Color Atlas of Nerve Biopsy Pathology [1 ed.] 0849316766, 9780849316760, 9781420039801

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COLOR ATLAS of

NERVE BIOPSY PATHOLOGY Shin J. Oh, M.D.

University of Alabama at Birmingham Department of Neurology Birmingham, Alabama

CRC Press Boca Raton London New York Washington, D.C.

Library of Congress Cataloging-in-Publication Data Oh, Shin J. Color atlas of nerve biopsy pathology / by Shin J. Oh p.; cm. Includes bibliographical references and index. ISBN 0-8493-1676-6 (alk. paper) 1. Nerves--Biopsy--Atlases. I. Title. [DNLM: 1. Nervous System--pathology--Atlases. 2. Biopsy--Atlases. 3. Nervous System Diseases--pathology--Atlases. WI 140 O36c 2001] RC409 .O46 2001 616.8'047'0222--dc21

2001025801 CIP

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from CRC Press LLC for such copying. Direct all inquiries to CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe.

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© 2002 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-1676-6 Library of Congress Card Number 2001025801 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper

Dedication This book is dedicated to my wife, Dr. Myung-Hi Kim Oh, Professor of Pediatrics, University of Alabama at Birmingham, my sons David and Michael, my daughter-in-law Bryn, and my grandson Braden, the newest addition to my family.

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Preface This book is based on my experience with approximately 2500 nerve biopsies collected during the past 30 years at the University of Alabama at Birmingham. Some of the cases that I did not have in my files were contributed by colleagues throughout the world. The nerve biopsy is now a well-established procedure in the regular practice of neurology, and its processing and interpretation have become integral parts of daily practice of pathology and neuropathology. The field of nerve biopsy pathology should, therefore, no longer be regarded as novel or exotic. This book should be useful in the everyday practice of pathologists and neuropathologists on the front lines of tissue diagnosis, as well as for neurologists who take a special interest in sural nerve biopsy pathology and neuromuscular diseases. I take great pride in the fact that this is the first nerve pathology book to introduce the diagnostic value of the extremely useful staining techniques of fresh-frozen sections. I have tried to provide all necessary practical knowledge regarding sural nerve biopsy pathology by means of a color atlas, which is based on commonly available frozen, paraffin, and semithin sections rather than on ultrastructural electron microscopy studies. The first five chapters present basic information on nerve biopsy, including the techniques of obtaining the nerve specimen, processing and staining methods of the biopsied nerve, and specific diagnostic pathological features. The next seven chapters present information on the nerve pathology of each disease in proportion to its commonness and importance for clinical practice from a biopsy standpoint. The clinicopathological correlation is introduced through the presentation of 46 cases which illustrate its value in the daily practice of neurology. I hope this book contains sufficient practical information on nerve pathology so that every practicing pathologist, neuropathologist, and neuromuscular disease specialist will find it an invaluable companion in his or her daily practice.

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Acknowledgments I thank my wife, Dr. Myung-Hi Kim Oh, for the steadfast encouragement and emotional support which she has provided me over a period of many years, from the conception of this book through its final writing. I also thank my administrative assistant, Dr. Mary Ward, for her masterful help in editing the manuscript, and my laboratory technologists, Judy Killian, Cheryl Snyder, Susan Lett, and Debbie Reynolds, for their superb technical assistance in the processing and staining of many hundreds of biopsied nerve specimens. In addition, I want to thank Drs. Yadollah Harati, Cheryl Palmer, and David Simpson, and Professors M.R.G. de Freitas, O.J.M. Nasmundo, N. Roertson, and Il Nam Sunwoo for providing color slides of their own cases. Finally, I thank Carol Hollander, Jonathan Pennell, and Judith Simon Kamin at CRC Press for their help in the production of this book.

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Author Born in Seoul, Korea, Dr. Shin J. Oh is professor of neurology and pathology at the University of Alabama at Birmingham. He serves as director of the Muscle/Nerve Histopathology Laboratory as well as director of the Department of Clinical Neurophysiology and the Electromyography and Evoked Potentials Laboratory. During his 30-year tenure at UAB, he established and brought the UAB Neuromuscular Disease Program to national and international prominence, published numerous articles, chapters, and abstracts, on electrodiagnosis and neuromuscular diseases, and trained more than 50 fellows, including many from Korea, Turkey, Japan, Poland, Colombia, and Brazil. He is the author of three EMG text books: Clinical Electromyography: Nerve Conduction Studies (1982 and 1993); Electromyography: Neuromuscular Transmission Study (1988); and Principles of Clinical Electromyography: Case Studies (1998). Dr. Oh serves on several medical advisory and journal review boards and, in recent years, has been an invited lecturer in Australia, Colombia, the Czech Republic, Korea, New Zealand, Turkey, and the United States. His interests include myasthenia gravis, Lambert–Eaton myasthenia syndrome, tarsal tunnel syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), vasculitic neuropathy, and nerve biopsy. His special interest in nerve biopsy led him to recognize early the subacute form of CIDP, chronic sensory demyelinating neuropathy, sensory Guillain–Barré syndrome, multifocal motor-sensory demyelinating neuropathy, and the diagnostic value of sural nerve biopsy in vasculitic neuropathy. Finally, this led him to write the classic paper on the diagnostic usefulness and limitation of sural nerve biopsy, which is the forerunner of this book.

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Table of Contents Chapter 1 General Concepts of Peripheral Neuropathy Classification of Peripheral Neuropathy Basic Pathological Mechanism Axonal Degeneration Wallerian Degeneration Dying-Back Axonal Degeneration Axonal Degeneration in Neuronopathy Secondary Axonal Degeneration Segmental Demyelination Secondary Segmental Demyelination Etiologies of Peripheral Neuropathy Types of Neuropathies Pattern of Involvement Polyneuropathy Mononeuropathy Multiplex Mononeuropathy Systemic Involvement Size of Nerve Fibers Symptoms and Signs Motor Nerve Dysfunction Sensory Nerve Dysfunction Autonomic Nerve Dysfunction Diagnostic Investigations Nerve Conduction Studies and Needle Electromyography Laboratory Studies References Chapter 2 The Nerve Biopsy Indication for the Nerve Biopsy Types of Nerve Biopsy Sural Nerve Biopsy Sequelae of Nerve Biopsy Biopsy of Other Nerves Superficial Peroneal Nerve Biopsy Superficial Radial Nerve Biopsy References Chapter 3 Histological Processing and Staining of the Biopsied Nerve Treatment of the Biopsied Nerve Immediate Care of the Biopsied Nerve

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Processing of the Nerve Paraffin Section Stainings Hematoxylin and Eosin Stain Modified Trichrome Stain Alkaline Congo-Red Stain Frozen Section Stainings Modified Trichrome Stain Hematoxylin and Eosin (H & E) Stain PASH (Periodic Acid Schiff and Hematoxylin) Stain Hirsch-Peiffer Cresyl-Violet Stain Alkaline Congo-Red Stain Processing of the Nerve for Semithin and Electron Microscopy Sections Processing and Embedding Procedure Semithin (0.5 – 1µm) Section Stainings Toluidine Blue and Basic Fuchsin Stain (Paragon Multiple Stain) Toluidine Blue Stain Other Stains Nerve Fiber Teasing Preparation of Nerve for Teasing General Guidelines for Teasing of Fibers Practical Tips for Teasing Fibers Preparing the Slide after Teasing Electron Microscope Study References Chapter 4 Normal Nerve: Histology Age-Related Changes in the Sural Nerve Biopsy References Chapter 5 Specific Diagnostic Pathological Features of Nerve Biopsy Vasculitis Amyloid Deposits Metachromatic Granules Polyglucosan Body Onion-Bulb Formation Inflammatory Cells and Segmental Demyelinatio Inflammatory Cells and Axonal Degeneration Noncaseating Granuloma Necrotizing (Caseating) Granuloma Giant Axons Tomacula Occlusion of Vasa Nervorum Malignant Cells IgM Deposits Segmental Demyelination Axonal Degeneration References

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Chapter 6 Vasculitic Neuropathy Vulnerability of the Peripheral Nerve to Vasculitic Neuropathy Clinical, Electromyographic, and Laboratory Features Diagnostic Sensitivity of Nerve and Muscle Biopsies Pathology of Vasculitic Neuropathy Pathogenesis of Vasculitic Neuropathy Systemic Necrotizing Vasculitides Polyarteritis Nodosa Churg–Strauss Syndrome (Allergic Granulomatosis) Wegener’s Granulomatosis Temporal (Giant Cell) Arteritis Vasculitis Associated with Connective Tissue Diseases Rheumatoid Arthritis Systemic Lupus Erythematosus (SLE) Sjögren’s Syndrome Hypersensitivity Vasculitis (HSV) Nonsystemic Vasculitic Neuropathy Vasculitis in Other Diseases Cases with Vasculitic Neuropathy Case 1: A Patient with Fever of Unknown Etiology for 1 Month Case 2: Numbness in the Right Foot in a Patient with Asthma Case 3: Numbness and Weakness in the Left Leg in a Patient with Endometrial Carcinoma Case 4: Hepatitis C, Cryoglobulinemia, and Vasculitic Neuropathy Case 5: Numbness and Pain in Legs with INH Treatment Case 6: High Sedimentation Rate in a Patient with Subacute Symmetrical Polyneuropathy Case 7: 3-Month History of Mononeuropathy Multiplex Case 8: Guillain–Barré Syndrome? Case 9: Progressive Multifocal Motor and Sensory Deficits over 3 Months References Chapter 7 Inflammatory Demyelinating Neuropathy Pathogenesis of Inflammatory Demyelinating Neuropathies Guillain–Barré Syndrome (Acute Inflammatory Demyelinating Polyneuropathy; AIDP) Variants of GBS Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) Multifocal Motor Neuropathy (MMN) Multifocal Motor Sensory Demyelinating Neuropathy (MMSDN) Chronic Sensory Demyelinating Neuropathy (CSDN) Cases of Inflammatory Demyelinating Neuropathy Case 1: Acute Motor Neuropathy with Axonal Neuropathy Case 2: Relapse of GBS Case 3: Acutely Developing "Ileus" Case 4: Subacute Sensory-Motor Neuropathy with 13 Negative Biopsies

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Case 5: Diffuse Areflexia in an MS Patient Case 6: Subacute Sensory-Motor Weakness after a Flu Vaccine Case 7: Uniform Slowing in the Nerve Conduction Study Case 8: Chronic Motor Weakness with Fasciculation and Hyperreflexia Case 9: Flail Arms for 3 Years Case 10: MMN with Sensory Deficits Case 11: Painful Sensory Neuropathy for 5 Years References Chapter 8 Immune-Mediated Neuropathies GM1 Antibody-Positive Neuropathy Anti-MAG Associated Neuropathy Neuropathy Associated with Anti-Hu (ANNA 1) Antibody Neuropathy with Monoclonal Gammopathy Polyneuropathy Associated with Monoclonal Gammopathy of Undetermined Significance (MGUS) Peripheral Neuropathy Associated with Osteosclerotic Myeloma (OSM) Peripheral Neuropathy Associated with Typical Multiple Myeloma (MM) Neuropathy Associated with Waldenström’s Macroglobulinemia (WM) Peripheral Neuropathy with Cryoglobulinemia Cases of Immune-Mediated Neuropathy Case 1: Numbness and Tingling Sensation in the Hands for 12 Years Case 2: Progressive Unsteady Gait for 5 Months in a Smoker Case 3: 2-Month History of Numbness of Hands and Feet in a 68-Year-Old Man Case 4: Progressive Sensory-Motor Neuropathy, Biclonal Gammopathy, Skin Discoloration, Pleural Effusion, and Hepatomegaly for 4 Years Case 5: Progressive Weakness of Legs for 6 Months in a Patient with History of Lymphadenopathy References Chapter 9 Neuropathies with Abnormal Deposits Amyloid Neuropathy Familial Amyloid Polyneuropathy (FAP) Nonfamilial Amyloid Neuropathy Pathology of Amyloid Neuropathy Metachromatic Leukodystrophy (Sulfatide Lipidosis; Arylsulfatidase Deficiency) Polyglucosan Body Neuropathy Fabry’s Disease (Alpha-Galactosidase-A Deficiency) Adrenomyeloneuropathy (AMN) Cases of Neuropathy with Abnormal Deposit Case 1: 6-Month History of Burning Dysesthesia in All Limbs and 4-Year History of Impotence Case 2: Delayed Walking and Hand Tremors in a 27-Month-Old Girl Case 3: A 2-Year History of Parkinsonism, Upper Motor Neuron Signs, and Peripheral Neuropathy

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References Chapter 10 Hereditary Neuropathies Hereditary Motor and Sensory Neuropathies (HMSN) HMSN Type I (Hypertrophic Form of the CMT Disease Including Roussy–Levy Syndrome) Roussy–Levy Syndrome Sex-Linked CMT HMSN Type II (Neuronal Type of CMT; CMT 2) HMSN Type III (Dejerine–Sottas Disease; DSA and DSB) Congenital Hypomyelination Neuropathy Autosomal Recessive CMT (CMT 4; CMT 4B) Hereditary Sensory Neuropathy Type I Hereditary Sensory Neuropathy (Hereditary Sensory Radicular Neuropathy of Denny–Brown; Dominant HSN; HSAN Type I) Type II HSN (Congenital Sensory Neuropathy; Recessively Inherited HSN; HSAN Type II) Hereditary Neuropathy to Pressure Palsy (HNPP) Giant Axonal Neuropathy (GAN) Friedreich’s Ataxia Cases with Hereditary Neuropathy Case 1: Hand-Shaking as an Initial Manifestation of Hereditary Neuropathy Case 2: CMT Patient with Conduction Block Case 3: Autosomal Recessive CMT with Focally Folded Myelin Case 4: 3-Year Worsening of Gait Difficulty, Present Since Early Childhood Case 5: Global Weakness and Sensory Loss in the Entire Left Arm in a Worker’s Compensation Case Case 6: A 31-Year-Old Woman with Numbness and Tingling Sensation in the Legs for 6 Months Case 7: Progressive Walking Difficulty for 19 Months in a Child With Insulin-Dependent Diabetes Mellitus References Chapter 11 Metabolic and Systemic Neuropathies Sarcoid Neuropathy Sensory Perineuritis Leprosy Lymphomatous Neuropathy Diabetic Neuropathy Diabetic Ophthalmoplegia Diabetic Amyotrophy (Diabetic Proximal Neuropathy) Diabetic Sensory Neuropathy Diabetic Polyneuropathy Uremic Neuropathy Alcoholic Neuropathy Hypothyroid Neuropathy

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Vitamin B12 Deficiency Neuropathy Pyridoxine-Induced Sensory Neuropathy Polyradiculoneuropathy in Lyme Disease AIDS Neuropathy Cases of Neuropathy Associated with Systemic Diseases Case 1: Subacute Symmetrical Polyneuropathy for 6 Months Case 2: Subacute Peripheral Neuropathy with White Matter Disease in the Brain MRI Case 3: Neuropathy in a Type I Diabetic with Many Microangiopathy Complications Case 4: 9 Months of Progressive Neuropathy in a 72-Year-Old Woman with Insulin-Dependent Diabetes Mellitus for 15 Years Case 5: Uremic Neuropathy in a Young Patient Whose Two Brothers Had Renal Problems Case 6: Subacute Neuropathy in a Chronic Alcoholic Patient Case 7: Paresthesia of Feet and Abdominal Colic at the Onset of Neuropathy References Chapter 12 Toxic Neuropathies Metal Neuropathies Arsenic Neuropathy Thallium Neuropathy Lead Neuropathy Cisplatinum Neuropathy Drug-Induced Neuropathy Neuropathy Due to Biological Toxins and Vaccines Diphtheritic Neuropathy Vaccine-Induced Neuropathy Toxic Neuropathy Due to Industrial and Environmental Agents Epidemic Toxic Inflammatory Neuropathies Spanish Toxic Oil Syndrome Eosinophilia–Myalgia Syndrome Cases of Toxic Neuropathies Case 1: Subacute Neuropathy in a 19-Year-Old Girl with Possible Anorexia Nervosa Case 2: Progressive Ascending Weakness in the Extremities and Numbness in the Toes for a Few Months Case 3: Subacute Progression of Weakness for 31/2 Months after Swine-Flu Vaccination Case 4: Guillian–Barré Syndrome Following Ingestion of an Unknown Amount of Antifreeze References Chapter 13 Interpretation of Nerve Biopsy References

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1

General Concepts of Peripheral Neuropathy

Peripheral neuropathy is one of the most common neurological disorders and refers to a disease involving the peripheral nerves, including motor, sensory, and autonomic nerves, with predominant clinical manifestations of weakness, loss of sensation, and muscle wasting. The frequency of peripheral neuropathy is not known, but it is a common feature of many systemic diseases. Diabetes is the most common cause of peripheral neuropathy in adults living in developed countries. Considering that 1.3% of the general population of the United States has diabetes mellitus and that roughly 25% of diabetic patients have peripheral neuropathy, peripheral neuropathy is considered a common disease. In fact, 1 out of 300 individuals has peripheral neuropathy associated with diabetes. This figure excludes other causes of peripheral neuropathy. The most important distinction between the central and peripheral nervous systems is the ability of the peripheral nerves to regenerate after disease or injury. Thus, the chance of clinical improvement is better in peripheral neuropathy than in any central nervous system diseases.

CLASSIFICATION OF PERIPHERAL NEUROPATHY There are two major anatomic components of the peripheral nerves — axons and myelin. Peripheral nerve axons are simply cytoplasmic extensions of the neurons. The axons are responsible for the maintenance and function of the peripheral nerves and derive most of the protein essential for this purpose from the neurons. Along the axons, membrane components, organelles, nutrients, and metabolic products are transported as axoplasm at different velocities in both directions.1 This system renders the axons extremely vulnerable to any metabolic changes in the neurons. Thus, severe damage to the neurons and disruption of proximal axonal integrity result in rapid degeneration of the entire distal portion of the axons. On the other hand, injury to the distal portion of the axons does not result in permanent damage to the neurons; the latter undergo transient swelling and breakdown of the endoplasmic reticulum (chromatolysis), but they usually survive and support regeneration of the damaged axons.2 The myelin of peripheral nerves is derived from the Schwann cells and is dependent on both the Schwann cells and the axons for its continued integrity. Myelin is responsible for the conduction of nerve action potentials along the nerves. This is due to saltatory conduction in myelinated fibers. Schwann cells envelop axons to form unmyelinated and myelinated fibers surrounded by basal lamina. A single Schwann cell occupies each myelinated internode, almost never associating itself with more than one axon.3 Damage to the axons results in the prompt breakdown of myelin but not of the Schwann cells. On the other hand, loss of myelin does not usually result in disruption of the axons. An axon denuded of several segments of myelin simply awaits Schwann cell division and remyelination before resuming normal impulse conduction.4 Depending on which component of the peripheral nerve is predominantly involved in the pathological process, peripheral neuropathy can be classified into two main categories: axonal degeneration and segmental demyelination (Table 1.1). There are also clear pathophysiological differences between axonal degeneration and segmental demyelination, as noted in Table 1.1.

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TABLE 1.1 Pathophysiology of Two Types of Peripheral Neuropathy Type

Axonal Neuropathy

Demyelinating Neuropathy

Primary lesion Pathological process Pathology by teasing preparation Regeneration: mechanism speed Nerve conduction: velocity

Axon Axonal degeneration Myelin ovoids

Myelin Demyelination Segmental demyelination

Axonal sprouting Slow

Remyelination Rapid

Mildly slow: above 30 m/sec Low amplitude

Markedly slow: below 30 m/sec Dispersion: conduction block

(++++)

(-) or (±)

Absent Arsenic, thallium, gold Alcoholic Nutritional Vasculitic Giant axonal Porphyric neuropathy Vitamin B12 Diabetic neuropathy Uremic neuropathy

Present in chronic form Guillain–Barré syndrome CIDP Hypertrophic Metachromatic Tomaculous Leprosy Multifocal motor neuropathy Diphtheric Charcot–Marie–Tooth 1 A

CMAP: Needle EMG: Fibrillation and positive sharp wave Fasciculation Examples:

Abbreviations: CMAP, Compound muscle action potential CIDP, Chronic inflammatory demyelinating polyneuropathy EMG, Electromyography

BASIC PATHOLOGICAL MECHANISM The peripheral nerves have a limited means of reacting to disease, i.e., axonal degeneration and segmental demyelination. It should be pointed out that an element of a minor process may coexist with the predominant process in almost all peripheral neuropathies.

AXONAL DEGENERATION The disease process affects axons primarily by producing axonal degeneration and secondarily by causing breakdown of the myelin sheath. Axonal degeneration is induced by three different mechanisms: (1) axonal degeneration distal to the site of transection of the nerve (Wallerian degeneration); (2) degeneration of the distal axons due to a metabolic derangement throughout the axon (dying-back degeneration; axonopathy) (Figure 1.1); and (3) axonal degeneration following morphologic or metabolic derangement in the neuron cell body (neuronopathy) (Color Figure 1.1).* Wallerian Degeneration The classical description of axonal degeneration following transection of a nerve was provided by Waller in 1850.5 When a nerve is totally transected, continuity of the axon is broken. As in all cells, the * Color insert figures.

