Soman (GD) Rat Model to Mimic Civilian Exposure to Nerve Agent: Mortality, Video-EEG Based Status Epilepticus Severity, Sex Differences, Spontaneously Recurring Seizures, and Brain Pathology

Abstract

Modeling a real-world scenario of organophosphate nerve agent (OPNA) exposure is challenging. Military personnel are premedicated with pyridostigmine, which led to the development of OPNA models with pyridostigmine/oxime pretreatment to investigate novel therapeutics for acute and chronic effects. However, civilians are not premedicated with pyridostigmine/oxime. Therefore, experimental models without pyridostigmine were developed by other laboratories though often only in males. Following OPNA exposure, prolonged convulsive seizures (CS) or status epilepticus (SE) are concerning. The duration and severity of CS/SE determine the extent of brain injury in survivors even after treating with medical countermeasures (MCM)/antidotes such as atropine, an oxime, and an anticonvulsant such as diazepam/midazolam. In this study, using a large mixed sex cohort of adult male and female rats, without pretreatment, we demonstrate severe SE lasting for >20 min in 82% of the animals in response to soman (GD,132 μg/kg, s.c.). Atropine sulfate (2 mg/kg, i.m.) and HI-6 (125 mg/kg, i.m.) were administered immediately following soman, and midazolam (3 mg/kg, i.m.) 1 h post-exposure. Immediate MCM treatment is impractical in civilian exposure to civilians, but this approach reduces mortality in experimental models. Interestingly, female rats, irrespective of estrous stages, had an average of 44 min CS (stage ≥ 3), while males had an average of 32 min CS during SE, starting from soman exposure to midazolam treatment. However, in telemetry device implanted groups, there were no significant sex differences in SE severity; males had 40 min and females 43 min of continuous CS until midazolam was administered. No animals died prior to midazolam administration and less than 5% died in the first week after soman intoxication. In telemetered animals, there was a direct correlation between EEG changes and behavioral seizures in real-time. In the long-term, convulsive spontaneously recurring seizures (SRS) were observed in 85% of randomly chosen animals. At 4-months post-soman, the brain histology confirmed reactive gliosis and neurodegeneration. The novel findings of this study are that, in non-telemetered animals, the SE severity following soman intoxication was significantly greater in females compared to males and that the estrous cycle did not influence the response.

DFP-Induced Status Epilepticus Severity in Mixed-Sex Cohorts of Adult Rats Housed in the Same Room: Behavioral and EEG Comparisons

“…Our previous DFP studies were conducted in male and female rats at different time points, with or without telemetry devices ( Gage et al, 2020 , 2022b ; Putra et al, 2020a , 2020b ). The published literature on other chemoconvulsants such as KA and pilocarpine models ( Tan and Tan, 2001 ; Scharfman and MacLusky, 2014 ; Smith et al, 2015 ), and our previous studies in the rat DFP model, led to the hypothesis that sex differences exist in SE severity in response to chemoconvulsant exposure.…”

“…The device consists of bipotential electrodes for single-channel recording. As described in our previous publications, we implanted the electrodes bilaterally, one on each side on the surface of the cortex over the dura mater, and the device was placed in a subcutaneous pouch ( Puttachary et al, 2016a ; Putra et al, 2020a , 2020b ; Sharma et al, 2021 ; Gage et al, 2022b ). Before surgery, the animals were administered an analgesic, buprenorphine (0.3 mg/kg, s.c.).…”

“…In this study, behavioral SE severity is defined as the duration (in minutes) of convulsive seizures (stage 3 and above) an animal spends between DFP and MDZ treatments. Animals were directly observed and scored, by two experimenters, on each minute for the seizure stage based on a modified Racine scale ( Racine, 1972 ) as described in our previous publications ( Putra et al, 2020a ; Gage et al, 2020 , 2022b ). In telemetry rats, in addition to the integrated vEEG acquisition, the seizures were also scored by direct observation (independent of vEEG).…”

“…We thought co-housing may have synchronized the estrous and yielded relatively consistent higher SE severity to soman challenge. However, the vaginal cytology did not provide evidence for estrous cycle synchronization at the time of soman exposure, and there was no correlation between the stages of the estrous cycle and SE severity ( Gage et al, 2022b ). We speculated that handling stress during the vaginal lavage might have reduced the seizure threshold in females in soman studies.…”

“…Recently, in a soman (GD, an OPNA) model, we surprisingly found that the female rats without telemetry device implantation had significantly higher SE-severity than the males at the same dose of soman (132 μg/kg, 1.2 LD 50 ) ( Gage et al, 2022b ). In that study, both males and females were housed in the same room, but in separate cages, for several days before soman exposure.…”