Cisplatin-induced DNA-platination in experimental dorsal root ganglia neuronopathy

The mechanism(s) and site(s) of the neurotoxic effect of cisplatin (CDDP) are still not entirely elucidated. A more detailed knowledge of these aspects of CDDP treatment might be useful to obtain a better understanding of the pathogenesis of its peripheral neurotoxicity, which is the dose-limiting side effect of CDDP. In the present study, the occurrence of CDDP-induced DNA-platination in dorsal root ganglia (DRG) of rats was evaluated in relation to DRG neuron pathological changes and CDDP-induced neuronopathy. Eight adult Wistar rats were treated with 2 mg/kg i.p. CDDP twice weekly for 9 times to induce sensory peripheral neuropathy. DNA-platination in specimens of DRG and kidney was measured immunohistochemically, with a polyclonal antibody (GPt) detecting CDDP-induced Pt-DNA adducts. Results were compared with those of untreated rats. Chronic CDDP-induced neurotoxicity, in a well described experimental model of chronic CDDP neurotoxicity in the Wistar rat, was confirmed by sensory DRG neuronopathy with secondary neuropathy, and demonstrated by reduced pain detection, decreased nerve conduction velocity in the tail nerve as well as morphological and morphometric changes in DRG neurons. Nuclear immunostaining for Pt-DNA adducts was observed in tubular cells of the kidney in 75% of the evaluated CDDP-treated rats, while in DRG cells CDDP-induced Pt-DNA adducts formation was found in 43% of the evaluated CDDP-treated rats. CDDP-induced DNA-platination was demonstrated in rat DRG neurons using a schedule of chronic CDDP administration which induced the onset of a sensory neuronopathy with secondary peripheral neuropathy. This finding further supports the hypothesis that CDDP is neurotoxic because it directly damages the DRG neurons.     

Pyridoxine-induced toxicity in rats: a stereological quantification of the sensory neuropathy

Excess ingestion of pyridoxine (vitamin B6) causes a severe sensory neuropathy in humans. The mechanism of action has not been fully elucidated, and studies of pyridoxine neuropathy in experimental animals have yielded disparate results. Pyridoxine intoxication appears to produce a neuropathy characterized by necrosis of dorsal root ganglion (DRG) sensory neurons and degeneration of peripheral and central sensory projections, with large diameter neurons being particularly affected. The major determinants affecting the severity of the pyridoxine neuropathy appear to be duration and dose of pyridoxine administration, differential neuronal vulnerability, and species susceptibility. The present study used design-based stereological techniques in conjunction with electrophysiological measures to quantify the morphological and physiological changes that occur in the DRG and the distal myelinated axons of the sciatic nerve following pyridoxine intoxication. This combined stereological and electrophysiological method demonstrates a general approach that could be used for assessing the correlation between pathophysiological and functional parameters in animal models of toxic neuropathy.     

Expression of sensory neuron antigens by a dorsal root ganglion cell line, F-11

The F-11 cell line is a fusion product of embryonic rat dorsal root ganglion (DRG) cells with mouse neuroblastoma cell line N18TG-2 (Platika, D., Boulos, M.H., Baizer, L. and Fishman, M.C., Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3499-3503). F-11 cells were uniformly labelled using a monoclonal antibody (RT-97) to the 200 kDa subunit of neurofilament protein, which labels a subpopulation of adult rat DRG neurons. F-11 cells did not stain for antigenic markers of fibroblasts or Schwann/satellite cells which are also present in DRG. Monoclonal antibodies that recognize cell surface carbohydrates have been shown to label subpopulations of DRG neurons. The stage-specific embryonic antigens SSEA-3 and SSEA-4, and the antigen recognized by B23D8, were expressed by some F-11 cells but not by the neuroblastoma parent of the hybrid cells. SSEA-3 was expressed by about 20% of the F-11 cells, whereas 40-60% expressed SSEA-4 or the antigen recognized by B23D8. The stability of F-11 cell subpopulations for sensory antigen expression was demonstrated by isolating single cells and growing the progeny as clonal lines. In some subclones, nearly 100% of the cells stably expressed SSEA-4 and/or B23D8, or failed to stain with anti-SSEA-4, anti-SSEA-3, or B23D8 over 12 passages. Other subclones were unstable for the expression of these antigens. This study demonstrates the derivation of the F-11 cell line from sensory neurons but also indicates that multiple phenotypes of varying stability are present in this line. This information is important for the use of this line as a model for DRG neurons.     

