Handbook of Clinical Neurology, Vol. 115 (3rd series)Peripheral Nerve DisordersG. Said and C. Krarup, Editors© 2013 Elsevier B.V. All rights reserved

Chapter 9

The nerve biopsy: indications, technical aspects, and contribution

ROSALIND KING* AND LIONEL GINSBERG

Department of Clinical Neurosciences, Institute of Neurology, University College London,Royal Free Campus, London, UK

INTRODUCTION: INDICATIONS

As is generally the case with neurological diagnosis,establishing the cause of a peripheral neuropathy restsprimarily on the patient’s history and physical examina-tion, supplemented, if necessary, by investigations.The latter are selected to be minimally invasive, at leastinitially, and typically may include blood and urine tests,chest x-ray, nerve conduction studies, and cerebrospinalfluid examination. Following this schema, nerve biopsyshould never be seen as a “screening” tool for identify-ing the cause of a neuropathy, but rather as an investiga-tion of last resort, to be used only when the initialapproach has been exhausted. The archetypal situationwhere a nerve biopsy is indicated would be in a patientwith an asymmetrical painful sensorimotor neuropathywhich is causing significant functional limitation. In thisexample, nerve biopsy may be the only way of reachinga definitive diagnosis of vasculitis. This applies particu-larly if the vasculitis is a tissue-specific process, withoutserological or other evidence of systemic involvement,as is found in one-quarter to one-third of patients withvasculitic neuropathy (Kissel et al., 2001). Other examplesof the usefulness of nerve biopsy are summarized inTable 9.1. Many of the conditions listed in this table canbe diagnosed by alternative means. However, a nervebiopsymay still be necessary to guide the clinician towardthe investigationwhichwill reveal the definitive diagnosis.For example, it is not uncommon for a patient with ahereditary neuropathy to have no family history, in whichcase nerve biopsy may be the first pointer to a geneticcause. The biopsy appearancesmay even direct the choiceof gene to be analysed. A final role for nerve biopsy, not

*Correspondence to: Dr. Rosalind King, Ph.D., F.R.C.Path., DeUniversity College London, Royal Free Campus, Rowland Hill St

(0)207 472 6829, Mobile: þ44(0)7725489013, E-mail: r.king@ucl.ac

to be underestimated, is in delineating the pathologicalfeatures of newly-described disease entities, and therebypotentially guiding individual patient management (see,for example, Staff et al., 2010).

The selective, and perhaps diminishing, use of nervebiopsy reflects the fact that it is an invasive procedure,with recognized complications (see below). Furthermore,the analysis of a nerve biopsy specimen is labour-intensive, expensive, and subject to errors of tissue han-dling and pathological interpretation. This analysisis therefore best conducted where specialist laboratoryfacilities are available. But even in ideal circum-stances, the diagnostic yield of a nerve biopsy may bedisappointing – the pathological findings merely con-firming a chronic axonal neuropathy, rather than reveal-ing specific features. Conversely, advances in moleculargenetics and immunology have generated less invasiveand more specific ways of diagnosing many hereditaryand autoimmune neuropathies respectively, withoutrecourse to nerve biopsy. Despite all these caveats, nervebiopsy remains an invaluable aid to diagnosis in carefullyselected patients. In general, these will be patients with aprogressive polyneuropathy, causing significant func-tional limitation, in whom no cause for the neuropathyhas been identified by less invasive means. The presenceof pain and asymmetry will increase the probability ofdiagnosing vasculitis. But biopsy should not be restrictedto patients with these features. Ultimately, it is a questionof weighing the risk of the biopsy (both in terms of harmto the patient and the possibility that the pathologicalfindings will be nonspecific) against the potential benefitof identifying a definite histological diagnosis, whichmay

partment of Clinical Neurosciences, Institute of Neurology,., London NW3 2PF, UK. Tel: þ44 (0)207 472 6364, Fax: þ44

.uk

Table 9.1

Usefulness of nerve biopsy

Disease Diagnostic Helpful Pathological features

CIDP Yes Demyelination, inflammationCMT1 Sometimes Yes Classic onion-bulbsCMT2B Yes

CMT4B Yes Focally folded myelinCMT4C Yes? Yes Very thin SC processesCMT4D Yes Large degenerated onion-bulbsFabry disease Yes Perineurial cell lipid inclusions

FAP Yes Amyloid, most cases TTRþveGAN Yes Numerous giant axons, secondary demyelinationHNPP Yes Tomaculous changes

HSNIV Yes Loss of unmyelinated and small myelinated fibersIgM paraproteinemia Yes Widely spaced myelinKrabbe disease Yes Specific lipid inclusions in SC of myelinated fibers

Leprosy Yes Mycobacterium lepraeLymphoma Yes Monoclonal lymphocytesMLD Yes Specific inclusions in SC

Primary amyloidosis Yes AmyloidToxic neuropathies Yes Sporadic giant axons, SC inclusionsVasculitis Sometimes Yes Cells in vessel wall, hemosiderin, fibrinoid necrosis

FAP¼ familial amyloid polyneuropathy, GAN¼giant axonal neuropathy, MLD¼metachromatic leukodystrophy, CMT¼Charcot–Marie–Tooth,

CIDP¼chronic inflammatory demyelinating polyneuropathy, HNPP¼hereditary neuropathy with liability to pressure palsies, HSNIV¼hereditary

sensory neuropathy type 4, SC¼Schwann cells, TTR¼ transthyretin.

156 R. KING AND L. GINSBERG

lead to treatment options. The remainder of this chapterwill focus on the technical aspects of the procedure (bothsurgical and in the laboratory), and on the interpretationof biopsy findings to maximize their contribution toneuropathy diagnosis.

