Peripheral Neuropathy In Humans Research Articles

Increase of MCP‐1 (CCL2) in myelin mutant Schwann cells is mediated by MEK‐ERK signaling pathway

Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in inherited demyelinating neuropathies. On the basis of the observation that upregulation of the Schwann cell-derived chemokine MCP-1 (CCL2) is a pathologically relevant mechanism for macrophage activation in mice heterozygously deficient for the myelin component P0 (P0+/-), we posed the question of the intracellular signaling cascade involved. By using western blot analysis of peripheral nerve lysates the MAP-kinases extracellular signal-regulated kinase 1/2 (ERK1/2) and MAP kinase/ERK kinase 1/2 (MEK1/2) showed an early and constantly increasing activation in P0 mutants. Furthermore, in nerve fibers from the P0+/- mutants, Schwann cell nuclei were much more often positive for phosphorylated ERK1/2 than in nerve fibers from wild type mice. In vitro experiments using the MEK1/2-inhibitor CI-1040 decreased ERK1/2-phosphorylation and MCP-1 expression in a Schwann cell-derived cell line. Finally, systemic application of CI-1040 lead to a decreased ERK1/2-phosphorylation and substantially reduced MCP-1-production in peripheral nerves of P0+/- mutant mice. Our study identifies MEK1/2-ERK1/2 signaling as an important intracellular pathway that connects the Schwann cell mutation with the activation of pathogenetically relevant macrophages in the peripheral nerves. These findings may have important implications for the treatment of inherited peripheral neuropathies in humans.

Arsenic-induced toxicity: effect on protein composition in sciatic nerve

Exposure to arsenic compounds may lead to skin and lung cancer and various disorders such as vascular disease and peripheral neuropathy in humans. Peripheral arsenic neurotoxicity has been demonstrated clinically and in electrophysiological studies. Patients intoxicated with arsenic show neurological symptoms in their feet and hands. These patients show significantly lower nerve conduction velocities (NCVs) in their peripheral nerves in comparison with controls. The mechanism of arsenic peripheral nervous system (PNS) toxicity, however, has never been described before. This is the first study to investigate the toxicity of arsenic on the PNS. Male Wistar rats were exposed to arsenite given as a single dose i.v. After sacrifice, sciatic nerves were excised and the protein composition was analysed. Protein analysis of sciatic nerves showed disappearance of neurofilament and fibroblast proteins in rats treated with arsenite doses of 15 and 20 mg/kg in comparison with the control groups. Some fibroblast protein bands had disappeared in the 20-mg/kg dose group. The analysed neurofilament-M and -L proteins decreased dose dependency over time. arsenic affects the composition of proteins in the rat sciatic nerve, especially the neurofilaments. The reduction of signals in Western blot analysis reveals changes in cytoskeletal composition, which may well lead to neurotoxic effects in vivo.

N,N-diethyldithiocarbamate produces copper accumulation, lipid peroxidation, and myelin injury in rat peripheral nerve.

Previous studies have demonstrated the ability of the dithiocarbamate, disulfiram, to produce a peripheral neuropathy in humans and experimental animals and have also provided evidence that N,N-diethyldithiocarbamate (DEDC) is a proximate toxic species of disulfiram. The ability of DEDC to elevate copper levels in the brain suggests that it may also elevate levels of copper in peripheral nerve, possibly leading to oxidative stress and lipid peroxidation from redox cycling of copper. The study presented here investigates the potential of DEDC to promote copper accumulation and lipid peroxidation in peripheral nerve. Rats were administered either DEDC or deionized water by ip osmotic pumps and fed a normal diet or diet containing elevated copper, and the levels of metals, isoprostanes, and the severity of lesions in peripheral nerve and brain were assessed by ICP-AES/AAS, GC/MS, and light microscopy, respectively. Copper was the only metal that demonstrated any significant compound-related elevations relative to controls, and total copper was increased in both brain and peripheral nerve in animals administered DEDC on both diets. In contrast, lesions and elevated F2-isoprostanes were significantly increased only in peripheral nerve for the rats administered DEDC on both diets. Autometallography staining of peripheral nerve was consistent with increased metal content along the myelin sheath, but in brain, focal densities were observed, and a periportal distribution occurred in liver. These data are consistent with the peripheral nervous system being more sensitive to DEDC-mediated demyelination and demonstrate the ability of DEDC to elevate copper levels in peripheral nerve. Additionally lipid peroxidation appears to either be a contributing event in the development of demyelination, possibly through an increase of redox active copper, or a consequence of the myelin injury.

