G. Caporaso

Myelinated nerve endings in human skin

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 Aβ fibers, whereas cutaneous terminations of small myelinated Aδ 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. Muscle Nerve, 2007

Evaluation of sudomotor function in diabetes using the dynamic sweat test

Background: 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. Methods: 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 cm2 were evaluated. Results: We observed a significant reduction of sweating in diabetic subjects as compared to controls; sweat gland density per cm2 (59.7 ± 18.6 vs 83.7 ± 17.3; p < 0.05) 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. Conclusions: 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.

Cutaneous innervation of the human face as assessed by skin biopsy

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.

A multi-center, multinational age- and gender-adjusted normative dataset for immunofluorescent intraepidermal nerve fiber density at the distal leg

Quantification of intraepidermal nerve fibers (IENFs) in skin biopsies is now the tool of choice to diagnose small fiber neuropathies. An adequate normative dataset, necessary to assess normality cutoffs, is available for brightfield microscopy but not for immunofluorescence.

Postganglionic sudomotor denervation in patients with multiple system atrophy

Objective: To evaluate postganglionic autonomic involvement in multiple system atrophy (MSA). Methods: 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. Results: 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/μm3, p < 0.001, in fingertip; 0.7 ± 0.2 vs 1.9 ± 0.5 nm/µm3, p < 0.001, in thigh; 0.6 ± 0.2 vs 1.8 ± 0.4 nm/µm3, p < 0.001, in leg). Conclusions: 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.

Epidermal innervation morphometry by immunofluorescence and bright-field microscopy

We investigated the agreement between simple indirect immunofluorescence (IF) and bright‐field immunohistochemistry (BFI) on free‐floating sections for intraepidermal nerve fiber density (IENFD) quantification. Fifty‐five healthy subjects and 63 patients with probable small fiber neuropathy (SFN) underwent two adjacent skin biopsies at the distal leg processed by IF and BFI technique. Agreement between IENFD pairs obtained by each method was assessed by Bland–Altman testing. The area under the curve of the receiving operating characteristics (ROC) curves was used to compare the discrimination ability. The diagnostic judgment was based on sex and age‐adjusted normative values. IF and BFI showed good correlation (r = 0.81), with a ratio of about 2:1 and a mean difference of 5.5 ± 3.0 IENF per millimeter between paired measures, as demonstrated by linear regression and Bland–Altman test analyses. The square root transformation confirmed a Poisson distribution of the data and a fixed bias between IF and BFI measurements. The ROC curves analysis demonstrated a striking overlap between IF and BFI (0.83 and 0.82; p = 0.72). The diagnosis of SFN disagreed in only 6.7% of cases when the judgment was based on a difference of >1 IENF from 5% cut‐off value. IF and BFI showed comparable diagnostic efficiency when referred to appropriate normative reference values.

Small nerve fiber involvement in CMT1A.

Objective: To assess the involvement of small nerve fibers in Charcot-Marie-Tooth type 1A (CMT1A). Methods: 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. Results: 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 (p < 0.05) and with tactile thresholds (p < 0.05). Sudomotor nerve fiber loss correlated with ENF density (p < 0.05) and sweating output (p < 0.001). Conclusions: 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.

Loss of cutaneous large and small fibers in naive and l-dopa–treated PD patients

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.

Ross syndrome A lesson from a monozygotic twin pair

Ross syndrome is a rare autonomic disorder described by Ross in 19581 and characterized by tonic pupil, hyporeflexia, and segmental anhidrosis. A postganglionic cholinergic nerve degeneration of unknown cause underlies this condition2 although erratic association with Sjögren3 and antinuclear antibody4 positivity has led some authors to hypothesize immunologic causes. Familial cases have not been reported.

Evidence of small fiber neuropathy in a patient with Ehlers-Danlos syndrome, hypermobility-type.

http://dx.doi.org/10.1016/j.clinph.2015.12.004 1388-2457/ 2015 International Federation of Clinical Neurophysiology. Joint hypermobility syndrome (JHS) and Ehlers–Danlos syndrome hypermobility type (EDS-HT) are two clinically overlapping heritable connective tissue diseases. Their clinical identity has been recently suggested and many researchers are now considering EDS-HT and JHS as one and the same (i.e. JHS/EDS-HT) (Castori et al., 2013). As the molecular basis of JHS/EDS-HT is largely unknown, the diagnosis remains based exclusively on clinical criteria. Generalized joint hypermobility, joint complications and skin hyperextensibility are the major features of JHS/EDS-HT and chronic pain, fatigue, headache and dysautonomia represent the major causes of patient disability (Castori et al., 2013; De Wandele et al., 2014). We studied morphology and function of cutaneous sensory and autonomic innervation in a 41 years old male subject with JHS/EDS-HT who arrived to our observation for an unbearable neuropathic pain (burning feet) and chronic fatigue. His clinical history retraced the 3 different phases, hypermobility, pain and stiffness, as classically described in typical JHS/EDS-HT disease course (Castori et al., 2013). In fact, since childhood he had been suffering of recurrent sprains due to an abnormal ligamentous laxity, followed by development of progressive musculoskeletal pain in adulthood. At the age of 38 years he started to complain of burning pain, unpleasant cold feeling and mechanical allodynia involving distal lower limbs. These symptoms worsened progressively leading to severe sleep disturbance and marked limitation in walking and in daily activities. He also reported post-prandial abdominal discomfort, constipation, gastro-esophageal reflux, tension-type headache and fatigue. For the complex clinical picture he underwent several clinical and instrumental investigations including cerebral and spinal MRI, all unremarkable. At neurological examination we found reduced deep tendon reflexes, diminished muscle bulk, tone and strength and impaired vibratory perception. Gait was slow, unsteady and impossible on heels and toes because of pain. Perception of pin-prick and light touchwas impairedwith a symmetrical ‘‘stocking and glove” distribution. Beighton score was 5 out of 9 (hypermobility: scoreP 5). Blood analysis showed normal hepatic, renal, endocrinological, immunologic and metabolic function. Electrodiagnostic evaluation (Supplementary Table S1) (not including needle electromyography, that was refused by the patient) showed only a slight reduction of sural nerve conduction velocity which was however above the normal cutoff after correction for skin temperature (Oh, 2003). Ewing’s standard cardiovascular reflex tests (Supplementary Table S2) were unremarkable. The peak to peak amplitude of the sympathetic skin responses (SSR) evoked at feet by electrical