Persistent neuropathology and behavioral deficits in a mouse model of status epilepticus induced by acute intoxication with diisopropylfluorophosphate.

Organophosphate (OP) nerve agents and pesticides are a class of neurotoxic compounds that can cause status epilepticus (SE), and death following acute high-dose exposures. While the standard of care for acute OP intoxication (atropine, oxime, and high-dose benzodiazepine) can prevent mortality, survivors of OP poisoning often experience long-term brain damage and cognitive deficits. Preclinical studies of acute OP intoxication have primarily used rat models to identify candidate medical countermeasures. However, the mouse offers the advantage of readily available knockout strains for mechanistic studies of acute and chronic consequences of OP-induced SE. Therefore, the main objective of this study was to determine whether a mouse model of acute diisopropylfluorophosphate (DFP) intoxication would produce acute and chronic neurotoxicity similar to that observed in rat models and humans following acute OP intoxication. Adult male C57BL/6J mice injected with DFP (9.5 mg/kg, s.c.) followed 1 min later with atropine sulfate (0.1 mg/kg, i.m.) and 2-pralidoxime (25 mg/kg, i.m.) developed behavioral and electrographic signs of SE within minutes that continued for at least 4 h. Acetylcholinesterase inhibition persisted for at least 3 d in the blood and 14 d in the brain of DFP mice relative to vehicle (VEH) controls. Immunohistochemical analyses revealed significant neurodegeneration and neuroinflammation in multiple brain regions at 1, 7, and 28 d post-exposure in the brains of DFP mice relative to VEH controls. Deficits in locomotor and home-cage behavior were observed in DFP mice at 28 d post-exposure. These findings demonstrate that this mouse model replicates many of the outcomes observed in rats and humans acutely intoxicated with OPs, suggesting the feasibility of using this model for mechanistic studies and therapeutic screening.

Combined treatment with diazepam and allopregnanolone reverses tetramethylenedisulfotetramine (TETS)-induced calcium dysregulation in cultured neurons and protects TETS-intoxicated mice against lethal seizures.

Tetramethylenedisulfotetramine (TETS) is a potent convulsant GABAA receptor blocker. Mice receiving a lethal dose of TETS (0.15 mg/kg i.p.) are rescued from death by a high dose of diazepam (5 mg/kg i.p.) administered shortly after the second clonic seizure (∼20 min post-TETS). However, this high dose of diazepam significantly impairs blood pressure and mobility, and does not prevent TETS-induced neuroinflammation in the brain. We previously demonstrated that TETS alters synchronous Ca(2+) oscillations in primary mouse hippocampal neuronal cell cultures and that pretreatment with the combination of diazepam and allopregnanolone at concentrations having negligible effects individually prevents TETS effects on intracellular Ca(2+) dynamics. Here, we show that treatment with diazepam and allopregnanolone (0.1 µM) 20 min after TETS challenge normalizes synchronous Ca(2+) oscillations when added in combination but not when added singly. Similarly, doses (0.03-0.1 mg/kg i.p.) of diazepam and allopregnanolone that provide minimal protection when administered singly to TETS intoxicated mice increase survival from 10% to 90% when given in combination either 10 min prior to TETS or following the second clonic seizure. This therapeutic combination has negligible effects on blood pressure or mobility. Combined treatment with diazepam and allopregnanolone also decreases TETS-induced microglial activation. Diazepam and allopregnanolone have distinct actions as positive allosteric modulators of GABAA receptors that in combination enhance survival and mitigate neuropathology following TETS intoxication without the adverse side effects associated with high dose benzodiazepines. Combination therapy with a benzodiazepine and neurosteroid represents a novel neurotherapeutic strategy with potentially broad application.