TheWldsMutation Delays Robust Loss of Motor and Sensory Axons in a Genetic Model for Myelin-Related Axonopathy

Mice deficient in the peripheral myelin component P0 mimic severe forms of inherited peripheral neuropathies in humans, with defective myelin formation and consequent axonal loss. We cross-bred these mice with the spontaneous mutant C57BL/Wld(s) typically showing protection from Wallerian degeneration because of fusion of the ubiquitination factor E4B (Ube4b) and nicotinamide mononucleotide adenylyltransferase (Nmnat) genes. We found that in the double mutants, the robust myelin-related axonal loss is reduced at 6 weeks and 3 months of age. Moreover, retrograde labeling from plantar nerves revealed an increased survival of motor axons. These motor axons appeared functionally active because both the amplitude of compound muscle action potentials and muscle strength were less reduced in the double mutants. At 6 months of age, reduction of axonal loss was no longer detectable in the double mutants when compared with littermates carrying the P0 null mutation only, although the Wld(s) gene was not reduced in its expression at this age. We conclude that myelin-related axonal loss is a process having some features in common with Wallerian degeneration. Introducing the Wld(s) gene would be a promising approach to delaying detrimental axonal loss in myelin disorders.

Alterations in the Arf6-regulated plasma membrane endosomal recycling pathway in cells overexpressing the tetraspan protein Gas3/PMP22.

Growth arrest specific 3 (Gas3)/peripheral myelin protein 22 (PMP22) is a component of the compact peripheral nerve myelin, and mutations affecting gas3/PMP22 gene are responsible for a group of peripheral neuropathies in humans. We have performed in vivo imaging in order to investigate in detail the phenotype induced by Gas3/PMP22 overexpression in cultured cells. Here we show that Gas3/PMP22 triggers the accumulation of vacuoles, before the induction of cell death or of changes in cell spreading. Overexpressed Gas3/PMP22 accumulates into two distinct types of intracellular membrane compartments. Gas3/PMP2 accumulates within late endosomes close to the juxtanuclear region, whereas in the proximity of the cell periphery, it induces the formation of actin/phosphatidylinositol (4,5)-bisphosphate (PIP(2))-positive large vacuoles. Gas3/PMP22-induced vacuoles do not contain transferrin receptor, but instead they trap membrane proteins that normally traffic through the ADP-ribosylation factor 6 (Arf6) endosomal compartment. Arf6 and Arf6-Q67L co-localize with Gas3/PMP22 in these vacuoles, and the dominant negative mutant of Arf6, T27N, blocks the appearance of vacuoles in response to Gas3/PMP22, but not its accumulation in the late endosomes. Finally a point mutant of Gas3/PMP22 responsible for the Charcot-Marie-Tooth 1A disease is unable to trigger the accumulation of PIP(2)-positive vacuoles. Altogether these results suggest that increased Gas3/PMP22 levels can alter membrane traffic of the Arf6 plasma-membrane-endosomal recycling pathway and show that, similarly to other tetraspan proteins, Gas3/PMP22 can accumulate in the late endosomes.

Mutations in the cytoplasmic domain of P0 reveal a role for PKC-mediated phosphorylation in adhesion and myelination.

