Unmyelinated Fiber Density Research Articles

Peripheral Nerve Regeneration Is Independent From Schwann Cell p75NTR Expression.

Schwann cell reprogramming and differentiation are crucial prerequisites for neuronal regeneration and re-myelination to occur following injury to peripheral nerves. The neurotrophin receptor p75NTR has been identified as a positive modulator for Schwann cell myelination during development and implicated in promoting nerve regeneration after injury. However, most studies base this conclusion on results obtained from complete p75NTR knockout mouse models and cannot dissect the specific role of p75NTR expressed by Schwann cells. In this present study, a conditional knockout model selectively deleting p75NTR expression in Schwann cells was generated, where p75NTR expression is replaced with that of an mCherry reporter. Silencing of Schwann cell p75NTR expression was confirmed in the sciatic nerve in vivo and in vitro, without altering axonal expression of p75NTR. No difference in sciatic nerve myelination during development or following sciatic nerve crush injury was observed, as determined by quantification of both myelinated and unmyelinated nerve fiber densities, myelinated axonal diameter and myelin thickness. However, the absence of Schwann cell p75NTR reduced motor nerve conduction velocity after crush injury. Our data indicate that the absence of Schwann cell p75NTR expression in vivo is not critical for axonal regrowth or remyelination following sciatic nerve crush injury, but does play a key role in functional recovery. Overall, this represents the first step in redefining the role of p75NTR in the peripheral nervous system, suggesting that the Schwann cell-axon unit functions as a syncytium, with the previous published involvement of p75NTR in remyelination most likely depending on axonal/neuronal p75NTR and/or mutual glial-axonal interactions.

Axonotmesis-evoked plantar vasodilatation as a novel assessment of C-fiber afferent function after sciatic nerve injury in rats

Quantitative assessment of the recovery of nerve function, especially sensory and autonomic nerve function, remains a challenge in the field of nerve regeneration research. We previously found that neural control of vasomotor activity could be potentially harnessed to evaluate nerve function. In the present study, five different models of left sciatic nerve injury in rats were established: nerve crush injury, nerve transection/suturing, nerve defect/autografting, nerve defect/conduit repair, and nerve defect/non-regeneration. Laser Doppler perfusion imaging was used to analyze blood perfusion of the hind feet. The toe pinch test and walking track analysis were used to assess sensory and motor functions of the rat hind limb, respectively. Transmission electron microscopy was used to observe the density of unmyelinated axons in the injured sciatic nerve. Our results showed that axonotmesis-evoked vasodilatation in the foot 6 months after nerve injury/repair recovered to normal levels in the nerve crush injury group and partially in the other three repair groups; whereas the nerve defect/non-regeneration group exhibited no recovery in vasodilatation. Furthermore, the recovery index of axonotmesis-evoked vasodilatation was positively correlated with toe pinch reflex scores and the density of unmyelinated nerve fibers in the regenerated nerve. As C-fiber afferents are predominantly responsible for dilatation of the superficial vasculature in the glabrous skin in rats, the present findings indicate that axonotmesis-evoked vasodilatation can be used as a novel way to assess C-afferent function recovery after peripheral nerve injury. This study was approved by the Ethics Committee for Laboratory Animals of Nantong University of China (approval No. 20130410-006) on April 10, 2013.

Bone marrow-derived mesenchymal stem/stromal cells reverse the sensorial diabetic neuropathy via modulation of spinal neuroinflammatory cascades

