skin biopsy in the management of peripheral neuropathy

632 http://neurology.thelancet.com Vol 6 July 2007

Review

Skin biopsy in the management of peripheral neuropathyClaudia Sommer, Giuseppe Lauria

Skin biopsy has been widely used in recent years for the investigation of small-calibre sensory nerves, including somatic unmyelinated intraepidermal nerve fi bres, dermal myelinated nerve fi bres, and autonomic nerve fi bres in peripheral neuropathies, with diff erent techniques for tissue processing and nerve fi bre assessment. Here, we review the techniques for skin biopsy, the processing and assessment of the biopsy sample, their possible uses in diff erent types of peripheral neuropathy, and their use in the follow-up of patients and in clinical trials. We also review the association between morphological measures of skin innervation and function and the limits of this method in the aetiological classifi cation of peripheral neuropathies.

IntroductionAlthough Paul Langerhans fi rst described nerve fi bres in the human epidermis in 1868, their existence was subsequently debated for more than a century.1 Only after the development of an antibody to protein gene product 9.5 (PGP 9.5)2 was it possible to easily and reproducibly stain nerve fi bres in skin.3–5 Skin biopsy has since gained widespread use in the investigation of small-diameter nerve fi bres in human epidermis and dermis. In particular, researchers have used this technique to investigate qualitatively and quantitatively intraepidermal nerve fi bres, and normative values have been reported for diff erent body regions and age groups.1,6–9 Laboratories in Europe and the USA have increasingly included skin biopsy in the diagnostic assessment or follow-up of patients with peripheral neuropathy. There are diff erent techniques for tissue processing and nerve-fi bre

assessment, including techniques for staining, quantifi cation of the intraepidermal and subepidermal nerve fi bres, and the use of diff erent antibodies to distinguish nerve-fi bre subtypes. Researchers have studied the association of skin innervation and physiological function, such as detection thresholds for temperature and pain. The investigation of skin innervation has also yielded some unexpected fi ndings—eg, in neuropathies previously thought to be of the large-fi bre type and in complex regional-pain syndrome.10–12

MethodologyMethods of skin biopsy assessmentSkin biopsy can be done at any site of the body, with a disposable punch, using sterile technique, and under local anaesthesia (lidocaine). A 3 mm diameter punch is commonly used with no need for sutures. In our experience of more than 1000 biopsy samples, there were no side-eff ects or complaints. Healing is usually complete within 1 week, and a barely visible scar usually remains (fi gure 1); however, informed consent is required, and information on the risk of bleeding, infection, and delayed healing must be provided. For diagnostic purposes, one skin biopsy is commonly done at a distal site on the leg (10 cm above the lateral malleolus) and a further biopsy taken at a proximal site on the thigh (20 cm below the iliac spine); thus, a proximal site and a distal site can be compared if a length-dependent process is suspected. Skin biopsy can also be done in other regions of the body (eg, face, trunk, or fi ngers).

Punch biopsy produces a sample of skin that includes the epidermis and the superfi cial (subpapillary and reticular) dermis. Immunostaining of 50 μm thick sections labels the diff erent structures (eg, nerve fi bres, sweat glands, blood vessels, and resident or infi ltrating cells). The most commonly used marker for nerve fi bres are antibodies against PGP 9.5: a form of ubiquitin carboxyl-terminal hydrolase (a cytosolic enzyme that removes ubiquitin) that is found mostly in neurons and accompanies the slow component of axonal transport. PGP 9.5 is widely distributed in the peripheral nervous system and is a non-specifi c panaxonal marker.13 Antibodies against specifi c components of the cytoskeleton (eg, microtubules and neurofi laments) and specifi c components of myelin (eg, myelin basic protein,

Lancet Neurol 2007; 6: 632–42

Department of Neurology, University of Würzburg,

Germany (C Sommer MD); Neuromuscular Diseases

Unit, National Neurological Institute Carlo Besta, Milan,

Italy (G Lauria MD)

Correspondence to:Claudia Sommer,

Neurologische Klinik der Universität, Josef-Schneider Str. 11, D-97080, Würzburg,

[email protected]

wuerzburg.de

A

B

Figure 1: Skin biopsy done with a 3 mm diameter punchBiopsy samples taken at the distal leg (A) and at the fi nger (B).

http://neurology.thelancet.com Vol 6 July 2007 633

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peripheral myelin protein 22, and myelin-associated glycoprotein) can also be used to investigate unmyelinated and myelinated cutaneous nerve fi bres.14 Moreover, intraepidermal nerve fi bres express transient receptor potential vanilloid receptor 1, which shows that they are nociceptors.15 Autonomic fi bres innervating sweat glands and blood vessels can be immunostained with antibodies against neuropeptides (eg, vasointestinal peptide, substance P, or calcitonin-gene-related peptide), whereas antibodies against collagen IV are used to mark the dermis–epidermis junction and blood vessels.