SURGICAL PROCEDURE

Deciding which nerve to sample depends on the patient’ssymptoms, signs, and neurophysiological findings, but,all else being equal, the sural nerve is preferred. It is themost frequently biopsied so the structure is better knownthan any other nerve. It is also reasonably easily accessi-ble and contains 8 to 13 fascicles at the ankle. Othernerves that may be biopsied include the superficial radialand dorsal cutaneous branch of the ulnar nerve at thewrist. The superficial peroneal nerve in the leg is occa-sionally used especially if a muscle biopsy is to be takenat the same time, as peroneus brevis can be sampledthrough the same incision. Combined nerve and musclebiopsy is said to increase diagnostic yield in vasculitis(Said et al., 1988) though the need for a simultaneousmuscle biopsy has more recently been questioned(Bennett et al., 2008). Skin biopsies are sometimes usedto help ascertain the cause of a neuropathy, particularlyif specific Schwann cell or axonal inclusions are suspected

or if a nerve biopsy is thought too invasive. The disadvan-tage of these biopsies is that only small myelinated fibersare sampled and their distribution in skin is very patchy,requiring several specimens (Kennedy et al., 2005).

Having decided on the nerve, the next decision iswhether to take a cross-section of the whole nerve orsample only a small part of it (full thickness versus“fascicular” biopsy). The sural nerve is often split intobranches of 3 or 4 fascicles. On theoretical grounds, tak-ing a branch should result in less sensory deficit than tak-ing the whole nerve, though this has not been confirmedby head-to-head comparison (Pollock et al., 1983). Theadvisability of sampling in this way depends on the sus-pected disease. For biopsies in which an inflammatoryprocess is likely, sampling is inappropriate as the lesionsmay be very small and patchy and pathology may varyconsiderably from one fascicle to the next as well as pro-ducing localized changes within a fascicle (Fig. 9.1).

The sural nerve is accessed via an incision midwaybetween the lateral malleolus and the Achilles tendon.The nerve trunk is identified by its white, glisteningappearance crossed with oblique dark and light bands(spiral bands of Nageotte) (Nageotte, 1922). Thesurgical approach to sural nerve biopsy is describedin detail elsewhere (Ginsberg et al., 2003) and summa-rized in Fig. 9.2.

Fig. 9.1. Sural nerve biopsy from a 64-year-old man with

chronic inflammatory demyelinating polyneuropathy. There is

considerable variation in fiber density with two fascicles being

much more severely abnormal than the other two. Scale

bar¼100 mm.

vein

A

B

nerve

Fig. 9.2. Sural nerve biopsy technique. The nerve will have

been selected on the basis of clinical and electrical findings.

Generally, the more severely affected side is biopsied. The

patient will have given written, informed consent. Under ster-

ile conditions, in an operating theatre or biopsy suite, the skin

over the biopsy site is cleaned and the surrounding area

draped. The site is infiltrated subcutaneously with lidocaine

(1%). A 4 cm incision is made longitudinally, midway

between the lateral malleolus and Achilles tendon (A). Thesubcutaneous tissue is exposed using cat’s paw retractors

and gently dissected to reveal the nerve, usually found poste-

rior and deep to a vein. The nerve is then isolated using

double-hooked retractors (B) and infiltrated with lidocaine,

using a fine needle proximal to the planned resection site.

With the nerve supported on a hook, the biopsy (at least

3 cm long) is taken with a sharp scalpel blade (No. 11) and

passed to a waiting technician on a damp swab. Finally, the

wound is closed with absorbable subcutaneous sutures and silk

stitches to the skin.

THE NERVE BIOPSY: INDICATIONS, TECHNICAL ASPECTS, AND CONTRIBUTION 157

Aftercare

It has been common practice to keep patients in hospitalovernight following a lower limb nerve biopsy. Althoughit has been traditionally recommended that the biopsiedleg should be rested for 24 hours, it is a moot pointwhether this is beneficial. Patients are advised to keepthe wound dry for 2 days and avoid strenuous activityfor 2 weeks.

Complications

Aswith any invasive procedure, there is the possibility ofwound infection or dehiscence, particularly if the patientis being treated with corticosteroids (Gabriel et al.,2000). There may also be reactions to the sutures anddressings. Especially after taking a full thickness nervebiopsy, there may be patchy sensory deficit on the foot.As there is considerable overlap from several nerves, thisis not usually significant and generally resolveswith timedue to collateral reinnervation (Theriault et al., 1998).

Fig. 9.3. Cutting artefact. This specimen was placed on a hard

Petri dish and cut in the direction of the arrow. Undue pressure

resulted in the annulate myelin sheaths being replaced by

dense solid circles. There is a clear demarcation between dam-

aged and undamaged regions. Scale bar¼50 mm.

L. GINSBERG

Formation of a traumatic neuroma on the proximal endof the sectioned nerve probably contributes to the persis-tent pain or paresthesiae at the biopsy site reported by upto 10% of patients. At least two professors of neurologywith expertise in peripheral neuropathy have undergonesural nerve biopsy themselves and have described theirexperiences in print (Ginsberg et al., 2003; Dyck et al.,2005). Their accounts are remarkably similar, particu-larly regarding the minimal long-term sequelae of theprocedure.

LABORATORY TECHNIQUES

Handling

Nerves are very fragile structures and readily damagedso they must be handled with great care and pinching ortwisting avoided. During and after removal the nervespecimen should be kept damp with normal saline. Itshould be placed on a gauze pad soaked in salineimmediately after excision. Myelin deteriorates rapidlyafter removal from the patient so the specimenmust be fixed or frozen at once. The nerve is kept asstraight as possible and cut into two or three pieces.One of these should be placed on a piece of thincard and fixed in a glutaraldehyde-containing solutionfor resin embedding and another fixed in neutralbuffered formalin (NBF) for paraffin embedding. Ifpossible a third segment should be frozen in liquid nitro-gen for cryostat sectioning. Sodium cacodylate(Karnovsky, 1965), phosphate, or piperazine-N-N’-bis(2-ethane sulfonic acid) (PIPES) (Baur and Stacey,1977) may be used to buffer the glutaraldehyde fixativeto pH 7.4. PIPES buffer is expensive but has the advan-tages of being nontoxic and very stable. Few antibodieswork on resin sections so it is always necessary topreserve part of the biopsy in NBF and/or liquidnitrogen for immunohistochemistry. Cutting the speci-men into suitable pieces for the different proceduresmust be done with a sharp scalpel blade using a side-to-side motion and avoiding downward pressure asmuch as possible. The results of careless cutting areshown in Fig. 9.3.

