Intraepidermal Nerve Fiber Density Research Articles

Neuronal Inflammation is Associated with Changes in Epidermal Innervation in High Fat Fed Mice.

Peripheral neuropathy (PN), a debilitating complication of diabetes, is associated with obesity and the metabolic syndrome in nondiabetic individuals. Evidence indicates that a high fat diet can induce signs of diabetic peripheral PN in mice but the pathogenesis of high fat diet-induced PN remains unknown. PURPOSE: Determine if neuronal inflammation is associated with the development of mechanical hypersensitivity and nerve fiber changes in high fat fed mice. METHODS: Male C57Bl/6 mice were randomized to a standard (Std, 15% kcal from fat) or high fat diet (HF, 54% kcal from fat) for 2, 4, or 8 weeks (n = 11-12 per group). Lumbar dorsal root ganglia were harvested and inflammatory mediators (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17, MCP-1, IFN-γ, TNF-α, MIP-1α, GMCSF, RANTES) were quantified. Hindpaw mechanical sensitivity was assessed using the von Frey test. Intraepidermal nerve fiber density (IENFD) and TrkA nerve fiber density were quantified via immunohistochemistry. RESULTS: After 8 weeks, HF had greater body mass (33.3 ± 1.0 vs 26.7 ± 0.5 g, p < 0.001), fasting blood glucose (160.3 ± 9.4 vs 138.5 ± 3.4 mg/dl, p < 0.05) and insulin (3.58 ± 0.46 vs 0.82 ± 0.14 ng/ml, p < 0.001) compared to Std. IL-1α, RANTES and IL-5 were higher in HF compared to Std after 2 and 4 weeks, respectively (IL-1α: 4.8 ± 1.3 vs 2.9 ± 0.6 pg/mg, p < 0.05; RANTES: 19.6 ± 2.2 vs 13.3 ± 1.2 pg/mg p < 0.05; IL-5: 5.8 ± 0.7 vs 3.1 ± 0.5 pg/mg, p < 0.05). IENFD and TrkA fiber density were also higher in HF vs Std after 4 weeks (IENFD: 39.4 ± 1.2 vs 32.2 ± 1.3 fibers/mm, p < 0.001; TrkA: 30.4 ± 1.8 vs 22.4 ± 1.3 fibers/mm). There were no significant differences in hindpaw sensitivity for Std vs HF. CONCLUSION: Increased inflammatory mediators preceded and accompanied an increase in cutaneous pain sensing nerve fibers in high fat fed mice but was not accompanied by significant mechanical allodynia. Diets high in fat may increase neuronal inflammation and lead to increased nociceptive nerve fiber density.

Neuritin Promotes Bone Marrow-Derived Mesenchymal Stem Cell Migration to Treat Diabetic Peripheral Neuropathy.

The purpose of this study is to explore the effect and mechanism of neuritin overexpression in the bone marrow on peripheral neuropathy in type 2 diabetic (db/db) mice. We analyzed the impact of bone marrow neuritin overexpression on diabetic peripheral neuropathy and migration of bone marrow mesenchymal stem cells in db/db mice. Antagonists were used to inhibit the stromal cell-derived factor (SDF)-1α/C-X-C chemokine receptor type 4 (CXCR4)-phosphoinositide 3-kinase (PI3K)/Akt signaling pathway in primary cultured bone marrow mesenchymal stem cells. Immunofluorescence, transmission electron microscopy, Oil Red O staining, and transwell migration assays were used. Bone marrow-specific overexpression of neuritin in db/db mice was successfully established. Overexpression of neuritin in the bone marrow ameliorated hyperglycemia, prevented diabetic peripheral neuropathy, protected the ultrastructure of the sciatic nerve and intra-epidermal nerve fiber density, and promoted Schwann cell proliferation and remyelination in the sciatic nerve. Moreover, it ameliorated fat accumulation, adipocyte number, and vascular and nerve densities; decreased glutamate content in serum and bone marrow; restored gradient SDF-1α contents between bone marrow, blood, and sciatic nerve; and promoted impaired diabetic bone marrow mesenchymal stem cell migration. Neuritin improves bone marrow mesenchymal stem cell migration via the SDF-1α/CXCR4-PI3K/Akt signaling pathway in vitro. Overexpression of neuritin in the bone marrow can locally ameliorate neuropathy in the bone marrow. This improves the migration capability of bone marrow mesenchymal stem cells and repairs diabetic peripheral neuropathy, at least partly by activating the PI3K/Akt pathway through the SDF-1α/CXCR4 axis.

