The roles of the necroptotic and excitotoxic pathways in diisopropyl fluorophosphate-induced neuronal necrosis

我们在这个项目中的目标是确定致痫的破坏性作用 大鼠脑内系统注射有机磷二异丙基氟磷酸盐(DFP)，并 确定程序性坏死的坏死性和排毒途径在DFP中的作用 诱导神经元坏死。对于具体目标1，我们将确定时间进程和程度 神经元坏死(苏木精-伊红[H&E]染色)和核移位 受体相互作用蛋白(K)-1(RIP1)、受体相互作用蛋白(K)-3(RIP3)和 免疫荧光显微镜下的混合谱系激酶结构域样蛋白(MLKL)，6，24和 给药后72h，大鼠背侧海马CA1-CA3区和门区注射DFP。为实现具体目标2，我们将 确定安定(目前的护理标准)是否与RIP1抑制剂中的任何一种联合使用 7-Cl-O-Necrostatin-1(7-Cl-O-NEC-1)或NMDA受体拮抗剂MK-801(地佐西平)，给予60 DFP注射后分钟，是否具有神经保护作用，以及7-氯-O-NEC-1是否减少核转位 RIP1、RIP3和MLKL在海马区24小时(或6小时或72小时，取决于特定的结果 目的1)注射DFP后。对于特定的目标3，我们将确定地西潘和安慰剂联合给药是否 DFP注射60min后7-氯-O-NEC-1和MK-801均提供大鼠海马区 神经保护比任何一种本身都要好。对于特定的目标4，我们将确定7-氯-O-NEC-1 和/或MK-801(取决于特定目标2和3的结果)提供更强的神经保护 与给药组相比，DFP组大鼠的空间学习和记忆能力更好 DFP诱发癫痫持续状态(SE)后3个月。我们将使用的方法是：(1)现场 4%磷酸盐缓冲多聚甲醛经心脏灌注固定大脑 和免疫荧光显微镜；(2)无偏估计的体视学光学分级仪 海马区CA1-3区和肺门区神经元60微米厚冠状切片的HE染色 光镜(特异性AIMS 1-4)或RIP1、RIP3和MLKL抗体用于免疫荧光 显微镜(特定目标2)；(3)亚细胞分离和蛋白质印迹作为单独的方法 确认DFP诱导的SE中RIP1、RIP3和MLKL的核转位程度(特异性 目的2)；(4)Barnes迷宫，检测DFP后2个月大鼠的空间学习记忆能力 注射，并将发现与背部剩余的正常神经元的数量相关联 海马区(特定目标4)。我们将对数据进行多因素方差分析和后置t检验， 使用合并标准差和α=0.05。我们将确定RIP1的抑制是否 神经保护和7-氯-O-NEC-1和MK-801均可提供最佳神经保护 如果这是真的，应该会影响接触有机磷后的护理标准。

Our objective in this project is to determine the destructive seizure-inducing effects of the systemically administered organophosphate diisopropyl fluorophosphate (DFP) on rat brain, and to determine the roles of the necroptotic and exitotoxic pathways of programmed necrosis in DFP- induced neuronal necrosis. For Specific Aim 1 we will determine the time course and degree of neuronal necrosis (using the hematoxylin and eosin [H & E] stain) and nuclear translocation of receptor-interacting protein (kinase)-1 (RIP1), receptor-interacting protein (kinase)-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL), using immunofluorescence microscopy, 6, 24 and 72 h after DFP administration in dorsal hippocampal CA1-CA3 and hilus. For Specific Aim 2 we will determine if co-administration of diazepam (the current standard of care) with either the RIP1 inhibitor 7-Cl-O-necrostatin-1 (7-Cl-O-nec-1) or the NMDA-receptor antagonist MK-801 (dizocilpine), given 60 min after DFP injection, are neuroprotective and if 7-Cl-O-nec-1 reduces nuclear translocation of RIP1, RIP3 and MLKL in the hippocampus 24 h (or 6 or 72 h, depending on the results of Specific Aim 1) after DFP injection. For Specific Aim 3 we will determine if co-administration of diazepam and both 7-Cl-O-nec-1 and MK-801 60 min after DFP injection provides greater hippocampal neuroprotection than either given by itself. For Specific Aim 4 we will determine if 7-Cl-O-Nec-1 and/or MK-801 (depending on the results of Specific Aims 2 and 3) provides greater neuroprotection and better spatial learning and memory in DFP-treated rats compared to those given vehicle two one months after DFP-induced status epilepticus (SE). The methods that we will use are (1) in situ transcardiac perfusion-fixation of the brain with 4% phosphate-buffered paraformaldehyde for light and immunofluorescence microscopy; (2) the stereological optical fractionator for unbiased estimates of hippocampal CA1-3 and hilar neurons after 60-µm-thick coronal sections are stained with H & E for light microscopy (Specific Aims 1-4) or RIP1, RIP3 and MLKL antibodies for immunofluorescence microscopy (Specific Aim 2); (3) subcellular fractionation and western blots as a separate approach to confirm the degree of nuclear translocation of RIP1, RIP3 and MLKL from DFP-induced SE (Specific Aim 2); and (4) the Barnes maze, to test rats’ spatial learning and memory two one months after DFP injection, and to correlate findings with the numbers of remaining normal neurons in dorsal hippocampus (Specific Aim 4). We will analyze the data with multi-factor ANOVA and post-hoc t-tests, using pooled standard deviations and α = 0.05. We will determine if inhibition of RIP1 is neuroprotective and if optimal neuroprotection is provided by both 7-Cl-O-nec-1 and MK-801 given together, which, if true, should influence the standard of care following organophosphate exposure.