Selective loss of small myelinated and unmyelinated fibers in Shy-Drager syndrome

Summary The number and sizes of myelinated and unmyelinated fibers in biopsied sural nerves in cases with Shy-Drager syndrome were studied in comparison with cases with olivopontocerebellar degeneration not having autonomic dysfunction. In Shy-Drager syndrome, there was a tendency for both small myelinated and unmyelinated fiber densities to be reduced in comparison with cases with olivopontocerebellar degeneration. Unmyelinated fibers more than 0.5 m in diameter were significantly reduced in Shy-Drager syndrome, a fact suggesting unmyelinated fiber degeneration. Multilamellated Schwann cell processes, isolated Schwann cell processes, and collagen pockets were more numerous and conspicuous in cases with Shy-Drager syndrome. It was concluded that unmyelinated fibers and small myelinated fibers in the peripheral nerves were involved selectively in Shy-Drager syndrome. The significance of the findings was discussed in terms of autonomic dysfunction observed clinically.     

Sural nerve biopsy findings in leprosy: a qualitative and quantitative light and electron microscope study in 4 treated cases of the lepromatous spectrum

Reports on biopsy findings in multifascicular nerves in lepromatous leprosy (LL) are rare and detailed morphometrical data are not available. In a case of early LL with normal electrodiagnostic findings in sural nerve, the present study revealed marked segmental de- and remyelination concomitant with the sequelae of considerable Wallerian degeneration of preferentially small myelinated fibers (MF) in spite of a normal number/nerve and density/mm2. Segmental de- and remyelination of several consecutive internodes in teased fibers suggests continuous bacterial spread via Schwann cells. In 2 more advanced LL-cases, nervous parenchyme was severely reduced, in a borderline lepromatous (BL) case obviously in part caused by cell infiltrates and granulomata. Distinct fascicle differences in MF-involvement were demonstrated by coefficients of variation of MF/mm2 and teased fiber preparations in LL, consistent with the hypothesis of initial focal spread of bacteria. Numbers and densities of endoneurial vessels were increased only in the later stages of LL. Enlargement of endoneurial area, due to different factors, was encountered except for the most severe LL-case with extensive endoneurial collagenization. Morphometric results were compared with those of other neuropathies. Intact and degenerating bacteria mostly in phagosomes of Schwann cells associated with unmyelinated axons and in macrophages were seen only in the early LL- and the BL-case. Sparse mononuclear cell infiltrates and small focal necrosis, present even in LL-cases, underline the complex pathogenesis of nerve fiber involvement.     

A morphometric study of human fetal sural nerve

Summary A morphometric study was performed on sural nerves from human fetuses at 15 to 36 weeks postovulation. There were no myelinated fibres at 15 and 16 weeks, but by 21 weeks there were 5,000/mm², rising to 25,000/mm² at 36 weeks. During the fetal period, the mean myelin lamellar count trebled and the g ratio (axon diameter: total fibre diameter) decreased from 0.90 to 0.75, although the axon diameter of myelinated fibres did not increase. The smallest myelinated axon diameter was 0.63 m, whereas the largest unmyelinated axon in a 1:1 relationship with a Schwann cell was 2.83 m, suggesting that axon size is unlikely to be the only stimulus for myelination. The density of unmyelinated axons that were the sole occupants of a Schwann cell fell considerably between 23 and 33 weeks, while the ratio of total unmyelinated axons to myelinated fibres decreased from 82:1 at 21 weeks to 6:1 at 36 weeks. Data for Schwann cell nuclear density and percentages of fibres cut through the nucleus are also presented.Supported by the Friedreich's Ataxia Group, and by grants from Ciba-Geigy Ltd., Basel and the London University Central Equipment Fund for the purchase of image analysis equipment     

Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves: quantitative microscopy and radioautography.

