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REVIEW ARTICLE

Assessment of Epidermal Nerve Fibers: A New Diagnosticand Predictive Tool for Peripheral Neuropathies

Gigi J. Ebenezer, MBBS, MD, Peter Hauer, BS, Christopher Gibbons, MD,

Justin C. McArthur, MBBS, MPH, and Michael Polydefkis, MD, MHS

AbstractToday, skin biopsies can play an important role in the diagnosis

of peripheral nerve disorders and have yielded another diagnostic

tool for the neurologist. One of the commonly reported neuro-

pathologic abnormalities observed in skin biopsies is a reduction of

epidermal nerve density. Analyzing the changes in the morphology

and density of epidermal nerves is of immense diagnostic and

prognostic value in peripheral neuropathies. These changes also

provide an assessment of disease progression and of tissue

responses to regenerative treatments. Combined with immunohis-

tochemical studies, newly evolved skin biopsy and epidermal count

techniques have the potential to provide significant information

about the pathogenesis of many peripheral nervous system diseases.

They have great potential for impacts on both research and clinical

approaches to treatment. Evolution of a standardized and validated

counting methodology and significant advances in procuring skin

biopsies have opened up a wide spectrum of applications that make

the technology easy to apply in practice. The application of this

technology may lead to early detection of many common peripheral

nerve diseases and an enhanced understanding of disease onset and

progression. In this article we review the state of current research

and clinical practice in the use of skin biopsies and epidermal nerve

densities.

Key Words: Dermis, Epidermis, Peripheral neuropathies, Protein

gene product 9.5 (PGP 9.5), Schwann cell, Skin biopsy.

INTRODUCTIONThe skin is the largest organ in the body, covering the

entire external surface of the human body. It serves as aprotective barrier, facilitates in sensory perception, immuno-logic surveillance, and thermoregulation and controls insen-sible fluid loss. It constantly regenerates throughout life asits outer layers are shed and are replaced by inner layers.

The thickness of the skin varies according to anatomiclocation, sex, and age of the individual.

The skin is densely innervated (Fig. 1A) and sensoryimpulses from it are transmitted to the brain by fibers ofprimary sensory neurons located in trigeminal and dorsalroot ganglia. These neurons are a heterogeneous populationand include both mechanoreceptors and nociceptors. Noci-ceptive neurons detect noxious thermal, mechanical, andchemical stimuli that evoke a painful sensation and can warnof impending bodily injury. The peripherally directed axonsof dorsal root ganglia neurons run in peripheral nerves,terminate in skin, and innervate a variety of cutaneousstructures, including sweat glands (Fig. 1B), hair follicles,Merkel cells, Meissner’s corpuscles, blood vessels and theepidermis (1, 2).

In the skin, the nerve bundles typically course throughthe dermis vertically and form horizontal subepidermalneural plexuses in the papillary dermis. From the papillarydermal plexuses these nerves travel vertically, lose theirSchwann cell investment at the dermoepidermal junction,penetrate the epidermal basement membrane, ascendbetween the keratinocytes, and terminate in the keratin orsuperficial layers of the stratum spinosum as free nerveendings (Fig. 1C).

Cutaneous innervation consists mainly of unmyeli-nated fibers, which account for approximately 90% of alldermal nerve fibers (3). The unmyelinated axons areorganized into Remak bundles, which contain Schwann cellsenveloping 1 or more unmyelinated axons and the profile isenclosed by continuous basal lamina. The individual Remakbundles in the dermis typically contain 2 to 3 axons (Fig. 1D).Larger and deeper dermal nerves have a complete perineu-rium with a basal lamina and junctional complexes. Manysmaller and more superficial nerves lack perineurium and aresurrounded by collagen bundles oriented in the long axis ofthe nerve fibers, resembling endoneurial collagen.

Morphologically, the sensory axons within the skin areclassified into myelinated (A-fibers) and unmyelinated (C-fibers) fibers. Neurophysiologically, they are categorized asA-C and C-fibers. A-C fibers are small, thinly myelinatedaxons subserving mechanoreceptors and thermal receptorsthat transduce and transmit pain (nociception). C-fibers arevery thin unmyelinated axons that slowly transmit nocicep-tion sensation. Cool threshold detection is mediated by A-Cfibers and C-fibers and warm sensation by C-fibers (4).Slow-conducting unmyelinated (C) afferents also signal light

1059J Neuropathol Exp Neurol � Volume 66, Number 12, December 2007

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Vol. 66, No. 12December 2007pp. 1059Y1073

From the Department of Neurology (GJE, PH, MP), Johns HopkinsUniversity, Baltimore, Maryland; Department of Neurology (CG), BethIsrael Deaconess Medical Center, Harvard Medical School, Boston, MA;and Departments of Neurology, Pathology and Epidemiology (JCM),Johns Hopkins University, Baltimore, Maryland.

Send correspondence and reprint requests to: Justin C. McArthur, MBBS,MPH, Interim Chair, Department of Neurology, Professor of Neurology,Pathology and Epidemiology, Johns Hopkins University, Meyer 6-113,600 N. Wolfe St., Baltimore MD 21287-7609; E-mail: jmcarth@jhmi.edu

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touch (5). Axonal conduction velocity depends on both themyelin thickness and nerve fiber diameter, and unmyelinatedC-fibers conduct impulses at about 1 m/s whereas myeli-nated A-fibers conduct at 40 to 1,000 m/s (2). Largemyelinated A> and AA fibers conduct vibration and lighttouch, and these fibers are not present in the epidermis (1).

Neurochemically, epidermal nerve endings can bebroadly categorized as either peptidergic or nonpeptidergic.Peptidergic axons densely express calcitonin gene-relatedpeptide (CGRP) along with other peptides including theneurotransmitter substance P. Nonpeptidergic afferents areidentified by their binding sites for the plant lectin IB4.Using genetically encoded axonal tracers expressed from theMrgrd locus, a subpopulation of small-diameter (presumedto be nociceptive, IB4+) nonpeptidergic epidermal axons thatexpress the molecular marker Mrgprd (Mrgprd+) have beenidentified in mice. These Mrgprd+ fibers course through the

stratum basalis and stratum spinosum and terminate in thestratum granulosum. In contrast, CGRP+ fibers terminate inthe stratum spinosum of the epidermis. Mrgprd+ fibers donot innervate any specific cutaneous sensory structure andthere is complete absence of Mrgprd+ fiber innervation in allother peripheral tissues. The central projections derived fromdistinct epidermal innervation zones terminate in adjacentlaminae in the dorsal spinal cord (6).