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FIGURE 1.1 Mechanism of axonal degeneration and regeneration. Axonal degeneration is induced either by a metabolic derangement either in the neuron cell body (motor neuronopathy) or throughout the axon (dying-back axonal degeneration) (early; arrows). Damage to the neurons and disruption of proximal axonal integrity result in rapid degeneration of the entire distal portion of axon, producing breakdown of the myelin sheath (late). Regeneration occurs with axonal sprouting. (Reproduced with permission from Oh, S.J., Diagnostic usefulness and limitations of the sural nerve biopsy, Yonsei Med. J., 1990;31; 2.)

part of the cytoplasm (axon) separated from the nucleus (neuron) gradually degenerates, producing axonal degeneration in the portion distal to the transection. The cardinal features of Wallerian degeneration are as follows: • It results from transection of an axon. • Paralysis and anesthesia in the distribution of the nerve are immediate, due to the conduction failure of the nerve impulse across the transected segment. • Axons and myelin sheaths degenerate distal to the site of transection. • Conduction over the distal segment fails in 3 or 4 days as the distal nerve becomes inexcitable. • Distal muscles undergo denervation atrophy. • Prominent fibrillation and positive sharp waves occur in distal muscles in 8–14 days. • Nerve cell chromatolysis may occur in severe cases. • A burst of Schwann cell proliferation takes place distal to transection. • Regeneration from the proximal stump begins early but proceeds slowly by the process of axonal sprouting at the rate of 2 or 3 mm per day. • Recovery is variable and depends upon (1) intactness of the neural tube (endoneurium, perineurium, and epineurium) — when the neural tube is intact, regeneration occurs spontaneously and is of excellent quality; (2) the proximo–distal site of injury — the more distal the lesion, the better the recovery; (3) the age of the individual — the younger the patient, the better the recovery; and (4) the closeness of approximation of the severed ends and the degree of adjacent soft-tissue injury — the closer the approximation of the severed ends and the less the degree of adjacent soft tissue injury, the better the recovery.

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Wallerian degeneration occurs following direct trauma to a nerve. In peripheral neuropathy, Wallerian degeneration occurs in vasculitic neuropathy. It is generally believed that in the vasculitides, the nerve fiber damage results from local ischemia severe enough to produce focal axonal damage and distal Wallerian degeneration.6

DYING-BACK AXONAL DEGENERATION Initially, a metabolic abnormality occurs throughout the axons (Color Figure 1.1 and Figure 1.1). Failure of axon transport results in degeneration of vulnerable distal regions of long or large-diameter axons.7 Degeneration appears to advance proximally toward the nerve cell body (dying-back). The clinical effect of this phenomenon is distal symmetrical polyneuropathy. The cardinal features of dying-back axonal degeneration are: • Metabolic abnormalities throughout the axon. • Initial distal axonal change. • Eventual axonal degeneration resembling Wallerian degeneration except that the early ultrastructural changes are spread over a much longer period.8 The myelin sheath breaks down concomitantly with the axon. Secondary demyelination and remyelination may occur more proximally, where the axon is still intact. • Normal or mildly slow conduction until it fails completely. The amplitudes of compound muscle action potential (CMAP) and compound nerve action potential (CNAP) are markedly reduced. • Denervation atrophy in distal muscles. • Prominent fibrillation or positive sharp waves in distal muscles. • Chromatolysis is sometimes present in severe cases. • More indolent and prolonged Schwann cell proliferation than in Wallerian degeneration.9 • Schwann cells and basal lamina tubes remaining in distal nerves and facilitating appropriate peripheral regeneration.

FIGURE 1.2 Mechanism of sensory neuronopathy and regeneration. Sensory neuronopathy is induced by metabolic derangement in the dorsal root ganglion (at onset). Degeneration of these cells is accompanied by fragmentation and phagocytosis of the peripheral-central processes (early). The Schwann cells remain; there is no axonal regeneration (late).

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• Recovery by axonal sprouting that reinnervates denervated muscles. • Slow recovery, proceeding at a rate of 2 or 3 mm per day, sometimes partial, depending on the basis of the neuropathy and its severity. The majority of metabolic and toxic neuropathies are due to this mechanism. Characteristically, the disease is insidious on onset, commences distally, and slowly proceeds toward the neuron cell body, resulting in symmetrical distal polyneuropathy. Axonal Degeneration in Neuronopathy In this process, the primary target of the disease process is in the nerve cell body. (Color Figure 1.1). Either the lower motor neurons or the primary sensory neurons may be affected. Thus, clinical manifestations depend on whether the affected neurons are motor or sensory. When the anterior horn cells are the target of disease, pure motor impairment is the consequence, as noted in poliomyelitis, motor neuron diseases, and the neuronal type of the Charcot–Marie–Tooth disease (HSMN Type II). When dorsal root ganglia cells are the target of disease, a pure sensory neuronopathy syndrome occurs, as in acute sensory neuropathy,10 herpes zoster, carcinomatous sensory neuropathy, and hereditary sensory autonomic neuropathy Type II (Color Figure 1.1 and Figure 1.2). The cardinal features are listed below: • Morphologic or metabolic abnormalities in the motor or sensory neurons. • Pathological changes appearing in the neuronal perikaryon, soon followed by axonal degeneration throughout the length of the axon. Clinically, widespread manifestation is the rule. • Axonal degeneration confined to the nerve that is controlled by the involved neurons: motor nerves in anterior horn cell diseases and sensory nerves in dorsal root ganglia diseases. • Eventual axonal degeneration resembling Wallerian degeneration, though the process is much slower. Myelin sheath breakdown concomitant with that of the axon. • Nerve conduction abnormality depending on the selective nerve cell loss. In anterior horn cell diseases, sensory nerve conduction is normal, and motor nerve conduction shows findings typical of axonal degeneration (motor neuronopathy pattern). In dorsal root ganglia diseases, normal motor nerve conduction and markedly abnormal sensory nerve conduction (sensory neuronopathy pattern), either absent CNAP or markedly reduced CNAP amplitude, are the rule. • In anterior horn cell diseases, prominent denervation atrophy and muscle weakness are the characteristic findings. In dorsal root ganglia diseases, loss of sensation and sensory ataxia reflecting the disappearance of sensory neurons are the usual findings. Sensory syndrome differs depending on the selective involvement of small or large cells in the dorsal root ganglia; pain and temperature are predominantly affected in small cell loss and proprioception is mainly affected in large cell loss. • Prominent fibrillation, positive sharp waves, and fasciculation in distal muscles in anterior horn cell diseases. • Regeneration occurring through collateral sprouting from surviving axons. However, recovery is usually poor.11 This is especially true in sensory neuronopathy.

SECONDARY AXONAL DEGENERATION It is well known that axonal degeneration occurs following severe primary demyelination in human neuropathies12,13 and in experimental demyelinating neuropathy.14 The most likely mechanism is that the Wallerian degeneration is initiated at sites of severe segmental demyelination.12 The electromyography

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test shows prominent fibrillation and positive sharp waves in addition to findings typically seen in segmental demyelination. In the Guillain–Barré syndrome, profuse fibrillations and positive sharp waves within the first four weeks of the illness, indicative of severe axonal degeneration, are associated with a prolonged recovery time and more pronounced residual deficits.15 In the entrapment neuropathies, axonal degeneration over the segment distal to the entrapment site is a well-known observation. This accounts for the minimal motor and sensory nerve conduction abnormalities distal to the entrapment site in entrapment neuropathies.

SEGMENTAL DEMYELINATION About 30 years after Waller’s classic description of axonal degeneration, Gombault16 described segmental demyelination in the nerves of a guinea pig with chronic lead intoxication (Color Figure 1.1 and Figure 1.3). Myelin sheath damage occurred in the internodal segments with sparing of axons. Each segment represented the length of one Schwann cell and its myelin sheath. The cardinal characteristics of segmental demyelination are: • Primary damage of the myelin sheath, leaving the axon intact. • Demyelination, usually beginning at the nodes of Ranvier. Segmental demyelination is induced by various mechanisms, including (1) metabolic damage of Schwann cells, as noted in diphtheric neuropathy; (2) telescoping (intussusception) of myelin, as noted in entrapment neuropathies; (3) edema formation within the myelin sheath, as noted in galactocerebroside neuropathy; and (4) peeling and engulfment of myelin by activated lymphocytes and macrophages, as noted in the Guillain–Barré syndrome. • Segmental demyelination, which may be diffuse, multifocal, or focal. • Conduction block or marked slowing of nerve conduction resulting from segmental demyelination. • Absent or rare fibrillation and positive sharp waves, but fasciculation not uncommon.

FIGURE 1.3 Mechanism of segmental demyelinaton and remyelination. Segmental demyelination is induced by metabolic damage of Schwann cells or peeling and engulfment by activated inflammatory cells (early). This process affects the myelin sheath producing primary segmental demyelination and leaving the axon intact (late). Remyelination occurs with myelination over demyelinated segment. (Reproduced with permission from Oh, S.J., Diagnostic usefulness and limitations of the sural nerve biopsy, Yonsei Med. J., 1990;31; 2.)

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• • • •

No denervation atrophy in muscles. Disuse atrophy occurs if paralysis is prolonged. Chromatolysis of the nerve cell body does not occur. Schwann cell proliferation is not as brisk as in Wallerian degeneration.9 The Schwann cell division and remyelination of the axon forms short internodes of thin myelin in the remyelination process. Once remyelination begins, rapid and usually complete recovery occurs. • In cases of repeated demyelination and remyelination processes, Schwann cells divide again and some of the daughter cells are unable to find a segment of axon to surround. They become detached and form a thin layer around the fibers. Thus, onion bulbs are formed.

SECONDARY SEGMENTAL DEMYELINATION This concept has recently been well documented by Dyck et al.17,18 They described segmental demyelination over many consecutive internodal segments along atrophic axons in Friedreich’s ataxia and uremic neuropathy, taking the view that segmental demyelination is the result of primary axonal degeneration. In contrast to primary demyelination, which tends to show a random distribution of segmental demyelination, secondary demyelination is characterized by segmental demyelination over many consecutive internodes. The needle EMG study shows findings typical of primary axonal degeneration.

ETIOLOGIES OF PERIPHERAL NEUROPATHY The most common known cause of peripheral neuropathy in the United States is diabetes mellitus, followed by chronic alcoholism, whereas in the world as a whole the most common cause is leprosy. For neurology patients, the most common cause of peripheral neuropathy is the Guillain–Barré syndrome (GBS). Despite extensive and costly evaluations, the causes of peripheral neuropathy remain unknown in a substantial number of cases. In 2 studies conducted in the 1980s, the causes were undetermined in only 13 to 24% of cases (Table 1.2). These figures were reported from centers where the sural nerve biopsy is used extensively to identify the cause of peripheral neuropathy. Compared with 52 to 70% in the 1960s, the frequency of unknown causes has decreased over the years. This decrease is due mainly to four factors: (1) greater sophistication of the electrophysiological study of differentiation between axonal neuropathy and demyelinating neuropathy; (2) classification of inflammatory neuropathies such as GBS as a known cause; (3) monoclonal neuropathy and paraneoplastic neuropathy are now known causes of some neuropathies, and (4) increasing use of the nerve biopsy in the work-up for peripheral neuropathy.

TYPES OF NEUROPATHIES Neuropathies can be categorized based on disease mechanisms and the size of involved nerve fibers, as discussed above, the pattern of involvement, and the clinical manifestation of diseases. All of these are helpful for diagnosis, for detection of the cause of neuropathy, and, eventually, for treatment.

PATTERN OF INVOLVEMENT The pattern of involvement can be either polyneuropathy, mononeuropathy multiplex, or mononeuropathy. This distinction is important because it provides the most helpful clinical clue as to the cause of the neuropathy.

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TABLE 1.2 Frequency of Unknown Causes in Peripheral Neuropathy Authors

Case Number 46

Unknown Cause (%) 70

Rose (1960)

80

56

GBS is listed as a known cause.

Prineas (1970)

278

14

GBS is listed as a known cause.

Dyck (1981)

205

24

Inflammatory neuropathy is listed as a known cause.

Fagius (1983)

91

74

Chronic inflammatory or hereditary neuropathies are listed as unknown causes.

519

13

Inflammatory neuropathy is listed as a known cause. All cases had sural nerve biopsy.

Mathews (1956)

McLeod (1984)

Comments GBS is listed as an unknown cause.

Polyneuropathy A polyneuropathy is a symmetrical, distal, usually ascending neuropathy due to involvement of the distal branches of nerves. Stocking-glove dysesthesia is the classic term describing the distribution of sensory impairment. Tingling, numbness, and pain, as well as sensory loss, occur in a symmetrical stocking or glove distribution in the feet or hands. Foot drop is common due to weakness of the lower leg muscles. Mixed sensorimotor polyneuropathy suggests nutritional neuropathy (due to alcoholism, beriberi, vitamin B deficiency, or pernicious anemia), metabolic neuropathy (caused by diabetes mellitus or uremia), and toxic neuropathy. Sensory polyneuropathy suggests a benign idiopathic sensory neuropathy, neuropathy related to diabetes or pernicious anemia, chronic sensory demyelinating neuropathy, and arsenic neuropathy. Sensory ataxic neuropathy is classically seen in paraneoplastic polyneuropathy or Sjögren’s neuropathy. Motor polyneuropathy suggests Guillain–Barré syndrome or chronic inflammatory demyelinating polyneuropath neuropathy (CIDP). Proximal neuropathy is rare and found mostly in inflammatory polyneuropathies such as GBS and CIDP. Cranial neuropathy can produce ophthalmoplegia, and swallowing and speech difficulty. GBS and Lyme disease frequently involve the facial nerves. Respiratory muscle weakness is rare but is one of the most dreadful symptoms of GBS because it is life threatening. Mononeuropathy Multiplex Mononeuropathy multiplex involves two or more nerves in more than one extremity, e.g., left ulnar neuropathy and right peroneal neuropathy. This is classically seen in vasculitic neuropathy. Two other causes of mononeuropathy multiplex are leprosy and diabetes mellitus. A rare cause of this disease is multifocal demyelinating neuropathy, including multifocal motor neuropathy (MMN) and multifocal motor-sensory demyelinating neuropathy (MMSDN). The detection of MMN is especially important because many patients may be misdiagnosed with amyotrophic lateral sclerosis (ALS). MMN is a treatable disease that responds to intravenous immunoglobulin (IVIG) treatment.

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Mononeuropathy The most common cause of mononeuropathy is entrapment neuropathy due to the compression of a nerve in an anatomically narrow area. The best example of this is carpal tunnel syndrome. Certain mononeuropathies are common to certain diseases, for example, femoral neuropathy and ophthalmoplegic neuropathy with pupil sparing in diabetes mellitus, recurrent or bilateral facial nerve palsy in sarcoidosis and Lyme disease, and radial nerve palsy in lead neuropathy.

SYSTEMIC INVOLVEMENT Systemic involvement deals with motor, sensory, autonomic, and mixed neuropathy. Pure motor and sensory neuropathies are described above. Most toxic and metabolic neuropathies are mixed motorsensory neuropathies.

SIZE OF NERVE FIBERS Large-fiber neuropathy is characterized by motor weakness and loss of vibration and position sense. Most neuropathies are large-fiber neuropathies and are thus easily detectable by the nerve conduction study, which usually tests the large fibers of nerves. Small-fiber neuropathy is a painful sensory neuropathy that occurs in diabetes, alcoholism, amyloidosis, leprosy, and AIDS.

SYMPTOMS AND SIGNS MOTOR NERVE DYSFUNCTION When motor nerves are affected, the primary manifestation is weakness. Muscle wasting may follow if the weakness persists. Distal leg weakness produces foot drop, causing patients to trip on their toes because they cannot fully flex their foot as they walk. Proximal leg weakness is most commonly reported as difficulty in getting out of a chair or climbing stairs. Weakness of the hands affects grip and fine coordination, such as that needed for writing or fastening buttons. Proximal weakness of the arms often causes difficulty with such routine chores as carrying groceries and brushing the teeth and hair. Occasionally, cramps or twitching (fasciculation) are described in motor nerve dysfunction. On examination, in addition to muscle weakness, muscle wasting and diminished tone may be seen in motor nerve dysfunction. Reflexes are also diminished or absent because the motor nerve is an efferent limb of the reflex arc. When the cranial nerves are involved, patients may have double vision due to paresis of the eye muscles, as well as swallowing and speech difficulty due to bulbar paresis. When the respiratory muscles are involved, breathing difficulty occurs. This is a life-threatening warning sign because respiratory dysfunction is the killer in peripheral neuropathy.

SENSORY NERVE DYSFUNCTION When sensory nerves are affected, the primary manifestation is abnormal sensation (dysesthesia). This complaint is usually reported as tingling, numbness, dead feeling (like a shot of novocaine), burning, or pain. In fact, pain is the most common complaint that brings patients with peripheral neuropathy to the physician. Patients with significant sensory loss in their feet may complain that they feel like they are walking on sand or are unsteady in a dark room because the visual input that usually compensates for the numbness is absent (sensory ataxia). On examination, decreased or absent pin-prick and temperature sensations are noted when small myelinated fibers are affected (small-fiber neuropathy). Loss of vibration or position sense is prominent when large myelinated fibers are affected (large-fiber neuropathy). Burn scars or unhealed ulcers are signs of severe sensory loss.

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Prominent sensory loss diminishes reflexes because the afferent limb of the reflex arc is sensory. When sensory loss becomes severe, patients may not perceive minor traumas and pressure and may, therefore develop trophic ulcers or arthritis (Charcot joint) without being aware of them. This is common in leprosy, diabetes, and amyloidosis.

AUTONOMIC NERVE DYSFUNCTION As subtle signs of autonomic nerve dysfunction, skin discoloration and hair loss are common findings in peripheral neuropathy. When autonomic nerve dysfunction is severe, the most common complaint is orthostatic manifestations (e.g., light-headedness or syncope on standing). Constipation or diarrhea due to bowel dysfunction, urinary retention caused by bladder dysfunction, and impotence are not uncommon findings. Evaluation of orthostatic blood pressure, measurement of the post-voiding residual in the bladder, and pupillary light response may aid in the identification of autonomic nerve dysfunction. Tonic pupils (large pupils without any light response) are commonly present in severe autonomic nerve dysfunction. Rarely, pseudo-obstruction of the gut occurs due to total paralysis of the gut muscles.

DIAGNOSTIC INVESTIGATIONS The first diagnostic step is to rule out other lower motor neuron diseases which can mimic peripheral neuropathy (Table 1.3) and confirm that the patient has a peripheral neuropathy. The major manifestations of neuropathy are muscle weakness, sensory loss to all modalities, weak or absent reflexes, and trophic changes, as described above. Among these, sensory impairment is the most important clue for peripheral neuropathy. Causes of generalized weakness include anterior horn cell diseases, disorders of the neuromuscular junction (myasthenia gravis), and myopathy. In these diseases, there should not be any sensory loss upon examination because sensory fibers are not damaged.