Cross-anastomoses and autogenous grafts of unmyelinated and myelinated nerves were examined by electron microscopy and radioautography to determine if Schwann cells are multipotential with regard to their capacity to produce myelin or to assume the configuration seen in unmyelinated fibres. Two groups of adult white mice were studied. (A) In one group, the myelinated phrenic nerve and the unmyelinated cervical sympathetic trunk (CST) were cross-anastomosed in the neck. From 2 to 6 months after anastomosis, previously unmyelinated distal stumps contained many myelinated fibres while phrenic nerves joined to proximal CSTs became largely unmyelinated. Radioautography of distal stumps indicated that proliferation of Schwann cells occurred mainly in the first few days after anastomosis but was also present to a similar extent in isolated stumps. (B) In other mice, CSTs were grafted to the myelinated sural nerves in the leg. One month later, the unmyelinated CSTs became myelinated and there was no radioautographic indication of Schwann cell migration from the sural nerve stump to the CST grafts. Thus, Schwann cell proliferation in distal stumps is an early local response independent of axonal influence. At later stages, axons from the proximal stumps cause indigenous Schwann cells in distal stumps from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously myelinated nerves become associated with unmyelinated fibres. Consequently, the regenerated distal nerve resembled the proximal stump. It is suggested that this change is possible because Schwann cells which divide after nerve injury reacquire the developmental multipotentiality which permits them to respond to aoxonal influences.     

Symmetric sarcoid polyneuropathy: analysis of a sural nerve biopsy

A 20-year-old woman with sarcoidosis had uveitis, subacute symmetric sensorimotor neuropathy, and noncaseous granulomas in biopsies of gastrocnemius muscle and sural nerve. Morphologic studies of the nerve confirmed the electrodiagnostic impression of an acute axonal and demyelinating neuropathy. Of 100 teased myelinated nerve fibers, 15% contained myelin ovoids and 24% demonstrated segmental demyelination. Quantitative analysis showed a reduction in the numerical density of myelinated fibers. By electronmicroscopy, unmyelinated fibers were largely spared. The exact mechanism of nerve fiber damage was not determined, but a local effect of the granuloma seemed likely, because most lesions were found in the endoneurium.     

Giant axonal neuropathy: a childhood disorder of microfilaments.

A sural nerve biopsy was performed on an 8-year-old boy with a chronic, slowly progressive polyneuropathy. Light and electron microscopy as well as teased nerve-fiber preparations demonstrated numerous giant axons filled with closely packed neurofilaments. Both myelinated and unmyelinated fibers were involved. Segmental demyelination, remyelination, and onion-bulb formation by multiple Schwann cell processes were observed, suggesting recurrent Schwann cell dysfunction. Abundant aggregates of cytoplasmic microfilaments occurred in Schwann cells, endothelial cells, perineurial cells, endoneurial fibroblasts, and endomysial fibroblasts. These findings support the proposal that giant axonal neuropathy is a generalized disorder of cytoplasmic microfilaments and that segmental demyelination occurs concomitantly with axonal and Schwann cell disease. The pathogenesis of this rare disorder is discussed with reference to experimental toxic neuropathies.     

Myelinated fibres in the human paravertebral sympathetic chain; quantitative studies on white rami communicantes

Myelinated fibres in the human sympathetic paravertebral chain were examined histologically and in single teased fibre preparations in various age groups in subjects dying from disorders not affecting primarily the autonomic nervous system. An increase in fibre density predominantly due to an increase in the number of small fibres was found in older subjects. A correlation between internodal length and fibre diameter was found but the internodes of sympathetic myelinated fibres are shorter for any given diameter than those found on fibres of comparable size in the sural nerve. A reduction in internodal lengths with advancing years was demonstrated. These observations are interpreted to show that Wallerian degeneration and segmental demyelination occur with increasing frequency in old age and that regeneration does not keep pace with successive degenerative events. The deterioration in function of the autonomic nervous system with advancing years may be attributed in part to the changes found in myelinated fibres in the paravertebral sympathetic chain.     

