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

Functional Electrical Stimulation (FES), is a tecnique that uses low-energy electrical pulses to artificially generate muscle contractions, in individuals with damages regarding the central nervous system. The application of FES in clinical environment involves both patients care and rehabilitation. Aim of this work is to introduce a clinical FES protocol for upper limbs rehabilitation, in order to assist and train the execution of complex movement, such as flexion- extension of wrist and fingers and palmar prehension. The new FES protocol has been tested on a cohort of five subjects with different upper limb neuromotor deficits, during their rehabilitation. The benefits deriving from the application of the new FES protocol have been evaluated by comparing specific quantitative electromyographic parameters assessed before and after the treatment. Results show effective improvements in performances of 4 patients out of 5.

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.

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.

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.

Meissner corpuscles are supposed to play a primary role in generating tactile responses. To verity this hypothesis, we combined electrophysiological and morphological methods. In a group of twelve patients affected by congenital or acquired neuropathies, electrical and tactile evoked potentials were near-nerve recorded along the median nerve. The density of Meissner corpuslces was calculated in the fingertip, exactly in the same area stimulated by the tactile probe. Using immunohistochemical techniques we determined in the skin specimens the density of normal and atrophic Meissner corpuscles and of myelinated nerve fibers per square millimeter. We demonstrated that tactile potentials depend exclusively on number of normal Meissner corpuscles, while electrical potentials are also correlated with atrophic receptors. Tactile stimulation is a more suitable method than electrical stimulation to discriminate between functional and not functional mechanoreceptive units.

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

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

Sensory symptoms in CMT1A are generally attributed to large fiber. However, an involvement of small fibers has been recently brought up. We performed an extensive clinical, morphological and functional study of cutaneous somatic and autonomic innervation in 10 patients affected by CMT 1A, in order to assess small fiber involvement in this condition. We used a 13 items questionnaire and QST to evaluate symptoms and function of somatic small fibers, DST, SSR and Ewing tests to evaluate autonomic function. Morphological analysis of cutaneous innervation included lENF density and sudomotor nerves assessment. Twenty age-and-sex-matched healthy subjects were used as controls for morphological data. All patients complained of at least 2 symptoms related to small fiber dysfunction. Nine out of ten patients had an increase in thermal/pain thresholds indicating an impairment of both C and A-delta fibers. DST revealed hypoidrosis in all patients (41.8 ± 22.8 vs 83.7 ± 17.3 sweat drops/cm 2 ). SSR amplitude at feet was significantly lower ( p p

Although the involvement of large myelinated sensory fibers in Friedreich's ataxia (FA) is well documented, an impairment of unmyelinated fibers has not been described. We demonstrate an involvement of cutaneous unmyelinated sensory and autonomic nerve fibers in FA patients. We performed a morphological and functional study of cutaneous nerve fibers in 14 FA patients and in a population of control subjects. We used immunohistochemical techniques and confocal microscopy applied to punch skin biopsies from thigh, distal leg, and fingertip, and compared the density of epidermal nerve fibers (ENFs) with the results of mechanical pain sensation and thermal and tactile thresholds performed on hand dorsum, thigh, distal leg, and foot dorsum. We observed in our patients a statistically significant loss of ENFs, a reduced innervation of sweat glands, arrector pilorum muscles and arterioles, and an impairment of thermal and tactile thresholds and mechanical pain detection.

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

Sensory potentials evoked by a physiological stimulus, tactile stimulation, can be recorded along a nerve. These responses express the function of the whole distal part of the sensory pathway, including mechanoreceptors and their afferent fibers, structures by-passed by the electrical stimulation. Using immunohistochemical procedures it is possible to investigate morphology and distribution of the neural structures presumably involved in the generation of tactile responses in digital glabrous skin. The mechanoreceptor density is higher in fingertip and in younger people. The area of the tactile potential tends to be smaller in older people and following the stimulation of the proximal phalanx with respect to the stimulation of the distal one, suggesting a correlation between tactile potential area and Meissner corpuscles' density. To assess the exact role of Meissner corpuscles in tactile potentials formation, we investigated in normal subjects the morphology, density and distribution of mechanoreceptors and their afferent myelinated fibers, and tactile and electrical responses. To correlate electrophysiological and morphological data we recruited a group of patients affected by congenital and acquired sensory neuropathies that underwent both skin biopsy and electrophysiological tests. We found a significant correlation between the area of the tactile potentials and the density of normal Meissner corpuscles and papillary myelinated fibers. These data support the hypothesis that Meissner corpuscles play a significant role in the generation of tactile potential. Therefore tactile stimulation is a reliable way to investigate the sensory pathway extreme termination.

We studied orthodromic sensory and motor conduction along median and ulnar nerves in 52 patients suspected for CTS (60 hands) and in 30 control subjects. Patients underwent electrophysiological examination before and up to 24 months after surgical decompression. We evaluated the following electrophysiological tests: 1. sensory nerve conduction velocity (SNCV) from digit III and V to wrist (dSNCV) and from palm to wrist (pSNCV) 2. pSNCV/dSNCV ratio in median nerve 3. median/ulnar pSNCV ratio 4. Distal latency (DL) 5. SNAP and CMAP amplitudes. In 3 patients, presurgical neurographic findings were normal. According to median dSNCV, the remaining 49 patients were classified into 3 groups: A = normal, B = mild slowing, C = marked slowing. In all B and C group patients, median pSNCV was significantly reduced. In group A, pSNCV was reduced in 16 patients (53%), pSNCV/dSNCV ratio was abnormal in 29 patients (97%), and median/ulnar pSNCV ratio in all subjects (100%). DL, SNAP, and CMAP amplitudes abnormalities were detected only in group B and C. Disease duration in the 3 goups was inversely correlated with SNCV and DL abnormalities. After surgery, neurographic findings returned within the normal range earlier in group A, then in group B; group C showed an incomplete recovery of digit and palm SNCV at 24 months. Median palm/digit SNCV and median/ulnar pSNCV ratios were the most sensitive tests, providing a reliable tool for the assessment of very early phase of CTS and for the subsequent surgery treatment.

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