Bright-fi eld immunohistochemistry and indirect immunofl uorescence with or without confocal microscopy are the two immunostaining methods most commonly used for the investigation of cutaneous innervation. Individual nerve fi bres crossing the dermis–epidermis junction are identifi ed and counted at high magnifi cation in at least three sections. The linear density of intraepidermal nerve fi bres per millimetre is calculated by measuring the length of the surface of each section by use of bespoke software. Semiquantitative or quantitative examination of dermal and sweat-gland innervation and qualitative analysis of the morphological features of epidermal-nerve and dermal-nerve fi bres are undertaken (fi gure 2).

Indirect immunofl uorescence can stain multiple antigens in the same neural structure (eg, axon and myelin sheath) or in diff erent structures (eg, mechanoreceptor and nerve fi bre, dermis–epidermis junction and nerve fi bres). By use of confocal laser microscopy, we can obtain a striking three-dimensional reconstruction of the section and quantify the intraepidermal nerve-fi bre density with computerised image analysis. Although this technique is complex and time consuming, it is particularly useful in the study of cutaneous receptors, sweat glands, and blood vessels (fi gure 3).16

A less-invasive method than punch biopsy can be used to investigate epidermal innervation. The blister technique is based on the use of a suction capsule that separates the epidermis from the dermis at the junction between the two.17 There is no need for local anaesthesia, the procedure does not cause bleeding, and the density of the intraepidermal nerve fi bres can be calculated using computerised image analysis after immunostaining the whole disk of epidermis. The advantage of this technique is that it enables the investigation of a much wider area of epidermis than the surface of the 3 mm diameter vertical sections obtained from punch biopsy; its disadvantage is that it does not give information on the innervation of the dermis or the morphology of intraepidermal nerve fi bres.

Normative values of intraepidermal nerve-fi bre densityBoth bright-fi eld immunohistochemistry and indirect immunofl uorescence have provided standard values of intraepidermal nerve-fi bre density in legs (table 1).18,24–27,122–124

Three large studies investigated the density of intraepidermal nerve fi bres in distal leg in healthy people by use of bright-fi eld immuno histochemistry, with similar results in each.6,9,18 One study quantifi ed the density of intraepidermal nerve fi bres in proximal thigh biopsies (mean 21·1 nerves per millimetre, [SD 10·4]), showing that the epidermal innervation density in humans is roughly 60% higher at the thigh than at the

A

B

Figure 2: Human skin stained for PGP 9.5Normal density of intraepidermal nerve fi bres (arrows) and normal appearance of dermal nerve bundles (arrowheads) in a biopsy from a healthy control (A). Severe loss of intraepidermal nerve fi bres and dermal nerve bundles in a patient with diabetic neuropathy (B). Note the large swellings of the intraepidermal nerve fi bre (arrow) refl ecting axonal degeneration

A B

C D

Figure 3: Measurement of the area of nerve fi bres in the subepidermal plexus (A, B) and innervating sweat glands (C, D)Original digital image (A) of intraepidermal and subepidermal nerve fi bres (bar=20 µm). Nerve fi bres (B) in white pseudocolor and area of interest (box) for morphometric determination of the area of the subepidermal plexus in relation to the area of interest. (C) Digital image of sweat gland innervation transferred to black and white image (bar=100 µm). The area of white structures (nerve fi bres) is measured in C; the complete area of the sweat gland complex is measured in D. Processed for fl uorescence microscopy with a Cy3-labelled secondary antibody to PGP 9.5.


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lower leg,6 although the reason for this proximal–distal gradient is unknown. The quantifi cation of linear intraepidermal nerve-fi bre density with bright-fi eld microscopy is a reliable method, with high interobserver and intraobserver agreement, and agreement among laboratories.19 This method signifi cantly correlated with stereological techniques of skin-nerve morphometry20,21 and with the quantifi cation of nerve-fi bres per area.22

The density of intraepidermal nerve fi bres at the distal leg is higher when evaluated with indirect immuno-fl uorescence than with bright-fi eld microscopy (33·0 nerves per millimetre [SD 7·9] vs 7·4 nerves per millimetre [SD 7·4]).23–27 One study estimated a linear density of 11·3 intraepidermal nerve fi bres per millimetre (SD 2·9) in the glabrous skin of the fi ngertips;8 in the same region, the density of myelinated endings was 59·0 per millimetre (SD 29·3).2

Whether normal values for skin innervation are age-dependent is unknown, although there is no age eff ect in the fi ngertips or abdomen.7,8 Ageing is probably associated with decreasing intraepidermal nerve-fi bre density in the distal leg,9,18,27–29 although the fi rst normative study did not report this fi nding.6