TABLE 1.3 Differential Clinical Features in Neuromuscular Disorders Anterior Horn Cell

Peripheral Nerves

Neuromuscular Junctions

Muscle

Involved area

Widespread

Distal

Proximal/ oculobulbar

Proximal

Motor or sensory impairment

Motor

Mixed

Motor

Motor

Reflexes

Weak/absent

Weak/absent

Normal

Normal

Other helpful signs

Fasciculation

Disease

Amyotrophic lateral sclerosis

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Myasthenic symptoms Peripheral neuropathy

Myasthenia gravis

Myopathy

The second step is to decide whether the patient has polyneuropathy, mononeuropathy multiplex, or mononeuropathy. This distinction is important because it will suggest the etiological diagnosis, as described above. The third step is to search for the cause of peripheral neuropathy. In many patients, the cause of peripheral neuropathy is obvious from the medical history and a brief examination, e.g., diabetes or chronic renal failure, and no further investigation is needed. Such diagnosis is easily made by the family physician or internist. However, in some patients, the cause is far from obvious, and further investigation is needed. This should be obtained from a complete history, including any history of drug use or exposure to toxins, and thorough general and neurological examinations. A partial guide to diagnosing peripheral neuropathy is given in Table 1.4. The tips found therein will suggest the need for special laboratory work-up to confirm the cause of neuropathy. This is normally handled by a neurologist. The temporal course of neuropathy varies according to the etiology. With trauma or ischemic infarction, the onset is sudden with the most severe symptoms occurring at the onset. This occurs with diabetic ophthalmoplegia or mononeuropathy in vasculitis. Inflammatory and some metabolic neuropathies have an acute (within a month) or subacute (1–3 months) course extending from days to months. GBS reaches its maximum deficit within four weeks of onset. A chronic course over weeks or months is the hallmark of most toxic and metabolic neuropathies as well as CIDP. A chronic, slowly progressive course over many years occurs with hereditary neuropathy and benign sensory neuropathy. Neuropathies with a relapsing and remitting course include CIDP, toxic neuropathy due to repeated exposure, and porphyria. A clinical assessment should include a careful past medical history, specifically looking for systemic diseases such as diabetes, chronic renal failure, or hypothyroidism that can be associated with neuropathy. Many medications can cause peripheral neuropathy, typically a distal symmetrical axonal sensory-motor neuropathy. Detailed inquiries about drug and alcohol use, as well as exposure to heavy metals and solvents, should be pursued (Table 1.5). Alcohol is one of the most frequently hidden causes of neuropathy. Glue sniffing or exposure to nitrous oxide as a recreational drug can also be a cause of neuropathy. All patients should be questioned regarding HIV risk factors, country of origin (leprosy), diet (vitamin B12 deficiency in a vegetarian), vitamin use (excessive vitamin B6), and the possibility of a tick bite (Lyme disease). Family history is extremely important in the workup of peripheral neuropathy. One study showed that in 42% of cases of peripheral neuropathy with unknown etiology, a hereditary cause was found after careful examination of family history and kin.19 Simply asking patients whether they have a family history of neuropathy is not enough. Instead, specific information should be sought, such as the presence of hammer toes, high arches, weak ankles, gait abnormalities, muscular dystrophy or even multiple sclerosis in the family that would suggest a long-standing or hereditary neuropathy. In Charcot–Marie–Tooth disease, high arches and hammer toes may be the only manifestation among family members. Sometimes, examining close family members is the only way to confirm hereditary neuropathy. The review of systems may provide clues regarding other organ involvement, as seen in rheumatoid diseases, or the presence of an underlying malignancy. A general examination may reveal another medical disease (e.g., diabetes, renal failure, rheumatoid diseases, hypothyroidism, or other autoimmune diseases) that could be the cause of the peripheral neuropathy. Many diseases, AIDS, Lyme disease, leprosy, and vasculitis have a sentinel marker for the disease upon examination (Table 1.4). Orthostatic hypotension without a compensatory rise in heart rate occurs when autonomic fibers are involved. Respiratory rate and vital capacity should be evaluated in GBS to assess for respiratory compromise. The presence of lymphadenopathy, hepatomegaly or splenomegaly, and skin lesions may provide evidence of systemic disease. Pale transverse bands in the nail beds (Mees’ lines) suggest arsenic poisoning. Alopecia may suggest thallium poisoning.

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TABLE 1.4 Helpful Tips in Etiological Diagnosis of Peripheral Neuropathy Family history, pes cavus, “stork-leg” Relapse Acute Alopecia, predominantly sensory neuropathy Painful ophthalmoplegia with sparing of pupil Gum lead line, wrist drop Anesthesic depigmented skin Angiokeratoma, sensory neuropathy Mees’ line, predominantly sensory neuropathy, hyperkeratosis of skin Femoral neuropathy Palpable thick nerves Charcot joint Trophic ulcer, insensitivity to pain Dysautonomia Kaposi sarcoma lymphadenopathy Erythema chronicum migrans, facial palsy Painful small-fiber neuropathy Proximal muscle weakness

Charcot–Marie–Tooth disease Chronic inflammatory demyelinating neuropathy, HNPP Guillain–Barré syndrome, acute intermittent porphyria Thallium neuropathy Diabetes mellitus Lead neuropathy Leprosy Fabry’s disease Arsenic neuropathy Diabetes mellitus Leprosy, Charcot–Marie–Tooth disease 1A Dejerinne–Sottas disease Diabetes mellitus, leprosy Diabetes mellitus, amyloidosis, leprosy, hereditary sensory neuropathy Diabetes mellitus, amyloidosis AIDS neuropathy Lyme disease Diabetes, alcoholism, AIDS, amyloid benign chronic sensory neuropathy Guillain–Barré syndrome, CIDP, diabetic amyotrophy

Source: Reproduced with permission from Oh, S.J., Clinical Electromyography: Case Studies, Williams & Wilkins, Baltimore, MD, 1998.

A thorough neurological evaluation is essential in the work-up of peripheral neuropathy to rule out other neurological disorders which mimic peripheral neuropathy, to confirm peripheral neuropathy, and to determine the type of neuropathy as discussed above. Funduscopic examination may show optic pallor, which is also a symptom of a vitamin B12 deficiency. The examination should include a search for fasciculation, which is the cardinal sign of anterior horn cell disease and a common sign of MMN and, sometimes, CIDP. Severe long-standing neuropathy can result in trophic changes including pes cavus (high arch foot), loss of hair and skin discoloration in affected areas, or ulceration. Unhealed scars are most prominent in diabetes, amyloid neuropathy, leprosy, and hereditary sensory neuropathy. Nerve thickening can be palpated in leprosy, hereditary motor sensory neuropathy (HMSN) Type I, and amyloid neuropathy.

NERVE CONDUCTION STUDIES AND NEEDLE ELECTROMYOGRAPHY The nerve conduction study (NCS) is the most essential part of the work-up in patients with a peripheral neuropathy.20 This study helps confirm peripheral neuropathy, determine the type of neuropathy, localize the site of lesion or entrapment, and follow the course of the disease. The nerve conduction study includes motor and sensory nerve conduction tests. Sensory nerve conduction is a more sensitive index than motor nerve conduction in the diagnosis of peripheral neuropathy.

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Nerve conduction is abnormal in peripheral neuropathy, but it is normal in myopathy and anterior horn cell disease. The nerve conduction study identifies the neuropathy in 76 to 80% of patients with diabetic neuropathy and in 81 to 100% of patients with the Guillain–Barré syndrome.20 It is important to remember that the NCS could be normal in a few patients with mild neuropathy of axonal degeneration. This is especially true in small-fiber neuropathy. In this case, the physician must rely on the needle EMG of distal muscles for evidence of the denervation process or other confirmatory tests for neuropathy such as a sweat test or skin biopsy. The nerve conduction study is also helpful in differentiating between axonal neuropathy and demyelinating neuropathy (Figures 1.4 to 1.6). The hallmark of nerve conduction abnormalities in axonal degeneration is a diminution of the amplitude of the CMAP and CNAP in the presence of normal or near-normal maximal nerve conduction velocity (NCV). On the other hand, the hallmark of nerve conduction abnormalities in demyelinating neuropathy are conduction block, abnormal temporal dispersion (dispersion phenomenon), and marked slowing in the NCV. The nerve conduction study can provide a certain pattern of abnormalities specific enough to be of value in localizing the lesions to specific parts of the nerve and in suggesting the nature of a neuropathy, as discussed above. The best example is the pure sensory neuronopathy pattern: the sensory nerve conduction is markedly abnormal, but the motor nerve conduction is completely normal. This pattern is pathognomonic of a sensory neuronopathy involving the dorsal root sensory ganglia. The NCS is also of some value in the follow-up evaluation of patients recovering from neuropathies, either under specific therapies or spontaneously. It is also of value in the study of families that have a hereditary neuropathy. This is especially true in the detection of asymptomatic cases of hereditary motor and sensory neuropathy I (hypertrophic type of the Charcot–Marie–Tooth (CMT) disease).

A

B

ankle

ankle 1000 µv

500 µv

5 msec 5 msec

knee

knee

C

D 2 µv 3 msec

2 µv 2 msec

FIGURE 1.4 CMAP in axonal neuropathy (arsenic neuropathy). (A) The amplitude of the CMAP in the peroneal nerve is markedly reduced. Terminal latency and motor NCVs are minimally abnormal. (B) Improved CMAP in the peroneal nerve 2 years later. (C) Markedly reduced amplitude and mild slowing of the sensory NCV (34.3 m/sec) over the finger-wrist segment of the median nerve. (D) Reduced amplitude and mild slowing in the sensory NCV (33.3 m/sec) over the finger-wrist segment of the ulnar nerve. (Reproduced with permission from Oh, S.J., Clinical Electromyography. Nerve Conduction Studies, Williams & Wilkins, Baltimore, 1993; 484.)

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A

8.5

B

20

100 µv 5 msec FIGURE 1.5 CMAP in demyelinating neuropathy. CMAP in segmental demyelination. This is from the posterior ibial nerve at the ankle (A) and the popliteal fossa (B) in a case of hypertrophic neuropathy. The reduced amplitude of the CMAP is due to a marked dispersion phenomenon (duration of the CMAP is 30 msec). Terminal latency is 8.5 msec. Motor NCV is 35.8m/sec. (Reproduced with permission from Oh, S.J., Clinical Electromyography. Nerve Conduction Studies, Williams & Wilkins, Baltimore, 1993; 486.)

27 msec C

17.5 msec B

7 msec 1000µv + 5 msec

A

FIGURE 1.6 Conduction block. Conduction block in segmental demyelination. Median motor nerve conduction in a case of CIDP. (A) Normal amplitude of the CMAP with wrist stimulation. (B) A dramatic reduction in amplitude of the CMAP with elbow stimulation. (C) CMAP with axillary stimulation. Conduction block is clearly seen between wrist and elbow stimulation. The dispersion phenomenon is also observed. The motor NCV is 21.9 m/sec over the wrist-elbow segment and 15.8 m/sec over the elbow-axilla segment. (Reproduced with permission from Oh, S.J., Clinical Electromyography. Nerve Conduction Studies, Williams & Wilkins, Baltimore, 1993; 487.)

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For the entrapment neuropathies, the NCS is the most definite diagnostic test, being positive in 91 to 98% of patients with carpal tunnel syndrome and in 95% of patients with ulnar neuropathy at the elbow. Fortunately, the localized pathology of entrapment neuropathy is segmental demyelination. The absence of CNAP or the slowing of sensory and mixed NCVs, as well as the slowing of motor NCVs in the involved segment, are the classical abnormalities. The needle EMG is very helpful in differentiating denervation process from myopathy and myotonia. In denervation, fibrillation and positive sharp waves (PSWs) are noted at rest. However, it is important to remember that they are not pathognomonic of the denervation process because they are also observed in patients with active myopathy, such as polymyositis. The motor unit potentials (MUPs) are either normal or increased in duration depending on the chronicity of the denervation. In chronic denervation, the collateral sprout from relatively normal axons may innervate denervated muscle fibers, producing high-amplitude and long-duration (HALD) MUPs. On maximal contraction of muscles, the MUPs are reduced in recruitment. In contrast, a different needle EMG pattern is seen in myopathy: MUPs are small in amplitude and short in duration (SASD MUPs, that is, small-amplitude short-duration MUPs) and there is excessive recruitment of MUPs on maximal contraction. In myotonia, the typical dive bomber sound is observed with the waxing and waning of abnormal potentials. The needle EMG is also helpful in identifying the activity of neuropathy. In active (ongoing) denervation, fibrillations and PSWs are prominent with increased polyphasic MUPs and reduced MUP recruitment. On the other hand, in inactive (usually chronic) denervation, fibrillations and PSWs are minimal together with HALD MUPs. In addition, the needle EMG is helpful in distinguishing axonal neuropathy from demyelinating neuropathy. Fibrillations and PSWs, electrophysiological hallmarks of axonal degeneration, are prominent in axonal neuropathy but are absent or scarce in demyelinating neuropathy. Fasciculation or myokymia is a more prominent finding in demyelinating neuropathy.

LABORATORY STUDIES Laboratory tests are most important in confirming the etiology of peripheral neuropathy. The firstline tests should be performed in all patients with suspected peripheral neuropathies (Table 1.5). They may reveal unsuspected causes of neuropathy such as diabetes, rheumatoid disease, vitamin B12 deficiency, hypothyroidism, and monoclonal gammopathy. Considering that all these neuropathies are treatable, it is important to search for these possible causes of neuropathy. The second-line tests are selected depending on the clinical impression, which is based on clinical, electrophysiological, and laboratory data. For example, if monoclonal gammopathy is found in the serum of a patient, then a metastatic bone survey and 24-hour urine immunoelectrophoresis by immunofixation are ordered to differentiate benign monoclonal gammopathy from malignant gammopathy. The spinal fluid evaluation is essential for the diagnosis of GBS, CIDP, and a few other neuropathies. An elevated total protein with fewer than five white blood cells is seen in inflammatory neuropathy (GBS and CIDP). Inflammatory cells are usually increased in AIDS and Lyme disease. Other studies useful in specific clinical contexts are cytology (lymphoma) and specific studies such as Lyme polymerase chain reaction and cytomegalovirus branches chain DNA (polyradiculopathy or mononeuritis multiplex in AIDS). CMT1A DNA duplication or hereditary neuropathy with liability to pressure palsy (HNPP) DNA deletion tests may confirm the specific type of hereditary neuropathy.

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TABLE 1.5 Laboratory Tests for Peripheral Neuropathy Test First-Line Tests CBC, sedimentation rate Renal and liver functions Rheumatoid profiles Blood sugar, fasting 2 hour post-brandial; HbA1C Serum B12 and folate level Thyroid functions Immunoelectrophoresis of serum protein by immunofixation test Second-Line Tests Porphobilinogen in urine Heavy metals in urine Arsenic in hair and nails Hepatitis B antigen Schilling test Antineutrophile cytoplasmic antibody Chest x-ray, cancer survey High CSF protein Increase cell in CSF Serum HIV antibody Serum Borrelia burgdorferi antibody Metastatic bone survey Anti-Hu antibody GM1 and MAG antibody CMT1A DNA duplication test HNPP DNA deletion test

Diagnostic Possibilities

Collagen disease, leukemia, vasculitis Uremic and hepatic neuropathy Collagen disease, vasculitis Diabetes Neuropathy with macrocytosis Hypothyroid neuropathy Dysproteinemia, monoclonal gammopathy lymphoma, amyloidosis

Acute porphyria Lead, arsenic, thallium, mercury Arsenic neuropathy Polyarteritis nodosa Vitamin B12 deficiency Wegener’s granulomatosis Carcinomatous neuropathy Guillain–Barré syndrome, chronic inflammatory demyelinating polyneuropathy Lyme disease, AIDS, paraneoplastic neuropathy AIDS neuropathy Lyme disease Sclerotic multiple myeloma Paraneoplastic neuropathy Autoimmune neuropathy CMT1A neuropathy HNPP

Source: Reproduced with permission from Oh, S.J., Clinical Electromyography: Case Studies, Williams & Wilkins, Baltimore, MD, 1998.

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REFERENCES 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20.

Schwartz, J.H., Axonal transport: components, mechanisms, and specificity, Ann. Rev. Neurosci., 2, 467, 1979. Price, D.L. and Proter, K.R., The response of ventral horn neurons to axonal transection, J. Cell Biol. 53, 24, 1972. Berthold, C.H., Morphology of normal peripheral axons, in Physiology and Pathobiology of Axons, Waxman, S.G., Ed., Raven Press, New York, NY, 1978. Raine, C.S., Pathology of demyelination, in Physiology and Pathobiology of Axons, Waxman, S.G., Ed., Raven Press, New York, NY, 1978. Waller, A.V., Experiments on the section of the glossopharyngeal and hypoglossal nerves of the frog, and observations of the alterations produced thereby in the structure of their primitive fibers, Phil. Trans. Roy. Soc. London B., 140, 423, 1850. Dyck, P.J., Conn, D.J., and Okazaki, H., Necrotizing angiopathic neuropathy. Three-dimensional morphology of fiber degeneration related to sites of occluded vessels, Mayo Clin. Proc., 47, 461, 1972. Spencer, P.S., Sabri, M.I., and Schaumburg, H.H., Does a defect of energy metabolism in the nerve fiber underlie axonal degeneration in polyneuropathies? Ann. Neurol., 5, 501, 1979. Weller, R.O. and Cervos-Navarro, J., Pathology of Peripheral Nerves, Butterworth & Co. Ltd., London, 1977. Asbury, A.K. and Johnson, P.C., Pathology of Peripheral Nerves, W.B. Saunders, Philadelphia, PA, 1978. Sternman, A.B., Schaumberg, H.H., and Asbury, A.K., The acute sensory neuronopathy syndrome: a distinct clinical entity, Ann. Neurol., 7, 354, 1980. Asbury, A.K. and Gilliatt, R.W., The clinical approach to neuropathy, in Peripheral Nerve Disorders, Asbury, A.K. and Gilliatt, R.W., Eds., Butterworth & Co. Ltd., London, 1984, 1. Asbury, A.K., Arnason, B.G., and Adams, R.D., The inflammatory lesion in idiopathic polyneuritis, Medicine, 48, 173, 1969. Dyck, P.J. et al., Chronic inflammatory polyradiculoneuropathy, Mayo Clin. Proc., 50, 621, 1975. Bradley, W.G. and Jennekens, F.G.I., Axonal degeneration in diphtheric neuroapthy, J. Neurol. Sci., 13, 415, 1971. Oh, S.J., Clinical Electromyography: Nerve Conduction Studies, 2nd ed., Williams & Wilkins, Baltimore, MD, 1993. Gombault, A., Contribution á l’étude anatomique de la névrite paraenchymateuse subaigué et chronique — névrite segmentaire péri-axile, Arch. Neurol., (French), 1, 11, 1880. Dyck, P.J., Johnson, W.J., Lambert, E.H., and O’Brien, P.C., Segmental demyelination secondary to axonal degeneration in uremic neuropathy, Mayo Clin. Proc., 46, 400, 1971. Dyck, P.J. and Lais, A.C., Evidence for segmental demyelination secondary to axonal degeneration in Friedreich’s ataxia, in Clinical Studies in Myology, Kakulas, B.K., Ed., Excerpta Medica, Amsterdam, 253, 1973. Dyck, P.J., Oviatt, K.F., and Lambert, E.H., Intensive evaluation of referred unclassified neuropathies yields improved diagnosis, Ann. Neurol., 10, 222, 1981. Oh, S.J., Clinical Electromyography. Nerve Conduction Studies, Williams & Wilkins, Baltimore, 1993.

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CHAPTER 1 Figure 1 Teasing nerve fibers. (1) axonal degeneration: arrows indicate row of myelin ovoids; (2) demyelination: arrows indicate demyelinated segments; (3) tomaculous change: a) thin arrows indicate a demyelinated segment; thick arrows indicate tomaculous change; b) enlarged tomaculous change; (4) giant axons: a) white arrows indicate rows of myelin ovoids. b) arrows indicate axons. (Reproduced with permission from Oh, S. J., Yonsei Med. J., 31, 16, 1990.)

2

The Nerve Biopsy

INDICATION FOR THE NERVE BIOPSY If the cause of a neuropathy is known by means of the clinical examination and laboratory tests, a nerve biopsy is not necessary. In many metabolic neuropathies, the patient’s history and laboratory tests are enough to make a definite causative diagnosis. These include diabetic, alcoholic, and uremic neuropathies. In such patients, the nerve biopsy is performed only to study the basic pathophysiology of neuropathy. Even in cases of GBS, the most common form of neuropathy seen by neurologists, the nerve biopsy is not indicated simply because the diagnosis can be made with certainty in most cases on the basis of the clinical, electrophysiological, and spinal fluid findings. The nerve biopsy is clearly indicated in two groups of patients: patients suspected of vasculitis and those with clinically significant peripheral neuropathy without known cause. The sural nerve biopsy is best indicated in patients suspected of having vasculitis, with or without the clinical features of neuropathy (Table 2.1).1 That is because the nerve is more commonly involved than other readily available biopsied tissues such as skin and muscle, and the diagnostic yield of the sural nerve biopsy is high in vasculitis.1 Peripheral neuropathy was reported in 52 to 60% of patients with vasculitis.2, 3 The nerve conduction test was crucial in those patients because it detected neuropathy in asymptomatic patients and because vasculitis was invariably found in the sural nerve when the nerve conduction was abnormal.1 In a recent study of the sural nerve biopsy conducted by our laboratory, we found a diagnostic sensitivity of 39% in 115 patients suspected of having vasculitic neuropathy.4 TABLE 2.1 Diagnostic Usefulness of the Sural Nerve Biopsy

Specific diagnoses Vasculitic neuropathy Hypertrophic neuropathy Inflammatory neuropathy Ischemic neuropathy Amyloid neuropathy Metachromatic neuropathy Sarcoid neuropapthy Leprosy Lymphoma Fabry’s disease Tomaculous neuropathy Amidarone Chronic inflammatory demyelinating polyneuropathy Hereditary neuropathy Total a

Oh (N = 385) 92 (24%) 46 (12%) 27 (7%) 12 (3%) 3 (0.8%) 2 (0.5%) 1 (0.3%) 1 (0.3%)

Midroni (N = 267) 43 (16%) 20 (7.5%) 4 (1.6%) 4 (1.6%)

46 (12%)

2 (0.8%) 1 (0.4%) 2 (0.8%) 1 (0.4%) 3 (1.2%) 3 (1.2%) 51 (19%)

35 (9%) 173 (45%)

12 (4.5%) 106 (40%)

GBS. b Demyelinating neuropathy. This is not necessarily CIDP.