Fixation and dehydration

Nerves for resin embedding need several hours if not daysfixation as a full thickness nerve biopsy may be up to5 mm thick and the epineurial fat also absorbs fixative.It is recommended that a large biopsy is post-fixed inbuffered osmium tetroxide overnight and, in this situa-tion, it is also helpful if 3% sodium iodate is added tothe solution as this slows the reaction down, so that theosmium is still active when it penetrates to the middleof the block (Dalley and Selinger, 1980). In addition, it

158 R. KING AND

has been shown that the preservation and staining ofmembranes (particularly important for myelin sheaths)is improved by the addition of 1.5% sodium ferricyanideto the solution (Langford and Coggeshall, 1980). Post-fixation dehydration is especially important for nervespecimens compared to other tissues and the slightesttrace of water left in the myelin sheath will result in local-ized distortion. This is particularly obvious in very thicklymyelinated fibers; thinnermyelin sheaths are easier to pre-serve well (Fig. 9.4A). Several hours and several changesare needed in absolute alcohol in order to remove all tracesof water. A suitable protocol is given in King (1999). Theparanodal myelin (Fig. 9.4B) and Schmidt–Lantermanincisures are particularly sensitive to fixation artefact(Fig. 9.4C). If the initial aldehyde fixation is too short,lipids in the myelin may be inadequately stabilized allow-ing movement in the tissue so that they are incorrectlylocalized by the osmium fixation (Fig. 9.4D). It is neces-sary to use an intermediary such as 1,2 epoxy propane(propylene oxide) to remove all the alcohol before infil-trating in epoxy resin. Other protocols such as those usingacetone dehydration give poorer preservation when usedon nerve biopsies.

Embedding in a good quality epoxy resin usuallygives best sections; it is helpful to use flat embeddingmoulds so that the orientation can be controlled.

Semithin sectioning

Sections are cut between 0.5 and 1 mm in thickness usingglass knives. Most information is obtained from trans-verse sections as these sample all the nerve fibers at

Fig. 9.4. Preservation artefacts. (A) The thickened tomaculous myelin characteristic of hereditary neuropathy with liability to

pressure palsies (HNPP) is difficult to preserve well; the small gaps (arrows) indicate inadequate dehydration. Scale bar¼5 mm.

(B) Paranodal myelin on large fibers is difficult to preserve adequately as shown here by the disruption of the lamellar structure in

this region (arrows), normal nerve. Scale bar¼1 mm. (C) This small fiberwas in the center of a large, traumatic neuroma.Due to the

size and density of the specimen, fixative penetration in 3 hours was inadequate, resulting in poor preservation of the Schmidt–

Lanterman incisure with loss of the structure of its component uncompacted Schwann cell membranes (asterisk). In addition, the

compacted myelin shows a different artefact; the regions marked by the arrows are denser than the central region. This is due to

poor penetration by an osmium fixative containing potassium ferricyanide. Scale bar¼1 mm. (D) Myelin figures in the axoplasm

resulting from inadequate primary fixation and very short dehydration times. Scale bar¼0.5 mm.

THE NERVE BIOPSY: INDICATIONS, TECHNICAL ASPECTS, AND CONTRIBUTION 159

one level. Longitudinal sections are also useful as,although they only show a small percentage of fibers,they allow examination of nodes of Ranvier and give abetter appreciation of the localization and frequencyof cytoplasmic inclusions.

As only highly alkaline stains work on resin sectionsthe range of stains available is severely limited. Differ-entiation of fascicular structures and contents is betterwith a double stain, such as thionine and acridine orange(Sievers, 1971) or basic fuchsin, than with a monochrome

stain such as p-phenylenediamine. Toluidine blue andmethylene blue give rather poor discrimination of neuralstructures and should be avoided.

Ultrathin sections

Best results are obtained for electron microscopy if greyto silver sections are cut with a diamond knife. A varietyof stains is available, most of which involve a uranylacetate solution followed by staining with a lead salt.

L

Methanolic uranyl acetate is a rather poor stain for colla-gen fibrils but it is therefore easier to distinguish the cel-lular components and any extracellular deposits (such asamyloid fibrils). Suitable protocols are given in King(1999).

Teasing

The preparation of specimens for fiber teasing is similarto that for resin embedding except that the use of propyl-ene oxide as an intermediary prior to infiltration withresin should be avoided as the myelinated fibers becomevery brittle. It is also advisable to tease in epoxy resinlacking accelerator as this avoids polymerization duringstorage prior to teasing and during the process itself. Theprepared nerve needs to be at least 1 cm long. Using agood quality dissecting microscope to view the speci-men, the perineurium is cut and fine needles used to sep-arate the endoneurial contents into bundles of fibers.These are then further subdivided until individual fiberscan be removed and laid singly, as straight as possible, ona drop of epoxy resin (containing accelerator) on a slide,coverslipped and polymerized. Assessing the consecu-tive internodes along one fiber cannot be done unless sin-gle fibers are examined. The process of removingindividual fibers without breaking them requires consi-derable practice, patience, and skill.

Histochemistry

On paraffin or frozen sections, staining for myelinsheaths and axons is more precise and reliable using spe-cific antibodies but some classic stains are still useful.The most often employed are Congo Red for amyloid,Perls’ iron stain for hemosiderin (as a marker ofblood vessel damage), and Wade–Fite stain for leprosybacilli. Other classical stains such as Sudan dyes forlipids and periodic acid–Schiff for glycogen are rarelyemployed now.

