-methylguanineDNA methyltransferase (MGMT), a DNA repair protein that directly and specifically repairs promutagenic DNA lesions by removing alkyl groups from the O6 position of guanine, is present in about half of grade II diffuse astrocytomas and may also be related to p53 alterations. Loss of MGMT expression could be an early change that facilitates TP53 mutations in astrocytomas.163

Prognosis The mean survival time after diagnosis is approximately 6–8 years, with marked individual variation.143,231 Five- and 10-year survival rates for diffuse fibrillary astrocytomas are 65 and 31 per cent, respectively.176 In addition to the extent of resection, the prognosis depends on whether or not the glioma progresses to a higher grade. Several reports indicate that the gemistocytic variant appears particularly prone to malignant progression,121,176,256 whereas protoplasmic forms and tumours with extensive microcystic change may follow a slower course, often with a history of chronic seizures.194 Proliferative potential correlates inversely with time to recurrence and survival in grade II astrocytomas, but does not allow a prediction of clinical outcome in individual cases. Higher Ki-67/MIB-1 labelling indices (>5 per cent) correlate with more rapid progression and shorter survival,101,155 but precise cut-off values are difficult to establish. Grade II astrocytomas with IDH mutations have a better prognosis than wild-type counterparts. Those with evidence of genetic changes more typical of high-grade astrocytomas, such as PTEN mutation, CDKN2A deletion or RB1 loss/mutation, may follow more aggressive courses.88

Anaplastic Astrocytoma Anaplastic astrocytomas are diffuse astrocytomas with mitotic activity, and are designated as grade III by the World Health Organization (WHO).143 They may arise following progression from a grade II diffuse astrocytoma, but are also frequently diagnosed at first biopsy, without indication of a lower grade precursor. The terms ‘malignant astrocytoma’ and ‘high-grade astrocytoma’ are also sometimes applied to this lesion, but should be avoided, because all diffuse astrocytomas (grades II–IV) are malignant and glioblastomas are also high grade.

Neuroimaging Anaplastic astrocytomas present as ill-defined intrinsic masses of low signal intensity on T1-weighted MRI and high signal intensity on T2-weighted and FLAIR MRI (Figure 27.7a). In contrast to grade II counterparts, partial contrast enhancement can be observed. However, the rim-like enhancement pattern typical of glioblastoma is not a feature. Grade III astrocytomas have higher rCBV than

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Anaplastic Astrocytoma  1649

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27.7 Anaplastic astrocytoma. (a) MRI demonstrates a large, T2-hyperintense lesion involving the left temporal and parietal lobes. (b) Large, partially cystic left temporal lobe mass. (c) Hypercellular invasive astrocytoma with mitotic activity.

grade II lesions and also show lower apparent diffusion coefficients and higher Cho/Cr and Cho/NAA ratios.4,9

Macroscopic Appearances The higher cell density of anaplastic astrocytomas produces a more discernible tumour mass that may appear more solid than grade II counterparts (Figure 27.7b). Nonetheless, there is infiltration without tissue destruction, often leading to enlargement of invaded structures. Macroscopic cysts are uncommon. Rapid tumour growth and peritumoral oedema may lead to mass shifts and increased intracranial pressure.

Microscopy Compared with grade II diffuse astrocytomas, there is increased cellular density, and individual cells contain enlarged, irregular, hyperchromatic nuclei (Figure 27.7c). Anaplastic astrocytomas often contain cells with scant cytoplasm (‘naked nuclei’), with glial processes merging with the surrounding neuropil. On cytologic preparations, the glial differentiation is usually more appreciable, with elongate cellular processes extending from individual neoplastic cells. Gemistocytic tumour cells may be present and are more common in anaplastic astrocytomas than in grade II lesions. Capillaries are lined by a single endothelial layer with frank microvascular proliferation, and necrosis is absent by definition. Immunoreactivity for GFAP and S-100 protein is typical. Mitotic activity is the defining feature (Figure 27.7c). The growth fraction, as determined by

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the antibodies Ki-67/MIB-1, is usually in the range of 4–10 per cent, but overlaps with grade II diffuse astrocytomas on one side and glioblastoma on the other.

Grading Anaplastic astrocytomas correspond to WHO grade III.143

Molecular Genetics IDH1 mutations are frequent (60–80 per cent) in grade III astrocytomas, with IDH2 less commonly implicated.177,264 These mutations are associated with ­prolonged survival in comparison to IDH wild-type anaplastic astrocytomas. ATRX mutations are present in 46 per cent and typically occur in combination with IDH and TP53 mutations.139 Anaplastic astrocytomas have TP53 mutation rates as grade II astrocytomas. Alterations of the RB pathway (CDKN2A/p16, CDK4, Rb1) and of the long arm of chromosome 19, however, are found more often in anaplastic astrocytomas, implicating these events in tumour progression. Allelic losses of chromosome 19q occur in 40–55 per cent.162,225 Typically, when the genetic events associated strongly with glioblastoma are found in cases of anaplastic astrocytoma, there is shortened survival, presumably because such cases represent incipient transformation to glioblastoma, sampling error or both. For instance, loss of CDKN2A, CDKN2B and RB1, or CDK4 gene amplification, are associated with worse prognosis in anaplastic astrocytomas.8 Chromosome

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1650  Chapter 27  Astrocytic Tumours

10 losses and EGFR amplifications are less common in anaplastic astrocytoma, yet the former may be accompanied by PTEN mutation and both are associated with shorter survival.226 In a series of 80 anaplastic astrocytomas, even simple gains of chromosome 7 were associated with shorter survival, independent of patient age.123

Prognosis The prognosis for patients with anaplastic astrocytoma, WHO grade III, lies between that of grade II and grade IV counterparts, with considerable survival ranges. Traditional estimates have been approximately 2–4 years. In a population-based study, the median survival time was only 1.6 years, with only 11 per cent of patients alive at 5 years after diagnosis.170 IDH mutant anaplastic astrocytomas are associated with improved survival.264 In one study, the median survival was 65 months for such cases, compared with 20 months for IDH wild-type anaplastic astrocytomas.264 This same study also demonstrated that IDH wild-type anaplastic astrocytomas had worse ­prognosis than IDH mutant GBMs.

Glioblastoma Glioblastoma is the most malignant neoplasm among the diffuse astrocytic tumours and is designated as WHO grade IV. They typically affect adults and are preferentially localized to cerebral hemispheres. In less than 10 per cent of cases,174 they develop from low-grade or anaplastic astrocytomas (‘secondary glioblastoma’). The rest arise de novo after a short clinical history, without evidence of a less malignant precursor lesion (‘primary glioblastoma’).

Incidence Glioblastoma accounts for approximately 12–15 per cent of all intracranial neoplasms,269 and 50–60 per cent of all astrocytic tumours. The incidence in the general population is two to four new cases per 100 000 for most European and North American countries.

Age and Gender Glioblastomas manifest at all ages, but preferentially affect adults, with a peak incidence between 40 and 70 years. In a review of 715 glioblastomas, approximately two-thirds fell into this age group.174 Primary glioblastomas typically develop in older patients (mean age 62 years), whereas secondary glioblastomas manifest in younger patients (mean age approximately 45 years).174 The overall male:female ratio is roughly 1.3:1, but primary glioblastomas are more frequent in males, whereas secondary glioblastomas are more common in females.174 In a series of 488 glioblastomas, 8.8 per cent were diagnosed in children.51

Site, Spread and Metastasis Glioblastomas preferentially affect the cerebral hemispheres (Figure 27.8a,b). Tumour infiltration is typically

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widespread, often extending deep into the basal ganglia and thalamus, and frequently to the opposite hemisphere (Figure 27.8c). Glioblastomas of the brain stem are less frequent and often affect children (Figure 27.8d), whereas the cerebellum and the spinal cord are rarer sites. Although occasional glioblastomas may appear deceptively circumscribed by neuroimaging and seem to be ‘shelled out’ surgically, these tumours are notorious for their extensive parenchymal invasion.30 The poor clinical prognosis is largely a result of this extensive spread, particularly along myelinated tracts, making complete resection impossible. Extension through the corpus callosum (Figure 27.8b,c) into the contralateral hemisphere is common, occasionally creating the image of a bilateral, symmetrical lesion (‘butterfly glioma’). Similarly, spread is observed in the internal capsule, fornix, anterior commissure and optic radiation, with these structures becoming enlarged and distorted. New masses may then arise at distant sites, thereby leading to the neuroradiological image of a multifocal glioblastoma. Most cases of multifocality on neuroimaging likely represent spread from an original lesion, followed by distant expansion at a second or third location.11,13,109 Despite their highly infiltrative growth pattern, glioblas­ tomas do not routinely invade the subarachnoid space and only rarely disseminate through the cerebrospinal fluid (CSF). Extension within and along perivascular spaces is another mode of infiltration, yet invasion through the vascular wall resulting in haematogenous spread to extraneural tissues is very rare in patients without previous surgical intervention; similarly, dural, venous sinus and bone penetration are exceptional.

Clinical Features Clinical history is usually less than 3 months, unless the neoplasm has developed from a lower-grade astrocytoma. Patients typically present with non-specific neurological symptoms, headache, personality changes and epilepsy, and sometimes focal signs.

Neuroimaging On CT scans, glioblastomas usually present as irregularly shaped lesions that are hypodense or isodense to brain and typically have a peripheral rim-like zone of contrast enhancement around a dark area of central necrosis. On contrast-enhanced MR images, the contrast-enhancing rim corresponds to the cellular and highly vascularized region that surrounds the central, hypointense area of necrosis (Figure 27.8a). The rim of intense contrast enhancement does not represent the outer tumour border, because infiltrating glioma cells are noted well outside this region and beyond a 2-cm margin.30 In T2-weighted images, this zone is broader and less well defined, and overlaps with surrounding vasogenic oedema. Advanced MR imaging including DWI, PWI and MRS are being increasingly used for the characterization of diffuse gliomas. Compared to lower-grade gliomas, glioblastomas have high rCBV, low apparent diffusion coefficients and high Cho/Cr and Cho/ NAA ratios.4,9

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Glioblastoma  1651

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27.8 Glioblastoma. (a) Contrast-enhanced, T1-weighted MR image of rim-enhancing left parietal lobe mass. (b) Large, haemorrhagic glioblastoma of the left frontal lobe with expansion of the corpus callosum. (c) Apparently multifocal glioblastoma involving the right temporal lobe, deep right hemispheric grey matter and corpus callosum. (d) Glioblastoma of the pons in a child (‘brain stem glioma’), with ventral extension wrapping around the basilar artery.

Macroscopic Appearances

Microscopy

Glioblastomas are poorly delineated with respect to adjacent brain. The cut surface shows heterogeneity with peripheral greyish tumour masses, yellowish necrosis and single or multiple haemorrhages (Figure 27.8b–d). The central necrosis may occupy as much as 90 per cent of the total tumour mass. Macroscopic cysts, if present, contain a turbid fluid and represent liquefied necrotic tumour tissue. Haemorrhages are usually small and dispersed throughout the neoplasm. However, extensive haemorrhages may occur and evoke stroke-like symptoms, which are sometimes the first clinical sign of tumour manifestation.

The histopathology of glioblastoma is highly variable and regional heterogeneity is found in nearly all cases. Although some lesions show a high degree of cellular density, nuclear pleomorphism and multiple patterns of differentiation, others have a high cell density, but are rather monotonous. The astrocytic nature of neoplastic cells can be appreciated morphologically, at least focally, in the majority of tumours, but can be difficult to recognize in others, owing to poor or metaplastic differentiation. Cytologic preparations are often helpful, because the astrocytic processes can be best appreciated using this technique. There are multiple variants, including gliosarcoma, giant cell glioblastoma, small

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1652  Chapter 27  Astrocytic Tumours

cell glioblastoma and glioblastoma with an oligodendroglioma component (Figure 27.9a–d). The combination of diffusely infiltrative astrocytoma cells, mitotic activity and microvascular proliferation and/ or necrosis is diagnostic (Figure 27.10a–d). The d ­ istribution of these key elements within the tumour is variable, but usually, large necrotic areas occupy the tumour centre, whereas viable tumour cells tend to accumulate in the periphery and infiltrate at the leading edge. The gradient from hypercellular neoplasm centrally to a low-density, infiltrative periphery is nearly pathognomonic, because non-glial neoplasms do not infiltrate as readily or with the same pattern (Figure 27.6a). Microvascular proliferation can be seen throughout the lesion, yet preferentially localizes around necrotic foci and at the peripheral infiltration zone. The infiltrating edges display the same propensity to create ‘secondary structures of Scherer’ as do lower-grade counterparts; subpial growth, perineuronal satellitosis and perivascular accumulation are all common findings. Few human neoplasms are as cytologically heterogeneous as glioblastomas. Although poorly differentiated,

fusiform, round or pleomorphic cells may prevail, more differentiated neoplastic astrocytes are usually discernible, at least focally and particularly in secondary ­ glioblastomas. The transition between areas with still recognizable astrocytic differentiation and highly anaplastic cells may be either gradual or abrupt. Some tumours are dominated by giant cells (Figure 27.9b), small monotonous anaplastic cells (Figure 27.9c), spindle-shaped cells (Figure 27.9a), large epithelioid cells, PNET-like foci, granular cells (Figure 27.6c) or lipidized cells, and therefore a host of other neoplasms may come into the differential diagnosis; immunohistochemistry may be helpful in ruling in a diagnosis of glioblastoma and in excluding other possibilities. Many of these differentiation patterns may be secondary in nature and do not necessarily designate a specific biological form of glioblastoma, especially when focal. Glioblastomas occasionally contain foci with glandular and ribbon-like epithelial (‘adenoid’) structures or even squamous epithelial-like cells, which may be well delineated from the remaining tumour areas. Others may

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27.9 Glioblastoma variants. (a) Gliosarcoma with spindled sarcomatous regions growing in fascicles and interspersed with c ­ ollagen. (b) Giant cell GBM with markedly pleomorphic enlarged cells. (c) Small cell glioblastoma with monomorphic cells growing in sheets with high mitotic rate. (d) GBM with oligodendroglioma component contains regions of astrocytic differentiation and oligodendroglial differentiation.

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Glioblastoma  1653



show mesenchymal changes, including cartilage and bone formation. Both epithelial and mesenchymal metaplastic changes are more common in the gliosarcoma variant. Significantly, genetic studies of histologically biphasic tumours have generally shown clonal genetic changes between the histologically distinct regions, including in oligoastrocytomas, gliosarcomas and glioblastomas with metaplastic changes. For instance, glial and sarcomatous,118 as well as glial and epithelial areas56,156 have the same TP53 mutations. In a recently described variant, glioblastoma with PNET-like foci, both components may share common features such as 10q loss, but the PNET foci may additionally show unique alterations, such MYCC or MYCN amplification.186 Proliferative activity is usually prominent, with numerous typical and atypical mitoses. The Ki-67/MIB-1 proliferation index varies considerably, but is generally greater than 5 per cent and may be very high (>50 per cent). Whereas the Ki-67/MIB-1 proliferative index is prognostic in grades II and III astrocytomas, its value as a prognostic marker in glioblastoma has not been adequately demonstrated.26 It is likely that biologic forces other than proliferation, such as hypoxia and angiogenesis, become more relevant to tumour behaviour at this level.

The cardinal diagnostic features of glioblastoma are microvascular proliferation and necrosis ­(Figure 27.10a–d). The biological underpinnings of these p ­henomena were previously discussed. Microvascular changes take on many morphologies including hypertrophy, hyperplasia of endothelial and pericytic cells, and endothelial layering within the lumen (Figure 27.10c). The most complex arrangements are glomeruloid bodies (Figure 27.10d), which are often arranged in a festoon-like pattern, typically immediately adjacent to regions of necrosis. The latter may extend over considerable distances, suggesting that they form a three-dimensional network, around the central necrotic core. Microvascular proliferation is a signpost for the presence of necrosis and either feature justifies classifying a high-grade astrocytoma as glioblastoma. The glomeruloid appearance is based on multilayered hyperplastic endothelial cells, which typically show mitotic activity and high MIB-1 labelling. In nearly all glioblastomas, intravascular thrombosis can be appreciated, both within larger calibre vessels extending into the neoplasm and within vessels near or within necrotic foci (Figure 27.10b). Neurosurgeons are well acquainted with the association of thrombosed blood vessels seen at surgery and the diagnosis of glioblastoma. It has been speculated that intravascular thrombosis either

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27

27.10 Glioblastoma. (a) Pseudo-palisading necrosis. (b) Foci of necrosis often show thrombosed vessels in their centres and around edges. (c) Vessels in GBM show endothelial hypertrophy, hyperplasia and multilayering. (d) Glomeruloid microvascular proliferation.

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1654  Chapter 27  Astrocytic Tumours

initiates or propagates hypoxia and necrosis and may be a fundamental property of this tumour type.208,236 Necrosis (Figure 27.10a,b) is found in nearly all glioblastomas, depending on the diagnostic criteria employed. Using the WHO criteria, in which glioblastoma can be diagnosed on the basis of either microvascular proliferation or necrosis, one study found necrosis in 88 per cent of glioblastomas.10 In some, necrosis comprises the vast majority of the tumour, creating diagnostic challenges for the pathologist. The most characteristic form, however, consists of multiple small, irregularly shaped necrotic foci, surrounded by radially oriented, small fusiform glioma cells – ‘­pseudo-palisading’ pattern (Figure 27.10a). The central area of such geographical necrosis sometimes consists of a fine fibrillar network in which neither viable nor necrotic glioma cells are identified. It has been suggested that these small foci of necrosis are growing outwardly and enlarging, eventually coalescing into larger zones that merge to form the central necrotic core of glioblastoma.208,236

Grading Glioblastomas are WHO grade IV.143

Immunohistochemistry and Differential Diagnosis Glioblastomas show variable staining for GFAP. In ­general, astrocyte-like tumour cells, in particular gemistocytes, are strongly positive, whereas small, undifferentiated cells may stain weakly or not at all. Giant cells stain variably, with marked differences in GFAP expression even among neighbouring cells. Although large portions may lack GFAP expression, wider sampling usually reveals at least occasional immunoreactive tumour cells. GFAP expression tends to decrease during glioma progression, but there is no indication that its extent is prognostic. Vimentin, S-100 protein and OLIG2 are usually expressed widely. The primary utility of GFAP immunohistochemistry is in the differential diagnosis of glioblastoma from other poorly differentiated malignancies. As mentioned earlier, the marked cytological heterogeneity of glioblastoma may bring metastatic carcinoma, sarcoma, melanoma, lymphoma or even anaplastic meningioma into the histological differential diagnosis. GFAP positivity is rare in other tumours and its presence therefore strongly supports a glioma diagnosis in the appropriate clinicopathological setting. Moreover, an immunohistochemical panel for epithelial, mesenchymal, melanocytic and lymphoid markers will typically exclude other diagnostic possibilities.

Molecular Genetics: Subtypes of Glioblastoma Two clinically distinctive subtypes of glioblastoma are now well established: primary glioblastoma arises de novo as a grade IV neoplasm, whereas the second­ ary glioblastoma arises following the progression of a lower-grade glioma. By definition, these clinically defined primary and secondary glioblastomas do not overlap.

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There is good correlation between these two clinical pathways and genetic alterations, because the majority of secondary glioblastomas have IDH mutations, also with frequent TP53 and ATRX mutations, whereas these mutations are much less frequent in de novo glioblastomas. EGFR gene amplification is rare in secondary glioblastoma and is relatively frequent in primary glioblastomas.174 However, true secondary glioblastomas are much less common than primary glioblastomas, accounting for only 5 per cent in a population-based study.174 A ­considerable number of primary, de novo glioblastomas have TP53 mutations.174,217 Thus, although TP53 mutations are more frequent (as a percentage) in secondary glioblastomas, the absolute number of TP53 mutations is greater in primary glioblastomas.

Molecular Genetics: Key Genetic Events As discussed earlier, glioblastoma may arise through alterations in multiple genetic pathways, yet there is strong convergence on particular sets of pathways that govern cell growth, cell death, invasion and angiogenesis. Genetic pathways clearly implicated in glioblastoma include those involving (1) p53 (TP53, MDM2/MDM4, ARF), (2) retinoblastoma (Rb1, cdk4, CDKN2A/p16) and (3) receptor tyrosine kinases (EGFR, PDGFR, MET, PI3-kinase, PTEN, Neurofibromatosis 1). These pathways are deregulated by particular genetic events, such as gene mutations, deletion and amplifications, but may also be influenced by other growth promoting and growth suppressing molecules. The EGFR gene is involved in the control of cell proliferation. It is amplified in about 40 per cent of glioblastomas, of which roughly 40 per cent include a truncated variant known as the vIII mutant,61 rarely with other mutations,61 and often together with neighbouring genes at 7p12.138 Only 8 per cent of secondary glioblastomas show EGFR amplification.174 EGFR protein is also overexpressed in primary glioblastomas: more than 60 per cent of cases versus 10 per cent in secondary glioblastomas.254 There is a close but imperfect correlation between gene amplification and overexpression. In particular, expression of the vIII mutant correlates closely with underlying EGFR amplification.1 Glioblastomas with EGFR amplification typically show simultaneous chromosome 10 loss.249 PDGFRA amplification is the second most frequent RTK amplification in glioblastoma. Most investigations using genome averaging techniques, such as array-based CGH, have found high level amplification in 10–15 per cent of cases.36 However, increased PDGF pathway activity has been documented in higher fractions, suggesting other mechanisms. A recent FISH-based analysis documented PDGFRA amplification in 23 per cent of adult and 39 per cent of paediatric GBM. In this study, both low- and highlevel amplification events could be detected, because FISH allows detection at the individual tumour cell level.189 Recent studies have noted that EGFR and PDGFRA amplifications occasionally occur within the same tumour.229,234 In most, EGFR and PDGFRA are not amplified within the same cells and the amplifications seem to be spatially distinct, with most EGFR amplification noted peripherally and most PDGFRA amplification present

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centrally.175,229 Signalling networks and drug-sensitive targets downstream of these RTKs would also be expected to vary with these amplification events, because EGFR and PDGFRA are powerful oncogenic stimuli in GBM.36,234 A key pathway that regulates cell cycle control and apoptosis, among other functions, involves p53, mdm2 and ARF.96 TP53 mutations were among the first genetic alterations identified in astrocytic brain tumours.42 In glioblastoma series, the reported frequencies vary considerably, with a mean of 25–30 per cent.174 TP53 mutations are uncommon in primary or de novo glioblastomas, whereas secondary glioblastomas have a high incidence (approximately 65 per cent), of which over 90 per cent are already present in the first biopsy.173,254,255 The incidence of p53 protein accumulation is observed more frequently than TP53 mutations,255 but is also significantly higher in secondary (>90 per cent) than in primary glioblastomas (70 have substantially improved outcomes.44,132 KPS is also an important prognostic factor for younger patients and other histologies, but the threshold KPS score varies. In general, patients with higher performance status show improved clinical outcomes. Numerous investigations have also indicated that the extent of surgical resection can have an impact on survival. In one large retrospective series, neurosurgical resection greater than 78 per cent based on neuroimaging assessment was associated with prolonged survival, with additional survival advantages noted with increasing extents of resection.211 Because the WHO allows the diagnosis of glioblastoma based on the histologic presence of either microvascular proliferation or necrosis, one study evaluated the prognostic effect of necrosis. In a series of 275 glioblastomas, the absence of necrosis (in 12 per cent of cases) was associated with younger age, less extensive surgical resection and a median survival of 12.5 months, compared to 10.9 months in those with tumour necrosis.10 Although it has been repeatedly demonstrated that the Ki-67/MIB-1 labelling

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index correlates with tumour grade, proliferative potential does not provide additional independent prognostic information once the glioblastoma histology is achieved. Many studies have attempted to correlate molecular alterations with prognosis in glioblastoma. Mutations in IDH1/2 are strongly associated with survival. One study demonstrated that glioblastomas with IDH mutations had a median survival of 31 months as compared to 15 months for IDH wild-type glioblastomas.264 This relationship is more complex, however, because most patients with IDH-mutated GBM are younger and have secondary GBM, which are both associated with improved survival as well. Another approach compares long- versus short-term GBM survivors for key molecular differences. A CGH study of 39 glioblastomas from patients who survived longer than three years and from 24 short-term survivors showed that 6q loss, 10q loss and 19q gain were associated with short survival times and loss of 19q with long survival times.34 Most investigations of prognostic molecular markers have not shown simple associations, but have suggested complex interplays between patient age, genetic parameters and possibly treatment response. Associations between older age and EGFR gene amplification,251 as well as younger age and TP53 mutation are strong; as mentioned earlier, age itself is a powerful prognostic factor.29 For example, in one report, EGFR amplification was an independent predictor of prolonged survival only in patients with glioblastoma who were older than 60 years of age.226 Furthermore, in addition to TP53 and EGFR alterations, the prognostic effects of CDKN2A deletions,12,105 also appear dependent on the age of the patient, being more pronounced in patients older than 70 years of age.12 Predictive molecular parameters, i.e. those that address response to therapy rather than prognosis, have drawn less attention in glioblastomas than in oligodendrogliomas, largely because measurable therapeutic responses are less common in glioblastomas. Most studies of astrocytic tumours have not found prognostic importance for 1p/19q loss,224 arguing against a role for 1p/19q testing in glioblas­ toma management.24 The demonstration that concomitant temozolomide treatment provided a 2-month survival advantage in patients with glioblastoma231 prompted correlative studies related to MGMT promoter methylation status.258 Glioblastomas with MGMT promoter methylation would be predicted to have better prognosis because the ability to repair DNA damage from alkylating agents is impaired in MGMT-deficient tumours. Indeed, such tumours were found to have improved overall survival and enhanced responses to temozolomide.85 Studies of glioblastoma patients who were treated in the era before the use of temozolomide indicate that GBMs with MGMT methylation also have improved survival following radiation alone, suggesting that this alteration may identify GBMs with a more general survival advantage.203 Thus, MGMT methylation is an important prognostic marker in GBM and appears to predict sensitivity to therapy. The advent of targeted molecular therapies provides potential for molecular markers as predictors of treatment response. Given the role of EGFR in tumorigenesis, a number of clinical trials have investigated small molecule inhibitors of EGFR for efficacy against glioblastoma.

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Unfortunately, only a small subset of patients have benefited.200 Others have investigated molecular determinants of response to EGFR inhibitors among this small group of responders. In a series of 41 patients with malignant glioma treated with erlotinib, eight responded to treatment and response was associated with EGFR expression and EGFR amplification (but not vIII mutant expression), ­particularly in the 29 patients with glioblastoma. Interestingly, none of the 22 tumours with high levels of phosphorylated PKB/ Akt responded to erlotinib treatment, suggesting further that PI3 kinase–PTEN pathway disruption predicted poor response.73 Similarly, another study found that only those tumours that had coexpression of both EGFRvIII and PTEN showed substantial response to EGFR inhibitors.150 In both studies, the suggestion is that downstream activation of Akt, due to PTEN loss or other mechanisms, renders EGFR antagonism ineffective. Gene expression profiling is a powerful manner to estimate prognosis of malignant gliomas. In one study, a set of approximately 70 genes separated glioblastomas into two groups that differed over four-fold in median duration of survival.135 Another study explored diagnostically difficult cases, in which glioblastoma was not easily distinguished from anaplastic oligodendroglioma pathologically and demonstrated that a model of about 19 differentially expressed genes was superior in estimating prognosis than standard histopathological classification.168 Large-scale gene expression profiles have been developed that identify a more aggressive form of glioblastoma, which has been referred to as the mesenchymal class, dominated by genes related to angiogenesis and mesenchymal cell differentiation.188 Upon recurrence of high-grade gliomas, many show conversion to the mesenchymal expression class, suggesting that it may represent an end-stage, highly malignant form. There have been additional efforts to develop a smaller panel of genes that could predict prognosis and be more clinically useful than large-scale gene expression platforms. One such study took the gene expression and clinical outcomes data from four large, independent GBM data sets and identified a group of nine genes that could be used on formalin-fixed material and reliably predict prognosis on multivariate analysis.43

Glioblastoma Variants Gliosarcoma Gliosarcoma, defined as a glioblastoma admixed with a ‘sarcomatous’ component, accounts for approximately 2 per cent of all glioblastomas.119,149 A preferential location in the temporal lobe has been observed.149 Gliosarcomas can arise as primary, recurrent or radiation-induced neoplasms.80,81 Clinical features are generally similar to those of classic glioblastoma with regard to age, gender, race, tumour size or use of adjuvant radiation therapy.119 The prognosis is dismal, with survivals similar to or slightly shorter than that of glioblastoma.119 In cases with a predominant sarcomatous component, the tumour appears as a well-demarcated hyperdense mass with homogeneous

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Glioblastoma Variants  1657

contrast enhancement. Those that are peripherally located and abut the dura may mimic a meningioma. A recent study indicates that this subset of gliosarcomas that mimic meningioma may have a better prognosis.79 Essential for the diagnosis of the gliosarcoma is a biphasic tissue pattern with areas displaying gliomatous or mesenchymal differentiation (Figure 27.9a). The glial portion usually resembles glioblastoma. Although the large majority have astrocytic differentiation, gliosarcomas have also been described as arising with oligodendroglial, PNET and ependymal differentiation.89,107,205 The mesenchymal areas may show the typical herringbone pattern of fibrosarcomas, with densely packed long bundles of spindle cells. Occasionally, they resemble features of a pleomorphic sarcoma. The sarcomatous component also often shows signs of malignant transformation, such as nuclear atypia, mitotic activity and necrosis. It forms a rich reticulin network, in which the sarcomatous areas are well demarcated from the glial elements. Some show additional lines of mesenchymal differentiation, such as cartilage, bone and muscle. Furthermore, epithelial metaplasia with clusters of keratinizing stratified epithelia and adenoid formations have been noted. The occasional occurrence of spindle cells within a glioblastoma does not justify the diagnosis of gliosarcoma. Moreover, the infiltration of otherwise typical glioblastoma into the overlying leptomeninges and dura may lead to the erroneous impression of gliosarcoma because of the incorporation of reactive and collagen-rich elements. Gliosarcomas show variable genetic aberrations similar to those occurring in glioblastomas, i.e. gain of chromosome 7, loss of chromosomes 10 and 17, deletions of the short arm of chromosome 9 and alterations of chromosome 3. TP53 mutations are common, as are PTEN mutations and CDKN2A deletions.18,178,199 In one series, however, no gliosarcomas showed EGFR amplification or overexpression.199 Only a small percentage of primary gliosarcomas (7.7 per cent) showed IDH1/2 mutations, similar to primary glioblastomas. More distinctive was the low percentage of MGMT promoter methylation in gliosarcoma (11.5 per cent) as compared to typical glioblastomas (35–45 per cent).127 Genetic analyses of gliosarcomas18,178 and comparison of the mutational pattern in the gliomatous and sarcomatous components have demonstrated that each have identical TP53 and PTEN mutations, suggesting a monoclonal origin.18,199 More recent investigations using high density genomic array techniques have demonstrated focal amplification of a chromosomal region, 13q13.3-q14.1, containing the genes STOML3, FREM2 and LHFP, ­exclusively within the sarcomatous component.159 The overexpression of these genes and proteins was confirmed predominantly in the sarcomatous elements, suggesting that additional genomic alterations in the sarcomatous component lead to its distinctive phenotype. The finding that transcription factors known to drive the epithelial-mesenchymal transition (EMT) in other forms of cancer, such as Twist and Slug, are expressed exclusively in the sarcomatous ­ elements of gliosarcoma indicate that these factors drive the mesenchymal transition.160

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1658  Chapter 27  Astrocytic Tumours

Giant Cell Glioblastoma This histological variant has a marked predominance of bizarre, multinucleated giant cells and, on occasion, an abundant stromal reticulin network (Figure 27.9b). The consistent expression of GFAP has firmly established its astrocytic nature and this is confirmed by the genetic profile.182 It accounts for approximately 1 per cent of all glioblastomas. The race and gender profiles are similar to conventional glioblastomas. However, giant cell g­lioblastomas tend to occur at a younger age (mean 51 years).118 Giant cell glioblastomas are more circumscribed than other glioblastomas and often located subcortically in the temporal and parietal lobe. As a result, they may mimic a metastasis on neuroimaging. If they abut the dura, they can be confused with meningioma by neuroimaging and intra-operatively. Giant cell glioblastomas are completely resected with greater frequency than other glioblastomas, most likely because of their peripheral location and their circumscription. There is also evidence indicating that the survival of patients with giant cell glioblastoma is longer than those with typical glioblastoma, perhaps because of in part the younger patient age and the improved likelihood of complete resection. However, even after correcting for other features associated with survival, the giant cell histology was independently prognostic.118 Importantly, the improved survival of giant cell glioblastoma has not been noted in the paediatric population.108 On microscopic examination, tumour cells may be extremely bizarre and up to 400 μm in diameter. The number of nuclei per cell varies from one to more than 20. They are often angulated, may contain prominent nucleoli and, on occasion, cytoplasmic inclusions. Atypical mitoses can be observed, but the overall proliferation rate is similar to that of ordinary glioblastomas. Expression of GFAP is highly variable, and perivascular lymphocyte cuffing may be present. Giant cell glioblastomas are characterized by frequent TP53 (75–90 per cent of cases) and PTEN mutations (33 per cent of cases), but typically lack homozygous CDKN2A deletion and EGFR amplification/overexpression.151,181,182 The primary diagnostic concern is pleomorphic xanthoastrocytoma (PXA), which is also a peripherally located, circumscribed neoplasm that contains large, bizarre tumour cells. However, PXA also usually contains more abundant xanthomatous cells, eosinophilic granular bodies, as well as a substantially lower proliferative rate.

Small Cell Glioblastoma Small cell glioblastoma is less clearly defined than gliosarcoma and giant cell glioblastoma, but is recognized as a pattern with distinctive genetic and clinical associations in the WHO classification.143 The tumour is characterized by monotonous, generally bland appearing cells with round-to-ovoid nuclei that are mildly hyperchromatic and resemble oligodendroglioma at lower magnifications (Figure 27.9c). The cell density is variable, but the nuclear:cytoplasmic ratio and mitotic index are high. The amount of small cell phenotype necessary for the diagnosis has not been established and foci of small

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cells can be noted in otherwise standard glioblastoma. Immunohistochemistry is helpful in the differential diagnosis, because these tumours are generally positive for GFAP, although the elongate processes may appear wispy or the stain may be mostly negative because of the presence of minimal cytoplasm. The small cell phenotype appears to correlate strongly with EGFR gene amplification. In a study of 79 glioblastomas, EGFR amplification was found in 14 of 21 (67 per cent) glioblastomas that were composed exclusively of small cells, in 8 of 25 (32 per cent) glioblastomas with both small cell and non – small cell areas, and in 3 of 33 (9 per cent) glioblastomas without small cells.31 The nosological position of ­so-called small cell astrocytomas lacking microvascular proliferation and necrosis is less clear, because they often behave like ­glioblastomas even when otherwise qualifying only for a WHO grade III designation.185 Small cell glioblastoma shares histological features with oligodendroglioma, including chicken-wire vasculature, haloes, p ­ erineuronal satellitosis and micro-calcifications, although typically lacking microcystic spaces and minigemistocytes. In contrast to oligodendrogliomas, EGFR amplification was present in 69 per cent, with 10q deletions in 97 per cent, but 1p and 19q losses were not present in one series.185 In another study of glioblastoma variants, none of the 45 small cell glioblastomas showed immunohistochemical evidence of IDH1 mutation.104

Glioblastoma with Oligodendroglioma Component Glioblastoma with oligodendroglioma component (GBM-O), WHO grade IV (‘grade IV oligoastrocytoma’), is essentially a high-grade oligoastrocytoma with necrosis.253 These tumours contain either admixed tumour cells with oligodendroglioma and astrocytoma differentiation or distinct regions containing these cell types (Figure 27.9d). GBM-O was only recently accepted following extensive clinicopathologic investigations of anaplastic oligoastrocytomas.152,227,247 Miller et al. studied the overall survival of 215 patients with anaplastic oligoastrocytoma as it related to age, gender, type of surgical procedure, necrosis and endothelial hyperplasia.152 A significantly shorter median survival was found for patients with anaplastic oligoastrocytomas with necrosis than for those without it. In contrast, the presence of endothelial proliferation was not found to be prognostically important in this subset. Thus, these data indicated that patients with anaplastic oligoastrocytomas with necrosis had shorter survival and that such tumours should be considered grade IV. In the fourth edition, the WHO classified GBM-O as grade IV.143 One recent investigation found that GBM-O accounts for 12 per cent of all glioblastomas.3 They were found to arise in younger patients than other forms of GBMs (50.7 versus 58.7 years, respectively) and were also more frequently secondary. Compared to other GBMs, they had a higher frequency of IDH1 mutations and a lower frequency of PTEN deletions. Survival was longer in GBM-O patients compared to conventional GBM, with median survivals of 16.2 and 8.1 months, respectively.

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Pilocytic Astrocytoma  1659



Most of the survival advantage appeared to be associated with younger age at presentation. Among patients with GBM-O, younger age at presentation and 1p deletion were most significant in conferring prolonged survival.

