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Neuropathy and related disabilities are the major medical consequences of leprosy, which remains a global medical concern. Despite major advances in understanding the mechanisms of M. leprae entry into peripheral nerves, most aspects of the pathogenesis of leprosy neuropathy remain poorly understood. Sensory loss is characteristic of leprosy, but neuropathic pain is sometimes observed. Effective anti-microbial therapy is available, but neuropathy remains a problem especially if diagnosis and treatment are delayed. Currently there is intense interest in post-exposure prophylaxis with single-dose rifampin in endemic areas, as well as with enhanced prophylactic regimens in some situations. Some degree of nerve involvement is seen in all cases and neuritis may occur in the absence of leprosy reactions, but acute neuritis commonly accompanies both Type 1 and Type 2 leprosy reactions and may be difficult to manage. A variety of established as well as new methods for the early diagnosis and assessment of leprosy neuropathy are reviewed. Corticosteroids offer the primary treatment for neuritis and for subclinical neuropathy in leprosy, but success is limited if nerve function impairment is present at the time of diagnosis. A candidate vaccine has shown apparent benefit in preventing nerve injury in the armadillo model. The development of new therapeutics for leprosy neuropathy is greatly needed. Supplementary Information The online version contains supplementary material available at 10.1007/s13311-021-01153-z.

Analysis of intraepidermal nerve fibers (IENFs) in skin biopsy samples has become a standard clinical tool for diagnosing peripheral neuropathies in human patients. Compared to sural nerve biopsy, skin biopsy is safer, less invasive, and can be performed repeatedly to facilitate longitudinal assessment. Intraepidermal nerve fiber analysis is also more sensitive than conventional nerve histology or electrophysiological tests for detecting damage to small-diameter sensory nerve fibers. The techniques used for IENF analysis in humans have been adapted for large and small animal models and successfully used in studies of diabetic neuropathy, chemotherapy-induced peripheral neuropathy, HIV-associated sensory neuropathy, among others. Although IENF analysis has yet to become a routine end point in nonclinical safety testing, it has the potential to serve as a highly relevant indicator of sensory nerve fiber status in neurotoxicity studies, as well as development of neuroprotective and neuroregenerative therapies. Recently, there is also interest in the evaluation of IENF via skin biopsy as a biomarker of small fiber neuropathy in the regulatory setting. This article provides an overview of the anatomic and pathophysiologic principles behind IENF analysis, its use as a diagnostic tool in humans, and applications in animal models with focus on comparative methodology and considerations for study design.

Objective To determine the utility of skin biopsies as a biomarker of disease severity in subjects with amyloid neuropathy. Methods Five groups of patients were studied: (1) transthyretin (TTR) familial amyloidotic polyneuropathy (FAP; n = 20), (2) TTR mutation carriers without peripheral neuropathy (TTR-noPN; n = 10), (3) healthy controls (n = 20), (4) diabetic neuropathy disease controls (n = 20), and (5) patients with light-chain (AL) amyloid (n = 2). All subjects underwent neurological examination and 3mm skin biopsies. Sections were stained with anti-PGP9.5, anti-TTR, and Congo red. Intraepidermal (IENFD), sweat gland (SGNFD), and pilomotor nerve fiber densities (PMNFD) were measured. Correlations between the amount of amyloid present (amyloid burden), fiber subtype, and Neuropathy Impairment Score in the Lower Limbs (NIS-LL) were evaluated. Results IENFD, SGNFD, and PMNFD were all significantly reduced in TTR-FAP patients versus healthy controls, whereas TTR-noPN subjects had intermediate reductions. Lower nerve fiber densities were associated with NIS-LL (p

Quantitative assessment of epidermal nerve fibers (ENFs) has become a widely used clinical tool for the diagnosis of small fiber neuropathies such as diabetic neuropathy and human immunodeficiency virus–associated sensory neuropathy (HIV-SN). To model and investigate the pathogenesis of HIV-SN using simian immunodeficiency virus (SIV)-infected Asian macaques, we adapted the skin biopsy and immunostaining techniques currently employed in human patients and then developed two unbiased image analysis techniques for quantifying ENF in macaque footpad skin. This report provides detailed descriptions of these tools and techniques for ENF assessment in macaques and outlines important experimental considerations that we have identified in the course of our long-term studies. Although initially developed for studies of HIV-SN in the SIV-infected macaque model, these methods could be readily translated to a range of studies involving peripheral nerve degeneration and neurotoxicity in nonhuman primates as well as preclinical investigations of agents aimed at neuroprotection and regeneration.