Findings in patients with neuropathiesQuantifi cation of nerve fi bresEvaluation of nerve fi bres in the skin has gained popularity because it enables the identifi cation of a neuropathological correlate to symptoms of small-fi bre neuropathy. In patients with painful or burning sensations in their feet but normal results on clinical examination and sensory-nerve conduction studies, skin biopsy can reveal loss of small-diameter nerve fi bres.30 By use of skin biopsy, the diagnosis of small-fi bre neuropathy can be made earlier and easier than with sural nerve biopsy. Comparative studies show that the density of intraepidermal nerve fi bres can be signifi cantly reduced, despite normal morphometry of small nerve fi bres on the ultrastructural

examination of a sural nerve biopsy.30 In about 25% of people with symptoms of a neuropathy, only skin biopsy could show small-fi bre neuropathy, which was not detected by nerve-conduction studies and sural nerve biopsy.31 When the diagnosis has been made, further examinations to assess the cause can be done and symptomatic treatment started.

Skin biopsy enables the separate investigation of small nerve fi bres with diff erent functions—namely, the somatic unmyelinated fi bres within the epidermis, and the autonomic fi bres innervating sweat glands and arrector pilorum muscles. Skin biopsy can help to detect both degeneration in somatic nerves in neuropathies that were otherwise regarded as exclusively autonomic, such as Ross syndrome,32 and subclinical autonomic impairment in people with painful neuropathies. The diagnosis of unknown autonomic dysfunction linked to small-fi bre neuropathy is important because of potentially life-threatening events caused by dysautonomia. The loss of autonomic nerve fi bres in sweat glands examined by morphometric quantifi cation was shown in Ross syndrome and in familial dysautonomia (Riley–Day syndrome).16,33 Skin biopsy enables the diff erentiation between intrinsic sweat gland abnormalities and sympathetic nerve degeneration.34

Skin biopsy can contribute to the assessment of the primary site of nerve pathology. In length-dependent axonal polyneuropathies, such as those occurring in diabetes, a reduction in the density of intraepidermal nerve fi bres is typically found in the distal rather than proximal leg, refl ecting the dying-back degenerative process. Conversely, in sensory neuronopathies caused by primary degeneration of dorsal root ganglia neurons, such as those associated with neoplasms or Sjögren’s syndrome, there is a pattern of non-length-dependent skin denervation.35

MorphologyMorphological changes in skin nerve fi bres might be an early sign of peripheral neuropathies. An increased density of large and diff use axonal swellings in intraepidermal nerve fi bres is associated with impaired heat–pain thresholds, the development of symptomatic neuropathy, and the progression of diabetic and HIV neuropathy.36–38 In patients with painful neuropathy but normal innervation density, such swellings might be an early marker of neuropathy; however, axonal swellings also occur in many healthy people,39 such that the relevance of the swellings has to be assessed in the context of other morphological and clinical fi ndings. In patients reporting symptoms, the swellings are commonly associated with degenerative changes in nerve bundles, seen as a weaker signal and fragmented appearance of the nerve fi bres after immunostaining with PGP 9.5.

Diagnostic abilityThe assessment of nerve fi bres in a skin biopsy can answer many diff erent questions, such as what is the

Normative values/mm (mean±SD)

Bright-fi eld IHC in distal leg6 13·8±6·7


Bright-fi eld IHC in distal leg9 12·4 ± 4·6



IF and confocal microscopy in distal leg124 18·6±6·5

IF and confocal microscopy24 10·3±6·21)

IF and confocal microscopy in distal leg25 17·4±3·2

IF in distal leg26 12·3±2·9

IF and confocal microscopy in distal leg27 32·3 ±5·9

Abbreviations: IHC, immunohistochemistry; IF, immunofl uorescence.1Data for young adults; values in elderly adults were lower.

Table 1: methods and locations for defi ning normative values of intraepidermal nerve fi bre density


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degree of involvement of skin innervation in diff erent types of neuropathy; or how much sweat gland innervation remains in an autonomic neuropathy? To answer this type of diagnostic question, the value of the method is self-evident, and only matters such as intra-rater and inter-rater reliability have to be considered. Another frequently asked question is whether small fi bre neuropathy is present? Assessment of the diagnostic ability (specifi city, sensitivity, and positive and negative predictive values) of the method against a gold standard is required. However, because a gold standard is not always available,40 as in small-fi bre neuropathy, and small-diameter fi bres are invisible on routine neurophysiological examination, in most studies the symptoms (eg, burning feet or distal pain and paraesthesias) are the benchmark against which the performance of skin biopsy evaluation is measured.