Morphometric evaluation of degenerative and regenerative changes in isoniazid-induced neuropathy

Morphometric studies of the pathologic changes were carried out on the peripheral nerves, spinal roots, and different levels of the Goll's tract in rats given isoniazid and killed 1, 2, 3, 4, 5, 6, 7, 14, and 30 days after intoxication. In teased fiber preparations, axonal degeneration was the main change present, and this was seen as early as day 2 in the peroneal and distal sural nerves. The frequency of myelinated fibers showing axonal degeneration was higher in the distal than the proximal sural nerve, and in the ventral than the dorsal root. In the group of rats killed on 5, 6, 7, and 14 days, a significant decrease of the myelinated fiber density was observed in the distal and proximal sural nerves, ventral root, and at the third cervical level of the Goll's tract. The degree of fiber degeneration was more severe in the distal than in the proximal sural nerve and in the third cervical than the fifth thoracic level of the Goll's tract. Preferential decrease of large myelinated fibers was noted in all the affected nerves. No definite abnormalities, however, were seen in nerve cells of the 6th lumbar spinal ganglia and anterior horn cells of the lumbar spinal cord on light microscopy. On 30 days, regeneration at varying degrees was discerned in all the affected nerves with significant increase of small myelinated fibers, particularly in the ventral root. The findings indicate that both centrally and peripherally directed myelinated axons are more affected in the distal than in the proximal segments while the neuronal cell bodies are spared. The spatio-temporal evolution of this pattern of change is compatible with the concept of the "dying back" process or central-peripheral distal axonopathy.     

Morphometric studies of normal sural nerves in children

Quantitative histologic studies of biopsies of normal sural nerves were performed on nine children aged 4 days to 17 years. Stereologic computerized procedures were used to determine total endoneurial area, size distribution and number of myelinated, unmyelinated fibers and Schwann cell nuclei per nerve and per square millimeter, and the ratio of myelin thickness to axonal diameter. There was an inverse linear relationship between the number of myelinated fibers per square millimeter and increasing age. A stronger correlation was found between the number of Schwann cell nuclei per nerve (P less than 0.01) and per square millimeter (P less than 0.001) and the logarithm of age. The slope of myelin thickness/axon diameter regression lines (P less than 0.001) changed with age in linear relationship (correlation coefficient: P less than 0.001). There were no age-dependent changes in the number and density of unmyelinated fibers, but the number of unmyelinated axons per Schwann cell subunit decreased with age. Size distribution histograms for myelinated fibers showed a unimodal profile in the newborn. A second peak at 6-7 micron appeared at age 3 months, shifting progressively to 9-11 micron at 14 years. The distribution of unmyelinated fibers was unimodal, with a peak around 0.8 micron, irrespective of age. There were marked individual variations in endoneurial area.     

Demyelinating peripheral neuropathy in Cockayne syndrome: a histopathologic and morphometric study.

The clinical and histopathological features of Cockayne syndrome in a 2-year-old girl are reported. Sural nerve biopsy revealed segmental demyelination and remyelination. The density of myelinated fibers, especially small ones, was decreased in comparison with an age-matched control. Although the total number of unmyelinated fibers showed no difference from that in the control, the number of small unmyelinated fibers was slightly increased. A study of teased fibers from the patient's nerve revealed that 1% of the fibers had segmental demyelination, and 7% showed remyelination. Ultrastructurally, demyelinated fibers were present sporadically. No degeneration of axons was evident. Our pathological and morphometric data for the sural nerve suggest the presence of primary demyelination in early childhood.     

Chlordecone intoxication in man. II. Ultrastructure of peripheral nerves and skeletal muscle.

Tremors, mental changes, opsoclonus, muscle weakness, gait ataxia, incoordination, and slurred speech developed in several employees in a Virginia chemical plant during the summer of 1974. Epidemiologic and clinical studies suggested that the chlorinated insecticide chlordecone (Kepone) was responsible. Severity of symptoms seemed directly related to dose and duration of exposure. Five sural nerve and six muscle biopsy specimens were examined by light microscopy and electronmicroscopy. The sural nerves were also evaluated by computerized morphometry, which showed considerable decrease in the number of unmyelinated fibers and lesser abnormalities of myelinated fibers. Compared with the nerves of the control subjects, those of patients may have had an increase in Reich and Elzholz bodies, and a modest increase in endoneurial collagen. There were occasional "collagen pockets," stacks of Schwann cell cytoplasmic membranes, redundant Schwann cell cytoplasmic folds, and fewer unmyelinated axons. The skeletal muscles contained increased amounts of lipofuscin and lipidlike droplets in subsarcolemmal areas and within intermyofibrillary spaces; the significance of this is unknown. Fiber size variability, type I predominance, and type grouping were present in three cases. All results strongly suggest that chlordecone is a neurotoxic agent predominantly affecting Schwann cells and unmyelinated fibers of peripheral nerves.     