Sweat glands in the dermis are highly immunoreactiveto pan-axonal marker protein gene product 9.5)(Fig. 1B). The majority are vasoactive intestinal polypep-tide+ and some are CGRP+ but are sparsely innervated bysubstance P-immunoreactive fibers (7, 8). Characteristically,these fibers are cholinergic sympathetic axons with a fewnoradrenergic fibers. Cholinergic/noradrenergic coexpres-sion, which is a mode of autonomic regulation, is a uniquefeature of the primate peripheral nervous system and has not

FIGURE 1. Double-stainedconfocal images of protein gene product 9.5 (PGP 9.5) with p75 and electron microscopic findings ofcutaneous nerves in human controls. (A) Double-stained confocal images of PGP 9.5 (red) staining axons (arrows) combinedwith p75 (green) staining Remak Schwann cells and perineurium (broken arrows). Overlapping colocalization is yellow. Thedermal nerve bundles course through the dermis and form horizontal bundles at the level of the papillary dermis. Scale bar =20 Km. (Used, by permission. First published in Brain 2007;130(10):2703Y2714, modified). (B) PGP 9.5-stained axons (red)

the epidermis, but the p75 staining for Schwann cells (broken arrow) stops at the dermoepidermal junction. Scale bar = 20 Km.(D) A Remak bundle with Schwann cell (Sch) wrapping around 3 unmyelinated axons (arrows); the profile is enclosed bycontinuous basal lamina (broken arrow) and surrounded by collagen bundles (C). Scale bar = 1 Km. (Used, by permission. Firstpublished in Brain 2007;130(10):2703Y2714, modified).

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9.5 (PGP

densely innervating the sweat glands (S). Scale bar = 20 Km. (C)PGP 9.5-stained epidermal nerve fibers (arrows)(red) extend into

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yet been identified in other portions of the human sympa-thetic nervous system (9). Dermal blood vessels areinnervated by both sensory and autonomic fibers (sympa-thetic and parasympathetic), and most of these are unmyeli-nated (Fig. 2A). The autonomic fibers lie significantly closerto the endothelial cell layer and smooth muscle cellscompared with the sensory fibers (10).

Neurotrophic factors play an important role in thegrowth and maintenance of these terminal nerve endings.C-fiber nociceptors respond to trophic factors: nerve growthfactor (NGF), neurotrophin 3, glial cell line-derivedneurotrophic factor, and brain-derived neurotrophic factor(11, 12). NGF binds to 2 classes of transmembrane proteinreceptors: tyrosine kinase family (high-affinity receptor) andp75 (the low-affinity and pan-neurotrophin receptor). Thep75 receptor can be activated even in the absence of tyrosinekinase A. Expression of both NGF and p75 are important inthe regeneration of peripheral nerves after injury (13, 14).NGF and its receptor, tyrosine kinase A, are important forsurvival and target innervation of small diameter sensoryneurons and sympathetic neurons during development (15).Expression of NGF is maintained in the adult, especially intarget tissues such as skin keratinocytes, indicating apossible role in maintenance and plasticity of these neuronsin adulthood. Previous experiments in rats using an NGFantiserum infusion addressed this question (16) by demon-strating that the levels of NGF in sympathetic and sensoryneurons after axotomy partly regulate subsequent changes inneuropeptide expression. Yet, it remains unclear whetherNGF is important for maintaining survival or innervation ofsensory and sympathetic neurons in the adult nervoussystem.

ANALYSIS OF SKIN BIOPSIES INCUTANEOUS NERVE

Large sensory and motor nerve fibers in peripheralnerve disorders are typically assessed using electrodiagnos-

tic methods or sural nerve biopsy. However, electrodiag-nostic methods are not useful in assessing unmyelinatednerve fibers simply because this population is Binvisible[ tonerve conduction velocity studies. Within the epidermis, theindividual unmyelinated axons are separated from each otheras they pass between the keratinocytes, and at the lightmicroscopic level it is possible to identify and count axonsusing immunocytochemical stains. Before the advent ofsensitive pan-axonal markers such as PGP 9.5 (17), silverstains were used but were too insensitive for practical use.For almost 15 years skin biopsy has been used as a reliabletool to assess the epidermal and dermal nerve fibers. Theskin can be sampled at several anatomical sites simul-taneously, and biopsies can be repeated serially over time(18Y23).

MethodologySampling of skin to analyze epidermal nerves is done

essentially by using 2 techniques: skin blister and skin punchtechniques.

Skin Blister TechniqueA suction capsule is placed over the skin and the

epidermis is separated from the dermis at the level of theepidermal basement membrane (24). The technique is leastinvasive, painless, and bloodless, but the procedure is time-consuming and the relationship of dermal nerves to epi-dermal nerves cannot be studied.

Skin Punch TechniqueCutaneous nerve evaluation using 3-mm skin punches

(8, 22, 25) is easy to perform, no sutures are required, andthe test leaves behind only a very small scar. The majoradvantages of skin biopsies are intraepidermal nerves fibers(IENFs) can be quantified, dermal innervation and morpho-logic features can be evaluated, and sections can be used forultrastructural studies, polymerase chain reaction analysis,and laser pressure catapulting studies. In addition, from skin

FIGURE 2. Confocal images of AIII-tubulin staining dermal nerve bundles. (A) A neurovascular bundle with a nerve bundle(arrow) stained by AIII-tubulin (green) and small branching fibers innervating a blood vessel (red). Scale bar = 20 Km. (B) Adermal nerve bundle (arrows) stained by AIII-tubulin (green). Schwann cell nuclei are blue. Scale bar = 10 Km.

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punches, epidermal sheets can be separated and subjected toprotein and mRNA analysis.