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Schröder (N = 5266) 1200 (23 %) 769 (15%) 124 (2.3%) 116 (2%) a 47 (0.9%) 22 (0.2%)

4 118

(2%)

830 (16%) b 273 (5%) 2303 (44%)

The reason for performing the sural nerve biopsy in neuropathy without known cause is obvious: it can often point to a definite diagnosis and provide other clinically helpful information in some patients. Even within this group, the nerve biopsy should be confined to patients with a clinically significant neuropathy, the treatment of which can be altered by the potential nerve biopsy finding. Under this guideline, patients with small-fiber neuropathy or mild non-progressive neuropathy are not likely candidates for nerve biopsy. Based on data obtained in 385 sural nerve biopsies performed over a 16-year period (1971–1986), we found clinically helpful or relevant information in 45% of cases5 (Table 2.1). Other investigators reported clinically helpful or relevant information in 27 to 44% of cases.6-8 Specific diagnoses were obtained in 24% of cases, diagnosis of chronic inflammatory demyelinating was confirmed in 12%, and hereditary neuropathy was diagnosed in 9% of cases. Among the specific diagnoses, vasculitic neuropathy was the most common form of neuropathy, accounting for 12% of 385 nerve biopsies. Once a specific diagnosis is made, it dictates the clinical management of the disorder. This is best exemplified in vasculitic neuropathy where steroid and cytotoxic agents are very helpful in inducing remission.9 In chronic inflammatory polyneuropathy, long-term steroid treatment, often over the course of many years, is required.10 Thus, it is essential to confirm such diagnoses with a nerve biopsy before steroids are administered. Confirmation of hereditary neuropathy is helpful in predicting the progression of disease and in genetic counseling of patients. This outlook has changed because of the easy availability of Charcot–Marie–Tooth (CMT) 1A and hereditary neuropathy with liability to pressure palsy (HNPP) DNA testing,11 but nerve biopsies were not clinically helpful in 55% of cases. In another series of tests, a specific diagnosis was made in 16 to 23% of cases, and nerve biopsies were helpful in 40 to 44% of cases.6,12 In the first prospective study of 50 cases, sural nerve biopsies altered the diagnosis in 14% of cases and affected management in 60% of cases.13 The diagnostic sensitivity of the sural nerve biopsy is analyzed in Table 2.2. It is important to recognize that specific diagnoses were made in only 24% of cases. In 55% of cases, the diagnosis of demyelinating or axonal neuropathy was made without further elucidation of any specific cause. In the latter cases, the nerve biopsy findings have to be correlated with the clinical information to reach a final diagnosis. This underlines the importance of exhaustive and detailed clinical examinations in the work-up of neuropathy.

TYPES OF NERVE BIOPSY There are two types of nerve biopsy: fascicular biopsy and whole biopsy. In fascicular biopsy, only a few fascicles of the nerve are biopsied in order to lessen permanent sensory loss and long-term dysesthesia.14 However, studies have shown that there is no significant difference in the areas of sensory loss 5 or more years after sural nerve biopsy in fascicular biopsy compared with whole nerve biopsy.15 Furthermore, fascicular biopsy may fail to show the vasculitic change in the perineurial space in cases TABLE 2.2 Diagnostic Sensitivity of the Sural Nerve Biopsy

Specific diagnosis Demyelinating neuropathy Axonal neuropathies Nonspecific findings Normal

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Oh (N = 385) 92 (24%) 132 (32%) 89 (23%) 62 (16%) 19 (5%)

Midroni (N = 267) 43 (16%)

27 (10%)

Schröder (N = 5266) 1200 (23%) 830 (44%) 1572 (30%)

of vasculitis because this is where splitting is done in fascicular biopsy.16 This is the most important disadvantage of fascicular biopsy since vasculitis is one of the prime indications for nerve biopsy. Therefore, the author has concluded that there is no justification for fascicular biopsy. Our laboratory routinely performs only whole nerve biopsy, which is practiced in most centers.

SURAL NERVE BIOPSY Biopsies of three different nerves have been described: the radial sensory nerve, the superficial peroneal nerve, and the sural nerve. The sural nerve biopsy is preferable for four reasons: (1) the nerve is easily identifiable and relatively protected from compression injury because it is located behind the lateral malleolus; (2) this nerve is purely sensory, thus producing no motor deficit following biopsy; (3) this nerve is liable to be affected by neuropathy because it is a distal branch of a long nerve; and (4) this nerve is easily tested electrophysiologically. The sural nerve biopsy is not recommended if the nerve conduction is completely normal because the diagnostic yield is small. This policy is based on our experience in a few cases in which normal nerve biopsy was found when the nerve conduction was normal in the sural nerve. One disadvantage of this nerve biopsy is that the sural nerve is not affected if the neuropathy is purely motor. However, in practice, this does not pose a major problem because sensory nerve conduction is often affected, even in a clinically identified motor neuropathy such as GBS or multifocal motor neuropathy.17,18 In a sural nerve biopsy, the patient is placed in the lateral decubitus position and a pillow is placed under the ankle to be biopsied. The skin incision is made under local anesthesia with 1% lidocaine behind the lateral malleolus and halfway between the posterior aspect of the Achilles tendon and the lateral malleolus. This skin incision is extended proximally for 4 to 5 cm, parallel to the Achilles tendon (Color Figure 2.1).* Under the incised skin, the lesser saphenous veins are usually seen. The whitish pearly sural nerve is identified medially under the lesser saphenous veins. When the sural nerve is touched by an instrument, the patient often feels a shooting electrical pain — a definite sign that the structure is the sural nerve. Both the nerve and the veins are superficial to the deep fascia. If the sural nerve is not found easily, the examiner may have gone too deep. Sometimes, a lesser saphenous vein is mistakenly identified as a sural nerve. This can be avoided by carefully inspecting the nerve prior to cutting it, observing the broad angles at which the vein branches in contrast to the narrow angles at which the nerve branches.19 If a vein is cut, the specimen will have a tiny hole through it. Once the sural nerve is identified, the nerve is anesthetized prior to cutting with a small amount of lidocaine a few millimeters proximal to the intended transection site in order to prevent pain when the nerve is cut. Nerve block is tested by gradual gentle clamping of the nerve with a tiny hemostat proximal to the site of transection. Once total anesthesia is achieved, the nerve is firmly clamped proximal to the transection site. This most likely reduces the likelihood of any potential post-biopsy neuralgia. Nevertheless, the patient should be warned that there may be a possible sharp pain at the moment the nerve is cut. Telling this to the patient will improve patient cooperation. Generally, the degree of pain is inversely proportional to the severity of the neuropathy.20 The proximal nerve is lifted gently and cut with sharp dissection distal to the hard clamping. A nerve segment at least 4 cm in length should be obtained with due care in order to avoid any unnecessary trauma to the nerve. The superficial fascia and skin incision is closed using interrupted mattress skin sutures with 4-0 coated vicryl sutures inside and 3-0 nylon sutures outside. An elastic bandage is applied locally to reduce the accumulation of blood and fluid. The patient may be up and about on the same day, but sitting with the leg in a dependent position for long periods, excessive walking, or running are discouraged. Local pain is controlled with mild narcotics. Sutures may be removed in 7 to 10 days. A narcotic painkiller and an antibiotic are prescribed for postoperative care.

* Color insert figures

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SEQUELAE OF NERVE BIOPSY Following the sural nerve biopsy, there is invariably a sensory loss over the lateral aspect of the foot corresponding to the sural nerve territory. This area gradually decreases in size, but a quarter-sized area remains permanently insensitive to pin-prick. Some immediate postoperative pain is not uncommon, having been observed in 30 to 50% of patients.21-23 However, pain gradually diminishes over time.23 Persistent pain has been reported in 19 to 25% of patients8, 23 for 2 years and in 6% of patients after more than 2 years.23 Serious reactions following the sural nerve biopsy are rare. Significant pain or paresthesia was noted in 10% of patients 1 year after the biopsy.21, 22, 24 Asbury and Connolly noted serious side-effects in only 2 of 103 patients: post-traumatic neuroma in 1 and pain in the other.19 Among 385 sural nerve biopsies performed in our laboratory, post-traumatic neuralgia lasted 1 year in 2 cases (0.5%) and delayed wound healing occurred in 4 cases (1%). In those 4 cases, steroids were administered for vasculitic neuropathy or chronic inflammatory demyelinating polyneuropathy immediately after the biopsy, contributing to delayed wound healing. Midroni and Bilbaro reported 2 patients who had significant wound infections and 1 who required resection of neuroma out of a total of 267 biopsies.12 Both patients with severe wound infections had a systemic vasculitis and were treated with steroids. We have not observed any troublesome side-effects in any of our cases 2 years after performing biopsies. In Pollock et al.’s series, there was no long-term pain or paresthesia in any of their cases 5 or more years after a nerve biopsy.15

BIOPSY OF OTHER NERVES SUPERFICIAL PERONEAL NERVE BIOPSY The superficial peroneal nerve is superficially located under the skin in the distal third of the leg and has two sensory branches, the medial (MDC) and intermediate dorsal cutaneous (IDC) nerves (Color Figure 2.1). Thus, this is an ideal site for the nerve biopsy. Kissel and Mendell recommended the following guidelines for biopsy of this nerve:25 The distance from the head of the fibula to the lateral malleolus is determined. This distance is divided into four and three equal segments. An incision is made between the lower one-third and one-quarter distal segmental markings at a point 1.5 to 2 cm anterior to the edge of the fibula (determined by firm palpation of the leg). The superficial peroneal nerve lies above the fascia and can be found in the subcutaneous tissue with minimal dissection, usually along the lateral edge of the fascia. In practice, the superficial peroneal nerve is not readily identifiable as stated because of its tiny size. Thus, the course of this nerve is usually mapped with the nerve conduction study prior to the biopsy. Following removal of the nerve, the fascia within the operative field is opened, revealing the peroneus brevis muscle which is easily accessible for muscle biopsy. Because of the advantage of obtaining the nerve and muscle biopsy under the same incision, Said et al. and Kissel and Mendell prefer the biopsy of this nerve for the diagnosis of vasculitic neuropathy.25, 26 In general, the sural nerve biopsy has a higher diagnostic yield for vasculitis (see Chapter 6). After the biopsy, the patient loses sensation over the territory of the medial or intermediate dorsal cutaneous branches on the dorsum of the foot, depending on which branch is biopsied.

SUPERFICIAL RADIAL NERVE BIOPSY The superficial radial nerve is superficially located under the skin in the distal fourth of the extensor surface of the forearm along the medial border of the radius (Color Figure 2.2). It is ideally located for biopsy. Often, it is buried under the cepahlic veins. In practice, it is not easy to identify this nerve because of its tiny size. Thus, the course of the radial nerve is normally mapped with the nerve

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conduction study prior to a biopsy in order to guarantee the success of the nerve biopsy. The same principle of identification of the nerve is applied as described above. After the biopsy, the patient loses sensation over the territory of the superficial radial sensory nerve, including the first web space.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Wees, S.J., Sunwoo, I.N. and Oh, S.J., Sural nerve biopsy in systemic necrotizing vasculitis, Am. J. Med., 71, 525, 1981. Frohnert, P.P. and Sheps, S.G., Long-term follow-up study of periarteritis nodosa, Am. J. Med., 43, 8, 1967. Cohen, R.D., Conn, D.L., and Ilstrup, D.M., Clinical features, prognosis, and response to treatment in polyarteritis, Mayo Clin. Proc., 55,1 46, 1980. Claussen, G.C., Thomas, D., Coyne, C., Våsques, LG., and Oh, S.J., Diagnostic value of nerve and muscle biopsy in suspected vasculitis cases, J. Clin. Neuromuscular, 1, 117, 2000. Oh, S.J., Diagnostic usefulness and limitations of the seural nerve biopsy, Yonsei Med. J., 31(1), 1, 1990. Schröder, M., Recommendations for the examination of peripheral nerve biopsies, Virchos Arch., 432, 199, 1998. Argov, X., Steiner, I., and Soffer, D., The yield of sural nerve biopsy in the evaluation of peripheral neuropathies, Acta Neurol. Scand., 79, 243, 1989. Neundörfer, B., Grahmann, F., Engelhart, A., and Harte, U., Postoperative effects and values of sural nerve biopsies: a retrospective study, Eur. Neurol., 30, 350, 1990. Fauci, A.S., Katz, P., Haynes, B.F., and Wolff, S.M., Cyclophosphamide therapy of severe systemic necrotizing vasculitis, New Eng. J. Med., 301, 235, 1979. Oh, S.J., Subacute demyelinating polyneuropathy responding to croticosteroid treatment, Arch. Neurol., 35, 509, 1978. Said, G., Indications and value of nerve biopsy, Muscle and Nerve, 22(12), 1617, 1999. Midroni, G. and Bilbaro, J.M., Biopsy Diagnosis of Peripheral Neuropathy, Butterworth-Heinemann, Boston, MA, 1995. Gabriel, C.M. et al., Prospective study of the usefulness of sural nerve biopsy, J. Neurol. Neurosurg. Psychiatry, 69, 442, 2000. Dyck, P.J. and Lofgren, E.P., Nerve biopsy. Choice of nerve, method, symptoms and usefulness, Med. Clin. North Am., 52, 885, 1968. Pollock, M., Nukada, H., Taylor, P., Donaldson, I., and Carrol, G., Comparison between fascicular and whole sural nerve biopsy, Ann. Neurol., 13, 65, 1983. Dyck, P.J., Conn, D.J., and Okazaki, H., Necrotizing angiopathic neuropathy. Three-dimensional morphology of fiber degeneration related to sites of occluded vessels, Mayo Clin. Proc., 47, 461, 1972. Oh, S.J., Clinical Electromyography, Nerve Conduction Studies, 2nd Ed., Williams & Wilkins, Baltimore, MD, 1993. Corse, A.M., Chaudhry, V., Crawford, T.O., Cornblath, D.R., Kuncl, R.W., and Griffin, J.W., Sensory nerve pathology in multifocal motor neuropathy, Ann. of Neurol., 39(3), 319, 1996. Asbury, A.K. and Connolly, E.S., Sural nerve biopsy: technical note, J. Neurosurg., 38, 391, 1973. Johnson, P.C., Diagnostic peripheral nerve biopsy, Barrow Neurological Institute Q., 1, 2, 1985. Perry, J.R. and Bril, V., Complications of sural nerve biopsy in diabetic versus non-diabetic patients, Can. J. Neurol. Sci., 21, 34, 1994. Solders, G., Discomfort after fascicular sural nerve biopsy, Acta Neurol. Scand., 77, 503, 1988. Flachenecker, P., Janka, M., Goldbrunner, R., and Toyka, K.V., Clinical outcome of sural nerve biopsy: a retrospective study, J. Neurol., 246(2), 93, 1999. Stevens, J.C., Lofgren, E.P., and Dyck, P.J., Biopsy of peripheral nerves, Peripheral Neuropathy, Vol. I, Dyck, P.J., Thomas, P.K., and Lambert, E.H., Eds., W.B. Saunders, Philadelphia, PA, 1975. Said, G., Lacroix-Ciaudo, C., Fujimura, H., Blas, C., and Faux, N., The peripheral neuropathy of necrotizing arteritis: a clinicopathological study, Ann. Neurol., 23, 461, 1988. Kissel, J.T. and Mendell, J.R., Vasculitic neuropathy, Neurol. Clin., 10(3), 761, 1992.

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CHAPTER 2 Figure 1 Sural nerve and superficial peroneal nerve in the anterior-lateral view of the ankle and dorsum of the foot. (Modified from J.C.B. Grant, An Atlas of Anatomy, 6th ed. Williams & Wilkins, Baltimore, 1972.)

CHAPTER 2 Figure 2 Superficial radial sensory nerve in the radial aspect of the wrist. (Modified from J.C.B. Grant, An Atlas of Anatomy, 6th ed. Williams & Wilkins, Baltimore, 1972.)

3

Histological Processing and Staining of the Biopsied Nerve

TREATMENT OF THE BIOPSIED NERVE Immediately after removal of the biopsy specimen, the nerve is gently straightened, stretched, and placed on a silicone pad in a dissecting dish for 15 minutes with pins at each end. This step is important for reducing contraction artifact. Asbury and Connolly stretched and applied the nerve to a thin strip of an index card for one minute prior to immersing it in fixatives.1 Dyck and Lofgren suspended the biopsied nerve in the fixative with a tiny weight at one end.2 The nerve is cut into 4 sections with a sharp razor, as described in Figure 3.1, to be processed for paraffin, frozen, semithin, and electronmicroscopic (EM) sections, and for fiber teasing. The piece closest to the transection site should be used for paraffin sections, and the most distal portion should be kept for frozen sections, with the midportions utilized for semithin and EM sections. This distribution is preferred because potential cutting artifacts are not that critical for paraffin sections or nerve fiber teasing, whereas artifact-free sections are essential for semithin and EM sections. All of our specimens for frozen sections are processed first in order to make a fast and definite diagnosis. The nerve specimen is frozen in isopentane cooled to -180°C in liquid nitrogen for 15 seconds. Rapid diagnosis is critical in cases of vasculitis since an immunosuppressive therapy should be instituted as soon as the diagnosis of vasculitis is made. In practice, the diagnosis of vasculitis can be made within 15 to 30 minutes after the biopsy.3 Metachromatic neuropathy can be diagnosed only with frozen sections since metachromatic granules are stained with cresyl-fast violet on frozen sections alone (Table 3.1). Other advantages of the frozen section are easy detection of myelin-digestion chambers and relative ease of preserving the longitudinal sections in a straight alignment. The latter 1.25 1.25

Neutral-buffered formalin

Paraffin section Teasing

H&E Modified trichrome Congo-red

4% glutaraldehyde

4 cm

Isopentane in -180°C liquid N 2

Semithin section Thin section for EM

Toluidine blue or Toluidine blue and basic fuchsin

FIGURE 3.1 Treatment of nerve biopsy specimens.

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1.5

Frozen section

H&E Modified trichrome PASH Congo-red Cresyl-fast-violet

is critical in recognizing segmental demyelination. These benefits are all achieved in sections stained with modified trichrome4 and Hemotoxylin and Eosin (H & E) stain with Harris hematoxylin.5 A rough estimate of the population of myelinated fibers is possible with modified trichrome staining on frozen sections, which shows the normal nerve fascicles filled with myelinated fibers (see Chapter 4). Paraffin sections are needed to identify amyloid by Congo-red staining and to delineate the detailed structures of cells and vessels (Table 3.1). In the past, when semithin EM sections were unavailable, the population of myelinated fibers, the distribution of the nerve fibers according to fiber diameter, and the relationship between the axon diameter and myelin diameter could be studied with paraffin sections stained with Kulschistky’s stain, which stains myelin black (see Chapter 4). Myelinated fibers are now stained red with modified trichrome on paraffin sections,6 giving an overview of the population of myelinated fibers and, sometimes, of myelin-digestion chambers in severe axonal neuropathy. The semithin EM section has been most commonly used for peripheral nerve pathology in recent years. This section is best for detailed study of the axon–myelin relationship, for identifying onion-bulb formations and clustering of regenerated fibers, and for calculating the density of myelinated fibers (Table 3.1). The semithin EM section is also the only reasonably sure means of detecting thinly myelinated fibers (remyelination). Nerve fiber teasing is superior for documenting segmental demyelination and also allows recognition of nerve fibers with myelin ovoids (axonal degeneration) in a quantitative manner (Table 3.1). With teasing of nerve fibers, one can study the relationship between internode length and fiber diameter. Teasing is not practical because of the time-consuming nature of the technique. However, teasing is the only way to recognize the nature of neuropathy in mild cases when studying other sections has not been informative. The electronmicroscopic study is essential for studying unmyelinated fibers because it is the only means of identifying unmyelinated fibers in the peripheral nerve (Table 3.2). Selective loss of unmyelinated fibers has been identified by such studies in amyloid neuropathy, Fabry’s disease, and small-fiber diabetic neuropathy. EM studies also played a pivotal role in recognizing the widely spaced myelin (WSM) in myelin associated glycoprotein (MAG)-positive neuropathy and uncompacted myelin lamellae (UML) in POEMS (polyneuropathy-organomegaly-endocrinopathy-M-protein-skin change). In rare storage diseases such as Krabbe’s disease, Battern–Kufs disease, adrenoleucodystrophy, Farber’s disease, Tangier’s disease, or Niemann–Pick disease, the EM study shows the distinct ultrastructural features of storage inclusion which are helpful in diagnosing such diseases.7,8 There are several excellent books and articles on this subject which readers can consult for more detailed information.

