Hydrogen sulfide (H S) is a highly neurotoxic gas. It is the second most common cause of gas-induced deaths. Beyond mortality, surviving victims of acute exposure may suffer long-term neurological sequelae. There is a need to develop countermeasures against H S poisoning. However, no translational animal model of H S-induced neurological sequelae exists. Here, we describe a novel mouse model of H S-induced neurotoxicity for translational research. In paradigm I, C57/BL6 mice were exposed to 765 ppm H S for 40 min on day 1, followed by 15-min daily exposures for periods ranging from 1 to 6 days. In paradigm II, mice were exposed once to 1000 ppm H S for 60 minutes. Mice were assessed for behavioral, neurochemical, biochemical, and histopathological changes. H S intoxication caused seizures, dyspnea, respiratory depression, knockdowns, and death. H S-exposed mice showed significant impairment in locomotor and coordinated motor movement activity compared with controls. Histopathology revealed neurodegenerative lesions in the collicular, thalamic, and cortical brain regions. H S significantly increased dopamine and serotonin concentration in several brain regions and caused time-dependent decreases in GABA and glutamate concentrations. Furthermore, H S significantly suppressed cytochrome c oxidase activity and caused significant loss in body weight. Overall, male mice were more sensitive than females. This novel translational mouse model of H S-induced neurotoxicity is reliable, reproducible, and recapitulates acute H S poisoning in humans.

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.

Hydrogen sulfide (H2S) is a highly neurotoxic gas. Acute exposure can lead to neurological sequelae among survivors. A drug for treating neurological sequelae in survivors of acute H2S intoxication is needed. Using a novel mouse model we evaluated the efficacy of cobinamide (Cob) for this purpose. There were two objectives: (1) to determine the dose–response efficacy of Cob and (2) to determine the effective therapeutic time window of Cob. To explore objective 1, mice were injected intramuscularly with Cob at 0, 50 or 100 mg/kg at 2 min after H2S exposure. For objective 2, mice were injected intramuscularly with 100 mg/kg Cob at 2, 15, and 30 min after H2S exposure. For both objectives, mice were exposed to 765 ppm of H2S gas. Cob significantly reduced H2S-induced lethality in a dose-dependent manner (P < 0.05). Cob-treated mice exhibited significantly fewer seizures and knockdowns compared with the H2S-exposed group. Cob also reversed H2S-induced weight loss, behavioral deficits, neurochemical changes, cytochrome c oxidase enzyme inhibition, and neurodegeneration in a dose- and time-dependent manner (P < 0.01). Overall, these findings show that Cob increases survival and is neuroprotective in a mouse model of H2S-induced neurological sequelae.

Acute exposure to high concentrations of HS causes severe brain injury and long-term neurological disorders, but the mechanisms involved are not known. To better understand the cellular and molecular mechanisms involved in acute HS-induced neurodegeneration we used a broad-spectrum proteomic analysis approach to identify key molecules and molecular pathways involved in the pathogenesis of acute HS-induced neurotoxicity and neurodegeneration. Mice were subjected to acute inhalation exposure of up to750 ppm of HS. HS induced behavioral deficits and severe lesions including hemorrhage in the inferior colliculus (IC). The IC was microdissected for proteomic analysis. Tandem mass tags (TMT) liquid chromatography mass spectrometry (LC-MS/MS)-based quantitative proteomics was applied for protein identification and quantitation. LC-MS/MS identified 598, 562, and 546 altered proteomic changes at 2 h, and on days 2 and 4 post-HS exposure, respectively. Of these, 77 proteomic changes were statistically significant at any of the 3 time points. Mass spectrometry data were subjected to Perseus 1.5.5.3 statistical analysis, and gene ontology heat map clustering. Expressions of several key molecules were verified to confirm HS-dependent proteomics changes. Webgestalt pathway overrepresentation enrichment analysis with Panther engine revealed HS exposure disrupted several biological processes including metabotropic glutamate receptor group 1 and inflammation mediated by chemokine and cytokine signaling pathways among others. Further analysis showed that energy metabolism, integrity of blood-brain barrier, hypoxic, and oxidative stress signaling pathways were also implicated. Collectively, this broad-spectrum proteomics data has provided important clues to follow up in future studies to further elucidate mechanisms of HS-induced neurotoxicity.

Acute exposure to high concentrations of H2S causes severe brain injury and long-term neurological disorders. The mechanisms of H2S-induced neurodegeneration are not known. To better understand the cellular and molecular mechanisms of H2S-induced neurodegeneration we used a broad-spectrum proteomic analysis approach to search for key molecules in H2S-induced neurotoxicity. Mice were subjected to acute whole body exposure of up to750 ppm of H2S. The H2S-treated group showed behavioral motor deficits and developed severe lesions in the inferior colliculus (IC), part of the brainstem. The IC was microdissected for proteomic analysis. Tandem mass tags (TMT) liquid chromatography mass spectrometry (LC-MS/MS)-based quantitative proteomics was applied for protein identification and quantitation. LC-MS/MS was able to identify 598, 562, and 546 altered proteomic changes for day 1 (2 h post H2S exposure), day 2, and day 4 of H2S exposure, respectively. Mass spectrometry data were analyzed by Perseus 1.5.5.3 statistical analysis, and gene ontology heat map clustering. Quantitative real-time PCR was used to confirm some of the H2S-dependent proteomics changes. Taken together, acute exposure to H2S induced behavioral motor deficits along with progressive neurodegeneration including disruption of several biological processes in the IC such as cellular morphology, energy metabolism, and calcium signaling. The obtained broad-spectrum proteomics data may provide important clues to elucidate mechanisms of H2S-induced neurotoxicity.HighlightsMice exposed to H2S recapitulated H2S-induced neurotoxicity manifested in humans.The IC in the mouse brain is the most sensitive to H2S-induced neurodegeneration.Proteomic expressions of key proteins were validated at transcription level.Several biological pathways were dysregulated by H2S exposure.