Mutations in P0 (MPZ), the major myelin protein of the peripheral nervous system, cause the inherited demyelinating neuropathy Charcot-Marie-Tooth disease type 1B. P0 is a member of the immunoglobulin superfamily and functions as a homophilic adhesion molecule. We now show that point mutations in the cytoplasmic domain that modify a PKC target motif (RSTK) or an adjacent serine residue abolish P0 adhesion function and can cause peripheral neuropathy in humans. Consistent with these data, PKCα along with the PKC binding protein RACK1 are immunoprecipitated with wild-type P0, and inhibition of PKC activity abolishes P0-mediated adhesion. Point mutations in the RSTK target site that abolish adhesion do not alter the association of PKC with P0; however, deletion of a 14 amino acid region, which includes the RSTK motif, does abolish the association. Thus, the interaction of PKCα with the cytoplasmic domain of P0 is independent of specific target residues but is dependent on a nearby sequence. We conclude that PKC-mediated phosphorylation of specific residues within the cytoplasmic domain of P0 is necessary for P0-mediated adhesion, and alteration of this process can cause demyelinating neuropathy in humans.

Differentiation of Disulfiram Effects on Central Catecholamines and Hepatic Ethanol Metabolism

Disulfiram is used in the treatment of chronic alcoholism, because of the unpleasant symptoms it provokes after ethanol intake. The underlying mechanism is believed to be the accumulation of acetaldehyde in the blood, due to inhibition of the liver aldehyde dehydrogenases. In addition, it is known that disulfiram also has some neurotoxic properties. The aim of our study was to investigate the relationship between the pharmacological and neurotoxicological properties of disulfiram with respect to the doses applied. Increasing doses of disulfiram (25, 50, 75, 100 and 150 mg/kg) were administered intraperitoneally to Wistar rats and the hepatic enzyme activities of alcohol and aldehyde dehydrogenases were measured. Also, in two brain subregions (midbrain and hypothalamus) the levels of noradrenaline, dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid were determined. The higher dose of disulfiram (150 mg/kg) produced lethal effects in all treated animals. Aldehyde dehydrogenase activities were inhibited by disulfiram in a dose-dependent way, while alcohol dehydrogenase was not affected at all. Concerning the levels of brain biogenic amines, disulfiram produced a significant reduction in noradrenaline and an increase in dopamine levels in both structures of the brain, in a dose-dependent way. However, the lowest dose applied (25 mg/kg) had no effects on brain catecholamines. It is known that high doses of disulfiram may cause severe encephalopathy and peripheral neuropathy in humans, which could be attributed to the impairment of the metabolism of brain biogenic amines, due to inhibition of dopamine-beta-hydroxylase. Our experimental data show that disulfiram affects the level of brain biogenic amines at dose levels higher than those inhibiting the activity of aldehyde dehydrogenase. Therefore, in clinical practice 'disulfiram reaction' could still be achieved with a low dosage regimen not producing neurotoxicity

Damage to the cytoskeleton of large diameter sensory neurons and myelinated axons in vincristine‐induced painful peripheral neuropathy in the rat

Vincristine, along with other antimitotic chemotherapeutic drugs, produces a peripheral neuropathy in humans that is accompanied by painful paresthesias, dysesthesias, and occasionally hypoesthesia, and by hyporeflexia (Holland et al. [1973] Cancer Res. 33:1258–1264; McLeod and Penny [1969] J Neurol Neurosurg Psychiatry 32:297–304; Postma et al. [1993] J Neurooncol. 15:23–27; Sandler et al. [1969] Neurology 19:367–374). Systemic administration of vincristine causes swelling of unmyelinated axons and disorientation of axonal microtubules (Tanner et al. [1998a1998a] J Comp Neurol. 395:481–492) at a time when it also produces allodynia and mechanical hyperalgesia (Aley et al. [1996] Neuroscience 73:259–265; Authier et al. [1999] Neuroreport 10:965–968) and enhanced responsiveness in C-fibers in the rat (Tanner et al. [1998b] J Neurosci. 18:6480–6491). Because slowing of A-fiber conduction velocities had also been demonstrated (Tanner et al. [1998b] J Neurosci. 18:6480–6491), and mechanical hyperalgesia can occur secondary to damage to large diameter sensory afferents (Basbaum et al. [1991] Can J Physiol Pharmacol. 69:647–651; Coggeshall et al. [1993] Pain 52:233–242; Woolf and Mannion [1999] Lancet 353:1959–1964), we sought to determine whether vincristine also induced ultrastructural changes in myelinated A-fibers. Moreover, since systemic treatment with vincristine did not cause profound microtubule depolymerization in the unmyelinated axons of the peripheral nerve, we hypothesized that the drug's effects may be more extensive in the cell body, because in the spinal ganglion, the blood-nerve barrier is less restrictive. We used quantitative ultrastructural methods to analyze the microtubule cytoskeleton in myelinated axons in the mid-shaft of the saphenous nerve and in the sensory ganglion cells. Vincristine induced swelling of the whole nerve and an increase in the cross-sectional areas of myelinated axons but no loss of myelinated axons. There was a significant decrease in axonal microtubules, as well as microtubule disorganization, in myelinated fibers from vincristine-treated rats. In the spinal ganglion, vincristine induced swelling of large diameter sensory neurons and a build-up of neurofilaments in the cell bodies and proximal axons, suggestive of impaired anterograde axonal transport. J. Comp. Neurol. 424:563–576, 2000. © 2000 Wiley-Liss, Inc.