Mirror peripheral neuropathy and unilateral chronic neuropathic pain: insights from asymmetric neurological patterns in leprosy.

Leprosy-related multiple mononeuropathy offers a pattern of impairment where neuropathy with and without neuropathic pain (NeP) are present in the same individual, thus allowing to investigate peripheral sensory and innervation in both conditions. This cross-sectional study collected data on clinical and neurological examination, pain assessment questionnaires, quantitative sensory test, and intraepidermal nerve fiber density of patients with leprosy and divided the cohort into 2 groups: with NeP (P+) and without NeP (P-). Furthermore, we assessed mirror body areas in the same NeP individuals with bilateral neuropathy also presenting unilateral NeP. Pain-free patients having unilateral neuropathy were controls. A total of 37 P+ and 22 P- patients were evaluated. Limb areas with NeP had signs of C-fiber dysfunction and hyperesthesia on quantitative sensory testing compared with limb areas having neuropathy without NeP. Skin denervation was found in all patients with leprosy. Comparisons of limbs with and without neuropathy and with and without NeP revealed that higher heat pain thresholds (HPTs) were associated with neuropathic pain areas, whereas less altered HPT was correlated with higher fiber density. Furthermore, a relationship was found between time of leprosy treatment termination and more intense neuropathy, expressed by HPT increasing 0.03°C each month. As expected, interindividual comparisons failed to show differences in intraepidermal nerve fiber density and subepidermal plexus areas between P+ and P- patients ( P = 0.2980, P = 0.9044; respectively). Higher HPT and lower mechanical detection threshold were related to NeP. This study pointed out the relevance of intraindividual comparisons including mirror areas when assessing local changes in peripheral NeP.

Increased Epidermal Nerve Growth Factor without Small-Fiber Neuropathy in Dermatomyositis.

Small-fiber neuropathy (SFN) is suggested to be involved in the pathogenesis of some types of autoimmune connective tissue diseases. SFN with a reduction in epidermal nerve fibers might affect sensory fibers and cause neuropathic symptoms, such as pruritus and pain, which are common in both dermatomyositis (DM) and cutaneous lupus erythematosus (CLE). Nerve growth factor (NGF) has been recognized as important in nociception by regulating epidermal nerve fiber density and sensitizing the peripheral nervous system. The present study aimed to investigate whether SFN was associated with the cutaneous manifestations of DM and CLE. We also investigated the relationship between SFN and axon guidance molecules, such as NGF, amphiregulin (AREG), and semaphorin (Sema3A) in DM and CLE. To explore the molecular signaling, interleukin (IL)-18 and IL-31, which have been implicated in the cutaneous manifestation and neuropathic symptoms in DM, were examined in keratinocytes. Our results revealed that intraepidermal nerve fiber density (IENFD) was unchanged in patients with DM, but significantly reduced in IENFD in patients with CLE compared with healthy control. Increased epidermal expression of NGF and decreased expression of Sema3A were demonstrated in patients with DM. Furthermore, IL-18 and IL-31 both induced the production of NGF from keratinocytes. Taken together, IL-18 and IL-31 mediated epidermal NGF expression might contribute to the cutaneous neuropathic symptoms in DM, while SFN might be important for CLE.

Sensory neuron dysfunction in orthotopic mouse models of colon cancer

Reports of neurological sequelae related to colon cancer are largely restricted to rare instances of paraneoplastic syndromes, due to autoimmune reactions. Systemic inflammation associated with tumor development influences sensory neuron function in other disease models, though the extent to which this occurs in colorectal cancer is unknown. We induced orthotopic colorectal cancer via orthotopic injection of two colorectal cancer cell lines (MC38 and CT26) in two different mouse strains (C57BL/6 and Balb/c, respectively). Behavioral tests of pain sensitivity and activity did not detect significant alterations in sensory sensitivity or diminished well-being throughout tumor development. However, immunohistochemistry revealed widespread reductions in intraepidermal nerve fiber density in the skin of tumor-bearing mice. Though loss of nerve fiber density was not associated with increased expression of cell injury markers in dorsal root ganglia, lumbar dorsal root ganglia neurons of tumor-bearing animals showed deficits in mitochondrial function. These neurons also had reduced cytosolic calcium levels in live-cell imaging and reduced spontaneous activity in multi-electrode array analysis. Bulk RNA sequencing of DRGs from tumor-bearing mice detected activation of gene expression pathways associated with elevated cytokine and chemokine signaling, including CXCL10. This is consistent with the detection of CXCL10 (and numerous other cytokines, chemokines and growth factors) in MC38 and CT26 cell-conditioned media, and the serum of tumor-bearing mice. Our study demonstrates in a pre-clinical setting that colon cancer is associated with latent sensory neuron dysfunction and implicates cytokine/chemokine signaling in this process. These findings may have implications for determining risk factors and treatment responsiveness related to neuropathy in colorectal cancer.

