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Facial-onset sensory-motor neuronopathy (FOSMN) first manifests with trigeminal sensory loss and pain, then spreads to bulbopontine and spinal motoneurons, sometimes with a fatal outcome.1,2

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.

Objective: To evaluate, by skin biopsy, dermal nerve fibers in 31 patients with 3 common Charcot-Marie-Tooth (CMT) genotypes (CMT1A, late-onset CMT1B, and CMTX1), and rarer forms of CMT caused by mutations in RAB7 (CMT2B), TRPV4 (CMT2C), and GDAP1 (AR-CMT2K) genes. Methods: We investigated axonal loss by quantifying Meissner corpuscles and intrapapillary myelinated endings and evaluated morphometric changes in myelinated dermal nerve fibers by measuring fiber caliber, internodal, and nodal gap length. Results: The density of both Meissner corpuscles and intrapapillary myelinated endings was reduced in skin samples from patients with CMT1A and all the other CMT genotypes. Nodal gaps were larger in all the CMT genotypes though widening was greater in CMT1A. Perhaps an altered communication between axons and glia may be a common feature for multiple forms of CMT. Internodal lengths were shorter in all the CMT genotypes, and patients with CMT1A had the shortest internodes of all our patients. The uniformly shortened internodes in all the CMT genotypes suggest that mutations in both myelin and axon genes may developmentally impede internode formation. The extent of internodal shortening and nodal gap widening are likely both important in determining nerve conduction velocities in CMT. Conclusions: This study extends the information gained from skin biopsies on morphologic abnormalities in various forms of CMT and provides insights into potential pathomechanisms of axonal and demyelinating CMT.

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.

Parkinson's disease is the second most common neurodegenerative disorder in the world. Assumed that gait dysfunctions represent a major motor symptom for the pathology, gait analysis can provide clinicians quantitative information about the rehabilitation outcome of patients. In this scenario, wearable inertial systems for gait analysis can be a valid tool to assess the functional recovery of patients in an automatic and quantitative way, helping clinicians in decision making. Aim of the study is to evaluate the impact of the short-term rehabilitation on gait and balance of patients with Parkinson's disease. A cohort of 12 patients with Idiopathic Parkinson's disease performed a gait analysis session instrumented by a wearable inertial system for gait analysis: Opal System, by APDM Inc., with spatial and temporal parameters being analyzed through a statistic and machine learning approach. Six out of fourteen motion parameters exhibited a statistically significant difference between the measurements at admission and at discharge of the patients, while the machine learning analysis confirmed the separability of the two phases in terms of Accuracy and Area under the Receiving Operating Characteristic Curve. The rehabilitation treatment especially improved the motion parameters related to the gait. The study shows the positive impact on the gait of a short-term rehabilitation in patients with Parkinson's disease and the feasibility of the wearable inertial devices, that are increasingly spreading in clinical practice, to quantitatively assess the gait improvement.

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

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.

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.

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.

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.

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.

We describe the occurrence of small fiber neuropathy in a patient affected by Chagas disease in the indeterminate phase. After the exclusion of all the possible etiologies of small fiber neuropathy, the disorder was considered related to Trypanosoma cruzi infection. Although a peripheral involvement has been described in Chagas disease, this is the first report of a selective involvement of small fibers.