Damage to the cytoskeleton of large diameter sensory neurons and myelinated axons in vincristine-induced painful peripheral neuropathy in the rat

Vincristine, along with other antimitotic chemotherapeutic drugs, produces a peripheral neuropathy in humans that is accompanied by painful paresthesias, dysesthesias, and occasionally hypoesthesia, and by hyporeflexia (Holland et al. [1973] Cancer Res. 33:1258-1264; McLeod and Penny [1969] J Neurol Neurosurg Psychiatry 32:297-304; Postma et al. [1993] J Neurooncol. 15:23-27; Sandler et al. [1969] Neurology 19:367-374). Systemic administration of vincristine causes swelling of unmyelinated axons and disorientation of axonal microtubules (Tanner et al. [1998a1998a] J Comp Neurol. 395:481-492) at a time when it also produces allodynia and mechanical hyperalgesia (Aley et al. [1996] Neuroscience 73:259-265; Authier et al. [1999] Neuroreport 10:965-968) and enhanced responsiveness in C-fibers in the rat (Tanner et al. [1998b] J Neurosci. 18:6480-6491). Because slowing of A-fiber conduction velocities had also been demonstrated (Tanner et al. [1998b] J Neurosci. 18:6480-6491), and mechanical hyperalgesia can occur secondary to damage to large diameter sensory afferents (Basbaum et al. [1991] Can J Physiol Pharmacol. 69:647-651; Coggeshall et al. [1993] Pain 52:233-242; Woolf and Mannion [1999] Lancet 353:1959-1964), we sought to determine whether vincristine also induced ultrastructural changes in myelinated A-fibers. Moreover, since systemic treatment with vincristine did not cause profound microtubule depolymerization in the unmyelinated axons of the peripheral nerve, we hypothesized that the drug's effects may be more extensive in the cell body, because in the spinal ganglion, the blood-nerve barrier is less restrictive. We used quantitative ultrastructural methods to analyze the microtubule cytoskeleton in myelinated axons in the mid-shaft of the saphenous nerve and in the sensory ganglion cells. Vincristine induced swelling of the whole nerve and an increase in the cross-sectional areas of myelinated axons but no loss of myelinated axons. There was a significant decrease in axonal microtubules, as well as microtubule disorganization, in myelinated fibers from vincristine-treated rats. In the spinal ganglion, vincristine induced swelling of large diameter sensory neurons and a build-up of neurofilaments in the cell bodies and proximal axons, suggestive of impaired anterograde axonal transport.