Up-regulation of apoptosis and regeneration genes in the dorsal root ganglia during cisplatin treatment

Cisplatin is an effective anti-neoplastic drug, but its use is dose-limited due to its association with severe peripheral neurotoxicity. The neurotoxic effect of cisplatin is believed to result from its accumulation in the dorsal root ganglia (DRG), although the mechanism is not completely understood. We used a rat model of cisplatin neurotoxicity to examine changes in gene expression in the DRG. The results indicate that cisplatin affects the expression of several genes associated with apoptosis (Cdkn1a, Ckap2, Bid3, S100a8, S100a9), inflammation (S100a8, S100a9, Cd163, Mmp9), and nerve growth and regeneration (Mmp9, Gfap, Fabp7). The differential regulation of some of these genes may directly contribute to the neurotoxic effect of cisplatin, while others are likely to be representative of the subsequent cellular response to contain damage and initiate recovery. As such, the identified genes may represent candidate processes and pathways that should be considered as targets for therapeutic intervention in cisplatin-induced neuropathy.     

On the neurotoxicity of glutaric, 3-hydroxyglutaric, and trans-glutaconic acids in glutaric acidemia type 1

Glutaric acidemia type 1 (GA1) is an autosomal recessively inherited deficiency of glutaryl-CoA dehydrogenase. Accumulating metabolites, 3-hydroxyglutaric (3-OH-GA), glutaric (GA), and trans-glutaconic (TG) acids, have been proposed to be involved in the development of the striatal degeneration seen in children with GA1 via an excitotoxic mechanism. We have studied the extent to which 3-OH-GA, GA, and TG are neurotoxic and whether neurotoxicity is caused by an excitotoxic mechanism in which 3-OH-GA, GA, or TG overactivates N-methyl-D-aspartate (NMDA) receptors. In cultured mouse neocortical neurons, all three compounds were weakly neurotoxic, possibly through activation of NMDA receptors. However, further studies in the rat cortical wedge preparation and with NMDA receptors expressed in Xenopus oocytes could not confirm an interaction of the compounds with NMDA receptors. It is concluded that the metabolites 3-OH-GA, GA, and TG are only weak neurotoxins and that the neurodegenerative cascade destroying the striatum in patients with GA1 involves mainly mechanisms other than excitoxicity.     

Carbon disulfide inhibits neurite outgrowth and neuronal migration of dorsal root ganglion in vitro

Carbon disulfide (CS?) is a neurotoxic industrial solvent and widely used in the vulcanization of rubber, rayon, cellophane, and adhesives. Although the neurotoxicity of CS? has been recognized for over a century, the precise mechanism of neurotoxic action of CS? remains unknown. In the present study, a embryonic rat dorsal root ganglia (DRG) explants culture model was established. Using the organotypic DRG cultures, the direct neurotoxic effects of CS? on outgrowth of neurites and migration of neurons from DRG explants were investigated. The organotypic DRG cultures were exposed to different concentrations of CS? (0.01 mmol/L, 0.1 mmol/L, 1 mmol/L). The number of nerve fiber bundles extended from DRG explants decreased significantly in the presence of CS? (0.01 mmol/L, 15.00 ± 2.61, p < .05; 0.1 mmol/L, 11.17 ± 1.47, p < .001; 1 mmol/L, 8.00 ± 1.41, p < .001) as compared with that in the absence of CS? (17.83 ± 2.48). The number of neurons migrated from DRG explants decreased significantly in the presence of CS? (0.01 mmol/L, 79.50 ± 9.40, p < .01; 0.1 mmol/L, 62.50 ± 14.15, p < .001; 1 mmol/L, 34.67 ± 7.58, p < .001) as compared with that in the absence of CS? (99.33 ± 15.16). And also, the decreases in the number of nerve fiber bundles and migrated DRG neurons were in a dose-dependent manner of CS?. These data implicated that CS? could inhibit neurite outgrowth and neuronal migration from DRG explants in vitro.     

