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Background: The role of peripheral phosphorylated-α-Synuclein (p-α-syn) deposition on nerve degeneration in synucleinopathies is still unknown. Objective: To assess the cutaneous neural distribution of p-α-Syn deposits and its correlation with clinical data and with morphology and function of cutaneous sensory and autonomic nerves in early Parkinson’s disease (PD) and multiple system atrophy-parkinson type (MSA-p). Methods: We recruited 57 PD (F/M = 21/36; age 63.5±9.4 years) and 43 MSA-p (F/M = 16/27; age 62.3±9.0 years) patients within 2 years from motor symptoms. We applied questionnaires and clinical scales, sensory thresholds, and sudomotor testing to assess severity of motor and non-motor involvement and sensory and autonomic dysfunction. We quantified, in skin biopsy from thigh, leg, and fingertip, epidermal, pilomotor, and sudomotor nerve fibers, Meissner corpuscles and intrapapillary myelinated endings and the neural distribution of p-α-syn deposits. Results: Compared to controls, we found a cutaneous denervation paralleling functional and clinical impairment. Sensory and autonomic denervation was more severe in MSA-p than in PD. Deposits of p-α-syn were found in the majority of patients, with no significant differences among sites in both groups. Higher occurrence of p-α-syn deposits in autonomic nerves differentiated (p

Objective The objective of this study was to evaluate patients with ganglionic acetylcholine receptor antibody (gAChR-Ab) positive autoimmune autonomic ganglionopathy using a multimodal testing protocol to characterize their full clinical phenotype and explore biomarkers to quantify immunotherapy response. Methods We conducted a cohort study of 13 individuals (7 women, 21-69 years of age) with autonomic failure and gAChR-Ab >100 pM identified between 2005 and 2019. From 2018, all patients were longitudinally assessed with cardiovascular, pupillary, urinary, sudomotor, lacrimal and salivary testing, and Composite Autonomic Symptom Score (COMPASS-31) autonomic symptom questionnaires. The orthostatic intolerance ratio was calculated by dividing change in systolic blood pressure over time tolerated on head-up tilt. Eleven patients received immunotherapy. Results At first assessment, all 13 patients had cardiovascular and pupillary impairments, 7 of 8 had postganglionic sudomotor dysfunction, 9 of 11 had urinary retention and xeropthalmia, and 6 of 8 had xerostomia. After immunotherapy, there were significant improvements in orthostatic intolerance ratio (33.3 [17.8-61.3] to 5.2 [1.4-8.2], p = 0.007), heart rate response to deep breathing (1.5 [0.0-3.3] to 4.5 [3.0-6.3], p = 0.02), pupillary constriction to light (12.0 [5.5-18.0] to 19.0 [10.6-23.8]%, p = 0.02), saliva production (0.01 [0.01-0.05] to 0.08 [0.02-0.20] g/min, p = 0.03), and COMPASS-31 scores (52 to 17, p = 0.03). Orthostatic intolerance ratio correlated with autonomic symptoms at baseline (r = 0.841, p = 0.01) and following immunotherapy (r = 0.889, p = 0.02). Immunofluorescence analyses of skin samples from a patient 32 years after disease onset showed loss of nerve fibers supplying the dermal autonomic adnexa and epidermis, with clear improvements following immunotherapy. Interpretation Patients with autoimmune autonomic ganglionopathy demonstrated objective evidence of widespread sympathetic and parasympathetic autonomic failure, with significant improvements after immunotherapy. Quantitative autonomic biomarkers should be used to define initial deficits, guide therapeutic decisions, and document treatment response. ANN NEUROL 2021;89:753-768.