Cardiomyopathy correlates to nerve damage in p.A117Slate‐onset transthyretin amyloid polyneuropathy

ObjectiveLate‐onset hereditary transthyretin amyloidosis with polyneuropathy (ATTRv‐PN) is often associated with heart involvement. Recent advances in cardiac imaging allow the detection of cardiac amyloidosis. This study aimed to explore cardiomyopathy by cardiac imaging and its clinical correlates with polyneuropathy in late‐onset ATTRv‐PN.MethodsPolyneuropathy was assessed by intraepidermal nerve fiber (IENF) density, nerve conduction study (NCS), autonomic function tests, quantitative sensory testing, and clinical questionnaires. Cardiomyopathy was evaluated by echocardiography, 99mTc‐pyrophosphate (PYP) single‐photon emission computed tomography (SPECT) imaging, cardiac magnetic resonance imaging (CMR), and serum Pro‐B‐type natriuretic peptide. Healthy controls and patients with Brugada syndrome were enrolled for comparison of CMR.ResultsFifty late‐onset ATTRv‐PN patients (38 men, 46 with p. A117S mutation), aged 63.7 ± 5.5 years, of polyneuropathy disability stage 1–4 were enrolled. All patients presented polyneuropathy in NCS, and 74.5% of patients had reduced IENF density in distal legs. All patients showed significant radiotracer uptake in the heart on 99mTc‐PYP SPECT imaging, and 87.8% of patients had abnormally increased left ventricular (LV) septum thickness on echocardiography. CMR showed longer myocardial native T1, larger extracellular volume, greater LV mass index, and higher LV mass to end‐diastolic volume ratio in ATTRv‐PN patients than healthy controls and patients with Brugada syndrome. These CMR parameters were associated with skin denervation, absent sympathetic skin responses, elevated thermal thresholds, worsened NCS profiles, and functional deficits of polyneuropathy.InterpretationLate‐onset ATTRv‐PN coexisted with cardiomyopathy regardless of the clinical severity of polyneuropathy. The cardiac amyloid burden revealed by CMR was correlated with pathophysiology and clinical disability of nerve degeneration.

Effects of Neural Mobilization on Sensory Dysfunction and Peripheral Nerve Degeneration in Rats With Painful Diabetic Neuropathy.

ObjectiveThis study aims to evaluate the effectiveness of neural mobilization (NM) in the management of sensory dysfunction and nerve degeneration related to experimental painful diabetic neuropathy (PDN).MethodsThis is a pre-clinical animal study performed in the streptozocin (STZ)-induced diabetic rat model. 3 groups were included; a treatment group of rats with PDN receiving NM under anesthesia (PDN-NM, n=10), a sham treatment group of rats with PDN that only received anesthesia (PDN-Sham, n=9) and a vehicle control group with nondiabetic animals (Vehicle, n=10). Rats in the PDN-NM and PDN-Sham groups received 1 treatment sessions on day-10, 12 and 14 after STZ injection, with a 48-hour rest period between sessions. Behavioral tests were performed using von Frey and Plantar tests. Evaluation for peripheral nerve degeneration was performed through measuring protein gene product 9.5-positive (PGP9.5+) intra-epidermal nerve fiber density (IENFD) in hind-paw skin biopsies. All measurements were performed by a blinded investigator.ResultsThe behavioral tests showed that a single NM session could reduce hyperalgesia which was maintained for 48 hours. The second treatment session further improved this treatment effect, and the third session maintained it. These results suggest that it requires multiple treatment sessions to produce and maintain hypoalgesic effects. Skin biopsy analysis showed that the PGP9.5+ IENFD was higher on the experimental side of the PDN-NM group compared to the PDN-Sham group, suggesting NM may mitigate the degeneration of peripheral nerves.ConclusionsThis study demonstrated that NM may be an effective method to manage experimentally induced PDN, potentially through mitigation of nerve degeneration.Further studies are needed to develop standardized protocols for clinical use.ImpactThese findings provide neurophysiological evidence for the use of NM in PDN and can form the basis for the development of physical therapy-based programs in clinics.

