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Diabetic polyneuropathy (DPN) is a common complication of diabetes and is often associated with neuropathic pain. The mechanisms underlying development and maintenance of painful DPN are largely unknown, and quantification of intraepidermal nerve fiber density from skin biopsy, one of the neuropathological gold standard when diagnosing DPN, does not differentiate between patients with and without pain. Identification of possible pain pathophysiological biomarkers in patients with painful DPN may increase our knowledge of mechanisms behind neuropathic pain. Animal models of painful DPN have been shown to have an increased density of peptidergic nerve fibers (substance P and calcitonin gene-related peptide). In this study, we performed a detailed skin biopsy analysis in a well-characterized group of DPN patients with primarily small fiber involvement, with and without pain, and in healthy controls and test for correlation between skin biopsy findings and pain intensity and quantitative sensory testing. We found that although there was no difference in intraepidermal nerve fiber density using protein gene product 9.5 between patients with and without pain, patients with pain had increased density of dermal peptidergic fibers containing substance P and calcitonin gene-related peptide compared with patients with painless DPN and healthy controls. Peptidergic nerve fiber density correlated with pain ratings in patients with pain (R = 0.33; P = 0.019), but not with quantitative sensory testing results. In this article, we show, for the first time in humans, an increased density of dermal peptidergic fibers in painful DPN. These findings provide new insight in the pathophysiological mechanisms of pain in diabetes and open the research towards new therapeutic targets.

AIM Progressive Supranuclear Palsy (PSP) is a progressive neurodegenerative tauopathy characterised by motor, behavioural and cognitive dysfunction. While in the last decade, sensory and autonomic disturbances as well as peripheral nerve involvement are well-recognised in Parkinson's Disease (PD), little is known in this regard for PSP. Herein, we aim to assess peripheral sensory and autonomic nerve involvement in PSP and to characterise possible differences in morpho-functional pattern compared to PD patients. METHODS We studied 27 PSP and 33 PD patients without electrophysiological signs of neuropathy, and 33 healthy controls (HC). In addition to motor impairment, evaluated by means of UPDRS-III and the PSP rating scale, all patients underwent clinical, functional and morphological assessment of sensory-autonomic nerves through dedicated questionnaires, sympathetic skin response, dynamic sweat test and skin biopsies. The analysis of cutaneous sensory and autonomic innervation was performed using indirect immunofluorescence and confocal microscopy. RESULTS PSP patients displayed a length-dependent loss of sensory and autonomic nerve fibres associated with functional impairment compared to HC and, overall, a more severe picture than in PD patients. The disease severity correlated with the loss of intraepidermal nerve fibre density in the leg of PSP patients (p

Aim: Small fibre neuropathy (SFN) diagnosis represents a challenge for neurologists. The diagnostic gold standard is intraepidermal nerve fibre (IENF) density, but in about 10–20% of patients with symptoms/signs and abnormalities on functional tests, it remains within normal range. We propose an adjunctive parameter to improve the efficiency of skin biopsy diagnosis. Methods: We recruited 31 patients with SFN symptoms/signs, normal nerve conduction study, abnormal quantitative sensory testing and normal IENF density. We also included 31 healthy controls and 31 SFN patients with reduced IENF density as control groups. Results: We measured the distance between consecutive IENFs in the three groups. Mean inter-fibre distances did not differ between patients with normal counts and healthy controls (66.7 ± 14.5 μm vs. 76.7 ± 13.4 μm; P = 0.052), while the relative standard deviation was significantly (P

In the last three decades the study of cutaneous innervation through 3 mm-punch-biopsy has provided an important contribution to the knowledge of small fiber somatic and autonomic neuropathies but also of large fiber neuropathies. Skin biopsy is a minimally invasive technique with the advantage, compared to sural nerve biopsy, of being suitable to be applied to any site in our body, of being repeatable over time, of allowing the identification of each population of nerve fiber through its target. In patients with symptoms and signs of small fiber neuropathy the assessment of IntraEpidermal Nerve Fiber density is the gold standard to confirm the diagnosis while the quantification of sudomotor, pilomotor, and vasomotor nerve fibers allows to evaluate and characterize the autonomic involvement. All these parameters can be re-evaluated over time to monitor the disease process and to evaluate the effectiveness of the treatments. Myelinated fibers and their receptors can also be evaluated to detect a “dying back” neuropathy early when nerve conduction study is still normal. Furthermore, the morphometry of dermal myelinated fibers has provided new insight into pathophysiological mechanisms of different types of inherited and acquired large fibers neuropathies. In genetic neuropathies skin biopsy has become a surrogate for sural nerve biopsy, no longer necessary in the diagnostic process, to study genotype–phenotype correlations.

Objective To describe morphological changes associated with degeneration and regeneration of large fibers in the skin using a model of chronic compression of the median nerve. Methods We studied cutaneous innervation in 30 patients with chronic compression of the median nerve at the wrist. Before surgery, we assessed the symptom severity and performed neurography, quantitative sensory testing, and analysis of nerve morphology and morphometry in skin biopsies from the third digit fingertip. Fifteen patients repeated all tests 12 months after the surgery. Thirty age- and sex-matched healthy subjects were included in the study. Results Clinical and neurophysiological basal assessment showed a moderate involvement of the median nerve. Quantitative sensory testing showed abnormal findings. The density of intraepidermal nerve fibers and intrapapillary myelinated endings was reduced. Myelinated fibers showed caliber reduction and nodal elongation. Meissner corpuscles had normal density but were located deeper in the dermis and their capsule appeared partially empty. During follow-up, patients exhibited a positive clinical and neurophysiological outcome. Quantitative sensory testing improved. Intraepidermal nerve fibers and intrapapillary myelinated endings remained unchanged, but the caliber of intrapapillary myelinated endings was increased. The neural component of the Meissner corpuscle filled the capsule of the mechanoreceptors that remained deeper in the dermis. The position of vasoactive intestinal peptide-immunoreactive fibers was more superficial compared to the basal assessment and controls. Interpretation We recognized and quantified the pathological changes associated with nerve degeneration and regeneration in skin and proposed new parameters that may increase the diagnostic yield of skin biopsy in clinical practice. Ann Neurol 2020;87:456-465.