Sustained elimination of leprosy as a global health concern likely requires a vaccine. The current standard, BCG, confers only partial protection and precipitates paucibacillary (PB) disease in some instances. When injected into mice with the T helper 1 (Th1)-biasing adjuvant formulation Glucopyranosyl Lipid Adjuvant in stable emulsion (GLA-SE), a cocktail of three prioritized antigens (ML2055, ML2380 and ML2028) reduced M. leprae infection levels. Recognition and protective efficacy of a single chimeric fusion protein incorporating these antigens, LEP-F1, was confirmed in similar experiments. The impact of post-exposure immunization was then assessed in nine-banded armadillos that demonstrate a functional recapitulation of leprosy. Armadillos were infected with M. leprae 1 month before the initiation of post-exposure prophylaxis. While BCG precipitated motor nerve conduction abnormalities more rapidly and severely than observed for control infected armadillos, motor nerve injury in armadillos treated three times, at monthly intervals with LepVax was appreciably delayed. Biopsy of cutaneous nerves indicated that epidermal nerve fiber density was not significantly altered in M. leprae-infected animals although Remak Schwann cells of the cutaneous nerves in the distal leg were denser in the infected armadillos. Importantly, LepVax immunization did not exacerbate cutaneous nerve involvement due to M. leprae infection, indicating its safe use. There was no intraneural inflammation but a reduction of intra axonal edema suggested that LepVax treatment might restore some early sensory axonal function. These data indicate that post-exposure prophylaxis with LepVax not only appears safe but, unlike BCG, alleviates and delays the neurologic disruptions caused by M. leprae infection., Leprosy: Working towards a safe and effective vaccine A leprosy vaccine candidate has been developed that raises immune responses against targets gleaned from naturally resistant individuals. Researchers from the United States and Japan, led by Malcolm Duthie, of Seattle’s Infectious Disease Research Institute, tested a Mycobacterium leprae vaccine candidate that generated immune responses mimicking those found in partially-resistant patients, and immune co-inhabitants of the severely infected. The candidate, dubbed LepVax, inhibited infection in mice and, when administered post-infection, delayed and mitigated nerve damage in armadillos. This contrasts with the current vaccine, BCG, which can precipitate leprosy symptoms when given after infection. This study also revealed that M. leprae infection can induce ‘silent’ pre-clinical nerve aberations. High-risk populations may already be infected with M. leprae, making safe and effective post-exposure prophylaxis a landmark step in combating both the individual and global burden of leprosy.

Background: Using a stereologic approach, the density of nerve fibers innervating sweat gland (SG) fragments in patients with diabetes mellitus (DM) and healthy controls using protein gene product (PGP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) was measured to determine which marker best detected differences between the groups. Factors associated with SG nerve fiber (SGNF) innervation were assessed and the change in SG innervation over a 1-year time period was determined. Methods: Ninety-two control subjects and 2 groups of subjects with DM totaling 97 were assessed in this cross-sectional study. Intraepidermal nerve fiber density and SG innervation were determined from leg skin biopsies that were immunohistochemically stained for ubiquitin hydrolase, VIP, and TH. Factors associated with SG innervation were assessed and 15 subjects were longitudinally followed for 1 year. Results: SGNF innervation was reduced in subjects with DM compared with controls. Lower SG innervation values were associated with increasing glycated hemoglobin A 1c , body mass index (BMI), men compared with women, and tobacco use, but not diabetes type or age. Sex, A 1c , and BMI remained significant in multivariate modeling. SG innervation measured by VIP+ fibers is a more sensitive marker for neuropathy than either PGP or TH. Fifteen subjects with DM followed for 1 year showed a significant decrease in SGNF innervation but not intraepidermal nerve fiber density. Conclusions: Stereologic measurement of SG innervation is feasible to assess postganglionic autonomic nerve fiber densities. SG innervation was reduced in subjects with DM compared with control subjects and was associated with sex, A 1c , and BMI in multivariate modeling. VIP+ SGNF is more severely reduced in DM than TH+ or PGP9.5+-based assessments. Progression of diabetic polyneuropathy was detected by SGNF over a 1-year time period.

