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

基本信息

批准号：
10618855
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
项目类别：
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/10618855
关键词：
Acetylcholine Acetylcholinesterase Inhibitors Acute Analysis of Variance Behavioral Brain Brain Injuries Calpain Cardiac 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 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 Proteins RIPK1 gene RIPK3 gene Rattus Receptor Activation Receptor Inhibition Research Personnel Role Seizures Severities Stains Status Epilepticus Terrorism Testing Thick Time Toxic effect Western Blotting antibody mimetics dizocilpine excitotoxicity fluorophosphate inhibitor light microscopy meter mixed lineage kinase 3 neuroprotection paraform postsynaptic neurons preservation receptor sample fixation spatial memory standard of care toxic organophosphate insecticide exposure

项目摘要

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.

我们在该项目中的目标是确定癫痫发作诱导的效果全身施用大鼠大脑上的有机磷酸二异丙磷酸二异丙基磷酸（DFP），然后确定编程坏死在DFP-的坏死和出口途径的作用诱导神经元坏死。对于特定目标1，我们将确定时间课程和程度神经元坏死（使用苏木精和曙红[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（MLKL），使用免疫荧光显微镜6、24和 DFP后72小时内海马CA1-CA3和Hilus。对于特定目标2，我们将确定地西epa（当前护理标准）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（Dizocilpine），给定60 DFP注射后的最小值是神经保护性的，如果7-CL-O-NEC-1减少了核易位 24 h（或6或72 h）在海马中的RIP1，RIP3和MLKL，取决于特定的结果目标1）注射DFP后。对于特定目的3，我们将确定地西ep和地西ep和 DFP注射后60分钟后，7-CL-O-NEC-1和MK-801均可提供更大的海马神经保护措施比本身给出的任何一个。对于特定目标4，我们将确定是否7-CL-O-NEC-1 和/或MK-801（取决于特定目标2和3的结果）提供了更大的神经保护作用与给定的车辆相比，DFP处理的大鼠的空间学习和记忆更好 DFP诱导状态癫痫持续状态（SE）几个月。我们将使用的方法是（1）原位大脑的心脏灌注固定，带有4％磷酸盐缓冲多聚甲醛的光和免疫荧光显微镜；（2）无偏估计的立体观念分级器 60 µm厚的冠状切片后，海马CA1-3和肺门神经元的含量用H＆E染色光学显微镜（特定目标1-4）或RIP1，RIP3和MLKL抗体用于免疫荧光显微镜（特定目标2）；（3）亚细胞分级和蛋白质印迹作为单独的方法确认来自DFP诱导的SE的RIP1，RIP3和MLKL的核转运程度（特定目标2）; （4）Barnes迷宫，以测试DFP后两个月后两个月测试老鼠的空间学习和记忆注射，并将发现与背侧剩余正常神经元的数量相关联海马（特定目标4）。我们将使用多因素方差分析和事后t检验分析数据，使用合并的标准偏差和α= 0.05。我们将确定RIP1的抑制是否为神经保护性且如果提供了7-CL-O-NEC-1和MK-801的最佳神经保护作用共同影响有机磷酸盐暴露后的护理标准。