856 Peripheral neuropathic changes in prurigo nodularis

Prurigo nodularis (PN) is a chronic inflammatory skin disease characterized by intensely pruritic and hyperkeratotic nodules of unknown etiology. PN is strongly associated with peripheral neuropathies, suggesting a role for neural dysregulation in its pathogenesis. Here we study components of the cutaneous peripheral neural system, including intraepidermal nerve fiber density (IENFD), mechanoreceptors, and blood vessels in lesional and nonlesional skin of 10 patients with PN and 10 matched healthy controls.

Identification and quantification of nociceptive Schwann cells in mice with and without Streptozotocin-induced diabetes

Specialized cutaneous Schwann cells (SCs), termed nociceptive SCs, were recently discovered. Their function is not fully understood, but they are believed not only to support peripheral axons in mouse skin by forming a mesh-like neural-glio networking structure in subepidermal area, but also contributing to transduction of mechanical sensation and neuropathic pain. Diabetic neuropathy (DPN) is one of the most common complication of diabetes, however, the mechanisms behind painful and painless DPN remain unclear. Using a mouse model of DPN, we want to investigate if there are quantitative differences in nociceptive SC density between the condition of hyperglycemia-induced sensory abnormalities and control condition and at which stage in the disease the damage occurs. Here, we developed a set of counting rules for nociceptive SCs based on immunofluorescent staining, and applied the method to quantify the density of nociceptive SCs in control mice (n = 10), mice with nociceptive hypersensitivity at early diabetic stage (n = 5), and mice with sensory hyposensitivity at late diabetic stage (n = 5) in the Streptozotocin (STZ) model of type 1 diabetes. Nociceptive SCs were identified as S100+/Sox10+/DAPI+ cells abutting to peripheral nerves, with the somas located within 25 µm depth in the subepidermal area and outside glands and large fiber bundles. Hypersensitive diabetic mice had decreased nociceptive SC density, despite having normal epidermal nerve fiber density, compared with age-matched control mice (P = 0.023). In contrast, there was a reduction in intraepidermal nerve fiber density but no difference in nociceptive SC density between hyposensitive diabetic mice and the age-matched control mice. This study provides a detailed description of how to identify and quantify nociceptive SC and demonstrates that nociceptive SC density declines before nerve fiber deterioration, which supports previous observations that nociceptive SCs are critical for maintenance of cutaneous sensory nerves.

460-P: Cellular Communication of Schwann Cell Subtypes in Metabolically-Acquired Neuropathy

The prevalence of the metabolic syndrome (MetS) is increasing worldwide due in part to the increased consumption of a Western diet. MetS is associated with peripheral neuropathy (PN) . PN develops in a cell non-autonomous manner with involvement of axons, Schwann cells (SC) and nerve tissue- infiltrating macrophages (TIM) . SC are divided into subtypes with different roles in nerve injury. Our aim in the current study was to determine how gene expression of different types of SC and macrophages are affected in MetS and how these cells communicate with one another. C57BL6/j male mice (6-weeks old) were fed either a standard diet (SD, 10% fat) or high fat diet (HFD, 60%) for 17 weeks. Metabolic and PN phenotyping were performed for all mice and SC were extracted from sciatic nerves for single cell RNA sequencing (scRNA-seq) . Cellular communication was determined using the CellChat tool. Mice on HFD had higher fasting blood glucose (FBG) , glycated hemoglobin (HbA1c) and decreased nerve conduction velocities and intraepidermal nerve fiber densities consistent with PN compared to mice on SD. Analysis of SC scRNA-seq data revealed unique RNA expression signatures for TIM and 4 subpopulations of SC denoted as myelinating SC (mySC) , non-myelinating SC (nmSC) , SC precursors (SCP) and regenerating SC. Inflammatory genes such as Ptprc and Il1b were significantly more expressed in both TIM and mySC in HFD mice compared to SD mice. CellChat analysis of cellular communication revealed more crosstalk between TIM and various types of SC in HFD animals and identified multiple metabolic and inflammatory pathways that were activated in HFD compared to SD mice. In conclusion, our study shows that there are many subtypes of SC that behave differently in MetS. Their communication with each other and with TIM strongly implicates these two cell types in the pathogenesis of metabolically acquired PN. Disclosure S.Eid: None. M.H.Noureldein: None. F.Mendelson: None. E.L.Feldman: None. Funding Funding was provided by the National Institutes of Health (NIH) (1R24082841 to ELF) , Novo Nordisk Foundation (NNF14OC0011633 to ELF) , the Nathan and Rose Milstein Research Fund (to SAE) , NeuroNetwork for Emerging Therapies at the University of Michigan (to SAE, MHN, and ELF) , and the A. Alfred Taubman Medical Research Institute (to SAE, MHN, and ELF) .

