Cornea: Fundamentals, Diagnosis and Management [4th Edition] 0323357571, 9780323357586, 9780323357579

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CORNEA

https://t.me/MedicalBooksStore

cornea Fourth Edition

Mark J. Mannis MD FACS Natalie Fosse Endowed Chair in Vision Science Research Professor and Chair Department of Ophthalmology & Vision Science University of California Davis Eye Center Sacramento, CA, USA Edward J. Holland MD Director of Cornea Cincinnati Eye Institute Professor of Ophthalmology University of Cincinnati Cincinnati, OH, USA

Edinburgh  London  New York  Oxford  Philadelphia  St Louis  Sydney  Toronto  2017

© 2017, Elsevier Inc. All rights reserved. First edition 1997 Second edition 2005 Third edition 2011 No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Csaba L Mártonyi and Mark Maio retain copyright for their original illustrations in Chapter 7. Michael E. Snyder retains copyright for his original figures and video clips in Chapter 145. Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: Print: 978-0-323-35757-9 E-book: 978-0-323-35758-6 Inkling: 978-0-323-35759-3 Printed in China Last digit is the print number:  9  8  7  6  5  4  3  2  1

The publisher’s policy is to use paper manufactured from sustainable forests

Content Strategist: Russell Gabbedy Content Development Specialist: Sharon Nash Content Coordinator: Joshua Mearns Project Manager: Joanna Souch Design: Christian Bilbow Illustration Manager: Emily Costantino Marketing Manager: Melissa Fogarty

Video Table of Contents Clip

Clip title

Video contributor/s

50.1

The Application of Cryopreserved Amniotic Membrane in the Treatment of Acute Stevens–Johnson Syndrome: Part 1

Darren G. Gregory

50.2

The Application of Cryopreserved Amniotic Membrane in the Treatment of Acute Stevens–Johnson Syndrome: Part 2

Darren G. Gregory

50.3

The Application of Cryopreserved Amniotic Membrane in the Treatment of Acute Stevens–Johnson Syndrome: Part 3

Darren G. Gregory

110.1

Penetrating Keratoplasty Using the Barron Trephine and Interrupted Sutures

Mauricio A. Perez, David S. Rootman

110.2

Penetrating Keratoplasty Using the Hanna Trephine and Interrupted Sutures

Mauricio A. Perez, David S. Rootman

110.3

Penetrating Keratoplasty Using the Slipknot Technique for Suturing

Clara C. Chan, Mauricio A. Perez

110.4

Penetrating Keratoplasty Using a Running Suture

Mauricio A. Perez, Allan R. Slomovic

111.1

An Intraoperative Suprachoroidal Hemorrhage During Penetrating Keratoplasty, which was Successfully Managed with the Assistance of a Cobo Temporary Keratoprosthesis

Michael C. Chen, Mark J. Mannis

112.1

Donor Preparation

Marjan Farid, Roger F. Steinert, Sumit Garg, Matthew Wade

112.2

Dissection and Suturing

Marjan Farid, Roger F. Steinert, Sumit Garg, Matthew Wade

112.3

Laser Incision

Marjan Farid, Roger F. Steinert, Sumit Garg, Matthew Wade

112.4

Femto DALK

Marjan Farid, Roger F. Steinert, Sumit Garg, Matthew Wade

117.1

Big Bubble DALK: A Video Presentation

Mohammad Anwar

118.1

Techniques of Anterior Lamellar Keratoplasty: Stromal Delamination

Luigi Fontana

118.2

Techniques of Anterior Lamellar Keratoplasty: ALTK 1

Luigi Fontana

118.3

Techniques of Anterior Lamellar Keratoplasty: ALTK 2

Luigi Fontana

118.4

Techniques of Anterior Lamellar Keratoplasty: Big Bubble

Luigi Fontana

118.5

Techniques of Anterior Lamellar Keratoplasty: Big Bubble Using a Cannula

Luigi Fontana

118.6

Techniques of Anterior Lamellar Keratoplasty: Donor Preparation

Luigi Fontana

119.1

Rupture of DM in a Keratoconus Patient

Shigeto Shimmura

120.1

Cannula Big-Bubble Technique, Bubble Test, and the New Opening of the Bubble

Vincenzo Sarnicola, Enrica Sarnicola, Caterina Sarnicola

120.2

Air-Viscobubble Technique (AVB)

Vincenzo Sarnicola, Enrica Sarnicola, Caterina Sarnicola

120.3

Layer-by-Layer Manual Dissection

Vincenzo Sarnicola, Enrica Sarnicola, Caterina Sarnicola

120.4

dDALK Rupture Management

Vincenzo Sarnicola, Enrica Sarnicola, Caterina Sarnicola

120.5

Subtotal Full Thickness Circular Cut of the Recipient Bed

Vincenzo Sarnicola, Enrica Sarnicola, Caterina Sarnicola

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Video Table of Contents

Clip

Clip title

Video contributor/s

120.6

Total Full Thickness Circular Cut of the Recipient Bed

Vincenzo Sarnicola, Enrica Sarnicola, Caterina Sarnicola

120.7

Excessive Trephination and Perforation

Vincenzo Sarnicola, Enrica Sarnicola, Caterina Sarnicola

120.8

Traumatic Postoperative DM Disinsertion

Vincenzo Sarnicola, Enrica Sarnicola, Caterina Sarnicola

126.1

Eye Bank Preparation of DMEK Graft Tissue Using a Modified SCUBA Technique

Mark A. Greiner, Gregory A. Schmidt, Kenneth M. Goins

130.1

Ultrathin DSAEK

Massimo Busin, Vincenzo Scorcia

131.1

DMEK Injectors

Michael D. Straiko

131.2

Peripheral Iridotomy

Kevin J. Shah, Michael D. Straiko, Mark A. Greiner

132.1

Torn Donor Graft During DMEK Graft Preparation Using SCUBA Technique

Dagny Zhu, Neda Shamie

134.1

DSEK Under Top Hat PKP with Laplace Phenomenon

Matthew T. Feng, Francis W. Price, Jr., Marianne O. Price

134.2

Suture Pull Through and Fixation

Matthew T. Feng, Francis W. Price, Jr., Marianne O. Price

136.1

RHCIII Implantation Surgery

May Griffith, Oleksiy Buznyk, Per Fagerholm

141.1

Primary Pterygium Excision and Conjunctival Autograft with Fibrin Glue

Donald T.H. Tan, Elaine W. Chong

141.2

Recurrent Pterygium Excision and Extensive Tenon’s Excision with Conjunctival Autograft Using Fibrin Glue

Donald T.H. Tan, Elaine W. Chong

143.1

How to Know the Orientation of the Amniotic Membrane

Jose L. Güell, Oscar Gris, Daniel Elies, Felicidad Manero, Merce Morral

143.2

Surgical Videos of Several Cases where Amniotic Membrane Transplantation was Performed

Jose L. Güell, Oscar Gris, Daniel Elies, Felicidad Manero, Merce Morral

143.3

The Modified Gundersen Approach Consists of Four Main Steps

Jose L. Güell, Oscar Gris, Daniel Elies, Felicidad Manero, Merce Morral

143.4

Two-Step Approach to Treat Unilateral Total Limbal Stem Cell Deficiency

Jose L. Güell, Oscar Gris, Daniel Elies, Felicidad Manero, Merce Morral

145.1

Iris Oversew

Michael E. Snyder, Jason H. Bell

145.2

Taco Down Fold of PCIOL for Insertion Behind 50-Series Iris Prostheses

Michael E. Snyder, Jason H. Bell

145.3

CustomFlex Iris Device Injection and Capsular Bag Implantation With Overfolding

Michael E. Snyder, Jason H. Bell

151.1

KPro Assembly and Surgery

Jose de la Cruz

155.1

Boston Keratoprosthesis Type 2 Surgical Techniques

Duna Raoof, James Chodosh

156.1

Stage 1 OOKP

Jean S.M. Chai, Donald T.H. Tan

156.2

Stage 2 OOKP

Jean S.M. Chai, Donald T.H. Tan

158.1

LR-CLAU

Edward J. Holland, Gary S. Schwartz, Sheraz M. Daya, Ali R. Djalilian, Clara C. Chan

158.2

KLAL

Edward J. Holland, Gary S. Schwartz, Sheraz M. Daya, Ali R. Djalilian, Clara C. Chan

158.3

CLAU-KLAL Modified Cincinnati Procedure

Edward J. Holland, Gary S. Schwartz, Sheraz M. Daya, Ali R. Djalilian, Clara C. Chan

160.1

Combined LR-CLAL/KLAL (Cincinnati Procedure) Followed by Penetrating Keratoplasty

Kevin J. Shah, Edward J. Holland, Mark J. Mannis

165.1

LASEK Azar Flap Technique

Dimitri T. Azar, Ramon C. Ghanem

166.1

Technique for Custom Femtosecond LASIK

Louis E. Probst

167.1

Femtosecond LASIK

Patricia B. Sierra, David R. Hardten

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Video Table of Contents

Clip

Clip title

Video contributor/s

169.1

Anterior Chamber Gas Bubbles Blocking the Tracking of the Pupil

Louis E. Probst, Clara C. Chan, Mario J. Saldanha

171.1

Small Incision Lenticule Extraction

Jodhbir Mehta

172.1

Post LASIK Ectasia – Intrastromal CXL and Ring

Adimara da Candelaria Renesto, Mauro Campos

173.1

Femtosecond Laser Astigmatic Keratotomy

Zaina Al-Mohtaseb, Leela V. Raju, Li Wang, Mitchell P. Weikert, Douglas D. Koch

174.1

Implantation of an Artisan Lens

Thomas Kohnen, Mehdi Shajari, Jose L. Güell, Daniel Kook, Rudy M.M.A. Nuijts

174.2

Implantation of a Toric Artisan Lens

Thomas Kohnen, Mehdi Shajari, Jose L. Güell, Daniel Kook, Rudy M.M.A. Nuijts

174.3

Implantation of an Artiflex Lens

Thomas Kohnen, Mehdi Shajari, Jose L. Güell, Daniel Kook, Rudy M.M.A. Nuijts

174.4

Implantation of an ICL

Thomas Kohnen, Mehdi Shajari, Jose L. Güell, Daniel Kook, Rudy M.M.A. Nuijts

174.5

Performing LASIK in a Patient with an Iris-Fixated Lens

Thomas Kohnen, Mehdi Shajari, Jose L. Güell, Daniel Kook, Rudy M.M.A. Nuijts

175.1

Implanting the Kamra Small-Aperture Inlay

Richard L. Lindstrom, Jay S. Pepose, John A. Vukich

Total video running time: 2 hours 41 minutes

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Preface The subspecialty of cornea and external disease has undergone significant transformation since the last edition of Cornea. New diagnostic technology has vastly improved our ability to detect disease. New medications and other therapeutic options have changed the treatment paradigm for many disorders. And new surgical techniques have had a remarkable impact on surgical outcomes. Not only do these new procedures provide better outcomes but, in addition, we can now offer surgical options earlier in the disease process. This edition of Cornea consists of numerous new chapters and extensive revisions of existing chapters, as well as many

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new surgical videos in order to keep pace with the dramatic innovation in our field. The editors and authors have strived to provide the most current material available for residents, fellows, clinicians, and researchers to help in the management of patients with cornea and external disease. We hope this edition will benefit patients for years to come. Mark J. Mannis Edward J. Holland

Acknowledgements This fourth edition of Cornea is the result of an extraordinary, coordinated effort by many talented individuals. We cannot thank our contributing authors and co-authors enough. We appreciate their excellent contributions of the most current information on the diagnosis and management of cornea and external disease. In addition, we thank these authors for adhering to the tight editorial requirements in order to keep this textbook current and on time. We have enjoyed our continued relationship with our publisher, Elsevier, who shares our goal: to provide the

highest quality medical textbook possible both in print and electronic format. Special thanks to Sharon Nash and Russell Gabbedy at Elsevier, who have motivated and guided us along this journey. Finally, we would like to thank our families, who continue to provide support and the considerable time needed to produce what we hope is an outstanding textbook.

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We dedicate this book to patients suffering from corneal blindness that we cannot help at this time but hope to be able to help in the future. Mark J. Mannis Edward J. Holland

Acknowledgement to the Founding Editors Jay H. Krachmer MD Mark J. Mannis MD FACS Edward J. Holland MD First published in 1997 Cornea established itself as the market-leading comprehensive title covering fundamentals, diagnosis, medical and surgical treatment of cornea and related external disorders. Continually used by practitioners and trainees throughout the world, what started out as a three volume book-set has now evolved into a complete multimedia resource delivering content in print, online,

ebook, and video formats. We acknowledge the founding editorial team who brought this project to fruition through their tireless efforts, expertise, and devotion that started out over 20 years ago. Elsevier

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List of Contributors Richard L Abbott MD Thomas W. Boyden Endowed Chair in Ophthalmology Health Sciences Professor Cornea and External Diseases UCSF Department of Ophthalmology Research Associate Francis I. Proctor Foundation San Francisco, CA, USA Chapter 115 Nisha R Acharya MD

Professor of Ophthalmology Director, Uveitis Service F.I. Proctor Foundation University of California, San Francisco San Francisco, CA, USA Chapter 101

Anthony J Aldave MD

Walton Li Chair in Cornea and Uveitis Chief, Cornea and Uveitis Division Director, Cornea and Refractive Surgery Fellowship The Jules Stein Eye Institute Los Angeles, CA, USA Chapter 70

Eduardo C Alfonso MD

Medical Director, Ocular Microbiology Laboratory Professor, Edward W D Norton Chair in Ophthalmology Bascom Palmer Eye Institute University of Miami Miami, FL, USA Chapter 80

Richard C Allen MD, PhD, FACS

Professor Section of Ophthalmology Department of Head and Neck Surgery University of Texas MD Anderson Cancer Center Houston, TX, USA Chapter 28

Zaina Al-Mohtaseb MD

Assistant Professor; Associate Residency Program Director Department of Ophthalmology Baylor College of Medicine Houston, TX, USA Chapter 173

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M Camille Almond MD

Mohammad Anwar FRCSEd, FRCOphth Senior Consultant Ophthalmic Surgeon Cornea and External Diseases Magrabi Eye Hospital Dubai, UAE Chapter 117

Shomoukh Al-Shamekh MD Research Fellow Department of Ophthalmology King Saud University Riyadh, Saudi Arabia Chapter 58

Penny A Asbell MD, FACS, MBA

Comprehensive Ophthalmology, Cornea, Refractive and Ocular Surface Disease Partner BayCare Eye Specialists Green Bay, WI, USA Chapter 103

Lênio Souza Alvarenga MD, PhD Country Medical Director-Brazil Roche Pharmaceuticals São Paulo, Brazil Chapter 42 Wallace LM Alward MD

Professor Frederick C. Blodi Chair in Ophthalmology Department of Ophthalmology and Visual Sciences University of Iowa Carver College of Medicine Iowa City, IA, USA Chapter 56

Renato Ambrósio Jr MD, PhD

Associate Professor of Post Graduation in Ophthalmology Universidade Federal de São Paulo & Pontific Catholic University of Rio de Janeiro Rio de Janeiro, Brazil Chapters 13; 168

Andrea Y Ang MBBS, MPH, FRANZCO Consultant Ophthalmologist Lions Eye Insitute Royal Perth Hospital Perth, Australia Chapter 54

Marcus Ang MBBS, MMED, MCI, FAMS,

FRCSEd Consultant Corneal and External Eye Disease Service Singapore National Eye Centre Singapore Chapter 127

Professor of Ophthalmology Director of Cornea and Refractive Services Director of the Cornea Fellowship Program Department of Ophthalmology Icahn School of Medicine at Mount Sinai New York, NY, USA Chapter 98

Dimitri T Azar MD

B.A. Field Chair of Ophthalmologic Research Professor and Head Department of Ophthalmology and Visual Science Illinois Eye and Ear Infirmary Chicago, IL, USA Chapters 164; 165

Irit Bahar MD, MHA Associate Professor Ophthalmology Department Rabin Medical Center Tel Aviv University Tel Aviv, Israel Chapter 52 Annie K Baik MD

Associate Professor Department of Ophthalmology University of California, Davis Sacramento, CA, USA Chapter 116

Neal P Barney MD Professor Department of Ophthalmology and Visual Sciences University of Wisconsin School of Medicine and Public Health Madison, WI, USA Chapter 47

List of Contributors

Brendan C Barry BA

Clinical Research Coordinator Ophthalmology Icahn School of Medicine at Mount Sinai New York, NY, USA Chapter 98

Joseph M Biber MD Cornea, Cataract, and Refractive Specialist Partner Horizon Eye Care Charlotte, NC, USA Chapters 76; 100

Allon Barsam MD, MA, FRCOphth

Andrea D Birnbaum MD, PhD

Director Cornea and Refractive Surgery Department of Ophthalmology Luton and Dunstable University Hospital UCL Partners UK Chapter 147

Rebecca M Bartow MD

Department of Ophthalmology Marshfield Clinic Marshfield, WI, USA Chapter 62

Assistant Professor of Ophthalmology Northwestern University Feinberg School of Medicine Chicago, IL, USA Chapters 101; 106

Kelley J Bohm BS Cornea Clinical Research Fellow Department of Ophthalmology Weill Cornell Medical College New York City, NY, USA Chapter 33

Jules Baum MD

Research Professor Department of Ophthalmology Tufts University School of Medicine Boston, MA, USA Chapter 41

Charles S Bouchard MD, MA John P. Mulcahy Professor and Chairman Department of Ophthalmology Loyola University Health System Maywood, IL, USA Chapter 5

Michael W Belin MD

Jay C Bradley MD

Professor of Ophthalmology and Vision Science University of Arizona Tucson, AZ, USA Chapters 13; 154; 163

West Texas Eye Associates Cornea, External Disease, & Refractive Surgery Lubbock, TX, USA Chapter 113

Jason H Bell MD

James D Brandt MD

Senior Resident University of Cincinnati University of Cincinnati Medical Center Cincinnati, OH, USA Chapter 145

Beth Ann Benetz CRA, FOPS

Professor Case Western Reserve University University Hospitals Case Medical Center Cleveland, OH, USA Chapter 14

Roger W Beuerman PhD Senior Scientific Director and Professor Singapore Eye Research Institute and Duke-NUS SRP Neuroscience and Behavioral Disorders Singapore Chapter 3

Professor & Vice-Chair of International Programs and New Technology Director, Glaucoma Service Department of Ophthalmology & Vision Science University of California, Davis Sacramento, CA, USA Chapter 116

Cat N Burkat MD, FACS Associate Professor Oculoplastic, Orbital, & Facial Cosmetic Surgery Department of Ophthalmology and Visual Sciences University of Wisconsin-Madison Madison, WI, USA Chapter 27 Massimo Busin MD

Professor of Ophthalmology “Villa Igea” Private Hospital Forli, Italy Chapter 130

Oleksiy Buznyk MD, PhD

Cornea and Oculoplastic Surgeon Department of Eye Burns, Ophthalmic Reconstructive Surgery, Keratoplasty and Keratoprosthesis Filatov Institute of Eye Diseases & Tissue Therapy of the NAMS of Ukraine Odessa, Ukraine Chapter 136

J Douglas Cameron MD, MBA Professor Departments of Ophthalmology and Visual Neuroscience and Laboratory Medicine and Pathology University of Minnesota School of Medicine Minneapolis, MN, USA Chapters 2; 38 Mauro Campos MD Professor Department of Ophthalmology and Visual Sciences Federal University of São Paulo São Paulo, Brazil Chapter 172 Emmett F Carpel MD

Adjunct Professor Department of Ophthalmology University of Minnesota Minneapolis, MN, USA Chief, Division of Ophthalmology Minneapolis VA Health Care System Minneapolis, MN, USA Medical Director and Chief of Staff Phillips Eye Institute Minneapolis, MN, USA Chapter 73

H Dwight Cavanagh MD, PhD, FACS Professor and Vice Chair Emeritus of Ophthalmology Medical Director, Transplant Services Center at UT Southwestern Medical Center Dallas, TX, USA Chapter 15 Jean SM Chai MBBS, FAMS, FRCSEd Consultant Corneal and External Eye Disease Service Singapore National Eye Centre Singapore Chapter 156 Winston Chamberlain MD, PhD

Associate Professor Department of Ophthalmology Casey Eye Institute Oregon Health and Science University Portland, OR, USA Chapter 17

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List of Contributors

Clara C Chan MD, FRCSC, FACS

Assistant Professor Department of Ophthalmology and Vision Sciences University of Toronto Toronto, ON, Canada Chapters 110; 153; 158; 169

Bernard H Chang MD

Private Practice Cornea Consultants of Nashville Nashville, TN, USA Chapter 87

Edwin S Chen MD

Cornea and Anterior Segment Scripps Memorial Hospital, La Jolla La Jolla, CA, USA Chapter 129

Michael C Chen MD Assistant Professor of Ophthalmology Penn State Eye Center Penn State Milton S. Hershey Medical Center Hershey, PA, USA Chapter 111 Neil Chen BSc

Clinical Intern Comite MD New York, NY, USA Chapter 98

Kenneth C Chern MD, MBA

Managing Partner, Peninsula Ophthalmology Group Associate Clinical Professor Department of Ophthalmology and Visual Sciences University of California, San Francisco and the Francis I. Proctor Foundation San Francisco, CA, USA Chapter 79

James Chodosh MD, MPH DG Cogan Professor of Ophthalmology Massachusetts Eye and Ear Infirmary Harvard Medical School Boston, MA, USA Chapters 150; 155 Elaine W Chong MBBS, MEpi, PhD, FRANZCO Consultant Ophthalmologist Royal Victorian Eye & Ear Hospital Melbourne, Victoria, Australia Chapter 141

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Mazen Y Choulakian MD, FRCSC

Assistant Professor of Ophthalmology Faculty of Medicine and Health Sciences Université de Sherbrooke Sherbrooke, QC, Canada Chapters 42; 61

Gary Chung MD

Private Practice Evergreen Eye Centers Federal Way, WA, USA Chapter 91

Joseph B Ciolino MD

Assistant Professor of Ophthalmology Department of Ophthalmology Massachusetts Eye and Ear Infirmary Boston, MA, USA Chapter 154

Jessica Ciralsky MD

Assistant Professor Department of Ophthalmology Weill Cornell Medical College New York, NY, USA Chapter 5

Maria Soledad Cortina MD

Assistant Professor of Ophthalmology University of Illinois Eye and Ear Infirmary Department of Ophthalmology and Visual Sciences Chicago, IL, USA Chapters 90; 165

Alexandra Z Crawford BA, MBChB Ophthalmology Registrar Department of Ophthalmology University of Auckland Auckland, New Zealand Chapter 94

Jose de la Cruz MD Assistant Professor Department of Ophthalmology University of Illinois Eye and Ear Infirmary Chicago, IL, USA Chapter 151 Mausam R Damani MD Cornea Fellow Department of Ophthalmology & Vision Science UC Davis Eye Center Sacramento, CA, USA Chapter 108

Paulo Elias C Dantas MD, PhD

Professor of Ophthalmology Department of Ophthalmology, Corneal and External Disease Service Santa Casa of São Paulo São Paulo, Brazil Chapter 84

Mahshad Darvish-Zargar MDCM, MBA, FRCSC Assistant Professor Department of Ophthalmology McGill University Montreal, QC, Canada Chapters 59; 62

Richard S Davidson MD

Professor of Ophthalmology and Vice Chair for Quality and Clinical Affairs Cataract, Cornea, and Refractive Surgery University of Colorado Eye Center University of Colorado School of Medicine Aurora, CO, USA Chapter 85

Sheraz M Daya MD, FACP, FACS, FRCS(Ed),

FRCOphth Medical Director Centre for Sight London, UK Chapters 153; 158

Ali R Djalilian MD Associate Professor of Ophthalmology Department of Ophthalmology and Visual Sciences University of Illinois at Chicago Chicago, IL, USA Chapters 33; 124; 153; 158; 159 Eric D Donnenfeld MD, FACS

Clinical Professor of Ophthalmology New York University Medical Center Ophthalmology New York, NY, USA Chapters 138; 147

Steven P Dunn MD Professor Department of Ophthalmology Oakland University William Beaumont School of Medical Rochester, MI, USA Chapter 48 Ralph C Eagle Jr MD

Director, Department of Pathology Wills Eye Hospital Philadelphia, PA, USA Chapter 18

List of Contributors

Allen O Eghrari MD

Assistant Professor of Ophthalmology Wilmer Eye Institute Johns Hopkins University School of Medicine Baltimore, MD, USA Chapter 11

Richard A Eiferman MD, FACS

Clinical Professor of Ophthalmology University of Louisville Louisville, KY, USA Chapter 138

Joseph A Eliason MD Clinical Professor of Ophthalmology Department of Ophthalmology Stanford University School of Medicine Palo Alto, CA, USA Chapter 30 Daniel Elies MD Cornea and Refractive Surgery Unit Instituto de Microcirugía Ocular (IMO) Barcelona, Spain Chapter 143 Per Fagerholm MD, PhD

Professor Emeritus Department of Clinical and Experimental Medicine – Ophthalmology Faculty of Health University of Linköping Linköping, Sweden Chapter 136

Marjan Farid MD

Associate Professor of Ophthalmology Director of Cornea, Cataract, and Refractive Surgery Gavin Herbert Eye Institute University of California, Irvine Irvine, CA, USA Chapters 112; 170

Asim V Farooq MD Fellow in Cornea and External Disease Department of Ophthalmology and Visual Sciences Washington University in St. Louis St. Louis, MO, USA Chapter 124 William J Faulkner MD

Director Urgents Clinic Cincinnati Eye Institute Cincinnati, OH, USA Chapter 9

Blake V Fausett MD, PhD Oculoplastics Fellow Cincinnati Eye Institute University of Cincinnati College of Medicine Department of Ophthalmology Cincinnati, OH, USA Chapter 28 Robert S Feder MD, MBA

Professor of Ophthalmology Chief Cornea/External Disease Northwestern University Feinberg School of Medicine Chicago, IL, USA Chapter 72

Vahid Feiz MD Private Practice California Eye Clinic Walnut Creek, CA, USA Chapter 21 Sergio Felberg MD

Professor of Ophthalmology Department of Ophthalmology, Corneal and External Disease Service Santa Casa of São Paulo São Paulo, Brazil Chapter 84

Denise de Freitas MD

Professor Department of Ophthalmology and Visual Sciences Paulista School of Medicine, São Paulo Hospital, Federal University of São Paulo (UNIFESP) São Paulo, Brazil Chapters 32; 123

Anat Galor MD, MSPH

Staff Physician, Associate Professor of Clinical Ophthalmology Bascom Palmer Eye Institute University of Miami Miami, FL, USA Chapter 80

Prashant Garg MD Consultant Ophthalmologist, Tej Kohli Cornea Institute Senior Ophthalmologist, Tej Kohli Cornea Institute, Kallam Anji Reddy Campus L V Prasad Eye Institute Hyderabad, India Chapters 82; 92 Sumit Garg MD

Assistant Professor Vice Chair of Clinical Ophthalmology Gavin Herbert Eye Institute University of California, Irvine Irvine, CA, USA Chapters 112; 170

Matthew T Feng MD Private Practice Price Vision Group Indianapolis, IN, USA Co-Medical Director Indiana Lions Eye & Tissue Transplant Bank Indianapolis, IN, USA Chapter 134

William G Gensheimer MD, Maj, USAF

Luigi Fontana MD, PhD Director Ophthalmic Unit Arcispedale Santa Maria Nuova – IRCCS Reggio Emilia, Italy Chapter 118

Elham Ghahari MD

Gary N Foulks MD

Emeritus Professor Department of Ophthalmology and Vision Science University of Louisville Louisville, KY, USA Chapters 31; 114

Chief of Cornea Service Warfighter Eye Center Malcolm Grow Medical Clinics and Surgery Center (MGMCSC) Joint Base Andrews, MD, USA Chapter 85

Cornea Research Fellow Department of Ophthalmology and Visual Sciences University of Illinois at Chicago Chicago, IL, USA Chapter 159

David B Glasser MD Assistant Professor Department of Ophthalmology Johns Hopkins University School of Medicine Baltimore, MD, USA Chapter 26

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List of Contributors

Kenneth M Goins MD

Steven A Greenstein MD

Kimberly K Gokoffski MD, PhD Resident Physician Department of Ophthalmology & Vision Science Univerisity of California, Davis School of Medicine Sacramento, CA, USA Chapters 4; 35

Darren G Gregory MD

Professor of Clinical Ophthalmology Corneal and External Diseases University of Iowa Hospitals and Clinics Iowa City, IA, USA Chapters 9; 126

Debra A Goldstein MD, FRCSC Professor Director, Uveitis Service Director, Uveitis Fellowship Department of Ophthalmology Northwestern University Feinberg School of Medicine Chicago, IL, USA Chapter 106 Jeffrey R Golen MD

Assistant Professor in Cornea, External Disease, and Refractive Surgery University of Virginia Department of Ophthalmology Charlottesville, VA, USA Chapter 79

Jose Gomes MD, PhD

Associate Professor & Director Anterior Segment & Ocular Surface Advanced Center Department of Ophthalmology Federal University of Sao Paulo (UNIFESP/EPM) Sao Paulo, SP, Brazil Chapter 157

John A Gonzales MD

Assistant Professor Department of Ophthalmology F.I. Proctor Foundation University of California, San Francisco San Francisco, CA, USA Chapter 101

John D Gottsch MD Professor of Ophthalmology Wilmer Eye Institute Johns Hopkins University School of Medicine Baltimore, MD, USA Chapter 11

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Cornea Fellow Cornea and Laser Eye Institute – Hersh Vision Group Teaneck, NJ, USA Department Ophthalmology Harvard Medical School Boston, MA, USA Chapter 148

Associate Professor Department of Ophthalmology University of Colorado School of Medicine Denver, CO, USA Chapter 50

Mark A Greiner MD Assistant Professor Cornea and External Diseases Department of Ophthalmology and Visual Sciences University of Iowa Carver College of Medicine Iowa City, IA, USA Chapters 9; 126; 131 May Griffith PhD

Professor of Regenerative Medicine Department of Clinical and Experimental Medicine Linköping University Linköping, Sweden Chapter 136

Oscar Gris MD Cornea and Refractive Surgery Unit Instituto de Microcirugía Ocular (IMO) Barcelona, Spain Chapter 143 Erich B Groos Jr MD Partner Cornea Consultants of Nashville Nashville, TN, USA Chapter 87 William D Gruzensky MD

Pacific Cataract and Laser Institute Olympia, WA, USA Chapter 45

Jose L Güell MD Director of Cornea and Refractive Surgery Unit IMO. Instituto Microcirugia Ocular of Barcelona Associate Professor of Ophthalmology UAB. Autònoma University of Barcelona Barcelona, Spain Chapters 143; 174

Frederico P Guerra MD

Medical Director of Centro Oftalmológico Jardim Icaraí Rio de Janeiro, Brazil Director of the Cornea Department of the Federal Hospital of Ipanema Rio de Janeiro, Brazil Chapter 168

Preeya K Gupta MD

Assistant Professor of Ophthalmology Cornea and Refractive Surgery Duke University Eye Center Durham, NC, USA Chapters 55; 114

M Bowes Hamill MD Professor of Ophthalmology Cullen Eye Institute Baylor College of Medicine Department of Ophthalmology Houston, TX, USA Chapters 93; 146 Kristin M Hammersmith MD

Assistant Surgeon Cornea Service Wills Eye Hospital Instructor, Thomas Jefferson Medical College Philadelphia, PA, USA Chapter 18

Pedram Hamrah MD, FACS

Director, Center for Translational Ocular Immunology Director, Anterior Segment Imaging, Boston Image Reading Center Cornea Service, New England Eye Center Tufts Medical Center Associate Professor, Department of Ophthalmology Tufts University School of Medicine Boston, MA, USA Chapter 124

Sadeer B Hannush MD Attending Surgeon Cornea Service, Wills Eye Hospital Department of Ophthalmology Sidney Kimmel Medical College of Thomas Jefferson University Medical Director Lions Eye Bank of Delaware Valley Philadelphia, PA, USA Chapters 109; 152

List of Contributors

David R Hardten MD Minnesota Eye Consultants Director of Research, Cornea, Refractive Surgery Department of Ophthalmology University of Minnesota & Minnesota Eye Consultants Minnetonka, MN, USA Chapter 167 David G Heidemann MD Department of Ophthalmology William Beaumont Hospital Royal Oak, MI, USA Chapter 48 Peter S Hersh MD

Clinical Professor Director, Cornea and Refractive Surgery Department of Ophthalmology Rutgers Medical School Newark, NJ, USA Chapter 148

Darren C Hill MD

Resident Physician, PGY1 Penn State College of Medicine Hershey, PA, USA Chapter 138

Ana Luisa Hofling-Lima MD Head Professor at Ophthalmology Department Escola Paulista de Medicina UNIFESP/EPM São Paulo, Brazil Chapter 149 Edward J Holland MD

Director, Cornea Services Cincinnati Eye Institute Professor of Clinical Ophthalmology University of Cincinnati Cincinnati, OH, USA Chapters 50; 53; 77; 108; 153; 157; 158; 159; 160

Gary N Holland MD

Professor of Ophthalmology Jack H. Skirball Chair in Ocular Inflammatory Diseases Cornea-External Ocular Disease Division / Uveitis Service Department of Ophthalmology David Geffen School of Medicine at UCLA UCLA Stein Eye Institute Los Angeles, CA, USA Chapter 65

Stephen Holland MD Resident Department of Ophthalmology Loyola University Chicago Stritch School of Medicine Maywood, IL, USA Chapter 103

David Huang MD, PhD Peterson Professor of Ophthalmology Department of Ophthalmology Casey Eye Institute Oregon Health and Science University Portland, OR, USA Chapter 17

Augustine R Hong MD

Jennifer I Hui MD, FACS Founder The Eyelid Institute (Palm Desert, CA) Assistant Professor Department of Ophthalmology Loma Linda University School of Medicine Loma Linda, CA, USA Chapter 29

Assistant Professor Department of Ophthalmology Washington University St. Louis, MO, USA Chapter 75

Marc A Honig MD

Clinical Instructor Wilmer Eye Institute, Johns Hopkins University School of Medicine Clinical Assistant Professor Department of Ophthalmology and Visual Sciences University of Maryland School of Medicine Baltimore, MD, USA Chapter 137

Christopher T Hood MD Clinical Assistant Professor Ophthalmology and Visual Sciences W.K. Kellogg Eye Center University of Michigan Medical School Ann Arbor, MI, USA Chapter 65 Eliza N Hoskins MD Cornea Consultant The Permanente Medical Group Walnut Creek, CA, USA Chapter 115 Joshua H Hou MD Assistant Professor Department of Ophthalmology and Visual Neurosciences University of Minnesota Minneapolis, MN, USA Chapter 2 Kimberly Hsu MD

Clinical Fellow Department of Ophthalmology University of Illinois Eye and Ear Infirmary Chicago, IL, USA Chapter 151

Andrew JW Huang MD, MPH Professor of Ophthalmology Department of Ophthalmology and Visual Sciences Washington University St. Louis, MO, USA Chapter 75

Alfonso Iovieno MD, PhD

Cornea and Ocular Surface Unit Arcispedale Santa Maria Nuova – IRCCS Reggio Emilia, Italy Chapter 118

Joseph D Iuorno MD

Commonwealth Eye Care Associates Richmond, VA, USA Chapter 91

W Bruce Jackson MD, FRCSC Professor Department of Ophthalmology University of Ottawa Eye Institute Ottawa, ON, Canada Chapter 164 Deborah S Jacobs MD

Medical Director Boston Foundation for Sight Needham, MA, USA Associate Professor of Ophthalmology Harvard Medical School Boston, MA, USA Chapter 97

Frederick A Jakobiec MD, DSc Henry Willard Williams Professor Emeritus of Ophthalmology and Pathology Former Chief and Chairman Department of Ophthalmology Massachusetts Eye and Ear Infirmary and Harvard Medical School Director, David Glendenning Cogan Laboratory of Ophthalmic Pathology Massachusetts Eye and Ear Infirmary Boston, MA, USA Chapters 36; 39

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List of Contributors

Bennie H Jeng MD, MS Professor and Chair Department of Ophthalmology and Visual Sciences University of Maryland School of Medicine Baltimore, MD, USA Chapters 65; 115 James V Jester PhD

Professor of Ophthalmology Gavin Herbert Eye Institute University of California, Irvine Orange, CA, USA Chapter 15

Madhura G Joag MD

Research Scholar Cornea Bascom Palmer Eye Institute Miami, FL, USA Chapter 20

David R Jordan MD, FRCSC Professor of Ophthalmology University of Ottawa Eye Institute Ottawa, ON, Canada Chapter 34 Raageen Kanjee MD Ophthalmology Resident Department of Ophthalmology University of Manitoba Winnipeg, MB, Canada Chapter 106 Carol L Karp MD

Professor of Ophthalmology Bascom Palmer Eye Institute University of Miami Miami, FL, USA Chapters 17; 20; 37

Stephen C Kaufman MD, PhD

Professor and Vice-Chairman of Ophthalmology Director of Cornea and Refractive Surgery State University of New York – Downstate Brooklyn and Manhattan, NY, USA Chapter 19

Jeremy D Keenan MD, MPH

Associate Professor of Ophthalmology Francis I. Proctor Foundation and Department of Ophthalmology University of California, San Francisco San Francisco, CA, USA Chapter 43

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Robert C Kersten MD, FACS

Shigeru Kinoshita MD, PhD

Stephen S Khachikian MD Cornea Fellow Department of Ophthalmology Albany Medical College Albany, NY, USA Chapter 163

Colin M Kirkness

Professor of Clinical Ophthalmology University of California, San Francisco San Francisco, CA, USA Chapter 27

Rohit C Khanna MD Director Gullapalli Pratibha Rao International Centre for Advancement of Rural Eye Care Consultant Ophthalmologist, Tej Kohli Cornea Institute L V Prasad Eye Institute Hyderabad, India Chapter 82 Timothy T Khater MD, PhD Cornea, External Disease, Cataract, & Refractive Surgery Specialist West Texas Eye Associates Lubbock, TX, USA Chapter 113 Eric J Kim BS

Research Fellow in Cataract and Refractive Surgery Department of Ophthalmology Baylor College of Medicine Houston, TX, USA Chapter 12

Michelle J Kim MD

Resident Physician Department of Ophthalmology Duke Eye Center Durham, NC, USA Chapter 55

Stella K Kim MD

Joe M. Green Jr. Professor of Clinical Ophthalmology Ruiz Department of Ophthalmology and Visual Science University of Texas Health, Medical School Houston, TX, USA Chapter 66

Terry Kim MD Professor of Ophthalmology Duke University School of Medicine Chief, Cornea and External Disease Service Director, Refractive Surgery Service Duke University Eye Center Durham, NC, USA Chapter 55

Professor and Chair Department of Frontier Medical Science and Technology for Ophthalmology Kyoto Prefectural University of Medicine Kyoto, Japan Chapter 135

Formerly Tennent Professor of Ophthalmology Department of Ophthalmology Faculty of Medicine University of Glasgow Glasgow, UK Chapter 60

Stephen D Klyce PhD, FARVO Adjunct Professor of Ophthalmology Icahn School of Medicine at Mount Sinai New York, NY, USA Chapter 12 Douglas D Koch MD

Professor and Allen Mosbacher, and Law Chair in Ophthalmology Cullen Eye Institute, Department of Ophthalmology Baylor College of Medicine Houston, TX, USA Chapter 173

Thomas Kohnen MD, PhD, FEBO Professor and Chair Department of Ophthalmology Goethe University Frankfurt, Germany Visiting Professor Cullen Eye Institute Baylor College of Medicine Houston, TX, USA Chapter 174

Noriko Koizumi MD, PhD Professor Department of Biomedical Engineering Doshisha University Kyotanabe, Japan Chapter 135 Daniel Kook MD, PhD

Smile Eyes Eye Clinic Munich Airport, Germany Chapter 174

Regis P Kowalski MS, M(ASCP) Professor Department of Ophthalmology School of Medicine University of Pittsburgh Pittsburgh, PA, USA Chapter 10

List of Contributors

Friedrich E Kruse MD

Professor and Chair of Ophthalmology Department of Ophthalmology University of Erlangen-Nuremberg Erlangen, Germany Chapter 133

Edward Lai MD

Assistant Professor of Ophthalmology Department of Ophthalmology Weill Cornell Medical College New York, NY, USA Chapter 5

Peter R Laibson MD Director Emeritus Cornea Department Wills Eye Hospital Philadelphia, PA, USA Chapter 69 Jonathan H Lass MD

Charles I Thomas Professor Department of Ophthalmology and Visual Sciences Case Western Reserve University University Hospitals Eye Institute Cleveland, OH, USA Chapter 14

Samuel H Lee MD Cornea and External Disease Sacramento Eye Consultants Sacramento, CA, USA Chapter 64 W Barry Lee MD, FACS

Medical Director, Georgia Eye Bank Cornea and Refractive Surgery Service Eye Consultants of Atlanta/Piedmont Hospital Atlanta, GA, USA Chapters 78; 128

Michael A Lemp MD

Clinical Professor of Ophthalmology Georgetown University and George Washington University Washington, DC, USA Chapters 3; 8; 31

Jennifer Y Li MD

Associate Professor Department of Ophthalmology & Vision Science University of California, Davis Sacramento, CA, USA Chapters 23; 89; 125

Yan Li PhD Research Assistant Professor Department of Ophthalmology Casey Eye Institute Oregon Health and Science University Portland, OR, USA Chapter 17 Thomas M Lietman MD Professor, Director of Francis I. Proctor Foundation Departments of Ophthalmology and Epidemiology and Biostatistics University of California, San Francisco San Francisco, CA, USA Chapter 43 Michele C Lim MD

Professor of Ophthalmology Vice Chair and Medical Director UC Davis Eye Center University of California, Davis School of Medicine Sacramento, CA, USA Chapter 116

Lily Koo Lin MD

Associate Professor Department of Ophthalmology & Vision Science University of California, Davis Health System Sacramento, CA, USA Chapters 4; 35

T Peter Lindquist MD

Associate Medical Director, Georgia Eye Bank SouthEast Eye Specialists Cornea, External Disease and Refractive Surgery Chattanooga, TN, USA Chapters 40; 128

Thomas D Lindquist MD, PhD Formerly Director, Cornea and External Disease Service Department of Ophthalmology Group Health Cooperative Bellevue, WA, USA Clinical Professor Department of Ophthalmology University of Washington School of Medicine Seattle, WA, USA Medical Director, SightLife Seattle, WA, USA Chapters 40; 44

Timothy P Lindquist MD Durrie Vision Overland Park, KS, USA Clinical Instructor Department of Ophthalmology University of Kansas Kansas City, KS, USA Chapter 44 Richard L Lindstrom MD Founder and Attending Surgeon, Minnesota Eye Consultants Adjunct Clinical Professor Emeritus University of Minnesota Department of Ophthalmology Associate Director Minnesota Lions Eye Bank Board Member University of Minnesota Foundation Visiting Professor UC Irvine, Gavin Herbert Eye Institute Irvine, CA, USA Chapter 175 David Litoff MD Kaiser Permanente Chief of Ophthalmology Assistant Clinical Professor Department of Ophthalmology University of Colorado Lafayette, CO, USA Chapter 24 Yu-Chi Liu MD, MCI Clinician Cornea and External Eye Disease Service Singapore National Eye Centre Singapore Chapter 171 Eitan Livny MD

Anterior Segment, Cornea and Cataract Specialist Department of Ophthalmology Rabin Medical Center Petach Tiqva, Israel Chapter 52

Lorena LoVerde MD

Associate Professor Department of Ophthalmology University of Cincinnati Cincinnati, OH, USA Chapter 157

Careen Y Lowder MD, PhD Staff, Cole Eye Institute Cleveland Clinic Foundation Cleveland, OH, USA Chapter 65

xxv

List of Contributors

Allan Luz MD, PhD Corneal Director of Hospital de Olhos de Sergipe Department of Ophthalmology and Visual Science Federal University of São Paulo São Paulo, Brazil Chapter 13 Marian S Macsai MD

Chief, Division of Ophthalmology NorthShore University HealthSystem Professor, University of Chicago Pritzker School of Medicine Glenview, IL, USA Chapters 139; 144

Mark Maio FOPS

President InVision, Inc Alpharetta, GA, USA Chapter 7

Jackie V Malling RN, CEBT

Chief Strategy Officer Saving Sight Kansas City, KS, USA Chapter 25

Amanda C Maltry MD

Assistant Professor Department of Ophthalmology and Visual Neuroscience University of Minnesota School of Medicine Minneapolis, MN, USA Chapter 38

Paramdeep S Mand MD Associate Department of Ophthalmology Kaiser Permanente Riverside, CA, USA Chapter 30 Felicidad Manero MD

Ophthalmology IMO (Instituto de Microcirugía Ocular) Barcelona, Spain Chapter 143

Mark J Mannis MD, FACS

Natalie Fosse Endowed Chair in Vision Science Research Professor and Chair Department of Ophthalmology & Vision Science University of California Davis Eye Center Sacramento, CA, USA Chapters 42; 51; 60; 70; 108; 111; 125; 142; 160

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Tova Mannis MD Clinical Fellow F.I. Proctor Foundation University of California, San Francisco San Francisco, CA, USA Chapter 60

Woodford S Van Meter MD

Carlos E Martinez MD, MS

Jay J Meyer MD, MPH Cornea and Anterior Segment Fellow Department of Ophthalmology New Zealand National Eye Centre University of Auckland Auckland, New Zealand Chapter 94

Chairman of Ophthalmology Long Beach Memorial Hospital Long Beach, CA, USA Chapter 12

Csaba L Mártonyi FOPS Emeritus Associate Professor Department of Ophthalmology and Visual Sciences University of Michigan Medical School Ann Arbor, MI, USA Chapter 7 Maite Sainz de la Maza MD, PhD Associate Professor Department of Ophthalmology Hospital Clinic of Barcelona Barcelona, Spain Chapter 100 Hall T McGee MD, MS Cornea & External Disease Specialist Everett & Hurite Ophthalmic Association Pittsburgh, PA, USA Chapter 6 Charles NJ McGhee MBChB, PhD, DSc, FRCS, FRCOphth, FRANZCO Maurice Paykel Professor and Chair of Ophthalmology Director, New Zealand National Eye Centre Department of Ophthalmology Faculty of Medical & Health Sciences University of Auckland Auckland, New Zealand Chapter 94 Jodhbir Mehta BSc, MBBS, FRCS(Ed) Associate Professor Corneal and External Disease Service Singapore National Eye Centre Singapore Chapter 171 David M Meisler MD Consultant, Cornea and External Diseases Cleveland Clinic Foundation Cleveland, OH, USA Chapter 65

Professor Department of Ophthalmology University of Kentucky School of Medicine Lexington, KY, USA Chapter 110

Shahzad I Mian MD Terry J. Bergstrom Professor Associate Chair, Education Residency Program Director Associate Professor University of Michigan Department of Ophthalmology Ann Arbor, MI, USA Chapter 95 Darlene Miller DHSc, MPH, CIC Research Associate Professor Department of Ophthalmology Bascom Palmer Eye Institute University of Miami Miller School of Medicine Miami, FL, USA Chapter 80 Naoyuki Morishige MD, PhD Associate Professor Department of Ophthalmology Yamaguchi University Graduate School of Medicine Ube, Japan Chapter 1

Merce Morral MD, PhD

Anterior Segment Diseases, Cornea, Cataract and Refractive Surgery Specialist Instituto Oftalmología Ocular (IMO) Barcelona, Spain Chapter 143

Majid Moshirfar MD, FACS Professor of Clinical Ophthalmology, Co-director of Cornea & Refractive Surgery Division Department of Ophthalmology University of California, San Francisco San Francisco, CA, USA Chapter 138 Adam Moss MD, MBA McCannel Eye Clinic Edina, MN, USA Chapter 121

List of Contributors

Asadolah Movahedan MD

Resident of Ophthalmology Department of Ophthalmology University of Chicago Chicago, IL, USA Chapter 124

Parveen Nagra MD Asssistant Surgeon Wills Eye Hospital Assistant Professor Jefferson Medical College Philadelphia, PA, USA Chapter 18 Afshan A Nanji MD, MPH Assistant Professor Department of Ophthalmology Casey Eye Institute Oregon Health and Science University Portland, OR, USA Chapter 17

Jacqueline Ng MD

Karen W Oxford MD

Lisa M Nijm MD, JD Medical & Surgical Director Warrenville EyeCare and LASIK Warrenville, IL, USA Assistant Clinical Professor of Ophthalmology University of Illinois Eye and Ear Infirmary Department of Ophthalmology and Visual Sciences Chicago, IL, USA Chapters 88; 108

David A Palay MD

Department of Ophthalmology Cornea Division University of California, Irvine Gavin Herbert Eye Institute Irvine, CA, USA Chapter 170

Ken K Nischal MD, FRCOphth

Leslie C Neems MD Ophthalmology Resident Physician Northwestern University Feinberg School of Medicine Chicago, IL, USA Chapter 72

Professor of Ophthalmology Director of Pediatric Ophthalmology Strabismus and Adult Motility UPMC Eye Center Children’s Hospital of Pittsburgh of UPMC University of Pittsburgh Pittsburgh, PA, USA Chapter 122

Kristiana D Neff MD

Teruo Nishida MD, DSc

Partner Carolina Cataract & Laser Center Charleston, SC, USA Chapter 53

J Daniel Nelson MD, FACS Professor of Ophthalmology University of Minnesota Associate Medical Director HealthPartners Medical Group Minneapolis, MN, USA Chapter 2 Jeffrey A Nerad MD

Partner, Cincinnati Eye Institute Professor of Ophthalmology University of Cincinnati Cincinnati, OH, USA Chapter 28

Marcelo V Netto MD, PhD Department of Ophthalmology University of São Paulo São Paulo, Brazil Medical Director Instituto Oftalmológico Paulista São Paulo, Brazil Chapter 168

Professor Emeritus Department of Ophthalmology Graduate School of Medicine Yamaguchi University Ube, Japan Chapter 1

M Cristina Nishiwaki-Dantas MD

Professor of Ophthalmology Department of Ophthalmology, Corneal and External Disease Service Santa Casa of São Paulo São Paulo, Brazil Chapter 84

Rudy MMA Nuijts MD, PhD Professor of Ophthalmology University Eye Clinic Maastricht Medical University Center Maastricht Maastricht, The Netherlands Chapter 174 Robert B Nussenblatt MD, MPH Formerly Chief, Laboratory of Immunology National Eye Institute, NIH Bethesda, MD, USA Chapter 101

Director, Cornea and Refractive Surgery Pacific Eye Associates Clinical Professor of Ophthalmology California Pacific Medical Center San Francisco, CA, USA Chapter 115

Associate Clinical Professor Department of Ophthalmology Emory University School of Medicine Atlanta, GA, USA Chapter 22

Sotiria Palioura MD, PhD

Instructor Department of Ophthalmology Bascom Palmer Eye Institute University of Miami Miller School of Medicine Miami, FL, USA Chapter 66

Deval R Paranjpe MD, ScB

Assistant Professor of Ophthalmology Drexel University College of Medicine Pittsburgh, PA, USA Chapter 60

Mansi Parikh MD Assistant Professor Casey Eye Institute Oregon Health and Sciences University Portland, OR, USA Chapter 56 Matthew R Parsons MD Chief Corneal Service Excel Eye Center Provo, UT, USA Chapter 88

Sirichai Pasadhika MD Director of Vitreoretinal Services Devers Eye Institute Legacy Health System Portland, OR, USA Affiliate Instructor Casey Eye Institute Oregon Health & Science University Portland, OR, USA Chapter 102 Dipika V Patel PhD, MRCOphth

Associate Professor Department of Ophthalmology New Zealand National Eye Centre University of Auckland Auckland, New Zealand Chapter 94

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List of Contributors

Charles J Pavlin MD, FRCS(Can)

Stephen C Pflugfelder MD

Eric S Pearlstein MD

Francis W Price Jr MD President Price Vision Group Indianapolis, IN, USA President of the Board Cornea Research Foundation of America Indianapolis, IN, USA Chapter 134

Formerly Professor, University of Toronto Department of Ophthalmology Mount Sinai Hospital Toronto, ON, Canada Chapter 16

Clinical Assistant Professor Department of Ophthalmology SUNY Downstate Medical Center Brooklyn, NY, USA Chapter 99

Jay S Pepose MD, PhD Professor of Clinical Ophthalmology and Visual Sciences Washington University School of Medicine St. Louis, MO, USA Chapter 175

Professor Ophthalmology Baylor College of Medicine Houston, TX, USA Chapter 33

Marianne O Price PhD, MBA Executive Director Cornea Research Foundation of America Indianapolis, IN, USA Chapter 134

Robert J Peralta MD Ophthalmic Plastic and Reconstructive Surgery Kaiser Permanente Oakland, CA, USA Chapter 6

Louis E Probst MD National Medical Director TLC The Laser Eye Centers Chicago, IL, USA Chapters 166; 169

Mauricio A Perez MD

Michael B Raizman MD

Cornea, Cataract & Refractive Surgery Department Clínica de Enfermedades de la Visión / Clínica Las Condes / Hospital Salvador / Fundación Imagina Volunteer Faculty University of Chile Santiago, Chile Chapters 110; 164

Victor L Perez MD Professor of Ophthalmology Walter G. Ross Chair in Ophthalmic Research Bascom Palmer Eye Institute University of Miami Miller School of Medicine Miami, FL, USA Chapters 49; 66 Alicia Perry BA Ophthalmic Consultants of Long Island New York, NY, USA Chapter 138 W Matthew Petroll PhD

Professor Department of Ophthalmology UT Southwestern Medical Center Dallas, TX, USA Chapter 15

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Ophthalmic Consultants of Boston Associate Professor of Ophthalmology Tufts University School of Medicine Boston, MA, USA Chapter 100

Leela V Raju MD

Eye Physicians and Surgeons Clinical Instructor New York Eye and Ear Infirmary Brooklyn, NY, USA Chapter 173

Gullapalli N Rao MD Chair L V Prasad Eye Institute Hyderabad, India Chapter 82

Duna Raoof MD Cornea, Cataract, and Refractive Surgery Specialist Harvard Eye Associates Laguna Hills, CA, USA Clinical Instructor Harbor-University of California Los Angeles Torrence, CA, USA Chapters 150; 155

Christopher J Rapuano MD

Professor, Department of Ophthalmology Sidney Kimmel Medical College at Thomas Jefferson University Chief, Cornea Service Wills Eye Hospital Philadelphia, PA, USA Chapters 18; 137; 140

Jagadesh C Reddy MD Assistant Ophthalmologist Tej Kohli Cornea Institute, Kallam Anji Reddy Campus L V Prasad Eye Institute Hyderabad, India Chapter 92 Ellen Redenbo CDOS, ROUB, CRA Imaging Center Supervisor Department of Ophthalmology UC Davis Eye Center Sacramento, CA, USA Chapter 16 James J Reidy MD

Associate Professor Vice Chair of Clinical Affairs Department of Ophthalmology and Visual Science University of Chicago Medicine and Biological Sciences Chicago, IL, USA Chapter 74

Charles D Reilly MD

Managing Partner Rashid, Rice, Flynn and Reilly Eye Associates Clinical Assistant Professor Department of Ophthalmology University of Texas Health Science Center San Antonio San Antonio, TX, USA Chapters 51; 161

Adimara da Candelaria Renesto MD

Fellow of Refractive Surgery Department of Ophthalmology and Visual Sciences Federal University of São Paulo Vision Institute IPEPO São Paulo, Brazil Chapter 172

Andri K Riau MSc

Research Associate Tissue Engineering and Stem Cell Group Singapore Eye Research Institute Singapore Chapter 171

List of Contributors

Lorena Riveroll-Hannush MD Clinical Director Oxford Valley Laser Vision Center Langhorne, PA, USA Cornea Service Asociación Para Evitar La Ceguera Hospital Dr. Luis Sánchez Bulnes Mexico City, Mexico Chapters 109; 152

Allison E Rizzuti MD

Clinical Assistant Professor Department of Ophthalmology State University of New York, Downstate Medical Center Brooklyn, NY, USA Chapter 19

Danielle M Robertson OD, PhD Associate Professor Department of Ophthalmology University of Texas Southwestern Medical Center Dallas, TX, USA Chapter 97

Shizuya Saika MD, PhD

Professor and Chairman Department of Ophthalmology Wakayama Medical University School of Medicine Wakayama, Japan Chapter 1

Mario J Saldanha DO, FRCS, FRCOphth Cornea, External Disease and Refractive Surgery Fellow Ophthalmology Toronto Western Hospital University of Toronto Toronto, ON, Canada Chapter 169

James J Salz MD Clinical Professor, Ophthalmoloygy University of Southern California Keck Medical School Los Angeles, CA, USA Chapter 162 Virender S Sangwan MD

Cornea Fellow Department of Ophthalmology Manhattan Eye, Ear, and Throat Hospital/ Northshore-LIJ Health System New York, NY, USA Chapters 139; 144

Dr. Paul Dubord Chair in Cornea Director, Center for Ocular Regeneration (CORE) Director, Srujana-Center for Innovation Kallam Anji Reddy Campus L V Prasad Eye Institute Hyderabad, India Chapter 92

David S Rootman MD, FRCSC

Caterina Sarnicola MD

Ashley Rohr MD

Professor, University of Toronto Toronto Western Hospital Toronto, ON, Canada Chapter 164

Resident University of Ferrara Ferrara, Italy Chapter 120

James T Rosenbaum MD

Enrica Sarnicola MD

Chief of Ophthalmology Devers Eye Institute Legacy Health System Portland, OR, USA Professor Departments of Ophthalmology, Medicine, and Cell Biology Casey Eye Institute Oregon Health & Science University Portland, OR, USA Chapter 102

Alan E Sadowsky MD Adjunct Assistant Professor Department of Ophthalmology University of Minnesota Fairview Medical Group Fridley, MN, USA Chapter 63

Resident University of Siena Siena, Italy Chapter 120

Vincenzo Sarnicola MD Director Private Practice “Clinica degli occhi Sarnicola” Grosseto, Italy Professor Department of Ophthalmology University of Siena Siena, Italy Chapter 120 Ibrahim O Sayed-Ahmed MD

Research Fellow Cornea Bascom Palmer Eye Institute Miami, FL, USA Chapter 20

Rony R Sayegh MD

Assistant Professor Department of Ophthalmology University Hospitals Case Medical Center Case Western Reserve University School of Medicine Cleveland, OH, USA Chapter 14

Gregory A Schmidt BS, CEBT Iowa Lions Eye Bank Coralville, IA, USA Chapter 126

Miriam T Schteingart MD Physician Andersen Eye Associates Saginaw, MI, USA Chapter 104

Ivan R Schwab MD, FACS Professor of Ophthalmology Department of Ophthalmology University of California, Davis Sacramento, CA, USA Chapters 64; 86 Brian L Schwam MD Vice President and Chief Medical Officer Johnson and Johnson Vision Care Jacksonville, FL, USA Chapter 100 Gary S Schwartz MD

Adjunct Associate Professor Department of Ophthalmology University of Minnesota School of Medicine Minneapolis, MN, USA Chapters 53; 77; 157; 158

Vincenzo Scorcia MD Associate Professor Department of Ophthalmology University of Magna Graecia Catanzaro, Italy Chapter 130 H Nida Sen MD, MHS

National Eye Institute National Institutes of Health Bethesda, MD, USA Chapter 101

