Allopregnanolone as an Adjunct Therapy to Midazolam is More Effective Than Midazolam Alone in Suppressing Soman-Induced Status Epilepticus in Male Rats

Abstract

Aims: Humans and animals acutely intoxicated with the organophosphate soman can develop sustained status epilepticus (SE) that rapidly becomes refractory to benzodiazepines. We compared the antiseizure efficacy of midazolam, a current standard of care treatment for OP-induced SE, versus combined therapy with midazolam and allopregnanolone (ALLO) in a rat model of soman-induced SE.

Methods: Soman-intoxicated male rats with robust seizure behavior and high-amplitude electroencephalographic (EEG) activity were administered midazolam (0.65 mg, i.m.) 20 min after seizure initiation and 10 min later either a second dose of midazolam or ALLO (12 or 24 mg/kg, i.m.). Seizure behavior and EEG were monitored for 4 h after treatment. Brains were collected at the end of the monitoring period for histological analyses.

Results: Animals receiving 2 doses of midazolam exhibited persistent SE. Sequential dosing with midazolam followed by ALLO suppressed electrographic seizure activity. The combination therapy also significantly reduced soman-induced neurodegeneration and neuroinflammation compared to 2 doses of midazolam. High but not low dose ALLO was associated with transitory and reversible respiratory compromise during the 1 h period after dosing.

Conclusions: Treatment with midazolam followed by ALLO was more effective than 2 doses of midazolam in suppressing benzodiazepine-refractory, soman-induced SE, and in mitigating its acute neuropathological consequences.

Keywords: benzodiazepine; chemical threat agents; neurosteroid; organophosphate; polytherapy; seizure.

FIGURE 1

(A) Adult male Sprague Dawley rats were implanted with EEG headpieces and allowed to recover for 5–7 d. On the day of experimentation, all animals were given the oxime HI‐6 (125 mg/kg, i.p.) 30 min before exposure to soman (121 μg/kg, s.c.) and treated with atropine methyl nitrate (AMN; 2 mg/kg, i.m.) 1 min after exposure. At 20 min after seizure onset, all animals received atropine sulfate (AS; 0.45 mg/kg, i.m), pralidoxime (2‐PAM; 25 mg/kg, i.m.), and midazolam (MDZ; 0.65 mg/kg, i.m.), and 10 min later, either a second injection of MDZ (0.65 mg/kg, i.m.) or allopregnanolone (ALLO) at either 12 or 24 mg/kg, i.m. EEG was recorded starting 30 min prior to continuing until 240 min after antiseizure drug administration. At the end of the recording period, animals were euthanized to harvest brain tissue for histological analyses of neurodegeneration and inflammation. (B) Placement of screws for EEG monitoring; one screw over each hemisphere at 4 mm posterior, +/− 4 mm mediolateral (ML); a third ground screw is placed over the cerebellum at approximately 10 mm posterior, 2 mm ML. (C) Kaplan–Meier curves showing survival during the 20 min period between soman administration and initial treatment with MDZ. (D) Survival in the treatment groups from time of initial treatment to end of observation period. (E) Mean ± SD arterial blood oxygen saturation (SpO₂) values as measured at various times during the observation period in the three treatment groups. SpO₂ levels below 90% (indicated by the horizontal dotted line) indicate hypoxemia. (F) Statistical analyses of SpO₂ data. Data are presented as mean differences (dot) with 95% confidence interval (bar) of average SpO₂ (%) between two groups (identified at the top of each box) (n = 4–15 animals per group). The horizontal line in each box corresponds to a difference of 0, which would indicate no difference between the two groups being compared. Confidence intervals that do not include 0 indicate a statistically significant difference between the two groups being compared; CI bars colored blue indicate significant differences that survived false discovery rate (FDR) correction.

FIGURE 2

(A) Representative EEG traces from an animal in each of the groups receiving: (1) MDZ + MDZ, (2) MDZ + ALLO 12 mg/kg, (3) MDZ + ALLO 24 mg/kg). (B–E) Samples of the EEG traces on an expanded time scale. Colored vertical dashed lines in (A) indicate positions of the samples shown in the colored boxes below each trace in panels (B–E). (B) Representative traces of baseline EEG activity prior to soman exposure. (C) Representative traces following soman exposure. (D, E) Representative traces at 2 time points after administration of the antiseizure drug pairs.