BackgroundDiabetic neuropathy (DN) is a frequent and debilitating manifestation of diabetes mellitus, to which there are no effective therapeutic approaches. Mesenchymal stem/stromal cells (MSC) have a great potential for the treatment of this syndrome, possibly through regenerative actions on peripheral nerves. Here, we evaluated the therapeutic effects of MSC on spinal neuroinflammation, as well as on ultrastructural aspects of the peripheral nerve in DN-associated sensorial dysfunction.MethodsC57Bl/6 mice were treated with bone marrow-derived MSC (1 × 106), conditioned medium from MSC cultures (CM-MSC) or vehicle by endovenous route following the onset of streptozotocin (STZ)-induced diabetes. Paw mechanical and thermal nociceptive thresholds were evaluated by using von Frey filaments and Hargreaves test, respectively. Morphological and morphometric analysis of the sciatic nerve was performed by light microscopy and transmission electron microscopy. Mediators and markers of neuroinflammation in the spinal cord were measured by radioimmunoassay, real-time PCR, and immunofluorescence analyses.ResultsDiabetic mice presented behavioral signs of sensory neuropathy, mechanical allodynia, and heat hypoalgesia, which were completely reversed by a single administration of MSC or CM-MSC. The ultrastructural analysis of the sciatic nerve showed that diabetic mice exhibited morphological and morphometric alterations, considered hallmarks of DN, such as degenerative changes in axons and myelin sheath, and reduced area and density of unmyelinated fibers. In MSC-treated mice, these structural alterations were markedly less commonly observed and/or less pronounced. Moreover, MSC transplantation inhibited multiple parameters of spinal neuroinflammation found in diabetic mice, causing the reduction of activated astrocytes and microglia, oxidative stress signals, galectin-3, IL-1β, and TNF-α production. Conversely, MSC increased the levels of anti-inflammatory cytokines, IL-10, and TGF-β.ConclusionsThe present study described the modulatory effects of MSC on spinal cord neuroinflammation in diabetic mice, suggesting new mechanisms by which MSC can improve DN.

Simultaneous Quantification of Unmyelinated Nerve Fibers in Sural Nerve and in Skin.

Peripheral polyneuropathies are common and their diagnosis may be challenging. We compared the results from sural-nerve and skin biopsies in 33 patients with a polyneuropathy and neuropathic pain examined in our hospital over a 6-year period. The biopsies were all from the same lower limb of each patient. Intraepidermal nerve fiber (IENF) densities in the skin were determined by fluorescence microscopy; unmyelinated fiber densities in sural-nerve biopsies (UFNB) were determined by electron microscopy. There was no correlation with age or gender in either biopsy type; there was a weak trend to correlation between UFNB density and IENF density, possibly because of the small sample size. The sensitivity of detection of quantitative abnormalities of unmyelinated fibers was better in the skin than in the nerves. Proximal and distal IENF densities were strongly correlated; and counts of UFNB were highly reproducible. Thus, quantification of unmyelinated fibers in sural-nerve and skin biopsies seem to be complementary. Sural-nerve biopsy may be required to confirm a specific diagnosis, to identify lesion mechanisms, and to devise therapeutic strategies, whereas skin biopsy seems to be more efficient in the follow-up of length-dependent polyneuropathies and in the diagnosis of neuropathic pain.

A reversible functional sensory neuropathy model

Small-fiber neuropathy was induced in young adult mice by intraperitoneal injection of resiniferatoxin (RTX), a TRPV1 agonist. At day 7, RTX induced significant thermal and mechanical hypoalgesia. At day 28, mechanical and thermal nociception were restored. No nerve degeneration in skin was observed and unmyelinated nerve fiber morphology and density in sciatic nerve were unchanged. At day 7, substance P (SP) was largely depleted in dorsal root ganglia (DRG) neurons, although calcitonin gene-related peptide (CGRP) was only moderately depleted. Three weeks after, SP and CGRP expression was restored in DRG neurons. At the same time, CGRP expression remained low in intraepidermal nerve fibers (IENFs) whereas SP expression had improved. In summary, RTX induced in our model a transient neuropeptide depletion in sensory neurons without nerve degeneration. We think this model is valuable as it brings the opportunity to study functional nerve changes in the very early phase of small fiber neuropathy. Moreover, it may represent a useful tool to study the mechanisms of action of therapeutic strategies to prevent sensory neuropathy of various origins.

