In vivo mechanisms of methamphetamine-induced neuronal necrosis

描述(由申请人提供)： 我们的总体目标是了解甲基苯丙胺(冰毒)是如何杀死神经元的，以及这种情况是否可以通过药物干预来改善或消除。我们有初步数据表明，冰毒诱导的亚临床重复电刺激(RESD)在与全身性癫痫相同的脑区产生嗜酸性神经元。由于57%患有RESD的小鼠没有癫痫发作的行为证据，因此EEG记录是强制性的。我们在这个项目中的具体目标是：(1)证明冰毒诱导的RESD与坏死的神经元有关，而不是像目前认为的那样与凋亡神经元有关；(2)确定冰毒诱导的RESD是否与Calain I激活、线粒体细胞色素c(Cytc)和溶酶体组织蛋白B和D(cath-B和cath-D)的核转位以及RESD诱导的神经元坏死有关；(3)证明在冰毒诱导的RESDs开始5分钟后用抗癫痫药物停止脑电惊厥可以抑制calain I的激活，起到神经保护作用，并防止或减少细胞色素c、cath-B和cath-D的核转位；以及(4)显示钙蛋白I的特异性抑制具有神经保护作用，可防止或减少Cyt c、Cath-B和Cath-D的核转位。我们将使用的方法包括：(1)连续脑电记录以识别RESD小鼠，直肠温度监测使小鼠保持在40.0℃和对照组小鼠给予37.0℃生理盐水；(2)使用光镜水平的苏木精-伊红(H&E)染色识别形态坏死的嗜酸性神经元；(3)用免疫荧光显微镜对嗜酸性神经元的7个脑区进行半定量评分，H&E染色和Cyt c、Cath-B和Cath-D的核易位；(4)对受损最严重的脑区(可能是海马区)的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有核的Cyt C、Cath-B和Cath-D的神经元的数量进行体视学评估；(4)荧光分光光度法检测Calain活性，并通过免疫印迹法检测calain底物？II-Spectrin的激活情况；(5)在甲基甲胺注射后肠外注射安定和苯巴比妥，以阻止脑电发作，我们预测这也将提供神经保护，抑制细胞色素C、Cath-B和Cath-D的激活和核转位；(6)注射钙蛋白酶I抑制剂MDL 28170，我们预测它将具有神经保护作用，并将防止或减少Cyt C、Cath-B和Cath-D的核转位；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，采用合并标准差的后组t检验，t检验为0.05。关于相关性，冰毒滥用是美国的一个主要健康问题(Cho&Melega 2002，Rawson等人)。2002年)。我们的目标是减少人类急性冰毒引起的脑损伤的发病率。我们的发现，冰毒诱导的小鼠亚临床RESD与广泛的神经元损伤有关，这一发现可能具有重要的翻译适用性。

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.