We explored associations between mitochondrial DNA (mtDNA) haplogroups, epidermal nerve fiber density (ENFD), and HIV-associated sensory neuropathy (HIV-SN) in a randomized trial of Thai patients initiating antiretroviral therapy (ART).The South East Asia Research Collaboration with Hawaii 003 study evaluated toxicity of nucleoside reverse transcriptase inhibitors (stavudine vs. zidovudine vs. tenofovir). We present secondary analyses of mtDNA haplogroups and ENFD changes.ENFD, peripheral blood mononuclear cell mitochondrial complex I and IV, and 8-oxo-deoxyguanine (8-oxo-dG) were quantified. Peripheral blood mononuclear cell mtDNA sequences were obtained for haplogroup determination. Multivariate regression of ENFD change was performed.Paired ENFD was available from 118 patients. Median age, CD4 cell count, and height at entry were 34 years, 172 cells/μl, and 162 cm, respectively. Major haplogroups included M (42%), F (21%), and B (16%). Baseline ENFD, CD4 cell count, randomized ART, and biomarkers did not differ by haplogroup. Haplogroup B patients were older (P=0.02) at baseline, and had an increase in median ENFD (+1.5 vs. -2.9 fibers/mm; P=0.03) and 8-oxo-dG break frequency (+0.05 vs. 0.00; P=0.05) compared to other haplogroups. In a multivariate model, haplogroup B was associated with increased ENFD (β=3.5, P=0.009) at week 24, whereas older age (P=0.02), higher baseline CD4 cell count, (P=0.03), higher complex I level (P=0.03), and higher ENFD (P0.001) at baseline were all associated with decreased ENFD. Three of the six HIV-SN cases were haplogroup B (P=0.05).Thai persons belonging to mtDNA haplogroup B had increased ENFD and 8-oxo-dG on ART, and were more likely to develop HIV-SN. These results suggest that mtDNA variation influences early oxidative damage and ENFD changes.

Peripheral neuropathy (PN) is the most frequent neurologic complication in individuals infected with human immunodeficiency virus (HIV). It affects over one third of infected patients, including those receiving effective combination antiretroviral therapy. The pathogenesis of HIV-associated peripheral neuropathy (HIV-PN) remains poorly understood. Clinical studies are complicated because both HIV and antiretroviral treatment cause damage to the peripheral nervous system. To study HIV-induced peripheral nervous system (PNS) damage, a unique simian immunodeficiency virus (SIV)/pigtailed macaque model of HIV-PN that enabled detailed morphologic and functional evaluation of the somatosensory pathway throughout disease progression was developed. Studies in this model have demonstrated that SIV induces key pathologic features that closely resemble HIV-induced alterations, including inflammation and damage to the neuronal cell bodies in somatosensory ganglia and decreased epidermal nerve fiber density. Insights generated in the model include: finding that SIV alters the conduction properties of small, unmyelinated peripheral nerves; and that SIV impairs peripheral nerve regeneration. This review will highlight the major findings in the SIV-infected pigtailed macaque model of HIV-PN, and will illustrate the great value of a reliable large animal model to show the pathogenesis of this complex, HIV-induced disorder of the PNS.

Objectives Distal leg epidermal nerve fibre density (ENFD) is a validated predictor of small unmyelinated nerve fibre damage and neuropathy risk in HIV infection. As pre-existing damage may increase the risk of neuropathy following antiretroviral (ARV) therapy, particularly when the regimen contains stavudine (d4T), we assessed the relationship between ENFD and various parameters including mitochondrial factors in HIV-infected Thai individuals naive to ARV therapy. Methods Distal leg and proximal thigh ENFDs were quantified in HIV-infected Thai individuals without neuropathy prior to randomization to a HIV clinical trial that focused on mitochondrial toxicity issues. We assessed their association with various clinical and immunovirological parameters as well as with peripheral blood mononuclear cell (PBMC) mitochondrial (mt) DNA copies/cell, oxidative phosphorylation (OXPHOS) complex I (CI) and complex IV (CIV) enzyme activities, and mt 8-oxo-deoxyguanine (8-oxo-dG) break frequencies. Results In 132 subjects, the median (interquartile range) ENFD (fibres/mm) values were 21.0 (16.2–26.6) for the distal leg and 31.7 (26.2–40.0) for the proximal thigh. By linear regression, lower CD4 count (P