Ultrastructural Morphometry of the Aortic Depressor Nerves and Extrinsic Renal Nerves: Similarities and Differences between Mice and Rats

Anatomical and physiological aspects involving rats and mice have shown similarities and differences between these experimental animal models. In cardiovascular physiology research, rats are being substituted by mice since mice are more susceptible to genetic manipulation. Nevertheless, little is known about mice normal anatomy and/or physiology to allow the correct interpretation of altered responses on genetically manipulated animals. We compared morphometric ultrastructural parameters of the aortic depressor nerve (ADN) and extrinsic renal nerve (SRN) between Wistar rats and C57BL/6J mice. After spontaneous activity recordings together with the arterial pressure pulse, ADN and SRN were dissected and prepared for light and transmission electron microscopy. Morphometry was performed with an image analysis software and took into consideration the fascicle area and diameter, myelinated and unmyelinated fiber number, density, area and diameter, myelin sheath area and the g-ratio. Comparisons were made for the same nerve between mice and rats and differences were considered significant when p<0.05. Both nerves were large in rats compared to mice, as were the myelinated fibers. On the other hand, unmyelinated fibers density, size and distributions were not different between species. These results suggest physiological differences on the fast conduction fibers between species, with possible different functional role. Morphological comparisons of the quantitative composition of peripheral nerves in different species and strains are very rare. Our study contributes to a morphological understanding of important nerves related to cardiovascular reflexes in two animal models. It also presents, for the first time, ultrastructural morphometric characteristics of the ADN in mice. This data provide a morphological basis for further studies involving functional investigations for reflex regulation of circulation in experimental model of hypertension and to improved cardiovascular system knowledge.

Unmyelinated Axons Show Selective Rostrocaudal Pathology in the Corpus Callosum After Traumatic Brain Injury

Axonal injury is consistently observed after traumatic brain injury (TBI). Prior research has extensively characterized the post-TBI response in myelinated axons. Despite evidence that unmyelinated axons comprise a numerical majority of cerebral axons, pathologic changes in unmyelinated axons after TBI have not been systematically studied. To identify morphologic correlates of functional impairment of unmyelinated fibers after TBI, we assessed ultrastructural changes in corpus callosum axons. Adult rats received moderate fluid percussion TBI, which produced diffuse injury with no contusion. Cross-sectional areas of 13,797 unmyelinated and 3,278 intact myelinated axons were stereologically measured at survival intervals from 3 hours to 15 days after injury. The mean caliber of unmyelinated axons was significantly reduced at 3 to 7 days and recovered by 15 days, but the time course of this shrinkage varied among the genu, mid callosum, and splenium. Relatively large unmyelinated axons seemed to be particularly vulnerable. Injury-induced decreases in unmyelinated fiber density were also observed, but they were more variable than caliber reductions. By contrast, no significant morphometric changes were observed in myelinated axons. The finding of a preferential vulnerability in unmyelinated axons has implications for current concepts of axonal responses after TBI and for development of specifically targeted therapies.

The spectrum of clinicopathological features in pure autonomic neuropathy

We assessed the clinicopathological features of nine patients with pure autonomic neuropathy, that is, neuropathy without sensory or motor deficits. The duration from symptom onset to diagnosis ranged from 1 month to 13 years. Of eight patients in whom serum antiganglionic acetylcholine receptor antibody was determined, four were positive. All patients who tested positive for this antibody manifested widespread autonomic dysfunction, with the exception of one patient who only experienced orthostatic hypotension. However, patients who were negative for the antiganglionic acetylcholine receptor antibody presented with partial autonomic failure. One of these patients had diffuse parasympathetic failure and generalized hypohidrosis but no orthostatic hypotension, which is clinically compatible with postganglionic cholinergic dysautonomia. Electron microscopic examination revealed a variable degree of reduction in unmyelinated fibers. Compared with normal controls, the patients had a significantly increased density of collagen pockets (p < 0.05). Additionally, the percentage of Schwann cell subunits with axons (out of the total number of Schwann cell subunits associated with unmyelinated fibers) was significantly decreased (p < 0.01). The density of unmyelinated fibers tended to decrease with increasing time between the onset of autonomic symptoms and biopsy (p < 0.05). In conclusion, the clinical and pathological features of pure autonomic neuropathy vary in terms of progression, autonomic involvement, presence of the antiganglionic acetylcholine receptor antibody, and loss of unmyelinated fibers.