BACKGROUND AND PURPOSE We aimed to test the clinical utility of the leg:thigh intraepidermal nerve-fiber (IENF) density ratio as a parameter to discriminate between length-dependent small-fiber neuropathy (SFN) and small-fiber sensory ganglionopathy (SFSG) in subjects with signs and symptoms of small-fiber pathology. METHODS We retrospectively evaluated thigh and leg IENF density in 314 subjects with small-fiber pathology (173 with distal symmetrical length-dependent SFN and 141 with non-length-dependent SFSG). A group of 288 healthy subjects was included as a control group. The leg:thigh IENF density ratio was calculated for all subjects. We used receiver operating characteristic curve analyses to assess the ability of this parameter to discriminate between length-dependent SFN and SFSG, and the decision curve analysis to estimate its net clinical benefit. RESULTS In patients with neuropathy, the mean IENF density was 14.8 ± 6.8/mm at the thigh (14.0 ± 6.9/mm in length-dependent SFN and 15.9 ± 6.7/mm in patients with SFSG) and 7.5 ± 4.5/mm at the distal leg (5.4 ± 3.2/mm in patients with length-dependent SFN and 10.1 ± 4.6/mm in patients with SFSG). The leg:thigh IENF density ratio was significantly (P < 0.01) lower in patients with length-dependent SFN (0.44 ± 0.23) compared with patients with SFSG (0.68 ± 0.28). The area under the curve of the receiver operating characteristic analysis to discriminate between patients with length-dependent SFN and SFSG was 0.79. The decision curve analysis demonstrated the clinical utility of this parameter. CONCLUSIONS The leg:thigh IENF ratio represents a valuable tool in the differential diagnosis between SFSG and length-dependent SFN.

Objective:To describe a second hereditary sensory autonomic neuropathy type VI (HSAN-VI) family harboring 2 novel heterozygous mutations in the dystonin (DST) gene and to evaluate their effect on neurons derived from induced pluripotent stem cells (iPSC).Methods:The family consisted of 3 affected siblings from nonconsanguineous healthy parents. All members underwent clinical and electrophysiologic evaluation and genetic analysis. Two patients underwent quantitative sensory testing (QST), cardiovascular reflexes, dynamic sweat test, and skin biopsy to evaluate somatic and autonomic cutaneous innervation and to get fibroblast cultures for developing iPSC-derived neurons.Results:Onset occurred in the first decade, with painless and progressive mutilating distal ulcerations leading to amputation and joint deformity. Sensation to pain, touch, and vibration was reduced. Autonomic disturbances included hypohidrosis, pupillary abnormalities, and gastrointestinal and sexual dysfunction. Nerve conduction studies showed a severe axonal sensory neuropathy. QST and autonomic functional studies were abnormal. Skin biopsy revealed a lack of sensory and autonomic nerve fibers. Genetic analysis revealed 2 pathogenic mutations in the DST gene affecting exclusively the DST neuronal isoform-a2. Neurons derived from iPSC showed absence or very low levels of DST protein and short and dystrophic neuritis or no projections at all.Conclusions:Unlike the previous HSAN-VI family, our description indicates that DST mutations may be associated with a nonlethal and nonsyndromic phenotype. Neuronal loss affects large and small sensory nerve fibers as well as autonomic ones. Induced-PSC findings suggest that dystonin defect might alter proper development of the peripheral nerves. Dystonin-a2 plays a major role in the HSAN-VI phenotype.

Introduction Intraepidermal nerve fiber (IENF) linear density is a reliable tool to detect small-fiber neuropathies. In immunofluorescence, the IENF count is performed typically by computer-assisted nerve tracing on confocal images. Alternatively, the count can be performed directly through the oculars of a standard epifluorescence microscope. We specifically compared measures obtained using the 2 methods. Methods We compared measures of IENF density in the same 50 skin samples using computer-assisted image analysis and direct count. Results There was excellent agreement between the 2 methods. Linear regression showed a slope between paired measures virtually equal to 1 (β = 0.99). Bland–Altman analysis showed a mean difference (offset) between the measures of 0.46 ± 0.91 fibers/mm. Conclusions Direct observation with epifluorescence microscopy proved as reliable as the more time-consuming 3-dimensional computer-assisted analysis of confocal digital images for determining IENF density. Muscle Nerve 51: 501–504, 2015

Objective:To study small and large fiber pathology in drug-naive and l-dopa–treated patients affected by Parkinson disease (PD) in early phases, before the occurrence of neuropathic electrophysiologic abnormalities.Methods:We enrolled 85 patients with idiopathic PD (male/female 49/36, age 61.3 ± 9.7 years) without electrophysiologic signs of neuropathy, including 48 participants naive to l-dopa treatment. All patients underwent clinical, functional, and morphologic assessment of sensory and autonomic nerves through dedicated questionnaires, quantitative sensory testing, sympathetic skin response, dynamic sweat test, and punch biopsies from glabrous and hairy skin. Sensory and autonomic innervation was visualized with specific antibodies and analyzed by confocal microscopy. Data were compared with those obtained from sex- and age-comparable healthy controls. In 35 patients, skin biopsies were performed bilaterally to evaluate side-to-side differences.Results:Intraepidermal nerve fiber density was lower in patients compared to controls in all the examined sites (p < 0.001). The loss was higher in the more affected side (p < 0.01). A loss of autonomic nerves to vessels, sweat glands, and arrector pili muscles and of Meissner corpuscles and their myelinated endings in glabrous skin was found (p < 0.001). Patients showed increased tactile and thermal thresholds, impairment of mechanical pain perception, and reduced sweat output (p < 0.001). The naive and l-dopa–treated groups differed only for Meissner corpuscle density (p < 0.001).Conclusions:Both large and small fiber pathology occurs in the early stages of PD and may account for the sensory and autonomic impairment. l-Dopa affects the 2 populations of fibers differently.