Pilocytic Astrocytoma The pilocytic astrocytoma is a slowly growing, c­ ircumscribed astrocytic neoplasm, predominantly of childhood, composed of a compact arrangement, well-differentiated tumour cells with highly elongate (‘hair-like’) glial ­processes. These tumours arise in stereotypic locations within the CNS and are associated with favourable prognosis.

optic gliomas, reduced visual acuity and visual field defects are most common. Hypothalamic involvement is typically associated with endocrine syndromes (e.g. diabetes insipidus, precocious puberty), electrolyte imbalance and autonomic dysregulation (e.g. hyperthermia). Astrocytomas of the brain stem typically cause cranial nerve deficits, whereas supratentorial tumours usually manifest with seizures or raised intracranial pressure.

Neuroimaging

More than 75 per cent of pilocytic astrocytomas occur in children and adolescents, with a peak incidence between 8 and 13 years. Tumours of the cerebral hemispheres and ­spinal cord tend to manifest at an older age than those of the optic nerve and chiasm, brain stem and cerebellum. Males and females are affected equally, independent of site.

CT scans usually reveal cystic, round to oval lesions with isodense to slightly hypodense signal, which enhance with contrast media. On MR imaging, tumours appear hypointense or isointense on T1- and hyperintense on T2-weighted images. The presence of a mural tumour nodule within a macroscopic cyst (Figure 27.11a) is particularly characteristic for cerebellar and supratentorial hemispheric tumours. Optic nerve tumours appear as fusiform enlargements (Figure 27.11b), whereas a more globular contour is often encountered in chiasmatic examples. Brain stem astrocytomas are usually dorsal and exophytic. Spinal lesions appear as fusiform intramedullary thickenings as a result of a longitudinal extension over several segments (‘pencil-shaped glioma’). Positron-emission tomography scans show a high rate of glucose utilization. MRS shows elevated choline and lactate peaks, with low NAA levels. These findings can be misleading because they are more typical of a high-grade neoplasms overall.

Sites

Macroscopic Appearances

The cerebellum is most frequently affected (Figure  27.11a), with cerebellar hemispheres (>80 per cent) more frequently affected than the vermis (20 per cent). Optic nerve (Figure 27.11b) and chiasmatic gliomas are second most common. Of the latter, a significant fraction involves the adjacent hypothalamus and third ventricle. Those of the cerebral hemispheres are less frequent, except for temporal lobe, in particular its medial portion. Tumours of the brain stem are next in frequency, followed by rarer locations, such as the infundibulum, hypothalamus (without optic chiasm) and spinal cord. In the brain stem, they tend to occupy the dorsal regions and pontomedullary junction, often with exophytic growth, in contrast to diffusely infiltrating fibrillary astrocytomas, which typically develop in the ventral pons.59 Some hypothalamic-chiasmatic lesions of infancy appear to be associated with leptomeningeal seeding and a poor outcome,183 but it is unclear whether such tumours constitute a distinct entity because this region predisposes to the more aggressive pilomyxoid variants.239 Pilocytic astrocytomas may occur simultaneously or subsequently at different CNS sites, usually in the setting of NF1.

In the cerebellum, pilocytic astrocytoma is well delineated and appears to expand rather than infiltrate adjacent brain structures. At other sites, the border with adjacent structures is less defined. The cut surface is greyish-pink and often shows mucoid degeneration, leading to the formation of cysts, a hallmark present in more than 80 per cent of cerebellar astrocytomas.97 In large cysts, the main tumour mass typically forms a mural nodule. The consistency is firm, unless there is extensive mucoid degeneration.

Incidence The pilocytic astrocytoma accounts for approximately 1.5 per cent of all intracranial tumours and has an incidence of 0.33 per 100 000.52 It is the most common low-grade brain tumour of childhood, accounting for 23.5 per cent in this age group. In adults, it comprises less than 1 per cent of brain tumours.

Age and Sex

Clinical Features In cerebellar astrocytomas, symptoms and signs of increased intracranial pressure (headache and papilloedema) prevail, together with ataxia, nausea and cranial nerve deficit. In

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Microscopy The histopathology of pilocytic astrocytomas varies considerably and the diagnosis is occasionally challenging. Typical, particularly of cerebellar lesions, is a biphasic pattern with compact highly fibrillated areas intermingled with loosely structured, micro-cystic tumour that displays a mucinous background (Figure 27.11c,d). In many instances, cells within the microcystic regions will be composed of cells that resemble oligodendroglioma, with round regular nuclei and perinuclear haloes. Although a biphasic pattern is classic, in some pilocytic astrocytomas one pattern will predominate. Those in the optic nerve typically have an exclusively more compact pattern, with abundant Rosenthal fibres. Furthermore, in the optic nerve, the connective tissue septa remain largely intact, but the fascicles are enlarged with nerve fibres being progressively replaced by tumour cells. Extension into the

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1660  Chapter 27  Astrocytic Tumours (a)

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27.11 Pilocytic astrocytomas. (a) Post-contrast T1-weighted magnetic resonance (MR) image showing characteristic enhancing mass associated with a cyst. (b) T2-weighted MR image of a pilocytic astrocytoma (‘optic glioma’) diffusely expanding the optic nerve. (c) Biphasic pattern with compact fibrillar and looser, microcystic areas. (d) Oligodendroglioma-like cells and microcysts are often seen in pilocytic astrocytoma. (e) Rosenthal fibres mainly involve compact regions. (f) Pilomyxoid astrocytoma contains cells with an angiocentric orientation and a myxoid background.

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Pilocytic Astrocytoma  1661



meninges further contributes to the marked, fusiform enlargement of the optic nerve. As its name suggests, the prevailing cell type in the pilocytic astrocytoma is elongated, unipolar or bipolar, with thin hair-like (Greek pilos, hair) processes packed with intermediate filaments, which often form parallel or interdigitating bundles. Although the pilocytic astrocytoma does not have a high cell density, the tumour cell processes produce a coarsely fibrillated, dense matrix. Piloid cells are strongly immunoreactive for GFAP. Focal mucoid degeneration is typical and associated with marked changes in morphology; cells become round or stellate, with or without discernible processes and lack GFAP gene expression, thus mimicking protoplasmic astrocytes or oligodendrocytes. This change is usually accompanied by micro-cyst formation. Highly characteristic of pilocytic astrocytomas are Rosenthal fibres and eosinophilic granular bodies. Rosenthal fibres are bright eosinophilic bodies, with a shape resembling a sausage, corkscrew or carrot (Figure 27.11e). Their presence is mostly restricted to compact areas and are generally rare in cases with extensive mucoid change. The number of Rosenthal fibres varies considerably, but in their absence a lesion should be diagnosed as pilocytic astrocytoma with caution. The expression of GFAP in Rosenthal fibres may be strong, intermediate or absent, and often confined to the periphery. More specific is the presence of αB-crystallin.242 Eosinophilic granular bodies are cytoplasmic inclusions that are also highly characteristic, although not specific for pilocytic astrocytoma. They appear as multiple ‘granular’ bright eosinophilic deposits that may share with Rosenthal fibres, immunoreactivity for GFAP and αB-crystallin.158 Their presence is most frequent in loose, micro-cystic areas. Pilocytic astrocytomas may display histological features that raise the spectre of malignancy, although they almost never assume a malignant character clinically. There may be a remarkable degree of nuclear pleomorphism and multinucleated giant cells may occur. Although mitoses are absent or rare and MIB-1 indices are generally less than 1 per cent, occasional pilocytic astrocytomas show increased proliferative activity, some of which often reflects inflammatory infiltrates.63 Microvascular proliferation may be extensive and often resembles glomerular tufts, although the more commonly found blood vessels are small and have thick, hyalinized walls. They are particularly prominent along the wall of tumour cysts and may occasionally resemble vascular malformations. As in malignant gliomas, their growth is due to secretion by neoplastic astrocytes of VEGF.129 Finally, necrosis may occur, but usually has an infarcted appearance and does not feature pseudo-palisading. Pilocytic astrocytomas have a tendency to invade the leptomeninges, particularly in optic gliomas and in patients with NF1. Neoplastic invasion of the meninges results from the extension of delicate cellular bridges from the cerebellar cortex through the pia mater into the subarachnoid space. Focal desmoplasia may form broad fibrocollagenous septa, but has also been observed in sulci adjacent to the tumour border.97 Meningeal invasion is not considered an aggressive feature and has been associated with improved survival in some studies. Seeding via CSF is uncommon.

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Grading Pilocytic astrocytomas correspond to WHO grade I.36 The rare malignant (anaplastic) pilocytic astrocytoma accounts for less than 2 per cent of all cases and has a biological behaviour similar to that of grade II or grade III astrocytomas.206

27

Differential Diagnosis The histological diagnosis of pilocytic astrocytoma can be challenging on occasion. The differential diagnosis with diffuse astrocytomas is most important, particularly in the brain stem and other sites affected by both astrocytoma types.59 Helpful in this distinction, particularly in small biopsies, is the use of neurofilament immunohistochemistry to highlight the axons of infiltrated neuropil. Axons are usually conspicuous in the background of diffuse astrocytic tumours, but absent or scant in pilocytic astrocytomas, except at the edges of some pilocytic lesions. Any slowgrowing lesion in the CNS, particularly in the brain stem and spinal cord, may produce Rosenthal fibres and thus mimic a pilocytic astrocytoma. This can be most misleading around a cystic cerebellar haemangioblastoma or around a suprasellar/third ventricular tumour, such as a craniopharyngioma; further searching for diagnostic features is therefore critical. Gangliogliomas may also include a pilocytic component; identification of neoplastic ganglion cells ensures a correct diagnosis. The dysembryoplastic neuroepithelial tumour (DNT) may be difficult to delineate from the pilocytic astrocytoma of the temporal lobe. The multinodularity of the DNT, its preferred cortical location and the presence of a specific glioneuronal elements make it a distinct lesion, even if some areas resemble pilocytic differentiation. Immunoreactivity for mutant IDH1 is helpful in distinguishing diffuse astrocytomas, because pilocytic astrocytomas will be negative, although lack of staining does not exclude the former, particularly in children where the majority are negative. In the appropriate clinical and radiological setting, alterations in BRAF, particularly BRAF gene fusion/ duplication, favours the diagnosis of pilocytic astrocytoma.

Histogenesis The cellular origin of pilocytic astrocytoma has remained enigmatic. It seems likely that they arise from a progenitorlike cell, via acquisition of specific genetic changes, particularly involving the BRAF and NF1 genes. In addition, the developmental transcription factor OLIG2, which is restricted to oligodendrocyte and spinal motor neuron formation during embryogenesis, is widely expressed in nonependymal gliomas, including pilocytic astrocytomas.136

Molecular Genetics Cytogenetic analyses of pilocytic astrocytomas have revealed either a normal karyotype or a variety of aberrations without a distinct pattern suggesting the loss of a particular tumour suppressor gene.146 Gains of chromosomes 5, 7 and 8 are the most frequent finding.262 Conventional CGH has shown a variety of changes, although none are either frequent or consistent.212,235

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In contrast, more recent array CGH studies have identified a low-level copy number gain of the BRAF gene on 7q34 in a large proportion of cerebellar pilocytic astrocytomas.146,187 BRAF is a proto-oncogene within the MAPK/ ERK signalling pathway and is a regulator of cellular differentiation, proliferation and migration. The BRAF copy gain is due to a tandem duplication producing a KIAA1549:BRAF fusion protein with loss of its Ras-binding domain and constitutive BRAF activity. BRAF fusions are present in 70 per cent of all pilocytic astrocytomas, but are rare in high-grade paediatric gliomas. These fusions are found in approximately 75 per cent of cerebellar, but only 55 per cent of non-cerebellar pilocytic astrocytomas.93 The frequency of BRAF-KIAA1549 fusions also appears to vary with patient age, decreasing in frequency with increasing age. Approximately 50 per cent of pilomyxoid astrocytomas contain BRAF fusions. The constitutively active V600E point mutation of BRAF is present in about 10 per cent of pilocytic astrocytomas, but is much less frequent in cerebellar tumours.216 The V600E is more common in other low-grade tumours in the differential, including 25 per cent of gangliogliomas and 80 per cent of pleomorphic xanthoastrocytomas. Thus, over 75 per cent of sporadic PAs have some sort of BRAF alteration. Less than 10 per cent of NF1-associated PAs contain a BRAF fusion or mutation, because NF1 loss similarly leads to activation of the MAPK/ERK signalling pathway. Those gliomas of childhood that have BRAF fusion events usually behave as a typical grade I pilocytic astrocytomas, whereas those without BRAF fusion are more likely to behave in a more aggressive fashion. Detection of BRAF fusion is feasible via several strategies, including reverse transcriptase polymerase chain reaction (PCR) analysis and FISH. Approximately 15 per cent of patients with NF1 develop pilocytic astrocytomas,130 particularly of the optic nerve, and up to one-third of patients with a pilocytic astrocytoma in this location fulfill the diagnostic criteria of NF1. Occasionally, sporadic pilocytic astrocytomas show a loss of chromosome 17q, including the region encoding the NF1 gene.250 Because the NF1 gene has tumour suppressor functions, loss of neurofibromin expression likely plays a tumorigenic role. However, screening of NF1 coding sequences, including the critical GRD region, has failed to detect mutations in sporadic cases.191 Immunohistochemically, ­ neurofibromin is overexpressed,191 but a study of eight pilocytic astrocytomas from six patients with NF1 showed loss of neurofibromin expression, with NF1 loss in two of four studied tumours.71 These results raise the possibility that the role of the NF1 gene may be different in NF1 associated versus sporadic pilocytic astrocytomas. Inactivation of the TP53 gene does not seem to play a role in pilocytic astrocytoma.126 Increased levels of the p53 protein may be found in pilocytic astrocytomas without TP53 mutations.126 Other alterations commonly involved in diffuse astrocytomas are also not frequently found in pilocytic astrocytomas. For example, a study of the CDNK2A, TP53, CDK4 and PTEN genes in 29 pilocytic astrocytomas showed only one TP53 mutation and one PTEN ­mutation.40 However, activation of the PI3-kinase/

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Akt pathway has been associated with aggressive clinical behaviour in pilocytic astrocytomas.207

Prognosis and Treatment In contrast to diffusely infiltrating astrocytomas, pilocytic astrocytomas do not have an intrinsic tendency to undergo malignant progression. Even after many years, recurrences usually display histopathological features similar to those of the primary lesion, without aggressive behaviour. Observed survival rates have been 100 per cent at 5 years and 95.8 per cent at 10 years after diagnosis.32 Nevertheless, exceptional cases of malignant recurrence from benign pilocytic astrocytoma have been reported.98 Occasionally, neuroradiological examination reveals multiple lesions (multicentric pilocytic astrocytoma). If association with NF1 is ruled out, metastatic spread via the CSF is the most likely cause. In the majority of cases reported, primary tumours were located in the hypothalamus and patients had undergone neurosurgical intervention followed by radiotherapy, chemotherapy or both.145,148 Meningeal spread may be associated with malignant progression, but it has also been observed in tumours with benign morphology.145,169,192 Pilocytic astrocytomas are considered most benign of the gliomas, with recurrence-free intervals of up to more than 20 years.60 Complete surgical resection is most effective but often impossible to achieve, particularly in midline structures, e.g. the optic chiasm, the hypothalamus and the brain stem. Radiotherapy may be carried out, particularly in the setting of subtotally resected but symptomatic tumours. Given adverse sequelae from radiation to the developing brain, however, chemotherapy has been advocated, particularly for progressing, surgically unresectable lesions. The recurrence-free survival of patients with pilocytic astrocytoma largely depends on whether or not complete surgical resection is possible. Histopathological criteria that allow a prediction of the clinical outcome have not been identified.63 Even in the rare cases of late malignant recurrence, the initial biopsy did not contain features i­ndicative of incipient malignant transformation.241

Variants of Pilocytic Astrocytoma The pilomyxoid astrocytoma (PMA), WHO grade II is typically a tumour of infancy239 and is considered a variant of pilocytic astrocytoma, with characteristic clinical, neuroimaging and pathologic features. PMAs most often arise in hypothalamic region, with symptoms referable to that site, including failure to thrive, developmental delay, vomiting and feeding difficulties. In older children, headaches, nausea and visual symptoms are more common.117 By MR imaging, they are well circumscribed, generally solid and homogeneously contrast-enhancing midline masses, mostly in the suprasellar and hypothalamic region. The histologic appearance of PMA is dominated by a hypercellular, monomorphous population of piloid cells that are typically embedded within a rich myxoid matrix and often display an angiocentric arrangement (Figure 27.11f). It has a compact architecture, with only a slight tendency for peripheral infiltration of adjacent brain. Individual

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Pleomorphic Xanthoastrocytomas  1663



tumour cells have elongate fibrillar processes, are moderate in size, and contain hyperchromatic nuclei with only modest nuclear pleomorphism. The mitotic index is typically low. The diagnosis of PMA is made only when this tissue pattern is predominant, because focal myxoid or angiocentric cell arrangement may be noted in typical pilocytic or infiltrating astrocytomas. Unlike ordinary pilocytic astrocytomas, PMA typically lack a biphasic appearance, do not contain Rosenthal fibres, and only exceptionally contain eosinophilic granular bodies. Immunohistochemically, PMAs label strongly and diffusely for GFAP and vimentin but are typically negative for the neuronal markers neurofilament and chromogranin. Synaptophysin immunoreactivity has been reported in a subset of PMAs, especially in a perivascular distribution.62 The MIB-1 labelling index is often around 5 per cent. The relation of PMA to pilocytic astrocytoma is still debated. Reports of hybrid tumours that contain components of both conventional pilocytic astrocytoma and PMA, as well as ‘maturation’ of the latter into the former on recurrence, suggest that the two tumours are related and form a spectrum. This concept is reflected in the new WHO classification, with PMA categorized as a variant of pilocytic astrocytoma. Nevertheless, PMAs are associated with a more aggressive clinical course, resulting in a WHO grade II designation.41,58,117 Anaplastic pilocytic astrocytoma refers to the rare examples that have undergone malignant transformation. In one systematic review of over 2000 pilocytic astrocytomas, 1.7 per cent were found to have anaplastic features, which included mitotic figures >4 per 10 HPF, moderate to severe nuclear atypia, hypercellularity or necrosis.206 Nearly a quarter of these arose in the setting of NF1 and 12 per cent arose following radiation therapy. Median overall and progression-free survivals after diagnosis for the entire study group were 24 and 14 months. Importantly, several histopathological features indicative of progression to anaplasia in diffuse astrocytomas are not associated with malignancy in the pilocytic astrocytoma, e.g. nuclear polymorphism, vascular proliferation and invasion of the meninges. The diagnosis of anaplastic (pilocytic) astrocytoma requires overt anaplasia with significant proliferative activity. Foci of necrosis are often present but not diagnostic in the absence of mitoses.241

Pleomorphic Xanthoastrocytomas Pleomorphic xanthoastrocytoma (PXA) is a rare, superficially located neuroepithelial neoplasm characterized by pleomorphic and lipidized cells, typically with a rich reticulin network.

Incidence, Age and Sex The PXA accounts for less than 1 per cent of astrocytic tumours.64,112 Without obvious predilection for either sex, these tumours typically manifest in children and young adults with a longstanding history of seizures. Two-thirds of patients are under the age of 20 years. Cases have been

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noted in the setting of NF1, including those with malignant features.161,210

Sites

27

The vast majority of tumours arise in superficial supratentorial locations, with extensive involvement of overlying subarachnoid space and a proclivity for the temporal lobe. Dural involvement is, however, exceptional.111,112 Neoplasms with the typical histopathological features of PXAs occasionally develop in other locations, including cerebellum137 and spinal cord;87 their biological behaviour potentially differing from supratentorial tumours.

Neuroimaging Neuroimaging typically demonstrates a cerebral cortical neoplasm with deep extension to the grey-white junction and superficial extension to the leptomeningeal surface. Both CT and MR images usually show clearly demarcated borders, cyst formation with proteinaceous contents and a mural nodule with strong contrast enhancement (Figure 27.12a). The tumour mass is often isointense to grey matter on T1-weighted and mildly hyperintense on T2-weighted MR imaging. Leptomeningeal contrast enhancement is seen occasionally.

Macroscopic Appearances PXA commonly have solid and cystic components. The cysts may be conspicuous, containing large amounts of dark golden, somewhat xanthochromic, serous fluid. The mural nodule has variable appearance, with a predominant yellow-orange hue that may be punctuated by focal haemorrhage. The tumours are usually firmer than the adjacent brain.

Microscopy The striking histopathological features are multinucleated, occasionally lipidized giant cells with bizarre, often hyperchromatic nuclei. In addition, there are small, round, polygonal and fusiform cells, often arranged in fascicular patterns (Figure 27.12b). As the name of the lesion suggests, cytological diversity is the rule. The astroglial nature is shown by GFAP immunoreactivity in plump polygonal cells without processes, and in the more fusiform cells arranged in interlacing bundles (Figure 27.12c). Cytoplasmic lipidization, especially in the large cells, may be prominent but is usually a minor feature and may be absent in some. Of particular importance is the presence of eosinophilic granular bodies, which are often numerous and an important clue to the diagnosis. Mitoses are infrequent. Perivascular lymphocytic infiltration is common. A reticulin-positive stroma is a hallmark of PXA, but is not universal. When present, it is most prominent in tumour involving the subarachnoid space. Reticulin deposition may delineate fascicles of cells or may envelop single tumour cells. Ultrastructural studies have demonstrated a basal lamina separating cells.113,261

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1664  Chapter 27  Astrocytic Tumours (a)

(b)

(c)

27.12 Pleomorphic xanthoastrocytoma. (a) Contrast-enhanced computed tomography (CT) scan showing superficial large cyst with an enhancing mural nodule. (b) Large, markedly pleomorphic cells with some lipidization; note also smaller cells and scattered lymphocytes. (c) Variable immunoreactivity for GFAP.

Although PXA appear well demarcated macroscopically, portions of the tumour invade the adjacent brain and perivascular spaces. The involvement of the superficial Virchow–Robin spaces may be striking in otherwise usual tumours. Several cases without a prominent reticulin stroma, but otherwise typical features, have also been described.111 Evidence of neuronal differentiation may be found in otherwise typical PXA, although as opposed to ganglioglioma, synaptophysin or neurofilament positive cells often do not resemble ganglion cells. A study of 40 cases found immunopositivity for GFAP and S-100 protein in 100 per cent of cases, class III beta-tubulin in 73 per cent, synaptophysin in 38 per cent, and neurofilaments and MAP2 in 8 per cent, but no staining for chromogranin A.66 Another report documented staining for neuronal markers (class III beta-tubulin, neuronal nuclear antigen, neurofilament protein, synaptophysin) at least focally in all nine tumours studied.147 Ultrastructural analyses have also demonstrated neuronal features in 20 per cent of tumours, including dense-core granules, microtubules and clear vesicles.90 Cases have also been reported in which PXA constitutes the glial component of a ganglioglioma or forms a combined collision-type tumour66,137 and tumours, particularly of the temporal lobe, have been observed in association with cortical dysplasia99,124 or arising in the setting of a prior cortical malformation.195 Thus, although the tumour is designated as an ‘astrocytoma’, neuronal differentiation is common.

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Grading Pleomorphic xanthoastrocytomas are designated WHO grade II. For lesions with significant mitotic activity (>5 mitoses per 10 high-power fields), areas of necrosis, or both, the designation ‘pleomorphic xanthoastrocytoma with anaplastic features’ has been proposed,64 but this variant has not yet been designated a particular grade.143

Differential Diagnosis The main differential diagnosis for PXA is that of malignant astrocytic tumours, such as giant cell glioblastoma. Most helpful in this setting is the presence of eosinophilic granular bodies, because these are rare in glioblastomas. Features such as reticulin deposition, xanthomatous change, relatively sharp borders and inflammation are not definitive, because other gliomas may occasionally show these findings. Documentation of neuronal differentiation may favour a diagnosis of PXA, but this is similarly not definitive, because more malignant gliomas are also showing positivity for neuronal markers on immunohistochemistry.

Histogenesis PXAs were originally suggested to arise from subpial astrocytes, because such cells elaborate basal lamina and would explain the superficial localization. The demonstration

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Subependymal Giant-Cell Astrocytomas  1665

of neuronal differentiation in some PXA suggests a more complex histogenesis.66,147,193 Moreover, the reported association with cortical dysplasia99,124,195 suggests a link to developmental dysregulation. It seems likely that PXA are derived from a cell of origin that is cortical and that has bipotential differentiation capability.

TSC is approximately 1/5000, and SEGAs occur in 5 to 15 per cent of TSC patients.259 In a large series of 345 welldocumented cases of TSC,221 histopathological examination confirmed that SEGAs were present in about 6 per cent of cases. Their true incidence is likely higher, because not all SEGAs warrant surgical intervention.

Cytogenetics and Molecular Genetics

Clinical Features

Complex karyotypes have been documented, with gains on chromosomes 3 and 7,213 as well as alterations of chromosome 1q.133,214 The activating BRAF mutation V600E has been documented in 60–70 per cent of PXA.48,216 However, the BRAF fusion that is typical of pilocytic astrocytomas is not seen in PXA.54 IDH1 and IDH2 mutations are also not typical.54,264 In two studies of a total of 14 PXAs, three tumours contained a TP53 missense mutation.157,179 In a larger series of 55 PXAs studied, two thirds of tumours were p53 immunonegative, and only a single TP53 mutation was detected.65 Another large series of 62 PXAs had only three tumours with TP53 mutation, and no tumours with homozygous CDKN2A deletion or amplifications of CDK4, MDM2 or EGFR.110 Although there are no definite tumorigenic associations with the NF1 gene, cases of PXA have been reported in NF1 patients.161,210

Patients with SEGA typically present with symptoms of hydrocephalus or acute haemorrhage. In some cases, it may be the first manifestation, but more frequently, patients have longstanding, antecedent seizures due to disease-related cortical tubers and have other evidence of TSC.

Prognosis A recent analysis of the Surveillance, Epidemiology, and End Results (SEER) database demonstrated 5- and 10-year overall survival rates of 75 and 67 per cent, respectively.184 On multivariate analysis, male gender and increasing age were associated with worse overall survival. The average interval to recurrence appears to be longer in cases lacking histological evidence of anaplasia.112,113,144261 However, malignant recurrence after an interval of 15 years has been documented.113 Increased mitotic rates may be predictive of tumour recurrence,144 but not necessarily of malignant progression. A study of 71 cases confirmed that mitotic index and extent of surgical resection appear to be the main predictors of recurrence-free survival.82

subependymal giant-cell astrocytomas Subependymal giant-cell astrocytomas (SEGAs) are WHO grade I neuroepithelial tumours that primarily develop within the setting of the tuberous sclerosis complex (TSC).143 Located in the wall of the lateral ventricles, they are of uncertain histogenesis and composed of large cells having both astrocytic and neuronal features.

Incidence, Age and Sex SEGAs typically present clinically within the first two decades of life (median age about 13 years, range 1–31 years) without any gender predilection. The incidence of

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27

Tumour Sites, Neuroimaging and Macroscopic Appearances The most common location of SEGA is in the wall of the lateral ventricles near the foramen of Monro. In the more anterior portion of the ventricle, smaller nodules of discrete subependymal hamartomas situated in the caudothalamic groove often coexist with a larger tumour. Neuroimaging shows a well-delineated, heterogeneous neoplasm with mixed hypodense and isodense regions on CT and mixed signal intensities on both T1- and T2-weighted MR imaging. The heterogeneity is due partly to the nodular and multicystic architecture of the tumours, which are variably calcified and have a marked but heterogeneous vascular component. Macroscopic examination of unfixed tissue reveals firm, tan-white nodules with multiple microcysts and occasional small or massive haemorrhages (Figure 27.13a).

Microscopy, Immunohistochemistry and Electron Microscopy SEGAs are characterized by an admixture of heterogeneous cell populations. Three major cell types are present, to varying degrees in different tumours, in a fibrillar background: small spindle-shaped or elongated cells often arranged in sweeping fascicles; intermediate-sized polygonal or ‘gemistocyte-like’ cells; and globoid or ganglion-like cells (Figure 27.13b). Nuclei are usually pleomorphic, round to oval, and contain finely granular chromatin with distinct, sometimes prominent, nucleoli. The spindle and polygonal cells are often randomly oriented, whereas in some areas the elongated cells appear to stream from the blood ­vessel walls in a manner reminiscent of ependymal pseudo-rosettes. Elongated cells commonly have broader, ­fibrillated cytoplasmic processes than is typical of ependymomas (Figure 27.13c). The polygonal cells may be conspicuous with eccentric nuclei and enlarged globoid cells often have eosinophilic, homogeneous cytoplasm. Giant cells, with a ‘ganglion cell-like’ appearance, are not uncommon and multinucleated cells can usually be found. The tumour microvasculature is often prominent, not uncommonly forming dilated channels with either thin or h ­ yalinized walls that may bleed, either spontaneously or after surgical manipulation. Calcification may be dense (Figure 27.13d). Some SEGAs have occasional mitoses, moderate atypia, focal necrosis or vascular hyperplasia, but such features do not connote more aggressive behaviour.

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1666  Chapter 27  Astrocytic Tumours (b)

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27.13 Subependymal giant-cell astrocytoma. (a) SEGA in the anterior lateral ventricle in a TSC patient; also note the cortical tubers. (b) Ganglioid and epithelioid cells within a fibrillar matrix. (c) Areas with more elongate and spindled cells intermingled among spans of dense fibrillarity. (d) Marked calcification.

GFAP expression is variably demonstrated in cellular processes and the cytoplasm of spindle, polygonal and ganglioid cells. S-100 protein is also typically present. A study of 20 cases showed immunoreactivity for class III β-tubulin (TUJ-1), a neuron-associated β-tubulin, in about 85 per cent of cases.140 Staining for TUJ-1 was found in the cytoplasm of most cell types; however, it was most apparent in the polygonal and ganglionlike cells. Epitopes for the medium and high molecular weight neurofilament proteins (NF-M/H) were less readily demonstrable; nevertheless, the patterns of the various phosphorylation-dependent epitopes in the cell bodies and processes were consistent with neuronal differentiation. Other studies have documented synaptophysin and neuron-specific enolase immunoreactivity.28 Some neoplastic

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cells express both glial- and neuron-associated antigens, suggesting a capacity for differentiation along glioneuronal, as well as neuroendocrine lineages.140 Although some TSC-associated lesions react with HMB-45, a melanoma marker, SEGA do not.72,115 Ultrastructural features of SEGA include cytoplasmic intermediate filaments within cellular processes, multiple mitochondria, ribosomes, microtubules, Golgi and endoplasmic reticulum profiles.28 Cells with secretory granules and rare synapse-like structures reflect neuronal differentiation.

Grading SEGA are designated WHO grade I.143

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References  1667



Treatment and Prognosis Symptomatic tumours are treated with resection. SEGAs are generally detected in patients with TSC who are imaged for other reasons. In this setting, depending on the size of the lesion, careful monitoring of tumour growth may be indicated rather than immediate surgery. These tumours have an almost uniformly favourable prognosis.220 Despite the potential for late recurrence, reported as late as 22 or 47 years after initial therapy,77,220 cases with malignant transformation have not been described, although rare cases may have drop metastases.237 Because the genetic alterations that lead to the development of SEGAs  – mutations in either TSC1 or TSC2 – are known to lead to upregulation of the mTOR pathway by neoplastic cells, clinical trials have evaluated the potential efficacy of an mTOR inhibitor, everolimus, for SEGA patients.122 Initial experience with everolimus has been positive, with patients showing marked reduction of SEGA volume and seizure frequency, suggesting that pharmacologic therapy may be a viable alternative when complete resection is not possible.

Histogenesis The histogenesis of SEGA is poorly understood. That these tumours are histologically identical to the subependymal nodules (‘candle gutterings’) that stud the ventricles in TSC patients strongly suggests that they are derived from these hamartomatous lesions.49 In this regard, their histogenesis is most likely related to other hamartomatous lesions in TSC. SEGA demonstrate variable glial (astrocytic/ependymal), neuronal or mixed

glial-neuronal differentiation on the basis of ultrastructural and immunohistochemical features.28 Therefore, SEGA may eventually be more properly classified as a glioneuronal, rather than purely astrocytic tumour.

27

Molecular Genetics SEGA is a major diagnostic criteria for TSC (see Chapter 44), an autosomal dominant disorder characterized by hamartomas and benign neoplasms of multiple organ systems.49,67,204 There are two related genes linked to TSC:45,190,204 TSC1 on chromosome 9q34 encodes hamartin, whereas TSC2 on chromosome 16p encodes tuberin.45,143,190 Tuberin and hamartin interact physically within the cell cytoplasm to form a tumour suppressor complex that inhibits the function of mTOR (mammalian target of rapamycin). Loss of function of either results in upregulation of mTOR and increased proliferative activity. Immunohistochemical and genetic analyses of SEGAs from seven patients with TSC (four with TSC1 mutations and three with TSC2 mutations) showed that tumour cells had high levels of phospho-S6K, phospho-S6 and phosphoStat3, indicating activation of the PTEN-mTOR pathway. In addition, five of six tumours also had biallelic mutations of TSC1 or TSC2.39 Inactivation of tuberin may also occur via phosphorylation, suggesting another possible mechanism for altering the pathway.78 Furthermore, expression of hamartin and tuberin tend to be mutually exclusive, suggesting a need to inactivate both copies of one or other of these molecules.57

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27

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28 28 Chapter

Oligodendroglial Tumours Guido Reifenberger

Oligodendroglioma....................................................................1673 Anaplastic Oligodendroglioma...................................................1681 Oligoastrocytoma......................................................................1685

Oligodendroglioma Oligodendroglioma is defined as a diffusely infiltrating lowgrade glioma composed of neoplastic cells morphologically resembling oligodendroglia. Most oligodendrogliomas carry an IDH1 or IDH2 mutation combined with deletion of chromosomal arms 1p and 19q.

Incidence, Age and Sex Distribution Oligodendroglioma accounts for approximately 2 per cent of all primary brain tumours and 6 per cent of all gliomas. For the United States of America, the overall annual incidence rate during the years 2004 to 2008 has been estimated as 0.28 per 100 000 persons.16 The incidence rate increased over time, in particular during the late 1990s and early 2000s, which has been attributed to a less stringent use of diagnostic criteria at that time; more recently, the incidence has stabilized at the rate given earlier.67 Oligodendrogliomas develop at any age, but the majority arise in adults with an incidence peak in the fourth and fifth decades of life.77 In children younger than 14 years of age, oligodendroglial tumours account for only 1.1 per cent of brain tumours.16 Oligodendrogliomas are more common in white than in black people, with a corresponding ratio of 2.43. Males are more often affected than females (male to female ratio: 1.26).16

Clinical and Radiological Features Seizures are the most common presenting symptom that is seen in approximately two thirds of the patients.63,78 Additional clinical symptoms include headache and other signs of increased intracranial pressure, focal neurological deficits and cognitive or mental changes. In older ­studies, patient histories were often long-standing with intervals greater than 5 years between the first symptom and the final diagnosis being common.75,104 Today, patient

Anaplastic Oligoastrocytoma������������������������������������������������������ 1687 Other Mixed Gliomas����������������������������������������������������������������� 1689 References�������������������������������������������������������������������������������� 1689

histories are usually shorter because computed tomography (CT) and magnetic resonance imaging (MRI) are widely available for diagnosis of patients with suspicious neurological signs and symptoms. On CT, oligodendrogliomas appear as hypo- or isodense, well-demarcated, frequently calcified masses most commonly located in the subcortical white matter with extension into the adjacent cortex. MRI typically reveals a T1-hypointense and T2-hyperintense lesion, which appears well-demarcated and shows little perifocal oedema (Figure 28.1). Some tumours demonstrate heterogeneity as a result of intratumoural haemorrhages and/or cystic degeneration. Gadolinium enhancement is commonly seen in anaplastic oligodendrogliomas. Contrast enhancement in low-grade tumours has been linked to less favourable prognosis.120 Perfusion and diffusion imaging may help to distinguish low-grade oligodendrogliomas from diffuse astrocytomas of WHO grade II, because regional cerebral blood volume (rCBV) and apparent diffusion coefficient (ADC) values are frequently higher in oligodendrogliomas.6 Positron emission tomography (PET) imaging with amino acid tracers often shows increased tumour/brain ratios in oligodendrogliomas as compared to diffuse astrocytomas.106 Tumour/brain ratios are higher in anaplastic as compared to low-grade oligodendroglial tumours.34 Paediatric oligodendrogliomas less frequently show calcification, contrast enhancement and oedema, when compared to adult counterparts.111 Oligodendrogliomas without 1p/19q deletions more often demonstrate mixed T1- and T2-weighted signal intensities.47,68 In addition, 1p/19q deletion appears to be associated with indistinct tumour borders on T1-weighted images, paramagnetic susceptibility and calcification.68 However, these findings were not confirmed by others.47 Recent studies indicate that elevated levels of 2-hydroxyglutarate, the oncometabolite aberrantly produced by mutant IDH1 and IDH2 proteins, can be readily detected by magnetic resonance spectroscopy,18 thereby allowing for the non-invasive identification 1673

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1674  Chapter 28  Oligodendroglial Tumours

extensive mucoid d ­ egeneration. Areas of m ­ acrocystic degeneration are not unusual. Non-therapy associated necrosis suggests ­anaplasia. Oligodendrogliomas are densely vascularized tumours and intratumoural haemorrhages are frequent.

Microscopy

28.1 Oligodendroglioma. Low-signal, non-enhancing, welldefined lesion on axial, T1-weighted, post-gadolinium MRI. Courtesy of Dr CC Penney, King’s Healthcare NHS Trust, London, UK.

of IDH1/2-mutant gliomas, including the vast majority of oligodendrogliomas.