Duloxetine alleviates oxaliplatin-induced peripheral neuropathy by regulating p53-mediated apoptosis.

Oxaliplatin (OXA) is a key platinum-based chemotherapeutic agent for treatment of metastatic colorectal cancer, but the side effects of acute and chronic neuropathies limit its clinical application. Duloxetine has been found to have the potential to prevent OXA-induced peripheral neuropathy in several studies, but the underlying mechanisms remain unclear. The purpose of this study was to evaluate the effects of duloxetine on OXA-induced peripheral neuropathy and to find the potential mechanisms. The neuropathic pain mice model was used to explore the role of duloxetine on OXA-induced peripheral neuropathy by measuring the change of thermal withdrawal latency (TWL), paw withdrawal threshold (PWT), and intraepidermal nerve fiber density (IENFD). Moreover, to explore molecular mechanisms, effects of duloxetine on OXA-induced changes in mRNA and protein expression of components of the p53-related pathways in cultured rat dorsal root ganglion (DRG) neurons were measured. In vivo, we found duloxetine treatment could significantly prevent the changes in the TWL, PWT to mechanical stimulation, and the IENFD of mice caused by OXA. In vitro, we found duloxetine notably inhibits the relative mRNA and protein expression levels of p53, Bax/Bcl2, caspase-3, and caspase-9 in DRG neurons, which may indicate duloxetine protected the DRG neuron by inhibiting p53-related pathways. These results suggest that duloxetine could alleviate the OXA-induced peripheral neuropathy. Duloxetine deserves further consideration as a potential protective agent against peripheral neuropathy.

Foretinib mitigates cutaneous nerve fiber loss in experimental diabetic neuropathy

Diabetes is by far, the most common cause of neuropathy, inducing neurodegeneration of terminal sensory nerve fibers associated with loss of sensation, paresthesia, and persistent pain. Foretinib prevents die-back degeneration in cultured sensory and sympathetic neurons by rescuing mitochondrial activity and has been proven safe in prospective clinical trials. Here we aimed at investigating a potential neuroprotective effect of Foretinib in experimental diabetic neuropathy. A mouse model of streptozotocin induced diabetes was used that expresses yellow fluorescent protein (YFP) in peripheral nerve fibers under the thy-1 promoter. Streptozotocin-injected mice developed a stable diabetic state (blood glucose > 270 mg/dl), with a significant reduction of intraepidermal nerve fiber density by 25% at 5 weeks compared to the non-diabetic controls. When diabetic mice were treated with Foretinib, a significantly greater volume of the cutaneous nerve fibers (67.3%) in the plantar skin was preserved compared to vehicle treated (37.8%) and non-treated (44.9%) diabetic mice while proximal nerve fiber morphology was not affected. Our results indicate a neuroprotective effect of Foretinib on cutaneous nerve fibers in experimental diabetic neuropathy. As Foretinib treated mice showed greater weight loss compared to vehicle treated controls, future studies may define more sustainable treatment regimen and thereby may allow patients to take advantage of this neuroprotective drug in chronic neurodegenerative diseases like diabetic neuropathy.

Small Fiber Pathology in CADASIL: Clinical Correlation With Cognitive Impairment.

