Background: Acute exposure to seizurogenic organophosphate (OP) nerve agents (OPNA) such as diisopropylfluorophosphate (DFP) or soman (GD), at high concentrations, induce immediate status epilepticus (SE), reactive gliosis, neurodegeneration, and epileptogenesis as a consequence. Medical countermeasures (MCMs—atropine, oximes, benzodiazepines), if administered in < 20 min of OPNA exposure, can control acute symptoms and mortality. However, MCMs alone are inadequate to prevent OPNA-induced brain injury and behavioral dysfunction in survivors. We have previously shown that OPNA exposure-induced SE increases the production of inducible nitric oxide synthase (iNOS) in glial cells in both short- and long- terms. Treating with a water soluble and highly selective iNOS inhibitor, 1400W, for 3 days significantly reduced OPNA-induced brain changes in those animals that had mild–moderate SE in the rat DFP model. However, such mitigating effects and the mechanisms of 1400W are unknown in a highly volatile nerve agent GD exposure. Methods: Mixed-sex cohort of adult Sprague Dawley rats were exposed to GD (132 μg/kg, s.c.) and immediately treated with atropine (2 mg/kg, i.m) and HI-6 (125 mg/kg, i.m.). Severity of seizures were quantified for an hour and treated with midazolam (3 mg/kg, i.m.). An hour post-midazolam, 1400W (20 mg/kg, i.m.) or vehicle was administered daily for 2 weeks. After behavioral testing and EEG acquisition, animals were euthanized at 3.5 months post-GD. Brains were processed for neuroinflammatory and neurodegeneration markers. Serum and CSF were used for nitrooxidative and proinflammatory cytokines assays. Results: We demonstrate a significant long-term (3.5 months post-soman) disease-modifying effect of 1400W in animals that had severe SE for > 20 min of continuous convulsive seizures. 1400W significantly reduced GD-induced motor and cognitive dysfunction; nitrooxidative stress (nitrite, ROS; increased GSH: GSSG); proinflammatory cytokines in the serum and some in the cerebrospinal fluid (CSF); epileptiform spikes and spontaneously recurring seizures (SRS) in males; reactive gliosis (GFAP + C3 and IBA1 + CD68-positive glia) as a measure of neuroinflammation, and neurodegeneration (especially parvalbumin-positive neurons) in some brain regions. Conclusion: These findings demonstrate the long-term disease-modifying effects of a glial-targeted iNOS inhibitor, 1400W, in a rat GD model by modulating reactive gliosis, neurodegeneration (parvalbumin-positive neurons), and neuronal hyperexcitability.

Sex is a biological variable in experimental models. In our previous diisopropylfluorophosphate (DFP) studies, female rats required a higher dose of DFP to achieve a somewhat similar severity of status epilepticus (SE) as males. In those studies, male and female rats were bought separately from the same vendor, housed in different rooms, and the DFP used was from different batches. We had also shown that surgery for epidural electrodes implantation reduces the threshold for SE. Our recent study in the soman (GD) model using a mixed-sex cohort of rats housed individually but in the same room showed that females achieved significantly higher SE severity than males for the same dose of GD. In this study, we demonstrate that housing the mixed-sex cohorts in the same room and treating them with DFP (4 mg/kg, s.c.) from the same pool, though from different batches, yielded reproducible SE severity in both sexes and both telemetry (surgery) and non-telemetry (non-surgery) groups. We conducted experiments in four mixed-sex cohorts of adult Sprague-Dawley rats. In females, the surgery for implanting the telemetry devices reduced the latency to convulsive seizure (CS) and increased SE severity compared to non-telemetry females. However, there were no sex differences in latency or SE severity within telemetry or non-telemetry groups. Once animals reached CS stage ≥3, they remained in CS stage in both sexes until midazolam was administered. Midazolam (3 mg/kg, i.m.) treatment 1-one-hour post-DFP significantly reduced epileptiform spikes in both sexes. The mortality was only 2% in 24 h. Irrespective of sex or stage of estrous cycle or surgery, the animals had continuous convulsive SE for ∼40 min. In telemetry rats, electrographic changes correlated with behavioral seizures. However, there was a significant difference in SE severity and the latency between directly-observed behavioral CS and EEG-based CS quantification in both sexes. Overall, these results suggest that housing both sexes in the same room and treating with DFP in a mixed-sex cohort from the same pool of reagents will minimize variability in SE severity. Such rigorous experiments will yield better outcomes while testing disease-modifying agents in epilepsy models.

