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.

Keywords: medical countermeasures; mixed-sex cohort; nerve agent; sex as a biological variable; status epilepticus severity; telemetry.

FIGURE 1

Experimental design and the relationship between estrous and convulsive seizures (CS) duration during status epilepticus (SE). (A) After randomization, grouping, and coding, three cohorts of animals were exposed to soman; first cohort had 24 rats (all males; nine rats from this group were used for implanting telemetry device at ∼3 months post-soman), second cohort had 50 rats without telemetry (25 rats per sex), and the third cohort had 35 rats with telemetry (17–18 rats per sex) and 10 rats without telemetry (eight males and two females). Each cohort of animals received soman (132 μg/kg, s.c.), HI-6 (125 mg/kg, i.m.), atropine sulfate (2 mg/kg, i.m.), and midazolam (3 mg/kg, i.m.) from the same pool of reagents prepared fresh on the day of the experiment. (B) Representative images showing vaginal cytology at each stage of estrous. Scale = 25 μm. (C) Percent of telemetry and non-telemetry animals at each stage of estrous. (D) Number of minutes in CS during SE for females in each stage of estrous. ANOVA or Kruskal–Wallis (n = 3–10).

FIGURE 2

Behavioral SE severity and latency to the onset of first CS comparison between sexes and telemetry versus non-telemetry animals. (A) Seizure stage overtime, latency to first CS and CS duration in non-telemetry males and females. (B) Seizure stage overtime, latency to first CS and CS duration in telemetry males and females. (C) Latency to first CS and CS duration comparison between non-telemetry and telemetry animals. Mixed measures ANOVA, or Mann–Whitney (n = 18–57). **p < 0.01.

FIGURE 3

Representative EEG traces and the corresponding images captured from integrated video-EEG system. (A) EEG signatures for seizure stages 1–5 with corresponding power and behavior. (B) Baseline (left) and SE EEG traces for a female animal. (C) Baseline (left) and SE EEG traces for a male animal. (D) Enlarged power spectrum.

FIGURE 4

Seizures (CS, convulsive seizure) detected on EEG and spike rate comparison between male and female rats during SE, and the immediate effect of midazolam (3 mg/kg, i.m.) on spike rate. (A) Spike rate over time between male and female animals. (B,C) Average spike rate during soman (B) and in the first 30 min after soman. (D) Total duration of seizures post soman. (E) SE duration comparison between behavioral (BEH) SE and EEG based SE in males and females.

FIGURE 5

Spontaneous recurrent seizures beginning 2 months post Soman. (A) Heatmap showing the incidence of SRS in male rats during the 35 days of continuous video-EEG observation. Telemetry device was implanted at ∼2 months post-SE. Six out of 7 severe SE rats developed SE. Two mild-SE rats did not show any SRS. Initial SE severity and the number of SRS episodes during the observation period is tabulated next to the heatmap. (B) Representative SRS episode and corresponding behaviors captured from integrated video-EEG system, corresponding powerbands are illustrated. R1–R6 represent rat number and the day in parenthesis indicate the SRS occurrence post-soman.

FIGURE 6

Representative images of amygdala (4 months post-soman) showing (A) reactive microgliosis (IBA1 + CD68, red IBA1 positive microglia, green/yellow CD68), (B) astrogliosis (GFAP, green), and (C) neurodegeneration (FJB + NeuN, green label FJB, red labeled cells NeuN). Examples of reactive and non-reactive astrocytes and microglia are shown in (D,E). T-test (n = 7) scale bar, 50 μm (A,B), 20 μm (D,E); field = 0.44 μm². *p < 0.05.