Macroscopy Oligodendrogliomas may arise anywhere in the central ­nervous system but most develop in the cerebral hemispheres. The frontal lobe is involved in approximately 50–65 per cent of patients. With decreasing frequencies, oligodendrogliomas manifest in the temporal, parietal and occipital lobes. Infiltrative growth involving more than one cerebral lobe or both hemispheres is not uncommon. Oligodendrogliomas are far less frequent in the basal ganglia, thalamus, brain stem or cerebellum. Primary oligodendrogliomas of the spinal cord are rare, accounting for only 1.5 per cent of all oligodendrogliomas and 2 per cent of all spinal cord tumours.32 Occasional patients present with primary leptomeningeal oligodendroglioma or diffuse leptomeningeal oligodendrogliomatosis, respectively. Patients with oligodendroglial gliomatosis cerebri have also been reported.109 Moreover, rare oligodendrogliomas arising in ovarian teratomas are on record.114 Macroscopically, the typical oligodendroglioma appears variably well-defined, grayish-pink and soft, growing in cerebral cortex and subcortical white matter. The affected gyrus is expanded and the gray-white matter junction is blurred. Oligodendrogliomas may focally infiltrate adjacent leptomeninges, sometimes with an accompanying desmoplastic ­reaction leading to a more firm, rubbery consistency of the superficial component. Calcifications are frequently present and may impart a gritty texture to the tumour, with occasional densely calcified lesions demonstrating intratumoural ‘stones’. Rare cases appear as very soft, ­ gelatinous masses due to

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Oligodendrogliomas are monomorphous, moderately cellular, diffusely infiltrating gliomas composed of cells with uniform, round to slightly oval nuclei and perinuclear halos on routinely formalin-fixed paraffin sections (Figure 28.2a). The nuclei are slightly larger than those of normal oligodendrocytes, with a chromatin pattern that is less coarse with small nucleoli evident in well preserved specimens. Occasional mitotic figures are still compatible with a lowgrade oligodendroglioma. The typical nuclear features are best demonstrated in tissue smears and in well-fixed, paraffin-embedded specimens. On smear preparations, oligodendroglioma cells have a small rim of cytoplasm and processes are sparse. In fixed tissue sections, cellular swelling and retraction of the delicate cytoplasmic processes produces the variable, but hallmark, perinuclear halos resulting in the characteristic ‘honeycomb’ or ‘fried egg’ appearance. However, in optimally preserved specimens, smear preparations, frozen sections and paraffin sections made from frozen tumour tissue this artefact is not present and the tumour cells have scant but distinct cytoplasm. There is a notable paucity of fibrillarity in solid regions of oligodendrogliomas, whereas the infiltrative edge incorporates background neuropil. This entrapped neuropil should not be confused with the inherent fibrillarity of astrocytic gliomas, or on immunohistochemistry, with its inherent synaptophysin positivity. On low-power examination of tissue sections, a striking feature of most oligodendrogliomas is their cellular uniformity. In this regard, the diagnosis of oligodendroglioma should be considered in the setting of any moderately hypercellular glioma with uniform nuclei. This may be particularly useful in frozen sections, where the presence of a hypercellular glioma without much nuclear pleomorphism, spindled cells or mitoses should prompt consideration of oligodendroglioma. However, pathologists should be reluctant to make a definite diagnosis of oligodendroglioma based on intraoperative frozen sections alone. The tumour cells are usually arranged in diffuse sheets, often conspicuously grouped by a branching capillary network. Some tumours contain rather distinct nodules that must be distinguished from those seen in dysembryoplastic neuroepithelial tumours. Nodular areas may show increased cellularity, which is not indicative of anaplasia as long as mitotic activity is low. Occasional tumours contain a spongioblastic pattern with parallel, palisaded rows of somewhat fusiform cells (Figure 28.2b), although this pattern is not specific as it may be encountered in several other tumor types. Extracellular mucin deposition and microcyst formation are prominent in some cases. Oligodendrogliomas can show varying ‘astrocytomalike’ features. For instance, tumour cells with a rim of glial fibrillary acidic protein (GFAP) positivity have been called ‘gliofibrillary oligodendrocytes’.41 Other neoplastic cells with eccentric, rounded, eosinophilic bellies of GFAP positive cytoplasm are referred to as ‘minigemistocytes’ or ‘microgemistocytes’. Neither of these cell types necessarily

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  Oligodendroglioma  1675 (a)

(b)

(c)

(d)

28

28.2 Oligodendroglioma. (a) The typical ‘honeycomb’ appearance: groups of cells, with regular, round nuclei and artificially swollen, empty-looking cytoplasm, bounded by distinct cell membrane. Haematoxylin and eosin (H&E). (b) Parallel rows of cells forming spongioblastoma-like palisades. H&E. (c) The delicate vascular pattern demonstrated by lectin histochemistry. Ulex europaeus antigen I. (d) Immunoreactivity for the R132H mutant form of IDH1 is common.

implies a transition to oligoastrocytoma. Similarly, hypertrophic reactive astrocytes are typically scattered throughout oligodendrogliomas and should not be confused with the neoplastic astrocytes in oligoastrocytomas. Individual cases of oligodendroglioma composed predominantly of GFAPnegative signet-ring cells have been reported.62 Rarely, eosinophilic granular cells are prominent.19 Individual tumours, including cases with 1p/19q-deletion, demonstrate evidence of neuronal or neurocytic differentiation, i.e. contain neoplastic cells with expression of neuronal markers and formation of neurocytic rosettes.81 Moreover, ganglioglioma-like foci have been reported in rare instances.80 Oligodendrogliomas have a characteristic delicate, branching, arcuate ‘chicken wire’ like vasculature (Figure 28.2c); when present, this pattern should prompt consideration of an oligodendroglioma, although this feature alone is not sufficiently specific. In some tumours, the capillary network subdivides the tumour tissue into smaller and larger lobules. Probably because of their dense vascularization, oligodendrogliomas have a tendency for intratumoural haemorrhages. Microvascular proliferation and necrosis are absent in low-grade oligodendroglioma. The infiltration patterns of oligodendroglioma ­parallel those of other diffuse gliomas, although ‘secondary structures of Scherer’ tend to be more pronounced. These ­‘secondary

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structures’ include perineuronal ­ satellitosis, perivascular aggregations and subpial spread. Oligodendrogliomas may also spread into the leptomeninges and, occasionally, grow primarily as leptomeningeal masses. Calcification is typical of oligodendroglioma, albeit not invariable. Calcification varies from minute particles to large, grossly visible ‘stones’, with intermediate-sized calcospherites being the most frequent. Calcospherites are irregular or spherical, sometimes lamellated calcifications. When large deposits are present, they are usually centrally localized. Minute calcifications are most common alongside or within the delicate vasculature.

Grading Oligodendrogliomas are slowly growing neoplasms that histologically correspond to World Health Organization (WHO) grade II.87 Various morphological features have been associated with higher malignancy and poor prognosis. These include nuclear atypia and pleomorphism, high cellularity, brisk mitotic activity, endothelial hypertrophy and microvascular proliferation, as well as necrosis. Collectively, these morphological features are similar to those commonly used for the grading of diffuse astrocytic gliomas. However, their individual impact on grading and their integration into an objective and reproducible grading

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1676  Chapter 28  Oligodendroglial Tumours

system of oligodendrogliomas is less established. A study by seven independent neuropathologists identified older patient age, high cellularity, presence of mitoses, endothelial hypertrophy and proliferation, and necrosis significantly associated with shorter survival on univariate analysis in a cohort of 124 patients.35 On multivariate analysis, however, only patient age and endothelial proliferation were independently associated with survival. The WHO classification separates oligodendroglial tumours into well-differentiated tumours of WHO grade II and anaplastic (malignant) tumours of WHO grade III.87,88 The value of the WHO grade as a significant predictor of survival has been confirmed in various studies.35,63,77,100 The WHO criteria for anaplasia in oligodendroglial tumours leave some room for subjectivity because neither the impact of the various parameters, i.e. ‘increased cellularity, marked cytological atypia, high mitotic activity, microvascular proliferation and necrosis’, nor the number of required parameters are precisely defined. As a rule, the histological diagnosis of an anaplastic oligodendroglioma should require either the presence of conspicuous endothelial proliferation and/or high mitotic activity. In borderline cases, immunostaining for MIB–1 and attention to clinical and neuroradiological features, such as rapid symptomatic growth and contrast enhancement, may provide helpful additional information. In addition to patient age, clinical performance score, contrast enhancement on neuroimaging and histological grading, there is increasing evidence that the expression of proliferation markers and, in particular, genetic alterations, such as IDH1/2 mutation and 1p/19q deletion, are important prognostic factors for patients with oligodendroglial tumours, in particular when patients are treated with radio- and/or chemotherapy. Thus, modern assessment of oligodendroglioma behaviour should consider clinical and neuroimaging findings, histological grading, proliferation indices and molecular genetic characteristics.

Immunohistochemistry Immunohistochemical studies may provide useful information for the classification and grading of oligodendroglial tumours. However, a specific and sensitive immunohistochemical marker of oligodendroglial tumours is still lacking. Nevertheless, certain immunohistochemical staining patterns may be helpful to distinguish oligodendrogliomas from most other brain tumour entities.

Differential Diagnostic Markers Most oligodendrogliomas stain strongly and uniformly with the monoclonal antibody against the IDH1 R132H mutant protein (Figure 28.2d), thereby distinguishing these tumours from non-neoplastic lesions and a variety of other ‘clear cell neoplasms’ of the central nervous system.15 However, absence of IDH1 R132H immunostaining does not exclude an oligodendroglioma because other, less common IDH1 mutations as well as IDH2 mutations are not detected by this antibody. In contrast to the majority of diffuse astrocytomas, oligodendrogliomas usually lack widespread nuclear p53 staining, a finding corresponding to the rarity of TP53 mutations in these tumours. In fact, TP53

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mutation and widespread nuclear p53 immunopositivity are mutually exclusive to 1p/19q deletion in gliomas.93 Oligodendrogliomas commonly show immunoreactivity for S–100 protein, CD57 (anti-Leu–7, HNK–1) and the microtubule-associated protein 2 (MAP2), the latter often in a characteristic perinuclear cytoplasmic ­immunostaining without significant process labelling.8 Unfortunately, immunoreactivity is not restricted to ­oligodendrogliomas but is also found in various other neuroectodermal tumours. Similarly, expression of the oligodendrocyte ­lineage-associated transcription factors OLIG1 and OLIG2 is found not only astrocytomas.93 The in oligodendrogliomas but also in ­ transcription factor Sox10, another key determinant of ­ ­oligodendroglial differentiation, is expressed in both oligodendrogliomas and astrocytic tumours.4 Immunohistochemical analyses for several proteins related either to myelin or to particular oligodendrocytic ­developmental stages have been performed on tissue sections of oligodendroglial tumours, but are generally not useful in diagnostic practice. The investigated markers include major protein components of central nervous system (CNS) myelin, such as myelin basic protein (MBP) and proteolipid protein (PLP), which were shown by some to be highly expressed in oligodendrogliomas.36 However, others did not identify MBP immunoreactivity in neoplastic oligodendrocytes.74 Immunoreactivity for myelinassociated glycoprotein (MAG) has been d ­ emonstrated in only occasional oligodendrogliomas.74 Membrane proteoglycans, neutral glycolipids, ­gangliosides and other proteins that are regulated during ­oligodendrocyte development are inconstantly or inconsistently expressed by neoplastic oligodendrocytes in routinely processed tissues. GFAP positive cytoplasmic processes are typical of astrocytomas but not oligodendrogliomas. However, as discussed earlier, both ‘gliofibrillary oligodendrocytes’ and ‘minigemistocytes’ are strongly GFAP positive and may predominate in some oligodendrogliomas.41,74 Vimentin is infrequently expressed in low-grade oligodendrogliomas but is more often found in anaplastic oligodendrogliomas.57 Dot-like immunoreactivity for epithelial membrane antigens (EMA) has been reported in a small fraction of oligodendrogliomas.57 Synaptophysin immunoreactivity due to residual neuropil is frequently observed in oligodendrogliomas, in particular at the infiltrating tumour borders. Such staining of tumourinfiltrated neuropil should not be mistaken as neuronal or neurocytic tumour differentiation. However, some oligodendrogliomas, including cases with combined losses of 1p and 19q, contain tumour cells with morphological features indicative of neuronal/neurocytic differentiation and immunohistochemical positivity for synaptophysin and other neuronal markers.81 Moreover, immunoreactivity for α–internexin, a proneural gene encoding a neurofilament interacting protein, is frequent in oligodendroglioma and has been proposed as a marker for 1p/19q-deleted tumours.26 Other authors confirmed frequent α–internexin positivity in oligodendrogliomas, but reported that immunoreactivity was regionally heterogeneous and not reliably linked to 1p/19q deletion.28

Proliferation Markers Ki–67 (MIB–1) has been widely studied in ­oligodendroglial tumours for tumour grading and prognostic ­ assessment. In general, the mean fraction of positive tumour cells is

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  Oligodendroglioma  1677

significantly higher in anaplastic o ­ligodendrogliomas as compared to low-grade oligodendrogliomas. Although independent review of the same set of MIB–1-stained slides from 30 oligodendrogliomas by six different pathologists revealed good interobserver agreement (concordance rate of 0.832),83 interlaboratory staining and scoring variability for MIB–1 constitutes a major obstacle in defining reproducible diagnostic cut-off values. Published data suggest that oligodendrogliomas with MIB–1 labelling indices above 5 per cent are more likely to behave aggressively with less favourable outcomes. For example, a study of 89 patients reported a 5-year survival rate of 83 per cent for patients whose tumours had labelling indices below 5 per cent ­compared with 24 per cent for patients with tumours with >5 per cent MIB–1 positive cells.22 Other authors also found statistically significant median survival differences based on a 5 per cent cut-off.20 Within the group of WHO grade II oligodendrogliomas, one report found no significant correlation between MIB–1 staining and survival124 whereas other authors reported that a MIB–1 index above 5 per cent was indicative of significantly shorter disease-free survival.89 In a series of 20 paediatric low-grade oligodendrogliomas, the MIB–1 labelling index was generally low and not associated with survival.9 In contrast, the MIB–1 index had a strong prognostic impact in anaplastic oligodendroglioma patients on univariate, but not multivariate, analyses, casting doubt that it represents an independent prognostic factor.84 Studies on the expression of other p ­ roliferation-associated antigens, such as the proliferating cell nuclear antigen (PCNA),89 topoisomerase II α (Ki–S1)58 and minichromosome maintenance 2 (MCM2) protein,124 also revealed associations with tumour grade and survival. However, none of these markers provide a significant advantage over MIB–1 in the routine diagnostic setting.

Electron Microscopy Oligodendroglioma cannot reliably be distinguished from other gliomas by means of electron microscopy because none of its ultrastructural features are entirely specific. Tumours are typically composed of small, round tumour cells showing a paucity of cytoplasmic intermediate glial filaments, which are otherwise common in normal and neoplastic astrocytes. Gliofibrillary oligodendrocytes and minigemistocytes contain cytoplasmic skeins or whorls of intermediate filaments, in contrast to the notably random distribution of short intermediate filaments in the gemistocytic astrocytoma tumour cells.60 The nucleus of oligodendroglioma cells is usually round to oval and contains abundant euchromatin. In comparison with other gliomas, cytoplasm is sparse and processes tend to be short and tapered. Variable numbers of randomly arranged microtubules may be observed, and there may be moderate amounts of endoplasmic reticulum, moderately to well-developed Golgi complex, and variable numbers of mitochondria. Signet-ring cells in oligodendroglioma have their cytoplasm filled with degenerating mitochondria and irregularly and widely dilated cisternae of rough endoplasmic reticulum containing granular material.62 In contrast, eosinophilic granular cells in oligodendrogliomas exhibit abundant autophagic type vacuoles and lysosomes containing electron-dense pleomorphic material.19

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Differential Diagnosis Several macrophage-rich lesions, including demyelinating disease, infarct and progressive multifocal leukoencephalopathy may mimic oligodendroglioma, in particular in small specimens with freezing artefacts. Cytological preparations from tissue smears most readily permit the identification of macrophages with their vacuolated cytoplasm and accompanying reactive astrocytes. Stains for myelin, e.g. Luxol fast blue, coupled with the periodic acid Schiff (PAS) reaction, allow the identification of myelin loss and ­infiltrating ­acrophage markers macrophages. Immunostains for m such as CD68, CD163, or HAM–56 also ­ facilitate the ­identification of macrophages. These cells often lie ­clustered around blood vessels in association with ­lymphocytes, but perivascular macrophages may also be present in otherwise typical oligodendrogliomas. Partial lobectomy specimens performed for intractable seizures not infrequently have seemingly increased numbers of oligodendrocytes. In such instances, however, the white matter is condensed with resultant crowding of oligodendroglial nuclei, whereas the accompanying astrogliosis further contributes to the increased cellularity. Similar histological features may be seen adjacent to arteriovenous malformations. However, in these situations, oligodendroglial atypia is ­usually lacking. Smear preparations also help to distinguish well-differentiated oligodendroglioma from gliosis, in that normal oligodendrocytes possess little discernible cytoplasm and no nuclear lobation, and reactive ­astrocytes typically have more open chromatin, ample cytoplasm, and symmetrically radiating tapered processes. Moreover, IDH1/2 mutations are generally absent in these non-neoplastic lesions, making the demonstration of such mutations by immunohistochemistry or DNA sequencing a powerful tool for the identification of oligodendroglial neoplasms. With respect to the differential diagnosis of neoplasms, the distinction of oligodendroglioma from astrocytomas, in particular diffuse and pilocytic subtypes, bears important clinical implications (Box 28.1). This differential diagnosis can be quite challenging. For example, a central pathology review of of low-grade oligodendrogliomas submitted for 1p deletion testing could confirm this diagnosis in only half of the cases.98 Differential diagnosis is particularly difficult in specimens lacking the characteristic honeycomb appearance, such as frozen sections or small, rapidly fixed paraffin specimens like stereotactic biopsy samples. In the absence of the typical perinuclear clearing, the distinction of oligodendroglioma from diffuse astrocytoma is largely based on the roundness and uniformity of nuclei. In addition, infiltration into the cortex and formation of secondary structures is more typical of oligodendrogliomas, but not specific. Overall GFAP and vimentin expression is usually less prominent in oligodendrogliomas than in fibrillary or gemistocytic astrocytomas. However, neither stain is specific. Strong nuclear immunoreactivity for p53 and loss of ATRX staining argue in favour of a diffuse astrocytoma, but lack of p53 and retained ATRX expression does not exclude this diagnosis.65 Some pilocytic astrocytomas mimic oligodendroglioma. In most instances, however, at least focally classic pilocytic features are present. In addition, typical clinical and neuroradiological features, such as paediatric presentation,

28

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1678  Chapter 28  Oligodendroglial Tumours BOX 28.1.  Diagnostic overview The histopathological differential diagnosis of oligodendroglioma involves the recognition of both reactive and neoplastic lesions. Concerning reactive lesions, oligodendrogliomas need to be distinguished from macrophage-rich processes such as demyelinating diseases or cerebral infarcts. The differential diagnosis towards neoplastic lesions includes a variety of ­different tumour types, in particular those that may present with clear cells, such as clear cell ependymoma, ­neurocytoma, dysembryoplastic neuroepithelial tumour (DNT), clear cell meningioma and metastastic clear cell carcinoma. Immunohistochemical analysis usually helps distinguish these entities. In particular, positivity for mutant IDH1 rules out nonneoplastic lesions and other clear cell tumours. It is also important to know if a biopsy comes from the temporal lobe because normal perineuronal oligodendrocytes may be numerous there and may mimic neoplastic perineuronal satellitosis. Freezing artefacts that cause loss of detail in chromatin structure, chromatin hyperchromasia and nuclear irregularity are problematic in low-grade ­oligodendrogliomas by eliminating salient nuclear features. Molecular genetics is also playing a growing role in differential diagnosis, particularly IDH1/2 mutation and 1p/19q deletion analyses (see text following).

location in the cerebellum, brain stem or spinal cord, and mural nodule/cyst formation argue in favour of pilocytic astrocytoma. In children, molecular analysis is of limited help because paediatric oligodendrogliomas usually lack IDH1/2 mutations and 1p/19q deletions.108 Moreover, BRAF gain/duplication or KIAA1549–BRAF fusion, i.e., characteristic aberrations in pilocytic astrocytomas, have also been detected in subsets of IDH1-mutant and 1p/19qdeleted oligodendroglial tumours.3,56 In paediatric patients, disseminated oligodendroglial-like leptomeningeal tumours have been suggested as a distinctive clinicopathologic entity.94 These tumours show widespread leptomeningeal spread, sometimes with an associated intraspinal mass. Histologically, they contain oligodendrogliallike tumour cells with frequent immunopositivity for OLIG2 and S–100 and variable GFAP and synaptophysin expression. In contrast to the vast majority of oligodendrogliomas, these tumours generally lack staining for the mutant IDH1 R132H protein and only rarely demonstrate 1p/19q deletions, although solitary 1p deletion is common. Ependymomas, particularly those of the clear cell type, may also be mistaken for oligodendroglioma. The scattered presence of perivascular pseudorosettes with elongated, often tapering processes, vimentin, as well as dot- or ringlike EMA immunoreactivity and the typical ultrastructural features of ependymomas are helpful. Sharp demarcation from adjacent brain is also typical. In addition, genetic analysis may be helpful because clear cell ependymomas lack IDH1/2 mutation15 and 1p/19q deletions.90 Dysembryoplastic neuroepithelial tumour (DNT) constitutes another important differential diagnosis of low-grade oligodendroglioma. DNT consists of patterned mucin-rich nodules with oligodendrocyte-like cells that often line capillaries or axonal bundles; they are largely limited to the cerebral cortex and may be associated with cortical dysplasia. Prominent intercellular mucin surrounds mature or occasionally dysmorphic neurons, which appear to ‘float’. The highly characteristic, complex nodules vary considerably in morphology, a minority being composed of astrocytic cells

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with pilocytic or other gliomatous features. The perineuronal satellitosis and subpial spread is not as prominent in DNT as in oligodendroglioma. Furthermore, these tumours neither show IDH1/2 mutations nor 1p/19q deletions.15,33 The distinction of oligodendroglioma from neurocytic tumours can pose considerable problems. The ­ finding of perinuclear halos, arborizing vasculature and not infrequent calcification in both tumours contributes to the p ­ossible confusion. However, unlike oligodendroglioma, the classic central neurocytoma is an intraventricular tumour that is typically attached to the septum pellucidum. This unique location combined with neurocytic rosettes, ­immunohistochemical reactivity for synaptophysin, lack of d ­ iffuse immunoreactivity for S–100 protein, and the characteristic neuronal ultrastructural features provide the d ­ efinitive diagnosis. Nevertheless, neurocytic tumours also arise outside the ventricular system, including the cerebral hemispheres.10 Such extraventricular neurocytomas may be very difficult to distinguish from oligodendroglioma, particularly as rare cases of diffusely infiltrating ‘oligodendrogliomas’ with deletion of 1p/19q ­ and clear evidence of neurocytic differentiation have been reported.81 Combined deletion of 1p and 19q, the genetic hallmark of oligodendroglioma, is rare in neurocytoma,33 as is IDH1/2 ­mutation.15 Thus, mutant IDH1 immunostaining and, if necessary, molecular testing for IDH1/2 mutations and 1p/19q deletions may distinguish these entities. Nuclear regularity, similar to oligodendrogliomas, may be noted in rare cases of cerebral neuroblastomas and other primitive neuroectodermal tumours of the central nervous system (CNS PNET). The differences in cellularity and mitotic activity, and the immunohistochemistry for neurofilaments and synaptophysin are features that distinguish them from oligodendrogliomas. Cerebellar liponeurocytoma also comes into the differential diagnosis. Distinguishing points are similar to those of neurocytoma, with the additional feature of lipidized cells that resemble adipocytes, and the preferential cerebellar location. Clear cell meningiomas rarely enter the differential diagnosis, but should be considered in the setting of a primarily leptomeningeal oligodendroglioma. The immunopositivity for epithelial membrane antigen, lack of mutant IDH1 expression, PAS–positive cytoplasm and presence of more classical meningothelial areas usually ensure an accurate diagnosis of clear cell meningioma.

Histogenesis The precise histogenesis of human oligodendrogliomas is still unknown. The immunocytochemical profile of oligodendroglioma cells is more reminiscent of immature glial cells than mature oligodendroglia. Furthermore, the finding that oligodendroglioma cells may occasionally express markers of other lineages, including astrocytic and neuronal antigens, might favour the hypothesis of an origin from ­neural stem or progenitor cells rather than mature oligodendrocytes. Nonetheless, it is important to stress that neoplastic development is not necessarily analogous to the highly regulated and co-ordinated normal glial development, and caution should be exercised in the histogenetic interpretation of immunohistochemical data using ‘­differentiation’ markers on p ­ rimary tumours. In transgenic mice, o ­ligodendrogliomas could be induced by expression of platelet-derived growth factor (PDGF) under

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  Oligodendroglioma  1679

the control of promoters for the nestin, GFAP or 2′,3′–cyclic nucleotide ­3′–phosphodiesterase (CNP) genes, suggesting that these neoplasms can arise from ­different ­stem/­precursor cell types, ­ including ­ oligodendrocyte ­ precursors.129 Oligodendrogliomas and oligoastrocytomas could also be induced in mice by ­combined expression of H–ras and epidermal growth factor receptor (EGFR) from the GFAP promoter, as well as EGFR from the S–100 beta ­promoter. More recent data on murine and human oligodendrogliomas suggest an origin from NG2–positive and a­ symmetric division-defective oligodendroglial progenitor cells in the cerebral white matter rather than from neural stem cells.82,107

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Molecular Genetics Deletions of Chromosomal Arms 1p and 19q Combined deletion of chromosomal arms 1p (Figure 28.3) and 19q constitutes the hallmark alteration in ­oligodendrogliomas and is found in up to 80 per cent of cases, typically on the background of IDH1 or IDH2 mutation and frequently associated with MGMT promoter ­methylation91 (Figure 28.4). Diagnostically, the d ­ emonstration of 1p/19q deletion may serve to confirm the diagnosis of oligodendroglioma versus other clear cell tumours. However, absence of detectable 1p and 19q deletion does not exclude an oligodendroglioma. In particular, paediatric oligodendrogliomas often lack these deletions but may represent a distinct entity.86 Combined 1p and 19q deletion is typically caused by an unbalanced t(1;19)(q10;p10) translocation, with the chromosomal breakpoints located close to the centromeres of both chromosomes, thus resulting in complete losses of one copy of 1p and 19q, respectively.45 Concerning oligodendroglioma-associated tumour suppressor genes on these ­chromosome arms, recent large-scale sequencing s­tudies have identified frequent mutations in the far-upstream ­element binding protein 1 (FUBP1) gene on 1p31.1 and the CIC (homologue of the Drosophila gene capicua) gene on 19q13.2.5,127 In fact, most oligodendrogliomas are genetically characterized by concurrent mutations in IDH1/2 and CIC as well as 1p/19q deletion,97,127 whereas most diffuse astrocytomas show concurrent mutations in IDH1/2, TP53 and ATRX (Jiao et al. 2012).65 FUBP1 mutations are restricted to a smaller subset of IDH1/2mutant and 1p/19q-deleted oligodendroglial tumours.97,127 Several other genes located on 1p or 19q have been suggested as ­ ­ oligodendroglioma-associated tumour suppressor gene candidates.92 For example, mutations or homozygous deletions of the cyclin-dependent kinase inhibitor 2C gene (CDKN2C) at 1p32 have been detected in a minor fraction of mostly anaplastic oligodendrogliomas. Other candidate genes on 1p include the calmodulinbinding transcription activator 1 gene (CAMTA1, 1p36), the chromodomain helicase DNA binding domain 5 gene (CHD5, 1p36), the DNA fragmentation factor subunit β gene (DFFB, 1p36), the adherens junction–associated protein 1 gene (AJAP1, 1p36.32), the transcriptional coactivator 4 gene (CITED4, 1p34), the peroxiredoxin 1 gene (PRDX1, 1p34), and the RAS homolog gene family gene DIRAS3 (1p31). Candidate tumour suppressor genes on 19q other than CIC include the p190RhoGAP gene (19q13.3), the myelin-related epithelial membrane

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28.3 Oligodendroglioma. Demonstration of 1p deletion by fluorescent in situ hybridisation (FISH) on interphase nuclei. Note two green signals from probes to the long arm of chromosome 1 (1q), but only a single red signal from the probe to the short arm of chromosome 1(1p). Courtesy of Dr Gayatry Mohapatra, Massachusetts General Hospital, Boston, MA, USA.

protein gene 3 (EMP3) at 19q13.3, ZNF342, a zinc-finger transcription factor gene at 19q13, and the maternally imprinted PEG3 gene at 19q13.4. However, in contrast to CIC, FUBP1 and CDKN2C, the other reported 1p or 19q genes are generally not inactivated by structural DNA alterations, such as point mutations or homozygous deletions, but show frequent epigenetic silencing due to aberrant promoter methylation. Hypermethylation of these and further genes located on other chromosomes is likely related to global changes in DNA methylation referred to as the Glioma CpG Island Methylator Phenotype (G–CIMP).76 This widespread aberration is particularly common in ­ oligodendroglial tumours but also present in IDH1/2-mutant diffuse ­ astrocytomas. G–CIMP has been ­mechanistically linked to reduced ­activity of certain α–ketoglutarate-dependent DNA and histone demethylases as a consequence of IDH1 or IDH2 ­mutation.113 In line with these findings, the MGMT gene promoter is hypermethylated in the vast majority of oligodendroglial tumours.71 At the transcriptional level, 1p/19q-deleted oligodendrogliomas typically display a proneural gene

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1680  Chapter 28  Oligodendroglial Tumours ��������� ����������� ��

IDH1 or IDH2 mutation ® gCIMP

t(1;19)(q10;p10)®1p/19q deletion CIC mutation, FUBP1 mutation MGMT promoter methylation

IDH1 or IDH2 mutation ® gCIMP

t(1;19)(q10;p10)®1p/19q deletion CIC mutation, FUBP1 mutation MGMT promoter methylation

�������� WHO grade II

TP53 mutation, ATRX mutation MGMT promoter methylation 7q gain, 17p loss

�������� WHO grade II

9p loss, CDKN2A/B, p14ARF homozygous deletion or promoter methylation RB1 promoter methylation, CDKN2C mutation or homozygous deletion various other genomic aberrations, e.g. losses of 10q and 18q and/or gains of 7, 8q and 19p

��������������WHO grade III

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28.4 Summary of important molecular aberrations associated with the initiation and progression of oligodendrogliomas and oligoastrocytomas (modified according to Riemenschneider and Reifenberger,93 with kind permission from Springer Science and Business Media).

expression signature,27 which again is linked to IDH1/2 mutation and G–CIMP.76,113 Chromosomal aberrations that are less frequent than 1p/19q deletion but still occur at more than random frequency include gains on chromosome 7 as well as losses on chromosomes 4, 6, 11p, 14, and 22q.92 In contrast to diffuse astrocytomas, losses on 17p and TP53 gene mutations are rare in oligodendrogliomas and mutually exclusive to 1p/19q losses. Nonetheless, the p53 pathway is frequently deregulated in oligodendrogliomas, e.g. by epigenetic silencing of the p14ARF gene, which encodes a negative regulator of p53 activity that binds to Mdm2 and inhibits the Mdm2mediated degradation of p53.92

Growth Factors and Receptors Both low-grade and anaplastic oligodendrogliomas frequently demonstrate increased expression of epidermal growth factor receptor (EGFR) mRNA and protein.92 The mechanisms causing upregulation of EGFR expression in these tumours are as yet unknown, because EGFR gene amplification is sufficiently rare that its presence strongly favours small cell glioblastoma over anaplastic oligodendroglioma.79 The PDGF/PDGFR growth factor/receptor system also is of paramount importance in oligodendrogliomas as indicated by the finding that gene transfer of PDGF to neural stem or progenitor cells can induce oligodendrogliomas in mice.2 Furthermore, both PDGF and PDGF receptors are frequently co-expressed in oligodendroglial tumours, suggesting auto- and/or paracrine growth stimulatory activities of this signalling pathway.24 Expression of vascular endothelial growth factor (VEGF) and its receptors has been documented in oligodendroglial tumours, with increased expression levels in anaplastic tumours.92

Prognostic Significance of Genetic Alterations The prognostic significance of genetic alterations in WHO grade II oligodendrogliomas is still unclear, largely because

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studies are usually based on small cohorts. In fact, low-grade gliomas are difficult to study because they rarely show dramatic radiological responses to therapy and require long follow-up times. In addition, IDH1 mutation, 1p/19q losses and MGMT promoter methylation are so common in WHO grade II oligodendrogliomas that it is difficult to identify larger numbers of tumours without such aberrations. Recent data suggest that none of these genetic alterations are prognostic in WHO grade II glioma patients who are not treated with radioor chemotherapy.38 Other authors reported that 1p/19q deletion is associated with significantly longer survival for patients with WHO grade II gliomas.55 Data on the prognostic role of IDH1 or IDH2 mutation in low-grade glioma patients are similarly controversial.38,55 A clinical trial on low-grade oligodendroglioma patients treated with temozolomide revealed that 1p/19q loss, MGMT promoter methylation and IDH1 mutation were associated with better therapeutic response and longer survival.53 Taken together, the favourable prognostic role of IDH1 mutation, 1p/19q losses and MGMT promoter methylation in low-grade oligodendroglioma patients is most evident when patients receive cytotoxic adjuvant treatment.

Aetiology and Genetic Susceptibility The aetiology of human oligodendrogliomas is still unclear. In rodents, oligodendroglioma can be induced by various carcinogens; in particular administration of ethylnitros­ ourea and methylnitrosourea in rats frequently causes glial tumours with the morphology of oligodendroglioma. However, a role of these carcinogens in human gliomas is unclear. Cranial irradiation clearly is a risk factor, as indicated by individual patients who developed oligodendrogliomas after radiation therapy for other tumours. Several studies proposed a role for viral infections, in particular the SV40, JC and BK papovaviruses, which can cause gliomas in experimental animals. In addition, viral DNA sequences and proteins have been detected in human

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  Anaplastic Oligodendroglioma  1681

oligodendrogliomas.23 However, other authors failed to detect any JC, BK or SV40 viral sequences in oligodendrogliomas.96 Therefore, it seems unlikely that these viruses play a causal role in human oligodendroglioma formation. The vast majority of oligodendrogliomas arise sporadically, i.e. in patients without an apparent cancer ­ predisposition. Recent single nucleotide polymorphism ­ (SNP) genotyping data indicate an association between a low-frequency SNP variant at 8q24.21 and the risk of oligodendroglioma, irrespective of IDH1/2 and 1p/19q status.46 Rare cases of oligodendroglioma in patients with a hereditary tumour syndrome have been documented, including one patient with hereditary breast and ovarian cancer,56 one child with retinoblastoma syndrome,1 a child with cafe-aulait spots, oligodendroglioma and rectal cancer on the basis of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome,69 and oligodendroglioma in identical twins with Ollier’s disease.17 Taylor et al.110 reported a patient with Turcot syndrome who developed two metachronous glioblastomas showing histological features of oligodendroglial differentiation. The patient’s sister also had a glioblastoma with oligodendroglial features.110 Familial clustering of oligodendroglial tumours has been documented in a few instances, including individual case reports on oligodendrogliomas in two brothers, in a mother and a daughter, in twin sisters, and in a father and a son, as well as polymorphous oligodendrogliomas in a brother and a sister.31 Additional studies identified familial oligoastrocytomas, including one study in identical twins, another study on two siblings with glioblastoma and oligoastrocytoma and an example of anaplastic oligoastrocytomas in two brothers with an oligodendroglioma in their maternal grandmother.31

Biological Behaviour and Prognosis WHO grade II oligodendrogliomas are slowly growing tumours usually associated with long postoperative survival. However, the vast majority of tumours recur locally and malignant progression on recurrence is not uncommon. A population-based study from Switzerland revealed a median overall survival (OS) of 11.6 years and a 10-year survival rate of 51 per cent for patients with WHO grade II oligodendroglioma.77 This was significantly longer than oligoastrocytoma patients (median OS: 6.6 years, 10-year survival: 49 per cent) or diffuse astrocytoma patients (median OS: 5.9 years, 10-year survival: 31 per cent). Data from the Central Brain Tumour Registry of the United States (CBTRUS) show 5- and 10-year survival rates of 79.25 per cent and 62.62 per cent, respectively, which again are longer than those of diffuse astrocytoma (47.58 per cent and 35.36 per cent) and mixed glioma (58.35 per cent and 45.42 per cent).16 Clinical parameters that have been associated with longer survival include younger age at diagnosis, frontal tumour location, presentation with seizures, high Karnofsky performance status, lack of contrast enhancement on neuroimaging and complete tumour resection.120 The optimal postoperative treatment of patients with WHO grade II oligodendroglioma is a matter of ongoing clinical investigations. Because late toxicity is a major concern for such patients with expected longterm survival, radio- and/ or chemotherapy is often deferred until tumour progression, in particular in young patients presenting with seizures

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only.120 On the other hand, patients with symptomatic residual and/or progressive tumours after surgery, anaplastic tumours or enhancing lesions should be treated. A randomized trial on 314 patients with low-grade a­ strocytomas oligodendrogliomas revealed that early radiotherand ­ apy after surgery lengthens the period without progression but does not affect overall survival.115 Radiotherapy could therefore be deferred for patients who are in good ­condition, ­provided they are carefully m ­ onitored. Another phase III trial compared radiotherapy versus ­radiotherapy plus procarbazine, CCNU, and vincristine (PCV) chemotherapy in 251 adult patients with low-grade gliomas.105 Progression-free survival, but not overall survival, was ­longer for patients receiving combined treatment. In addition, ­ combined treatment ­ provided a ­ survival benefit in patients alive beyond two years, ­suggesting a delayed benefit for chemotherapy. A phase II study also suggested a role of upfront ­chemotherapy with t­ emozolomide for patients with ­progressive low-grade ­oligodendroglioma.53 Radiotherapy or chemotherapy with either PCV or temozolomide is usually administered to patients with recurrent tumours ­showing histological and/or clinical progression.120

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Anaplastic Oligodendroglioma Anaplastic oligodendroglioma is defined by the WHO classification of tumours of the central nervous system as an oligodendroglioma with either focal or diffuse histological features of anaplasia and less favourable prognosis .88

Incidence, Age and Gender Distribution Anaplastic oligodendroglioma accounts for approximately 0.6 per cent of all primary brain tumours and 30 per cent of the oligodendroglial tumours; the adjusted annual incidence rate has been estimated as 0.12 per 100 000 population.16 The vast majority develop in adults, peaking between 45 and 50 years of age, i.e. approximately 7–8 years later on average than low-grade oligodendroglioma.77 In children, anaplastic oligodendrogliomas are very rare. Caucasians are more commonly affected than black people, as indicated by incidence rates of 0.13 versus 0.05 per 100 000 persons, with rates slightly higher for males (0.14 per 100 000) than for females (0.11 per 100 000).16

Clinical and Radiological Features Anaplastic oligodendroglioma either may develop de novo or secondarily via progression from a low-grade oligodendroglioma. Patients with secondary tumours are younger than their de novo counterparts.63 The preoperative history of patients with de novo tumours is usually short, but clinical symptoms and signs do not significantly differ from those of other primary anaplastic gliomas. Epilepsy is the most common presenting symptom.63 In patients with secondary forms, the mean time to progression from low-grade oligodendroglioma has been estimated at 6.6 ± 4.2 years.77 Other authors reported a slightly shorter interval of 72 months, but a similar survival time from WHO grade III diagnosis in primary and secondary anaplastic oligodendrogliomas.63 However,

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1682  Chapter 28  Oligodendroglial Tumours

previous resection for lower grade tumour has also been reported as a prognostically favourable factor.120 On neuroimaging, anaplastic oligodendrogliomas may show heterogeneous patterns owing to the variable presence of necrosis, cystic degeneration, intratumoural ­haemorrhages and calcification. The majority of tumours demonstrate contrast enhancement on CT and MRI, which can be either patchy or homogeneous.47,63 However, lack of contrast enhancement does not exclude an anaplastic oligodendroglioma. Ring enhancement is uncommon and associated with less favourable prognosis.13

Macroscopy The vast majority of anaplastic oligodendrogliomas are located in the cerebral hemispheres with a preference for the frontal lobe, followed by the temporal lobe. The macroscopic appearance does not allow for a reliable distinction from low-grade oligodendrogliomas, although anaplastic oligodendrogliomas often show a more heterogeneous cut surface due to haemorrhages, cyst formations and areas of necrosis (Figure 28.5a).

