Cyclosporin A retards the wallerian degeneration of peripheral mammalian axons.

The distal (anucleate) segments of mammalian peripheral axons typically undergo complete Wallerian degeneration within 1-3 days after severance from their cell bodies, unlike invertebrates and lower vertebrates, where anucleate axons do not degenerate for weeks to months. This rapid Wallerian degeneration in mammals could be due to a more efficient immune system and/or to differences in calcium-dependent pathways relative to invertebrates and lower vertebrates. To suppress the immune system and to inhibit calcium-dependent pathways in axons, we gave daily subcutaneous injections of cyclosporin A (CsA: 10 mg/kg) to Sprague-Dawley rats for 7 days before, and 5 days after, severing their right ventral tail nerves. To confirm that CsA suppressed the immune system, white blood cell density was measured in CsA-treated and in non-treated rats. Our data showed that the number of surviving anucleate myelinated axons at 5 postoperative days in CsA-treated rats was significantly higher than the number in non-treated rats. Anucleate unmyelinated axons in the ventral tail nerve also exhibited better survival in CsA-treated rats than in nontreated rats. These results are consistent with the hypothesis that the immune response and/or calcium-dependent pathways play important roles in the rapid Wallerian degeneration of anucleate mammalian axons.

Glycosaminoglycans in nerve injury: II. Effects on transganglionic degeneration and on the expression of neurotrophic factors.

Injury to the sciatic nerve leads to the transganglionic degeneration of sensory axons and to the induction of neurotrophins and p75 nerve growth factor receptor synthesis by the denervated Schwann cells. Sciatic nerve axotomy caused a marked loss of substance P and of met-enkephalin in the lumbar cord. Substance P immunostaining and pre-proenkephalin mRNA expression were reduced in the dorsal horn layers I and II ipsilaterally to the lesion. Treating rats with low doses (0.25 mg/kg) of heparin or COS 8, a natural glycosaminoglycan mixture with low anticoagulant activity, the peptide loss was prevented and the content increased of about 50% above control values. The effects of COS 8 treatment were also evident on Schwann cells. COS 8 augmented the increase of nerve growth factor, brain-derived neurotrophic factor, and NT-3 mRNA expression in the distal stump of the axotomized sciatic nerve. Therefore, it can be concluded that glycosaminoglycans neuroprotective effects on lesioned sensory axons might have been mediated by the dramatic promotion of neurotrophin synthesis. Although the in vitro studies (Lesma et al.: J Neurosci Res, 1996) suggested also a likely direct effect as extracellular matrix components that is not mediated by trophic factors.

Microglial response to N-methyl-D-aspartate-mediated excitotoxicity in the immature rat brain.

The intracerebral injection of N-methyl-D-aspartate (NMDA) has been proposed as a model for hypoxic-ischemic insult in the immature brain. In this light, the aim of this study was to describe the time course of the microglial reaction in the areas undergoing primary degeneration at the site of intracortical NMDA injection as well as in areas undergoing secondary anterograde and/or retrograde degeneration. Fifty nanomoles of NMDA were injected in the sensorimotor cortex of 6-day-old rats. After survival times ranging from 10 hours to 28 days, cryostat sections were stained for routine histology and for the demonstration of microglial cells by means of tomato lectin histochemistry. The areas affected by primary degeneration caused by the intracortical injection of NMDA were the neocortex, the hippocampus, and the rostral thalamus. Secondary degeneration (retrograde and anterograde) was observed in the ventrobasal complex of the thalamus. The cortical lesion also caused Wallerian degeneration of the cortical descending efferents as observed in the basilar pons. Microglial reactivity in all these areas was present at 10 hours postinjection and was restricted to the areas undergoing neuronal or axonal degeneration. Reactive microglial cells were stained intensely and showed a round or pseudopodic morphology. At 3 days, an apparent increase in the number of tomato lectin-positive cells was observed in the areas undergoing neuronal death. By 7 days after the injection, the lesion became nonprogressive, and by 14 and 28 days, microglial cells showed moderate lectin binding and a more ramified morphology.

