Establishing a Dexamethasone Treatment Regimen To Alleviate Sulfur Mustard-Induced Corneal Injuries in a Rabbit Model.

Sulfur mustard (SM) is an ominous chemical warfare agent. Eyes are extremely susceptible to SM toxicity; injuries include inflammation, fibrosis, neovascularization (NV), and vision impairment/blindness, depending on the exposure dosage. Effective countermeasures against ocular SM toxicity remain elusive and are warranted during conflicts/terrorist activities and accidental exposures. We previously determined that dexamethasone (DEX) effectively counters corneal nitrogen mustard toxicity and that the 2-hour postexposure therapeutic window is most beneficial. Here, the efficacy of two DEX dosing frequencies [i.e., every 8 or 12 hours (initiated, as previously established, 2 hours after exposure)] until 28 days after SM exposure was assessed. Furthermore, sustained effects of DEX treatments were observed up to day 56 after SM exposure. Corneal clinical assessments (thickness, opacity, ulceration, and NV) were performed at the day 14, 28, 42, and 56 post-SM exposure time points. Histopathological assessments of corneal injuries (corneal thickness, epithelial degradation, epithelial-stromal separation, inflammatory cell, and blood vessel counts) using H&E staining and molecular assessments (COX-2, MMP-9, VEGF, and SPARC expressions) were performed at days 28, 42, and 56 after SM exposure. Statistical significance was assessed using two-way ANOVA, with Holm-Sidak post hoc pairwise multiple comparisons; significance was established if P < 0.05 (data represented as the mean ± S.E.M.). DEX administration every 8 hours was more potent than every 12 hours in reversing ocular SM injury, with the most pronounced effects observed at days 28 and 42 after SM exposure. These comprehensive results are novel and provide a comprehensive DEX treatment regimen (therapeutic-window and dosing-frequency) for counteracting SM-induced corneal injuries. SIGNIFICANCE STATEMENT: The study aims to establish a dexamethasone (DEX) treatment regimen by comparing the efficacy of DEX administration at 12 versus 8 hours initiated 2 hours after exposure. DEX administration every 8 hours was more effective in reversing sulfur mustard (SM)-induced corneal injuries. SM injury reversal during DEX administration (initial 28 days after exposure) and sustained [further 28 days after cessation of DEX administration (i.e., up to 56 days after exposure)] effects were assessed using clinical, pathophysiological, and molecular biomarkers.

Dermal Exposure to Vesicating Nettle Agent Phosgene Oxime: Clinically Relevant Biomarkers and Skin Injury Progression in Murine Models.

Phosgene oxime (CX), categorized as a vesicating chemical threat agent, causes effects that resemble an urticant or nettle agent. CX is an emerging potential threat agent that can be deployed alone or with other chemical threat agents to enhance their toxic effects. Studies on CX-induced skin toxicity, injury progression, and related biomarkers are largely unknown. To study the physiologic changes, skin clinical lesions and their progression, skin exposure of SKH-1 and C57BL/6 mice was carried out with vapor from 10 µl CX for 0.5-minute or 1.0-minute durations using a designed exposure system for consistent CX vapor exposure. One-minute exposure caused sharp (SKH-1) or sustained (C57BL/6) decrease in respiratory and heart rate, leading to mortality in both mouse strains. Both exposures caused immediate blanching, erythema with erythematous ring (wheel) and edema, and an increase in skin bifold thickness. Necrosis was also observed in the 0.5-minute CX exposure group. Both mouse strains showed comparative skin clinical lesions upon CX exposure; however, skin bifold thickness and erythema remained elevated up to 14 days postexposure in SKH-1 mice but not in C57BL/6 mice. Our data suggest that CX causes immediate changes in the physiologic parameters and gross skin lesions resembling urticaria, which could involve mast cell activation and intense systemic toxicity. This novel study recorded and compared the progression of skin injury to establish clinical biomarkers of CX dermal exposure in both the sexes of two murine strains relevant for skin and systemic injury studies and therapeutic target identification. SIGNIFICANCE STATEMENT: Phosgene oxime (CX), categorized as a vesicating agent, is considered as a potent chemical weapon and is of high military and terrorist threat interest since it produces rapid onset of severe injury as an urticant. However, biomarkers of clinical relevance related to its toxicity and injury progression are not studied. Data from this study provide useful clinical markers of CX skin toxicity in mouse models using a reliable CX exposure system for future mechanistic and efficacy studies.