Alcoholic neuropathy: Clinical,electrophysiological, and biopsy findings

Nerve conduction and biopsy findings from the sural nerve in 37 patients with alcohokic neuropathy were compared with findings in 6 patients who had neuropathy associated with postgastrectomy malnutrition. Half the patients with alcoholic neuropathy had both muscle weakness and sensory loss, half had only sensory impairment, but all had electromyographic signs of denervation. Only half the patients, with or without muscle weakness, had signs of malnutrition. In alcoholics, sural nerve conduction velocity was slowed to at most 60% of normal, correlating with loss of large fibers. These findings, together with a marked reduction in amplitude of the sensory potentials, are consistent with axonal loss. Myelinated fiber counts showed loss of small and large fibers in most nerves, retaining a bimodal distribution. Signs of regeneration were rare. Segmental demyelination was found in only 0.3% of teased fibers. Electron microscopy confirmed axonal degeneration of myelinated and unmyelinated fibers. Neuropathy after gastrectomy malnutrition was clinically similar to alcoholic neuropathy. Conduction velocities were slower than expected from the diameter of the largest myelinated fibers, however, and teased fibers showed segmental demyelination. The findings are against alcoholic neuropathy being due to malnutrition and suggest a toxic action on peripheral nerve.     

A subset of Schwann cells in peripheral nerves contain a 50-kDa protein antigenically related to astrocyte intermediate filaments

Antisera raised to the astrocyte intermediate filament structural protein stained elements in the peripheral nerves of several species. These elements were not associated with myelinated nerve fibers, were more common in splenic and vagus nerves than in the sciatic nerve, and persisted after nerve transection. In teased nerve preparations antigen-positive cells appeared to be the Schwann cells that surround small diameter, unmyelinated axons. Absorption of the antiserum with purified rat spinal cord 50-kDa protein or with bovine splenic nerve cytoskeletal extract blocked the reaction with CNS astrocyte processes or with PNS nerve fibers. Immunoblots of cytoskeletal preparations of bovine splenic nerve or rat sciatic nerve showed that the antigen from peripheral nerves comigrated at 50 kDa with antigen from bovine or rat spinal cord or cultured rat astrocytes. The CNS and PNS 50-kDa proteins from bovine tissues were subjected to limited digestion with Staphylococcus aureus protease V8. After separation on SDS-gels, antigenic peptides were detected by immunoblotting. The pattern of antigenic peptides for the CNS and PNS proteins were identical. We conclude that Schwann cells associated with nonmyelinated axons contain a cytoskeletal protein that is the same size and has the same peptide map as the major structural protein of astrocyte intermediate filaments.     

Unmyelinated nerve fiber degeneration in chronic inflammatory demyelinating polyneuropathy

To determine whether unmyelinated nerve fibers escape degeneration as one might expect in an immune response exclusively directed at myelin, we performed a morphometric examination of unmyelinated axons and myelinated nerve fibers in sural nerve biopsy specimens of 14 patients with a chronic inflammatory demyelinating polyneuropathy (CIDP) and of 12 age-matched normal controls. The numbers of unmyelinated axons, myelinated nerve fibers, denervated Schwann cell units and collagen pockets were quantified and related to the clinical and electrophysiological data of the patients with CIDP. In 4 patients with a rapid onset of the neuropathy and a highly elevated CSF protein, the numbers of both unmyelinated axons and myelinated nerve fibers were decreased equally. In 8 patients we found that the unmyelinated axons were relatively spared compared with the loss of myelinated nerve fibers. In these patients, however, the presence of denervated Schwann cell units and of collagen pockets was increased. We conclude that unmyelinated nerve fibers are affected in patients with CIDP.     