Microscopy According to the WHO classification, the histological ­features that separate anaplastic oligodendroglioma from low-grade oligodendroglioma include high ­ cellularity, nuclear pleomorphism and hyperchromasia, moderate

to high mitotic activity, microvascular proliferation and necrosis.88 Anaplastic ­oligodendrogliomas usually show a number of these features. Unfortunately, it is not possible to equate anaplastic oligodendroglioma with the presence of any one of the above histological ­­­­characteristics, although it has been argued that endothelial proliferation and brisk ­ itoses/10 HPF) are of particular mitotic activity (at least 6 m importance.35 In general, anaplastic oligodendrogliomas are cellular, mitotically active, diffusely infiltrating gliomas composed of neoplastic cells showing morphological features ­indicative of oligodendroglial cells, i.e. rounded nuclei, perinuclear halos in routinely processed tissue samples and few cellular processes. In comparison to low-grade oligodendroglioma, there is often increased cytoplasm, more open chromatin and increased nucleolar prominence. The characteristic vascular pattern of branching capillaries is often still recognizable, although additional microvascular proliferation is usually obvious. Focal microcalcifications are frequent. Rare cases demonstrate marked desmoplasia.48 Gliofibrillary oligodendrocytes and minigemistocytes (Figure 28.5b) are more common in anaplastic oligodendrogliomas but are not of prognostic relevance. Occasional tumours show marked cellular pleomorphism including multinucleated giant cells. In addition, rare anaplastic oligodendrogliomas may present with a sarcomatous ­ component.95 Infiltration of the cerebral cortex is often associated with the formation of secondary structures, in particular perineuronal satellitosis.

(a)

(b)

(c)

(d)

28.5 Anaplastic oligodendroglioma. (a) Large tumour with haemorrhage, necrosis and cysts, involving the frontal lobe and spreading to the corpus callosum. (b) Minigemistocytes with eccentric nuclei and eosinophilic cytoplasm. (c) Pseudopalisading necrosis. (d) Immunohistochemistry for GFAP demonstrating positivity in a subset of tumour cells. Courtesy of Dr Arie Perry, UCSF, San Francisco, CA, USA.

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  Anaplastic Oligodendroglioma  1683

Some anaplastic oligodendrogliomas feature necrosis, including rare cases with pseudopalisades resembling those of glioblastoma (Figure 28.5c). As long as the tumour shows the typical cytological and histological hallmarks of oligodendroglioma, the WHO classification recommends classifying these tumours as anaplastic oligodendroglioma of WHO grade III.88 In contrast to patients with malignant oligoastrocytomas, the presence of necrosis did not have an adverse prognostic effect in anaplastic oligodendroglioma patients.70

Grading Anaplastic oligodendrogliomas correspond to WHO grade III.

Immunohistochemistry The immunohistochemical profile of anaplastic oligodendrogliomas is similar to that of low-grade oligodendrogliomas, including frequent positivity for IDH1 R132H and MAP2. GFAP-immunoreactive cells such as gliofibrillary oligodendrocytes and microgemistocytes are occasionally numerous (Figure 28.5d). Vimentin immunopositivity is also more common in anaplastic than in low-grade oligodendrogliomas. The MIB–1 labelling index is significantly higher in anaplastic oligodendrogliomas as compared to their low-grade counterparts, reflecting the more rapid tumour growth. Several studies suggested a MIB–1 index of 5 per cent as a cut-off value to distinguish two groups of oligodendroglial tumours with significantly different outcome.20,89 A study based on a prospective phase III trial revealed a strong prognostic impact of the MIB–1 index on univariate analysis, but no independent influence on multivariate analysis.84

Differential Diagnosis Similar to low-grade oligodendrogliomas, the differential diagnosis includes a variety of other clear cell tumour entities. Occasional anaplastic oligodendrogliomas may superficially resemble metastatic clear cell carcinomas. Unlike the latter, which are solid and sharply demarcated, oligodendrogliomas are infiltrative. Although immunohistochemistry readily permits their distinction, caution is recommended because there is a misleading cross-reactivity with certain broad-spectrum cytokeratin antibodies and GFAP. By contrast, stains for low molecular weight cytokeratin are negative in ­ oligodendrogliomas and therefore reliably exclude metastatic carcinoma. In a fraction of anaplastic oligo­ dendrogliomas, the differential diagnosis with anaplastic oligoastrocytoma may be problematic and associated with considerable interobserver variability. Moreover, it is important to separate anaplastic oligodendrogliomas from malignant small cell astrocytic neoplasms, i.e. ‘small cell ­astrocytoma/glioblastoma’, which behave much more aggressively, akin to conventional glioblastomas.11,79 Molecular genetic analyses can be helpful to solve this differential diagnosis because small cell astrocytic tumours lack 1p/19q deletion but often d ­ emonstrate EGFR amplification and chromosome 10 losses. Conversely, IDH1 (R132H) immunoreactivity and IDH1/2 mutations are found in most anaplastic oligodendrogliomas, but not in small cell glioblastomas.52

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Molecular Genetics Anaplastic oligodendrogliomas share with low-grade oligodendrogliomas the frequent mutations in IDH1 or IDH2, detectable in up to 80 per cent of cases (Box 28.2).39 1p and 19q codeletions are found in 50–70 per cent, with frequent CIC mutation in 1p/19q-deleted tumours and additional FUBP1 mutations in a subset of cases.97 Anaplastic oligodendrogliomas display a higher degree of genomic instability as indicated by aberrations ­involving genes or chromosomal segments from several other chromosomes. These include homozygous ­deletions on 9p21 involving the tumour suppressor genes CDKN2A, p14ARF and CDKN2B in up to one third of anaplastic oligodendrogliomas. Such deletions are ­particularly common in anaplastic oligodendrogliomas w ­ ithout 1p and 19q losses, but may also be present in 1p/19q-deleted cases. Other chromosomal imbalances detected at more than random frequency include deletions on 4q, 10q and 18q as well as gains on 7, 8q and 19p.112 PTEN ­mutations are rare in anaplastic oligodendrogliomas.99 Large-scale sequencing efforts revealed individual mutations in a large variety of genes, with recurrent changes affecting the PIK3CA, NOTCH1, NOTCH2 and HDAC2 genes in a study of seven tumours.5 Whole exome sequencing of ten 1p/19q-deleted oligodendroglial tumours, including three anaplastic oligodendrogliomas, also revealed mutations across the genome, with the IDH1/2, CIC and NOTCH1 genes most commonly affected.127 Gene amplifications targeting the EGFR, PDGFRA, CDK4, MDM4, MYC or MYCN are uncommon (generally 3 cm), poorly demarcated masses, with invasion into adjacent brain parenchyma and meninges.47,62 Tumoural calcification is peripheral, giving rise to the ‘exploded’ calcification appearance.30 On MRI, PB are heterogeneous, with the solid portion appearing hypo- to iso-intense on T1-weighted images and iso- to mildly hyperintense relative to the cortex on T2-weighted images. They demonstrate heterogeneous enhancement on post-contrast images.83 Craniospinal dissemination is frequent, but metastases outside the CNS are exceptional.13,21

Macroscopic Examination PB are poorly demarcated, soft or gelatinous, grey-pink tumours. Haemorrhagic and necrotic areas may be present. The tumours often destroy the pineal gland, bulge into the posterior third ventricle and compress the colliculi and the aqueduct.

Microscopy PB are composed of densely packed small cells with scant cytoplasm, hyperchromatic, round to oval nuclei (Figure 33.1e) and high nuclear/cytoplasmic ratios. The tumour lacks any obvious lobular architecture or pineocytomatous rosettes. However, PB often contain Homer Wright (neuroblastic) and/or Flexner–Wintersteiner (retinoblastic) rosettes. Large cell/anaplastic features, as seen in medulloblastomas, can be present. PB occasionally show evidence of advanced photoreceptor differentiation, with the formation of ‘fleurettes’. Rare tumours contain cells with melanin pigment. Mitotic figures are frequent. Apoptotic bodies and areas of necrosis may be prominent, the latter sometimes being associated with microcalcifications. Vessels are usually thin walled, but focal endothelial proliferation may be seen.

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  Pineal Parenchymal Tumours  1759

Immunohistochemistry A dot-like or diffuse immunoreactivity for SYN is common in PB.39,56 NSE is also expressed, whereas immunoreactivity for NFPs and/or chromogranin A is inconsistent and tends to be restricted to individual tumour cells.39,56 PB can contain cells with immunoreactivity for photoreceptor ­markers, such as retinal S-antigen.69 GFAP and PS100 immunoreactivity is rare.39 The Ki-67 LI is high (>20 per cent).26

Electron Microscopy In line with ultrastructural findings for other CNS PNETs, PB contain poorly differentiated cells with a few short processes lacking the bulbous terminations typical of PC.38,66 Scant microtubules and occasional dense-core vesicles may be observed. However, specialized structures, such as paired twisted filaments, vesicle-crowned rodlets or synaptic junctions are absent.

Differential Diagnosis The differential diagnosis includes embryonal tumours that can seed or infiltrate the pineal region, in particular, medulloblastoma, CNS PNET and primitive appearing gliomas. PB must also be distinguished from higher-grade PPTID. The distinction hinges upon degrees of cellularity, atypia, mitotic activity and necrosis. In adults, metastatic small-cell carcinoma can be distinguished by their immunoreactivity for epithelial markers, such as cytokeratins. Malignant germ cell tumours can resemble PB on imaging, but the two can usually be easily distinguished histologically.

Molecular Genetics Conventional and CGH cytogenetic studies have shown frequent numerical and structural abnormalities.6,54,76,87,88 The karyotypes are mostly near-diploid, but hypertetraploidy has been reported. Recurrent aberrations include gains of 1q, 5p, 5q, 6p and 14q and losses of chromosomes 20 and 22, as well as isochromosome 17q (i[17q]), or unbalanced gain of 17q. In cytogenetic reports of 13 PB, i[17q] or unbalanced 17q gain was described in four cases, including two tumours and two cell lines.6,54 In contrast, CGH studies have shown that most PB do not exhibit 17q gain.76,77 Thus, whether PB are genetically related to medulloblastomas remains unclear. In vitro, PB cell lines showed enhanced N-myc expression in the absence of MYCN gene amplification, in contrast to the situation in medulloblastomas.42,43 No TP53 mutations were demonstrated in four PB.91 The RB1 gene has not been studied for genetic alterations in sporadic PB.54,88 However, studies on knock-out mice indicate that the simultaneous inactivation of p53 and Rb results in an increased rate of PB.94 Interestingly, patients with familial retinoblastoma syndrome have an increased risk of developing PB, a condition known as ‘trilateral retinoblastoma’.18,44 PB that arise in this setting have a more aggressive course than sporadic cases.70 However, no specific abnormalities of chromosome 13q in the region of the Rb gene have been found in the latter.6,76 One case of PB associated with familial adenomatous polyposis has been reported as a potential variant of Turcot’s syndrome type 2 (see Chapter 44).29 This case remains isolated, and

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no association has been made between APC gene mutation and PB, in contrast to the firm association between mutation of this gene and medulloblastomas.

33

Biological Behaviour and Prognosis PB are locally invasive and tend to disseminate via the cerebrospinal fluid (CSF), requiring aggressive treatment. In adults, stereotactic biopsy or open surgery is usually followed by adjuvant radiotherapy and chemotherapy.53,55,85,86 Nevertheless, the prognosis is poor, as indicated by the median survival time of 16 months and 10 per cent 5-year survival rate.21 A slightly better median overall survival has been reported for adult patients, ranging from 25.7 to 77 months.53,55 The extent of disease at diagnosis, the histology (PB versus PPTID) and the degree of residual disease after initial treatment are independent prognostic factors in adult patients.55 The prognosis in infants younger than 18 months is even more dismal.37 In children treated with radiotherapy and chemotherapy, a 3-year progression-free survival rate of 61 per cent has been reported, albeit commonly with severe developmental side effects in children younger than 9 years of age.37 In children pineoblastomas appear to do slightly better than CNS PNETs.12,89

Pineal Parenchymal Tumour of Intermediate Differentiation Synonyms and Historical Overview PPTIDs are cellular tumours with variable architectural ­features. First proposed in 1993,79 they were subsequently recognized by the World Health Organization (WHO).

Incidence, Age and Sex Distribution The relative frequency of PPTID varies greatly in the literature, reflecting the difficulties in establishing reproducible definitional criteria. In carefully defined series, PPTIDs represent 20 to 62 per cent of PPTs.3,21,79 They occur mostly in adults, with a peak incidence in young adults (20–40 years of age). Most present as localized disease, with CSF dissemination less common than for PB.21,80

Clinical and Radiological Features The clinical signs are similar to those of other pineal region tumours.21 Imaging findings are similarly non-specific. PPTIDs are heterogeneously hypointense on T1-weighted and hyperintense on T2-weighted images; they are contrast enhancing. Cystic areas may be seen.45,83

Macroscopic Examination The gross appearance of PPTIDs is similar to that of PC, with no gross evidence of necrosis.

Microscopy Histologically, PPTIDs are variable.39 One pattern shows endocrine-like lobular/diffuse architecture with isomorphic tumour cells containing round nuclei and clear cytoplasm (Figure 33.1f). Another phenotype, the ‘transitional variant’, has mixed lobular/diffuse areas, in addition to regions with

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1760  Chapter 33  Neuroepithelial Tumours of the Pineal Region

Grading of PPT

PC-like morphology. Finally, some rare tumours may present with a biphasic pattern combining the typical features of PC and PB. Neoplastic cells have less cytoplasm than in PC, but it is still visible on standard staining. Nuclear atypia is moderate. Mitotic activity is usually present, but may vary considerably. Foci of necrosis or vascular proliferation have been reported in subsets of PPTID tumours, although they lack the primitive ‘small blue cell’ appearance of PB.39 A pleomorphic variant may be encountered in low-grade PPTIDs.24

Classification of PPT is difficult, especially when using small biopsy samples. PC and pleomorphic PC are welldifferentiated tumours and correspond to WHO grade I.63 PB correspond to WHO grade IV. PPTID may correspond to WHO grade II or III, but the grading criteria described in the prior section have yet to be validated. The Ki-67 LI may also be a useful tool.3,26

Immunohistochemistry

Histogenesis of PPT

In PPTID, expression of neuronal markers is variable. Cytoplasmic SYN labelling is usually diffuse, but of variable intensity. NFP can be expressed in a variable number of cells, and this variability has been used together with morphology and proliferation to discriminate low-grade from high-grade PPTID. Chromogranin A may be seen in PPTID, especially those with a lobulated architecture.39

PPTs are thought to originate from cells of pineocytic lineage. Pineocytes have photosensory and neuroendocrine functions and constitute the major cell population within the normal pineal gland.60 They develop from neuroepithelial precursors in the roof of the diencephalon and are ontogenetically related to retinal photoreceptor cells. Pineocyte development depends crucially on the homeobox transcription factor Otx2, because Otx2 knock-out mice lack these cells.64 Otx2 expression is maintained in adult pineocytes and may control the expression of genes associated with phototransduction and melatonin synthesis.73 Although a study has demonstrated amplification and frequent overexpression of the OTX2 gene in medulloblastomas,5 a role for aberrant Otx2 expression in PPT remains to be established. PB probably originate from these same neuroepithelial precursors, a hypothesis based on ultrastructural and immunohistochemical similarities between PB cells and cells of the developing pineal gland,38,59,60 as well as similarities with retinoblastomas and the common occurrence of both tumour types in patients with trilateral retinoblastoma.44

Electron Microscopy PPTIDs display short cytoplasmic processes and rare bulbous endings. In lobulated PPTID, the organelles characteristic of neurosensory/photoreceptor differentiation are usually absent, but the cell processes contain oriented microtubules, clear and dense-cored vesicles.

Differential Diagnosis PPTID should not be mistaken for neurocytoma or oligodendroglioma. The clear cells and sheet-like growth patterns of some oligodendrogliomas and neurocytomas, as well as the presence of fibrillary areas or perivascular neuritic processes in the latter, may lead to misdiagnosis, but PPTIDs do not express OLIG2 or NeuN. PPTIDs are distinguished from PC and PB on the basis of histopathological criteria, mitotic activity and NFP expression. More particularly, high-grade PPTIDs need to be distinguished from PB, which, in contrast, presents a higher cellular density and small cells with scant cytoplasm and hyperchromatic nuclei.

Molecular Genetics One CGH study of PPTID showed overlap with PB in terms of chromosome 22 loss and similar proportions of chromosomal imbalances per case (5.3 for PPTID and 5.6 for PB).76

Biological Behaviour and Prognosis The clinical outcome for patients with PPTID is highly variable, so the WHO classification does not assign a definite grade to these neoplasms. Other studies have proposed that intermediate tumours can be subdivided histologically into two grades associated with significantly different outcomes: grade II for tumours with 50 per cent) should show the defining morphology before the tumour is reflexively ‘upgraded’.109

Grading and Prognosis Based on histological criteria discussed later, the 2007 WHO classification provides a grading system for estimating meningioma prognosis.136 The high-grade variants are associated with significantly increased risks of recurrence and death (see Table 36.1 and later). However, because the majority of meningiomas are considered benign, the risk of recurrence is often influenced more by the site and

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Tumours of Meningothelial/Arachnoidal Cells  1807

TABLE 36.1  Meningiomas with a greater likelihood of recurrence and/or aggressive behaviour Type of meningioma

WHO grade

Atypical

II

Clear cell

II

Choroid

II

Brain invasive (but not anaplastic)

II

Anaplastic (malignant)

III

Papillary

III

Rhabdoid

III

Any histological subtype or grade with high proliferative index WHO, World Health Organization.

the surgeon than histological subtype, because extent of surgical removal is highly correlated with recurrence rates. When operative mortality is excluded, meningioma patients treated with complete resection do not seem to suffer any excess mortality when compared with age- and sex-matched controls.77,177 On the other hand, there is a small but statistically significant increased risk with subtotal resection. Additionally, estimated 5- and 10-year recurrence rates increase from 5 and 25 per cent with gross total to 40 and 61 per cent with subtotal resections, respectively.177 On multivariate analyses from large series, negative prognostic v­ ariables besides extent of resection include higher histologic grade, male gender, young patient age (90 per cent of positive CSF samples.7 However, CSF cytology, and flow cytometry in the case of haematologic malignancies, may be negative, necessitating a presumptive diagnosis based on clinicoradiologic features.6 Metastases to the skull and dura occur in 8–9 per cent of patients.36 Among all metastatic carcinomas, those arising in the breast are distinctive in their affinity for the dura, accounting for approximately half of all dural-based metastases.26 These skull/dura tumours may access the CNS via communications between the external and internal vertebral venous plexuses1 where bidirectional flow through a valveless system is possible.6 Additional common histologic types to involve dura and skull are prostate carcinoma and multiple myeloma.1 Vertebral osseous metastases are also common, in which the ventral elements (vertebral bodies) are more frequently involved compared to the dorsal e­ lements (pedicles and laminae).6 Epidural soft tissues of the spine may be invaded by local spread of tumour out of involved vertebrae, paravertebral soft tissues, or lymph nodes. In contrast, ­primary CNS tumours of cord parenchyma only exceptionally

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General Pathologic ­C onsiderations  1921

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43

43.1 Magnetic resonance imaging of CNS metastatic disease: the pathologist's ‘gross anatomy’. MR imaging permits exquisite delineation of the specific pattern of central nervous system (CNS) metastatic disease (‘gross anatomy’) in living patients, particularly with respect to the anatomic compartment(s) involved, which has important treatment ramifications. Illustrated here are nine patterns of metastasis, ordered from superficial to deep: (a) Cranial bone metastasis to the calvarium with intra- and extra-cranial extension; (b) dural metastasis; (c) subarachnoid space (leptomeningeal) metastasis (‘leptomeningeal carcinomatosis’, ‘carcinomatous meningitis’). Note the extension of contrast-enhancing tumour as finger-like projections into the sulci, in contrast to the purely dural metastatic pattern seen in (b), in which subarachnoid space involvement, and hence sulcal extension, is absent; (d) dural metastasis with subsequent leptomeningeal extension (note the sulcal involvement) and invasion of the underlying cerebral initially via the perivascular (Virchow–Robin) spaces and ultimately leading to ‘blooming’ of large parenchymal metastatic nodules with centrally necrotic (hypodense) core; (e) miliary metastasis, with innumerable (literally hundreds) of punctate, contrast-enhancing foci, primarily superficially located in the cerebral cortex but also involving the deep grey matter (basal ganglia and thalamus); (f) classic CNS metastases to the grey–white junction, which exhibit ring enhancement with central necrosis (hypointense) and are surrounded by vasogenic oedema (visualized as mild hypointensity on this T1-weighted sequence); (g) isolated metastasis to the midbrain tectum. Although the vast majority of solitary metastases are found in the cerebral hemisphere, any part of the CNS may be affected, often with specific localizing presenting symptoms; (h) isolated metastasis to the choroid plexus (glomus choroideum) of the lateral ventricle. Among primary site carcinomas, renal cell carcinoma in particular exhibits a tendency for choroid plexus metastasis; (i) isolated metastasis to the pituitary gland (sella turcica), with suprasellar extension mimicking pituitary macroadenoma.

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1922  Chapter 43  Metastatic Disease

extend through leptomeninges and dura to involve the epidural space.4,25 Spinal epidural metastases are most common with carcinomas of the prostate, breast, kidney and lung, as well as non-Hodgkin lymphoma,36 with the thoracic spine most frequently involved.26 There are a few noteworthy clinicopathologic points that distinguish ­epidural metastasis from intramedullary metastasis. First, the clinical presentation of epidural metastasis often reflects cord and nerve root compression, leading to findings of extremity weakness and sensory loss.26,36 Second, epidural metastases rarely penetrate through the spinal dura to invade cord parenchyma. This is unlike leptomeningeal metastatic disease, in which invasion into parenchyma is common.16 Third, compressive myelopathy is a significant complication of epidural metastases that is often progressive and irreversible.25 These patients may present acutely with features of paraparesis and paraplegia, with incontinence occurring later in the course.6 Histologic examination shows myelopathy, with white matter spongiosis and vacuolation in ascending/ descending tracts that may progress to cavitation and parenchymal necrosis.25 This may result from compression of radicular arteries or veins by tumour at affected cord levels.25

Macroscopic Features of Metastasis The macroscopic features of metastatic tumours reflect their preoperative imaging attributes. Prototypical parenchymal metastases are well demarcated, with the epicentre commonly located at the cortical grey–white junction in the vascular distribution territory of the middle cerebral artery, particularly in the arterial border zone area (‘watershed zone’).4,10,26 The tendency of metastases to localize to the grey–white junction has been attributed to narrowing of the calibre and acute branching of cortical vessels at this site.10 Larger tumours usually exhibit a necrotic centre, which correlates with preoperative MR imaging findings. Cystic tumours with prominent myxoid material may be seen in various types of metastatic adenocarcinoma.25 Metastatic melanoma exhibits a more variable appearance grossly, ranging from fleshy pink-tan in amelanotic, non-­ haemorrhagic examples, to dark red-black in tumours with haemorrhage and/or high melanin content.36 Leptomeningeal involvement by melanoma may be reflected by thickening and/or red-purple to black-brown discoloration of the meninges. Secondary changes in tumour metastases may be associated with high morbidity and mortality; chief among these is acute i­ntratumoral haemorrhage, which, through acute mass effect, may lead to herniation, secondary brain stem (Duret) haemorrhage and death.15 Intratumoral haemorrhage is also common in secondary leukaemia, with multiple parenchymal haemorrhages seen in patients with blast crisis.26 Even in the absence of a dominant tumour mass, emboli of tightly cohesive tumour cells (e.g. squamous cell carcinoma) may cause multifocal parenchymal infarcts distal to points of vessel occlusion.25 Metastases to the brain stem, fourth ventricle choroid plexus and cerebellum may result in significant compression of ­vasomotor and respiratory centres in the pons and medulla.15

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Microscopic Features of Metastasis Cytologic specimens from metastases often reveal c­ ohesive tissue fragments of pleomorphic epithelial-appearing cells with prominent nucleoli in a background of blood and necrotic debris.3 Cell cohesion on cytologic preparations reflects the presence of junctional complexes and desmosomes.8 Not all metastases appear high-grade; mildly pleomorphic or even bland nuclear features may be seen with metastatic melanoma, and prostatic, renal cell and breast carcinomas.3 Frozen and formalin-fixed paraffin-embedded (FFPE) histologic sections of metastases typically show a relatively sharply circumscribed ‘pushing’ border4 with gliotic adjacent parenchyma. A less common microscopic pattern is parenchymal infiltration by small cohesive groups of tumour cells or, rarely, even single cells;3 this feature may be exhibited by some cases of SCLC or melanoma.4 Prominent vascular proliferation is often seen in the neuropil adjacent to metastases, especially with renal cell carcinoma.3,36

Differential Diagnosis Patient history and imaging are critical in the setting of metastatic tumour. In some cases, clinical features do not resolve the differential diagnosis and additional ancillary studies (immunohistochemical, molecular) are required. Moreover, even in patients with a known cancer history and disseminated systemic disease, a primary CNS tumour, such as ­glioblastoma, or a non-neoplastic mimic of metastatic disease (e.g. abscess, tumefactive demyelinating disease, subacute infarct, lobar haemorrhage) may arise independently, and this possibility must always be entertained.16,27 Reviews of the microscopic and immunohistochemical features of primary CNS tumours and of metastases from different primary sites with diagnostic algorithms are available,10,29 but these rapidly become outdated secondary to the continual advance in biomarker discovery and clinical implementation; there is no substitute for vigilant ­monitoring of the current literature.

Miliary Metastasis Miliary metastasis, termed ‘carcinomatous encephalitis’ by Madow and Alpers in 1951,4,17,25,30 constitutes a unique form of CNS metastatic involvement that has been described in the setting of several different primary tumours, including lung carcinoma,13 melanoma, and cancer of unknown primary (see later under CUP, p. 1924).13 Miliary metastasis is characterized by dozens to hundreds of minute micrometastases distributed throughout the brain, with a distinct proclivity for the middle and upper layers of the cerebral cortex, rather than for the grey–white junction as seen in classical cerebral metastasis.14,30 Non-contrast CT imaging may fail to identify the minute foci of tumour and MR imaging (T2-weighted and/or T1-weighted with gadolinium) is far more sensitive. The characteristic pattern of miliary metastasis is innumerable, superficially located, punctate enhancing nodules (Figures 43.1 and 43.2). This pattern can mimic neurocysticercosis,2 toxoplasmosis or

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Miliary Metastasis  1923

43

43.2 Histologic patterns of central nervous system (CNS) metastatic disease: Anatomic compartments. (a–c) Dural metastasis from breast adenocarcinoma with secondary invasion of underlying subarachnoid space, perivascular Virchow–Robin spaces and cerebral parenchyma. (a) Solitary dural metastasis often mimics meningioma, including the presence of tapering lateral extensions (‘dural tail’), as seen in this example. Also note the focal ‘fuzzy’ extension into the deep cerebral parenchyma, which corresponded to perivascular invasion microscopically. (b) Dural metastasis (same case as panel [a]) showing the thicker part of the dural tail. Note the reactive sclerosis with abundant collagen deposition. There was no invasion of the underlying subarachnoid space in this area. (c) Dural metastasis (same case as panel [a]) showing focal invasion into the subarachnoid space with sulcal spread. (d–f) Leptomeningeal melanomatosis. (d) Coronal T1-weighted image with contrast shows diffuse involvement of the subarachnoid space of the convexity and sulci by melanoma. (e) Gross photo (same case as panel [d]) showing diffuse leptomeningeal melanomatosis, identical to the antemortem MRI findings. (f) Leptomeningeal melanomatosis (same case as panel [d]) is seen filling the subarachnoid space overlying the cerebellum with spread into the sulci between the folia and early invasion along the perivascular Virchow Robin spaces. (g–l) Miliary metastasis. (g–i) Multiple punctate metastases primarily localized to the superficial cerebral and cerebellar cortex ([g,h] sagittal T1-weighted images with contrast; [i] gross specimen photograph). (j–l) At increasing magnification, miliary metastasis displaying the characteristic anatomic localization with an epicentre in cortical lamina III (j), and anatomic involvement confined to the perivascular (k, l) and subpial (l) compartment. In panel (l), the pia has artefactually separated from the underlying glia limitans, expanding the subpial space. Note that a layer of tumour cells remains adherent to the inner surface (subpial space side) of the pia; the subarachnoid space, seen here as the small triangular spaces on either side of a venule, is not involved by tumour, which explains the absence of subarachnoid space spread pattern on the antemortem magnetic resonance imaging studies (g, h).

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1924  Chapter 43  Metastatic Disease

Cryptococcus meningitis.30 Presenting symptoms in miliary metastasis may be very atypical compared to the usual focal motor and/or sensory complaints seen with ordinary metastasis, and include such dramatic presentations as rapid onset dementia, mutism or visual hallucinations.31 Microscopically, miliary metastasis foci are seen as minute intracortical tumour deposits characteristically confined to the perivascular–subpial space compartment, with the smallest foci epicentred in cortical lamina III. Subsequent enlargement of individual metastatic foci occurs by centrifugal perivascular spread, including into the contiguous subpial space,24 but typically not into the subarachnoid space. The perivascular/subpial space, which has received relatively little attention in the neuropathology literature, is a well-documented anatomic compartment5,12,35,37 for which miliary metastasis is a striking example of clinical relevance. Although involvement of the grey–white junction, white matter, deep grey nuclei, pineal gland, mammillary bodies and meninges can be seen30 in miliary metastasis, it is the involvement of the perivascular/ subpial space compartment that is distinctive. One hypothesis24 postulates an affinity between tumour cells and the pia, including the arteriolar pial sheath and the pial membrane, as the critical pathophysiologic feature common to all tumours that present with miliary metastasis; however, the pathobiology underlying this distinctive tropism has yet to be elucidated, and this represents but one of the many intriguing and clinically important areas of CNS metastatic disease begging for additional investigative scrutiny.

Cancer of Unknown Primary In approximately 10 per cent of brain metastases, a primary site is not identified even after comprehensive clinical evaluation, yielding a working clinical diagnosis of CUP. CUP is defined as a malignant tumour, most commonly a carcinoma, for which no primary site has been identified after an exhaustive clinical (e.g. medical history, physical examination), laboratory (e.g. urinalysis, occult blood in stool), serologic, endoscopic34 and imaging search.28,32 The imaging search typically entails computed tomography (CT) scan of the chest, abdomen and pelvis, and/ or 18-fluorodeoxyglucose positron emission tomography (FDG-PET). Serologic work-up typically includes, among others, prostate-specific antigen and other cancer antigen markers (e.g. CA 19-9, CA-125) and alpha-fetoprotein.22 The clinical CUP work-up does not ensure localization of a very small (or, in the case of melanoma, regressed) primary site tumour, with even FDG-PET scanning having a tumour detection rate of only approximately 50 per cent in this setting.22 The majority of CUPs are adenocarcinomas or poorly differentiated carcinomas (approximately 80 per cent),28 with the remainder largely consisting of squamous cell carcinoma (10 per cent) and neuroendocrine carcinoma (5 per cent).32 For brain metastases specifically, CUP most commonly occurs in patients with a median age of 51–55 years, with a slight male predilection.28 More recently, molecular profiling approaches to the characterization of CUP for diagnostic and treatment

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purposes have been explored. One operating assumption of this approach is that the gene expression pattern of a metastasis reflects that of the primary tumour.22 The earliest studies used gene expression microarrays to predict primary site (‘tissue of origin’). Subsequently, more limited gene panels amenable to FFPE tissue testing, such as quantitative reverse transcriptase PCR (qRT-PCR),20 were explored. Most recently, microRNA expression assays have been used to identify primary site-specific alterations.9 Determinants of normal development, microRNAs are short, 21–23 nucleotide in length, non-translated RNA molecules that regulate gene expression at the mRNA level.22 The resulting mRNA degradation or inhibition in tumour cells may be oncogenic.22 As with other gene expression studies, a variety of clustering algorithms have been used to determine the similarities of microRNA expression across tumours from a primary site, develop classifiers, and ‘assign’ a tumour one of a finite number of tumour diagnoses,34 typically after ‘training’ the algorithm on test data sets of known primary site malignancies.11 These studies have shown that microRNA expression profiling can assign a primary site in approximately 80 per cent of CUP, including CUP for which immunohistochemical evaluation is non-informative or equivocal. Determination of primary site in CUP through immunophenotyping and/or molecular profiling will likely become increasingly prevalent as more precisely targeted agents tailored to specific primary site malignancies yields efficacious results. This CUP treatment approach is based on the tenet that even poorly differentiated metastases respond better to a primaryspecific therapeutic regimen, as opposed to the empiric platinum or taxane-based chemotherapy that has historically been the mainstay for CUP.28 The traditional binary algorithm approach using immunophenotypic profiling may be supplemented by molecular profiling in diagnostically refractory cases to provide a more confident estimate of tumour origin and allow the oncologist to pursue optimal therapy. For example, in the setting of a brain metastasis, this might include molecular profiling of a poorly differentiated carcinoma with an otherwise non-informative immunophenotyping work-up. Conversely, molecular profiling of CUP may also be of use when immunohistochemical findings do strongly suggestive of a specific anatomic origin, e.g. a CK7−, CK20+, CDX2+ metastatic adenocarcinoma, when there is an absence of confirmatory clinical, endoscopic and radiologic evidence for a colorectal primary.34 This therapeutic approach has been used for poorly differentiated CUP presenting in a variety of body sites in which molecular characterization identifies a ‘colorectal profile’. Even in the absence of a confirmed colorectal primary, patients with a CUP so identified respond better to site-specific chemotherapy compared to empiric chemotherapy.33 This may not be a universally applicable assumption, however. By definition, all CUPs, regardless of site of origin, exhibit in common the property of early metastasis, and thus share this important aspect of tumour biology, the pathophysiological basis for which is an area requiring additional investigation.

