A histomorphometric study of unmyelinated fibers of the fibular nerve in Wistar rats.

There are few histomorphometric studies on the unmyelinated fibers of the fibular nerve in rats, and the number of experimental studies using this nerve has been increasing in the last years. Sixty-two percent of the endoneurial area from 10 fibular nerves of adult Wistar rats was scanned by electron microscopy, and digitized. The total number of unmyelinated axons (1.882 ± 271) was significantly lesser, and their axon diameters (0.2 µm to 2.8 µm) significantly higher than that determined in previous studies. The histogram peaked at 1 µm. The differences could be due to the nerve sampled area, the number and the age of the animals evaluated, and the laboratory techniques used. This study brings new and referential data to be used in experimental investigations involving histomorphometric evaluation of the rat fibular nerve.

A histomorphometric study of unmyelinated fibers of the fibular nerve in Wistar rats / Histomorfometria das fibras amielínicas do nervo fibular em ratos Wistar

ABSTRACT There are few histomorphometric studies on the unmyelinated fibers of the fibular nerve in rats, and the number of experimental studies using this nerve has been increasing in the last years. Sixty-two percent of the endoneurial area from 10 fibular nerves of adult Wistar rats was scanned by electron microscopy, and digitized. The total number of unmyelinated axons (1.882 ± 271) was significantly lesser, and their axon diameters (0.2 µm to 2.8 µm) significantly higher than that determined in previous studies. The histogram peaked at 1 µm. The differences could be due to the nerve sampled area, the number and the age of the animals evaluated, and the laboratory techniques used. This study brings new and referential data to be used in experimental investigations involving histomorphometric evaluation of the rat fibular nerve.

RESUMO Embora o nervo fibular de ratos venha sendo incluído progressivamente em maior número de estudos experimentais nos últimos anos, há poucos estudos a respeito das suas fibras amielínicas. Os nervos fibulares de 10 ratos Wistar adultos foram avaliados através de microscopia óptica e eletrônica. Varredura sistemática através de microscopia eletrônica de transmissão das áreas fasciculares da porção distal no nervo foi realizada. Em média, 62% da área endoneural foi digitalizada. O número total de axônios amielínicos encontrados (1.882 ± 271) foi significativamente menor e as medidas dos diâmetros axonais (0,2 µm a 2,8 µm) maiores do que o determinado em estudos prévios. O pico do histograma foi constituído por fibras de 1µm. As diferenças podem ser devidas à amostragem de maior área endoneural, ao número e à idade dos animais avaliados, e as técnicas laboratoriais utilizadas. Os dados obtidos podem ser considerados referenciais para o nervo fibular de ratos Wistar adultos.

Motor neuronopathy in Chediak-Higashi syndrome.

Chediak-Higashi syndrome is a rare autosomal recessive disease characterized by partial oculocutaneous albinism, recurrent pyogenic infections and the presence of giant granules in many cells such as leucocytes (hallmark of the disease). Neurological symptoms are rare. We describe two sisters who presented the same phenotype of slowly progressive motor neuronopathy (with Babinski sign in one patient); biopsy of the sural nerve showed an abnormal endoneurial accumulation of lipofuscin granules. We discuss these two observations and compare them with the few case reports of neuropathy in Chediak-Higashi syndrome.

A clinical and neuropathological study of Chinese patients with diabetic peripheral neuropathy.

OBJECTIVE: To examine whether the neuropathological and metabolic changes of peripheral nerves are correlated to clinical features in diabetes mellitus type 2 patients with peripheral neuropathy. METHODS: 147 type 2 diabetic patients with signs/symptoms of diabetic peripheralneuropathy (DPN) aged 53.4 ± 12.3 years and 134 healthy volunteers aged 55.5 ± 11.7 years were investigated for fasting plasma glucose (FPG), hemoglobin A1C (HbA1c), and red blood cell sorbitol (RBC sorbitol) in addition to nerve conduction velocity (NCV). Among the 147 diabetic patients, 10 patients underwent superficial peroneal nerve biopsy for light and electron microscopy. RESULTS: In the experimental group, the levels of HbA1c and RBC sorbitol showed significant increase compared with the controlled group, whereas motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) both showed decline and SNCV decreased at a greater extent. Morphologically, there were various degrees of nerve fiber loss, associated with axon degeneration and capillary luminal narrowing in 10 patients undergone nerve biopsy. CONCLUSION: The metabolic change of sorbitol, the consequently observed changes in NCV and histopathology of peripheral nerves are positively correlated with the duration of diabetes and overall level of blood glucose.

