Rationale: Organophosphate nerve agents (OPNA) pose threat to both the military and civilians’ safety. OPNA exposure induces cholinergic crisis and status epilepticus (SE) because of irreversible inhibition of acetylcholinesterase. If not adequately treated immediately, SE-induced brain injury leads to an increased risk of epilepsy, severe brain pathology, and long‐term behavioral deficits due to the activation of disease promotors such as nitro-oxidative stress, neuroinflammation, and progressive neurodegeneration. Currently available medical countermeasures (MCMs) failed to alleviate these pathologies. In this study, we hypothesized that the modification of these disease promoters can be achieved using the novel drug 1400W, a highly selective inhibitor of
inducible nitric oxide synthase (iNOS/NOS-II), in addition to MCMs, in a rat Soman (GD) model.
Methods: Sprague Dawley rats (7-8 weeks, mixed-sex) were used in this study. One group (N=32) was implanted with telemetry devices for continuous EEG recording for 4 months to monitor spontaneously recurring seizures (SRS). The other group without devices (non-telemetry, N=50) were used for behavioral testing (5-6 weeks post-soman). The animals were challenged with a single dose of soman (1.2 LD
50, 132 mg/kg, SC), followed immediately by HI-6 (125 mg/kg, IM), atropine sulfate (2 mg/kg, IM), and one hour later by midazolam (MDZ, 3 mg/kg, IM). SE was scored and animals were randomized, grouped, and coded. An hour post-MDZ, animals were injected with vehicle or 1400W (20 mg/kg, IM, twice daily for the first three days and a single dose/day for the next 11 days) Vehicle/1400W group had both sexes and SE severity was matched. Behavioral tests were conducted to assess the effects of 1400W on motor coordination (rotarod), learning and memory (Novel Object Recognition (NOR)]. All animals were euthanized with pentobarbital (100 mg/kg) at the end of each study. Serum biochemical assays such as nitrite, ROS, and glutathione and ELISA for cytokines (IL-1β, IL-6, IL-10, TNF-α, & MCP1) were conducted.
Results: Females, irrespective of the stages of estrous cycle, had severe SE ( >40 min) than males (~30 min). However, in the telemetered group, both sexes had severe SE ( >40 min). 1400W significantly reduced SRS frequency and duration, and epileptiform spikes in males. Interestingly, vehicle-treated females had lower number of SRS and confounded the real effects of 1400W. Soman-induced motor dysfunction and learning and memory were significantly mitigated by 1400W in mixed-sex cohort. Soman-induced peripheral inflammatory cytokines (IL-1β, TNF-α, IL-6, and MCP-1), nitrite and ROS generation, and imbalanced GSH:GSSG ratio were significantly mitigated by 1400W. Cerebrospinal fluid cytokines were also mitigated by 1400W. No sex differences were observed in behavioral or biochemical parameters.
Conclusions: 1400W treatment reduces soman-induced seizure burden (in males), behavioral dysfunction, nitrooxidative stress, and key proinflammatory cytokines. Overall, 1400W can be a potential disease-modifying agent, in combination with MCM, in OPNA model.
Funding: This project is supported by the NIH/NINDS (1U01 NS117284).