Progressive Supranuclear Palsy (PSP) is a rare neurodegenerative disease characterized by intraneuronal accumulation of hyperphosphorylated Tau. Because PSP is strongly linked biochemically and genetically to tau protein abnormalities there is a growing interest in new tau-directed therapies and in the search of new biomarkers able to monitor disease progression. We studied cutaneous innervation in PSP in order to identify new biomarkers of nerve degeneration trough a minimally invasive technique such as skin biopsy. We performed 3 mm punch biopsies from thigh, leg and fingertip of 12 PSP patients (mean age = 68.4 ± 7.8). Samples were processed with indirect immunofluorescence using a large panel of antibodies to visualize and quantify sensory and autonomic cutaneous nerves. Results were compared with those obtained from a population of age and sex matched controls (mean age = 69 ± 3.3). In PSP patients, compared to controls, we found a severe length-dependent loss of intraepidermal nerve fibers associated to a severe loss of autonomic vasomotor, sudomotor and pilomotor nerves. Moreover, we observed a marked loss of Meissner corpuscles and intrapapillar myelinated endings in glabrous skin. The pattern of nerve degeneration appeared different compared to other neurodegenerative disorders. Skin may provide useful neuropathogical biomarkers to study PSP progression.

We reported a sensory cutaneous denervation more pronounced on the more affected side in Parkinson’s disease (PD) 1 . Our aim is to evaluate if autonomic function is also asymmetrically affected. We investigated sudomotor function on both legs of ten patients affected by idiopathic PD by the dynamic sweat test (DST) 2 . After pilocarpine by iontophoresis we evaluated: (1) density of activated sweat glands per cm 2 (SGD); (2) mean sweat production/gland/minute (MSPG); (3) mean sweat production/skin surface/minute (MSPS). We observed an impairment of sweating function in all our patients on both sides. SGD per cm 2 was 39.9 ± 15.9 (more affected side) and 53.5 ± 15.2 (less affected side) vs. 83.7 ± 17.3 in control subjects (p

To assess cutaneous sensory and autonomic nerves and the vascular bed in amyotrophic lateral sclerosis (ALS).We enrolled 41 patients (M = 20, aged 63.5 ± 11.8 years), and 41 age- and gender-matched healthy volunteers (M = 20, aged 63.5 ± 11.8 years). Disease severity and sensory and autonomic symptoms were scored using dedicated rating scales. Skin biopsies obtained from thigh, leg and fingertip were processed using indirect immunofluorescence. Intraepidermal nerve fibres, Meissner corpuscles (MCs), intrapapillary myelinated endings, cholinergic and noradrenergic pilomotor nerves and dermal vessels were quantified on confocal images. Intraepidermal nerve fibres, pilomotor nerves and vessels were also assessed on distal leg skin samples of 10 spinal cord injury patients to compare our findings with those of a chronic hypomobility condition.Compared to healthy controls skin biopsies showed: (i) non-length-dependent loss of intraepidermal nerve fibres (P0.01) and loss of MCs (P0.01); (ii) reduced (P0.01) density of pilomotor nerves involving cholinergic and noradrenergic fibres and (iii) a reduced (P0.01) vascular bed. Autonomic nerve and dermal vessel densities were higher in patients with higher disease progression rate (P0.01). Moreover, we observed signs of nerve regeneration coexisting with nerve degeneration and increased complexity of the dermal vessels. In patients with posttraumatic spinal cord injury, the density of intraepidermal nerve fibres, pilomotor nerves and of the vascular bed did not differ from controls (P0.05).We demonstrated a cutaneous sensory and autonomic denervation in ALS and a previously undescribed relationship between autonomic and vascular involvement that appeared to be linked to the disease progression rate.

OBJECTIVE: To assess the involvement of the peripheral nervous system in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) by means of immunofluorescence and confocal analysis of punch skin biopsies. METHODS: We recruited 14 unrelated patients with CADASIL (M/F = 9/5; age 53.9 ± 10.5 years) and 52 healthy controls (M/F = 31/21; age 53.8 ± 9.8). Patients underwent clinical and neuroradiologic assessment. Three-millimeter punch skin biopsies were taken from the fingertip, the thigh, and the distal leg and processed using indirect immunofluorescence and a panel of primary antibodies to mark vessels and sensory and autonomic nerve fibers. Intraepidermal nerve fibers (IENF), Meissner corpuscles (MC), and sudomotor, vasomotor, and pilomotor nerves were assessed using confocal microscopy. RESULTS: In patients, compared to controls, we found a severe loss of IENF at the distal leg (p < 0.01), at the thigh (p < 0.01), and at the fingertip (p < 0.01) with a non-length-dependent pattern and a loss of MC (p < 0.01). A severe sudomotor, vasomotor, and pilomotor nerve fiber loss was found by semiquantitative evaluation. Along with nerve loss, a severe derangement of the vascular bed was observed. In our patient population, sensory and autonomic denervation did not correlate with age, sex, type of mutation, or MRI involvement. CONCLUSIONS: We found an involvement of the peripheral nervous system in patients with CADASIL through the assessment of cutaneous somatic and autonomic nerves. The neurovascular derangement observed in the skin may reflect, although to a lesser extent, what happens in the CNS.

