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

项目摘要 化学品威胁是一个主要的公共卫生问题，CounterACT认识到有必要确定新的化学品威胁。 医疗对策（MCM），改善公共卫生应对大规模伤亡事件， 化学威胁。有机磷酸盐（OP）试剂由于其在工业中的广泛可用性而受到特别关注。 常见的杀虫剂及其在化学神经毒剂中的应用。OP引起的癫痫是一种严重的后果 OP-中毒和治疗这种并发症的最佳药物尚不清楚。主要差距包括： 在大规模伤亡之前对人类进行系统临床评估的机会，现有 药物，这可能无法控制癫痫发作或兴奋性中毒，并恶化镇静;和缺乏方向， 发现新的MCM。为了解决这些差距，我们提出了一个斑马鱼模型的特点OP诱导 使用毒死蜱-氧磷（CPO）1 --一种常见OP杀虫剂的活性代谢物， 毒死蜱-并推进其用于高通量化合物筛选，以确定新的MCMs， OP诱导的癫痫发作和兴奋性毒性。在目标1中，我们将描述一个幼斑马鱼模型的CPO诱导的 捕捉和优化参数的识别新的对策。在目标1A中，我们将确认 使用电生理学（EEG）以及基于钙的非侵入性测量的癫痫活动 荧光。此外，我们将评估存活率和兴奋性毒性，如通过细胞凋亡的负担所证明的。 使用TUNEL测定和时间密集性较低的吖啶橙子（AO）测定来检测癫痫发作后的细胞。在Aim 1B中， 我们将通过以下方法确定用于检测CPO诱导癫痫发作的新对策的理想测定参数： 确定现有对策的最大耐受剂量，随后是CPO 剂量反应实验，以确定导致CPO诱导的癫痫发作减少25-50%的条件 对比对照组。选择这一目标是为了检测现有对策的效果， 为检测上级对抗措施提供动态范围。在目标1C中，我们将量化镇静 作为常规AED中抗癫痫活性的函数，通过确定几种AED的ED 50， CPO诱导的癫痫发作;这些数据将与AED在视觉惊吓中的镇静作用进行比较 反应（VSR）和声学惊吓反应（ASR）测定。最后，在目标2中，我们将实现一个整体 生物化合物筛选抗OP诱导的癫痫发作和兴奋性毒性的新型MCM，并优先考虑命中 基于排序配对稳健严格标准化平均差（SSMD*）和最小镇静效应。 总的来说，这一探索性的R21是响应多方面的反ACT计划，并提供 一个框架，为整个生物体在体内发现新的对策，对CPO，可能是 扩展到其他OP代理。

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.