Processing and ImmunohistochemistryMost laboratories use either a PLP (paraformaldehyde,

lysine, and periodate) or Zamboni (2% paraformaldehydeand picric acid) fixative, as tissues fixed in formalin tend toproduce a fragmented appearance of nerve fibers (22, 26).PGP 9.5 is a cytoplasmic neuronal marker commonly used toinvestigate small caliber sensory fibers in skin biopsies, andanti-PGP 9.5 targets ubiquitin carboxyl-terminal esteraseL1, an enzyme found exclusively and ubiquitously in neu-rons (27). Other markers are antibodies against specific cyto-skeletal filaments (Fig. 2B), axonal membrane, and epitopes(28, 29).

Fifty-micrometer-thick sections are cut perpendicularto the epidermis to visualize both dermis and epidermisusing a freezing sliding microtome. At least 3 sections of 50Km from each biopsy are evaluated using bright field lightmicroscopy. Thick sections allow the visualization of

cutaneous nerves, as continuous wavy bundles and as singlenerves.

QUANTITATION OF EPIDERMAL NERVESThis involves 2 distinct steps: counting the number

of IENFs under light microscope at 40� magnification,and then measuring the length of the epidermis along theupper margin of the stratum corneum. The linear IENFdensity is calculated and expressed as the number of fibersper millimeter of epidermal length (26, 30). Two differentcounting rules have evolved: 1) counting only the number ofnerve fibers crossing the epidermal basement membrane(Fig. 3A), and 2) counting that includes isolated nervefragments in epidermis that do not cross the basementmembrane (31, 32) (Fig. 3B). We prefer to use the lattercount rule as it provides the advantage of including theisolated fragments in the count and a higher IENF numberwith less error is likely to be obtained. This lower errorbecomes critically important in evaluating neuropathic siteswhere only a few fibers are present. Measuring the length of

FIGURE 3. (A) Counting rule 1. Nerves are shown in black and basement membrane is dark gray. A) Count nerve as it crossesthe basement membrane of the epidermis. B) Nerves that branch after crossing the basement membrane are counted as a singleunit. C) Nerves that split below the basement membrane are counted as separate units. D) Nerves that appear to branch withinthe basement membrane are counted as separate units. E) Nerve fragments that do cross the basement membrane are counted.F) Nerve fibers that approach the basement membrane but do not cross it are not counted. G) Nerve fragments in epidermis thatdo not cross the basement membrane in the section are not counted. (B) Counting rule 2. Nerves are shown in black andbasement membrane is dark gray. A) Count nerve as it crosses the basement membrane of the epidermis. B) Nerves that branchafter crossing the basement membrane are counted as a single unit. C) Nerves that split below the basement membrane arecounted as separate units. D) Nerves that appear to branch within the basement membrane are counted as separate units.E) Nerve fragments that do cross the basement membrane are counted. F) Nerve fibers that approach the basement membranebut do not cross it are not counted. G) Nerve fragments in epidermis that do not cross the basement membrane in the sectionare counted. First published in Eur J Neurol 2005;12:747Y58, modified.

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epidermis using both computerized image software and amicroscope intraocular lens ruler has produced similarresults (33). IENF density can also be derived simply bydividing the number of IENFs by 3 mm, and this result hascorrelated significantly with IENF density measured byimage analysis (34). Very good correlation exists betweenthe various numerical methods (35). The European Feder-ation of Neurological Studies task force has establishedguidelines and rules for counting and reporting IENFs(31, 32).

With strict counting rules and intensive training, a highdegree of inter- and intrarater reliability has been achievedin IENF density estimation. Density variations amongadjacent sections within the same site are minimal, and ahigh degree of correlation of IENF densities between 2punches at the same anatomical site has been demonstrated(26, 36). When the lowest 5th percentile of the normativerange in the distal leg is used as a cut-off value to identifynormal, the technique had a diagnostic efficiency of 88%, apositive predictive value of 75%, and a negative predictivevalue of 90% for the detection of small fiber sensoryneuropathy (26). Although this study used formalin fixation,IENF density differences between formalin and PLP fixationhave not been appreciative (37). In contrast, estimation ofIENF using confocal microscopy is expensive and laborintensive. Fifty- to 100-Km-thick sections are used forconfocal studies (23, 38, 39). As the IENF density dependson the optical sections created by the user, wide variations inresults occur, and normative data have not yet beenestablished using this technique.

IENF Density As a Diagnostic ToolIENF density normative data are available for only a

few anatomical sites: proximal thigh, distal thigh, distal leg,distal forearm, trunk and heel. Three major studies haveshown that in healthy subjects, IENF density is typicallyhigher in the proximal thigh than the distal leg with a normalproximal to distal length-dependent gradient in innervation(26, 37, 40) (Table, Fig. 4A, B).

In normal individuals, higher IENF densities are foundin very young individuals (G20 years). The effect of gender,age, and height on distal leg innervation has been inves-tigated by several groups. A study that focused on subjectsaged 16 to 82 years (26) did not find an effect of increasingage other than subjects in the youngest decile (16Y20 years)having higher densities. Other studies of healthy controlsubjects have reported a mild inverse, gender-adjustedrelationship between IENF density and age with a decreaseof 0.6 to 1.8 fibers/mm per decade (37, 40). It appears thatexcluding the extremes of age, the effect of age onepidermal innervation is modest, if any. Similarly, therehas been variation in the role that gender plays with somestudies reporting no effect (26) and others finding mildincreases in epidermal innervation among females (37, 40).No significant relationship of race, height, or weight onIENF density has been reported (26, 37). Interestingly,height, presumably as a surrogate for axonal length, is a riskfactor for the development of diabetic neuropathy and hasbeen linked to reduced rates of IENF regeneration rates (48).

The epidermal nerve fiber density measure has shown highspecificity and reliability and published data in the normalpopulation could serve as a good baseline for detecting smallfiber sensory neuropathy (26, 40, 49, 50). Studies haveestimated IENF in the forearm, trunk, and heel, but thesedata have not been consistent (22, 41, 51).

In patients with sensory neuropathy patients the IENFdensity is consistently and significantly lower than that incontrols (Fig. 4C, D) (20, 23, 39, 50, 52Y54). The IENFdensity measurements were found to be more sensitive thansensory nerve conduction studies for diagnosing small fibersensory neuropathy (19, 23, 42, 43, 50, 55).