Clinical trials for muscular dystrophy molecular treatment require multiple sampling of skeletal muscle to monitor protein rescue. This practice is invasive and could raise ethical problems. A less invasive tool to obtain sequential muscle sampling is necessary. Using indirect immunofluorescence, we evaluated muscle protein expression in myofiber bundles included in 2-2.5-mm punch skin biopsies from the perioral region from 6 healthy subjects and 6 patients with genetically defined forms of muscular dystrophy. Large intradermal bundles of orbicularis oris muscle were constantly present in skin biopsies. They showed a typical muscular antigenic pattern in controls and the expected protein defect in muscular dystrophy patients. These results demonstrate the feasibility of muscular protein expression analysis using skin biopsy. We propose this minimally invasive technique to follow-up the response to genetic or conventional therapies in muscular dystrophies and to confirm the diagnosis in some special clinical conditions.

Vestibular-Evoked-Myogenic-Potentials (VEMPs) represent a muscular response following an intense acoustic stimulation. The cVEMPs explore a reflex arc (from saccule to the neck musculature) and usually are only recorded from sternocleidomastoideus (SCM). 1 They are non-invasive, well-tolerated, simple-to-perform and very useful in several diseases. The normal response is a biphasic complex P13/N23. We propose a more extensive protocol. We recruited 31 subjects (mean age ± SD:50.8 ± 19.0). VEMPs were recorded symmetrically and simultaneously through surface electromyography from SCM, splenius (SPL), trapezius (TRP) and orbicularis oculi (OO). The sensitivity was 20 μ V, band pass filter 20–2000 Hz, tone acoustic stimulus was monaural and binaural at 500 Hz, rate at 5.3 Hz. Three runs of 150 sweeps were averaged. The patients remained in clinostatic position with anterior neck flexion. Like the SCM cVEMP, the SPL and TRP cVEMP was characterized by a biphasic wave of muscle activity that began at ∼13 ms. Responses were consistent and repeatable. VEMPs latencies and amplitude showed a higher inter-reliability for the four muscles. The protocol offer a complementary target for cVEMP assessments. The simultaneous recording of oVEMPs and cVEMPs provide the evaluation of the ascending and descending vestibular pathways, allowing an extensive overview of brainstem function.

Anhidrosis occurs in the majority of multiple system atrophy (MSA) patients but the underlying site of lesion is not well established. We describe three patients with long-standing MSA and anhidrosis diagnosed on the basis of a thermoregulatory sweating test. In biopsies of anhidrotic skin, immunofluorescence analysis disclosed a well preserved postganglionic sudomotor innervation in all three patients supporting the hypothesis of a preganglionic nerve fiber lesion underlying their anhidrosis. Postganglionic sudomotor fiber integrity was also confirmed by normal electrodermal responses in one patient, whereas such responses and microneurographically detectable skin sympathetic nerve activity were absent in the other two MSA patients, suggesting a functional inactivity of structurally intact postganglionic sympathetic skin fibers.

We used immunohistochemical techniques and confocal microscopy to study the morphometry of myelinated nerve endings in glabrous and hairy skin. A total of 30 healthy volunteers took part in this study designed to assess the possibility of obtaining reliable information on myelinated fibers using samples of hairy skin and to determine whether differences exist between myelinated terminations from different sites. We obtained consistent information on cutaneous myelinated terminations using hairy as well as glabrous skin samples. Myelinated endings from hairy and glabrous skin differ in density and distribution. However, from a comparison of our findings with data from nerve biopsy studies, we conclude that all cutaneous myelinated terminations are thinner terminal branches of large myelinated A beta fibers, whereas cutaneous terminations of small myelinated A delta fibers lose their myelin before entering the dermis and become indistinguishable from C-fiber terminations. The classic criteria, based on fiber size, used to distinguish myelinated fiber subgroups in sensory nerves are therefore not suitable for identifying myelinated terminations in the skin.

The morphology of cutaneous sensory and autonomic innervation in human trigeminal territory is still unknown. The aim of this study is to describe facial cutaneous innervation using skin biopsy. This new tool could be useful in understanding the mechanisms underlying several facial pain conditions. In 30 healthy subjects, we quantified epidermal nerve fibers (ENFs) and dermal myelinated fibers (MFs) in V1, V2 and V3, using indirect immunofluorescence and confocal microscopy applied to 2-mm punch skin biopsies from areas adjacent to the eyebrow, upper and lower lip. Using selective markers, we also evaluated the distribution of peptidergic, cholinergic and noradrenergic fibers. Facial skin appeared abundantly innervated and rich in annexes. The ENF density decreased and the MF density increased, moving from the supraorbital to the perioral skin. Noradrenergic sudomotor fibers were particularly and constantly expressed compared with other body sites. Distribution of vasoactive intestinal peptide-immunoreactive (VIP-ir) fibers appeared peculiar for their constant presence in the subepidermal neural plexus - in close contact, but without colocalization with calcitonin gene related peptide (CGRP) and substance P (Sub-P)-ir fibers. Finally, in perioral skin samples, we observed striated muscle fibers with their motor nerves and motor endplates. Our work provides the first morphological study of human facial cutaneous innervation, highlighting some unique features of this territory. Quantification of unmyelinated and myelinated fibers on 2-mm punch biopsies appeared to be feasible and reliable. Facial skin biopsy may be a new approach with which to study and to better characterize facial pain syndromes.