Anatomy of corpus callosum in prenatally malnourished rats

The effect of prenatal malnutrition on the anatomy of the corpus callosum was assessed in adult rats (45-52 days old). In the prenatally malnourished animals we observed a significant reduction of the corpus callosum total area, partial areas, and perimeter, as compared with normal animals. In addition, the splenium of corpus callosum (posterior fifth) showed a significant decrease of fiber diameters in the myelinated fibers without changing density. There was also a significant decrease in diameter and a significant increase in density of unmyelinated fibers. Measurements of perimeter's fractal dimensions from sagittal sections of the brain and corpus callosum did not show significant differences between malnourished and control animals. These findings indicate that cortico-cortical connections are vulnerable to the prenatal malnutrition, and suggest this may affect interhemispheric conduction velocity, particularly in visual connections (splenium).

Dorsal cutaneous branch of the ulnar nerve: a light and electron microscopy histometric study

This study describes the normal morphology and morphometry of the dorsal cutaneous branch of the ulnar nerve (DCBU) in humans. Fourteen nerves of eight donors were prepared by conventional techniques for paraffin and epoxy resin embedding. Semiautomatic morphometric analysis was performed by means of specific computer software. Histograms of the myelinated and unmyelinated fiber population and the G-ratio distribution of fibers were plotted. Myelinated fiber density per nerve varied from 5,910 to 10,166 fibers/mm(2) , with an average of 8,170 ± 393 fibers/mm(2) . The distribution was bimodal with peaks at 4.0 and 9.5 µm. Unmyelinated fiber density per nerve varied from 50,985 to 127,108, with an average of 78,474 ± 6,610 fibers/mm(2) , with a unimodal distribution displaying a peak at 0.8 µm. This study thus adds information about the fascicles and myelinated and unmyelinated fibers of DCBU nerves in normal people, which may be useful in further studies concerning ulnar nerve neuropathies, mainly leprosy neuropathy.

A morphometric study of the vagus nerve in amyotropic lateral sclerosis with circulatory collapse

Amyotrophic lateral sclerosis (ALS) shows peculiar abnormalities of the autonomic nervous system, including sympathetic hyperactivity, which might result in sudden death. In general, the sympathetic hyperactivity could be caused by disruption of vagal inhibition. Our objective was to evaluate the vagus nerve morphometrically in autopsy cases of ALS with sympathetic hyperactivity and circulatory collapse (CC). We investigated 10 autopsied ALS patients, six of whom had exhibited autonomic storms or CC. We also examined 10 patients without ALS as controls, and one patient with Guillain-Barré syndrome (GBS) who died from CC, for comparison. After obtaining the visceral branch of the left vagus nerve at necropsy, we analyzed the density of the myelinated and unmyelinated fibers, and the fiber diameter distribution for each fiber. Results showed that the densities of both myelinated and unmyelinated fibers in ALS patients with or without CC were not significantly different from those in control patients. In contrast, the GBS patient showed marked reduction in the whole myelinated and large unmyelinated fiber density. In conclusion, the autonomic storms or CC due to sympathetic hyperactivity in ALS could not be ascribed to the deafferentation of the baroreflex, and more central neural pathophysiology should be investigated.