Entrapment syndrome of multiple nerves in graft-versus-host disease.

INTRODUCTION: Peripheral nerve entrapment syndromes are associated with hereditary neuropathy with liability to pressure palsies and a variety of rheumatic and endocrinological diseases. METHODS: We report a patient with entrapment syndromes of multiple nerves associated with chronic graft-versus-host-disease (GVHD) after allogeneic hematopoietic stem cell transplantation. Nerve ultrasound, histology, and ultrastructural changes were assessed. RESULTS: The 51-year-old man had developed severe deep dermal sclerosis due to chronic GVHD with a progressive polyneuropathy and entrapment syndromes of multiple nerves. Pre-stenotic enlargement was shown by nerve ultrasound. Histology demonstrated fibrosis of the epineurium with scarce infiltration of macrophages. Electron microscopy demonstrated alterations of the myelin sheaths and marked depletion of normal-sized myelinated nerve fibers. CONCLUSIONS: In addition to polyneuropathy, chronic GVHD can be associated with peripheral nerve entrapment syndromes and should be added to the differential diagnosis of compressive neuropathies.

An update on nerve biopsy.

Indications for nerve biopsy have decreased during the last 20 years. For the most part, this is a result of progress in the application of molecular biologic diagnostic testing for genetic peripheral neuropathies (PNs) and the increasing use of skin biopsy. The latter is primarily used to evaluate small-fiber PN, although it rarely discloses the specific etiology of a PN. Nerve biopsies are usually performed on either the sural or the superficial peroneal nerve, the latter in combination with removal of portions of the peroneus brevis muscle. The definite diagnosis of vasculitic lesions can be readily established on small paraffin-embedded nerve biopsy samples, although in some cases, the characteristic lesions are only apparent in muscle specimens. Other nerve specimens are routinely fixed in buffered glutaraldehyde and prepared for semithin sections and electron microscopy; frozen specimens are used for immunofluorescence studies. Electron microscopy is of great value in some cases of chronic inflammatory demyelinating polyneuropathies, monoclonal gammopathy, and storage diseases. Because more than 30 genes may be involved in genetic PNs, analysis of nerve lesions can direct the search for mutations in specific genes. Electron microscopy immunocytochemistry is mandatory in some cases of monoclonal dysglobulinemia. Thus, nerve biopsy is still of value in specific circumstances when it is performed by trained physicians and examined in a laboratory with expertise in nerve pathology.

Sex-related differences in recovery of cutaneous nociception after end-to-side nerve repair in the rat.

Sex-related differences in the recovery of cutaneous nociception after end-to-side nerve repair were examined in rats. Recovery of nociception in the dorsal foot was determined by skin pinch test 19 weeks after the proximal end of the distal stump of the transected peroneal nerve was sutured to the side of the adjacent intact sural nerve (end-to-side nerve coaptation). Axon sprouts in the recipient peroneal nerve were counted by light and electron microscopy. Recovery of nociception due to axon sprouting through the end-to-side coaptation was found in 87% of females and in 60% of males. The area of nociception was not significantly different (P=0.59) between females and males (13+/-8% and 11+/-9%, respectively). The number of myelinated axons in the recipient peroneal nerve (but not of unmyelinated axons) was significantly larger (P=0.028) in females (median=512, 25th and 75th percentiles: 467 and 594) than in males (median=322, 25th and 75th percentiles: 239 and 468). The majority of these axons in females and males were thin fibres, and recipient nerves in both groups were responsive to nerve pinch test. In conclusion, collateral sprouting of thin myelinated nociceptive axons into the end-to-side coapted nerve is more abundant in female than in male rats. However, recovery of cutaneous mechano-nociception due to sprouting of these axons was not different between the two sexes. Possible reasons are discussed.