To characterize the regenerative pattern of cutaneous nerves in simian immunodeficiency virus (SIV)-infected and uninfected macaques, excisional axotomies were performed in nonglabrous skin at 14-day intervals. Samples were examined after immunostaining for the pan-axonal marker PGP 9.5 and the Schwann cell marker p75 nerve growth factor receptor. Collateral sprouting of axons from adjacent uninjured superficial dermal nerve bundles was the initial response to axotomy. Both horizontal collateral sprouts and dense vertical regeneration of axons from the deeper dermis led to complete, rapid reinnervation of the epidermis at the axotomy site. In contrast to the slower, incomplete reinnervation previously noted in humans after this technique, in both SIV-infected and uninfected macaques epidermal reinnervation was rapid and completed by 56 days postaxotomy. p75 was densely expressed on the Schwann cells of uninjured nerve bundles along the excision line and on epidermal Schwann cell processes. In both SIV-infected and uninfected macaques, Schwann cell process density was highest at the earliest timepoints postaxotomy and then declined at a similar rate. However, SIV-infection delayed epidermal nerve fiber regeneration and remodeling of new sprouts at every timepoint postaxotomy, and SIV-infected animals consistently had lower mean epidermal Schwann cell densities, suggesting that Schwann cell guidance and support of epidermal nerve fiber regeneration may account for altered nerve regeneration. The relatively rapid regeneration time and the completeness of epidermal reinnervation in this macaque model provides a useful platform for assessing the efficacy of neurotrophic or regenerative drugs for sensory neuropathies including those caused by HIV, diabetes mellitus, medications, and toxins.

Objectives—Antiretroviral Toxic Neuropathy is associated with dideoxy-nucleoside reverse transcriptase inhibitor use in HIV, possibly due to mitochondrial toxicity. Acetyl-L-Carnitine (ALC) has been linked to symptomatic improvement in ATN. We present an open-label single-arm pilot study to evaluate change in intra-epidermal nerve fiber (IENF) density and mitochondrial DNA (mtDNA) copies/cell among subjects treated with 3000mg ALC daily. Methods—Punch skin biopsies were examined at baseline and 24 weeks after therapy. Participants reported neuropathic symptoms using the Gracely Pain Intensity Score. Neurological examinations were completed. Results—Twenty-one subjects completed the study. ALC was generally well-tolerated. The IENF density did not change among cases completing 24 weeks of ALC therapy, with median (90% confidence interval (CI)) IENF changes of −1.70 (−3.50, ∞), p=0.98 and 2.15 (−0.10, ∞), p=0.11 for the distal leg and proximal thigh, respectively. Fat mtDNA copies/cell did not change with therapy. Improvements in neuropathic pain (p

Objective:To demonstrate the relationship between epidermal nerve fiber density (ENFD) in the leg and the phenotype of HIV-associated distal sensory polyneuropathy (HIV-DSP) in a multicenter prospective study (ACTG A5117).Methods:A total of 101 HIV-infected adults, with CD4 cell count 3and who had received antiretroviral therapy (ART) for at least 15 consecutive weeks, underwent standardized clinical and electrophysiologic assessment. All 101 subjects were biopsied at the distal leg (DL) and 99 at the proximal thigh (PT) at baseline. ENFD was assessed by skin biopsy using PGP9.5 immunostaining. Associations of ENFD with demographics, ART treatment, Total Neuropathy Score (TNS), sural sensory nerve action potential (SNAP) amplitude and conduction velocity, quantitative sensory testing (QST) measures, and neuropathic pain were explored.Results:ENFD at the DL site correlated with neuropathy severity as gauged by TNS (p< 0.01), the level of neuropathic pain quantified by the Gracely Pain Scale (GPS) (p= 0.01) and Visual Analogue Scale (VAS) (p= 0.01), sural SNAP amplitude (p< 0.01), and toe cooling (p< 0.01) and vibration (p= 0.02) detection thresholds. ENFD did not correlate with neurotoxic ART exposure, CD4 cell count, or plasma HIV-1 viral load.Conclusions:In subjects with advanced HIV-1 infection, epidermal nerve fiber density (ENFD) assessment correlates with the clinical and electrophysiologic severity of distal sensory polyneuropathy (DSP). ENFD did not correlate with previously established risk factors for HIV-DSP, including CD4 cell count, plasma HIV-1 viral load, and neurotoxic antiretroviral therapy exposure.