Sustained blockade of neurotrophin receptors TrkA, TrkB and TrkC reduces non-malignant skeletal pain but not the maintenance of sensory and sympathetic nerve fibers

Current therapies for treating skeletal pain have significant limitations as available drugs (non-steroidal anti-inflammatory drugs and opiates) have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomysin receptor kinase A (TrkA) has recently become an attractive target for inhibition of adult skeletal pain. Here we explore whether sustained administration of a selective small molecule Trk inhibitor that blocks TrkA, TrkB and TrkC kinase activity with nanomolar affinity reduces skeletal pain while allowing the maintenance of sensory and sympathetic neurons in the adult mouse. Twice-daily administration of a Trk inhibitor was begun 1 day post fracture and within 8 h of acute administration fracture pain-related behaviors were reduced by 50% without significant sedation, weight gain or inhibition of fracture healing. Following administration of the Trk inhibitor for 7 weeks, there was no significant decline in the density of unmyelinated or myelinated sensory nerve fibers, sympathetic nerve fibers, measures of acute thermal pain, acute mechanical pain, or general neuromuscular function. The present results suggest that sustained administration of a peripherally selective TrkA, B and C inhibitor significantly reduces skeletal pain without having any obvious detrimental effects on adult sensory and sympathetic nerve fibers or early fracture healing. As with any potential therapeutic advance, understanding whether the benefits of Trk blockade are associated with any risks or unexpected effects will be required to fully appreciate the patient populations that may benefit from this therapeutic approach.

Effects of 4-Methylcatechol on Skin Reinnervation: Promotion of Cutaneous Nerve Regeneration After Crush Injury

We assessed the effects of treatment with 4-methylcatechol (4MC), a known inducer of nerve growth factor, on peripheral nerve regeneration by analyzing cutaneous and muscular reinnervation in mice after sciatic nerve crush injury. At 3 months postinjury, the skin innervation index was significantly higher in the 4MC group than the control group (p=0.0002); there was also increased unmyelinated fiber density (p=0.0042) and unmyelinated fibers/Remak bundle (p = 0.001) in sural nerves, indicating unmyelinated nerve fiber regeneration. These changes were accompanied by increases of transcripts for nerve growth factor (p = 0.0026) and glial cell line-derived neurotrophic factor (p=0.03) in the 4MC group. In contrast, muscle innervation indices were similar in both groups and were higher than the skin innervation index (p < 0.0001). The regeneration of myelinated nerve fibers, as assessed by fiber density, diameter and g ratio analyses in sural nerves, and amplitudes of muscle action potential in sciatic nerves, was similar in both groups. Taken together, these data suggest that 4MC specifically promoted the regeneration of unmyelinated nerve fibers and reinnervation of the skin by increasing the expression of nerve growth factor and glial cell line-derived neurotrophic factor.

Sympathetic skin response in Parkinson's disease.

Sudomotor function in 83 patients with Parkinson's disease (PD) was evaluated using the sympathetic skin response (SSR) and sweat response to intradermal acetylcholine (ACh) injection. The incidence of abnormal SSRs (36.1%) increased, and the size of the response decrease with the severity of the illness. Neither the incidence of abnormal SSRs nor the amplitudes of the responses were influenced by levodopa or an anticholinergic agent. The SSR therefore can be used to evaluate the sudomotor efferent pathway in PD patients. In all the patients who had no SSR response, the local sweat response to ACh showed a reduced number of excitable sweat glands and low sweat volume. One patient, whose local sweat response to ACh was markedly impaired, had unmyelinated and acetylcholinesterase-positive fiber densities that were in the normal range in his biopsied sural nerve. The abnormal sweat response to ACh is considered to reflect the dysfunction of postganglionic sympathetic fibers in PD patients.

Ultrastructural Morphology and Morphometry of Phrenic Nerve in Rats

Despite numerous literature reports on the morphometry of the myelinated fibers of phrenic nerves in rats, a systematic study of the longitudinal and lateral symmetry of the unmyelinated fibers morphometry is not available. In this study, we have undertaken ultrastructural and morphometric studies of the phrenic nerve in adult rats, assessing two different levels (proximal and distal) from both right and left sides. Phrenic nerves of adult male Wistar rats were prepared for epoxy resin embedding and transmission electron microscopy. Morphometric analysis was performed with the aid of computer software, which took into consideration the unmyelinated fiber number, density, area, and diameter, as well as ratio between myelinated and unmyelinated fibers, and the percentage of the fascicular area occupied by the myelinated and unmyelinated fibers. Comparison of data from proximal and distal segments on the same side and from the same levels between sides was performed. Differences were considered significant when P < 0.05. The most important finding is that morphometric parameters of the phrenic nerve unmyelinated fibers in adult rats are both longitudinally and laterally symmetric. This study adds important morphometric information about the unmyelinated fibers of the phrenic nerves in adult rats for proximal and distal levels on both sides of the animal.