Nociceptor Hyper-Responsiveness during Vincristine-Induced Painful Peripheral Neuropathy in the Rat

Neuropathic pain accompanies peripheral nerve injury after a wide variety of insults including metabolic disorders, traumatic nerve injury, and neurotoxic drugs. Chemotherapy-induced neuropathic pain, caused by drugs such as vincristine and taxol, occurs in cancer patients who receive these drugs as antineoplastic agents. Although a variety of remediations have been attempted, the absence of knowledge concerning mechanisms of chemotherapy-induced neuropathic pain has hindered the development of treatment strategies. Vincristine, a widely used chemotherapeutic agent, produces painful peripheral neuropathy in humans and mechanical hyperalgesia in rats. To test the hypothesis that alterations in C-fiber nociceptor function occur during vincristine-induced painful peripheral neuropathy, we performed in vivo extracellular recordings of single neurons from the saphenous nerve of vincristine-treated rats. Forty-one percent of C-fiber nociceptors were significantly hyper-responsive to suprathreshold mechanical stimulation. As a population, these mechanically hyper-responsive nociceptors also had significantly greater responses to suprathreshold heat stimulation; however, heat hyper-responsiveness was found only in a subset of these nociceptors and was never detected in the absence of mechanical hyper-responsiveness. In addition, mean conduction velocities of A-fibers and C-fibers in vincristine-treated rats were significantly slowed. Mean heat and mechanical activation thresholds of C-fiber nociceptors, their distribution among subclasses, and the percentage of spontaneously active neurons in vincristine-treated rats were not statistically different from controls. Vincristine does not, therefore, cause generalized impairment of C-fiber nociceptor function but rather specifically interferes with mechanisms underlying responsiveness to suprathreshold stimuli. Furthermore, vincristine-induced nociceptor hyper-responsiveness may involve alterations specifically in mechanotransduction in some nociceptors and alterations in general cellular adaptation mechanisms in others.

Microtubule disorientation and axonal swelling in unmyelinated sensory axons during vincristine-induced painful neuropathy in rat

Neuropathic pain accompanies peripheral nerve injury following a variety of insults including metabolic disorders, traumatic injury, and exposure to neurotoxins such as vincristine and taxol. Vincristine, a microtubule depolymerizing drug, produces a peripheral neuropathy in humans that is accompanied by painful paresthesias and dysesthesias (Sandler et al., [1969] Neurology 19:367-374; Holland et al. [1973] Cancer Res. 33:1258-1264). The recent development of an animal model of vincristine-induced neuropathy provides an opportunity to investigate mechanisms underlying this form of neuropathic pain. Systemic vincristine (100 microg/kg) produces hyperalgesia to mechanical stimuli during the second week of administration, which persists for more than a week (Aley et al. [1996] Neuroscience 73:259-265). To test the hypothesis that changes in microtubule structure in nociceptive sensory neurons accompany vincristine-induced hyperalgesia, we analyzed unmyelinated axons in saphenous nerves of vincristine-treated rats. This study constitutes the first quantitative ultrastructural analysis of the cytoskeleton of unmyelinated axons in peripheral nerve during neuropathic hyperalgesia. There was no evidence of unmyelinated fiber loss or a decrease in the number of microtubules per axons. There was, however, a significant decrease in microtubule density in unmyelinated axons from vincristine-treated rats. This decrease in microtubule density was due to a significant increase in the cross-sectional area of unmyelinated axons, suggesting swelling of axons. In addition, vincristine-treated axons had significantly fewer microtubules cut in cross-section and significantly more tangentially oriented microtubules per axon compared to controls. These results suggest that vincristine causes disorganization of the axonal microtubule cytoskeleton, as well as an increase in the caliber of unmyelinated sensory axons.

Ultrastructure of neuromuscular junction in vacor-induced diabetic rats.

ObjectivesRodenticide Vacor causes a severe peripheral neuropathy in humans.Electrophysiologic studies on a peripheral motor nerve-skeletal system of Vacor-treated rat showed decreased amplitude of muscle action potential without conduction velocity abnormalities. The ultrastructural studies of the neuromuscular junction were performed to clarify the anatomic site of the Vacor-induced peripheral neuropathy in male Wistar rats.MethodsAfter oral administration of a single dose of Vacor, 80 mg/kg of body weight, to the experimental animals, neuromuscular junctions within the interosseous muscles of the hind foot were observed in time.ResultsNo axon terminal change was noted until 24 hours after the administration of Vacor. Remarkable loss of presynaptic vesicles and swollen endoplasmic reticulum in the axon terminal were developed at 3 days after Vacor treatment. Progressive degenerative changes consisting of marked loss of presynaptic vesicles, focal disruption of membrane in the axon terminal with disappearance of the number of the damaged axon terminal appeared, and flattening of postsynaptic folds was also seen.ConclusionsThese results suggest that degenerative changes in axon terminal at neuromuscular junction may contribute to the peripheral neuropathy developed in the early phase of Vacor poisoning.