Activation and proliferation of murine microglia are insensitive to glucocorticoids in Wallerian degeneration.

Activation and proliferation of microglia are commonly described in the central nervous system after a wide range of insults, but the mechanisms that regulate their phenotype in vivo are still poorly understood. We have studied the effect that adrenalectomy and dexamethasone treatment have on the proliferation and activation of microglia during Wallerian degeneration of the optic nerve in BALB/c mice. We found that the onset and rate of microglia proliferation is independent of glucocorticoids. There was an increase in F4/80-positive cells 3 days after optic nerve crush, with a peak at 7 days, both in the optic nerve and its target, the superior colliculus. The numbers of F4/80-positive cells remained high up to 3 weeks after crush, the longest time point examined. We also found that up-regulation of F4/80 and the complement receptor type 3 and expression of major histocompatibility complex class II antigens were not affected by adrenalectomy or dexamethasone treatment. These observations show that, unlike microglia in vitro or peripheral macrophages, microglia do not readily respond to glucocorticoids, which could indicate a lack of or reduced expression of glucocorticoid receptor in these cells.

Differential expression of interleukin-10 mRNA in Wallerian degeneration and immune-mediated inflammation of the rat peripheral nervous system.

Interleukin-10 (IL-10) is a potent immunosuppressant cytokine which downregulates MHC class II antigen expression and inflammatory cytokine production. In this study we localized mRNA for IL-10 in the rat peripheral nervous system (PNS) by nonradioactive in situ hybridization using a digoxygenin-labeled riboprobe specific for rat IL-10. IL-10 mRNA was expressed by some Schwann cells (SCs) in the normal sciatic nerve. During Wallerian degeneration, SCs strongly expressed IL-10 mRNA between days 2 and 4 after transection. By day 14 only occasional cells were positive for IL-10 mRNA. The vast majority of ED1-positive macrophages were IL-10 negative after axotomy. Contrastingly, infiltrating macrophages expressed IL-10 mRNA conincident with beginning clinical recovery in experimental autoimmune neuritis (EAN), the rat model of human Guillain-Barré syndrome. Our data suggest that SCs provide a constitutive immunosuppressant system in the PNS. In EAN additional macrophage-derived IL-10 may be important for the resolution of the T cell-mediated immune response.

The rate of Wallerian degeneration in cultured neurons from wild type and C57BL/WldS mice depends on time in culture and may be extended in the presence of elevated K+ levels.

Wallerian degeneration of severed axons is delayed in C57BL/WldS mice. We have examined this further in cultured sympathetic, sensory and CNS neurons using superior cervical ganglion (SCG), dorsal root ganglion (DRG) and cerebellar granule neurons respectively from neonatal mice. We found that the time taken for the neurites to degenerate depends upon the length of time in culture before cutting, reaching a maximum by approximately 7 days when C57BL/WldS neurites survive for > 6 days after axotomy. The onset of degeneration could also be extended in SCG and DRG neurites from wild type C57BL/6J mice. After 7 days in culture these neurites normally degenerate within approximately 12-16 h of axotomy, but in the presence of raised K+ (50 mM) degeneration often did not begin until a further 2 days had lapsed. Under similar conditions degeneration of neurites from C57BL/WldS mice was also found to be further delayed, extending survival from approximately 5-6 days to > 7 days. The L-type Ca2+ channel blockers nifedipine (5 microM) and verapamil (10 microM) both blocked the effect of raised [K+], although not completely. Thapsigargin, which raises cytoplasmic [Ca2+], and the cAMP analogue 8-(4-chlorophenyl-thio)cAMP were also able to delay degeneration, but only when added 24 h prior to axotomy. These results show that it is possible to influence the course of Wallerian degeneration and that increases in levels of cytoplasmic Ca2+ can protect neurites from its onset.