Sensory neuropathiesIn the diagnosis of sensory neuropathies, intraepidermal nerve-fi bre density is a marker of sensory axonal integrity because it is highly reproducible and its reliablility is not aff ected by the severity of neuropathy.6,19 Skin biopsy with linear quantifi cation (counting intraepidermal nerve-fi bre density per length of epidermis) of intraepidermal nerve fi bres has a high predictive value for sensory neuropathy. However, the value of data on the diagnostic yield of skin biopsy in sensory neuropathies is diminished because the early studies did not distinguish between those patients with mixed small-fi bre and large-fi bre neuropathy and those with suspected small-fi bre neuropathy. For the diagnosis of a mixed neuropathy, the gold standard would be pathological values in nerve-conductions studies. In a study of 20 patients by McArthur and colleagues, nerve conduction was normal in four, abnormal in eight, and not done in eight.30 Compared with a group of 98 healthy individuals, the researchers report a sensitivity of 45% and a specifi city of 97% in this mixed group.6 The low sensitivity is most probably a result of the heterogeneity of the group, which might have included patients with dysaesthesias without manifest neuropathy and patients with mainly large-fi bre involvement. In 35 patients with sensory neuropathy and pathological nerve-conduction studies in sensory nerves, the sensitivity of intraepidermal nerve-fi bre quantifi cation was 80% and the specifi city was 95%. Another study, which included only patients with symptoms of small-fi bre neuropathy,18,22 reported a sensitivity of 90% and a specifi city of 95%. Investigators have analysed patients with and without abnormal nerve conduction, separately, and compared the diagnostic effi cacy of skin biopsy for evaluating intraepidermal nerve-fi bre density in these subpopulations. Of 75 patients with symptoms of small-fi bre neuropathy, those with concomitant large-fi bre involvement had lower intraepidermal nerve-fi bre density and more pronounced abnormities on quantitative sudomotor axonal refl ex test than those without.41 Of

Sensory symptoms (pain, burning, parasthesia, hypoesthesia)

Consider laboratory andCSF examination andnerve biopsy

Consider MRI or CT scanConsider skin biopsyor quantative sensorytesting

Consider skin biopsy,quantative sensorytesting, quantitativesudomotor axon al reflextext, or autonomic tests

Small fibre neuropathyMixed (large and small fibre) neuropathy

Nerve conduction studies

Sensory polyneuropathy Multiplex neuropathy Sensory mononeuropathy Radiculopathy

Signs of small fibreimpairment (thermal andpinprick sensory loss,hyperalgesia, allodynia,dysautonomia)

Signs of large fibreimpairment (light touchor proprioceptive sensoryloss, absent or reduceddeep tendon reflexes,allodynia)

Clinical examination

Full work-upaccording to publishedrecommendations¹²⁶

Figure 4: The investigation of sensory neuropathy

Diagnoses

Reduced intraepidermal nerve fi bre density23 Diabetic neuropathy

Reduced intraepidermal nerve fi bre density49,52 Reduced glucose tolerance

Non-length-dependent reduction of intraepidermal nerve fi bre density10

Sensory neuronopathies

IgM deposits on dermal myelinated fi bres53 Anti-MAG-neuropathy

Reduced intraepidermal nerve fi bre density73 Guillain–Barré syndrome

Myelin pathology and altered expression of gene encoding myelin54,55

Charcot–Marie–Tooth disease

Loss of epidermal and dermal innervation25 Friedreich ataxia

Reduced intraepidermal nerve fi bre density60 Fabry’s disease

Reduced intraepidermal nerve fi bre density26 Sarcoidosis

Non-length-dependent reduction of intraepidermal nerve fi bre density58

Coeliac disease

Reduced intraepidermal nerve fi bre density and evidence of vasculitis57,68

Systemic lupus erythematosus

Loss of sweat gland innervation; with or without loss of intraepidermal nerve fi bres16,125

Ross syndrome

Loss of intraepidermal nerve fi bres, sweat gland, and deep dermal innervation33

Familial dysautonomia

Innervation of skin and sweat glands absent83 CIPA

Abbreviations: IENF, intraepidermal nerve fi bers; MAG, myelin-associated glycoprotein; CIPA, congenital insensitivity to pain with anhidrosis.

Table2: examples of fi ndings for skin innervation in diff erent etiological categories


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99 patients with clinical symptoms of painful sensory neuropathy compared with 37 healthy volunteers, sensitivity and specifi city were calculated from receiver-operated curve analysis;42 by combining patients with and without additional large-fi bre involvement, and with a cut-off of 8·8 fi bres per millimetre, sensitivity was 79% and specifi city 82%. In the patients with only pure small-fi bre neuropathy, sensitivity was 77% and specifi city 79%, whereas, in patients with additional large-fi bre involvement, sensitivity was 76% and specifi city 81%. The area occupied by the subepidermal plexus was quantifi ed morphometrically; this measure detected pure, small-fi bre neuropathy with a specifi city of 78% and a sensitivity of 79%, and neuropathy with additional large-fi bre involvement with a specifi city of 95% and a sensitivity of 89%. The combination of raised intraepidermal nerve-fi bre density and raised subepidermal-nerve-plexus density increased the sensitivity to 86% for pure small-fi bre neuropathy and to 100% for mixed-fi bre neuropathy.42 Thus, a thorough analysis of skin innervation is a highly sensitive tool for sensory neuropathy. However, as with every other diagnostic method, some people will have borderline values and, in practice, skin-biopsy data should be judged in the context of clinical and other fi ndings (table 2, fi gure 4).

