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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.

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.

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.

We compared the pathological changes in cutaneous axons and Schwann cells of individuals with nerve transection to the changes in patients with chronic neuropathies. Following axotomy there was segmentation of axons in the epidermis and dermis on the first day, and loss of axons from the skin was virtually complete by Day 11. Epidermal and small superficial dermal axons were lost before larger caliber and deeper dermal axons. Within the first 50 days following nerve transection, the denervated Schwann cells in the dermis were easily identified by their markers p75 and S100, but by 8 months they had largely disappeared. The chronic neuropathy patients had distally predominant fibre loss, with greater loss of epidermal and dermal fibres in the distal regions of the leg than proximal regions. Several patients had large axonal swellings, often alternating with axonal attenuation, even in regions with normal or nearly normal fibre densities. By electron microscopy the swellings contained accumulations of mitochondria and other particulate organelles as well as neurofilaments. These swellings are likely to represent predegenerative changes in sites of impaired axonal transport, and previous data indicate that the swellings presage fibre loss in the subsequent months. Some of the severely denervated regions had remaining Schwann cells, as judged by immunocytochemistry and by electron microscopy, but others lacked Schwann cells. By analogy with animal experiments, these regions are likely to have had more prolonged denervation. The distribution of axonal loss, the axonal swellings and the changes in Schwann cells all have implications for the design of clinical trials of agents intended to protect cutaneous innervation and to promote regeneration of cutaneous axons in peripheral neuropathies.

Human immunodeficiency virus (HIV)-induced peripheral neuropathy is the most common neurologic complication associated with HIV infection. In addition to virus-mediated injury of the peripheral nervous system (PNS), treatment of HIV infection with combination antiretroviral therapy (cART) may induce toxic neuropathy as a side effect. Antiretroviral toxic neuropathy is clinically indistinguishable from the sensory neuropathy induced by HIV; in some patients, these 2 processes are likely superimposed. To study these intercurrent PNS disease processes, we first established a simian immunodeficiency virus (SIV)/pigtailed macaque model in which more than 90% of animals developed PNS changes closely resembling those seen in HIV-infected individuals with distal sensory neuropathy. To determine whether cART alters the progression of SIV-induced PNS damage, dorsal root ganglia and epidermal nerve fibers were evaluated in SIV-infected macaques after long-term suppressive cART. Although cART effectively suppressed SIV replication and reduced macrophage activation in the dorsal root ganglia, PGP 9.5 immunostaining and measurements of epidermal nerve fibers in the plantar surface of the feet of treated SIV-infected macaques clearly showed that cART did not normalize epidermal nerve fiber density. These findings illustrate that significant PNS damage persists in SIV-infected macaques on suppressive cART.

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.

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.

Recent studies identified an association between genetic variants in the lymphotoxin-alpha (LTalpha) gene and leprosy. To study the influence of LTalpha on the control of experimental leprosy, both low- and high-dose Mycobacterium leprae foot pad (FP) infections were evaluated in LTalpha-deficient chimeric (cLTalpha(-/-)) and control chimeric (cB6) mice. Cellular responses to low-dose infection in cLTalpha(-/-) mice were dramatically different, with reduced accumulation of CD4(+) and CD8(+) lymphocytes and macrophages and failure to form granulomas. Growth of M. leprae was contained for 6 months, but augmented late in infection. In contrast, tumor necrosis factor knockout and tumor necrosis factor receptor 1 knockout FPs exhibited extensive inflammatory infiltration with an increase in M. leprae growth throughout infection. Following high-dose infection, cB6 FP induration peaked at 4 weeks and was maintained for 12 weeks. Induration was not sustained in cLTalpha(-/-) FPs that contained few lymphocytes and no granulomas. There was a reduction in the expression levels of inflammatory cytokines, chemokines, and chemokine receptors, including nitric oxide synthase 2, vascular cell adhesion molecule, and intercellular cell adhesion molecule. Furthermore, cLTalpha(-/-) popliteal lymph nodes contained a higher proportion of naïve CD44(lo)CD62L(hi) T cells than cB6 mice, suggestive of reduced T cell activation. Therefore, both LTalpha and tumor necrosis factor are essential for the regulation of the granuloma, but they have distinctive roles in the recruitment of lymphocytes and maintenance of the granulomatous response during chronic M. leprae infection.

Background Glutamate excitotoxicity is thought to be involved in the pathogenesis of neurodegenerative disease. One potential source of glutamate is N-acetyl-aspartyl-glutamate (NAAG) which is hydrolyzed to glutamate and N-acetyl-aspartate (NAA) in a reaction catalyzed by glutamate carboxypeptidase (GCP). As a result, GCP inhibition is thought to be beneficial for the treatment of neurodegenerative diseases where excess glutamate is presumed pathogenic. Both pharmacological and genetic inhibition of GCP has shown therapeutic utility in preclinical models and this has led to GCP inhibitors being pursued for the treatment of nervous system disorders in human clinical trials. Specifically, GCP inhibitors are currently being developed for peripheral neuropathy and neuropathic pain. The purpose of this study was to develop a pharmacodynamic (PD) marker assay to use in clinical development. The PD marker will determine the effect of GCP inhibitors on GCP enzymatic activity in human skin as measure of inhibition in peripheral nerve and help predict drug doses required to elicit pharmacologic responses. Methods GCP activity was first characterized in both human skin and rat paw pads. GCP activity was then monitored in both rodent paw pads and sciatic nerve from the same animals following peripheral administration of various doses of GCP inhibitor. Significant differences among measurements were determined using two-tailed distribution, equal variance student's t test. Results We describe for the first time, a direct and quantifiable assay to evaluate GCP enzymatic activity in human skin biopsy samples. In addition, we show that GCP activity in skin is responsive to pharmacological manipulation; GCP activity in rodent paws was inhibited in a dose response manner following peripheral administration of a potent and selective GCP inhibitor. Inhibition of GCP activity in rat paw pads was shown to correlate to inhibition of GCP activity in peripheral nerve. Conclusion Monitoring GCP activity in human skin after administration of GCP inhibitors could be readily used as PD marker in the clinical development of GCP inhibitors. Enzymatic activity provides a simple and direct measurement of GCP activity from tissue samples easily assessable in human subjects.