IMMEDIATE CARE OF THE BIOPSIED NERVE The procedure for caring for the nerve immediately after biopsy is as follows. The nerve is stretched gently and secured with a pin at each end on a silicon pad in a dissecting dish or on a waxed plate for 15 minutes. The nerve specimen is cut into three pieces: 1.5 cm for the frozen section, 1.25 cm for the semithin and EM sections, and 1.25 cm for paraffin sections and nerve teasing (Figure 3.1). For teasing, a nerve fixed in 4% glutaraldehyde can also be used.

PROCESSING OF THE NERVE Processing of the nerve for the frozen sections takes place as follows. The nerve specimen is cut into two pieces, one-third for the transverse section and two-thirds for the longitudinal section. The nerve

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TABLE 3.1 Advantages and Disadvantages of Tissue Sections Section Type Frozen section

Advantage Rapid diagnosis Population of myelinated fibers (modified trichrome) Detection of myelin digestion chambers (modified trichrome) Cresyl-fast-violet stain for metachromatic material Oil red O stain for lipid Relative ease of preserving the longitudinal sections straight for segmental demyelination Immunofluorescent studies Paraffin section Details of cells and anatomical structure Semithin section Population of myelinated fiber Detection of thinly myelinated fibers Detection of clustering of regenerated fibers Detection of onion-bulb formation Axon–myelin relationship EM section The only test for the unmyelinated fibers Widely spaced myelin (WSM) Uncompacted myelin lamellae (UML) Schwann cell inclusions Macrophage-induced demyelination or axonal change Teasing fiber Best method for differential diagnosis for axonal neuropathy vs. demyelinating neuropathy

Disadvantage Details of cells are not clear

Artifact is unavoidable Details of cells are not clear

Special training

Too much time

TABLE 3.2 Diagnosis by the Ultrastructural EM Study Pathological Features Loss of unmyelinated fibers Macrophage mediated demyelination Widely spaced myelin (WSM) Uncompacted myelin lamella (UML) Schwann cell inclusions and demyelination Tuffstone inclusions Needle-like inclusion of GLDb Lipid inclusions Banana body Pi body-like cytosomes Lysosomal inclusions (myelinoid bodies) Schwan cell inclusion and axonal degeneration Lipid storage in perineurium a b

Diagnosis Small-fiber neuropathya Inflammatory demyelinating neuropathy MAG/IgM neuropathy POEMS neuropathy Metachromatic leucodystrophy Krabbe’s disease Nieman pick Farber’s disease Adrenoleukodystrophy Toxic neuropathies due to Amidarone, perhexiline, chloroquine Fabry’s disease

Amyloidosis, Fabry’s disease, small-fiber diabetic neuropathy. GLD, globoid cell leucodystrophy.

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specimen is then oriented correctly for the transverse and longitudinal sections on the OCT medium and covered with the OCT medium. The nerve must be frozen in a -180°C isopentane solution cooled in liquid nitrogen for 15 seconds and then cut at 10 µm by the cryostat using an antiroller plate. The cut sections are picked up on glass slides. The sections are stained with H & E, modified trichrome, PASH, cresyl-fast violet, and Congo-red stains. For paraffin sections, the nerve is processed in the following way. The nerve is fixed in a neutral buffered formalin solution (Formalde-Fresh 10% solution from Fisher Scientific Co.; cat. #SF94-4).* It is then cut into two pieces, one-third for the transverse section and two-thirds for the longitudinal section. Sections are cut at 5 µm, except for Congo-red stain, which should be cut at 8 to 10 µm, and are then stained with H & E, modified trichrome, and Congo-red stains. When processing the nerve for semithin sections, the nerve is fixed in buffered 4% glutaraldehyde solution** for 24 hours and is then dehydrated, osmicated, and embedded in resin. The nerve is cut at 1 µm by the EM microtome for the transverse sections and, if possible, for the longitudinal sections.

PARAFFIN SECTION STAININGS HEMATOXYLIN AND EOSIN STAIN Deparaffinize and hydrate slides to water. Stain in Harris hematoxylin (modified Harris hematoxylin from Richard Allen Co., cat. # 72711) for 5 minutes. Wash in warm running tap water for 10 minutes. Differentiate in acid alcohol (0.5–1% HCl in 70% alcohol) by 2 dips. Rinse in tap water. Dip in ammonia water for 2 minutes and rinse in tap water. Place in eosin Y (eosin Y from Richard Allen Co., cat. #7111) for 2 minutes and rinse in tap water. Dehydrate in alcohol, clear in xylene, and coverslip using permount. The results appear as follows: Nuclei — dark blue Eosinophil granules — bright orange red Thick elastic fibers — deep pink Myelin — pink Red blood cells — bright orange red Cytoplasm — pink Collagen — light pink

MODIFIED TRICHROME STAIN 9 Deparaffinize and hydrate slides to water. Place in Bouin’s fixative (LabChem Inc. LC, cat. #117902) for 30 minutes at 56°C at room temperature. Wash well in running water to remove all yellow color. Stain nuclei with Gill’s hematoxylin† (Surgipath Gill’s II formula, cat. #01520) for 5 minutes. Wash in tap water for 2 minutes. Stain in Gomori’s trichrome solution†† (Richard Allen Co., cat. #88031) for 30 minutes to 1 hour. Rinse in 0.5% acetic water for 20 seconds. If the stain is too dark, decolorize in * Neutral buffered formalin solution: 40% formaldehyde, 100 ml; distilled water (DW), 900 ml; acid sodium phosphate monohydrate, 4 gm; anhydrous disodium phosphate, 6.5 gm. ** 4% glutaraldehyde solution: Sorensen’s phosphate buffer, 2.5 ml; DW, 7.5 ml; 8% glutaraldehyde, 10 ml. For Sorensen’s phosphate buffer, consult a later section of this book. † Gill’s hematoxylin solution: DW, 730 ml; ethylene glycol, 250 ml; hematoxylin, anhydrous powder (C.I. 75290), 2 gm; sodium iodate, 0.2 gm; aluminum ammonia sulfate, 17.6 gm; glacial acetic acid, 20 ml. †† Gomori’s trichrome solution: Fast Green FCF, 0.3 gm; Chromotrope 2 R, 0.6 gm; phosphotungstic acid, 0.6 gm; glacial acetic acid, 1 ml; DW, 100 ml. Dissolve above ingredients in glass beaker using the magnetic stirrer until all ingredients are dissolved. Adjust pH to 3.4 with 0.l N HCl or NaOH. Store at room temperature.

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1% acetic water plus 0.7% phosphotungstic acid solution. Dehydrate, clear in xylene, and coverslip, using permount. Results appear as follows: Myelin — red Connective tissue — green Nuclei — blue

ALKALINE CONGO-RED STAIN Sections are cut at 8 to 10 µm. Deparaffinize and hydrate slides to water. Stain nuclei in Harris hematoxylin for 5 minutes. Wash with tap water for 31/2 minutes. Rinse in distilled water for 2 minutes. Differentiate in acid alcohol solution for 30 seconds. Wash in tap water for 21/2 minutes, and then rinse in distilled water for 2 minutes. Dip in 50% alcohol for 30 seconds. Stain in buffered Congo-red solution* for 1 hour. Dehydrate, clear in xylene, and coverslip, using permount. Sections are examined using crossed Polaroid filters; red-stained amyloid deposits show bright green birefringence. Results appear as follows: Nuclei — blue Amyloid — deep pink to red Elastic fiber — pink to red

FROZEN SECTION STAININGS MODIFIED TRICHROME STAIN 10,11 Stain in Gill’s hematoxylin for 5 minutes. Wash in warm running water to remove excess stain. Stain in Gomori’s trichrome stain for 20 minutes. Wash in warm running water until clear. Do not overrinse. Dehydrate in graded alcohols and xylene. Mount in permount. The results are as follows: Myelin — red Axon — green Nuclei — dark blue

HEMATOXYLIN AND EOSIN (H & E) STAIN Stain in Harris hematoxylin (modified Harris hematoxylin from Richard Allen Co., cat. #72711) for 5 minutes. Wash in warm running tap water for 10 minutes. Place sections in 0.5% ammonium water for 2 minutes. Rinse in warm water for 5 minutes. Stain in eosin for 1 to 2 minutes. Wash in warm running tap water until water is clear. Dehydrate in graded alcohols and xylene. Mount in permount. Results appear as follows: Nuclei — dark blue Myelin — purple Cells with basophilia — varying shades of blue * Congo-red solution: Congo-red C.I. 22120, 0.5 gm; buffered solution at pH 10, 50 ml; absolute alcohol, 50 ml. Dissolve the Congo-red in the buffer solution. Then add the absolute alcohol. This solution is stable for 6 months at room temperature. Alkaline buffer solution, pH 10.0. 0.1 M glycine (7.51 gm in 1000 ml DW), 30 ml; 0.1 M NaCl (NaCl 5.85 gm in 1000 ml DW), 30 ml; 0.2 M sodium hydroxide (4 gm of sodium hydroxide in 1000 ml DW), 40 ml.

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PASH (PERIODIC ACID SCHIFF AND HEMATOXYLIN) STAIN Put the section in Carnoy’s fixative* for 5 minutes. Wash until clear with distilled water. Put in 0.5% periodic acid** for 5 minutes. Wash until clear with distilled water. Put in Schiff’s solution† (Sigma, cat. #S-5133) for 10 minutes. Wash in warm, running tap water for 10 minutes. Counterstain in Gill’s hematoxylin for 5 minutes. Wash in warm running tap water for 10 minutes. Dehydrate in graded alcohols and xylene. Mount in permount. Results appear as follows: PAS positive sustances such as amyloid, basal laminae, polyglucosan body — red or magenta Nuclei — dark blue

HIRSCH–PEIFFER CRESYL-VIOLET STAIN Stain for 3 minutes in 1% aqueous cresyl-fast violet acetate.†† Blot the sections dry after rinsing in water. Dehydrate, clear, and mount. Results appear as follows: Sulphatide — golden brown Normal myelin — purple

ALKALINE CONGO-RED STAIN See the Congo-red stain for the paraffin section. Cut at 10 µm. Follow the Congo-red staining procedure for the paraffin section with two differences: begin at step 2 and stain in buffered Congo-red stain for 45 minutes. Sections are examined using crossed Polaroid filters; red-stained amyloid deposits show bright green birefringence. Results are as follows: Nuclei — blue Amyloid — deep pink to red Elastic fiber — pink to red Myelin — blue-purple

PROCESSING OF THE NERVE FOR SEMITHIN AND ELECTRON MICROSCOPY SECTIONS Always fix the nerve in 4% glutaraldehyde for at least 24 hours before dehydration and embedding.

PROCESSING AND EMBEDDING PROCEDURE Put tissue into Sorensen’s phosphate buffer‡ for 10 minutes, three times, each time in a new buffer solution. Place sample in a 1:1 mixture of 1% osmium tetroxide and the phosphate buffer for 2 hours and * Carnoy’s fixative: absolute alcohol, 60 ml; chloroform, 30 ml; glacial acetic acid, 10 ml. Mix all together in a dry glass bottle and store in glass bottle at room temperature. ** 0.5% periodic acid: periodic acid, 0.5 g; DW, 100 ml. Dissolve all in a glass bottle and store at room temperature. † Schiff’s solution: basic fuchsin, 1 g; DW, 200 ml; l N HCl, 20 ml; anhydrous Na bisulfite, 1 g. To dissolve basic fuchsin in DW in a glass flask, boil with stirring. Cool to 50°C and filter. Add HCl and cool to 250°C. Add Na bisulfite very carefully. Keep in the dark for 2 days. Filter and store in dark bottle in refrigerator. †† 1% aqueous cresyl-fast violet acetate solution: cresyl-fast acetate, 1 g; DW, 100 ml. Mix with the aid of low heat, filter, and store in the cabinet. Solution is good for 6 months. ‡ Sorensen’s phosphate buffer: A solution: 17.6 gm sodium phosphate monobasic in 500 ml DW. B solution: 28.4 gm sodium phosphate dibasic in 500 ml DW. For 100 ml of Sorensen’s phosphate buffer: 13.0 ml A solution and 87 ml B solution.

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30 minutes. Wash tissue with distilled water. Pour on and off. Place in 50% alcohol, 70% alcohol, and 80% alcohol for 10 minutes each. Place in 95% alcohol for 10 minutes twice. Place in 100% alcohol for 10 minutes, 3 times. Place in a 1:1 mixture of 100% alcohol and propylene oxide solution for 10 minutes, 4 times. Fix in a 1:1 mixture of propylene oxide and Spurr resin for at least 6 hours. Tissue can stay in this solution for up to 24 hours. Place in 100% Spurr resin (Electronmiscroscopy Science, Spurr Resin Kit 49001). Tissue should stay in this solution for at least 24 hours but can stay indefinitely. Embed in 100% Spurr resin and place into a 65°C oven for 24 hours.

SEMITHIN (0.5–1 µm) SECTION STAININGS After embedding, cut the nerve at 1 µm with the EM microtome for transverse sections, which are the most important. Once the transverse cut is made, attempt to cut the nerve for longitudinal sections. Often, it is not easy to cut the nerve for the longitudinal section. Always cut tissue at a slant to maintain the correct orientation.

TOLUIDINE BLUE AND BASIC FUCHSIN STAIN (PARAGON MULTIPLE STAIN) Pick up the sections on a drop of distilled water on a clean glass slide. Use a saturated chloroform swab to remove wrinkles from the sections. Gently wave the swab back and forth until the sections spread. Do not touch sections with swab. Heat gently to dry. With the aid of low heat on a hot plate, flood sections with Paragon multiple stain solution** and sprinkle sodium borate lightly over staining. Staining is complete when a green sheen covers the staining surface. Wash well in warm water and flush slides with absolute alcohol. Store slides in an upright position in a slide spacer. Do not mount to avoid any wrinkling. Results appear as follows: Collagen — magenta to pink Cytoplasm — blue Nuclei — dark blue Myelin — very dark blue to black

TOLUIDINE BLUE STAIN Everything is the same as above except a 1% toluidine blue solution† is used. The results appear as follows: Collagen — pale blue Cytoplasm — pale blue Nuclei — dark blue Myelin — very dark blue to black

** Paragon multiple stain: Toluidine blue, 1.095 gm; basic fuchsin, 0.405 gm; 50% alcohol, 150 ml. Add all ingredients together, stir well, and filter before use. Store at room temperature. † 1% toluidine blue solution: 1 gm toluidine blue in 100 ml DW.

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OTHER STAINS Thionin and acridine orange, thionine and basic fuchsin, methyline blue and basic fuchsin,10 and p-Phenylenediamine11 stains can also be used for staining the semithin section. Details of the staining procedure are available in the referenced literature.

NERVE FIBER TEASING For the teasing of nerve fibers, the nerve is fixed in a neutral buffered formalin solution (Formalde-Fresh 10% solution from Fisher Scientific Co.; cat. # SF94-4)* in our laboratory. We prefer formalin fixation because we can choose the time of teasing at our convenience. Other laboratories prefer fixation in glutaraldehyde.17, 11,** The teasing of one nerve fiber from beginning to end requires a minimum of 3 days. Thus, it is always important to plan ahead for the teasing of nerve fibers.

PREPARATION OF NERVE FOR TEASING Find the original sample and pour on two changes of Sorensen’s phosphate buffer for one hour each. With a pair of forceps, place most of the original sample in the second container. Pour just enough 1% osmic acid in the second container to cover the sample. Place a lid on the container, and write the number of pieces in the container on the lid. Any fat in the sample will begin to dissolve, sometimes causing the osmic acid solution to become opaque. Without knowing how many pieces are in the solution, it may be impossible to determine whether all samples have been removed. Allow the tissue to sit in the container for 36 to 48 hours. It is virtually impossible to overstain the sample with osmic acid, so it is better to allow as much time as is practical than to remove the sample after only 24 hours. We do not recommend more than 48 hours, however, because the nerve becomes too hard to tease. After 48 hours, fill another container with diluted glycerol. Remove the stained samples from the original container, and place them in the water/glycerol mixture.† Allow the container to sit for at least 24 hours. The samples can be stored in glycerol indefinitely, and, in general, the longer they sit there, the softer and, thus, easier to tease they will be.

GENERAL GUIDELINES FOR TEASING OF FIBERS Two slides are placed on the stage of a dissecting microscope. One serves as a work surface on which the intact nerve rests in a pool of glycerin, and the other receives the teased individual fibers. With a pair of curved, pointed forceps (such as jeweler’s forceps), strip off the softened epineurium and perineurium onto lightly glycerinated glass slides under a dissecting microscope into several bundles of fascicles. Under higher magnification, subdivide the bundles of fascicles into smaller-sized bundles until single myelinated fibers can be identified. Gently pull a single fiber or a few fibers from the parent strand with forceps and drag them onto the receiving slide in a thin trail of glycerin. After five fibers have been placed on the receiving slide, apply a cover slip (see Figure 3.2).

* Neutral buffered formalin solution: 40% formaldehyde, 100 ml; DW, 900 ml; acid sodium phosphate monohydrate, 4 gm; anhydrous disodium phosphate, 6.5 gm. ** Asbury’s glutaraldehyde fixation: The nerve is fixed for one hour in 0.1 M phosphate-buffered 3.6% glutaraldehyde. After two 15 minute buffer washes, the nerve is immersed in 0.1 M phosphate-buffered 2% osmium tetroxide for 4 to 6 hours. After two further washes, the tissue is placed in 66% glycerin in water for at least 12 hours and is then stored in 100% glycerin at 4°C. Material can be held this way for 6 months or more without recognizable tissue alteration. † Glycerol and DW mixed in a ratio of 1:1 by volume.

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FIGURE 3.2 From left to right and from top to bottom, consecutive steps in fiber teasing: a fascicle of nerve, fixed in glutaraldehyde or formalin and osmium tetroxide, lying in pool of glycerin on glass slide; proximal ends are grapsed and fascicles are pulled apart; epineurium and perineurium are stripped off; strands of fibers are pulled apart; from separated strands of nerve, a single teased fiber is slide onto an adjacent slide as described in the text; teased fibers in place under cover slip. (With permission from Dyck, P.J., Peripheral Neuropathy, Dyck, P.J., Thomas P.K., and Lambert E.H. Eds., W.B. Saunders, Philadelphia, 1975.)

PRACTICAL TIPS FOR TEASING FIBERS* It is not necessary to place a minute drop of glycerol between the slides when transferring the fiber from one slide to another. In fact, this drop has a tendency to slip through the crack between the slides and ruin the microscope stage. In any case, glycerol is likely to get on the stage, which can be cleaned off with alcohol if it becomes bothersome. Note that the stage can be scratched, just like a pair of glasses. Therefore, use only a soft cloth like Kleenex to clean the stage. The forceps can leave the surface of the slide while holding a fiber. It is not important that the tip of the forceps touch the stage, but the far end of the nerve fiber must touch the stage, thus pulling the fiber straight while it is being dragged from one slide to another. In practice, it is virtually impossible to tease a truly single fiber from any nerve sample. A single fiber is very fragile and is liable to break as it is transferred from one slide to another. Instead, it is more practical to collect groups of two or three fibers as one fiber and then transfer this group intact to the second slide. Remember, however, that a group of more than a few fibers will be almost impossible to interpret, as one will not be able to trace the individual fibers under a light microscope. It is virtually impossible to randomly sample fibers in the manner outlined in this chapter without a lot of experience with this technique, and it is not worth the effort. A simple, easier method for * These tips were prepared by Dr. David Oh, who worked on nerve-teasing as an undergraduate student project.

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random selection is this: for each slide, pull one thick strand off the entire sample, and then tease off five fibers from various parts of the strand (this will involve splitting the strand into many smaller sections, because fibers must be obtained from the middle as well as the edge of the thick strand). Repeat this process with new slides until you have 11 slides (50 fibers, with 1 slide left over just in case). Since each fiber is actually a group of 2 or 3 (see above), the total number of fibers ready for analysis will usually exceed 100. Be careful about concentrating too much on the most visible fibers in the sample. Demyelinated fibers are relatively difficult to see and tease, but must be considered in any valid analysis. Therefore, in difficult situations (on the 5–20 fiber level), use the “right hand rule.” Grab the fibers on the right regardless of their visibility or condition. Using this rule leads to a very random sampling of fibers in any situation. Make sure you are using the sharpest pair of forceps available.

PREPARING THE SLIDE AFTER TEASING When preparing the slide, first place a drop of Surgipath Acrytol or similar mounting medium on the cover slip. Use a very small drop, as a large drop will spread out too quickly, dislodging the nerve fibers and ruining the slide. Then align the cover slip so the center of the drop is over the center of the fiber, and drop it on the slide. Do not press on the cover slip, as this, too, will dislodge the fibers. Label the slide and let it sit by itself for 24 hours. Finally, seal the edge of the slip with your fingernail polish.

ELECTRON MICROSCOPE STUDY For the ultrastructural electron microscope study of a nerve, one has to follow standard procedures and techniques. The general guidelines on this subject are given by King.10

REFERENCES 1. 2.