Retinal injury mouse model and pathophysiological assessment of the effect of arsenical vesicants.

The eye is ten times more vulnerable to chemical warfare agents than other organs. Consistently, exposure to vesicant arsenical lewisite (LEW) manifests significant corneal damage leading to chronic inflammation, corneal opacity, vascularization, and edema, culminating in corneal cell death. However, despite the progress has made in the research field investigating arsenical-induced pathogenesis of the anterior chamber of the eye, the retinal damage resulted from exposure to arsenicals has not been identified yet. Therefore, we investigated the effects of direct ocular exposure (DOE) to LEW and phenylarsine oxide (PAO) on the retina. DOE to arsenicals was conducted using the vapor cap method at the MRIGlobal facility or an eye patch soaked in solutions with different PAO concentrations at UAB. Animals were assessed at 1, 3, 14, and 28 days postexposure. Results of the study demonstrated that both arsenicals cause severe retinal damage, activating proinflammatory programs and launching apoptotic cell death. Moreover, the DOE to PAO resulted in diminishing ERG amplitudes in a dose-dependent manner, indicating severe retinal damage. The current study established a prototype mouse model of arsenical-induced ocular damage that can be widely used to identify the key cellular signaling pathways involved in retinal damage pathobiology and to validate medical countermeasures against the progression of ocular damage.

Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson's disease.

Characterization of the key cellular targets contributing to sustained microglial activation in neurodegenerative diseases, including Parkinson's disease (PD), and optimal modulation of these targets can provide potential treatments to halt disease progression. Here, we demonstrated that microglial Kv1.3, a voltage-gated potassium channel, was transcriptionally upregulated in response to aggregated α-synuclein (αSynAgg) stimulation in primary microglial cultures and animal models of PD, as well as in postmortem human PD brains. Patch-clamp electrophysiological studies confirmed that the observed Kv1.3 upregulation translated to increased Kv1.3 channel activity. The kinase Fyn, a risk factor for PD, modulated transcriptional upregulation and posttranslational modification of microglial Kv1.3. Multiple state-of-the-art analyses, including Duolink proximity ligation assay imaging, revealed that Fyn directly bound to Kv1.3 and posttranslationally modified its channel activity. Furthermore, we demonstrated the functional relevance of Kv1.3 in augmenting the neuroinflammatory response by using Kv1.3-KO primary microglia and the Kv1.3-specific small-molecule inhibitor PAP-1, thus highlighting the importance of Kv1.3 in neuroinflammation. Administration of PAP-1 significantly inhibited neurodegeneration and neuroinflammation in multiple animal models of PD. Collectively, our results imply that Fyn-dependent regulation of Kv1.3 channels plays an obligatory role in accentuating the neuroinflammatory response in PD and identify Kv1.3 as a potential therapeutic target for PD.

Alleviation of methyl isocyanate-induced airway obstruction and mortality by tissue plasminogen activator.

Methyl isocyanate (MIC, "Bhopal agent") is a highly reactive, toxic industrial chemical. Inhalation of high levels (500-1000 ppm) of MIC vapor is almost uniformly fatal. No therapeutic interventions other than supportive care have been described that can delay the onset of illness or death due to MIC. Recently, we found that inhalation of MIC caused the appearance of activated tissue factor in circulation with subsequent activation of the coagulation cascade. Herein, we report that MIC exposure (500 ppm for 30 min, nose-only) caused deposition of fibrin-rich casts in the conducting airways resulting in respiratory failure and death within 24 h in a rat model (LC90-100 ). We thus investigated the effect of airway delivery of the fibrinolytic agent tissue plasminogen activator (tPA) on mortality and morbidity in this model. Intratracheal administration of tPA was initiated 11 h post MIC exposure and repeated every 4 h for the duration of the study. Treatment with tPA afforded nearly 60% survival at 24 h post MIC exposure and was associated with decreased airway fibrin casts, stabilization of hypoxemia and respiratory distress, and improved acidosis. This work supports the potential of airway-delivered tPA therapy as a useful countermeasure in stabilizing victims of high-level MIC exposure.