The repair effects of Achyranthes bidentata extract on the crushed common peroneal nerve of rabbits.

In this study the effect of the Achyranthes bidentata root aqueous extract on regeneration of the crushed common peroneal nerve in rabbits by using a combination of electrophysiological assessment and histological aspect were investigated. The examined functional and morphological parameters suggest that A. bidentata extract could accelerate peripheral nerve regeneration in a dose-dependent manner (10-20 microg/kg, i.v.).

Ingrowth of sensory axons into an end-to-side coapted nerve stump after donor nerve crush in the rat.

BACKGROUND: Target innervation through an end-to-side (ETS) nerve coaptation depends on axonal sprouting from the donor nerve. Terminal axonal sprouting in a partially denervated target tissue is more extensive from a crushed donor nerve than from an intact donor nerve. We hypothesized that axonal sprouting into an ETS coapted recipient nerve could be stimulated by crushing the donor nerve. METHOD: Twenty-seven rats were randomised into 3 groups. In all, the distal stump of the transected peroneal nerve was sutured to the side of the sural nerve in place of the epineural window. The control group received no additional treatment. In the experimental groups, the sural donor nerve was crushed either 8 mm proximal (proximal crush group) or 8 mm distal to the coaptation site (distal crush group). Sixteen weeks after the surgery, histomorphometric analysis of the recipient peroneal nerve stump 4 mm distal to the coaptation site was performed. FINDINGS: The number of myelinated axons in the recipient peroneal nerve stump was 758 +/- 247 in the control group, 503 +/- 246 in the distal crush group and 211 +/- 96 in the proximal crush group. The differences between the groups were statistically significant (p < 0.05). The majority of myelinated axons were thin myelinated axons and the frequency distribution of their cross-sectional areas was similar in all groups. CONCLUSION: Contrary to our expectations, a significantly lower number of myelinated axons were present in recipient nerves in the proximal and distal crush groups than in the control group. This suggests that sensory axon ingrowth into an ETS coapted nerve cannot be enhanced by crushing the donor nerve.

Which myelinated sensory axons sprout into an end-to-side coapted peripheral nerve in the rat?

BACKGROUND: The high-threshold sensory afferents, which express trkA, are predominantly involved in terminal collateral sprouting in the skin of adult mammals. We explored which sensory axons are capable of sprouting into the end-to-side coapted nerve in the rat. METHOD: The distal stump of the transected peroneal nerve was sutured to the side of the uninjured sural nerve. After 36 weeks, sprouting of sensory axons into the end-to-side coapted nerve was assessed by the electrophysiologic measurements of compound action potential and by counting the myelinated axons. The neurons in the dorsal root ganglia (DRG) L5 whose axons sprouted into the end-to-side coapted nerve were retrogradely labelled by the fluorescent dye Fluorogold. The expression of trkA in sprouting DRG neurons was investigated by immunohistochemistry. FINDINGS: Predominantly thin myelinated axons were found in the end-to-side coapted peroneal nerve. Their mean conduction velocity (CV) was between the average CVs of the Adelta and Abeta fibres in the normal sural nerves. About 90% of the sprouting DRG neurons were small and medium sized, and about 10% were large. About 85% of sprouting DRG neurons was immunoreactive to trkA, but the rest were not. CONCLUSIONS: Mostly the high-threshold sensory afferents sprouted into the end-to-side coapted nerve, which resembles the collateral sprouting of sensory axons in the skin. However, our results suggest that also some low-threshold mechanoreceptors can sprout after the end-to-side nerve repair.

Zone of traction injury of the common peroneal nerve.