Boris Severinsky OD, MOptom Contact Lens Service Department of Ophthalmology Hadassah University Hospital Jerusalem, Israel Chapter 97

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List of Contributors

Kevin J Shah MD

Staff Surgeon Department of Ophthalmology Cole Eye Institute, Cleveland Clinic Foundation Cleveland, OH, USA Chapters 77; 131; 160

Kavitha R Sivaraman MD Fellow, Cornea and External Disease Bascom Palmer Eye Institute University of Miami Miller School of Medicine Miami, FL, USA Chapter 37

Anna M Stagner MD

Mehdi Shajari MD

Craig A Skolnick MD

Christopher E Starr MD

Neda Shamie MD

Allan R Slomovic MSc, MD, FRCS(C)

Department of Ophthalmology Goethe University Frankfurt, Germany Chapter 174

Cornea, Refractive and Cataract Surgeon Advanced Vision Care Los Angeles, CA, USA Chapter 132

Brett Shapiro MD

Attending Ophthalmologist Kaiser Permanente, Hawai’i Region Wailuku, Maui, HI, USA Chapter 21

Raneen Shehadeh-Mashor MD

Ophthalmologist, Corneal specialist Department of Ophthalmology, Bnai Zion Medical Center Institute – Technion Haifa, Israel Chapter 57

Shigeto Shimmura MD, PhD

Associate Professor Department of Ophthalmology Keio University School of Medicine Tokyo, Japan Chapter 119

Thomas S Shute MD, MS

Department of Ophthalmology and Visual Sciences Washington University School of Medicine St. Louis, MO, USA Chapter 75

Patricia B Sierra MD Cornea, Cataract and Refractive Surgery Sacramento Eye Consultants Sacramento, CA, USA Chapter 167 Francisco Bandeira e Silva MD

Post-graduation Student Department of Ophthalmology Paulista School of Medicine Federal University of São Paulo São Paulo, Brazil Chapter 149

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President Skolnick Eye Institute Jupiter, FL, USA Chapter 96

Marta and Owen Boris Endowed Chair in Cornea and Stem Cell Research Professor of Ophthalmology University of Toronto Research Director, Cornea Service University Health Network President, Canadian Ophthalmological Society Toronto Western Hospital Toronto, ON, Canada Chapters 52; 57

Michael E Snyder MD

Board of Directors, Cincinnati Eye Institute Chair, Clinical Research Steering Committee Volunteer Faculty, University of Cincinnati Cincinnati, OH, USA Chapter 145

Renée Solomon MD

Private Practice New York, NY, USA Chapter 138

Sarkis H Soukiasian MD Assistant Professor of Ophthalmology Tufts University School of Medicine Director, Corneal and External Disease Department of Ophthalmology Lahey Health Burlington, MA, USA Chapter 41 Luciene Barbosa de Sousa MD

Head of Cornea Section Federal University of São Paulo São Paulo, Brazil Chapter 70

Sathish Srinivasan FRCSEd, FRCOphth, FACS Consultant Corneal Surgeon Joint Clinical Director Department of Ophthalmology University Hospital Ayr Ayr, Scotland, UK Chapter 57

Ophthalmic Pathology Fellow David Glendenning Cogan Laboratory of Ophthalmic Pathology Massachusetts Eye and Ear Infirmary Boston, MA, USA Chapters 36; 39

Associate Professor of Ophthalmology Director, Cornea, Cataract & Refractive Surgery Fellowship Director, Refractive Surgery Service Director, Ophthalmic Education Weill Cornell Medical College New York Presbyterian Hospital New York, NY, USA Chapter 33

Roger F Steinert MD

Irving H Leopold Professor Chair of Ophthalmology Professor of Biomedical Engineering Director, Gavin Herbert Eye Institute University of California Irvine, CA, USA Chapters 112; 170

Bazil TL Stoica MD

Oculoplastic Fellow Department of Ophthalmology University of Ottawa Ottawa, ON, Canada Chapter 34

Michael D Straiko MD Associate Director of Corneal Services Devers Eye Institute Legacy Health System Portland, OR, USA Chapter 131 Alan Sugar MD Professor and Vice-Chair Ophthalmology and Visual Sciences W.K. Kellogg Eye Center University of Michigan Medical School Ann Arbor, MI, USA Chapter 95 Joel Sugar MD

Professor and Vice-Head Ophthalmology and Visual Sciences Illinois Eye and Ear Infirmary University of Illinois College of Medicine Chicago, IL, USA Chapters 67; 90

Christopher N Ta MD

Professor Byers Eye Institute at Stanford School of Medicine Palo Alto, CA, USA Chapter 46

List of Contributors

Khalid F Tabbara MD

Adjunct Professor Department of Ophthalmology Wilmer Institute Johns Hopkins University Baltimore, MD, USA Medical Director The Eye Center Riyadh, Saudi Arabia Chapter 105

Donald TH Tan FRCSG, FRCSE, FRCOphth, FAMS Arthur Lim Professor in Ophthalmology Ophthalmology and Visual Sciences Academic Clinical Program, Duke-NUS Graduate Medical School, Singapore Singapore National Eye Centre Singapore Chapters 127; 141; 156 Maolong Tang PhD Research Assistant Professor Department of Ophthalmology Casey Eye Institute Oregon Health and Science University Portland, OR, USA Chapter 17 Joseph Tauber MD Tauber Eye Center Kansas City, MO, USA Chapter 107

Shabnam Taylor MD

Resident Physician Department of Ophthalmology University of California, Davis Sacramento, CA, USA Chapter 89

Mark A Terry MD

Director, Corneal Services, Devers Eye Institute Professor, Clinical Ophthalmology Oregon Health Sciences University Portland, OR, USA Chapter 129

Howard H Tessler MD

Professor Emeritus of Ophthalmology University of Illinois at Chicago Chicago, IL, USA Chapters 104; 106

Theofilos Tourtas MD Consultant Ophthalmic Surgeon Corneal and External Disease Service Department of Ophthalmology University of Erlangen-Nuremberg Erlangen, Germany Chapter 133

Elias I Traboulsi MD, MEd Professor Cole Eye Institute Cleveland Clinic Lerner College of Medicine Case University Cleveland, OH, USA Chapter 58

David D Verdier MD Clinical Professor Department of Surgery, Ophthalmology Division Michigan State University College of Human Medicine Grand Rapids, MI, USA Chapter 110

William Trattler MD

Laura A Vickers MD Chief Resident Duke University Eye Center Durham, NC, USA Chapter 114

Matthew GJ Trese MA Clinical Research Intern Department of Ophthalmology and Visual Sciences Kellogg Eye Center, University of Michigan Ann Arbor, MI, USA Chapter 95

Ana Carolina Vieira MD, PhD

Director of Cornea Center for Excellence in Eye Care Miami, FL, USA Chapter 162

David T Tse MD, FACS

Professor of Ophthalmology Dr Nasser Ibrahim Al-Rashid Chair in Ophthalmic Plastic Orbital Surgery and Oncology Bascom Palmer Eye Institute Miami, FL, USA Chapter 29

Elmer Y Tu MD

Professor of Clinical Ophthalmology Department of Ophthalmology and Visual Science University of Illinois Eye and Ear Infirmary Chicago, IL, USA Chapter 81

Pravin K Vaddavalli MD Consultant Ophthalmologist Tej Kohli Cornea Institute LV Prasad Eye Institute Hyderabad, India Chapter 82 Felipe A Valenzuela MD Clinical Fellow Department of Ophthalmology Bascom Palmer Eye Institute University of Miami Miller School of Medicine Miami, FL, USA Chapter 49 Gary A Varley MD

Cincinnati Eye Institute Cincinnati, OH, USA Chapter 9

Cornea and External Diseases Specialist Department of Ophthalmology State University of Rio de Janeiro Rio de Janeiro, Brazil Chapters 86; 142

Jesse M Vislisel MD

Fellow Department of Ophthalmology and Vision Science University of Iowa Iowa City, IA, USA Chapter 9

An Vo MD

Cornea and External Disease Fellow Department of Ophthalmology Icahn School of Medicine at Mount Sinai New York, NY, USA Chapter 98

Rosalind C Vo MD

Associate Physician Southern California Permanente Medical Group Los Angeles, CA, USA Clinical Instructor UCLA David Geffen School of Medicine Los Angeles, CA, USA Chapter 70

John A Vukich MD

Clinical Adjunct Assistant Professor of Ophthalmology and Visual Sciences University of Wisconsin Madison School of Medicine Madison, WI, USA Chapter 175

Matthew Wade MD Assistant Professor of Ophthalmology Gavin Herbert Eye Institute University of California Irvine, CA, USA Chapters 112; 170

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List of Contributors

Jay C Wang MD Ophthalmology Resident Department of Ophthalmology Massachusetts Eye and Ear Infirmary Boston, MA, USA Chapter 154

Jessica E Weinstein MD

Steven E Wilson MD, FARVO Professor of Ophthalmology Cole Eye Institute Cleveland Clinic Cleveland, OH, USA Chapter 168

Li Wang MD, PhD Associate Professor Department of Ophthalmology Baylor College of Medicine Houston, TX, USA Chapter 173

Jayne S Weiss MD

Elizabeth Yeu MD Assistant Professor of Ophthalmology Eastern Virginia Medical School Virginia Eye Consultants Cornea, Cataract, Anterior Segment and Refractive Surgery Norfolk, VA, USA Chapter 163

George O Waring III MD, FACS, FRCOphth Formerly Founding Surgeon InView Atlanta, GA, USA Chapter 5

George O Waring IV MD, FACS Assistant Professor of Ophthalmology Director of Refractive Surgery Adjunct Assistant Professor of Bioengineering Medical University of South Carolina Clemson University College of Engineering and Science Storm Eye Institute Charleston, SC, USA Chapter 161 Michael A Warner MD

Clinical Instructor Oregon Health and Sciences University Portland, OR, USA Chapters 36; 39

Mitchell P Weikert MD Associate Professor Department of Ophthalmology Baylor College of Medicine Houston, TX, USA Chapters 12; 173

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Resident Physician Department of Ophthalmology Louisiana State Health Sciences Center New Orleans, LA, USA Chapter 71

Professor and Chair, Department of Ophthalmology Associate Dean of Clinical Affairs, Herbert E Kaufman, MD Endowed Chair in Ophthalmology Professor of Pharmacology and Pathology Director, Louisiana State University Eye Center of Excellence Louisiana State University School of Medicine New Orleans, LA, USA Chapters 68; 71

Julia M Weller MD

Charles Q Yu MD Assistant Professor Illinois Eye and Ear Infirmary University of Illinois Chicago Chicago, IL, USA Chapter 46

Kirk R Wilhelmus MD, PhD

Dagny Zhu MD Resident Physician Department of Ophthalmology University of Southern California Los Angeles, CA, USA Chapter 132

Cornea Fellow Department of Ophthalmology University of Erlangen-Nuremberg Erlangen, Germany Chapter 133

Professor Emeritus Department of Ophthalmology Baylor College of Medicine Houston, TX, USA Chapter 83

Samantha Williamson MD

Clinical Fellow Department of Ophthalmology University of Illinois Eye and Ear Infirmary Chicago, IL, USA Chapters 67; 151

Mohammed Ziaei MBChB (Hons), FRCOphth Corneal Fellow Moorfields Eye Hospital London, UK Chapter 147

Part i

Basic Science: Cornea, Sclera, Ocular Adnexa Anatomy, Physiology and Pathophysiologic Responses





Chapter 1  Cornea and Sclera: Anatomy and Physiology Teruo Nishida, Shizuya Saika, Naoyuki Morishige

Key Concepts • • • •

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The cornea and sclera form the outer shell of the eye. The principal physiological role of the cornea is to allow external light to enter the eye and to contribute to its focusing on the retina. Transparency and refractive power are, thus, essential for this function. The cornea consists of the epithelium, Bowman’s layer, stroma, Descemet membrane, and endothelium. The functions of the corneal epithelium are regulated by various biologically active agents such as growth factors, cytokines, and chemokines in tear fluid, whereas those of the endothelium are regulated by factors in aqueous humor. Dynamic homeostasis of the corneal epithelium is maintained by the generation of new epithelial cells from limbal stem cells, centripetal cell movement, differentiation of basal cells into wing cells and then superficial cells, and cell desquamation via apoptosis. The corneal stroma is composed primarily of extracellular matrix, predominantly type I collagen and proteoglycans. Collagen fibrils are homogeneous in diameter and are aligned at a constant distance to maintain tissue transparency. Keratocytes are the resident cells of the stroma, and although relatively few in number, they play important roles in the maintenance of stromal structure through synthesis and secretion as well as degradation of collagen and proteoglycans. The cornea is one of the most sensitive tissues in the body as a result of its abundant sensory nerve endings. Neural regulation is, thus, another important factor in the maintenance of corneal structure and function. Morphogenesis of the eye is achieved by cell lineages of various origins including the surface and neural ectoderm during embryonic development. Characterization of the development of ocular tissues during embryogenesis is important for understanding the pathogenesis of congenital anomalies of the cornea and anterior eye segment.

Introduction The avascular cornea is not an isolated tissue. It forms, together with the sclera, the outer shell of the eye, occupying

one-third of the ocular tunic. Although most of both the cornea and sclera consists of dense connective tissue, the physiological roles of these two components of the eye shell differ. The cornea serves as the transparent “window” of the eye that allows the entry of light, whereas the sclera provides a “dark box” that allows the formation of an image on the retina. The cornea is exposed to the outer environment, whereas the opaque sclera is covered with the semitransparent conjunctiva and has no direct exposure to the outside. The differences in the functions of the cornea and sclera reflect those in their microscopic structures and biochemical components (Fig. 1.1). Interwoven fibrous collagen is responsible for the mechanical strength of both the cornea and sclera, protecting the inner components of the eye from physical injury and maintaining the ocular contour.1 The corneal epithelium forms an effective mechanical barrier as a result of the interdigitation of cell membranes and the formation of junctional complexes such as tight junctions and desmosomes between adjacent cells. Together with the cellular and chemical components of the conjunctiva and tear film, the corneal surface protects against potential pathological agents and microorganisms. The smooth surface of the cornea contributes to visual clarity. The regular arrangement of collagen fibrils in the corneal stroma accounts for the transparency of this tissue.2,3 Maintenance of corneal shape and transparency is critical for light refraction, with the cornea accounting for more than two-thirds of the total refractive power of the eye. A functionally intact corneal endothelium is important for maintenance of stromal transparency as a result of regulation by the endothelium of corneal hydration. The cornea thus plays a central role in vision as a result of its transparency and refractive power, and it maintains the eye shell. Each part of the cornea contributes to its transparency and shape, and its anatomy is closely related to its physiology and function.

Anatomy and Physiology Structure of the cornea and sclera The ocular surface is composed of the cornea, conjunctiva, lacrimal glands, and other adnexa. The outermost portion of the cornea and conjunctiva is an epithelium that directly

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CHAPTER 1 Cornea and Sclera: Anatomy and Physiology

Chapter Outline Introduction Anatomy and Physiology Development of the Anterior Eye Segment

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Fig. 1.1  Anatomy of the human cornea. (A) Slit lamp microscopic view of the cornea. (B) Histology of the cornea showing the epithelium (1), Bowman’s layer (2), stroma (3), Descemet membrane (4), and endothelium (5).

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faces the environment. The anterior corneal surface is covered by tear fluid, which protects the cornea from dehydration and helps to maintain a smooth epithelial surface. Tear fluid contains many biologically important ions and molecules, including electrolytes, glucose, immunoglobulins, lactoferrin, lysozyme, albumin, and oxygen. Moreover, it contains a wide range of biologically active substances such as histamine, prostaglandins, growth factors, and cytokines.4 The tear film thus serves not only as a lubricant and source of oxygen and nutrients for the corneal epithelium but also as a source of regulatory factors required for epithelial maintenance and repair. The posterior surface of the cornea is bathed directly by the aqueous humor. The highly vascularized limbus, which is thought to contain a reservoir of pluripotent stem cells, constitutes the transition zone between the cornea and the sclera or conjunctiva. The structure and function of the corneal epithelium and endothelium are, therefore, regulated by biologically active factors present in tear fluid and aqueous humor, respectively. The sclera, a tough and nontransparent tissue, shapes the eye shell, which is approximately 24 mm in diameter in the emmetropic eye. The sclera is the continuation of the corneal stroma and does not directly face the environment. The anterior part of the sclera is covered with the bulbar conjunctiva and Tenon’s capsule, which consists of loose connective tissue and is located beneath the conjunctiva (Fig. 1.2). The nontransparency of the sclera prevents light from reaching the retina other than through the cornea, and, together with the pigmentation of the choroid and retinal pigment epithelium, the sclera provides a dark box for image formation. The scleral spur is a projection of the anterior scleral stroma toward the angle of the anterior chamber and is the site of insertion for the anterior ciliary muscle. Contraction of this muscle thus opens the trabecular meshwork. At the posterior pole of the eyeball, where the optic nerve fibers enter the eye, the scleral stroma is separated into outer and inner layers. The outer layer fuses with the sheath of the optic nerve, dura, and arachnoid, whereas the inner

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Fig. 1.2  Histology of the human sclera. (A) Hematoxylin-eosin staining of a cross-section of the sclera. Blood vessels (asterisks) are largely restricted to the episclera (upper region of section). (B) Highermagnification view of the conjunctiva and episclera as well as of stromal fibroblasts (arrows) in the sclera. Bar, 100 µm.

layer contains the sieve-like structure of the lamina cribrosa. The rigidity of the lamina cribrosa accounts for the susceptibility of retinal nerve fibers to damage during the development of chronic open-angle glaucoma. The sclera contains six insertion sites of the extraocular muscles as well as the inputs of arteries (anterior and posterior ciliary arteries) and outputs of veins (vortex veins) that circulate blood through the uveal tissues.

CHAPTER 1 Cornea and Sclera: Anatomy and Physiology

Dimensions and optical properties of the cornea The anterior corneal surface is convex and aspheric (Fig. 1.1), and it is transversely oval as a result of scleralization superiorly and inferiorly. The adult human cornea measures 11 to 12 mm horizontally and 9 to 11 mm vertically. It is approximately 0.5 mm thick at the center, with the thickness increasing gradually toward the periphery, where it is about 0.7 mm thick.5 The curvature of the corneal surface is not constant, being greatest at the center and smallest at the periphery. The radius of curvature is between 7.5 and 8.0 mm at the 3 mm central optical zone of the cornea, where the surface is almost spherical. The refractive power of the cornea is 40 to 44 diopters, constituting about two-thirds of the total refractive power of the eye. The optical properties of the cornea are determined by its transparency, surface smoothness, contour, and refractive index of the tissue.3 If the diameter of (or the distance between) collagen fibrils in the corneal stroma becomes heterogeneous (as occurs in fibrosis or edema), incident light rays are scattered randomly and the cornea loses its transparency. Given that the spherocylindrical surface of the cornea has both minor and major axes, changes in corneal contour caused either by pathological conditions such as scarring, thinning, or keratoconus or by refractive surgery render the surface regularly or irregularly astigmatic. The total refractive index of the cornea is determined by the sum of refraction at the anterior and posterior interfaces as well as by the transmission properties of the tissue. The refractive indices of air, tear fluid, corneal tissue, and aqueous humor are 1.000, 1.336, 1.376, and 1.336, respectively. The refractive power of a curved surface is determined by the refractive index and the radius of curvature. The refractive

power at the central cornea is about +43 diopters, being the sum of that at the air–tear fluid (+44 diopters), tear fluid–cornea (+5 diopters), and cornea–aqueous humor (−6 diopters) interfaces. Most keratometry and topography measurements assume a standard refractive index of 1.3375.

Corneal epithelium The corneal and conjunctival epithelia are continuous and together form the ocular surface. They are both composed of nonkeratinized, stratified, squamous epithelial cells. The thickness of the corneal epithelium is approximately 50 µm, which is about 10% of the total thickness of the cornea (Fig. 1.1), and it is constant over the entire corneal surface. The corneal epithelium consists of five or six layers of three different types of epithelial cells: superficial cells, wing cells, and columnar basal cells, the latter of which adhere to the basement membrane adjacent to Bowman’s layer (Figs 1.1, 1.3 and 1.4). Although their characteristics differ, both corneal and conjunctival epithelia cooperate to provide the biodefense system of the anterior surface of the eye.6,7 The presence of junctional complexes between adjacent corneal epithelial cells prevents the passage of external agents into the deeper layers of the cornea. Both cell–cell and cell–matrix interactions are important for maintenance of the normal stratified structure and physiological functions of the corneal epithelium.8 The characteristics of the different types of junctional complexes present in the corneal epithelium (Figs 1.4 and 1.5) are summarized in Table 1.1. Tight junctions (zonula occludens) are present mostly between cells of the superficial cell layers and provide a highly effective barrier to prevent the penetration of tear fluid and its chemical constituents.

Table 1.1  Characteristics of the various types of corneal epithelial cells Shape

Layers Size

Superficial Flat 2–4 cells Microvilli Microplicae

Wing cells

Winglike processes

2–3

Basal cells

Columnar

Monolayer

40–60 µm in diameter 4–6 µm thick at the nucleus 2 µm thick at the periphery

18–20 µm high 8–10 µm in diameter Flat at posterior surface

Mitotic Interdigitation Junctional activity complexes

Cytoplasmic Keratin Microfilaments Microtubules organelles (actin)

−

Entire surface

Desmosomes Tight junctions Adherens junctions

Sparse

+

+

+/−

−

Entire surface

Desmosomes Gap junctions Adherens junctions

Sparse

+++

+

+/−

+

Apical surface

Desmosomes Gap junctions Adherens junctions Hemidesmosomes

More than superficial cells Large numbers of glycogen granules Prominent mitochondria and Golgi apparatus

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+

+

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Desmosomes and adherens junctions are present in all layers of the corneal epithelium, whereas gap junctions, which allow the passage of small molecules between cells, are present in wing cells and basal cells; and hemidesmosomes are localized to basal cells. After damage to the corneal epithelium, actively migrating epithelial cells no longer manifest junctional complexes in the wounded region lacking a basement membrane. Re-establishment of the continuity of the corneal epithelium is accompanied by the synthesis of basement membrane proteins and reconstruction of the basement membrane and by the reassembly of the various types of junctional apparatus, suggesting that the presence of the basement membrane may be required for re-formation of cell–cell junctions in the corneal epithelium (Fig. 1.5).9 In corneal epithelial cells, intermediate filaments of the cytoskeleton are formed by specific types of acidic (type I) and basic (type II) keratin molecules. Basal cells of the corneal epithelium express keratin 5/14, like basal epidermal cells of the skin. However, keratin 3/12 (64-kDa keratin) is specifically expressed in the epithelium of the cornea, not being found in that of the conjunctiva or in the epidermis.10,11 Deletion of the keratin 12 gene results in fragility of the corneal epithelium in mice.12 In humans, genetic

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Fig. 1.3  Confocal biomicroscopy of the human cornea.   (A–C) Superficial, wing, and basal cell layers of the corneal epithelium, respectively. (D) Subepithelial nerve plexus. (E) Shallow layer of the stroma, containing a high density of polygonal keratocytes. (F) Mid-layer of the stroma, containing thick, nonbranching nerve fibers. (G) Deep layer of the stroma, containing   a lower density of keratocytes.   (H) Amorphous appearance of Descemet membrane.   (I) Endothelium, comprising hexagonal endothelial cells of uniform size.

mutation of the keratin 12 gene is responsible for Meesmann dystrophy of the corneal epithelium.13 Replacement of most organs or tissues by transplantation from a genetically nonidentical individual is associated with an immune response that may lead to rejection. In contrast, the cornea is “immune privileged,” a characteristic that is critical for the success of corneal transplantation. Dendritic Langerhans cells, specialized macrophages derived from the bone marrow that are implicated in antigen processing, are abundant at the periphery of the corneal epithelium but are not present in the central region of the normal cornea.14,15 These cells express human leukocyte antigen (HLA) class II molecules and are thought to function in the afferent arm of the ocular immune response by presenting antigens to T lymphocytes.16,17 Injury to the central cornea results in the rapid migration of peripheral Langerhans cells to the damaged area.

Limbal stem cells and lineage of corneal epithelial cells Corneal epithelial cells are renewed continuously to maintain the normal layered structure of the epithelium in a

CHAPTER 1 Cornea and Sclera: Anatomy and Physiology

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Fig. 1.4  Transmission electron microscopy of the human corneal epithelium. (A) The epithelium comprises five or six layers of epithelial cells. The electron-dense cell is about to undergo desquamation. (B) Basal cells. Note the numerous junctional complexes. (C) Basement membrane and anterior portion of Bowman’s layer. Note hemidesmosomes at the basal surface of the epithelial cells. (D) Interdigitation and junctional complexes at the lateral surface of basal epithelial cells. (E) Gap junction at the lateral surface of basal cells.

process characterized by dynamic equilibrium (Fig. 1.6). Only the basal cells of the corneal epithelium proliferate, with the daughter cells instead differentiating into wing cells and subsequently into superficial cells and gradually emerging at the corneal surface.18 The differentiation process requires about 7 to 14 days, after which the superficial

cells undergo desquamation into the tear film.19 Mechanical friction associated with blinking, ultraviolet radiation, and hypoxia induce apoptosis (programmed cell death) and desquamation of corneal epithelial cells.20–22 Thoft and Friend proposed that an equilibrium, represented by the equation X + Y = Z, exists between the proliferation of basal epithelial

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G AF

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Cx

Cx

Cx

AF

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AF

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PG KF

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DP I/II

7H6

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KF

zo-1

zo-1 AF

AF

zo-2 7H6

Ca2+

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Dsg/Dsc

DP I/II KF

AF

AF

AF

P120 β-ctn α-ctn P120

AF N AF

E-cad E-cad

a-ctn β-ctn AF

Dsg/Dsc AF

Fig. 1.5  Intercellular junctions in the corneal epithelium. Upper panels: (A–D) Transmission electron micrographs of the human corneal epithelium. Scale bars, 50 nm. Middle panels: (E–J) Immunofluorescence micrographs of the rat corneal epithelium stained with antibodies to the indicated proteins. Scale bars, 20 µm. Lower panels: (K–N) Schematic representation of intercellular junctions in the corneal epithelium. GJ, gap junction; TJ, tight junction; DS, desmosome; AJ, adherens junction; Cx43, connexin 43; Oc, occludin; Dsg 1+2, desmogleins 1 and 2; E-cad, E-cadherin; c-AMP, cyclic adenosine monophosphate; Cld, claudin; zo-1 and -2, zonula occludens–1 and –2; 7H6, 7H6 antigen; AF, actin filament;   Dsc, desmocollin; DP I/II, desmoplakin I or II; PG, plakoglobin; KF, keratin filament; α- and β-ctn, α- and β-catenin; P120, P120 catenin. (Modified from Suzuki K, et al. Cell–matrix and cell–cell interactions during corneal epithelial wound healing. Prog Retin Eye Res 22:113–33, 2003. Copyright Elsevier.)

cells and their differentiation into superficial cells (X), the centripetal movement of peripheral epithelial cells (Y), and epithelial cell loss from the corneal surface (Z).23 This X, Y, Z hypothesis explains well the dynamic equilibrium of epithelial cells in the cornea. Given the continuous desquamation of surface epithelial cells, it is essential that new epithelial cells be supplied not only by mitosis of basal cells but also by the emergence of epithelial cells from the periphery. The function of stem cells is to replenish cells lost in normal or damaged tissue. The asymmetric division of each stem cell generates a new stem cell and a transit amplifying cell that initially proliferates and then gives rise to terminally differentiated cells. As in other tissues, the existence of stem cells to maintain homeostasis of the corneal epithelium has been postulated.24–28 Whereas keratin 3/12 (64-kDa keratin) is expressed in all layers of corneal epithelial cells, it is present only in the suprabasal epithelial cells at the limbus.10 The presence of slowly cycling cells in the basal cell layer at the limbus was demonstrated by cell labeling with [3H]thymidine,29 and basal cells at the limbus were

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found to have a higher mitotic potential than those of the central cornea in vitro.30 Centripetal movement of corneal epithelial cells has also been well documented.31–36 These observations suggested that stem cells for corneal epithelial cells reside at the limbus, the transitional zone between the cornea and conjunctiva.24,28,37 The palisades of Vogt, richly vascularized papillae at the transition zone between the cornea and conjunctiva, have been identified as the likely location of limbal stem cells for corneal epithelial cells.38,39 These structures are able to provide a protective environment for stem cells as well as to supply them with growth factors, extracellular matrix (ECM), and neural signals to maintain their nature as stem cells. Limbal epithelial crypts, anatomic structures that extend from the palisades of Vogt, have been proposed to be the actual site of the stem cell niche.40 The putative stem cells in the basal layer of the limbus have unique characteristics in that they cycle slowly and therefore retain DNA labels such as [3H]thymidine, they are poorly differentiated with primitive cytoplasm, they have a high proliferative potential without undergoing maturation, they are small with a high

CHAPTER 1 Cornea and Sclera: Anatomy and Physiology

Conjunctiva

Limbus

Cornea

Transient amplifying cells Limbal stem cells

Terminally differentiated cells Basal cells

Z

Centripetal movement X

Y

Palisades of Vogt Limbus ZXY X: Proliferation of basal cells

Y: Centripetal movement of cells (supply from limbal stem cells)

Z: Cell loss from the surface (desquamation via apoptosis)

Fig. 1.6  Lineage of corneal epithelial cells. Stem cells thought to reside in the basal cell layer at the limbus proliferate asymmetrically to yield a daughter stem cell and a transit amplifying cell, the progeny of which move centripetally toward the center of the cornea to become basal corneal epithelial cells. These newly generated basal cells proliferate symmetrically and then differentiate consecutively into wing cells and superficial cells, the latter of which undergo apoptosis and consequent desquamation.

nucleus-to-cytoplasm ratio, they are capable of generating a large number of differentiated progeny, and they reside in close contact with a subset of mesenchymal niche cells.41,42 Although many markers for limbal stem cells have been proposed,38,43 there is no single positive marker that distinguishes these cells. The expression of p63 (a marker of cell proliferative ability), α-enolase, keratin 19, and the hepatocyte growth factor (HGF) receptor has been shown to be higher in the limbal epithelium than in the corneal epithelium. The transporter protein ABCG2 is also expressed specifically in the basal layer of the conjunctival epithelium at the fornix area. Although no direct evidence for the existence of limbal stem cells has been obtained to date, ABCG2 appears to be the most promising surface marker for the identification of such cells. Limbal stem cell deficiency has been recognized as a complex corneal disorder resulting from functional or structural loss of the limbus. Deficiency of limbal stem cells has been suggested to lead to impairment of corneal epithelial homeostasis in individuals with aniridia, inflammatory disorders of the ocular surface such as Stevens–Johnson syndrome, or severe alkali burn of the ocular surface.44 No medical treatment for limbal stem cell deficiency is currently available. Transplantation of stem cells is a potential approach to the treatment of limbal stem cell deficiency. Such an approach requires sorting of limbal stem cells from explants of limbus tissue, however, given that the stem cells

are thought to constitute less than 1% of cells in the basal layer of the limbus. The lack of a definitive stem cell marker has thus impeded the sorting process. Sorting based on the presence of stem cell-associated markers (ABCG2, vimentin, keratin 19) and the absence of differentiation markers (keratin 3/12, connexin 43, involucrin) might be the best current approach.38

Layered structure of the corneal epithelium Superficial cells The surface of the corneal epithelium contains two to four layers of terminally differentiated superficial cells. In contrast to the epidermis of the skin, the corneal epithelium is not normally keratinized, although it may become so under pathological conditions such as vitamin A deficiency. These cells are flat and polygonal with a diameter of 40–60 µm and a thickness of 2–6 µm (Table 1.1). Their surface is covered with microvilli.45 Given that superficial cells are well differentiated, they do not proliferate. Numerous glycoprotein (mucin) and glycolipid molecules are embedded in the cell membrane of epithelial cells. Mucins include both membrane-bound and secreted molecules, with the former in humans including MUC1, MUC4, and MUC16, all of which have been detected in surperficial epithelial cells of the cornea and conjunctiva.46 In mice, MUC16 is expressed in the conjunctiva but not in the

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cornea.47 These glycoproteins and glycolipids form floating particles in the cell membrane that are collectively termed the glycocalyx and which confer hydrophilic properties on the anterior surface of the superficial epithelial cells. The glycocalyx interacts with the mucinous layer of the tear film and helps to maintain the layered structure of the latter.48 Loss either of the glycocalyx of corneal epithelial cells or of goblet cells in the conjunctival epithelium results in tear film instability and the mucin-deficiency form of dry eye. The superficial cells of the corneal epithelium are joined by desmosomes, adherens junctions, and tight junctions (Figs 1.4 and 1.5), which prevent the passage of substances through the intercellular space. Examination of fluorescein penetration into the corneal stroma with a fluorophotometer provides a measure of the barrier function of the corneal epithelium.49

Wing cells Beneath the superficial cells lie two or three layers of wing cells, so called because of their characteristic wing-like shape. Wing cells are in an intermediate state of differentiation between basal and superficial cells and are rich in intracellular tonofilaments composed of keratin (Table 1.1). The cell membranes of adjacent wing cells are interdigitated. Numerous desmosomes, adherens junctions, and gap junctions are present between wing cells (Fig. 1.5).

Basal cells The single layer of columnar basal cells of the corneal epithelium rests on the basement membrane. Basal cells, unlike superficial and wing cells, possess mitotic activity, and they differentiate consecutively into wing and superficial cells (Table 1.1). Neighboring basal cells interdigitate laterally and are joined by desmosomes, gap junctions, and adherens junctions (Fig. 1.5). The posterior surface of basal cells is flat and abuts the basement membrane. Basal cells adhere to the basement membrane via hemidesmosomes that are linked to anchoring fibrils of type VII collagen (Fig. 1.4).50 The anchoring fibrils penetrate the basement membrane and course into the stroma, where they form anchoring plaques together with type I collagen, a major component of the stroma. The adherens junctions are present at the lateral surface of the basal cells of the corneal epithelium and are thought to mediate cell–cell interaction.51

A

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Members of the integrin family of cell surface receptors for ECM proteins exist as heterodimers of α and β subunits.52 The integrin α5β1 heterodimer, which is the major receptor for fibronectin, is present at the surface of basal cells in the normal corneal epithelium.53 All epithelial cells undergoing active migration after debridement of the corneal epithelium express integrin α5β1.54

Basement membrane As in epithelia in other parts of the body, basal cells of the corneal epithelium are anchored to a basement membrane. The presence of the basement membrane between the basal epithelium and the underlying stroma fixes the polarity of epithelial cells. Ultrastructurally, the basement membrane, which is 40–60 nm thick, is composed of a pale layer (the lamina lucida) immediately posterior to the cell membrane of the basal epithelial cells as well as an electron-dense layer (the lamina densa) (Fig. 1.4). Type IV collagen and laminin are major components of the basement membrane (Fig. 1.7).55 The basement membranes of the corneal and conjunctival epithelia contain different type IV collagen chains, although the functional relevance of this difference is unknown. Whereas collagen α5 (IV) is present in the corneal basement membrane, collagen α2 (IV) is present in the conjunctival basement membrane (as well as in the amniotic membrane).56

Physiology of the corneal epithelium Maintenance of corneal structure is crucial for the physiological roles of this tissue in refraction and biodefense. A smooth epithelium, a transparent stroma, and a functioning endothelium are all essential for clear vision. The cornea is vulnerable to various chemical or biological agents as well as to physical events in the outside world. It is therefore equipped with an active maintenance system responsible for renewal of the corneal epithelium and wound healing. The widespread application of corneal surgery, including keratoplasty and refractive surgery, has necessitated a more detailed understanding of the cellular and molecular biology of corneal wound healing. In most parts of the body, wound healing is initiated by the extravasation of blood constituents that accompanies disruption of blood vessels. The

D

Fig. 1.7  Immunofluorescence analysis of the expression of matrix proteins in the rat corneal epithelium. (A) Type I collagen. (B) Type IV collagen. (C) Laminin. (D) Fibronectin. Bar: 50 µm.

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CHAPTER 1 Cornea and Sclera: Anatomy and Physiology cornea, however, is an avascular tissue. The mechanism of wound healing in the cornea thus differs from that elsewhere in the body.

response to injury. Such changes are under the overall control of growth factors and cytokines.

Fibronectin-integrin system

Epithelial movement

The fibronectin-integrin system plays a central role in corneal epithelial wound healing.62 Fibronectin provides a provisional matrix during the first phase of epithelial wound healing. It appears at the newly exposed corneal surface soon after epithelial or stromal injury,63,64 and epithelial cells then attach to and spread over the fibronectin matrix in an integrin-dependent manner.53 The integrin family has been shown to include 24 different α subunits and nine different β subunits, with the selective combination of these α and β subunits determining the specificity of binding to ECM proteins. The integrin subunits α2, α3, α5, α6, αv, β1, β4, and β5 have been detected in the human cornea.65 The binding of integrins α5β1, αvβl, and αIIβ3 to fibronectin is mediated by the RGD sequence. The appearance and disappearance of the integrin β1 chain and fibronectin during corneal epithelial wound healing are well coordinated (Fig. 1.8).54

Injury to the corneal surface is not uncommon and results in an epithelial defect, the rapid resurfacing of which is required for restoration of the continuity of the corneal epithelium. Repair of epithelial defects occurs in three distinct phases characterized by epithelial cell migration, proliferation, and differentiation, resulting in restoration of the stratified structure of the epithelium. Epithelial migration is thus the initial step in the resurfacing of epithelial defects.57 Trauma to the corneal epithelium induces the sliding and migration of the remaining epithelial cells adjacent to the injury site toward the defective area.58–61 Dynamic changes in cell–cell and cell–matrix (fibronectin-integrin system) interactions, upregulation of hyaluronan (hyaluronic acid), and modulation of the ECM by newly expressed proteolytic enzymes play important roles in these two types of epithelial cell movement in

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Fig. 1.8  Changes in the localization of the integrin β1 chain, fibronectin, and laminin after a nonpenetrating incision in the rat cornea. Phase-contrast microscopy (A, E, I, M, Q) and immunofluorescence microscopy for the integrin β1 chain (B, F, J, N, R), fibronectin (C, G, K, O, S), and laminin (D, H, L, P, T) are shown for the intact rat cornea (A–D) as well as at 12 hours (E–H), 1 day (I–L), 1 week (M–P), and 1 month (Q–T) after incision. Immediately after the incision, fibronectin was detected at the surface of the V-shaped defect in the stroma. Epithelial cells expressing the integrin β1 subunit then began to migrate over and to fill in the defect. With the exception of that in basal cells, expression of the integrin β1 chain in epithelial cells was downregulated coincident with the completion of wound healing. The abundance of fibronectin at the interface between the new epithelium and the stroma also markedly decreased at this time.

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The integrin α6β4 heterodimer is a component of hemidesmosomes and is not related to fibronectin-mediated cell adhesion and migration. In response to wounding of the corneal epithelium, hemidesmosomes in the basal cell layer are disassembled. They eventually reappear after migration of the remaining epithelium has resulted in a recovering of the denuded area.66

Hyaluronan Hyaluronan is also recognized as a biological signaling molecule and, like fibronectin, plays an important role in inflammation and wound healing.67 Unlike other glycosaminoglycans, a core protein for hyaluronan binding has not yet been identified. Hyaluronan is not present in the normal cornea, but it is transiently expressed in the rabbit cornea during wound healing.68 These observations suggest that hyaluronan might play a role in the late stages of corneal wound healing. Exogenous hyaluronan also increases the rate of corneal epithelial wound healing. The administration of hyaluronan eyedrops thus promotes corneal epithelial wound closure after epithelial debridement in rabbits69 and in diabetic rats.70,71

Proteolytic enzymes Proteolytic enzymes also play an important role in wound healing. Cellular motility thus depends not only on the interaction of cells with the underlying ECM but also on the termination of such interaction by degradation of matrix proteins. Proteases, including plasminogen activator, have been detected in tear fluid.72,73 Mechanical wounding induces upregulation of urokinase-type plasminogen activator at both the protein and mRNA levels in corneal epithelial cells, suggesting that this protease may contribute to epithelial cell migration by degrading fibronectin during corneal epithelial wound healing.74,75 Matrix metalloproteinases (MMPs) are also upregulated in the migrating corneal epithelium.76

Cytokines and growth factors The roles of various cytokines and growth factors in the regulation of corneal epithelial migration have also been investigated.77 In general, these molecules modulate corneal epithelial wound healing by regulating the various healingrelated systems described above.4,78 Epidermal growth factor (EGF) was first isolated from the mouse submaxillary gland as a factor that stimulates eye opening and incisory tooth eruption in newborn mice.79 This 53–amino acid polypeptide is a potent stimulator of proliferation in a variety of cell types, including corneal epithelial cells.80,81 EGF is synthesized in lacrimal glands82,83 and is present in tear fluid.84,85 It influences the physiology of the corneal epithelium and promotes corneal epithelial wound closure in animals.86 The continuous exposure of the corneal epithelium to EGF present in tear fluid suggests that the stimulatory effect of this growth factor on epithelial cell proliferation must be counteracted if the normal thickness and function of the epithelium are to be maintained. In addition to its stimulatory effect on cell proliferation, EGF exerts a variety of other actions in corneal epithelial cells, including promotion of cell adhesion to a fibronectin matrix.87,88

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Corneal epithelial cells express transforming growth factor (TGF)–β1.83 Endogenous TGF-β also promotes corneal epithelial cell migration.89 The stimulatory effects of EGF on corneal epithelial cell proliferation, attachment to fibronectin, and migration are modulated by TGF-β.90,91 Although TGF-β alone inhibits corneal epithelial cell proliferation, it has no effect on cell attachment to a fibronectin matrix in the absence of EGF. Basic fibroblast growth factor (bFGF) is another polypeptide growth factor that stimulates the proliferation of various cell types of mesodermal or neuroectodermal origin.92 FGF binds to heparan sulfate and is thus present in the basement membrane of the corneal epithelium.93 The application of recombinant human bFGF was shown to accelerate corneal epithelial wound closure in rabbits.94 Given that FGF receptors are expressed in corneal stromal cells, but not in corneal epithelial cells, this latter effect might be mediated indirectly.95 FGF is also a strong angiogenic factor in the cornea.96 Platelet-derived growth factor (PDGF) has been detected in tear fluid.97 Rabbit corneal epithelial cells also express both the β and α type of receptors for PDGF.98 Interleukins are cytokines that regulate the function of the immune system, inflammation, and other reactions of tissue to external stimuli.99 They modulate the activities of immune or inflammatory cells both locally in tissue as well as systemically in the circulation and in bone marrow. Although 35 members of the interleukin family (IL-1 to IL-35) have been identified to date, the roles of many of these proteins in corneal wound healing remain to be investigated. For example, the corneal epithelium expresses IL-1, and exogenous IL-1 promotes the healing of corneal epithelial wounds.100 Corneal epithelial cells also express IL-6.101 Exposure of rabbit corneal epithelial cells in culture to IL-6 resulted in a marked increase in the number of cells that attached to a fibronectin matrix. IL-6 stimulates the expression of integrin α5β1 in corneal epithelial cells, suggesting that this cytokine may regulate corneal epithelial migration through modulation of the fibronectin-integrin system as well as through increase of cell proliferation.102–105

Stroma of the cornea and sclera Bowman’s layer An acellular, membrane-like zone known as Bowman’s layer, or Bowman’s membrane, is detectable by light microscopy at the interface between the corneal epithelium and stroma in humans and certain other mammals (but not in rodents). Given that this structure, which is 12 µm thick, is not a membrane but rather a random arrangement of collagen fibrils and proteoglycans, the term Bowman’s layer is preferable. The collagen fibrils in Bowman’s layer are primarily collagen types I and III. The diameter of these fibrils is 20–30 nm, which is smaller than that of the collagen fibrils present in the corneal stroma (22.5–35 nm). Bowman’s layer is considered to be the anterior portion of the corneal stroma. The anterior surface of this layer, which faces the basement membrane, is smooth. Given that the collagen fibrils in Bowman’s layer are synthesized and secreted by stromal keratocytes, they appear continuous with those in the stroma.

CHAPTER 1 Cornea and Sclera: Anatomy and Physiology Biological functions originally attributed to Bowman’s layer are now thought to be mediated by the basement membrane. Bowman’s layer does not regenerate after injury. Recent clinical experience with excimer laser photoablation demonstrates that a normal epithelium is formed and maintained even in the absence of Bowman’s layer. Furthermore, many mammals do not have a Bowman’s layer but still exhibit a well-organized epithelial structure. The physiological role of Bowman’s layer therefore remains unclear.

Structure of the stroma The stroma constitutes the largest portion, more than 90%, of the thickness of the cornea. The peripheral portion of the cornea connects to the anterior sclera at the limbus, where the tissue loses its transparency. Many characteristics of the cornea, including its physical strength, stability of shape, and transparency, are largely attributable to the anatomic and biochemical properties of the stroma. The uniform arrangement and continuous slow turnover (production and degradation) of collagen fibrils in the stroma are essential for corneal transparency. The sclera is also composed mostly of collagen fibrils and other matrix macromolecules, but nonuniformity in the arrangement of these fibrils accounts for its lack of transparency.106 The thickness of the scleral stroma ranges from approximately 0.5 to 1.0 mm depending on the area, with the exception of the sites of insertion for the rectus muscle, where the sclera is thinnest. The toughness of the scleral stroma is essential for its role as a container of the intraocular tissues. Scleral fibroblasts are embedded within the collagen lamellae.

A

5 µm

Cells The cellular components (predominantly keratocytes) occupy only 2–3% of the total volume of the corneal stroma,107 with the remaining portion comprising mostly the ECM components collagen and proteoglycans. Keratocytes are thought to turn over about every two to three years. The spindle-shaped keratocytes are scattered among the lamellae of the stroma (Fig. 1.9). These cells extend long processes, and the processes of neighboring cells are connected at their tips by gap junctions (Fig. 1.10).108 The three-dimensional network structure of keratocytes can be observed by light microscopy in flat preparations of the corneal stroma, by confocal biomicroscopy, and, after digestion of stromal collagen, by scanning electron microscopy (Fig. 1.10).109 Keratocytes are similar to fibroblasts and possess an extensive intracellular cytoskeleton, including prominent actin filaments. Keratocytes are thus quiescent in the normal cornea but are readily activated and undergo transformation into myofibroblasts, which express α–smooth muscle actin, in response to various types of insult to the stroma.110,111 Myofibroblasts produce ECM, collagen-degrading enzymes, MMPs, and cytokines for stromal tissue repair, and their ability to contract contributes to wound closure. In addition to keratocytes, bone marrow-derived cells are present in the corneal stroma. Approximately 6% of cells that reside in the normal human corneal stroma express the hematopoietic cell marker CD45,112 and 7.58% of stromal

B

1 µm

Fig. 1.9  Transmission electron microscopy of the human corneal stroma. (A) A keratocyte localized between stromal lamellae. (B) A higher-magnification view showing a keratocyte in relation to collagen fibrils coursing in various directions.

cells were found to express this marker in a mouse bone marrow transplantion model.113 In response to infection or injury, however, many inflammatory cells infiltrate the corneal stroma from the limbal vessels surrounding the cornea. Although scleral fibroblasts are not as well characterized as keratocytes, they are thought to be similar to fibroblasts in other parts of the body. As in the corneal stroma, a slow

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1

A

B

10 µm

Fig. 1.10  Electron microscopy of the corneal stroma. (A) Lamellar structure of collagen fibrils and electron-dense gap junctions (1) between the cellular processes of keratocytes in the human cornea.   (B) Three-dimensional view of keratocytes in the rat cornea after digestion of collagen. Note the cellular network formed by keratocytes.

turnover of collagen fibrils by scleral fibroblasts is required for connective tissue homeostasis. Matrix degradation by scleral fibroblasts is promoted by prostaglandin derivatives, which accounts in part for the increase in uveoscleral outflow of aqueous humor and the reduction in intraocular pressure induced by such drugs.114 Activation of scleral fibroblasts by external stimuli, such as injury or surgery, also results in their transdifferentiation into myofibroblasts and consequent tissue fibrosis.

Collagen Collagen constitutes more than 70% of the dry weight of the cornea. Collagen in the corneal stroma is mostly type I, with smaller amounts of types III, V, VI, XII, and XIV also

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present.115–122 Proteoglycans are distributed among the major collagen fibrils. Both the mean diameter of collagen fibrils and the mean distance between such fibrils in the corneal stroma are relatively homogeneous and are less than half of the wavelength of visible light (400–700 nm). This anatomic arrangement is thought to be responsible for the fact that scattering of an incident ray of light by each collagen fibril is canceled by interference from other scattered rays,3,123 allowing light to pass through the cornea. If the diameter of or the distance between collagen fibrils becomes heterogeneous (as occurs in fibrosis or edema), incident rays are scattered randomly and the cornea loses its transparency. Procollagen molecules are secreted into the extracellular space by keratocytes, after which the pro-peptides at both ends are cleaved to yield the mature collagen molecules. The collagen molecules self-assemble into fibrils with a diameter of 10–300 nm, and these fibrils subsequently further assemble into collagen fibers.123 Individual collagen fibrils in the corneal stroma can be observed by transmission electron microscopy (Fig. 1.9). As mentioned above, both the diameter of (22.5–35 nm) and distance between (41.4 ± 0.5 nm) collagen fibrils in the corneal stroma are highly uniform, with this regular arrangement being a major determinant of corneal transparency.120,124–126 At high magnification, each collagen fibril exhibits a characteristic cross-striation pattern with a periodicity of 67 nm. In the corneal stroma, the collagen fibrils align with the same orientation to form about 300 lamellae. Each lamella courses parallel to the surface of the cornea from limbus to limbus. The collagen lamellae in the human corneal stroma vary in width from 0.5 to 250 µm and in thickness from 0.2 to 2.5 µm, and they interweave with each other at various angles.120,127,128 They change direction as they course from the center to the outer zone of the cornea.126 Three-dimensional analysis by second harmonic generation imaging microscopy has revealed that the anatomic characteristics of collagen lamellae are not uniform throughout the normal human corneal stroma (Fig. 1.11).128 Whereas lamellae at the anterior stroma manifest an interwoven structure with an angle of approximately 21° relative to Bowman’s layer, those in the middle and posterior regions of the stroma are parallel.128–130 The width of collagen lamellae also gradually increases from the anterior to posterior stroma.128 Given that dense and interwoven collagen lamellae confer a more rigid structure compared with parallel lamellae, this organization of stromal collagen lamellae is thought to contribute to maintenance of anterior stromal curvature. The collagen lamellae immediately below Bowman’s layer adhere evenly to it. The width of collagen lamellae adherent to Bowman’s layer is slightly larger than that of those located immediately posteriorly,128,130 likely facilitating strong adherence. Similar adherence of collagen lamellae to Descemet membrane in the posterior region of the stroma has not been observed. The turnover of collagen molecules in the cornea is slow, requiring two to three years.131 The histological features of the scleral stroma are similar to those of the corneal stroma, with the scleral stroma also being composed largely of major collagen fibrils and proteoglycans.132 The collagen types detected in the scleral stroma are also similar to those in the corneal stroma. In contrast,

CHAPTER 1 Cornea and Sclera: Anatomy and Physiology

A

B

C

D

Fig. 1.11  Second harmonic generation (SHG) imaging microscopy of the human cornea. Note the difference in alignment of collagen fibers in anterior (A), middle (B), and posterior (C) regions of the stroma. Reconstructed projection image of collagen lamellae in the normal corneal stroma are also shown (D). Note that interwoven collagen lamellae were observed at the anterior stroma. Bar: 50 µm.

the matrix components present in the spaces between the major collagen fibrils in the scleral stroma differ from those in the corneal stroma. This difference in the noncollagenous matrix largely accounts for the difference in ultrastructure between the cornea and sclera. Whereas the collagen fibrils in the corneal stroma are highly uniform in diameter, those in the scleral stroma range in diameter from 25 to 250 nm. Furthermore, whereas collagen fibrils are arranged regularly with a relatively uniform interfiber distance in the corneal stroma, the distance between collagen fibrils in the scleral stroma varies. The ECM of the scleral stroma, including both collagen and noncollagenous components, is produced by the stromal fibroblasts.

Proteoglycans Proteoglycans, the major matrix components located in the spaces among major collagen fibrils in the stroma of the cornea and sclera, are composed of a core protein and glycosaminoglycan chains and are thought to modulate collagen fibrillogenesis.133,134 Glycosaminoglycans comprise repeating disaccharide units and play important roles regardless of the core protein to which they are attached. The functions of proteoglycans can thus be considered from the points of view of both the core protein and glycosaminoglycans. With the exception of hyaluronan, the glycosaminoglycans of the corneal stroma are present in the form of

proteoglycans. The most abundant glycosaminoglycan in the cornea is keratan sulfate,135 constituting about 65% of the total glycosaminoglycan content. The remaining glycosaminoglycans include chondroitin sulfate and dermatan sulfate. Glycosaminoglycans have the ability to absorb and retain large amounts of water. In terms of core proteins, the corneal stroma contains lumican, keratocan, and mimecan (osteoglycin) as keratan sulfate proteoglycans as well as decorin and biglycan as chondroitin sulfate or dermatan sulfate proteoglycans (Table 1.2).136,137 These core proteins are members of the family of small leucine-rich proteoglycans (SLRPs), which contain a common central domain consisting of about 10 leucine-rich repeats.138 They first accumulate as low-sulfate glycoproteins in the embryonic stroma and subsequently bind glycosaminoglycans to form proteoglycans typical of the adult cornea. Although the roles of specific proteoglycans in the maintenance of corneal transparency or shape under physiological conditions or in the development of corneal haziness under pathological conditions remain unclear, spontaneous mutation of a core protein gene has provided some insight. Mutation of the keratocan gene was recently shown to result in cornea plana, an anomaly characterized by abnormal corneal curvature, but it did not affect the transparency of the corneal stroma.139,140 Recent studies with transgenic or knockout mice have also provided insight into the roles of proteoglycan core proteins. A lumican-null mouse was shown to undergo age-dependent opacification of the corneal stroma.141,142 Transmission electron microscopy revealed an irregular arrangement of collagen fibrils in the posterior stroma of these animals, indicating that lumican is required for the regular organization of collagen fibrils and that its dis­ tribution is not uniform throughout the thickness of the stroma.123 Similar to the effect of keratocan gene mutation in humans,140 keratocan-deficient mice show a change in the shape of the eye shell, but the transparency of the corneal stroma is not affected.139 These observations indicate that two major keratan sulfate proteoglycans (those based on lumican or keratocan) do not similarly influence collagen fibril organization in the corneal stroma.138 Mice lacking decorin exhibit abnormal collagen fibrillogenesis in the tail tendon but not in the corneal stroma,143 indicating that decorin may not play an important role in maintenance of corneal stromal transparency, despite its abundance in the stroma. Such genetically modified mice not only shed light on the functions of specific molecules but also provide models of human genetic disorders of the cornea. The main difference between the proteoglycan composition of the sclera and that of the cornea is the absence of keratocan, a specific marker of keratocyte differentiation,137 in the sclera. However, this difference alone does not explain the lack of uniformity in the size and arrangement of collagen fibrils in the sclera. The eyeball of lumican-null mice is larger than that of wild-type animals, whereas that of keratocan-deficient mice is smaller.139,141,142 Although humans with a mutated lumican gene have not yet been described, recent studies suggest that high myopia due to an increased axial length of the eye globe is associated with lumican gene polymorphisms.144 The relative amounts of proteoglycan components in the sclera are changed in an animal model

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Table 1.2  Glycosaminoglycans and proteoglycan core proteins in the cornea Glycosaminoglycan

Size (kDa)

Constituent disaccharide

Heparan sulfate

5–12

N-acetylgalactosamine, glucuronic acid

Heparin

6–25

N-acetylgalactosamine, glucuronic acid

Dermatan sulfate

15–49

N-acetylgalactosamine, iduronic acid

Chondroitin 4,6-sulfate

5–50

N-acetylgalactosamine, glucuronic acid

Keratan sulfate

4–19

N-acetylgalactosamine, galactose

Hyaluronan

4–8000

N-acetylgalactosamine, glucuronic acid

Core protein

Glycosaminoglycan

Function

Lumican

Keratan sulfate

Interaction with corneal epithelial cells

Keratocan

Keratan sulfate

Mutation causes cornea plana

Mimecan

Keratan sulfate

Unknown

Decorin

Chondroitin sulfate or dermatan sulfate

Wound healing

In the normal cornea, proteoglycans are synthesized by stromal keratocytes. They are transiently synthesized by corneal epithelial cells during the early phase of wound healing.

of myopia.145 Recent studies thus suggest that matrix components of the cornea or sclera play specific roles in regulation of the shape or size of the eye shell.

Physiology of the stroma ECM and stromal repair Structural and biochemical homeostasis of the ECM in the corneal stroma is maintained by a balance in the synthesis and degradation of ECM components by keratocytes. In response to corneal injury, keratocytes transdifferentiate into myofibroblasts and actively produce matrix components for healing of the injured stroma, with each newly expressed macromolecule appearing to play an important role in the repair process. During infectious ulceration of the corneal stroma, enzymes that degrade the ECM of the stroma are released by both host cells and the infecting bacteria. Furthermore, pseudomonal elastase degrades collagen directly as well as promoting collagen degradation by keratocytes, in part via activation of pro-MMPs.146 There thus appear to be at least three different pathways for the degradation of stromal collagen fibrils in individuals with infectious corneal ulceration: (1) direct degradation by bacterial collagenase, (2) degradation by MMPs released from keratocytes (or myofibroblasts) and activated by bacterial factors such as elastase, and (3) degradation by proteases released from infiltrated inflammatory cells.

factors and thereby modulate the behavior of cells in the healing corneal stroma. Each cytokine or growth factor activates signal transduction pathways that regulate the expression of specific genes that contribute to the inflammatory response. Targeting of such regulation at the ligand or signaling level may provide new strategies for treatment of woundrelated pathology. TGF-β is thought to play a key role in the healing of the corneal stroma.147,148 It is expressed by both epithelial cells and stromal cells (keratocytes or scleral fibroblasts) as well as by inflammatory cells that activate stromal cells and promote their transdifferentiation into myofibroblasts. Myofibroblasts contribute not only to wound repair but also to post-injury stromal scarring in the cornea and sclera as a result of the overproduction of matrix components. Blockade of TGF-β signaling effectively reduces the fibrogenic reaction and consequent scarring and opacification in a mouse model of corneal alkali burn.147,148 The proinflammatory cytokine tumor necrosis factor (TNF)–α is also upregulated in response to injury.149 TNF-α induces various effects in the cornea under pathological conditions such as injury, allergy, and infection.150–153 However, the complete loss of TNF-α in the cornea of knockout mice results in enhancement of post-alkali burn inflammation, suggesting that the role of TNF-α in the cornea might depend on the specific condition.154

Endothelium

Cytokines and growth factors

Descemet membrane

Both keratocytes and infiltrated cells, such as lymphocytes, neutrophils, and macrophages, secrete cytokines and growth

Descemet membrane, the basement membrane of the corneal endothelium, gradually increases in thickness from

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CHAPTER 1 Cornea and Sclera: Anatomy and Physiology birth (3 µm) to adulthood (8–10 µm) in humans. Histological analysis reveals it to be stratified into a thin (0.3 µm), nonbanded layer adjacent to the stroma, an anterior banded zone (2–4 µm), and a posterior, amorphous, nonbanded zone (>4 µm), the latter of which can represent up to twothirds of the thickness of the membrane and is laid down over time.155 Descemet membrane is composed primarily of collagen types IV and VIII and laminin156 but also contains fibronectin.63,64 Type VIII collagen, which is produced by the corneal endothelium, forms a hexagonal lattice quite different from the structure of type IV collagen in the basement membrane. Collagen fibers in the stroma are continuous with those in Bowman’s layer but not with those in the Descemet membrane. The Descemet membrane adheres tightly to the posterior surface of the corneal stroma and reflects any change in the shape of the stroma. Rupture of the Descemet membrane by physical stress, such as compression birth injury, results in the penetration of aqueous humor into the corneal stroma and consequent stromal edema. The Descemet membrane does not regenerate after endothelial cells re-cover the ruptured area. Diseases such as Fuchs dystrophy are associated with an atypical striated pattern of collagen deposition in the Descemet membrane.157 A patient with early-onset Fuchs dystrophy was found to harbor a mutation in COL8A2,158 which encodes the α2 chain of type VIII collagen.