Ross syndrome is described as a rare disorder of sweating associated with areflexia and tonic pupil. Since Ross's first description in 1958, approximately 40 cases have been described. We assessed the involvement of cutaneous innervation in 12 subjects with Ross syndrome using quantitative sensory testing, sweating assessment and immunohistochemical study of anhidrotic and hyperhidrotic skin. This evaluation was repeated over time in 4 out of 12 subjects. In addition, we enrolled four subjects with Holmes-Adie syndrome (areflexia and tonic pupil) to investigate similarities between the two conditions. We found in Ross patients a complex and progressive involvement of cutaneous sensory and autonomic innervation underlying the impairment of heat production and heat dissipation through both loss of sweating and loss of cutaneous blood flow regulation. In Holmes-Adie subjects we found a mild impairment of sweating without thermoregulatory problems. The persistence of a sudomotor vasoactive intestinal peptide-immunoreactive (VIP-ir) innervation, although deranged and poor, definitely differentiated Holmes-Adie from Ross patients. Ross syndrome is a progressive and complex disorder of thermoregulation difficult to differentiate from the probably pathogenetically related Holmes-Adie syndrome. Sweating assessment and skin biopsy are suitable tools to define a boundary between them. Owing to the large number of Ross patients observed in only 5 years, and to the long and complex medical history of most of them, doubts arise on the effective rarity of this condition, and we warn family doctors and other specialists, besides neurologists, to become aware of this complex disorder.

A severe sensory neuropathy, mostly involving large myelinated sensory fibers, characterizes Friedreich ataxia since the early onset. Recently, an involvement of unmyelinated nerve fibers has been observed studying cutaneous innervation by means of immunohistochemical techniques and confocal microscopy. Quantification of epidermal nerve fibers (ENFs) by skin biopsy has been extensively used in the last decade to evaluate small-fiber neuropathies. This minimally invasive procedure, repeatable over time, allows to monitor a neuropathic process. An even less invasive technique is the biopsy of the only epidermis, obtained removing the roof of suction skin blister, sparing basal membrane and vessels. Skin blister, beside a diagnostic tool in sensory neuropathies, is a wound model to study nerve regeneration in human epidermis. The aim of our research was to define the regenerative capability of cutaneous nerve fibers in patients affected by Friedreich's ataxia evaluating time-course and patterns of nerve fibers regeneration. Six patients (4F, 2M, age 19–34) affected by genetically determined Friedreich ataxia were included in our study. In each subject we induced 5 couples of 2 mm blisters on the forearm. At different time points (1–2–3–4 weeks after blister induction) one blister was removed by 3 mm punch skin biopsy and one was reblistered (3-mm blister) and the roof removed. The larger size of these biopsies and blisters allowed at including normal skin immediately adjacent to the blister wound. Epidermal nerve fibers were visualised using antibodies to the pan-neuronal marker protein gene product 9.5 (PGP 9.5) on frozen sections from skin biopsies and on the whole blister roofs. Quantification of ENFs was performed on skin sections using digitised images acquired by confocal microscopy (CARV) and Neurolucida software. Blister allowed to have a panoramic and global view of the regenerative process especially of the collateral extension of axons from adjacent normal epidermis, while biopsy allowed to observe the sprouting from the subepidermal neural plexus. Both these phenomena resulted markedly evident in all FA subjects indicating a normal capability of nerve fibers to regenerate. Blister wound probably induces the release of nerve growth factors stimulating nerve regeneration as in normal subjects. At least for the short period of observation (four weeks) nerve fibers maintained this capability challenging the noxa patogena that leads to their loss.

To evaluate postganglionic autonomic involvement in multiple system atrophy (MSA).We quantified sudomotor innervation in skin biopsy of 29 patients with MSA (19 male and 10 female; age 60.0 ± 7.7 years) and 29 age- and sex-matched healthy subjects. Samples were obtained from thigh and leg and, in 20 out of the 29 cases, also from fingertip. Dysautonomic complaints were evaluated by SCOPA-AUT, a self-administered questionnaire. Sudomotor function was evaluated in a subgroup of patients by the silastic imprint test. Skin samples were processed by indirect immunofluorescence using pan-neuronal and selective cholinergic markers. Total length of sudomotor nerves was measured on digital confocal images using a semiautomated morphometric approach.Measurements of sudomotor nerve density (total length of nerve per volume of glandular tissue) favorably correlated to values obtained using a stereologic unbiased method. Sudomotor nerve density was lower in patients compared to controls in all the examined sites (0.9 ± 0.2 vs 1.9 ± 0.4 nm/μm(3), p0.001, in fingertip; 0.7 ± 0.2 vs 1.9 ± 0.5 nm/µm(3), p0.001, in thigh; 0.6 ± 0.2 vs 1.8 ± 0.4 nm/µm(3), p0.001, in leg).Our data support the hypothesis that postganglionic impairment occurs in MSA and may contribute with the coexisting degeneration of central structures to the development of dysautonomic disorders in this condition.

In fibromyalgia (FM), reduced habituation of laser-evoked potentials (LEPs) suggests a dysfunction of pain processing at a central level. In this study, we aimed to further examine the nociceptive pathways at the peripheral to the central level in a large group of FM patients by means of LEPs and skin biopsy, in light of healthy controls findings and main clinical features. One hundred and ninety-nine FM patients and 109 age- and sex-matched controls were submitted to LEPs by the dorsum of the right hand and the skin over the right chest and knee tender point stimulation. Skin biopsy was performed in 21 randomly selected FM patients and 60 age- and sex-matched controls. The mean N2-P2 amplitude was reduced in the whole FM group, with normal or even increased values in patients with migraine as comorbidity and reduced values in other patients including those presenting with distal sensory deficits. All patients had reduced N2-P2 habituation in respect to controls. In the FM group, LEPs habituation was correlated with pain at tender points and bad quality of life. Epidermal fiber density was significantly reduced in FM patients versus controls, and correlated with N2-P2 amplitude by the hand and chest tender-point stimulation. Dysfunction in the nociceptive system at both the central and peripheral levels may concur to explain phenotypical eterogeneity and clinical symptom complexity in fibromyalgia.