Asbury, A.K. and Connolly, E.S., Sural nerve biopsy: technical note, J. Neurosurg., 38, 391, 1973. Dyck, P.J. and Lofgren, E.P., Nerve biopsy. Choice of nerve, methods, symptoms, and usefulness, Med. Clin. North Am., 52, 885, 1968. 3. Oh, S.J., The nerve conduction and sural nerve biopsy helpful in rapid diagnosis of vasculitis, Neurology, 35 (S1), 240, 1985. 4. Harati, Y. and Matta, K., Gomori trichrome stain, Arch. Neurol., 36, 454, 1979. 5. Oh, S.J., Diagnostic usefulness and limitations of the sural nerve biopsy, Yonsei Med. J., 31(1), 1, 1990. 6. Grunnet, M.L., Gomori’s trichrome stain. Its use with myelin sheaths, Arch. Neurol., 35, 692, 1978. 7. Dyck, P.J., Karnes, J., Lais, A., Lofgren, E.P., and Stevens, J.C., Pathologic alterations of the peripheral nervous system of humans, in Peripheral Neuropathy, Dyck, P.J., Thomas, P.K., Lambert, E.H., and Bunge, R., Eds.,W.B. Saunders, Philadelphia, PA, 1984, 760. 8. Midroni, G. and Bilbao, J.M., Biopsy Diagnosis of Peripheral Neuropathy, Butterworth-Heinemann, Boston, MA, 1995. 9. Engel, W.K. and Cunningham, G.C., Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy sections, Neurology, 13, 919, 1963. 10. King, R., Atlas of Peripheral Nerve Biopsy, Arnold, London, UK, 1999. 11. Asbury, A.K. and Johnson, P.C., Pathology of Peripheral Nerve, W.B. Saunders, Philadelphia, PA, 1978.

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4

Normal Nerve: Histology

Grossly, the sural nerve looks like a pearly white cord and measures 2 to 3 mm in diameter. Thus, it resembles angel-hair pasta. It is usually adhered to some loose adipose tissue. In general, the superficial peroneal and radial nerves are smaller than the sural nerve in diameter. There are three compartments in the nerve: the epineurium, perineurium, and endoneurium. Five to fifteen nerve fascicles are usually present in the sural nerve (Color Figure 4.1),* surrounded and bound by connective tissue in the epineurium (Color Figures 4.2 and 4.3). The epineurium makes up approximately one-half of the cross-section area of the nerve. The most important structures in the epineurium are arterioles and venules because these are the vessels most often involved in vasculitic neuropathy. One or two arterioles are found in the epineurium, and their diameters range from 30 to 300 µm. Pacinian corpuscles are rarely observed in the epineurium. Midroni et al. observed this in only 3 of nearly 700 consecutive cases. Apparently, a few mononuclear cell infiltrates were found around the vessels in the epineurium of normal nerves.1,2 Dyck stated that it is not always easy to decide whether the degree of perivascular infiltration is abnormal.1 Again, one has to judge such findings in correlation with the clinical findings. Other cell types normally seen in the epineurium include fibroblasts and mast cells. The perineurium separates the endoneurium of the nerve fascicle from the epineurium. The endoneurium contains nerve fibers, Schwann cells, and blood vessels, together with bundles of endoneurial collagen fibers oriented longitudinally along the nerve fascicles. Ninety percent of the cell nuclei in the endoneurium belong to Schwann cells; the rest of the cells are mainly fibroblasts and capillary endothelium. Occasional mast cells are also present in the endoneurium. A regular light microscope does not reliably detect and identify scattered lymphocytes in normal nerves. Thus, if scattered lymphocytes are definitely observed under the light microscope, this should be interpreted as abnormal. A few recent studies have found a few leukocytes in normal nerves using Leukocyte Common Antigen (LCA) immunohistochemical staining.3,4 There were no immunopositive T- or Bcells.5 As a practical guideline, Midroni stated that a few (three to four on cross-section) LCA-positive cells randomly dispersed throughout the endoneurium of an average fascicle do not necessarily indicate abnormality.2 However, cuffing around an endoneurial vessel is always regarded as a significant marker of inflammation. Total endoneurial area in the distal sural nerve ranges from 0.65 to 1.26 mm 2.6 Myelinated fibers and their Schwann cells account for 24 to 36% of this total cross-sectional area, and unmyelinated fibers and their Schwann cells account for 11 to 12%. Eighty percent of the Schwann cells are associated with nonmyelinated axons. The nonmyelinated fibers are nearly four times as numerous (approximately 30,000 per square millimeter of nerve) as the myelinated fibers (average 8000 per square millimeter). Nonmyelinated fibers have a range of 0.5 to 3.0 µm in a unimodal distribution but are reliably demonstrated only by electron microscopy. Myelinated fibers have a range of external diameter (axon plus myelin sheath) of 2 to 17 µm and show bimodal distribution with peaks at 5 µm and 13 µm. The thickness of the myelin sheath is proportional to axon diameter in the semithin sections (Color Figure 4.4) and Kultschitzky’s stained paraffin sections (Color Figure 4.5). As a rough guide, the ratio of the diameter of an axon without myelin to that of a fully myelinated axon, called the G-ratio, is normally 0.5 to 0.7. Most histologically normal axons over 3 µm in diameter should have a myelin sheath. If there is no myelin sheath in axons over 3 µm in diameter, one can interpret them as denuded axons (demyelinated axons). This G-ratio is not applicable in the frozen or paraffin * Color insert figures. © 2002 CRC Press LLC

sections because the axon is not mostly visible, and axons are smaller in diameter when visible (Color Figures 4.6 and 4.7). In the frozen and paraffin sections, myelinated fibers fill the entire area of the nerve fascicle (Color Figures 4.8–4.11). Frozen and paraffin sections tend to predominantly show the large-diameter fibers. Sometimes axons can be identified in the center (Color Figures 4.6 and 4.7). On the other hand, in semithin sections, myelinated fibers of varying diameter can easily be seen in transverse sections of the normal sural nerve (Color Figure 4.12). In semithin sections, one can easily recognize the separation of myelinated fibers and two populations of myelinated fibers. Cylindrical hyaline bodies (Renault bodies) occur in the endoneurium (Color Figures 4.13–4.15) as a normal variant and should not be interpreted as abnormal. Renault bodies appear round or ellipsoid in cross-section and are 30 to 200 µm in diameter, lightly eosinophilic, and lightly stained with toluidine blue and Alcian blue, but not with PAS or Congo-red stains. Renault bodies are found in approximately 2 to 7.5% of sural nerve biopsies.2,7,8

AGE-RELATED CHANGES IN THE SURAL NERVE BIOPSY Age-related changes occur at childhood and old age (Figure 4.1). In view of the smaller endoneurial area at birth, fiber density is clearly highest at this time,9 though the total number of myelinated fibers in the sural nerve at birth is smaller, roughly half of the adult value.10 Thus, myelinated fibers are densely packed, and intervening endoneurial collagen forms compact bundles with little space between adjacent collagen fibrils. Unmyelinated fibers contain 10 or more axons. Axon diameter and myelin thickness are below adult values at birth, but the G-ratio is above normal, indicating relative hypomyelination.11,12 Thus, to the inexperienced eye, normal nerves at birth may look like demyelinating neuropathy compared to the normal myelinated population for adults. With increasing age, there is an obvious increase in the size and separation of myelinated fibers.10,13 These changes are most marked during the first few years. During this period there is a progressive increase in the thickness of the myelin sheath in relation to the axon diameter, but a few large fibers have relatively thin myelin sheaths. By the age of 5, axon diameters reach adult values, with final adjustments in myelin thickness continuing for at least 10 years. Midroni recommended 10 years of age as a convenient but rough guideline for the age at which the human sural nerve reaches histologic maturity. The increase in myelin sheath thickness and axon diameter appears complete by the second decade. During the next three or four decades, the amount of endoneurial collagen increases slightly, but there is only occasional evidence of axonal degeneration or demyelination.10 Changes due to aging are obvious from about 60 years of age; there is an obvious reduction in the density of myelinated fibers.10,14,15 The depletion is most prominent for large myelinated fibers.10 In one study of 79 sural nerves, the average large myelinated fiber density decreased by almost 46% from the third to the ninth decade.14 Fascicles contained an occasional degenerating axon as well as scattered regeneration clusters and remyelinated fibers. Also present in moderate numbers were fine- or medium-sized axons with disproportionately thick myelin sheaths. The amount of endoneurial collagen increased, and individual collagen fibrils appeared to be more widely separated. Many unmyelinated fibers showed banding of Schwann cell processes associated with loss of axons. From the sixth decade onward, the vasa nervorum showed increasing reduplication of the endothelial and pericytic basement membranes, but the membranes did not appear to be thickened. In older subjects there was obvious thickening of the perineurial basement membranes. It is, therefore, obvious that one has to be careful in interpreting the nerve biopsy from older patients. Unless the abnormality is obvious, one should not interpret the findings as abnormal in this age group. In patients with minimal abnormalities, changes could well be due to aging and, thus, clinical correlation is essential.

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FIGURE 4.1 Age-related changes. Transverse sections of sural nerves at 5 months (A) 10 years (B) 30 years (C) and 67 years (D). With increasing age there is a reduction in the density of myelinated fibers, an increase in axonal caliber and myelin sheath thickness, and an increase in the amount of endoneurial collagen. In (D) there are scattered fibers with inappropriately thin sheaths, probably remyelinated, myelin sheath irregularities, and clusters of regenerated fibers (arrows). Bar 25 µm. (With permission from Jacobs, J.M. and Love, S., Qualitative and quantitative morphology of human sural nerve at different ages. Brain, 1985, 108:900–901.)

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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Dyck, P.J., Pathologic alterations of the peripheral nervous system of man, in Peripheral Neuropathy, Dyck, P.J., Thomas, P.K., and Lambert, E.H., Eds., W.B. Saunders, Philadelphia, PA, 1975, 296. Midroni, G. and Bilbao, J.M., Biopsy Diagnosis of Peripheral Neuropathy, Butterworth-Heinemann, Boston, MA, 1995. Kerkoff, A. et al., Inflammatory cells in the peripheral nervous sytem in motor neuron disease, Acta Neuropathol., 85, 560, 1993. Hanovar, M. et al., A clinicopathological study of the Guillain–Barré syndrome: nine cases and literature review, Brain, 114, 1245, 1991. De la Monte, S.M. et al., Peripheral neuropathy in the acquired immunodeficiency syndrome., Ann. Neurol. 23, 485, 1988. Behse, F., Morphometrric studies on the human sural nerve, Acta Neurol. Scand., S132, 1, 1990. Bergouignan, F.X. and Vital, C., Occurrence of Renault bodies in a peripheral nerve, Arch Pathol. Lab. Med., 108, 330, 1984. Weis, J., Alexianu, M.E., Heide, G., and Schroder, J.M., Renault bodies contain elastic fiber components, J. Neuropathol. Exp. Neurol., 52, 444, 1993. Ouvier, R.A., McLeod, J., and Conchin, T., Morphometric studies of sural nerve in childhood, Muscle and Nerve, 10, 47, 1987. Jacobs, J.M. and Love, S., Qualitative and quantitative morphology of the human sural nerve at different ages, Brain, 108, 897, 1985. Schroder, J.M., Bohl, J., and Brodda, K., Changes of the ratio between myelin thickness and axon diameter in the human developing sural nerve, Acta Neuropathol., 84, 416, 1992. Ferriere, G., Denef, J.F., Rodriguez, J., and Guzzeta, F., Morphometric studies of normal sural nerve in children, Muscle and Nerve, 8, 697, 1983. Schellens, R.L.L.A. et al., A statistical approach to fiber diameter distribution in human sural nerve, Muscle and Nerve, 16, 1342, 1993. Tohgi, H., Tsukagoshi, H., and Toyokura, Y., Quantitative changes with aging in normal sural nerves, Acta Neuropathol., 38, 213, 1977. O’Sullivan, D.J. and Swallow, M., The fiber size and content of the radial and sural nerves, J. Neurol. Neurosurg. Psychiat., 31, 464, 1968.

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CHAPTER 4 Figure 1 Five fascicles in a sural nerve. Each fascicle is filled with red myelinated fibers. Frozen section. Modified trichrome stain. (40 × magnification.)

CHAPTER 4 Figure 3 Three compartments in a normal nerve: epineurial space (ep), perineurium (p), endoneurial space (en); subperineurial space (sps); arteriole (a). Each fascicle is filled with red myelinated fibers. This is normal. Paraffin section. Modified trichrome stain. (100 × magnification.)

CHAPTER 4 Figure 2 Three compartments in a nerve: epineurial space (EP), perineurium (P), and endoneurial space (EN); arteriole (A); vein (V). This nerve had ten fascicles, four of which are visible here. Semithin section. Toluidine blue/basic fuchsin. (100 × magnification.)

CHAPTER 4 Figure 4 Normal sural nerve. G-ratio in one myelinated fiber (arrow) is 0.6. Semithin section. Toluidine blue and basic fuchsin. (400 × magnification.)

CHAPTER 4 Figure 5 Normal sural nerve. G-ratio in one myelinated fiber (arrow) is 0.5. Paraffin section. Kultschitzky’s stain. (400 × magnification.)

CHAPTER 4 Figure 6 Normal sural nerve. Axon is visible as a dot in the center of myelinated fibers. Axon is rarely visible in the paraffin section; lm means largediameter fiber; sm means small-diameter fiber; a stands for axon. Paraffin section. Modified trichrome stain. (400 × magnification.)

CHAPTER 4 Figure 7 Normal sural nerve. Axon (a) is somewhat larger in the frozen section than the paraffin section. Again, the axon is rarely visible in the frozen section; sm means small myelinated fiber and lm means large myelinated fiber. Frozen section. Modified trichrome stain. (400 × magnification.)

CHAPTER 4 Figure 8 Entire nerve fascicle is filled with red myelinated fibers in normal nerve. Frozen section. Modified trichrome stain. (100 × magnification.)

CHAPTER 4 Figure 10 Normal sural nerve. Entire nerve fascicle is filled with red large-diameter myelinated fibers in the longitudinal cut. Frozen section. H & E stain (200 × magnification.)

CHAPTER 4 Figure 9 Normal sural nerve. Myelinated fibers are stained as purple. Frozen section. H & E stain (200 × magnification.)

CHAPTER 4 Figure 11 Normal sural nerve. Entire nerve fascicle is filled with red large-diameter myelinated fibers in the longitudinal cut. Curved distortion (arrow) of the specimen is unavoidable in the paraffin section. Modified trichrome stain. (200 × magnification.)

CHAPTER 4 Figure 12 Normal sural nerve. Varying diameter fibers are clearly identifiable in the semithin section. Semithin section. Toluidine blue and basic fuchsin. (200 × magnification.)

CHAPTER 4 Figure 13 Renault’s body (double arrow) in the endoneurial space. Semithin section. Toluidine blue and basic fuchsin. (200 × magnification.)

CHAPTER 4 Figure 14 Renault’s body (arrow) in the endoneurial space in the longitudinal cut. Frozen section. Modified trichrome. (200 × magnification.)

CHAPTER 4 Figure 15 Four Renault bodies (arrows) in each nerve fascicle in the transverse cut. Frozen section. H & E stain. (100 × magnification.)

5

Specific Diagnostic Pathological Features of Nerve Biopsy

There are some pathological features that are diagnostic of the specific neuropathy in the nerve biopsy. These will be discussed here in the order of specificity. More detailed information on specific pathlogical features is available in the chapters dealing with the specific subjects.

VASCULITIS Vasculitis in the sural nerve biopsy is diagnostic of vasculitic neuropathy and vasculitis. Vasculitis is histologically characterized by the intramural infiltration of inflammatory cells and fibrinoid necrosis of vessel walls. Vasculitis is usually observed in small arterioles in perineurial or epineurial spaces. Peripheral neuropathy is common in systemic vasculitides. Neuropathy is present in 60% of cases of polyarteritis nodosa and in 64% of cases of the Churg–Strauss syndrome.1,2 Vasculitis tends to involve medium- and small-sized arteries in many systemic vasculitides. Since the vasa nervorum in the peripheral nerve fall directly into the spectrum of small-sized arteries and arterioles, it is not surprising that peripheral neuropathy is a common manifestation of systemic vasculitides. As discussed above, whole nerve biopsy should be performed in suspected cases of vasculitic neuropathy. The sural nerve biopsy should be done before any steroid treatment is initiated. It is necessary to cut multiple sections from different levels of the specimen since vasculitis is multifocal and segmental. It has been our repeated experience that only a few sections of the biopsied nerve show the diagnostic change. To render a definite diagnosis of vasculitic neuropathy, the unmistakable histological features of vasculitis must be present: active, inactive, or healed necrotizing changes and infiltration of inflammatory cells within the vessel wall (Color Figures 5.1 and 5.2).* Perivascular infiltration of inflammatory mononuclear cells without intramural necrosis or cellular infiltration is an early and mild change in vasculitis.3 This alone is not enough to diagnose vasculitis because similar effects are observed in inflammatory neuropathies. However, there are some histological features which are helpful in differentiating these disorders: in vasculitic neuropathies, axonal degeneration is the predominant finding, whereas in inflammatory neuropathy, segmental demyelination and endoneurial inflammatory cells are typical findings. Thus, the diagnosis of probable vasculitis is made when perivascular infiltrations of inflammatory cells are present together with axonal degeneration if the clinical findings are compatible with vasculitis.4 Various patterns of degeneration of fibers are noted, ranging from central fascicular degeneration to selective nerve fascicular degeneration depending upon the severity of the neuropathy. Central fascicular degeneration is typical of ischemic neuropathy and is seen in vasculitic neuropathy.5 Selective nerve fascicular degeneration has been observed predominantly in vascular neuropathy. Any combination of these changes may be found in a single sural nerve biopsy in cases of vasculitic neuropathy. In recent years, nonsystemic vasculitic neuropathy (NSVN) has been reported. In this disorder, vasculitis is confined to the peripheral nerve, sparing other organs. Thus, a nerve biopsy is critical. Without nerve biopsy, vasculitis cannot be reliably differentiated from other rapidly progressive neuropathies because many cases of NSVN appear symmetrical and serological markers are usually absent. There are two ideas about nature of NSVN: it is either an organ-specific vasculitis6,7 vs. a mild form of systemic vasculitis.8,9 * Color insert figures.

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Rarely, vasculitic neuropathy is reported in association with cancer (paraneoplastic vasculitic neuropathy),10-12 Lyme disease,13,14 AIDS,15,16 sarcoidosis, Hepatitis C, and diabetes mellitus.

AMYLOID DEPOSITS Amyloid deposits in the nerve biopsy are diagnostic of amyloid neuropathy and amyloidosis. The nerve biopsy is the diagnostic test of choice in any suspected cases of amyloid neuropathy. The combined nerve and muscle biopsy is recommended because, in rare cases, amyloid is positive in the muscle biopsy whereas it is negative in the nerve biopsy. The hallmark of amyloid neuropathy is amyloid in the nerve. Amyloid is histochemically Congored positive and green birefringent after Congo-red with polarized light (Color Figures 5.3 and 5.4). Thus, Congo-red staining of a biopsy specimen which is then examined by polarizing microscopy is the single best procedure for the diagnosis of amyloid.17 Using fresh-frozen sections, Trotter and Engel were able to demonstrate amyloid quickly and clearly using crystal-violet stain in biopsied muscles in ten cases of amyloid neuropathy, whereas amyloid deposits were rarely observed in the biopsied nerves.18 Crystal-violet staining is used on frozen sections to screen for amyloidosis, but the presence of amyloidosis is either confirmed or ruled out on paraffin sections with Congo-red stain in every biopsied nerve. Three patterns of amyloid deposits are found in the peripheral nerve: (1) amyloid deposit in extraneural connective tissue, (2) widespread endoneurial amyloid deposit, and (3) amyloid deposit within the walls of vasa nervorum both in epineurial and endoneurial spaces. The predominant nerve degeneration in amyloid neuropathy is axonal degeneration, involving smaller diameter fibers.

METACHROMATIC GRANULES The presence of metachromatic granules in the nerve is diagnostic of metachromatic neuropathy and metachromatic leukodystrophy (MLD). Metachromatic leukodystrophy is a rare autosomal recessive disorder characterized by the accumulation of galactosyl-3-sulfate (sulfatide) in the brain, kidney, gallbladder, and peripheral nerve. Four forms of MLD have been recognized: late infantile, juvenile, adult, and multiple sulfatase deficiency. The enzyme, arylsufatase A, is deficient in the first three forms. Its assay in blood leucocytes and cultured skin fibroblasts is used as a standard diagnostic test. The nerve biopsy constitutes a rapid and reliable procedure for the diagnosis of MLD when biochemical assay is not possible. Metachromatic granules are demonstrable in all cases. For demonstration of metachromatic granules, the biopsied nerve should be stained on frozen sections since metachromatia is best demonstrable with acidified cresyl-violet stain (Color Figures 5.5 and 5.6).19 Metachromatic granules are accumulated in the perinuclear cytoplasm of Schwann cells, within macrophages, and in the vicinity of endoneurial capillaries. These metachromatic granules are stained brown instead of purple or blue with cresyl-violet or toluidine blue. They are also PAS-positive and methyl-blue positive. These metachromatic granules are demonstrated in all forms of MLD, including multiple sulfatase deficiency.