Inhibitory effects of thalidomide on cellular proliferation, endoneurial edema and myelin phagocytosis during early wallerian degeneration.

In addition to the well-known teratogenic effect of thalidomide, previous studies have revealed mild immunosuppressive properties and, more recently, an anti-angiogenic activity. To find out more about the specificity of these effects we studied the influence of orally administered thalidomide on Wallerian degeneration in rats. Wallerian degeneration is a potent experimental model for studying reproducible cell proliferation in vivo. Examination of distal nerve segments of transected sciatic nerves from rats that had been treated with thalidomide (2 x 250 mg/kg per day) revealed a significant reduction of endoneurial cell counts at 10-15 days after surgery compared to that seen in controls. This effect was not statistically significant, at a very early stage of Wallerian degeneration, i.e., at 5 days after transection of the nerve. Subperineurial edema and phagocytosis was also reduced, although this was not statistically significant. This apparently nonspecific inhibitory effect of thalidomide during early Wallerian degeneration shown in the present study should be investigated further for its possible relationship to other previously established inhibitory activities of thalidomide, especially its immunosuppressive effect in man.

Transient immunohistochemical labelling of rat retinal axons during Wallerian degeneration by a monoclonal antibody to neurofilaments.

Immunohistochemical labelling with the monoclonal antibody SMI32 to non-phosphorylated epitopes on neurofilament proteins of high molecular weight class was low in rat central optic fibers of controls. After unilateral transection of optic nerve, a strong, transient increase of labelling with SMI32 occurred in degenerating fibers of optic tract at 2 and 4 days, which then declined at 8 and remained low at 21 days. Consequently, immunostaining with SMI32 may serve as a positive marker for degenerating fibers in rat optic system.

Sensory and motor neuropathy caused by excessive ingestion of vitamin B6: a case report.

We describe a patient who developed a severe sensory and a mild motor neuropathy. This syndrome was due to massive and prolonged ingestion of vitamin B6 (10 g daily for 5 years). To our knowledge this is the first published case of motor neuropathy caused by chronic abuse of vitamin B6.

Quantitative analysis of neuronal damage induced by tri-ortho-cresyl phosphate in Wistar rats.

A quantitative analysis of neuronal damage was performed on the fasciculus gracilis (FG) of the cervical spinal cord in male Wistar rats that received orally a single dose of tri-ortho-cresyl phosphate (TOCP) at 1500 mg/kg. FG tissues were sampled at 1, 2, and 3 weeks after treatment and examined histopathologically. Wallerian degeneration of myelinated nerve fibers was observed in FG at 2 weeks. Morphological changes were most evident at 3 weeks after treatment and the number of fibers was reduced. Ultrastructurally, axonal swelling due to the accumulation of cytoplasmic contents was observed near the node of Ranvier in the affected animals, indicating paranodal degeneration. Axonal atrophy and swelling in organophosphorus-induced delayed neuropathy (OPIDN) were evaluated quantitatively using a computer-assisted image analyzer. Morphometric examinations on semi-thin sections and frozen sections stained with Nauta's method were demonstrated to be useful for objective evaluation of OPIDN in the rat.

Demyelination, and remyelination by Schwann cells and oligodendrocytes after kainate-induced neuronal depletion in the central nervous system.