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Adams RD, Victor M. Intracranial neoplasms. In: Principles of neurology, 4th edn. New York: McGraw-Hill, 1989: 516–53 Bhushan C. ‘Miliary’ metastatic tumors in the brain. Case report. J Neurosurg 1997; 86: 564–6. Burger PC. Smears and frozen sections in surgical neuropathology: a manual. Baltimore, MD: PB Medical, 2009 Burger PC, Scheithauer BW, Vogel FS. Surgical pathology of the nervous system and its coverings, 4th edn. New York: Churchill Livingstone, 2002 Cerase A, Vallone IM, Muccio CF, et al. Regression of dilated perivascular spaces of the brain. Surg Radiol Anat 2010; 32: 555–61 Chamberlain MC. Neoplastic meningitis and metastatic epidural spinal cord compression. In: Lacy J, Baehring JM eds. Central nervous system malignancies, vol. 26. Philadelphia: WB Saunders, 2012: 917–32 Cibas ES. Cerebrospinal fluid. In: Cytology: diagnostic principles and clinical correlates, 3rd edn. Philadelphia: Elsevier, 2009: 171–96 Dickersin GR. Large cell undifferentiated neoplasms. In: Diagnostic electron microscopy: a text/atlas. New York: Igaku-Shoin, 1988: 26–79 Ferracin M, Pedriali M, Veronese A, et al. MicroRNA profiling for the identification of cancers with unknown primary tissue-of-origin. J Pathol 2011; 225: 43–53 Fuller GN. Epithelial, neuroendocrine, and metastatic lesions. In: Perry A, Brat DJ eds. Practical surgical neuropathology: a diagnostic approach, 1st edn. Philadelphia: Churchill Livingstone, 2010: 287–314 Greco FA, Oien K, Erlander M, et al. Cancer of unknown primary: progress in the search for improved and rapid diagnosis leading toward superior patient outcomes. Ann Oncol 2012; 23: 298–304 Hutchings M, Weller RO. Anatomical relationships of the pia mater to cerebral blood vessels in man. J Neurosurg 1986; 65: 316–25 Iguchi Y, Mano K, Goto Y, et al. Miliary brain metastases from adenocarcinoma of the lung: MR imaging findings with clinical

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and post-mortem histopathologic correlation. Neuroradiology 2007; 49: 35–9 Inomata M, Hayashi R, Kambara K, et al. Miliary brain metastasis presenting with calcification in a patient with lung cancer: a case report. J Med Case Rep 2012; 6: 279 Itabashi HH. Forensic neuropathology: a practical review of the fundamentals. Boston: Elsevier Academic Press, 2007 Keogh BP, Henson JW. Clinical manifestations and diagnostic imaging of brain tumors. In: Lacy J, Baehring JM eds. Central nervous system malignancies, vol. 26. Philadelphia: WB Saunders, 2012: 733–56 Madow L, Alpers BJ. Encephalitic form of metastatic carcinoma. AMA Arch Neurol Psychiatry 1951; 65: 161–73 McKeever PE. The brain, spinal cord, and meninges. In: Mills SE ed. Sternberg's diagnostic surgical pathology, vol. 1, 5th edn. Philadelphia: Lippincott Williams & Wilkins, 2010: 351–448 McWilliams RR, Giannini C, Hay ID, et al. Management of brain metastases from thyroid carcinoma: a study of 16 pathologically confirmed cases over 25 years. Cancer 2003; 98: 356–62 Monzon FA, Koen TJ. Diagnosis of metastatic neoplasms: molecular approaches for identification of tissue of origin. Arch Pathol Lab Med 2010; 134: 216–24 Morita A, Meyer FB, Laws ER, Jr. Symptomatic pituitary metastases. J Neurosurg 1998; 89: 69–73 Natoli C, Ramazzotti V, Nappi O, et al. Unknown primary tumors. Biochim Biophys Acta 2011; 1816: 13–24 Nussbaum ES, Djalilian HR, Cho KH, Hall WA. Brain metastases. Histology, multiplicity, surgery, and survival. Cancer 1996; 78: 1781–8 Ogawa M, Kurahashi K, Ebina A, et al. Miliary brain metastasis presenting with dementia: progression pattern of cancer metastases in the cerebral cortex. Neuropathology 2007; 27: 390–5 Okazaki H. Fundamentals of neuropathology: morphologic basis of neurologic disorders, 2nd edn. New York: Igaku-Shoin, 1989 Parisi JE, Mena H, Scheithauer BW. CNS tumors (excluding pituitary, PNET, and embryonal tumors). In: Nelson JS, Mena

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H, Parisi JE, Schochet SS eds. Principles and practice of neuropathology, 2nd edn. New York: Oxford University Press, 2003: 298–382 Patchell RA, Tibbs PA, Walsh JW, et al. A randomized trial of surgery in the treatment of single metastases to the brain. New Engl J Med 1990; 322: 494–500 Pavlidis N, Pentheroudakis G. Cancer of unknown primary site. Lancet 2012; 379: 1428–35 Pekmezci M, Perry A. Neuropathology of brain metastases. Surg Neurol Int 2013; 4(Suppl 4): S245–55 Ribeiro HB, Paiva TF, Jr, Mamprin GP et al. Carcinomatous encephalitis as clinical presentation of occult lung adenocarcinoma: case report. Arq Neuropsiquiatr 2007; 65: 841–4 Rivas E, Sanchez-Herrero J, Alonso M, et al. Miliary brain metastases presenting as rapidly progressive dementia. Neuropathology 2005; 25: 153–8. Taylor MB, Bromham NR, Arnold SE. Carcinoma of unknown primary: key radiological issues from the recent National Institute for Health and Clinical Excellence guidelines. Br J Radiol 2012; 85: 661–71 Varadhachary GR, Talantov D, Raber MN, et al. Molecular profiling of carcinoma of unknown primary and correlation with clinical evaluation. J Clin Oncol 2008; 26: 4442–8 Varadhachary GR, Spector Y, Abbruzzese JL, et al. Prospective gene signature study using microRNA to identify the tissue of origin in patients with carcinoma of unknown primary. Clin Cancer Res 2011; 17: 4063–70 Weller RO. Microscopic morphology and histology of the human meninges. Morphologie 2005; 89: 22–34 Wesseling P, von Deimling A, Aldape KD. Metastatic tumours of the CNS. In: Louis D, Ohgaki H, Wiestler O, Cavenee W eds. WHO Classification of Tumours of the Central Nervous System, 4th ed. Geneva: Distributed by WHO Press, World Health Organization; 2007: 309 Zhang ET, Inman CB, Weller RO. Interrelationships of the pia mater and the perivascular (Virchow–Robin) spaces in the human cerebrum. J Anat 1990; 170: 111–23

43

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44 Chapter

Hereditary Tumour Syndromes Arie Perry

Introduction..............................................................................1926 Neurofibromatosis....................................................................1926 Neurofibromatosis 1.................................................................1926 Neurofibromatosis 2.................................................................1931 Schwannomatosis....................................................................1934 Von Hippel–Lindau Disease.......................................................1934 Tuberous Sclerosis....................................................................1935 Li–Fraumeni syndrome.............................................................1937

Introduction The nervous system is affected in many hereditary tumour syndromes, with the most important manifestations listed in Table 44.1. Most are autosomal dominant and are typically caused by the inactivation of a tumour suppressor gene, so that a growth suppressing gene is either inherited in a mutant form from a parent or mutated in the germline. Therefore, with the exception of somatic mosaics that carry a mutation in only a portion of their body, the patient carries a germline mutation in all cells; once the second allele is inactivated (somatic mutation), tumourigenesis ensues. Approximately half of cases in common syndromes, such as neurofibromatosis 1 (NF1) and tuberous sclerosis, arise sporadically, without a family history, as new germline mutations. Very rarely, two syndromes may occur in the same individual or family, e.g. combined NF1 and tuberous sclerosis5,131 or von Hippel–Lindau with neurofibromatosis118a or multiple endocrine neoplasia.92 The study of these syndromes has proved essential in the identification of tumour suppressor genes involved in nervous system neoplasia. Indeed, more tumour suppressors have been identified through investigations of these rare syndromes than through studies of the more common, sporadic tumours. Nonetheless, these same syndromic tumour genes are also involved in the sporadic counterparts. For instance, although neurofibromatosis 2 (NF2) is an uncommon condition, the NF2 gene is inactivated in the majority of sporadic schwannomas and meningiomas, both common human tumours. The correlation between genetic defects in hereditary syndromes and sporadic tumours, however, is not exact; for instance, although germline defects in DNA mismatch repair genes underlie some of the glioblastomas

Cowden Disease.......................................................................1938 Turcot Syndrome......................................................................1939 Gorlin-Goltz Syndrome..............................................................1939 Rhabdoid Tumour Predisposition Syndrome..............................1940 Carney Complex.......................................................................1940 DICER1 Syndrome.....................................................................1940 Other Hereditary Brain Tumour Syndromes...............................1941 References...............................................................................1941

arising in Turcot’s syndrome, evidence of DNA mismatch repair is only rarely noted in sporadic glioblastomas.

Neurofibromatosis The term neurofibromatosis (NF) historically encompassed a spectrum of syndromes: NF1 (von Recklinghausen’s disease, peripheral NF), NF2 (central, bilateral acoustic NF), NF3 (overlapping NF1 and NF2), NF5 (segmental NF), NF6 (café-au-lait lesions only), NF7 (late-onset NF), NF8 (gastrointestinal NF) and NF9 (NF with Noonan syndrome).39,95 Of these, however, only NF1 and NF2 are distinct. Most cases previously considered NF3 to NF9 are nosologically related to either NF1 or NF2 on the basis of molecular genetics;22,104 for instance, many regionally localized examples are now recognized as either a third genetic disorder, schwannomatosis or mosaic/segmental forms of NF1 or NF2.

Neurofibromatosis 1 The most common genetic disorder, NF1, predisposes to hyperplasias, hamartomas, malformations and neoplasms of multiple organs that vary by patient age and disease severity.99 Malignancies are nearly three-fold as common in NF1 patients than in the general population, represent the leading cause of death, and reduce life expectancy by an average of 10–15 years.85

Diagnostic Criteria A definitive diagnosis of NF1 is made when ≥2 of the following are present: (1) ≥6 café-au-lait macules (≥1.5 cm in

1926

��������������

���������

��������������

22q12.2

NF2

Neurofibromatosis 2

9q34

16p13

17p13

TSC1

TSC2

TP53

Tuberous sclerosis

Li–Fraumeni

3q25

VHL

von Hippel–Lindau

SMARCB1

22q11.2

17q11.2

NF1

Neurofibromatosis 1

Schwannomatosis INI1/ (± rare forms of hSNF5/ familial meningiomas) BAF47/

Chromosome

Gene

Syndrome

p53: transcriptional regulator regulating cell cycle arrest and apoptosis during DNA damage

Tuberin: complexes with hamartin to regulate PI3K-mTOR pathway

Hamartin: complexes with tuberin to regulate PI3K-mTOR pathway

pVHL: regulates hypoxia-inducible factors, such as HIF, VEGF, and erythropoeitin

INI1/hSNF5/BAF47/ SMARCB1: SWI/SNF chromatin-remodelling complex subunit

Merlin: member of protein 4.1 family thought to mediate signals between cell membrane and actin ­cytoskeleton

Neurofibromin: KRAS ­suppressor

­ ajor Protein and m functions

Astrocytomas including glioblastoma, medulloblastoma/PNET, choroid plexus papilloma, choroid plexus carcinoma

Subependymal giant-cell tumour, subependymal nodules, cortical tubers, radial white matter migration lines, transmantle cortical dysplasia, other malformations and dysplasias, including vascular aneurysms

Haemangioblastomas of brain, spinal cord, or nerve roots; paraganglioma; brain involvement from regional spread or metastatic disease of other primaries (see other organs)

Non-vestibular, non-dermal schwannomas. Rarely, meningiomas and/or unilateral vestibular schwannoma

Bilateral schwannoma (VIIIth nerve), meningioma, ependymoma, glial microhamartomas, non-­vestibular schwannomas, meningioangiomatosis, peripheral neuropathies, cerebral calcifications

Neurofibromas (­especially plexiform), MPNST, learning disabilities, cerebral arteriopathies, ‘optic glioma’, and pilocytic, pilomyxoid, and diffuse astrocytomas; less commonly, ganglioglioma, dysembryoplastic neuroepithelial tumour, rosette-forming glioneuronal tumour

Nervous system

Table 44.1  Synopsis of hereditary tumour syndromes involving the nervous system

Malignant melanoma; cutaneous carcinomas or sarcomas (rare)

Cutaneous angiofibromas (‘adenoma sebaceum’), hypomelanotic macules, subungual fibromas, shagreen patches

None

Rare e ­ xamples of dermal neurofibroma reported

Café-au-lait spots (rare), hairy plaque-like lesions, cutaneous schwannoma. Less often neurofibroma

Café-au-lait spots, axillary freckling (virtually pathognomonic)

Skin

Major manifestations

Continued

Breast cancer, bone and soft-tissue sarcoma, adrenocortical carcinoma, leukaemia, other carcinomas, Wilms tumour

Cardiac rhabdomyomas, hamartomatous gastrointestinal polyps, cysts of the kidney, angiomyolipoma of the kidney, lymphangioleiomymatosis of the lung, retinal hamartomas, bone cysts, dental enamel pits, gingival fibromas

Retinal angiomatosis, renal cysts and carcinoma, epidydimal cystadenoma, pancreatic microcystic adenoma, pancreatic neuroendocrine tumours, phaeochromocytoma, endolymphatic sac tumour, rare extraneural haemangioblastomas

None

Cataract, retinal hamartoma, epiretinal membranes

Lisch nodules of the iris (virtually pathognomonic), pulmonary stenosis, phaeochromocytoma, osseous lesions, rhabdomyosarcoma, CML, ALL, lymphomas, JXG, GIST, duodenal carcinoid, breast carcinomas, melanoma, medullary thyroid carcinoma, glomus tumour

Other organs

Neurofibromatosis 1  1927

44

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��������������

10q23.2

5q21 Multiple loci

9q22.3

22q11.2

17q24

14q32.13

17q21.2

10q24.32

PTEN

APC

Mismatch repair genes

PTCH

INI1/ hSNF5/ BAF47/ SMARCB1

PRKAR1A

DICER1

SMARCE1

SUFU

Cowden

Turcot type 2

Turcot type 1

Gorlin-Goltz (naevoid basal cell carcinoma) syndrome

Rhabdoid tumour predisposition syndrome

Carney complex

DICER1 syndrome

Familial spinal/clear cell meningioma*

Familial ­meningioma*

SMARCE1; SWI/SNF chromatin-remodelling complex subunit

SMARCE1; SWI/SNF chromatin-remodelling complex subunit

Member of RNaseIII family, involved in generating miRNAs that regulate gene expression

PRKAR1A: regulates cyclic AMP signalling

INI1/hSNF5/BAF47/ SMARCB1: SWI/SNF chromatin-remodelling complex subunit

Patched: regulates sonic hedgehog pathway

Multiple intracranial meningiomas

Spinal meningiomas of clear cell type

CNS primitive neuroectodermal tumours including pineoblastoma, pituitary blastoma

Psammomatous melanotic shwannoma, somatotrophic pituitary adenoma

Atypical teratoid/rhabdoid tumour, schwannoma,* meningioma*

Medulloblastoma, dural calcifications, other CNS and skull malformations, meningioma,* glioblastoma*

Glioblastoma

Medulloblastoma, pineoblastoma

APC: regulates β-catenin pathway DNA mismatch repair enzymes

Dysplastic gangliocytoma of the cerebellum (Lhermitte–Duclos), macrocephaly. Possibly meningioma, pseudotumour cerebri, dural AVM, subcutaneous neuromas, ganglioneuromas, neurofibromas, oral neuromas, granular cell tumour, myasthenia gravis

Nervous system

PTEN: regulates PI3KmTOR pathway

­ ajor Protein and m functions

Not known

Not known

None

Spotty pigmentation, myxomas, epithelioid blue naevi

None

Basal cell carcinomas, palmar/plantar pits

NF1-like manifestations

None

Multiple tricholemmomas, fibromas

Skin

Major manifestations

Not known

Not known

Pleuropulmonary blastoma, cystic nephroma, embryonal rhabdomyosarcoma, ovarian. Sertoli-Leydig cell tumour, intraocular medulloepithelioma, Wilms tumour, and multinodular goiter

Cardiac myxoma, pigmented nodular adrenocortical dysplasia, fibromyxoma of breast, ductal breast adenoma, large cell calcifying Sertoli cell tumor, thyroid carcinoma, osteochondromyxoma

Malignant rhabdoid tumours of kidney and soft tissue, chondrosarcoma*

Jaw cysts, ovarian tumours, dysmorphic features, skeletal anomalies

Hereditary non-polyposis colorectal cancer, NF1-like manifestations, leukaemias/lymphomas

Familial adenomatous polyposis

Breast, thyroid, endometrial, renal and colorectal carcinomas, multiple hamartomas, gastrointestinal polyps/ ganglioneuromatosis, fibrocystic breast disease, lipomas, uterine leiomyomas, oral papillomas

Other organs

ALL, acute lymphocytic leukaemia; AVM, arteriovenous malformation; CML, chronic myelogenous leukaemia; GIST, gastrointestinal stromal tumour; JXG, juvenile xanthogranuloma; MPNST, malignant peripheral nerve sheath tumour; PNET, primitive neuroectodermal tumour; VEGF, vascular endothelial growth factor.

*Based on limited data.

Chromosome

Gene

Syndrome

Table 44.1  Synopsis of hereditary tumour syndromes involving the nervous system (Continued)

1928  Chapter 44  Hereditary Tumour Syndromes

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Neurofibromatosis 1  1929



post-pubertal or ≥0.5 cm in pre-pubertal individuals); (2) ≥2 neurofibromas of any type or ≥1 plexiform neurofibromas; (3) freckling of armpits or groin; (4) pilocytic astrocytoma of optic pathway (‘optic glioma’); (5) ≥2 Lisch nodules (iris hamartomas); (6) dysplasia/absence of the sphenoid bone or dysplasia/thinning of long bone cortex; (7) first-degree relative with NF1.43 By adulthood, about 95 per cent of patients manifest café-au-lait spots as lightly pigmented, flat, approximately symmetrical cutaneous patches. These may be present at birth, as the first manifestation, and increase in size and number until puberty. A related cutaneous lesion that usually develops later in about half of cases is the formation of freckles within intertriginous zones of the axilla or groin. About 90 per cent will develop Lisch nodules by adulthood. Skeletal lesions primarily involve dysplasias of the skull, spine and long bones, including macrocephaly, absence of the sphenoid, vertebral scalloping, scoliosis, pseudoarthrosis (mostly tibia or other long bones after non-union fractures) and long bone thinning.120 Optic gliomas affect 1.5 to 7.5 per cent of the NF1 paediatric group and are mostly indolent.63 On T2-weighted magnetic resonance (MR) sequences, up to 93 per cent of affected children between 4 and 10 years of age have focal increases in signal intensity referred to as ‘unidentified bright objects’ (UBOs), corresponding to vacuolated myelin in the basal ganglia, brainstem and cerebellum. These are typically transient, but relatively characteristic and may be particularly helpful in diagnosing NF1 in young children who have not yet developed other pathognomonic features.21 Apparent diffusion coefficients are also elevated in children with NF1, both in the UBOs and even in normal appearing white matter, suggesting higher water content.119 Lastly, white matter volumes are often increased, contributing to the megalencephaly commonly noted and further supporting the presence of a myelinopathy in NF1.19 Whether or not these alterations correlate with the learning deficits often encountered in NF1 patients remains uncertain. Systemic tumours also occur in association with NF1 (Table 44.1), accounting for considerable morbidity and mortality.138

Nervous System Tumours Tumours of the nervous system associated with NF1 include: (1) multiple neurofibromas at any site, particularly (a)

cutaneous and paraspinal examples; (2) the hallmark plexiform neurofibroma, most often affecting larger, deep nerves; (3) malignant peripheral nerve sheath tumours (MPNSTs); (4) optic pathway pilocytic and pilomyxoid ­astrocytomas; (5) other astrocytoma subtypes, WHO grades II–IV; (6) glioneuronal neoplasms, such as ganglioglioma, dysembryoplastic neuroepithelial tumour, and rosette-forming glioneuronal tumour.85 The neurofibromas that affect NF1 patients are pathologically similar to sporadic neurofibromas, with some notable exceptions. The presence of paraspinal neurofibromas, particularly multiple tumours, is essentially diagnostic and often associated with significant peripheral neuropathy.24 Furthermore, both ‘rope-like’ expansions of multiple deep peripheral nerve fascicles by plexiform neurofibroma and massive diffuse appearing soft tissue neurofibromas are virtually restricted to NF1 (Figure 44.1a–d).99 Malignant transformation of nerve sheath tumours in NF1 patients is a major cause of morbidity and mortality, with plexiform neurofibromas being most prone. Lifetime risks of developing MPNST have been estimated at 8–13 per cent,85 with patients harbouring large germline deletions at even greater risk.23 Additionally, there are 3- and 20-fold increased risks of malignant transformation to MPNST in NF1 patients with subcutaneous and plexiform neurofibromas, respectively.85 Such NF1 associated MPNSTs represent 10–50 per cent of cases, with the remainder considered de novo. These tumours may be histologically identical to sporadic MPNSTs (Figure 44.1e), but there is a greater tendency for malignant Triton tumour (MPNST with rhabdomyosarcomatous differentiation), glandular MPNST and nerve sheath tumour with angiosarcoma in NF1.134 Five-year survival is also worse in NF1associated (16–38 per cent) than sporadic MPNST (42–57 per cent), possibly because of later presentations and difficulties in clinically distinguishing malignant from benign tumours in NF1.85 Optic gliomas have an incidence of 1.5–20 per cent in NF1 patients, most commonly presenting in the first 6 years of life;64,85 they show classic features of pilocytic astrocytoma, although with greater propensity towards leptomeningeal invasion and meningothelial hyperplasia than sporadic counterparts (Figure 44.1f,g).103 These are often remarkably indolent tumours; many resolve

44

(b)

44.1 Representative tumours encountered in NF1 patients. See next page for legend.

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1930  Chapter 44  Hereditary Tumour Syndromes (c)

(d)

(e)

(f )

(g)

(h)

44.1 (Continued ) Representative tumours encountered in NF1 patients. Plexiform neurofibroma (a–d), showing expansion of multiple nerve fascicles (a, c: EMA highlighting perineurium), ­formation of tactile-like bodies (a: upper left, b), and a subset of S-100 protein positive Schwann cells (d). Intraneural malignant ­peripheral nerve sheath tumour (MPNST) with non-specific fibrosarcoma-like spindle cell proliferation (e). Optic glioma with infiltration between the normal collagenous septae of the optic nerve simulating diffuse astrocytoma (f), but with presence of Rosenthal fibres elsewhere (g), consistent with pilocytic astrocytoma. Glioblastoma with focus of necrosis (h).

without therapy. NF1-associated pilocytic astrocytomas are frequently in the cerebellum and brain stem, especially the medulla. Patients also have increased risks of developing malignant astrocytomas,44 particularly in those over 10 years of age or those whose optic gliomas have been treated with irradiation; such tumours are often highly aggressive (Figure 44.1h). In contrast, some seemingly

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diffuse parenchymal lesions appear to resolve spontaneously. The expansive pontine lesions in NF1 patients that resemble pontine gliomas on neuroimaging often follow a ­ surprisingly indolent course.123 However, symptomatic presentation, adult onset, and extra-optic location appear independently associated with decreased survival in NF1 patients with central nervous system (CNS) tumours.42

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Neurofibromatosis 2  1931



Molecular Genetics and Biology of Disease NF1 is an autosomal dominant disorder with an estimated incidence of 1 in 3000. Half, however, have no family history, representing new mutations.102 Penetrance is nearly 100 per cent by 5 years of age,85 but a definitive determination is difficult, owing to intrafamilial variability of manifestations and the number of new mutations. Linkage studies demonstrate that the majority of mutations occur in the parental germline. The high mutation rate, estimated at 1 per 10 000 alleles per generation, may relate to the large size of the NF1 gene, which is located on chromosome 17q11.2, spans about 350 kb of genomic DNA and contains 59 exons. This large size has frustrated extensive mutation screening, although over 500 pathogenic mutations have now been identified, most being kindred s­pecific.85 No hotspots have been identified; however, a common 1.5 Mb microdeletion involving the entire NF1 gene is seen in 5–10 per cent of patients and is generally associated with more severe phenotype, including facial dysmorphism, mental retardation, developmental delay, increased neurofibroma burden and enhanced risk of MPNST.23 The NF1 transcript is approximately 13 kb long and includes three alternatively spliced isoforms (exons 9a, 23a and 48a), thought to reflect tissue-specific and differentiation-associated regulation.102 The gene encodes a widely expressed 220–250 kDa protein, designated neurofibromin. One major functional role for neurofibromin appears to be related to its activity as a RAS GTPase activator protein (GAP), as suggested by sequence homology to the catalytic subunits of a variety of GAP and by functional assays with the GAP-related domain (GRD). Neurofibromin loss may specifically activate the KRAS, but not the HRAS isoform, thereby activating various downstream signaling cascades and mitogenic mediators, such as AKT, ERK1/2, RAF, PI3K, mTOR and S6K.102 There is also evidence for growth-­ regulatory functions outside the neurofibromin GRD. Inactivation of the wild-type NF1 allele with loss of neurofibromin expression is considered a critical early event in tumourigenesis. It was therefore surprising that genetically engineered mouse models with conditionally knocked out NF1 in either Schwann cells or astrocytes do not form neurofibromas or optic gliomas respectively. In both cases, a heterozygous Nf1± state of non-neoplastic elements (similar to the human condition) was required for tumour formation.9,137 These data suggest that growth factors secreted by mast cells, microglia or other NF1 haploinsufficient stromal elements are needed for a permissive tumourigenic environment, possibly progressing through a hyperplastic proliferation initially.102,114 By the time neurofibromas transform to MPNSTs, numerous additional alterations have taken place, including the loss of other tumour suppressor genes, such as TP53 and CDKN2A/ p16 and overexpression of various oncogenes, growth factors, growth factor receptors, and chemokines, such as EGFR, HER-2/neu, neuregulin-1 (NRG1), topoisomerase IIα, hepatocyte growth factor (HGF), c-Met, PDGF and CXCR4.15,76,136

Neurofibromatosis 2 NF2 (synonyms: bilateral acoustic neurofibromatosis or central neurofibromatosis) is an autosomal dominant

��������������

syndrome with high penetrance (0.95), an estimated incidence of 1 in 25 000 and prevalence of 1 in 60 000.26 Over half are due to de novo gene mutations and therefore, have no family history; of those, 20–30 per cent represent somatic mosaics, often with too few mutation-bearing cells in the blood to be detected by standard techniques. Life expectancy is reduced considerably.

44

Diagnostic Criteria Patients with NF2 develop hyperplastic/hamartomatous lesions and benign Schwann cell (schwannomas and schwannosis; less commonly, neurofibromas), meningothelial (meningiomas and meningioangiomatosis), and glial (ependymomas and glial hamartomas) proliferations. In addition, NF2 patients are susceptible to posterior lens opacities/cataracts, cerebral calcifications and peripheral neuropathies, the latter resulting not only from schwannomas, but from also tumourlets, neurofibromalike reactive proliferations, onion bulb formation, and functional deficits related to NF2 haploinsufficiency.8,109 Bilateral VIIIth nerve schwannomas are pathognomonic. However, given that many patients have no family history and present with other manifestations initially, the Manchester criteria have been devised to increase diagnostic sensitivity without significant loss in specificity. Other diagnostic criteria include: (1) a first-degree relative with NF2 and either a unilateral vestibular schwannoma or two of the following: meningioma, schwannoma, glioma (ependymoma), neurofibroma, posterior subcapsular lens opacity; (2) unilateral vestibular schwannoma and any two additional lesions listed in point (1); (3) ­multiple meningiomas and unilateral vestibular schwannoma or any two of the following: schwannoma, glioma, neurofibroma, cataract.10 The mean age of onset, related to symptoms from VIIIth nerve schwannomas is about 22 years. Although NF2 may have manifestations in the paediatric population, its onset typically does not extend beyond the sixth decade. Café-au-lait spots can occur but are usually fewer than six; superficial plaque-like cutaneous or nodular subcutaneous lesions (typically schwannomas and only rarely neurofibromas) occur in 68 per cent and cataracts in about 38 per cent of cases.100 The latter may be among the first detectable lesion in the paediatric group.105 Death is often due to the disease and may be as early as the fourth decade, with worse prognosis associated with younger age at diagnosis, numerous meningiomas, and truncating NF2 mutations.26 There is, however, marked variability with two ends of the severity spectrum: the Gardner (mild) and Wishart (severe) variants, based on the number and types of tumours. The mild form commonly presents with bilateral vestibular schwannomas in adults, whereas the severe form often presents during childhood with other tumour types, including meningiomas.87 There is a tendency for these variants to cluster within families and to segregate with distinct types of NF2 gene mutations, such that nonsense and frameshift mutations are more often seen in patients with the severe phenotype. Because of the complex multiorgan manifestations, NF2 patients and their families do better with both quality and quantity of life issues when receiving care at specialized multidisciplinary centres.27

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1932  Chapter 44  Hereditary Tumour Syndromes

Vestibular Schwannomas The most common and diagnostic neuroimaging features in NF2 are bilateral vestibular schwannomas. When small, they may require thin MR cuts through the internal auditory meatus for detection; without such detailed neuroimaging, NF2 cannot be excluded. Vestibular schwannomas in NF2 are histologically similar to sporadic schwannomas, except NF2 vestibular schwannomas tend to be multicentric, have a multinodular (‘cluster of grapes’) appearance, demonstrate more entrapped nerve fibres, and more often display a mosaic pattern (mixed positive and negative nuclei) of INI1 expression.45,86 In rare cases, irradiation may be necessary to control tumour growth, although this option should be considered with caution because secondary radiation-induced malignancies are more common in NF2 than in the general population.26

Peripheral Schwannomas In addition to the vestibular nerves, schwannomas may arise at any site of the body in patients with NF2. Other cranial nerves may be affected, such as the Vth, VIIth and XIIth, as well as paraspinal and cutaneous nerves. Unlike the deep-seated neurofibromas of NF1, however, peripheral schwannomas rarely involve deep nerves in a plexiform manner or undergo malignant change. Paraspinal schwannomas appear to arise from minute precursor neoplasms, dubbed ‘tumourlets’, that may stud the rootlets and proximal nerves.26,113 Peripheral NF2-associated schwannomas are histologically identical to sporadic schwannomas, although the former similarly appear more infiltrative, with entrapped axons evident using neurofilament immunohistochemistry.130 Also, the presence of a mosaic pattern of INI1 expression favours a syndromic (NF2 or schwannomatosis) schwannoma.86 Lastly, there is a link between plexiform cutaneous schwannomas and NF2;62 indeed, most tumours diagnosed as ‘plexiform neurofibromas’ in NF2 patients turn out to be plexiform schwannomas on review (Figure 44.2a,b). In contrast the majority of deep­ seated plexiform schwannomas have no association with either NF1 or NF2, although their increased cellularity, proliferative activity and large size may be misinterpreted as MPNST.3,135

Schwannosis Schwannosis is a non-neoplastic proliferation of Schwann cells that occurs in both NF2 and non-NF2 patients. It is typically found in the spinal cord, either at the dorsal root entry zones or in the parenchyma of the cord, but is most commonly noted in NF2 patients. In non-NF2 patients, schwannosis occurs in response to infarction or trauma, although such inciting events are not usually identifiable in NF2 patients. Clonality studies have to be performed, although a reactive process is favoured.

Meningiomas Multiple meningiomas are also a common feature of NF2 and represent a major source of morbidity and mortality. They occasionally precede the detection of vestibular schwannomas, being the presenting feature in 20–30 per

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cent of patients.26 Evidence suggests that fibroblastic or transitional meningiomas are more common in NF2 patients. However, the full range of both benign and aggressive subtypes is seen. Additionally, larger series suggest an increased incidence of atypical and malignant subtypes (Figure 44.2c,d),87 although it is unclear whether this might represent a selection bias given that slow-growing meningiomas are less likely to be resected. Furthermore, whereas NF2 accounts for only a small fraction of adult meningiomas, up to 40 per cent of paediatric meningioma patients ultimately fulfill criteria for NF2. Therefore, it is important to screen children with meningioma for this possibility, particularly if multiple tumours are present.

Meningioangiomatosis Meningioangiomatosis is an uncommon plaque-like ­cerebral mass characterized by meningothelial and fibroblastic proliferations in association with angiomatous microvascular formation (Figure 44.2e).88 These masses may develop in the meninges, cortex or both. The relative composition of meningothelial and vascular components varies among lesions such that they may appear principally as a vascular malformation or as a primary meningothelial/fibroblastic lesion. Low or absent MIB-1 labelling and lack of meningioma-associated genetic alterations suggest a hamartomatous aetiology. The adjacent brain may manifest a spectrum of reactive and degenerative changes. Importantly, in NF2 patients, these lesions are often multiple, associated with adjacent glial microhamartomas, and are incidental findings; in contrast, sporadic meningioangiomatosis is typically single and symptomatic, presenting with seizures. Some may be associated with an overlying meningioma, although genetic data suggest that most such cases actually represent meningiomas with extensive perivascular spread, mimicking meningioangiomatosis.88

Gliomas The association of NF2 with gliomas is infrequent, with the exception of spinal ependymomas. These are typically indolent, well-demarcated, intramedullary masses that are amenable to surgical excision. They nearly always affect the cervical cord, but may be multicentric. The occurrence of other gliomas, usually low-grade pilocytic astrocytomas, has been reported in NF2 patients, but some of these may represent misdiagnoses of tanycytic ependymomas.59,121

Glial Microhamartomas These circumscribed clusters of cytologically atypical cells with S-100 immunoreactivity are common in the molecular and deeper layers of the cerebral cortex, basal ganglia, thalamus and cerebellum (Figure 44.2f). They are pathognomonic of NF2 and do not resemble the hamartomas seen in other neurological diseases, such as tuberous sclerosis.88,132

Molecular Genetics and Biology of Disease The NF2 gene is located on chromosome 22q12.2 and appears to act as a classic tumour suppressor.8,26 It

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Neurofibromatosis 2  1933

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44.2 Representative lesions in NF2 patients. Cutaneous plexiform schwannoma (a,b), with involvement of multiple fascicles (a) and foci showing Verocay bodies (b). Meningioma with aggressive features includeing spontaneous necrosis (c) and high Ki-67 labelling index (d). Meningioangiomatosis with markedly hyalinized vascular proliferations and thin ribbons of epithelioid meningothelial cells (e). Glial microhamartomas consisting of clusters of bizarre-appearing astrocytes in the cortex (f).

encompasses about 110 kb, including 16 constitutive and one alternatively spliced exons. The NF2 gene is expressed in most tissues, including the nervous system, where it is present at high levels during development. The gene product, designated merlin or schwannomin, is a member of the protein 4.1 family of cytoskeletal-associated proteins,

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impacting PI3 kinase/Akt, Raf/MEK/ERK and mTOR signaling pathways.26 Two merlin isoforms exist, with only isoform 1 thought to have tumour suppressor qualities.8 Point mutations are most common, with the majority of mutations leading to truncated protein products; large deletions, duplications, and insertions may be more difficult to

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1934  Chapter 44  Hereditary Tumour Syndromes

detect. Mutations resulting in abnormal protein expression lead to a more severe phenotype than the mutations/large deletions resulting in complete merlin loss or retention.100 Using current multimodality approaches, mutations are detectable in nearly all familial and 59 per cent of sporadic NF2 cases.58,128 The similarity of merlin to the 4.1 protein family of cytoskeletal-associated proteins suggests that it may play a role in mediating communication between surface membrane signalling and the cytoskeleton matrix, with its normal role in contact-dependent inhibition being a key tumourigenic function.100 Indeed, merlin interacts with transmembrane molecules, such as CD44, in addition to having actin-binding sites. Through its interaction with a variety of other submembrane proteins, such as Na+-H+ exchanger-regulatory factor (NHE-RF), merlin probably acts to transmit growth and motility signals to the underlying cytoskeleton. In this manner, the absence of merlin in NF2-associated tumours leads to their growth potential. Conditional knockout mouse models of the NF2 gene in Schwann cells have similarly yielded examples of schwannosis and schwannomas.35

Schwannomatosis Schwannomatosis, sometimes referred to as the third form of neurofibromatosis, is characterized by the development of multiple, mostly non-vestibular, non-dermal schwannomas in the absence of other NF2 features. The true ­incidence is difficult to determine, although it may be as common as NF2, accounting for up to 2.4–5 per cent of all schwannoma resections.70 Familial (inherited) cases only account for 15–20 per cent, with the majority representing sporadic (non-inherited) examples.90 Segmental forms involving a single limb or region of the spine represent up to a third of cases. Patients often present with localized and sometimes debilitating chronic pain, a feature not frequent in typical NF2.100 Notably, patients with schwannomatosis do not have germline NF2 mutations, although somatic mutations are involved in tumourigenesis. Although when originally described, neither vestibular schwannomas nor meningiomas were allowed, rare occurrences of both have now been described,75,90,111,124 complicating the distinction from NF2. Nonetheless, bilateral vestibular schwannomas are still definitional of NF2 and are therefore, not allowed in schwannomatosis. Additionally, rare examples including cutaneous neurofibromas have also been reported.98 Diagnostic criteria for schwannomatosis are still developing, but the diagnosis can currently be made either molecularly or clinically. The latter require (1) ≥2 non-intradermal schwannomas, one with pathological confirmation, but no evidence of bilateral vestibular schwannomas on high-quality magnetic resonance imaging (MRI); (2) one pathologically confirmed schwannoma or intracranial meningioma and an affected first-degree relative or (3) a possible diagnosis if there are two or more nonintradermal tumours, but none are pathologically proven schwannomas, especially in association with chronic pain.90 A cut-off age of 30 years has also been proposed to exclude bilateral vestibular schwannomas (and therefore NF2).100 The schwannomas generally resemble their non-syndromic counterparts, but more frequently

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feature prominent myxoid changes, intraneural growth, peritumoural oedema and mosaic INI1 immunoreactivity.