POLYGLUCOSAN BODY Many polyglucosan bodies in the nerve biopsy are diagnostic of polyglucosan body disease (PGBD): such as adult polyglucosan body disease (APGBD), Lafora’s disease, and Type IV glycogenosis, if the typical clinical constellations of such diseases are present. A nerve biopsy is the diagnostic test of choice in any suspected case of APGBD. The hallmark of APGBD is the presence of a polyglucosan body in the central and peripheral nervous sytems (Color Figures 5.7 and 5.8). Polyglucosan body (PGB) is a generic name referring to Lafora body, corpora amylacea, and all other similar structures.

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A polyglucosan body is stained pale blue with the modified trichrome stain, basophilic with H & E, metachromatic with toluidine blue, and strongly positive with PAS before and after amylase and with iodine.Typically, the bodies are intra-axonal, round, range from 5 to 70 µm in diameter, and usually occur in myelinated fibers. In the nerve, many huge distended axons with polyglucosan bodies and thin myelin sheaths have been observed in all studied cases.20-23 Teased nerves show a “string of beads” appearance because of an ellipsoid dilatation of an axon due to a polyglucosan body and axonal degeneration. One or two PGBs in the nerve biopsy are a nonspecific finding without any pathological implication. Thus, many polyglucosan bodies are required for diagnosing PGBD. Because PGBs have been reported in other neuropathies, the clinical constellation of APGBD is required for the diagnosis of this disease.

ONION-BULB FORMATION The pathological hallmark of hypertrophic neuropathy is onion-bulb formation (Color Figures 5.9 and 5.10). This term refers to the concentric laminated layers surrounding the nerve fiber as viewed in the transverse section. These concentric layers of flat cell processes are arranged primarily around demyelinated or normal myelinated fibers. Most cell processes are Schwann cells. They are surrounded by basement membrane, and some contain nonmyelinated axons. In electron microscopy, these laminated layers represent the intertwined and attenuated Schwann cell processes. Though onion-bulb formation is discernable in the frozen section, it is best detected in the semithin section. When advanced, it is detectable even in the paraffin section. One way to identify onion-bulb formation in the paraffin section is to look for an increased number of Schwann cell nuclei. In advanced cases, onion-bulb formation is usually associated with prominent endoneurial and subperineurial spaces, a decreased number of myelinated fibers, and thin myelin. Thickening of the nerve may occur in hypertrophic neuropathy, due in part to the increased collagen content and cellularity of the nerve bundle. There is also often an increase in mucosubstance in the endoneurium. In severe cases, the enlarged nerves are palpable through the skin, and at biopsy they may appear grey and gelatious macroscopically due to the large amounts of endoneurial mucosubstance.24 Pathogenetically, onionbulb formation is indicative of repeated demyelination and remyelination.25 Thus, hypertrophic neuropathy itself is indicative of demyelinating neuropathy. The presence of onion-bulb formation is diagnostic of hypertrophic neuropathy. Thus, hypertrophic neuropathy represents a pathological diagnosis observed in many clinical entities. Among these, the hypertrophic type of the Charcot–Marie–Tooth disease (hereditary motor sensory neuropathy [HMSN] type I) is best known. In Roussy–Levy syndrome, Dejerinne–Sottas disease (HMSN type III), congenital hypomyelinative neuropathy, and Refsum’s disease (HMSN type IV), onion-bulb formation is the most prominent finding in the biopsied nerve. In CIDP, onion-bulb formation is seen in 11 to 43% of cases.26,27 Onion-bulb formation is also observed in hypertrophic mononeuropathy, which is characterized by focal enlargement of a single peripheral nerve.28 Hypertrophic mononeuropathy is different from generalized hypertrophic polyneuropathy because of the following characteristics: (1) it is sporadic; (2) only one site is involved; (3) it can be adequately excised and does not recur; and (4) it lacks systemic extraneural manifestations.28

INFLAMMATORY CELLS AND SEGMENTAL DEMYELINATION The presence of inflammatory cells in the nerve fibers and segmental demyelination is diagnostic of inflammatory demyelinating neuropathy. In inflammatory demyelinating neuropathy, the inflammatory cells in the endoneurial space are specific to this type of neuropathy, which is the type of neuropathy most commonly encountered in the practice of neurology. Inflammatory neuropathy is classified into two main categories: acute and chronic.

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Acute inflammatory demyelinating polyneuropathy (AIDP), better known as the Guillain-Barré syndrome (GBS), is a well-known entity. In contrast to the ubiquitous presence of inflammatory cells in the peripheral nerve in the autopsy series in GBS, inflammatory cells are unfortunately not commonly observed in the nerve biopsy.29 Inflammatory cells were observed in 41% of the cases in our series and in 33% of cases in the study performed by Prineas.30 The presence of inflammatory cells in the endoneurial space is the most specific finding indicative of inflammatory neuropathy (Color Figure 5.11). Inflammatory cells are distinctly mononuclear, composed of both small and large lymphocytes. Plasma cells are scattered among the lymphocytes. In some cases, perivascular infiltration of lymphocytes is seen only in the epineurial space. The most consistent finding in the sural nerve biopsy in GBS is segmental demyelination, as demonstrated by our series and Prineas’s study.33 This is best observed in the teased fibers, semithin sections, and longitudinal sections of the frozen section (Color Figure 5.12). Chronic inflammatory demyelinating polyneuropathy (CIDP) is considered a separate clinical entity on the basis of subacute progression of polyneuropathy, marked nerve conduction abnormalities, a high rate of relapse, and responsiveness to steroid treatment.26,27,31,32 There are two forms of CIPD: the monophasic form and the relapsing form.26 The diagnosis of CIDP is based on the typical clinical features such as subacute progression of polyneuropathy, high CSF protein, and marked nerve conduction abnormalities indicative of demyelinating neuropathy. The sural nerve biopsy is recommended for all patients with this disorder for the reasons as described in Chapter 7. The pathological hallmark of CIDP is primary demyelination, the most constant finding in the sural nerve biopsy (Color Figure 5.12). The presence of inflammatory cells, an expected feature in inflammatory neuropathy, is a rare occurrence, observed in about 20 to 30% of cases. When present, inflammation is not as prominent a feature as in GBS.33 Usually, perivascular infiltration in the epineurial space is more common than endoneurial infiltration of cells. AIDP and CIDP were reported as the most frequently observed neuropathies in acquired immune deficiency syndrome (AIDS) due to the HIV virus.15,34,35 CIDP was also reported in a single case of HTLV I myelopathy, another retroviral disease.36 CIDP is a well-known feature of many dysproteinemic neuropathies associated with osteosclerotic myeloma, benign monoclonal gammopathy, and Waldenström’s macroglobulinemia.

INFLAMMATORY CELLS AND AXONAL DEGENERATION The presence of inflammatory cells in the nerve fibers and axonal neuropathy is diagnostic of inflammatory axonal neuropathy. In inflammatory axonal neuropathy, inflammatory cells are characteristically observed in the epineurial space (Color Figures 5.13 and 5.14). This is in contrast to inflammatory demyelinating neuropathy, in which endoneurial inflammatory cells are known to occur more specifically. Inflammatory axonal neuropathy is classically observed in vasculitic neuropathy. Certainly, in vasculitic neuropathy, definite pathological evidence of vasculitis is required to make the diagnosis. However, in roughly one-third of patients with vasculitic neuropathy, definite pathological features of vasculitis were lacking.37,38 In those cases, the diagnosis of probable vasculitic neuropathy was made on the basis of inflammatory axonal neuropathy. This is especially true in nonsystemic vasculitic neuropathy. In Dyck’s series, inflammatory axonal neuropathy was the most common finding in the sural nerve biopsy in nonsystemic vasculitic neuropathy.39 Inflammatory axonal neuropathy has also been observed in paraneoplastic neuropathy, sensory perineuritis, toxic oil syndrome, and eosinophilic myalgic syndrome.

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NONCASEATING GRANULOMA The presence of noncaseating granuloma in the nerve is diagnostic of sarcoid neuropathy and sarcoidosis once leprosy has been ruled out by the acid fast baccilus (AFB) stain. In sarcoidosis, microscopic granulomata were found in muscle in up to 60% of patients with active sarcoidosis, while peripheral nerve involvement was less than 1% in sarcoidosis.40,41 Thus, a muscle biopsy is the procedure of choice for diagnosis of sarcoidosis if skin or lymph node biopsy is not diagnostic. Sarcoid granuloma is classically a noncaseating granuloma consisting of epithelioid cells, Langhan’s giant cells, and lymphocytes (Color Figure 5.15). No organisms are found in sarcoid granuloma. Noncaseating granuloma has been observed primarily in the epi- and perineurial spaces.42-44 Granuloma in the endoneurium was reported in only one case.45 Granulomatous periangitis and panangitis were observed in the epi- and perineurial spaces in four cases.42-44 In practice, the combined muscle and nerve biopsy is recommended in patients clinically suspected of sarcoid neuropathy for two reasons: the diagnostic yield is high in muscle biopsy, as described above, and granuloma was not always observed in biopsied nerves possibly because of the sampling error. In three of four patients with sarcoid neuropathy, in our series, the sural nerve biopsy did not show classical granuloma.

NECROTIZING (CASEATING) GRANULOMA Necrotizing granulomatous neuropathy is diagnostic of neuropathy secondary to leprosy. Leprosy is an infectious disease caused by Mycobacterium leprae and characterized by its skin and peripheral nerve lesions. Mycobacterium leprae is the only bacterium which invades peripheral nerves in man and animals. It is classified into two polar types, tuberculoid and lepromatous, and a borderline (dimorphous, intermediate) type possessing some characteristics of each polar type. In addition, there is an indeterminate type of mycobacterium which has not established itself into any of the three types mentioned above. The pathological features in a nerve are different according to the type of leprosy involved.46,47 In indeterminate leprosy, the nerve shows lymphocytic infiltration in the endoneurial and perineurial space. In tuberculoid leprosy, noncaseating or caseating granulomatous lesions are the most prominent features. Granuloma can be found in the epi- and perineurial spaces as well as in the endoneurial space. Caseation may occur and produce large abscesses within the nerve. With healing, the nerve shows fibrosis and hyalization in the endoneurium and thick perineurial and epineurial sheaths. Bacilli are scanty and, when present, are almost always in the nerve. In lepromatous leprosy, the perineurial and endoneurial infiltration of macrophages and Schwann cells with AFB bacilli (foamy cells) and inflammatory cells is the cardinal feature (Color Figure 5.16). Massive bacilli are found in these foamy cells. In severe cases, the epineurium may be infiltrated by huge numbers of foamy cells, especially around blood vessels. With time, endoneurial fibrosis occurs. Intraneurial microabscesses may be present in either type, especially during an attack of erythema nodosum. In dimorphous leprosy, granuloma and endoneurial foamy cells are present. In all of these cases, the pathological diagnosis of leprosy should be made on the demonstration of acid-fast bacilli in the nerve using the Fite method (Color Figure 5.17).48 In a majority of cases, the diagnosis of leprosy is usually made by observation of typical skin lesions and the presence of acid-fast bacilli from the skin smear or the skin biopsy. The nerve biopsy is imperative for the diagnosis of primary neuritic leprosy in which neuropathy is the sole clinical manifestation without typical skin lesions or a positive skin smear. In those cases, the skin biopsy from anesthesic areas may fail to show histological changes suggestive of leprosy.49 In 77 patients with peripheral neuropathy without any known causes in a leprosy-endemic area, Jacob and Mathai were able to confirm leprosy in 49.4% of cases by performing a nerve biopsy of the cutaneous nerve

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near the neurological deficit: the superficial radial sensory nerve in patients with “glove” anesthesia and the superficial peroneal or sural nerve in patients with “stocking” anesthesia.49 This study clearly shows the important diagnostic role of the biopsy of the cutaneous nerve in primary neuritic leprosy.

GIANT AXONS Giant axons in the nerve are diagnostic of giant axonal neuropathy and certain toxic neuropathies. Giant axonal neuropathy is seldom familial and is classically accompanied by sensory neuropathy and curly hair.50,51 Giant axons have been reported in certain toxic neuropathies: glue-sniffer’s neuropathy, huffer’s neuropathy, and toxic neuropathy induced by n-hexane, methyl n-butyl ketone, acrylamide, and disulfiram.52-57 N-hexane and methyl n-butyl ketone are widely used as solvents and as components of lacquers, glues, and glue and lacquer thinners. Huffer’s neuropathy is peripheral neuropathy due to “huffing” of lacquer thinner. Thus, glue-sniffer’s neuropathy and huffer’s neuropathy are, in essence, due to inhalation of n-hexane or methyl n-butyl ketone. In disulfiram neuropathy, carbon disulfide, a metabolite of disulfiram, is responsible for giant axons. Giant axonal swelling represents a focal mass of neurofilaments surrounded by thin myelin. Swelling ranges from two to three times the original diameter of the fibers and is usually associated with increased paranodal gap (Color Figures 5.18 and 5.19). The axons may reach a diameter of 50 µm but typically range from 20 to 30 µm. Giant axonal swelling is best seen as “green swollen axons” in the transerve sections with the modified trichrome stain on frozen sections and as “swollen axons” in semithin sections. Axonal degeneration is the predominant feature of these neuropathies.

TOMACULA Tomacula (Latin for sausages) in the nerve biopsy are diagnostic of tomaculous neuropathy. In 1975, Madrid and Bradley coined this term in four patients: two with recurrent familial brachial plexus neuropathy, one with a pressure-sensitive neuropathy, and one with a chronic distal sensorimotor neuropathy predominantly affecting the arms.58 Tomacula refer to the focal sausage-shaped swellings of myelin sheaths, best seen in the teased nerves. However, tomacula can easily be detected on the frozen sections as red sausage-shaped swollen myelin in the longitudinal sections and red swollen myelin in the transverse sections. There is no accompanying axonal swelling (Color Figures 5.20 and 5.21). Tomacula measured up to 27 µm in diameter and from 80 to 250 µm in length in Madrid and Bradley’s cases.58 Within the tomacula, the myelin sheath had an increased number of lamellae, two or three times the normal number in the thickest myelin sheath of a normal nerve.71 Tomaculous neuropathy was first described in 1975 by Behse et al. in six patients with hereditary neuropathy with liability to pressure palsies (HNPP).59 So far, all nerve biopsies from patients with hereditary pressure neuropathy and recurrent familial mononeuropathy or brachial plexus neuropathy have exhibited tomaculous neuropathy.60-63 This neuropathy has also been described in a few cases of HMSN I (CMT 1A), HMSN with myelin outfolding (CMT 4B), IgM paraproteinemic neuropathy, and CIDP.64 Segmental demyelination is the unform finding in these cases. Onion-bulb formation is seen in some cases. Tomaculous neuropathy represents demyelinating neuropathy and is most commonly and typically seen in HNPP and familial recurrent brachial plexopathy.

OCCLUSION OF VASA NERVORUM Occlusion of the small arterioles and capillaries in the nerve is diagnostic of ischemic neuropathy and is observed in diabetic neuropathy, vasculitic neuropathy, and arteriosclerotic neuropathy (Color Figure 5.22).

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Recent reports suggest ischemia as one possible factor in the pathogenesis of diabetic polyneuropathy.65,66 In contrast, ischemia does seem to be important in the pathogenesis of diabetic ophthalmoplegia and proximal asymmetrical diabetic neuropathy.67-69 In vasculitic neuropathies, occlusion of the arterioles may occur due to endothelial and intramural inflammation and proliferation, as discussed above. Ischemia may be responsible for ischemic neuropathy due to severe arteriosclerosis.70 Small arterioles and capillaries in the perineurial and epineurial spaces show occlusion due to extensive fibrotic thickening and hyalization (Color Figure 5.14). In vasculitis, inflammatory cells may be present (Color Figures 5.1 and 5.2). Because of the anatomical distribution of the blood supply, ischemia produces degeneration of nerve fibers in a certain section of nerves, producing central fascicular degeneration (depopulation of fiber in the center of a fascicle) and selective nerve fascicular degeneration (depopulation of fibers in one or two fascicles). Thus, central fascicular degeneration and selective nerve fascicular degeneration are used as the histological markers of ischemic neuropathy.

MALIGNANT CELLS The presence of maligant cells in the nerve fiber is indicative of lymphomatous neuropathy (Color Figure 5.23). This is because neoplastic neuropathy is essentially confined to hematological malignancy including lymphoma, lymphomatous granulomatosis, leukemia, and myeloma. The infiltrating cells have all the characteristics of malignant cells: mitotic figures, pleomorphism, and atopia. A diffuse massive infiltration of all peripheral nerve compartments is most typical of lymphomatous neuropathy. In this type of neuropathy, a tendency toward perivascular cuffing commonly occurs, and, sometimes, a striking angiocentricity of the tumor cells is present, as typically observed in lymphomatoid granulomatosis. When malignant cells are seen in a nerve biopsy, B- and T-cell markers can confirm a lymphoid malignancy. The presence of a monoclonal population of infiltrative cells is inferred when the vast majority of the cells belong to a single lymphocyte subset in the bone marrow or peripheral blood cells. That is because the amount of tissue required for the immunotyping of cells is not available on a routine nerve specimen, and thus, an immunotyping of monoclonality is done with bone marrow or peripheral blood cells by using flow cytometry.

IgM DEPOSITS IgM deposits have been the most important exception to the general lack of diagnostic usefulness of immunohistochemical and immunofluorescent techniques in nerve biopsy (Color Figure 5.24). IgM deposits in the myelin sheath or endoneurium are diagnostic of IgM paraproteinemic neuropathy, including anti-MAG neuropathy. IgM deposits in myelin sheaths are specific for IgM-associated neuropathy, being positive in 40 to 80% of patients with this neuropathy, usually in the presence of antiMAG activity.71 This was not reported in IgG- or IgA-associated neuropathies. Endoneurial deposits of IgM are also specific for IgM-associated neuropathy in that they have been reported only in several cases of Waldenström’s macroglobulinemia and a few cases of IgM MGUS neuropathy.72 Usually, in patients with endoneurial IgM deposits, the nerve lesions are mainly axonal, and anti-MAG activity is usually absent.73 IgM deposits can be demonstrated by either immunofluorescent staining on the frozen sections or immunohistochemical staining on the paraffin sections.

SEGMENTAL DEMYELINATION Segmental demyelination in the nerve is diagnostic of demyelinating neuropathy (Color Figure 5.25). The classic example of demyelinating neuropathy is inflammatory neuropathy, either acute or chronic. In inflammatory neuropathy, inflammatory cells are often present in the nerve to make this

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diagnosis possible. Another example is hereditary hypertrophic neuropathy. However, there are many demyelinating neuropathies which are neither inflammatory nor hereditary. In those cases, segmental demyelination is the sole finding in the nerve without any histological clue for the exact etiology. Thus, the etiology for neuropathy should be sought by conducting other tests. Segmental demyelination can best be observed in teased nerves (Color Figure 5.26). Demyelination can also be diagnosed by a thin myelin sheath in proportion to axon diameter in the semithin sections, onion-bulb formation, or tomaculous change. In the longitudinal cuts of frozen sections, segmental demyelination can rarely be observed when the nerve is well stretched and the cut plane is uniformly flat. Segmental demyelination in the nerve is diagnostic of nonspecific demyelinating neuropathy if other histological features diagnostic of specific neuropathies are lacking.

AXONAL DEGENERATION Axonal degeneration in the nerve is diagnostic of axonal neuropathy. Nutritional, alcoholic, vitamin deficiency, and most toxic neuropathies are the best examples. In these neuropathies, there is no histological feature indicative of a specific diagnosis, which should be made on the basis of other findings. Axonal degeneration can best be diagnosed by the presence of myelin-digestion chambers in the frozen sections and myelin ovoids in teased nerves (Color Figures 5.27 and 5.28). Axonal degeneration is indirectly diagnosed by the presence of giant axons in the nerve and is also expressed by small clusters of small axons with thin myelin (axonal regeneration). This is most readily observed in the semithin transverse sections and represents repeated axon degeneration and regeneration.74 In smoldering axonal degeneration, axon atrophy may be the sole finding indicative of axonal degeneration. Axon atrophy is best observed with electron microscopy by smaller axon diameter in proportion to normal myelin thickness. Except for giant axonal and vasculitic neuropathies, most axonal neuropathies do not have any characteristic histological features in the nerve indicative of etiology. Thus, in these neuropathies, etiology should be sought by conducting other tests.