A

B

Endothelial cells A single layer of corneal endothelial cells covers the posterior surface of the Descemet membrane in a well-arranged mosaic pattern (Fig. 1.12). In humans, these cells are uniformly 5 µm in thickness and 20 µm in width and are polygonal (mostly hexagonal) in shape. The uniformity of endothelial cell size has been evaluated by statistical analysis based on photographs taken by a wide-field specular microscope.159 In young adults, the cell density is about 3500 cells/mm2. The coefficient of variation (standard deviation/mean) for cell area is a clinically valuable marker and is about 0.25 in the normal cornea. An increase in the variability of cell area is termed polymegethism. Another morphometric parameter of the state of the endothelium is hexagonality. In the normal healthy cornea, about 70–80% of endothelial cells are hexagonal. However, endothelial damage can result in a decrease in the hexagonality value and an increase in the variability of cell area (Fig. 1.12). Deviation from hexagonality is referred to as pleomorphism. Corneal endothelial cells contain a large nucleus and abundant cytoplasmic organelles, including mitochondria, endoplasmic reticulum, free ribosomes, and Golgi apparatus (Fig. 1.12), suggesting that they are metabolically active. The endothelial cells interdigitate and contain various junctional complexes, including zonula occludens, macula occludens, and macula adherens. The interconnected endothelial cell

C Fig. 1.12  Corneal endothelium observed by scanning (A) and transmission electron microscopy (B) of the rabbit. Immunofluorescent microscopy (C) of rabbit endothelial cells stained for ZO-1 (green) and nucleus (red). Note each endothelial cell is bound by tight junctions. Specular microscopic images of the human corneal endothelial cells of normal (D) and early stages of bullous keratopathy (E). Cell density is lowered in bullous keratopathy.

D

E

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layer provides a leaky barrier to aqueous humor. In addition, gap junctions allow the transfer of small molecules and electrolytes between the endothelial cells.

Physiology of the endothelium Loss of or damage to corneal endothelial cells will result in increased imbibition of water by the corneal stroma. The endothelial cells contain ion transport systems that counteract the imbibition of water into the stroma. An osmotic gradient of Na+ is present between the aqueous humor (143 mEq/L) and the stroma (134 mEq/L). This gradient results in the flow of Na+ from the aqueous humor to the stroma and in a flux of K+ in the opposite direction. The Na+- and K+-dependent ATPase and the Na+/H+ exchanger are expressed in the basolateral membrane of corneal endothelial cells. Carbon dioxide also diffuses into the cytoplasm of these cells and, together with water, generates bicarbonate ions (HCO3−) in a reaction catalyzed by carbonic anhydrase. The HCO3− then diffuses or is transported into the aqueous humor. Coupled with this movement of HCO3− is a flux of water across endothelial cells into the aqueous humor.160 Given that this ion transport system is partially dependent on cellular energy, cooling of the cornea results in its thickening and in it becoming opaque. The return of the cornea to normal body temperature, however, results in the restoration of its normal thickness and clarity in a phenomenon known as temperature reversal. Corneal endothelial cells essentially do not proliferate in humans, monkeys, and cats, but they do divide in rabbits. Endothelial cell density in the healthy human cornea decreases with age.161 It is important that corneal endothelial cells are protected and preserved as much as possible during surgery and during inflammation in the anterior chamber. Endothelial cells are targeted by immune and inflammatory cells that appear in the anterior chamber in association with corneal graft rejection or conditions such as anterior uveitis. Furthermore, inflammatory cytokines disturb the pumping function of endothelial cells and thereby promote stromal edema. Steroids and insulin have been found to increase Na+, K+-dependent ATPase activity in corneal endothelial cells.162 The loss of endothelial cells for any reason results in enlargement of the remaining neighboring cells and their spreading to cover the defective area, without an increase in cell number. The indexes based on specular microscopy fluctuate as endothelial damage is resurfaced by the migration and enlargement of the remaining endothelial cells. The coefficient of the variation of cell area is the most sensitive index of corneal endothelial dysfunction, whereas hexagonality is a good index of the progress of endothelial wound healing.

Innervation The cornea is one of the most highly innervated and sensitive tissues in the body. The density of nerve endings in the cornea is thus about 300 to 400 times greater than that in the skin.163,164 Most of the sensory nerves in the cornea are derived from the ciliary nerves of the ophthalmic branch of the trigeminal nerve. The long ciliary nerves provide the perilimbal nerve ring. Nerve fibers penetrate the cornea in

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the deep peripheral stroma radially and then course anteriorly, forming a terminal subepithelial plexus.165 The nerve fibers lose their myelination within a short distance of their point of entry into the cornea, penetrate the Bowman’s layer, and terminate at the wing cell level of the epithelium. Loss of the superficial corneal epithelium results in exposure of the nerve endings and consequent severe ocular pain. Slit lamp microscopy allows observation of nerve fibers in the corneal stroma. The fibers are especially prominent at the corneal periphery, where their diameter is relatively large. Laser-scanning confocal biomicroscopy has revealed networks of fine nerve fibers (subepithelial nerve plexuses) in or below the basal cell layer of the corneal epithelium.164,166 The diameter of these nerve fibers increases with distance from the anterior corneal surface (Fig. 1.3). Histochemical studies have revealed the presence of various neurotransmitters, including substance P, calcitonin gene-related peptide, neuropeptide Y, vasoactive intestinal peptide, galanin, methionine-enkephalin, catecholamines, and acetylcholine, in the cornea.167–174 The cornea thus contains peptidergic, sympathetic, and parasympathetic nerve fibers. Sensory nerves release neuropeptides that modulate local cell behavior and inflammation. The loss of corneal sensation often results in breakdown of corneal integrity. Trigeminal denervation is associated with a reduction in the abundance of substance P in the cornea.175 Sectioning of the trigeminal nerve results in trophic or degenerative changes in the cornea as well as in the depletion of substance P.176–178 Substance P may therefore contribute to maintenance of the corneal epithelium. Degeneration or dysfunction of sensory nerves (trigeminal nerve branches) in the cornea can result in delayed healing of corneal injuries and the development of neurotrophic ulcer or neurotrophic keratopathy, one of the most refractory corneal disorders.179 Persistent corneal epithelial defects or delayed epithelial wound healing are frequently observed in individuals with a reduced corneal sensation, such as those infected with herpes simplex or herpes zoster viruses as well as those with trigeminal nerve damage due to intracranial disorders or diabetes mellitus. Topical anesthetics, which are potentially drugs of abuse, also impair corneal epithelial migration in an organ culture system.180 Furthermore, frank corneal ulceration has been shown to develop in anesthetized eyes. These various observations thus implicate neural regulation in maintenance and repair of the corneal epithelium. The physiological role of corneal innervation in corneal epithelial wound healing remains to be fully clarified, however. Substance P is thought to regulate various physiological processes, including plasma extravasation, vasodilation, and the release of histamine from mast cells.181–184 Exposure of rabbit corneal epithelial cells in culture to the combination of substance P and insulin-like growth factor–1 (IGF-1) resulted in a marked increase in the number of cells that attached to a fibronectin matrix.185 The combination of substance P and IGF-1 also synergistically promotes corneal epithelial migration, with neither agent alone having an effect on this process. Furthermore, the administration of eyedrops containing IGF-1 and either substance P or a tetrapeptide derived from its carboxyl terminus has been shown to be an effective treatment for persistent corneal epithelial

CHAPTER 1 Cornea and Sclera: Anatomy and Physiology defects in individuals with neurotrophic keratopathy or diabetic neuropathy.186–188 Nerve growth factor (NGF), first discovered by LeviMontalcini in the early 1950s,189 is a polypeptide that stimulates the regeneration of peripheral nerve fibers.190 Eye drops containing NGF also promote resurfacing of persistent corneal epithelial defects in animals and humans.190–194 Although the role of calcitonin gene-related peptide in homeostasis of the corneal epithelium remains unknown, the finding that this peptide stimulates the proliferation of keratinocytes suggests that it might also modulate corneal epithelial cell behavior.195 Members of the family of transient receptor potential (TRP) cation channels have been shown to modulate inflammation and tissue repair by promoting the secretion of neuropeptides in nonocular tissues.196 Studies of mutant mice with deletions in genes for members of this family have revealed that loss of specific channels inhibits corneal inflammation in response to alkali exposure197,198 or retards corneal epithelial wound healing.199 The short and long posterior ciliary nerves, which are branches of the trigeminal nerve, penetrate the sclera and provide fine sensory branches to the scleral stroma. In addition, nerve fibers are also present in the episclera.200 These fibers include those of vasodilator and vasoconstrictor nerves and are thought to regulate blood flow and volume in the episcleral vessels for modulation of episcleral venous pressure and outflow facility. Cells in the scleral spur are also thought to contribute to the regulation of intraocular pressure. Axons of presumably parasympathetic origin are present in the scleral spur of humans. On the other hand, cholinergic innervation of scleral spur cells appears to be rare or absent.201

Vascular system The cornea is one of the few avascular tissues in the body. Although the normal cornea does not contain blood vessels, factors derived from the blood play important roles in corneal metabolism and wound healing. The anterior ciliary artery, which is derived from the ophthalmic artery, forms a vascular arcade in the limbal region that anastomoses with vessels derived from the facial branch of the external carotid artery. The cornea is thus supplied with blood components by both internal and external carotid arteries. In certain pathological conditions, new vessels enter the corneal stroma from the limbus and result in a loss of corneal transparency. In contrast to the cornea, the episclera is highly vascularized. The episcleral vasculature shows a specialized morphology characterized by the absence of capillaries, numerous arteriovenous anastomoses, and a muscle-rich venous network, which is thought to play an important role in the regulation of intraocular pressure. Such vascularization is also apparent in the loose connective tissue of Tenon’s capsule. The scleral stroma contains few blood vessels, with the exception of the input and output of the vessels of the choroidal circulation.

Oxygen and nutrient supply Corneal epithelial and endothelial cells are metabolically active. Cellular activities require ATP as an energy source,

with catabolism of glucose by glycolysis and the citric acid cycle generating ATP under aerobic conditions. A supply of glucose and oxygen is thus essential to maintain the normal metabolic functions of the cornea.202–205 The cornea is supplied with glucose by diffusion from the aqueous humor. In contrast, oxygen is supplied to the cornea primarily by diffusion from tear fluid, which absorbs oxygen from the air. Direct exposure of tear fluid to the atmosphere is thus essential for oxygenation of the cornea. Disruption of the oxygen supply to the cornea, such as that resulting from the wearing of contact lenses with low gas permeability, can lead to corneal hypoxia and consequent stromal edema.206–208 Closure of the eyelids during sleep also reduces the amount of oxygen that reaches the cornea. Corneal metabolism therefore changes from aerobic to anaerobic (with consequent accumulation of lactate) during sleep.209

Development of the Anterior Eye Segment Characterization of the development of ocular tissues during embryogenesis is important for understanding the pathogenesis of congenital anomalies of the cornea and anterior eye segment (Fig. 1.13).210,211 Morphogenesis of the eye is achieved by cell lineages of various origins including the surface and neural ectoderm during embryonic development. Epithelial cells of the cornea are derived from the epidermal ectoderm, whereas keratocytes, scleral fibroblasts, and endothelial cells are of neural crest (neuroectodermal) origin. The surface ectoderm above the neuronal optic cup invaginates to form the crystalline lens. After the lens vesicle has separated from the surface ectoderm, the epithelium on the immature lens differentiates into the corneal epithelium. Neural crest–derived mesenchymal cells migrate in the space between the lens and primitive corneal epithelium and develop into the corneal stroma, endothelium, iris, and trabecular meshwork. Many anomalies of the anterior eye segment result from impaired differentiation of these neural crest-derived tissues. The surface ectoderm above the optic cup invaginates during the fifth week of gestation in humans, and the primitive corneal epithelium develops junctional complexes by the sixth week. Most scleral fibroblasts differentiate from neural crest cells that surround the optic cup during the sixth week. Mesodermal cells also contribute to development of the sclera and the extraocular muscles. The neural crest cell-derived mesenchyme migrates into the space between the primitive corneal epithelium and lens vesicle in three waves during the seventh week. The first wave of migration results in formation of the corneal endothelium and trabecular endothelium; the second wave of cells differentiates into keratocytes; and the third wave gives rise to the iris. During the eighth week, the keratocytes form five to eight layers of collagen lamellae and the corneal endothelium starts to form the Descemet membrane. Defects in the migration of neural crest–derived mesenchymal cells are responsible for anomalies of the cornea and anterior eye segment including Peters anomaly. Several genes, including those encoding TGF-β2 and the transcription factor FOXC, have been implicated in the differentiation of neural crest cells into the primitive corneal stroma in mice.212

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A

B Fig. 1.13  Ocular histology of C57BL/6 mouse embryos as revealed by hematoxylineosin staining. (A) The lens vesicle has separated from the surface ectoderm, which will develop into the corneal epithelium, in an embryo at E12.5. Neural crest cells, which will form the corneal stroma, are present between the lens capsule and surface ectoderm. (B) The corneal endothelium has separated from the lens capsule to form the anterior chamber in an embryo at E18.5. The ocular surface is covered with the eyelids, which are fused to each other. A few cells are apparent in the vitreous cavity. (C) Stratification of the corneal epithelium is not well developed at E18.5, but the endothelium has matured. The density of keratocytes is higher than that observed in the adult mouse cornea. Bars: 500 µm (A, B) and 50 µm (C).

C

The spaces among collagen fibrils in the embryonic corneal stroma become occupied by proteoglycans that are formed as a result of the binding of glycosaminoglycan chains to previously accumulated core proteins. Studies with mice have shown that the composition of proteoglycans in the corneal stroma changes markedly during embryonic development.213 Even by the sixth month of gestation, the human cornea is still not fully mature. The epithelium has only three or four layers of cells, and keratan sulfate proteoglycans continue to accumulate. By the seventh month, however, the cornea is well developed, with the epithelium consisting of four or five layers with readily recognizable basal, wing, and superficial cells. The stroma is also almost fully developed at this time, with the accumulation of keratan sulfate proteoglycans among collagen fibrils being virtually complete. Hyaluronan is a major glycosaminoglycan in the corneal stroma during the early stages of embryonic development, but its abundance declines concomitantly with the increase in that of keratan sulfate, chondroitin sulfate, and dermatan sulfate, giving rise to a glycosaminoglycan composition similar to that of the adult stroma.214

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Recent advances in transgenic and gene knockout technologies in mice have provided important insight into the role of specific genes in the development and homeostasis of corneal tissue as well as into congenital anomalies in humans.214,215 Interpretation of such studies also depends on an understanding of the normal process of eye development in the mouse (Fig. 1.13). The surface ectoderm invaginates into the optic cup at embryonic day (E) 10.5 in mice. At E12.5, the primitive lens has already separated from the surface ectoderm, which will become the corneal epithelium, and the neural crest-derived mesenchyme has begun to migrate into the space between the primitive corneal epithelium and lens. In contrast to the human embryo, the neural crest-derived cells migrate into this space in a single wave. At E14.5, the embryo has already developed the epithelium, stroma, and endothelium of the cornea, and at E18.5 the corneal stroma has increased in thickness as a result of the synthesis of matrix macromolecules. Eyelid morphogenesis is orchestrated by signaling networks activated by growth factors and other morphogenesis- related molecules.216 The palpebral conjunctiva develops in

CHAPTER 1 Cornea and Sclera: Anatomy and Physiology association with eyelid morphogenesis during embryonic development. The upper and lower eyelids fuse to each other between E14.5 and E16.5; the eyelids separate and the eyes reopen after birth, and the corneal epithelium then undergoes final maturation.217

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CHAPTER 1 Cornea and Sclera: Anatomy and Physiology

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Immunohistochemical evidence by anti-collagen antibodies characterized by immunoelectroblotting. Am J Pathol 1984;116:417–26. Birk DE, Fitch JM, Linsenmayer TF. Organization of collagen types I and V in the embryonic chicken cornea. Invest Ophthalmol Vis Sci 1986;27: 1470–7. Yue BY, Sugar J, Schrode K. Collagen staining in corneal tissues. Curr Eye Res 1986;5:559–64. Komai Y, Ushiki T. The three-dimensional organization of collagen fibrils in the human cornea and sclera. Invest Ophthalmol Vis Sci 1991; 32:2244–58. Doane KJ, Yang G, Birk DE. Corneal cell-matrix interactions: type VI collagen promotes adhesion and spreading of corneal fibroblasts. Exp Cell Res 1992;200:490–9. Drubaix I, Legeais JM, Malek-Chehire N, et al. Collagen synthesized in fluorocarbon polymer implant in the rabbit cornea. Exp Eye Res 1996; 62:367–76. Hassell JR, Birk DE. The molecular basis of corneal transparency. Exp Eye Res 2010;91:326–35. Giraud JP, Pouliquen Y, Offret G, et al. Statistical morphometric studies in normal human and rabbit corneal stroma. Exp Eye Res 1975;21: 221–9. Akhtar S, Bron AJ, Salvi SM, et al. Ultrastructural analysis of collagen fibrils and proteoglycans in keratoconus. Acta Ophthalmol 2008;86: 764–72. Meek KM, Boote C. The organization of collagen in the corneal stroma. Exp Eye Res 2004;78:503–12. Radner W, Zehetmayer M, Aufreiter R, et al. Interlacing and cross-angle distribution of collagen lamellae in the human cornea. Cornea 1998; 17:537–43. Morishige N, Shin-Gyou-Uchi R, Azumi H, et al. Quantitative analysis of collagen lamellae in the normal and keratoconic human cornea by second harmonic generation imaging microscopy. Invest Ophthalmol Vis Sci 2014;55:8377–85. Morishige N, Petroll WM, Nishida T, et al. Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals. J Cataract Refract Surg 2006;32:1784–91. Morishige N, Takagi Y, Chikama T, et al. Three-dimensional analysis of collagen lamellae in the anterior stroma of the human cornea visualized by second harmonic generation imaging microscopy. Invest Ophthalmol Vis Sci 2011;52:911–15. Meek KM, Fullwood NJ. Corneal and scleral collagens − a microscopist’s perspective. Micron 2001;32:261–72. Ihanamaki T, Pelliniemi LJ, Vuorio E. Collagens and collagen-related matrix components in the human and mouse eye. Prog Retin Eye Res 2004;23:403–34. Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 1998;67:609–52. Ho LT, Harris AM, Tanioka H, et al. A comparison of glycosaminoglycan distributions, keratan sulphate sulphation patterns and collagen fibril architecture from central to peripheral regions of the bovine cornea. Matrix Biol 2014;38:59–68. Funderburgh JL. Keratan sulfate: structure, biosynthesis, and function. Glycobiology 2000;10:951–8. Funderburgh JL, Corpuz LM, Roth MR, et al. Mimecan, the 25-kDa corneal keratan sulfate proteoglycan, is a product of the gene producing osteoglycin. J Biol Chem 1997;272:28089–95. Liu CY, Shiraishi A, Kao CW, et al. The cloning of mouse keratocan cDNA and genomic DNA and the characterization of its expression during eye development. J Biol Chem 1998;273:22584–8. Kao WW, Liu CY. Roles of lumican and keratocan on corneal transparency. Glycoconj J 2002;19:275–85. Liu CY, Birk DE, Hassell JR, et al. Keratocan-deficient mice display alterations in corneal structure. J Biol Chem 2003;278:21672–7. Pellegata NS, Dieguez-Lucena JL, Joensuu T, et al. Mutations in KERA, encoding keratocan, cause cornea plana. Nat Genet 2000;25:91–5. Chakravarti S, Magnuson T, Lass JH, et al. Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican. J Cell Biol 1998;141:1277–86. Saika S, Shiraishi A, Liu CY, et al. Role of lumican in the corneal epithelium during wound healing. J Biol Chem 2000;275:2607–12. Danielson KG, Baribault H, Holmes DF, et al. Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. J Cell Biol 1997;136:729–43. Lin HJ, Wan L, Tsai Y, et al. The association between lumican gene polymorphisms and high myopia. Eye (Lond) 2010;24:1093–101. Paluru PC, Scavello GS, Ganter WR, et al. Exclusion of lumican and fibromodulin as candidate genes in MYP3 linked high grade myopia. Mol Vis 2004;10:917–22.

146. Nagano T, Hao JL, Nakamura M, et al. Stimulatory effect of pseudomonal elastase on collagen degradation by cultured keratocytes. Invest Ophthalmol Vis Sci 2001;42:1247–53. 147. Saika S. TGF-beta signal transduction in corneal wound healing as a therapeutic target. Cornea 2004;23:S25–30. 148. Saika S, Yamanaka O, Sumioka T, et al. Fibrotic disorders in the eye: Targets of gene therapy. Prog Retin Eye Res 2008;27:177–96. 149. Brenner MK. Tumour necrosis factor. Br J Haematol 1988;69:149–52. 150. Hong JW, Liu JJ, Lee JS, et al. Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea. Invest Ophthalmol Vis Sci 2001;42:2795–803. 151. Keadle TL, Usui N, Laycock KA, et al. IL-1 and TNF-alpha are important factors in the pathogenesis of murine recurrent herpetic stromal keratitis. Invest Ophthalmol Vis Sci 2000;41:96–102. 152. Dekaris I, Zhu SN, Dana MR. TNF-alpha regulates corneal Langerhans cell migration. J Immunol 1999;162:4235–9. 153. Planck SR, Rich LF, Ansel JC, et al. Trauma and alkali burns induce distinct patterns of cytokine gene expression in the rat cornea. Ocul Immunol Inflamm 1997;5:95–100. 154. Saika S, Ikeda K, Yamanaka O, et al. Loss of tumor necrosis factor alpha potentiates transforming growth factor beta-mediated pathogenic tissue response during wound healing. Am J Pathol 2006;168:1848–60. 155. Johnson DH, Bourne WM, Campbell RJ. The ultrastructure of Descemet’s membrane. I. Changes with age in normal corneas. Arch Ophthalmol 1982;100:1942–7. 156. Fitch JM, Birk DE, Linsenmayer C, et al. The spatial organization of Descemet’s membrane-associated type IV collagen in the avian cornea. J Cell Biol 1990;110:1457–68. 157. Bourne WM, Johnson DH, Campbell RJ. The ultrastructure of Descemet’s membrane. III. Fuchs’ dystrophy. Arch Ophthalmol 1982;100: 1952–5. 158. Biswas S, Munier FL, Yardley J, et al. Missense mutations in COL8A2, the gene encoding the alpha2 chain of type VIII collagen, cause two forms of corneal endothelial dystrophy. Hum Mol Genet 2001;10: 2415–23. 159. Hodson SA, Sherrard ES. The specular microscope: its impact on laboratory and clinical studies of the cornea. Eye (Lond) 1988;2(Suppl.):S81–97. 160. Bonanno JA. Molecular mechanisms underlying the corneal endothelial pump. Exp Eye Res 2012;95:2–7. 161. Laule A, Cable MK, Hoffman CE, et al. Endothelial cell population changes of human cornea during life. Arch Ophthalmol 1978;96: 2031–5. 162. Hatou S. Hormonal regulation of Na+/K+-dependent ATPase activity and pump function in corneal endothelial cells. Cornea 2011;30(Suppl. 1):S60–6. 163. Rozsa AJ, Beuerman RW. Density and organization of free nerve endings in the corneal epithelium of the rabbit. Pain 1982;14:105–20. 164. Muller LJ, Marfurt CF, Kruse F, et al. Corneal nerves: structure, contents and function. Exp Eye Res 2003;76:521–42. 165. Hogan MJ, Alvarado JA, Weddell JE. Histology of the human eye. Philadelphia: WB Saunders; 1971. 166. Oliveira-Soto L, Efron N. Morphology of corneal nerves using confocal microscopy. Cornea 2001;20:374–84. 167. Jones MA, Marfurt CF. Peptidergic innervation of the rat cornea. Exp Eye Res 1998;66:421–35. 168. Tervo K, Tervo T, Eranko L, et al. Substance P-immunoreactive nerves in the human cornea and iris. Invest Ophthalmol Vis Sci 1982;23: 671–4. 169. Lehtosalo JI, Substance P-like immunoreactive trigeminal ganglion cells supplying the cornea. Histochemistry 1984;80:273–6. 170. Marfurt CF, Murphy CJ, Florczak JL. Morphology and neurochemistry of canine corneal innervation. Invest Ophthalmol Vis Sci 2001;42: 2242–51. 171. Stone RA, Kuwayama Y, Terenghi G, et al. Calcitonin gene-related peptide: occurrence in corneal sensory nerves. Exp Eye Res 1986;43: 279–83. 172. Stone RA. Neuropeptide Y and the innervation of the human eye. Exp Eye Res 1986;42:349–55. 173. Ueda S, del Cerro M, LoCascio JA, et al. Peptidergic and catecholaminergic fibers in the human corneal epithelium. An immunohistochemical and electron microscopic study. Acta Ophthalmol Suppl 1989;192: 80–90. 174. Stone RA, Tervo T, Tervo K, et al. Vasoactive intestinal polypeptide-like immunoreactive nerves to the human eye. Acta Ophthalmol (Copenh) 1986;64:12–18. 175. Unger WG, Butler JM, Cole DF, et al. Substance P, vasoactive intestinal polypeptide (VIP) and somatostatin levels in ocular tissue of normal and sensorily denervated rabbit eyes. Exp Eye Res 1981;32:797–801.

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176. Mishima S. The effects of the denervation and the stimulation of the sympathetic and the trigeminal nerve on the mitotic rate of the corneal epithelium in the rabbit. Jpn J Ophthalmol 1957;1:65–73. 177. Kahl BF, Reid TW. Substance P and the eye. Prog Retin Eye Res 1995;14: 473–504. 178. Terenghi G, Zhang SQ, Unger WG, et al. Morphological changes of sensory CGRP-immunoreactive and sympathetic nerves in peripheral tissues following chronic denervation. Histochemistry 1986;86:89–95. 179. Nishida T, Yanai R. Advances in treatment for neurotrophic keratopathy. Curr Opin Ophthalmol 2009;20:276–81. 180. Bisla K, Tanelian DL. Concentration-dependent effects of lidocaine on corneal epithelial wound healing. Invest Ophthalmol Vis Sci 1992;33: 3029–33. 181. Pernow B. Substance P. Pharmacol Rev 1983;35:85–141. 182. McGillis JP, Organist ML, Payan DG. Substance P and immunoregulation. Fed Proc 1987;46:196–9. 183. Payan DG. Neuropeptides and inflammation: the role of substance P. Annu Rev Med 1989;40:341–52. 184. Wallengren J, Hakanson R. Effects of substance P, neurokinin A and calcitonin gene-related peptide in human skin and their involvement in sensory nerve-mediated responses. Eur J Pharmacol 1987;143: 267–73. 185. Nakamura M, Chikama T, Nishida T. Up-regulation of integrin alpha 5 expression by combination of substance P and insulin-like growth factor-1 in rabbit corneal epithelial cells. Biochem Biophys Res Commun 1998;246:777–82. 186. Brown SM, Lamberts DW, Reid TW, et al. Neurotrophic and anhidrotic keratopathy treated with substance P and insulinlike growth factor 1. Arch Ophthalmol 1997;115:926–7. 187. Chikama T, Fukuda K, Morishige N, et al. Treatment of neurotrophic keratopathy with substance-P-derived peptide (FGLM) and insulin-like growth factor I. Lancet 1998;351:1783–4. 188. Morishige N, Komatsubara T, Chikama T, et al. Direct observation of corneal nerve fibres in neurotrophic keratopathy by confocal biomicroscopy. Lancet 1999;354:1613–14. 189. Levi-Montalcini R. The nerve growth factor 35 years later. Science 1987; 237:1154–62. 190. Rask CA. Biological actions of nerve growth factor in the peripheral nervous system. Eur Neurol 1999;41(Suppl. 1):14–19. 191. Lambiase A, Rama P, Bonini S, et al. Topical treatment with nerve growth factor for corneal neurotrophic ulcers. N Engl J Med 1998;338: 1174–80. 192. Lambiase A, Pagani L, Di Fausto V, et al. Nerve growth factor eye drop administrated on the ocular surface of rodents affects the nucleus basalis and septum: biochemical and structural evidence. Brain Res 2007;1127: 45–51. 193. Lambiase A, Manni L, Bonini S, et al. Nerve growth factor promotes corneal healing: structural, biochemical, and molecular analyses of rat and human corneas. Invest Ophthalmol Vis Sci 2000;41:1063–9. 194. Bonini S, Lambiase A, Rama P, et al. Topical treatment with nerve growth factor for neurotrophic keratitis. Ophthalmology 2000;107: 1347–51. 195. Roggenkamp D, Kopnick S, Stab F, et al. Epidermal nerve fibers modulate keratinocyte growth via neuropeptide signaling in an innervated skin model. J Invest Dermatol 2013;133:1620–8.

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196. Vay L, Gu C, McNaughton PA. The thermo-TRP ion channel family: properties and therapeutic implications. Br J Pharmacol 2012;165: 787–801. 197. Okada Y, Reinach PS, Shirai K, et al. TRPV1 involvement in inflammatory tissue fibrosis in mice. Am J Pathol 2011;178:2654–64. 198. Okada Y, Shirai K, Reinach PS, et al. TRPA1 is required for TGF-beta signaling and its loss blocks inflammatory fibrosis in mouse corneal stroma. Lab Invest 2014;94:1030–41. 199. Sumioka T, Okada Y, Reinach PS, et al. Impairment of corneal epithelial wound healing in a TRPV1-deficient mouse. Invest Ophthalmol Vis Sci 2014;55:3295–302. 200. Selbach JM, Buschnack SH, Steuhl KP, et al. Substance P and opioid peptidergic innervation of the anterior eye segment of the rat: an immunohistochemical study. J Anat 2005;206:237–42. 201. Tamm ER, Koch TA, Mayer B, et al. Innervation of myofibroblast-like scleral spur cells in human monkey eyes. Invest Ophthalmol Vis Sci 1995; 36:1633–44. 202. Aguayo JB, McLennan IJ, Graham C Jr, et al. Dynamic monitoring of corneal carbohydrate metabolism using high-resolution deuterium NMR spectroscopy. Exp Eye Res 1988;47:337–43. 203. Gottsch JD, Chen CH, Aguayo JB, et al. Glycolytic activity in the human cornea monitored with nuclear magnetic resonance spectroscopy. Arch Ophthalmol 1986;104:886–9. 204. Riley MV. Glucose and oxygen utilization by the rabbit cornea. Exp Eye Res 1969;8:193–200. 205. Weissman BA, Fatt I, Rasson J. Diffusion of oxygen in human corneas in vivo. Invest Ophthalmol Vis Sci 1981;20:123–5. 206. Holden BA, Sweeney DF, Vannas A, et al. Effects of long-term extended contact lens wear on the human cornea. Invest Ophthalmol Vis Sci 1985; 26:1489–501. 207. Ichijima H, Ohashi J, Cavanagh HD. Effect of contact-lens-induced hypoxia on lactate dehydrogenase activity and isozyme in rabbit cornea. Cornea 1992;11:108–13. 208. Thoft RA, Friend J. Biochmical aspects of contact lens wear. Am J Ophthalmol 1975;80:139–45. 209. Sack RA, Beaton A, Sathe S, et al. Towards a closed eye model of the pre-ocular tear layer. Prog Retin Eye Res 2000;19:649–68. 210. Graw J. Genetic aspects of embryonic eye development in vertebrates. Dev Genet 1996;18:181–97. 211. Sevel D, Isaacs R. A re-evaluation of corneal development. Trans Am Ophthalmol Soc 1988;86:178–207. 212. Kim JE, Han MS, Bae YC, et al. Anterior segment dysgenesis after overexpression of transforming growth factor-beta-induced gene, beta igh3, in the mouse eye. Mol Vis 2007;13:1942–52. 213. Quantock AJ, Young RD. Development of the corneal stroma, and the collagen-proteoglycan associations that help define its structure and function. Dev Dyn 2008;237:2607–21. 214. Saika S, Liu CY, Azhar M, et al. TGFbeta2 in corneal morphogenesis during mouse embryonic development. Dev Biol 2001;240:419–32. 215. Kao WW, Xia Y, Liu CY, et al. Signaling pathways in morphogenesis of cornea and eyelid. Ocul Surf 2008;6:9–23. 216. Xia Y, Karin M. The control of cell motility and epithelial morphogenesis by Jun kinases. Trends Cell Biol 2004;14:94–101. 217. Zieske JD. Corneal development associated with eyelid opening. Int J Dev Biol 2004;48:903–11.

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Basic Science: Cornea, Sclera, Ocular Adnexa Anatomy, Physiology and Pathophysiologic Responses





Chapter 2  The Conjunctiva: Anatomy and Physiology Joshua H. Hou, J. Daniel Nelson, J. Douglas Cameron

Key Concepts • • • • • • •

The conjunctival and limbal epithelia are derived from surface ectoderm. The mucocutaneous junction at the lid margins is the junction between hydrophobic (unwettable) epidermis and hydrophilic (wettable) conjunctiva. The corneoscleral limbus is a unique niche environment that supports proliferation and differentiation of corneal stem cells. Injury to the limbus and loss of corneal stem cells results in conjunctivalization of the cornea. Conjunctival goblet cells secrete MUC5AC, the primary constituent of the tear mucin layer. Conjunctival epithelial cells express membrane-bound mucins (MUC1, MUC4, MUC16) which confer wettability to the ocular surface. The conjunctiva modulates the volume, osmolarity, and electrolyte concentration of the tear film.

The conjunctiva is a mucous membrane that is vital to the health of the ocular surface and the eye as a whole. It provides extensive coverage of the ocular surface from the upper and lower lid margins to the corneal limbus. Its unique anatomy allows unrestricted movement of the eye and facilitates normal lid function. The conjunctiva also plays critical roles in the production of both the aqueous and mucous components of the tear film. It further plays a major role in ocular surface immunology as the main reservoir of ocular surface lymphoid tissue and other antimicrobial agents which protect the surface. The corneal limbus at the junction of the conjunctiva and cornea is a crucial and unique microenvironment that maintains and promotes differentiation of corneal epithelial stem cells. Abnormalities of the conjunctiva and limbus may lead to restriction of ocular movement, lid malposition, deficiency of the tear film, decreased host resistance to ocular surface infection, and loss of corneal epithelial integrity and clarity.

Embryology The conjunctival epithelium is derived from surface ectoderm and becomes distinguishable from adjacent limbalcorneal epithelium as early as the seventh week of human fetal development.1 Outgrowth and apposition of the neuralectoderm derived optic vesicle to the overlying surface ectoderm results in activation of PAX6 gene expression within the overlying surface ectoderm cells. This triggers elongation of the majority of the surface ectodermal cells within the apposition area, resulting in formation of a lens placode. The lens placode cells then invaginate and separate from the surface ectoderm to form the lens vesicle. The small cohort of residual nonelongated, PAX6 expressing ectodermal cells on the embryonic surface in turn differentiate into conjunctival and limbal-corneal epithelium. The surrounding surface ectodermal cells which lack PAX6 expression continue through a default differentiation pathway to become the epidermis of the lids (Fig. 2.1).1 Limbal-corneal epithelium then undergoes a further, remarkable segregation of stem cells and early precursor cells to the anatomic limbus. Subsequent expression of stem cell markers such as ABCG2 and loss of gap junctions in the limbal basal epithelium; and expression of tissue specific cytokeratins in corneal and conjunctival epithelium lead to phenotypically distinct corneal, limbal, and conjunctival epithelium.1 At eight weeks the eyelids form from folds of the surface ectoderm and fuse together. The conjunctival epithelium develops within the lid folds from surface ectoderm along the posterior surface of the lids and around the developing cornea. Budding of the epithelium in the conjunctival fornices forms the lacrimal gland superotemporally and accessory lacrimal glands of Wolfring and Krause in the inferior and superior fornices (12 weeks). The caruncle arises as a sequestration of the medial lower eyelid to accommodate the development of the nasolacrimal duct.2

Anatomy The conjunctiva extends from the mucocutaneous junction (MCJ) at the margin of the eyelids to the corneoscleral

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CHAPTER 2 The Conjunctiva: Anatomy and Physiology

Chapter Outline Embryology Anatomy Histology Vascular Supply Lymphatic Drainage Nerve Supply Normal Flora Physiology of the Conjunctiva

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 Surface ectoderm

Optic vesicle

Lens placode invagination

Lens placode

PAX6 expression

Neural ectoderm A

B

C Epidermis

Residual PAX6 surface ectoderm Lens

Conjunctival epithelium Corneal epithelium Fornix

D

E

Fig. 2.1  Conjunctival embryogenesis. Apposition of the optic vesicle to overlying surface ectoderm results in localized PAX6 expression (A) and induction of the lens placode (B). After invagination of the lens placode (C), residual PAX6+ surface ectoderm (D) differentiates into conjunctival, limbal, and corneal epithelium (E).

limbus. The conjunctiva covering the posterior surface of the eyelid is known as palpebral conjunctiva while the conjunctiva covering the surface of the globe is known as bulbar conjunctiva. Redundant conjunctiva at the transition between the palpebral and bulbar conjunctiva forms the fornices superiorly, inferiorly, and temporally, and an extendible plica medially. The redundancy of the conjunctiva in the fornices and medially at the plica allows for independent movement of the eye and eyelids. The larger superior fornix is maintained by fine smooth muscle slips passing from the deep surface of the levator palpebrae muscle to insert into the conjunctiva. These effectively prevent the superior forniceal conjunctiva from prolapsing down and blocking vision during upward gaze. The temporal conjunctiva is attached by fine fibrous slips to the lateral rectus tendon, which maintains the position of the conjunctiva during horizontal gaze. A true fornix does not exist medially except in adduction. Instead, a crescentshaped fold of conjunctiva known as the plica semilunaris is formed medially, with its free lateral border lying 3−6 mm temporal to the caruncle when the eye is in primary position. Fine fibrous strips from the medial rectus tendon insert deep into the plica and caruncle. With contraction of the medial rectus and adduction of the eye, these slips tighten and form a medial cul-de-sac, approximately 2−3 mm in depth. The mean total surface area for the adult conjunctival sac, including the cornea, is 16 cm2 per eye (Fig. 2.2). The epithelium contains goblet cells, Langerhans cells, and dendritic melanocytes. The substantia propria, or conjunctival stroma, is highly vascularized and may contain nonstriated muscle, sympathetic nerves, cartilage, and fatty tissue. The caruncle measures 4–5 mm horizontally and 3–4 mm vertically and is located at the most medial aspect of the interpalpebral fissure. The caruncle is composed of pilosebaceous units, accessory lacrimal gland tissue, fibrofatty tissue, occasional smooth muscle fibers, and eccrine glands. Deep to the caruncle there may be several large sebaceous glands

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10 mm

12–14 mm

7 mm

8 mm Fig. 2.2  Geography of the fornices. Distance from the corneoscleral limbus to the fornix.

without cilia, similar to meibomian glands, which open onto the surface. The MCJ is the transition from the keratinized skin of the eyelid margin to the nonkeratinized skin of the palpebral conjunctiva. Physiologically, this also correlates with the transition from the hydrophobic (unwettable), air-exposed cutaneous surface of the lid to hydrophilic (wettable), fluidcovered conjunctival mucous membrane. The MCJ also demarcates and defines the anterior apex of the tear meniscus, which terminates at the unwettable eyelid skin. The MCJ is not stationary, but rather can move depending on the position of the tear film meniscus. Nasally, the MCJ runs anterior to the lacrimal puncta, which ensures that the puncta remains bathed within the tear meniscus. Just posterior to the MCJ, a physiological line of vital-dye (lissamine green, rose Bengal, fluorescein, etc.) stainable conjunctival epithelium known as Marx’s line is found along the upper and lower margins in normal subjects of all ages.3 Anterior displacement of the MCJ and widening of Marx’s line can occur with natural aging or dry eye conditions. Posterior

CHAPTER 2 The Conjunctiva: Anatomy and Physiology

2 3 1

1

Fig. 2.3  Histologic section of tarsal conjunctiva showing the pseudocrypts of Henle (1).

displacement of the MCJ can occur in cases of conjunctival cicatrization.3 Just anterior to the MCJ lie the meibomian gland orfices of the upper and lower lids. Their location anterior to the MCJ ensures that lipid excreted from the meibomian glands can pool as a reservoir on the hydrophobic skin surface of the eyelid margin before being spread over the ocular surface during a blink.3 Meibomian glands of the eyelid are seen easily through the transparent palpebral conjunctiva as yellow lobulated structures separated by vascular arcades in the tarsus of the upper and lower eyelids running perpendicular to the eyelid margin. The tarsal conjunctiva is tightly adherent to the substance of the tarsus in order to present a smooth surface to interface with the anterior corneal surface. Consequently, there is no accessible subconjunctival tissue plane for dissection of the tarsal conjunctiva. Along the tarsal surface, 2 mm posterior to the lid margin, lies a shallow subtarsal groove less than 1 mm deep that runs parallel to the eyelid margin for most of the length of the tarsus. Between the eyelid margin and the tarsal groove are multiple ridges and grooves that communicate with goblet cell-lined invaginations of the conjunctival epithelium (the pseudocrypts of Henle) (Fig. 2.3). Few crypts are present at birth; most develop at puberty. By age 50 years the crypts are found in about one-third of specimens.2 The crypts are more numerous in the nasal conjunctiva and around the plica. Accessory lacrimal glands are located in the forniceal conjunctiva (glands of Krause) and in the palpebral conjunctiva above or within the tarsus (glands of Wolfring) (Fig. 2.4). The bulbar conjunctiva is smoother and more loosely adherent to underlying tissues than the tarsal conjunctiva. Near the limbus, the bulbar conjunctiva blends with the underlying Tenon’s capsule and episclera. At the corneoscleral limbus there is a series of fibrovascular ridges perpendicular to the corneal margin (palisades of Vogt) which are more prominent superiorly and inferiorly and contain corneal stem cells.

Fig. 2.4  Histologic section through the superior tarsus demonstrating the glands of Wolfring (1), lymphocytes in the adenoid layer (2), and pseudocrypts of Henle (3).

Histology The conjunctival surface is composed of two layers, the stratified epithelial layer and its underlying stroma (substantia propria). The stratified nonkeratinizing epithelium varies in thickness and appearance from the eyelid margin to the limbus. Unlike any other stratified squamous epithelium, goblet cells are dispersed among and attached to neighboring epithelial cells within the conjunctiva.

Mucocutaneous junction At the MCJ of the eyelid margin, there is an abrupt transition from the keratinized, stratified squamous epithelium of the eyelid skin to the nonkeratinized, stratified squamous epithelium of the marginal conjunctiva. The marginal conjunctiva then extends posteriorly for approximately 2 mm around the crest of the posterior lid margin before terminating at the subtarsal fold. Here, the stratified squamous, nonkeratinized epithelium transitions into the columnar/ cuboidal epithelium characteristic of the remaining palpebral conjunctiva overlying the tarsus (Fig. 2.5).3

Palpebral and forniceal conjunctiva The palpebral conjunctival epithelium thickens from two to three cell layers thick over the superior tarsus to four to five cell layers thick over the inferior tarsus. The palpebral conjunctival epithelium tends to be more cuboidal in nature, whereas the forniceal conjunctival epithelium is more columnar. Near the eyelid margin, the stratified cuboidal/ columnar epithelium of the conjunctiva also contains more tonofilaments. Conjunctival subepithelial cysts often arise from invaginated areas of palpebral conjunctival epithelium or crypts that have closed off (pseudocrypts of Henle). These cysts are lined by surface epithelial cells and contain mucin secreted from goblet cells.

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2

2

1 1 1

Fig. 2.5  Histologic section through the upper eyelid. Meibomian glands (1) and the mucocutaneous junction (2) can be seen. (Courtesy of Amy Maltry, MD, Assistant Professor of Ophthalmology, University of Minnesota.)

Bulbar conjunctiva The bulbar conjunctival epithelium consists of six to nine layers of stratified nonkeratinizing squamous epithelial cells arranged in an irregular fashion in contrast to the more regularly arranged corneal epithelium. Ultrastructurally, cytoplasmic tonofilaments are present and form dense bundles. Additionally, cytoplasmic organelles similar to those found in corneal epithelium are also present but are more abundant. The basal and intermediate epithelial cells of the conjunctiva contain more abundant and larger mitochondria compared to corneal epithelium, suggesting a higher level of oxidative metabolism. Conjunctival epithelial cellular membranes show marked infoldings. Furthermore, there is incomplete interdigitation between adjacent cells and fewer desmosomes present between adjacent cells in the conjunctiva compared to in the cornea. This configuration produces wide intercellular spaces in which antibodies and other plasma constituents and inflammatory cells from underlying vessels can accumulate. Both infectious and topically applied substances can gain access to these intercellular spaces which facilitate the absorption of topical substances into the subconjunctival capillaries and systemic circulation. The bulbar conjunctival epithelium is attached to a thin basement membrane by relatively few hemidesmosomes. The basement membrane is discontinuous in some places, allowing wandering cells, such as lymphocytes, dendritic melanocytes, and Langerhans cells which may be seen in the suprabasal region of the epithelium, access to the conjunctival stroma. Apically, a glycocalyx is secreted from mucin-containing intraepithelial vesicles (Fig. 2.6).4 These intraepithelial vesicles release their contents by fusing with the apical membrane thereby forming the glycocalyx, which consists of transmembrane mucins (MUC1, MUC4, MUC16). The longchain glycoprotein molecules maintain tear film stability by

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Fig. 2.6  Electron micrograph of the conjunctiva showing the microvilli (1) and glycocalyx (2, inset).

conferring wettability to the hydrophobic epithelium surface and anchoring the soluble mucin produced by the goblet cells (MUC5AC) to the conjunctival surface.5 Although superficial conjunctival epithelial cells in normal subjects do show variable amounts of IgA and IgG reactivity, the conjunctival basement membrane zone (BMZ) does not have immunoreactivity to any immunoglobulins, complement components, or albumin. BMZ immunoreactivity to IgM, IgD, and IgE is pathological and may be seen in patients with mucous membrane pemphigoid with ocular involvement. Fibrinogen is normally found at the BMZ and can serve as a positive control when processing conjunctival specimens for immunoreactivity.6

The corneoscleral limbus The corneoscleral limbus anatomically resides at the junction between the conjunctiva and the cornea and is characterized by radially oriented undulation in the epithelium and underlying stroma known as the palisades of Vogt. These critical structures are the sole reservoir for limbal stem cells which sustain the self-renewal and regenerative properties of the corneal epithelium during normal cellular turnover and injury. Limbal stem cells are characterized by their small cell size with high nuclear to cytoplasmic ratio, their slow-cycling (thus label-retaining) nature, their high proliferative potential in cultures, and their lack of terminal differentiation markers such as cytokeratin 3 and 12, and connexin 43, which are found in normal corneal epithelium.7 Significant research looking for definitive stem cell markers is ongoing, but protein expression of putative markers, p63α and ABCG2, has been used to identify stem cells and their very early progeny.7,8 Loss of limbal stem cells can occur with any form of damage to the corneoscleral limbus. Causes of limbal stem cell loss include chemical or thermal burns, Stevens−Johnson syndrome, mucous membrane pemphigoid, multiple surgeries, radiation, contact lens hypoxia, or genetic disorders such as aniridia. Subsequent limbal stem cell deficiency can result in proliferation of conjunctival epithelium and goblet cells

CHAPTER 2 The Conjunctiva: Anatomy and Physiology

3 4 2

1

Fig. 2.7  Histologic section demonstrating the conjunctival (1), limbal (2), and corneal (3) epithelium, and Bowman’s membrane (4).

over the corneal surface. Conjunctivalization of the cornea leads to chronic inflammation, vascularization, and scarring and is a major cause of corneal blindness.7 Conjunctival epithelium on the corneal surface is unstable and often results in recurrent or persistent epithelial defects which can lead to sterile corneal stromal melts or infectious corneal ulcers. Conventional corneal transplantation fails in all cases of severe limbal stem cell deficiency as the host is unable to provide corneal epithelium to the graft. The limbal failure leads to persistent epithelial defects, conjunctivalizaiton of the cornea, increased inflammation and subsequent graft rejection. However, advances in autogenic and allogenic limbal transplantation, with or without ex vivo expansion of the limbal stem cell population, have yielded promising results as alternative therapy.9,10 Similar to the palpebral eyelid margin, there is a gradual transition at the limbus from the stratified, nonkeratinized columnar epithelium of the conjunctiva to the stratified, nonkeratinized squamous epithelium of the cornea (Fig. 2.7). There are seven to ten layers of cells at the limbus, which have cell-to-cell and cell-to-substrate attachments similar to those of the cornea. The limbal stem cells and their immediate progeny, known as early transient amplifying cells, are located in the basal layer of the limbal epithelium within the palisades of Vogt. Recent studies have suggested that limbal stem cells may be specifically concentrated within microstructures known as limbal crypts or around microstructures known as focal stromal projections at the base of the palisades of Vogt.11,12 Progressive differentiation of transient amplifying cells into terminal corneal epithelial cells occurs as the cells migrate centripetally over the corneal surface and superficially within the corneal epithelium.7 The microenvironment around the limbal stem cells, known collectively as the limbal niche, is known to be crucial for supporting the self-renewal and differentiation of limbal stem cells. Research into what constitutes the limbal niche is ongoing and will be important for future development of techniques for ex vivo expansion of limbal stem cells. To date, proposed components of the limbal niche include mesenchymal cells of the underlying limbal stroma, the unique basement membrane of the limbal epithelium

(unlike corneal epithelial basement membrane, limbal epithelial basement membrane contains α1, α2, α5 chains of type IV collagen and is fenestrated), the underlying nerve and vascular plexus of limbal stroma, melanocytes in the limbal epithelium, and Langerhans and suppressor T cells in the limbal epithelium.7,8,11 Melanocytes within the basal layer of the limbal epithelium are believed to play an important role in protecting the limbal stem cells from UV damage. The extensive innervation and vascularity of the limbal stromal is thought to play a role in providing nutrients to the limbal stem cell population.8,10 Further research into the roles of other proposed components of the limbal niche will be important for future advancements in limbal reconstruction.

Conjunctival goblet cells Goblet cells are unicellular, mucin-secreting glands that are found within the conjunctival epithelium and account for approximately 5% to 10% of conjunctival basal cells.13 These cells may occur singly or in clusters and are the primary source of MUC5AC, the large soluble mucin found in the tear film.14,15 Though goblet cells may secrete other proteins, no other mucins have been identified in goblet cells.16 They are apocrine in nature, with all secretory granules secreted once the cell has been activated.16 The cysteine-rich domains at the N and C termini of MUC5AC make it a very viscous, gel-forming mucus which allows it to serve as a scaffold for the mucin layer of the tear film.14 In combination with the transmembrane mucins, MUC1, MUC4, and MUC16, which are expressed by conjunctival and corneal epithelium, MUC5AC plays an important role in stabilizing the tear film and facilitating even distribution of tears over the ocular surface.16,17 Together, these mucins function to protect, hydrate, and lubricate the ocular surface. They are also involved in cell signaling and may be deficient from the ocular surface in conditions associated with dry eye or inflammation.16,18 Overall, conjunctival goblet cell density ranges between 1000 and 56 000 cells/mm2, with goblet cells more concentrated in the palpebral conjunctiva than the bulbar conjunctiva and more concentrated inferonasally.19 Goblet cell density decreases near the limbus with small patches of perilimbal conjunctiva nasally and temporally completely devoid of goblet cells.20 The density of goblet cells is influenced by age (peaks in young adulthood) and other environmental factors such as humidity, temperature, and pollution.20,21 The density of goblet cells is further affected by ocular diseases such as keratoconjunctivitis sicca, ocular pemphigoid, Stevens–Johnson syndrome, and chemical injuries.19,22,23 Loss of goblet cells is an early sign of ocular surface squamous metaplasia.24 Goblet cell density is decreased in mucous membrane pemphigoid but increased in atopic keratoconjunctivitis.25,26 Deficiency in vitamin A, an important factor for conjunctival differentiation, results in goblet cell loss and subsequent conjunctival keratinization (squamous metaplasia).24,27 Histochemical studies of conjunctiva have demonstrated that goblet cells are innervated by both sympathetic and parasympathetic nerves.28,29 However, the release of secretory mucins by goblet cells appears to be primarily mediated

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1

1

The deeper, fibrous layer of the conjunctival substantia propria contains vessels, lymphatics, and nerves. Capillaries arise from the anterior ciliary arteries, which are branches of the ophthalmic artery, and drain into the episcleral venous plexus. Lymphatics drain into the episcleral plexus, which joins the drainage system of the eyelids draining into the submandibular and preauricular lymph node systems.

Vascular Supply

Fig. 2.8  Photomicrograph of conjunctival epithelium with periodic acid–Schiff-positive staining goblet cells (1).

by parasympathetic stimulation.16 The neurotransmitters acetylcholine and vasoactive intestinal peptide (VIP) are known mediators of parasympathetic goblet cell stimulation.30 While sensory nerves do innervate conjunctival squamous epithelial cells, they do not appear to innervate conjunctival goblet cells.16 Ultrastructural studies of goblet cells have shown that the nucleus and cytoplasmic organelles are displaced toward the basal aspect of the cells while mucin packets are located apically. This accounts for the cell’s goblet-like appearance (Fig. 2.8). Although tonofilaments are present, they are not highly differentiated. Tight junctions adhere goblet cells to adjacent epithelial cells.

Substantia propria The conjunctiva rests on fibrovascular connective tissue of variable thickness and density known as the substantia propria. In the tarsal conjunctiva, the substantia propria is thin, compact, and firmly attached to the tarsus. In the fornices, it is thick and loosely attached to the globe and orbital septum. The substantia propria extends temporally behind the canthus and nasally to the semilunar fold. At the limbus it is thin and compact, and merges with Tenon’s fascia and episcleral tissues. The substantia propria can be divided into superficial and deep layers. The superficial layer of the substantia propria consists of loose, interconnected connective tissue. This layer is not present at birth and begins to form at 8 to 12 weeks of age. In adults there is additionally a 50- to 70 mm thick layer of lymphocytes (adenoid layer), which is more prominent inferiorly. In normal, noninflamed conjunctiva, there are no true follicles with germinal centers; however, conjunctival lymphocytes can be stimulated by viral or chlamydial infections or by a toxic reaction to certain topical medications to form follicles with reactive germinal centers. Follicles tend to elevate the conjunctival epithelium, producing a round, fish-egg-like mound. In contrast, papillae form from a reactive, histamine-mediated vascular reaction. Papillae are characterized by chronic inflammatory cells (lymphocytes and plasma cells) and the presence of a central vascular core.31

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The palpebral conjunctiva and lids share a common arterial blood supply that arises from terminal branches of the ophthalmic artery: the dorsal, nasal, frontal, supraorbital, and lacrimal arteries. The facial, superficial, temporal, and infraorbital branches from the facial artery provide supplemental blood supply. In the bulbar conjunctiva, branches from the anterior ciliary arteries, which are a continuation of the muscular branches supplying the rectus muscles, form a superficial marginal plexus at the limbus, giving rise to the terminal vessels of the peripheral arcades and the palisades of Vogt. Branches of the bulbar anterior ciliary arterial system anastomose in the fornices with recurrent vessels from the palpebral conjunctiva. Conjunctival vessels maintain their superficial position near the limbus while a deeper circulation furnishes blood supply to the peripheral corneal arcades, iris, and ciliary body. Inflammatory processes of the conjunctiva result in prominence of the superficial vessels, which increases as you move away from the limbus. Inflammatory processes of the cornea, iris, or ciliary body result in prominence of the deep vessels, which increases as you move toward the limbus. Clinically, this process manifests as a coronal or ciliary flush.32 Most conjunctival capillaries are fenestrated, though some of the deeper vessels are not.32 Fenestration allows more rapid passage of luminal contents under inflammatory conditions. After intravenous injection of fluorescein, conjunctival vessels can be seen to leak in a time and concentration sequence similar to that of the choroidal capillaries. The vessels at the palisades of Vogt may be more competent and leak less than conjunctival vessels elsewhere.32 Conjunctival inflammation, infections, irritation, or severe intraorbital inflammation can cause conjunctival capillaries to leak plasma proteins faster than the fluid can pass between the epithelial cells. This process causes thickening of the epithelium and chemosis of the conjunctiva.33 Venous drainage from the palpebral conjunctiva joins the post-tarsal veins of the eyelids and the deep facial branches of the anterior facial vein and pterygoid plexus. The bulbar conjunctival veins drain into the episcleral venous plexus, which drains into the intrascleral plexus. Wind, cold, heat, and endocrine changes associated with menstruation and early pregnancy can cause dilation and engorgement of the venous circulation.34

Lymphatic Drainage The conjunctiva contains a rich anastomotic network of lymphatic channels that drain into the episcleral lymphatic plexus. Many small, irregular lymphatic channels arise 1 mm peripheral to the limbus and anastomose to form larger

CHAPTER 2 The Conjunctiva: Anatomy and Physiology collecting channels in the deep layer of the substantia propria. Occasionally, these can be seen as irregular, dilated, sausage-shaped channels (lymphangiectasia). Lymphatics of the conjunctiva join the lymphatics of the eyelids and drain medially to the submandibular lymph node and laterally to the preauricular (intraparotid) lymph node system.