Small fiber pathology has been demonstrated in Parkinson Disease (PD). To verify if it is among the early non-motor features of PD, unrelated to Levodopa therapy, we assessed function and morphology of cutaneous sensory and autonomic nerves in 32 consecutive drug-naive PD patients (M/F = 16/16; age 57.3 ± 11.1 years) and 64 age and sex matched controls. Fifty-micron-thick sections of skin samples from thigh, leg and fingertip were processed by indirect immunofluorescence techniques. Digital images were acquired using a non-laser confocal microscope. Density of intrapapillar myelinated endings (IME), Meissner’s corpuscles (MC) and epidermal nerve fibers (ENFs) was calculated. Sudomotor, pilomotor and vasomotor nerves were assessed semi-quantitatively. Further evaluation included sural nerve conduction study, sympathetic skin response, quantitative sensory testing and dynamic sweat test. Nerve conduction was normal in all patients. Morphological analysis showed: a significant loss (p

Cold-induced sweating syndrome type 1 (CISS1) is a rare disorder characterized by profuse sweating in a cold environment, determined by mutations in cytokine receptor–like factor 1 ( CRLF1 ).1 Its pathogenesis is not fully understood. It has been demonstrated in vitro that CRLF1 may be involved in inducing differentiation from a noradrenergic to a cholinergic transmitter phenotype. During development, catecholaminergic and cholinergic neurotransmission is required for the induction of secretory function, whereas cholinergic transmission becomes crucial for the maintenance of the secretory responsiveness.2 The abnormal sweat response in CISS1 could be also related to alterations in temperature signals acting on hypothalamus and preoptic areas.1 We report clinical, molecular, skin biopsy, and temperature monitoring data of an Italian boy with CISS1. ### Case report. A 16-year-old boy had come to our observation at 1 year of age. His parents were healthy and unrelated. A brother, with facial dysmorphisms and hypotonia, had died at age 3 months from bronchopulmonary infection. Our patient was hypotonic at birth and experienced severe feeding difficulties. He had elongated face, high-arched palate, weak cry, large pinnae, short hands, tapering fingers, clinodactyly, and reduced tendon reflexes. Concentric needle EMG, nerve conduction velocity (NCV) studies (ulnar, median, peroneal, tibial, and sural nerves), and repetitive nerve stimulation testing excluded a …

Our aim was to study peripheral nerve involvement in CADASIL, a hereditary cerebral microangiopathy due to mutations in the Notch 3 gene. We studied skin innervation in 14 patients with CADASIL (6 women, 8 male, mean age 57.6 ± 5.4) by means of 3 mm-punch biopsy from thigh, distal leg and fingertip. Samples were processed using indirect immunofluorescence techniques and an extensive panel of primary antibodies to mark both myelinated and unmyelinated somatic and autonomic nerve fibers. Vascular and neural signs of remodeling were present in all patients. We found, compared to 40 age and sex matched controls, a lower density of ENF/mm in patient thigh (12.7 ± 4.3 versus 23.4 ± 3.8), leg (6.7 ± 1.3 versus 13.3 ± 3.0) and fingertip (1.8 ± 0.8 versus 6.9 ± 3.1). In fingertip we found also a marked loss of Meissner corpuscles with a lower density/mm2 (8.3 ± 2.2 versus 27.3 ± 7.4). In addition autonomic innervation appeared severely affected with aspect of structural derangement of dermal annexes. Vascular bed appeared very abnormal with dilated and complex vessels. The involvement of last endings of sensory and autonomic nerve fibers is part of the neuropathological picture of CADASIL.

Our aim was to evaluate if autonomic impairment in Parkinson disease (PD) differently affects the primarily compromised side. Fourteen PD patients underwent bilateral sympathetic skin response (SSR) recording. The response was elicited on palms and soles following standard procedures. Patients underwent also sudomotor output evaluation by dynamic sweat test (DST), a technique able to assess postganglionic sudomotor function by evaluating gland density and sweat production. A significant ( p

The study of sudomotor function represents a useful tool to evaluate autonomic disorders. Currently available tests allow either the measurement of sweat output from the whole body or selected small skin locations over time, or quantification of the number and size of sweat drops at a fixed time after stimulation. We devised a dynamic sweat test (DST) that measures at the same time sweat gland density, distribution of active glands, and sweat rate, and applied it to the evaluation of sudomotor function in diabetes.The DST was used to evaluate sweating in the forearm of 14 asymptomatic diabetic subjects and 14 age- and sex-matched healthy controls. Distal leg was also tested in 7 patients and 7 controls. The formation of the imprint of pilocarpine-induced sweating was recorded by a digital video camera through a cornstarch-powdered transparent tape used as a contrast-enhancing device. Mean sweat output per gland and per skin area and sweat gland density per cm(2) were evaluated.We observed a significant reduction of sweating in diabetic subjects as compared to controls; sweat gland density per cm(2) (59.7 +/- 18.6 vs 83.7 +/- 17.3; p0.05) and mean sweat output (nL/min) per gland (4.7 +/- 0.7 vs 8.3 +/- 2.7; p = 0.01) and per skin area (261 +/- 100 vs 645 +/- 296; p = 0.01) were reduced in the lower limb. Values for the forearm were not significantly different from controls.Dynamic sweat test is an easy-to-perform, informative method to study sudomotor function. It provides the ability to detect subtle functional changes occurring in the early stages of diabetic neuropathy.

We assessed the involvement of cutaneous innervation in two subjects with a molecularly confirmed diagnosis of spinobulbar muscular atrophy (SBMA) using antidromic nerve conduction studies, quantitative sensory testing, and sweat tests, as well as immunohistochemical techniques and confocal microscopy of glabrous and hairy skin biopsy. Both patients showed a marked reduction in amplitude of sensory action potentials and moderate or severe abnormalities of tactile thresholds and mechanical pain perception. A severe reduction of sweat drops on the Silastic imprint test and a widespread loss of small myelinated and unmyelinated fibers in hairy skin were also observed. Fiber loss involved either somatic or autonomic fibers and did not show any distal-proximal gradient. These results, together with loss of Meissner corpuscles and their large myelinated afferent fibers in glabrous skin, confirmed the extensive involvement of sensory neurons of large and small size and revealed an autonomic skin denervation in SBMA.