The history of functional motor recovery after reconstruction of traction injury to the common peroneal nerve is poor, regardless of technique and regardless of the experience of the surgeon doing the reconstruction. The hypothesis tested is that the failure of functional motor recovery after common peroneal nerve traction injury is because the zone of injury extends beyond the visible region of peroneal nerve in continuity injury and into the muscle entry zone of the motor nerve terminations. The opportunity arose to examine pathologically this suspected distal zone of injury in one patient. Histology from this patient was compared with that from a similar zone in a leg amputated for vascular indications. With a peroneal traction injury severe enough to cause disruption of the nerve, histology using Masson trichrome stain for collagen, neurofilament and S-100 stain for nerve fibers and Schwann cells demonstrated collagen deposition between the peroneal nerve and the muscle. This fibrosis was not seen in the in the same location from the amputation specimen. These findings were corroborated by electron microscopy of the myoneural junction in both specimens. It is concluded that stretch/traction injury zone extends into the myoneural junction, preventing otherwise successful neural regeneration through nerve grafts to reinnervate muscle. This suggests that with a distal zone of injury extending into the myoneural junction region, peroneal motor function may be better achieved by direct neurotization than with nerve grafting.

Comparable myelinated nerve pathology in feline and human diabetes mellitus.

The occurrence of diabetic neuropathy in cats provides an opportunity to study the development and treatment of neurological complications not present in diabetic rodent models, where few pathological alterations are evident. The present study further defines pathological alterations in nerve biopsies from 12 cats with spontaneously occurring diabetes mellitus. Peroneal nerve biopsies displayed concurrent injury to both Schwann cells and axons of myelinated fibers that was remarkably similar to that present in human diabetic neuropathy. In addition to demyelination, remyelination (constituting 20-84% of the total myelinated fiber population) was indicated by fibers with inappropriately thin myelin sheaths. Unlike our previous investigations, striking axonal injury was apparent, and consisted of dystrophic accumulations of membranous debris or neurofilaments, as well as degenerative fiber loss resulting in a 50% decrease in myelinated fiber density. In spite of extensive fiber loss, regenerative clusters were apparent, suggesting that axonal regeneration was not completely frustrated. These data highlight the potential utility of feline diabetic neuropathy as a model that faithfully replicates the nerve injury in human diabetes mellitus.

Nerve regeneration with the use of a poly(l-lactide-co-glycolic acid)-coated collagen tube filled with collagen gel.

AIM: The aim of this study was to develop a novel artificial nerve conduit and to evaluate its efficiency based on the promotion of peripheral nerve regeneration in rabbits. MATERIAL AND METHODS: The nerve conduit was made of a poly (l-lactide-co-glycolic acid)-coated collagen tube filled with collagen gel. The conduits were implanted into a 15 mm gap in the peroneal nerves of five rabbits. On the contralateral side, the defects were bridged with collagen-filled vein grafts. RESULTS: Twelve weeks postoperatively nerve regeneration was superior to the vein graft in the PLGA-coated collagen tube, both morphologically and electrophysiologically. CONCLUSION: The results indicate the superiority of the PLGA-coated collagen tube over vein grafts. Furthermore, they show that entubulation repair with this type of tube can support nerve regeneration over a nerve gap distance of at least 15 mm.

Peripheral nerve lesions associated with a dominant missense mutation, E33D, of the lamin A/C gene.

Some mutations of the lamin A/C gene may be responsible for a combination of distinct phenotypes, such as muscular dystrophy and peripheral neuropathy. We describe muscle and peripheral nerve lesions in a patient with a dominant lamin A/C missense mutation, E33D. Myopathic and neurogenic patterns coexisted on muscle biopsy specimens, whereas the peripheral nerve presented a mixture of axonopathy and Schwann cell hypertrophy. A few abnormal nuclei were found in muscle fibers and Schwann cells. Our morphological findings in this case attest to the predominant axonal damage, but suggest possible involvement of Schwann cells in neuropathies related to laminopathies.

Type grouping in skeletal muscles after experimental reinnervation: another explanation.