NADPH oxidase (NOX) is a primary mediator of superoxides, which promote oxidative stress, neurodegeneration, and neuroinflammation after diisopropylfluorophosphate (DFP) intoxication. Although orally administered mitoapocynin (MPO, 10 mg/kg), a mitochondrial-targeted NOX inhibitor, reduced oxidative stress and proinflammatory cytokines in the periphery, its efficacy in the brain regions of DFP-exposed rats was limited. In this study, we encapsulated MPO in polyanhydride nanoparticles (NPs) based on 1,6-bis(p-carboxyphenoxy) hexane (CPH) and sebacic anhydride (SA) for enhanced drug delivery to the brain and compared with a high oral dose of MPO (30 mg/kg). NOX2 (GP91^phox) regulation and microglial (IBA1) morphology were analyzed to determine the efficacy of MPO-NP vs. MPO-oral in an 8-day study in the rat DFP model. Compared to the control, DFP-exposed animals exhibited significant upregulation of NOX2 and a reduced length and number of microglial processes, indicative of reactive microglia. Neither MPO treatment attenuated the DFP effect. Neurodegeneration (FJB+NeuN) was significantly greater in DFP-exposed groups regardless of treatment. Interestingly, neuronal loss in DFP+MPO-treated animals was not significantly different from the control. MPO-oral rescued inhibitory neuronal loss in the CA1 region of the hippocampus. Notably, MPO-NP and MPO-oral significantly reduced astrogliosis (absolute GFAP counts) and reactive gliosis (C3+GFAP). An analysis of inwardly rectifying potassium channels (K_ir4.1) in astroglia revealed a significant reduction in the brain regions of the DFP+VEH group, but MPO had no effect. Overall, both NP-encapsulated and orally administered MPO had similar effects. Our findings demonstrate that MPO effectively mitigates DFP-induced reactive astrogliosis in several key brain regions and protects neurons in CA1, which may have long-term beneficial effects on spontaneous seizures and behavioral comorbidities. Long-term telemetry and behavioral studies and a different dosing regimen of MPO are required to understand its therapeutic potential.

Acute exposure to the organophosphate nerve agent (OPNA) soman (GD) triggers severe and persistent brain injury characterized by seizures, oxidative stress

Acute exposure to organophosphate nerve agents such as soman (GD) induces status epilepticus (SE) that progresses to chronic network dysfunction, neuroinflammation, and epilepsy, yet the signaling mechanisms sustaining this transition remain unclear. We tested whether combined targeting of nitro-oxidative stress and Src family tyrosine kinase (SFK) signaling using 1400W (iNOS inhibitor) and saracatinib (SAR), respectively, could disrupt disease progression in a translational rat model. Adult mixed-sex Sprague-Dawley rats were exposed to soman and treated with standard medical countermeasures followed by vehicle, monotherapy, or dose-reduced combination therapy- optimized through pharmacokinetic analyses. Longitudinal behavioral testing, resting-state fMRI, and video-EEG were integrated with histological, molecular, and systemic assessments. Soman exposure produced robust SE without sex differences and resulted in persistent behavioral deficits, disrupted functional connectivity, epileptiform activity, neuroinflammation, and neurodegeneration. While monotherapies provided partial benefit, combination therapy consistently produced the most robust effects, improving motor and fear-associated learning and memory, restoring limbic network connectivity, reducing epileptiform spike burden, shifting seizure phenotype toward moderate non-convulsive events, attenuating gliosis, neurodegeneration, and proinflammatory gene expression. Systemic nitro-oxidative stress was normalized, and long-term treatment was well tolerated without hematological or organ toxicity. These findings identify that convergent iNOS and SFK signaling drives chronic soman neuropathology, with dual inhibition therapy superior to monotherapy.