Nerve Supply Proper sensory innervation of the conjunctiva is essential to maintain its health and ultimately the health of the eye. The conjunctiva is richly supplied with free nerve endings that arise from the lacrimal, supraorbital, supratrochlear, and infraorbital branches of the ophthalmic branch of the trigeminal nerve (V1). The threshold for tactile conjunctival sensitivity is 100 times higher than that of the center of the cornea. This is likely due to the fact that there is lower innervation density and that the nerve endings are further away from the surface and less exposed to stimuli in the conjunctiva than in the cornea. The conjunctiva is least sensitive in the perilimbal area and most sensitive along the marginal palpebral conjunctiva. Pain can occur with inflammation, epithelial defects, hypoxia, and osmotic shock, all of which can stimulate or cause deformation of the nerve endings. The most common sensations are foreign body sensation, burning, and itching. The conjunctiva is also capable of low-threshold temperature sensitivity.35 Animal studies in rats have identified multiple neuropeptides within conjunctival nerve fibers, including neuro­ peptide Y, vasoactive intestinal peptide (VIP), histidine, isoleucine, helospectin, substance P, and calcitonin generelated peptide. The superior cervical ganglion contributes the most to conjunctival innervation via neuropeptide Y-containing sympathetic nerve fibers. VIP-containing parasympathetic nerve fibers, which are known to mediate goblet cell secretion, arise from the sphenopalatine ganglion. Substance P-containing fibers which mediate conjunctival sensation travel to the trigeminal ganglion.36 In humans, the accessory lacrimal glands of Zeiss and Wolfring, the glands of Moll, and goblet cells are innervated by VIP-containing nerve fibers.37

Normal Flora The conjunctiva is well protected from infection due to multiple defensive mechanisms. The mechanical sweeping of the lids and the presence of antimicrobial factors, such as lysozyme and lactoferrin, in the tear film are both important ocular surface defense mechanisms. Additionally, the ability of antibodies and inflammatory cells to migrate across the conjunctival epithelium from both the indigenous lymphocytic population and the systemic circulation further contributes to conjunctival resistance to infectious disease. The normal conjunctival flora is relatively consistent worldwide and is often quite similar between fellow eyes of any given patient.38 Overall, the chance of culturing a given bacteria from the conjunctiva of one eye is two to ten-fold higher if the bacteria was also found in the fellow eye. Similarly, there is significant concomitance between the normal flora of the conjunctiva and the normal flora of the adjacent

eyelids. Organisms cultured from the conjunctival sac are almost always found on the adjacent lids as well, though only 50% of organisms cultured from the eyelids are also present in the conjunctival sac.38 The ocular surface of healthy individuals supports a relatively small population of bacteria under normal conditions. Under conditions of dry eye, bacteria are more frequently isolated from the conjunctival surface, suggesting a change in normal flora.39 The most common bacteria cultured from the ocular surface are coagulase negative staphylococci, with Staphylococcus epidermidis being the most common isolate. Using more sensitive techniques for detecting bacteria such as molecular cloning and DNA sequencing, a greater diversity of conjunctival bacteria can be isolated from normal subjects. Previously identified bacteria using these methods include Corynebacterium, Propionibacterium, Rhodococcus erythropolis, Klebsiella species, and Erwinia species.39 Further studies of conjunctival flora in adults compared to children have shown that adults tend to have a greater number of aerobic and anaerobic bacterial species while children tend to have more Streptococcus species.40 In contrast to bacteria, fungal colonization of the ocular surface is less common but can occur. In one study, up to 10% of adults, 5% of children, and 1% of infants were found to have positive conjunctival fungal cultures.41

Physiology of the Conjunctiva Unlike the cornea, whose primary function is to provide a clear refractive window for the eye, the conjunctiva serves multiple functions in maintaining the health of the ocular surface. Grossly, the conjunctiva provides a barrier to exogenous infectious agents and foreign bodies and allows free rotation of the globe. At a cellular level, the conjunctiva plays key roles in maintaining the tear film and supporting the health of the corneal epithelium. Overall, the conjunctiva occupies 17 times more surface area than the cornea and is more permeable than the cornea, highlighting its importance to the homeostasis of the ocular surface.42 The conjunctiva plays an important role in modulating the volume, osmolarity, and electrolyte concentrations of the tear film.43 Human conjunctival epithelium is water permeable and expresses both aquaporin 3 and aquaporin 5.44 Through active transport of Cl− molecules and coupled trans­ epithelial fluid movement, the conjunctiva is able to actively secrete fluid in the stroma-to-mucosal direction. It is estimated based on conjunctival surface area that conjunctival epithelium fluid secretion may reach rates of 50 μL/h. Such rates would be sufficient to supply basal tear production even in the absence of accessory lacrimal gland secretion.43,44 Physiological fluid secretion across the conjunctival epithelium is regulated by nerves, growth factors, and other small molecules such as the P2Y2 agonists UTP and ATP.45 Therapeutic modulation of conjunctival fluid secretion may yield effective new treatments for dry eyes in the future. The conjunctiva is also an important source of tear mucins, as noted above. The glycocalyx of the ocular surface, formed by membrane bound mucins MUC1, MUC2, and MUC16, plays a critical role in conferring wettability to the otherwise hydrophobic cell membranes of ocular surface epithelium. Soluble MUC5AC is believed to play a role in

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increasing tear film viscosity and stability and restoring wettability to the ocular surface after epithelial injury.5 In addition to secretion of electrolytes, water, and mucin into the tear film, the conjunctiva also plays an important role in absorption of electrolytes, water, and other compounds from the tear film. The conjunctival epithelium actively absorbs Na+ from the tear film and also expresses transport elements for glucose, K+, Cl−, and HCO3.45 It further plays an important role in the absorption of ophthalmic drugs applied to the ocular surface.46 Under pathological conditions such as inflammation or following application of substances that increase vascular permeability, there is leakage of plasma, electrolytes, water, and proteins from the conjunctiva, which can alter the composition of the tear film. The limbal corneal epithelium is also critical for the health of the corneal epithelium. The limbus, with its limbal stem cells, acts as a barrier that prevents migration of conjunctival epithelial cells on to the cornea.47 Loss of limbal stem cells results in conjunctivalization of the cornea. Conjunctivalization of the cornea, in turn, results in vascularization and scarring of the cornea.48 Conjunctival epithelium over the corneal stroma is not stable, does not tolerate trauma well, and is prone to epithelial defects. Conjunctival epithelium differs significantly from corneal epithelium in both gross and histologic appearance and in biochemical function (Table 2.1). The cornea is a clear, regular, refracting and reflecting surface without blood vessels. The conjunctiva, in contrast, is translucent, irregular, and vascularized. The cornea is devoid of goblet cells while the conjunctiva has numerous goblet cells. The corneal epithelium is five to six layers thick with an orderly progression from basal to wing to superficial cells on an avascularized stroma, whereas the conjunctiva consists of six to nine layers of cells arranged in an irregular fashion on a vascularized stromal bed. The corneal epithelial cells maintain and require large stores of glycogen for epithelial wound healing while the conjunctiva does not.49 Overall, conjunctival epithelium demonstrates greater dependence on glycolytic and

Table 2.1  Comparison of conjunctiva and corneal anatomy, histology, and physiology Characteristic

Conjunctiva

Cornea

Clarity

Translucent

Clear

Epithelium

6–9 less orderly layers

5–6 orderly layers

Goblet cells

Present

Absent

Stromal bed

Vascular

Avascular

Source of nutrition

Conjunctival vessels, tear film

Anterior chamber, tear film

Glycogen content

Low

High

Dependence on glycogen

Low

High

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tricarboxyacetic acid (TCA) cycle activity while corneal epithelium demonstrates greater dependence on hexose monophosphate shunt activity.50 Nutrition for the cornea, which comes from the tear film and aqueous humor, must diffuse across a great distance through the corneal epithelium, stroma, and endothelium. In contrast, conjunctival nutrition comes directly from nearby blood vessels and the overlying tear film.

References 1. Wolosin JM, Budak MT, Akinci MA. Ocular surface epithelial and stem cell development. Int J Dev Biol 2004;48:981–91. 2. Spencer W, Zimmerman L, editors. Conjunctiva. Philadelphia: WB Saunders; 1985. 3. Bron AJ, Yokoi N, Gaffney EA, et al. A solute gradient in the tear meniscus. I. A hypothesis to explain Marx’s line. Ocul Surf 2011;9:70–91. 4. Dilly P, Makie I. Surface changes in the anesthetic conjunctiva in man with special reference to the production of mucin from non-goblet cell source. Br J Ophthalmol 1981;65:833–42. 5. Bron AJ, Tiffany JM, Gouveia SM, et al. Functional aspects of the tear film lipid layer. Exp Eye Res 2004;78:347–60. 6. Foster C, Dutt J, Rice B, et al. Conjunctival epithelial basement membrane zone immunohistology: normal and inflamed conjunctiva. Int Ophthalmol Clin 1994;34:209–14. 7. Li W, Hayashida Y, Chen YT, et al. Niche regulation of corneal epithelial stem cells at the limbus. Cell Res 2007;17:26–36. 8. Huang M, Wang B, Wan P, et al. Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells. Cell Tissue Res 2015;359:547–63. 9. Dua HS, Miri A, Said DG. Contemporary limbal stem cell transplantation – a review. Clin Experiment Ophthalmol 2010;38:104–17. 10. Holland EJ, Mogilishetty G, Skeens HK, et al. Systemic immunosuppression in ocular surface stem cell transplantation: Results of a 10-year experience. Cornea 2012;31(6):655–61. 11. Ordonez P, Di Girolamo N. Limbal epithelial stem cells: role of the niche microenvironment. Stem Cells 2012;30(2):100–7. 12. Dua HS, Shanmuganathan VA, Powell-Richards AO, et al. Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche. Br J Ophthalmol 2005;89:529–32. 13. Thoft R, Friend J. Ocular surface evaluation. In: Francois J, Brown S, Itoi M, editors. Proceedings of the symposium of the International Society for Corneal Research (Doc Ophthalmol Proc Series 20). The Hague: Junk, The Netherlands; 1980. 14. Jumblatt M, McKenzie R, Jumblatt J. MUC5AC is a component of the human precorneal tear film. Invest Ophthalmol Vis Sci 1999;40:43–9. 15. Berry M, Ellingham R, Corfield A. Membrane-associated mucins in normal human conjunctiva. Invest Ophthalmol Vis Sci 2000;41:398–403. 16. Dartt D. Regulation of mucin and fluid secretion by conjunctival epithelial cells. Prog Retin Eye Res 2002;21:555–76. 17. Sweeney DF, Millar TJ, Raju SR. Tear film stability: a review. Exp Eye Res 2013;117:28–38. 18. Blalock T, Spurr-Michaud S, Tisdale A, et al. Release of membraneassociated mucins from ocular surface epithelia. Invest Ophthalmol Vis Sci 2008;49:1864–71. 19. Ralph R. Conjunctival goblet cell density in normal subjects and in dry eye syndromes. Invest Ophthalmol Vis Sci 1975;14:299–302. 20. Kessing S. Mucous gland system of the conjunctiva. Acta Ophthalmol (Copenh) 1968;95(Suppl.):1–133. 21. Waheed M, Basu M. The effect of air pollutants on the eye. I. The effects of an organic extract on the conjunctival goblet cells. Can J Ophthalmol 1970;5:226–30. 22. Allansmith M, Baird G, Greiner G. Density of goblet cells in vernal conjunctivitis and contact lens associated giant papillary conjunctivitis. Arch Ophthalmol 1981;99:884–5. 23. Nelson J, Wright J. Conjunctival goblet cell densities in ocular surface disease. Arch Ophthalmol 1984;102:1049–51. 24. Tseng S, Hirst L, Maumenee A, et al. Possible mechanisms for the loss of goblet cells in mucin-deficient disorders. Ophthalmology 1984;91: 545–52. 25. Thoft R, Friend J, Kinoshita S, et al. Ocular cicatricial pemphigoid associated with hyperproliferation of the conjunctival epithelium. Am J Ophthalmol 1984;98:37–42. 26. Roat M, Ohji M, Hunt L, et al. Conjunctival epithelial cell hypermitosis and goblet cell hyperplasia in atopic keratoconjunctivitis. Am J Ophthalmol 1993;116:456–63.

CHAPTER 2 The Conjunctiva: Anatomy and Physiology 27. Rao V, Friend J, Thoft R, et al. Conjunctival goblet cells and mitotic rate in children with retinol deficiency and measles. Arch Ophthalmol 1987; 105:378–80. 28. Diebold Y, Rios J, Hodges R, et al. Presence of nerves and their receptors in mouse and human conjunctival goblet cells. Invest Ophthalmol Vis Sci 2001;42:2270–82. 29. Dartt D, McCarthy D, Mercer H, et al. Localization of nerves adjacent to goblet cells in rat conjunctiva. Curr Eye Res 1995;14:993–1000. 30. Rios J, Ghinelli J, Hodges R, et al. Role of neurotrophins and neurotrophin receptors in rat conjunctival goblet cell secretion and proliferation. Invest Ophthalmol Vis Sci 2007;48:1543–51. 31. Kessing S. On the conjunctiva papillae and follicles. Acta Ophthalmol (Copenh) 1966;44:846–51. 32. Goldberg M, Bron A. Anatomy and angiography of the palisades of Vogt. Trans Am Ophthalmol Soc 1982;80:201–6. 33. Lockard I, Debacker H. Conjunctival circulation in relation to circulatory disorders. J S C Med Assoc 1967;63:201–6. 34. Landsman R, Douglas RG, Dreishpoon G, et al. The vascular bed of the bulbar conjunctiva in the normal menstrual cycle. Am J Obstet Gynecol 1953;66:988–98. 35. Burton H, editor. Somatosensory features of the eye. 9th ed. St Louis: Mosby; 1987. p. 71–100. 36. Elsas T, Edvinsson L, Sundler F, et al. Neuronal pathways to the rat conjunctiva revealed by retrograde tracing and immunochemistry. Exp Eye Res 1994;58:117–26. 37. Seifert P, Spitznas M. Vasoactive intestinal peptide (VIP) innervation of the human eyelid glands. Exp Eye Res 1999;68:685–92. 38. Allansmith M, Osler H, Butterwoth M. Concomitance of bacteria in various areas of the eye. Arch Ophthalmol 1969;82:37–42. 39. Graham J, Moore J, Jiru X, et al. Ocular pathogen or commensal: A PCRbased study of surface bacterial flora in normal and dry eyes. Invest Ophthalmol Vis Sci 2007;48:5616–23.

40. Singer T, Isenberg S, Apt L. Conjunctival anaerobic and aerobic bacterial flora in paediatric versus adult subjects. Br J Ophthalmol 1988;72: 448–51. 41. Hammeke J, Ellis P. Mycotic flora of the conjunctiva. Am J Ophthalmol 1960;49:1174–8. 42. Watsky M, Jablonski M, Edelhauser H. Comparision of conjunctival and corneal surface areas in rabbit and human. Curr Eye Res 1988;7:519–31. 43. Li Y, Kuang K, Yerxa B, et al. Rabbit conjunctival epithelium transports fluid, and P2Y22 receptor agonists stimulate Cl− and fluid secretion. Am J Physiol Cell Physiol 2001;281:C595–602. 44. Oen H, Cheng P, Turner HC, et al. Identification and localization of aquaporin 5 in the mammalian conjunctival epithelium. Exp Eye Res 2006;83:995–8. 45. Candia OA. Electrolyte and fluid transport across corneal, conjunctival and lens epithelia. Exp Eye Res 2004;78:527–35. 46. Yang J, Ueda H, Kim K, et al. Meeting future challenges in topical ocular drug delivery: Development of an air-interfaced primary culture of rabbit conjunctival epithelial cells on a permeable support for drug transport studies. J Control Release 2000;65:1. 47. Tseng S. Concept and application of limbal stem cells. Eye 1989;3: 141–57. 48. Thoft R, Friend J, Murphy H. Ocular surface epithelium and corneal vascularization. Invest Ophthalmol Vis Sci 1979;18:85–92. 49. Thoft R, Friend J. Biochemical transformation of regenerating ocular surface epithelium. Invest Ophthalmol Vis Sci 1977;16:14–20. 50. Baum B. A histochemical study of corneal respiratory enzyme. Arch Ophthalmol 1963;70:59.

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Basic Science: Cornea, Sclera, Ocular Adnexa Anatomy, Physiology and Pathophysiologic Responses





Chapter 3  Tear Film Roger W. Beuerman, Michael A. Lemp

Key Concepts • • • • •

The tear film is an important component of the visual system and an accessible source of components in health and disease. The tear film has a complex structure which is metastable between blinks in normal subjects. The tear film is a source of proteins, lipids, and electrolytes, the levels of which are altered in disease states, e.g. dry eye disease and other ocular and systemic diseases. Considerable research has been conducted on components, shedding light on the potential for tear sampling as a diagnostic tool and guide for therapeutic intervention. A new platform for tear collection and analysis of tear osmolarity has been developed and is in clinical use for the diagnosis of dry eye disease. The development of measurements of other tear components augurs well for a new generation of tear diagnostics.

Overview and Function The tear film is a complex composite whose components have multiple sources, which include the lacrimal gland, meibomian glands, goblet cells, and accessory lacrimal glands of the ocular surface. Additional secretory contributions come from the ocular surface, which contains several types of embedded tissues, such as the glands of Krause, Moll, and Wolfring, whose structure is very similar to that of the main lacrimal gland.1,2 The base of the tear film is the outer surface membrane of the corneal or conjunctival epithelial cells. The membrane of the corneal cells is striking in appearance. The membrane is thrown into folds called microplicae or microvilli and the membrane leaflet touching the tear film is very osmophilic. It is usually assumed that the reason for the elaborate folds and filaments extending into the tear film is to aid adherence of the tear film. However, the tear film extends over the conjunctiva as well, and the outer surface of those cells does not show the same elaborations to the extent seen for the corneal cells (Fig. 3.1). Both structures serve to increase the surface area, presumably

32

aiding in tear–epithelial adhesion. The tears have been reviewed often and there are classic texts that detail the wellaccepted concepts of the tears; in the present work the emphasis will be on the evolving concepts of the tears.3–5 The functions of the tear film include lubrication, protection from disease, nutrition of the cornea, and a critical role in the optical properties of the eye.6 In fact, the crisp optical (corneal) reflex commonly seen in clinical or casual photographs of the eye provides evidence of the mirror-like quality of the optical function of this surface and an indication that the tear film is intact. Normal tear volume is around 6 µL and production is about 1.2 µL/minute with a turnover rate of about 16% per minute.7 Precorneal tear film is a metastable structure between blinks allowing for clear vision; this limited stability is compromised in dry eye disease, leading to optical image degradation between blinks.8 Although early studies separated the tear film into three discernable layers, structural rigidity changed with time, and the tear layers are considered to be more of a continuum with the lipid layer most anterior to the aqueous and mucin components. The aqueous layer contains electrolytes (sodium, potassium, calcium, magnesium, chloride, phosphate and bicarbonate), proteins/peptides (such as enzymes, growth factors, cytokines), small molecule metabolites such as amino acids, urea, glucose, and lactate.3,9 Normal tears contain 6–10 mg/mL total proteins.5 Tears are a dilute protein solution and the composition of the tears is different than that of serum in the electrolyte and protein content. Chloride and potassium are higher in the tears (tears, 120 mEq/L and 20 mEq/L; serum, 102 mEq/L and 5 mEq/L, respectively) and glucose concentration is lower in the tears (about 2.5 mg/100 mL) compared to plasma (85 mg/L). The osmotic pressure of the tears in normal eyes ranges between 280 and 305 mOsm/L, whereas in plasma the normal value is about 6 atm.3,4

Tear Layer Thickness Although its importance may not be immediately apparent, the thickness of the tear layer has received a great deal of attention. It is of interest to know the volume of the tears over the surface of the eye, particularly the cornea, as it is a reservoir for drugs that have been delivered by either topical or systemic routes for penetration into the eye. As a major

CHAPTER 3 Tear Film

Chapter Outline Overview and Function Tear Layer Thickness Tears Reflect the Health of the Ocular Surface Control of Tear Secretion Analytical Methods Peptide Components of Tears

32.e1

CHAPTER 3 Tear Film interference patterns could be used to detect changes in the lipid layer and that a dry eye patient was deficient in this regard.11 Recent studies have reported that the lipid layer of the tears is thinner in many patients with dry eye disease.12 However, the controversy has continued. Making use of innovative methods, additional thickness values are still being offered. Using reflection spectra of the human tear film, it was found that there were no oscillations that compared to Prydal’s measurements or earlier estimates. Rather, the results of this study suggested a tear film of about 3 µm.13 A study of the mouse tear film using a microelectrode technique found the tear film to be about 7 µm.14 In infants, the lipid layer was found to be thicker than in adults, which may be a response to a thinner aqueous layer.15 A

Tears Reflect the Health of the Ocular Surface As an extracellular fluid, the tears contain molecules of immune origin, proinflammatory molecules and growth factors among many others, and present studies have examined their levels as an aspect of the health of the ocular surface and the person.16–23 Examination of the levels of IgE and IgA antibodies to grass and tree allergens in serum and tears in a series of patients showed the same specificity for many allergens.24 Cytokines and chemokines have been examined in both normal conditions and as a response to environmental stress.25,26 Growth factors, EGF, TGF-β1, and TGF-β2, are present in normal tear fluids and have been associated with corneal wound healing.27 Rapid, reliable analyses of tear properties such as tear osmolarity and individual protein components have become clinically useful quantitative biomarkers for the diagnosis of eye diseases such as dry eye disease and conjunctivitis.28–31 Sampling of tears, however, presents certain challenges and almost certainly affects results.5

B Fig. 3.1  As shown in the transmission electron micrographs of the surface cell layer of the cornea (A) and conjunctiva (B), the outer membrane of these epithelial cells is thrown into folds that will increase the total surface area in contact with the tears. However, the membrane of the corneal epithelial cells shows the additional specializations of very dense osmium staining, and fine filaments that radiate into the tear layer. This difference in surface articulations between cornea and conjunctiva is similar for humans, nonhuman primates, and rabbits. Both tissues are from human material fixed at 2–3 hours postmortem. The cornea (A, × 51 587) is from a 66-year-old individual, and the bulbar conjunctiva (B, × 50 000) from an 86-year-old.

risk to vision, the lack of a sufficient amount of tears is the primary problem in aqueous-deficient dry eye. The contact lens industry has been interested in the thickness of the tear layer as contact lenses need the support of the tear layer for both optical placement and comfort. From earlier studies, the thickness of the tear layer was found to be about 7–8 µm.6 Studies by Prydal using confocal microscopy and interferometry, however, estimated tear film thickness to be over 40 µm.10 The use of lipid interference patterns to monitor the lipid component of tears has produced interesting new insights and it has been found that several orders of

Control of Tear Secretion Control of the tears and hence the activity of the tears has recently been suggested to be under constant neural regulation: a somewhat different concept from the more traditional one postulating that only reflex tears are a result of neural activity and normal tears were the results of intrinsic lacrimal gland activity. Obviously, either too little or too much aqueous secretion will present a problem with visual function. Thus, there is a means for the ongoing homeostatic regulation of the ocular surface, which is under a control mechanism whose components include afferent nerves from the cornea and other ocular surface tissues, central nervous system relay nuclei, and efferent nerves which comprise the autonomic innervation to secretory tissues whose products contribute to the tear film (Fig. 3.2).32–33 This mechanism is suggested to supply a relatively constant level of neural signals that precisely meter the amount of tears secreted by the main lacrimal gland, but also may mediate lipid production by the meibomian glands and mucin secretion from the goblet cells.34 The accessory lacrimal glands have been shown to have local innervation, but it is not known if these nerves are included in the homeostatic mechanism or if secretion from the accessory

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Lacrimal gland

Pterygopalatine ganglion

Protective reflexes

Goblet cells

CNS

Accessory lacrimal glands

Irritation

Ocular surface

Remove irritation Fig. 3.2  The small sensory nerve endings located just below the epithelial surface of the cornea, lid margin, and conjunctiva constantly respond to drying and temperature change as well as contact and chemical changes, by sending intensity-coded neural signals to the spinal trigeminal nucleus located in the brain stem. A multisynaptic pathway to the preganglionic parasympathetic nuclei in the superior salivatory nucleus forms the output to the secretory tissue. An irritation to the ocular surface gives rise to a large neural input, which provides the neural signal for reflex tearing. The loop is reset when the irritation is removed by copious tearing. For simplicity, some of the components of the neural pathways such as the trigeminal ganglion, sympathetic nerves, and meibomian glands, have been omitted.

Fig. 3.3  Transmission electron micrograph of the accessory lacrimal gland of the tarsal conjunctiva of the upper eyelid from a nonhuman primate (× 6000). The basic secretory morphology is very similar to that of the orbital lacrimal gland. Small unmyelinated axons (asterisks) are seen adjacent to the basal aspect of the acinar cells.

lacrimal glands can be stimulated by transmitter release as part of the lacrimal reflex (Fig. 3.3). Interruption of the neural pathway by different means such as LASIK or anesthesia of the cornea decreases tear flow.35,36

Analytical Methods The tears are an attractive source for sampling due to their accessibility, rich content, and largely acellular structure. There are, however, challenges to sampling tears. Tear samples are often collected using a glass capillary tube applied to the inferior marginal tear strip. Since the entire

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volume of minimally stimulated tears (ordinary environmental stimulation) is around 7 µL and only about 2–3 µL are in the inferior marginal tear strip, and there is minimal exchange between the marginal tear strip and other compartments of tears, collection of more than 2 µL samples at a single sampling implies reflex tearing.37 However, for patients with tear insufficiency glass capillary procedures except with the smallest volume, fire-polished tubes, 1−2 µL, are difficult, and collection times may require 3−5 minutes, increasing the opportunity for inadvertent contact with tissue. Patients are generally more hesitant to accept the capillary procedure. Therefore many studies find the Schirmer Type I method useful.5,29,32 This may lead to great variability in individual tear components, creating variances in the measured levels depending on the degree of induced reflex tearing.38,39 Attempts to measure glucose, for example, have been plagued by large differences which are thought to be due to influx of glucose from serum across the conjunctiva induced by sampling stimulation.16 Useful insights regarding tear composition have been published based on microliter sample sizes using high sensitivity and resolution. Gel based techniques have prompted more detailed studies of event-related changes and disease associated changes in tear composition. Qualitative and quantitative techniques have included one- and two-dimensional polyacrylamide gel electrophoresis (PAGE), isoelectric focusing (IEF), crossed immunoelectrophoresis, enzyme-linked immunosorbent assay (ELISA), and highpressure liquid chromatography (HPLC).37–40 However, these methods are limited due to the large volume of tears needed for analysis requiring sample pooling, the time required to perform the analysis, as well as the inability to critically evaluate a large number of tear proteins simultaneously. Furthermore, 2D-PAGE is limited in its ability to analyze small proteins, extremely acidic or basic proteins, or hydrophobic proteins. Mass spectrometry has largely replaced older gel based methods to study tear proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, which provides accurate mass weights, was used to study tear proteins before and after corneal wound healing, and to map tear protein profiles.19,41 Surface-enhanced laser desorptiontime of flight (SELDI-TOF) ProteinChip technology utilizes affinity surfaces to retain adherent proteins based on their physical or chemical characteristics, which is then followed by direct analysis using TOF-MS.42,43 SELDI-TOF is more sensitive and requires smaller amounts of tear samples (2–3 µL) than 2D-PAGE (Fig. 3.4). More recently, high pressure liquid chromatography (HPLC) has been used as a reproducible and effective method for peptide and protein separation and identification when coupled with mass spectrometry (LC/ MS/MS).30,44 Importantly, these methods offer an unbiased approach with a high degree of accuracy and reproducibility. Large antibody arrays are used frequently as they provide excellent accuracy and reproducibility for cytokines; however, these require a predetermined set of probes.25,26

Peptide Components of Tears Current clinical interest in tear proteins focuses on the discovery of new protein biomarkers that are correlated with a

CHAPTER 3 Tear Film Cellular component Proton-transporting two-sector ATPase complex 4% Golgi apparatus 4% Secretory granule 5%

Cytoplasmic vesicle 25%

Cell cortex 5%

Fig. 3.4  Gene ontology analysis of human tear proteins identified from this study based on DAVID tool. (A) Cellular compartment; (B) biological process; (C) molecular function. (From Zhou L, Zhao SZ, Koh SW, et al. In-depth analysis of the human tear proteome. J Proteomics 2012, 75, 3877–3885, Fig. 5.)

Protein-lipid complex 5%

Cell fraction 6% Ribonucleoprotein complex 14% Lysosome 10% A

Extracellular region 10%

Cytoskeleton 10%

Biological process Protein complex assembly and biogenesis 7% Cellular carbohydrate catabolic process 15%

Regulation of apoptosis 7% Cytoskeleton organization 7%

Proteolysis 13% Protein oligomerization 7%

Immune response 7% Protein transport and localization 12% B

Response to inorganic substance 8%

Cofactor metabolic process 9%

Cellular component organization and biogenesis 8%

Molecular function Lipase activity 7% GTP binding 8% Enzyme inhibitor activity 30% Protein binding 9%

Intra-molecular oxidoreductase activity 9%

Ligase activity 12%

Antioxidant activity 14%

C

Peptidase activity 12%

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disease and can be used for patient stratification, diagnosis and response to treatment. A wide range of molecular types including proteins/peptides, lipids, small molecule metabolites, and electrolytes have been identified in the tears.5,30 The tear proteome has now been extended to more than 1500 proteins and peptides linked to specific genes using mass spectrometry.45 Peptides and proteins in the tear film include a heterogeneous variety of bioactive molecules, including a wide range of growth factors with multicellular targets and neuropeptides (Table 3.1 and Fig 3.4). There has been a longstanding interest in molecules which augment corneal wound healing, as well as understanding how some of these growth factors may complicate wound healing by stimulating scar formation. Epidermal growth factor (EGF) has been shown to stimulate migration of corneal epithelial cells in tissue culture. However, it was found that EGF is a naturally occurring component of the tears.20,46–60 The presence of cytokines and chemokines in diseases of the ocular surface has long been of interest. Dry eye has been the subject of many proteomic studies; however, more recently these have relied on the use of antibody bead assays as the levels remain below the sensitivity of even the most advanced mass spectrometers.25,26,64–70 Changes in the tear levels of immunoglobulins have been of clinical interest for some time, particularly associated with allergic conjunctivitis.72–75 However, these studies have generally used more conventional methods including ELISA assays and flow cytometry. However, matrix metalloproteinases have also been associated with degradative diseases such as vernal conjunctivitis and they have held an interest in wound healing for some time.76–79 The confirmation of the presence of MMP9 has come from several sources. The role of antimicrobial peptides in tears was an early area of interest due to their potentially beneficial role in infectious disease of the cornea. It has become clear that these peptides have a number of properties in addition to their antimicrobial properties and may in fact be active in the corneal wound response.80–86 Antimicrobial peptides form the system of innate immunity of the ocular surface and are evolutionarily ancient. These naturally occurring antibiotics act against a wide range of viruses, bacteria, and fungi; however, recently these have been suggested to directly participate in wound healing. The tears have been shown to have lysozyme, lactoferrin, and both α- and β-defensins. PMNs which synthesize defensins are not found in great numbers in normal tears, but following stress or injury of the ocular surface these are abundant in tears and clearly contribute to the protein milieu.85 The defensin family of peptides has received a great deal of attention as there is considerable evidence for multifunctionality of antimicrobial and wound healing effects. Defensins are a family of small, cationic antimicrobial peptides containing an average of 35 amino acids with molecular weights around 3–4 kDa. Various eye diseases have been associated with an increase in the tear levels of immune based molecules and proinflammatory molecules. Thus, cytokines and chemokines have been determined in dry eye patients, following contact lens wear and in conjunctivitis and changes have been shown, but it has been difficult to develop clear associations.25,26,87,88

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Table 3.1  Functional peptides of tears References

Association

Growth factors Epidermal growth factor (EGF)

20, 25, 26, 46−49

Epithelial wound healing, stress Tear concentration higher than saliva or serum

Transforming growth factor alpha (TGF-α)

50−51

Wound response

Transforming growth factor beta-1 (TGF-β1)

52−55

Wound response. Found in normal tears, increases after wounding

HGF, FGF-2, VEGF, PDGF-ββ

55−60

Wound response, refractive surgery

Neuropeptides Substance P, CGRP

19, 61−63

Wound healing, neurogenic inflammation

Interleukins, chemokines and cytokines IL-4, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, CCL1, GM-CSF, G-CSF

25, 26, 64−70

Vernal conjunctivitis, elevation of IL-1 in dry eye patients, contact lens wear, ocular allergy

Immunoglobulins IgA, IgE, IgG(1–4) and complement

71−75

Allergic conjunctivitis, contact lens wear

Proteases MMP-1, MMP-3, MMP-9, TIMP-1, cathepsin, alpha2macroglobulin

76−79

Role in pterygium migration and vernal keratoconjunctivitis, protection of the ocular surface

Antimicrobial peptides Lysozyme, lactoferrin, α and β defensins, phospholipase A2

80−86

Increases in infections and after ocular surface surgery, wound healing, may decrease in dry eye

Proinflammatory peptides S100 peptides A4, A8, A9

5, 45, 87, 88

Dry eye, pterygium, chronic use of glaucoma medication

Pterygium and dry eye both show upregulation of several members of the S100 class of proteins which are able to interact with macrophages as signaling molecules.5,30 The association of S100 proteins with the long-term use of preserved glaucoma medication suggests an inflammatory component of the response likely originating from the conjunctiva epithelium.

CHAPTER 3 Tear Film A functional classification of identified human tear proteins from the results of a study of the normal human tear proteome45 shows entries in three Gene Ontology categories, cellular component, biological process, and molecular function (Fig. 3.4). For the cellular component, the top five subcategories were cytoplasmic (25%), nucleus (14%), extracellular (10%), cytoskeleton (10%) and lysosome (10%). A classification based on biological process revealed that these proteins were mainly involved in cellular carbohydrate catabolic process (15%), proteolysis (13%), protein transport and localization (12%), cofactor metabolic process (9%), cellular component organization and biogenesis (8%), responses to inorganic substance (8%), immune response (7%), protein oligomerization (7%), cytoskeleton organization (7%), regulation of apoptosis (7%) and protein complex assembly and biogenesis (7%). For molecular function, several major subcategories were enzyme inhibitor activity (30%), antioxidant activity (14%), peptidase activity (12%), ligase activity (12%), intra-molecular oxidoreductase activity (9%), protein binding (9%), GTP binding (8%) and lipase activity (7%). Tear research should continue at the present intensity of interest over the next several years and, with the ability to measure a wide range of peptides in a single small volume sample, the diagnostic value of tears may become a reality for diseases such as keratoconus, immune disease, allergies, and in some systemic diseases as well. The dynamic nature of the tears as a response mediator has been demonstrated by the clinical utility of being able to differentiate between laser treatments and the effects of chronic glaucoma medication. As the tear proteome has been presented, it is now feasible to provide absolute concentrations of hundreds of relevant tear proteins in a clinical study. Mass spectrometry has become much faster and more sensitive to approach clinical utility. Progress in this field may depend on the development of new methods of sampling which collect small samples without inducing reflex tearing and nano-assay techniques for quantitative analysis. Identification of proteins which are not normally present in tears, e.g. IgE in ocular allergy, presents a binary approach which may not be as dependent on quantitative assays. Another approach is to measure properties of the whole of the tear film rather than individual components, e.g. tear stability and tear concentration of electrolytes or its surrogate, tear osmolarity. Tear film break-up time is a measure of the stability of the tear film; a platform for sampling nanoliter quantities of tear atraumatically and measurement of tear osmolarity on samples of 50 nL size has been developed and has been in use in clinical practice in North America, Europe, and the Middle East since 2010. This platform is currently being developed to measure a number of cytokines as markers of disease without prior sample processing and for use in the clinical setting.89,90–92

Acknowledgments Supported by the following grants: NEI EY 12416 and EY 02377, Research to Prevent Blindness (US National Institutes of Health) and NMRC proto-PG002, NMRC-IBG NMRC/ TCR/R1018 (Singapore).(RB)

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60.

61. 62. 63. 64. 65.

66. 67. 68. 69. 70. 71.

72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86.

healing following photorefractive keratectomy. Curr Eye Res 1997;16: 825–31. Tuominen IS, Tervo T, Teppo AM, et al. Human tear fluid PDGF-BB, TNFalpha, and TGF-beta 1 vs corneal haze and regeneration of corneal epithelium and subbasal nerve plexus after PRK. Exp Eye Res 2001;72: 631–41. Fujishima H, Takeyama M, Takeuchi T, et al. Elevated levels of substance P in tears of patients with allergic conjunctivitis and vernal conjunctivitis. Clin Exp Allergy 1997;27:372–8. Yamada M, Ogata M, Kawai M, et al. Decreased substance P concentrations in tears from patients with corneal hypesthesia. Am J Ophthalmol 2000;129:671–2. Merrtaniemi P, Ylatupa S, Partanen P, et al. Increased release of immunoreactive calcitonin gene-related peptide (CGRP) in tears after excimer laser keratectomy. Exp Eye Res 1995;60:659–65. Leonardi A, DeFranchis G, Zancanaro F, et al. Identification of local Th2 and Th0 lymphocytes in vernal conjunctivitis by cytokine flow cytometry. Invest Ophthalmol Vis Sci 1999;40:3036–40. Uchio E, Ono SY, Ikezawa Z, et al. Tear levels of interferon-gamma, interleukin (IL)-2, IL-4 and IL-5 in patients with vernal keratoconjunctivitis, atopic keratoconjunctivitis and allergic conjunctivitis. Clin Exp Allergy 2000;30:103–9. Thakur A, Willcox MD. Contact lens wear alters the production of certain inflammatory mediators in tears. Exp Eye Res 2000;70:255–9. Solomon A, Dursun D, Liu Z, et al. Pro-and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease. Invest Ophthalmol Vis Sci 2001;42:2283–92. Malecaze F, Simorre V, Chollet P, et al. Interleukin-6 in tear fluid after photorefractive keratectomy and its effects on keratocytes in culture. Cornea 1997;16:580–7. Schultz CL, Kunert KS. Interleukin-6 levels in tears of contact lens wearers. J Interferon Cytokine Res 2000;20:309–10. Cook EB, Stahl JL, Lowe L, et al. Simultaneous measurement of six cytokines in a single sample of human tears using particle-based flow cytometry: allergics vs. non-allergics. J Immunol Methods 2001;254:109–18. Baudoin C, Bourcier T, Brignole F, et al. Correlation between tear IgE levels and HLA-DR expression by conjunctival cells in allergic and nonallergic chronic conjunctivitis. Graefe’s Arch Clin Exp Ophthalmol 2000;238: 900–14. Eperon S, Berguiga M, Ballabeni P, et al. Total IgE and eotaxin (CCL11) contents in tears of patients suffering from seasonal allergic conjunctivitis. Graefes Arch Exp Ophthalmol 2014;252:359–67. Maurya RP, Brushan P, Singh VP, et al. Immunoglobulin concentration in tears of contact lens wearers. J Ophthalmic Vis Res 2014;9:320–3. Mimura T, Usui T, Mori M, et al. Specific tear IgE in patients with moderate to severe autumnal allergic conjunctivitis. Int Arch Allergy Immunol 2011;156:381–6. Mimura T, Usui T, Miya T, et al. Relation between total tear IgE and severity of acute seasonal allergic conjunctivitis. Curr Eye Res 2012;371: 864–70. Leonadi A, Brun P, Abatangelo G, et al. Tear levels and activity of matrix metalloproteinase (MMP)-1 and MMP-9 in vernal keratoconjunctivitis. Invest Ophthalmol Vis Sci 2003;44:3052–8. Sack RA, Beaton A, Sathe S, et al. Towards a closed eye model of the preocular tear layer. Prog Retin Eye Res 2000;19:649–68. Di Girolamo N, Wakefield D, Coroneo MT. Differential expression of matrix metalloproteinases and their tissue inhibitors at the advancing pterygium head. Invest Ophthalmol Vis Sci 2000;41:4142–9. Sobrin L, Liu Z, Monroy DC, et al. Regulation of MMP-9 activity in human tear fluid and corneal culture supernatant. Invest Ophthalmol Vis Sci 2000;41:1703–9. Nevalainen TJ, Aho HJ. Peuravuori H: secretion of group 2 phospholipase A2 by lacrimal glands. Invest Ophthalmol Vis Sci 1994;35:417–21. Qu XD, Lehrer RI. Secretory phospholipase A2 is the principal bactericide for staphylococci and other Gram-positive bacteria in human tears. Infect Immun 1998;66:2791–7. Haynes RJ, Tighe PJ, Dua HS. Antimicrobial defensin peptides of the human ocular surface. Br J Ophthalmol 1999;83:737–41. Paulsen FP, Pufe T, Schaudig U, et al. Detection of natural peptide antibiotics in human nasolacrimal ducts. Adv Exp Med Biol 2001;42: 2157–63. Caccavo D, Pelligrino NM, Altamura M, et al. Antimicrobial and immunoregulatory functions of lactoferrin and its potential therapeutic application. J Endotoxin Res 2002;8:403–17. Zhou L, Huang LQ, Beuerman RW, et al. Proteomic analysis of human tears: defensin expression after ocular surface surgery. J Proteome Res 2004;3:410–16. Murphy CJ, Foster BA, Mannis MJ, et al. Defensins are mitogenic for epithelial cells and fibroblasts. J Cell Physiol 1993;155:408–13.

CHAPTER 3 Tear Film 87. Wong TT, Zhou L, Tong L, et al. Proteomic profiling of inflammatory signaling molecules in the tears of patients on chronic glaucoma medication. Invest Ophthalmol Vis Sci 2011;52:7358–91. 88. Boehm N, Funke S, Wiegand N, et al. Alterations in the tear proteome of dry eye patients – a matter of the clinical phenotype. Invest Ophthalmol Vis Sci 2013;54:2385–92. 89. Saleh GM, Hussain B, Woodruff SA, et al. Tear film osmolarity in epiphora. Ophthal Plast Reconstr Surg 2012;28:338–40.

90. Sullivan BD, Whitmer D, Nichols KK, et al. An objective approach to dry eye severity. Invest Ophthalmol Vis Sci 2010;51:6125–30. 91. Lemp MA, Bron AJ, Baudouin C, et al. Tear osmolarity in the diagnosis and management of dry eye disease. Am J Ophthalmol 2011;151(5): 792–8. 92. Sullivan BD, Crews LA, Sonmez B, et al. Clinical utility of objective tests for dry eye disease: variability over time and implications for clinical trials and disease management. Cornea 2012;31(9):1000–8.

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Basic Science: Cornea, Sclera, Ocular Adnexa Anatomy, Physiology and Pathophysiologic Responses

Chapter 4  Eyelids and the Corneal Surface Lily Koo Lin, Kimberly K. Gokoffski

Key Concepts • • • • •

The eyelids function to cover, cleanse, and lubricate the eye. The eyelid skin is the thinnest in the body and allows for unrestricted movement. The tarsal plates provide structural stability to the eyelids. The orbicularis oculi is important for eyelid closure and the involuntary blink reflex. The conjunctiva, meibomian glands, and lacrimal glands produce secretions that make up the tear film.

Introduction An understanding of eyelid anatomy and function is important to achieve ocular surface health. The eyelids are a thin, complex, and dynamic structure, whose primary function is to cover and protect the ocular surface of the eye. With every blink, the eyelids cleanse and lubricate the globe and, in doing so, maintain optical visual clarity of the cornea. They serve as both a physical and immunological barrier against infection. In addition, the eyelids also serve as an important facial aesthetic subunit, playing an essential role in facial expression. It is for these reasons that the eyelids are not only critical for vision but also for quality of life. The upper and lower eyelids join at the medial and lateral canthi. The average aperture of the eyelids is about 30 mm in horizontal width, and 10 mm in vertical height. The peak of the upper eyelid is slightly nasal to the pupillary axis, and the lowest point of the lower lid rests just lateral. In general, the upper lid covers 1−3 mm of the upper cornea and the lower lid rests at or near the inferior limbus. The upper lid crease measures about 6−10 mm from the eyelid lash line. The eyelid is often compartmentalized into the anterior and posterior lamellae. The anterior layer is composed of the eyelid skin and orbicularis oculi. The posterior layer consists of the tarsal plate and palpebral conjunctiva. The gray line

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is considered the junction of the anterior and posterior lamellae.1,2 The eyelid structures starting from the most superficial are skin, eyelid protractors, orbital septum, orbital fat, eyelid retractors, tarsus, and conjunctiva. Pathology affecting the eyelid apposition to the globe can have detrimental consequences for the ocular surface.

Anatomy Eyelid skin The skin of the eyelid is the thinnest and most pliable on the human body, ranging from 500 to 1000 µm in thickness. This allows for movement of the eyelid with minimal resistance. It is thinnest near the lid margin and thickest near the orbital rims. Because of its physical characteristics and constant dynamic movement, the eyelid skin is more prone to laxity than other facial skin, and replacement of eyelid skin may pose a challenge in finding donor sites with comparably thin skin. Histologically, the skin of the eyelid is composed of keratinized stratified squamous epithelium overlying a basement membrane and subcutaneous connective tissue. The skin becomes mucosa, or nonkeratinized, at the mucocutaneous junction which is adherent to the posterior tarsus (Fig. 4.1).3 The dermis of the eyelid is nearly nonexistent as the eyelid skin is attached to the underlying muscle by way of loose collagenous fibers interspersed with an elastic fiber network. The subcutaneous tissue of the eyelid is unique, as it contains no fat. Sparse glands and hair follicles, when present, are contained in the dermis. The skin is more adherent at the eyelid crease, canthal angles, and eyelid margin. The eyelids are relatively hairless. Eyelashes are a relatively sparse, specialized type of hair, which may serve as sensory structures causing a reflex eyelid closure when dust or foreign bodies hit them. There are approximately 100 eyelashes in the upper eyelid and 50 in the lower eyelid.4 In addition, eyelashes serve an important role for eyelid esthetics. Their limited length, increased shaft diameter, and unique curvature set them apart from any other hair on the body. A variety of conditions both dermatologic and systemic, as well as trauma, can result in eyelid scarring, retraction,

CHAPTER 4 Eyelids and the Corneal Surface

Chapter Outline Introduction Anatomy

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CHAPTER 4 Eyelids and the Corneal Surface

Orbital fat Gland of Krause

Orbicularis oculi – orbital portion Orbital septum

Müller's muscle Levator palpebrae superioris aponeurosis

Orbicularis oculi – preseptal portion

Conjunctival crypt

Orbicularis oculi – pretarsal portion

Superior arterial arcade Gland of Wolfring Tarsus Submuscular areolar tissue space Meibomian gland Inferior arterial arcade

Pilosebaceous apparatus Sweat gland Gland of Zeis Gland of Moll Orbicularis oculi – Riolan's muscle Cilium

Fig. 4.1  Magnified view of the upper eyelid and margin.

poor closure, and trichiasis, which can lead to problems with the ocular surface.

Eyelid muscles: protractors Orbicularis oculi The main protractor of the eyelid is the orbicularis oculi. Its functions include eyelid closure, blink, drainage of tears, and meibomian gland secretion. It acts as a sphincter, depressing the brow and upper eyelid while elevating the lower lid and cheek. The orbicularis oculi is a striated muscle that lies directly beneath the skin and extends from the lid margin to beyond the superior and inferior orbital rims. The orbicularis originates from and into the canthal tendons. It is densely adherent to the tarsal plates and innervated by the facial nerve. It is the reason why facial nerve palsy can lead to lagophthalmos and incomplete blink. The orbicularis is divided into three concentric portions: pretarsal, preseptal, and orbital. The orbital is under voluntary control, preseptal under voluntary and involuntary, and the pretarsal orbicularis primarily under the involuntary blink reflex.3,5 The pretarsal deep origins are located on the posterior lacrimal crest, with superficial origins on the anterior limb of the medial canthal tendon. Horner muscle is a branch of the pretarsal orbicularis that encircles both canaliculi and contributes to the lacrimal pump.6 The muscle of Riolan consists of marginal fibers of the orbicularis, and forms the “gray line” at the eyelid margin. It functions to hold the lacrimal punctum against the sclera for proper drainage of tears7 and its contraction facilitates expression of meibomian gland secretions.8 Disruption of the orbicularis oculi, whether due to myopathy, facial nerve palsy, trauma, or chemo-denervation (botulinum toxin) may lead to eyelid laxity, ectropion, lagophthalmos, and incomplete blink, and subsequently ocular surface dysfunction.

Eyelid muscles: retractors The eyelid retractors open the eye. The upper retractors are the levator palpebrae superioris, Müller’s muscle, and frontalis. The lower retractors are the capsulopalpebral muscle and the inferior tarsal/palpebral muscle.

Upper lid retractor: levator palpebrae superioris The levator palpebrae superioris is the primary retractor of the upper eyelid. It originates on the orbital roof near the apex, in front of the optic foramen and is located anterior to the superior rectus muscle. It is innervated on its undersurface by the third cranial nerve. The levator muscle passes along with the superior rectus muscle through the posterior orbit, and is loosely attached to the rectus muscle by intramuscular septa. At the point where the superior rectus attaches to the globe, the levator muscle transitions to a fascial sheet known as the levator aponeurosis. The levator aponeurosis is a fan-like structure that divides anteriorly and posteriorly. The anterior layer becomes contiguous with the orbital septum, and travels through the orbicularis oculi to insert onto subcutaneous tissue, forming the upper eyelid crease. The posterior layer lies anterior to Müller’s muscle and attaches to the anterior aspect of the tarsal plate.6 The levator muscle is 40 mm long, and the aponeurosis is 14−20 mm in length. Whitnall’s ligament is a condensation of elastic fibers of the anterior sheath of the levator muscle. It is located between the levator aponeurosis and muscle. It acts as the main suspensory ligament of the upper eyelid, a support ligament for the fornix, and as a check ligament for the levator complex. Whitnall’s ligament is thought to transfer the vector of force of the levator muscle from anteriorposterior to superior-inferior. The medial horn of the aponeurosis attaches near the trochlea and superior oblique tendon medially, on the posterior lacrimal crest. The lateral horn runs through the lacrimal gland laterally, dividing it into the orbital and palpebral lobes. It attaches 10 mm above the lateral tubercle on the inner lateral orbital wall. The lateral horn is stronger than the medial, and it accounts for lateral flare in thyroid lid retraction.9

Upper lid retractor: Müller’s muscle Just deep to the levator aponeurosis lies the Müller’s muscle. The Müller’s muscle is composed of smooth muscle fibers and has sympathetic innervation. It inserts upon the superior tarsal border and is adherent to the underlying conjunctiva. At rest, it is responsible for approximately 1–2 mm of vertical elevation of the upper eyelid, whereas at times of stress, its contraction can create a look of surprise or shock.10 Interruption of sympathetic input such as in Horner syndrome causes a mild ptosis.

Upper lid retractor: frontalis The frontalis muscle acts to lift the brows and is a weak retractor of the upper eyelid. Elevation of the brow can elevate the eyelid by 2 mm. The absence of frontalis muscle over the lateral tail-end of the brow accounts for age-related

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temporal brow hooding. The superior division of the facial nerve (frontal nerve) innervates the frontalis.

Lower lid retractors The lower lid retractors serve to depress the eyelid in downgaze and maintain the upright position of the lower eyelid. The capsulopalpebral fascia in the lower eyelid is analogous to the levator in the upper eyelid. The capsulopalpebral fascia is fibromuscular tissue that originates from the sheath of the inferior rectus muscle, divides as it encircles the inferior oblique, and fuses with the sheath of the inferior oblique. The two portions then join to form Lockwood’s ligament, where it is joined by smooth muscle fibers and the orbital septum. Lockwood’s ligament acts as a suspensory sling or “hammock” for the globe and anchors the inferior conjunctival fornix. It is composed of a fibrous condensation of the capsulopalpebral fascia, Tenon’s capsule, intramuscular septa, check ligaments, and fibers from the inferior rectus sheath. It attaches medially to the medial canthal tendon and laterally onto Whitnall’s tubercle.11 The inferior tarsal or palpebral muscle is analogous to Müller’s muscle in the upper eyelid and is also sympathetically innervated. It runs between the capsulopalpebral fascia and conjunctiva. It starts at Lockwood’s ligament to extend onto the inferior tarsal border. As Horner syndrome causes mild ptosis of the upper eyelid, it causes “inverse or reverse ptosis” of the lower lid, raising it by 1−2 mm. The lower lid structures are less defined than in the upper eyelid, and often collectively referred to as lower lid retractors. The lower lid retractors also are comprised of muscle extending from the inferior rectus. Dysfunction of the eyelid retractors can result from a variety of mechanisms. If the etiology affects the upper retractors, this typically will manifest as ptosis or mechanical ptosis. Surgical overcorrection of these conditions can lead to lid retraction and lagophthalmos. Thyroid eye disease is the most common cause of both upper and lower eyelid retraction. Lid retraction can lead to exposure of the ocular surface. Eyelid retraction can also occur after the recession of the vertical rectus muscles, due to the close proximity of the superior rectus and levator and the inferior rectus to the capsulopalpebral fascia. Disinsertion of the lower lid retractors results in entropion. The keratinized skin and inverted lashes of entropion can cause mechanical trauma to the corneal surface.

Orbital septum The orbital septum serves as an anatomical boundary between the preseptal tissues and the orbit. The orbital septum is thin fibrous tissue that originates from the arcus marginalis of the orbital rim and fuses with the lid retractors. The upper eyelid septum fuses with the levator aponeurosis superior to the tarsal plate. The lower lid septum fuses with the capsulopalpebral fascia at the inferior tarsal border. The septum lies anterior to orbital fat and does not contribute to the movement of the eyelid. Controversy exists as to whether age-related attenuation of the orbital septum is responsible for steatochalasis of the upper and lower eyelids.12

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Orbital fat The orbital fat lies posterior to the septum and the two serve as a barrier between the orbit and the eyelid, and can limit the spread of infection and hemorrhage. The upper eyelid has two fat compartments, the larger preaponeurotic central fat pad and the medial fat pad, separated by the trochlea. The medial fat pad is pale white in color, and is associated with the medial palpebral artery and infratrochlear nerve. Due to the medial fat pad proximity to the trochlea, superior oblique palsy and Brown syndrome have been reported following excision during upper blepharoplasty.13 The larger central fat pad is more yellow in color due to increased carotenoid content14 and extends laterally over the lacrimal gland. Its location overlying the aponeurosis is an important surgical landmark. There is a thin fat layer in the upper eyelid between the orbicularis oculi and orbital septum. This layer of fat can be thicker and give a fuller appearance to the eyelid in certain ethnicities and may contribute to the absence of the eyelid crease in certain Asian eyelids. The lower eyelid traditionally has three distinct fat compartments and lies anterior to the capsulopalpebral fascia. The inferior oblique muscle separates the medial and central fat pads. The central and lateral fat compartments are separated by the arcuate expansion of Lockwood’s ligament. There may be anatomic variation in the number of fat compartments.15

Tarsus The tarsal plates are composed of firm, densely packed collagen fibers. They provide structural stability to the eyelids and serve as a platform upon which the orbicularis muscle and eyelid retractors insert. The tarsal plates are rigidly attached to the periosteum medially and laterally by way of the canthal tendons, forming a tarsoligamentous sling upon which the forces of the protractors and the retractors act. The tarsal plates measure approximately 25–30 mm in horizontal length and about 1 mm in thickness. The vertical length of the tarsal plates is generally 10 mm centrally in the upper eyelid and 4 mm in the lower eyelid (Fig. 4.2).

Meibomian glands The tarsal plates contain the meibomian glands. These sebaceous glands produce meibum, which mostly consists of lipids, and is responsible for the oil portion of the tear film. The oil layer is important for stabilizing the tear film, promoting uniform distribution over the ocular surface, and slowing evaporation of the aqueous layer. Meibum also aids in trapping tears between the lid margin and globe, and also forms an airtight seal of the ocular surface with lid closure. Meibum is biochemically complex and different than sebum. More than 90 proteins have been identified in meibomian gland secretions.16,17 The meibomian glands in the upper eyelid are both greater in number (approximately 40−50 versus 20−25) and longer in length in the upper eyelid compared to the lower eyelid.3 This may explain the increased production of lipid

CHAPTER 4 Eyelids and the Corneal Surface

Orbital fat Levator palpebrae superioris muscle Superior rectus muscle

Orbital septum Orbicularis oculi muscle Müller's muscle Levator palpebrae superioris aponeurosis Tarsus Meibomian gland Skin Conjunctiva Tarsus

Inferior rectus muscle Inferior oblique muscle

Capsulopalpebral fascia Orbital septum Müller's muscle Orbicularis oculi muscle

Orbital fat

Fig. 4.2  Sagittal section of the upper and lower eyelid.

material by glands of the upper eyelid relative to the lower eyelid. Production of lipid is dependent on hormonal, neuronal, and vascular factors.18 The meibomian glands are a frequent site of chronic granulomatous inflammation of the lids, and rarely undergo malignant transformation into sebaceous cell carcinomas.19 Meibography studies show that meibomian gland loss increases with age, with significant drop out occurring after age 40.20 Meibomian gland dysfunction or posterior blepharitis is regarded as one of the main causes of dry eye syndrome, one of the more prevalent eye conditions, and is a frequent cause of visits to an eye care provider. The meibomian gland orifices are obstructed by thick waxy secretions, decreasing meibum outflow and ultimately resulting in an unstable tear film. Furthermore, bacterial lipases can degrade the secretions, resulting in free fatty acids, which irritate the corneal surface and cause keratopathy. Contributing factors to meibomian gland dysfunction include age, hormones. Chronic inflammatory conditions such as acne rosacea and ocular cicatricial pemphigoid often affect the meibomian glands and can lead to severe ocular surface problems. Chronic inflammation and trauma can case a lash follicle to arise from a meibomian gland leading to acquired distichiasis and ocular surface pathology.9

Conjunctiva The conjunctiva is the mucous membrane that lines the inner surface of the eyelids, reflects in the fornix, and lines the anterior surface of the globe. It is adherent at the limbus, and redundant in the fornices. Its main function is to lubricate the eye. Histologically, it is composed of a nonkeratinized stratified epithelium. The underlying substantia propria, or stroma, is richly vascularized and contains numerous immune defense cells. The conjunctiva is continuous with the eyelid skin through the mucocutaneous junction at the lid margin, with the corneal epithelium at the corneoscleral limbus, and with the respiratory mucosa through the lacrimal puncta. The marginal mucosa of the conjunctiva is responsible for spreading the tear film.21 At the superior and inferior fornices, the conjunctiva is adherent to underlying structures via attachments to fibrous extensions from the superior and inferior rectus muscles, respectively. These attachments maintain the shape and integrity of the fornices and prevent prolapse of the conjunctival tissue into the lid aperture. The epithelium of the conjunctiva is interspersed with goblet cells or specialized columnar mucin-producing epithelial cells. Goblet cells are more numerous in the inferior

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palpebral conjunctiva and posterior lid margins.22 Goblet cells of the conjunctiva and the lacrimal acinar cells contribute to mucin, an integral part of the tear film. Mucins are complex molecules that create a sticky base layer of protection on the ocular surface. The conjunctiva also contains lacrimal accessory glands of Wolfring and Krause within the submucosal tissue. These contribute to the aqueous layer of the tear film. The majority of the accessory glands are along the superior tarsal border and upper lid fornix, and a few are in the inferior fornix.23

Lacrimal glands The lacrimal glands, with the main gland located in the superotemporal orbit in the lacrimal fossa of the frontal bone, and the small accessory glands in the conjunctiva, promote a homeostatic ocular surface via tear secretion. The lacrimal glands produce the aqueous layer of the tear film, composed of salts, water and proteins like lactoferrin and lipcalin, with moisturizing and antimicrobial activity. The main gland contains acini, ducts, and myoepithelial cells. The acini have two cell types, aqueous producing serous cells producing most of tear volume, as well as mucin secreting acini, which are smaller in number. Myoepithelial cells produce IgG and IgA antibodies. The orbital lobe of the gland has interlobular ducts that connect to main excretory ducts, which join into ducts of the palpebral lobe, which secrete tears into the upper fornix. Lacrimal gland dysfunction can occur with systemic diseases such as Sjögren, leading to keratoconjunctivitis sicca.23,24

Eyelid margin The lid margin measures 2 mm wide. The posterior aspect of the margin is the mucocutaneous junction. Just anterior to this are the meibomian gland orifices. The gray line is the section of pretarsal orbicularis (Riolan) and is located between the meibomian gland orifices and ciliary follicles or lash line. A complex interplay of signaling molecules maintains the mucocutaneous junction.25 Chronic inflammation, as in the case of blepharitis, can lead to disruption of these distinct transitions as well as effacement and anterior migration of the mucocutaneous junction.3,7 The lid margin contains three types of glandular structures: meibomian glands, the glands of Zeis, and the glands of Moll. Meibomian glands are specialized holocrine sebaceous glands embedded within the tarsal plates. They have multiple acini that secrete lipid material into a central duct.8 The glands of Zeis are holocrine sebaceous glands and are lined by epithelium continuous with the eyelash follicle.26 The glands of Moll are apocrine sweat glands, which empty into the follicle of the eyelash or directly onto the surface of the lid margin. These glands are less numerous than the glands of Zeis and have the potential of forming cystic tumors (sudoriferous cysts).27

Canthal tendons The medial and lateral canthal tendons are extensions of the orbicularis and attach to the periosteum, creating a sling-like

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structure to support the lid margin and maintain apposition of the eyelids against the globe. The medial canthal tendon is a broad tendon that divides and inserts onto both the anterior lacrimal crest and the posterior lacrimal crest, surrounding the lacrimal sac.28 The medial canthal tendon and the orbicularis form the lacrimal pump. Although the anterior attachments are stronger than the posterior, the posterior attachments are thought to be more critical with respect to eyelid apposition against the globe. The lateral canthal tendon has a superior crus arising from the superior tarsus and an inferior crus arising from the inferior tarsus. These fuse at the lateral border of the tarsal plates to join the lateral retinaculum, which attaches to the lateral orbital tubercle (Whitnall’s tubercle) on the inner aspect of the lateral orbital rim.11 The lateral canthus inserts 2 mm higher than the medial canthus.3

Lacrimal drainage system The lacrimal drainage system is responsible for the outflow of tears. The puncta are located on the posterior aspect of the eyelid margin, medial to the ciliary border, on the medial upper and lower lids. The puncta connect to the canaliculi, which are surrounded by orbicularis. More often the upper and lower canaliculi join into a common canaliculus before entering the lacrimal sac. The sac is protected by the bony lacrimal fossa, and surrounded by the medial canthal tendon. In dacryocystitis, the lacrimal sac will distend inferiorly, but not superior to the medial canthal tendon. The lacrimal sac narrows into the nasolacrimal duct, which passes within an osseous portion for 15 mm before exiting under the inferior turbinate in the nose.9 Obstruction at any level along this system can lead to epiphora. Stasis of tears within an obstructed canaliculus or lacrimal sac from a nasolacrimal duct obstruction can lead to infection. Canaliculitis and dacryocystitis can cause chronic conjunctivitis and disruption of the ocular surface.