Findings in specifi c neuropathiesDiabetic neuropathyIn patients with diabetic neuropathy, low density of intraepidermal nerve fi bres was fi rst described by Levy and colleagues43,44 and confi rmed by Kennedy and colleagues23 and many other studies. The extent of epidermal denervation rises with the duration of diabetes but is not correlated with haemoglobin A1c concentrations,27,28 suggesting that intraepidermal nerve-fi bre density is an independent indicator of neuropathy progression. One study found low intraepidermal nerve-fi bre density in a subgroup of patients with high concentrations of erythrocyte aldose reductase.45 Neurotrophin concentrations in skin biopsy samples have occasionally been studied, with the fi ndings of raised neurotrophin-3 concentration and low concentrations of nerve growth factor.46,47 Furthermore, impaired glucose tolerance might be associated with nerve degeneration, as shown by skin-biopsy samples.48,49 In 40% of patients with small-fi bre neuropathy diagnosed only by skin biopsy, oral glucose-tolerance testing revealed an otherwise unknown impaired glucose tolerance.

Skin biopsy can also detect axon regeneration in human and experimental diabetic neuropathy. Diabetes induces early impairment of small nerve fi bres, which manifests as a slower regeneration rate of intraepidermal nerve fi bres, even in the absence of clinical and neurophysiological evidence of neuropathy.50 In diabetic truncal neuropathy, dermatome denervation and later reinnervation follows clinical recovery and indicates

postganglionic damage rather than a radiculopathy.51 In a small study in patients with impaired glucose tolerance, diet and exercise induced a signifi cant increase in intraepidermal nerve-fi bre density, correlated with an improvement in pain and sural sensory action-potential amplitude.52 Thus, the main indication for skin biopsy in the clinical management of diabetic neuropathy is follow-up under treatment.

Demyelinating neuropathiesThe ultrastructure and expression of the gene encoding myelin do not diff er between the dermal nerve fi bres and the sural nerves. This similarity will make it possible to show the abnormalities in immune-mediated and hereditary demyelinating neuropathies that can otherwise only be detected in sural nerve biopsy samples. In neuropathy associated with antibodies against myelin-associated glycoprotein, deposits of IgM were seen on dermal myelinated fi bres.53 Similarly, in Charcot–Marie–Tooth disease and related neuropathies, typical neuropathological changes were seen in biopsy from glabrous skin, which might help to investigate genotype–phenotype associations.54,55 Further studies are needed before skin biopsy can be recommended for the assessment of demyelinating neuropathies in clinical practice.

Neuropathy associated with systemic diseasesThe small-fi bre degeneration in acquired and genetic diseases, including sarcoidosis, systemic lupus erythematosus, Sjögren’s syndrome, coeliac disease, and Friedreich’s ataxia,25,26,56–60 can be seen in skin biopsies. Small-fi bre neuropathy is also a feature of Fabry’s disease,60 where the pathognomonic inclusions can be detected in skin.61 The diagnosis of small-fi bre neuropathy in these disorders enables the proper recognition and understanding of patients’ symptoms and the appropriate treatment for neuropathic pain.

Infectious and infl ammatory neuropathiesBecause there is obvious skin involvement in leprosy, this was the fi rst infectious neuropathy investigated with skin biopsy. Of 100 skin biopsy samples from patients with lepromatous, tuberculoid, and indeterminate leprosy, the number of cutaneous nerves and nerve endings that were immunoreactive for neurofi laments and PGP 9.5 was low, mostly in patients with tuberculoid disease.4 Only a few specimens from patients with indeterminate leprosy had immunoreactivity to the neuropeptides substance P and calcitonin gene-related peptide. Infl ammatory cells, which are abundant in the epineurium of dermal nerves, are not involved in the destruction of nerve fi bres;62 indeed, the loss of dermal innervation is not spatially related to infl ammatory cells.63 Of 28 patients with diff erent subtypes of leprosy, the unaff ected sites had normal skin innervation in the subepidermal regions, but intraepidermal nerve fi bres


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were absent in most patients. The fall in nerve fi bres in the subepidermis correlated with the rise in thermal-detection thresholds and a reduction in the cutaneous fl are response.64

In HIV-associated sensory neuropathy, the intra-epidermal nerve-fi bre density was lower than that in a published control population, and low intraepidermal nerve-fi bre density in the distal leg correlates with lower CD4 counts and higher plasma HIV RNA concentrations.65 Many intraepidermal nerve-fi bre swellings in the distal leg and low intraepidermal nerve-fi bre density were associated with a shorter time to symptomatic neuropathy in patients with HIV.37,66

Although sural nerve biopsy is the gold standard for the diagnosis of vasculitic neuropathy, skin biopsies can be helpful. Vasculitis and eosinophilia were detected around dermal nerves in a high percentage of patients with Churg Strauss syndrome,67 and the presence of vasculitis correlated with reduced intraepidermal nerve-fi bre density in patients with systemic lupus erythematosus.68 However, the use of skin biopsy in infectious and infl ammatory neuropathies is restricted to research and clinical trials.