Type grouping signifies clustering of muscle fibres of the same metabolic type, and is a frequent finding in reinnervated muscles. To elucidate the mechanism behind it, the rat sciatic nerve was either autografted or grafted with hollow synthetic nerve grafts. Twelve weeks later the number and fibre area of the type I and type II muscle fibres in the gastrocnemic and anterior tibial muscles were determined after ATP-ase staining. The number and diameter of peroneal nerve fibres distal to the grafts were measured, and the number of Aalpha-nerve fibres was derived. Nearly all nerve and muscle morphometrical parameters changed equally in both experimental groups. However, type grouping occurred frequently only after autografting, whereas the number of nerve fibres and the number of Aalpha-nerve fibres increased in this group. Hence type grouping cannot be explained by increased intramuscular sprouting subsequent to a decrease in the number of innervating nerve fibres, as previously presumed. Regenerating axons branch along their course through the peripheral nerve. We propose that the probability of the occurrence of type grouping is related to the dispersion of sibling branches in the nerve. In the autograft, emerging branches are kept together by Schwann cell basal lamina scaffolds, in contrast to the hollow synthetic nerve grafts where the emerging branches become dispersed. Thus, in muscles reinnervated after autografting, the probability that nerve branches that arrive at a specific muscle territory are sibling branches is greater than after hollow tube grafting. Consequently, the probability that type grouping will occur is greater.

Amyloid neuropathy: a retrospective study of 35 peripheral nerve biopsies.

We performed a retrospective study of 35 peripheral nerve biopsies (PNBs) with amyloid deposits in the endoneurium. In every case, nerve lesions were studied on paraffin-embedded fragments (PEFs) and by ultrastructural examination (USE). In addition, muscle fragments were taken and embedded in paraffin. Immunohistochemistry was performed with anti-transthyretin (TTR) serum on 19 nerve and 15 muscle PEFs. Direct immunofluorescence with anti-light-chain sera was performed on frozen nerve fragments in 19 cases. Endoneurial amyloid deposits were easily identified on routine PEF in 26 cases, after Congo red or thioflavine staining in three, and by USE in six. A dramatic myelinated fiber loss was evidenced in 34 cases (77-2970 per mm2), and features of axonal degeneration were present in every case. Segmental demyelination was observed in 10 cases. A mutation in the TTR gene was present in 14 cases, with Met30 mutation in 10 and Ala49 in four members of the same family. Amyloid deposits were strongly marked by the anti-TTR serum in 11 other cases, twice in the endoneurium, five around muscle fibers, and four in both locations. In eight patients, light-chain positivity was evidenced in endoneurial deposits, lambda in six and kappa in two. Two other patients with monoclonal gammopathy did not present any light-chain fixation. In 17 cases, amyloidosis was disclosed by PNB and 13 had a TTR pathology; eight of them, over 65 years old, correspond to a late-onset form of familial amyloid polyneuropathy which is an underdiagnosed condition.

Experimental study on the regeneration of peripheral nerve gaps through a polyglycolic acid-collagen (PGA-collagen) tube.

We have developed a bioabsorbable polyglycolic acid (PGA) tube filled with collagen sponge (PGA-collagen tube) as a nerve connective guide, and compared its effectiveness with that of autograft in terms of nerve regeneration across a gap. The PGA-collagen tube was implanted into 24 beagle dogs across a 15-mm gap in the left peroneal nerve. The right peroneal nerve was reconstructed with the autograft harvested from the left side, as a control. After the surgery, the connective tissue extended from both cut ends in the PGA-collagen tube and connected again at the center. Pathologically, the collagen sponge in the tube provided adequate scaffolding for nerve tissue extension, and the nerve tissue reconnected within 3 weeks. Electrophysiologically, muscle-evoked potentials (MEPs) and compound nerve action potentials (CNAPs) were detected 18 days after the surgery. For up to 6 months postsurgery, CNAPs and somatosensory-evoked potentials (SEPs) on the PGA-collagen side had a shorter latency and larger peak voltage than those on the autograft side. The myelinated axons on the PGA side were larger in diameter than those on the autograft side. It is suggested that the PGA-collagen tube has the potential to be an effective alternative to conventional autografting for the repair of some peripheral nerve defects.