Vascular supply The eyelids have a rich vascular supply beneficial for healing and preventing infection. The arterial supply arises from the internal carotid artery and the ophthalmic artery and its branches. The two systems anastomose throughout the upper and lower eyelids, and form the marginal arcades. The upper eyelid also has an additional peripheral arcade. The upper marginal arcade is located 2 mm superior to the margin along the tarsus. The peripheral arcade lies superior to the tarsal border, on the anterior face of Müller’s muscle. The lower arcade is at the inferior tarsal border.9 Venous drainage of the eyelids is through the anterior facial and superior temporal veins into the external jugular system and through the ophthalmic vein into the cavernous sinus and internal jugular system.29,30

Lymphatic drainage Traditionally, the lateral aspect of the upper and lower eyelids and conjunctiva were thought to drain into the preauricular nodes, while the medial third of the upper and

CHAPTER 4 Eyelids and the Corneal Surface medial two-thirds of the lower eyelids and conjunctiva were thought to drain into the submandibular nodes.1 Lympho­ scintigraphy studies, however, suggest that the preauricular lymph node basin is the primary site of eyelid lymphatic draining.31,32

Nerves Sensory innervations of the eyelid are provided by the fifth cranial nerve (CN V). The ophthalmic division (VI) branches include the supraorbital, supratrochlear, infratrochlear, nasociliary, and lacrimal. The supraorbital nerve supplies the upper lid, forehead, and scalp. The supratrochlear supplies the superior portion of the medial canthus and upper lid, conjunctiva, and forehead. The infratrochlear provides sensory innervation to the skin of the inferior medial canthus and lateral nose, conjunctiva, caruncle, and lacrimal sac. The lacrimal nerve supplies the lacrimal gland, lateral upper lid, and conjunctiva. The infraorbital nerve (V2) supplies the skin and conjunctiva of the lower lid, lower aspect of the nose, and upper lip. The zygomaticofacial nerve (V2) supplies the skin of the lateral lower eyelid. Motor innervations are provided by cranial nerves III, VII, and sympathetic fibers. The facial nerve (CN VII) innervates the muscles of facial expression: orbicularis oculi, frontalis, procerus, and corrugator supercilii. The levator is innervated by CN III and Müller’s muscle is sympathetically innervated.9

Blink reflex and tear flow Spreading of the tear film, inducing meibomian gland secretion, and tear drainage depend on the blink reflex. Involuntary or reflex closure responds to somatosensory, acoustic, or light stimulation.33 Basal and reflex tearing is also stimulated by sensory input from external and internal factors. Blinking and tearing, both integral to ocular surface health, are intimately connected in complex sensory circuits involving the eyelid anatomy.23

References 1. Kikkawa DO, Vasani SN. Ophthalmic facial anatomy. In: Chen WP, editor. Oculoplastic surgery: the essentials. New York: Thieme; 2001. 2. Newman MI, Spinelli HM. Reconstruction of the eyelids, correction of ptosis, and canthoplasty. In: Thorne CH, Beasley RW, Aston SJ, editors. Grabb & Smith’s plastic surgery. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2007. 3. Wolfley DE. Eyelids. In: Krachmer JH, Mannis MJ, Holland EJ, editors. Cornea. 2nd ed. Philadelphia: Elsevier; 2005. 4. Nerad JA, Chang A. Trichiasis. In: Chen WP, editor. Oculoplastic surgery: the essentials. New York: Thieme; 2001. 5. Humphrey T. Some correlations between the appearance of human fetal reflexes and the development of the nervous system. Prog Brain Res 1964;4:93–135.

6. Kakizaki H, Zako M, Miyaishi O, et al. The lacrimal canaliculus and sac bordered by the Horner’s muscle form the functional lacrimal drainage system. Ophthalmology 2005;112:710–16. 7. Lipham WJ, Tawfik HA, Dutton JJ. A histologic analysis and threedimensional reconstruction of the muscle of Riolan. Ophthal Plast Reconstr Surg 2002;18:93–8. 8. Mansour A. Meibomian gland secretion. Orbit 1988;7:201–9. 9. Lin LK. Eyelid anatomy and function. In: Holland EJ, Mannis MJ, Lee WB, editors. Ocular surface disease: Cornea, conjuctiva, and tear film. Philadelphia: Saunders Elsevier; 2012. 10. Felt DP, Frueh BR. A pharmacologic study of the sympathetic eyelid tarsal muscles. Ophthal Plast Reconstr Surg 1988;4:15–24. 11. Bedrossian EH Jr. Surgical anatomy of the eyelids. In: Della Rocca RC, Bedrossian EH, Arthurs BP, editors. Ophthalmic plastic surgery: decision making and techniques. New York: McGraw-Hill Professional; 2002. 12. Camirand A, Doucet J, Harris J. Anatomy, pathophysiology, and prevention of senile enophthalmia and associated herniated lower eyelid fat pads. Plast Reconstr Surg 1997;100:1535–46. 13. Wilhelmi BJ, Mowlavi A, Neumeister MW, et al. Upper blepharoplasty with bony anatomical landmarks to avoid injury to trochlea and superior oblique muscle tendon with fat resection. Plast Reconstr Surg 2001;108: 2137–40. 14. Sires BS, Saari JC, Garwin GG, et al. The color difference in orbital fat. Arch Ophthalmol 2001;119:868–71. 15. Oh CS, Chung IH, Kim YS, et al. Anatomic variations of the infraorbital fat compartment. J Plast Reconstr Aesthet Surg 2006;59:376–9. 16. Butovich IA. The Meibomian puzzle: combining pieces together. Prog Retin Eye Res 2009;28:483–98. 17. Tsai PS, Evans JE, Green KM, et al. Proteomic analysis of human meibomian gland secretions. Br J Ophthalmol 2006;90:372–7. 18. McCulley JP, Shine WE. Meibomian gland function and the tear lipid layer. Ocul Surf 2003;1:97–106. 19. Khan JA, Doane JF, Grove AS Jr. Sebaceous and meibomian carcinomas of the eyelid. Recognition, diagnosis, and management. Ophthal Plast Reconstr Surg 1991;7:61–6. 20. Den S, Shimizu K, Ikeda T, et al. Association between meibomian gland changes and aging, sex, or tear function. Cornea 2006;25:651–5. 21. Mastrota KM. The conjunctiva and dry eye. Contact Lens Spectrum 2009. Available at: . 22. Knop N, Korb DR, Blackie CA, et al. The lid wiper contains goblet cells and goblet cell crypts for ocular surface lubrication during the blink. Cornea 2012;31:668–79. 23. Abelson MB, Kelley N, McLaughlin J. Bringing the focus to the aqueous. Rev Ophthalmol 2012;19:42–5. 24. Hirayama M, Ogawa M, Oshima M, et al. Functional lacrimal gland regeneration by transplantation of a bioengineered organ germ. Nat Commun 2013;4:2497. 25. Liu S, Li J, Tan DT, et al. The eyelid margin: a transitional zone for 2 epithelial phenotypes. Arch Ophthalmol 2007;125:523–32. 26. Honavar SG, Shields CL, Maus M, et al. Primary intraepithelial sebaceous gland carcinoma of the palpebral conjunctiva. Arch Ophthalmol 2001; 119:764–7. 27. Jordan DR. Common eyelid lumps and bumps. Insight: A Quarterly Report For Health Care Professionals Delivering Eye Care 1997;1–2. 28. Poh E, Kakizaki H, Selva D, et al. Anatomy of medial canthal tendon in Caucasians. Clin Experiment Ophthalmol 2012;40:170–3. 29. Snell R, Lemp M. The orbital blood vessels. Hoboken, NJ: Wiley-Blackwell; 1997. 30. Hayreh SS. Orbital vascular anatomy. Eye 2006;20:1130–44. 31. Echegoyen JC, Hirabayashi KE, Lin KY, et al. Imaging of eyelid lymphatic drainage. Saudi J Ophthalmol 2012;26:441–3. 32. Nijhawan N, Marriott C, Harvey JT. Lymphatic drainage patterns of the human eyelid: assessed by lymphoscintigraphy. Ophthal Plast Reconstr Surg 2010;26:281–5. 33. Aramideh M, Ongerboer de Visser BW. Brainstem reflexes: electrodiagnostic techniques, physiology, normative data, and clinical applications. Muscle Nerve 2002;26:14–30.

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Chapter 5  A Matrix of Pathologic Responses in the Cornea Jessica Ciralsky, Edward Lai, George O. Waring III†, Charles S. Bouchard

Key Concepts • • • • • • •

Four regions of the cornea are: 1) epithelium, 2) subepithelial zone (epithelial basement membrane, Bowman’s layer, superficial stroma), 3) stroma, 4) endothelium and Descemet membrane. Six corneal responses are: 1) defects and their repair, 2) fibrosis and vascularization, 3) edema and cysts, 4) inflammation and immune responses, 5) deposits, 6) proliferation. There are two distinct types of immune response to antigens: 1) innate, 2) adaptive. The major types of immunocompetent cells include cells of the lymphoid system, mononuclear phagocytic system, myeloid system, and auxiliary cells. Although the immune response is usually protective, tissue injury may occur from an exuberant immune reaction (hypersensitivity reactions). Regulation of the immune response is complex and several immunoregulatory phenomena occur in the anterior segment. The eye has its own mucosa-associated lymphoid tissue (MALT), the eye-associated lymphoid tissue (EALT).

Anatomical Regions of the Cornea

3. Stroma 4. Endothelium and Descemet membrane. A spectrum of pathologic processes can disrupt the structure of these four zones and interfere with corneal function. However, the cornea can generate only a limited number of responses to these insults. These responses can be grouped into six distinct categories, although there is some overlap among them (see Fig. 5.1): 1. Defects and their repair 2. Fibrosis and vascularization 3. Edema and cysts 4. Inflammation and immune responses 5. Deposits 6. Proliferation. In this section, the authors describe the patterns of tissue response that characterize each zone and provide representative clinicopathologic examples, as originally presented by Waring and Rodrigues1 and elaborated by Freddo and Waring2 (Fig. 5.2).

General Pathologic Responses of the Cornea 1.  Defects and their repair Defects are a partial or complete absence of a portion of corneal tissue. A defect is acute if it appears suddenly, recurrent if it appears repeatedly, and chronic if it persists.

The cornea is bound by two cellular layers, the epithelium and the endothelium. Each layer rests on a basement membrane: the epithelial basement membrane and the Descemet membrane, respectively. Sandwiched between these cellular layers is Bowman’s layer, a thin layer of acellular connective tissue, and the stroma, a thicker, cellular layer of connective tissue. For the purpose of discussing pathologic responses in the cornea, we can divide the cornea into four regions (Fig. 5.1):

2.  Fibrosis and vascularization

1. Epithelium 2. Subepithelial zone a. Epithelial basement membrane b. Bowman’s layer c. Superficial stroma

3.  Edema and cysts

†

Deceased

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Fibrosis and vascularization are part of the normal repair process in connective tissues. In most tissues, these processes are beneficial; in the cornea, however, they lead to stromal scarring with opacification and disruption of optical function. Because the normal cornea is avascular, the appearance of blood vessels in the cornea is always abnormal.

Edema and cysts are grouped together for simplicity and because they often resemble each other clinically. When edema (i.e. excess fluid in or between cells) occurs, the normal architecture is disrupted, leading to opacification. The edema can be diffuse (stromal edema) or focal (epithelial

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea

Chapter Outline Anatomical Regions of the Cornea General Pathologic Responses of the Cornea Specific Pathologic Responses of the Cornea Pathologic Responses of the Corneal Epithelium The Pathologic Responses of the Subepithelial Zone Pathologic Responses of the Corneal Stroma Pathologic Responses of the Corneal Endothelium and Descemet Membrane The Immune Response: Components and Reactions in the Eye Soluble Mediators/Receptors of Inflammation Tissue Components of the Ocular Immune System The Cell-Mediated Immune (CMI) Response The Humoral (Antibody-Mediated) Immune Response Anterior Chamber Associated Immune Deviation (ACAID) Immune Hypersensitivity Reactions

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CHAPTER 5 A Matrix of Pathologic Responses in the Cornea bullae). Corneal cystic areas are focal collections of fluid or solid material without an epithelial lining.

4.  Inflammation and immune responses Inflammation and immune reactions result from a variety of insults that can lead to reversible or irreversible changes. In general, three basic steps are involved: (1) an inciting

Four zones of cornea Epithelium

Six types of pathologic responses

Edema cysts Inflammation immune response Deposits

Descemet membrane endothelium

Inflammation and immune response

Deposits

Specific Pathologic Responses of the Cornea A summary of the six pathologic responses in the four corneal zones is presented (Fig. 5.2) with representative disorders occupying each box of this matrix. The amount of functional deficit inflicted by a disease process depends on the type, duration, severity, and location of the insult as well as the cornea’s ability to repair and restore normal structure and function.

Proliferation

Fig. 5.1  For the purpose of discussing pathologic responses in the cornea, we can divide the cornea into four regions. Each zone can manifest six types of responses. (Modified from Waring GO 3rd, Rodrigues MM. Patterns of pathologic response in the cornea. Surv Ophthalmol 1987; 31:262. Copyright Elsevier 1987.)

Corneal layer

Abnormal types or amounts of material can be deposited in the cornea from exogenous or endogenous sources, corneal dystrophies and degenerations, and autologous breakdown products.

There are three basic types of abnormal proliferative responses: (1) abnormalities of growth and maturation; (2) ectopic migration; and (3) corneal stem cell deficiency, leading to conjunctivalization of the corneal surface.

Fibrosis vascularization

Stroma

5.  Deposits

6.  Proliferation

Defects

Subepithelial zone

pathologic event; (2) a host cellular and humoral inflammatory and/or immune response; and (3) a repair process. These three steps are beneficial when they contain and control the pathologic process, but can be harmful if they lead to corneal damage.

Proliferation

Defects

Fibrosis and vascularization

Edema and cysts

Epithelium

Subepithelial zone

Stroma

Endothelium and Descemet membrane

Fig. 5.2  This matrix of the four corneal zones and six types of pathologic responses can include almost all corneal diseases, as demonstrated by the listed examples. (Modified from Waring GO 3rd, Rodrigues MM. Patterns of pathologic response in the cornea. Surv Ophthalmol 1987; 31:262. Copyright Elsevier 1987.) Continued

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PATHOLOGIC RESPONSES OF THE CORNEA Examples of Each Represented by Drawings

Corneal Layer

Fibrosis and Vascularization

Defects 4

1

2

none 2

Epithelium

1

3

1. Neurotrophic keratopathy 2. Herpes simplex dendrite 3. Recurrent erosion 4. Punctate epithelial keratopathy

1. Microcystic edema, bulla 2. Cysts in epithelial basement membrane dystrophy

None

3

1 2

2

2 1

Subepithelial zone

Edema and Cysts

1

1. Pterygium 2. Salzmann’s nodular degeneration 3. Pannus

1. Foreign body 2. Keratoconus

1. Basement membrane folds 2. Subepithelial bulla

2 2 1

1

Stroma

1. Terrien degeneration 2. Sterile stromal ulcer

1. Vascularization and scarring 2. Avascular scar

2

Endothelium and Descemet membrane

Fig. 5.2, Continued

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1

1. Focal damage during surgery 2. Birth trauma

Stromal edema

3 2 1

1. Posterior collagenous layer 2. Posterior polymorphous dystrophy 3. Cornea guttata

Endothelial edema

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea

PATHOLOGIC RESPONSES OF THE CORNEA Examples of Each Represented by Drawings

Corneal Layer

Inflammation and Immune Responses

Deposits

Proliferation

3

2

3

2 1

Epithelium

2

1

1. Zoster dendrite 2. Thygeson’s superficial punctate keratitis

1

1. Hudson-Stähli line 2. Crystals 3. Amiodarone or chloroquine

2

2 1

1. Corneal intraepithelial neoplasia 2. Facet 3. Keratinization

2

3 1

1

Subepithelial zone 1. Phlyctenulosis 2. Adenovirus punctate keratitis

1. Maps and 2. Fingerprints in epithelial basement membrane dystrophy Bowman’s layer does not proliferate

1. Spheroidal degeneration 2. Calcific band keratopathy 3. Reis-Bücklers’ dystrophy 1

2 1

4

1

5

2

2

3

Stroma

1. Suppuration in herpes simplex keratitis 2. Immune ring

2

Endothelium and Descemet membrane

1. Vessels with lipid leakage 2. Corneal arcus 3. Crystals in gammopathy 4. Granular dystrophy 5. Lattice dystrophy

1 2

1

1. Allograft rejection line 2. Keratic precipitates

1. Fibrous ingrowth 2. Dermoid

1. Corneal arcus 2. Wilson’s disease

2 1

1. Spread in ICE syndrome 2. Hypertrophic cells

Fig. 5.2, Continued

Pathologic Responses of the Corneal Epithelium 1.  Defects and their repair The normal corneal epithelium is continuously replaced every four to seven days, a process that involves:

(1) differentiation of the basal cells toward the surface; pathologic example: epidermalization and keratinization in vitamin A deficiency; (2) centripetal movement of limbal and peripheral cells; pathologic example: chemical damage of the limbal epithelium; (3) desquamation of epithelial cells from the surface; pathologic example: extended-wear

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soft contact lenses interfering with normal desquamation. Among the causes of epithelial defects are corneal abrasions, focal foreign bodies, neurotrophic keratopathy, and sloughing of cells in recurrent erosion. Healing of the corneal epithelium involves four major stages: sliding of cells to cover the defect, mitosis of cells to restore normal thickness, attachment of cells to the basement membrane, and remodeling to establish normal architecture.3,4 Four factors are required to re-establish normal epithelial integrity: a normal basement membrane, vitamin A, normal tear film, and intact sensory innervation.

2.  Fibrosis and vascularization Because the corneal epithelium lacks connective tissue, it is not subject to fibrosis or vascularization. However, either process can occur beneath the epithelium and may consequently affect epithelial repair.

A

3.  Edema and cysts The epithelium takes on a cystic appearance when edema develops within or between the cells (e.g. endothelial dysfunction) or when changes in epithelial maturation create small, debris-filled cystic spaces (e.g. Cogan microcysts in epithelial basement membrane degeneration (EBMD)). Even mild changes can reduce visual acuity if they create an irregular surface that diffracts and scatters light.

Epithelial edema There are two common causes of epithelial edema: endothelial dysfunction and epithelial hypoxia and trauma. When fluid lifts cells from the basement membrane, bullae appear. The epithelial sheet is held together by desmosomal connections (Fig. 5.3). Contact lens-induced edema can be caused by epithelial hypoxia, hypercapnia, trauma due to improper fitting or overwear, or a combination of these. Intracellular edema results when the compensatory abilities of the epithelium are exceeded. Cysts can result from accumulation of rapidly multiplying (e.g. Meesmann dystrophy) or degenerating (e.g. EBMD) epithelial cells. In recurrent epithelial erosions, chronically regenerating epithelium often manifests as clusters of clear, pinpoint microcysts in the area of a previous erosion.

4.  Inflammation and immune responses In corneal allograft rejection, the donor epithelium may be attacked by sensitized cytotoxic T lymphocytes. This is a specific response to foreign antigens (e.g. the human leukocyte antigens [HLAs] in epithelial cells) and appears clinically as a serpentine line that spreads from the graft–host margin toward the center of the transplant. Epithelial healing often keeps pace with cell death and may make epithelial rejection a passing, asymptomatic phenomenon.

5.  Deposits Epithelial deposits can be divided into four categories based on their origin: (1) elements, (2) drugs (3) systemic diseases and (4) corneal dystrophies and degenerations.

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B Fig. 5.3  Corneal edema. (A) Diffuse corneal edema with epithelial bullous elevations from a spontaneous break in Descemet membrane and endothelium in keratoconus (corneal hydrops). (B) Histopathology shows an epithelial bulla with fluid separation of corneal epithelium from Bowman’s layer (asterisks). (A and B, from Leibowitz HM, Waring GO, III. Corneal Disorders. Clinical Diagnosis and Management 2nd edn, Philadelphia, W.B. Saunders Company, 1998. Copyright Elsevier 1998.)

Elements The most common intraepithelial deposit is iron; hemosiderin pigment is deposited in lysosomes of the basal epithelial cells in a linear pattern (Fig. 5.4).

Drugs5 Numerous systemically administered drugs can accumulate in the epithelium; the most common being the antiarrhythmic drug amiodarone, which causes a characteristic whorl-like pattern. The severity of the deposits is directly proportional to the total drug dose. Generally, most of the corneal deposits disappear when the drug is withdrawn. Epithelial deposits from systemic diseases seldom reduce visual acuity. Exceptions include certain inherited metabolic

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea

Eyelid closure (Hudson-Stähli)

Keratoconus (Fleischer)

Pterygium

Filtering bleb

Salzmann’s nodular degeneration

Focal, elevated corneal scar B

Keratoplasty

Radial keratotomy

Irregular corneal scar

A

C Fig. 5.4  (A) Nine types of iron lines in the corneal epithelium. (B) Irregular epithelial iron line with whorl-shaped brownish deposits in Fuchs dystrophy with chronic corneal edema and an irregular epithelial surface. (C) Histopathology demonstrates dark stain of iron deposits in the basal layer of corneal epithelium. (A, from Steinberg EB, Wilson LA, Waring GO 3rd, et al. Stellate iron lines in the corneal epithelium after radial keratotomy. Am J Ophthalmol 1984; 98:416. B and C, from Leibowitz HM, Waring GO, III. Corneal Disorders. Clinical Diagnosis and Management. 2nd edn. Philadelphia, W.B. Saunders Company, 1998. Copyright Elsevier 1998.)

disorders (e.g. mucopolysaccharidosis type VI-A, Maroteaux− Lamy, and the sphingolipidosis of Fabry’s disease), multiple myeloma, and cystinosis.

Corneal dystrophies and degenerations Corneal dystrophies rarely produce deposits within the epithelium. Meesmann epithelial dystrophy is the exception.

6.  Proliferation The epithelium manifests a full spectrum of growth and maturation disorders, including hyperplasia, metaplasia, and dysplasia-neoplasia. Because the epithelium conforms to the contour of the underlying basement membrane and stroma, its thickness varies. Areas of thinning occur over elevations (e.g. over Salzmann’s nodules or keratoconic cones),6 whereas areas of thickening occur when epithelium fills in defects (e.g. a facet) . These adjustments in epithelial thickness preserve a smooth corneal surface to help maintain optimal optical function, but the mechanisms involved are unknown. Metaplasia from a normal to an abnormal keratin- forming epithelium can occur in severe ocular inflammation (e.g. Stevens−Johnson syndrome).

The epithelium is the only layer of the cornea that can become neoplastic, giving rise to squamous cell carcinoma, predominantly at the limbus where the stem cells are located.7 The full spectrum of changes, from mild dysplasia through carcinoma in situ, is termed “intraepithelial neoplasia.” It commonly appears as a gray intraepithelial sheet advancing onto clear cornea but can also appear as a raised limbal mass (Fig. 5.5). Ectopic migration can be seen after perforating, accidental or surgical trauma. In these cases, proliferating corneal epithelium can invade the anterior chamber through a fistula and form a cyst or a downgrowth sheet. Epithelium can also proliferate, such as ingrowth under a laser in situ keratomileusis (LASIK) flap.

The Pathologic Responses of the Subepithelial Zone 1.  Defects and their repair The epithelial basement membrane and Bowman’s layer are both acellular and have different healing responses. The basement membrane is secreted by the basal epithelial cells and, therefore, can be regenerated or produced in an excess

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A

B

Fig. 5.5  (A) Corneal intraepithelial neoplasia. A flat limbal mass extends onto the cornea as a gray, opaque sheet with a sharply marginated, fimbriated leading edge. (B) Histopathology demonstrates thickened epithelium with loss of normal maturation and basilar neoplastic cells. (A, from Leibowitz HM, Waring GO, III. Corneal Disorders. Clinical Diagnosis and Management. 2nd edn, Philadelphia, W.B. Saunders Company, 1998. Copyright Elsevier 1998. B, from Waring GO 3rd, Roth AM, Ekins MB. Clinical and pathological description of 17 cases of clinical intraepithelial neoplasia. Am J Ophthalmol 1984; 97:547.)

or altered form. Bowman’s layer, however, does not regenerate. A defect in Bowman’s layer fills with fibroblasts and connective tissue, creating a permanent scar.

2.  Fibrosis and vascularization Neither the epithelial basement membrane nor Bowman’s layer can become fibrotic or vascularized. Fibrous or vascular tissues can spread between the two layers as either an avascular or vascular pannus.

Subepithelial avascular fibrosis Patches of avascular fibrous tissue appear beneath the epithelium as a nonspecific response (e.g. advanced Fuchs endothelial dystrophy). The subepithelial haze that can occur four to eight weeks after photorefractive keratectomy (PRK) is another example. This haze corresponds to a layer of subepithelial collagen and proteoglycans8 that remodels over time, Salzmann’s nodular degeneration is a distinctive type of avascular subepithelial fibrosis. The grayish “stuck-on” lesions consist of hyaline and basement membrane material and accumulate between Bowman’s layer and the thinned but continuous epithelium.

Subepithelial vascular fibrosis Subepithelial vascular fibrosis involves three basic cells: leukocytes, proliferating vascular endothelial cells, and active fibroblasts that secrete an extracellular connective tissue matrix. This process occurs (1) after mild inflammation (e.g.

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hypoxia beneath an extended-wear soft contact lens) in which a very fine sheet of fibrovascular tissue slowly migrates from the limbus, (2) during chronic inflammation (e.g. trachomatous eyelid scarring) where a progressive dense pannus spreads centrally, as well as (3) after severe inflammation (e.g. alkali burns) in which a thick layer of exuberant fibrovascular tissue can progress across the entire cornea (Fig. 5.6).

3.  Edema and cysts Subepithelial edema arises from endothelial dysfunction, as described above. Diffuse stromal edema can throw the epithelial basement membrane into folds.

4.  Inflammation and immune responses Damage to the subepithelial zone results from an inflammatory (innate immune) response. This is usually characterized by an epithelial defect and a focal white superficial infiltrate that damages Bowman’s layer and the superficial stroma. This response can be seen with severe infection or trauma. Subepithelial infiltrates can also be caused by antigens and toxins (e.g. acute adenoviral keratoconjunctivitis) that pass through intact epithelium into Bowman’s layer and superficial stroma, where they can elicit immune and inflammatory responses. This may cause focal areas of infiltration and edema, generally in the absence of concurrent epithelial defects. Subepithelial infiltrates can also be seen following penetrating keratoplasty as an immune response, reflecting a mild form of allograft rejection.

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea

A Fig. 5.6  (A) Image showing subepithelial vascular fibrosis in an alkali burn of the cornea. (B) Histopathology of subepithelial vascular fibrosis demonstrates thickened epithelium, diffuse fibrosis, and vessels (v). (From Leibowitz HM, Waring GO, III. Corneal Disorders. Clinical Diagnosis and Management, 2nd edn. Philadelphia, W.B. Saunders Company, 1998. Copyright Elsevier 1998.)

B

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A

B

Fig. 5.7  Epithelial basement membrane degeneration. (A) An irregular, gray maplike pattern of epithelial basement membrane. (B) Histopathology demonstrates basement membrane duplication (asterisks) within the epithelium, trapping epithelial cells that show thickening and degeneration (the basis of the map figure, clinically) and focal cyst-like formation (white area) which create “Cogan microcysts.” (A and B, from Leibowitz HM, Waring GO, III. Corneal Disorders. Clinical Diagnosis and Management. 2nd edn. Philadelphia, W.B. Saunders Company, 1998. Copyright Elsevier, 1998.)

5.  Deposits Deposits in the epithelial basement membrane, such as silver granules from topical medications, are seldom visible clinically. Topical and systemic drugs rarely accumulate beneath the epithelium. One exception is epinephrine, which historically was seen within and below the epithelium as adrenochrome pigment. Superficial, iron-containing foreign bodies can deposit a rust ring in Bowman’s layer and superficial stroma. Systemic diseases rarely leave deposits selectively in Bowman’s layer. A form of amyloid is deposited beneath the epithelium in primary, gelatinous, droplike dystrophy. Reis−Bücklers dystrophy produces deposition of fine, curled filaments that replace Bowman’s layer. Calcium can be deposited in Bowman’s layer in band keratopathy, a degenerative process that gradually progresses within the palpebral fissure as a chalk-white plaque. Peripheral, arcuate calcific anterior stromal deposits may result from hypercalcemia.

6.  Proliferation of the epithelial basement membrane The basal corneal epithelial cells can secrete exuberant amounts of basement membrane, both beneath and within the epithelium. This excess tissue appears in primary epithelial disorders (e.g. EBMD9) (Fig. 5.7), as a nonspecific response (e.g. Salzmann’s degeneration), or as a manifestation of systemic diseases (e.g. diabetes mellitus).

Pathologic Responses of the Corneal Stroma The structural integrity, tensile strength, and contour of the cornea are derived primarily from stromal collagen, predominantly type 1.

1.  Defects and their repair Stromal defects may occur acutely after accidental or surgical trauma and are repaired according to the principles of

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normal corneal wound healing. Stromal defects also occur from ulceration due to microbial invasion, where repair requires elimination of the microorganism and control of the inflammation. Chronic defects are often progressive and fall into three categories: (1) stromal thinning without ulceration; (2) sterile stromal ulceration; and (3) congenital posterior corneal defects (see Endothelium). Stable or progressive thinning of the stroma without epithelial ulceration or stromal inflammation occurs in ectatic disorders (e.g. keratoconus (Fig. 5.8)) keratoglobus, pellucid marginal degeneration), and Terrien marginal degeneration. Significant stromal thinning alters the corneal curvature, which leads to instability and vision loss. In general, a total thickness of >400 µm is probably necessary to preserve corneal integrity and normal contour. The more common causes of stromal loss include alkali burns, autoimmune disease, and infection. These insults may set off a chain of destructive inflammatory and enzymatic events10 that result in a persistent, sterile, sharply demarcated stromal defect that may progress to a descemetocele and corneal perforation.

2.  Fibrosis and vascularization Two of the most common and nonspecific pathologic processes that opacify the stroma are fibrosis (scarring) and vascularization. Wound healing in the corneal stroma occurs slowly, presumably because the tissue is avascular, and its rate decreases with age. The resultant scar tissue is weaker than the normal stroma, as evidenced by traumatic dehiscence of penetrating keratoplasty wounds many years after surgery. There are three basic phases in stromal wound healing: (1) the destructive phase involves removal of abnormal tissues by polymorphonuclear (PMN) leukocytes and macrophages, aided by collagenases and proteoglycanases; (2) the synthetic phase involves closure of the wound through synthesis of new collagen and proteoglycans by stromal fibroblasts; and (3) the remodeling phase involves the transformation of an initial scar into a clearer structure, more

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea

A

Fig. 5.8  Keratoconus. (A) The red fundus reflection highlights the central cone, giving an oil droplet appearance. (B) Histopathology of the central cornea in keratoconus. Left side of the figure shows normal corneal thickness paracentrally. Right side of the figure shows stromal thinning with focal breaks in Bowman’s layer (between arrowheads). (A and B, from Leibowitz HM, Waring GO, III. Corneal Disorders. Clinical Diagnosis and Management. 2nd edn. Philadelphia, W.B. Saunders Company, 1998. Copyright Elsevier, 1998.)

closely resembling normal cornea, through changes in the organizational structure of collagen. If the destructive phase is not contained, melting of the corneal stroma can lead to corneal perforation. If the synthetic phase is inhibited by drugs (e.g. corticosteroids) or disease (e.g. rheumatoid arthritis (RA)), healing can be delayed and wound strength may be decreased. In contrast, if the synthetic phase proceeds uncontrolled, visually significant scars can result. Within hours of anterior stromal wounding, a fibrin clot fills the defect. Fluid from the tears and aqueous humor produces swelling of the adjacent stroma and PMNs migrate into the wound from the tear film. Then, keratocytes (K) at the edge of the wound die, and the epithelium migrates toward the wound. Epithelial–stromal interactions are important in corneal wound healing. The healing epithelium elaborates cytokines (e.g. interleukin [IL]-1, tissue growth factor [TGF]-β), which stimulate stromal keratocytes to transform into fibroblasts and myofibroblasts and to secrete extracellular matrix. This can be seen after PRK,11 where the healing epithelium is in direct contact with the underlying stroma (without Bowman’s layer). However, the same excimer laser photoablation done under a flap of anterior cornea (LASIK) does not elicit haze, presumably because of an absence of epithelial–stromal interaction except at the edge of the flap.12

Stromal fibrosis Alterations in the regular alignment of collagen fibrils cause scattering of light and appear as a stromal opacity. The severity, duration and extent of healing typically determine the degree of corneal scarring.

B

The pattern of scarring is usually not diagnostic, but some processes leave characteristic scars. Bacterial and fungal keratitis usually creates a focal, sharply demarcated scar. Alkali burns often leave diffuse, opaque, marbleized scars, and syphilitic interstitial keratitis leaves deep stromal scars with ghost vessels and lipid deposits.

Stromal vascularization Vascularization of the corneal stroma is a nonspecific pathologic response; the location and number of vessels reflect the location and severity of the inflammatory response.13 Stromal vessels can reduce vision in three ways: (1) they disrupt the normal stromal architecture; (2) they allow leakage of lipid; and (3) they increase the potential for allograft rejection in corneal transplantation. Typically, the clinician tries to prevent stromal vascularization to preserve vision. Stromal vessels characteristically grow at three levels: (1) subepithelial and superficial stroma vessels in response to superficial corneal disease); (2) in the middle layers of the stroma (in response to chronic inflammation); and (3) in the deep stroma (in eyes with keratouveitis). Blood vessels that invade the stroma arise from superficial conjunctival vessels, deep scleral vessels, or iris vessels, when the iris is in contact with the cornea. These vessels spread along the collagen lamellar planes but do not grow in an anterior–posterior direction unless a scar is present. In inflammatory conditions, the pattern of the vessels often follows that of the leukocytic infiltrate. Triangular tufts grow toward focal inflammation and in penetrating keratoplasty, a ring of vessels surrounds the host–graft junction. Generally, no single pattern characterizes a particular disease with a few exceptions (e.g. superior limbal pannus

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of trachoma and the 360-degree limbal tufts from excessive soft contact lens wear). Stromal vessels dilate during active inflammation and shrink to endothelium-lined tubes without blood flow (i.e. ghost vessels) when inflammation subsides; these vessels can refill with blood if inflammation recurs or ischemia develops.

3.  Edema and cysts Edema of the stroma is a common clinical sign,14 while epithelial-lined cysts of the corneal stroma only occur rarely. Edema of the corneal stroma occurs when its water content rises above the normal 78%. In most cases, corneal stromal edema results from disruption of endothelial or epithelial pump functions and manifests as an increase in corneal thickness. Fluid accumulates in the glycosaminoglycans of the stroma, altering the regular arrangement of collagen fibrils. Clinically, stromal edema appears as a gray, ground-glass haze that varies from a fine, diffuse granularity to a dense, gray opacity, with clear, cyst-like lakes of fluid sometimes present. As the stroma swells, the anterior curvature of the cornea remains fixed, whereas the more elastic Descemet membrane is displaced posteriorly, developing folds (corneal striae). Disruption of the endothelium is the most common cause of stromal edema. It occurs frequently in: (1) surgical trauma, (2) Fuchs endothelial dystrophy, (3) severe iridocyclitis, herpes simplex immune stromal keratitis, and acute angleclosure glaucoma, and (4) when there is a defect in the Descemet membrane and absent endothelium in the same location (e.g. hydrops in keratoconus). Stromal edema usually remains confined to the area in which the endothelial or epithelial damage has occurred, presumably because the functioning endothelium in the other areas continues to pump fluid from the stroma (e.g. edema often remains adjacent to a cataract incision). With corneal edema, “cystic” spaces are fluid-filled lacunae without an epithelial lining caused by extreme stromal edema (e.g. pressure induced interface keratitis (PIIK15) after LASIK.

4.  Inflammation and immune responses Numerous infections, immunologic diseases, and traumatic disorders lead to the aggregation of leukocytes in the corneal stroma resulting in a faint, gray-brown, granular appearance. When leukocytes congregate, they create foci of yellowwhite suppuration. If corneal damage secondary to leukocytic infiltration is severe, the stroma thickens with edema and pus, becomes gelatinous, and begins to melt. The destructive activity of leukocytes usually is balanced by fibrosis with or without vascularization. Histopathologically, leukocytes migrate along stromal lamellae and congregate with varying density. Bacteria, particularly Gram-negative bacteria such as Pseudomonas aeruginosa, cause severe stromal suppuration and destruction due to the secretion of proteolytic enzymes from both the PMNs and the bacteria. Immune-based stromal inflammation is more complicated and can result from deposition of antigen–antibody complexes and complement-mediated hypersensitivity (e.g.

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RA) as well as from alteration of stromal cell surface antigens through previous exposure to an infectious agent, such as herpes simplex virus. Herpes simplex immune stromal keratitis is probably mediated by deposition of viral antigens into the stroma and subsequent immune complex hypersensitivity with migration of PMNs and lymphocytes. This forms a centripetally migrating immune ring (Wessely ring). In contrast, herpes simplex disciform endotheliitis more likely represents a delayed-type hypersensitivity response prompted by herpes simplex virus-induced modification of membrane surface expression in corneal stromal cells or by damage to the underlying endothelium.

5.  Deposits Stromal deposits disrupt image formation in proportion to their central location and density.

Topical and systemic drugs Few drugs accumulate in the stroma. Gold is the exception; it can accumulate in the cytoplasm of keratocytes and appear as myriad, fine, round, ash-like particles (ocular chrysiasis).16

Ocular diseases Retained stromal foreign bodies, such as wood, if not removed surgically, are expelled as part of the inflammatory response. Other less reactive foreign bodies, such as nylon sutures, may lead to stromal scarring. Lipid deposits in the cornea, which occur from leakage from stromal blood vessels, are common.17 Stromal keratocytes are capable of synthesizing lipids. Lipid precursors can leak from vessels and may be absorbed by the keratocytes, which synthesize and secrete cholesterol and fatty acids. These deposits vary from refractile crystals at the tip of a vessel to a full-thickness stromal mass. Corneal arcus is the most common lipid deposition in the cornea and is considered a normal change of aging unless it appears before the mid-30s, when it is suggestive of hyperlipoproteinemia.18 Blood staining of the cornea occurs following hyphema, particularly in the setting of a persistent intraocular pressure (IOP) rise or damage to the corneal endothelium.

Systemic diseases In certain systemic diseases, nonimmune deposits can appear in the corneal stroma, either because the keratocytes are involved in an inherited metabolic disorder (e.g. the mucopolysaccharidoses) or because the corneal stroma is a repository for abnormal, circulating substances (e.g. globulin crystals in multiple myeloma19). Central corneal lipid deposits are found as part of rare genetic disorders of high-density lipoprotein (HDL) metabolism such as lecithin-cholesterol acyltransferase (LCAT) deficiency. In the mucopolysaccharidoses, corneal deposits of excess dermatan sulfate and keratan sulfate create a diffuse, groundglass appearance.

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea

Deposits from dystrophies and degenerations of the stroma Deposits of abnormal substances or abnormal amounts of normal substances can create opacities in corneal stromal dystrophies,20 such as amyloid in lattice dystrophy or polymorphic amyloid degeneration, phospholipids in granular dystrophy, glycosaminoglycans in macular dystrophy, and lipid in Schnyder central crystalline dystrophy.

6.  Proliferation Stromal proliferation usually occurs in the peripheral cornea and can be congenital or acquired. The congenital type includes dermoid choristomas, which are histologically normal tissues in an abnormal location. The acquired type includes connective tissue elements of the stroma that proliferate at a surgical or accidental wound without vascularization. This can occur anteriorly when a defect in Bowman’s layer persists and allows stromal outgrowth that appears as a flat, gray plaque with a feathered leading edge. Proliferation of the corneal stroma posteriorly, through a keratoplasty21 wound or along a keratoprosthesis,22 can form a thick gray layer, a retrocorneal membrane.

Pathologic Responses of the Corneal Endothelium and Descemet Membrane 1.  Defects (and their repair) Normal adult endothelial cell density is approximately 2500 cells/mm2 and normal cell size is approximately 250 µm. Defects in the endothelium can occur alone or in combination with defects in the Descemet membrane. In either case, aqueous humor rushes through the defect into the corneal stroma, producing stromal and epithelial edema that persists until a functioning endothelial monolayer re-establishes itself.

Defects in the endothelium Defects in the endothelium may occur acutely (after trauma or surgery (DMEK, DSEK)) or chronically (as in Fuchs dystrophy). The wounded corneal endothelium repairs itself primarily through limited migration and hypertrophy and minimally through cell division.23 The corneal endothelium does not divide under normal circumstances but can be stimulated by injury. The regenerative potential of the endothelium in children is substantial but decreases with age. After injury, only cells adjacent to the defect participate directly in wound healing; those farther from the site retain their normal configuration, although peripheral endothelium may be a source of regenerative cells. Stromal edema resolves when the endothelial monolayer and barrier and pump functions are re-established. In normal corneas, 48–90% of endothelial cells are hexagonal. Alterations in endothelial cell size and shape occur during healing, decreasing the number of hexagonal cells. Variation in cell size and shape reflects the severity of

the damage. Enlarged cells represent those that spread out to cover the defect, while smaller cells represent those desquamating or those resultant from mitotic division. There is poor correlation between endothelial cell size and function. Acute damage to the endothelium is most commonly surgically induced (e.g. cataract extraction or endothelial keratoplasty techniques that involve stripping host Descemet and endothelium or folding the donor disk).24 Chronic diseases of the endothelium, such as Fuchs endothelial dystrophy25 and pseudophakic corneal edema,26 cause a progressive loss of endothelial cells. As cells are lost, the remaining cells progressively enlarge and flatten to maintain a continuous covering over Descemet membrane. If cell loss continues, however, the capacity of the remaining cells to maintain corneal deturgescence is exceeded and corneal decompensation results, with stromal and epithelial edema and sometimes scarring. After penetrating keratoplasty, endothelial cell density drops for about five years and then becomes relatively stable.27 After endothelial keratoplasty (DSAEK and DMEK), three year and one year endothelial cell density drops are comparable to those of penetrating keratoplasty, respectively.28,29

Defects in Descemet membrane Descemet membrane has less tensile strength than fullthickness stroma, and thus may break with corneal stretching. These defects can enlarge by retraction and coiling of Descemet membrane along the edge of the break as seen in acute corneal hydrops. For example, birth forceps injury compresses the globe vertically, stretching Descemet membrane horizontally and creating vertical or oblique breaks. Elevated IOP in infantile glaucoma stretches the cornea, creating serpentine or circular breaks in Descemet membrane. In all of these disorders, Descemet membrane can separate from the overlying stroma to form a ledge or strand in the anterior chamber. Because most of these disorders occur in younger patients, the corneal endothelium can repair and cover the defect, usually through production of a thick subendothelial fibrillar matrix. The retracted, coiled ends of the ruptured Descemet membrane do not reapproximate, even when endothelial continuity is re-established.30 Congenital, focal defects in Descemet membrane and the endothelium are present in most cases of Peters anomaly, ranging from a slight indentation to an excavation that reaches Bowman’s layer.

2.  Fibrosis and vascularization posterior to Descemet membrane Endothelial cells, like stromal keratocytes, can transdifferentiate into epithelium-like cells (e.g. posterior polymorphous dystrophy (PPMD)). The endothelium and Descemet membrane contain no connective tissue and therefore do not develop classic fibrosis or vascularization. However, when the endothelium is damaged or diseased, it secretes a layer of abnormal fibrillar tissue (the posterior collagenous layer), which may lead to visual loss (Fig. 5.9).

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 Insult

Normal endothelium

Damaged endothelium

Transformed endothelium

abnormalities in the anterior portion of the nonbanded layer, indicating congenital etiology. Vascularization does not occur in Descemet membrane or in the PCL. Certain thick, retrocorneal membranes can become vascularized, but most have adherent iris.

3.  Edema and cysts

Posterior collagenous layer

Recovery and/or regeneration

Fig. 5.9  A variety of endothelial insults transform corneal endothelial cells to fibroblast-like cells that secrete extracellular matrix on the posterior surface of the original Descemet membrane, forming a posterior collagenous layer. Normal endothelial morphology can recover, depending on the severity and duration of the insult. (From Leibowitz HM, Waring GO, III. Corneal Disorders. Clinical Diagnosis and Management, 2nd edn. Philadelphia. W.B. Saunders, 1998. Copyright Elsevier, 1998.)

Edema of the endothelium is usually associated with decreased endothelial function and overlying stromal edema. True cysts do not occur in these layers. Accumulated fluid within and between endothelial cells forms dilated spaces, creating a dewdrop, beaten-metal appearance (“pseudo-guttata”).34 Because Descemet membrane is a compact tissue readily permeable to water and contains only small amounts of glycosaminoglycans, it does not become edematous. However, Descemet membrane can be displaced from the posterior stroma by edema forming a posterior bulge. PPMD is characterized by focal, round, small lesions that resemble a group of vesicles or blisters. However, these are not true vesicles but are small pits in the posterior stroma lined by a thin Descemet membrane.35

4.  Inflammation and immune responses Posterior collagenous layer (PCL) Clinically, this tissue appears as a gray sheet at the level of Descemet membrane. This tissue has been described by various names in more than 30 different corneal disorders including: corneal guttae in Fuchs endothelial dystrophy and the gray “thickened Descemet membrane” in pseudophakic corneal edema. The posterior collagenous layer’ (PCL) is the preferred name.31 With light microscopy, the periodic acid–Schiff (PAS) stain demonstrates the original uniform Descemet membrane, with the PCL behind it, consisting of multiple lamellae of varying thickness and staining. Immunohistochemistry identified five different collagen types and proteoglycans in the abnormal layers.32

Using the PCL to date the onset of endothelial or Descemet membrane disease with transmission electron microscopy Under normal conditions, Descemet membrane thickens throughout life, increasing from approximately 3 µm at birth to approximately 18 µm by age 90. When viewed by transmission electron microscopy, the anterior banded portion of the Descemet membrane is present at birth. The posterior, homogeneous, nonbanded layer is produced and thickens throughout life. The multiple lamellae of the PCL that result from disease in or trauma to the endothelium accumulate as a historical record.31 By noting where abnormalities exist, one can infer whether the process is congenital or acquired. For example, in corneas affected by ICE syndrome, the layers of normal banded and nonbanded Descemet membrane are present, bounded posteriorly by abnormal posterior collagenous layers, indicating an acute onset of the endothelial disorder in adulthood.33 PPMD, on the other hand, demonstrates

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The endothelium indirectly becomes involved in inflammatory processes in disorders such as microbial keratitis and iridocyclitis. Vasodilatory and chemotactic factors bring it into contact with leukocytes, forming keratic precipitates (KP). KP form a variety of patterns, including (1) a nonspecific spattering on the posterior cornea (e.g. ankylosing spondylitis), (2) a focal aggregation (e.g. herpes simplex disciform endotheliitis), and (3) a central, inferior, elliptical or triangular pattern (e.g. sarcoid uveitis). The endothelium also becomes directly involved in inflammatory processes in disorders such as herpetic disease and allograft rejection reactions, in which antigens on the endothelial cell surface stimulate an immune reaction (e.g. endothelial rejection line (Khodadoust) on the donor).36 PMNs and mononuclear leukocytes bind to the endothelial cell surface through specialized cell surface receptors, penetrate between the cells, and then migrate between Descemet membrane and the endothelium. If the inflammatory process is mild to moderate or is appropriately treated, the leukocytes migrate back into the anterior chamber, and the endothelial monolayer recovers function. If the inflammatory process is more severe or is inadequately treated, endothelial cells show increasing vacuolization, separation from Descemet membrane, desquamation into the anterior chamber, and death. Descemet membrane is remarkably resistant to proteolytic enzymes. It resists destruction in the presence of severe keratitis, iridocyclitis, and endophthalmitis, and acts as a barrier that prevents the passage of leukocytes and most microorganisms.37 Fungi are an exception; many elaborate enzymes enable them to penetrate Descemet membrane. After severe stromal melting, Descemet membrane, bulging anteriorly as a descemetocele, may persist as the only intact structure in the cornea. Inflammation directed specifically at Descemet membrane is rare.

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea

5.  Deposits Topical and systemic drugs Drugs and elements deposit in Descemet membrane. Although seldom used today, prolonged topical administration of silver-containing medications (e.g. Argyrol) historically was the most common source of Descemet deposits. Melanin pigment selectively deposits in or on the endothelium.

Ocular and systemic diseases In systemic disease, the most common deposit in Descemet membrane is copper (e.g. Kayser−Fleischer ring in Wilson’s disease). Clinically, the copper accumulates in peripheral Descemet membrane, initially in superior and inferior arcs that coalesce to form a 360-degree greenish brown deposit. The deposition of melanin pigment in or on the endothelium can occur from several different sources within the eye. Endothelial cells phagocytose pigment in disorders such as pigment dispersion syndrome, creating a fine dusting of the posterior surface of the cornea (Krukenberg’s spindle). Iris stromal melanocytes and pigment epithelial cells can migrate over the posterior cornea, particularly in areas where there has been endothelial damage or where iris adhesions are present. They can form a faint, brownish membrane with dendriform-shaped cells or sharply marginated, rounded patches of dark-brown pigment, respectively. Pigmented macrophages also may be found in the endothelium but generally are not visible clinically. Other types of material that can deposit on the endothelial surface include: lymphocytes and PMNs (KP), red blood cells in hyphema, tumor cells in lymphoproliferative disorders, white flakes in pseudoexfoliation syndrome, and pieces of lens cortex or capsule after cataract extraction.

Corneal dystrophies and degenerations The most common material deposited in Descemet membrane is lipid (corneal arcus). Among corneal dystrophies, only macular dystrophy produces deposits in the endothelium and Descemet membrane.38

6.  Proliferation The production of excess basement membrane and collagenous tissue by the endothelium (PCL) is discussed above. There are no neoplastic or dysplastic disorders of the endothelium. Endothelial cells are capable of transforming into both fibroblast-like and epithelium-like cells. Patches of keratincontaining, epithelium-like cells occupy the posterior cornea in disorders such as PPMD,39 and ICE syndrome. Despite minimal regenerative capacity, the endothelium can proliferate over the surface of the trabecular meshwork, iris, and vitreous under specific circumstances, especially in children. When this occurs, the endothelium itself is not visible but the basement membrane it produces (ectopic Descemet membrane) is visible and often described as a glass or hyaline membrane. The process is sometimes referred to

as endothelialization or descemetization of the anterior chamber and can result in glaucoma and distortion of the iris and pupil (e.g. ICE syndrome).31

The Immune Response: Components and Reactions in the Eye Overview The ocular immune response involves a complex set of interactions between local and systemic immunocompetent and parenchymal cells that communicate through specialized cell surface receptors and soluble mediators to protect the delicate functional and structural integrity of the eye from a variety of insults. Although the immune response is usually protective, tissue injury may occur from an exuberant immune reaction (e.g. hypersensitivity reactions).40 Regulation of the immune response is also complex, and several immunoregulatory phenomena occur in the anterior segment, including immune privilege, immune tolerance, and autoimmunity.41 The immune system has primary (thymus, spleen, and bone marrow) and secondary (lymph nodes and mucosaassociated lymphoid tissue (MALT)) tissue components. The eye has its own MALT, the eye-associated lymphoid tissue (EALT). There are two distinct types of immune response to antigens.40 The first is the innate or natural response, which is the first line of defense against molecular components found only in microorganisms. It activates and instructs adaptive immune responses, regulates inflammation, and mediates “immune homeostasis” (balance between proinflammatory and anti-inflammatory processes). It is rapid in onset (minutes) and lacks memory. Cells of the innate immune system use immune-recognition receptors (pattern recognition receptors (PRRs, such as toll-like receptors (TLR) and intracellular PRRs)), to recognize specific pathogen- associated molecular patterns (PAMPs) on microorganisms. Each PAMP is characteristic of a specific group of microbes. PRRs can be secreted or circulating (e.g. antimicrobial peptides (AMPs), defensins, collectins, lectins, and pentraxins). This triggers inflammation mediated by cellular elements (e.g. neutrophils, monocytes, natural killer [NK] cells, and dendritic cells (DC)) and soluble factors (e.g. antimicrobial peptides (AMPs), complement, lysozyme, and inflammatory mediators).41 The second response is the adaptive or acquired immune response that has both humoral (antibody) and cell- mediated immune (CMI) pathways. This occurs over a longer time frame (hours, days) and changes over time. There are three important components comprising the adaptive response: 1) cells (B and T lymphocytes); 2) soluble components (antibodies, cytokines); and 3) receptors. The adaptive immune response has three phases. The initial afferent phase involves antigen recognition followed by antigen presentation by antigen presenting cells (APCs) to a host T lymphocyte. The second phase involves antigen processing, activation of lymphocytes (B and T cells) through a two signal model followed by differentiation and proliferation of specific effector lymphocytes. In the last phase,

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Acute inflammation

Goblet cells Stratified epithelium Lacrimal gland

PMN

Ig’s Growth factors Cytokines

Neuropeptides

Inflammatory stimulus Trauma Antigens UV light Hyperosmolar stress

TNFR

Epithelium

HLA-DR ICAM-1 B defensins HBD 1,2 CD59 DAF

TLR2,4,5

MMPs

Growth factors Angiogenic factors Stroma

PMN-2 PMN-3

Extended survival up to 5 days due to IFN-γ

IFN-γ IL-2

Cap3 MPO IL-8 IL-12 IL-6 TNF IL-18 IL-10

Elastin Cathepsins ROS (H2O2) MMPs 8,9,25 TLR IL-1

Neuropeptides

PMN-1

IL-IR IL-1αβ TNF-γ IL-6 IL-8 MIP2, KC RANTES MIP-2γ

MMP-9

Neutrophil chemotaxis and maturation

Macrophage

IFN-γ

Keratocytes

CXCR2

CXCR1

Limbal vessels

Activated dendritic cells

L-selectin PECAM-1 ICAM-1 E-selectin P-selectin VCAM-1 (late)

NK cell

Fig. 5.10  Acute inflammation: Following an acute stimulus, cells release IL-1, IL-6, IL-8, and TNF-α, which stimulates the migration of limbal Langerhans cell into the central cornea. These cytokines also upregulate ICAM-1, E-selectin, L-selectin, PECAM-1 on the vascular endothelium of the limbus, and facilitate PMN (PMN-1) infiltration. Complement becomes activated and local receptors regulate the complement response. Specific growth factors and angiogenic factors are also released. Keratomalacia may result from IL-1-stimulated IL-8 release and activation of PMNs (PMN-2), which release metalloproteinase (MMP) and other lysozomal enzymes that cause corneal ulceration. IFN-γ and IL-2 release may extend PMN (PMN-3) and recruitment of NK cells.

mature specialized cells interact with their specific target antigens. Subsequent exposure to antigen generates a more aggressive (anamnestic) response through the activation of memory cells that have been sensitized to that specific antigen.40 In this section, we will focus on basic principles of the body’s immune response and special features of the ocular immune response.41–43 Figure 5.10 illustrates the cascade of events for acute inflammation and Figure 5.11 for chronic inflammation. Table 5.1 lists major soluble mediators and receptors of inflammation.

Cells of inflammation and the immune response The major types of immunocompetent cells include lymphocytes (B, T, and non-B, non-T cells), cells of the mononuclear phagocytic system (monocytes and macrophages), cells of the myeloid system (polymorphonuclear leukocytes), and auxiliary cells (APCs, dendritic cells (DC), platelets, and endothelial cells).40

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Cells of the lymphoid system Lymphocytes, which cannot be distinguished morphologically, are defined by their development, cellular products, and characteristic cell membrane receptors. They are named based on “clusters of differentiation” (CD) designations.40,41

B lymphocytes B lymphocytes constitute 5–15% of the circulating lymphocytes and are primarily responsible for the humoral (antibody) arm of the adaptive immune response. B cells manufacture a large number (20 000 to 200 000) of specific immunoglobulins, which are expressed on their cell surface. There are five subclasses of B lymphocytes: IgG, IgA, IgM, IgE, IgD. Most human B cells in the peripheral blood express IgM and IgD. The receptor for the Fc portion of IgG is also expressed on B cells. Additionally, major histocompatibility class (MHC) II antigens are located on most B cells and provide the “antigen-presenting” capacity of these cells.