Excitotoxins are thought to kill neurons while sparing afferent fibers and axons of passage. The validity of this classical conclusion has recently been questioned by the demonstration of axonal demyelination. In addition, axons are submitted to a profound alteration of their glial environment. This work was, therefore, undertaken to reassess axonoglial interactions over time after an excitotoxic lesion in the rat. Ultrastructural studies were carried out in the ventrobasal thalamus two days to 18 months after neuronal depletion by in situ injections of kainic acid. In some cases, lemniscal afferents were identified by using anterograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase from the dorsal column nuclei. Two and four days after kainate injection, numerous dying axons displaying typical signs of Wallerian degeneration were observed in a neuropile characterized by the loss of neuronal somata and dendrites, an increase in number of microglia/macrophages and the disappearance of astrocytes. Ten and 12 days after kainate injection, degenerating axons were no longer observed although myelin degeneration of otherwise unaltered axons was ongoing with an accumulation of myelin remnants in the neuropile. At 16 and 20 days, the demyelination process was apparently complete and axons of different diameters were sometimes packed together. One and two months after kainate injection, the axonal environment changed again: remyelination of large-caliber axons occurred at the same time as reactive astrocytes, oligodendrocytes and numerous Schwann cells appeared in the tissue. Schwann cell processes surrounded aggregates of axons of diverse calibers, ensheathed small ones and myelinated larger ones. Axons were also remyelinated by oligodendrocytes. Horseradish peroxidase-labeled lemniscal afferents could be myelinated by either of the two cell types. After three months, the neuropile exhibited an increase in number of hypertrophied astrocytes and the progressive loss of any other cellular or axonal element. At this stage, remaining Schwann cells were surrounded by a glia limitans formed by astrocytic processes. These data indicate that although excitotoxins are sparing the axons, they are having a profound and complex effect on the axonal environment. Demyelination occurs over the first weeks, accompanying the loss of astrocytes and oligodendrocytes. Axonal ensheathment and remyelination takes place in a second period, associated with the reappearance of oligodendrocytes and recruitment of numerous Schwann cells, while reactive astrocytes appear in the tissue at a slightly later time. Over the following months, astrocytes occupy a greater proportion of the neuron-depleted territory and other elements decrease in number.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropathologic findings in young male rats in a subchronic oral toxicity study using triethyl lead.

This study was undertaken to ascertain the neuropathologic effects of low level exposure of triethyl lead (3EL) to young male rats. Groups of 20 male Sprague-Dawley weanling rats were given 3EL at 0, 0.05, 0.10, 0.20, 0.50, and 1.00 mg/kg body wt for 91 days, 5 days/week by oral gavage. Lead acetate (PbHOAC) was given at 200 mg/kg body wt/day as a positive control. Animals (five or six) were perfused with glutaraldehyde following barbiturate anesthesia at the termination of the experiment. These animals and the remaining members of the group received a thorough gross and microscopic postmortem examination. Sections of the central, peripheral, and autonomic nervous systems were examined and lesions scored. No lesions were noted in the brain, but randomly distributed light microscopic changes of spinal cord Wallerian degeneration were noted to increase in a dose responsive manner (rho = 0.48; p < 0.01), with 3EL administration. Ultrastructural examination of selected sections of the lumbosacral nerves, revealed lesions characterized by reduced neurofilaments and neurotubules, and irregular lamellated axoplasmic dense bodies in all animals receiving lead. Organolead was only detected in animals receiving 3EL, but lead cations were detected in all lead-treated animals. The brain lead levels of 1.00 mg/kg/day and 200 mg Pb acetate positive control animals were equivalent. As distinctive ultrastructural lesions were seen in all rats treated with 3EL, we suggest that the no observed adverse effect level (NOAEL) for 3EL be lowered to less than 0.05 mg/kg/day.(ABSTRACT TRUNCATED AT 250 WORDS)

Presynaptic terminal loss from alpha-motoneurones following the retrograde axonal transport of diphtheria toxin.

