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

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.

Nerve agents are amongst the deadliest chemicals known to man and continue to pose a significant societal threat. Nerve agents function via inhibition of acetylcholinesterase and cause seizures, which can progress to protracted, recurring seizures. Recurrent seizures can cause irreparable damage to the brain through extensive neuronal cell death. The first line treatment for nerve agent exposure includes benzodiazepines; however, these are sometimes ineffective. The greater the delay between exposure and treatment, for example on the battlefield, the lower the likelihood of treatment effectiveness. Consequently, alternative treatment options are required. Current methods used for testing new treatments are reliant on the use of rodents and the procedures are invasive, costly and time consuming. As such, more ethically and economically favourable methods for the testing of such treatments are needed. We propose using non-protected larval zebrafish as a non-mammalian replacement model. Non-protected larval zebrafish have already been shown to be responsive to a range of seizure-inducing pharmacologies, and are amenable to non-invasive higher throughput testing strategies. Our overall aim is to build on techniques developed during previous NC3Rs-funded research at Portsmouth and Exeter Universities to provide the defence research community with a non-protected larval zebrafish model that can be used to identify novel therapeutics to treat nerve agent-induced seizures. We will modify a technique in which seizures are visualised using light sheet microscopic Ca2+ imaging in a 4dpf transgenic zebrafish, to allow single z-plane assessment of neural activity on a standard confocal microscope. We will then refine behavioural models of seizure activity to create high throughput assays for the accurate quantification of convulsive behaviour. The new technology will be transferred to Dstl, where the direct relevance for the study of nerve agents will be definitively assessed.