感觉性神经元神经病的实验病理研究

目的:建立一成功率高,感觉症状确实的感觉性神经元神经病的动物模型。并同时观察不同剂量维生素 B6对于大鼠感觉性神经系统的作用。方法:50只雌性Wistar大鼠分别给予600mg／kg(1周或2周),400mg／kg(4周), 200mg／kg(4周或8周)与100mg／kg(4周或8周)维生素B6,腹腔注射,每天一次。取其后根神经节(DRG),周围神经及脊髓作光镜与电镜分析。染色方法有HE,Luxol Fast Blue,Masson三色与银浸染色。半薄切片用甲苯胺兰染色,超薄切片用醋酸双氧铀与枸橼酸铅染色。结果:腰段DRG受累最重,其次为颈段、胸段。大剂量组导致感觉性神经元神经病,动物步态不稳,甚至瘫痪。DRG细胞体体积减少或坏死,伴以轴突萎缩与崩解,并可见吞噬细胞吞噬现象。小剂量组动物无异常临床表现,DRG神经元病变轻微,但存在轴突萎缩与变性。电镜下发现近端突与胞体均有细胞骨架异常。脊髓后束中薄束比楔束病变重。结论:多种因素包括用药时间、用药剂量及不同亚群神经元的药物敏感性不同,均可影响维生素B6神经毒性的最终表现。     

P2X3-mediated peripheral sensitization of neuropathic pain in resiniferatoxin-induced neuropathy

Patients suffering from sensory neuropathy due to skin denervation frequently have paradoxical manifestations of reduced nociception and neuropathic pain. However, there is a lack of satisfactory animal models to investigate these phenomena and underlying mechanisms. We developed a mouse system of neuropathy induced by resiniferatoxin (RTX), a capsaicin analog, and examined the functional significance of P2X3 receptor in neuropathic pain. From day 7 of RTX neuropathy, mice displayed mechanical allodynia (p<0.0001) and thermal hypoalgesia (p<0.0001). After RTX treatment, dorsal root ganglion (DRG) neurons of the peripherin type were depleted (p=0.012), while neurofilament (+) DRG neurons were not affected (p=0.62). In addition, RTX caused a shift in neuronal profiles of DRG: (1) increased in P2X3 receptor (p=0.0002) and ATF3 (p=0.0006) but (2) reduced TRPV1 (p=0.036) and CGRP (p=0.015). The number of P2X3(+)/ATF3(+) neurons was linearly correlated with mechanical thresholds (p=0.0017). The peripheral expression of P2X3 receptor in dermal nerves was accordingly increased (p=0.016), and an intraplantar injection of the P2X3 antagonists, A-317491 and TNP-ATP, relieved mechanical allodynia in a dose-dependent manner. In conclusion, RTX-induced sensory neuropathy with upregulation of P2X3 receptor for peripheral sensitization of mechanical allodynia, which provides a new therapeutic target for neuropathic pain after skin denervation.     

Neuroprotective effects of dopamine D2 receptor agonist on neuroinflammatory injury in olfactory bulb neurons in vitro and in vivo in a mouse model of allergic rhinitis