This study investigated the cutaneous small fiber pathology of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and its clinical significance, that is, the NOTCH3 deposition in cutaneous vasculatures and CNS neurodegeneration focusing on cognitive impairment. Thirty-seven patients with CADASIL and 59 age-matched healthy controls were enrolled to evaluate cutaneous small fiber pathology by quantitative measures of intraepidermal nerve fiber density (IENFD), sweat gland innervation, and vascular innervation. Cognitive performance of patients with CADASIL was evaluated by a comprehensive neuropsychological assessment, and its association with small fiber pathology was tested using multivariable linear regression analysis adjusted for age and diabetes mellitus. We further assessed the relationships of IENFD with cutaneous vascular NOTCH3 ectodomain (NOTCH3ECD) deposition and biomarkers of neurodegeneration including structural brain MRI measures, serum neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), tau, and ubiquitin carboxy-terminal hydrolase L1. Patients with CADASIL showed reduced IENFD (5.22 ± 2.42 vs 7.88 ± 2.89 fibers/mm, p = 0.0001) and reduced sweat gland (p < 0.0001) and vascular (p < 0.0001) innervations compared with age-matched controls. Reduced IENFD was associated with impaired global cognition measured by Mini-Mental State Examination (B = 1.062, 95% CI = 0.370-1.753, p = 0.004), and this association remained after adjustment for age and diabetes mellitus (p = 0.043). In addition, IENFD in patients with CADASIL was associated with mean cortical thickness (Pearson r = 0.565, p = 0.0023) but not white matter hyperintensity volume, total lacune count, or total microbleed count. Reduced IENFD was associated with cutaneous vascular NOTCH3ECD deposition amount among patients harboring pathogenic variants in exon 11 (mainly p.R544C) (B = -0.092, 95% CI = -0.175 to -0.009, p = 0.031). Compared with those with normal cognition, patients with CADASIL with cognitive impairment had an elevated plasma NfL level regardless of concurrent small fiber denervation, whereas only patients with both cognitive impairment and small fiber denervation showed an elevated plasma GFAP level. Cutaneous small fiber pathology correlates with cognitive impairment and CNS neurodegeneration in patients with CADASIL, indicating a peripheral neurodegenerative process related to NOTCH3ECD aggregation.

Characterization of somatosensory neuron involvement in the SOD1G93A mouse model

SOD1G93A mice show loss of cutaneous small fibers, as in ALS patients. Our objective is to characterize the involvement of different somatosensory neuron populations and its temporal progression in the SOD1G93A mice. We aim to further define peripheral sensory involvement, analyzing at the same time points the neuronal bodies located in the dorsal root ganglia (DRG) and the distal part of their axons in the skin, in order to shed light in the mechanisms of sensory involvement in ALS. We performed immunohistochemical analysis of peptidergic (CGRP), non-peptidergic (IB4) fibers in epidermis, as well as sympathetic sudomotor fibers (VIP) in the footpads of SOD1G93A mice and wild type littermates at 4, 8, 12 and 16 weeks of age. We also immunolabeled and quantified neuronal bodies of IB4, CGRP and parvalbumin (PV) positive sensory neurons in lumbar DRG. We detected a reduction of intraepidermal nerve fiber density in the SOD1G93A mice of both peptidergic and non-peptidergic axons, compared with the WT, being the non-peptidergic the fewest. Sweat gland innervation was similarly affected in the SOD1G93A mouse at 12 weeks. Nonetheless, the number of DRG neurons from different sensory populations remained unchanged during all stages. Cutaneous sensory axons are affected in the SOD1G93A mouse, with non-peptidergic being slightly more vulnerable than peptidergic axons. Loss or lack of growth of the distal portion of sensory axons with preservation of the corresponding neuronal bodies suggest a distal axonopathy.

Pathological changes of the sural nerve in patients with familial episodic pain syndrome.

Familial episodic pain syndrome type 3 (FEPS3) is an inherited disorder characterized by the early-childhood onset of severe episodic pain that primarily affects the distal extremities. As skin biopsy has revealed a reduction in intraepidermal nerve fiber density and degeneration of the unmyelinated axons, it remains unclear whether FEPS3 patients have pathological changes in the peripheral nerve. The clinical features of patients with FEPS3 were summarized in a large autosomal dominant family. Sural nerve biopsies were conducted in two patients. Whole exome sequencing (WES) was performed in the index patient. Sanger sequencing was used to analyze family co-segregation. Fourteen members exhibited typical and uniform clinical phenotypes characterized by length-dependent and age-dependent severe episodic pain affecting the distal extremities, which can be relieved with anti-inflammatory medicine. The WES revealed a heterozygous mutation c.665G > A (p.R222H) in the SCN11A gene, which was co-segregated with the clinical phenotype in this family. A sural biopsy in patient V:1, who was experiencing episodic pain at 16years old, showed normal structure, while the sural nerve in patient IV:1, whose pain attack had completely diminished at 42years old, displayed a decrease of the density of unmyelinated axons with the axonal degeneration. The clinical phenotype of FEPS3 showed distinctive characteristics that likely arise from dysfunctional nociceptive neurons that lack detectable pathological alterations in the nerve fibers. Nevertheless, long-term dysfunction of the Nav1.9 channel may cause degeneration of the unmyelinated fibers in FEPS3 patient with pain remission.