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea Epithelium

Release of angiogenic factors

Keratocyte

Basement membrane

β-FGF

Stroma

PDGF

Type 1 collagen

Neovascularization

Fibroblast Macrophage

TGF-β

Epithelium tears

Myofibroblast Anti-inflammatory Aberrant collagen production (types VI, VIII) Wound contraction TNF, IL-1

NK cell Fibrosis

Proinflammatory

Fig. 5.11  Chronic immune/inflammatory response: In the chronic phases of immune/inflammatory responses, infiltrating lymphocytes (NK cells) release IFN-γ, which stimulates an upregulation of ICAM-1 and HLA-DR coexpression on the corneal stroma/endothelium, thereby providing the mechanism for HLA-DR-dependent cell-mediated cytotoxicity. Macrophages may play either a proinflammatory or anti-inflammatory role depending on the cytokines released. Specific immune responses through antigen processing (between the macrophage and T lymphocyte) result in the production of Th1 or Th2 lymphocytes. The Th1 responses result in release of IL-2, IFN-γ associated with viral infections, graft rejection, and dry eye. Th2 responses result in the release of IL-4, IL-5, and IL-13. These are associated with allergic and parasitic reactions. In addition, keratocytes through the action of bFGF and PDGF transform into fibroblasts and through TGF-β become myofibroblasts producing aberrant collagen (types V, VIII) leading to stromal scarring. Angiogenic factors also result in abnormal stromal vascularization.

T lymphocytes T lymphocytes make up 65–85% of peripheral blood lymphocytes and direct the cell-mediated arm of the adaptive immune response.40 T cells develop from cell precursors within the thymus. During intrathymic differentiation, the repertoire of T-cell antigen receptor (TCR), the definitive marker for T lymphocytes, specificities are generated. Each TCR is also associated with the CD3 or T-cell differentiation antigen. T cells have MHC class II HLA-DR surface antigens. Four major functional subsets of helper T cells have been characterized: Th1, Th2, Th17, and Treg. These are differentiated by their unique patterns of cytokine secretion. Their regulation of immune responses is shown in Figure 5.12. The first subset of helper T cells, Th1 cells, manufactures TNF-β and IFN-γ. IFN-γ increases the production of IL-12 by DC and macrophages, and via positive feedback stimulates production of IFN-γ in helper T cells, thereby promoting the Th1 profile. The second type, Th2 cells, secretes IL-4, IL-5, IL-6, IL-10, and IL-13. Th2 also promotes its own profile through IL-4’s action on helper T cells to promote production of Th2 cytokines and through IL-10’s inhibition of multiple cytokines. The third type, Th17 cells, produces IL-17, IL-6, IL-22, and G-CSF. They are distinct from Th1 and Th2 cells; excessive amounts are thought to play a key role in chronic infection, allergy and autoimmune disease. IL-22 may play a role in mucosal immunity. The fourth type, Treg cells, plays a critical role in the establishment and

maintenance of tolerance and “suppressing” immune responses. They may contribute to disease processes through abnormal inhibition of protective immune responses (such as in certain malignancies).41 Cytotoxic T cells (Tc) express the CD8 co-receptor for MHC class I. They carry the TCR-2 receptor, participate in cell destruction reactions, and destroy viral infected cells and foreign allogeneic cells. Cytotoxicity occurs primarily through granule exocytosis (which initiates apoptosis) and expression of the Fas ligand. They also secrete cytokines including IFN-gamma and TNF. Gamma-delta T cells are a distinct class of lymphocyte expressing the TCR1 form of the TCR and are CD4 and CD8 negative. The exact function is unknown, although they are expanded in a variety of infections.

Null lymphocytes Natural killer (NK) cells are a heterogeneous population of non-T, non-B, granular, nonadherent, nonphagocytic lymphocytes that participate in the innate immune response and are found in the peripheral blood, spleen, and lymph nodes. They represent 10–15% of circulating lymphocytes and function in immune surveillance, destroying cells without prior sensitization or interaction with APCs. NK cells kill tumor cells, viral-infected cells, and xenogeneic cells. NK cells release IFN-γ, TNF-α, and IL-1 and other cytotoxic factors.40

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Table 5.1  Soluble mediators and receptors of inflammation (important examples) Group

Example

Source/cell

Target/ligand/action

Adhesion molecule

Intercellular adhesion molecule 1 (ICAM-1)

Endothelial cells (EC)

Lymphocyte function associated antigen 1 (LFA-1) Promote leukocyte recruitment

Very late antigen 1 (VLA-1)

T cells

Collagen, fibronectin, laminin

Vascular cell adhesion molecule (VCAM)

Endothelial cells (EC) Macrophage (MΦ)

Very late antigen 4 (VLA4)

Platelet endothelial cell adhesion molecule (PECAM)

T cells

Endothelial cells (EC) Platelets

Fas ligand (Fas L)

Many cells

Fas ligand receptor (FasR) Apoptosis Cytotoxic T-cell activity Corneal immune privilege

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL)

T cells

Apoptosis Death receptors DR4 (TRAIL-RI) and DR5 (TRAIL-RII).

Mucosal adressin cell adhesion molecule 1 (MAdCAM-1)

T lymphocytes (T)

Lymphocyte Peyer’s patch HEV adhesion molecule 1 (LPAM-1 or integrin α4β7)

P-selectin

Endothelial cells (EC)

White blood cells (WBC)

E-selectin

Endothelial cells (EC)

White blood cells (WBC)

L-selectin

White blood cells (WBC)

Endothelial cells (EC)

Chemokine ligand 5 (CCL5), Regulated in activation of normal T cells expressed and secreted (RANTES)

T cells Basophils (BΦ) Eosinophils (EΦ)

Chemotactic for T cells, eosinophils, basophils Activation of natural killer (NK) cells

CXCR1,2

Natural killer (NK) cells, basophils (BΦ)

IL-8

Interleukin 8 (IL-8)

Fibroblasts Corneal epithelial cells (EpC)

Corneal neovascularization Attract neutrophils

Eosinophil chemotactic factor

Mast cells (MC)

Attract eosinophils

Neutrophil chemotactic factor

Mast cells (MC)

Attract neutrophils

Eotaxin

Eosinophils (EΦ)

Attract eosinophils

Macrophage migration inhibiting factor (MIF)

T cells

Cell-mediated immunity (CMI) Immunoregulation Inflammation

Platelet activating factor (PAF)

Mast cell (MC)

Vasodilatation Increase permeability

Fibrin (factor Ia)

Fibrinogen

Clotting Inflammation

Thrombin (factor XIII)

Endothelial cells

Converts fibrinogen to fibrin

Fibrinogen (factor I)

Liver

Fibrin precursor

Laminin

Basal epithelial cells

Integrins

Fibronectin

Macrophages (MΦ)

Chemokines

Chemotactic factors

Clotting and fibrinolytic factors

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CHAPTER 5 A Matrix of Pathologic Responses in the Cornea

Table 5.1  (Continued) Group

Example

Source/cell

Target/ligand/action

Complement

Complement factor C5a

Hepatocytes, APC

Anaphylatoxin Histamine release from mast cells Neutrophil chemotaxis

Complement factor C3a

Macrophages

Chemotaxis Anaphylatoxin

Complement factor C3b (opsonin)

Opsonize bacteria by macrophage (MΦ)

Decay accelerating factor (DAF)

Corneal epithelial cells (EpC)

Complement regulation prevents the assembly of the C3bBb complex

Colony stimulating factors

Granulocyte-macrophage colonystimulating factor (GM-CSF)

Macrophages (MΦ), fibroblasts (FB)

Macrophage activation

Cytokines

Interleukin 1 (IL-1α, β)

Corneal epithelial cells (EpC) Macrophages (MΦ), Langerhans cells (LC)

T-cell stimulation, metalloproteinase induction, Adhesion molecule expression

Interleukin 2 (IL-2)

T helper 1 cells (Th1) Natural killer (NK) cells Keratocytes

T: proliferation and lymphokine secretion Th2: induces interferon gamma (IFN-γ) secretion

Interleukin 4 (IL-4)

T helper 2 cells (Th2) Natural killer (NK) cells Mast cells (MC)

Increase IgE Decrease proinflammatory cytokines Suppress T helper 1 (Th1)

Interleukin 6 (IL-6) (IFN-β2)

T helper 2 cells (Th2) Macrophages (MΦ) Dendritic cells (DC) Mast cells (MC)

T-cell activation B-cell Ig secretion Macrophages (MΦ) differentiation

Interleukin 10 (IL-10)

T helper 2 cells (Th2) Macrophages (MΦ) Mast cells (MC)

Th1: inhibit IL-2, IL-3, interferon gamma (IFN-γ) synthesis Th1: inhibit DTH Macrophage (MΦ): inhibit TNF, IL-1, IL-12 production

Interleukin 12 (IL-12)

Macrophages (MΦ) Dendritic cells (DC)

T helper 1 (Th1) differentiation Interferon gamma (IFN-γ) production

Interleukin 18 (IL-18)

Macrophages (MΦ)

Cell-mediated immunity (CMI) Inflammation

Tumor necrosis factor alpha (TNF-α)

T cells Macrophages (MΦ) Mast cells (MC)

T-cell stimulation Matrix metalloproteinase (MMP) induction Adhesion molecule expression

Interferon gamma (IFN-γ)

T cells Natural killer (NK) cells

HLA-DR expression Activation of: T cells, natural killer (NK) cells, macrophages (MΦ)

Interferon alpha (IFN-α) (14 subtypes)

Macrophages (MΦ) Leukocytes

Innate immune response (virus) IFN-α receptor (IFNAR)

Leukotriene B4

Mast cells (MC)

Promotes inflammation and breakdown blood–ocular barriers

Leukotriene C4

Eosinophils (EΦ)

Increase capillary permeability

Prostaglandin D2 (PGD2)

Mast cells (MC)

Vasodilatation

Interleukin 1 receptor (IL-1R)

Eicosanoids

Continued

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Table 5.1  (Continued) Group

Example

Source/cell

Target/ligand/action

Growth factors

Vascular endothelial factor (VEGF-A, B, C, D)

RPE and neurosensory retinal cells

Angiogenesis Lymphangiogenesis Macrophage chemotaxis Vasodilation

TGF-β

Many cells

Fibroblast proliferation Collagen synthesis Decrease matrix metalloproteinases Decrease T-cell proliferation Decrease proinflammatory cytokines

TGF-α

Macrophages (MΦ)

Epithelial growth Neural cell development

Nerve growth factor (NGF)

B cells T cells Fibroblasts

Nerve proliferation and development

Basic fibroblast growth factor (bFGF)

Basement membrane Vascular subendothelial matrix

Angiogenesis

Epidermal growth factor (EGF)

Macrophages Platelets

Epidermal growth factor receptor (EGFR) Epithelial migration

Platelet derived growth factor (PDGF)

Platelets

Angiogenesis

Immunoglobulin A (IgA)

B lymphocytes

Mucosal immunity

Immunoglobulin D (IgD)

Immature B cells

B-cell activation

Immunoglobulin E (IgE)

B lymphocytes

Allergy, type I reactions Binds to Fc receptors on mast cells (MC)

Immunoglobulin G (IgG)

B lymphocytes

Ig2a fixes complement

Immunoglobulin M (IgM)

B lymphocytes

Complement activation

Kinin forming system

Bradykinin

Vascular endothelial cells

Increase vascular permeability Vasodilator

Leukocyte oxidants

Hydrogen peroxide

Polymorphonuclear cells

Oxidizes free radicals

Neuropeptides

Substance P

Neural cells

Inflammation and pain neurokinin 1 receptor (NK1-receptor, NK1R)

Alpha melanocyte stimulating hormone (α-MSH)

Pituitary cells

Suppresses inflammation and T-cell responses

Collagenase (MMP-1,8,13,18)

Keratocytes (K) Corneal epithelial cells (EpC) Polymorphonuclear cells

Degrade collagen and stromal matrix

Membrane type matrix metalloproteinases (MMP, 14–17)

Keratocytes (K)

Activate progelatinase A

Gelatinases (matrix metalloproteinases [MMP] 2,9)

Keratocytes (K)

Native type IV, V, VII collagens Fibronectin

Immunoglobulins

Proteases/enzymes

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CHAPTER 5 A Matrix of Pathologic Responses in the Cornea

Table 5.1  (Continued) Group

Example

Source/cell

Target/ligand/action

Matrilysins (matrix metalloproteinases [MMP] 7,12,19,20)

Keratocytes (K)

Gelatins Fibronectin Elastin

Stromelysins (matrix metalloproteinases [MMP] 3,10,11)

Keratocytes (K)

Proteoglycans, fibronectin, serine proteinase inhibitors

Cathepsins (A and B)

Lysosomes

Protease activity

Tryptase

Mast cell (MC)

Complement activation

Peroxidase

Eosinophil (EΦ)

Epithelial cytotoxicity

Lysozyme

Macrophages (MΦ) Lacrimal acinar cells (AC)

Degrade bacterial cell walls

Vasoactive amines

Histamine

Mast cell (MC)

Dilate blood vessels

Other

MBP

Eosinophil (EΦ)

Mast cell degranulation

Heparin

Mast cell (MC)

Anticoagulation

Cationic protein

Eosinophil (EΦ)

Epithelial cytotoxicity

Lactoferrin

Lacrimal acinar cells

Monocytes, macrophages, PMN Antimicrobial activity Binds divalent cations

Cells of the myeloid system Macrophages and the mononuclear phagocytic system The mononuclear phagocyte system primarily consists of monocytes and macrophages (MΦ). Macrophages are bone marrow-derived cells located throughout the body. They develop from a myeloid progenitor cell, enter the bloodstream as monocytes, and migrate into various tissues as macrophages. Macrophages are the pre-eminent APC. They serve as a link between the innate and adaptive immune responses, actively participating in innate immune responses through phagocytosis of foreign material. They also mediate the initiation and effector phases of immune responses, influence lymphocyte responses to antigen, and stimulate T lymphocytes directly. Macrophages produce a variety of important secretory factors (including proteases, collagenases, prostaglandins, and oxygen metabolites) and release monokines (including IFN-α, IL-1, IL-6, and TNF-α).40

Dendritic cells Dendritic cells (DC) are specialized APCs within the mononuclear phagocytic system. DCs initiate a variety of immune responses, including antigen recognition and processing. Dendritic cells migrate between tissues and home to specific T-cell-dependent areas of lymphoid structures through

specialized cell surface adhesion molecules (β2 integrins). Dendritic cells are found in nonlymphoid tissues including the epithelium of the skin, ocular surface, iris, ciliary body, and other mucosal epithelia.42,43

Langerhans cells Langerhans cells (LC), also part of the mononuclear phagocytic system, are bone marrow-derived DCs found in the thymus, lymph nodes, and epithelial layers of skin, oral cavity, esophagus, nasopharynx, cervix, conjunctiva, and cornea.42,43 They have been extensively studied for their capacity to present antigen to T lymphocytes and trigger T-cell proliferative responses.

Other cells of the myeloid system Polymorphonuclear leukocytes The myeloid system is composed of erythrocytes, platelets, and granulocytes (also called PMN leukocytes). Granulocytes represent 60–70% of circulating white cells, are relatively short lived, and are divided into three categories: neutrophils, basophils, and eosinophils. They participate in the innate immune response by migrating into tissues at sites of inflammation and releasing mediators.40 The neutrophil is the first cell type to appear at sites of inflammation and infection. Neutrophils possess cytoplasmic granules that contain a variety of enzymes including myeloperoxidase, acid and alkaline phosphatases, and

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Ag

Ag

Stimulatory Inhibitory

APC

(Allergen?) Antigen presentation

(Antigen?)

(IL-6, IL-23, TGF-β)

(IL-4) IL-10 TH2

TDTH

Antigen presentation (IL-12, IFN-α)

IL-2

TH1

IFN-γ

IL-2

TC

3 IL- 4 IL-

IL-5

?

IL-6 IL-17

IL-2

IFN-γ

NK

IL-2 γ IFN

IL-4 IL-5 IL-13 B

EΦ

MC

TH17

?

Effector cells

IL-2

K

IgG

IgE

A D C C

IgG2a MC

Ag

MΦ EΦ

IgM IgA

Complement

Soluble mediators Fig. 5.12  Cytokine regulation of the acquired immune response. Hypothetical activation pathways of proposed Th1, Th2 and Th17 cells leading to effector cell stimulation are depicted. Hypothetical stimulatory pathways (solid arrows) and inhibitory effects (dashed arrows) are shown. Th1 cells release IFN-γ, which inhibits Th2 cells. Th2 cells release IL-10, which may inhibit IFN-γ production and APC activation of Th1 cells. Th17 cells produce IL-17, IL-6 and G-CSF. Allergens seem to preferentially activate Th2 cells, which stimulate IgE-mediated allergic responses (including IgE Fce receptors). IL-3 and IL-4 release would also activate mucosal mast cells, and IL-5 stimulates eosinophil proliferation. Activation of Th1 cells would also be inhibited. On the other hand, APCs seem to present antigens more to the Th1 side of the immune response. These cells release IL-2 and IFN-γ, mediating CTL and macrophage activation and IgG2a production. Antibody-dependent, cell-mediated cytotoxicity (ADCC) and delayed-type hypersensitivity (DTH) responses are also mediated by this pathway. This response is characteristic for responses to intracellular (viral, parasitic) antigens. (Adapted from Roitt IM, Brostoff J, Male DK, eds. Immunology, London, 1993, Mosby; Niederkorn JY, Li XY. Invest Ophthalmol Vis Sci 1995; 35:S817.)

lysozyme. They phagocytose organisms and degrade them through lysosomal enzymes. Adhesion molecules on their cell surfaces regulate their migration out of the vascular compartment and into tissue.

phils circulate in the peripheral blood, have life spans of several days, migrate to sites of inflammation, and possess a wide variety of cell surface receptors for adhesion molecules.

Eosinophils

Mast cells

Eosinophils represent 2–5% of peripheral leukocytes. They possess intracellular granules and have the capacity to activate many other cells including basophils, neutrophils, and platelets. Eosinophils release eosinophil major basic protein (MBP) that induces the production of IL-8 by other eosinophils, macrophages, and T cells.40 Eosinophils are phagocytic and participate in the ingestion of antigen and antibody complexes. They can present antigen through their cell surface MHC class II antigen.

Mast cells play active roles in both innate and adaptive immune responses.40,41 Mast cells are present only in mucosal epithelia and connective tissue and have life spans of months. They have receptors for IL-4 and IL-6 and release TNF-α, IL-3 to -6, -10, -13, -16, VEGF, and GM-CSF (Table 5.1). They participate in all four types of hypersensitivity responses.

Basophils

Adhesion molecules

Basophils make up less than 0.5% of all circulating leukocytes. Basophils release cytokines IL-4 and IL-13 and have cell surface receptors for cytokines IL-1 to IL-5.40 Baso-

Adhesion molecules are cell-surface glycoproteins located on circulating and fixed cells that regulate cell–cell interactions and cellular contact with intercellular matrix proteins

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Soluble Mediators/Receptors of Inflammation

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea such as collagen and fibronectin. Adhesion molecules participate in antigen presentation, migration of leukocytes to inflammatory sites, lymphocyte homing to specific tissues, and adherence of immunocompetent cells to target cells. Three groups of adhesion molecules exist: selectins, integrins, and immunoglobulins.40 Blocking the expression of these molecules may inhibit inflammatory processes.

Cytokines Cytokines are intercellular peptides or glycopeptides secreted by immune and nonimmune cells40,41 that act on other hematopoietic cells to modulate immune and inflammatory responses. Cytokines include interleukins, TNF, chemokines, colony-stimulating factors, interferons, and growth factors (Table 5.1). Cytokines can act synergistically and many function as part of a complex cascade of cytokine responses between cells. Different cytokines can act on the same cell type to mediate similar effects (redundancy). The effects of each cytokine also may depend on the specific target cell (pleio­ tropism). Because cytokines depend on a variety of factors, the effects of the cytokine network are determined to a large extent by the local environment. Their synthesis is highly regulated, especially in the cornea.40,41,43

Chemokines Chemokines are small secreted molecules with chemoattractant and cytokine properties.40,41,44 They have four families: CXC (α), CC (β), XC (γ), and CX3C (δ) (see Table 5.1). Chemokines can be divided into two classes. Constitutive chemokines (e.g. SDF-1, TARC) are expressed in primary and secondary lymphoid organs and regulate lymphocyte traffic in physiologic conditions. Inducible chemokines (e.g. RANTES, IL-8) have a role in inflammatory responses. Chemokines also have redundancy: most receptors interact with multiple chemokines and most chemokines bind to most receptors.40

Complement The complement system is an integral component of the innate immune system. It involves a set of proteins numbered C1 to C9 that interact in a cascade-like fashion determined by a series of enzymatic steps. This complex system is a potent mechanism for initiating and amplifying the inflammatory host response to bacteria and foreign antigens; it also participates in types II and III hypersensitivity responses.40 Both recognition and effector pathways promote the inflammatory response, assist in immune complex formation, and alter the plasma membrane of cells leading to cell death. Three major functions of complement are opsonization of bacteria/immune complexes, target cell lysis, and activation of phagocytosis. Three major pathways can activate the complement cascade: classical, lectin, and alternative. The classical pathway is activated by IgG or IgM bound to a specific target whereas the other two pathways are immunoglobulin independent. The lectin pathway uses PRRs and the alternative pathway uses factors such as the Fab area of immunoglobulin complexes (IgA, IgE, IgG).41

Formation of C3 convertase is a critical step in all pathways since it stimulates the formation of C3b (the opsonin component) and C4b, which bind to cell membranes. The final common pathway of the complement system is cell destruction by osmotic lysis, which is mediated by the formation of membrane attack complex (MAC) (factors C5– C9). C5a, the chemoattractant component, serves to recruit other inflammatory cells. In the eye, activation of the complement cascade must be controlled to focus on foreign targets and not on host cells. This process is regulated by complement-regulatory proteins (CRPs) (see Fig. 5.10). There are three principal locations of CRPs: fluid phase (e.g. C1-INH); cell membranes (decay accelerating factor (DAF), membrane cofactor protein (MCP), and CD59); and matrix (decorin).40 All cell membrane regulatory proteins are expressed differentially in the normal human cornea (see Fig. 5.10). C1-7 and factors B and P have also been identified in the cornea.

Tissue Components of the Ocular Immune System Mucosa-associated immune system (MALT) The eye-associated lymphoid tissue (EALT) consists of conjunctiva, lacrimal gland, and the lacrimal drainage system. EALT is part of the common mucosal secretory immune system of the body which consists of a distinct network of mucosa-associated lymphoid tissue (MALT) located in the gut (GALT), bronchus (BALT), and other sites throughout the body.45,46 Because mucosa-associated lymphocytes actually recirculate throughout the different sites, the various components actually compose a distinct lymphoid system.

The lacrimal functional unit (LFU) The lacrimal gland, tear film, ocular surface epithelium, eyelids, and the interconnecting innervation comprise a complex functional unit, which modulates the homeostasis of the ocular surface.44,46–48 The local immune pathways are determined by a wide variety of factors including the products of the lacrimal gland. In the normal lacrimal gland, the predominant lymphocytic cell type in the lymphocytic aggregates of the interstitium is the plasma cell (IgA and IgD), and in the interstitium away from the lymphoid aggregates, Tc cells.

The Cell-Mediated Immune (CMI) Response Major histocompatibility complex40 The major histocompatibility complex (MHC) gene, found on human chromosome 6p21.31, codes for cell membrane glycoproteins important in immune regulation. MHC is divided into three regions: class I, II, and III. Class I code for HLA-A, HLA-B, and HLA-C antigens found on all nucleated cells. Class I molecules present peptides from endogenous antigens to CD8+ T cells. Class II code for HLA-DP, HLADQ, and HLA-DR. These antigens are present on several important immunocompetent cells including monocytes,

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macrophages, DC, and B lymphocytes. These molecules present peptides from exogenous antigens to CD4+ T cells. Class II antigen expression may be stimulated by certain cytokines such as IFN-γ. Class II antigens help regulate the immune response through interactions between lymphocytes and APCs. The class III region contains genes that code for molecules of the complement system, inflammation, and other system functions.

Antigen presentation and T-cell activation40 Antigen processing is a complex sequence of events between a T lymphocyte and an APC that involves antigen recognition, antigen uptake, intracellular processing, and finally, presentation to resting Th lymphocytes. Activated Th cells then interact with and sensitize other cells to bring about immune responses. Although macrophages, monocytes, and DC are the most important APCs, other parenchymal cells may be stimulated by IFN-γ to acquire antigen-presenting capacity. The activated Th cell then stimulates the differentiation and clonal expansion of a variety of committed antigen-specific effector cells through the secretion of a variety of cytokines including IL-2 to IL-6, IFN-γ, and TNF-β. IL-2 stimulates antigen-responding cytotoxic T cells (Tc) to mediate direct tissue destruction and cells of delayed-type hypersensitivity to mediate DTH responses. IL-2, IL-4, and IL-5 also stimulate B cells to produce memory cells and antibody-producing plasma cells.

Cell-mediated immune response Cell-mediated immune (CMI) responses can be T-cell dependent or T-cell independent.40 T-cell-independent responses constitute the innate immune response and include phagocytosis by PMNs, complement-mediated cell destruction, and cytotoxic activity of NK cells and macrophages. T-celldependent responses are more complex and may involve different subsets of T cells. The mechanism by which specific pathways are selected is unknown (see Fig. 5.12). Th cells contribute to the differentiation and proliferation of effector cells and the final mechanism of target cell/antigen destruction/elimination. The main effector cell pathways take place via cytotoxic lymphocytes (Tc, NK, K), and via mast cells and eosinophils through antigen-specific IgE. In antibody- dependent cell-mediated cytotoxicity (ADCC), cytotoxic cells that possess the Fc receptor for IgG on their cell membrane mediate cell destruction through the release of cytotoxic cytokines (TNF-α, TNF-β, IFN-γ). Complement also may play a role in this mechanism. Finally, lymphokine-mediated macrophage activation also occurs through Tdth cells. If the CMI response fails to effectively eliminate the target cell, then the localization of T cells, immune complexes, macrophages, and PMNs may lead to chronic inflammation and granuloma formation.

The Humoral (Antibody-Mediated) Immune Response B-cell activation occurs when antigen binds antibodies located on its plasma membrane. After contact with antigen, Ig actively migrates within the cell membrane to form a

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“cap,” which is then either internalized or shed. B cells respond to specific antigenic stimulation, with help from T helper (Th) cells, by blastogenic transformation. This transformation is associated with increased protein and DNA synthesis, antibody synthesis, and finally differentiation into plasma and memory cells. Plasma cells are immunoglobulin “factories” that manufacture a specific antibody. Memory cells are previously sensitized cells that manufacture a specific antibody. They account for the more rapid and effective immune response following re-exposure to antigen.

Immunoglobulins Characteristics of immunoglobulins40 Antibodies are immunoglobulins (Ig) produced by B lymphocytes in response to antigenic stimulation. They play a key role, together with the TCRs, in providing the characteristic speci­ ficity of the adaptive immune response. Immunoglobulins are composed of four polypeptide chains linked together by disulfide bonds. Two of the chains are longer and are termed heavy (H) chains; the two shorter chains are termed light (L) chains. Similar amino acid sequences are termed constant (C) regions, while dissimilar sequences are variable (V). A small quite variable sequence, termed the hypervariable region, is associated with the antigen-binding portion of the immunoglobulin. Antibody-mediated immunity requires noncovalent contact between the antigen and antibody. There are five classes of immunoglobulins in humans: IgG, IgA, IgM, IgE, and IgD. IgG is the principal antibody of the secondary immune response, accounting for ~75% of the serum Ig. It fixes complement (IgG2a) and plays an important role in mediating inflammation and fighting infection through types II, III, and IV hypersensitivity reactions. It also functions in the cytotoxic arm of the immune response through ADCC by binding to Fc receptors on macrophages, NK cells, mast cells, and basophils. IgA is the next most common serum Ig accounting for 15–20% of circulating Ig molecules. IgA functions primarily in opsonization, neutralization of toxins, and agglutination. IgA can be differentiated as serum IgA or secretory IgA (sIgA). sIgA contains a secretory component, synthesized by lacrimal glandular epithelial cells, that protects the Ig from proteolysis by enzymes usually found on the mucosal surface. sIgA is the predominant Ig found in external secretions such as tears, saliva, milk, and secretions from the respiratory and digestive tracts. Therefore it participates in the peripheral surveillance system of MALT where there is frequent exposure to a wide variety of foreign antigens. IgM is the largest Ig and is composed of five Ig molecules. It constitutes 5–10% of the total serum Ig. IgM is the predominant Ig formed after initial exposure to antigen and plays a dominant role in agglutination, complement fixation, and cytolysis. Because of its size and structure, IgM has a high antigen-combining capacity and does not migrate across the placenta. IgE is an important mediator of anaphylactic responses and ocular allergy. The IgE molecule is fixed to mast cells and basophils through their Fc receptor. In an immune response to allergen, Th2 cells respond by releasing IL-4,

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea which promotes an isotype switching to IgE production (see Fig. 5.12). After binding with antigen, IgE mediates the type I hypersensitivity immune response characterized by histamine and vasoactive mediator release.

Anterior Chamber Associated Immune Deviation (ACAID) There are many physiological and regulatory phenomena which provide “immune privilege” to the anterior segment including anterior chamber associated immune deviation (ACAID).41 The blood–ocular barrier, located at the tight junctions of the ciliary epithelium of the ciliary body, physically provides a barrier to cellular infiltration. Soluble factors also inhibit a variety of immunological processes including: (1) T-cell proliferation; (2) IFN-γ production by Th1 cells; (3) proinflammatory factors secreted by macrophages; (4) NK cell activity; (5) DTH response; and (6) infiltrating cells by FasL. ACAID is an unusual systemic immune response. Following foreign antigen injection into the anterior chamber of the eye, a signal is produced that communicates with the immune system through the spleen. A series of events then occurs with the following important features: (1) an inhibition of systemic DTH; (2) an inhibition of a complementfixing antibody response; (3) the maintenance of a normal cytotoxic T-cell and humoral immune response; and (4) the capacity to adoptively transfer ACAID through antigen- specific splenic suppressor T cells, both CD4+ and CD8+, to immunologically naive recipients. As DTH and complement fixing antibodies would generate intense local immunogenic inflammation, the eye has developed a mechanism to reduce this type of immune response. Two cell populations are responsible for ACAID. The first are CD4+ cells that produce increased amounts of IL-10 and decreased amounts of IFN-γ. These Th1-type cells are termed “afferent suppressor cells.” These cells are required to generate a second population of CD8+ cells that inhibit the expression of DTH responses and are termed “efferent suppressor cells.”

Immune Hypersensitivity Reactions When the adaptive ocular immune response occurs in an excessive or inappropriate form and results in damage to ocular tissue, it is termed a hypersensitivity response. In 1968, Gell and Coombs described four classic types of hypersensitivity response.49 Many clinical entities probably result from a combination of mechanisms.

Type I hypersensitivity response (atopic, allergic reactions) After exposure to antigen, an APC presents antigen to Th2 causing the release of cytokines IL-4 and 5, which stimulate the (excessive) antigen-specific synthesis of IgE antibodies by B lymphocytes. IL-3 and IL-4 stimulate the proliferation of FceRI+ mucosal mast cells. Treg cells also play an important role in the response.50 After secondary exposure to

antigen, eosinophils and mast cells with antigen-specific IgE respond by bridging two immunoglobulin molecules. An aggregation of receptors in the membrane then causes a rapid membrane-coupled activation of adenylate cyclase, which leads to the increase in cyclic adenosine monophosphate (cAMP) and degranulation of preformed mediators of inflammation and allergy from storage granules. Newly synthesized mediators are also generated. Mast cells and basophils release a variety of mediators. Preformed mediators include amines (histamine or serotonin), proteoglycans (heparin or chondroitin sulfate), and many different neutral proteases, including aryl sulfatase. Newly formed mediators are usually produced following an IgE-mediated activation and include arachidonic acid metabolites, prostaglandins (PGD2), products of the cyclooxygenase (thromboxanes) and lipooxygenase pathways (leukotriene C4, D4, B4), and cytokines (TNF-α, IL-3 to IL-6, IL-10, IL-13, and VEGF). The release of vasoactive mediators results in the familiar clinical signs of chemosis, vascular injection, itching, and increases in local (tear) IgE levels.

Type II (cytotoxic) hypersensitivity response A type II hypersensitivity response results from complementfixing antibodies (IgG1, IgG3, or IgM), which bind to endogenous (acetylcholine receptor in myasthenia gravis) or exogenous (microbes) membrane antigens. Cell damage is mediated by several mechanisms: 1) through phagocytic effector cells (macrophages, neutrophils, eosinophils, NK cells) binding via their Fc receptor and releasing enzymes (proteolytic and collagenolytic) and in turn, damaging “bystander” tissue, when the target tissue is too large to be engulfed by the phagocyte; 2) through antibody-dependent cell cytoxicity (ADCC) where NK cells cause direct target cell damage through nonspecific binding of antibody to their Fc receptor and release of proteolytic enzymes; and 3) through the activation of complement by antibodies resulting in the deposition of the C5b-9 MAC. C3b can also bind to target cells and mediate membrane damage via the C3b receptor on phagocytic cells. (Table 5.1).

Type III hypersensitivity response (immune complex) In the type III hypersensitivity response, soluble antigen– antibody complexes bind complement and either become deposited into blood vessels or the antigen combines with the antibody in the extracellular space. PMNs and phagocytes are attracted into the tissue and directly or indirectly destroy it. The same complement-fixing antibodies in the type II response (IgG and IgM) participate. Antigen–antibody complexes are normally eliminated through the mononuclear phagocyte system (larger complexes). This system may become overloaded, resulting in the deposition of complexes into tissues, typically with intermediate-sized complexes. Persistent antigen exposure to specific body sites may generate a systemic circulating antibody response with local deposition. Increase in vascular permeability, either through vasoactive amine release or previous damage to the endothelium, is necessary for complex deposition.

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Type IV (delayed-type hypersensitivity [DTH]) response Type IV reactions are mediated by macrophages and antigenspecific T lymphocytes. They are unique in that they are reactions to fixed, rather than soluble, antigens. These include infectious agents, tumors, and foreign grafts. Antigen is presented to T cells by an APC, which then migrates to lymphoid tissue where it presents to resting T lymphocytes. Once activated, these antigen-specific sensitized cells respond by direct cytotoxic attack or through the release of cytokines leading to macrophage chemotaxis and activation. It requires about 48 hours to elicit a maximum response through antigen-specific T cells. Tc and Tdth cells directly attack the target cell. Macrophages also participate in the elimination of the antigen or organism. Three types of type IV hypersensitivity responses are currently recognized: contact hypersensitivity, tuberculin-type hypersensitivity, and granulomatous hypersensitivity. Corneal allograft rejection results from this process as well.

References 1. Waring GO 3rd, Rodrigues MM. Patterns of pathologic response in the cornea. Surv Ophthalmol 1987;31:262–6. 2. Leibowitz HM, Waring GO III. Corneal disorders. Clinical diagnosis and management. 2nd ed. Philadelphia: WB, Saunders Company; 1998. p. 154–200. 3. Ambrósio R Jr, Kara-José N, Wilson SE. Early keratocyte apoptosis after epithelial scrape injury in the human cornea. Exp Eye Res 2009;89: 597–9. 4. Lagali NS, Germundsson J, Fagerholm P. The role of Bowman’s layer in anterior corneal regeneration after phototherapeutic keratectomy: a prospective, morphological study using in-vivo confocal microscopy. Invest Ophthalmol Vis Sci 2009;50:4192–8. 5. Fraunfelder FW. Corneal toxicity from topical ocular and systemic medications. Cornea 2006;5:1133–8. 6. Reinstein DZ, Archer TJ, Gobbe M. Corneal epithelial thickness profile in the diagnosis of keratoconus. J Refract Surg 2009;25:604–10. 7. Hamam R, Bhat P, Foster CS. Conjunctival/corneal intraepithelial neoplasia. Int Ophthalmol Clin 2009;49:63–70. 8. Dawson DG, Grossniklaus HE, McCarey BE, et al. Biomechanical and wound healing characteristics of corneas after excimer laser keratorefractive surgery: is there a difference between advanced surface ablation and sub-Bowman’s keratomileusis? J Refract Surg 2008;24:S90–6. 9. Itty S, Hamilton SS, Baratz KH, et al. Outcomes of epithelial debridement for anterior basement membrane dystrophy. Am J Ophthalmol 2007;144: 217–21. 10. Hsu JK, Johnston WT, Read RW, et al. Histopathology of corneal melting associated with diclofenac use after refractive surgery. J Cataract Refract Surg 2003;29:250–6. 11. Kremer I, Kaplan A, Novikov I, et al. Patterns of late corneal scarring after photorefractive keratectomy in high and severe myopia. Ophthalmology 1999;106:467–73. 12. Meltendorf C, Burbach GJ, Ohrloff C, et al. Intrastromal keratotomy with femtosecond laser avoids profibrotic TGF-β1 induction. Invest Ophthalmol Vis Sci 2009;50:3688–95. 13. Lee P, Wang CC, Adamis AP. Ocular neovascularization: an epidemiologic review. Surv Ophthalmol 1998;43:245–69. 14. Levenson JE. Corneal edema: cause and treatment. Surv Ophthalmol 1975;20:190–204. 15. Nordlund ML, Grimm S, Lane S, et al. Pressure-induced interface keratitis: a late complication following LASIK. Cornea 2004;23(3): 225–34. 16. Singh AD, Puri P, Amos RS. Deposition of gold in ocular structures, although known, is rare. A case of ocular chrysiasis in a patient of rheumatoid arthritis on gold treatment is presented. Eye (Lond) 2004;18: 443–4. 17. Barchiesi BJ, Eckel RH, Ellis PP. The cornea and disorders of lipid metabolism. Surv Ophthalmol 1991;36:1–22.

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18. Fernandez AB, Keyes MJ, Pencina M, et al. Relation of corneal arcus to cardiovascular disease (from the Framingham Heart Study data set). Am J Cardiol 2008;103:64–6. 19. Nakatsukasa M, Sotozono C, Tanioka H, et al. Diagnosis of multiple myeloma in a patient with atypical corneal findings. Cornea 2008;27: 249–51. 20. Waring GO 3rd, Rodrigues MM, Laibson PR. Corneal dystrophies: I. Dystrophies of the epithelium, Bowman’s layer and stroma. Surv Ophthalmol 1978;23:71–122. 21. Sbarbaro JA, Eagle RC, Thumma P, et al. Histopathology of posterior lamellar endothelial keratoplasty graft failure. Cornea 2008;27:900–4. 22. Stacy RC, Jakobiec FA, Michaud NA, et al. Characterization of retrokeratoprosthetic membranes in the Boston type 1 keratoprosthesis. Arch Ophthalmol 2011;129(3):310–16. 23. Waring GO 3rd, Bourne WM, Edelhauser HF, et al. The corneal endothelium. Normal and pathologic structure and function. Ophthalmology 1982;89:531–90. 24. Mehta JS, Por YM, Poh R, et al. Comparison of donor insertion techniques for Descemet stripping automated endothelial keratoplasty. Arch Ophthalmol 2008;126:1383–8. 25. Afshari NA, Pittard AB, Siddiqui A, et al. Clinical study of Fuchs’ corneal endothelial dystrophy leading to penetrating keratoplasty: a 30-year experience. Arch Ophthalmol 2006;124:777–80. 26. Waring GO 3rd. The 50-year epidemic of pseudophakic corneal edema. Arch Ophthalmol 1989;107:657–9. 27. Patel SV, Hodge DO, Bourne WM. Corneal endothelium and postoperative outcomes 15 years after penetrating keratoplasty. Trans Am Ophthalmol Soc 2004;102:57–65, discussion 65–66. 28. Price MO, Gorovoy M, Price FW Jr, et al. Descemet’s stripping automated endothelial keratoplasty: three-year graft and endothelial cell survival compared with penetrating keratoplasty. Ophthalmology 2013;120(2): 246–51. 29. Gorovoy IR, Gorovoy MS. Descemet membrane endothelial keratoplasty postoperative year 1 endothelial cell counts. Am J Ophthalmol 2015;159(3): 597–600. 30. Waring GO, Laibson PR, Rodrigues MM. Clinical and pathologic alterations of Descemet’s membrane: with emphasis on endothelial metaplasia. Surv Ophthalmol 1974;18:325–68. 31. Waring GO. Posterior collagenous layer of the cornea: ultrastructural classification of abnormal collagenous tissue posterior to Descemet’s membrane in 30 cases. Arch Ophthalmol 1982;100:122–34. 32. Dogru M, Kato N, Matsumoto Y, et al. Immunohistochemistry and electron microscopy of retrocorneal scrolls in syphilitic interstitial keratitis. Curr Eye Res 2007;32:863–70. 33. Alvarado JA, Murphy CG, Maglia M, et al. Pathogenesis of Chandler’s syndrome, essential iris atrophy and the Cogan−Reese syndrome. II: Estimated age at disease onset. Invest Ophthalmol Vis Sci 1986;27: 873–82. 34. Krachmer JH, Schnitzer JI, Fratkin J. Cornea pseudogutta: a clinical and histopathologic description of endothelial cell edema. Arch Ophthalmol 1981;99:1377–81. 35. Mazzotta C, Baiocchi S, Caporossi O, et al. Confocal microscopy identification of keratoconus associated with posterior polymorphous corneal dystrophy. J Cataract Refract Surg 2008;34:318–21. 36. Olsen TW, Hardten DR, Meiusi RS, et al. Linear endotheliitis. Am J Ophthalmol 1994;117:468–74. 37. Vemuganti GK, Garg P, Gopinathan U, et al. Evaluation of agent and host factors in progression of mycotic keratitis: a histologic and microbiologic study of 167 corneal buttons. Ophthalmology 2002;109: 1538–46. 38. Santos LN, Fernandes BF, de Moura LR, et al. Histopathologic study of corneal stromal dystrophies: a 10-year experience. Cornea 2007;26: 1027–31. 39. Jirsova K, Merjava S, Martincova R, et al. Immunohistochemical characterization of cytokeratins in the abnormal corneal endothelium of posterior polymorphous corneal dystrophy patients. Exp Eye Res 2007;84: 680–6. 40. Delves PJ, Martin SJ, Burton DR, et al., editors. Roitt’s essential immunology. 11th ed. New York: Wiley-Blackwell; 2006. 41. Niederkorn JY, Kaplan HJ, editors. Immune response and the eye. 2nd. revised edition. Basel: Karger AG; 2007. 42. Zierhut M, Rammensee H-G, Streilein JW, editors. Antigen-presenting cells and the eye. New York: Informa Health Care; 2007. 43. Hendricks R. Interaction of angiogenic and immune mechanisms in the eye. Semin Ophthalmol 2006;21:37–40. 44. Pflugfelder SC, Beuerman RW, Stern ME, editors. Dry eye and ocular surface disorders. New York: Informa Health Care; 2004. 45. Knop E, Knop N. The role of eye-associated lymphoid tissue in corneal immune protection. J Anat 2005;206(3):271–85.

CHAPTER 5 A Matrix of Pathologic Responses in the Cornea 46. Steven P, Gebert A. Conjunctiva-associated lymphoid tissue – current knowledge, animal models and experimental prospects. Ophthalmic Res 2009;42:2–8. 47. Stern ME, Gao J, Siemasko KF, et al. The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res 2004;78(3): 409–16.

48. Zierhut M, Stern ME, Sullivan DA, editors. Immunology of the lacrimal gland, tear film and ocular surface. New York: Informa Health Care; 2005. 49. Gell PGH, Coombs RRA, editors. Clinical aspects of immunology. Oxford: Blackwell; 1968. 50. Palomares O, Yaman G, Azkur AK, et al. Role of Treg in immune regulation of allergic diseases. Eur J Immunol 2010;40(5):1232–40.

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Section 1

Basic Evaluation of the Cornea and External Eye

Chapter 6  Examination of the Lids Robert J. Peralta, Hall T. McGee

Key Concepts • • • • • •

An anatomically and physiologically normal eyelid is vital in maintaining the health of the eye. Meticulous examination of the tear film yields valuable information in the diagnosis and treatment of dry eye. Anterior eyelid examination may reveal trichiasis, an often frustrating malady that may result in severe symptoms, chronic inflammation, and corneal scarring. Examination of the posterior eyelid may reveal significant meibomian gland dysfunction (MGD) which can alter the mucocutaneous junction. Meibomian gland expression is an important part of the eyelid examination and is helpful in distinguishing seborrheic from obstructive MGD. Meibography provides information on meibomian gland structure and may be a valuable clinical tool in the treatment of MGD.

General Principles The health of the ocular surface is dependent on an ade­ quately positioned and properly functioning eyelid. A sys­ tematic approach such as that outlined in Box 6.1 is helpful but should be modified as necessary.

History of the Patient Symptoms of eyelid disease may be vague and nonspecific. While history of disease onset, duration, severity, exacerba­ tion, localization, and previous treatments is being obtained, it is helpful to observe unconscious behaviors such as eye rubbing, scratching, or wiping away excess tears. These behaviors may be more indicative of the true malady, espe­ cially if the patient is having difficulty verbalizing a chief complaint. Furthermore, important observations on orbicu­ laris oculi function can be made by observing the strength and rate of blinking.

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Dermatologic Examination Examination of the facial and adnexal skin is best done with fairly bright, diffuse, indirect light. Dark exam rooms and harsh lighting may distort the color and translucency of tissues. Many patients are unaware of dermatologic condi­ tions that may be clearly apparent to clinicians, thus clinical photos are helpful educational tools. Many conditions affect the periorbital skin—focal or diffuse, local or systemic, congenital, infectious, inflamma­ tory, and neoplastic. The full breadth of the discussion is beyond the scope of this chapter, but several entities are discussed below. Contact dermatitis of the eyelids is quite common and is associated with other ocular allergies.1 The periocular skin may be erythematous, edematous, and scaly. A history of lotions, creams, topical medications, or other exacerbants should be sought. Atopic dermatitis can result in keratocon­ junctivitis and may also present with thickened, scaly, ery­ thematous, and fissured periocular skin. Patients may be aware of lesions elsewhere on their body, but may not have associated their dermatitis with their eye condition. Rosacea is a common dermatologic condition that affects up to 10% of the population and most commonly in those of northern European origin. Rosacea dermatitis is character­ ized by malar flushing, telangiectasias, papules, pustules, sebaceous gland hypertrophy, and rhinophyma. Bacterial infections may be focal (hordeola, chalazia), diffuse (preseptal cellulitis), or potentially fatal (orbital cel­ lulitis). Cutaneous malignancies are commonly seen on the periorbital skin, accounting for upwards of 18% of all eyelid lesions.2 Basal cell carcinoma is by far the most frequent (86%), followed by squamous cell carcinoma (7%) and seba­ ceous carcinoma (3%).2

Eyelid Position Alteration of eyelid position and function can lead to expo­ sure keratopathy, which may go unnoticed by patients.3 Evaluation of eyelid position starts with the measurement of margin reflex distance (MRD). MRD1 is the distance from the central light reflex, congruent with the visual axis, to the upper eyelid margin. Conversely, MRD2 is the distance from

CHAPTER 6 Examination of the Lids

Chapter Outline General Principles History of the Patient Dermatologic Examination Eyelid Position Tear Meniscus and Puncta Anterior Eyelid Posterior Eyelid Meibomian Gland Expression Mucocutaneous Junction Meibomian Gland Imagery

72.e1

CHAPTER 6 Examination of the Lids Margin Reflex Distance (MRD)

Box 6.1 A recommended order for examination of the eyelids  

• Take history and observe patient’s unconscious behavior and habits. • Examine face and eyelids in ambient lighting. • Examine tear meniscus and puncta with slit lamp prior to administration of drops or dyes.

Normal MRD1 = 4 mm MRD2 = 5 mm Palpebral fissure = 9

• Examine anterior and posterior eyelid. • Express the meibomian glands.

A

• Re-examine mechanical properties of the lids. • Instill dye (typically fluorescein, also lissamine green or rose Bengal). • Use slit lamp again to identify the mucocutaneous junction and its position relative to the meibomian gland orifices. • Consider imaging studies as appropriate (typically for research purposes).

the central light reflex to the lower eyelid margin (Fig. 6.1). Together, they comprise the interpalpebral fissure (IPF) height. Measurement of MRD along with IPF provides a more accurate clinical picture than measurement of IPF alone (Fig. 6.1). Eyes with a larger IPF have a greater surface area. Because tear evaporation rate is correlated to surface area, patients with a larger IPF are more susceptible to dry eye symptoms. Both eyelid malposition and decreased force of contrac­ ture contribute to lagophthalmos. Forceful lid closure on exam may mask subtle degrees of incomplete lid closure. In these instances, it may be helpful to wait for one minute with the lids closed to mitigate forced lid closure. Normal eyelid position is dependent on appropriate hori­ zontal and vertical tension mediated by the lateral canthal tendon and lower eyelid retractors, respectively. Pulling the lid directly away from the ocular surface tests displacement, while pulling the lid inferiorly tests the ability of the lid to “snap back” into position. Although most abnormalities of lid tension are due to increased laxity, occasionally abnor­ malities due to increased tension are seen, such as superior limbic keratoconjunctivitis. Increased horizontal laxity predisposes the lid to involu­ tional ectropion (Fig. 6.2). Concomitant vertical laxity (via dehiscence of the lower lid retractors) predisposes the lid to involutional entropion (Fig. 6.3) as contraction of the pre­ tarsal orbicularis oculi fibers forces the lower lid margin inward. In the absence of horizontal laxity, spastic entropion can occur due to vigorous contraction of the pretarsal orbi­ cularis. The examination for ectropion and entropion is important as symptoms are often nonspecific.4 Ectropion may present insidiously with tearing, redness, irritation, tear film abnormalities, dry eye, and conjunctival keratiniza­ tion. Entropion may present more acutely with pain, foreign body sensation and photophobia due to ocular surface contact. Floppy eyelid syndrome (FES) results in excessive eyelid elasticity and usually presents with mucous discharge,

Upper lid ptosis MRD1 = 2 mm MRD2 = 5 mm Palpebral fissure = 7 B

Upper lid retraction MRD1 = 7 mm MRD2 = 5 mm Palpebral fissure = 12 C

Upper lid ptosis and lower lid retraction MRD1 = 1 mm MRD2 = 8 mm Palpebral fissure = 9

D

*Note palpebral aperture measurement is the same for examples A and D. Fig. 6.1  Measurement of margin reflex distance (MRD1 and MRD2) along with the interpalpebral fissure (IPF) provides a much clearer view of the clinical picture. Note how IPF is equal in examples A and D. Whereas A is “normal,” the eye depicted in D presents with upper lid ptosis and lower lid retraction. (Courtesy of Jeffrey A. Nerad MD. From Nerad JA, Techniques in Ophthalmic Plastic Surgery, 2010, Elsevier Inc. Page 31. Figure 2.5.)

chronic irritation, papillary conjunctivitis, and keratopathy. Symptoms may be worse in the morning and patients may not be cognizant of any associated eyelid disease.5–8 FES patients are commonly obese and frequently report snoring or sleep apnea. Histological studies have demonstrated decreased tarsal elastin.10,11 Markedly increased upper and lower eyelid laxity may be seen. Examination involves placing both thumbs on the superotemporal orbital rims and drawing the upper eyelid superotemporally. FES is diagnosed when the lid stretches excessively, often to the superior orbital rim, and the tarsal plate everts, exposing the palpebral conjunctiva. Treatment is aimed at correcting lid laxity and excising redundant tissue.

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Section 1

Basic Evaluation of the Cornea and External Eye

Fig. 6.2  Involutional ectropion of the lower eyelid due to increased horizontal lid laxity. Patients may present with tearing, redness, irritation, tear film abnormalities, and dry eye.

Fig. 6.4  Trichiasis from marginal entropion can be seen in blepharitis as well as other scarring diseases of the conjunctiva (mucous membrane pemphigoid, Stevens–Johnson syndrome, drug induced cicatrizing conjunctivitis, chemical or thermal injury).

Punctal position and patency are important for normal tear drainage. Punctal ectropion, even with a well-positioned central eyelid, prevents access to the nasolacrimal system and can lead to epiphora. Puncta may be scarred from a variety of conjunctival diseases (pemphigoid, chemical injury, blepharitis) or as a treatment for dry eye.18,19

Anterior Eyelid

Fig. 6.3  Involutional entropion of the lower eyelid due to increased horizontal and vertical lid laxity. Symptoms of pain, foreign body sensation, and photophobia are typically more acute than those seen in ectropion.

Tear Meniscus and Puncta The slit lamp exam should begin with the lamp off and just enough ambient light to measure the the tear meniscus. Manipulation of the eyelids should be avoided. Slit lamp illumination can then be turned on to assess reflex tearing. Patients with a small tear meniscus who are unable to gener­ ate reflex tears are much more likely to have difficulty with dry eye.9,12,15–17 Foamy tears generally indicate meibomian gland dysfunction (MGD). An ocular surface interferometer (LipiView, Tearscience Inc.) has been developed to quantify the tear film lipid layer thickness. Studies suggest that decreased lipid layer thickness may correlate with obstruc­ tive MGD.13,14

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The anterior lamella comprises the skin and orbicularis oculi muscle. The eyelid should first be examined in ambient light with attention to color, transparency, induration, and other general characteristics. Lesions suspicious for a cutaneous malignancy should be examined with the biomicroscope. Potentially malignant characteristics such as a nodular pearly consistency, ulceration, induration, irregular borders, suspicious telangiectasias, madarosis, and loss of lid archi­ tecture are more easily seen with magnification. Examination of the lashes is most readily performed with the biomicroscope. Length, number, and absence of lashes should be noted. Particular attention should be paid to the presence of trichiasis—posteriorly misdirected lashes (Fig. 6.4). This is commonly due to conditions that cause poste­ rior lamella shortening, such as blepharitis, mucous mem­ brane pemphigoid, Stevens–Johnson syndrome, chemical burns, and drug-induced cicatrizing conjunctivitis. Trauma is also a common cause, since soft tissue scarring distorts eyelash orientation. Less commonly this is due to epiblepha­ ron (Fig. 6.5), a congenital condition in which redundant skin and muscle override the lid margin and force the eye­ lashes against the eye. Rarely is this due to distichiasis— growth of lashes from the meibomian gland orifices. Trichiasis may be a difficult problem that can lead to severe keratopathy, inflammation, and corneal scarring.20

CHAPTER 6 Examination of the Lids

Fig. 6.5  Epiblepharon may cause trichiasis as an extra roll of skin and muscle overrides the lid margin and pushes the lashes towards the ocular surface.

Fig. 6.6  Vascularization and hypertrophy along the lid margin alters the normal contours and obscures landmarks.

The lashes should also be examined for signs of inflam­ mation, infestation, or infection. Collarettes are mucous debris and desquamated epithelium adherent to the lash base and are a nonspecific sign of inflammation. Phthirus pubis are easily seen, whereas Demodex mites are smaller and more difficult to identify.21–24 Infectious processes may occur and are usually evident by swelling and pus at the lash base. Such hordeola of the lash follicles may be associated with a more generalized bacterial infection the eyelid.25,26

Posterior Eyelid The posterior lamella comprises the tarsus and conjunctiva. An uninflamed eyelid has a square edge and fine capillaries.27 Inflammatory and infectious stimuli may induce rounding of the posterior lid margin.27 Atrophy of the lid margin may result in hypervascularity as deeper vessels become visible. Although relatively nonspecific, these changes are often associated with obstructive MGD, rosacea, and infections (Fig. 6.6). Chalazia are indicative of obstructive MGD and

Fig. 6.7  Lipid expressed with digital pressure on an eyelid with seborrheic meibomian gland dysfunction reveals semitransparent liquid of increased volume.

commonly cause lid scarring. Resolution of chalazia may result in notching and trichiasis. Allergic processes may cause thickening of the conjunc­ tiva and chronic changes to the lid margin. In severe cases, deep furrows develop in the skin and conjunctiva and may become secondarily infected or ulcerated.28 The openings of the meibomian glands should be inspected carefully for signs of chronic disease. Periglandular atrophy renders the glands more evident as the lid margin recedes around the keratinized duct.29 Hyperkeratinization of the ductal epithelium may partially or completely occlude the meibomian gland orifices.30–32 Partial occlusion from hyperkeratinization may augment obstruction from dry and hardened inflammatory debris and exacerbate obstruc­ tive MGD. Chronic aging changes also occur and are exac­ erbated by the effects of long-term obstructive MGD and dry eye.33

Meibomian Gland Expression Meibomian gland expression is an important part of the lid examination.33,34 With the patient in upgaze, firm sustained pressure (via a finger or cotton tipped applicator) is applied to the lower eyelid inferior to the lid margin until meibo­ mian gland excreta is seen. Approximately 20–25 meibo­ mian glands are typically present in the lower lid; two or three can be compressed at one time. The entire lid margin should be examined and the volume and viscosity of the excreta noted. The volume can be recorded as the diameter of the lipid dome that forms after several seconds of pressure. Normal diameter is 0.5–0.7 mm. Diameters of 0.8 mm or larger are associated with increased lipid volume and are diagnostic of seborrheic MGD (Fig. 6.7). Decreased lipid volumes or inex­ pressible glands are associated with obstructive MGD. Mei­ bomian gland lipid production may also be measured by evaluating the area of increased transparency on a paper

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Examining and Imaging the Cornea and External Eye

Section 1

Basic Evaluation of the Cornea and External Eye

strip placed against the meibomian orifices, a technique termed “meibometry.”35,36 The viscosity and opacity of the expressed meibomian lipid are important signs of eyelid disease. Normal lipid flows easily and remains transparent at body temperature. Sebor­ rheic MGD is associated with increased lipid opacity. Obstruc­ tive MGD demonstrates increased lipid viscosity and opacity. The most viscous lipid will emerge slowly like toothpaste, and will be totally opaque with a white or light yellow tint (Fig. 6.8).33 Although typically associated with obstructive MGD, this may also be seen in rosacea.34 The differences in the consistency of meibomian excreta have been attributed to variations in lipid composition.37

In cases of infection, meibomian glands may be tender and may yield pus on expression. This may be difficult to distinguish from staphylococcal blepharitis.38 Although Staphylococcus and Streptococcus organisms are typically responsible, there is evidence that different strains of bacte­ ria may be involved.39 Culturing the eyelid for antibiotic sensitivity may be helpful, but because of the ubiquity of these organisms, the clinical significance is equivocal.39–41 The relative contribution of bacterial overgrowth, infection, bacterial toxins, and abnormal immune responses towards the development of blepharitis and meibomian gland dys­ function is a subject of controversy.42,43 In practice, this dis­ tinction is moot as current regimens employ strategies that reduce both infection and inflammation.42,44 It is important, however, to recognize the presence of meibomian gland disease to direct appropriate treatment.

Mucocutaneous Junction The mucocutaneous junction is the confluence of the kera­ tinized squamous epithelium of the skin and the nonkera­ tinized squamous epithelium of the conjunctiva. Normally just posterior to the meibomian gland orifices, visualization is aided by mucosal staining with lissamine green, rose Bengal or fluorescein (the Marx line).27,45,46 Anterior displace­ ment of the mucocutaneous junction relative to the meibo­ mian gland orifices may correlate with MGD, although this has been debated.27,45

Meibomian Gland Imagery Meibography is a noninvasive in vivo study of the gross and microscopic structure of meibomian glands that provides valuable adjunctive information in the evaluation and treat­ ment of MGD. Studies on infrared (IR) photography of the meibomian glands date back to the late 1970s.47 In 1994, Mathers et al. introduced video IR meibography with resolu­ tion approximately equal to that of IR film.47,48 Contact meibography involves direct application of a light probe for eversion and transillumination of the eyelid (Fig. 6.9). Less invasive noncontact meibography techniques

Fig. 6.8  Meibomian gland expression from a lid with obstructive meibomian dysfunction showing thickened and opaque lipid (toothpaste).

A

B

Fig. 6.9  Meibomian gland imagery. (A) Transillumination of a normal eyelid showing evenly spaced glands. (B) An infrared image with transillumination of the lower lid showing loss of glands.