Morphologic ChangesMorphologic changes are seen in both epidermal and

dermal nerves in peripheral neuropathy and sometimes inotherwise healthy individuals from local effects of trauma.Although some small-sized swellings, beading, and varicos-ities may be present in the leg skin biopsy of normalindividuals, attenuation of fibers, large globular and fusiform-shaped swellings (Fig. 5A), dystrophic changes, and tortuousand increasingly complex branching are some of the morpho-logic changes that have been described in the skin biopsies ofpatients with neuropathy (3, 18, 22, 42, 44, 56, 57). Swellingswere more numerous in the calf of patients with small fiberneuropathy and also tended to be more numerous in diabeticthighs (57).

The higher density of swellings in the distal leg ofneuropathic patients correlates well with impaired heat-painthreshold, development of symptoms, and progression ofdisease (42, 44). These axonal swellings are thought topresage the subsequent loss of axons; that is, large swellingsserve as predictive markers of nerve fiber degeneration. In astudy of 28 patients with sensory complaints of unknownetiology, large axonal swellings on the initial skin biopsysubsequently showed a decline in IENF density on repeatedbiopsies. This decline was not observed in patients withoutnerve fiber swellings or in those with smaller nerve fiberswellings (18).

TABLE. Intraepidermal Nerve Fiber Density: Normative Rangefrom Different Laboratories

Author andReference Number No. of Subjects

PTENF/mm

Distal LegENF/mm

McArthur et al (26) 98 21.1 T 10.4 13.8 T 6.7

Omdal et al (36) 15 Not done 12.2 T 3.8

Umapathi et al (37) 84 20.4 T 6.9 11.7 T 4.1*

GLransson et al (40) 106 Not done 12.4 T 4.6Pan et al (41) 55 Not done 12.970 T .284

Lauria et al (42) 15 22.6 T 4.8 14.4 T 3.0

Shun et al (43) 38 Not done 9.359 T 3.466

Herrmann et al (44) 19 21.6 T 5.4 16.6 T 5.3

22.8 T 6.9 15.0 T 5.0†

Lauria et al (46) 15 24.37 T 5.80 14.46 T 4.45

*, Male.†, Female.ENF, epidermal nerve fiber; PT, proximal thigh.

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Ultrastructural examination of skin biopsies frompatients with neuropathy have shown that dilated cytoskele-tal organelles, disintegration of axoplasmic organelles,watery axoplasm, and accumulation of abnormal mitochon-dria within axons and Schwann cells contribute to theformation of axonal swellings in terminal nerve endings(Fig. 5B). Pathologic changes were similar in all types ofsensory neuropathic cases examined without any specificrecognizable differentiating features. Thus, nerve fiberswellings are now considered to represent a predegenerativechange, and the presence of larger swellings at the lowerlimb can be used as a predictive marker to assess theprogression of neuropathy. Further studies are needed todescribe a methodology to quantify the swellings.

INTRAEPIDERMAL NERVE FIBERS DENSITY INPERIPHERAL NERVE DISEASE

Diabetic NeuropathyIt is estimated that the global prevalence of diabetes

mellitus in people of all ages will increase from 2.8% in theyear 2000 to 4.4% in 2030. The total number of people withdiabetes is projected to rise from 171 million in 2000 to 366million in 2030 (58). Epidemiologic studies suggest that

45% to 60% of all diabetic patients develop neuropathy (59,60). Abnormalities on nerve conduction studies (NCS) orelectromyographic changes were found in 18% to 25% ofpatients at the time of diabetes diagnosis (61).

Direct metabolic effects due to hyperglycemia, oxida-tive injury, mitochondrial dysfunction, and disruption ofaxonal transport by advanced glycation end products havebeen implicated in the pathogenesis of neuropathy (62, 63).

A number of epidermal nerve quantification studieshave been completed in diabetic patients and in experimentaldiabetic animals. IENF densities are reduced in type 2diabetic patients compared with those in age-matchedhealthy controls and correlate with changes in warm sensorydetection thresholds and the amplitude of the sural sensoryaction potential. The extent of denervation increases with theduration of type 2 diabetes (43). There is an inversecorrelation between IENF density and severity of neuropathy(20, 64), with progressively decreasing densities from prox-imal to distal sites (39). IENF density is also lower in thosepresenting with neuropathic pain compared with thosepresenting without such pain (64). Differences betweenpeople with type I versus type II diabetes have not beenidentified although severity of IENF loss has been linked tothe severity of glucose dysmetabolism (50).

FIGURE 4. Skin biopsies immunostained with PGP 9.5 showing length-dependent innervation of skin in the lower limb of ahealthy adult and in a patient with neuropathy. (A) PGP 9.5-stained section showing epidermal nerve fibers (arrow) in the distalleg of a healthy adult. Scale bar = 50 Km. (B) PGP 9.5-stained section showing epidermal nerve fibers (arrows) in the proximalthigh of a healthy adult. Scale bar = 50 Km. (C) PGP 9.5- and p75-stained section showing complete absence of epidermal fibersin the distal leg in a patient with chronic neuropathy. Axons = red; Schwann cells = green. Scale bar = 20 Km. (D) PGP 9.5-stained section showing an occasional epidermal nerve fiber (arrows) in the proximal thigh of a patient with chronic neuropathy.Axons = red; Schwann cells = green. Scale bar = 20 Km.

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Increased incidence of impaired glucose tolerance isseen in neuropathy patients, particularly in those with painand sensory loss and is more common in patients withapparently idiopathic polyneuropathy than in the age-matched general population (61, 65). IENF density isreduced even in patients with neuropathy who have impairedglucose tolerance neuropathy with normal NCS (66). Theneuropathy associated with impaired glucose tolerance isgenerally milder than the neuropathy associated withdiabetes mellitus. Small nerve fibers are prominentlyaffected and may be the earliest detectable sign of neuro-pathy in glucose dysmetabolism (45, 50, 67, 68).

By using a human model of IENF regeneration, it hasbeen shown that reduced rates of nerve regeneration occur in

diabetic subjects without any evidence of neuropathy (29),providing a rationale to include subjects without overtneuropathy in trials of regenerative agents. Pancreas trans-plantation has not produced the expected major beneficialeffects on small nerve fibers, probably because of thereduced regeneration capacity of patients with advanceddiabetes. Severe depletion of epidermal and dermal fiberswas noted in both lower limbs during the post-transplantperiod (69). To register nerve fiber regeneration, prolongedobservation periods will probably be necessary, and IENFquantification can be used to assess the progress of poly-neuropathy during post-transplant follow up.