Transcriptomic profile analysis of brain inferior colliculus following acute hydrogen sulfide exposure.

Hydrogen sulfide (H2S) is a gaseous molecule found naturally in the environment, and as an industrial byproduct, and is known to cause acute death and induces long-term neurological disorders following acute high dose exposures. Currently, there is no drug approved for treatment of acute H2S-induced neurotoxicity and/or neurological sequelae. Lack of a deep understanding of pathogenesis of H2S-induced neurotoxicity has delayed the development of appropriate therapeutic drugs that target H2S-induced neuropathology. RNA sequencing analysis was performed to elucidate the cellular and molecular mechanisms of H2S-induced neurodegeneration, and to identify key molecular elements and pathways that contribute to H2S-induced neurotoxicity. C57BL/6J mice were exposed by whole body inhalation to 700 ppm of H2S for either one day, two consecutive days or 4 consecutive days. Magnetic resonance imaging (MRI) scan analyses showed H2S exposure induced lesions in the inferior colliculus (IC) and thalamus (TH). This mechanistic study focused on the IC. RNA Sequencing analysis revealed that mice exposed once, twice, or 4 times had 283, 193 and 296 differentially expressed genes (DEG), respectively (q-value < 0.05, fold-change> 1.5). Hydrogen sulfide exposure modulated multiple biological pathways including unfolded protein response, neurotransmitters, oxidative stress, hypoxia, calcium signaling, and inflammatory response in the IC. Hydrogen sulfide exposure activated PI3K/Akt and MAPK signaling pathways. Pro-inflammatory cytokines were shown to be potential initiators of the modulated signaling pathways following H2S exposure. Furthermore, microglia were shown to release IL-18 and astrocytes released both IL-1ß and IL-18 in response to H2S. This transcriptomic analysis data revealed complex signaling pathways involved in H2S-induced neurotoxicity and may provide important associated mechanistic insights.

Broad spectrum proteomics analysis of the inferior colliculus following acute hydrogen sulfide exposure.

Acute exposure to high concentrations of H2S 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 H2S-induced neurodegeneration we used a broad-spectrum proteomic analysis approach to identify key molecules and molecular pathways involved in the pathogenesis of acute H2S-induced neurotoxicity and neurodegeneration. Mice were subjected to acute inhalation exposure of up to750 ppm of H2S. H2S 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-H2S 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 H2S-dependent proteomics changes. Webgestalt pathway overrepresentation enrichment analysis with Panther engine revealed H2S 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 H2S-induced neurotoxicity.

Midazolam Efficacy Against Acute Hydrogen Sulfide-Induced Mortality and Neurotoxicity.

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.

Cobinamide is effective for treatment of hydrogen sulfide-induced neurological sequelae in a mouse model.

Hydrogen sulfide (H2 S) is a highly neurotoxic gas. Acute exposure can lead to neurological sequelae among survivors. A drug for treating neurological sequelae in survivors of acute H2 S intoxication is needed. Using a novel mouse model we evaluated the efficacy of cobinamide (Cob) for increasing survival of, and reducing neurological sequalae in, mice exposed to sublethal doses of H2 S. 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 H2 S exposure. For objective 2, mice were injected intramuscularly with 100 mg/kg Cob at 2, 15, and 30 min after H2 S exposure. For both objectives, mice were exposed to 765 ppm of H2 S gas. Cob significantly reduced H2 S-induced lethality in a dose-dependent manner (P < 0.05). Cob-treated mice exhibited significantly fewer seizures and knockdowns compared with the H2 S-exposed group. Cob also reversed H2 S-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 H2 S-induced neurological sequelae.

Characterizing a mouse model for evaluation of countermeasures against hydrogen sulfide-induced neurotoxicity and neurological sequelae.