Molecular Genetics and Biology of Disease The gene considered responsible for schwannomatosis is the SMARCB1 (INI1/hSNF5/BAF47) gene on chromosome 22q11.2, centromeric to the NF2 gene.49 This gene is involed in the ATP-dependent SWI/SNF chromatin remodelling complex and functional alterations are thought to lead to secondary transcriptional and epigenetic changes.90 The protein product interacts with the E1 protein of human papilloma virus 18 and the EBNA-2 protein of Epstein– Barr virus, as well as with cellular proteins MYC, MLL (HXR), GADD34 and AKT. It is also involved in HIV-1 viral replication. Studies suggest that tumour suppressor activities include: (1) the induction of G1 arrest; (2) mitotic arrest; (3) inhibition of aneuploidy and (4) the induction of senescence. Germline SMARCB1 mutations are detectable in 40–50 per cent of familial and 8–10 per cent of sporadic schwannomatosis cases, where a four-hit, threestep model has now been touted.90 The latter starts with a germline mutation (step 1, hit 1), followed by a variably sized chromosome 22q deletion that includes both SMARCB1 and NF2 (step 2, hits 2 and 3), culminating in a somatic mutation of the remaining NF2 allele (step 3, hit 4). This same model has also been implicated in rare familial meningiomas.17 Given that there is considerable overlap between sporadic schwannomatosis and mosaic NF2, genetic analysis of both SMARCB1 and NF2 genes in multiple tumours from the same individual is sometimes required to definitively distinguish these two possibilities.90 A molecular diagnosis can be made by (1) ≥2 pathologically proven schwannomas or meningiomas with genetic studies showing loss of heterozygosity (LOH) for chromosome 22 and two different NF2 mutations but a common SMARCB1 mutation or (2) one pathologically proven schwannoma or meningioma and a germline SMARCB1 pathogenic mutation.90 Given that germline SMARCB1 mutations are also responsible for rhabdoid predisposition syndrome (RPS), it remains unclear which additional factors predispose to these very different syndromes. Because, in contrast to RPS, the germline mutations in schwannomatosis tend be nontruncating, it has been postulated that the resulting milder phenotype underlies the differences. Nonetheless, it is interesting to note that combined manifestations of both syndromes are encountered rarely.90,116

Von Hippel–Lindau Disease This autosomal dominant tumour syndrome manifests in many tissues, including the CNS, eye, kidney, adrenal medulla, pancreas, middle ear and epididymis. The incidence is estimated at 1 in 36 000 live births, up to 20 per cent representing new mutations, with penetrance over 90 per cent by 65 years of age.53,127 As with the neurofibromatoses, it is a complex disorder with significant morbidity and mortality, which is most effectively treated at multidisciplinary specialty centres.

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Tuberous Sclerosis  1935

Diagnostic Criteria

Molecular Genetics and Biology of Disease

Von Hippel–Lindau (VHL) disease is defined by the presence of retinal angiomatosis (25–60 per cent), CNS haemangioblastomas (44–72 per cent), phaeochromocytoma (10–20 per cent), pancreatic cysts (35–70 per cent), renal cysts and renal cell carcinomas (RCC; 25–60 per cent), endolymphatic sac tumours (10 per cent), and in males, bilateral papillary cystadenoma of the epididymis (25–60 per cent). Families with and without pheochromocytomas are broadly divided into type 2 and type 1 variants of VHL, respectively. The type 2 category is further stratified into 2A (haemangioblastomas, low risk of RCC), 2B (haemangioblastomas, high risk of RCC) and 2C (phaeochromocytomas only). The minimal diagnostic criteria are (1) a family history plus CNS haemangioblastoma, phaeochromocytoma or clear cell RCC, (2) ≥2 CNS haemangioblastomas or (3) one CNS haemangioblastoma plus one of the visceral tumours mentioned earlier.66 Most lesions can be identified by computed tomography (CT) or MR imaging in patients known to be at risk, but ophthalmological screening for retinal angiomatosis is considered the least invasive early detection approach. The mean ages at clinical manifestation are 22 for endolymphatic sac tumour, 25 for retinal angiomatosis, 33 for CNS haemangioblastoma, 30 for phaeochromocytoma and 39 years for RCC.66 Until recently, life expectancy was estimated at 50 years, with death commonly resulting from complications of RCC and CNS haemangioblastoma; however, modern screening and surveillance programmes have led to earlier detection, improving morbidity and mortality figures considerably.127

The VHL tumour suppressor gene maps to chromosome 3p25. The 852 nucleotide coding sequence is divided into three highly conserved exons and encodes a 213 amino-acid, 30-kDa protein (pVHL) that is widely expressed in both fetal and adult tissues.53 A second 19-kDa isoform results from internal translational initiation at codon 54. Both isoforms have tumour suppressor activity, with dysfunction leading to developmental arrest.127 The pVHL protein forms oligomeric complexes with several cellular proteins, including the elongin B and C subunits, Cul2 and Rbx1. Elongin is a transcription factor that regulates mRNA elongation, particularly during pauses in transcription. Even in the earliest tumour precursors, VHL protein dysfunction leads to hypoxia inducible factor (HIF) accumulation and activation, which then increases expression of VEGF, erythropoietin (Epo), nitric oxide synthase and glucose transporter 1.127 In normoxic conditions, the pVHL complex targets HIF for degradation by ubiquitination, whereas in the setting of hypoxia or loss of pVHL function, HIF levels increase. The subsequent overexpression of angiogenic downstream targets, such as VEGF and PDGF-B, likely account for the hypervascularity of VHL-associated tumours and represent attractive candidates for targeted therapies, along with TGFα and EGFR, which are also overexpressed in haemangioblastomas and RCCs. In addition to its interactions with HIF, alternative functions of pVHL relate to regulation of extracellular matrix, cytoskeletal stability, cell cycle control and cellular differentiation.53 Mutations of the VHL gene occur in affected family members, as well as in roughly 30 per cent of sporadic CNS haemangioblastomas and 50 per cent of clear cell RCCs.53,66 Promoter region hypermethylation is found in 10–20 per cent of sporadic RCC, but not in haemangioblastomas. In VHL patients, the gene is typically affected by insertions, deletions or missense mutations with subsequent loss of the wild-type allele in tumours. Genotype–phenotype associations exist, such that the vast majority of families with phaeochromocytomas (VHL type 2) harbour missense mutations, whereas most type 1 families demonstrate deletions or nonsense mutations. Molecular testing now enables detection of a germline mutation in blood from virtually all affected families, with the exception of rare mosaic patients.33,66 The mechanism of somatic loss of the wild-type allele in tumours and benign cysts is highly variable, ranging from small deletions to monosomy 3.36 Because even preneoplastic lesions, known as tumourlets or developmentally arrested structural elements (DASE), demonstrate biallelic inactivation, it is likely that additional genetic or epigenetic events are necessary for neoplastic transformation.127 Another common denominator in these early lesions is the presence of haemangioblast progenitor cells, which may also represent the origin of the neoplastic ‘stromal cells’ in haemangioblastoma.

Central Nervous System Manifestations Solitary or multiple haemangioblastomas are hallmarks of VHL and are seen in 80 per cent of patients.127 As in sporadic haemangioblastomas, in VHL most are located infratentorially. In a study of 1921 CNS hemangioblastomas from 225 patients, tumours were localized to cerebellum (45 per cent), spinal cord (36 per cent), cauda equina (11 per cent), brain stem (7 per cent), supratentorial region (1 per cent) and nerve roots (0.3 per cent).67 Roughly half remained stable over time, although the other half grew in a saltatory (most common), exponential or linear pattern; however, it was difficult to predict future behaviour. Rare extraneural examples have also been reported.20,83 During the course of disease, additional haemangioblastomas may develop, either at the same site or remotely. Symptoms most often correlate with cyst enlargement. CNS haemangioblastomas may produce sufficient erythropoietin to induce polycythaemia. In rare cases, VHL patients develop other brain neoplasms, including cerebellar astrocytoma,78 medulloblastoma/PNET11 and meningioma.51 In addition, papillary middle ear tumours, known as endolymphatic sac tumours, are relatively common and may involve the cerebellopontine angle with resultant hearing loss, tinnitus and vertigo.52 Lastly, CNS metastases are encountered, most commonly from RCC, but occasionally from phaeochromocytoma, paraganglioma or pancreatic neuroendocrine tumour.127

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44

Tuberous Sclerosis Tuberous sclerosis complex (TSC) is an autosomal dominant disease with high penetrance, in which hamartomatous lesions involving multiple organ systems develop at different

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1936  Chapter 44  Hereditary Tumour Syndromes

TSC affects tissues derived from all three embryonal germ layers and invariably involves the CNS with various dysplastic and benign neoplastic lesions. The presence of multiple facial angiofibromas (previously known as ‘adenoma sebaceum’) and subungual fibromas (‘Koenen tumours’) are characteristic cutaneous lesions; however, hypomelanotic macules (‘ashleaf spots’) are most common and appear earliest. Shagreen patches and fibrous plaques on the forehead are also typical, but not specific. TSC was once defined by a classic triad of adenoma sebaceum, seizures and mental retardation. However, revised diagnostic criteria are more accurate and cite major features as facial angiofibromas or forehead plaques, subungual/periungual fibroma, hypomelanotic macules (at least three), shagreen patch (connective tissue naevus), retinal nodular hamartomas, tubers, subependymal nodules, subependymal giant cell astrocytoma (SEGA), cardiac rhabdomyoma, and lymphangiomyomatosis and/or renal angiomyolipoma.40,97 Minor diagnostic features include dental enamel pits, hamartomatous rectal polyps, bone cysts, cerebral white-matter migration lines, gingival fibromas, non-renal hamartomas, retinal achromic patch, multiple renal cysts and ‘confetti’ skin lesions. Definite TSC can be diagnosed with two major or one major plus two minor features. Probable TSC requires one major and one minor feature, although the designation of possible TSC requires a major feature or two or more minor features. Dental examination may be particularly helpful, because enamel pits/craters and gingival fibromas are seen in nearly all TSC patients.72 RCC, angiomyolipomas, oncocytomas, high-grade astrocytomas and chordomas have also been associated.6,61 Angiomyolipomas occur in approximately 80 per cent of patients, representing the leading cause of death due to hemorrhage.82 Neurological manifestations vary considerably, but are clinically apparent in about 90 per cent. Seizure disorders (including infantile spasms) often manifesting within the first year of life (60–90 per cent), mental retardation (45 per cent) and autism (25 per cent) are most common and are roughly correlated with the location, number and size of tubers, although data also suggest functional deficits in tuber-free cortex.25,82 Progressive signs of elevated intracranial pressure (ICP) often herald the presence of a SEGA near the foramen of Monro.

seen in 8–15 per cent.40,50 In infants, they are T1-bright and T2-dark compared to unmyelinated white matter, although in older patients, they are mostly T1-hypodense to isodense and hyperintense on T2-weighted and FLAIR sequences, with variable calcification, the latter best detected on CT scan. Positron emission tomography (PET) scans reveal increased N-acetylaspartate (NAA) and myo-inositol, consistent with neuronal reduction, gliosis and the presence of immature neurons. Grossly, they are mushroom-shaped, firm cortical expansions (‘tuber’ reflecting potato-like consistency) limited to one or two gyri (Figure 44.3a), although more diffuse examples manifest as hemimegalencephaly. Cortical tubers and radially oriented heterotopias are composed of enlarged, atypical and disorganized glial, neuronal and mixed glioneuronal elements (e.g. neuronomegaly, balloon cells), with astrocytosis and variable calcification (Figure 44.3b). Demonstrably epileptogenic tubers are often excised for seizure control.84 Dysplastic cells abnormally regulate gamma-aminobutyric acid (GABA) and may overexpress glutamate transporters, such as EAAC1, potentially contributing to epilepsy.25,40 A potential diagnostic pitfall in a subset includes increased proliferative indices and astrocytoma-like histology.29 In the developing fetus, such dysplastic cells are often found in deep white matter, suggesting a migrational defect as part of the lesion. Distinctive CD34immunoreactive cells are also seen in a subset of tubers, possibly representing a progenitor cell (Figure 44.3c). The subependymal nodules and SEGAs are also characterized histologically by atypical, enlarged glioneuronal cells, and often by dystrophic calcification (Figure 44.3d). The smaller ‘candle guttering’ lesions are seen in about 80 per cent of patients, grossly resemble wax drippings, line the lateral ventricular walls, but only become symptomatic if they enlarge to form SEGA (5–10 per cent of patients), with hydrocephalus resulting from obstruction of the foramen of Monro. In contrast to the subependymal nodules, SEGAs are >1 cm in size and are usually contrast enhancing on MR imaging. They are composed of polygonal, epithelioid, gemistocytic or spindle-shaped cells with marked pleomorphism, abundant pink cytoplasm, eccentric nuclei, prominent nucleoli and nuclear pseudo-inclusions, often forming sweeping fascicles or ependymoma-like perivascular pseudo-rosettes (Figure 44.3d). Whereas resection is the treatment of choice, medical therapy with everolimus or other mTOR inhibitors represents a successful alternative for nonresectable cases and those with residual/recurrent masses following surgery.18,32 Retinal astroglial hamartomas are present in about 50 per cent and may undergo calcification and cystic degeneration. However, visual impairment only accompanies macular involvement or secondary haemorrhage.

CNS Manifestations

Molecular Genetics

The hallmark lesions are focal cortical dysplasias or tubers, typically centred around the corticomedullary junction, along with subependymal nodules, SEGA, radial white matter migration lines, transmantle cortical dysplasia, and less often, other malformations, such as agenesis of the corpus callosum, schizencephaly, cerebellar dysplasias and vascular aneurysms.40 Cortical tubers are usually detectable by MRI, most being supratentorial, with cerebellar examples

Two genes have been identified.73 The first, designated TSC1, resides on 9q34 and encodes a 8.6-kb transcript for a 1164 amino-acid hydrophilic protein, hamartin, with no clear homology to known vertebrate proteins. The second, designated TSC2, maps to 16p13.3 and encodes a 5.5-kb transcript for a 1807 amino-acid protein, tuberin, with partial homology to the Rap1 activator Rap1-GAP. Over 1000 mutations have been identified, with TSC2 mutations

stages of the disease.25,40,82 TSC is the second most common hereditary tumour syndrome of the nervous system, with an incidence of 1 in 6000. Roughly 70 per cent occur sporadically as new cases and about 2 per cent are thought to be mosaic.

Diagnostic Criteria

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Li–Fraumeni Syndrome  1937

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44.3 Central nervous system (CNS) lesions in patients with tuberous sclerosis. Multiple tubers are grossly evident as foci of cortical pallor and blurring of the corticomedullary junctions in this seizure resection specimen (a). Microscopically, there are numerous balloon cells with glassy eosinophilic cytoplasm, neuronomegaly (arrow), and microcalcifications (b). Distinctive CD34positive cells with highly ramified cellular processes were evident, potentially representing progenitor cells (c). Similar dysmorphic glioneuronal cells are seen in subependymal giant cell astrocytoma; ependymoma-like perivascular pseudorosettes (right) are also common (d).

roughly five times as common as TSC1 mutations in sporadic cases, but with equal representation in familial examples.77 Mutations are detectable in 80–90 per cent of TSC patients, nearly all TSC1 mutations being truncating, although 20 per cent of TSC2 mutations are missense. TSC2 mutations may be associated with a more severe phenotype, particularly in terms of CNS disease.77 In tubers and SEGAs, classic second hits are often absent, although targeting of other genes in the pathway, epigenetic mechanisms and haploinsufficiency alone may play roles.25,40,47 Both proteins are widely expressed in human tissues. Hamartin and tuberin form heterodimers via coiled-coil regions and function together at a critical point in the PI3K signalling pathway. The tuberin–hamartin complex normally inhibits Rheb (a Ras-like GTPase) and mTOR, with these downstream regulators overexpressed in TSC. TSC1 and TSC2 proteins function in cell body size, proliferation, dendritic arborization, axonal outgrowth and targeting, neuronal migration, cortical lamination and spine formation.77,82 This correlates well with the observed pathology including giant cells, an immature glioneuronal phenotype,

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proliferative potential and migrational defects. Most studies have found remarkably similar genotypic and phenotypic features among the giant cells of SEGA, tubers and focal cortical dysplasia type IIb, suggesting that these morphologically similar cell types arise from a single CNS progenitor, most likely orginating from the periventricular region. Progress has also been made using animal models, including Eker rats that harbour a natural TSC2 mutation and genetically engineered mice.25 For example, conditional knockout of Tsc1 in astrocytes produces not only astrocytic proliferation, but also abnormal neuronal migration and differentiation, as well as a distinct clinical phenotype that includes seizures.122 A knockout model involving Tsc2 in Purkinje cells potentially models autism.94

Li–Fraumeni Syndrome Li–Fraumeni syndrome (LFS) is an autosomal dominant condition in which affected family members are prone to develop a wide variety of neoplasms. Although bone and

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1938  Chapter 44  Hereditary Tumour Syndromes

soft tissue sarcomas, breast cancer, adrenocortical carcinoma (ACC), brain tumours and leukaemia remain the classical hallmarks, further studies reveal a wider spectrum, including cancers of the lung, colorectum, stomach, prostate, ovary, pancreas, as well as lymphoma, melanoma and choroid plexus carcinoma (Table 44.1).71,81 Penetrance is less than 20 per cent in children, but 100 per cent by age 70, with earlier onset in women, mostly due to breast cancer. The responsible gene in most families is the TP53 gene, a tumour suppressor that is mutated somatically in many sporadic human cancers. Conversely, the majority of families with TP53 germline mutations meet diagnostic criteria for Li–Fraumeni syndrome.56

Diagnostic Criteria The diagnostic criteria used to identify LFS are (1) a proband with a sarcoma before 45 years of age; (2) at least one first-degree relative with any tumour before age 45 and (3) a ­second- (or first-) degree relative with cancer before age 45 or a sarcoma at any age. A modification of this definition designated Li–Fraumeni-like syndrome (LFL) is ­ defined by (1) a proband with any childhood tumour or a sarcoma, brain tumour or adrenocortical tumour under age 45; (2) a first- (or second-) degree relative with a typical LFS tumour at any age and (3) an additional first- (or second-) degree relative with any cancer under 60 years of age. Lastly, in order to guide which individuals should be referred for genetic testing, the Chompret criteria include (1) a proband with a tumour in the LFS spectrum before age 46 and at least one first- or second-degree relative with an LFS tumour (except breast cancer if the proband has breast cancer) before age 56 or with multiple tumours; (2) a proband with multiple tumours (except multiple breast tumours), two of which are in the LFS spectrum, the first of which occurred before age 46 or (3) a proband who is diagnosed with childhood ACC or choroid plexus tumour, irrespective of family history.71,118 In a review of 475 tumours in 91 reported families with TP53 germline mutations, breast carcinomas were most frequent, accounting for 24 per cent of all tumours, followed by bone sarcomas (12.6 per cent), brain tumours (12.0 per cent) and soft-tissue sarcomas (11.6 per cent).56 ACCs are uncommon (3.6 per cent) but, if occurring in children, are nearly pathognomonic of LFS. Given the increased risk of secondary malignancies, irradiation should be avoided if at all possible.71

Central Nervous System Manifestations Of a total of 475 tumours reported in families with TP53 germline mutations, 57 were located in the nervous system. Of those classified histopathologically, 69 per cent were astrocytic (diffuse astrocytoma, anaplastic astrocytoma and glioblastoma), followed by medulloblastomas and other CNS PNETs (17 per cent). This corresponds to the observation that in sporadic brain tumours, TP53 mutations occur preferentially in astrocytic tumours and less frequently in medulloblastomas. As in sporadic brain tumours, the age of patients with CNS tumours due to TP53 germline mutations shows a bimodal distribution, with a first peak in childhood and a second peak in the third to fourth decades.

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Molecular Genetics and Biology of Disease The highly conserved TP53 tumour suppressor gene, which maps to 17p13, encodes the p53 protein with five conserved domains, an N-terminal acidic domain and a C-terminal oligomerization domain. It binds in a sequencespecific manner to DNA acting as a transcriptional regulatory element. The biological functions of p53 are manifold and include growth arrest through suspension of cell-cycle progression from G1- to S-phase and induction of apoptosis as a response to DNA damage. An international IARC website has been created for cataloguing the large number of familial and sporadic TP53 mutations, which is updated annually (http://p53.iarc.fr). Germline TP53 mutations are detected in roughly 70 per cent of LFS, 40 per cent of LFL and 30 per cent of Chompret criteria positive individuals, the most common types being missense mutations (75 per cent), followed by small 1–4 bp deletions resulting in a frameshift (10 per cent).71,81 A similar spectrum of somatic mutations is encountered in sporadic neoplasms. Additionally, about 80 per cent of all paediatric ACC patients carry a germline TP53 mutation, most often involving mutations at codons 152 or 158.71 A number of genotype–phenotype associations exist. For instance, most LFS/LFL families with brain tumours have missense mutations in the DNA-binding loop that contacts the minor groove of DNA, whereas those with null phenotype mutations present with significantly earlier onset brain tumours.81 In contrast, LFS/LFL families lacking TP53 mutations rarely develop brain tumours. Lastly, families with mutations in the central core domain typically suffer more cancers and earlier onsets. Similar to somatic mutations, TP53 germline mutations are most commonly in exons 5–8, with clusters at codons 248, 273, 245, 175 and 182. However, as many as 27 per cent of germline and 14 per cent of somatic mutations fall outside these regions. Tumours often show a loss of the wild-type allele, consistent with the classic two-hit model. Of interest, LFS associated astrocytomas often develop a rare form of IDH1 gene mutation (R132C), in contrast to the common R132H mutation seen in sporadic counterparts.129 In LFS-associated medulloblastomas, CTNNB mutations and MYCN gene amplifications may be more common, although not many tumours have been studied to date.89

Cowden Disease Cowden disease (CD; or multiple hamartoma syndrome) is an autosomal dominant disorder featuring a wide range of benign and malignant tumours. Adult onset Lhermitte– Duclos disease (LDD; dysplastic gangliocytoma of the cerebellum) is now considered pathognomonic. Other ­ features include mucocutaneous lesions (facial trichilemmomas, acral keratoses, papillomatous papules, mucosal lesions), as well as major (macrocephaly (≥97th percentile); breast, thyroid and endometrial cancers), and minor (benign thyroid lesions, IQ ≤75, gastrointestinal (GI) hamartomas, fibrocystic breast disease, lipomas, fibromas, renal cell carcinoma or other genitourinary (GU) tumours, uterine leiomyomas, and GU malformations) diagnostic criteria.28,96 Colorectal carcinomas have been more recently

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reported in roughly 16 per cent of patients.96 Additionally, meningioma, pseudotumour cerebri, dural arteriovenous malformation, subcutaneous neuromas, ganglioneuromas, neurofibromas, oral neuromas, granular cell tumour, and myasthenia gravis have all been reported, although it is not entirely clear whether these associations are beyond chance.28 Roughly half are familial and half are sporadic. In the absence of a family history, classic mucocutaneous lesions, two major criteria, one major and three minor criteria, or four minor criteria are necessary for diagnosis. In patients with at least one family member involved, the diagnosis requires mucocutaneous lesions, one major criterion, two minor criteria, or a history of Bannayan–Riley–Ruvalcaba syndrome (BRRS). The cumulative risk of developing any cancer and/or LDD by age 60 is 56 per cent for men and 87 per cent for women;79 there is an 81 per cent lifetime risk for breast cancer in women and 32 per cent risk of LDD overall.96 In the CNS, LDD is the major manifestation. Most patients with LDD either eventually fulfill diagnostic features of CD or harbour at least one other characteristic feature. Interestingly, a subset with childhood onset LDD neither acquire other features nor have the typical germline mutations of CD. CD is caused by germline mutations in the PTEN (MMAC1/TEP1) gene on chromosome 10q23.2, evident in roughly 80 per cent of patients, with an additional 10 per cent showing mutations in the promoter region. Nearly 100 different point, nonsense, frame shift, splice site, missense and deletion/insertion germline mutations have been reported.28 Related disorders with similar germline mutations include BRRS (macrocephaly, lipomatosis and pigmented macules of the glans penis), proteus syndrome, proteus-like syndrome and CD-like syndrome (not fully meeting the diagnostic criteria of CD). PTEN encodes a molecule with phosphatase activity and homology to tensin and auxilin that functions in several cell signalling pathways, including the AKT-mTOR-S6K pathway involved in cell size regulation as also implicated in TSC. There are also at least two microRNA modifiers, miR-19a and miR-21 that may play a biologic role in determining phenotype.28 As a tumour suppressor, PTEN is widely involved in sporadic malignant tumours, including malignant gliomas. Notably, however, patients with CD are not predisposed to malignant gliomas, perhaps because PTEN inactivation is a progression-associated, rather than an early event in sporadic glioma tumourigenesis. Biallelic inactivation is suspected in LDD: the large dysplastic neurons in most examples lose PTEN immunoreactivity and strongly express downstream regulators, such as phospho-AKT and phospho-S6K.2 Interestingly, conditional Pten knockout mice develop cerebellar lesions virtually identical to human LDD and this phenotype is rescued using an mTOR inhibitor.60

Turcot Syndrome Also known as brain tumour-polyposis syndrome, Turcot syndrome (TS) is a rare autosomal dominant or recessive disease characterized by combined tumours of the colon and brain.46 The majority of patients have TS type 2, with colonic disease identical to familial polyposis (FAP). Accordingly,

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Gorlin-Goltz Syndrome  1939

these patients have germline defects in the APC gene on chromosome 5q21, which is implicated in FAP. In these kindreds, the predominant CNS lesions are medulloblastomas, which develop at an incidence nearly 100-fold higher than the general population.46 However, APC mutations and chromosomal 5q loss are rare in sporadic medulloblastomas.93 Rare pineoblastomas have also been reported.34 In contrast, a smaller number of TS type 1 families feature colonic disease similar to hereditary non-polyposis colorectal carcinoma (HNPCC), and have germline defects in DNA mismatch repair (MMR) genes (e.g. MLH1, PMS2), often yielding microsatellite instability (MSI) in associated neoplasms. In these families, glioblastoma is the primary CNS manifestation. The risk of gliomas appears greater in patients with MSH2 than with MLH1 mutations.68 In addition, PMS2 mutations are rare, but associated with a particularly severe phenotype, including gliomas. Studies suggest that the giant cell variant of glioblastoma and gliosarcomas may be overrepresented, but that associated survival times can be surprisingly long.69 Furthermore, patients with homozygous MMR gene mutations often display NF1-like manifestations (café-au-lait spots, axillary freckling, neurofibromas, tibial pseudoarthrosis), one potential explanation being coexistent NF1 mutations resulting from the global mismatch repair and genomic instability.4,68 These rare patients often die of childhood leukaemias or lymphomas, rather than brain tumours. Data also suggest a possible link between TS type 1 and MuirTorre syndrome, the latter similarly associated with germline MMR gene mutations.41,57 Finally, it is important to note that cases do not always fall neatly into the FAP or HNPCC phenotype, with rare families having both medulloblastomas and glioblastomas.

44

Gorlin-Goltz Syndrome This autosomal dominant condition, also referred to as naevoid basal cell carcinoma syndrome (NBCCS), has an estimated prevalence of up to 1 in 60 000.55 It is diagnosed by the presence of two major or one major and two minor criteria.38,54 Major criteria are ≥2 basal cell carcinomas or a basal cell carcinoma under 20 years of age, medulloblastoma, odontogenic keratocysts of the jaw, ≥3 palmar or plantar pits, lamellar calcification of the falx cerebri, characteristic rib anomalies, ovarian fibroma, phalangeal flame-shaped radiolucencies, brachymetacarpaly in all four limbs, and a first-degree relative with NBCCS. Minor features include spina bifida, brachymetacarpaly in at least one limb, hypertelorism or telcanthus, and frontal bossing. Additionally, neuroimaging studies have demonstrated increased frequencies of falx calcification (79 per cent), bridging of the sella (68 per cent), tentorial calcification (20 per cent), abnormal frontal sinus aeration (18 per cent), asymmetric or dilated ventricles (24 per cent), cavum septum pellucidum (19 per cent), cerebral atrophy (10 per cent), agenesis/dysgenesis of the corpus callosum (10 per cent) and meningiomas (5 per cent).54 Cystic mesenchymal hamartomas involving the skull are occasionally misinterpreted as metastatic medulloblastoma.38 Patients are particularly hypersensitive to therapeutic irradiation and may develop secondary carcinomas. Less commonly, other CNS tumours may be radiation-associated, possibly including meningioma, astrocytoma, oligodendroglioma, and

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1940  Chapter 44  Hereditary Tumour Syndromes

craniopharyngioma. As a result, cranial CT scans are discouraged. Roughly 2–5 per cent of NBCCS patients develop medulloblastoma and the male to female ratio is 3:1, with mean age of onset being 2 years, as opposed to 7 years in sporadic counterparts.54,55 The possibility of NBCCS should therefore be explored in any medulloblastoma patient under 5 years of age. The vast majority belong to the desmoplastic/nodular subtype and follow a more indolent course than conventional counterparts. For this reason and the hypersensitivities already discussed, irradiation should be avoided. As with other complex syndromes, multidisciplinary care is critical for optimal management.55 The gene responsible for NBCCS is the PTCH1 gene on chromosome 9q22.3, although 20–40 per cent represent de novo (sporadic) mutations.55 Germline mutations are detectable in the rest, most of which result in a truncated protein.38 The PTCH gene product functions in a growth signalling pathway that involves the sonic hedgehog (SHH) protein and the smoothened (SMO) protein. Accordingly, PTCH and SMO mutations have been detected in sporadic medulloblastomas; however, germline alterations leading to NBCCS have only been detected in the PTCH gene.

Rhabdoid Tumour Predisposition Syndrome This complex familial disorder is usually characterized by germline mutations of the INI1/hSNF5/BAF47/SMARCB1 gene on 22q11.2, predisposing patients to the formation of malignant rhabdoid tumours (MRT) of the kidney and soft tissue, as well as to atypical teratoid/rhabdoid tumours (AT/RT) and, to a lesser extent, schwannomas, meningiomas and chondrosarcomas later in life.7,13,14,30,107 In very rare examples, germline mutations for other SWI/SNF chromatin remodelling members are implicated, notably the SMARCA4/BRG1 gene on 19p13.2.108,133 Most AT/RTs occur in children under 4 years of age, roughly a third being associated with germline mutations and up to 60 per cent in those presenting during the first 6 months of life.13,14 Given these high rates of germline mutations, genetic screening is probably warranted in all newly diagnosed patients. Nevertheless, only a minority have a ­positive family history, with occasional parents being silent carriers. New mutations and gonadal mosaicism are thought to be the most likely explanations for this finding. Sporadic AT/RTs similarly demonstrate biallelic INI1 inactivation and loss of protein expression in the vast majority. In comparison, however, the familial forms present at earlier ages with more extensive disease and shorter survival times.13,14 Smarcb1 heterozygote mouse models develop rhabdoid tumours in 5–35 per cent, as well as other often highly aggressive tumour types.90 Lastly, rare families with both rhabdoid tumour predisposition syndrome (RPS) and schwannomatosis have been reported, providing some link between these two disorders.116

Carney Complex This autosomal dominant disorder is considered a form of multiple endocrine neoplasia (MEN) in that two or more endocrine tumours are often present. However, it

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is considerably more complex with additional manifestations. Approximately 70 per cent of cases are familial.100 The diagnosis requires at least two of the following: spotty pigmentation (70–75 per cent), cutaneous or mucosal myxomas, ­cardiac myxoma (potentially fatal), fibromyxoma of the breast, Cushing syndrome associated with primary pigmented nodular adrenocortical dysplasia (25–30 per cent), acromegaly or gigantism from a growth hormone adenoma (10 per cent), large cell calcifying Sertoli cell tumour (often bilateral; 50 per cent of men), thyroid carcinoma, psammomatous melanotic schwannoma (PMS in 8 per cent; potentially malignant), blue naevi (especially epthelioid), ductal breast adenoma, osteochondromyxoma, an affected first-degree relative or a mutation of the PRKAR1A gene. Neuropathological features include psammomatous melanotic schwannoma (10 per cent) (Figure 44.4) and c­ erebral infarcts associated with emboli from cardiac myxomas. Additionally, pituitary abnormalities are relatively frequent and recent data suggest that somatomammotroph hyperplasia precedes growth hormone adenomas.115 The PRKAR1A tumour suppressor gene (representing the CNC1 locus) maps to chromosome 17q24.2 and encodes the R1α regulatory subunit (RI-A) of protein kinase A, the main mediator of cyclic AMP signalling. Over 100 mutations in the RI-A subunit have been detected, mostly leading to R1A haploinsufficiency and involving about 60 per cent of CNC patients.101 Most mutations are base substitutions, small deletions/insertions, or rearrangements and are either patient or family specific.100 PRKAR1A mutations are found in 80 per cent of familial and 37 per cent of sporadic CNC patients. Those with mutations present earlier and more often have myxomas, skin lesions, thyroid and gonadal tumours. Exonic mutations are more often associated with acromegaly, cardiac myxoma, lentigines and PMS. Important downstream tumourigenic mediators involve Wnt signalling and cell cycle dysregulation.101 Other disease-causing genes have yet to be identified, including a CNC2 locus on chromosome 2p16.

DICER1 Syndrome Also known as familial pleuropulmonaryblastoma (PPB) tumour predisposition, this syndrome is associated with the development of multiple developmental and neoplastic lesions, especially PPB, cystic nephroma (CN), ovarian SertoliLeydig cell tumour and multinodular goiters.31 Congenital malformations such as pulmonary sequestration and transposition of the great arteries (TGA) are also described rarely. Nonetheless, the spectrum of associated alterations continues to expand, including occasional intracranial manifestations, such as CNS primitive neuroectodermal tumour (PNET), pineoblastoma and pituitary blastoma, the latter typically presenting as Cushing disease in infants under 2 years of age with a histologically distinctive pituitary mass characterized by proliferation of developmentally arrested corticotroph cells.16,31,106,110 Over 40 different germline mutations have been identified in the DICER1 gene on 14q32.13. Despite the germline alteration, family history is often negative or inconclusive.107 The DICER1 gene transcribes a member of the RNaseIII family, cleaving precursor molecules into small mature double-stranded noncoding RNAs (miRNAs)

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References  1941

(a)

(b)

(c)

(d)

44

44.4 Psammomatous melanotic schwannoma in a patient with Carney complex. Like ordinary schwannomas, Verocay bodies were seen focally (a). Unlike conventional schwannomas, there was melanin pigment (b–d), psammoma bodies (b, arrows), lipidization (c) and foci showing malignant cytologic features (d).

that regulate gene expression in a wide variety of pathways, particularly during early development. Although homozygous knockout is embryologically lethal, hemizygous knockout animal models yield phenotypes resembling the human counterpart.