We aimed to examine biomechanical and neuroautonomic adaptation to blood volume displacement induced by tilt test in patients with previous inferoapical/inferolateral (IA-IL) or basal/apical septal (BS-AS) myocardial infarction (MI). Twenty-four patients with heart failure (HF) and previous IA-IL MI and 30 patients with HF and previous BS-AS MI were enrolled. All patients underwent head-up tilt test, radionuclide ventricular function monitoring (VEST), sympathovagal balance evaluation, and chronotropic 25-dose isoproterenol infusion test (CD25). Physiopathological reactions to stress-tests were assessed in both groups. Follow-up lasted 36 mo. IA-IL patients showed lower stroke volume (SV), cardiac output (CO), and left ventricle ejection fraction (LVEF) compared with BS-AS. End-diastolic volume decreased in IA-IL group (F = 3.1, P = 0.043) more than in BS-AS group during tilt test. The time trend of end-systolic volume, SV, CO, LVEF, and peak filling rate were similar in the two groups. Norepinephrine (IA-IL supine→tilting 499.5 (SD:28.8)→719.3 (SD:41.5) pg/mL vs. BS-AS supine→tilting 533.9 (SD:33.3)→768.8 (SD:47.9) pg/mL; P < 0.001) and epinephrine plasma concentrations (IA-IL supine→ tilting 125.7 (SD:9.8)→193.7 (SD:9.6) pg/mL vs. BS-AS supine→ tilting 118.8 (SD:8.9)→191.7 (SD:10.2) pg/mL; P < 0.001) increased in both groups. Low-to-high frequencies ratio significantly increased in IA-IL and decreased in BS-AS patients. CD25 was similar in IA-IL and BS-AS patients (IA-IL = 4.6 (SD:0.94), BS-AS = 5.0 (SD:1.06) mg; P = 0.79). CD25 predicted all-cause mortality (hazard ratio 1.48, 95% confidence interval 1.32–1.67; P < 0.0001) after adjusting for age/heart rate. In conclusion, patients with ischemic HF show abnormal biomechanical adaptation to volume displacement and compensatory sympathetic overdrive. The association of reduced b-adrenergic sensitivity and sympathetic denervation in such patients warrants for careful therapeutic choices. NEW & NOTEWORTHY The adaptation to volume displacement induced by tilt test was assessed in patients with heart failure and previous inferoapical/inferolateral or basal/apical septal myocardial infarction. The responsiveness of cardiac muscle to sympathetic nervous system stimulation predicts the mortality in patients with ischemic heart failure and may represent a useful tool for clinicians in the general assessment of this kind of patients.

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.

IntroductionCardiovascular autonomic failure and neurogenic orthostatic hypotension (nOH) are common and disabling features of Parkinson’s disease (PD) and multiple system atrophy (MSA). Compared to PD, MSA is considered a primarily central alpha-synucleinopathy.AimTo characterise the relationship between cardiovascular autonomic failure and cutaneous autonomic and somatic innervation in early PD and MSA.MethodsFifty-five patients (37 PD, 18 MSA) within 2 years of motor onset underwent: 1) cardiovascular autonomic testing 2) plasma catecholamines 3) distal leg skin biopsies for quantification of intraepider- mal (IENF), pilomotor (PNF), and sudomotor nerve fibres (SNF), and 4) COMPASS-31 autonomic symptom questionnaires.Results75% with MSA and 36% with PD had OH ≥20/10mmHg. Supine noradrenaline was significantly higher in MSA vs PD with OH (264[241-304] vs 158[154-190] pg/ml, P=.03). Compared to patients without OH, patients with OH had significantly reduced cutaneous innervation (3.1[1.3-6.0] vs 7.4[5.3-8.7] IENF/mm, P=.008; 2.8[0-8.2] vs 11.8[6.5-23.4] PNF/mm, P=.007; and 0.9[0.1-1.4] vs 1.6[1.4-1.8] nm SNF/µm3,P=.002), with no significant differences between MSA and PD with OH subgroups. Markers of cardiovas- cular autonomic failure, including severity of OH, correlated significantly with cutaneous denervation (IENF: ρ=-0.47, P=.007; PNF: ρ=-0.51, P=.004; SNF: ρ=-0.51, P=.006), and patient symptoms (COMPASS-31: ρ=0.57, P=.002).ConclusionCardiovascular autonomic failure was associated with cutaneous denervation in both MSA and PD. Postganglionic denervation may contribute to the pathophysiology of cardiovascular autonomic failure and nOH, and influence response to therapeutic agents for nOH.

