Gigi J. Ebenezer

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

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.

Correlates of epidermal nerve fiber densities in HIV-associated distal sensory polyneuropathy

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 <300 cells/mm3 and 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.

The histological diagnosis of leprosy type 1 reactions: identification of key variables and an analysis of the process of histological diagnosis

Background: Type 1 leprosy reactions (T1R) are a major inflammatory complication of leprosy affecting 30% of patients with borderline leprosy, but there has been no diagnostic evaluation of the histological diagnosis of this entity. Methods: In a prospective study based in India, skin biopsies were taken from 99 patients with clinically diagnosed T1R and 52 non-reactional controls. These were assessed histologically by four histopathologists whose assessments were then compared. Results: Reactions were under-diagnosed, with 32–62% of clinically diagnosed reactions being given a histological diagnosis. The pathologists showed good specificities (range 72% to 93%) but much poorer sensitivities (range 42% to 78%). The most commonly reported histological features of TIR were cell maturity, oedema and giant cells. Five key variables were identified that the pathologists used in diagnosing a reaction: intra-granuloma oedema, giant cell size, giant cell numbers, dermal oedema and HLA-DR expression. A predictive model for the diagnosis of T1R was developed using stepwise logistic regression analysis, with clinical diagnosis of reaction as an outcome, and then identification of the key variables that each pathologist used in making the diagnosis of T1R. 34–53% of the variation between pathologists could be accounted for. The four pathologists used a similar diagnostic model and for all of them their estimations of epithelioid cell granuloma oedema, dermal oedema, plasma cells and granuloma fraction were significant variables in the diagnosis of T1R. Each pathologist then added in variables that were specific to themselves. Conclusions: This study has identified T1R as being under-diagnosed in comparison with clinical assessments. Key variables for diagnosing T1R were established. This comparative masked study highlights the need for such studies in other inflammatory conditions.

Lymphotoxin-α and TNF Have Essential but Independent Roles in the Evolution of the Granulomatous Response in Experimental Leprosy

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.

Peripheral Nerve Toxic Effects of Nitrofurantoin

OBJECTIVE To investigate the role of skin biopsy in nitrofurantoin peripheral neuropathy. DESIGN We describe the clinical features and skin biopsies of 2 cases of non-length-dependent small-fiber neuropathy/ganglionopathy attributable to nitrofurantoin. SETTING Clinical evaluation and skin biopsies were performed at a tertiary teaching hospital in Baltimore, Maryland. PATIENTS A 59-year-old woman with disabling generalized dysesthesia and a 53-year-old woman with progressive burning pain in the perineum and extremities. MAIN OUTCOME MEASURES Slow or incomplete recovery and possibly irreversible damage. RESULTS The neuropathy was neither dose dependent nor associated with impaired renal function. Results from nerve conduction studies were normal. Skin biopsies revealed distinctive morphologic changes with clustered terminal nerve swellings without evidence of nerve fiber degeneration. CONCLUSIONS These distinct morphologic changes associated with nitrofurantoin have not been previously reported to our knowledge. Skin biopsy appears to be helpful in confirming the diagnosis in these patients.

Acetyl-l-carnitine and nucleoside reverse transcriptase inhibitor-associated neuropathy in HIV infection.

Antiretroviral toxic neuropathy (ATN) is associated with dideoxynucleoside reverse transcriptase inhibitor use in patients infected with HIV, possibly as a result of 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 changes in intra‐epidermal nerve fibre (IENF) density and mitochondrial DNA (mtDNA) copies/cell among subjects treated with 3000 mg ALC daily.

Collateral sprouting of human epidermal nerve fibers following intracutaneous axotomy

Abstract  Despite the clinical need, there are no therapeutic compounds available to promote peripheral nerve regeneration. In part, this may be due to a lack of sensitive measures of nerve growth. Here, we describe a novel approach of measuring collateral sprouting of epidermal nerve fibers (ENF) in human subjects and describe the effect of the neuroimmunophilin ligand timcodar dimesylate on collateral nerve sprouting. The objective of this study was to describe a model of intracutaneous axotomy and evaluate the ability of timcodar dimesylate to accelerate human cutaneous nerve regeneration through collateral sprouting. Subjects were randomized to receive placebo, 12.5 or 50 mg/day timcodar dimesylate in a prospective, two‐center, double‐blind, placebo‐controlled trial. A 3‐mm distal thigh punch skin biopsy was performed at baseline, and a 4‐mm overlapping concentric biopsy was taken after 56 days of treatment. Biopsies were processed to visualize ENF, and the collateral sprouting distance (CSD) was measured. Sixty‐two subjects completed the trial, and the CSD was measurable in 52. The CSD (mean ± SEM) was 474.5 µm ± 38.3, 473.4 µm ± 28.4, and 450.8 µm ± 26.5 for the placebo, low and high dose groups, respectively (p = 0.84). The baseline ENF density was associated with the CSD (p = 0.02). Collateral sprouting was efficiently measured using an intracutaneous axotomy model and suggests a collateral sprouting rate of 8.5 µm/day in healthy subjects. The model was consistent across treatment groups and had a low coefficient of variation. Timcodar dimesylate treatment was safe over an 8‐week period but did not improve collateral sprouting among healthy subjects.

Altered cutaneous nerve regeneration in a simian immunodeficiency virus / macaque intracutaneous axotomy model

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. J. Comp. Neurol. 514:272–283, 2009.

Factors influencing sweat gland innervation in diabetes

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 A1c, body mass index (BMI), men compared with women, and tobacco use, but not diabetes type or age. Sex, A1c, 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, A1c, 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.

Mechanisms of nerve injury in leprosy

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.