Although it is generally believed that diabetic neuro-pathy is due to chronic hyperglycemia, experience frompatients with insulinoma and experimental studies show thatperiods of hypoglycemia from overcontrol of glucose levelsmay also cause neuropathy in diabetic patients. The plantarnerves of diabetic eu-/hypoglycemic BB/Wor rats treatedwith insulin implants exhibited a distinct neuropathy, whichis accompanied by mild alterations in the epidermalinnervation of plantar skin and a more obviously abnormalnerve terminal pattern in plantar muscle (70).

Human Immunodeficiency Virus-AssociatedSensory Neuropathies

Various types of peripheral neuropathies have knownto be associated with human immunodeficiency virus (HIV)infection, and 10% to 35% of adults with AIDS are known todevelop HIV-associated sensory neuropathies (HIV-SN) (71,72). Two subtypes are manifest: one predominantly asso-ciated with HIV infection (HIV-associated distal sensoryneuropathy), and the other associated with specific neuro-toxic antiretroviral drugs (antiretroviral toxic neuropathy).IENF density is typically reduced in a length-dependentmanner in adult patients with HIV infection who have neuro-pathic signs independent of neuropathic symptoms (44).

Follow-up of HIV-infected medically asymptomatic,non-neuropathic individuals over a period of time has shownthat a lower leg IENF density, a higher cooling threshold,and a higher heat pain threshold for minimal pain werefound to be associated with a greater risk of transition tosymptomatic HIV-associated distal sensory neuropathy.There is a longitudinal association between leg IENF andquantitative sensory testing, and serial small fiber evaluationin chronic neuropathies can be useful in predicting patientsat risk for transition to HIV-associated distal sensoryneuropathy (73, 74). Decreased IENF density was associatedwith higher levels of neuropathic pain, higher plasma HIVRNA levels, and lower CD4 counts (54).

In subjects with advanced HIV, IENF density corre-lates with severity of neuropathy severity and with the levelof neuropathic pain quantified by both the Gracely PainScale and the Visual Analogue Scale. IENF density at thedistal leg also correlates with sural amplitude, but IENF atboth distal leg and proximal thigh correlated negatively withtoe cooling detection threshold. In contrast to the study byPolydefkis et al (54), IENF density from patients receivinghighly active antiretroviral therapy was not associated withexposure to neurotoxic antiretroviral drugs, CD4 cell count,

FIGURE 5. Light and electron microscopic findings of cuta-neous nerves in chronic neuropathy patients. (A) PGP 9.5-stained skin section showing dermal axons with globularaxonal swellings bar = 50 Km. (B) Remak

both Schwann cell and axoplasm (Ax) containing abnormalmitochondria (broken arrows). Scale bar = 1 Km.

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bundle containing denervated Schwann cell (Sch, arrow) andScale(arrows).

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or plasma log HIV-RNA level. These findings suggest that inthis group of patients with advanced HIV, the abovepreviously established risk factors for HIV and HIV-SN areno longer determinants of the severity of HIV-SN in thecurrent highly active antiretroviral therapy era (74).

In a prospective cohort study HIV-infected patientswere examined at 2 different geographic locations toemphasize demographic differences. Reduced vibrationthresholds and epidermal nerve fiber densities had thehighest diagnostic efficiency of the laboratory indicators ofneuropathy examined, but were found to be relativelyinsensitive in isolation (75). IENF density in longitudinalstudies can be used to identify risk factors for neuropathyprogression and to monitor epidermal nerve fiber regener-ation in clinical trials of regenerative agents.

Sensory neuropathy also occurs in a significantproportion of HIV-infected pediatric patients (76, 77).Twenty-eight HIV-seropositive patients aged 5.6 to 18.2years were compared with 48 HIV-seronegative subjectsaged 6.5 to 17.8 years. NCS and quantitative sensory testingdid not significantly differ between patients and controlsubjects, but HIV-seropositive patients as a group showed ahigher IENF density than control subjects. This paradoxicalincrease in innervation indicates age-specific differences inthe expression of HIV-SN and the difficulty in establishingthe presence of peripheral neuropathy in the pediatric andadolescent group (C. Luciano, unpublished observation). Re-generation studies using human models of collateral (excisionmodel) and regenerative (capsaicin model) sprouting showedreduced rates of epidermal nerve fiber regeneration. Thisabnormality was more pronounced in those with evidence ofbaseline neuropathy but was also observed in those withnormal peripheral nerve function and no neuropathy symp-toms. The rate of regeneration was highly dependent upon thebaseline distal thigh IENF density but was independent ofCD4 cell count, HIV viral load, HIV duration, gender, race, orapolipoprotein E4 status. Regeneration was not furtherreduced in those subjects exposed to dideoxynucleosidemedications. Swellings were not a prominent feature amongsubjects in that study, suggesting that reductions in regener-ation may be the earliest change that occurs. These findingsalso imply that there is a progression from the introduction ofan insult such as infection with HIV to development of areduced regeneration rate followed by morphologic changesand finally the establishment of peripheral neuropathy. In thiscontext, an improvement in regeneration would be the earlieststep toward the improvement of nerve function in the settingof a regenerative neuropathy study (78).

To date, no agents have been shown to be effective inspeeding neuronal regeneration in humans although atherapeutic agent, acetyl-L-carnitine, which is vital formitochondrial dysfunction, has been reported to enhanceepidermal innervation in HIV-SN (79). A trial of recombi-nant human erythropoietin is now underway because of itspresumed neuroprotective properties (80, 81).

Idiopathic Small Fiber Sensory NeuropathyPatients with a clinical diagnosis of possible idiopathic

small fiber neuropathy often present with painful burning

feet, but on clinical examination show normal strength,proprioception, tendon reflexes, and normal electrophysio-logic responses. These patients often have a reduced IENFdensity at the calf with normal proximal nerve fiberdensities, indicating that idiopathic small fiber neuropathyis typically a length-dependent process (19, 82). Skin biop-sies can be useful to distinguish neuropathies from radicu-lopathies, which typically do not produce epidermal nervechanges because the damage occurs proximal to the dorsalroot ganglion. Follow-up skin biopsies of patients withidiopathic small fiber sensory neuropathy with durationsranging from 12 to 28 months show a decrease of IENFdensity in the leg, indicating the progressive destruction ofthese fibers in the disease (42).