Background and ObjectivesSudomotor impairment has been recognized as a key feature in differentiating Parkinson disease (PD) and multiple system atrophy–parkinsonian type (MSA-P), with the latter characterized by diffuse anhidrosis in prospective study, including patients in late stage of disease. We aimed to evaluate morphologic and functional postganglionic sudomotor involvement in patients with newly diagnosed MSA-P and PD to identify possible biomarkers that might be of help in differentiating the 2 conditions in the early stage.MethodsOne hundred patients with parkinsonism within 2 years from onset of motor symptoms were included in the study. At the time of recruitment, questionnaires to assess nonmotor, autonomic, and small fiber symptoms were administered, and patients underwent postganglionic sudomotor function assessment by the dynamic sweat test and punch skin biopsy from the distal leg. Skin samples were processed for indirect immunofluorescence with a panel of antibodies, including noradrenergic and cholinergic markers. The density of intraepidermal, sudomotor, and pilomotor nerve fibers was measured on confocal images with dedicated software. A follow-up visit 12 months after recruitment was performed to confirm the diagnosis.ResultsWe recruited 57 patients with PD (M/F 36/21, age 63.5 ± 9.4 years) and 43 patients with MSA-P (M/F 27/16, age 62.3 ± 9.0 years). Clinical scales and questionnaires showed a more severe clinical picture in patients with MSA-P compared to those with PD. Sweating output and intraepidermal, pilomotor, and sudomotor nerve densities, compared to controls, were lower in both groups but with a greater impairment in patients with MSA-P. Pilomotor and sudomotor nerve density correlated with sweating function and with nonmotor clinical symptoms. A composite sudomotor parameter defined as the arithmetic product of sweat production multiplied by the density of sudomotor fibers efficiently separated the 2 populations; the receiver operating characteristics curve showed an area under the curve of 0.83.DiscussionDynamic sweat test and the quantification of cutaneous autonomic nerves proved to be a sensitive morpho-functional approach to assess the postganglionic component of the sudomotor pathway, revealing a more severe involvement in MSA-P than in PD early in the disease course. This approach can be applied to differentiate the 2 conditions early.Classification of EvidenceThis study provides Class II evidence that postganglionic sudomotor morpho-functional assessment accurately distinguish patients with PD from patients with MSA-P.

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.

The aim of this study was to report a method that quantifies axon reflex sweating from individual sweat glands with nanoliter precision. Measurement of the axon reflex is generally expressed as a single variable (e.g., the flare area or total sweat volume). High-definition videography enables precise measurement of sweating from single, axon reflex-stimulated sweat glands (SGs). The sudomotor axon reflex was activated in healthy subjects and subjects with peripheral neuropathy by iontophoresis of 10% acetylcholine. Sweating was simultaneously imaged for 5 min in a 2.5-cm2 area of iodine-coated skin to one side of the stimulus, using a customized high-resolution camera with starch-coated transparent tape over a rigid viewing screen. A second video then imaged the directly stimulated sweating. The indirect sweat response was quantified in terms of sweat gland number and distance from the stimulation site (radius), sweat rate per gland, and total sweat. Fifty-two healthy control and twenty subjects with neuropathy underwent testing at the foot, calf, thigh, and hand. Normal ranges were calculated for SG density, mean sweat rate per SG, and total sweat volume. Neuropathy subjects demonstrated reduced sweating, and values differed between body sites. The described method precisely measures the total and individual sweat output of hundreds of SGs in response to a standard, axon reflex-mediated stimulus, and quantifies alterations in axon reflex sweating seen in peripheral neuropathy.

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.

There is a need for quantitative, precise assessment of small fiber peripheral nerve function. We tested a customized camera device and protocol designed to quantify secretions of individual sweat glands (SGs). Testing was performed on 178 healthy controls and 20 neuropathy subjects. Sweating was stimulated on a 2.25 cm2 skin area by iontophoresis of pilocarpine. The camera imaged sweat from 50 to 400 sweat ducts. We calculated secretion rate of individual SGs, total sweat volume, and number of secreting SGs at four body sites. Neuropathy subjects were tested at the two distal sites to demonstrate the device's capability to detect abnormal sudomotor function. Normal ranges were calculated for each body site. Neuropathy subjects had lower sweat rates per SG, lower total sweat, and lower SG density. The normal values decreased with advancing age, were lower in females, and differed between body sites. There was good agreement with repeat testing. The device provides reliable, precise quantitative measures of sweat secretion from single SGs for characterization of sudomotor nerve function in healthy control subjects and in subjects with known peripheral neuropathy. The test combines the capabilities of existing tests of sudomotor function while providing additional capabilities.

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.

The identification of sensitive and specific biomarkers in a CR setting is still an open question, also considering that not all of the circulatory and urinary biomarkers have been evaluated for their possible role in CV-risk prevention.

To improve patient care and help clinical research, the Neuropathic Pain Special Interest Group of the Italian Neurological Society appointed a task force to elaborate a consensus statement on pharmacoresistant neuropathic pain. The task force included 19 experts in neuropathic pain. These experts participated in a Delphi survey consisting of three consecutive rounds of questions and a face-to-face meeting, designed to achieve a consensus definition of pharmacoresistant neuropathic pain. In the three rounds of questions, the participants identified and described the main distinguishing features of pharmacoresistance. In the face-to-face meeting the participants discussed the clinical features determining pharmacoresistance. They finally agreed that neuropathic pain is pharmacoresistant when "the patient does not reach the 50% reduction of pain or an improvement of at least 2 points in the Patient Global Impression of Change, having used all drug classes indicated as first, second, or third line in the most recent and widely agreed international guidelines, for at least 1 month after titration to the highest tolerable dose." Our consensus statement might be useful for identifying eligible patients for invasive treatments, and selecting patients in pharmacological trials, thus improving patient care and helping clinical research.