Immunohistochemistry

This can be performed on either paraffin or cryostat sec-tions. Probably the most important of the panel of anti-bodies employed are those needed to differentiatelymphocytes into T (CD3þve) and B cells (CD20þve).In addition, antibodies against macrophages, myelinbasic protein, neurofilaments, and epithelial membraneantigen (EMA) (for perineurial cells) should be included.Additional antibodies may be required after preliminaryexamination of the results. In particular, if amyloiddeposits are seen, antibodies against transthyretin willdiscriminate between acquired amyloid and the mostcommon hereditary cause. Further cellular immunophe-notyping is required if a large group of cells is found thatare all positive for CD20. B cells in vasculitis are usually

160 R. KING AND

mixed with T cells and neurolymphomatosis should beconsidered if this is not the case.

Morphometry

Computer-assisted image analysis can be useful quanti-fying myelin thickness. It may also be helpful in deter-mining myelinated fiber loss and assessing anypreferential involvement of fibers of specific calibers;visual assessment is not always reliable (see below).Quantification of unmyelinated fibers requires electronmicroscopy and is of little help in diagnosis due to thedifficulty in differentiating between normal unmyelin-ated axons and small regenerative sprouts from dam-aged myelinated fibers.

INTERPRETATION

Examining the resin sections at low magnification willenable assessment of inter- and intrafascicular variation.There is some normal variation in fiber populationbetween fascicles of varying sizes with small fasciclesoften having a lower fiber density; marked variationbetween fascicles is unlikely in the inherited neuropathies.

In an appropriately stained preparation, the mostobvious attribute of a nerve section is the density ofmye-linated fibers. This must be assessed cautiously; the eyeis attracted to the largest myelinated fibers and a casualobserver may fail to appreciate the relative proportionsof large and small diameter fibers. In a normal suralnerve, there should be of the order of four times as manysmall as large fibers and also at least four times as manyunmyelinated axons as myelinated ones (the latter ratiocan only be assessed by electron microscopy). If onlymedium and small sized fibers are present, the absenceof the largest fibers may not be appreciated. Conversely,if the number of very small fibers is reduced, this may bemissed unless a conscious effort is made to focus onthem. Morphometric studies are useful in this context.Plotting the numbers or frequency of fibers of differentdiameters and comparing this plot to an age-matchednormal control will show if a particular myelinated fibercategory is affected (Fig. 9.5A). A scatterplot of g ratio(axon diameter:fiber diameter) will emphasize the fre-quency of abnormal myelin thickness. The scatter cloudwill extend toward the top of the distribution when thereare increased numbers of thinlymyelinated fibers (with alarger g ratio) (Fig. 9.5B) and, conversely, there will bemore fibers below the normal range when there is anincrease in myelin thickness.

At higher magnification, details of the myelinatedfibers can be examined and whether or not their axonsappear normally stained or are absent, dense, or other-wise abnormal.

On a resin section, demyelinated and unmyelinatedaxons are very pale so if there appear to be extensive

. GINSBERG

H

regions between fibers containing few cellular compo-nents, a paraffin or frozen section stained for neurofila-ments to show axons or electron microscopy is needed.The definition of nuclear chromatin may be poor on resin

THE NERVE BIOPSY: INDICATIONS, TEC

A

B

C

24

20

16

12

8

4

02 4 6 8

Fibre diameter (microns)

Freq

uenc

y (%

)g

ratio

10 12 14 16

0

0.2

0.4

0.6

0.8

1

20 4 6 8

Fibre diameter (microns)

10 12 14 16

g ra

tio

0

0.2

0.4

0.6

0.8

1

20 4 6 8

Fibre diameter (microns)

10 12 14 16

Control

Patient

Fig. 9.5. Morphometry. (A) Bar chart of fiber sizes comparing

patient with a recovered vasculitis (black bars; same case as in

Fig. 9.6) with a normal nerve (white bars). The large fibers have

been lost and replaced by smaller regenerated fibers resulting in

only a slight reduction in fiber density to 6577/mm2 compared to

the control whose density was 7491/mm2. (B) Scatterplot ofmyelin thickness of the same case showing an increase in the

numbers of thinly myelinated fibers, more than 50% have a g

ratio greater than 0.7 (marked with a black line). (C) Scatterplotof the same normal nerve as in 5A. Here only 20% have a g ratio

greater than 0.7 (line). There is also a higher proportion of

thickly myelinated fibers than in the patient’s nerve.

sections and basal laminae cannot be resolved by lightmicroscopy. This means that it is difficult to distinguishbetween various types of inflammatory cells and it isnot possible to distinguish Schwann cells that are not asso-ciated with axons from small lymphocytes. Hematoxylinand eosin (H&E) stains on frozen or paraffin sections giveonly limited information about myelinated nerve fibersbut are valuable for visualizing cellular infiltration.

NICAL ASPECTS, AND CONTRIBUTION 161

DIAGNOSTIC FINDINGS

Light microscopy

DEMYELINATION VERSUS DEGENERATION

Axonal degeneration can be identified by dense axons,loss of annulate structure, or dense myelin inclusionsin denervated Schwann cells. Regeneration is most easilyidentified by the presence of tight clusters of small mye-linated fibers (Fig. 9.6). However, there is often only onemyelinated axon sprout and ascertaining the presence ofsmaller unmyelinated axons in the same cluster requireselectron microscopy.