Small and large fibers are not evenly distributed withincutaneous nerves, and this inconsistency increases thecomplexity of morphometric quantification of nerve fibersin biopsies such as those of the sural nerve. One studycomparing sural nerve biopsy to skin biopsy demonstratedthat a reduction in IENF density was the only evidence forsmall fiber neuropathy in 23% of patients (55). In 1 largestudy skin biopsy was found to be more sensitive than aquantitative sudomotor axon reflex test or quantitativesensory testing in diagnosing small fiber neuropathy (23).In another study with painful feet of unknown cause, anexcellent correlation was observed between results of thequantitative sudomotor axon reflex test, cooling abnormal-ities, and loss of IENFs (83). Although sural sensory nerveaction potentials have served as the traditional electro-physiologic marker of distal sensory polyneuropathy, inpatients with suspected distal sensory polyneuropathy whohave normal NCS measuring both medial plantar sural nerveaction potentials and small sensory fibers will serve ascomplementary tools in evaluation of distal SN (23, 84, 85).

LeprosyLeprosy is a chronic granulomatous disorder affecting

multiple peripheral nerves and skin. Cutaneous nerve fibersare affected in all types of leprosy (86, 87), resulting inanesthetic or hypoesthetic skin lesions. In a large multi-centric cohort study in multibacillary patients, unmyelinatedC-fibers were found to be more frequently affected thansmall myelinated AC fibers and NCS, and warm and coldthreshold measurements have emerged as the most promis-ing tools for early detection of leprosy (88). One of themajor challenges in leprosy control programs is to diagnoseindeterminate skin lesions, relapses, and dermatologic con-ditions that may mimic leprosy. Although no attempt hasbeen made to quantify epidermal nerve fibers in leprosy skinlesions, a large study evaluated cutaneous innervation andneuropeptides using immunohistochemical methods. Neuro-peptide immunoreactivity was seen in only 14% of theindeterminate leprosy specimens and was completely absentin other types of leprosy, highlighting the diagnosticsignificance of early disappearance of neuropeptide immu-noreactivity in leprosy lesions (89). Because the 3-mmpunch technique is simple to perform, nontechnical staff canbe trained in the quantification technique, and IENF densityof skin lesions could serve as an additional tool along with

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skin smear examination and NCS to increase the diagnosticefficiency of detecting neuropathy in leprosy controlprograms.

Chemotherapy-Induced Peripheral NeuropathyChemotherapeutic agents produce both acute neuro-

toxicity and chronic neuropathy (90, 91). A number ofeffective chemotherapeutic agents, including platinumcompounds, taxanes, and vinca alkaloids produce length-dependent axonal injury and even worsen preexisting neu-ropathy (92). In an experimental rat model of paclitaxel- andvincristine-evoked painful peripheral neuropathies, a partialdegeneration of the sensory innervation of the epidermisand upregulation of PGP 9.5 in epidermal Langerhans cellshave been demonstrated in glabrous skin of the plantarhind paw (93). IENF density with total neuropathy scorealong the course of chemotherapy could help to predict thecourse and severity of chemotherapy-induced peripheralneuropathy.

Pyridoxine-Induced ToxicityExcess pyridoxine (vitamin B6) ingestion causes a

dose-dependent peripheral neuropathy characterized bynecrosis of dorsal root ganglion and degeneration of longmyelinated fibers in the sciatic nerve. In experimental rats aswell as in human subjects lower doses of pyridoxine alsoresult in development of small fiber involvement as theearliest and predominant abnormality, and higher doses leadto involvement of both large and small fibers (94Y96). Wehave observed 1 patient who developed a sensory neuro-pathy after consuming excessive amounts of pyridoxine.Serial skin biopsies demonstrated epidermal denervation thatrecovered over a period of several months (V. Choudhry,personal communication, 2007).

SKIN BIOPSY IN NON-LENGTH-DEPENDENTNEUROPATHIES

Sensory GanglionopathiesIn patients with sensory ganglion degeneration,

symptoms of ataxia and proprioceptive sensory losspredominate and the distribution of sensory impairment iswidespread and asymmetric (97Y99). A study comparedbiopsies from patients with ganglionopathy and axonalneuropathy with those from normal control subjects. Skinbiopsies at the proximal thigh and the distal leg in patientswith axonal neuropathies showed significantly lower valuesat the distal site of the leg, confirming the length-dependentloss of cutaneous innervation. In the patients with ganglion-opathy, in contrast, the degeneration of small-diametersensory fibers was seen equally in all locations (46). Itshould be noted that a severe length-dependent neuropathywill also show denervation at all sites and cannot be clearlydifferentiated from ganglionopathy on a neuropathologicbasis alone.

Postherpetic NeuralgiaHerpes zoster is caused by reactivation of latent

varicella zoster virus within the sensory ganglia, resulting

in destruction and degeneration of the respective spinal andperipheral axons (100). Several pathologic processes for thecause of postherpetic neuralgic pain have been suggested.IENF was examined in chronic postherpetic neuralgia-affected skin, and the loss of cutaneous innervation inverselycorrelated with allodynia. It is thought that survivingcutaneous primary afferent nociceptors spontaneouslybecame active and sensitized and contributed to postherpeticneuralgic pain and allodynia (101). Some have suggestedthat degeneration of primary afferent neurons causes centralhyperactivity (102). These studies expressed epidermaldensity as an average number of epidermal neurites/mm2

of skin surface area (101), a method of counting that isgenerally not used for other neuropathies.

SKIN BIOPSY IN INFLAMMATORYDEMYELINATING NEUROPATHIES

Guillain-Barre SyndromeGuillain-Barre Syndrome (GBS) or acute inflamma-

tory demyelinating polyneuropathy is an acute inflammatoryneuropathy, and, according to various clinical, neurophysio-logic, and pathologic studies large-diameter myelinatednerves are affected. Axonal degeneration of motor andsensory nerves has been described in the demyelinatingform of GBS (103). Skin biopsy from the distal leg ofpatients with the demyelinating form of GBS was inves-tigated for the involvement of small sensory fibers. IENFdensity was reduced in 55% of the cases in comparison withage- and gender-matched controls, and the values correlatedwith functional disabilities. These findings suggest that GBSis not a purely large-fiber neuropathy but that small-fibersensory and autonomic neuropathies exist in a significantproportion of patients with GBS (104).