The development of patient-specific induced pluripotent stem cells (iPSCs) offered interesting insights in modeling the pathogenesis of Charcot-Marie-Tooth (CMT) disease and thus we decided to explore the phenotypes of iPSCs derived from a single CMT patient carrying a mutant ATP1A1 allele (p.Pro600Ala). iPSCs clones generated from CMT and control fibroblasts, were induced to differentiate into neural precursors and then into post-mitotic neurons. Control iPSCs differentiated into neuronal precursors and then into post-mitotic neurons within 6-8 days. On the contrary, the differentiation of CMT iPSCs was clearly defective. Electrophysiological properties confirmed that post-mitotic neurons were less mature compared to the normal counterpart. The impairment of in vitro differentiation of CMT iPSCs only concerned with the neuronal pathway, because they were able to differentiate into mesendodermal cells and other ectodermal derivatives. ATP1A1 was undetectable in the few neuronal cells derived from CMT iPSCs. ATP1A1 gene mutation (p.Pro600Ala), responsible for a form of axonal CMT disease, is associated in vitro with a dramatic alteration of the differentiation of patient-derived iPSCs into post-mitotic neurons. Thus, the defect in neuronal cell development might lead in vivo to a decreased number of mature neurons in ATP1A1-CMT disease.

Congenital insensitivity to pain (CIP) is a rare autosomal recessive disorder presenting with a spectrum of clinical features caused by mutations in different genes. A 10-year-old girl with CIP, hyposmia and hypogeusia, and her unaffected twin and parents underwent next generation sequencing of SCN9A exons and flanking splice sites. Transcript analysis from whole blood successfully assayed the effect of the mutation on the mRNA splicing by polymerase chain reaction amplification on cDNA and Sanger sequencing. We identified the novel splicing variant c.1108-2A>G compound with the p.Arg896Gln (c.2687G>A) missense mutation previously described in a homozygous patient. The new intronic variant was predicted to induce exon 10 skipping. Conversely, SCN9A mRNA assay demonstrated its partial deletion with a loss of 46 nucleotides causing a premature stop codon in position p.Gln369 (NP_002968). Genetic analysis showed that the two variants were biallelic, being the mother and brother heterozygous carriers of the missense mutation, and the father heterozygous for the splicing mutation. Skin biopsy showed lack of Meissner's corpuscles, loss of epidermal nociceptors and normal autonomic organ innervation. We report a novel splicing mutation and provide clues on its pathogenic effect, broadening the spectrum of genotypes and phenotypes associated to CIP.

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

To assess the involvement of small nerve fibers in Charcot-Marie-Tooth type 1A (CMT1A).We used indirect immunofluorescence and confocal microscopy on punch biopsies from glabrous (fingertip) and hairy (thigh and leg) skin of 20 unrelated patients with CMT1A to quantify somatic and autonomic nerve fibers. In particular, we quantified epidermal nerve fibers (ENF), Meissner corpuscles (MC), intrapapillary myelinated endings (IME), and sudomotor nerves. We correlated morphologic data with findings from quantitative sensory testing, sudomotor output, sympathetic skin response, and cardiovascular reflexes. A control population of healthy age- and sex-matched controls was included with a matching ratio of 1:2.We found a length-dependent loss of ENFs that worsened with aging. We also observed a loss of MCs, IMEs, and sudomotor nerves. The loss of ENF at distal leg correlated with the increase in heat-pain thresholds (p0.05) and with tactile thresholds (p0.05). Sudomotor nerve fiber loss correlated with ENF density (p0.05) and sweating output (p0.001).We demonstrated through morphologic, physical, and psychophysical testing that small somatic and autonomic fibers are abnormal and cause symptoms in patients with CMT1A. Awareness of such symptoms by the clinician could lead to better treatment.

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.

To describe a second hereditary sensory autonomic neuropathy type VI (HSAN-VI) family harboring 2 novel heterozygous mutations in the dystonin (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.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 theUnlike the previous HSAN-VI family, our description indicates that

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

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.

There is a need for quantitative, precise assessment of small fiber peripheral nerve function. We tested a customized camera device and protocol designed to quantify secretions of individual sweat glands (SGs). Testing was performed on 178 healthy controls and 20 neuropathy subjects. Sweating was stimulated on a 2.25 cm

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.