76

CHAPTER 6 Examination of the Lids have been developed involving biomicroscope-mounted and hand held devices.49,50 Both techniques most commonly utilize IR meibography. Newer technologies (laser confocal microscopy [LCM] and optical coherence tomography [OCT]) provide valuable structural and volumetric information previously only avail­ able via ex vivo studies. In obstructive MGD, IR meibogra­ phy demonstrates gland enlargement, duct dilation and gland dropout. In addition, LCM meibography demonstrates increased acinar unit diameter, decreased acinar unit density, periglandular inflammation, and fibrosis.51 OCT meibogra­ phy also provides volumetric information but is still under development. Meibography has great potential as a diagnostic tool but is limited by the lack of a widely accepted standardized grading system.52 The meiboscore and meibograde methods are two promising candidates. In the meiboscore method, meibographs of the upper and lower eyelid are quantified by the degree of glandular dropout. A score of 0 is given to a lid with no missing glands. Scores of 1 to 3 are assigned based on the relative area of gland loss: (1) for 66%. The scores are summed by lateral­ ity for a total score of 0 to 6 per eye. Although methodical, this fails to account for changes in gland architecture that may precede dropout. These “pre-dropout” stages are better incorporated in the meibograde method. In this method, meibographs are assessed for gland distortion, shortening and dropout on a scale of 0 to 3 also based on the area involved (similar to the meiboscore method), resulting in a total score of 0 to 18 per eye.53

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

References 1. Fonacier L, Luchs J, Udell I. Ocular allergies. Curr Allergy Asthma Rep 2001;1(4):389–96. 2. Deperez M, Uffer S. Clinicopathologic features of eyelid skin tumors. A retrospective study of 5504 cases and review of literature. Am J Dermatopath 2009;31(3):256–62. 3. Cosar CB, Cohen EJ, Rapuano CJ, et al. Tarsorrhaphy: clinical experience from a cornea practice. Cornea 2001;20(8):787–91. 4. Vallabhanath P, Carter SR. Ectropion and entropion. Curr Opin Ophthalmol 2000;11(5):345–51. 5. Madjlessi F, Kluppel M, Sundmacher R. [Operation of the floppy eyelid. Symptomatic cases require surgical eyelid stabilization]. Klin Monatsblatt Augenheilkde 2000;216(3):148–51. 6. Culbertson WW, Tseng SC. Corneal disorders in floppy eyelid syndrome. Cornea 1994;13(1):33–42. 7. van den Bosch WA, Lemij HG. The lax eyelid syndrome. Br J Ophthalmol 1994;78(9):666–70. 8. Boulton JE, Sullivan TJ. Floppy eyelid syndrome and mental retardation. Ophthalmology 2000;107(11):1989–91. 9. Doughty MJ, Laiquzzaman M, Button NF. Video-assessment of tear meniscus height in elderly Caucasians and its relationship to the exposed ocular surface. Curr Eye Res 2001;22(6):420–6. 10. Schlötzer-Schrerhardt U, Stojkovic M, Hofmann-Rummelt C, et al. The pathogenesis of floppy eyelid syndrome: involvement of matrix metal­ loproteinases in elastic fiber degradation. Ophthalmology 2005;112(4): 694–794. 11. Netland PA, Sugrue SP, Albert DM, et al. Histopathologic features of the floppy eyelid syndrome. Involvement of tarsal elastin. Ophthalmology 1994;101(1):174–81. 12. Yaylali V, Ozyurt C. Comparison of tear function tests and impression cytology with the ocular findings in acne rosacea. Eur J Ophthalmol 2002;12(1):11–17. 13. Finis D, Pischel N, Schrader S, et al. Evaluation of lipid layer thickness measurement of the tear film as a diagnostic tool for Meibomian gland dysfunction. Cornea 2013;32(12):1549–53. 14. Eom Y, Lee JS, Kang SY, et al. Correlation between quantitative measure­ ments of tear film lipid layer thickness and meibomian gland loss in

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patients with obstructive meibomian gland dysfunction and normal con­ trols. Am J Ophthalmol 2013;155(6):1104–10. Tomlinson A, Blades KJ, Pearce EI. What does the phenol red thread test actually measure? Optom Vis Sci 2001;78(3):142–6. Tsubota K, Kaido M, Yagi Y, et al. Diseases associated with ocular surface abnormalities: the importance of reflex tearing. Br J Ophthalmol 1999; 83(1):89–91. Yokoi N, Kinoshita S, Bron AJ, et al. Tear meniscus changes during cotton thread and Schirmer testing. Invest Ophthalmol Vis Sci 2000;41(12): 3748–53. McNab AA. Lacrimal canalicular obstruction associated with topical ocular medication. Aust NZ J Ophthalmol 1998;26(3):219–23. Sakol PJ. Tearing: lacrimal obstructions [Review]. Pa Med 1996;99(Suppl.): 99–104. Lehman SS. Long-term ocular complication of Stevens–Johnson syn­ drome. Clin Pediatr 1999;38(7):425–7. Key JE. A comparative study of eyelid cleaning regimens in chronic blepharitis. CLAO J 1996;22(3):209–12. Demmler M, de Kaspar HM, Mohring C, et al. Blepharitis. Demodex folliculorum-associated pathogen spectrum and specific therapy. Ophthalmologe 1997;94(3):191–6. Junk AK, Lukacs A, Kampik A. Topical administration of metronidazole gel as an effective therapy alternative in chronic Demodex blepharitis – a case report. Klin Monatsblatt Augenheilkd 1998;213(1):48–50. Burkhart CN, Burkhart CG. Oral ivermectin therapy for phthiriasis pal­ pebrum. Arch Ophthalmol 2000;118(1):134–5. Kiratli HK, Akar Y. Multiple recurrent hordeola associated with selective IgM deficiency. J AAPOS 2001;5(1):60–1. Lederman C, Miller M. Hordeola and chalazia. Pediatr Rev 1999;20(8): 283–4. Hykin PG, Bron AJ. Age-related morphological changes in lid margin and meibomian gland anatomy. Cornea 1992;11(4):334–42. Inoue Y. Ocular infections in patients with atopic dermatitis. Int Ophthalmol Clin 2002;42(1):55–69. Bron AJ, Benjamin L, Snibson GR. Meibomian gland disease. Classifica­ tion and grading of lid changes. Eye 1991;5(Pt 4):395–411. Jester JV, Rife L, Nii D, et al. In vivo biomicroscopy and photography of meibomian glands in a rabbit model of meibomian gland dysfunction. Invest Ophthalmol Vis Sci 1982;22(5):660–7. Robin JB, Jester JV, Nobe J, et al. In vivo transillumination biomicroscopy and photography of meibomian gland dysfunction. A clinical study. Ophthalmology 1985;92(10):1423–6. Jester JV, Rajagopalan S, Rodrigues M. Meibomian gland changes in the rhino (hrrhhrrh) mouse. Invest Ophthalmol Vis Sci 1988;29(7): 1190–4. Mathers WD, Shields WJ, Sachdev MS, et al. Meibomian gland dysfunc­ tion in chronic blepharitis. Cornea 1991;10(4):277–85. Mathers WD, Lane JA, Sutphin JE, et al. Model for ocular tear film func­ tion. Cornea 1996;15(2):110–19. Chew CK, Jansweijer C, Tiffany JM, et al. An instrument for quantifying meibomian lipid on the lid margin: the Meibometer. Curr Eye Res 1993;12(3):247–54. Chew CK, Hykin PG, Jansweijer C, et al. The casual level of meibomian lipids in humans. Curr Eye Res 1993;12(3):255–9. Shine WE, McCulley JP. Association of meibum oleic acid with meibo­ mian seborrhea. Cornea 2000;19(1):72–4. Groden LR, Murphy B, Rodnite J, et al. Lid flora in blepharitis. Cornea 1991;10(1):50–3. Dougherty JM, McCulley JP. Bacterial lipases and chronic blepharitis. Invest Ophthalmol Vis Sci 1986;27(4):486–91. Dougherty JM, McCulley JP. Comparative bacteriology of chronic blepha­ ritis. Br J Ophthalmol 1984;68(8):524–8. McCulley JP, Dougherty JM, Deneau DG. Classification of chronic bleph­ aritis. Ophthalmology 1982;89(10):1173–80. Pflugfelder SC, Karpecki PM, Perez VL. Treatment of blepharitis: most recent clinical trials. Ocul Surf 2014;12(4):273–84. Jackson WB. Blepharitis: current strategies for diagnosis and manage­ ment. Can J Ophthalmol 2008;43(2):170–9. Dougherty JM, McCulley JP, Silvany RE, et al. The role of tetracycline in chronic blepharitis. Inhibition of lipase production in staphylococci. Invest Ophthalmol Vis Sci 1991;32(11):2970–5. Yamaguchi M, Kutsuna M, Uno T, et al. Marx line: fluorescein staining line on the inner lid as indicator of meibomian gland function. Am J Ophthalmol 2006;141(4):669–75. Bron AJ, Yokoi N, Gaffney EA, et al. A solute gradient in the tear meniscus: I. A hypothesis to explain Marx’s line. Ocul Surf 2011;9(2): 70–91. Wise RJ, Sobel RK, Allen RC. Meibography: A review of techniques and technologies. Saudi J Ophthalmol 2012;26(4):349–56.

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48. Mathers WD, Daley T, Verdick R. Video imaging of the meibomian gland [letter]. Arch Ophthalmol 1994;112(4):448–9. 49. Arita R, Itoh K, Inoue K, et al. Noncontact infrared meibography to docu­ ment age-related changes of the meibomian glands in a normal popula­ tion. Ophthalmology 2008;115(5):911–15. 50. Arita R, Itoh K, Maeda S, et al. A newly developed and noninvasive mobile pen-shaped meibography system. Cornea 2013;32(3):242–7. 51. Matsumoto Y, Sato E, Ibrahim O, et al. The application of in vivo laser confocal microscopy to the diagnosis and evaluation of meibomian gland dysfunction. Mol Vis 2008;14:1263–71.

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52. Matsumoto Y, Shigeno Y, Sato EA, et al. The evaluation of the treatment response in obstructive meibomian gland disease by in vivo laser confocal microscopy. Graefe’s Arch Clin Exp Ophthalmol 2009;247(6): 821–9. 53. Call CB, Wise RF, Hansen MR, et al. In vivo examination of meibomian gland morphology in patients with facial nerve palsy using infrared meibography. Ophthal Plast Reconstr Surg 2012;28(6):396–400.

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Chapter 7  Slit Lamp Examination and Photography Csaba L. Mártonyi, Mark Maio

Key Concepts •

Isolation of the layer or entity to be viewed is the key to slit lamp biomicroscopy. • Techniques of biomicroscopic examination include diffuse illumination, broad beam illumination, optical section, indirect illumination, red reflex illumination, specular reflection, and sclerotic scatter. • Slit lamp photography employs the same modes of illumination with modifications (such as the fill light) necessary for accurate photographic documentation.

“On August 3, 1911, Alvar Gullstrand presented his first rudimentary model of the slit lamp … and explained its optics and applications.”1 An occasion of tremendous significance to ophthalmology had taken place: Gullstrand had introduced a device with the potential to advance the understanding of the eye and its problems as profoundly as did the direct ophthalmoscope 50 years earlier. This chapter will deal primarily with techniques of examination (all applicable to the photographic process as well) and will address special considerations required for photo­ documentation in Section II: Photography, below.

Section I: Examination “The examination of the eyes is begun after establishing the history of the case. In making this examination, too much stress can not be laid upon the necessity of proceeding systematically, since otherwise important matters can very readily be overlooked. We first examine the patient with regard to his general physical condition as well as with regard to the expression of his countenance, and then, in observing the eyes themselves, proceed gradually from the superficial parts – lids, conjunctiva, and cornea – to the deeper portions.”2 The ideal examination includes a careful, highly dynamic analysis of all structures, using each applicable form of illumination. The result should be a fully detailed, threedimensional mental image of the segments of the eye. Although many abnormalities are easily identified, some of

subtle expressivity cannot be ruled out without having exercised fully the capabilities of the slit lamp. In the absence of clear clinical signs, with only vague symptomatology reported, the examiner must exhaust all possibilities. After the abnormality is identified, additional information about its severity, extent, or particular characteristics can be gleaned through observation under all forms of illumination. The importance of a dynamic approach cannot be sufficiently stressed. Observing the eye in static light deprives the examiner of much of the available information. The process of examining the cornea in direct and indirect retroillumination from the iris, for instance, requires a scan of the cornea from one limbus to the other. This motion itself will reveal information that may otherwise go unnoted. It enhances the dimensional qualities of the information observed and results in a more accurate and complete impression of the extent and severity of the abnormality present. Similarly, observing the motion of the eye and the eyelids can provide important clues to normal or abnormal function.3 Establishing a routine protocol will minimize the time required to complete an examination and provides a fail-safe measure to ensure its completeness. As the examiner gathers experience, an individualized routine emerges. Type of practice and attendant patient population may be influencing factors in establishing a protocol. When circumstances permit, steps of an examination that may cause somewhat greater discomfort (e.g. eversion of the upper eyelid, application of vital dyes) may be best carried out toward the end of the routine to help ensure the patient’s ability to cooperate throughout the examination.

The instrument The principle underlying the slit lamp biomicroscope is isolation of the layer or object to be viewed. This instrument provides precise and modifiable illumination plus magnification with which to isolate, and thereby make visible, fine detail (Fig. 7.1). Composed of two primary components, the biomicroscope and the slit illuminator, today’s slit lamp is both highly efficient and accommodating. The addition of available accessories provides for an impressive array of functions. Most biomicroscopes consist of a parallel, Galilean telescope design. Utilizing optical changers, interchangeable

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Chapter Outline Section I: Examination Section II: Photography

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A

A

B Fig. 7.2  (A) Diagrammatic representation of the slit lamp illuminator, biomicroscope objective lens, and fill light. (B) Chemical injury to the cornea seen in optical section combined with diffuse illumination from the fill light. (A, Redrawn from Mártonyi CL, Bahn CF, Meyer RF. Clinical slit lamp biomicroscopy and photo slit lamp biomicrography. Ann Arbor: Time One Ink, Ltd; 1985. © Csaba L Mártonyi. B, Mártonyi CL. Landscapes of the eye: images from ophthalmology. A Photographic Exhibit, 1993. © Csaba L Mártonyi.)

B Fig. 7.1  (A) Slit illuminator and biomicroscope. (B) The magnified optical section is the most important capability of the slit lamp biomicroscope. (A, Redrawn from Mártonyi CL, Bahn CF, Meyer RF. Clinical slit lamp biomicroscopy and photo slit lamp biomicrography. Ann Arbor: Time One Ink, Ltd; 1985. B, From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

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oculars, or both, these instruments produce an effective range of magnification with excellent resolution. Many offer optional beam splitters to accommodate one-to-one teaching or to accept a digital camera for real-time display or recording for later use (Fig. 7.2). The slit beam delivery system is basically a projector, with the slit aperture as the actual “object” focused on a plane corresponding to the focal length of the biomicroscope. The foremost prowess of the slit lamp is its ability to create a focused, well-delineated, narrow slit beam that forms an optical section in transparent and translucent tissue. Not restricted to a single configuration, however, this beam is

CHAPTER 7 Slit Lamp Examination and Photography highly malleable through the use of simple controls that dictate its size and shape. It finds many additional applications in its various forms. The biomicroscope and the illuminator are mounted on a common axis in a co-pivotal arrangement. This arrangement facilitates the parfocal (biomicroscope and slit beam are focused on the same plane) and isocentric (the slit beam is centered in the field of view) relationships essential for practical function. A departure from these relationships can be purposely created for certain techniques of examination; otherwise, the absence of isocentricity or parfocality indicates a faulty condition requiring adjustment or repair.

Forms of illumination While there are only a few basic forms of illumination, most have variable uses and are highly effective in specific applications. This chapter concentrates on those forms of illumination and techniques of examination and photography that specifically address the eyelids, conjunctiva, cornea, sclera, and iris. Other structures are mentioned as their examination may be helpful in establishing a diagnosis of conditions involving the principal structure under consideration. Techniques of illumination are broadly divided into direct and indirect forms. Direct illumination, as the term implies, describes any situation where the beam of light is directed to strike the principal subject area. Direct illumination may be diffused or focal. Indirect illumination techniques use a secondary surface that reflects light onto the principal subject area or light transmitted through tissue from an area of adjacent illumination.

Direct illumination Diffuse illumination Diffuse illumination facilitates simultaneous observation of large areas at low magnification. The area surrounding the eyes, the eyelids, conjunctiva, sclera, cornea, and iris can be quickly reviewed for gross abnormalities. Initiating the slit lamp examination in this manner generates an early, overall impression and provides a unifying matrix for the more isolating magnifications and forms of illumination to follow. With the slit illuminator set at its largest aperture and the diffuser in place, the illuminator is rotated through its arc of travel from side to side. The effect is to create alternating axial and tangential illumination. Tangentially applied light, even when diffused, produces highlights and shadows and enhances the visibility of many changes. As shadows and highlights wax and wane with the oscillating illumination, alterations from normal topography become exaggerated and more readily apparent. A subtle presentation of molluscum contagiosum, for example, possibly hidden by cilia, may elude detection in static light, but may become quite obvious through the motion of the illuminator and the biomicroscope. Abnormalities of the lashes, such as collarettes, scales, and broken or missing lashes, are well enhanced with this approach. Because of shadows cast by cilia and foreign matter and the generally translucent nature of such deposits, static light may not provide adequate discrimination. The dynamic travel of light, however, animates shadows

Box 7.1 Examples of conditions seen in diffuse illumination  

Sclerocornea

Pterygium

Band keratopathy

Follicles

Trichiasis

Hypopion

Distichiasis

Ectropion

Pinguecula

Corneal pannus

Papillae

Blepharitis

Hordeolum

Arcus senilis

Entropion

Xanthelasma

Megalocornea

Chalazion

Lagophthalmos

Hyphema

Poliosis

and cascades highlights to fully dimensionalize and identify even mild expressions of various conditions (Fig. 7.3). Focal alterations in skin color (e.g. hyperemia, hyperpigmentation, or hypopigmentation) also present more readily under diffused and dynamically altered light. Focal illumination tends to isolate an area with a proportionate loss of perspective. Additionally, the brightness of focal illumination, with its inherent contrast, makes slight differences in color difficult to appreciate. Another factor to limit the usefulness of focal illumination in this application is the possibly enhanced reflectivity of the skin of the eyelids. Secretions from resident sebaceous glands can engender considerable specular reflections, greatly limiting a view beyond the episurface. As stated earlier, the initial survey of the conjunctiva, sclera, cornea, and iris in diffuse illumination provides a useful introduction to the overall condition (Fig. 7.4). Many abnormalities are easily visualized with this technique (Box 7.1). Findings such as conjunctival injection, follicles, papillae, chemosis, membranes/pseudomembranes, and scarring are recognizable in diffused light and should prompt examination with additional forms of illumination. The inferior and superior palpebral conjunctivae and much of the fornices can be given a preliminary review in the same manner. Tangentially applied diffuse illumination, with increased magnification, is an excellent technique for initial examination of these surfaces (Fig. 7.5). Inspection in diffuse light often provides the first indication of abnormalities present in the cornea. Gross opacification or changes that affect its topography present with little coaxing. After such an overview, further investigation can continue with more selective illumination and magnification.

Broad-beam illumination The term broad-beam illumination is variably used and highly subject to interpretation. It can vary from a beam width of 1 mm to its full size of approximately 11 mm. In this discussion a flexible width is assumed. As with the recommended oscillation of the slit illuminator, a dynamically altered

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1

A

B Fig. 7.3  (A) Illuminated from the left, the diminutive cilium and the white excrescence at its base (1) are clearly visible. The tangentially applied   light diminishes specular reflections and enhances contrast for good visualization of subtle findings. (B) Illuminated from the front, specular reflections abound, contrast is minimized, and the essential information   is greatly diminished (1). (C) Axial illumination and a slightly altered perspective preclude visualization of the abnormality altogether. Without the views shown in A and B, the examination would be quite incomplete. (© CL Mártonyi, WK Kellogg Eye Center, University of Michigan.)

C

Fig. 7.4  Staphylococcal keratoconjunctivitis and blepharitis are presented in diffuse illumination. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

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Fig. 7.5  Trachoma with linear scarring seen in diffuse illumination. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

CHAPTER 7 Slit Lamp Examination and Photography

Box 7.2 Examples of conditions seen in broad-beam illumination  

Corneal vascularization

Corneal scars

Basement membrane dystrophy

Lisch nodules

Reis-Bücklers dystrophy

Keratic precipitates

Schnyder crystalline dystrophy

Granular dystrophy

Terrien marginal dystrophy

Iris atrophy

Amiodarone vortex dystrophy

Pterygium

Prominent corneal nerves

Band keratopathy

Salzmann’s nodular degeneration

Macular dystrophy

Posterior embryotoxon

Arcus senilis

A

B Fig. 7.6  (A) Keratic precipitates are well visualized within a moderate, direct beam. (B) Vitreous presenting in the anterior chamber is illuminated with a broad beam of a much higher intensity than would be used for tissue of greater reflectivity. (A, © CL Mártonyi, WK Kellogg Eye Center, University of Michigan, B, From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

beam width is also beneficial. In this application, the beam is intended only as a source of bright, focal illumination, with the width adjusted to maximize information within the area under study. As the light strikes tissue interfaces, it is reflected, refracted, transmitted, scattered, and absorbed in a highly variable fashion. Thus, a given width of beam, with its corresponding overall intensity, may provide good information to confirm a particular entity, but may overpower findings associated with another. A beam width of 2–3 mm can provide a good starting point (Fig. 7.6). Although width is an important factor in the beam’s efficiency in specific applications, its intensity also affects its usefulness. A beam that is too bright will produce scatter, reducing the examiner’s ability to discriminate. Conversely, a beam intensity that is too “gentle” (in deference to patient comfort, for example) may preclude detection of mild departures from the normal.

Fig. 7.7  Broad-beam illumination demonstrating a luxated, mature lens nucleus in the anterior chamber. The tangentially applied light dramatizes dimension and minimizes reflections from overlying surfaces. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

As various tissues are examined, suspected alterations from the normal will either be confirmed or will prompt the examiner to use more or less light in their continued pursuit. As a general rule, forms of illumination should be exaggerated in both directions beyond the ideal setting, especially with the use of broad-beam illumination. The beam should be narrowed to the point of diminished width and then increased in width beyond the ideal setting to the point at which information loss occurs once again. Only by testing these limits will optimum size and intensity become apparent. Many conditions are seen best using broad-beam illumination (Box 7.2). All changes that are fairly opaque and reflect or absorb considerable amounts of light can be visualized easily. The light should be applied tangentially for maximum effectiveness. Topographic changes will become dramatically sculpted by the raking light. Additionally, oblique illumination will obviate the dazzling specular reflections resulting from axial lighting (Fig. 7.7). Tangentially applied broad-beam illumination is one of the most

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Fig. 7.8  Broad-beam illumination of the iris and anterior lens surface in Rieger’s syndrome. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.) A

effective forms for examining the iris surface (Figs 7.8–7.10) and certain changes within the lens (Fig. 7.11). When no abnormalities are seen with this technique, more selective forms of illumination are indicated. The absence of findings under broad-beam illumination should never encourage the conclusion that abnormalities are not present. However useful in the applications described above, broad-beam illumination can be quite counterproductive to the detection of many alterations of subtle expression.

Optical section This narrowest slit beam is, in effect, a fine blade of light that makes possible the virtual serial sectioning of transparent tissues in the living eye. The tangential presentation of these “light slices” facilitates an essentially cross-sectional view of the cornea and lens, even though these structures are largely parallel with the plane of observation. The sharply focused light is completely confined to the optical section, reducing scatter and maximizing contrast between the illuminated section and the dark, unilluminated surround. The result is a clear, basically uncompromised view of the tissue within the beam. As the slit beam is projected from an increasingly lateral position (away from the axis of the biomicroscope), the greater angular presentation has the effect of increasing the distance between the anterior and posterior surfaces of the structure under study. This increase serves to clarify intrastructural relationships and localization of abnormalities within. This capability represents the most selective and most isolating manner in which such tissue may be illuminated and observed (Box 7.3). For maximum effectiveness, the light intensity is set to maximum, and the slit beam is diminished in width to a point just before the optical section loses structural integrity. The thinner the beam, the more selective the optical section, thus producing finer delineation of information within that section. Beam width, however, should never be reduced to the point at which information is compromised because of light loss.

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B Fig. 7.9  (A) and (B) This iris lesion is de-emphasized by the diffusing axial reflection of light and the lack of highlights and shadows to demonstrate its dimensional nature. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

The transparency of the cornea, coupled with its propensity for both primary and secondary expressions of numerous diseases, makes it the most important component of the eye to section with light. Although remarkably transparent, normal corneal tissue is sufficiently relucent to reflect the narrow slit beam and articulate the optical section. A high-magnification view of the optical section will produce excellent discrimination of the substantial corneal layers. Beginning with the tear film, each layer may be selectively examined for departures from the normal (Fig. 7.12). The normal tear film will “flow” dynamically within the slit beam following each blink of the eyelids. Its reflectivity will

CHAPTER 7 Slit Lamp Examination and Photography

A

B

Fig. 7.10  (A) and (B) A tangentially applied beam will obviate the reflections inherent in axial illumination and will dimensionalize the subject with the highlights and shadows it creates. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

Box 7.3 Examples of conditions seen in optical section  

Edema

Lens opacities

Stromal opacities

Dellen

Marginal dystrophy

Microcysts

Kayser-Fleisher ring

Bullae

Fuchs dystrophy

Ectatic changes

Corneal pannus

Anterior chamber depth

Epithelial defect

Tear film deficiency

Corneal infiltrates

Corneal thinning

Furrow dystrophy

alter with the amount of refreshed, protective lipid on its surface, but will maintain a constant thickness and a smooth anterior face. The epithelium is seen as a line of nonreflectance or greatly diminished reflectance between reflections from the tear film and Bowman’s layer, which is contiguous with (and largely indistinguishable from) the anterior-most reflection from the stroma. The stroma itself is quite transparent. By encroaching on the zone of specular reflection, however, its reflectance can be enhanced considerably for better visualization of its structure. The optical section will terminate with the heightened reflection from the endothelium. As corneal transparency is lost to disease or injury, an increased amount of light is reflected. (The condition of a sclerotic, totally opaque cornea represents the extreme, or terminal, end of this transmission spectrum. In this condition, much of the light is reflected by the surface, limiting visual access to deeper layers. In such cases, moderate amounts of illumination will be considerably more informative, and a thorough examination will necessitate the use of indirect techniques, as discussed later in this chapter.) An inadequate tear film will present as a compromise to the normally smooth, unbroken reflection of the beam (Fig. 7.13). An edematous epithelium will become dimensional in the optical section and reflect increasing amounts of light (Fig. 7.14). Focal density changes will be isolated to the anterior, mid, or posterior cornea (Figs 7.15 and 7.16). Descemet membrane will become visible when abnormal mechanical forces alter its normal topography (Fig. 7.17). The endothelium will reflect increased amounts of light when affected by changes such as Fuchs dystrophy (Fig. 7.18). Within the visual axis, relatively mild expressions of tissue compromise can cause notable symptoms. Determining exact location and distribution is significant for arriving at a diagnosis. As the cornea is scanned in optical section, the relationship of the anterior to the posterior surface is also observed for changes in normal thickness and curvature. Ectatic changes, such as keratoconus or keratoglobus, will become readily apparent (Fig. 7.19). Focal elevations or depressions also become obvious as the slit beam deviates toward or away from the light source (Fig. 7.20). The narrow slit beam is important to apply to all ocular surfaces. A thorough scan of the lid margins, the bulbar and palpebral conjunctiva, the plica, caruncle, and corneal limbus will provide confirmatory information or add to what was gleaned using the modalities described earlier. The narrow slit beam is most effective in detecting topographic changes in these structures (Fig. 7.21). Follicles, papillae, or other dimensional alterations are well stated in this manner. All tissue will demonstrate some penetration by the beam. The degree of penetration is variably limited by the optical density of the tissue under study. Although actual penetration may be minimal, the information produced can be valuable. Of equal or greater importance is the indirect, proximal illumination simultaneously achieved (see following section entitled Indirect Illumination). The relationship between the cornea and iris is also evaluated with a narrow slit beam. By projecting the light from a moderate angle and observing the distance between the reflections from the cornea and the iris, a good estimate of

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B

C

Fig. 7.11  (A) and (B) Metallic foreign body in a clear lens, isolated with a moderate, tangentially applied beam. (C) “Christmas tree” cataract shown in great clarity due to the unilluminated surfaces in front and behind the subject, facilitated by tangential illumination. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

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1 2 3 4 5

Fig. 7.13  The fragmented reflection from the corneal surface indicates an inadequate tear film, an uneven epithelial surface, or both. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

Fig. 7.12  Optical section through a normal cornea demonstrating its principal layers. (1) Tear film, (2) epithelium, (3) anterior stroma with high density of keratocytes, (4) posterior stroma with lower density of keratocytes, (5) posterior layer (Descemet membrane and endothelium). (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

anterior chamber depth can be obtained. Observing this relationship at the limbus provides information regarding the grade of the angle.5 A completely closed segment of the angle is indicated by a contiguous presentation of corneal and iris reflections (Fig. 7.22). In a similar fashion, anterior synechiae may present as focal areas of contact between the reflected beams at the posterior corneal surface. This condition is confirmed by observing the slit beam coursing up the side of the tented iris tissue to make contact with the reflection from the posterior corneal surface (Fig. 7.23).

Tyndall light/anterior chamber cells and flare Based on the Tyndall phenomenon, pinpoint illumination is maximally effective at isolating aqueous cells and flare.

Fig. 7.14  The normally nonreflective epithelium is visible when edematous. The reflection is contiguous with the reflections from the tear film and the stroma. Two small edema clefts are seen in optical section. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007 © Csaba L Mártonyi.)

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Fig. 7.15  Anterior to mid-stromal deposits are seen in a patient with dystonia. (Copyright Mártonyi CL, WK Kellogg Eye Center, University of Michigan.)

A

Fig. 7.17  The normally nonreflective Descemet membrane layer is made visible by the reflection of light from its disturbed architecture in pseudophakic bullous keratopathy. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

B

Fig. 7.16  (A) and (B) Two images demonstrating the right-to-left, anterior-to-posterior track of a penetrating foreign body. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

The anterior chamber is considered optically empty, as its contents do not reflect sufficient light to express the beam in the normal state. The cells and protein that present in response to local inflammation, therefore, can be seen readily when isolated within the well-defined, narrow tunnel of light produced by pinpoint illumination. A small, round beam of high intensity is directed tangentially through the anterior chamber, and the focal point of the light (and the biomicroscope) is swept through the aqueous to determine the presence and density of cells and

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Fig. 7.18  Abnormal amounts of light are seen reflected from the endothelial layer in Fuchs dystrophy. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

CHAPTER 7 Slit Lamp Examination and Photography Fig. 7.19  Central thinning is obvious in this optical section of keratoconus. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

A

B

C

D

Fig. 7.20  (A) Aphakic bullous keratopathy with the large bulla well described by the slit beam. (B) Chemical injury with central loss of epithelium and limbal vascularization. (C) An iris lesion, clearly seen as elevated by the contour of the slit beam. (D) The deviating slit beam indicates a focal elevation of the iris by a ciliary body cyst. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

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Fig. 7.21  The slit beam deviates toward the source, confirming elevations in the bulbar conjunctiva. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

flare. For maximum contrast, when conditions permit, cells and flare should be observed against the dark background of a dilated pupil, while minimizing the light striking the iris. Although the “pinpoint” or “pencil of light” configuration represents the most discriminating technique1 (Fig. 7.24), the standard grading system used to describe the concentration of cells and flare assumes the use of a beam approximately 1 × 3 mm in size. The number of actual cells seen simultaneously within that beam is the stated degree of the condition. The amount of abnormal protein is determined by the examiner’s impression of the reflectivity (or Tyndall effect) of the aqueous. The degree of expression of these two conditions is stated in terms of one to four-plus cells and/or flare (Fig. 7.25).

Specular reflection The bright, mirrored reflections of light sources are considered regular, or specular, reflections, as opposed to the more common irregular reflections whereby most objects are seen.1 Specular reflections are subject to Snell’s law of optics, which states that the angle of reflection equals the angle of incidence. That suggests a level of difficulty associated with the location of such a reflection that is simply not present when examining the eye. On the curved ocular surfaces, specular reflections are easily elicited. The convex cornea and the high reflectivity of its anterior-most layer, the tear film, combine to make such reflections more or less everpresent companions during the course of an examination. While at times annoying, these reflections are, in fact, of great value for gathering information about the condition of the eye. As the eye is viewed in direct illumination, the zone of specular reflection is studied as an expression of surface

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B Fig. 7.22  (A) A very shallow anterior chamber is evidenced by the proximity of reflections from the iris and the corneal endothelium. Superiorly, contiguous reflections indicate an area of closed angle.   (B) An area of the angle closed by an iris lesion. (A, From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi. B, © CL Mártonyi, WK Kellogg Eye Center, University of Michigan.)

integrity. This evaluation should be performed under a fairly low light level, so as to diminish scatter and make visible detail within the zone of specular reflection. The area of specular reflection is not only a mirror image of the source but also faithfully mirrors the topographic condition of the surface on which it rests. Therefore, a compromised corneal surface will produce an abnormal reflection of the light source. Broken or granular reflections may indicate an inadequate tear film, the presence of foreign material, or a compromise to underlying tissue expressed as an alteration from

CHAPTER 7 Slit Lamp Examination and Photography

Fig. 7.25  A dramatic expression of four-plus aqueous cells in a case of endophthalmitis. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.) Fig. 7.23  Iris tissue adherent to the posterior corneal surface in an eye that, following successful penetrating corneal transplantation, suffered a penetrating foreign body injury. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

A

B Fig. 7.24  (A) and (B) Pinpoint illumination of cells and flare, recorded with the ISO setting at 1000. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

normal topography. A greatly diminished or irregular reflection is always a clear sign of abnormality (Fig. 7.26). The most important application of the specular reflection is in the evaluation of the corneal endothelium. While not expressly difficult, this technique may prove initially challenging. The reflectivity of the endothelial surface is so much lower than that of the tear film layer that the specular reflection may not be appreciated even when present. An angular difference of 30 to 40 degrees between the slit illuminator and the biomicroscope will make the task easier by producing greater separation between the two reflections. Moving the incident beam laterally across the face of the cornea will elicit the bright specular reflection from the tear film layer. By observing the adjacent area on the side opposite the light source, the more demure endothelial reflection is seen. Moving the biomicroscope forward approximately 0.5 mm will bring into focus the endothelial layer and cellular detail should become apparent. To obtain a clear view of endothelial cells, especially those that populate the uncompromised, young cornea, a magnification of 25× to 40× is required (Fig. 7.27). When such levels of magnification are not available, the endothelium can still be evaluated with this technique. The reflection is observed for continuity and uniform intensity as the light is played across the endothelium. When conditions such as guttae are present, the homogeneity of the reflection is interrupted (Fig. 7.28). In severe expressions of disease-related endothelial compromise, such as advanced Fuchs dystrophy, the reflection may become totally altered from the normal. Coalesced guttae may cause it to appear patchy or quite dark overall and, even when viewed at high magnification, information about individual cell borders may not be present (Fig. 7.29). In such conditions, even specular micrography may fail to produce satisfactory information regarding cell morphology or density. The specular reflection can also be used to examine the surface of the conjunctiva and the anterior and posterior surfaces of the lens.

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A

A

B Fig. 7.26  (A) Specular reflections faithfully reproduce the shapes of light sources on the surface of a normal tear film layer. (B) The specular reflections are altered from normal by the condition of interstitial keratitis. (A, From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi. B, © Csaba L Mártonyi.)

Indirect illumination Proximal illumination In proximal illumination, the light is directed to strike an area just adjacent to the area to be examined. The principal subject, therefore, is illuminated by light transmitted through tissue. The effect is one of retroillumination from deeper layers. It is remarkably effective for observing subsurface changes in tissue of sufficient opacity to prevent light penetration to the desired level with direct illumination (Fig. 7.30). Similarly, proximal illumination can facilitate location and determination of size and shape of an imbedded foreign

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B Fig. 7.27  (A) The bright specular reflection from the tear film layer is easily seen. The reflection from the endothelium is found just adjacent on the side opposite the light source. (B) At 40× magnification, even the small cells of this young, healthy endothelium are appreciable.   (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

body or one obscured by soft tissue reaction. It also can be helpful in gathering additional information about abnormalities that are apparent in direct focal illumination. By observing conjunctival or skin alterations with this modality, the specular reflections produced by direct illumination are eliminated, and another, valuable perspective is obtained in what becomes a form of retroillumination (Fig. 7.31).

CHAPTER 7 Slit Lamp Examination and Photography The benefits of proximal illumination are probably exploited more frequently than may be realized. A scan of the conjunctiva in direct focal illumination (e.g. with a narrow slit beam) includes making use of the information seen in the adjacent area of proximal illumination. Proximal illumination may not be the conscious goal of the examiner, but information from this zone is nonetheless gleaned. In fact, without it, the examination would be incomplete. When high magnification is used in observations by proximal illumination, the subject area may become sufficiently decentered to make viewing cumbersome. In such cases, the slit illuminator must be decentered from its normal isocentric position to permit centration of the principal subject area within the field of view.

Sclerotic scatter

Fig. 7.28  The normally smooth specular reflection is altered by low- or off-axis reflections. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

Specifically applicable to the cornea, sclerotic scatter permits the illumination of the entire cornea against a largely unilluminated background. An intense beam of moderate size is directed at the corneoscleral junction. The light travels the breath and width of the cornea by total internal reflection. In the normal cornea, this light passes through the stroma undisturbed and is visible only as a ring of light at the limbus, where it intersects, and is reflected by, the sclera. The brightest portion of this ring of light is located directly opposite the source. The normal cornea itself will appear unilluminated. Because of the extreme degree to which the cornea must be decentered to accommodate illumination of the limbus, the slit illuminator must be disengaged from its normal, isocentric relationship with the biomicroscope to allow centration of the cornea within the field of view (Fig. 7.32). In the abnormal cornea, the light that is axially reflected or refracted makes the abnormality visible. The degree to which this light is visible depends on the optical density and other characteristics of the abnormality and the size and intensity of the incident light beam (Fig. 7.33). Sclerotic scatter is quite remarkable for its sensitivity to subtle change while yielding information over a large area of distribution (Box 7.4). That combination is not possible with most forms of illumination. The ever-present compromise of “area versus detail” limits each application.4 Generally, the larger the area of simultaneous illumination, the more light that will be scattered, producing a corresponding loss of fine detail. However, in sclerotic scatter, as the cornea is illuminated by a source that is comparatively small (the size of the beam directed at the limbus), this technique is not strictly subject to this limitation. In reality, sclerotic scatter provides a simultaneous view of a large expanse

Box 7.4 Examples of conditions seen in sclerotic scatter  

Fig. 7.29  Severe Fuchs dystrophy preventing a view of endothelial cells. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

Corneal foreign bodies

Interstitial keratitis

Corneal edema

Granular dystrophy

Keratic precipitates

Radial keratotomy scars

Verticillata

Hydrops

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A

B

D

C Fig. 7.30  (A) and (B) Direct, broad-beam illumination of a Cardona keratoprosthesis is ineffective in demonstrating the flange of the device by light reflected by the sclerotic corneal tissue. (C) and (D) Proximal illumination transmits light behind the flange, creating a light background against which the flange is easily seen. Additionally, the material of the flange “pipes” the light to make its contours quite visible. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

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A A

B B Fig. 7.31  The size, shape, and density of an eyelid nevus are better appreciated when viewed in both direct (A) and proximal (B) illumination. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

of cornea, making it useful in identifying certain disease entities through recognition of a characteristic, overall pattern. In certain instances, the incidental light that falls on the iris, especially one of light pigmentation, may become a significant negative factor. Thus, a condition of subtle expression (e.g. cornea verticillata in Fabry’s disease) can be best appreciated against the dark background of a dilated pupil (Fig. 7.34).4

Direct and indirect retroillumination from the iris Representing two distinct forms from the standpoint of how they function, direct and indirect retroillumination from the iris are most informative when used together. This combined technique is the most important to the thorough

Fig. 7.32  A, The beam is decentered to facilitate centration of the cornea in the biomicroscope. (B) The light at the corneoscleral junction illuminates the cornea by total internal reflection. The normal cornea will not reflect the light along the viewing axis and remains dark against an essentially dark background. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

examination of the cornea. It actually produces three types of illumination with corresponding zones of information. With the beam applied tangentially, the area of the cornea observed against the directly illuminated iris (direct retroillumination from the iris) demonstrates alterations that are chiefly opaque. The zone of cornea that falls on either side of the illuminated background, i.e. cornea that lies in front of unilluminated iris or pupil (the zone of indirect retroillumination from the iris), demonstrates primarily refractile changes and changes of low optical density. Of greatest importance is the interface between light and dark backgrounds, where the most subtle changes may be seen. Abnormalities that both refract and reflect light become most dimensional in this zone between light and dark backgrounds.

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Fig. 7.33  Multiple fiberglass foreign bodies are seen over a wide distribution with the technique of sclerotic scatter. The light color (low optical density) of these particles makes them excellent candidates for viewing in what becomes, in effect, darkfield illumination. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

A

Fig. 7.34  The subtle cornea verticillata in Fabry’s disease is best seen in sclerotic scatter against the dark background of a well-dilated pupil. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

Technically, this juncture is an interface rather than a true zone. Because the biomicroscope (and therefore the slit beam) is focused at the level of the cornea, however, the interface is formed by divergent rays, resulting in a blurred image at the level of the iris. Because this blurred image of the interface appears to occupy space by virtue of its broader appearance, it becomes a zone in the practical sense (Fig. 7.35). The entire cornea can thus be examined for both subtle and obvious alterations. The beam of light is applied tangentially and moved across the cornea while observing the three zones simultaneously, with particular attention

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B Fig. 7.35  (A) and (B) The combination of direct and indirect retroillumination from the iris produces remarkable detail of subtle corneal findings, as seen in this example of lattice dystrophy. The   zone of interface between light and dark backgrounds is the most informative. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

CHAPTER 7 Slit Lamp Examination and Photography

Box 7.5 Examples of conditions seen in direct and indirect retroillumination from the iris  

Lattice dystrophy

Corneal infiltrates

Corneal foreign bodies

Early edema

Meesmann dystrophy

Filaments

Map-dot-fingerprint

Microcysts

Cornea farinata

Fuchs dystrophy

Descemet folds

Corneal scars

Keratic precipitates Thygeson’s superficial punctate keratitis

paid to the interface of light and dark backgrounds.4 To ensure that all information about the cornea has been gathered with this modality, the scan should be repeated, with the light applied from both the temporal and nasal sides. Many entities are easily visualized and identified in this manner (Box 7.5). Lattice dystrophy, with its characteristic signature, is seen in all three zones of illumination (see Fig. 7.35). Folds in the Descemet membrane are best seen in indirect retroillumination from the iris, but close to the interface of light and dark backgrounds (Fig. 7.36). The classic, bubble-like microcysts characterizing Meesmann dystrophy are most dimensionally described within the interface zone (Fig. 7.37). Figure 7.38 demonstrates the effective use of the illuminated iris to delineate overlying dense corneal foreign bodies and Figure 7.39 shows the effectiveness of indirect retroillumination from the iris in highlighting subtle, translucent corneal foreign bodies against a dark pupil and unilluminated iris.

A

Retroillumination from the fundus Using the light reflected by the retinal pigment epithelium, the anterior vitreous, lens, and cornea may be examined in retroillumination. The slit lamp illuminator is placed into an axial position with the biomicroscope and the light is introduced through a dilated pupil to illuminate the fundus. With modest excursions of the illuminator to either side of center, the optimum position is established when the greatest retroillumination effect is achieved. A large pupil is required for maximum effectiveness. After configuring a moderately sized beam, the entire instrument can be moved from side to side to facilitate examination of most of the cornea. Decentering the slit to one side of the available pupil and then to the other facilitates a serial, uncompromised view of both sides of the subject stratum, and the area under study remains centered in the biomicroscope. Leaving the iris unilluminated ensures maximum visibility of the abnormality. Light striking the iris causes scatter and reduces the effectiveness of this valuable form of illumination. One major advantage of this modality is that it produces excellent delineation of subtle changes over a wide area of distribution. In that regard, it is similar to sclerotic scatter. The principal difference between the two is that sclerotic scatter produces dark field illumination (objects illuminated

B Fig. 7.36  (A) and (B) Folds in the Descemet membrane are primarily refractile and are best seen against a dark background directly adjacent to the illuminated background. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

against a dark background), whereas retroillumination from the fundus is a brightfield technique (objects silhouetted against a bright background). Dark field excels at demonstrating changes that primarily reflect light, and brightfield produces best contrast for opaque changes and those that are refractile (Box 7.6). Much of the cornea or lens may be visualized simultaneously, limited only by the size of the pupil and shallow depth of field. The classic findings in fingerprint dystrophy are beautifully displayed in this form of illumination (Fig. 7.40). Similarly, many lens changes are most easily identified in this manner. Cataract formation and subluxation of the lens are dramatically demonstrated

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Fig. 7.37  Meesmann dystrophy is effectively demonstrated in indirect retroillumination from the iris. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

Box 7.6 Examples of abnormalities seen in retroillumination from the fundus  

Lattice dystrophy

Map-dot-fingerprint dystrophy

Pseudoexfoliation

Lens vacuoles

Keratic precipitates

Cataract

Corneal scars

Corneal rejection lines

Meesmann dystrophy

B Fig. 7.38  (A) Direct retroillumination from the iris. (B) Opaque corneal foreign bodies silhouetted against an illuminated, lightly pigmented iris. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

(Fig. 7.41). The essentially colorless lens and cornea are transformed into structures that are seen in additional contrast by virtue of the color reflected by the retinal pigment epithelium.

demonstrate even subtle expressions of transillumination (Fig. 7.43).4

Transillumination of the iris

The peripheral cornea cannot be examined without the use of a gonioscope. Indirect lenses provide the ideal view with a choice of mirror angles and reduced light scatter. Once the lens is placed, the optical section can provide information regarding the posterior surface of the cornea and the condition of the angle. A wider beam, which is conformed to the area under study, is useful for the simultaneous view of a larger area to assess structural relationships further. Confining the beam to the zone of immediate attention

Iris transillumination is a simple extension of the technique just described (Fig. 7.42). An important difference is the optimum pupil size. A completely dilated pupil is counterproductive to iris transillumination; a pupil size of 2–3 mm is ideal. Through such an opening, a moderate beam of high intensity can be introduced to illuminate the fundus. In that presentation, the iris is still sufficiently attenuated to

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The peripheral cornea (gonioscopy)

CHAPTER 7 Slit Lamp Examination and Photography

A

A

B Fig. 7.39  (A) Indirect retroillumination from the iris. (B) Fiberglass particles in the cornea seen in indirect retroillumination from the iris. The light particles are nicely contrasted against the dark pupil. An increase in illumination intensity was required. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

minimizes distracting reflections that inevitably reduce contrast and detail (Fig. 7.44). The anterior chamber angle is extremely reflective in most eyes, and moderate amounts of light are suggested for its examination (Fig. 7.45).

Vital dyes Vital dyes provide information important to a complete ocular examination. Their application is essential to

B Fig. 7.40  (A) and (B) Epithelial fingerprint dystrophy is best visualized in retroillumination from the fundus. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

determine the condition of the corneal and conjunctival epithelium. Because these dyes can cause irritation (rose Bengal, in particular) and because their presence may interfere with the assessment of deeper layers of the cornea, they may be best used toward the end of the examination. Fluorescein is a good indicator of contact lens fit. With the blue exciter filter in place and the light intensity sufficiently increased, such information is easily obtained (Fig. 7.46). Similarly, tear film break-up time can be ascertained

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Fig. 7.41  A traumatically subluxated lens is seen with blood on its posterior surface, well demonstrated in retroillumination from the fundus. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

A

Fig. 7.42  Cataract formation and iris atrophy, resulting from a contusion injury, are simultaneously observed in retroillumination from the fundus and transillumination of the iris. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

B Fig. 7.43  (A) and (B) Information regarding transmission defects of the iris in pigment dispersion syndrome is maximized by projecting a small, round beam through a partially dilated pupil. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi. B, Copyright Mártonyi CL: WK Kellogg Eye Center, University of Michigan.)

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Fig. 7.45  The 67-degree mirror produces a view that provides better perspective for this presumed (by history) caterpillar hair in the angle. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

A

1

2

3

B Fig. 7.44  (A) View of the peripheral cornea through a three-mirror lens. (B) A prominent Schwalbe’s line (1) is seen, with adherent iris strands (2). Haab striae (3) of the cornea prevent an optimum view in this case of Axenfeld syndrome. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

(Fig. 7.47). Conditions that disturb the normal tear film also can be detected6 or confirmed with this same technique (Fig. 7.48). To determine the presence of epithelial compromise, the dyes can be used individually or mixed and applied in combination. The irritation caused by rose Bengal may warrant the use of a topical anesthetic before instillation. The cornea and conjunctiva should be examined for signs of staining with both white and blue light (Fig. 7.49). Because devitalized epithelium stains with rose Bengal and areas that are de-epithelialized stain with both rose Bengal and fluorescein, the subtle areas of rose Bengal staining may be better appreciated when viewed in a matrix of fluorescein. Since rose Bengal absorbs much of the incident blue light, its consequently dark appearance contrasts well with a brightly fluorescing background (Fig. 7.50). Lissamine green dye is a useful alternative to rose Bengal in certain applications.7 Rose Bengal can be irritating to the

Fig. 7.46  Fluorescein pooled under a poorly fitting contact lens. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

patient and has antiviral properties that may adversely affect results of viral cultures obtained after dye instillation. Lissamine green, a synthetic organic dye with a staining pattern similar to that of rose Bengal, is apparently well tolerated and has not been shown to exhibit antiviral properties. Lissamine green, when applied to conditions where rose Bengal staining loses definition due to the presence of new or engorged vessels, will produce better contrast against such a dominantly red background (Fig. 7.51) The color of the iris will influence the effectiveness of both rose Bengal and lissamine green (Fig. 7.52). The patient should blink repeatedly to help differentiate pooling from staining. Areas of staining become apparent as they move with the eye. Pooled dye appears somewhat static

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Fig. 7.47  Tear film break-up in a normal eye. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

A

B Fig. 7.48  Tear film interrupted by changes in lattice dystrophy. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

by comparison. A definitive differentiation can be made only by rinsing the pooled dye. A particularly dry eye will certainly require rinsing before an accurate assessment can be made regarding actual staining.

The Seidel test The Seidel test is used to determine corneal or conjunctival patency. When the escape of aqueous is suspected, fluorescein dye is applied directly to the site of suspected leakage. When present, escaping aqueous dilutes the fluorescein as it flows down the surface of the eye. The rate of dilution is the indicator of the dynamic of the positive test. The application of concentrated fluorescein results in a dark, nonfluorescing background, against which the diluted and now brightly fluorescing dye is highly visible (Figs 7.53 and 7.54).8

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Fig. 7.49  (A) An obviously compromised cornea stained with rose Bengal and fluorescein, seen in white light. (B) Corneal filaments stained with fluorescein seen in a recently transplanted cornea. (A, From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi. B, Copyright Mártonyi CL, WK Kellogg Eye Center, University of Michigan.)

Section II: Photography While a fully equipped photo slit lamp biomicroscope (PSL) may still be the ideal for documenting the full range of information seen during an examination, the continued development of digital add-on systems for the clinical slit lamp offers impressive capabilities in image capture with increasing simplicity and economy. Post capture processing in an imaging program can further enhance the utility of the final image, effectively expanding the photographic range of the clinical slit lamp.

CHAPTER 7 Slit Lamp Examination and Photography

A

Fig. 7.50  A case of dendritic keratitis, stained with rose Bengal and fluorescein. Rose Bengal absorbs the blue light, resulting in a dark signature against the fluorescing surround. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

The instrument The following additional options will greatly improve imaging capability: BEAM SPLITTER: A beam splitter provides the necessary coaxial view shared by the examiner and the camera back and ensures complete control over the image before it is recorded. Beam splitters will divert from 50% to 85% of the light to the camera to ensure satisfactory exposures with most forms of illumination. As more light is diverted to the camera back, less remains for the examiner. Some beam splitters may be toggled in and out of the viewing axis to provide maximum viewing illumination before and after the image has been recorded. ILLUMINATION: Electronic flash, an integral part of the PSL, produces light of high intensity at an effective duration of exposure of approximately 1ms, an interval that will arrest the motion of the eye at high magnifications. Newer LED light sources can also produce good results. FILL LIGHT: The fill light is an additional source of diffuse illumination, common to the PSL. While not standard on a clinical slit lamp, it may be added to most. It is an important accessory that can improve the quality of images dramatically. It provides partial compensation for the loss of the dynamic, three-dimensional character of an examination by contributing the important element of perspective in situations that call for limited direct focal illumination. The addition of diffused, overall light places isolated elements into context. In a single image, the fill light provides overall, general information about the eye, and the slit beam is used to highlight specific changes in the cornea (see Fig. 7.2). The

B Fig. 7.51  (A) Rose Bengal staining of a squamous cell lesion with reduced contrast against the engorged conjunctival vessels. (B) A similar lesion stained with lissamine green shows greater contrast. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi. Images courtesy of Timothy J. Bennett, CRA, FOPS.)

fill light should be set to approximately one half of the intensity of the slit illuminator to provide the necessary contrast between the diffusely illuminated background and the bright, narrow slit beam.

Preparing for photography The basic protocol for photo documentation is essentially the same as for slit lamp biomicroscopy. One begins with an overview and proceeds to isolate further with illumination and magnification the salient features of the condition under consideration. Techniques of illumination that produce specific information over a wide area of distribution should be considered whenever applicable. The most utilized images are those that present findings in recognizable context.

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B

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D

Fig. 7.52  (A) Punctate rose Bengal staining in superior limbic keratoconjunctivitis and (B) The same eye stained with lissamine green. (C) Herpes simplex keratitis stained with rose Benagal and (D) the same eye stained with lissamine green, which appears to produce reduced contrast against this lightly pigmented iris. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi. Images courtesy of Timothy J. Bennett, CRA, FOPS.)

A Fig. 7.53  A Seidel-positive filtering bleb. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

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B

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Fig. 7.54  (A) The Seidel test. The fluorescein is applied directly to the site of suspected compromise to demonstrate leakage of aqueous through a perforated cornea. (B) and (C) Moments later, the pattern progresses to demonstrate the highly dynamic flow of aqueous.   (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

CHAPTER 7 Slit Lamp Examination and Photography Essential detail, however, should not be compromised in an attempt to include everything in a single view, and the fill light should not be used in conjunction with indirect forms of illumination. While such photographs are often used to obviate the need for multiple images, the results will always be compromised. Although the fundamental principles of clinical slit lamp biomicroscopy and photo slit lamp biomicrography are essentially the same, additional considerations are necessary for successful photo documentation. Chief among these considerations is the conscious awareness that slit lamp illumination, by its very nature, is a compromise. The larger the area of simultaneous illumination, the less fine detail is seen. Conversely, the more detail elicited by selective illumination, the more out of context that information will be. During a dynamic, three-dimensional examination of the eye, these limitations have little effect on the process of gathering information. The result of a thorough examination is a complete mental image of the condition of the eye. By comparison, a static, two-dimensional photograph is not only deprived of the elements of motion and the third dimension, but is also limited to a single moment of such an examination. As such, it is amazing how effective a single photograph can be. Several components have been discussed as essential to imaging. Additional factors must be considered to produce consistently accurate and pleasing photographs. Correct mechanical focus, format, centration, control of artifacts, and optimum exposure are elements that combine to reproduce visual impressions most accurately.

Focus The maintenance of a sharp image in the biomicroscope is a continuous element of a dynamic slit lamp examination. Focus is a perpetual, flowing transition as the slit beam is played over the gently curving surfaces of the eye. In a practical sense, there are no specifically individual images, but rather a compendium of infinite, transitional views that produce an aggregate impression. Each photograph, however, is but a single slice of that examination. Therefore, preparations for photo documentation must include the selection of the most informative single view (or the first in a series of views) and the perception of its appearance as a static, two-dimensional image. To ensure a sharp image, precise mechanical focus of the biomicroscope at the time of exposure is critical. The view seen in the biomicroscope is an aerial image. An aerial image is suspended in space rather than projected onto a flat, immovable plane (as the focusing screen of a single lens reflex camera). Because the view through the biomicroscope would be unacceptably diffused by such a focusing screen, it is not used. The aerial image system, therefore, is the most practical in such an application, but not without limitation. With the image literally floating in air, the mechanical position of the biomicroscope (and the camera back) can be unwittingly altered from its correct focal distance through simple accommodation. Although seen as a sharp image by the examiner, the resultant photograph may be so unsharp as to be unusable. To produce a sharp image on the digital sensor, a specific protocol is followed.

To facilitate the correct focus of the biomicroscope, a “cross-hair” reticle is used in one ocular for reference. To use the reticle correctly, the ocular must be adjusted for the user’s refractive error. That is best achieved by first turning the eyepiece adjustment to its maximum plus setting. Then, while the user looks through the eyepiece, with accommodation completely relaxed, the setting is adjusted toward the minus side while observing the reticle. This rotation toward the minus should be relatively brisk and should result in a sharp impression of the reticle at or near the user’s refractive error, or at zero for an emmetrope. This exercise should be repeated until consistent results are achieved. The image in the biomicroscope should be treated as an object at infinity and viewed with the accommodation completely relaxed. The reticle is always used in the ocular that shares the image with the camera back.

The photographic format The circular field seen in the biomicroscope must be reduced to a corresponding rectangle within that circle. The areas beyond the rectangle, which had been used for examining the eye, must now be disregarded and the intended picture area confined to the photographic format. As a guide, an optional reticle eyepiece including the rectangular outline is an ideal solution.

Centration The need to ensure that the principal subject is in the center of the photographic field seems obvious. Nevertheless, this element is frequently compromised. The most common cause may be a momentary disregard of the rectangular format in favor of the full, circular field seen in the oculars. Additionally, when the isocentric relationship between the slit illuminator and the biomicroscope is left intact when using indirect forms of illumination, the subject area becomes decentered in favor of the isocentric incident beam. The beam must always be decentered to allow centration of the principal subject area when indirect forms of illumination are used.

Control of artifacts To be effective, a photograph must first include the principal subject area. Of equal importance is the elimination of unwanted elements that either obscure the desired detail or distract attention from the principal subject. The most distracting artifacts of illumination are the ubiquitous specular reflections seen during the course of an examination. They are of little concern as they come and go, because their effect is nullified by their momentary presentation during the dynamic examination. In a static photograph, however, such artifacts can considerably compromise an otherwise excellent image. When final adjustments are made preparatory to making an exposure, it is important to view the image only through the ocular that shares that image with the camera back. By closing the opposite eye, the monocular image, as seen by the camera, will be much more manageable in terms of minimizing artifacts and maximizing desirable elements before its capture.

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The final image The final image is a product of the elements listed above and, of great importance, good exposure. Good exposure results from correct white balance, media sensitivity, intensity of illumination, subject reflectivity, and duration of exposure.