Objective The aim of this study was to characterize sympathetic adrenergic innervation of the skin in healthy subjects using dopamine β hydroxylase (DβH) as a specific marker for noradrenergic fibers. Methods Sympathetic adrenergic innervation of human skin was studied in 10 healthy subjects by indirect immunofluorescence and confocal microscopy applied to punch skin biopsies. Noradrenergic fibers were identified both in glabrous and hairy skin using DβH antibody. Results DβH immunoreactive fibers were mainly localized in arteriovenous anastomoses, arrector pilorum muscles, and arterioles, whereas few adrenergic fibers were found around sweat glands. Interpretation Our description of sympathetic adrenergic innervation of human skin aims to improve the diagnostic ability of skin biopsy to detect selective autonomic nervous system disorders. Ann Neurol 2006;59;376–381

Generalised anhidrosis (GA) shows a uniform clinical picture whether the pathogenesis involves intrinsic abnormalities of sweat glands or postganglionic sympathetic cholinergic nerve dysfunction. We describe two patients who presented intolerance to heat and anhidrosis. In the first patient, symptoms started at 33 years of age, and were associated with absent tendon reflexes and a mydriatic right pupil unreactive to light. The other patient had been unable to sweat since birth. GA was diagnosed on the basis of clinical findings and thermoregulatory tests. Microneurography and morphological analysis of the skin and its innervation disclosed a different lesion site underlying GA in the two patients, and distinguished between a postganglionic autonomic nerve fibre lesion and sweat gland dysfunction.

We used immunohistochemistry and confocal microscopy applied to fingertip punch biopsy to study glabrous skin innervation in 14 healthy subjects. In addition to epidermal nerve fibers, we quantified mechanoreceptors and their myelinated afferents. Using digital images and dedicated software, we calculated caliber, internodal and nodal length, and G-ratio of the last four internodes of the myelinated endings. In our skin samples, we found a mean density of 59.0 +/- 29.3 myelinated endings per square millimeter with a mean diameter of 3.3 +/- 0.5 microm and an internodal length of 79.1 +/- 13.8 microm. These findings indicate that Abeta fibers undergo drastic changes in their course from the nerve trunk to the target organ, with repeated branching and consequent tapering and shortening of internodal length. Our work demonstrates that skin biopsy can give information on the status of large myelinated endings as well as unmyelinated sensory and autonomic nerves. Since distal endings are primarily involved in distal axonopathy, skin biopsy can be more suitable than sural nerve biopsy to detect early abnormalities. In addition to diagnostic applications, this technique allows clarification of the mode of termination of Abeta fibers and their relationship with mechanoreceptors, leading to relevant electrophysiological speculations.

The assessment of epidermal nerve fiber density (ENF) in skin biopsy has been accepted as an objective and reliable technique to diagnose SFN. Bright-field microscopy and immunofluorescence with or without confocal microscopy, are currently used to quantify ENF. The objective of this study was to evaluate the agreement between the two techniques of immunofluorescence ENF analysis, with and without confocal microscopy. Twenty biopsies were obtained from distal leg of controls and neuropathic patients and immunostained for immunofluorescence according to standard procedures. Four sections double stained with PGP and COLIV were analyzed at immunofluorescence microscope and ENF along the entire section length counted directly throughout the oculars. Epidermal length was measured by means of an intraocular calibrated grid. We assessed the accuracy of this method by calculating, inter and intra-rater reliability. ENF were then counted in the same samples using confocal images and Neurolucida software. ENFs were traced on a stack of 16 consecutive 2 μm optical sections following published rules. We found a significant agreement between the two methods (p Our study demonstrates that the two techniques are comparable, and that the direct count through the oculars is a reliable and faster method for routinary quantification.

The assessment of autonomic nervous system (ANS) in hereditary sensory autonomic neuropathy (HSAN) type II is still lacking. To this aim we investigated the ANS involvement in a new family with HSAN II phenotype. Three siblings from non consanguineous healthy parents were clinically and electrophysiologically evaluated. Two of them underwent quantitative sensory testing (QST), cardiovascular and dynamic sweat tests and skin biopsy. Genetic testing for WNK1 and FAM134B mutations is ongoing. Onset occurred in the first decade, with impaired pain sensitivity and progressive distal mutilating ulceration with amputation and joint deformity. Autonomic disturbances included hypohidrosis with heat intolerance, pupillary abnormalities and chronic diarrhoea. Nerve conduction studies showed a severe axonal sensory neuropathy. Sympathetic skin response was absent. QST was altered for both painful and nonpainful sensory modalities. Autonomic functional studies detected the impairment of both sudomotor and cardiovascular systems. Skin biopsy revealed a lack of epidermal and dermal sensory and autonomic nerve fibers with complete denervation of sweat glands and arteriovenous anastomoses. Our findings expand the HSAN II phenotype providing pathological and functional evidence of a widespread ANS impairment. The complete sensory and arteriovenous denervation accounts for the severe sensory loss and distal ulcerations in HSAN type II.

Objective To evaluate postganglionic autonomic and somatic nerve fiber involvement in a patient with chronic autoimmune autonomic ganglionopathy. Design Case report. Setting Department of Neurological Sciences, University Federico II of Naples. Patient A patient with a 16-year history of severe autonomic failure and a high nicotinic acetylcholine receptor antibody titer underwent an extensive laboratory evaluation. Main Outcome Measures Evaluation of sympathetic and parasympathetic functions and sural nerve and skin biopsies. Results Clinical and laboratory evaluations showed the involvement of cardiovascular, pupillary, sudomotor, gastrointestinal, and bladder functions. Sudomotor function study and skin biopsy findings revealed postganglionic autonomic damage. Moreover, sural nerve and skin biopsy specimens provided clear evidence of somatic nerve fiber involvement. Conclusions We demonstrated postganglionic autonomic damage that could be related to a prolonged and severe impaired synaptic transmission and we report, for the first time to our knowledge, a somatic nerve fiber involvement in autoimmune autonomic ganglionopathy.