Small-fi bre neuropathiesSmall-fi bre neuropathies are a subtype of sensory neuropathies that exclusively or predominately aff ect small diameter (A-delta and C) nerve fi bres. Small-fi bre neuropathy might be associated with an underlying disease (eg, diabetes or the early stages of amyloid neuropathy) or can be an idiopathic form. There is no consensus for the diagnosis of small-fi bre neuropathy, although the most generally accepted defi nition is a sensory neuropathy with paraesthesias (abnormal sensations) that are typically painful (ie, dysaesthesias), along with abnormal fi ndings of small-fi bre function in at least one of the following: neurological examination, specialised neurophysiological testing, or skin biopsy.69 In this context, specialised neurophysiological testing involves electrodiagnostic methods targeting distal nerve fi bres,70 quantitative sensory testing, and the quantitative sudomotor axonal refl ex test. The defi nition can be expanded to possible (one component present), probable (two present), or defi nite (all present) small-fi bre neuropathy.

The most common presentation is the sensation of burning feet. On examination, reduced thermal and pain sensations might be found, particularly in the feet, but these are not present in all cases. Low vibration sense in the toes is suggestive of possible impairment of the most distal large nerve fi bres; but this sense should be normal in the ankles, apart from where there is concomitant large-fi bre neuropathy. Because standard nerve-conduction studies are typically normal in small-fi bre neuropathy, the assessment of skin biopsy samples can aid diagnosis. Accordingly, the investigation of intraepidermal nerve fi bres is useful for the diagnosis

of small-fi bre neuropathy.17,23,25,26,38,56,58,68–75,71 Quantitative sensory testing and quantitative sudomotor axonal refl ex test are also useful for diagnosis of small-fi bre neuropathy, with less sensitivity than skin biopsy. With an overall sensitivity of skin biopsy analysis of 80%, and a low correlation between intraepidermal nerve fi bres and quantitative sensory testing fi ndings in some studies, up to 20% of patients with suspected small-fi bre neuropathy might have abnormal quantitative sensory test results but normal skin-biopsy fi ndings.

Morphology and functionRelation to sensory nerve conductionThe amplitude of sensory-nerve action potentials reveals the integrity of large diameter fi bres. Accordingly, concordance between sural nerve action-potential amplitude and intraepidermal nerve-fi bre density was found in patients with mixed neuropathy, although skin biopsy was more sensitive than nerve-conduction studies for the diagnosis of small-fi breneuropathy.24,28,31,41,48,72 One study described a parallel reduction in intraepidermal nerve-fi bre density and medial plantar sensory-nerve action potential amplitude in patients with painful neuropathy but normal sural nerve conduction.70 This fi nding suggests that the subclinical involvement of most distal large fi bres might occur in small-fi bre neuropathy. Alternatively, some mild distal neuropathies that aff ect both large and small fi bres might not be picked up by routine studies of sural nerve conduction.

Relation to psychophysical and autonomic testsQuantitative sensory testing is an extension of the usual neurological examination of somatosensory function. The assessment of detection thresholds and pain thresholds for diff erent sensory modalities, such as heat, cold, touch, and vibration, provides information on the diff erent fi bre classes: heat and cold thresholds give information about the function of small-diameter fi bres; vibration and touch detection thresholds give information about large-fi bre function. Because the fi bres conveying warm sensation are in the C-fi bre range, and cold sensation is transmitted by A-delta fi bres, a correlation between thermal thresholds and intraepidermal nerve-fi bre density would be expected, at least in small-fi bre neuropathy. In diabetic neuropathy, intraepidermal nerve-fi bre density is inversely correlated with temperature detection and pain thresholds, with the strongest correlation with warm detection thresholds.27,28 In patients with Guillain–Barré syndrome, low intraepidermal nerve-fi bre density is associated with raised warm detection threshold.73 Some researchers found no correlation of quantitative sensory testing parameters and intraepidermal nerve-fi bre density.24,72,74 In an experimental study with capsaicin denervation, intraepidermal nerve-fi bre density only correlated with heat pain if a small thermode was used for quantitative


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sensory testing,75 suggesting that the lack of correlation of these measures in some studies might have methodological causes.