The numbers of unmyelinated and myelinated axons in normal and regenerated rat saphenous nerves

Counts have been made of the numbers of unmyelinated and myelinated axons in the proximal and distal stumps of regenerated rat saphenous nerves and from equivalent sites in normal nerves. In the proximal part of normal nerves there were averages of 1 045 myelinated axons and 4 160 unmyelinated ones. Regenerated nerves contained the same number of myelinated axons in their proximal stumps but there was a 40% reduction in the unmyelinated axon count. In the distal stumps of these nerves the myelinated axon count had increased by an average of 620; this comes about because some regenerated myelinated axons support more than one process in the distal stump. In contrast, the number of unmyelinated axons was reduced further, from a mean of 2 476 in the proximal stump to one of 2 219.

The sizes of Schwann cell units in the normal and regenerated nerves were also noted. Schwann cell units in the proximal and distal stumps of the regenerated nerves were smaller than those in the normal ones.

These changes associated with unmyelinated axons in regenerated nerves are likely to contribute to the sensory, vasomotor and sudomotor abnormalities that sometimes occur after peripheral nerve injury and regeneration.     

The normal sural nerve in man

Summary A combined light and electron microscope study of the normal sural nerve in 7 people aged 15–59 years is reported. Qualitative and quantitative studies of the Schwann cells and fibroblasts, myelinated and unmyelinated fibres are made in isolated fascicles.Schwann cells predominate over fibroblasts in the ratio of about 9-1. Most Schwann cells, almost 80%, are attached to unmyelinated fibres. Factors influencing the densities of these cells per cross sectional area are discussed.Some ultrastructural features of the myelinated fibres are described and their numbers per sq.mm and frequency distribution of their sizes are produced. An indirect method is proposed for assessing the mean internodal length for earch of the myelinated fibre size populations in cross sections of fascicles of normal nerves by estimating the proportion of myelinated segments cut through their nucleus.The ultrastructure of unmyelinated fibres is described and the identification of axons of extreme diameter is discussed. Their densities and size frequency histograms are the first to be reported in man by systematic electron microscope studies. The average ratio of unmyelinated to myelinated fibre density is about 3.7:1 though it varies in the fascicles of the different individuals.The implications of axonal diameter in the presence of myelin are commented on.British Council Scholar on leave of absence from Escuela de Medicina, Universidad Austral, Valdivia, Chile.     

Quantification of myelinated endings and mechanoreceptors in human digital skin

We used immunohistochemistry and confocal microscopy applied to fingertip punch biopsy to study glabrous skin innervation in 14 healthy subjects. In addition to epidermal nerve fibers, we quantified mechanoreceptors and their myelinated afferents. Using digital images and dedicated software, we calculated caliber, internodal and nodal length, and G-ratio of the last four internodes of the myelinated endings. In our skin samples, we found a mean density of 59.0 +/- 29.3 myelinated endings per square millimeter with a mean diameter of 3.3 +/- 0.5 microm and an internodal length of 79.1 +/- 13.8 microm. These findings indicate that Abeta fibers undergo drastic changes in their course from the nerve trunk to the target organ, with repeated branching and consequent tapering and shortening of internodal length. Our work demonstrates that skin biopsy can give information on the status of large myelinated endings as well as unmyelinated sensory and autonomic nerves. Since distal endings are primarily involved in distal axonopathy, skin biopsy can be more suitable than sural nerve biopsy to detect early abnormalities. In addition to diagnostic applications, this technique allows clarification of the mode of termination of Abeta fibers and their relationship with mechanoreceptors, leading to relevant electrophysiological speculations.     

