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（METH）如何杀死神经元，以及是否可以通过药物干预改善或消除。我们有初步的数据，甲基苯丙胺诱导的亚临床重复电图癫痫放电（RESDs）产生嗜酸性神经元在相同的大脑区域的全面发作。由于57%的RESD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）显示MET诱导的RESD与坏死有关，而不是目前认为的凋亡神经元;（2）确定MET诱导的RESD是否与钙蛋白酶I激活和线粒体细胞色素c（cyt c）和溶酶体组织蛋白酶B和D的核转位有关（cath-B和cath-D）以及RESD诱导的神经元坏死;（3）显示在METH诱导的RESD发作后5分钟用抗癫痫药物停止电描记癫痫发作抑制钙蛋白酶I活化，具有神经保护作用并防止或减少细胞色素c的核转位，cath-B和cath-D;和（4）表明钙蛋白酶I的特异性抑制具有神经保护作用，并阻止或减少细胞色素c、cath-B和cath-D的核转位。我们将使用的方法包括（1）连续EEG记录以识别具有RESD的小鼠，并监测直肠温度以使小鼠在40.0 <$1.0 <$C下给予METH，对照小鼠在37.0 <$1.0 <$C下给予生理盐水;（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（3）用免疫荧光法对嗜酸性神经元的7个脑区进行半定量评分，并用H & E染色和cyt c、cath-B和cath-D的核转位;（4）对受损最严重脑区嗜酸性神经元和正常神经元数量的体视学估计（可能是海马）和正常神经元的数量以及显示核细胞色素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钙蛋白酶底物<$II-血影蛋白的蛋白质印迹，以记录钙蛋白酶I激活;（5）METH给药后再给予安定和苯巴比妥以终止电描记癫痫发作，我们预测这也将提供神经保护作用，并抑制calpain I激活和cyt c、cath-B和cath-D的核转位;（6）注射钙蛋白酶I抑制剂MDL 28170，我们预测其将具有神经保护作用，并将阻止或减少cyt-c、cath-B和cath-D的核转位;和（6）统计分析，使用偏差的多因素分析和多因素方差分析，采用合并标准差的事后t检验，且Δ为0.05。就相关性而言，METH滥用是美国的一个主要健康问题（Cho & Melega 2002，罗森等人2002）。我们的目标是减少急性METH中毒引起的人类脑损伤的发病率。我们的发现，在小鼠中，MET诱导的亚临床RESD与广泛的神经元损伤相关，这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.