Color balance and sensitivity Digital cameras provide for easy adjustment to match the color temperature of various light sources. This process is called white balance and should be set following the instructions for the camera back used. It will ensure accurate color reproduction in captured images. Sensitivity refers to the responsiveness of the digital sensor to light, stated in ISO numbers. Low ISO settings will provide better image quality when there is sufficient light for a good exposure. When the captured image is too dark and the light intensity is already set to maximum, the ISO should be increased sufficiently to obtain good results.

Subject reflectivity and exposure

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Subject reflectivity is an element of great influence. In recording conditions by direct illumination, levels of subject reflectivity simply require correspondingly opposite adjustments in light intensity for a good exposure. In certain applications of indirect illumination, however, the maximum output of the slit illuminator will be required and, in some cases, an increase in the ISO setting as well. Some systems adjust the ISO setting automatically when compensation is necessary, but in response only to the brightest area in the field of view. A manual override may be necessary for good exposure in indirect forms of illumination.

Duration of exposure When using electronic flash, the duration of exposure is dictated by the 1ms duration of the flash. The intensity and short duration of light provides good exposure while arresting the natural movement of the subject eye. Clinical slit lamps, however, are not usually equipped with electronic flash and must rely on the continuous but much lower intensity of the slit illuminator and the accessory fill light. To compensate for the lower light levels, the duration of exposure is increased, but only marginally to avoid blurring the image. Trial images, instantly displayed, will quickly establish parameters for each instrument and examiner.

Diffuse illumination Diffuse illumination is required to show large areas simultaneously at low magnification. Diffusing the light from the slit illuminator, used in conjunction with the fill light (when available), will produce even illumination of the external eye (Fig. 7.55). Such overviews are useful for demonstrating the general condition of the eye and serve as introductions for more isolated views.

Broad-beam illumination When diffused light causes too much scatter or when a specific element of a condition must be emphasized, a broad

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B Fig. 7.55  (A) Two sources of diffuse illumination. (B) A case of phakolytic glaucoma is seen in dual, diffuse illumination. (A, From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi. B, © CL Mártonyi, WK Kellogg Eye Center, University of Michigan.)

beam, without fill light, can be used. Beam size is important to the outcome. Although desirable from the standpoint of an all-inclusive photograph, when beam size is enlarged to include too much, the results are often compromised. The effect of illumination must be observed carefully while dynamically altering beam width to determine the optimum setting (see Figs 7.6–7.8). Exposure is not a problem because this form of illumination returns the largest percentage of the light available. Magnification should be increased to make best use of the photographic format. The presentation of the beam should be as oblique as possible. Axial light is reflected axially from surfaces overlying the condition or object to be photographed and further reduces dimensional information within the subject area (see Fig. 7.9). The tangential presentation of light, however, facilitates illumination of the subject without overlying reflections to diffuse information and simultaneously enhances its topography (see Fig. 7.10). This form can also

CHAPTER 7 Slit Lamp Examination and Photography be used to isolate abnormalities of high reflectance in transparent tissue. When photographing an object within the lens or the anterior vitreous, a well-dilated pupil is necessary to accommodate a sufficiently tangential presentation of light to obviate reflections from overlying surfaces (see Fig. 7.11).

Optical section Easily applied at the clinical slit lamp, the optical section remains a challenge to reproduce photographically. The primary problem is insufficient light especially at higher magnifications. Most digital imaging systems are capable of adequately exposing a thin slit beam in relatively clear cornea at lower magnifications. Nevertheless, the most refined sectioning capabilities of the slit beam may have to be sacrificed in the interest of exposure. A wider than optimum beam may have to be used as the standard; however, the beam should not be widened to the point at which the optical sectioning capability is lost. Rather than settling for a beam too wide, one should consider increasing the ISO setting. A short series of test exposures will determine the best compromise between slit width and intensity of illumination. Photographs of the optical section can convey precise information regarding the condition of the cornea and other structures (see Figs 7.12−7.23).

Combined direct focal and diffused illumination This type of illumination produces one of the most informative single images of the eye. It combines the narrow slit beam discussed above with diffuse illumination from the fill light. The fill light is responsible for mean exposure and, therefore, the slit beam is used at an intensity that technically constitutes an overexposure. This relationship is necessary to demonstrate adequate background information and a sufficiently brilliant slit beam to highlight information within the section. The best images are achieved when the slit beam is approximately four times brighter than the fill light. This combination is excellent for portraying conditions of the cornea and also applicable to numerous other situations (see Figs 7.19−7.23).

Tyndall light/anterior chamber cells and flare Documentation of this condition is one of the most challenging tasks in slit lamp photography. Although cells and flare are easily visualized, their low reflectivity makes it difficult to obtain an adequate exposure. Even “four-plus” expressions will require maximum viewing illumination in combination with a high ISO setting.4 The beam should be configured into a small spot or “pencil of light” to produce maximum isolation.1 With a fairly tangential presentation of the beam at moderate magnification, useful images can be achieved. For maximum contrast and best results, the focal point of the beam should be placed over the dark, unilluminated pupil (see Fig. 7.24).

epithelium (or more accurately, the tear film layer), there is more than adequate light with which to obtain a good exposure. Photographs should be taken at high magnifications (25× to 40×) to show cellular detail. Critical focus is essential (see Fig. 7.27).

Proximal illumination All indirect forms of illumination require decentration of the slit beam for the maintenance of a centered principal subject area. Although it may be unnecessary for most of a slit lamp examination, decentration of the incident beam is essential to the success of each slit lamp photograph. Proximal illumination poses quite a challenge in achieving good exposure. The amount of light by which such changes are visible represents a small percentage of the light used to directly illuminate the adjacent area. For this reason, the exposure is frequently underestimated. The area of direct illumination must be dramatically overexposed as compared with the area of indirect illumination. This is an unavoidable by-product of this technique, which the photographic process exaggerates beyond the visual impression. In some cases, the distraction factor of this zone of overexposure may be tempered by increasing magnification to the exclusion of the directly illuminated area. The important goal, however, is to use sufficient incident light to adequately expose the principal subject area. Although somewhat variable because of differences in tissue reflectivity and absorption, an increase in light intensity is required4 (see Figs 7.30 and 7.31).

Sclerotic scatter Sclerotic scatter can produce information over a wide expanse of cornea in a single photograph. It is best used to delineate alterations that are of low optical density. This technique requires a complete decentration of the slit beam to permit centration of the cornea in the final image (see Fig. 7.32). It also requires the maximum light output from the slit illuminator. Obvious conditions, such as the bright foreign bodies seen in Figure 7.33, are easily exposed. More subtle entities, such as the verticillate pattern in Fabry’s disease may require a setting of 400–800 ISO. Dilating the pupil ensures a sufficiently dark background to provide the contrast necessary for good visualization (see Fig. 7.34).

Direct retroillumination from the iris A beam of moderate width is projected onto the iris to create a light background against which opaque changes are visible. When the condition presents in an eye with a light iris, the exposure appropriate to the documentation of that iris in direct illumination is sufficient. When the condition presents against a dark iris, an increase in light intensity is required. The beam should be wide enough to create an adequate background, without directly illuminating the corneal condition (see Fig. 7.38).

Specular reflection

Indirect retroillumination from the iris

Although the reflection from the endothelium is much less intense when compared to the reflection from the

The photography involved in this technique is somewhat more challenging because the light available to illuminate

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the abnormal condition is but a small portion of the light striking the iris (see Fig. 7.39). With increased pigmentation of the iris, light loss also increases, requiring a greater adjustment in exposure. Many changes are visible in this form of illumination. Alterations that are primarily refractile are especially well described in this manner. Such changes are the most striking at the interface of light and dark backgrounds, demanding careful attention to exposure (see Figs 7.35–7.37). As a general rule, the level of illumination used for direct illumination of the iris should be increased to provide good exposure of refractile changes at the interface generated by a light iris, and set even higher when the condition is being photographed at the interface created by the surface of a darkly pigmented iris.

Retroillumination from the fundus Exposure is generally not a problem. With a well-dilated pupil and clear media, excellent images of corneal or lenticular changes can be captured. The slit beam is configured into a short rectangle or, when possible, into a half-moon shape to permit maximum illumination of the background (the retinal pigment epithelium) without allowing the light to strike the iris. The light beam is then displaced to the side of the pupil that causes the least compromise to the information to be recorded. A considerable decentration of the beam is necessary to maintain centration of the pupillary area (see Figs 7.40 and 7.41). When necessitated by the presentation of the condition, the beam is moved to the other side of the pupil for additional photographs to provide complete documentation. In the absence of optimum conditions, coupled with a heavily pigmented retinal pigment epithelium, exposure may need to be increased considerably. When maximum retroillumination is required, the eye is rotated to cause the incident beam to strike the optic nerve head, producing more intense retroillumination from that highly reflective surface.

instances, a moderate underexposure produces a more saturated image containing more detailed information (see Fig. 7.44). Previewing the image with only the eye that shares the image with the digital camera will help in managing unwanted reflections from the flat, anterior surface of the contact lens.

Vital dyes Vital dyes are best photographed in direct, broad-beam illumination. Of great importance is the removal of excess dye before photodocumentation. Thus, only actual staining is recorded. This is important to the accurate recording of rose Bengal, lissamine green and fluorescein staining. Rose Bengal and lissamine green staining is easily photographed (see Figs 7.51 and 7.52), requiring only minor adjustments from the normal exposure used for broad-beam illumination. Since both dyes delineate subtle areas of epithelial compromise, care must be taken to preserve the subtle nature of the information. The light beam is applied tangentially to avoid obscuring information with overlying specular reflections. In most cases, a slightly lower level of illumination will better express subtle focal staining. Fluorescein is best viewed and photographed under blue light (approximately 480 nm) used to excite the dye to fluorescence. Because the blue filter diminishes overall light intensity, a higher level of illumination is required, depending on the actual filter used. For most applications, only an excitation filter is required (see Figs 7.46–7.48, 7.49B and 7.50). Combining rose Bengal and fluorescein can add a further dimension to such coverage (see Fig. 7.50). For photographs that demonstrate extremely subtle staining, a barrier filter (approximately 520 nm) is added to provide adequate discrimination.9 An additional increase in exposure may be necessary.

The Seidel test

Figure 7.42 required midrange intensity of illumination, whereas Figure 7.43 required the maximum light output of the slit illuminator.

To produce photographs of this technique, the blue filter is placed over the light source, which is set to produce full, broad-beam illumination, and the exposure is set for routine fluorescein photographs. The fluorescein is applied directly to the suspected site of leakage and the image captured as the diluted fluorescein cascades down the surface of the eye (see Figs 7.53 and 7.54).8

The peripheral cornea

Techniques specific to keratoconus

Photography of the peripheral cornea requires the same basic techniques as photography of the filtration angle. Both areas are imaged simultaneously, and the delineation of the principal subject area is largely a matter of focus. The best access to this area is provided with the most oblique mirror in the Goldmann three-mirror lens, set at an angle of 59 degrees. In this application, the more oblique view provides better access to this zone of the anterior chamber (see Fig. 7.44). For some conditions, the 67-degree mirror produces a better overview, providing an enhanced perspective (see Fig. 7.45). Because of the highly reflective nature of this area, the danger of using too much light is much greater than using too little. As findings in this zone are frequently subtle, it is doubly important to avoid overexposing the image. In some

Documenting the Fleischer ring

Transillumination of the iris

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The Fleischer ring may be documented by using the blue filter described earlier for exciting fluorescein. In this case, the blue light is absorbed by the iron line, delineating the conus and causing it to appear dark in the resultant photograph. It is only effective, however, when the iron line is seen against a lightly pigmented iris (Fig. 7.56).

Munson’s sign Munson’s sign is a simple and graphic means of demonstrating the abnormal corneal outline. By asking the patient to look down, the lid margin conforms to the cornea’s horizontal profile, boldly revealing the condition (Fig. 7.57).

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Fig. 7.56  The iron line (1), or Fleischer ring, seen in keratoconus can be enhanced with blue light. Because the basis for greater visibility is rendering the ring darker through its absorption of the incident blue light, this technique is effective only in eyes with lightly pigmented irides. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

Fig. 7.58  A corneal profile of mild keratoconus seen against the illuminated nasal bridge. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

Further reading For a more complete description of the slit lamp examination and photography techniques detailed in this chapter, see reference 4.

References Fig. 7.57  Munson’s sign in keratoconus. (From Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. © Csaba L Mártonyi.)

A vertical profile can be documented by turning the patient’s head in the chin rest assembly sufficiently to obtain a temporal view. By directing a moderate beam of light to strike the nasal bridge behind the cornea, a light background is produced, against which the condition is presented in a dramatic and pleasing manner (Fig. 7.58).

1. Berliner ML. Biomicroscopy of the eye, vol. 1. New York: Paul B. Hoeber; 1949. 2. Fuchs E. Textbook of ophthalmology. New York: D. Appleton & Co; 1892. 3. Arffa RC. Grayson’s diseases of the cornea. 3rd ed. St Louis: Mosby; 1991. 4. Mártonyi CL, Bahn CF, Meyer RF. Slit lamp: examination and photography. Sedona: Time One Ink, Ltd; 2007. 5. Van Herick W, Schaffer RN, Schwartz A. Estimation of width of angle of the anterior chamber. Am J Ophthalmol 1969;68:626–9. 6. Shahinian L. Corneal valance: a tear film pattern in map-dot-fingerprint corneal dystrophy. Ann Ophthalmol 1984;16:567–71. 7. Bennett TJ, Miller GE. Lissamine green dye-An alternative to Rose Bengal in photo slit-lamp biomicrography. J Ophthal Photo 2002;24(2):74–7. 8. Romanchuck KG. Seidel’s test using 10% fluorescein. Can J Ophthalmol 1979;14:253–6. 9. Justice J Jr, Soper JW. An improved method of viewing topical fluorescein. Trans Am Acad Ophthalmol Otolaryngol 1976;81:927–8.

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Chapter 8  Tear Film Evaluation Michael A. Lemp

Key Concepts • • • •

• •

New technologies have become available which expand our clinical capabilities to better diagnose dry eye disease (DED), to assess its severity and response to treatment. A great number of tear proteins have been identified, many of which are genetic markers and others relate to inflammation on the ocular surface. It is now possible to measure tear osmolarity in a clinical setting. Tear osmolarity values over 308 mOsm/L are considered indicative of DED and values over 328 mOsm/L are seen in severe disease. Standard diagnostic tests do not correlate with each other, particularly in mild-moderate disease but become more uniformly positive in severe disease. There are new ways of visualizing the meibomian glands, and these methodologies have documented an increase in incomplete blinking in DED. New imaging devices employing OCT have allowed clinicians to measure features of the ocular surface previously not seen. DED is characterized by a loss in visual function with instability of the tear film and early breakdown between blinks with effects on higher order aberrations and light scattering.

The tear film is a critical component in maintaining the health of the ocular surface and as a pathway for repair. Oxygen captured from the atmosphere during the day and from the capillaries of the conjunctiva lining the upper lid during sleep supplies the cornea and conjunctiva, supporting the cellular turnover and maturation necessary to maintain a clear cornea for vision.1 The tear film, moreover, provides an exit pathway for cellular debris, metabolic waste products, microbes, and other particulate matter in the tear film via drainage through the nasolacrimal ducts.2 In addition, the ocular surface and the tear-producing structures (lacrimal glands, meibomian glands of the eyelid, and mucin-producing cells of the conjunctiva and the mucosal lining of the nasolacrimal ducts) are linked by a neural pathway forming an integrated functional unit that

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regulates epithelial cell turnover in health and epithelial repair processes in response to trauma or pathophysiological processes.3,4 The tear film contains water, electrolytes, proteins (which form cytokines directing epithelial cell activities), mucins, sugars, and other water-soluble substances. Overlying this aqueous phase produced primarily by the main and accessory lacrimal glands (and to a lesser extent by the conjunctiva) is a thin lipid layer. The lipid is produced by the meibomian glands of the eyelids and serves to stabilize the tear film, retard evaporative tear loss, and prevent contamination of the ocular surface with skin lipids.5,6 The ocular surface is covered by a mucin layer consisting of two parts: a thin membrane-associated mucin produced by the epithelial cells and a thicker mucin blanket, the product of the goblet cells of the conjunctiva.7 Mucin serves to render the epithelial cells wettable by aqueous tears and interacts with the overlying lipid layer to stabilize the tear film. In dry eye disease there are qualitative and quantitative alterations in the volume, composition, and structure of the tear film. In this chapter we will consider examination techniques and clinical tests designed to aid in the diagnosis of dry eye disease. The last five years have seen the emergence of a number of new diagnostic technologies and the further development of others, opening a new era of understanding of the dynamic aspects of the lacrimal functional unit and the pathophysiology of ocular surface disease processes.

General Inspection Gross examination of the ocular adnexa can reveal significant structural changes important in the pathogenesis of dry eye disease. Alterations in the eyelid structure and function can be observed with bright natural or artificial light. The eyelids should approximate the ocular surface, and the upper lid travel over two-thirds of the cornea with each blink. Interpalpebral fissure widths vary greatly but an excessively wide interpalpebral fissure, e.g. in thyroid eye disease, is associated with increased evaporative tear loss.8 Trichiasis, ectropion, or entropion can interfere with normal tear film dynamics, and incomplete closure of the lids can lead to localized areas of drying on the ocular surface. Bell’s phenomenon, in which the cornea rotates upward on lid closure, ensures protection for the corneal surface. About 5% of the

CHAPTER 8 Tear Film Evaluation

Chapter Outline General Inspection Slit Lamp Examination Tear Stability Tear Production Tear Composition and Characteristics Meibomian Gland Structure and Excreta Tear Clearance Tests Staining of the Ocular Surface Tests of Visual Function Conclusion

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CHAPTER 8 Tear Film Evaluation normal population will have an absent or deficient Bell’s reflex. This can be estimated by asking the patient to close the eye while holding the lid and observing the cornea. A deficient Bell’s reflex can lead to exposure keratopathy.

Tear Stability

Examination of the inferior marginal tear strip can yield information about the volume of tears present on the ocular surface. The tear strip is a line of tears just above the lower lid (Fig. 8.1). It is normally about 0.5 mm in width and has a concave upper aspect. When this strip is thin or discontinuous, it is evidence of deficient aqueous tear volume. The tear strip is better visualized by fluorescein staining but care must be taken not to flood the surface by overwetting the fluorescein strip; it should be just barely moistened. While thinning of the marginal tear strip is a relatively late sign of aqueous tear deficiency (ATD), attention to this area can yield valuable information. Another feature frequently seen in dry eye is increased debris in the tear film. Bits of mucus, fragments of sheets of sloughed epithelial cells, and other foreign material trapped in the tear film are suggestive of delayed tear clearance seen in dry eye.9 Examination of the ocular surface with the slit lamp can also reveal alterations in the morphology of the conjunctiva such as redundant folds in the bulbar conjunctival epithelium (conjunctivochalasis). This finding, while not specific for DED, has been reported to be seen in patients with dry eye.10 There are a variety of objective tests of tear film characteristics and function. Although most of these have some clinical utility, some have been relegated to a research setting or have not gained wide clinical acceptance. This chapter will confine itself to objective tests which are either in wide clinical use or are developing into rapidly accepted clinical tools of such importance that they may become essential elements of routine clinical examination.

In dry eye disease the tear film is unstable, resulting in an abnormally rapid break-up of the pre-corneal tear film between blinks.11,12 After tears are surfaced by the action of the lids, a meta-stable tear film is established. Over time (usually 10–30 seconds) the tear film thins, leading to the development of randomly distributed dry spots in the precorneal tear film (Fig. 8.2). The interval between the last complete blink and the appearance of the first random dry spot is the break-up time (BUT). This is generally measured after a small amount of fluorescein has been instilled or a slightly moistened fluorescein strip has been applied to the superior aspect of the bulbar conjunctiva. A wide slit lamp beam with the cobalt blue filter is used to scan the cornea; the patient is instructed to blink several times and then not blink. A hand-held timer is used to measure the seconds until the appearance of the first randomly distributed dry spot in the fluorescein-stained pre-corneal tear film. This is repeated several times and averaged. Values of less than 10 seconds are considered abnormal.13 There are non-fluorescein (noninvasive) measurements of BUT that employ reflective devices with a grid projected onto the corneal surface.14 These values are slightly higher and require equipment not widely available. BUT is a measure of the stability of the tear film; abnormally low values are seen in aqueous tear-deficient and in evaporative dry eye. Abnormal BUT values are reflective of a tear film abnormality but do not specify the type of dry eye. The tear film will break up rapidly over an underlying epithelial irregularity such as superficial punctate keratopathy. The presence of corneal staining will result in a rapid BUT that is not necessarily evidence of an intrinsic tear abnormality but rather the epitheliopathy. BUT has been criticized as being quite variable in an individual.15 This inter-test variability is probably due to the method with which the test is performed, variations in blink patterns, and the dynamics of tear production and flow. Consistent results below 10 seconds are, however, pathognomonic of a pathologically unstable tear film. More recently, a newer method of measuring and recording tear film break-up has been developed and is being used in clinical drug trials.16 In this

Fig. 8.1  Fluorescein-stained marginal tear strip.

Fig. 8.2  Fluorescein-stained tear film break-up.

Slit Lamp Examination

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method, a quantified amount of sodium fluorescein solution 1% is instilled into the conjunctival sac, blinking occurs and the appearance of the first randomly occurring corneal dry spot is video-recorded with a timer recording the time in 0.1 second increments. Three measurements are recorded and the timing measured by three independent observers. The authors have reported a new reference value for this technique. Values below 7 seconds are considered abnormal and reflective of the presence of dry eye disease.16 The same authors have combined their BUT measurements with an assessment of the blink rate, which is calculated by dividing 60 by the number of observed blinks per second. The ratio of the tear film BUT over the inter-blink interval (IBI) is referred to as the Ocular Protection Index (OPI): OPI = BUT/ IBI. Values below 1 are characteristic of tear film instability and dry eye disease. Because of a lack of reproducibility of BUT and unavoidable subjective judgments required, different approaches to more objective measurement of tear stability have been developed. These include the use of videokeratography to document tear film break-up. The onset of the first recorded pre-corneal dry spot and its enlargement patterns have been reported.17 The use of this technology with specific analytical programs in videokeratography devices has demonstrated the clinical utility of the technology, but new referent values are still in development. Another recently described objective measure of tear stability involves tear osmolarity and the inter-eye differences. This will be described in a later section.

Tear Production The most widely used test to measure aqueous tear production is the Schirmer test. In this test a standardized size strip of filter paper is inserted over the lower lid margin into the cul-de-sac, usually in the temporal one-third of the lid (Fig. 8.3). The patient is instructed to close the eyes and the strip is removed at 5 minutes; the extent of wetting of the strip is measured. Values below 5.5 mm of wetting are diagnostic of aqueous tear deficiency.18 This test is performed both with and without the use of topical anesthesia. The so-called Schirmer II (with anesthesia) has been purported to measure “basal” tear secretion, i.e. nonstimulated tears.19

Fig. 8.3  Schirmer strip in position on the temporal one-third of the lower lid.

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It has been demonstrated that, even with anesthesia of the cornea and conjunctiva, tear secretion is driven by sensory stimuli, e.g. the lids, lashes, air currents, and light.20 The whole concept of “basal” or unstimulated tears has been called into question. A Schirmer I (without anesthesia) has become the generally accepted method for assessing aqueous tear production. This test has been criticized for its variability.21 Differences in performance of the test will greatly influence the sensory stimuli. The Schirmer test, however, is a useful estimate of aqueous tear production because of its ease of performance, wide availability, and low cost. As aqueous tear-deficient dry eye disease progresses and the lacrimal glands lose their ability to respond to sensory stimuli or the sensory receptors on the ocular surface are compromised, results of the Schirmer test become more consistent. Serially consistent Schirmer I results below 5 mm of wetting at 5 minutes are highly suggestive of dry eye disease. An alternative method of measuring aqueous tear production has been proposed – the phenol red test.22 This involves the use of a special cotton thread that has been impregnated with a dye – phenol red. The thread is inserted over the inferior lid margin into the temporal conjunctival sac. At the end of 15 seconds, the dye, which is pH sensitive, turns color from yellow to orange, indicating the length of the thread wetted by tears. This test has been reported to be less uncomfortable and more specific in the diagnosis of aqueous teardeficient dry eye disease.23

Tear Composition and Characteristics Of the more than 500 proteins that have been identified in tears, several have been used as surrogate measures of aqueous tear production, i.e. lysozyme and lactoferrin. Lysozyme was first of interest because of its antibacterial activity. It has been demonstrated that tear lysozyme levels are decreased in aqueous tear-deficient dry eye disease.24 Lysozyme is one of the principal protein components of tears. Its measurement is based on the enzyme’s ability to lyse a suspension of the bacterium, Micrococcus lysodeikticus. When this suspension is placed in an agar gel, a tear sample is collected by micropipette and placed in a well in the suspension containing gel. The plate is incubated and the area of lysis noted. The larger the area of lysis, the greater the concentration of the enzyme. This method is not used very often because of lack of availability of the plates, cost, and the lack of specificity of the results. Decreased tear lysozyme levels are also seen in a number of inflammatory conditions.25 Of more recent interest is the tear protein lactoferrin, which also possess antibacterial activity.26 In addition, it has a protective effect on the corneal and conjunctival epithelium.27 Previously, an assay was based on a commercial solidphase ELISA methodology but more recent reports of a colorimetric analysis of microvolumes of tears have shown good diagnostic utility. In this method (Touch MicroAssay), a micropipette is used to collect a small volume of tears, which is then transferred to a cell where the tears are exposed to a reactive reagent that is colorimetrically tagged; the resultant sample is read in a commercially available colorimeter. Clinical experience has, however, shown tear lactoferrin levels to be scattered

CHAPTER 8 Tear Film Evaluation over a broad area.28,29 Decreased lactoferrin secretion in aqueous tear-deficient dry eye disease would be expected to be counterbalanced by the tear-concentrating effects seen in both aqueous deficient and evaporative dry eye disease, yielding variable results. Ways to compensate for the increased tear concentration characteristic of dry eye disease might improve the diagnostic value of this marker for aqueous tear-deficiency dry eye disease.

Tear ferning It has been observed that tear samples dried on a slide and examined under a microscope display a crystalline pattern of tear mucin. In aqueous tear deficiency, this pattern resembles ferns. A grading system has been developed and this test has been reported to have greater specificity and sensitivity than the Schirmer test, particularly for more severe forms of dry eye disease.30

Tear osmolarity In dry eye disease, the tear film is in a hyperosmolar state. This is true for both aqueous deficient and evaporative dry eye disease.31 Tear film osmolarity has been measured using freezing point depression and vapor pressure changes. Unfortunately, these methods have been limited primarily to a research setting, owing to their complexity, the high operator skill required and, most importantly, the need for relatively large volumes of tears, which necessitates stimulating tears for collection, and even this is insufficient in many dry eye subjects.32 A large, recently published meta-analysis of the literature over the last 25 years identifies tear hyperosmolarity as the single diagnostic test with the highest accuracy in identifying patients with dry eye disease.33 The advent of a new technology requiring tear samples of less than 50 nanoliters that measures tear osmolarity easily and quickly in a clinical setting promises to provide a new practical diagnostic test suitable for clinical use.34 A recent report suggests very high sensitivity and specificity and positive predictive values for this TearLab technology, in the diagnosis of dry eye disease. Over the past 15 years there have been over 170 peer-reviewed publications on the performance and clinical utility of measuring tear osmolarity for the diagnosis of DED. The large majority of these publications have found that this is a valuable tool not only in the diagnosis of DED but also in assessing disease severity and response to treatment. The data collected in these studies has added greatly in our understanding of the characteristics of DED, particularly of the close connection between tear osmolarity and tear film stability. While several publications have reported that tear osmolarity readings do not correlate with other objective measures of the disease e.g. Schirmer test, corneal staining, or tear break-up time, further research has documented that none of these tests correlate well with each other.35 It is now clear that both an elevated tear osmolarity and tear instability are hallmarks of DED.36 In addition, tear osmolarity is the only objective test that correlates in a linear manner with DED over the entire spectrum of disease severity.37 This does not mean that the other tests have no value but rather that they provide independent information and that most of them are not abnormal early in the disease

process. They may become more positive in more severe disease when the homeostatic control of the lacrimal functional unit (tears and ocular surface) is lost. While there are unilateral cases of DED, particularly with lid abnormalities, the overwhelming number of DED cases are bilateral, and DED should be viewed as a bilateral disease. It is important to test both eyes. In DED these values frequently differ (over 8 mOsm/L) due to transient compensatory mechanisms e.g. increased aqueous tear secretion in evaporative DED. The higher figure is the appropriate one to use; note both numbers to determine the dynamic range of the variability. This latter number is an objective measurement of the tear film stability.37 Not only does the higher number become lower in response to effective treatment, but the difference between the two eyes decreases. The most useful reference value for distinguishing normal from dry eye patients is 308 mOsm/L.38 Tear osmolarity does not distinguish between the two major subtypes of DED i.e. aqueous tear deficiency and evaporative dry eye. These can be distinguished by slit lamp examination and appropriate treatment initiated. The accurate measurement of tear osmolarity has not only provided us with a diagnostic tool of high clinical value but has acted as a probe to understand DED better, improve assessment of disease severity, and provide a guide for appropriate therapy and to gauge response to treatment.39–41 Reliance on symptoms alone is inadequate and misleading, since many patients with DED do not report symptomatology.

Meibomian Gland Structure and Excreta The meibomian glands of the eyelid number between 20 and 25 in each lid. They secrete a lipid mixture that is discharged onto the tear film: excretion of the lipid is effected primarily through the muscular contraction associated with blinking. Meibomian gland lipid forms the top layer of the tear film and both stabilizes the tear film and retards evaporative tear loss.42 Evaluation of the functional status of these glands involves slit lamp inspection of the lid margin and an estimate of the quantity and quality of the excreted lipid (meibum). Evidence of altered meibomian gland structure includes increased vascularity of the lid margin, plugging of the orifices, and loss of orifices.43 Increased vascularity of the lid margins occurs with advancing age and is not, by itself, a reliable indicator of meibomian gland disease. Meibum can be evaluated by pressing against the lower lid with a finger about 1 mm below the lid margin. In the normal subject, it will be possible to express some lipid from about two-thirds of the glands at a given time. This excretion is normally fluid and clear. Lack of expression from the glands, and/or alteration in the character of the excretion, is critical in the diagnosis of meibomian gland dysfunction (MGD). As the disease process advances, the excretion will vary from turbid to coagulated (toothpaste-like). Such meibum is pathognomonic of MGD. A grading scale for assessing severity of meibomian gland dysfunction has been developed for use in clinical trials but is equally suitable for clinical practice.43,44 Another method of assessing meibomian gland dysfunction involves transillumination of the eyelid. Using an examining muscle light placed inside the lower eyelid (after

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topical anesthesia), it is possible to visualize the outline of the glandular structure. This visualization can be enhanced and recorded with the use of infrared film.44,45 The normal pattern is that of branching ductules coming off a central vertical core. Obliteration of this structure is evidence of chronic inflammation and glandular dysfunction.

Tear Clearance Tests Coincident with a decrease in aqueous tear production, there is a decrease in tear turnover, which is defined as the rate at which newly secreted tears reside within the tear film before they are lost either to evaporation or drainage through the lacrimal punctae and the nasolacrimal ducts. Tear volume and turnover are most accurately measured by dye dilution studies. In this methodology, a small amount of fluorescein dye is instilled into the tear film and the concentration of the dye is measured over time. Special fluorophotometers have been built to accurately measure dilution of the dye as new tears enter the tear film and old tears exit. Expense has limited the availability of this methodology. An alternative, inexpensive method of semiquantitatively grading fluorescein dilution has been proposed and is in use.46 In this method (Fluorescein Clearance Test [FTC]), 5 µL of 1% fluorescein dye is instilled into the tear film. The patient is asked to blink to distribute the dye and serial 1-minute Schirmer tests are performed every 10 minutes. Initially, the staining of the paper strip with the dye will be intense. Persistent staining (beyond 10 minutes) indicates delayed tear clearance (DTC). A combined use of the Schirmer II test with the FTC has been proposed.47 This tear function index (TFI) is the ratio of the value of the Schirmer test over the tear clearance rate. The use of the TFI in the diagnosis of dry eye disease is reported to demonstrate a specificity of 91% and a sensitivity of 79%. It should be noted that this index refers to aqueous deficiency dry eye only and not evaporative dry eye.

Staining of the Ocular Surface The normal ocular surface does not take up water-soluble dyes instilled into the tear film. With disruption of the mucin coating protecting the surface epithelial cells and/or damage to the epithelial cell walls, water-soluble dyes will diffuse into the surface cells. The three most commonly used dyes are fluorescein, rose Bengal (RB), and lissamine green (LG). Fluorescein, which stains damaged epithelial cells, is best visualized on the corneal surface. A 1% solution or a filter paper strip impregnated with fluorescein is used to introduce the dye into the tear film. The patient is instructed to blink to distribute the dye. The ocular surface is scanned using the broad beam of the slit lamp with the cobalt blue filter (or Wratten 47 blue filter). The extent and intensity of the stain are assessed. There are a number of grading scales, including the Van Bijsterveld,26 NEI/Industry Workshop,48 and Oxford systems.49 The NEI/Industry Workshop grading system has the advantage of collecting data on five discrete subareas of the cornea separately, e.g. the central cornea. Staining of the conjunctiva is seen when there are disruptions in the protective mucin coating; RB and LG are used. A 1% solution of either is instilled into the tear film, and the

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patient is asked to blink. The surface of the conjunctiva can be viewed within 10 seconds with RB but one should wait at least 2–3 minutes before viewing LG stain, and low light should be used. RB is more irritating to the patient and LG is gaining wider acceptance for this reason. Staining of the ocular surface is evidence of ocular surface damage and is characteristic of more severe dry eye. Mechanisms of ocular surface staining are discussed in a recent review article.50

Tests of Visual Function Recently, attention has been directed to optical aberrations that have been identified in patients with DED. Although severe dry eye with significant staining of the central cornea has long been known to reduce visual acuity, recent studies have demonstrated that even in the absence of significant central corneal staining, the instability which is characteristic in all forms of dry eye disease results in rapid break-up of the tear film between blinks, compromising image quality. This effect on visual acuity is not captured during ordinary Snellen chart measurement of acuity because the patient can blink, momentarily improving vision. More rapid break-up occurs within 3 seconds of a blink in many dry eye patients, reducing their inter-blink acuity to levels of 20/60 or less.51 Recent work has developed two instruments to detect these changes. In one, the tear stability analysis system (TSAS), serial videokeratographic images are collected each second between blinks.52 In another approach, a functional visual acuity (FVA) device has been developed which measures visual acuity by way of rapid presentation of optotypes. Both of these technologies promise to add to our armamentarium of diagnostic technologies in the near future.53

Conclusion The various objective methods of examining the tear film can provide useful information, diagnosing, classifying, and grading the severity of dry eye.

References 1. Holly FJ, Lemp MA. Tear physiology and dry eyes: review. Surv Ophthalmol 1977;22:69–87. 2. Doane EMG. Blinking and the mechanics of the lacrimal drainage system. Ophthalmology 1981;88:844–51. 3. Stern ME, Beuerman RW, Fox RI, et al. The pathology of dry eye: the interaction between ocular surface and lacrimal glands. Cornea 1998;17: 584–9. 4. Paulsen FP, Schaudig U, Thale AB. Drainage of tears: impact on the ocular surface and lacrimal system. Ocul Surf 2003;1(4):180–91. 5. Holly FJ. Formation and stability of the tear film. Int Ophthalmol Clin 1973;13:73–96. 6. Lozato PA, Pisella PJ, Baudouin C. The lipid layer of the lacrimal tear film: physiology and pathology. J Fr Ophtalmol 2001;24(6):643–58. 7. Watanabe H. Significance of mucin on the ocular surface. Cornea 2002; 21(2 Suppl. 1):S17–22. 8. Khurana AK, Sunder S, Ahluwalia BK, et al. Tear film profiles in Graves’ ophthalmopathy. Acta Ophthalmol (Copenh) 1992;70:346–9. 9. Pflugfelder SC, Solomon A, Stern ME. The diagnosis and management of dry eye: a twenty-five year review. Cornea 2000;19(5):644–9. 10. Meller D, Tseng SC. Conjunctivochalasis: literature review and possible pathophysiology. Surv Ophthalmol 1998;43(3):225–32. 11. Norn MS. Desiccation of the precorneal tear film. I. Corneal wetting time. Acta Ophthalmol (Copenh) 1969;47:865–80. 12. Lemp MA, Holly FJ. Recent advances in ocular surface chemistry. Am J Optom Arch Am Acad Optom 1970;47:669–72.

CHAPTER 8 Tear Film Evaluation 13. Lemp MA, Hamill JR. Factors affecting tear film breakup in normal eyes. Arch Ophthalmol 1973;89:103–5. 14. Mengher LS, Bron AJ, Tonge SR, et al. A non-invasive instrument for clinical assessment of the pre-corneal tear film stability. Curr Eye Res 1985;4:1–7. 15. Vanley GT, Leopold IH, Gregg TH. Interpretation of tear film breakup. Arch Ophthalmol 1977;95:445–8. 16. Abelson M, Ousler G, Nally L. Alternate reference values for tear film break-up time in normal and dry eye populations. Lacrimal Gland, Tear Film, and Dry Eye Syndromes 3 Part B. Adv Exp Med Biol 2002;506: 1121–5. 17. Goto T, Zheng X, Okamoto S, et al. Tear film stability analysis system: introducing a new application of videokeratography. Cornea 2004;23(8 Suppl.):865–70. 18. van Bijsterveld OP. Diagnostic tests in sicca syndrome. Arch Ophthalmol 1969;82:10–14. 19. Jones LT. The lacrimal secretory system and its treatment. Am J Ophthalmol 1966;62:47–60. 20. Jordan A, Baum J. Basic tear flow, does it exist? Ophthalmology 1980;87: 920–30. 21. Clinch TE, Benedetto DA, Felberg NT, et al. Schirmer’s test: a closer look. Arch Ophthalmol 1983;101:1383–6. 22. Hamano H, Hori M, Hamano T, et al. A new method for measuring tears. CLAO J 1983;9:281–9. 23. Asbell PA, Chiang B, Li K. Phenol-red thread test compared to Schirmer’s test in normal subjects. Ophthalmology 1987;94(Suppl.):128. 24. Regan E. The lysozyme content of tears. Am J Ophthalmol 1950;33: 600–5. 25. Sapse AT, Bonavida B. Preliminary study of lysozyme levels in subjects with smog eye irritation. Am J Ophthalmol 1968;66:79. 26. van Bijsterveld OP. The Sjogren syndrome and tear function profile. Adv Exp Med Biol 1998;438:949–52. 27. Shimmura S, Shimoyama M, Hojo M, et al. Reoxygenation injury in a cultured corneal epithelial cell line protected by the uptake of lactoferrin. Invest Ophthalmol Vis Sci 1998;39:1346–51. 28. Foulks GN. Personal communication. 29. Albach KA, Lauer M, Stolze HH. Diagnosis of KCS in rheumatoid arthritis. Ophthalmologe 1994;91(2):229–34. 30. Masmali AM, Purslow C, Murphy PJ. The tear ferning test: a simple clinical technique to evaluate the ocular tear film. Clin Exp Optom 2014;97(5): 399–406. 31. Gilbard JP, Farris RL, Santamaria HJ. Osmolarity of tear film microvolumes in keratoconjunctivitis sicca. Arch Ophthalmol 1978;96:677–81. 32. Nelson JD, Wright JC. Tear film osmolarity determination: an evaluation of potential errors in measurement. Curr Eye Res 1986;5(9):677–81. 33. Tomlinson A, Khanal S, Ramesh K, et al. Tear film osmolarity determination of a referent value for dry eye diagnosis. Invest Ophthal Vis Sci 2006; 47(10):4309–15.

34. Tomlinson A, McCann L, Pearce I. Comparison of human tear film osmolarity measured by electrical impedance and freezing point depression. Cornea 2010;29(9):1036–41. 35. Sullivan BD, Crews LA, Messmer EM, et al. Correlations between commonly used objective signs and symptoms for the diagnosis of dry eye disease: Clinical implications. Acta Ophthalmogica 2014;92(2):161–6. 36. Potvin R, Makari S, Rapuano C. Tearfilm osmolarity and dry eye: a review of the literature. Clin Ophthalmol 2015;9:2039–47. 37. Sullivan BD, Whitmer D, Nichols KK, et al. An objective approach to dry eye disease severity. Invest Ophthalmol Vis Sci 2010;51:6125–30. 38. Lemp MA, Bron AJ, Baudouin C, et al. Tear osmolarity in the diagnosis and management of dry eye disease. Am J Ophthalmol 2011;151(5): 792–8. 39. Sullivan BD. Challenges in using signs and symptoms to evaluate new biomarkers of dry eye disease. Ocul Surf 2014;2(1):2–3. 40. Bron AJ, Tomlinson A, Foulks GN, et al. Rethinking dry eye disease: a perspective on clinical implications. Ocul Surf 2014;12(Suppl 2):S1–31. 41. Foulks GN. Challenges and pitfalls in clinical trials of treatments for dry eye. Ocul Surf 2003;1:20–30. 42. Driver PJ, Lemp MA. Meibomian gland dysfunction. Surv Ophthalmol 1996;40:343–67. 43. Bron AJ, Benjamin L, Snibson GR. Meibomian gland disease. Classification and grading of lid changes. Eye 1991;5:395–411. 44. Foulks GN, Bron AJ. Meibomian gland dysfunction: a clinical scheme for description, diagnosis, classification and grading. Ocul Surf 2003;1(4): 107–26. 45. Mathers WD, Shields WJ, Sachdev MS, et al. Meibomian gland dysfunction in chronic blepharitis. Cornea 1991;10:277–85. 46. Macri A, Rolando M, Pflugfelder S. A standardized visual scale for evaluation of tear fluorescein clearance. Ophthalmology 2000;107:1338–43. 47. Xu KP, Yagi Y, Toda I, et al. Tear function index: a new measure of dry eye. Arch Ophthalmol 1995;113:84–8. 48. Lemp MA. Report of the National Eye Institute/Industry Workshop on Clinical Trials in Dry Eyes. CLAO J 1995;21(4):231–32. 49. Lemp MA, Baudouin C, Baum J, et al. The definition and classification of dry eye disease: Report of the Definition and Classification Subcommittee of the International Dry Eye Workshop (2007). Ocul Surf 2007;5(2): 75–92. 50. Bron AJ, Argüeso P, Irkec M, et al. Clinical staining of the ocular surface: mechanisms and interpretations. Prog Retin Eye Res 2015;44:36–61. 51. Goto E, Ishida R, Kaido M, et al. Optical aberrations and visual disturbance associated with dry eye. Ocul Surf 2006;4(4):207–13. 52. Kojima T, Ishida R, Dogru M, et al. A new noninvasive tear stability analysis system for the assessment of dry eyes. Invest Ophthalmol Vis Sci 2004;45:1369–74. 53. Ishida R, Kojima T, Dogru M, et al. The application of a new continuous functional visual acuity measurement system in dry eye syndromes. Am J Ophthalmol 2005;139:253–8.

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Chapter 9  Corneal Diagnostic Techniques Mark A. Greiner, William J. Faulkner, Jesse M. Vislisel, Gary A. Varley, Kenneth M. Goins

Key Concepts • • • • • •

Vital dye stains, applied carefully and selectively to the cornea and ocular surface, can resolve characteristic patterns that aid in the diagnosis and treatment of a broad variety of conditions. Corneal pachymetry is an essential tool for surgical planning, disease diagnosis and management, and monitoring of corneal endothelial cell function before and after corneal transplantation. Ultrasound pachymetry remains the gold standard for measuring central corneal thickness. Optical coherence tomography and Scheimpflug imaging can provide detailed measurements of corneal thickness regionally and by corneal layer. Corneal esthesiometry, assessed prior to instillation of topical anesthetic drops, can provide vital diagnostic and prognostic information about the functional health of the cornea. Corneal sensitivity may be the most reliable test of long-term corneal compromise, and defects in corneal sensation can adversely affect the cornea.

Corneal diagnostic techniques are specialized methods of examination that may involve simple or complex aids to yield valuable information for the diagnosis and treatment of ocular disease. These techniques are commonly but selectively used, depending on the patient’s history and the goals of the examination. External examination of the lids, slit lamp biomicroscopy techniques,and tear film evaluation usually precede other diagnostic tests and have been discussed in previous chapters. Techniques discussed here are corneal staining, pachymetry, and tests for corneal sensation.

Corneal Staining Corneal stains are diagnostic tools to assess the integrity of superficial cell layers of the cornea and ocular surface. These may be the most commonly performed tests in routine slit lamp biomicroscopy. Characteristic staining patterns aid in the diagnosis and management of corneal and external

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disease. Staining should be documented, noting depth and extent. Descriptions may specify micropunctate (small dots), macropunctate (larger dots), or coalescent (a patch). Depth may be limited to the epithelium or include stroma. Fluorescein and rose Bengal are the most common dyes used to evaluate the ocular surface. Both are halide derivatives of the hydroxyxanthene family. The addition of seven halogen atoms (three iodide, four chloride) to the hydroxyxanthene skeleton is responsible for the photophysical differences of rose Bengal. The spectroscopic absorption undergoes a red shift that contributes to rose Bengal’s characteristic color. Feenstra and Tseng have demonstrated that the original concepts of fluorescein and rose Bengal staining have not been entirely correct.1 While both dyes can stain living cells, rose Bengal does so more effectively and is intrinsically toxic. However, a healthy pre-corneal tear film will block rose Bengal staining of healthy and damaged cells. The lack of a healthy tear film in keratoconjunctivitis sicca explains the usefulness of rose Bengal staining in that disorder. Cell degeneration or death increases membrane permeability to both dyes, but rose Bengal diffusion into the stroma is limited. Its usefulness is recognized in the evaluation of keratoconjunctivitis sicca, epithelial dendrites, and dysplastic or neoplastic lesions (Fig. 9.1). Because of the fluorescence property of fluorescein, examination of a fluorescein-stained cornea is enhanced by the use of a cobalt (blue) filter along with a yellow (Wratten #12) barrier filter. Conjunctival staining, otherwise often difficult to appreciate, becomes more visible. Fluorescein staining of healthy cells is limited, but fluorescein diffuses rapidly into the intercellular spaces or stroma when disruption of cell– cell junctions occurs. This diffusion property is responsible for the need to examine the cornea very soon after fluorescein is applied. Fine details of fluorescein staining may be lost after as little as 2–3 minutes (Fig. 9.2). One technique for visualizing staining (according to Korb) is as follows: instill a drop, have the patient blink three times, and wait 1–2 minutes. This is enough time for the stain to penetrate damaged epithelial cells but also leach out of the tear film.2 Pathologic conditions such as diabetes mellitus, as well as some medications, may increase epithelial permeability.3 As noted earlier, cell degeneration or death increases membrane permeability to both dyes, and given time, fluorescein will also stain dead cells.1 These properties of fluorescein dye are

CHAPTER 9 Corneal Diagnostic Techniques

Chapter Outline Corneal Staining Pachymetry Esthesiometry Summary

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A

B

Fig. 9.3  Cornea with depressed area two weeks after removal of a foreign body. (A) Demonstrates fluorescein in the depressed area. (B) After removal of fluorescein tear film with a wisp of cotton, the epithelium is found to be intact. Fig. 9.1  Ocular surface squamous neoplasia stained with rose Bengal. (Courtesy of EyeRounds.org and the University of Iowa.)

A

B

Fig. 9.2  Cornea stained with fluorescein strip demonstrating a herpes simplex dendrite. (A) Photo taken immediately after application of stain. (B) Photo taken three minutes later.

responsible for its usefulness in the various forms of epithelial defect, evaluating the status of pre-corneal tear film, in fitting contact lenses, in detection of aqueous humor leakage, and measuring epithelial or endothelial permeability. The technique of applying stain to the cornea can influence the information gathered for fluorescein. The clinician quickly learns “less is more.” A very small amount of concentrated dye yields much better diagnostic information than a full drop. In fact, a full drop may overwhelm the cornea and mask subtle findings. Therefore, dye strips may be more useful, as well as more sanitary, than corresponding solution. Placing a small drop in the middle or proximal end of the strip and letting it run down to the end provides a small but highly concentrated volume of corneal stain. Gross evidence of epithelial discontinuity can be seen easily after the instillation of dye but must be distinguished from pooling. Pooling of the fluorescein-impregnated tear film occurs in a depressed or irregular area of the cornea. The easiest method to distinguish pooling from staining uses a wisp of cotton from a cotton-tipped applicator or surgical sponge. In an anesthetized cornea, without blinking, the cotton wisp is used to absorb the fluorescein tear film in the area of concern. If the epithelium is intact, the pool of fluorescein will be removed and no staining in the base will be found (Fig. 9.3).

Fig. 9.4  Lissamine green staining in a 33-year-old non-contact lens wearer with severe keratoconjunctivitis sicca. (Courtesy of EyeRounds. org and the University of Iowa.)

Assessing the ocular surface with fluorescein dye staining is one of the most valuable examination techniques in assessing dry eye. Others include fluorescein tear break-up time (BUT), Schirmer test, and examination of the meibomian glands and their secretions. The monitoring and assessment of staining can be greatly enhanced by the use of a grading scale and standardized dye instillation and evaluation techniques. At least three grading systems (the van Bijsterveld system, the Oxford system, and a standardized version of the NEI/Industry Workshop system) are in current use or discussed in the International Dry Eye WorkShop (DEWS) report.4 Part of the difficulty in ocular surface disease diagnosis is the common scenario of a mismatch in the signs and symptoms. Indeed, the repeatability of both fluorescein and rose Bengal staining has been found to be poor.5 In contrast, the repeatability of serial Schirmer testing was moderate, repeatability of tear BUT was substantial, and repeatability of subjective symptoms (dryness and grittiness) was moderate to high. No single diagnostic test is a gold standard for diagnosis, but various combinations of tests have been recommended and shown to be more valid. Another biological stain used commonly is lissamine green (Fig. 9.4). At least as effective in evaluating the ocular surface as rose Bengal, Manning et al. showed that lissamine

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Diffuse Early bacterial Viral Medicamentosus

Inferior Staphylococcal blepharoconjunctivitis Trichiasis

Interpalpebral Keratitis sicca Photokeratopathy Exposure Inadequate blink

Superior Superior limbic keratitis Vernal conjunctivitis TRIC

Contact lens overwear

Mechanical abrasion Trichiasis

Fig. 9.5  Staining patterns of the cornea and conjunctiva in various disease states. TRIC, trachoma and inclusion conjunctivitis. (Reprinted with permission from Pavan-Langston D, ed. Manual of ocular diagnosis and therapy. Boston: Little Brown; 1991.)

green was better tolerated than rose Bengal by patients. Mean sensation score was significantly lower and duration of symptoms after dye instillation was shorter.6 Effects of lissamine green and rose Bengal were compared on proliferating human corneal epithelial (HCE) cells in vitro. Rose Bengal stained normal proliferating HCE cells and adversely affected HCE cell viability, unlike lissamine green, which demonstrated neither of these characteristics.7 When lissamine green is used, a relatively large volume (10–20 mL) is necessary to view staining maximally. A red barrier filter (Wratten #25) on the slit lamp will enhance staining. Characteristic corneal staining patterns may occur with corneal infections, inflammation, toxic changes, degenerative changes, allergic conditions, and ocular surface dryness. Staining may be diffuse, regional, or focal depending on the underlying cause. Noting both the location and the pattern of corneal staining will aid in diagnosis and management of corneal diseases (Fig. 9.5). For example, linear staining in the superior third of the cornea is typically found with a foreign body on the superior tarsal conjunctiva.8 Linear staining in a contact lens wearer indicates a foreign body beneath the lens. Superior bulbar conjunctival staining is characteristic of superior limbic keratoconjunctivitis. While some prefer to indicate any corneal staining as superficial punctate keratitis (SPK), it is more helpful to

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Fig. 9.6  Diffuse punctate epithelial erosions viewed with cobalt light in a dry eye patient. (Courtesy of EyeRounds.org and the University of Iowa.)

describe corneal staining precisely. Minute focal defects, visualized as small green dots at the slit lamp after fluorescein instillation, are best described as punctate epithelial erosions (PEE) (Fig. 9.6). While these are often the earliest stage of tear film instability or desiccation, they are also found in some infectious disorders. Epithelial lesions with focal inflammatory infiltrates within the epithelium have punctate staining but also have areas of negative stain and are known as punctate epithelial keratitis (PEK). Finally, subepithelial infiltrates (SEI) are deep to the epithelium and do not stain. Complete evolution of these staining patterns (PEE → PEK → SEI) occurs typically in some cases of adenoviral conjunctivitis and on occasion in herpes simplex, herpes zoster, chlamydia, and rosacea keratitis. Negative staining patterns may provide as much information as positive staining of the cornea. Negative staining refers to an elevated or irregular area of the cornea with intact epithelium in which the normal fluorescein tear film quickly dissipates. These patterns have diagnostic and therapeutic implications in cases of recurrent corneal erosion and anterior basement membrane dystrophy. Negative staining may also demonstrate elevated areas of the cornea that may be contributing to irregular astigmatism including corneal scars, Salzmann’s degeneration, or corneal striae after laserassisted in situ keratomileusis (LASIK).

Pachymetry Pachymetry, the measurement of corneal thickness, has become routine and is increasingly important in ophthalmic practice. Refractive surgeons invariably use central corneal thickness (CCT) in planning surgery,9 as adequate thickness is key in reducing the risk of developing postrefractive ectasia. Corneal thickness influences intraocular pressure (IOP) and glaucoma specialists have demonstrated that corneal thinning is an independent risk factor for development of glaucomatous optic neuropathy.10 Therefore, pachymetry is performed as a standard in glaucoma consultation. While corneal thickness is an indirect measurement of the endothelial pump function, it is also affected to a lesser degree by IOP.

CHAPTER 9 Corneal Diagnostic Techniques Pachymetry is an important indicator of corneal health but varies widely in “normal” patients. The thinnest part of the cornea is usually located about 1.5 mm temporal to the center of the cornea.11 Rapuano et al.12 measured 303 normal corneas and found a range of 410 to 625 µm, with a mean thickness of 515 µm in the central cornea. In the paracentral region, thickness varied from 522 µm inferiorly to 574 µm superiorly. In the peripheral zone, thickness was 633 µm inferiorly and 673 µm superiorly. No significant differences were noted in readings between right or left eyes, males or females, time of day, month of year, or systemic medication use.12 Paracentral and peripheral, but not central, measurements tended to become thinner with age, but this trend was not statistically significant.11 Since the absolute central value can vary significantly and still be “normal,” the relationship of central, midperipheral, and peripheral corneal thickness is important and should remain constant. The central area (within a 4 mm optical zone) is typically thinner than the midperipheral cornea (4–9 mm optical zone), which is thinner than the peripheral cornea (outside a 9 mm optical zone). Therefore, a cornea with a central thickness greater than the midperipheral thickness should be considered suspicious for endothelial dysfunction or midperipheral corneal thinning, regardless of absolute values. In fact, a patient with early endothelial compromise may have a CCT equal to the midperipheral corneal thickness. Corneal pachymetry is useful in determining the health of a cornea transplant. When performed serially, corneal pachymetry is useful in monitoring the functional health of the corneal endothelium in corneal transplants, including the progress of corneal deturgescence after keratoplasty (Fig. 9.7). Corneal pachymetry is also useful in determining and monitoring abnormalities in corneal structure and/or function, including disorders characterized by corneal thinning such as keratoconus and pellucid marginal corneal degeneration, and by corneal thickening including endothelial dysfunction due to Fuchs endothelial corneal dystrophy and herpetic disciform keratitis. One of the most common uses of corneal pachymetry is in assessing the extent of functional impairment of the endothelial pump in patients with Fuchs endothelial

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dystrophy or previous intraocular surgery before a planned intraocular procedure. If the IOP is normal, epithelial edema develops when the stroma has swollen about 40%, to a corneal thickness greater than 700 µm. If, however, swelling is only 20% or pachymetry demonstrates corneal thickness greater than 640 µm, the risk of corneal decompensation after cataract surgery is significant.13 In contact lens wear, corneal edema and hypoxia can develop in daily wear, extended wear, and therapeutic lens patients. Corneal swelling averages 4% during eye closure, 9–10% with extended wear lenses, 11–14% during sleep, and up to 18% with contact lens wear. Corneal striae become visible at 4–8%, folds are seen at 11–12% swelling, and loss of transparency can occur at greater than 20% swelling.14 High altitude causes a significant increase in CCT in healthy volunteers with normal corneas.15 Techniques for measuring CCT include optical pachymetry, ultrasound pachymetry, confocal microscopy, ultrasound biomicroscopy, optical ray path analysis or scanning slit corneal topography, and optical coherence tomography.16 Optical methods of pachymetry were first described as early as 1951 by Maurice and Giardini.17 Donaldson18 and Mishima19 also described manual slit lamp techniques to view the tear film or anterior corneal surface and the endothelial surface of the cornea. The Mishima−Hedbys fixation device for the Haag-Streit slit lamp reduced alignment problems. Various equations were used to calculate corneal thickness. Variables in these equations were the cornea’s refractive index and the anterior radius of curvature. These variables, along with the subjective nature of optical pachymetry readings, led to imprecise measurements and further investigation.12 Because of its subjective endpoints, the accuracy of optical pachymetry is partly dependent on the skill of the examiner. Advantages, however, include relatively low cost and noncontact technique.20 Some specular microscopes designed to evaluate the corneal endothelial cell count also measure corneal thickness using electromechanical devices. These were designed to measure central and apical readings only. The measurement derived is based on the distance from the posterior surface of the tear film to the posterior surface of the

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Fig. 9.7  Scheimpflug corneal thickness maps demonstrating (A) corneal edema preoperatively in a patient with Fuchs endothelial corneal dystrophy, (B) deturgescence one month after Descemet membrane endothelial keratoplasty (DMEK), and (C) pachymetric stability one year after DMEK. (Courtesy of the University of Iowa.)

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PART ii

Examining and Imaging the Cornea and External Eye

Section 1

Basic Evaluation of the Cornea and External Eye

Descemet membrane, thus inducing an error of as much as 20 or 30 µm. In the contact mode, corneal touch is involved and compression may be another source of error.20 Another optical method of measuring corneal thickness utilizes rotating Scheimpflug cameras to obtain corneal tomography images, available on devices such as the Pentacam (Oculus, Wetzlar, Germany) and Galilei (Ziemer, Port, Switzerland). Scheimpflug CCT measurements are highly reproducible and highly correlated with other measures of central corneal thickness but may not always be interchangeable with ultrasound pachymetry.21,22 An advantage of Scheimpflug imaging is the display of pachymetric values in a map that facilitates evaluation of regional changes in corneal thickness. Since its introduction in 1980, ultrasound technology has improved tremendously. Early units were more expensive, difficult to use, variable, and subject to alignment errors. Salz et al.23 compared optical pachymetry with three ultrasonic pachymeters and concluded that optical pachymetry had more intersession variation, significant intraobserver variation, and significant right–left thickness differences. Ultrasound pachymetry is not without disadvantages. Topical anesthesia is necessary due to direct contact with the cornea. Contact may be undesirable in the early postoperative period. The handheld nature of the ultrasound probe limits measurement accuracy.24 Sources of error in pachymetry may be “systematic” or inherent in the methods used in the procedure. Stucchi et al.25 studied several factors including repeated measurements, drying of the cornea, patient positioning, and marking. Repeated measurements of the same corneal point showed small variability (