High-throughput in vivo discovery of novel countermeasures against organophosphate-induced seizure and status epilepticus using zebrafish

项目总结 化学品威胁是一个主要的公共卫生问题，反化学武器组织认识到有必要确定新的 改善公共卫生应对重大伤亡事件的医疗对策(MCM) 化学威胁。有机磷(OP)试剂因其在 常见农药及其在化学神经毒剂中的应用。OP诱发的癫痫是一种严重的后果 目前尚不清楚OP中毒的原因和治疗这种并发症的最佳药物。主要差距包括有限的 在大规模伤亡之前对人类进行系统临床评估的机会，现有的缺点 可能无法控制癫痫发作或兴奋毒性并恶化镇静的药物；以及缺乏指导 发现新的MCM。为了解决这些差距，我们建议描述一个斑马鱼模型的OP诱导 常见有机磷农药的活性代谢物毒死蜱-氧磷(CPO)1的癫痫发作和兴奋毒性， 毒死蜱--并推进其在高通量化合物筛选中的应用，以寻找新的抗病毒死蜱 OP诱发的癫痫发作和兴奋性毒性。在目标1中，我们将描述CPO诱导的斑马鱼幼体模型。 抓取和优化参数，以识别新的对策。在目标1A中，我们将确认 电生理(EEG)和基于钙的非侵入性测量的癫痫活动 荧光。此外，我们还将评估存活率和兴奋性毒性，以细胞凋亡的负担为证据。 使用TUNEL法和时间密集度较低的丫啶橙(AO)法检测癫痫发作后的细胞数量。在目标1B中， 我们将确定理想的检测参数，用于检测针对CPO诱导的癫痫的新对策 确定现有对策下CPO对斑马鱼幼体的最大耐受量 剂量-反应实验确定导致CPO诱导的癫痫发作减少25%-50%的条件 与控制组的对抗。选择此目标是为了检测现有对策的效果，同时 为检测优势对策提供动态范围。在目标1C中，我们将量化镇静 作为常规抗癫痫药物中抗癫痫活性的函数，通过建立几种抗癫痫药物的ED50作为响应 这些数据将与AEDs在视觉惊吓中的镇静作用进行比较 反应(VSR)和声学惊厥反应(ASR)分析。最后，在目标2中，我们将实现一个 抗OP诱发癫痫和兴奋性毒性的新型MCMS的生物体复合筛选和HITS优先 基于排序配对稳健严格标准化均值差(SSMD*)和最小镇静效果。 总而言之，这个探索性的R21响应于中和程序的多个方面，并提供 在体内发现针对CPO的新对策的整体框架可能是 扩展到更多的操作人员。

PROJECT SUMMARY Chemical threats are a major public health concern and CounterACT recognizes the need to identify new medical countermeasures (MCMs) that improve the public health response to mass casualty events related to chemical threats. Organophosphate (OP) agents are of particular concern due to their wide availability in common pesticides and their use in chemical nerve agents. OP-induced seizures are a serious consequence of OP-intoxication and best agents for treating this complication are unclear. Major gaps include limited opportunities for systematic clinical evaluations in humans prior to mass casualty, drawbacks to existing medication, which may not control seizures or excitotoxicity, and worsen sedation; and lack of direction in discovering new MCMs. To address these gaps, we propose to characterize a zebrafish model of OP-induced seizures and excitotoxicity using chlorpyrifos-oxon (CPO)1 – an active metabolite of the common OP pesticide, chlorpyrifos -- and to advance its use for high-throughput compound screening to identify novel MCMs against OP-induced seizures and excitotoxicity. In Aim 1, we will characterize a larval zebrafish model of CPO-induced seizure and optimize parameters for the identification of novel countermeasures. In Aim 1A, we will confirm seizure activity using both electrophysiology (EEG) as well as noninvasive measures based on calcium fluorescence. In addition, we will assess survival and excitotoxicity as evidenced by the burden of apoptotic cells following seizures using TUNEL assay and less time-intensive acridine orange (AO) assay. In Aim 1B, we will identify the ideal assay parameters for detecting novel countermeasures to CPO-induced seizures by identifying the maximum tolerated dose in larval zebrafish of existing countermeasures followed by a CPO dose-response experiment to identify the conditions that result in 25-50% reduction in CPO-induced seizures versus control. This target is chosen to allow detection of the effect of existing countermeasures while providing dynamic range for the detection of superior countermeasures. In Aim 1C, we will quantify sedation as a function of anti-seizure activity in conventional AEDs, by establishing ED50 for several AEDs in response to CPO-induced seizures; these data will be compared to the sedative effect of AEDs in the visual startle response (VSR) and acoustic startle response (ASR) assays. Finally, in Aim 2, we will implement a whole organism compound screen for novel MCMs against OP-induced seizure and excitotoxicity, and prioritize hits based on ranked paired robust strictly standardized mean difference (SSMD*) and least sedating effects. Taken together, this exploratory R21 is responsive to multiple facets of the CounterACT program and provides a framework for whole organism in vivo discovery of novel countermeasures against CPO that may be extended to additional OP agents.