Quantification of epidermal nerve fiber density (ENFD) is a validated tool to confirm the clinical diagnosis of small fiber neuropathy.1 Several groups have published normative ENFD data in healthy controls.2-6 We assessed ENFD values in healthy ethnic Thais and report that values are significantly higher when compared with normative ENFD data from the United States. Following local Institutional Review Board approval, distal leg and proximal thigh skin punch biopsies were performed on healthy Thai subjects free of neuropathic symptoms (persistent pain, tingling, or numbness of lower extremities) and signs (diminished or absent ankle reflexes, decreased vibratory, pin or temperature sensation, or contact allodynia). Subjects were excluded if they were pregnant or had disease or medication use known to cause neuropathy, including HIV, diabetes, vitamin B12 deficiency, excessive alcohol intake, or hepatitis C infection. Skin punch biopsy and processing were performed in strict adherence to guidelines of the Cutaneous Nerve Laboratory at Johns Hopkins (http://www.hopkinsmedicine.org/neurology_neurosurgery/specialty_areas/cutaneous_nerve_lab/), with on-site training in Bangkok. and quality control performed by Johns Hopkins. Slides of 50-mM-thick PGP9.5 immunostained sections were examined at Johns Hopkins for specimen and staining quality, and the number of unmyelinated nerve fibers per millimeter of length of epidermis was quantitated using established counting rules.2,7,8 All slides were blinded and clinical information masked. One author (G.J.E.) counted 95% of biopsies. Results were then compared with U.S. normative data using the same lab and counting techniques.2 Forty-nine healthy ethnic Thai subjects (21 men/28 women) were studied. The ages ranged from 25 to 44 years, with a mean (SD) of 33.2 (4.8) and median of 32. The mean (SD) and median height and body mass index (BMI) were 162.1 (6.2) and 161 cm, and 22.1 (2.4) and 22.0, respectively. The reference paper on U.S. normative range for ENFD used 98 healthy controls with an age range of 13–82 years, 76% of whom were white and 18% black.2 Height and BMI were not reported. Results are shown in Table 1. The mean (SD) distal leg ENFD in our Thai subjects was 30.4 (10.4) mm (fifth percentile 16.5 mm) compared with 13.8 (6.7) mm (fifth percentile 3.8 mm) in the reference paper, with proximal thigh ENFD in Thais of 47.1 (12.5) mm (fifth percentile 27.0 mm) compared with reference values of 21.1 (10.4) mm (fifth percentile 5.2 mm). The thigh/distal leg ratios were similar. Table 1 Proximal thigh and distal leg epidermal nerve fiber density in Thai healthy controls compared to healthy controls in the United States* using 2-sample t-test. In summary, our study found approximately 2–3× higher ENFD in healthy controls in Thailand compared with healthy subjects in the United States. Previous studies that have included Asians have not reported ethnic differences,3,4 but the credibility of our analysis is strengthened by the use of the same technique and laboratory that reported U.S. norms. The limitations include the 14-year difference between comparisons, and lack of height, BMI, and other relevant information to assess potential confounders. We conclude that substantial differences in ENFD may exist by ethnicity, and further investigation into the role of ethnicity is warranted.

