Midazolam Efficacy Against Acute Hydrogen Sulfide-Induced Mortality and Neurotoxicity

Abstract

Hydrogen sulfide (H₂S) is a colorless, highly neurotoxic gas. It is not only an occupational and environmental hazard but also of concern to the Department of Homeland Security for potential nefarious use. Acute high-dose H₂S exposure causes death, while survivors may develop neurological sequelae. Currently, there is no suitable antidote for treatment of acute H₂S-induced neurotoxicity. Midazolam (MDZ), an anti-convulsant drug recommended for treatment of nerve agent intoxications, could also be of value in treating acute H₂S intoxication. In this study, we tested the hypothesis that MDZ is effective in preventing/treating acute H₂S-induced neurotoxicity. This proof-of-concept study had two objectives: to determine whether MDZ prevents/reduces H₂S-induced mortality and to test whether MDZ prevents H₂S-induced neurological sequelae. MDZ (4 mg/kg) was administered IM in mice, 5 min pre-exposure to a high concentration of H₂S at 1000 ppm or 12 min post-exposure to 1000 ppm H₂S followed by 30 min of continuous exposure. A separate experiment tested whether MDZ pre-treatment prevented neurological sequelae. Endpoints monitored included assessment of clinical signs, mortality, behavioral changes, and brain histopathological changes. MDZ significantly reduced H₂S-induced lethality, seizures, knockdown, and behavioral deficits (p < 0.01). MDZ also significantly prevented H₂S-induced neurological sequelae, including weight loss, behavior deficits, neuroinflammation, and histopathologic lesions (p < 0.01). Overall, our findings show that MDZ is a promising drug for reducing H₂S-induced acute mortality, neurotoxicity, and neurological sequelae.

Keywords: Acute toxicity; Hydrogen sulfide; Neurodegeneration; Neurotoxicity; Translational model.

Fig. 1

a Treatment paradigm to determine the prophylactic efficacy of MDZ. b MDZ prevented H₂S-induced mortality by 90%. The survival data between H₂S + saline and H₂S + MDZ groups is significantly different (p < 0.05, log-rank test, n = 10). c MDZ prevented H₂S-induced seizure activity in mice. The seizure data between H₂S + saline and H₂S + MDZ groups is significantly different (p < 0.05, log-rank test, n = 10). Asterisks (***p < 0.001) indicate statistically significant difference between H₂S + saline and the H₂S + MDZ groups

Fig. 2

a Treatment paradigm to determine the efficacy of MDZ for treatment of H₂S-induced neurotoxicity given during H₂S exposure. b Following MDZ treatment, no more mice died compared to about 20% survival for saline-treated mice. The survival curve between H₂S + saline and H₂S + MDZ groups is significantly different (p < 0.0001, log-rank test, n = 20). c MDZ abolished H₂S-induced seizure activity in mice (n = 20). Asterisks (*p < 0.05, **p < 0.01, ***p < 0.001) indicate statistically significant difference between H₂S + saline and the H₂S + MDZ groups

Fig. 3

a Summary treatment paradigm of H₂S-induced neurological sequelae in mice prophylactically treated with MDZ. b MDZ completely prevented seizure activity and knockdown (c) consistently during the entire exposure period (n = 5). Seizure and knockdown were presented as percentage to breathing air control group. Seizure and knockdown data were not statistically analyzed due to the possibility of multiple seizure and knockdown from same mice during repeated exposure to H₂S. d Mice exposed to H₂S and injected with saline lost statistically significant more weight compared to the breathing air controls injected with saline. MDZ prophylactically prevented H₂S-induced weight loss (n = 5). e MDZ prevented H₂S-induced motor deficits (n = 5). Graphs are represented as mean values. *p < 0.05, **p < 0.001, ***p < 0.001, two-way ANOVA followed by Bonferroni’s post-test between H₂S + saline and H₂S + MDZ groups

Fig. 4

Photomicrographs of lesions in the thalamus and inferior colliculus of mice prophylactically treated with MDZ prior to H₂S. Note the pallor and loss of neurons in the thalamus of the saline/H₂S-exposed mouse. Note that brain tissue of the H₂S + MDZ group is similar to that of the breathing air + saline group. Severe changes in the inferior colliculus of the H₂S + saline-exposed mice include marked vacuolization of the neuropil, degeneration and loss of neurons, and prominent glial response. Graphs are represented as mean values. Asterisks (****p < 0.0001, ***p < 0.001) indicate a significant difference between H₂S + saline and the H₂S + MDZ groups

Fig. 5

Representative photomicrographs of immunohistochemical staining of the inferior colliculus demonstrating expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte activation, and inducible nitric oxide synthase (iNOS), a marker for neuroinflammation. Note the increased expression of GFAP and iNOS (brown chromogen deposition) in the brain of the saline/H₂S group, while levels of these markers in the brains of MDZ-treated animals have less immunostaining, suggesting less inflammation the MDZ-treated group

Fig. 6

MDZ concentration in the brain. Note the significantly higher MDZ concentration in mice exposed to high concentration of H₂S compared to those without H₂S exposure. Graphs are represented as mean values. ****p < 0.0001, ANOVA followed by Bonferroni’s post-test between H₂S + MDZ and the breathing air + MDZ group