Overloaded Endoplasmic Reticulum–Golgi Compartments, a Possible Pathomechanism of Peripheral Neuropathies Caused by Mutations of the Peripheral Myelin Protein PMP22

Nonconservative point mutations of the peripheral myelin protein 22 (PMP22) are associated with Charcot-Marie-Tooth type 1A disease, the most common inherited peripheral neuropathy in humans, and with the Trembler J (TrJ) and Trembler (Tr) alleles in mice. We investigated the intracellular transport of wild-type PMP22 and its TrJ and Tr mutant forms in Schwann cells and in a non-neuronal cell line. In contrast to wild type, mutant proteins were not inserted into the plasma membrane and accumulated in the endoplasmic reticulum and Golgi compartments. Coexpression of each mutant with wild-type PMP22 confirmed the different intracellular distribution of the mutant forms, indicating that neither the TrJ nor Tr protein has a dominant-negative effect on the cellular distribution of wild-type PMP22. Accumulation of PMP22 immunoreactivity in the cell body of myelinating Schwann cells was also observed in nerve biopsies obtained from CMT1A patients carrying the TrJ point mutation. We propose that impaired trafficking of mutated PMP22 affects Schwann cell physiology leading to myelin instability and loss.

Physiological characterization of Taxol-induced large-fiber sensory neuropathy in the rat.

The cancer chemotherapeutic agent Taxol (paclitaxel) causes a dose-related peripheral neuropathy in humans. We produced a dose-dependent large-fiber sensory neuropathy, without detrimental effects on general health, in mature rats by using two intravenous injections 3 days apart. Tests of other dosing schedules demonstrated the dependence of the severity of the neuropathy and of animal health on both the dose and the frequency of dosing. Pathologically, severe axonal degeneration and hypomyelination were observed in sections of dorsal roots, whereas ventral roots remained intact. Electrophysiologically, H-wave amplitudes in the hindlimb and amplitudes of predominantly sensory compound nerve action potentials in the tail were reduced. These effects persisted for at least 4 months after treatment. Motor amplitudes were not affected, but both motor and sensory conduction velocities decreased. The ability of rats to remain balanced on a narrow beam was impaired, indicating proprioceptive deficits. Muscle strength, measured by hindlimb and forelimb grip strength, and heat nociception, measured by tail-flick and hindlimb withdrawal tests, were not affected by Taxol. This model of Taxol-induced neuropathy in mature rats, with minimal effects on general health, parallels closely the clinical syndrome observed after Taxol treatment in humans.

A Transgenic Mouse Model for Human Hereditary Neuropathy with Liability to Pressure Palsies

Mutations in the gene encoding peripheral myelin protein 22 (PMP22) account for several inherited peripheral neuropathies in humans. We now show that transgenic mice expressing antisense PMP22 RNA exhibit modestly reduced levels of PMP22 together with a phenotype that is reminiscent of hereditary neuropathy with liability to pressure palsies (HNPP), a human disease caused by a 1.5-Mb deletion of a chromosome 17 region that contains thePMP22gene. Transgenic antisense homozygotes display a striking movement disorder and a slowing of nerve conduction that worsens with age. Morphological analysis of peripheral nerves demonstrates that a subset of axons have thickened myelin sheaths and tomacula in young adults, with significant myelin degeneration detected in older animals. Together with other recent work, these data suggest that dosage of thePMP22gene alone underlies the pathophysiology observed in HNPP and related disorders.