Chronic Inflammatory DemyelinatingPolyneuropathy

When IENF densities and thermal thresholds inpatients with chronic inflammatory demyelinating polyneu-ropathy were tested, patients with chronic inflammatorydemyelinating polyneuropathy showed lower IENF densitythan that of control subjects. The low IENF density andelevated thermal threshold were associated with autonomicsymptoms. Patients with chronic inflammatory demyelinat-ing polyneuropathy have small-fiber sensory and autonomicneuropathies in addition to the immunologic inflammation inlarge diameter myelinated fibers (105).

SarcoidosisSarcoidosis is a chronic progressive granulomatous

multisystem disorder that may affect virtually any part of thenervous system, and patients can present with peripheralnerve pain and fatigue (106, 107). Patients with sarcoidosiswith normal nerve conduction studies had shown reducedtemperature sensitivity and low IENF in the leg comparedwith control subjects. Small fiber sensory neuropathy occursin sarcoidosis, and reduced IENF may be the only detectableabnormality in patients with sarcoidosis presenting withperipheral nerve pain (53).

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Hepatitis C InfectionThe most frequent form of hepatitis C infection

(HCV)-associated neuropathy is distal sensory or sensori-motor polyneuropathy (108). Most cases occur in associationwith cryoglobulinemia (109) and vasculitis (110). A multi-centric study in an unselected, untreated referral populationwith HCV infection has shown that electrophysiologicperipheral neuropathy occurs in 15.3% of patients (111).Coinfection with HIV and HCV has become increasinglyprevalent and distal symmetric polyneuropathy is the mostcommon form of peripheral neuropathy in HIV and HCVinfections. However, in a cohort study of 147 HIV-infectedadults, HCV was not found to be significantly associatedwith HIV-related sensory neuropathies (75).

Vasculitic NeuropathyVasculitis is one of the rare causes of peripheral

neuropathy, and clinical manifestations include motor weak-ness, reduced sensitivity, and neuropathic pain. Smallmyelinated nerves were shown to be more susceptible toischemia than larger myelinated fibers in experimentallyinduced ischemia in Wistar rats (112). Patients with motorand sensory impairments have shown significant IENFreduction, demonstrating that the depletion and degenerationof small-diameter sensory nerves in vasculitis occur asfrequently as those of large-diameter nerves (113, 114).

SKIN BIOPSY IN AUTOIMMUNE DISEASESSkin biopsies have played an important role in

identifying small fiber neuropathy in chronic inflammatoryautoimmune diseases.

Sjogren SyndromeSensory neuropathy with prominent ataxia and gan-

glionitis are well-recognized forms of neuropathy associatedwith Sjogren syndrome. Sural biopsies show a wide range inloss of both large and small myelinated and unmyelinatedaxons (98, 115). A study of 20 consecutive patients withSjogren neuropathy showed that 60% of the subjects hadnonpatchy symptoms and a non-length-dependent pattern ofepidermal nerve fiber loss, suggesting that patients with thisdisorder commonly have a small-fiber sensory neuronopathyrather than a Bdying-back[ axonopathy (116). This hypoth-esis has been further confirmed in a population-based studyin a small group of patients (117).

Systemic Lupus ErythematosusThe prevalence of peripheral neuropathy in systemic

lupus erythematosus (SLE) varies from 5% to 27%, and it ischaracterized by a mild sensory or sensorimotor neuropathy(47, 118, 119). Pure small-diameter nerve fiber neuropathyoccurs in SLE with a significant reduction in IENF density.Disease activity, disease duration, or routine hematologic,biochemical, or immunologic variables did not influence thenumber of IENFs in patients with SLE (36, 120).

The degree of loss of small-diameter nerve fibersamong patients with these chronic inflammatory autoim-mune diseases varies. Sixty patients with SLE, 61 patientswith primary Sjogren syndrome, and 52 patients with

rheumatoid arthritis were compared with 106 healthysubjects. Densities were significantly less in patients withSLE compared with patients with rheumatoid arthritis orhealthy subjects. Nerve fiber densities were also reduced inpatients with primary Sjogren syndrome compared withhealthy subjects. Only 8 patients (13%) with SLE, 2 patients(3%) with primary Sjogren syndrome, and 2 patients (4%)with rheumatoid arthritis have shown densities below thelower reference limit of 3.4 fibers/mm (121). Despite thelower average mean values, very few patients actually meeta diagnosis of small fiber neuropathy.

Celiac DiseaseCeliac disease is an autoimmune disease of the small

intestine and the gastrointestinal symptoms result fromingestion of gluten in genetically susceptible people. Neuro-logic complications are estimated to occur in 10% ofaffected patients (122), with sensory neuropathy being oneof the most frequently reported neurologic abnormalities(123). Despite the association between celiac disease andsmall fiber neuropathy, a definitive causal relationship hasnot been established. Interestingly, in patients presentingwith asymmetric numbness and paresthesias, low IENFdensities occur at the thigh, forearm, and distal leg,suggesting a widespread involvement of small fibers (124).Detection of anti-gliadin antibodies can be useful indiagnosis (125).

Friedreich AtaxiaThis is an autosomal recessive cardioneurodegener-

ative disorder resulting from an inability to produce theprotein frataxin. It is due to extensive trinucleotide repeatexpansions in the first intron of the gene encoding frataxin,resulting in defective transcriptions and protein deficiency(126). Patients with Friedreich ataxia have been evaluated byskin biopsy and have shown significant loss of epidermalfibers, reduced autonomic innervation to sweat glands,arrector pilorum muscles, and arterioles in parallel withtheir clinical findings of impaired thermal sensitivity, tactilethresholds, and decreased mechanical pain detection (127).