All patients with leprosy have some degree of nerve involvement. Perineural inflammation is the histopathologic hallmark of leprosy, and this localization may reflect a vascular route of entry of Mycobacterium leprae into nerves. Once inside nerves, M. leprae are ingested by Schwann cells, with a wide array of consequences. Axonal atrophy may occur early in this process; ultimately, affected nerves undergo segmental demyelination. Knowledge of the mechanisms of nerve injury in leprosy has been greatly limited by the minimal opportunities to study affected nerves in man. The nine-banded armadillo provides the only animal model of the pathogenesis of M. leprae infection. New tools available for this model enable the study and correlation of events occurring in epidermal nerve fibers, dermal nerves, and nerve trunks, including neurophysiologic parameters, bacterial load, and changes in gene transcription in both neural and inflammatory cells. The armadillo model is likely to enhance understanding of the mechanisms of nerve injury in leprosy and offers a means of testing proposed interventions.

Leprosy (also known as Hansen's Disease) is a chronic infectious disease caused by Mycobacterium leprae that primarily targets the peripheral nervous system; skin, muscle, and other tissues are also affected. Other than humans, nine-banded armadillos (Dasypus novemcinctus) are the only natural hosts of M. leprae, and they are the only laboratory animals that develop extensive neurological involvement with this bacterium. Infection in the armadillo closely recapitulates many of the structural, physiological, and functional aspects of leprosy seen in humans. Armadillos can be useful models of leprosy for basic scientific investigations into the pathogenesis of leprosy neuropathy and its associated myopathies, as well as for translational research studies in piloting new diagnostic methods or therapeutic interventions. Practical and ethical constraints often limit investigation into human neuropathies, but armadillos are an abundant source of leprotic neurologic fibers. Studies with these animals may provide new insights into the mechanisms involved in leprosy that also might benefit the understanding of other demyelinating neuropathies. Although there is only a limited supply of armadillo-specific reagents, the armadillo whole genomic sequence has been completed, and gene expression studies can be employed. Clinical procedures, such as electrophysiological nerve conduction testing, provide a functional assessment of armadillo nerves. A variety of standard histopathological and immunopathological procedures including Epidermal Nerve Fiber Density (ENFD) analysis, Schwann Cell Density, and analysis for other conserved cellular markers can be used effectively with armadillos and will be briefly reviewed in this text.

The skin is innervated by small sensory and autonomic fibers. In the epidermis, sensory fibers are present as unmyelinated C fibers that terminate as free nerve endings. The determination of epidermal nerve fiber (ENF) density using the immunohistochemical method is a powerful tool that provides insight into a population of nerve fibers that is prominently altered in small fiber neuropathy. The superficial location of epidermal nerve fibers allows repeated sampling of these nerves in a relatively noninvasive fashion, and in sites that cannot be assessed through conventional electrodiagnostic techniques. These features have allowed investigators to diagnose diabetic neuropathy earlier in the course of disease. ENF density holds promise as a biomarker for neuropathic pain and is a sensitive indicator of neuropathic progression. Finally, the ability to injure these fibers in a standardized fashion has led to novel measures of human axonal regeneration that may provide a more sensitive ruler by which to assess promising regenerative compounds in clinical trials.

Peripheral nerves and blood vessels travel together closely during development but little is known about their interactions post-injury. The SIV-infected pigtailed macaque model of human immunodeficiency virus (HIV) recapitu- lates peripheral nervous system pathology of HIV infection. In this study, we assessed the effect of SIV infection on neurovascular regrowth using a validated excisional axot- omy model. Six uninfected and five SIV-infected macaques were studied 14 and 70 days after axotomy to characterize regenerating vessels and axons. Blood vessel extension pre- ceded the appearance of regenerating nerve fibers suggest- ing that vessels serve as scaffolding to guide regenerating axons through extracellular matrix. Vascular endothelial growth factor (VEGF) was expressed along vascular silhou- ettes by endothelial cells, pericytes, and perivascular cells. VEGF expression correlated with dermal nerve (r00.68, p0 0.01) and epidermal nerve fiber regrowth (r00.63, p00.02). No difference in blood vessel growth was observed between SIV-infected and control macaques. In contrast, SIV- infected animals demonstrated altered length, pruning and arborization of nerve fibers as well as alteration of VEGF expression. These results reinforce earlier human primate findings that vessel growth precedes and influences axonal regeneration. The consistency of these observations across human and non-human primates validates the use of the pigtailed-macaque as a preclinical model.