Demyelination is most convincingly shown by findinglarge axons with no resolvable myelin sheath. Electronmicroscopy is again usually necessary to confirm this, assections cut through a node of Ranvier may appear verysimilar at light level (see Chapter 2, Fig. 2.6C). In abnormalnerves, there may be large, pale Schwann cell cytoplasmicprocesses that can be difficult to distinguish from demye-linated axons by light microscopy (Fig. 9.7A). When aSchwann cell contains myelin debris, its associated axonis usually relatively easy to discriminate, but, even in thissituation, it is not possible to be completely confident thata pale round structure is an axon. Completely bare axonsare easy tomiss (Fig. 9.7B). When a fiber undergoes demy-elination, the original Schwann cell basal lamina persistsfor some time around the demyelinated axon. Initially,the axon may be bare apart from this, but Schwann cellsrapidly multiply and surround it prior to remyelination.During this process, new collagen fibrils are laid downand separate the demyelinated axon from adjacent struc-tures; this helps to discriminate a true demyelinated fiberfrom other profiles of similar appearance.

Identifying remyelination can be difficult. Myelinsheaths that are inappropriately thin for axon diametercan arise as a result of either remyelination or regenera-tion. Sometimes a regenerative cluster will be seen inwhich all the fibers are thinly myelinated, in which caseinterpretation is easier. However, the possibility of confu-sion arises when a thinly myelinated fiber lies singly with-out extra Schwann cell processes around it (Fig. 9.6). Themost reliable way of distinguishing between remyelina-tion and regeneration is with teased fiber preparations(see below). When there have been several episodes ofdemyelination and remyelination, excess Schwann cells

Fig. 9.6. This micrograph is of the same case as in Fig. 9.5 and

shows that most fibers have inappropriately thin myelin for

fiber diameter; some of these are clearly clusters of regenerat-

ing fibers (arrows) but other single, thinly myelinated fibers

(arrowhead) could equally well be the result of remyelination.

Scale bar¼10 mm.

162 R. KING AND L

are formedduring each cycle. These redundant cells encir-cle the parent fibers and form onion-bulb structures(Fig. 9.8A). The excessive cellularity may lead to palpablehypertrophy of the whole nerve. Classical onion bulbs aremuch easier to see by light microscopy than completelydemyelinated axons, but where the Schwann cell pro-cesses are mobile and short-lived, only the basal lamina

A

Fig. 9.7. (A) High-power light microscopy shows some ambiguou

elinated axons, or abnormal Schwann cell processes (arrows). The

resulted from either regeneration or remyelination (arrowheads). S

7A shows a Schwann cell containing myelin debris associated with

whose myelin is now in the process of degenerating. Another pale p

only associated with some small Schwann cell processes and basa

remains, forming basal laminal onion-bulbs that are lesswell defined at light level (Fig. 9.8B).

. GINSBERG

INTRACELLULAR INCLUSIONS

Polyglucosan bodies are dense axonal inclusionsthat often completely fill the axon. They aremetachromatic on resin sections (see below), positive withperiodic acid–Schiff reagent, and stain specifically withLugol’s iodine. The occasional polyglucosan body in a fas-cicle is of no diagnostic significance and probably relatedto aging (Thomas et al., 1980) but larger numbers togetherwith myelinated fiber degeneration and regeneration areassociated with a specific disease (adult polyglucosanbody disease) (Vos et al., 1983).

Leprosy bacilli can be seen in all constituent cells ofthe nerve in cases of lepromatous leprosy when paraffinsections, stained with Wade–Fite stain, are examinedwith� 100 objective and oil immersion (but see below).

EXTRACELLULAR DEPOSITS

Amyloid is seen on an H&E stained section as a solidpink area. With Congo Red, it is brighter pink and whenviewed with polarizing filters shows a green birefrin-gence. On resin sections stained with thionine or tolui-dine blue, it is pale to mid blue–purple (Fig. 9.9A). Infamilial amyloid neuropathy, the deposits are clearlyidentified by anti-transthyretin antibodies (Fig. 9.9B),but other types of amyloid may bemore difficult to stainimmunochemically.

B

s structures that could be demyelinated axons, enlarged unmy-

re are also some very thinly myelinated fibers that could have

cale bar¼10 mm. (B) Another region of the same section as in

a round pale profile (arrow) that is most likely to be the axon

rofile (arrowhead) is probably a completely demyelinated axon

l lamina. Scale bar¼10 mm.

A B

Fig. 9.8. (A) Resin section of a case of Charcot-Marie-Tooth disease type 1A due to a duplication of the gene for PMP22. There are

numerous onion-bulbs consisting of Schwann cell processes circumferentially arranged around a central myelinated fiber, most of

which have a normal myelin sheath; occasional fibers are demyelinated (arrow) and some central fibers have been replaced by a

regenerative cluster (arrowheads). Scale bar¼10 mm. (B) In this sural nerve section, CMT1A is related to an amino acid substi-

tution in the PMP22 gene. The fibers are very small and have very thin myelin sheaths. Occasional fibers have no myelin at all

(arrow). All fibers are surrounded by onion-bulbs that are mainly composed of basal lamina (arrowheads). Scale bar¼10 mm.

THE NERVE BIOPSY: INDICATIONS, TECHNICAL ASPECTS, AND CONTRIBUTION 163

Immunoglobulin deposits in the perineurium andendoneurium are usually less well defined than amyloidand stain for IgG or IgM.

BLOOD VESSELS

Endoneurial blood vessels may be ensheathed by exten-sive basal laminal reduplication in several diseases andold age. This can be particularly striking in diabeticneuropathy and the neuropathy associated with IgMgammopathy.

Abnormalities of epineurial blood vessels are readilyseen on resin sections. These include cellular infiltration,though immunohistochemistry is required to character-ize the inflammatory cells (Fig. 9.10A). Structuralchanges associated with vasculitis are also detectable,e.g., recanalization of obliterated vessels (Fig. 9.10B),and the presence of fibrinoid necrosis.