Axonal degeneration and regeneration in sensory roots in a genital herpes model

Summary In a mouse model of genital herpes simplex virus type 2 (HSV-2) infection, roots of the lower spinal cord were examined 5 days to 6 months after inoculation. Using immunoperoxidase methods on paraffin sections, viral antigen was found in sensory ganglia, their proximal roots and distal nerves on days 5 and 6 after infection. In Epon sections, most mice had focal sensory root abnormalities in lower thoracic, lumbar or sacral levels. At days 7 and 10, lesions showed chiefly nerve fiber degeneration, particularly of large myelinated fibers. At 2 weeks, lesions contained relatively large bundles of small unmyelinated fibers with immature axon-Schwann cell relationships. From 3 to 6 weeks, lesions again contained many more small unmyelinated fibers than normal but, in increasing proportions, axons in bundles were isolated from their neighbors by Schwann cell cytoplasm, and Schwann cells having 11 relationships with axons showed mesaxon or thin myelin sheath formation. At later times, the proportion of small unmyelinated axons decreased in parallel with increased numbers of small myelinated axons. By 6 months, affected roots showed a relative reduction in large myelinated fibers, increased proportions of small myelinated fibers and Schwann cell nuclei. Numbers of unmyelinated fibers were reduced relative to 3- to 6-week lesions. Axonal degeneration and regeneration appears to be the chief pathological change in sensory roots in this model. If regenerated fibers arise from latently infected neurons, then establishment of latency is not a relatively silent event, but is associated with major long-lasting, morphologically detectable effects.     

Nerve regeneration through the cryoinjured allogeneic nerve graft in the rabbit

Summary To examine whether the 34-cm-long allogeneic basal lamina tubes of Schwann cells serve as conduits for regenerating axons in rabbits, allogeneic saphenous nerve, which had been predenervated and pretreated by freezing, were transplanted from Japanese White rabbits (JW) to New Zealand White rabbits (NW). Animals were killed 1, 2, 6, 8, and 14 weeks after transplantation, and the cytology at the mid-portion of the grafts was examined by electron microscopy. The distal portion of the host saphenous nerves was also examined 14 weeks after grafting. Myelin sheath debris was phagocytosed by macrophages, while the basal lamina of Schwann cells were left intact in the form of tubes. Regenerating axons were first found in such basal lamina tubes 2 weeks after grafting, and gradually increased in number. Host Schwann cells accompanied the regenerating axons behind their growing tips, separating them into individual fibers and forming thin myelin sheaths on thick axons by 6 weeks after grafting. Regenerating nerves were divided into small compartments by new perineurial cells. Newly formed blood vessels were situated outside the compartment 8 weeks after grafting. The percentage of myelinated fibers in the regenerating nerves was roughly 10% at 8 weeks and 30% at 14 weeks after grafting. The diameter of the regenerating axons, both myelinated and unmyelinated, was less than that of normal axons at all the stages examined. Numerous regenerating axons, some of which were fully myelinated, were found at the site 10 mm distal to the distal end of the graft 14 weeks after grafting. These results indicate that the Schwann cell basal lamina tubes of cryoinjured allogeneic nerves can serve as conduits for regenerating nerves in the 34-cm-long graft in the rabbit.     

Neurofibromatosis xenografts. Contribution to pathogenesis

We transplanted Schwann cells of 3 patients with neurofibromatosis from neurofibromas, sural nerve, and from a malignant schwannoma into sciatic nerves of immunoincompetent mice. Three and six months later, the grafts and distal nerve segments contained normal myelinated fibers. After rendering host animals immune competent again, neurofibroma and malignant schwannoma Schwann cells were rejected, but grafts retained normally myelinated fibers indicating that these were of mouse origin. Sural nerve Schwann cells from a neurofibromatosis patient were rejected also leaving naked axons in the grafted segments showing that human Schwann cells from the sural nerve of one patient had invested and myelinated the regenerating mouse axons. The nature of putative signals passing between axons and Schwann cells might be elucidated by the combination of human and animal cells in immunoincompetent host nerves. Hypothetical signals for myelination of mouse axons were normally received by sural nerve Schwann cells of a patient with neurofibromatosis, but not by Schwann cells from neurofibromas or malignant schwannomas.