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37. Claussen, G.C., Thomas, D., Goyne, C., Vázquez, L.G., and Oh, S.J., Diagnostic value of nerve and muscle biopsy in suspected vasculitis cases, J. Clin. Neuromusc. Dis., 1, 117, 2000. 38. Oh, S.J., Vasculitic neuropathy, in Vasculitis, Ball, G.V. and Bridges, S.L. Eds., Oxford University Press, UK, 2001, (in press). 39. Dyck, P.J., Benstead, T.J., Conn, D.L., Stevens, J.C., and Windebank, A.J., Non-systemic vasculitic neuropathy, Brain, 110, 843, 1987. 40. Silverstein, A. and Siltzbach, L.E., Muscle involvement in sarcoidosis, Arch. Neurol., 21, 235, 1969. 41. Delany, P., Neurological manifestations in sarcoidosis: review of the literature with a report of 23 cases, Ann. Intern. Med., 87, 336, 1977. 42. Oh, S.J., Sarcoid polyneuropathy: a histologically proved case, Ann. Neurol., 7, 178, 1979. 43. Vital, C., Aubertin, J., Ragnault, M., Amigues, H., Mouton, L., and Bellance, R., Sarcoidosis of the peripheral nerve: a histological and ultrastructural study of two cases, Acta Neuropathol., 58, 111, 1982. 44. Gellacit, G., Gilbertoni, M., Mancini, A., Nemci, R., Volpi, G., Merelli, E., and Vacca, G., Sarcoidosis of the peripheral nerve: clinical, electrophysiological and histological study of two cases, Eur. Neurol., 23, 459, 1984. 45. Nemi, R., Balassi, G., Latove, N., Sherman, W.H., Olarte, M.R., and Hays, A.P., Symmetric sarcoid polyneuropathy: analysis of a sural nerve biopsy, Neurology, 31, 1217, 1981. 46. Weddell, A.G.M. and Pearson, J.M.H., Leprosy; histopathologic aspects of nerve involvement, in Topics on Tropical Neurology, Hohnbrook, R.W., Ed., F.A. Davis Co., Philadelphia, PA, 1975, 17. 47. Sabin, T.D. and Swift, T.R., Leprosy, in Peripheral Neuropathy, Dyck, P.J., Thomas, P.K., and Lambert, E.H., Eds., W.B. Saunders Co., Philadelphia, PA, 1975, 1166. 48. Fite, G.L., Cambre, P.J., and Turner, M.H., Procedure for demonstrating lepra bacilli in paraffin sections, Arch. Pathol., 43, 624, 1947. 49. Jacbo, M. and Mathi, R., Diagnostic efficacy of cutaneous nerve biopsy in primary neuritic leprosy, Int. J. Lepr., 56, 56, 1988. 50. Berg, B.O., Rosenberg, S.H., and Asbury, A.K., Giant axonal neuropathy, Pediatrics, 49, 894, 1972. 51. Jones, M.Z., Nigro, M.A., and Bare, P.S., Familial “giant axonal neuropathy,” J. Neuropathol. Exp. Neurol., 38, 324, 1979. 52. Korobkin, R. et al., Glue-sniffing neuropathy, Arch. Neurol., 32, 158, 1975. 53. Oh, S.J. and Kim, J.M., Giant axonal swelling in “Huffer’s” neuropathy, Arch. Neurol., 33, 583, 1976. 54. Ansbacger, K.E., Bosche, E.P., and Cancilla, P.A., Disulfiram neuropathy: a neurofilamentous distal axonopathy, Neurology, 32, 424, 1982. 55. Allen, N., Mendell, J.R., Billmaier, D.J., Fontaine, R.E., and O’Neill, J., Toxic polyneuropathy due to methyl n-butyl ketone. An industrial outbreak, Arch. Neurol., 32, 209, 1975. 56. Rizzujto, W., Terzian, H., and Galiazzo-Rizzuto, S., Toxic polyneuropathies in Italy due to leather cement poisoning in shoe industries. A light and electron microscopic study, J. Neurol. Sci., 31, 343, 1977. 57. Davenport, J.G., Farrell, D.F., and Sumi, S.M., ‘Giant axonal neuropathy’ caused by industrial chemicals: neurofilamentous axonal masses in man, Neurology, 26, 919, 1976. 58. Madrid, R. and Bradley, W.G., The pathology of neuropathies with focal thickening of the myelin sheath (Tomaculous Neuropathy). Studies on the formation of the abnormal myelin sheath, J. Neurol. Sci., 25, 415, 1975. 59. Behse, F., Buchthal, F., Carlsen, F., and Knappeis, G.G., Hereditary neuropathy with liability to pressure palsies; electrophysiological and histopathological aspects, Brain, 95, 777, 1972. 60. Earl, C.J., Fullerton, P.M., Wakerfield, G.S., and Schutta, H.S., Hereditary neuropathy, with liability to pressure palsies; a clinical and electrophysiological study of four families, Q. J. Med., 33, 481, 1964. 61. Fewings, J.D., Blumbergs, P.C., Mukherjee, T.M., and Hallpike, J.F., Tomaculous neuropathy: Hereditary predisposition to pressure palsies, Aust. N.Z. J. Med., 15, 598, 1985. 62. Meier, C. and Moll, C., Hereditary neuropathy with liability to pressure palsies: report of two families and review of the literature, J. Neurol., 228, 73, 1982. 63. Pellissier, J.F. et al., Neuropathies tomaculaires: etude histolopathologique et correlations electrocliniques dans 10 cas, Rev. Neurol., 143, 263, 1987. 64. Sanders, S., Ourrier, R.A., McLeod, J.G., Nicholson, G.A., and Pollard, J.D., Clinical syndromes associated with tomacula or myelin swellings in sural nerve biopsy, J. Neurol. Neurosurg. Psychiatry, 68, 483, 2000.

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65. Dyck, P.J., Karnes, J.L., O’Brien, P., Okazaki, H., Lais, A., and Engelstad, J., The spatial distribution of fiber loss in diabetic polyneuropathy sugggests ischemia, Ann. Neurol., 19, 440, 1986. 66. Johnson, P.C., Doll, S.C., and Cromery, D.W., Pathogenesis of diabetic neuropathy, Ann. Neurol., 19, 450, 1986. 67. Asbury, A.K., Aldredge, H., Hershberg, R., and Fisher, C.M., Oculomotor palsy in diabetes mellitus: a clinico-pathologic study, Brain, 93, 555, 1970. 68. Dreyfus, P.M., Hakim, S., and Adams, R.D., Diabetic ophthalmoplegia, Arch. Neurol. Psychiatry, 77, 337, 1957. 69. Raff, M.C., Sangalang, V., and Asbury, A.K., Ischemic mononeuropathy multiplex associated with diabetes mellitus, Arch. Neurol., 18, 487, 1968. 70. Eames, R.A. and Lange, L.S., Clinical and pathological study of ischemic neuropathy, J. Neurol. Neurosurg. Psychiat., 30, 215, 1967. 71. Young, K.B. et al., The clinical spectrum of peripheral neuropathies associated with benign monoclonal IgM, IgG and IgA paraproteinemia. Comparative, clinical, immunological, and nerve biopsy findings, J. Neurol., 238, 383, 1991. 72. Vital, A. and Vital, C., Immunoelectron identification of endoneurial IgM deposits in four patients with Waldenström’s macroglobulinemia: a specific ultrastructural pattern related to the presence of cryoglobulin in one case, Clin. Neuropathol., 12, 49, 1993. 73. Dubas, F., Pouplard-Barthelaix, A., Delestre, F., and Emile, J., Polyneuropathies avec gammapathies monoclonale IgM. 12 cas, Rev. Neurol., 143, 670, 1987. 74. Schroeder, J.M., Die Hyperneurotisation buengnerscher baender bei der experimenellen isoniazid-neuropathie: phasenkonstrast und electronmikroskopische Untersuchungen, Virchoes Arch. Zellpath., Abt B, 1, 131, 1968.

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CHAPTER 5 Figure 1 Active vasculitis. Fibrinoid necrosis and intramural infiltration of mononuclear cells of intimal and muscular layers of arterioles in the epineurial space. Almost total occlusion of vessels due to intimal thickening. Paraffin section. H & E stain. (200 × magnification.)

CHAPTER 5 Figure 2 Inactive vasculitis. Intramural red blood cells and mononuclear inflammatory cells in a thickened arteriole in the epineurial space. Paraffin section. H & E stain. (400 × magnification.)

CHAPTER 5 Figure 3 Amyloid. Congo-red material (arrow) in the wall of tiny vein in the epineurial space. Paraffin section. Alkaline Congo-red stain. (200 × magnification.)

CHAPTER 5 Figure 4 Bright apple-green birefringence of Congo-red material under the polarizing filter. Paraffin section. Alkaline Congo-red stain. (200 × magnification.)

CHAPTER 5 Figure 5 Metachromatic granules are stained as dirty yellow in the perivascular area in the endoneurial vessel. Dirty yellow granules represent metachromasia. The normal color for this stain is blue. Frozen section. Thionine stain. (400 × magnification.)

CHAPTER 5 Figure 7 Polyglucosan body (blue arrow): pearly body with laminated rings and greenish core. Red arrow indicates myelin digestion chambers in one myelinated fiber. Paraffin section. Modified trichrome stain. (400 × magnification.)

CHAPTER 5 Figure 6 Scattered metachromatic granules in the endoneurial space are stained brown instead of normal purple color. Frozen section. Cresylfast violet stain. (400 × magnification.)

CHAPTER 5 Figure 8 Diffuse loss of myelinated fibers. One axon contains a large polyglucosan body (approximately 30 µm) with a round profile in transverse section. It has a laminated appearance with a slightly denser core. The surrounding myelin sheath is thinned. Semithin section. Toulidine blue and acridine orange. (600 × magnification.) (Courtesy of Dr. N.P. Robertson, University Hospitals of Wales, Cardiff, Wales.)

CHAPTER 5 Figure 9 Onion-bulb formations (OBF) are visible around every red myelinated fiber. Thin lines surrounding myelinated fibers represent proliferated Schwann cell processes. There is about a 50% loss of myelinated fibers. Frozen section. Modified trichrome. (200 × magnification.)

CHAPTER 5 Figure 11 Scattered mononuclear inflammatory cells in the endoneurial space (double arrows). Perivascular collections of inflammatory cells in the endoneurial space (arrow). These are typically seen in inflammatory demyelinating neuropathy. Paraffin section. H & E stain. (200 × magnification. )

CHAPTER 5 Figure 10 Onion-bulb formation (OBF) is recognized by many fine lines around nerve fibers with varying myelin thickness. Red arrow indicates denuded axon (demyelination) and blue arrow indicates OBF without any recognizable axon. Some fibers with OBF have more than one Schwann cell nucleus. Semithin section. Toluidine blue. (400 × magnification.)

CHAPTER 5 Figure 12 Remyelinated fibers in chronic inflammatory demyelinating polyneuropathy. About 50% of myelinated fibers are remyelinated fibers (blue arrow) characterized by a thin myelin sheath in proportion to axon diameter. Red arrow indicates normal myelinated fibers. Yellow arrow indicates thinly myelinated fibers with two Schwann cell nuclei and tiny OBF. Semithin section. Toluidine blue. (400 × magnification.)

CHAPTER 5 Figure 13 Perivascular lymphocytes in the epineurial space in a case of vasculitic neuropathy. Paraffin section. H & E stain. (200 × magnification. )

CHAPTER 5 Figure 14 Myelin-digestion chambers (arrows) in the longitudinal cut. Frozen section is indicative of axonal degeneration. Frozen section. Modified trichrome. (200 × magnification.)

CHAPTER 5 Figure 15 Noncaseating granuloma (arrow) with many epithelioid cells and mononuclear inflammatory cells in the subperineurial space. Arrow head indicates another granuloma in the subperineurial space. No granuloma or inflammatory cell is present in the endoneurium itself in the middle. Paraffin section. H & E stain. (200 × magnification.)

CHAPTER 5 Figure 16 Foamy cells in the endoneurium, diagnostic of leprosy (arrows). The entire field is replaced by fibrosis. No myelinated fiber is noted. Semithin section. Toluidine blue stain. (Courtesy of Professor I. Sunwoo, Yonsei University Medical School, Seoul, Korea.)

CHAPTER 5 Figure 17 Mycobacterium leprae. Many acid-fast bacilli (bright red rods or globs) in the foamy cells. Paraffin section. Wade-Fite stain. (Courtesy of Dr. Y. Harati, Baylor Medical College, Houston, TX.)

CHAPTER 5 Figure 19 Giant axon. The dark line represents an axon in the nerve fiber (red arrow). The giant axon (blue arrow) is characterized by a hazelcolored center marked as a dark line. The diameter of the giant axon is 10 times that of the axon. Paraffin section. Glees and Marsland’s silver stain. (400 × magnification.)

CHAPTER 5 Figure 18 Three giant axons are visible in one nerve fascicle. Normally, the axon is stained green as a dot in the middle of red myelinated fibers. The giant axon is easily identified here as a green center surrounded by thin red myelin. Giant axons are three times larger in diameter than normal fibers. The population of myelinated fibers is minimally decreased. Frozen section. Modified trichrome. (200 × magnification.) (With permission from Oh, S.J, Yonsei Med. J., 31, 20, 1990.)

CHAPTER 5 Figure 20 Tomaculous formation. Red sausage-like myelin swelling (red arrow) represents tomaculous formation near the node of Ranvier. The diameter is twice that of normal myelinated fibers. There is also a moderate decrease in the population of myelinated fibers. (With permission from Oh, S.J., Yonsei Med. J., 31, 22, 1990.)

CHAPTER 5 Figure 21 Tomaculous formation with thick myelin layers is noted in the center of the figure. The axon area is extremely small due to thick myelinated layers. Semithin section. Toluidine blue stain. (400 × magnification.)

CHAPTER 5 Figure 23 Malignant cells. Many atypical cells are scattered in the epi- and endoneurial spaces of the nerve. Intramural infiltration of atypical cells is obvious (arrowhead). The infiltrating cells have all the characteristics of malignant cells: mitotic figures, pleomorphism, and atopia. This patient had lymphomatous neuropathy caused by natural killer cell lymphoma. Paraffin section. H & E stain. (200 × magnification.)

CHAPTER 5 Figure 22 Occlusion of a tiny arteriole in the subperineurial space in a case of diabetic neuropathy. Near occlusion of an endoneurial arteriole due to arteriosclerotic endothelial thickening. There are a few scattered red myelinated fibers, indicating a decreased population of myelinated fibers. Paraffin section. Gomori trichrome (200 × magnification.)

CHAPTER 5 Figure 24 IgM deposits in the myelin sheaths in a patient with MAG-positive neuropathy. Immunofluorescent stain for IgM outlines all myelinated fibers. Frozen section. Immunofluorescent stain for IgM. (200 × magnification.)

CHAPTER 5 Figure 26 Segmental demyelination (A and B) and paranodal demyelination (C and D) in the teased nerve. CHAPTER 5 Figure 25 Demyelination and remyelination. Blue arrow indicates one denuded axon (demyelinated fiber), and red arrow indicates one thinly myelinated fiber (remyelinated fiber). Inflammatory cells are scattered in the endoneurial space. There is also a decrease in the population of myelinated fibers. Semithin section. Toluidine blue and basic fuchsin. (200 × magnification.)

CHAPTER 5 Figure 27 Axonal degeneration. Moderate (60%) loss of myelinated fibers. Many myelin digestion chambers (empty spaces, ghost fibers) are noted in the endoneurium, indicating axonal degeneration. Frozen sections. Modified trichrome. (100 × magnification.)

CHAPTER 5 Figure 28 Axonal degeneration. All myelinated fibers are undergoing myelin breakdown indicative of axonal degeneration. Semithin section. Toluidine blue. (400 × magnification.)

6

Vasculitic Neuropathy

Peripheral neuropathy is common in many systemic necrotizing vasculitides (SNV) (Table 6.1) and, thus, is an important clinical manifestation of SNV. When peripheral neuropathy is caused by necrotizing vasculitis, it is called vasculitic neuropathy. Peripheral neuropathy is due to ischemic damage of nerves as a result of occlusion of blood vessels associated with an inflammatory process in the vessel walls in the vasa nervorum. This can occur either as a manifestation of multisystem involvement in SNV or as an independent disease process such as nonsystemic vasculitic neuropathy. Although vasculitic neuropathy is relatively rare, recognizing it is important because it is potentially treatable.

VULNERABILITY OF THE PERIPHERAL NERVE TO VASCULITIC NEUROPATHY The hallmark of all SNVs is “vasculitis,” or inflammation and necrosis of the blood vessels. Vasculitis tends to involve medium- and small-sized arteries in SNV. Since the vasa nervorum in the peripheral nerve fall directly into the spectrum of small-sized arteries and arterioles, it is not surprising that peripheral neuropathy is a common manifestation of SNV. The vessels responsible for vasculitic neuropathy are predominantly the arterioles of the epineurium, which typically have a diameter ranging from 30 to 300 µm.1,2 Peripheral nerves have a rich anastomotic blood supply through two functionally distinct vascular systems: the extrinsic system composed of the epineurial vessels and regional arteries, arterioles, and venules, and the intrinsic system of the longitudinal microvessels within the fascicles themselves. These two systems are linked by a complex network of interconnecting vessels that provide high blood flow in the baseline state.3,4 This rich blood supply, along with the capacity of nerves to function relatively well with anaerobic metabolism, makes peripheral nerves relatively resistant to ischemia. Only with extensive involvement of the vasa nervorum does ischemic damage occur in the nerves. On the other hand, some characteristics of the endoneurial vessels actually render nerves susceptible to ischemia. The endoneurial capillaries are larger and more widely spaced, particularly in the central fascicular regions, than in other tissues.5,6 In addition, endoneurial vessels have a poorly developed smooth muscle layer3 and, thus, peripheral nerves have almost no capability to autoregulate blood flow and are susceptible to the small changes in perfusion pressure that may occur with a vasculitic process. Because of this vulnerability, a central fascicular fiber loss occurs in vasculitic neuropathy and is regarded as a typical pathological feature of ischemic neuropathy.1

CLINICAL, ELECTROMYOGRAPHIC, AND LABORATORY FEATURES In addition to the signs and symptoms of neuropathy, there are usually features of an underlying associated systemic disease in vasculitic neuropathy due to SNV. Common systemic symptoms are fever, anorexia, weight loss, and fatigue.7,8 Skin rash and ulcers, myalgia, arthralgia, Raynaud’s phenomenon, and photosensitivity are less common systemic manifestations. Systemic symptoms have been observed in 82 to 94% of cases.7,9 Systemic symptoms usually develop at the same time as vasculitic

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neuropathy and may precede vasculitic neuropathy by a mean duration of 24 weeks. In nonvasculitic neuropathy, systemic features are absent by definition.8,10,11 Peripheral neuropathy in SNV is manifested in various forms: mononeuropathy, plexus neuropathy, mononeuropathy multiplex, asymmetrical polyneuropathy, and symmetrical polyneuropathy.7,12 Mononeuropathy multiplex has been regarded as the classic clinical manifestation in vasculitic neuropathy and as the most common neurological abnormality in polyarteritis nodosa (PAN).12 In Ford and Siekert’s series, 54% of patients had mononeuropathy multiplex.13 In Gullivan et al.’s recent series, mononeuropathy multiplex was reported in 70% of 182 cases of PAN.14 Cohen, Conn, and Ilstrup even used mononeuropathy multiplex as a criterion in the diagnosis of polyarteritis nodosa.15 Since mononeuropathy multiplex can be due to multiple causes, especially with the introduction of multifocal motor and motor-sensory demyelinating neuropathies, this practice is no longer justifiable. In fact, multifocal demyelinating neuropathy is more common than vasculitic mononeuropathy multiplex in our clinic. In 1981, our study showed that symmetrical and asymmetrical polyneuropathies are common.7 Since then, it has been well accepted that there are three main patterns of neuropathy (mononeuropathy multiplex, asymmetrical polyneuropathy, and symmetrical polyneuropathy), though the relative frequency varies from study to study. A recent review of the reports on the frequency of these three patterns confirmed our initial finding that polyneuropathy (asymmetrical or symmetrical) is more common, observed in 55% of cases.12 The classical pattern of mononeuropathy multiplex was seen in only one-third of patients. Recognition of this concept is important because vasculitic

TABLE 6.1 Frequency of Vasculitic Neuropathy or Peripheral Neuropathy in Systemic Diseases Diseases

Prevalence

Frequency of Neuropathy (%)

Primary vasculitic diseasesa Polyarteritis nodosa Churg–Strauss syndrome Wegener’s granulomatosis Giant cell artertis

Rare Rare Rare Common in elderly

52–60 64–69 11–21 5–14

Rheumatoid diseasesb Rheumatoid arthritis Systemic lupus erythematosus Sjögren’s syndrome Progressive systemic sclerois

Common Common Common Uncommon

10 2–21 10–15 1.5

Other conditions with vasculitis Crygolobulinaemia Malignancy HIV infection Lyme disease

Rare Common Variable Variable

50 Rare 2