Ultrastructural changes

GENERAL

Early myelinated fiber degeneration/regeneration isshown by the presence of bands of B€ungner (B€ungner,1891). These are formed by the basal laminal tubesleft behind by the degeneration of the original fibers.In the early stages, all remnants of Schwann cells andaxonsmay be removed leaving just the basal lamina. This

is not resolvable by light microscopy. The tube then actsas a conduit for regeneration of new axonal sprouts andtheir accompanying Schwann cells. If the damage to thefiber was caused by trauma, and the continuity of thesebasal laminal tubes has been disrupted, regenerationwill be muchmore difficult as there are no guides to leadthe sprouts to the correct end organ. Furthermore, fibro-blast reduplication forms scar tissue that hinders axonaloutgrowth.

The termRemak fiber is used for unmyelinated axonsand their associated Schwann cell processes. These canonly be resolved by electron microscopy as the largestof the unmyelinated fiber population is only about2.5 mm in diameter and the Schwann cell processes aresimilarly small. Care should be taken to avoid confusinglarge normal unmyelinated axons with small demyeli-nated ones (Fig. 9.11A); there is considerable overlap indiameter between myelinated and unmyelinated axons.In man, there are normally between one and four axonsin each Schwann cell unit, with smaller numbers beingmore common. Larger numbers of axons signify abnor-mality and very small (0.1 mm) axons imply regenerationof unmyelinated fibers. In bands of B€ungner left bydegeneration of myelinated nerve fibers, the Schwanncell processes are approximately circular in cross-section, but when unmyelinated axons degenerate, thesurrounding Schwann cell ensheathment collapses toform flattened sheets (Fig. 9.11B). These are a useful

Fig. 9.9. (A) In this resin section of a radial nerve, there are irregular extracellular endoneurial amyloid deposits that stain bluewith

thionine (arrows). Scale bar¼50 mm. (B) The same patient as A; staining with a monoclonal antibody against transthyretin shows

large deposits (arrows). Scale bar¼50 mm.

Fig. 9.10. (A) Sural nerve from a case of vasculitis; there is a large group of cells staining with CD22 (B cells) surrounding and

partially obliterating three small blood vessels (arrows). Serial sections stained with CD4 showed many T cells in the same area.

Scale bar¼20 mm. (B) This resin section from another case of vasculitis shows two recanalized small arterioles (arrows).

The presence of numerous small vessels suggests angiogenesis. Scale bar¼20 mm.

164 R. KING AND L. GINSBERG

marker of unmyelinated fiber degeneration. So-calledcollagen pockets where bundles of fibrous collagenreplace axonsmay also be encountered; significant num-bers of these imply abnormality of the Remak fibers.

As previously noted, electron microscopy is the defin-itive method for identifying demyelination (Fig. 9.11C)and, in particular, distinguishing it from the appearance

of a normal node of Ranvier in transverse section. Closeultrastructural examination shows the normal nodal axo-nal dense undercoating, reduction in neurofilament den-sity, and Schwann cell nodal processes around the axon(see Chapter 2, Fig. 2.6C). Other fibers that appear by lightmicroscopy to be demyelinatedmay be found also to havesuffered axonal degeneration when examined by electron

A B

C D

E F

Fig. 9.11. Ultrastructural studies. (A) Several normal Remak fibers containing one or two axons (*). Scale bar¼2 mm. (B) Flat-tened sheets of Schwann cell cytoplasm that have resulted from unmyelinated fiber degeneration (arrow). There is also a normal

unmyelinated fiber (*). Scale bar¼0.5 mm. (C) Transverse section through a demyelinated fiber (*). The basal laminal sheath of

the original myelinated fiber is still present (arrow). Scale bar¼1 mm. (D) Degeneration (or poor fixation or postmortem artefact)

of unmyelinated axons often causes an increase in diameter. This large pale axon (*) has lost most of the normal cytoplasmic

contents, as can be seen by comparison with the adjacent unmyelinated and myelinated fibers. There is a very small axon (arrow)

in the same Schwann cell process, additional evidence that this is an abnormal unmyelinated not demyelinated axon. Scale

bar¼1 mm. (E) The space left by a degenerated myelinated fiber is defined by small Schwann cell processes (arrows) and basal

lamina (arrowheads) containing only flocculent material. There is no axolemma or normal axonal components. Scale bar¼1 mm.

(F) Abnormally large and pale Schwann cell process (*) that could be mistaken for a demyelinated fiber by light microscopy.

The profiles arrowed are possibly small regenerating axon sprouts. Scale bar¼1 mm.

THE NERVE BIOPSY: INDICATIONS, TECHNICAL ASPECTS, AND CONTRIBUTION 165

microscopy (Fig. 9.11D and E) and abnormal Schwann cellprocesses may mimic demyelination until examined byelectron microscopy (Fig. 9.11F).

SPECIFIC DIAGNOSTIC PATHOLOGICAL CHANGES

Myelin stripping. The identification of macrophageprocesses invading the myelin sheath of an intact axonand removing lamellae distinguishes immune-mediated

demyelination from primary demyelination and can onlybe seen by electron microscopy.

Widely spaced myelin. In this feature, spacing of themyelin lamellae is increased from about 14 nm to 24 nm(see Chapter 2, Fig. 2.2B). This abnormality is seen in about50% of cases of neuropathy associated with IgM globuli-nemia and is almost pathognomonic. Attempts to createsimilar myelin spacing by altering the osmolality and pH

L

of the fixative failed to generate appearances that could beconfused with those found in IgM paraproteinemic biop-sies (King and Thomas, 1984).

Focally folded myelin sheaths. Abnormal myelinexcrescences are particularly related to Charcot–Marie–Tooth (CMT) disease type 4B, caused by muta-tions in the myotubularin related genes MTMR2(Houlden et al., 2001) and MTMR13. In this disease,the myelin sheath is also abnormally thin. Similar,although usually less striking, abnormalities may alsobe found in the CMT diseases associated with mutationsin the genes for myelin protein zero (MPZ), early growthresponse gene 2 (EGR2), and periaxin (PRX).