Regeneration of unmyelinated and myelinated sensory nerve fibres studied by a retrograde tracer method.

Regeneration of myelinated and unmyelinated sensory nerve fibres after a crush lesion of the rat sciatic nerve was investigated by means of retrograde labelling. The advantage of this method is that the degree of regeneration is estimated on the basis of sensory somata rather than the number of axons. Axonal counts do not reflect the number of regenerated neurons because of axonal branching and because myelinated axons form unmyelinated sprouts. Two days to 10 weeks after crushing, the distal sural or peroneal nerves were cut and exposed to fluoro-dextran. Large and small dorsal root ganglion cells that had been labelled, i.e., that had regenerated axons towards or beyond the injection site, were counted in serial sections. Large and small neurons with presumably myelinated and unmyelinated axons, respectively, were classified by immunostaining for neurofilaments. The axonal growth rate was 3.7 mm/day with no obvious differences between myelinated and unmyelinated axons. This contrasted with previous claims of two to three times faster regeneration rates of unmyelinated as compared to myelinated fibres. The initial delay was 0.55 days. Fewer small neurons were labelled relative to large neurons after crush and regeneration than in controls, indicating that regeneration of small neurons was less complete than that of large ones. This contrasted with the fact that unmyelinated axons in the regenerated sural nerve after 74 days were only slightly reduced.

Demyelinating peripheral neuropathy associated with hemophagocytic lymphohistiocytosis. An immuno-electron microscopic study.

We report the case of an 11-year-old male who developed subacute diffuse polyradiculoneuropathy, associated with digestive symptoms and Epstein-Barr virus infection. Parental consanguinity was present. The laboratory findings including bone marrow smear were consistent with hemophagocytic lymphohistiocytosis (HLH). Electrophysiological study of peripheral nerves revealed an intense and diffuse demyelinating process. The histological nerve lesions were severe and purely demyelinating. Most axons were intact. There was a diffuse infiltration of the nerve parenchyma by mononuclear cells. Immuno-electron microscopic study evidenced entry of macrophages into Schwann cell cytoplasm with dissociation of myelin sheaths. This boy died several months after the onset of the neuropathic symptoms. HLH is a rare genetic or acquired disorder in childhood characterized by abnormal immune activation, which induces an uncontrolled inflammatory response with sustained hyperactivation of T lymphocytes and macrophages. Only very rare cases of peripheral nerve involvement have been described in HLH. This is the first case showing that peripheral nerves, as other viscera, may be destroyed by the macrophagic infiltration, which characterizes HLH.

Assessment of the neurologic effects of dietary deficiencies of phenylalanine and tyrosine in cats.

OBJECTIVE: To determine the neurologic effects of reduced intake of phenylalanine and tyrosine in black-haired cats. ANIMALS: 53 specific pathogen-free black domestic shorthair cats. PROCEDURE: Cats were fed purified diets containing various concentrations of phenylalanine and tyrosine for < or = 9 months. Blood samples were obtained every 2 months for evaluation of serum aromatic amino acid concentrations. Cats were monitored for changes in hair color and neurologic or behavioral abnormalities. Three cats with neurologic deficits underwent clinical and electrophysiologic investigation; muscle and nerve biopsy specimens were also obtained from these cats. RESULTS: After 6 months, neurologic and behavioral abnormalities including vocalization and abnormal posture and gait were observed in cats that had received diets containing < 16 g of total aromatic amino acid/kg of diet. Electrophysiologic data and results of microscopic examination of muscle and nerve biopsy specimens from 3 cats with neurologic signs were consistent with sensory neuropathy with primary axonal degeneration. Changes in hair color were detected in cats from all groups receiving < 16 g of phenylalanine plus tyrosine/kg of diet. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that chronic dietary restriction of phenylalanine and tyrosine in cats may result in a predominantly sensory neuropathy. In cats, the long-term nutritional requirement for phenylalanine and tyrosine appears to be greater for normal neurologic function than that required in short-term growth experiments. Official present-day recommendations for dietary phenylalanine and tyrosine in cats may be insufficient to support normal long-term neurologic function.