Other Hereditary Brain Tumour Syndromes Pituitary adenomas are common endocrine neoplasms that may be seen in a number of hereditary syndromes,12,48,80 including some that have already been discussed (see Chapter 41). In rare examples, patients with familial retinoblastoma develop pineoblastomas or other central PNETs

(‘trilateral retinoblastoma’) associated with inactivation of the RB gene.91 Patients with one form of progeria, Werner’s syndrome, have been reported to have a slightly higher likelihood of developing meningiomas. Also, a prostate and brain cancer susceptibility gene (CAPB) has been localized to chromosome 1p36.125 A number of families have been described with isolated predisposition to brain tumours, particularly malignant gliomas, or with predispositions to brain and systemic neoplasms that do not fall clearly into a known syndrome.126 Some of these are associated with specific high-risk single nucleotide polymorphisms.37,65 Rare CDKN2A germline mutations have also been described.117 Lastly, familial meningiomas have been described outside the setting of NF2 or schwannomatosis; rare associations have included germline mutations of SUFU and SMARCE4.1,112

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with the formation of malignant neurofibrosarcomas in von Recklinghausen neurofibromatosis. Proc Natl Acad Sci U S A 1990;87:5435–9. Merker VL, Esparza S, Smith MJ, Stemmer-Rachamimov A, Plotkin SR. Clinical features of schwannomatosis: a retrospective analysis of 87 patients. Oncologist 2012;17:1317–22. Mo W, Chen J, Patel A, et al. CXCR4/ CXCL12 mediate autocrine cell-cycle progression in NF1-associated malignant peripheral nerve sheath tumors. Cell 2013;152:1077–90. Napolioni V, Moavero R, Curatolo P. Recent advances in neurobiology of tuberous sclerosis complex. Brain Dev 2009;31:104–13. Ng HK, Tse JY, Poon WS. Cerebellar astrocytoma associated with von HippelLindau disease: case report with molecular findings. Br J Neurosurg 1999;13:504–7. Nieuwenhuis MH, Kets CM, MurphyRyan M, et al. Cancer risk and genotypephenotype correlations in PTEN hamartoma tumor syndrome. Fam Cancer 2014;13:57–63. Nunes VS, Souza GL, Perone D, Conde SJ, Nogueira CR. Frequency of multiple endocrine neoplasia type 1 in a group of patients with pituitary adenoma: genetic study and familial screening. Pituitary 2014;17:30–7. Olivier M, Goldgar DE, Sodha N, et al. Li-Fraumeni and related syndromes: correlation between tumor type, family structure, and TP53 genotype. Cancer Res 2003;63:6643–50. Orlova KA, Crino PB. The tuberous sclerosis complex. Ann N Y Acad Sci 2010;1184:87–105. Panelos J, Beltrami G, Capanna R, Franchi A. Primary capillary hemangioblastoma of bone: report of a case arising in the sacrum. Int J Surg Pathol 2010;18:580–3. Pascual-Castroviejo I. Neurosurgical treatment of tuberous sclerosis complex lesions. Childs Nerv Syst 2011;27:1211–9. Patil S, Chamberlain RS. Neoplasms associated with germline and somatic NF1 gene mutations. Oncologist Review 2012;17:101–16. Patil S, Perry A, Maccollin M, et al. Immunohistochemical analysis supports a role for INI1/SMARCB1 in hereditary forms of schwannomas, but not in solitary, sporadic schwannomas. Brain Pathol 2008;18:517–9. Perry A, Giannini C, Raghavan R, et al. Aggressive phenotypic and genotypic features in pediatric and NF2-associated meningiomas: a clinicopathologic study of 53 cases. J Neuropathol Exp Neurol 2001;60:994–1003. Perry A, Kurtkaya-Yapicier O, Scheithauer BW, et al. Insights into meningioangiomatosis with and without meningioma: a clinicopathologic and genetic series of 24 cases with review of the literature. Brain Pathol 2005;15:55–65. Pfaff E, Remke M, Sturm D, et al. TP53 mutation is frequently associated with CTNNB1 mutation or MYCN amplification and is compatible with longterm survival in medulloblastoma. J Clin Oncol 2010;28:5188–96. Plotkin SR, Blakeley JO, Evans DG, et al. Update from the 2011 International Schwannomatosis Workshop: From

genetics to diagnostic criteria. Am J Med Genet A 2013;161:405–16. 91. Plowman PN, Pizer B, Kingston JE. Pineal parenchymal tumours: II. On the aggressive behaviour of pineoblastoma in patients with an inherited mutation of the RB1 gene. Clin Oncol (R Coll Radiol) 2004;16:244–7. 92. Probst A, Lotz M, Heitz P. Von HippelLindau’s disease, syringomyelia and multiple endocrine tumors: a complex neuroendocrinopathy. Virchows Arch A Pathol Anat Histol 1978;378:265–72. 93. Raffel C. Medulloblastoma: molecular genetics and animal models. Neoplasia 2004;6:310–22. 94. Reith RM, McKenna J, Wu H, et al. Loss of Tsc2 in Purkinje cells is associated with autistic-like behavior in a mouse model of tuberous sclerosis complex. Neurobiol Dis 2012;51:93–103. 95. Riccardi V. Neurofibromatosis: phenotype, natural history and pathogenesis, 2nd edn. Baltimore, MD: Johns Hopkins University Press, 1992. 96. Riegert-Johnson DL, Gleeson FC, Roberts M, et al. Cancer and Lhermitte–Duclos disease are common in Cowden syndrome patients. Hered Cancer Clin Pract 2010;8:6. 97. Roach ES, DiMario FJ, Kandt RS, Northrup H. Tuberous Sclerosis Consensus Conference: recommendations for diagnostic evaluation. National Tuberous Sclerosis Association. J Child Neurol 1999;14:401–7. 98. Rodriguez FJ, Scheithauer BW, George D, et al. Superficial neurofibromas in the setting of schwannomatosis: nosologic implications. Acta Neuropathol 2011;121:663–8. 99. Rodriguez FJ, Folpe AL, Giannini C, Perry A. Pathology of peripheral nerve sheath tumors: diagnostic overview and update on selected diagnostic problems. Acta Neuropathol 2012;123:295–319. 100. Rodriguez FJ, Stratakis CA, Evans DG. Genetic predisposition to peripheral nerve neoplasia: diagnostic criteria and pathogenesis of neurofibromatoses, Carney complex, and related syndromes. Acta Neuropathol 2012;123:349–67. 101. Rothenbuhler A, Stratakis CA. Clinical and molecular genetics of Carney complex. Best Pract Res Clin Endocrinol Metab 2010;24:389–99. 102. Rubin JB, Gutmann DH. Neurofibromatosis type 1 – a model for nervous system tumour formation? Nat Rev Cancer 2005;5:557–64. 103. Rubinstein LJ. Pathological features of optic nerve and chiasmatic gliomas. Neurofibromatosis 1988;1:152–8. 104. Ruggieri M. The different forms of neurofibromatosis. Childs Nerv Syst 1999;15:295–308. 105. Ruggieri M, Iannetti P, Polizzi A, et al. Earliest clinical manifestations and natural history of neurofibromatosis type 2 (NF2) in childhood: a study of 24 patients. Neuropediatrics 2005;36:21–34. 106. Sabbaghian N, Hamel N, Srivastava A, et al. Germline DICER1 mutation and associated loss of heterozygosity in a pineoblastoma. J Med Genet 2012;49:417–9. 107. Schiffman JD, Geller JI, Mundt E, et al. Update on pediatric cancer predisposition syndromes. Pediatr Blood Cancer 2013;60:1247–52.

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1944  Chapter 44  Hereditary Tumour Syndromes 108. Schneppenheim R, Fruhwald MC, Gesk S, et al. Germline nonsense mutation and somatic inactivation of SMARCA4/ BRG1 in a family with rhabdoid tumor predisposition syndrome. Am J Hum Genet 2010;86:279–84. 109. Schulz A, Baader SL, Niwa-Kawakita M, et al. Merlin isoform 2 in neurofibromatosis type 2-associated polyneuropathy. Nat Neurosci 2013;16:426–33. 110. Slade I, Bacchelli C, Davies H, et al. DICER1 syndrome: clarifying the diagnosis, clinical features and management implications of a pleiotropic tumour predisposition syndrome. J Med Genet 2011;48:273–8. 111. Smith MJ, Kulkarni A, Rustad C, et al. Vestibular schwannomas occur in schwannomatosis and should not be considered an exclusion criterion for clinical diagnosis. Am J Med Genet A. 2012;158A:215–9. 112. Smith MJ, O’Sullivan J, Bhaskar SS, et al. Loss-of-function mutations in SMARCE1 cause an inherited disorder of multiple spinal meningiomas. Nat Genet 2013;45:295–8. 113. Stemmer-Rachamimov AO, Ino Y, Lim ZY, et al. Loss of the NF2 gene and merlin occur by the tumorlet stage of schwannoma development in neurofibromatosis 2. J Neuropathol Exp Neurol 1998;57:1164–7. 114. Stemmer-Rachamimov AO, Louis DN, Nielsen GP, et al. Comparative pathology of nerve sheath tumors in mouse models and humans. Cancer Res 2004;64: 3718–24. 115. Stergiopoulos SG, Abu-Asab MS, Tsokos M, Stratakis CA. Pituitary pathology in Carney complex patients. Pituitary 2004;7:73–82. 116. Swensen JJ, Keyser J, Coffin CM et al. Familial occurrence of schwannomas and malignant rhabdoid tumour associated with a duplication in SMARCB1. J Med Genet 2009;46:68–72. 117. Tachibana I, Smith JS, Sato K, et al. Investigation of germline PTEN, p53, p16(INK4A)/p14(ARF), and CDK4 alterations in familial glioma. Am J Med Genet 2000;92:136–41. 118. Tinat J, Bougeard G, Baert-Desurmont S, et al. 2009 version of the Chompret criteria for Li Fraumeni syndrome.

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J Clin Oncol 2009;27:e108–9; author reply e10. 118a. Tishler PV. A family with coexistent von Recklinghausen’s neurofibromatosis and von Hippel-Lindau’s disease. Diseases possibly derived from a common gene. Neurology 1975 Sep;25(9):840–4. 119. Tognini G, Ferrozzi F, Garlaschi G, et al. Brain apparent diffusion coefficient evaluation in pediatric patients with neurofibromatosis type 1. J Comput Assist Tomogr 2005;29:298–304. 120. Tsirikos AI, Saifuddin A, Noordeen MH. Spinal deformity in neurofibromatosis type-1: diagnosis and treatment. Eur Spine J 2005;14:427–39. 121. Ueki K, Sasaki T, Ishida T, Kirino T. Spinal tanycytic ependymoma associated with neurofibromatosis type 2 – case report. Neurol Med Chir (Tokyo) 2001;41:513–6. 122. Uhlmann EJ, Wong M, Baldwin RL, et al. Astrocyte-specific TSC1 conditional knockout mice exhibit abnormal neuronal organization and seizures. Ann Neurol 2002;52:285–96. 123. Ullrich NJ, Raja AI, Irons MB, Kieran MW, Goumnerova L. Brainstem lesions in neurofibromatosis type 1. Neurosurgery 2007;61:762–6; discussion 6–7. 124. van den Munckhof P, Christiaans I, Kenter SB, Baas F, Hulsebos TJ. Germline SMARCB1 mutation predisposes to multiple meningiomas and schwannomas with preferential location of cranial meningiomas at the falx cerebri. Neurogenetics 2012;13:1–7. 125. Verhage BA, Aben KK, Witjes JA, et al. Site-specific familial aggregation of prostate cancer. Int J Cancer 2004;109:611–7. 126. von Koch CS, Gulati M, Aldape K, Berger MS. Familial medulloblastoma: case report of one family and review of the literature. Neurosurgery 2002;51:227–33; discussion 33. 127. Vortmeyer AO, Falke EA, Glasker S, Li J, Oldfield EH. Nervous system involvement in von Hippel-Lindau disease: pathology and mechanisms. Acta Neuropathol 2013;125:333–50. 128. Wallace AJ, Watson CJ, Oward E, Evans DG, Elles RG. Mutation scanning of the NF2 gene: an improved service based on meta-PCR/sequencing, dosage analysis,

and loss of heterozygosity analysis. Genet Test 2004;8:368–80. 129. Watanabe T, Vital A, Nobusawa S, Kleihues P, Ohgaki H. Selective acquisition of IDH1 R132C mutations in astrocytomas associated with LiFraumeni syndrome. Acta Neuropathol 2009;117:653–6. 130. Wechsler J, Lantieri L, Zeller J, et al. Aberrant axon neurofilaments in schwannomas associated with phacomatoses. Virchows Arch 2003;443:768–73. 131. Wheeler PG, Sadeghi-Nejad A. Simultaneous occurrence of neurofibromatosis type 1 and tuberous sclerosis in a young girl. Am J Med Genet A 2005;133:78–81. 132. Wiestler OD, von Siebenthal K, Schmitt HP, Feiden W, Kleihues P. Distribution and immunoreactivity of cerebral micro-hamartomas in bilateral acoustic neurofibromatosis (neurofibromatosis 2). Acta Neuropathol (Berl) 1989;79:137–43. 133. Witkowski L, Lalonde E, Zhang J, et al. Familial rhabdoid tumour ‘avant la lettre’ – from pathology review to exome sequencing and back again. J Pathol 2013;231:35–43. 134. Woodruff JM. Pathology of tumors of the peripheral nerve sheath in type 1 neurofibromatosis. Am J Med Genet 1999;89:23–30. 135. Woodruff JM, Scheithauer BW, KurtkayaYapicier O, et al. Congenital and childhood plexiform (multinodular) cellular schwannoma: a troublesome mimic of malignant peripheral nerve sheath tumor. Am J Surg Pathol 2003;27:1321–9. 136. Wu J, Patmore DM, Jousma E, et al. EGFR-STAT3 signaling promotes formation of malignant peripheral nerve sheath tumors. Oncogene 2014;33:173–80. 137. Zhu Y, Ghosh P, Charnay P, Burns DK, Parada LF. Neurofibromas in NF1: Schwann cell origin and role of tumor environment. Science 2002;296:920–2. 138. Zoller ME, Rembeck B, Oden A, Samuelsson M, Angervall L. Malignant and benign tumors in patients with neurofibromatosis type 1 in a defined Swedish population. Cancer 1997;79:2125–31.

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  Syndromes Associated with Antibodies to Intracellular Neuronal Antigens  1945

45 45 Chapter

Paraneoplastic Syndromes Marc K Rosenblum

Introduction..............................................................................1945 Syndromes Associated with Antibodies to Intracellular Neuronal Antigens...................................................................................1945

Introduction ‘Paraneoplastic’ is a designation reserved for tumour-associated neurologic disorders that cannot be ascribed to compression or infiltration of the nervous system by tumour cells or attributed to the metabolic derangements, unwanted effects of therapy, disturbances of coagulation or opportunistic infections that potentially complicate the course and management of systemic neoplasia. Though rare, paraneoplastic phenomena compel attention because these frequently constitute the first manifestations of otherwise occult tumours. Paraneoplasia, furthermore, looms large in the differential diagnosis of certain symptom complexes that, in turn, have come to be associated with offending cancers of relatively restricted types. Thus, 60–70 per cent of patients developing the Lambert–Eaton myasthenic syndrome harbour small-cell carcinomas of the lung,13 whereas over 50 per cent of women presenting with subacute pancerebellar dysfunction will be found to have adenocarcinomas of mullerian or mammary duct origin.66 Paraneoplastic injury may affect any division of the central or peripheral (including autonomic) neuraxis and often proves a greater threat to the patient than its inciting tumour, which is often relatively confined on discovery and which may remain in abeyance even as neurological symptoms progress to devastating disability. Investigations conducted over the last several decades have demonstrated that many paraneoplastic neurological disorders are attributable to an immune attack, provoked by the tumoural expression of native neuronal antigens, that comes to be misdirected against the nervous system.58 Surveyed here are the more prevalent and best characterized of this autoimmune group, the members of which have come to be defined by their association with antibodies to specific ‘onconeural’ antigens. These antibodies can be divided into two broad classes depending on whether the target is an intracellular or cell membrane-associated/ extracellular epitope. Antibodies of the first type are more tightly correlated with underlying neoplastic disease, but the evidence amassed to date indicates that these do not

Syndromes Associated with Antibodies to Synaptic and Other Cell Membrane–Associated Neuronal Proteins..........................1950 References...............................................................................1951

suffice to cause nervous system injury. Antibody-depleting strategies are typically of no benefit to affected patients, as both experimental and neuropathological studies (reviewed later) implicate cell-mediated cytotoxic mechanisms. On the other hand, both favourable responses to antibody depletion and experimental models implicate autoantibodies to cell membrane-associated and extracellular neuronal antigens as directly pathogenic agents in nervous system injury.

Syndromes Associated with Antibodies to Intracellular Neuronal Antigens Hu Antigen The most prevalent of paraneoplastic neurological disorders in the intracellular neuronal antigen group is a syndrome of potentially widespread injury to the central and peripheral neuraxes associated with high-titre anti-Hu20,39 (a.k.a. antineuronal nuclear autoantibody type 1)54 antibodies. The offending tumour in over 75 per cent of cases is a smallcell carcinoma of the lung, the most common presenting manifestation (and dominant clinical feature in many cases) being peripheral sensory loss that involves all modalities and progresses inexorably to crippling deafferentation over a few weeks or months. Some 70–80 per cent of patients develop evidence of central nervous system (CNS) injury as well, this often being multifocal and including (singly or in any combination) bulbar and cerebellar dysfunction, limbic encephalopathy (seizures, disturbances of cognition, affect and short-term memory) and myelopathy (particularly a paralyzing lower motor neuron syndrome). Autonomic damage may produce gastrointestinal pseudo-obstruction, urinary retention, impotence, severe orthostatic hypotension and life-threatening cardiac arrhythmias. Treatment of the underlying neoplasm, plasmapheresis and immunosuppressive regimens usually fail to effect neurologic improvement. 1945

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1946  Chapter 45  Paraneoplastic Syndromes

Predominantly of IgG1 subtype and found in both the serum and cerebrospinal fluid (CSF) of symptomatic patients, anti-Hu antibodies identify a family of highly conserved RNA-binding proteins that are involved in the post-transcriptional regulation of gene expression and that are integral to neuronal differentiation and maintenance.41 In addition to labelling the nuclei and, to a lesser extent,

perikarya of neurons throughout the central and peripheral nervous systems,4,19 anti-Hu antibodies consistently label small-cell carcinomas of the lung (including those unassociated with paraneoplastic disease) – a phenomenon reflecting their ubiquitous expression of the major immunogen, HuD, in non-mutated form (Figure 45.1a–c).15,56 The triggering mechanism of injurious immune reactions

(c)

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SCLC tumour

200– 92– 69– 46–

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30– 21– 14–

1 N

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45.1 Paraneoplastic sensory neuropathy/encephalomyelitis. (a) Expression of Hu antigen in central nervous system and smallcell lung carcinomas. Western blot study demonstrating bands in the 35–40 kDa region on assay of purified cortical neuronal protein preparations (lanes 2 and 3) or small-cell lung cancer extracts (lanes 5 and 6) against anti-Hu IgG from patients with paraneoplastic sensory neuropathy/encephalomyelitis. Normal human IgG (lanes 1 and 4) does not yield such bands. (b) Hu expression is concentrated in neuronal nuclei, as shown in this immunohistochemical study of normal human cerebral cortex. Perikaryal labelling is also apparent. (Immunohistochemistry for Fab GLN 495 recombinant anti-Hu.) (c) Small-cell carcinoma of the lung showing anti-Hu immunoreactivity. (Immunohistochemistry for Fab GLN 495 recombinant anti-Hu.) (d) Paraneoplastic sensory neuropathy. Note the dorsal root ganglion cell surrounded by small lymphocytes. A residual nodule of Nageotte marks the adjacent ganglion cell bed. (e) The selective pallor of the dorsal columns demonstrated in these spinal cord sections reflects advanced dorsal root ganglion cell loss with secondary degeneration of ascending sensory fibres.

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  Syndromes Associated with Antibodies to Intracellular Neuronal Antigens  1947

in affected patients is unclear, with only 15–20 per cent of subjects with small-cell lung cancers developing Hu seropositivity and only a fraction of these suffering paraneoplastic consequences.18,36 Studies have found HuD-specific CD8+ T-lymphocytes to be normal components of the T-cell repertoire in mice, which display a high level of immune tolerance to this antigen.29 A patient in the subacute phase of the anti-Hu syndrome, on the other hand, was found to harbour circulating HuD-specific T-cells of classic, CD8+ cytotoxic type that could not be detected in neurologically normal controls.73 Two chronically impaired patients in this study had, instead, HuD-specific ‘type 2’ CD8+ lymphocytes sharing properties of CD4+ type (Th2) helper cells. Having attenuated cytolytic capacity, T-cells of this kind were speculated to downregulate cytotoxic T-cell activity following the initial nervous system assault and to possibly augment anti-Hu IgG production. Production of antibody alone does not suffice to cause neurological disease in the experimental setting.79 The clinical manifestations of the anti-Hu syndrome reflect an inflammatory attack on neurons.7,20,45 Lymphocytes flood the dorsal root ganglia of patients with sensory neuropathy, surrounding ganglion cells and sometimes appearing to invade their degenerating or necrotic perikarya (Figure 45.1d). Nests of reactive satellite cells known as nodules of Nageotte come to mark, in tombstone-like fashion, the positions formerly occupied by ganglion cells, the end stage being a ‘burned out’ ganglion devoid of neuronal elements but deceptively free of inflammatory invaders. The consequences of advanced ganglion cell extinction may be appreciated at autopsy as atrophy of the posterior spinal roots and pallor of the dorsal columns of the spinal cord (Figure 45.1e). A comparable sequence of events is observed in affected myenteric plexi and CNS, where lymphocytes cuff regional blood vessels, migrate into the neuropil, converge on neurons and participate, along with histiocytes, in the formation of microglial or ‘neuronophagic’ nodules. Neuronal loss, highly variable in extent, is typically accompanied by striking astrogliosis. Immunopathological analyses support a primary role for cytotoxic T-cells in anti-Hu-associated neuronal damage.7,45 Although B- and T- (predominantly CD4+) lymphocytes cuff intraparenchymal blood vessels, the lymphoid elements that infiltrate the neuropil and surround neurons are principally CD8+ T-cells that have been shown to express the TIA-1 component of cytotoxic granules.7 Upregulated regional expression of the ICAM-1 intercellular adhesion molecule may contribute to a pro-inflammatory environment and augment lymphocyte-neuron interactions in dorsal root ganglia and the CNS of affected patients.7 This cited study could not marshal evidence for antibody-mediated complement activation. Of note, the Hu-expressing neoplasms associated with paraneoplastic sensory neuropathy and encephalomyelitis are often occult and localized. Neurological complaints precede the diagnosis of cancer in over 70 per cent of patients, most of whom have small-cell lung carcinomas that not only are limited to the thorax upon discovery but often remain so through the course of their illnesses.20,39,54 A majority die of neurological complications. This may not reflect early tumour detection alone, as an unexpectedly low incidence of extrathoracic metastasis, enhanced treatment

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responsiveness and improved survival seem to characterize small-cell lung cancers deriving from a subset of patients having low-titre anti-Hu seropositivity unattended by paraneoplastic phenomena.18,36 ‘Spontaneous’ regression of small-cell lung carcinoma in the setting of Hu-seropositive paraneoplastic disease has been documented,26 as has growth inhibition of Hu-expressing neuroblastoma cells in mice following vaccination with HuD-encoding DNA.14 The presence within syndrome-associated tumours of lymphocytes that include recombinant HuD-binding elements83 further suggests that immune reflexes, while wreaking havoc on the nervous system, serve to check the growth and spread of these neoplasms.

45

Nova Proteins/Ri Antigen High-titre anti-Ri55 or antineuronal nuclear autoantibody type 253 antibodies are associated with an encephalomyelitic syndrome occurring mainly in patients with small-cell carcinomas of the lung and mammary adenocarcinomas.55,68,70 As additional autoantibodies to other onconeural antigens (e.g. Hu, CRMP-5, calcium channel proteins, etc.) may be found, a wide variety of neurologic manifestations can be encountered in anti-Ri seropositive patients. Brain stem and cerebellar involvement are particularly common, the most distinctive features of the anti-Ri syndrome including a relatively high frequency of opsoclonus, myoclonus, jaw dystonia and laryngospasm. Anti-Ri antibodies identify RNA-binding 55 and 80kDa proteins, termed Nova 1 and Nova 2, that regulate premRNA alternative splicing in neurons25 and that are coexpressed in tumour cells and the nuclei of neuronal subpopulations restricted to the CNS.35 Autopsy studies have demonstrated that anti-Ri-associated neurologic dysfunction reflects a potentially widespread encephalomyelitic process in which the cerebellum, brain stem and spinal cord are targets of particularly severe injury.10,42,70,71 Purkinje cell loss, destruction of bulbar (especially pontine) and spinal cord neurons, ventrolateral spinal tract degenerations, accompanying astrogliosis, microglial nodule formation and lymphocytic infiltrates composed principally of CD8+ cytotoxic/suppressor T-cells with cuffing of regional blood vessels by mixed T- and B-cell populations have all been documented. Ocular movement abnormalities may specifically reflect involvement of the pontine paramedian reticular formation.42,70 Complement deposition and natural killer cell infiltration were identified in one case.42 The absence of conspicuous inflammatory change in some affected regions of the CNS may simply reflect disease chronicity.

Collapsin Response-Mediator Protein-5 IgG antibodies to the 62kDa collapsin-response mediatorprotein-5 (CRMP-5), member of a protein family that mediates axonal guidance and axon–Schwann cell interactions12 among other functions, define a paraneoplastic neurologic disorder associated with small-cell carcinomas of the lung (over 75 per cent of cases) and occasionally with thymomas and tumours of other types.17,46,94 The antigen is expressed widely within the central and peripheral neuraxes, localizing to synapse-rich neuropil and some neuronal perikarya, as well as the retina, where expression is found by

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1948  Chapter 45  Paraneoplastic Syndromes

photoreceptor and ganglion cell elements.17 Clinical manifestations commonly include peripheral and autonomic neuropathy, cerebellar dysfunction, cognitive impairment and cranial neuropathy (particularly loss of olfaction and taste as well as visual loss due to optic neuritis and retinitis).17 Chorea and other movement abnormalities referable to basal ganglionic injury may be encountered in this setting94 as may progressive myelopathy.46 Autopsy examination of an affected patient with encephalomyelopathy, optic neuritis and retinitis demonstrated lymphoid infiltrates of predominantly CD8+ T-cell type within the optic nerves in association with nerve fibre and myelin loss.17 Microglial activation, isolated microglial nodules and perivascular lymphoid cuffing (again principally by CD8+ T-cells) were also widely distributed in the CNS, being most prominent in the mesial temporal lobes, brain stem, cerebellum and spinal cord. Spinal roots, dorsal root ganglia and peripheral nerves exhibited mild loss of myelinated axons.

Cdr2 Protein/Yo Antigen High serum and CSF titres of an IgG antibody initially designated anti-Yo66 and subsequently termed anti-Purkinje cell cytoplasmic antibody type 153 are strongly associated with a subacutely evolving and relentlessly progressive syndrome of paraneoplastic cerebellar dysfunction.60,66 Evidence of bulbar, spinal cord and peripheral nervous system injury may also be encountered in this setting. Over 95 per cent of affected patients are women, the great majority of whom harbour adenocarcinomas of mullerian (usually ovarian) or mammary origin. Neurologic complaints typically precede the identification of these neoplasms, which tend to be relatively confined on discovery and which may be minute.40,66 Treatment of the underlying tumour and immunosuppressive therapy benefit only a small minority of patients and many succumb to complications of nervous system damage rather than progression of neoplastic disease.60,66,74 Anti-Yo antibodies characteristically yield a major band at 52 or 62 kDa and minor band at 34 kDa on Western blotting against purified Purkinje cell proteins (Figure 45.2a), immunohistochemical preparations demonstrating perikaryal distribution of the antigen in Purkinje cells (Figure 45.2b). Yo expression by select neuronal subpopulations in the human cerebral cortex, brain stem, spinal cord, dorsal root ganglia and autonomic plexi has been demonstrated.1,4,56 Shared cytoplasmic expression of the immunogen – a 52 kDa leucine zipper protein that is designated cdr2 and that displays c-Myc binding with resultant inhibition of c-Myc transcriptional activity63 – is a constant feature of syndrome-associated cancers, but has also been detected in mammary and ovarian carcinomas unattended by neurologic dysfunction.27 It has been suggested that Her 2 overexpression, a regular feature of associated breast cancers, acts to facilitate immune responses to cdr2.75 Anti-Yo antibodies have not been shown to cause nervous system injury. Although these could, in theory, interfere with the down-regulatory binding of cdr2 to c-Myc, resulting in excess c-Myc activation and consequent neuronal apoptosis,63 anti-Yo IgG does not suffice to produce neurologic disease in animals34,77,84-86 and is occasionally demonstrable in cancer patients without neurologic manifestations.30 The detection of Yo peptide/cdr2–specific cytotoxic

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T-lymphocytes in the blood of affected subjects3,87 and recovery of activated T-cells from their CSF2 support a role for cell-mediated immune responses in this disorder. In vitro data indicate that dendritic cells bearing apoptotic tumour cell fragments to lymph nodes could initiate cdr2specific T-cell activation.72 At autopsy, the hallmark of anti-Yo-associated cer­ ebellar dysfunction is Purkinje cell loss that affects all portions of the cerebellum, which is severe and may be total. (Figure 45.2c).1,60,66,81,91 Variable thinning of the granule cell layer may be encountered and reactive Bergmann astrogliosis is the rule. The appearance in most cases is that of a non-inflammatory degeneration but this likely reflects the end-stage, ‘burned out’ nature of the process. In some cases, microglial nodules have been identified in the Purkinje cell layer1,81 and in one example cytotoxic T-lymphocytes with polarized granzyme B-labelling granules were noted to surround residual, MHC class I-expressing Purkinje cells.1 Bulbar inflammation is not uncommonly found, even in the absence of brain stem signs, and cytotoxic T-cell infiltrates may be widely distributed in the brain, spinal cord and dorsal root ganglia.1,60 The gracile, cuneate, spinocerebellar and corticospinal tracts may exhibit pronounced rarefaction, vacuolar change and myelin loss.81,91 Carcinomas provoking anti-Yo-associated neurological injury are typically infiltrated by lymphocytes in large numbers, these including cytotoxic T-cells,1 and may also harbour prominent plasmacellular infiltrates.66,91 Thus, immune mechanisms may act to restrain their growth.

Ma Antigens Paraneoplastic syndromes having prominent components of limbic and bulbar encephalitis, hypothalamic dysfunction and cerebellar injury are associated with antibodies to the Ma (also termed paraneoplastic Ma (PNMA)) protein family.21,76,92 Patients seropositive against Ma2 (42 kDa) alone are usually men with underlying testicular germ cell tumours, whereas women with carcinomas of varying origin (including breast, lung, colon and ovary) predominate among a group of patients having antibodies to Ma1 (40 kDa) as well.76 Neurological responses to anti-cancer therapy and immunosuppression may be seen in the former group. Ma1 and Ma2 are co-expressed by syndromeassociated neoplasms and neurons throughout the nervous system, Ma1 expression also being a feature of testicular germ cells.21 Applied to tissue sections of CNS, patient sera yield neuron-specific immunolabelling in a dot-like pattern that is predominantly nuclear and that includes labelling of nucleoli.76 Speculations regarding Ma protein function include roles in RNA transcription76 and apoptosis.78 As has been the case in other paraneoplastic neurological disorders associated with immune responses to intracellular antigens, biopsy and autopsy studies of Ma-seropositive patients have consistently demonstrated infiltration of clinically affected CNS structures by lymphoid elements with variable degrees of microglial nodule formation, gliosis and neuronal loss (the last especially prominent at brain stem and cerebellar levels in post-mortem specimens).5,21,82,92 Immunophenotyping of the lymphoid infiltrates observed has shown these to be composed overwhelmingly of T-cells and to be heavily dominated by CD8+ cytotoxic

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  Syndromes Associated with Antibodies to Intracellular Neuronal Antigens  1949 (a)

(b)

45

68 CDR 62

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CDR 34 30 21

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45.2 Paraneoplastic cerebellar degeneration. (a) Western blot demonstrating 62 and 34 kDa bands apparent when anti-Yo IgG from an afflicted patient is applied to purified Purkinje cell proteins (lane 2). Normal IgG does not produce corresponding bands (lane 1). (b) The perikaryon of this Purkinje cell is selectively labelled by anti-Yo IgG in a characteristically coarse, ‘tigroid’ fashion. Immunohistochemistry for anti-Yo. (c) Typical of anti-Yo-associated paraneoplastic cerebellar degeneration is the diffuse loss of Purkinje cells and reactive Bergmann astrogliosis shown in this autopsy specimen of affected cerebellar cortex.

T-lymphocytes.21 The surrounding of neurons by granzyme B-expressing cytotoxic T-cells has been depicted.6 In further support of cell-mediated immune mechanisms as affecting Ma-associated CNS injury is a rat model in which adoptive transfer of CD4+ T-helper cells specifically sensitized to autologous Ma1 induced meningoencephalitic inflammatory alterations (though unassociated with neurologic dysfunction) whereas immunization with recombinant Ma1 prompted anti-Ma1 antibody production but not neuropathologic abnormalities.65

Amphiphysin Antibodies to amphiphysin, a 128 kDa presynaptic protein that acts to inhibit neurotransmission through the endocytosis of synaptic vesicles released at axon terminals, are associated with a paraneoplastic syndrome having, as its hallmark, muscle stiffness, rigidity and spasms affecting the neck, trunk and limbs.61,62,69 The inciting neoplasm is typically a small-cell carcinoma of lung or adenocarcinoma of the breast. Whereas women with underlying mammary cancers are usually seropositive for anti-amphiphysin

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alone, patients with small-cell carcinomas often evidence circulating antibodies to other onconeural antigens such as Hu, CRMP-5 and voltage-gated calcium channel proteins. The latter may contribute to accompanying clinical manifestations that can include encephalopathy, cerebellar dysfunction and neuropathies. Though the anti-amphiphysin antibody-associated disorder is often referred to as ‘stiff man’ or ‘stiff person’ syndrome, the classic stiff man syndrome occurring in patients with antibodies to the 65 kDa isoform of glutamic acid decarboxylase (GAD65) differs in its far less frequent involvement of the cervical musculature and upper extremities, common occurrence in complex with extraneural autoimmune disease (especially type 1 diabetes mellitus) and only exceptional association with neoplasia.61,62 Adduced in support of a directly pathogenic role for antibodies to amphiphysin in neurologic injury are the observations that some afflicted patients benefit from antibody-depleting plasmapheresis93 and the induction of stiff man syndrome-like disorders in rats by passive transfer of human anti-amphiphysin IgG.33,80 Intrathecally administered anti-amphiphysin IgG was found in one such model to

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1950  Chapter 45  Paraneoplastic Syndromes

co-localize with several presynaptic antigens and to result in reduced presynaptic GABAergic inhibition, the authors also reporting the internalization of this IgG by cultured hippocampal neurons.33 Autopsy examinations of amphiphysin-seropositive patients without potentially confounding autoantibodies of other types, although limited to isolated cases, suggest, however, that cell-mediated immune reflexes are also activated in this setting.69,93 Major findings, principally observed within the pons, medulla and spinal cord, have included perivascular cuffing by lymphocytes of both B- and T-cell types with neuroparenchymal infiltration primarily by CD8+ T-cells and accompanying neuronal loss, gliosis and microglial nodule formation. Deposition of the terminal component of complement C5b9 was observed in one case.93

Syndromes Associated with Antibodies to Synaptic and Other Cell Membrane– Associated Neuronal Proteins N-Methyl-d-Aspartate Receptors By far the most prevalent disorder in this group is an encephalitis associated with antibodies of predominantly IgG1 and IgG3 subtypes to an extracellular epitope localized to the N-terminal domain of the N-methyl-d-aspartate receptor (NMDAR) NR1 subunit.22,24 These antibodies produce a pattern of neuropil immunolabelling that is particularly concentrated in the hippocampal region when applied to sections of rodent brain. The target is one of a group of ionotropic glutamate receptors. Often unassociated with underlying neoplastic disease, anti-NMDAR paraneoplastic encephalitis principally affects women (80 per cent of cases) and the inciting tumour is typically an ovarian teratoma. Approximately 70 per cent of patients experience a virus infection-like prodrome of fever, headache and, in some cases, vomiting, diarrhoea or upper respiratory tract symptoms. This is shortly followed by psychiatric manifestations (anxiety, insomnia, manic hyper-religiosity, bizarre behaviour, delusions or hallucinations), short-term memory loss and language deterioration ranging from reduced verbal output with echolalia to frank mutism. The affected then develop movement abnormalities (limb and orofacial dyskinesias, choreoathetosis, dystonia, rigidity and opisthotonic posturing) and decline into catatonia and coma attended by disturbances of autonomic function and breathing. Remarkably, antibody-depleting immunotherapy and tumour resection completely reverse or substantially alleviate neurologic dysfunction in 75–80 per cent of patients. Neuropathological and immunopathological assessment of paraneoplastic anti-NMDAR encephalitis has been limited.22,57,89 Prominent findings at autopsy have included gliosis, conspicuous microglial proliferation and IgG deposition predominantly affecting the hippocampi, basal forebrain, basal ganglia and cervical spinal cord. Reduced immunoexpression of NMDAR NR1 characterized the hippocampal regions of two autopsied patients.43 Modest infiltration of the leptomeninges, perivascular spaces and neuroparenchyma by lymphocytes of T and B types as well

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as plasma cells has been documented, but complement deposition has not been seen and lymphocytes expressing cytotoxic T-cell markers (TIA-1, perforin, granzyme B, Fas/ Fas ligand) have been absent or constituted only about 1 per cent of inflammatory cells present.57,89 Neuronal loss was conspicuous in Sommer’s sector of the hippocampus in one case, but was not otherwise striking.89 Examination of teratomas from affected patients has shown these to consistently harbour central nervous system-type tissues that include NMDAR-expressing neuronal elements and to manifest infiltration by T-lymphocytes, macrophages and, in smaller numbers, B-cells and plasma cells.89 Complement deposition in neuron-containing regions of these tumours has also been documented.57 Anti-NMDAR encephalitis emerges from the clinical and pathologic studies cited previously as differing fundamentally from paraneoplastic syndromes associated with antibodies to cytoplasmic and nuclear neuronal proteins (such as Yo and Hu, respectively) in its responsiveness to antibody-depleting therapeutic strategies and a pathogenesis that does not seem to depend on the cytotoxic T-cell-mediated destruction of target neuronal populations. Additional evidence for the direct pathogenicity of anti-NMDAR antibodies includes experimental observations that the exposure of rodent hippocampal neurons to the CSF or purified IgG of affected patients causes a reduction of synaptic NMDAR clusters and protein that is titre-proportional, selective (i.e. sparing of other synaptic components), reversed on antibody removal and associated with specific dampening of excitatory NMDAR-mediated post-synaptic currents.23,43 NMDAR cluster reduction appears to reflect internalization following capping and bivalent IgG antigenic cross-linking.43