Neuronal Dysfunction Associated with Colorectal Cancer Augments Sensitivity to Chemotherapy-induced Neuropathic Pain

Neuropathic pain is a common and debilitating side effect of many cancer treatments. The platinum-based chemotherapeutic agent oxaliplatin is widely used in treatment regimens for colorectal cancer, but its side effects (including numbness, tingling or sharp, stabbing, 'electrical' pain) are so severe as to become dose limiting in a subset of patients. We set out to determine if the systemic immune system alterations in colon cancer could influence subsequent sensitivity to oxaliplatin therapy. Using two orthotopic mouse models of colon cancer, we perform immunohistochemistry, behavioral analysis, functional calcium imaging, plasma cytokine/chemokine array analysis and mitochondrial functional capacity to determine the extent to which cancer-associated inflammation drives neuronal dysfunction in response to chemotherapy. We found that two distinct colorectal cancer cell lines in two immunocompetent mouse strains induced impaired mitochondrial function in dorsal root ganglion neurons in vitro. This was accompanied by a significant reduction in intra-epidermal nerve fiber density in the plantar hindpaw skin. Multi-electrode array and ratiometric calcium imaging show abnormalities in spontaneous firing and cytosolic calcium concentration in dorsal root ganglion neurons. Finally, we show that tumor-bearing mice show a greater degree of tactile and cold sensitivity in response to a sub-maximal dose of oxaliplatin, despite there being no overt symptoms of neuropathy in tumor-bearing mice that did not receive oxaliplatin. We conclude that colorectal cancer induces 'sub-clinical' neuronal injury that predisposes to neuropathic pain hypersensitivity upon subsequent treatment with oxaliplatin. Future experiments will focus on identifying the soluble factor(s) responsible, and the generalizability of this phenomenon. Grant support from Rita Allen Foundation.

Role of skin punch biopsy in diagnosis of small fiber neuropathy-A review for the neuropathologist.

Over the last three decades, skin punch biopsy has become the gold standard for diagnosis of small fiber neuropathies, including autonomic neuropathies commonly seen in diabetics, patients with HIV, and children with hereditary sensory autonomic neuropathies and toxin-induced neuropathy. Clinical, biochemical, electrophysiological tests are inconclusive, making it difficult to diagnose and initiate treatment. A skin punch biopsy is easy to perform in the outpatient clinic, is highly sensitive, and provides an objective diagnosis. Importantly, it helps avoid performing invasive nerve biopsy in patients with small fiber neuropathy, thereby preventing complications such as non-healing of the biopsy site, which is common in these patients. Secondly, the greatest advantage of skin punch biopsies is that they can be repeated any number of times, unlike a nerve biopsy, and are useful to evaluate disease progression and therapeutic response. More recently, its use has been expanded to the diagnosis of large fiber neuropathies, inherited demyelinating neuropathies, etc., obviating the need for a nerve biopsy. The European Federation of Neurological Societies has published guidelines for evaluation to ensure uniformity with regard to the site of biopsy, processing, and quantification. The evaluation of the skin biopsy involves morphometric assessment of the intraepidermal nerve fiber density using PGP 9.5 immunostained sections by bright-field microscopy. This review focuses on the practical aspects of skin punch biopsy and its utility for the practicing pathologist.

