In vivo mechanisms of methamphetamine-induced neuronal necrosis

甲基苯丙胺诱导神经元坏死的体内机制

• 批准号: 8540280
• 负责人: DENSON G FUJIKAWA
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8540280
• 关键词: Acute; Antiepileptic Agents; Apoptotic; Behavioral; Biological Assay; Brain; Brain Injuries; Brain region; Calpain; Calpain I; Cathepsins B; Cell Death; Cerebrum; Cessation of life; Chronic; Cognitive; Corpus striatum structure; Cysteine Protease; Data; Diazepam; Dose; Electroencephalography; Electrons; Enzymes; Generalized seizures; Goals; Health; Heat-Shock Proteins 70; Hematoxylin and Eosin Staining Method; Hippocampus (Brain); Human; Immunofluorescence Microscopy; In Vitro; Ingestion; Injection of therapeutic agent; Intervention; Light; Lithium; Membrane; Methamphetamine; Methods; Microscopic; Mitochondria; Molecular; Monitor; Morbidity - disease rate; Mus; Necrosis; Nerve; Neurons; Normal saline; Nuclear; Nuclear Translocation; Phenobarbital; Pilocarpine; Rattus; Research; Sahara; Seizures; Spectrin; Staining method; Stains; Status Epilepticus; Study Section; Subclinical Seizures; Temperature; Testing; Toxic effect; Transmission Electron Microscopy; United States; Western Blotting; Work; base; cytochrome c; in vivo; inhibitor/antagonist; killings; methamphetamine abuse; neuroprotection; prevent; public health relevance; rectal

DESCRIPTION (provided by applicant): Our overall objective is to understand how methamphetamine (METH) kills neurons, and whether this can be ameliorated or eliminated with pharmacologic intervention. We have preliminary data that METH-induced subclinical repetitive electrographic seizure discharges (RESDs) produce acidophilic neurons in the same brain regions as generalized seizures. Since 57% of mice with RESDs have no behavioral evidence of seizures, EEG recording is mandatory. Our Specific Aims in this project are (1) to show that METH-induced RESDs are associated with necrotic, not apoptotic neurons, as is currently believed; (2) to determine if METH-induced RESDs are associated with calpain I activation and nuclear translocation of mitochondrial cytochrome c (cyt c) and lysosomal cathepsins B and D (cath-B and cath-D) as well as RESD-induced neuronal necrosis; (3) to show that stopping electrographic seizures with antiepileptic drugs 5 min after the onset of METH-induced RESDs inhibits calpain I activation, is neuroprotective and prevents or reduces nuclear translocation of cyt c, cath-B and cath-D; and (4) to show that specific inhibition of calpain I is neuroprotective and prevents or reduces nuclear translocation of cyt c, cath-B and cath-D. The methods that we will use include (1) continuous EEG recording to identify mice with RESDs and rectal temperature monitoring to keep mice given METH at 40.0 ¿ 1.0¿C and control mice given normal saline at 37.0 ¿ 1.0¿C; (2) use of the hematoxylin and eosin (H & E) stain at the light-microscopic level to identify dead (acidophilic) neurons with confirmation of morphological necrosis with transmission electron microscopy; (3) semi-quantitative scoring of 7 brain regions for acidophilic neurons with the H & E stain and nuclear translocation of cyt c, cath-B and cath-D with immunofluorescence microscopy; (4) stereological estimate of the numbers of acidophilic and normal neurons in the most damaged brain region (probably hippocampus) and the numbers of normal neurons and those showing nuclear cyt c, cath-B and cath-D; (4) fluorometric calpain activity assays and western blots of ¿II-spectrin, a calpain substrate, to document calpain I activation; (5) parentera administration of diazepam and phenobarbital after METH administration to stop electrographic seizures, which we predict will also provide neuroprotection and inhibit calpain I activation and nuclear translocation of cyt c, cath-B and cath-D; (6) injection of the calpain I inhibitor MDL 28170, which we predict will be neuroprotective and will prevent or reduce nuclear translocation of cyt c, cath-B and cath-D; and (6) statistical analysis, using multifactorial analysis of devianc and multi- factorial ANOVA, with post-hoc t-tests with pooled standard deviations, and an ¿ of 0.05. With respect to relevance, METH abuse is a major health problem in the United States (Cho & Melega 2002, Rawson et al. 2002). Our goal is to reduce morbidity from brain damage caused by acute METH toxicity in humans. Our finding that METH-induced subclinical RESDs in mice are associated with widespread neuronal damage could have important translational applicability.

描述（由申请人提供）： 我们的总体目标是了解甲基苯丙胺 (METH) 如何杀死神经元，以及是否可以通过药物干预来改善或消除这种现象。我们有初步数据表明，甲基苯丙胺诱导的亚临床重复性电图癫痫放电 (RESD) 在同一大脑区域产生嗜酸神经元。由于 57% 的 RESD 小鼠没有癫痫发作的行为证据，因此我们在该项目中的具体目标是 (1) 表明这一点。 METH 诱导的 RESD 与坏死而非凋亡神经元相关，正如目前所认为的那样；(2) 确定 METH 诱导的 RESD 是否与钙蛋白酶 I 激活以及线粒体细胞色素 c (cyt c) 和溶酶体组织蛋白酶 B 的核转位相关； D（cath-B 和 cath-D）以及 RESD 诱导的神经元坏死；(3) 表明抗癫痫药物可以阻止电图癫痫发作METH 诱导的 RESD 发生后 5 分钟，抑制钙蛋白酶 I 激活，具有神经保护作用，并防止或减少细胞色素 c、cath-B 和 cath-D 的核转位；(4) 表明钙蛋白酶 I 的特异性抑制具有神经保护作用，并且防止或减少 cyt c、cath-B 和 cath-D 的核易位。我们将使用的方法包括 (1) 连续脑电图记录以识别患有 RESD 的小鼠，并监测直肠温度以保持。小鼠给予 40.0 的冰毒 ¿ 1.0¿ C和对照小鼠在37.0 ¿下给予生理盐水1.0¿ C；（2）在光学显微镜水平上使用苏木精和伊红（H＆E）染色来识别死亡（嗜酸）神经元，并通过透射电子显微镜确认形态坏死；（3）对7个脑进行半定量评分嗜酸性神经元的 H 和 E 染色区域以及细胞色素 c、cath-B 和 cath-D 的核易位，以及免疫荧光显微镜检查 (4) 的体视学估计；受损最严重的大脑区域（可能是海马）中嗜酸性神经元和正常神经元的数量以及正常神经元和显示核细胞色素 c、cath-B 和 cath-D 的神经元的数量；（4）荧光钙蛋白酶活性测定和 ¿ II-血影蛋白，一种钙蛋白酶底物，用于记录钙蛋白酶 I 的激活；(5) 在给予 METH 后肠外给予地西泮和苯巴比妥以阻止电图癫痫发作，我们预测这也将提供神经保护并抑制钙蛋白酶 I 激活和细胞色素 c 的核转位， cath-B 和 cath-D；(6) 注射钙蛋白酶 I 抑制剂 MDL 28170，我们预测其具有神经保护作用，并可防止或减少细胞核易位cyt c、cath-B 和 cath-D；以及（6）统计分析，使用偏差的多因素分析和多因素方差分析，以及带有汇总标准差的事后 t 检验和 ¿就相关性而言，冰毒滥用是美国的一个主要健康问题（Cho & Melega 2002，Rawson 等人 2002）。我们的目标是减少因冰毒急性中毒而导致的发病率。 METH 诱导的小鼠亚临床 RESD 与广泛的神经元损伤相关，可能具有重要的转化适用性。