Available evidence indicates that dopamine D2 receptor modulates the neurotoxic effects induced by glutamate. However, neurotoxicity mediated by AMPA-subtype glutamate receptor has rarely been studied in the olfactory bulb. This study mainly explores the neuroprotective effects of dopamine D2 receptor agonist on AMPA receptor-mediated neurotoxicity in the olfactory bulb in a mouse model of allergic rhinitis (AR) with olfactory dysfunction (OD). In our study, we found that AR with OD was closely associated with increased surface expression of the AMPA receptor GluR1, reduced surface expression of GluR2, and apoptosis damage in the olfactory bulb in vivo. Quinpirole (a dopamine D2 receptor agonist) improved olfactory function in mice, ameliorated apoptosis injury in the olfactory bulb but not in the olfactory mucosa, and inhibited the internalization of GluR2-containing AMPA receptor in vitro and in vivo. In addition, phosphorylation plays a crucial role in the regulation of AMPA receptor trafficking. Our results showed that quinpirole reduced the phosphorylation of GluR1 S845 and GluR2 S880 in olfactory bulb neurons in vitro, but it had no obvious effect on GluR1 S831. Therefore, dopamine D2 receptor agonist may inhibit the phosphorylation of GluR1 S845 and GluR2 S880, thereby reducing AMPA receptor-mediated neurotoxicity and alleviating neurotoxic injury to the olfactory bulb caused by AR.     

Neurotoxicity of oxaliplatin and cisplatin for dorsal root ganglion neurons correlates with platinum-DNA binding

Cisplatin has been in use for 40 years, primarily for treatment of ovarian and testicular cancer. Oxaliplatin is the only effective treatment for metastatic colorectal cancer. Neurotoxicity occurs in up to 30% of patients and is dose-limiting for both drugs. The neuropathy is characterized by selective sensory loss in the extremities. Cisplatin treatment is associated with high levels of Pt-DNA binding and apoptosis of dorsal root ganglion (DRG) neurons. In this study, we directly compared the effects of oxaliplatin on DRG in vitro. Compared with cisplatin, oxaliplatin formed fewer Pt-DNA adducts following 6, 12, 24, and 48h (0.007ng Pt/mug DNA, 0.012ng/microg, 0.011ng/microg, 0.011ng/microg versus 0.014ng/microg, 0.022ng/microg, 0.041ng/microg, 0.030ng/microg), respectively. These findings closely correlated with data on cell survival where equimolar concentrations of oxaliplatin induced less cell death than cisplatin. Oxaliplatin-induced DRG death was associated with the morphological characteristics of apoptosis defined by 4'-6-diamidino-2-phenylindole and annexin/propidium iodide staining. Death was completely inhibited by the caspase inhibitor z-VAD-fmk. Our results demonstrate that both compounds cause apoptosis of DRG neurons but compared to cisplatin, oxaliplatin forms fewer Pt-DNA adducts and is less neurotoxic to DRG neurons in vitro.     

Pyruvate and lactate protect striatal neurons against N-methyl-d-aspartate-induced neurotoxicity

A sustained release of glutamate contributes to neuronal loss during cerebral ischaemia. Using cultured mouse striatal neurons, we observed that glucose deprivation, which occurs in this pathological process, enhanced the N-Methyl-D-aspartate (NMDA)- or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced neurotoxicity. The end products of glycolysis, lactate and pyruvate, strongly protected neurons from these neurotoxic effects. The neuroprotective effect of pyruvate (which is more prominent in the absence of glucose) was not related to its ability to react with H2O2 by a decarboxylation process. Pyruvate and L-lactate strongly counteracted the deep decrease in the neuronal ATP content induced by NMDA, indicating that they might protect striatal neurons by rescuing cellular energy charge. Addition of MK-801 after the NMDA withdrawal completely protected neurons, suggesting that NMDA neurotoxicity resulted from a delayed NMDA receptor activation probably linked to a delayed release of an endogenous agonist in the extracellular medium. The strong accumulation of extracellular glutamate which was found in both sham and NMDA-treated cultures was markedly decreased by pyruvate. Thus, pyruvate might also exert its protecting activity by decreasing the delayed accumulation of glutamate which seemed to be neurotoxic only after a preexposure of neurons to NMDA.     