Small fiber pathology is part of the clinical picture of PD. Cross-sectional studies have shown no correlation between intraepidermal nerve fiber (IENF) density and patient disease duration and severity. However, it is not known whether IENF density changes over time. We aimed to assess the progression of small fiber pathology in PD patients along the disease course. We assessed epidermal innervation at time 0 and after 1–9 years (mean 3.5) in 17 PD patients (M/F = 11/6, age = 64.4 ± 15.3) leg on punch biopsies. Patients were assessed in a rehabilitative setting. They underwent yearly 30-days hospitalizations and were trained to continue a home-personalized program of physical exercises between successive evaluations. Quantitative sensory testing and sudomotor assessment were also performed. Overall, no difference ( p = 0.12) between the first and the last IENF density (8.7 ± 4.0 vs. 10.3 ± 4.9) assessment was found. However, the IENF density variation correlated with the disease duration ( r = 0.71; p The small fiber pathology associated to PD can improve to some extent by optimizing patient care, at least in the first stages of disease.

“The bumps” is a sensitive test to quantify tactile threshold of fingerpads. We tested its ability to detect distal axonal degeneration in peripheral neuropathies. The bumps device is a checkerboard-like surface divided into squares. Each square includes 5 colored circles, one of which contains a round bump (height range 2.5–14 μ m) to identify using the fingerpad. For each subject, we defined the height of the lowest bump such that it and the next 2 highest were detected. Functional data were compared to the density of Meissner corpuscles (MC) in fingertip skin biopsies processed and quantified according to standard procedures. Study population included 86 subjects (M/F = 51/35; age = 52.3 ± 15.1) affected by peripheral neuropathy and 15 subjects (M/F = 9/6; age = 50.4 ± 13.9) affected by CNS pathology as control group. Average threshold in the neuropathy group was 7.0 ± 4.4 μ m, average MC density was 9.3 ± 7.3/mm2. Average threshold in the CNS pathology group was 7.4 ± 5.1 μ m and average density of MC was 10.5 ± 6.8/mm2. Digital acuity threshold correlated with the density of Meissner corpuscles in the neuropathy group (r = 0.353; p The Bumps is a sensitive and easy-to-use tool, suitable to detect degeneration of sensory endings in peripheral neuropathies.

Sensory disturbances are part of the clinical picture of Parkinson's disease. Abnormalities in sensory processing, through a basal ganglia involvement, are thought to be responsible for the sensory dysfunction since sensory nerve conduction velocity (NCV) is usually normal. However, NCV does not examine small fibres or terminal endings of large sensory fibres, whereas skin biopsy is more suitable for these purposes. To evaluate peripheral sensory nerves in Parkinson's disease, we studied cutaneous free and encapsulated sensory nerve endings in 18 patients and 30 healthy controls using 3-mm punch biopsies from glabrous and hairy skin. Ten patients had additional skin biopsies from the contralateral side. Further evaluation included NCV and Quantitative Sensory Testing. Parkinson's disease patients showed a significant increase in tactile and thermal thresholds (P < 0.01), a significant reduction in mechanical pain perception (P < 0.01) and significant loss of epidermal nerve fibres (ENFs) and Meissner corpuscles (MCs) (P < 0.01). In patients with bilateral biopsies, loss of pain perception and ENFs was higher on the more affected side (P < 0.01). We found evidence suggesting attempts at counteracting degenerative processes as increased branching, sprouting of nerves and enlargement of the vascular bed. Morphological and functional findings did not correlate with age or disease duration. Disease severity correlated with loss of MCs and reduction in cold perception and pain perception. We demonstrated a peripheral deafferentation in Parkinson's disease that could play a major role in the pathogenesis of the sensory dysfunction.

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.

We studied the effect of chronic compression of median nerve at wrist on cutaneous innervation. Two-mm punch biopsies were obtained from third fingertip in 40 patients (32 female and 8 male, age range 30–74 years) with carpal tunnel syndrome. Patients with diabetes and other metabolic or endocrinological disorders or with electrophysiological findings of a diffuse neuropathy were excluded. By means of immunohistochemical techniques and confocal microscopy we counted epidermal nerve fibers, Meissner corpuscles and myelinated papillary endings. All patients underwent electrophysiological study and quantitative sensory testing using Semmes-Weinstein monofilaments for tactile threshold and Medoc TSA II Neurosensory analyzer for thermal threshold evaluation. Morphological and functional data were compared with age and sex matched controls. We observed abnormalities of epidermal nerve fibers, mechanoreceptors and their myelinated afferents in all patients. These abnormalities ranged from mild to marked and appeared more severe in patients with a longer disease duration and a slower conduction velocity along the median nerve.

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.