Lipid inclusions. With electron microscopy, the spe-cific periodicities and morphologies of lipid storageinclusions in Schwann cell cytoplasm can be seen. Therelevant diseases includemetachromatic leukodystrophy(Duckett et al., 1975; Thomas et al., 1977), Krabbe’s glo-boid cell leukodystrophy (Bischoff and Ulrich, 1969;Thomas et al., 1984), Farber leukodystrophy (Vitalet al., 1976), and Tangier disease (Kocen et al., 1973;Ferrans and Fredrickson, 1975; Gibbels et al., 1985).Nerve biopsy is no longer needed to diagnose these con-ditions, but it is theoretically possible that the inclusionsmay be encountered accidentally. Similarly there are spe-cific lipid inclusions in the perineurial and vascular endo-thelial cells in Fabry disease (Bischoff et al., 1968; Kocenand Thomas, 1970).

Neurofilaments. Giant axonal neuropathy is associ-ated with abnormality of the axonal neurofilamentsand has occasionally been found unexpectedly on nervebiopsy. The extreme enlargement of the axons often trig-gers secondary demyelination and onion-bulb forma-tions may be found around the giant axons as a resultof continuing changes in axon diameter. The axonalabnormalities may have some similarities to those seenwith solvent abuse, but in the latter case fiber involve-ment is usually patchy (King et al., 1993).

Leprosy bacilli. Bacilli are only just visible by lightmicroscopy and even using specific stains for acid-fastbacilli, may be difficult to find, especially when thereare few bacilli in the nerve. Electron microscopy showsthem clearly, particularly asmost will frequently be deadand very electron dense. On unsupported sections, theymove in the tissue creating clear spaces which renderidentification even easier.

Polyglucosan bodies. By electron microscopy thesecan be seen to have a specific ultrastructure of finecurved filaments. Small deposits may be encounteredthat are not recognizable by light microscopy.

Amyloid. Very small deposits of amyloid do not showup convincingly on standard light microscopy, and either

166 R. KING AND

thioflavin T is needed, which gives a bright blue fluores-cence, or electron microscopy to show the typicalstraight nonbranching tubular fibrils of 8 nm in diame-ter. They may superficially resemble oxytalan fibrils(an elastic fiber component; see Chapter 2 for moredetail) but are slightly smaller in diameter, less electrondense, and do not have the longitudinal periodicity of thelatter.

Extracellular fibrin deposits. Electron microscopyallows the identification of deposits of fibrin in the endo-neurium that are too small to be seen by light micros-copy. They are electron dense and have a finelongitudinal periodicity. Their presence implies damageto the blood nerve barrier and finding fibrin wouldsuggest a vasculitic process.

NONSPECIFIC ABNORMALITIES

Schwann cell basal lamina. Reduplication of thebasal lamina is thought usually to indicate change inthe size or shape of a cell, as a new basal lamina is depos-ited to accommodate these changes. This is seen to thegreatest extent in CMT disease type 1A due to a pointmutation of the PMP-22 gene in what was formerlycalled Dejerine-Sottas disease. Light microscopy of resinsections only shows a pale staining ensheathment of thenerve fibers (Fig. 9.8B) but electron microscopy enablesthis to be resolved into layers of basal lamina in a concen-tric arrangement. These layers are usually paired andresult from temporary Schwann cell processes that haveextended around the parent axon and then retreated leav-ing their basal lamina behind. Occasional fibers showingthese changes are also seen in CMT disease type 4C.

The persistence of the parent Schwann cell basallamina after degeneration and regeneration of the orig-inal fiber seems particularly common in diabetic neurop-athy and may be related to glycation of the basal lamina(King et al., 1989).

Uncompactedmyelin.A complete failure in the com-paction of the Schwann cell membranes to form amyelinsheath results in concentric layers of membrane withoutformation of a major dense line. This abnormality hasbeen reported as particularly common in POEMS syn-drome but may be found in many neuropathies (Vitalet al., 1994). Myelin in Schmidt–Lanterman incisuresand the paranode is also uncompacted and may be con-fused with true uncompacted myelin. The distinction iseasier in longitudinal sections where it may be seen thatthe affected myelin layers in true uncompacted myelinare parallel to the axon rather than running obliquelyas in a Schmidt–Lanterman incisure.

Intramyelinic edema.This is seen as large clear spacesin themyelin sheath and implies an instability of themye-lin structure. Experimental work in rats has shown it to

. GINSBERG

Fig. 9.12. Individual teased nerve fibers; in all cases nodes of

Ranvier are marked with arrows. (A) A single normal inter-

node. (B) Recent axonal degeneration; the myelin sheath has

degenerated into a series of myelin ovoids of varying sizes.

It is not possible to determine the position of the original nodes.

(C) Series of equally short internodes that have resulted from

myelinated fiber regeneration. (D) Demyelination. This inter-

node is completely devoid of myelin. Debris has already been

removed. (E) Remyelination; a series of thinly myelinated

internodes of varying length replacing one normal internode

and lying between normally myelinated internodes. Scale

bar¼100 mm.

HNICAL ASPECTS, AND CONTRIBUTION 167

be an early age-related change (Krinke et al., 1981;Mitsumori et al., 1981). This is probably also the casein man where demyelination may be found in normalnerves after the age of about 55 years.

Luse bodies. These are endoneurial deposits of fibrouslong spacing collagen and may be found attached to theSchwann cell basal lamina in neurofibromatosis. Similarstructures may be found in the perineurium where theyare possibly related to abnormality of the endoneurialmilieu (see Chapter 2). They have been encounteredmostoften in chronic inflammatory demyelinating polyneuro-pathy (CIDP), CMT1A, and diabetic neuropathy.

Reich granules. Although it has been reported thatthere is an increase in Reich granules in diabetic neurop-athy, these inclusions are commonly encountered, par-ticularly in older patients, and are of little diagnostichelp (see below and Chapter 2).