In the last decade, applying multiple immunolabeling with highly specific antibodies and confocal microscopy to skin biopsies, it has been possible to study extensively cutaneous innervation. This technique has proved to be reliable and even more sensitive than sural nerve biopsy to investigate mild dying back neuropathies with only a very distal involvement of nerve fibers. So far it has been used mainly on hairy skin to evaluate small fiber anomalies in congenital, acquired and idiopathic sensory neuropathies. Compared to hairy skin, glabrous skin has a peculiar characteristic: it is rich in mechanoreceptors and myelinated Aβ fibers. To study these structures we selected a group of healthy volunteers and recruited a group of patients affected by hereditary or acquired sensory neuropathies. Skin biopsy was performed using a three mm disposable punch in all subjects on fingertip. We stained the cutaneous samples with a panel of antibodies identifying different neural structures such as the axon, the myelin sheath, the Schwann cell, and the Meissner corpuscle capsula. Three-D digitized images were acquired using a confocal microscope. We studied Meissner and intrapapillar myelinated nerve fiber density and using a dedicated software we measured in normal and neuropathic subjects area and density of innervation of Meissner corpuscles and thickness, internodal and Ranvier node length of the sensory fibers approaching the mechanoreceptors.

Ross syndrome is described as a very rare disorder characterized by segmental anhydrosis, hyporeflexia and tonic pupils. We observed 8 subjects (6 males and 2 females; age range 31–57 years) affected by Ross syndrome in the last 4 years. They had suffered heat intolerance for at least 7 years with severe impairment of thermoregulation before diagnosis of Ross syndrome was defined. All patients underwent neurological examination, electrophysiological study, quantitative sensory testing, Minor test, silastic imprint test, sympathetic skin response, cardiovascular reflexes and 3 mm punch biopsies from fingertip, thigh, leg and from residual hyperhydrotic areas. Skin samples were cut in 80-micron sections and incubated with a panel of primary antibodies and then with secondary antibodies conjugated with fluorophores Cy2, Cy3, Cy5 to visualize neural and vascular structures. Different primary antibodies were used to distinguish myelinated and sensory and autonomic unmyelinated fibers. Digital images were obtained using a non-laser confocal microscope (CARV, Atto Biosciences, Rockville MD, USA) and analyzed by dedicated image analysis software (Neurolucida, Microbrightfield, Willistone VT, USA). As we previously described in 3 patients (Perretti et al., Clin Neurophysiol, 2003), besides a lack of cholinergic sudomotor fibers we found a milder involvement of unmyelinated and myelinated sensory fibers. These morphological findings were in keeping with a moderate subclinical impairment of tactile and thermal thresholds and mechanical pain detection. Sensory nerve conduction study was normal indicating a very distal involvement of sensory nerve endings. This study confirms our previous observation that Ross syndrome is a degenerative process involving progressively, besides cholinergic autonomic fibers, sensory unmyelinated and myelinated nerve fibers.

Evaluation of epidermal nerve fiber (ENF) density by skin biopsy is nowadays considered as the gold standard for the diagnosis of small fiber neuropathies. ENFs show in normal subjects quite a regular distribution between the keratinocytes while in patients with small fiber neuropathies we can find, besides a loss of ENFs, evident anomalies of their distribution. Different patterns of nerve fiber dispersion can express different pathophysiological processes. The aim of this study was to define a tool capable of describing the distribution of ENF in the epidermis. For this purpose, we measured the distance between subsequent epidermal fibers on skin samples of healthy and diabetic subjects. We calculated the standard deviation of the interfiber length as a parameter (the dispersion index – DI) to evaluate the variability of the distance between consecutive ENFs. We selected thigh skin samples from five healthy subjects (age range 30–50 years) and five age and sex matched diabetic patients. Samples were processed using immunohistochemical techniques. Sections were PGP 9.5 and Collagen IV double stained to show nerve fibers and basement membrane. One randomly selected section for each subject was acquired for its entire length (usually seven 20x confocal images obtained from a z-stack of 16 two micron optical sections) using a confocal microscope (CARV, Atto Biosciences, Rockville MD, USA). We measured the distance between consecutive ENFs along the entire epidermal length, using dedicated software (ScionImage, Scion Corporation Frederick, Maryland USA). In diabetic patients the DI resulted significantly higher than in normal subjects. We suggest that DI along with the ENF density can be useful to better characterize epidermal innervation.

To evaluate small fiber function in Friedreich's Ataxia (FA), we performed in 7 patients pin-prick, thermal thresholds, and sweat test. All tests were performed in four different sites: hand dorsum, anterior thigh, lateral distal leg, and foot dorsum. The same subjects underwent 3 mm punch skin biopsy from fingertip, anterior thigh, and lateral distal leg. We used a thin needle mounted on a calibrated nylon wire for the pin-prick test, and a Medoc 2001 TSA system for thermal threshold assessment. Sweat test was performed using a silicon mold after stimulation with pilocarpine by iontophoresis. Skin specimens, cut into 100-μm-thick sections, were double-stained using primary antibodies specific for collagen and nervous fibers and secondary antibodies labeled with Cy3 and Cy5 fluorophores. Tridimensional digitized images were obtained from z-series of 2-μm-thick optical sections acquired with a confocal microscope. We found in all patients in the more distal sites definite signs of functional impairment of the small fibers. These data correlated with the skin innervation morphological findings that showed, in the same sites, a sensible loss of small fibers regarding both the epidermal free endings and the subepidermal neural plexus. Less severe morphological abnormalities were found in the proximal sites. The large fiber neuropathy in FA is well documented. Our data show a length-dependent involvement of small fibers in the pathological process.