Other Ionotropic Glutamate Receptors Infrequently, ionotropic glutamate receptors other than NMDARs are implicated in paraneoplastic CNS dysfunction but the GluR1 and GluR2 subunits of the α-amino3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and the B1 subunit of the gamma-aminobutyric acid-B (GABA-B) receptor are targets of antibody responses that have been associated with limbic encephalitis.48,50 Anti-AMPA receptor limbic encephalitis has been seen in patients with lung carcinomas of both small and non-smallcell types, thymomas and mammary adenocarcinomas. The antibodies preferentially label those brain regions richest in GluR1/2 and GluR2/3 receptors, including the CA3-CA1 zones of the hippocampus, amygdala, cerebellum, caudate, putamen and cerebral cortex, causing a reversible decrease in AMPA receptor clusters through receptor cross-linking and internalization. Such patients may harbour antibodies to other neuronal antigens (e.g. CRMP-5, GAD65 and voltage-gated calcium channel-associated proteins), complicating treatment as well as interpretation of the scant neuropathological data available. One autopsied patient with coexisting SOX1 and N-type calcium channel antibodies had only mild perivascular lymphocytic cuffing, foci of lymphocytic infiltration of the hippocampus (mainly involving CA4) and only rare microglial nodules.48 A second fatal case, involving a patient who also harboured CRMP-5 antibodies, was characterized by perivascular and interstitial infiltrates dominated by CD8+ cytotoxic T-lymphocytes,

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  References  1951

astrogliosis and microglial nodules principally localized to the hippocampi.48 The neuropathologic substrate of anti-GABA-B receptor limbic encephalitis, seen mainly in the setting of small-cell lung cancer and also potentially complicated by production of other antineuronal antibodies, is yet to be elucidated. Anti-GABA-B receptor antibodies block receptor function, but do not appear to cause receptor internalization.51

Tr Antigen; Delta/Notch-like Epidermal Growth Factor-Related Receptor IgG autoantibodies to an antigen designated Tr are powerful markers of paraneoplastic cerebellar dysfunction occurring in complex with Hodgkin’s lymphoma,8,9,37 but have also been detected in select patients with cerebellar degeneration and lymphomas of non-Hodgkin type.9 Anti-Tr IgG diffusely labels the cytoplasm and proximal dendrites of Purkinje cells, producing, as well, a punctate pattern of immunoreactivity in the overlying molecular layer.37 In rat studies, Tr expression appeared localized to Purkinje cell perikarya and dendrites, as well as the cytosol and outer surfaces of the endoplasmic reticulum in molecular layer neurons.38 Recent investigations, however, have provided evidence that the principal, if not sole, Tr immunogen resides in the extracellular domain of the Delta/Notch-like epidermal growth factor-related receptor (DNER),28 which may mediate neuronal-glial interactions through Notch signalling.32 The trigger of anti-Tr-associated cerebellar injury is unclear. Only exceptionally has a syndrome-associated Hodgkin’s lymphoma been shown to label with anti-Tr IgG8 and preliminary studies failed to detect DNER protein within tumour biopsies from Tr-seropositive patients.28 Severe loss of Purkinje cells and Bergmann gliosis with only sparse infiltration of the cerebellum by CD3-labelling T-lymphocytes and CD68-labelling histiocytes/microglia have been documented at autopsy,8 but the role of anti-Tr antibodies in this picture is obscure. Exposure to anti-Trpositive sera in vitro reportedly yielded endogenous labelling of Purkinje cells and hippocampal neurons but failed to affect obvious morphological alterations in these cells.28 Impaired cerebellar development and function are features of a DNER knockout mouse model.88

Voltage-Gated Potassium Channel– Interacting Proteins Proteins that interact with voltage-gated potassium channels (VGKCs) have also been implicated as targets of pathogenic antibody formation, including leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein 2 (CASPR2), though only a minority of affected

patients have demonstrable neoplastic disease. Limbic encephalitis, brief tonic-myoclonic seizures and hyponatremia have been associated with anti-GLI1 antibodies in patients having small-cell carcinoma of the lung, thymoma and other epithelial neoplasms,49 whereas neuromuscular hyperexcitability, dysautonomia and encephalopathy have been recorded in thymoma-related anti-CASPR2 cases.44 Paraneoplastic examples of these immunotherapy-responsive syndromes have not been subject to neuropathologic study, though three tumour-unassociated autopsy cases of ‘VGKC antibody-associated’ limbic encephalitis probably corresponding to the anti-LGI1 syndrome have been communicated.31,47,64 Findings included variable degrees of T-lymphocytic infiltration, astrogliosis and microglial proliferation in the amygdaloid nuclei and hippocampi, one report describing perivascular lymphoid infiltrates dominated by CD20+ B-cells47 and two describing neuronal loss in these regions.31,47

45

Other Membrane Channel and Receptor Proteins Antibodies to voltage-gated calcium channels cause the Lambert–Eaton myasthenic syndrome, most of the offending neoplasms in this setting being small-cell carcinomas of lung origin.13 Antibody-mediated blockage of calcium influx prevents quantal acetylcholine release, producing weakness and electrophysiological disturbances that remit on removal of the offending immunoglobulins from patient sera and that are replicated in animals by passive antibody transfer.52 Myasthenic syndromes may also be encountered in patients with thymoma and antibodies to muscle-associated acetylcholine receptors,90 whereas patients with a variety of neoplasms (particularly adenocarcinomas of mammary, prostatic, pulmonary and gastrointestinal types) can manifest dysautonomia, peripheral neuropathy and encephalopathy in association with antibodies to neuronal/ganglionic acetylcholine receptors.59 Thymic epithelial neoplasms and carcinomas of diverse (principally breast) origin have been documented in optic neuritis and transverse myelitis patients with astrocytic aquaporin-4 water channel antibodies of the type that characterize neuromyelitis optica.67 Isolated reports describe progressive muscle rigidity, brain stem dysfunction and stimulus-sensitive myoclonus (the ‘progressive encephalomyelitis with rigidity and myoclonus’ or PERM syndrome) in one patient with thymoma and antibodies to glycine receptors16 and a second harbouring an undifferentiated carcinoma that presented in the mediastinum and was associated with antibodies to gephyrin, which interacts with glycine and GABA receptors in the postsynaptic membrane component of inhibitory synapses.11

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45

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46 Chapter

CNS Reactions to Anti-neoplastic Therapies Arie Perry

Introduction..............................................................................1954 Radiation Necrosis and Other Forms of Radiation Injury............1954 Therapy-Induced Leukoencephalopathies and Vasculopathies..1956

Introduction The management of brain tumour patients poses ­formidable challenges because the same therapies used to enhance ­ survival can cause severe central nervous system (CNS) toxicities.37 Unfortunately, as therapeutic options increase over time, so do the frequencies of neurotoxicity. Potential surgical complications are mostly acute in nature and consist of haemorrhage, vascular damage, infarcts, coagulopathies, malignant cerebral oedema with herniation and post-operative infection. In contrast, serious side effects of radiation and chemotherapy, such as radiation necrosis, chemotherapy-associated leukoencephalopathy and secondary neoplasms, are typically more subacute to chronic in nature. Therapy-induced peripheral neuropathies and pituitary changes are also common, but are covered in Chapter 24, Diseases of Peripheral Nerve, and Chapter 41, Pituitary and Suprasellar Tumours, respectively. Individual predisposing factors remain poorly understood. Nonetheless, it is clear that the developing nervous system is particularly vulnerable, especially to the effects of radiation.38 Other known risk factors include the specific therapeutic modality and dosage, combined radiochemotherapy, genetic background and idiosyncratic predilections.21,38,51,58

Radiation Necrosis and Other Forms of Radiation Injury Injury to the nervous system is a common complication of radiation therapy, with acute, early delayed and late delayed forms.52 The acute and early delayed forms are thought to primarily represent blood–brain barrier disruption and cerebral oedema, with neurologic deficits being mostly transient and reversible. In contrast, the late delayed effects occur months to years after therapy

Therapy-Induced Secondary Neoplasms...................................1959 Genetic Predispositions to Therapeutic Neurotoxicity.................1960 References...............................................................................1960

and are often irreversible, sometimes fatal injuries. In adults, the incidence of radiation necrosis after conventional doses for CNS therapy ranges from 5–24 per cent and is rarely encountered with cumulative doses of standard fractionated radiation under 50–60 Gy to the brain or 45 Gy to the spinal cord.37 However, a nonnecrotizing form of encephalopathy with neurobehavioural manifestations is even more common and is seen with lower dosages as well. Some data suggest that neuroprotective agents, such as lithium, have the potential to reduce this complication.23 Although less severe than radiation necrosis and not life threatening, this form of encephalopathy tends to progress over many years and significantly alters quality of life. Individual risk factors for radiation damage are inadequately understood, but may include superimposed vascular disorders due to diabetes, hypertension and old age. Paediatric patients are even more vulnerable and generally, the younger the patient, the more susceptible they are to radiationinduced neurotoxicity. Long-term survivors of irradiated childhood brain tumours often suffer ­moderate to marked cognitive deficits, learning disabilities, h ­ ormonal deficits, growth retardation and psychomotor retardation. For instance, in an autopsy study of 22 paediatric glioma patients treated with radiation, there were foci of demyelination in 7, necrosis in 6 and cortical atrophy in 4 cases, respectively.34 Clinically, the need to distinguish radiation necrosis from tumour recurrence is common and critical, because the two are managed quite differently. On routine magnetic resonance (MR) imaging, a ‘soap bubble’ or ‘Swiss cheese-like’ interior favours radiation necrosis, although this sign is neither sufficiently sensitive nor specific.26 Other examples display features that are indistinguishable from high-grade glioma. Newer radiologic modalities, such as diffusion weighted imaging (DWI), perfusion studies, proton magnetic resonance spectroscopy (H-MRS), and positron emission tomography (PET) have further enhanced the clinical distinction, although false positives

1954

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  Radiation Necrosis and Other Forms of Radiation Injury  1955

and negatives remain common. Therefore, tissue diagnosis remains the gold standard. The clinical, radiographic and histological features that favour treatment effects or tumour progression are summarized in Table 46.1. Most commonly, however, there is a mixture of both; nonetheless, some data suggest that the ratio of the two may provide additional prognostic information.13,18,56,63 Specimens composed predominantly of radiation necrosis indicate a favourable response to therapy, whereas cellular tumour specimens with little necrosis suggest minimal cytotoxic effects.

Gross Pathology Radiation effects and radio-necrosis may involve any CNS site, although cerebral white matter commonly bears the brunt of the injury. Subcortical U-fibres are mostly spared, although lesions may extend into adjacent cortex or deep grey matter. Depending on the relative proportions of necrosis, vasculopathy/haemorrhage, oedema and gliosis, lesions may appear as firm, ill-defined, glioma-like masses or soft, friable, infarct-like regions of cystic degeneration (Figure 46.1a). Grey-white junctions may be blurred, with necrotic foci showing yellow to tan–brown discolouration. Areas of dystrophic calcification appear white and chalky. Superimposed petechial haemorrhages may produce a variegated appearance similar to that of glioblastoma, although more advanced cases show marked white matter loss and cerebral atrophy with hydrocephalus ex vacuo, rather than mass effect.

Histopathology and Proposed Mechanisms of Injury Vascular changes are prominent in radiation injury and likely play an important pathogenic role. As one of the few actively proliferating sites in the brain, it is not surprising that the endothelial cell is particularly susceptible to radiation damage. Early disruptions in the blood–brain barrier are likely responsible for the vasogenic oedema seen in the transient acute and early delayed, often steroid responsive forms of radiation toxicity.53 In contrast, more permanent forms of endothelial damage likely account for many of the classic changes of late delayed radiation injury seen in roughly 3–24 per cent of patients, including coagulative necrosis, thrombosis, haemorrhage, telangiectasia, vascular fibrosis/hyalinization with luminal stenosis and necrotising vasculopathy (Figure 46.1b), all of which facilitate hypoxic injury, white matter damage and parenchymal necrosis.20,53 Other common findings include dystrophic calcifications and ­variable macrophage-rich infiltrates. Occasionally, neurodegenerative changes in the adjacent cortex or malformation-like vasculature are seen. Besides endothelial damage, other proposed mechanisms of radiation damage include coagulopathies and immune mechanisms, such as autoimmune vasculitis. The disseminated form of white matter damage from whole brain irradiation is known as radiation leukoencephalopathy.7,41 Although it is generally assumed that the radiation effects from newer modalities (e.g. stereotactic radiosurgery) are essentially identical to those of conventional external beam therapy, there is considerably less experience with

46

Table 46.1  Changes favouring therapy effects* vs recurrent/progressive tumour Clinical feature

Therapy effects

Tumour recurrence/progression

Patient symptoms

No new symptoms

Worsening neurological status

Standard MRI

Swiss cheese or soap bubble patterns, limited mass effect

New enhancement outside 80%% isodose line, multifocality, corpus callosum spread, subependymal disease

DWI/ADC/DTI

High ADC values, low fractional anisotropy

Restricted DWI, low ADC, high fractional anisotropy

DSC

rCBV 2.6

H–MRS

Low NAA, low Cr, Cho/Cr and NAA/Cr ratios

Increased Cho, high Cho/Cr and NAA/Cr ratios

PET

Metabolically inactive

Metabolically active

Necrosis

Large infarct like zones with hypocellular edges and dystrophic calcifications

Large or microscopic foci with hypercellular or pseudopalisading edges

Blood vessels

Telangiectatic Hyalinized Angionecrotic

Microvascular proliferation with enlarged and multilayered endothelial lining

Adjacent brain parenchyma

Rarefied or vacuolated, pale, and gliotic with vascular changes listed earlier

Nearly normal or infiltrated by individual tumour cells

Cytologic atypia

Bizarre bubbly nuclei and abundant cytoplasm

High N/C ratio

Mitotic figures

Rare

Frequent (if tumour is high grade)

Imaging feature

Histologic feature

*Given overlapping features, ‘therapy effects’ refers to changes seen with radiation, chemoradiation or pseudoprogression. ADC, apparent diffusion coefficient; Cho, choline; Cr, creatine; DSC, dynamic, susceptibility-weighted, contrast-enhanced MR imaging; DTI, diffusion tensor imaging; DWI, diffusion weighted imaging; NAA, N–acetyl aspartate; rCBV, relative cerebral blood volume; H–MRS, proton magnetic resonance spectroscopy; PET, positron ­emission tomography (fluorine–18 fluorodeoxyglucose is most common but may be less sensitive and specific than 11C–methionine, 3,4–dihydroxy–6–18F–fluoro–L–phenylalanine, O– (2–18F–fluoroethyl)–L–tyrosine and 3’–deoxy–3’–18F–fluorothymidine forms of PET).

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1956  Chapter 46  CNS Reactions to Anti-neoplastic Therapies (a)

(b)

(c)

(d)

46.1 (a) Autopsy specimen from a patient with radiation leukoencephalopathy displaying predominantly white matter foci of radiation necrosis, cystic degeneration and atrophy. (b) Example of radiation necrosis with large zone of coagulative necrosis and extensive vasculopathy, including fibrinoid vascular necrosis and markedly hyalinized vessel walls. (c) Marked radiation-induced cytologic atypia in an irradiated, previously classic appearing oligodendroglioma, characterized by increased quantities of eosinophilic cytoplasm and large bizarre nuclei with bubbly chromatin. These changes resemble astrocytic differentiation, although in this case, both the original tumour and the post-radiation recurrence showed the 1p and 19q chromosomal co-deletions typical of oligodendroglioma. (d) Case of disseminated necrotizing leukoencephalopathy with prominent white matter vacuolation, foci of necrosis, diminished numbers of oligodendrocytes, and collections of swollen, dystrophic axons, many of which are calcified.

such post-therapy biopsies. Generally, observed pathological changes have been similar to those of conventional external beam radiotherapy, although the severity is often exaggerated and findings may be seen beyond high dose margins.41 Foci of paucicellular, congealed fibrin-rich tissue are common. Viable tumour may be scant or totally absent and the peritumoural brain is often highly gliotic, sometimes with inflammation and considerable radiation-induced atypia. Radiation-induced cytologic atypia and its potential for mimicking cancer is more commonly emphasised in other parts of the body. Nevertheless, it can be dramatic in the CNS as well, involving tumour cells, endothelial cells and adjacent brain parenchymal cells, such as reactive astrocytes and even neurons. Such changes can pose substantial diagnostic challenges in terms of glioma classification and grading. For example, because both increased nuclear pleomorphism and the accumulation of eosinophilic cytoplasm may be seen, oligodendroglial neoplasms may appear ‘astrocytoma-like’ after therapy (Figure 46.1c). In general, isolated cytomegalic cells with bizarre appearing bubbly nuclei are more likely to be radiation-induced, particularly if they are seen in hypocellular regions. Nevertheless, it is sometimes impossible to distinguish radiation from true atypia. Furthermore, when this

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increased pleomorphism is coupled with radiation necrosis and telangiectatic vessels that may resemble the microvascular proliferation of glioblastoma, a previously radiated low-grade glioma can easily be overgraded if the radiation changes are not recognized as such. Therefore, one should use strict diagnostic criteria for endothelial hyperplasia (multilayering of hypertrophic cells) and spontaneous tumour necrosis (e.g. pseudopalisading pattern) before upgrading a previously irradiated low-grade glioma. In contrast, an increased mitotic or proliferative index is not a radiation effect and is therefore a reliable indicator of tumour progression in this setting.

Therapy-Induced Leukoencephalopathies and Vasculopathies Radiation The vulnerability of both endothelial cells and oligodendrocytes to radiation injury likely accounts for the radiation leukoencephalopathy seen in some patients. However,

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  Therapy-Induced Leukoencephalopathies and Vasculopathies  1957

the risk of encephalopathy is enhanced about 3-fold20,53 and the timing is shortened when chemotherapy is added, one study finding roughly a fourth of glioma patients with radiographic evidence of ‘progression’ after combined radiation/temozolomide therapy showing early (3–20 weeks post-therapy) radiation necrosis (also see section on Pseudoprogression and Pseudoresponse).61 Pure radiation leukoencephalopathy is rarely seen today, as a result of the therapeutic shifts towards more localized forms of radiation over whole brain irradiation. Nonetheless, the clinical and radiologic features are essentially identical to those encountered with chemoradiation, despite a few histopathologic differences. Injury of small to medium sized vessels with superimposed oligodendroglial and stem cell/ progenitor cell toxicities are thought to account for this leukoencephalopathy. The histopathology shows a spectrum of changes ranging from myelin pallor and gliosis to demyelination to coagulative necrosis, typically superimposed on the classic radiation-induced vascular changes already described. Arterial infarcts, vascular malformations and large vessel vasculopathies are less common complications, but have also been reported, including aneurysm formation, haemorrhage, arterial dissection, accelerated atherosclerosis and thrombosis.37

Methotrexate and Other Chemotherapeutic Agents White matter toxicity is a potentially serious complication of chemotherapy. This is particularly true for regimens including methotrexate, although drugs of virtually all mechanistic categories have been implicated, including temozolomide, vincristine, lomustine (CCNU), carmustine (BCNU), melphalan, fludarabine, cytarabine (Ara–C), 5–fluorouracil (5–FU), levamisole, doxorubicin, cyclophosphamide, ifosfamide, thioTEPA, oxaliplatin, paclitaxel, vinblastine and cisplatin.51 The frequency of pure chemotherapy-induced cognitive dysfunction in learning, memory, information processing speed and executive function is increasingly reported, especially in breast cancer patients where no confounding CNS factors exist; estimated frequencies range widely from 15–80 per cent.51,58 Synergistic or additive effects are also seen with combined chemotherapies and because this is the current direction of medical oncology, it is likely that neurotoxicities will continue to increase in the future. Commonly disrupted processes include neurogenesis, gliogenesis and maintenance of myelin fibre integrity, with damage to neural progenitor cells thought to be particularly important in delayed neurotoxic disorders. Oxidative stress, inflammation, angiogenic changes, apoptotic dysregulation, altered transcriptional expression and histone acetylation likely play important roles as well. Potentially, neuroprotective agents may be useful in reducing neurotoxicity in the future, although some may potentially also reduce therapeutic efficacy and further study is therefore needed.51 Additionally, because molecularly targeted strategies may also affect critical signalling in progenitor cells, there is growing concern over potential long term CNS effects using these newer agents. In many cases, a mild and reversible form of injury is seen acutely, although in up to a third, deficits persist for

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months to years.21 Other clinical manifestations include reversible posterior leukoencephalopathy (see later), subcortical dementia, incontinence, gait disturbances, strokelike episodes, and a cerebellar syndrome, with white matter pathology predominating.58 The risk of serious or permanent damage appears to be greatest when methotrexate is combined with radiation therapy and therefore, it is difficult to dissect the precise contribution of each modality to this type of pathology. Nevertheless, methotrexate and other drugs have occasionally been shown to cause the same leukoencephalopathy in the absence of radiation, particularly in cases with intrathecal or intraventricular administration.27,30 Additional evidence for its pathogenic role comes from occasional examples where the injury is exacerbated around a leaky or misplaced ventriculostomy tube used to administer cerebrospinal fluid (CSF) methotrexate.37 The mechanisms of chemotherapy neurotoxicity are elusive, but may include direct toxic effects on axons, oligodendrocytes and progenitor cells, as well as secondary immunologic reactions, oxidative stress and microvascular injury.5,7,35 Risk factors for chemotherapy toxicity are also poorly understood, but likely depend on treatment modalities, nutritional deficiencies, patient age, cognitive reserve, co-morbidities and idiosyncratic predispositions, including genetic variability in multi-drug resistance pumps, DNA repair mechanisms, telomere maintenance, cytokine regulation, neurotransmitter activity, Alzheimer disease risk factors (e.g. ApoE alleles), and hormone status/endocrine therapy.21,51,58 A fairly wide spectrum of pathologic changes may be seen. In some, the pathology appears to be partially or entirely reversible, whereas others suffer permanent, potentially fatal deficits. For example, an acute asymptomatic form of white matter disease is commonly detected radiologically in medulloblastoma and supratentorial primitive neuroectodermal tumour (PNET) patients receiving multiagent chemotherapy regimens, with or without concomitant radiation.14,47 In most, lesions are transient, although there is increased risk of subsequent neurocognitive deficits, suggesting that permanent damage occurs in a subset. Because such cases are rarely biopsied, however, the pathogenesis of this milder form of leukoencephalopathy is unclear. A disruption of the blood–brain barrier with oedema is suspected. In slightly more severe examples, myelin pallor, vacuolation, axonal spheroids, modest macrophage infiltrates and gliosis are common.33,37 The most dramatic examples of chemotherapy-induced leukoencephalopathy present with miliary, small rounded to large confluent foci of predominantly non-inflammatory demyelination or, more commonly, frank white matter necrosis. Rubinstein and colleagues coined the term disseminated necrotizing leukoencephalopathy (DNL) for this severe, progressive and often fatal form that they and others described originally in children with metastatic meningeal acute lymphoblastic leukaemia (ALL) treated with high-dose methotrexate based chemotherapy and whole brain irradiation.45,46 Similar examples have subsequently been reported both in adults and patients with other tumour types.37 This complication has also been encountered in cases of meningeal carcinomatosis,16 which makes sense given that DNL is mostly encountered with whole brain irradiation. Lastly, a similar pathology has

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1958  Chapter 46  CNS Reactions to Anti-neoplastic Therapies

been reported in glioma patients treated with intra-arterial BCNU, either in combination with brain irradiation or less often, administered in isolation.44 However, the latter pathology is generally limited to the vascular distribution of drug administration and shows a greater degree of vasculopathy than classic DNL. Grossly, DNL is characterized by small, discrete, greybrown foci of softened oedematous white matter, a chalky to friable consistency and petechial or ring-shaped haemorrhages in larger coalescent lesions. The pathology is most prominent in cerebral white matter, but can also affect the cerebellum and brain stem. Microscopically, lesions range from ill-defined zones of myelin pallor, oligodendrocyte loss and prominent spongiosis to discrete rounded foci of coagulative necrosis with numerous microcalcifications, a surprising paucity of inflammation and collections of markedly swollen axons with dystrophic calcifications (Figure 46.1d). Ultrastructurally, dystrophic axons are stuffed with mitochondria, autophagic vacuoles and microfilaments, with this prominent axonopathy considered one of the key distinguishers of DNL from pure radiation leukoencephalopathy. Only rare cases of DNL have included superimposed features of radiation damage, such as fibrinoid ­ vascular necrosis, vascular hyalinization, telangiectasias and fibrinous exudates. In a series of 185 primary CNS lymphoma patients treated at Memorial Sloan–Kettering Cancer Center, the estimated 5-year incidence of DNL was 24 per cent.35 Clinically, it was characterized by a rapidly progressive subcortical dementia, similar in type to arteriosclerotic encephalopathy (Binswanger’s disease), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and normal pressure hydrocephalus. Symptoms begin shortly after therapy to many months later, including irritability, somnolence, ataxia, memory deficits, apathy, psychomotor retardation and psychiatric disturbances. Over time, cognitive deficits progress to frank dementia, with motor and autonomic deficits increasingly apparent. Most progress further to seizures, coma and death, often within a few months of onset. Neuroimaging studies of DNL may be unremarkable at presentation, but later reveal variably calcified, progressively confluent white matter lesions. On computed tomography (CT), the calcifications are among the earliest findings, followed by low density lesions and contrast enhancing masses that may be difficult to distinguish from tumour recurrence or progression. MR imaging is more sensitive in early lesions, with white matter pathology showing decreased T1-weighted signal intensity and increased signal on T2-weighted and fluid attenuated inversion recovery (FLAIR) studies.28,35 Large advanced lesions commonly demonstrate contrast enhancement that may mimic tumour initially, although cerebral atrophy and hydrocephalus ex vacuo typically develop as the disease progresses.

Pseudoprogression and Pseudoresponse These two terms have been introduced to reflect increasing challenges in accurately distinguishing tumour progression and therapeutic responses from radiological mimics using current chemoradiation approaches. For instance, since the 2005 publication by Stupp, et al. of their phase III trial

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showing a survival benefit for combined radiation and temozolomide chemotherapy in glioblastoma patients,55 this has become the standard of care.11,20,53 In early followup imaging (2–6 months), roughly half develop contrast enhancing lesions consistent with tumour progression using standard MacDonald criteria.31 On follow-up imaging of patients continuing this therapeutic regimen however, 28–64 per cent show stabilization, improvement or resolution,11 a process termed ‘pseudoprogression’. As such, this is now considered an exaggerated favourable response to therapy and correlates well with the finding that patient survival is improved in pseudoprogression when compared with true progression. This phenomenon appears to be even more commonly associated with tumours displaying MGMT hypermethylation.2,9,36 One study suggested that glioblastomas with high proliferative indices (Ki–67 labelling >20 per cent) are also more likely to undergo pseudoprogression.39 The mechanism of pseudoprogression remains poorly understood, but is sometimes explained as transient disruptions in blood-brain barrier or endothelial function, with or without associated inflammation.10,20,53 However, this premise conflicts with the nearly invariable reports of necrosis in the rare cases undergoing biopsy.18,56,61,63 Similarly, the proposed notion that pseudoprogression affects the tumour while radiation necrosis affects adjacent brain parenchyma seems overly simplistic, as simultaneous changes involving both are often seen histologically. Therefore, it is possible that despite perceived clinical differences, pseudoprogression and chemoradiation necrosis represent a single spectrum, with a preferable all-­ encompassing term being ‘therapy effects’. Features favouring treatment effects or progression are ­summarized in Table 46.1, although none are completely reliable. The great conundrum for oncologists is that in the setting of a worrisome post-therapy imaging change, they must wait until the next MRI to determine whether the current therapy was actually working (pseudoprogression) or whether a true progression occurred, in which case an earlier switch in therapy might have been warranted. As such, repeat surgery for pseudoprogression vs progression has become more common. Unfortunately, even with tissue examination, this question may be challenging, because most samples contain a combination of both viable tumour and treatment effects. As such, additional studies are sorely needed to better predict patient outcome based on posttherapy findings. In contrast, the term pseudoresponse refers to apparent ‘normalization’ of tumour associated vasculature, such that seemingly dramatic responses to enhancing foci on MR imaging occur despite a lack of reduction in tumour burden.10,11,19,20 This phenomenon is mostly seen with ­anti-angiogenic therapy, such as the VEGF inhibitor, bevacizumab. Further complicating interpretations, the reduction in contrast enhancement is accompanied by marked expansion of the T2-weighted and FLAIR abnormalities, sometimes resulting in a gliomatosis-like pattern. Given these ­pitfalls and the lack of reliability of post-contrast ­measurements alone, the MacDonald criteria were replaced by the response assessment in neuro-oncology (RANO) criteria in 2010 in order to obtain more accurate response determinations in the setting of anti-angiogenic therapy.20,53,59

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  Therapy-Induced Secondary Neoplasms  1959

Posterior Reversible Encephalopathy Syndrome (PRES) The term reversible posterior leukoencephalopathy syndrome was first coined by Hinchey, et al. in 199617 and refers to a disorder presenting with acute cortical blindness, headaches, mental status changes and sometimes seizures, typically associated with malignant hypertension and T2-weighted/FLAIR MRI signal abnormalities in the occipital and posterior parietal lobes. Given that changes may be irreversible, lesions may not be limited to the posterior circulation and that grey matter may also be involved, some have advocated alternate names, such as posterior reversible encephalopathy (PRES) or occipital–parietal encephalopathy.50,54 PRES has been associated with a wide variety of inciting factors, including the use of high-dose corticosteroids and various single or multiagent chemotherapy regimens.60 The latter have included cisplatin, cytarabine, cyclophosphamide and methotrexate. Symptoms and signs may develop during therapy or be delayed for days to weeks. In most cases, the neurotoxicity is completely reversible, although the importance of controlling the hypertension in order to prevent permanent damage has been emphasized. The mechanism of toxicity is also poorly understood, although radiologic studies, particularly those utilizing diffusion weighted imaging with apparent diffusion coefficient measurements, are consistent with vasogenic oedema. This pattern differs from that of the cytotoxic oedema encountered in classic cerebral infarcts and could explain the reversible nature of most examples. Proposed mechanisms have included endothelial damage with blood–brain barrier disruption, transient episodes of hypertension overloading the autoregulatory capabilities of the posterior circulation, and electrolyte imbalances with hypomagnesaemia.37,50,54 Because PRES is predominantly a clinicoradiological diagnosis, few descriptions of the neuropathology exist. However, a biopsied example found evidence of vasogenic oedema without overt vascular damage or infarct.50 A rare fatal example found dilated perivascular spaces with proteinaceous exudates, macrophages, fibrinoid necrosis and acute haemorrhage reminiscent of acute hypertensive encephalopathy.24 Even so, the permanent deficits occasionally encountered suggest that ischemic damage is possible in severe cases or those in whom diagnostic delay leads to inadequate control of the hypertension.

Therapy-Induced Secondary Neoplasms Given that both radiation and chemotherapy induce DNA mutations, it is not surprising that a potential complication is therapy-induced secondary neoplasms. In fact, as oncology becomes more successful at prolonging the survival of cancer patients, secondary neoplasms are increasingly encountered (see25,32 for reviews). Epidemiological data links the risk of secondary brain tumours with ionizing radiation much more than with chemotherapy, although given their mutagenic properties, it is likely that the latter increases risk as well, either alone or in combination with radiation.

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Cahan’s criteria for a radiation-induced neoplasm state that the tumour must differ histologically from the original lesion being treated and should arise within the irradiation field over a sufficient latency period, typically years.4 In the CNS, the most common secondary tumours are meningiomas, nerve sheath tumours, pituitary adenomas, gliomas, sarcomas and embryonal neoplasms, with latency periods varying greatly, but typically presenting years to decades after therapy.25,32 The greatest risks have been associated with irradiation during childhood, suggesting that there are critical windows of enhanced susceptibility, perhaps associated with active CNS development and growth in general, increased proliferation rates in various progenitor cell types or an increased number of progenitor cells overall in comparison to adult brains. The data also suggest a dosage effect, such that greater exposures are associated with shorter latency periods and higher risks of malignancy. For instance, the British Childhood Cancer Survivor Study of nearly 18 000 paediatric cancer survivors found subsequent primary neoplasms to be 4 times greater than expected, with the most frequently observed second neoplasms at a median follow-up of 24.3 years being located in the CNS.42 Additional epidemiologic data come from follow-up studies of paediatric patients receiving CNS irradiation for ALL or other common childhood malignancies, Israeli immigrants treated with radiation for tinea capitis between 1948 and 1960, and atomic bomb survivors from Hiroshima and Nagasaki.43,48,62 For example, Ron and colleagues estimated relative risks among nearly 11 000 children treated with low-dose scalp irradiation (mean 1.4–1.8 Gy) for tinea capitis as 9.5 for meningiomas, 2.6 for gliomas and 18.8 for nerve sheath tumours (mostly schwannomas) when compared with non-irradiated controls from the general population.43 The relative risk of developing a meningioma increased to nearly 20 for patients treated with doses of roughly 2.5 Gy.48 Similarly, the latency period is inversely associated with dosage, such that those with greater exposures present earlier, potentially developing tumours as soon as 12 months after therapy.5 In studies of atomic bomb survivors, meningiomas, schwannomas and pituitary tumours were most common and there was generally an inverse relationship between the patient’s distance from the epicentre of the blast and the relative risk of tumour development.62

46

Pathology and Genetics Although some have emphasized features such as nuclear atypia and vascular hyalinization as being common in radiation-induced brain tumours, the histology of secondary neoplasms is usually indistinguishable from sporadic counterparts. Nevertheless, there may be genetic and biologic differences, most notably in meningiomas. There has been a debate as to whether or not radiation-induced meningiomas (RIMs) are more aggressive as a group, with increased numbers of high grade examples.8,15,57 Although it is clear that most reported examples are both histologically and biologically benign, most data now support this hypothesis, particularly in patients that received high-dose (>20 Gy) radiation at a young age. Additionally, the RIMs are more likely to present with multicentric disease, unusual histologic subtypes and calvarial involvement, often with associated alopecia, atrophy and poor vascularization of previously irradiated

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1960  Chapter 46  CNS Reactions to Anti-neoplastic Therapies

scalp. In contrast to sporadic counterparts, RIMs are less likely to harbour alterations of the NF2 gene, instead being characterized genetically by frequent losses and structural alterations of chromosome 1.8,29,40 Lastly, there is evidence to suggest that genotypic patterns may be able to predict higher or lower predispositions for RIMs in individual patients.12 Radiation-induced gliomas (RIGs) are also similar to their sporadic counterparts, with high-grade astrocytomas encountered most frequently.3,49 However, these tumours characteristically present at younger ages of onset than sporadic glioblastomas, especially for patients irradiated during childhood. Genetically, Brat and colleagues examined nine examples for alterations of the TP53, p16, MTAP, PTEN, KRAS and EGFR genes, finding similar alterations to those of sporadic gliomas, particularly de novo glioblastomas.3 One potential difference was the lack of PTEN mutations in the RIGs; however, the numbers of cases examined were insufficient for statistical significance. Additionally, an expression profiling and Western blot study comparing RIGs and paediatric glioblastomas found a less complex pattern in the former with increased expression levels of ErbB3, Sox10 and platelet-derived growth factor receptor– proteins.6

Therapy-Induced Malignant Transformation Recent data suggest that temozolomide chemotherapy can induce a characteristic ‘hypermutator phenotype’ associated with malignant progression of lower grade gliomas to glioblastoma.22 It remains unclear whether or not irradiation can promote or enhance the rate of malignant transformation within benign or low-grade primary CNS tumours. Malignant progression in such tumours may be mediated by additional mutations, which could conceivably be facilitated by irradiation. This hypothesis is difficult to prove in tumours that normally undergo malignant progression even in the absence of therapy. However, there is some support for this notion in benign tumour types that almost never progress otherwise, such as pilocytic astrocytoma, ganglioglioma, dysembryoplastic neuroepithelial tumour and schwannoma.37

Nevertheless, such examples remain rare. Considerably fewer data are available regarding the risks of secondary neoplasms or malignant transformation induced by newer modalities of radiation therapy, although examples are increasingly being reported as experience is gained, particularly with various forms of stereotactic neurosurgery, such as gamma knife.25,37

Genetic Predispositions to Therapeutic Neurotoxicity Given that familial cancer predisposition syndromes are caused by germline mutations in tumour suppressor genes, patients with these syndromes may be even more susceptible than other patients to the mutagenic effects of radiation and chemotherapy (see Chapter 44, Hereditary Tumour Syndromes). Examples include neurofibromatosis 1 (NF1) patients developing malignant peripheral nerve sheath tumours (MPNST) or other sarcomas within radiation fields.25,32,37 Because MPNSTs are uncommon in the head and orbital regions where optic pathway gliomas have been radiated, it seems unlikely that such cases are coincidental, although admittedly such patients are already at risk for this tumour type. NF1 patients also appear at greater risk for developing post-irradiation strokes and other vasculopathies, especially moyamoya syndrome. Similarly, increased risks of radiation-induced sarcomas have been reported in children with Li–Fraumeni syndrome and familial retinoblastoma.25,37 Lastly, secondary glial, meningothelial and neural (e.g. MPNST) malignancies have been reported in NF2 patients receiving radiosurgery for vestibular schwannomas.1 Although the tumour types that have developed within the radiation fields in these patients have often been the types to which the patients are already at risk, such studies nevertheless suggest an increased susceptibility to the transforming effects of radiation. This could be true for various forms of chemotherapy as well, although there are currently no clear data to prove this.

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