Fabry DiseaseFabry disease is an X-linked recessive disorder caused

by deficiency of >-galactosidase that results in accumulationof neutral glycosphingolipids (principally ceramide trihexo-side) primarily in endothelial cells (128) of blood-brain/nerve barriers within the central or peripheral nervoussystems (129). In the peripheral nervous system it producesa painful small fiber neuropathy (130Y132). Patients withFabry disease may have reduced IENF density at distalbiopsy sites with relatively preserved proximal nerve fiberdensity (133). Twenty-five heterozygous male subjects withFabry disease did not show an increase in epidermalinnervation density after >-galactosidase A (agalsidase->)enzyme replacement at 6 months. After an additional yearof therapy, there was a significant reduction in IENFdensity in the patient group as a whole, perhaps attributableto the declining glomerular filtration rate and the effects ofuremia on peripheral nerves. Thermal thresholds remained

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unchanged. This study suggests that longer treatmentperiods may be needed for nerve fiber regeneration orthat enzyme replacement therapy alone is not sufficient toreverse the pathology of this disease (134).

Congenital Insensitivity to Pain With AnhydrosisCongenital insensitivity to pain with anhydrosis or

hereditary sensory neuropathy type IV (135) is a very raredisorder and clinical manifestations include mental retarda-tion, congenital analgesia that results in self-mutilation,multiple scars, fractures, and anhidrosis (136). Ultrastruc-tural and morphometric studies of sural nerves show loss ofboth unmyelinated and small myelinated fibers (137, 138).Skin biopsy examination in a 10-year-old girl has shownextensive loss of epidermal nerve fibers and denervation ofdermal adnexal structures including sweat glands andarrector pili (138).

Abnormalities in Dermal InnervationGlabrous skin is richly innervated by myelinated fibers

in the dermis and morphologically these fibers appearsimilar to the myelinated fibers in sciatic and sural nerves.Quantification of myelinated fibers and mechanoreceptors inglabrous skin in normal subjects has shown that thecutaneous segments of AA fibers undergo repeated branch-ing and shortening of internodal length during their coursefrom the nerve trunk (139).

Charcot-Marie-Tooth disease is caused by mutationsin myelin proteins and evaluation of myelinated dermalfibers in skin has revealed pathologic abnormalities inaxolemmal molecular architecture (140, 141). Patients withanti-myelin-associated glycoprotein neuropathy have shownIgM deposits on dermal myelinated fibers with a greaterprevalence at the distal site of extremities. These findingshighlight the use of glabrous skin in identifying abnormal-ities in inherited demyelinating and chronic demyelinatingneuropathies (142).

Many studies have shown reduced sweat gland inner-vation in patients with peripheral neuropathy (20, 22, 105,128, 139, 143). A few researchers have tried differentmethods to quantify and measure sweat gland innervation.Decreased nerve fiber length around sweat glands in diabeticpatients (52), reduced innervation in patients with Rosssyndrome (144, 145), and familial dysautonomia (146) havebeen demonstrated. Larger studies with validation of thequantification methods in both healthy control subjects andin diseased conditions are needed for quantification ofdermal innervation in sweat glands.

USE OF SKIN BIOPSIES IN HUMANREGENERATION STUDIES OF SENSORY NERVES

As the epidermis is highly innervated by small sensorynerves, skin biopsies provide easy accessibility to study theeffect of chemical, thermal, and mechanical trauma on theregeneration and degeneration of sensory nerves. Two typesof nerve injury models have evolved: an Bincision model[ inwhich, following transection of subepidermal plexes, thesprouting epidermal axons outside the incision line completethe reinnervation by 30 to 75 days. In the Bexcision model[

removal of the incised cylinder of skin resulted in adenervated area, and no complete reinnervation of epidermiswas seen even after 23 months (147). The effect of atherapeutic compound, neuroimmunophilin ligand timcodardimesylate, on collateral nerve sprouting was measuredusing the excision model. Though the compound did notimprove collateral sprouting, the model was consistentacross treatment groups and had a low coefficient ofvariation (148).

Application of capsaicin, an ingredient of hot chillipepper, to the skin produces a reproducible chemicalaxotomy of the epidermal nerves. Capsaicin can be admin-istered intradermally or topically. Serial biopsies haveshown that nerve regeneration after topical capsaicin isfaster than after intradermal delivery (149, 150). ThisBcapsaicin model[ has been used to study the degenerative(150) and regenerative properties of epidermal fibers (29)after chemical axotomy. The rate of regeneration of IENFwas lower in diabetic subjects irrespective of the presence orabsence of neuropathy, indicating that abnormalities inperipheral nerve function are present early in diabetes, evenbefore signs or symptoms develop (29). In a randomized,double-blind, placebo-controlled trial, the effect of timcodardimesylate, a putative regenerative neurophilin compound,on the regeneration of epidermal fibers after capsaicin nerveinjury was investigated. There was no difference in theregeneration rate between the treatment and placebo arms,but the baseline IENF density and the height and race of theparticipants influenced the rate of regeneration (48). Thesemodels hold potential for use in clinical trials to study theeffect of regenerative drugs and growth factors on sensorynerve fibers.

CONCLUSIONSSkin biopsy analysis of nerve fibers is a technique that

has opened a new window to help visualize the previouslyinaccessible small unmyelinated fibers. Over the last 2decades there has been a dramatic increase in our under-standing of cutaneous innervation, leading to improveddiagnostic and therapeutic interventions. The EuropeanFederation of Neurological Societies Task Force has for-mulated guidelines for IENF quantification (32) and sum-marized the utility of the technique. Normative data fromhealthy subjects matched for age and gender and fromappropriate anatomical sites should be used to interpretneuropathy. Quality control measures must be followed at alllevels to ensure the optimal examination of skin biopsies.The laboratory techniques are not automated, and artifacts orerrors can potentially be introduced at the stage of section-ing, staining, or interpreting. Intra- and interobserver ratingsand quality control between laboratories should be periodi-cally assessed. Skin biopsies could become a critical tool forstudies of axon-Schwann cell interactions that are vital forthe survival and maintenance of axons. Our data suggest thatSchwann cells are lost during prolonged denervation andalso in chronic peripheral neuropathies (3). In neuroregener-ative trials it is vital to identify group of patients who retainSchwann cells and who are most likely to respond toregenerative drugs. Identifying sites with low or absent

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IENF or with more normal IENF would help in identifyingpatients who would progress and/or benefit from therapeuticintervention.

ACKNOWLEDGMENTSThe authors thank Barbara Freeman and Christopher

Wright for expert technical assistance.

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