OTHER NONSPECIFIC ULTRASTRUCTURAL

ABNORMALITIES

Crystalline structures, possibly related to cholesterolmetabolism (Hedley-Whyte, 1973), may be seen inSchwann cell cytoplasm in a wide range of neuropathies.Apart from a possible association with axonal loss ordamage, they are of no help diagnostically. Similarly,the presence of Reich granules in Schwann cells associ-ated with unmyelinated axons means that these Schwanncells were once part of a myelinated fiber unit, so indi-cating that the original axon has been lost.

Calcium deposits may lie in the extracellular spacebetween perineurial laminae. These have been reportedas particularly common in diabetic neuropathy butmay be encountered in many other diseases (Anzil andPalmucci, 1983; King et al., 1988).

Teasing

Teasing out individual myelinated nerve fibers from afascicle requires special technical preparation. This is adifficult and time-consuming procedure but is the onlyway of convincingly differentiating between regenera-tion and remyelination (Fig. 9.12). The theoretical basisfor the different patterns seen in teased fiber prepara-tions is as follows. A damaged nerve fiber (Fig. 9.12B)regenerates by producing one or more sprouts fromthe last intact portion of the fiber. During this process,Schwann cells multiply extensively and, as the axonaloutgrowths extend and increase in diameter, each ofthe larger ones will acquire a Schwann cell in a recapit-ulation of the initial process of development. However,whereas initially nerves grow as organisms grow, so thatinternodal length extendswith limb growth, this is not thecase in regeneration. In this situation, the internodallength remains at the initial Schwann cell length of about

THE NERVE BIOPSY: INDICATIONS, TEC

200 mm. There may be some remodeling but a series ofevenly short internodes remains as a marker of axonalregeneration for some considerable time. This can beclearly seen on teased nerve preparations (Fig. 9.12C).As the thickness of the myelin sheath is almost directlyproportional to the internodal length (Arbuthnott et al.,1980), the myelin on these short internodes will be inap-propriately thin for fiber diameter. A different situationoccurs when a fiber suffers segmental demyelinationand myelin is lost from one or a few segments(Fig. 9.12D). This deficit is repaired by Schwann cell divi-sion and results in the replacement of each original inter-node by a few new internodes with shorter internodallength and thinner myelin sheath, lying between inter-nodes of normal length and myelination (Fig. 9.12E).During remyelination, a new sheath develops in a reca-pitulation of the initial process of myelination. Hence,early in the process of remyelination, inappropriatelythin sheaths are seen but these may mature to be very lit-tle different from normal as remodeling reduces thenumber of new internodes. The situation may be some-what different in some hereditary demyelinating dis-eases where myelin of normal thickness is never formed.

Artefacts, pitfalls, and distractions

Interpreting a suboptimally preserved specimen is notonly difficult but may lead to completely erroneous con-clusions as the very early stage of fiber breakdown

L

(Wallerian degeneration) is morphologically very similarto mechanical specimen damage.

As already mentioned, in the paranode and Schmidt–Lanterman incisures the myelin sheath is not compactedand these regions are difficult to preserve well, possiblydue to the absence of a major dense line which binds thelamellar structure tightly together. Without the majordense line, the myelin sheath is just concentric layersof Schwann cell membrane. These layers are very sus-ceptible to fixation artefact, especially incorrectly

168 R. KING AND

A

C

Fig. 9.13. (A) In this section there is a Pacinian corpuscle close to aouter layers of the perineurium (arrowhead). Scale bar¼100 mm. (Bof fibroblasts and connective tissue. A mast cell lies just subperineu

be seen by high-power light microscopy and tend to stain metachr

inappropriately thinly myelinated fiber that could have resulted f

Scale bar¼10 mm. (D) Polyglucosan bodies (arrow) show up w

Scale bar¼10 mm.

preserved lipids that are often re-deposited asmyelin fig-ures. It is particularly difficult to preserve the paranodalregion of large diameter fibers well.

In addition to poorly preserved nerves, there may alsobe structures that appear unusual or abnormal but are, infact, normal components of peripheral nerves. Amongthose most commonly encountered are Pacinian corpus-cles (encapsulated nerve endings, vibration receptors)(Fig. 9.13A), Renaut bodies (Fig. 9.13B), Reich granules(Fig. 9.13C), and polyglucosan bodies (Fig. 9.13D). Mast

. GINSBERG

B

D

small nerve fascicle (arrow) and, as is often the case, sharing the

) This fascicle contains twoRenaut bodies (arrows) that consist

rially (arrowhead). Scale bar¼50 mm. (C) Reich granules canomatically (arrows). This section also shows an example of an

rom either remyelination or axonal regeneration (arrowhead).

ell on resin sections and often have a targetoid appearance.

HNICAL ASPECTS, AND CONTRIBUTION 169

cells are also frequently encountered in the endoneurium(Fig. 9.13B).

It is always wise to keep in mind the possibility of arte-factual changes when interpreting “abnormal” structures.It can be very difficult to distinguish postmortem changesfrom genuine pathological abnormalities. Delayedfixation can allow movement of lipid so that the bilayerstructure of the plasma membrane appears as a stringof vesicles and the myelin sheath is replaced by honey-comb structures. As similar effects can also be foundin acute degeneration, one must have complete confi-dence in the optimal preservation of the tissue beforejumping to any conclusions about their significance.

THE NERVE BIOPSY: INDICATIONS, TEC

CONCLUSIONS

The great majority of patients with a peripheral neuropa-thy do not require a nerve biopsy to establish its cause. Theprocedure should be reserved for those with a progressiveneuropathy of unknown etiology (despite thorough nonin-vasive investigation), which is causing significant func-tional disability. Nerve biopsy is best performed inspecialist centers, where particular techniques are mostlikely to be available, e.g., immunohistochemistry andelectron microscopy, and where there will be expertisein interpretation and avoidance of artefacts. With thesecaveats, it remains a useful diagnostic investigation fora significant minority of neuropathy patients.

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