The overall goal of Project 2 is to identify effective therapeutic strategies for mitigating the delayed neurological sequelae of acute intoxication with the GABA antagonist tetramethylenedisulfotetramine (TETS) or organophosphorus (OP) cholinesterase inhibitors. In the context of accidental or terrorist release of these nerve poisons, it is likely that emergency medical support will not be available within the first 30-60 min after seizures begin, at which time the neuropathological processes that promote brain damage and ultimately persistent functional deficits have been set in motion. The specific objectives of Project 2 are to: (1) develop in vivo rodent models for assessing persistent neurological damage subsequent to seizures induced by TETS or the OPs diisopropylfluorophosphate (DFP) and parathion, with a focus on developing in vivo imaging modalities that can be used in preclinical and clinical studies to longitudinally monitor progressive neurological effects of intoxication and the efficacy of candidate therapeutics;and (2) identify effective therapeutic strategies for mitigating neurological damage following acute intoxication with these chemical threat agents. Project 2 will test the hypothesis that compounds that are more effective post-exposure anticonvulsants than diazepam will be more effective in mitigating the neurological damage following TETS- and OP-induced seizures than current medical countermeasures and that neuroprotection will be significantly enhanced by combining anticonvulsants with therapeutics that attenuate neuroinflammation and/or stabilize Ca channels. Neurological damage will be assessed up to 28 days post-exposure using: (1) histological and biochemical indices of neuropathology, inflammation/oxidative stress (Core A) and dysregulated calcium signaling (Project 3);(2) behavioral testing of cognitive and emotional behavior and telemetry EEG video monitoring of spontaneous recurrent seizures (conducted in collaboration with Project 1);and (3) in vivo imaging of neuroinflammation by positron emission tomography (PET) and functional connectivity by resting state functional magnetic resonance imaging (fcMRI). Pharmacological tools will include therapeutics already approved for use in humans or currently undergoing clinical trials (referred to as Tier 1 candidates) to facilitate translation of positive findings in rodent models to human studies as well as novel compounds identified in mechanistic screening studies in Core B and Project 3 (referred to as Tier 2 candidates). Anticonvulsants tested in Project 2 will include diazepam and lead compounds identified by Project 1 to terminate TETS- and DFP-induced seizures when administered at times post-exposure when diazepam has lost efficacy. Candidate lead compounds include AMPA receptor (AMPA-R) antagonists. Pharmacological tools for attenuating neuroinflammation and/or stabilizing Ca2+ channels will be the most promising compounds identified in mechanistic screening studies in Core B and Project 3. Lead candidates include the ryanodine receptor (RyR) antagonist dantrolene (Tier 1 therapeutic), inhibitors of soluble epoxide hydrolase (sEH), phosphodiesterase (PDE) and cyclooxygenase (COX) alone or in combination (Tier 2 therapeutics), and the KCa3.1 inhibitor TRAM-34 (Tier 2 therapeutic). Dosing paradigms will be informed by preliminary pharmacokinetic studies in Core B;optimization studies performed in Project 3 to evaluate timing, dose, and drug combinations;and determination by Core A of ADME and brain levels of therapeutic candidates and toxicants. Across all Aims, study design and data analyses will be developed in consultation with Core C. The most effective therapeutic strategies for enhancing neuroprotection in the DFP model will be assessed for neuroprotective efficacy against parathion and live nerve agent (soman). By comparing the efficacy of novel therapeutic approaches across models of acute TETS and OP intoxication, we hope to identify a convergent therapeutic strategy for protecting against diverse classes of chemical threat agents.
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