Identification and Characterization of a cDNA and the Structural Gene Encoding the Mouse Epithelial Membrane Protein-1

The PMP22/EMP/MP20 gene family includes four closely related proteins, peripheral myelin protein-22 (PMP22), epithelial membrane protein-1 (EMP-1), epithelial membrane protein-2 (EMP-2), and epithelial membrane protein-3 (EMP-3), which share amino acid identities ranging from 33 to 43%. In addition, the lens-specific membrane protein MP20 represents a more distant relative. Functionally, this family of proteins is likely to play important roles in the control of cell proliferation, cell differentiation, and cell death. In particular, mutations affecting thePMP22gene are responsible for various hereditary peripheral neuropathies in humans and mice. We report the isolation and characterization of a mouse EMP-1 cDNA and the correspondingemp-1gene. Mouse EMP-1 displays 93% amino acid identity to rat EMP-1 and 39% identity to mouse PMP22. The cDNA-predicted EMP-1 protein contains four putative membrane-associated domains and can beN-linked glycosylatedin vitro.EMP-1 is encoded by a single-copy gene with the positions of introns exactly conserved betweenemp-1andPMP22,corroborating the hypothesis that both genes belong to the same family. Computer-predicted structural domains of EMP-1 are partially mirrored by the exon/intron structure ofemp-1.Most interestingly, exon 4, which covers the potential second transmembrane domain, a small intracellular loop, and half of the third transmembrane domain, encodes the most highly conserved regions between the EMP-1 and PMP22 proteins and is also remarkably conserved in the MP20 gene, indicating some shared functional significance for this module in the PMP22/EMP/MP20 family.

NEUROTOXICITY OF MISONIDAZOLE IN RATS FOLLOWING INTRAVENOUS ADMINISTRATION

Misonidazole is a hypoxic cell radiosensitizer that induces a peripheral neuropathy in humans after exceeding a schedule-dependent cumulative threshold dose. Clinical studies of misonidazole have been conducted using oral administration, whereas most other radiosensitizers have been administered intravenously. Since route of exposure can potentially influence the toxicity of xenobiotics, the objective of this study was to assess the neurotoxicity of misonidazole in rats following intravenous dosing using a battery of routine clinical, neurofunctional, biochemical, and histopathologic screening methods. Male Sprague–Dawley rats were administered intravenous doses of misonidazole at 0 (vehicle control), 100, 200, 300, or 400mgkg −1once per day, 5 days per week, for 2 weeks. Animals were evaluated for neurofunctional and pathological changes following termination of treatment (Days 15–17) and at the end of a 4 week observation period (Days 43–45). During the dosing phase, hypoactivity, salivation, rhinorrhea, chromodacryorrhea, rough pelage and ataxia were observed at 400mgkg −1, and body weight gain of the 300 and 400mgkg −1groups was significantly decreased relative to the vehicle controls by 24% and 49%, respectively. Corresponding reductions in food consumption were 8% and 23%, respectively. Although most 400mgkg −1animals appeared normal on Day 15 prior to the neurofunctional evaluations, rotorod testing precipitated a number of clinical signs including: ataxia, impaired righting reflex, excessive rearing, tremors, vocalization, circling, head jerking, excessive sniffing and hyperactivity. All of these animals recovered and appeared normal from Day 17 through study termination. There were no treatment-related effects on motor activity, acoustic startle response, rotorod performance, forelimb group strength, toe and tail pinch reflexes, tibial nerve β-glucuronidase activity or tail nerve conduction velocity. Although hindlimb grip strength of the 400mgkg −1group was significantly decreased by 17% relative to the vehicle controls on Day 15, this finding appeared related to the reduced food consumption and body weight gain in these animals. No microscopic changes were detected in peripheral nerves. Necrosis and proliferation of fibrillary astrocytes (gliosis) were seen in the cerebellum and medulla of the 400mgkg −1animals on Day 16. Gliosis in these same brain regions was observed in the 300 and 400mgkg −1groups on Day 44. The results show that intravenous administration of misonidazole to rats causes dose-limiting central nervous system toxicity without effects on peripheral nervous tissue. The lack of peripheral neurotoxicity was most likely due to a combination of several interrelated factors including route of administration, duration and intensity of the dosing regimen, and total cumulative dose.

