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的破坏性的诱导作用， 在大鼠脑上全身施用有机磷酸二异丙基氟磷酸盐（DFP），以及 确定DFP中程序性坏死的坏死性和外向毒性途径的作用- 诱发神经元坏死。对于具体目标1，我们将确定 神经元坏死（使用苏木精和伊红[H & E]染色）和细胞核转位。 受体相互作用蛋白（激酶）-1（RIP 1）、受体相互作用蛋白（激酶）-3（RIP 3）和 混合谱系激酶结构域样蛋白（MLKL），使用免疫荧光显微镜，6，24和 在背侧海马CA 1-CA 3和门中DFP给药后72 h。具体目标2我们将 确定地西泮（当前标准治疗）与RIP 1抑制剂联合给药 7-Cl-O-necrostatin-1（7-Cl-O-nec-1）或NMDA受体拮抗剂MK-801（地佐环平），给予60 如果7-Cl-O-nec-1减少DFP注射后10分钟的核转位， RIP 1、RIP 3和MLKL在海马24小时（或6或72小时，取决于特异性免疫组织化学的结果）中的表达。 目的1）DFP注射后。对于特定目标3，我们将确定是否同时给予地西泮和 注射DFP后60分钟，7-Cl-O-nec-1和MK-801均能增强海马神经元的功能， 比任何一种都要好。对于特定目标4，我们将确定7-Cl-O-Nec-1 和/或MK-801（取决于特定目的2和3的结果）提供更大的神经保护 与给予载体21的大鼠相比，给予DFP的大鼠的空间学习和记忆能力更好 DFP诱导的癫痫持续状态（SE）后3个月。我们将使用的方法是（1）原位 经心脏灌注-用4%磷酸缓冲多聚甲醛固定大脑， 和免疫荧光显微镜;（2）无偏估计的体视光学分级仪 60 μ m厚的冠状切片后，海马CA 1 -3和门神经元的H & E染色， 光学显微镜（特异性目的1-4）或RIP 1、RIP 3和MLKL抗体用于免疫荧光 显微镜（特定目标2）;（3）亚细胞分级分离和蛋白质印迹作为一种单独的方法， 证实RIP 1、RIP 3和MLKL从DFP诱导的SE的核转位程度（特异性 目的（2）和（4）巴恩斯迷宫，检测DFP后2 ~ 1个月大鼠的空间学习记忆能力 注射，并将结果与背侧中剩余的正常神经元的数量相关联， 海马（具体目标4）。我们将用多因素方差分析和事后t检验分析数据， 使用合并标准差和α = 0.05。我们将确定RIP 1的抑制是否 神经保护作用，如果7-Cl-O-nec-1和MK-801均提供最佳神经保护作用， 如果这是真的，应该会影响有机磷酸盐暴露后的护理标准。