Small fiber involvement is independent from clinical pain in late-onset Pompe disease

BackgroundPain occurs in the majority of patients with late onset Pompe disease (LOPD) and is associated with a reduced quality of life. The aim of this study was to analyse the pain characteristics and its relation to a small nerve fiber involvement in LOPD patients.MethodsIn 35 patients with LOPD under enzyme replacement therapy without clinical signs of polyneuropathy (19 females; 51 ± 15 years), pain characteristics as well as depressive and anxiety symptoms were assessed using the PainDetect questionnaire (PDQ) and the hospital anxiety and depression scale (HADS), respectively. Distal skin biopsies were analysed for intraepidermal nerve fiber density (IENFD) and compared to age- and gender-matched reference data. Skin biopsies from 20 healthy subjects served as controls to assure validity of the morphometric analysis.ResultsPain was reported in 69% of the patients with an average intensity of 4.1 ± 1.1 on the numeric rating scale (NRS; anchors: 0–10). According to PDQ, neuropathic pain was likely in one patient, possible in 29%, and unlikely in 67%. Relevant depression and anxiety symptoms occurred in 31% and 23%, respectively, and correlated with pain intensity. Distal IENFD (3.98 ± 1.95 fibers/mm) was reduced in 57% of the patients. The degree of IENFD reduction did not correlate with the durations of symptoms to ERT or duration of ERT to biopsy.ConclusionsPain is a frequent symptom in treated LOPD on ERT, though a screening questionnaire seldom indicated neuropathic pain. The high frequency of small nerve fiber pathology in a treated LOPD cohort was found regardless of the presence of pain or comorbid risk factors for SFN and needs further exploration in terms of clinical context, exact mechanisms and when developing novel therapeutic options for LOPD.

Corneal Confocal Microscopy Identifies People with Type 1 Diabetes with More Rapid Corneal Nerve Fibre Loss and Progression of Neuropathy.

There is a need to accurately identify patients with diabetes at higher risk of developing and progressing diabetic peripheral neuropathy (DPN). Fifty subjects with Type 1 Diabetes Mellitus (T1DM) and sixteen age matched healthy controls underwent detailed neuropathy assessments including symptoms, signs, quantitative sensory testing (QST), nerve conduction studies (NCS), intra epidermal nerve fiber density (IENFD) and corneal confocal microscopy (CCM) at baseline and after 2 years of follow-up. Overall, people with type 1 diabetes mellitus showed no significant change in HbA1c, blood pressure, lipids or neuropathic symptoms, signs, QST, neurophysiology, IENFD and CCM over 2 years. However, a sub-group (n = 11, 22%) referred to as progressors, demonstrated rapid corneal nerve fiber loss (RCNFL) with a reduction in corneal nerve fiber density (CNFD) (p = 0.0006), branch density (CNBD) (p = 0.0002), fiber length (CNFL) (p = 0.0002) and sural (p = 0.04) and peroneal (p = 0.05) nerve conduction velocities, which was not related to a change in HbA1c or cardiovascular risk factors. The majority of people with T1DM and good risk factor control do not show worsening of neuropathy over 2 years. However, CCM identifies a sub-group of people with T1DM who show a more rapid decline in corneal nerve fibers and nerve conduction velocity.

Nonpermissive Skin Environment Impairs Nerve Regeneration in Diabetes via Sec31a.

Although the microenvironment for peripheral nerve regeneration is permissive, such a mechanism is defective in diabetes, and the molecular mediators remain elusive. [Correction added on May 11, 2022, after first online publication: In the preceding sentence, "is ok" was changed to "is defective".] This study aimed to (1) investigate the relationship between skin innervation and collagen pathology in diabetic neuropathy and to (2) clarify the molecular alterations that occur in response to hyperglycemia and their effects on axon regeneration. We addressed this issue using two complementary systems: (1) human skin from patients with diabetic neuropathy and to (2) a coculture model of human dermal fibroblasts (HDFs) with rat dorsal root ganglia neurons in the context of intrinsic neuronal factor and extrinsic microenvironmental collagen and its biosynthetic pathways. In diabetic neuropathy, the skin innervation of intraepidermal nerve fiber density (IENFd), a measure of sensory nerve degeneration, was reduced with similar expression of a growth associated protein 43, a marker of nerve regeneration. In contrast, the content and packing of collagen in the diabetic skin became more rigid than the control skin. Sec31a, a protein that regulates the collagen biosynthetic pathway, was upregulated and inversely correlated with IENFd. In the cell model, activated HDFs exposed to high-glucose medium enhanced the expression of Sec31a and collagen I through the activation of transforming growth factor β, a profibrotic molecule. Sec31a upregulation impaired neurite outgrowth. This effect was reversed by silencing Sec31a expression and neurite outgrowth was resumed. The current study provides evidence that Sec31a plays a key role in inhibiting nerve regeneration in diabetic neuropathy. ANN NEUROL 2022;91:821-833.