Congenital Insensitivity to Pain with Anhidrosis in Taiwan: A Morphometric and Genetic Study

Congenital insensitivity to pain with anhidrosis (CIPA) is characterized by insensitivity to pain, anhidrosis, recurrent hyperpyrexia, mild mental retardation, and self-mutilating behavior. We report 2 brothers, aged 20 and 18 years, who suffered from phenotypes of CIPA. Both brothers had a branch site mutation in intron 7 (IVS7-33 T→A) of the neurotrophic tyrosine kinase receptor type 1 gene. The electrophysiological studies showed no significant abnormal findings in sensory evoked potentials, motor evoked potentials to transcranial magnetic stimulation, or heart rate variations; sympathetic skin responses were absent. Morphometric study of their sural nerve histopathology revealed normal myelinated fiber density, 8,082 fibers/mm² and 5,637 fibers/mm²(normal 6,141 ± 421); decreased unmyelinated fiber density, 2,537 fibers/mm² and 2,211 fibers/mm² (normal 28,578 ± 8,669); increased axon size, 4.41 ± 1.59 µm and 5.33 ± 1.48 µm (normal 3.73 ± 1.45), and increased axon diameter (A)/myelin thickness (M) ratio (A/M), 3.47 ± 1.42 and 2.70 ± 1.07 (normal 2.49 ± 0.93). Scatterplot analysis of the G ratio (axon diameter:fiber diameter) did not show consistent results in the relationship between axon size and myelin thickness. In conclusion, the neuropathy of our CIPA patients included a marked reduction of small myelinated and unmyelinated fibers and a relatively increased axon size. This is the first CIPA family encountered in Taiwan.

Painful diabetic neuropathy (pdn): a morphological study of capsaicin receptor distribution

Capsaicin receptor TRPV1 is a non‐selective ligand‐gated channel activated by different noxious stimuli. Previous studies suggested that an increased density of TRPV1 positive axons in the skin could be involved in the pathogenesis of neuropathic pain. To investigate the expression of TRPV1 in PDN patients, skin biopsies from 6 patients with PDN and 10 controls, and sural nerve biopsies from 2 patients with PDN and 2 controls were studied using the polyclonal anti‐human TRPV1 antibody. Skin nerve fibers widely expressed TRPV1 immunoreactivity. The density of TRPV1 positive dermal and intra‐epidermal nerve fibers (IENF) did not differ from that obtained using the PGP 9.5 antibody (the standard marker for IEFN recognition). Confocal analysis demonstrated that TRPV1 and anti unique‐tubulin‐1 antibody (TuJ1) co‐localized in all axons. In PDN patients IENF density was significantly reduced, but no difference in immunostaining was detectable using TRPV1, PGP 9.5 and TuJ1 antibodies. Sural nerve unmyelinated fibers and few small‐myelinated fibers were intensely stained by TRPV1. DPN patients disclosed a severe reduction in unmyelinated fiber density, but residual fibers were recognized by TRPV1. These data suggest that TRPV1 is widely expressed in skin and sural nerve unmyelinated axons, but its expression is not increased in PDN. Other mechanisms, such as changes in proximal axon or neuron excitability, are more likely involved in the pathogenesis of neuropathic pain in diabetic neuropathy.

Nerve pathology in the type 1 diabetic dog: effects of treatment with sulindac.