Etoposide- and BMY-40481-induced sensory neuropathy in mice.

The effects of high toxic doses of the anticancer drugs, etoposide and its phosphate derivative, BMY-40481, on the nervous system of female CD-1 mice were examined by light microscopy (LM) and transmission electron microscopy. Mice were euthanatized 4 wk following a single iv injection of either 0, 50, 100, or 150 mg/kg of BMY-40481 or 44 or 88 mg/kg of etoposide. Mice treated with 100 or 150 mg/kg of BMY-40481 or 88 mg/kg of etoposide had clinical symptomology of progressive ataxia, impaired righting reflex, and splaying and paresis of fore- and hindlimbs at day 8. Similar, dose-related LM changes were observed with both drugs at all doses and consisted of degeneration of dorsal root ganglion cells and axonal degeneration of their distal and proximal processes in peripheral nerves, dorsal spinal roots, and dorsal funiculi of spinal cord. Axonal degeneration was characterized by LM as shrinkage, swelling, and fragmentation of axon cylinders accompanied by secondary demyelination. Degenerative changes in ganglion cell bodies included eccentric nuclei, cytoplasmic vacuolation, central chromatolysis, and peripheral clumping of Nissl's bodies. Ultrastructurally, ganglion cell bodies had focally extensive dilation of the rough endoplasmic reticulum, mitochondrial swelling, increased numbers of phagolysosomes and prominent aggregations of microfilaments (globular filamentous bodies). Ultrastructural axonal changes occurred primarily in large, myelinated fibers and consisted of axonal swelling or loss, thinning of myelin sheaths, and a decrease in the number of organelles. This is the first report of etoposide-related sensory neuropathy in laboratory rats, a model that my be useful for the study of etoposide-related peripheral neuropathy in humans.

Increased nitric oxide synthase immunoreactivity in rat dorsal root ganglia in a neuropathic pain model

In rats, tight ligation of L5 and L6 spinal nerves produces symptoms of thermal hyperalgesia and mechanical allodynia, mimicking the symptoms which characterise painful peripheral neuropathies in humans. Immunoreactivity for nitric oxide synthase (NOS) was investigated in lumbar (L1, L4, L5 and L6) dorsal root ganglia from naive controls and from rats surviving for 3, 7, and 14 days after unilateral ligation of the L5 and L6 spinal nerves. Quantitative analysis revealed significant increases in the percentage of NOS-immunoreactive cell profiles in L5 and L6 ganglia on the operated side at all time points, with the number of labelled profiles increasing with time following ligation, but L1 and L4 ganglia were unaffected. These findings suggest that nitric oxide may have a role in the generation and/or maintenance of neuropathic pain.

The molecular genetics of myelination: an update.

Recent molecular genetic studies have provided new insights into the structure and function of 2 of the major integral membrane proteins of myelin--the proteolipid protein (PLP) and protein zero (P0)--and have uncovered a third such protein--PMP22/gas3. The rumpshaker mouse has been shown to carry a point mutation in the PLP gene that uncouples a deleterious effect on CNS myelin assembly, which these mice exhibit, from oligodendrocyte degeneration and cell death, which they do not. The developmental importance of the P0 protein in PNS myelination has been dramatically demonstrated by the analysis of loss-of-function mutations engineered through the expression of antisense RNA and through the insertional inactivation of the P0 gene by homologous recombination in embryonic stem cells and the generation of P0-deficient mice. The cloned promoter of the P0 gene has been shown to drive quantitative, Schwann cell-specific expression of heterologous genes in transgenic mice. The PMP22/gas3 gene, previously cloned from fibroblast cell lines, has been found to encode an axonally regulated Schwann cell protein that is assembled into PNS myelin. Importantly, this gene appears to be the target of mutations that result in the Trembler alleles in mice, and in Charcot-Marie-Tooth disease Type 1a, the most common inherited peripheral neuropathy in humans.