FIGURE 3

(A) The percentage of animals that remained in SE as determined by behavioral seizure scores presented as a function of time after treatment (tx) with MDZ + MDZ (black solid line), MDZ + ALLO 12 mg/kg (red dashed line), MDZ + ALLO 24 mg/kg (blue dashed line) (n = 6–13 per group). * Indicates a significant difference between groups as determined by Fisher Exact test (p < 0.05). (B) The percentage of time animals were in SE as determined using behavioral seizure scores following treatment with antiseizure drug, calculated as time to SE termination plus time in SE after recurrence as applicable. Data are presented as violin plots, with each individual point within the plot representing a single animal treated with MDZ + MDZ (black), MDZ + ALLO 12 mg/kg (magenta) or MDZ + ALLO 24 mg/kg (cyan) (n = 6–13 per group). * Indicates a significant difference between groups as determined by Kruskal‐Wallis non‐parametric one‐way ANOVA (p < 0.05). (C) The percentage of animals that experienced recurrence of SE following termination of seizure activity with ALLO 12 mg/kg (magenta; n = 5) or ALLO 24 mg/kg (cyan; n = 13). No animals in the MDZ + MDZ group experienced termination of seizure activity; therefore, the MDZ + MDZ group is not included in panel (C). * Indicates a significant difference between groups as determined by Fisher Exact test (p < 0.05). Antiseizure treatment effects on (D and E) change in spike rate and (F and G) absolute EEG power over the recording period in animals treated with MDZ + MDZ (black lines), MDZ + ALLO 12 mg/kg (magenta lines), or MDZ + ALLO 24 mg/kg (cyan lines). (D and F) For each animal, data were normalized to baseline and are presented as the mean ± SD (shaded regions) (n = 6–13 per group). The times at which soman and antiseizure treatments (Tx) were administered are indicated by vertical dotted lines. (E and G) Geometric mean ratio (GMR, dot) and 95% confidence interval (CI, bars) of (E) spike rates and (G) absolute power between two treatment groups (identified at the top of each box) (n = 6–13 per group). The horizontal line in each box corresponds to a GMR of 1.0, which would indicate the geometric mean of the parameter was identical in the two groups being compared. Confidence intervals that do not include 1.0 indicate a statistically significant difference between the two groups being compared; CI bars colored blue indicate the significant difference survived false discovery rate (FDR) correction.

FIGURE 4

The density of degenerating neurons as identified by Fluoro‐Jade C (FJC) labelling was quantified in the somatosensory cortex, hippocampus, thalamus, piriform cortex/amygdala 4 h after exposure to soman and treatment with MDZ followed by MDZ or ALLO at 12 or 24 mg/kg. (A) Representative photomicrographs of FJC staining in the piriform cortex. (B) Geometric mean ratio (filled circles) with 95% confidence interval (CI, bar) of FJC labeled cells/mm² (n = 6 animals per group). Confidence intervals that do not include 1.0 (indicated by the horizontal bar) indicate a statistically significant difference between the two groups being compared; bars colored blue indicate significant differences that survived false discovery rate (FDR) analysis.

FIGURE 5

Reactive astrogliosis was evaluated by quantifying immunoreactivity of GFAP and S100β in the somatosensory cortex, hippocampus, thalamus and piriform cortex/amygdala at 4 h after exposure to soman and treatment with MDZ alone or MDZ + ALLO at 12 or 24 mg/kg. (A) Representative photomicrographs of the piriform cortex immunostained for GFAP (magenta) and S100β (cyan) to identify astrocytes; sections were counterstained with DAPI to identify cell nuclei. (B–D) Mean difference (dot) or Geometric mean ratio (GMR, dot) with 95% confidence interval (CI, bar) of (B) percent area of regions expressing GFAP, (C) percent area of regions expressing S100β, and (D) percent area expressing both GFAP and S100β (n = 6 animals per group). The difference between groups did not vary by brain region, so overall estimates of group differences are presented. The horizontal line in each box corresponds to a mean difference of 0 or a GMR of 1.0, which would indicate no difference between the groups. Confidence intervals that do not include 1.0 indicate a statistically significant difference between the two groups being compared; CI bars colored blue indicate the significant difference survived false discovery rate (FDR) correction.

FIGURE 6

Microgliosis was evaluated by quantifying immunoreactivity of IBA1 and CD68 in the somatosensory cortex, hippocampus, thalamus and piriform cortex/amygdala 4 h after exposure to soman and treatment with MDZ alone of MDZ + ALLO at 12 or 24 mg/kg. (A) Representative photomicrographs of the piriform cortex immunostained for IBA1 (magenta) to identify microglia and CD68 (cyan) to identify phagocytic cells; sections were counterstained with DAPI to identify cell nuclei. (B–D) Geometric mean ratio (GMR, filled circles) with 95% confidence interval (CI) of (B) the percentage of total cells expressing IBA1, (C) the percentage of total cells expressing CD68, and (D) the percentage of IBA1 expressing cells also expressing CD68 (n = 6 animals per group). (B and D) The difference between groups did not vary by brain region, so overall estimates of group differences are presented. The horizontal line in each box corresponds to a GMR of 1.0, which would indicate the geometric mean of the parameter was identical in the two groups being compared. Confidence intervals that do not include 1.0 indicate a statistically significant difference between the two groups being compared; CI bars colored blue indicate the significant difference survived false discovery rate (FDR) correction.