4-Hydroxy-2-Nonenal Induces Mitochondrial Dysfunction and Aberrant Axonal Outgrowth in Adult Sensory Neurons that Mimics Features of Diabetic Neuropathy

Modification of proteins by 4-hydroxy-2-nonenal (4-HNE) has been proposed to cause neurotoxicity in a number of neurodegenerative diseases, including distal axonopathy in diabetic sensory neuropathy. We tested the hypothesis that exposure of cultured adult rat sensory neurons to 4-HNE would result in the formation of amino acid adducts on mitochondrial proteins and that this process would be associated with impaired mitochondrial function and axonal regeneration. In addition, we compared 4-HNE-induced axon pathology with that exhibited by neurons isolated from diabetic rats. Cultured adult rat dorsal root ganglion (DRG) sensory neurons were incubated with varying concentrations of 4-HNE. Cell survival, axonal morphology, and level of axon outgrowth were assessed. In addition, video microscopy of live cells, western blot, and immunofluorescent staining were utilized to detect protein adduct formation by 4-HNE and to localize actively respiring mitochondria. 4-HNE induced formation of protein adducts on cytoskeletal and mitochondrial proteins, and impaired axon regeneration by approximately 50% at 3 μM while having no effect on neuronal survival. 4-HNE initiated formation of aberrant axonal structures and caused the accumulation of mitochondria in these dystrophic structures. Neurons treated with 4-HNE exhibited a distal loss of active mitochondria. Finally, the distal axonopathy and the associated aberrant axonal structures generated by 4-HNE treatment mimicked axon pathology observed in DRG sensory neurons isolated from diabetic rats and replicated aspects of neurodegeneration observed in human diabetic sensory neuropathy.     

Neurotoxic catecholamine metabolite in nociceptors contributes to painful peripheral neuropathy

The neurotoxic effects of catecholamine metabolites have been implicated in neurodegenerative diseases. As some sensory neurons express tyrosine hydroxylase and monoamine oxidase (MAO), we investigated the potential contribution of catecholamine metabolites to neuropathic pain in a model of alcoholic neuropathy. The presence of catecholamines in sensory neurons is supported by capsaicin-stimulated epinephrine release, an effect enhanced in ethanol-fed rats. mRNA for enzymes in dorsal root ganglia involved in catecholamine uptake and metabolism, dopamine beta-hydroxylase and MAO-A, were decreased by neonatal administration of capsaicin. Ethanol-induced hyperalgesia was attenuated by systemic and local peripheral administration of inhibitors of MAO-A, reduction of norepinephrine transporter (NET) in sensory neurons and a NET inhibitor. Finally, intradermal injection of 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), a neurotoxic MAO-A catecholamine metabolite, produced robust mechanical hyperalgesia. These observations suggest that catecholamines in nociceptors are metabolized to neurotoxic products by MAO-A, which can cause neuronal dysfunction underlying neuropathic pain.     

Overexpression of human alpha-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the appearance of intracytoplasmic inclusions called Lewy bodies (LB) in dopamine neurons in the substantia nigra and the progressive loss of these neurons. Recently, mutations in the alpha-synuclein gene have been identified in early-onset familial PD, and alpha-synuclein has been shown to be a major component of LB in all patients. Yet, the pathophysiological function of alpha-synuclein remains unknown. In this report, we have investigated the toxic effects of adenovirus-mediated alpha-synuclein overexpression on dopamine neurons in rat primary mesencephalic cultures and in a rat dopaminergic cell line - the large T-antigen immortalized, mesencephalon-derived 1RB3AN27 (N27). Adenovirus-transduced cultures showed high-level expression of alpha-synuclein within the cells. Overexpression of human mutant alpha-synuclein (Ala(53)Thr) selectively induced apoptotic programmed cell death of primary dopamine neurons as well as N27 cells. The mutant protein also potentiated the neurotoxicity of 6-hydroxydopamine (6-OHDA). By contrast, overexpression of wild-type human alpha-synuclein was not directly neurotoxic but did increase cell death after 6-OHDA. Overexpression of wild-type rat alpha-synuclein had no effect on dopamine cell survival or 6-OHDA neurotoxicity. These results indicate that overexpression of human mutant alpha-synuclein directly leads to dopamine neuron death, and overexpression of either human mutant or human wild-type alpha-synuclein renders dopamine neurons more vulnerable to neurotoxic insults.     

