A non-protected larval zebrafish model for the investigation of novel strategies to protect against nerve agent-induced toxicity and seizures

神经毒剂是人类已知的最致命的化学物质之一，并继续构成重大的社会威胁。神经毒剂通过抑制大脑中影响神经系统的化学物质来发挥作用。因此，神经毒剂会导致大脑癫痫发作，从而导致严重的大脑损伤，甚至死亡。有几种药物被用作神经毒剂中毒的初始治疗，但这些药物有时无效，本身可能是有害的。此外，暴露于神经毒剂和提供治疗之间的延迟越长，例如在战场上，这些治疗有效的可能性就越低。因此，需要新的更好的治疗方案来防止神经毒剂的影响。目前用于测试治疗神经毒剂中毒的新药有效性的方法在很大程度上依赖于啮齿动物的使用。这样的实验既缓慢又昂贵，通常涉及严格的程序，如在大脑中植入电极和暴露于神经毒剂。需要开发更高吞吐量的方法来确定新的治疗方法，这些方法在伦理上也比目前可用的方法更有利。无保护的受精后4天(DPF)斑马鱼幼体可能被证明是无价的，因为它们已被证明对一系列诱发癫痫的药物有反应，并且可以快速且容易地进行大量测试。来自朴茨茅斯大学和埃克塞特大学的科学家将在以前NC3R资助的工作的基础上，将无保护的幼虫斑马鱼癫痫模型转移到DSTL，在那里这些方法可以用来确定神经毒剂中毒的新治疗方法。帕克博士是一名斑马鱼行为专家，他将开发4dpf幼虫癫痫发作的行为测量方法。许多方案是帕克博士在伦敦玛丽女王学院(Pi Caroline Brennan)为NC3Rs项目拨款工作期间开发的。温特博士是使用先进的成像技术检查斑马鱼癫痫发作期间大脑的专家，其中一些技术是在正在进行的NC3RS学生期间开发的。他的团队将专注于开发评估4dpf斑马鱼大脑中癫痫发作活动的方法，以了解预测哺乳动物效果的模型相关性。DSTL的终端用户将利用这种方法来识别和开发神经毒剂中毒的新治疗方法。DSTL的同事们将在国际国防研究界推动这一方法的更广泛采用，并将斑马鱼作为评估化学毒性的模型。这种方法可以取代大量用于测试对抗神经毒剂毒性的新疗法的啮齿动物，从而使啮齿动物的总体使用量减少约75%。有限的啮齿动物实验将仅用于证实目的。因此，我们的方法可以防止每年在这些严重的方案中使用至少1500只全球啮齿动物。除了直接替代啮齿动物外，在未受保护的斑马鱼幼体中产生的数据也可用于完善剩余的啮齿动物研究，以确保使用适当的无毒剂量。在升级为啮齿动物模型之前，还将通过识别和排除任何具有不良特性的推定治疗来进行改进。DSTL积极参与许多国际研究合作，包括与欧洲国家的双边安排以及澳大利亚、加拿大、英国和美国之间的一项重要的跨国协议--CBR谅解备忘录(MOU)。科恩博士将利用这些安排以及他作为CBR谅解备忘录下一项预测毒理学任务的英国负责人的身份，分享该项目的数据和方法，支持其成果，并影响其他国家的计划，以鼓励采用这项技术。

Nerve agents are amongst the most deadly chemicals known to man and continue to pose a significant societal threat. Nerve agents function by inhibiting chemicals in the brain that affect the nervous system. Thus, nerve agents cause seizures in the brain which can lead to severe brain damage and even death. Several drugs are used as initial treatments for nerve agent poisoning, but these are sometimes ineffective and can themselves be harmful. In addition, the greater the delay between exposure to the nerve agent and the provision of treatment, for example on the battlefield, the lower the likelihood of the effectiveness of these treatments. Consequently, new and better treatment options are needed to protect against the effects of nerve agents. Current methods used for testing new drug effectiveness for the treatment of nerve agent poisoning are largely reliant on the use of rodents. Such experiments are slow and costly, and usually involve severe procedures, such as the surgical implantation of electrodes in the brain and exposure to nerve agents. There is a requirement to develop higher throughput methods for identifying novel treatments, that are also more ethically favourable than those currently available. The non-protected 4-days post-fertilisation (dpf) larval zebrafish could prove invaluable as they have been shown to be responsive to a range of seizure-inducing drugs and can be tested quickly and easily in large numbers.Scientists from the universities of Portsmouth and Exeter will build on previous NC3Rs-funded work to transfer a non-protected larval zebrafish seizure model to Dstl, where the methods can be used to identify novel treatments for nerve agent poisoning. Dr Parker is a zebrafish behavioural expert, and will develop behavioural measures of seizures in 4dpf larvae. Many of the protocols were developed during Dr Parker's work on an NC3Rs project grant at Queen Mary, London (PI Caroline Brennan). Dr Winter is an expert in examining the brain during seizures in zebrafish using advanced imaging techniques, some of which have been developed during an ongoing NC3Rs studentship. His team will focus on developing approaches assessing seizure activity in the 4dpf zebrafish brain to understand model relevance for predicting effects in mammals. The end users at Dstl will utilise this approach for the identification and development of novel treatments for nerve agent poisoning. Dstl colleagues will promote the wider uptake of this approach, and the zebrafish as a model for assessing chemical toxicity, within the international defence research community. This approach could replace a significant number of rodents used in testing novel treatments against nerve agent toxicity, thus reducing overall rodent use by an estimated 75%. Limited rodent experiments would remain only for confirmatory purposes. Our approach could therefore prevent the yearly global use of at least 1500 rodents in these severe protocols. In addition to the direct replacement of rodents, the data generated in non- protected zebrafish larvae can also be used to refine remaining rodent studies to ensure that appropriate non-toxic doses are used. Refinement will also result from the identification and ruling out of any putative treatments with undesirable properties prior to escalation to rodent models. Dstl actively participates in a number of international research collaborations including bilateral arrangements with European countries and an important multinational agreement between the Australia, Canada, the UK and the USA, the CBR Memorandum of Understanding (MOU). Dr Kearn will use these arrangements and his position as UK lead for a predictive toxicology task under the CBR MOU to share data and methodologies from this project, champion its outputs and influence other Nations' programmes to encourage uptake of this technology.