Intercostal motoneurones intoxicated following intraneural injection of diphtheria toxin exhibited a progressive dilatation and fragmentation of Nissl body rough endoplasmic reticulum (rER), coupled with two different forms of presynaptic terminal response. Firstly, terminal dysjunction without prior degeneration, and secondly, Wallerian-type degeneration. Dysjunction was attributed to a toxin-related failure by the motoneurones to maintain postsynaptic site structure. Degeneration was considered to arise from toxicity in presynaptic neurones, either neighbouring motoneurones or local interneurones. Morphometry revealed that by 8 days, intoxicated motoneurones exhibited a 33% loss in terminal frequency, a 15% loss in residual presynaptic membrane, and a 43% loss in overall presynaptic input. The concomitant loss of synaptic sites was greater that the overall loss of presynaptic membrane, indicating a toxin-related deficiency of the maintenance of postsynaptic sites. Analyses of the relationship between changes in terminal numbers and the development of Nissl body abnormality in the postsynaptic motoneurone identified three groups of motoneurones: (i) those with normal presynaptic input and normal neuronal Nissl body rER; (ii) those showing a dramatic loss of presynaptic input and a marked dilatation and fragmentation of Nissl bodies; and (iii) neurones exhibiting a maintained or further loss of presynaptic input coupled with extreme dilatation and fragmentation of Nissl body rER with loss of Nissl body structure. These changes are discussed in context with the known molecular action of diphtheria toxin.

A contribution to the scientific assessment of degenerative and regenerative processes of peripheral nerve fibers following axonotmesis under the systemic administration of vitamins B1, B6 and B12--light and electron microscopy findings of the saphenous nerve in the rabbit.

The present study investigates the de- and regenerative changes in the saphenous nerve of the rabbit following systemic treatment with a combination of the vitamins B1, B6 and B12 and a control group that was treated with physiological saline solution. Cold lesion of the nerve, which led to an optimal axonotmesis, was used to cause a secondary degeneration. After 4, 10 and 21 days the nerves were removed and investigated by light and electron microscopy. The morphological results show that the number of regenerating axons is higher and that of degenerating axons lower in the group treated with the given doses of the vitamins than in the comparable control group. Statements as to the metabolic processes and vitamins from which the better regeneration results are due are as yet not possible. Further investigations with the individual vitamins are necessary. Clinical indications of neurotoxicity due to the dose levels used were not observed in any of the cases.

Changes in composition of endoneurial and perineurial fatty acids during glycerol-induced Wallerian degeneration and regeneration in the sciatic nerve of the adult rat.

Intraneural injection of pure glycerol induces Wallerian degeneration with subsequent regeneration. In agreement with other reports, we observed an increase in endoneurial polyunsaturated fatty acids 8 days after the glycerol injection. Levels then fell until day 30. After a period of 5 months, there was an increase in C18:2(n-6) in the intrafascicular tissue, concomitant with a marked fall in this fatty acid in the remaining extrafascicular perineurium. The rise in C18:2(n-6) in endoneurium correlated with infiltration of this tissue by perineurial cells. Interactions between perineurium and endoneurium during nerve regeneration are discussed.

Peripheral neurotoxicity of 2-chloroprocaine and bisulfite in the cat.

The responses of peripheral nerves less than 6 hr after and 2 weeks after exposure to 2-chloroprocaine (2-CP) and bisulfite (BS) were studied in cats using electrophysiologic techniques and by light and electron microscopy. Three percent 2-CP with 0.07% or 0.2% BS was not toxic to peripheral nerves. Three percent 2-CP with 1.2% BS and 1.2% BS (pH 2.8) alone had an acute effect on the peripheral nerves but no chronic effect. Ten percent BS (pH 2.8) caused Wallerian degeneration. However, at pH 4.8, no chronic toxicity was observed. Using conductivity measurements, it was shown that 2-CP and BS, when mixed, form an ion pair in solution that lowers the effective concentration of BS.

Peripheral neuropathy associated with high-dose Ara-C therapy.

Central nervous system toxicity associated with high-dose cytosine arabinoside (Ara-C) therapy (HD Ara-C) is well known. The authors report the case of a severe isolated peripheral polyneuropathy due to HD Ara-C. Electrophysiologic changes and histologic observations were consistent with axonal degeneration and scattered destruction of myelin sheaths. This observation emphasizes the need for careful complete neurologic evaluation for patients receiving HD Ara-C treatment.