Researchers using the quantitative sudomotor axonal-refl ex test have investigated whether intraepidermal nerve-fi bre density correlates with sweat output. The rationale behind this comparison is that patients with painful neuropathy frequently have autonomic symptoms. Hence, intraepidermal nerve fi bre and sweat output might decrease concomitantly. In patients with painful neuropathy, quantitative sudomotor axonal-refl ex was related to intraepidermal nerve-fi bre density, but only in a subgroup of patients with reduced quantitative sudomotor axonal refl ex test values.76 No correlation was found between quantitative sudomotor axonal refl ex test and intraepidermal nerve-fi bre density in a group of patients with painful, burning sensations in the feet.24 In patients with either Guillain–Barré syndrome or chronic infl ammatory demyelinating polyradiculoneuropathy, the subgroups with autonomic symptoms have lower intraepidermal nerve-fi bre density.73,77 Thus, at least in some diagnostic groups, the loss of intraepidermal nerve fi bres might be an indicator of dysfunction of autonomic systems.

Intraepidermal nerve-fi bre density and neuropathic painWhether a peripheral neuropathy is painful or not depends on several variables, some of which have not yet been fully explored.78 One factor is the involvement of nociceptive C-fi bres. Because intraepidermal nerve fi bres are capsaicin responsive79 and express transient receptor potential vanilloid receptor 1,15 at least a large part of them are nociceptors. Therefore, quantifying them should show the degree of C-fi bre involvement in a neuropathy and greater pathology—ie, loss of intraepidermal nerve fi bre might be associated with more pain. However, C-fi bres can be aff ected by a pathological process or sensitised by infl ammatory mediators without the structure being lost,80 for example, in the concept of the irritable nociceptor in postherpetic neuralgia.81

In a study of 60 patients with HIV-associated neuropathy, those patients classifi ed as having severe pain had a lower density of intraepidermal nerve fi bres in the distal leg than those with low to moderate pain. A similar fi nding was reported in 38 patients with diabetes, with and without pain.82 Patients with pain had lower intraepidermal nerve-fi bre densities; however, within the group of patients with pain, the intensity did not correlate with intraepidermal nerve-fi bre density. Similarly, another study of HIV-associated neuropathy found only a trend toward an inverse correlation of intraepidermal nerve-fi bre density with pain intensity.66

Patients with neuropathy can have persistent pain, despite complete skin denervation in distal leg regions, showing that the generator of pain is proximal.15 The situation is diff erent in congenital insensitivity to pain with anhidrosis (hereditary sensory neuropathy type IV),

where lack of skin nerves83 suggests a complete lack of nociceptors.83,84 Thus, our current understanding of the role of intraepidermal nerve-fi bre density and neuropathic pain is that the most severe loss of intraepidermal nerve fi bres suggests more severe neuropathy, and the presence of intact proximal nervous structures is associated with a higher risk of neuropathic pain. By contrast, an increase in intraepidermal nerve-fi bre density as a sign of recovery from a neuropathy is associated with a reduction in pain. Whether the loss of intraepidermal nerve fi bres itself is causally related to the pain or is just an indicator of a more proximal pathology is unknown.

Skin biopsy in follow-up of neuropathy and in nerve regenerationFollow-upBecause skin biopsy is a minimally invasive method, it can be repeated to monitor the spontaneous course of a neuropathy or the eff ect of an intervention—eg, dermatome reinnervation in diabetic truncal neuropathy, and nerve regeneration in steroid-responsive sensory neuropathy.51,85 Patients with diabetes have reduced ability to regenerate nerves, as shown with skin biopsy.50 The presence of large and diff use axonal swellings is related to the development of symptomatic neuropathy in asymptomatic people and the progression of neuropathy in patients with diabetes or HIV.36–38 In experimental studies in human beings, capsaicin leads to a recovery of the superfi cial skin innervation sensory loss for thermal stimuli and pain, and regeneration happens within weeks, as does recovery of sensation.79, 86

Clinical trialsMeasurement of skin innervation has been used as an outcome measure in trials of therapy for diabetic neuropathy.52,87-89 In patients with impaired glucose tolerance, participation in a diet and exercise programme is associated with signifi cant increases in intraepidermal nerve-fi bre density related to improvements in pain and action-potential amplitude in sural nerves.52 Sprouting of nerve fi bres after skin biopsy was observed in a larger, concentric biopsy but no eff ect of timcodar dimesylate was detected.90 Ongoing trials have incorporated skin-biopsy data as an outcome measure—eg, the ascorbic acid Charcot–Marie–Tooth disease 1A trial.91

Experimental studiesEvidence that skin biopsy is the most sensitive measure of a change in the severity of neuropathy was strengthened by studies in experimental diabetic neuropathy with neuroprotective agents.92,93 Intraepidermal nerve-fi bre density is related to an improvement in the results of behavioural and nerve-conduction tests. Other animal studies have used intraepidermal nerve-fi bre quantifi cation as a measure of axon degeneration and regeneration—eg, in models of chemotherapy-induced or diabetes-induced neuropathy;94–97 in nerve injury


Review

models;98–100 in the assessment of neuroprotective drugs;101 and in the study of transgenic mice.102,103 Therefore, quantifi cation of the intraepidermal nerve fi bres in the footpads of mice and rats gives an objective assessment of axon integrity and is a reliable outcome measure in preclinical trials on neuroprotection or regeneration in the peripheral nervous system.