Dose-dependent expression of neuronopathy after experimental pyridoxine intoxication

We examined the sequence of nervous system abnormalities that resulted when rats were given excess amounts of vitamin B6 (pyridoxine). High doses of pyridoxine (1,200 or 600 mg/kg/d) for 6 to 10 days caused a neuronopathy with necrosis of dorsal root ganglion (DRG) sensory neurons, accompanied by centrifugal axonal atrophy and breakdown of peripheral and central sensory axons. Large diameter neurons with long processes and large cytoplasmic volumes were especially affected. Smaller doses (300 to 150 mg/kg/d) for up to 12 weeks had minor effects on DRG neurons, but produced a neuropathy with axonal atrophy and degeneration. Guinea pigs given 1,800 mg/kg/d developed sensory neuronopathy, whereas mice given similar or higher doses did not have neuropathologic abnormalities. Multiple factors including rate of administration, differential neuronal vulnerability, and species susceptibility have bearing on the final expression of pyridoxine neurotoxicity.     

Cytotoxic effect of glutamate and its agonists on mouse hippocampal neurons

The neurodegenerative action of the excitatory amino acid neurotransmitter (glutamate) and its exogenous (N-methyl-D-aspartate, kainate) or endogenous (quinolinate) analogues were studied on cultures of dissociated nerve cells from the embryonal mouse hippocampus. The exposure of primary cultures for 3-6 h to these excitotoxins showed that neurons were vulnerable to both glutamate and all tested agonists which induced the swelling and vacuolization of neuronal bodies accompanied by degeneration of their dendrites. This process terminated by complete cell destruction. The neurotoxic effect of glutamate (1 mM) was not suppressed by a competitive NMDA receptor antagonist (D, L-2-amino-5-phosphonovalerate, 0.3 mM) and was only slightly prevented by gamma-D-glutamylglycine (3mM). The protective action of the latter was more evident in the presence of lower glutamate concentration (0.5 mM). The excitotoxic effect of N-methyl-D-aspartate (0.1 mM) or quinolinate (0.5mM) was almost completely blocked by both antagonists. In contrast, D, L-2-amino-5-phosphonovalerate failed to protect hippocampal neurons from damage induced by kainate while partial antagonism of kainate neurotoxicity was observed with gamma-D-glutamylglycine. These finding suggest that glutamate neurotoxicity may be derived, mainly, from the non-NMDA type(s) of glutamate receptor present on hippocampal cell membranes with a low effectiveness to suppress this effect by selective competitive NMDA antagonist. Possible involvement of glutamate receptor(s) in the early dendritic outgrowth of hippocampal neurons and in the process of neuronal "cell death" is discussed.     

D1 but not D4 Dopamine Receptors are Critical for MDMA-Induced Neurotoxicity in Mice

MDMA, an addictive psychostimulant-consumed worldwide, has the ability to induce neurotoxic effects and addiction in laboratory animals and in humans through its effects on monoaminergic systems. MDMA-induced neurotoxicity in mice occurs primarily in dopaminergic neurons and does not significantly affect the serotonergic system. As the neurotoxic effects of MDMA in mice involve excessive dopamine (DA) release, DA receptors are highly likely to play a role in MDMA neurotoxicity, but the specific dopamine receptor subtypes involved have not previously been determined definitively. In this study, dopamine D1 and D4 receptor knock-out mice (D1R^?/? and D4R^?/?) were used to determine whether these receptors are involved in MDMA neurotoxicity. D1R inactivation attenuated MDMA-induced hyperthermia, decreased the reduction of dopamine and dopamine metabolite levels, and protected against dopamine terminal loss and reactive astrogliosis as determined in the striatum, 7 days after MDMA treatment. In sharp contrast, inactivation of D4R did not prevent hyperthermia or the neurotoxic effects of MDMA. Altogether, these results indicate that D1R, but not D4R, plays a significant role in the dopaminergic striatal neurotoxicity observed after exposure to MDMA.