The purpose of this study was to define pathological abnormalities in the peripheral nerve of a large animal model of long-duration type 1 diabetes and also to determine the effects of treatment with sulindac. Detailed morphometric studies were performed to define nerve fiber and endoneurial capillary pathology in 6 control dogs, 6 type 1 diabetic dogs treated with insulin, and 6 type 1 diabetic dogs treated with insulin and sulindac for 4 years. Myelinated fiber and regenerative cluster density showed a non-significant trend toward a reduction in diabetic compared to control animals, which was prevented by treatment with sulindac. Unmyelinated fiber density did not differ among groups. However, diabetic animals showed a non-significant trend toward an increase in axon diameter (p < 0.07), with a shift of the size frequency distribution towards larger axons, which was not prevented by treatment with sulindac. Endoneurial capillary density and luminal area showed a non-significant trend toward an increase in diabetic animals, which was prevented with sulindac treatment. Endoneurial capillary basement membrane area was significantly increased (p < 0.05) in diabetic animals, but was not prevented with sulindac treatment. We conclude that the type 1 diabetic dog demonstrates minor structural abnormalities in the nerve fibers and endoneurial capillaries of the sciatic nerve, and treatment with sulindac ameliorates some but not all of these abnormalities.

DENSITY OF SYMPATHETIC AXONS IN SURAL NERVE BIOPSIES OF NEUROPATHY PATIENTS IS RELATED TO PAINFULNESS

In this study, differences of unmyelinated nerve fiber density in sural nerve biopsy material from patients suffering from neuropathies of unknown origin with (n = 14) or without pain (n = 13) were analyzed. Immunocytochemistry was applied to differentiate afferent sensory and efferent sympathetic nerve fibers. All patients were evaluated for deficits of small fiber function with thermotesting, quantitative sudomotor‐axon reflex‐testing and testing of painfulness of mechanical stimuli before performing the biopsy. No difference was found between patients with and without pain concerning clinical deficits or results in any of the neurophysiological examinations. There were also no histopathological differences concerning the density of afferent C‐fibers. However, absolute and relative density of efferent sympathetic nerve fibers was significantly higher in patients with painful neuropathy (P &lt; 0.001), although none of the patients demonstrated clinical sympathetic abnormalities. We conclude that an imbalance between afferent and sympathetic nerve fiber density in the periphery may contribute to neuropathic pain even in those patients without obvious clinical autonomic disturbances.

A FAMILY WITH AUTOSOMAL DOMINANT MUTILATING NEUROPATHY NOT LINKED TO EITHER 3q13‐q22 OR 9q22 LOCI

The clinical separation of CMT2 from HSAN I may be difficult in some kindreds in which the sensory and motor symptoms and deficits are approximately alike. The genetic studies of CMT2 families are also controversial: one form of CMT2 was shown to map on chromosome 3q13‐q22 and named CMT2B; the HSAN I locus was mapped to 9q22.1. We describe a family with an autosomal dominant inheritance in which at least three members, belonging to three generations, developed a progressive neuropathy that combined limb weakness, wasting, and severe distal sensory loss leading to prominent mutilating changes. The onset was in late childhood, with progressive weakness in the lower limbs and later in the hands, resulting in a severe paralysis in the feet in one patient. Sensory disturbances were pronounced in 2 patients, and led to poorly healing ulcerations with osteomyelitis and amputations in one foot and mutilating lesions of both hands. Electrophysiological investigation revealed an axonopaty with consistent motor damage. Sural nerve biopsy showed a reduction in the density of both myelinated and unmyelinated fibers, with regenerating clusters.Linkage analysis using 5 microsatellite markers within to the critical 9q22 region was performed. Lod scores of this family calculated by LINKAGE package excluded association to this locus. We also performed linkage studies with chr. 3q13‐q22 markers associated to the CMT2b locus. Lod scores excluded this locus as well as responsiblity of the familial phenotype. The severity of motor involvement would suggest classifying the disorder of this family as a form of HSMN II rather than HSAN, indicating that a new locus is involved in the pathogenesis of this disorder.