Midazolam-Resistant Seizures and Brain Injury after Acute Intoxication of Diisopropylfluorophosphate, an Organophosphate Pesticide and Surrogate for Nerve Agents

Abstract

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 currently considered as a new anticonvulsant for nerve agents. Here, we characterized the time course of protective efficacy of midazolam (0.2-5 mg/kg, i.m.) in rats exposed to DFP, a chemical threat agent and surrogate for nerve agents. Behavioral and electroencephalogram (EEG) seizures were monitored for 24 hours after DFP exposure. The extent of brain injury was determined 3 days after DFP exposure by unbiased stereologic analyses of valid markers of neurodegeneration and neuroinflammation. Seizures were elicited within ∼8 minutes after DFP exposure 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 astrocytes in the hippocampus, amygdala, and other brain regions. Midazolam controlled seizures, neurodegeneration, and neuroinflammation when given early (10 minutes) after DFP exposure, but it was less effective when given at 40 minutes or later. Delayed therapy (≥40 minutes), a simulation of the practical therapeutic window for first responders or hospital admission, was associated with reduced seizure protection and neuroprotection. 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. Thus, novel anticonvulsants superior to midazolam or adjunct therapies that enhance its efficacy are needed for effective treatment of refractory SE.

Antiseizure and neuroprotective effects of delayed treatment with midazolam in a rodent model of organophosphate exposure

“…These data are consistent with those of other reports that have shown little or no effect of MDZ on neuronal death in delayed treatment (≥40 minutes) of NA‐induced SE . In contrast, treatment with MDZ at 10 to 20 minutes appears to be effective at reducing neuronal death . Our analysis of the change in power in the gamma band for up to 20 hours from the time of treatment suggests that the observed neuronal damage may be affected by the reemergence of epileptiform activity hours after treatment, which is similar to what has been previously shown by Wu et al …”

“…Although MDZ did provide neuroprotection when all of the data were grouped together, the lack of effect of MDZ on neuronal loss at individual delays and the small effect of MDZ when these data were grouped together emphasize the need to focus on new treatments that target the molecular pathways that would reduce seizure‐induced neuronal death when administered after electrographic SE has been suppressed with drugs like MDZ. These data are consistent with those of other reports that have shown little or no effect of MDZ on neuronal death in delayed treatment (≥40 minutes) of NA‐induced SE . In contrast, treatment with MDZ at 10 to 20 minutes appears to be effective at reducing neuronal death .…”

“…Studies reporting a time-dependent decrease in the efficacy of benzodiazepines have typically compared much shorter delay times for benzodiazepine administration, ranging from a few minutes to 30 to 40 minutes as the longest intervals (ie, our shortest delay time was 30 minutes). For instance, in a study with a treatment paradigm that was very similar to ours, Wu et al observed a reduction in efficacy of MDZ in treatment of DFP-induced SE when treatment delay was increased from 10 to 40 minutes, 40,41 but they also found that MDZ treatment still partially suppressed DFP-induced SE when given after delays of 60 and 120 minutes. Our dose of MDZ (2.0 mg/ kg, IM) was similar to the above-mentioned studies, 32,39,40 although these authors used a slightly different analysis of the EEG (spikes/10 minutes) and a behavioral measure (seizure stage).…”

“…For instance, in a study with a treatment paradigm that was very similar to ours, Wu et al observed a reduction in efficacy of MDZ in treatment of DFP-induced SE when treatment delay was increased from 10 to 40 minutes, 40,41 but they also found that MDZ treatment still partially suppressed DFP-induced SE when given after delays of 60 and 120 minutes. Our dose of MDZ (2.0 mg/ kg, IM) was similar to the above-mentioned studies, 32,39,40 although these authors used a slightly different analysis of the EEG (spikes/10 minutes) and a behavioral measure (seizure stage). Therefore, treatment with MDZ at 10 minutes was far more effective than at the delay times of 40 and 60 minutes, but by both measures these authors found that MDZ treatment still had some effect on seizures at 40 and 60 minutes.…”

“…13,39,40 In contrast, treatment with MDZ at 10 to 20 minutes appears to be effective at reducing neuronal death. 40,46 Our analysis of the change in power in the gamma band for up to 20 hours from the time of treatment suggests that the observed neuronal damage may be affected by the reemergence of epileptiform activity hours after treatment, which is similar to what has been previously shown by Wu et al 40 The primary goal of the CNS Program is to provide a screening assay available to any participant who is interested in generating data toward application for grant funding from the CounterACT program. As such, the scope of the work here is limited to the development of a medium-throughput assay that somewhat models a real-life exposure scenario, and not open to traditional academic scientific endeavor.…”