Abstracts

Rationale: Organophosphates (OP) such as the pesticide diisopropylfluorophosphate (DFP) and the nerve agent sarin are lethal chemicals that induce seizures, status epilepticus (SE), and brain damage. Midazolam, a benzodiazepine modulator of synaptic GABA-A receptors, is being considered as a new anticonvulsant for nerve agents. There is illustrious evidence of benzodiazepine resistance or refractory SE after OP intoxication.  Here, we characterized the time-course of protective efficacy of midazolam in rats exposed to DFP, a credible pesticide threat agent and chemical surrogate for nerve agents.   Methods: The surrogate DFP model was used for OP intoxication in rats. Rats were given a standard antidote regimen (atropine methylnitrate and 2-PAM) after DFP. Midazolam (2 mg/kg, im) was given at 10, 40, 60 or 120 min after DFP exposure. Behavioral and EEG seizures were monitored for 24 h after DFP exposure. The extent of brain injury was determined 3 days after DFP by stereological analysis of fluoro-jade B(+) neurons, neuronal nuclei(+) principal neurons, parvalbumin(+) interneurons, as well as GFAP(+) and IBA1(+) cellular neuroinflammation. Results: Seizures were elicited within ~8 min after DFP that progressively developed into persistent SE lasting for hours. DFP exposure resulted in massive neuronal injury or necrosis, neurodegeneration of principal cells and interneurons, and neuroinflammation as evident by extensive activation of microglia and astrocytosis in the hippocampus, amygdala and other brain regions. Midazolam controlled seizures, neurodegeneration, and neuroinflammation when given early (10 min), but it was less effective when given at 40 min or later. Delayed therapy (>40 min), a simulation of practical therapeutic window for first responders or hospital admission, was associated with reduced seizure protection and variable neuroprotection that was time- and brain region-dependent. Conclusions: These results strongly reaffirm that the DFP-induced seizures and brain damage are progressively resistant to delayed treatment with midazolam, confirming the benzodiazepine refractory SE after OP intoxication.   Funding: Supported by NIH CounterACT grants U01 NS083460 & R21 NS099009.