Limitations of the methodIn the assessment of most neuropathies, skin biopsy cannot identify the cause. This limitation is particularly true if vasculitis is suspected, in which case nerve biopsy is warranted for the histological confi rmation of the diagnosis. In infl ammatory neuropathies, such as Guillain–Barré syndrome and chronic infl ammatory demyelinating polyradiculoneuropathy, when neuro-pathological examination is needed because of an atypical presentation, nerve biopsy should be done. Furthermore, skin innervation might be normal in demyelinating neuropathies. Finally, amyloid deposits are only rarely detectable in skin but are commonly seen in nerve biopsy; thus, in patients with a neuropathy that includes more than just small fi bres, and in which the cause cannot be detected by non-invasive methods, nerve biopsy is warranted and can not be replaced by assessment of skin innervation.

Some patients with a burning sensation in their feet might have normal fi ndings in clinical neurophysiology, normal or abnormal fi ndings in quantitative sudomotor axonal refl ex test, and normal fi ndings in skin biopsy. Although skin biopsy does not support small-fi bre neuropathy in these patients, normal intraepidermal nerve-fi bre density counts can not exclude a functional disturbance in C-fi bres.

OutlookNew and unexpected fi ndingsAs analysis of intraepidermal nerve fi bres becomes more widely used, abnormal fi ndings are reported in unexpected diseases. For example, burning-mouth syndrome, a disorder thought of as idiopathic and commonly attributed to psychological causes, is associated with a reduction in epithelial nerve fi bres in the tongue, relating this disorder to the small-fi bre neuropathies.104 Complex regional pain syndrome is another such disorder; two groups independently detected a reduction in epidermal innervation in patients with complex regional pain syndrome.11,12 Whether the loss of small fi bres in this disorder is primary or secondary is unknown; however, this fi nding adds new information about its pathophysiology. In erythro-melalgia, a disorder characterised by episodic burning pain and redness in the limbs that is aggravated by heat, some patients have low epidermal innervation, and the autosomal dominant forms are caused by mutations in the gene encoding a sodium channel (SCN9A) that is expressed in nociceptors.105–107 In restless legs syndrome,

skin biopsy enabled subclinical sensory neuropathy exclusively involving small nerve fi bres and with distinct clinical features to be shown.108 A study in non-human primates showed not only nerve fi bre loss but also substantial innervation remodelling during the progression of type II diabetes.109

New markers and diff erentiation of fi bre subtypesDetailed analysis of the innervation of dermal receptive endings with sophisticated techniques might provide an additional means for the early diagnosis and follow-up of sensory neuropathies.8,110

Several investigators have used specifi c antibodies to identify receptors or nerve-fi bre subtypes—eg, antibodies to neuropeptides,111,112 transient receptor potential vanilloid receptor 1,15 opioid receptors,113 or NMDA receptors.114 To distinguish C-fi bre subtypes morphologically and correlate them with the fi bre subtypes identifi ed by single-fi bre recording would greatly advance our understanding of the pathophysiological processes in nerve degeneration.115,116

A related, and largely unexplored, topic is the role of intraepidermal nerve fi bres in pruritus. Although beyond the scope of this article, there is a lot written about cutaneous innervation in animal experiments.

Beyond morphologySkin biopsy is amenable to the extraction of mRNA, and RT-PCR has only recently been used in the study of neuropathies. The technique might also aid the search for local cofactors implicated in the generation of pain in small-fi bre pathology; preliminary data lend support to the altered expression of proinfl ammatory cytokines in the painful skin of patients with small-fi bre neuropathy.117 Reliable methods for protein extraction from skin biopsy samples have recently been developed,118 whereby, in future studies, immunoassays might complement the semiquantitative, morphometrical data derived from immunohistochemistry.

Neurotrophin concentrations in skin biopsy samples have only been studied occasionally44,45 but certainly have potential in the study of the pathophysiology of the various disorders. The role of neurotrophins in the crosstalk among cutaneous innervation, structural cells, and immune cells in skin homoeostasis is being investigated;119 furthermore, the role of keratinocytes in sensory perception is also being investigated.120

In patients with hereditary neuropathies, expression of the genes encoding myelin has been measured in skin biopsy samples.121 Immunoelectron microscopy showed the expected rise in myelin protein PMP22 in patients with Charcot–Marie–Tooth disease 1A and decrease in patients with hereditary neuropathy with liability to pressure palsies. Expression of the genes encoding myelin was shown in Schwann cells from skin with RT-PCR. Overall, the use of skin biopsy in the diagnostic assessment of neuropathies is only beginning.


Review

ContributorsCS and GL both did literature searches and contributed equally to the writing of the manuscript.

Confl icts of interestWe have no confl icts of interest.

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skin biopsy in the management of peripheral neuropathy

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