EXCITOTOXIN EFFECTS ON SPINAL CORD IN VITRO

体外兴奋毒素对脊髓的影响

• 批准号: 3414065
• 负责人: JOHN R DELFS
• 金额: $ 14.98万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3414065
• 关键词: acetylcholinesterase; aspartate; cholinergic receptors; degenerative motor system disease; dosage; excitatory aminoacid; glutamate receptor; histochemistry /cytochemistry; inhibitor /antagonist; kynurenate; laboratory rat; lactate dehydrogenases; neural degeneration; neurotoxins; neurotransmitter receptor; somatostatin; spinal cord; stimulant /agonist; synapses; tissue /cell culture

Excitatory amino acids (EAAs) acting at specific membrane receptors may promote neuron degeneration or death in many nervous system diseases and have been specifically implicated in pathogenesis of motor neuron diseases. EAA-mediated damage to anatomically and histochemically defined neurons can be examined in organotypic roller tube cultures of postnatal rat spinal cord (OTC-SCs). Our studies show damage to ventral and dorsal horns in OTC-SCs by the excitotoxin, N-methyl-D-aspartic acid (NMDA). This projects examines actions of NMDA and other EAA agonists on cholinergic markers in spinal cord in vitro using the OTC-SC system. First, OTC-SCs will be treated with NMDA, with or without specific antagonists. Damage to spinal cord cholinergic systems will be assessed by histological and morphometric analysis after staining cultures for acetylcholinesterase activity (AChE) and choline acetyltransferase (CHAT) - immunoreactivity, and by biochemical analysis of ACHE and CHAT activities. Lactate dehydrogenase efflux from cultures will be examined to obtain an overall assessment of neuronal damage. Next, dose-response curves and temporal aspects of the effects will be determined. Third, cultures will be treated with the EAA agonists, kainic acid or quisqualic acid. Using specific NMDA-receptor antagonists or non-NMDA- receptor antagonists, the extent to which damage is mediated through different EAA receptor subtypes will be determined. In OTC-SCs, damage by L-glutamate compared with kainate or NMDA is less than in many culture systems, reflecting more closely its effect in vivo. Therefore, the fourth specific aim is to investigate two possible mechanisms for the decreased effect of glutamate: the role of glutamate uptake and the role of kynurenic acid as an endogenous EAA antagonist. Fifth, we will determine whether a potent somatostatin analogue, SMS 201- 995, can alter the toxic effects of NMDA, L-glutamate, or other excitotoxins on neurons in OTC-SCs. The long-term objective of this research is to understand better the role of excitotoxins in spinal neuron degeneration and death.

作用于特定膜受体的兴奋性氨基酸(EaAs)可能 在许多神经系统疾病中促进神经元退化或死亡 已明确与运动神经元疾病的发病机制有关。 EAA介导的解剖学和组织化学定义的神经元损伤可以 出生后大鼠脊髓器官型滚管培养中的检测 电源线(OTC-SCS)。我们的研究显示腹角和背角在 OTC-SCs由兴奋性毒素N-甲基-D-天冬氨酸(NMDA)诱导。这个项目 检测NMDA和其他EAA激动剂对胆碱能标记物的作用 采用OTC-SC系统进行脊髓体外培养。 首先，OTC-SCs将接受NMDA治疗，无论是否有特定的 对抗者。脊髓胆碱能系统的损害将通过以下方法进行评估 培养物染色后的组织学和形态计量学分析 乙酰胆碱酯酶活力(AChE)和胆碱乙酰转移酶(ChAT)- 免疫反应性，AChE和ChAT活性的生化分析。 从培养物中排出的乳酸脱氢酶将被检测以获得 对神经元损伤进行全面评估。接下来，剂量-反应曲线和 影响的时间方面将被确定。 第三，培养物将用EAA激动剂、红藻氨酸或 准奎奎酸。使用特定的NMDA受体拮抗剂或非NMDA- 受体拮抗剂，损伤的程度是通过 不同的EAA受体亚型将被确定。 在OTC-SCs中，L-谷氨酸的损伤比红藻氨酸或N-甲基-D-天冬氨酸要小 比在许多培养系统中更能反映其在体内的作用。 因此，第四个具体目标是调查两种可能的 谷氨酸作用减弱的机制：谷氨酸的作用 犬尿酸作为内源性EAA拮抗剂的摄取和作用。 第五，我们将确定一种强有力的生长抑素类似物，SMS201- 可以改变N-甲基-D-天冬氨酸、L-谷氨酸等的毒性作用 兴奋性毒素对OTC-SCs神经元的影响。 这项研究的长期目标是更好地理解 兴奋性毒素在脊髓神经元退化和死亡中的作用。