Diabetic complications and vascular disease are closely intertwined. Diabetes mellitus is a well-established risk factor for both large and small vessel vascular changes, and conversely other vascular risk factors confer increased risk for diabetic complications such as peripheral neuropathy, nephropathy and retinopathy. Furthermore, axons and blood vessels share molecular signals for purposes of navigation, regeneration and terminal arborizations. We examined blood vessel, Schwann cell and axonal regeneration using validated axotomy models to study and compare patterns and the relationship of regeneration among these different structures. Ten subjects with diabetes mellitus complicated by neuropathy and 10 healthy controls underwent 3 mm distal thigh punch skin biopsies to create an intracutaneous excision axotomy followed by a concentric 4-mm overlapping biopsy at different time points. Serial sections were immunostained against a pan-axonal marker (PGP9.5), an axonal regenerative marker (GAP43), Schwann cells (p75) and blood vessels (CD31) to visualize regenerating structures in the dermis and epidermis. The regenerative and collateral axonal sprouting rates, blood vessel growth rate and Schwann cell density were quantified using established stereology techniques. Subjects also underwent a chemical ‘axotomy’ through the topical application of capsaicin, and regenerative sprouting was assessed by the return of intraepidermal nerve fibre density through regenerative regrowth. In the healed 3 mm biopsy sites, collateral and dermal regenerative axonal sprouts grew into the central denervated area in a stereotypic pattern with collateral sprouts growing along the dermal–epidermal junction while regenerative dermal axons, blood vessels and Schwann cells grew from their transected proximal stumps into the deep dermis. Vessel growth preceded axon and Schwann cell migration into the denervated region, perhaps acting as scaffolding for axon and Schwann cell growth. In control subjects, Schwann cell growth was more robust and extended into the superficial dermis, while among subjects with diabetes mellitus, Schwann tubes appeared atrophic and were limited to the mid-dermis. Rates of collateral (P = 0.0001), dermal axonal regenerative sprouting (P = 0.02), Schwann cell migration (P < 0.05) and blood vessel growth (P = 0.002) were slower among subjects with diabetes mellitus compared with control subjects. Regenerative deficits are a common theme in diabetes mellitus and may underlie the development of neuropathy. We observed that blood vessel growth recapitulated the pattern seen in ontogeny and preceded regenerating nerve fibres, suggesting that enhancement of blood vessel growth might facilitate axonal regeneration. These models are useful tools for the efficient investigation of neurotrophic and regenerative drugs, and also to explore factors that may differentially affect axonal regeneration.

Today, 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 neuropathologic 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 immunohistochemical 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.

Objective: To explore the association between specific nucleoside reverse transcriptase inhibitors and sensory neuropathies (SNs) and define the modifying roles of hepatitis C (HCV), vitamin B 12 deficiency, and impaired glucose tolerance. Methods: The authors conducted a prospective cohort study of 147 HIV-infected adults at two sites chosen to emphasize demographic differences. Standardized assessments included detailed antiretroviral histories, neurologic examinations, skin biopsies for epidermal nerve quantitation, and quantitative sensory testing. Results: There were significant differences between subjects at Johns Hopkins University (JHU) and Monash University (MU) in gender, race, HIV transmission route, and HCV seroprevalence. Symptomatic SN was present in 49% at JHU and 55% at MU (χ 2 = 4.02, p = 0.134) and was significantly more common in those at least age 40 than younger patients (odds ratio [OR] = 2.87, 95% CI = 1.27, 6.49). After adjusting for site, age, and CD4 cell count, exposure to didanosine (ddI) or stavudine (d4T) was associated with an significantly increased likelihood of symptomatic SN (ddI: OR = 3.21, 95% CI: 1.56, 6.60; d4T: OR = 7.66, 95% CI: 2.89, 20.33). Plasma HIV RNA, lactate, and HCV were not associated with SN. Quantitative vibratory testing identified neuropathy with a positive predictive value of 76% and epidermal nerve fiber densities 59%. Conclusions: Exposure to stavudine and didanosine was significantly associated with a heightened risk for symptomatic sensory neuropathy. Reduced vibration thresholds and epidermal nerve fiber densities had the highest diagnostic efficiency of the laboratory indicators of neuropathy examined, but were relatively insensitive in isolation.