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

坏死性凋亡和兴奋性毒性途径在氟磷酸二异丙酯诱导的神经元坏死中的作用

• 批准号: 10454773
• 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: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10454773
• 关键词: Acetylcholine; Acetylcholinesterase Inhibitors; Acute; Analysis of Variance; Behavioral; Brain; Brain Injuries; Calpain; Caspase; Caspase Inhibitor; Cations; Cell Culture Techniques; Cell Fractionation; Cells; Cessation of life; Cholinergic Receptors; Color; Data; Diazepam; Dorsal; Dose; Epilepsy; Evolution; Glutamates; Goals; Hematoxylin and Eosin Staining Method; Hilar; Hippocampus (Brain); Immunofluorescence Microscopy; In Situ; Injections; Learning; Lithium; MK801; Measures; Memory; Memory impairment; Methods; Military Personnel; Morphology; Muscarinics; N-Methyl-D-Aspartate Receptors; NMDA receptor antagonist; Necrosis; Neurological emergencies; Neurons; Neuroprotective Agents; Nitric Oxide Synthase Type I; Nuclear Translocation; Optics; Organophosphates; Pathway interactions; Perfusion; Phosphate Buffer; Phosphotransferases; Pilocarpine; Poly(ADP-ribose) Polymerases; Protein Kinase; Proteins; RIPK1 gene; RIPK3 gene; Rattus; Receptor Activation; Research Personnel; Role; Seizures; Severities; Stains; Status Epilepticus; Terrorism; Testing; Thick; Time; Toxic effect; Western Blotting; antibody mimetics; dizocilpine; excitotoxicity; fluorophosphate; inhibitor; light microscopy; neuroprotection; paraform; postsynaptic neurons; preservation; receptor; sample fixation; spatial memory; standard of care; toxic organophosphate insecticide exposure

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.

我们在这个项目中的目标是确定破坏性癫痫发作诱发作用 向大鼠脑部全身施用有机磷酸酯二异丙基氟磷酸酯（DFP），并 确定 DFP 中程序性坏死的坏死性凋亡和退出毒性途径的作用 对于具体目标 1，我们将确定诱导神经坏死的时间进程和程度。 神经元坏死（使用苏木精和伊红 [H & E] 染色）和核易位 受体相互作用蛋白（激酶）-1 (RIP1)、受体相互作用蛋白（激酶）-3 (RIP3) 和 混合谱系激酶结构域样蛋白 (MLK​​L)，使用免疫荧光显微镜，6、24 和 在背侧海马 CA1-CA3 和肺门中 DFP 给药后 72 小时，我们将进行特定目标 2。 确定地西泮（目前的护理标准）是否与 RIP1 抑制剂共同给药 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，具有神经保护作用，并且如果 7-Cl-O-nec-1 减少 海马中的 RIP1、RIP3 和 MLKL 24 小时（或 6 或 72 小时，具体取决于特定结果） 目标 1) 在注射 DFP 后，对于具体目标 3，我们将确定是否同时服用地西泮和 DFP 注射后 60 分钟，7-Cl-O-nec-1 和 MK-801 均提供更大的海马 对于特定目标 4，我们将确定 7-Cl-O-Nec-1 是否具有神经保护作用。 和/或 MK-801（取决于特定目标 2 和 3 的结果）提供更好的神经保护 与给予载体二一的大鼠相比，DFP 治疗的大鼠具有更好的空间学习和记忆能力 DFP 诱发癫痫持续状态 (SE) 后几个月我们将使用的方法是 (1) 原位。 经心灌注-用 4% 磷酸盐缓冲多聚甲醛固定大脑以进行光照 和免疫荧光显微镜；（2）用于无偏估计的体视光学分馏器 60 µm 厚的冠状切片用 H & E 染色后的海马 CA1-3 和肺门神经元 光学显微镜（具体目标 1-4）或用于免疫荧光的 RIP1、RIP3 和 MLKL 抗体 显微镜检查（具体目标 2）；(3) 亚细胞分级分离和蛋白质印迹作为分离方法 确认 DFP 诱导的 SE 中 RIP1、RIP3 和 MLKL 的核转位程度（具体 目标 2); 和 (4) Barnes 迷宫，测试 DFP 两个月后大鼠的空间学习和记忆能力 注射，并将结果与​​背侧剩余正常神经元的数量相关联 海马体（具体目标 4）我们将通过多因素方差分析和事后 t 检验来分析数据， 使用汇总标准差和 α = 0.05 我们将确定 RIP1 的抑制是否有效。 神经保护作用，如果 7-Cl-O-nec-1 和 MK-801 都能提供最佳的神经保护作用 在一起，如果属实，应该会影响有机磷暴露后的护理标准。