Hydrogen sulfide (H2 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 H2 S poisoning. However, no translational animal model of H2 S-induced neurological sequelae exists. Here, we describe a novel mouse model of H2 S-induced neurotoxicity for translational research. In paradigm I, C57/BL6 mice were exposed to 765 ppm H2 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 H2 S for 60 minutes. Mice were assessed for behavioral, neurochemical, biochemical, and histopathological changes. H2 S intoxication caused seizures, dyspnea, respiratory depression, knockdowns, and death. H2 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. H2 S significantly increased dopamine and serotonin concentration in several brain regions and caused time-dependent decreases in GABA and glutamate concentrations. Furthermore, H2 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 H2 S-induced neurotoxicity is reliable, reproducible, and recapitulates acute H2 S poisoning in humans.

Acute hydrogen sulfide-induced neuropathology and neurological sequelae: challenges for translational neuroprotective research.

Hydrogen sulfide (H2 S), the gas with the odor of rotten eggs, was formally discovered in 1777, over 239 years ago. For many years, it was considered an environmental pollutant and a health concern only in occupational settings. Recently, however, it was discovered that H2 S is produced endogenously and plays critical physiological roles as a gasotransmitter. Although at low physiological concentrations it is physiologically beneficial, exposure to high concentrations of H2 S is known to cause brain damage, leading to neurodegeneration and long-term neurological sequelae or death. Neurological sequelae include motor, behavioral, and cognitive deficits, which are incapacitating. Currently, there are concerns about accidental or malicious acute mass civilian exposure to H2 S. There is a major unmet need for an ideal neuroprotective treatment, for use in the field, in the event of mass civilian exposure to high H2 S concentrations. This review focuses on the neuropathology of high acute H2 S exposure, knowledge gaps, and the challenges associated with development of effective neuroprotective therapy to counteract H2 S-induced neurodegeneration.

Improved Tissue-Based Analytical Test Methods for Orellanine, a Biomarker of Cortinarius Mushroom Intoxication.

Orellanine (OR) toxin is produced by mushrooms of the genus Cortinarius which grow in North America and in Europe. OR poisoning is characterized by severe oliguric acute renal failure, with a mortality rate of 10%-30%. Diagnosis of OR poisoning currently hinges on a history of ingestion of Cortinarius mushrooms and histopathology of renal biopsies. A key step in the diagnostic approach is analysis of tissues for OR. Currently, tissue-based analytical methods for OR are nonspecific and lack sensitivity. The objectives of this study were: (1) to develop definitive HPLC and LC-MS/MS tissue-based analytical methods for OR; and (2) to investigate toxicological effects of OR in mice. The HPLC limit of quantitation was 10 µg/g. For fortification levels of 15 µg/g to 50 µg/g OR in kidney, the relative standard deviation was between 1.3% and 9.8%, and accuracy was within 1.5% to 7.1%. A matrix-matched calibration curve was reproduced in this range with a correlation coefficient (r) of 0.97-0.99. The limit of detection was 20 ng/g for LC-MS/MS. In OR-injected mice, kidney OR concentrations were 97 ± 51 µg/g on Day 0 and 17 ± 1 µg/g on termination Day 3. Splenic and liver injuries were novel findings in this mouse model. The new tissue-based analytical tests will improve diagnosis of OR poisoning, while the mouse model has yielded new data advancing knowledge on OR-induced pathology. The new tissue-based analytical tests will improve diagnosis of OR poisoning, while the mouse model has yielded new data advancing knowledge on OR-induced pathology.

Glia maturation factor expression in hippocampus of human Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the presence of neuropathological lesions containing amyloid plaques (APs) and neurofibrillary tangles (NFTs) associated with neuroinflammation and neuronal degeneration. Hippocampus is one of the earliest and severely damaged areas in AD brain. Glia maturation factor (GMF), a known proinflammatory molecule is up-regulated in AD. Here, we have investigated the expression and distribution of GMF in relation to the distribution of APs and NFTs in the hippocampus of AD brains. Our immunohistochemical results showed GMF is expressed specifically in the vicinity of high density of APs and NFTs in the hippocampus of AD patients. Moreover, reactive astrocytes and activated microglia surrounds the APs and NFTs. We further demonstrate that GMF immunoreactive glial cells were increased at the sites of Tau containing NFTs and APs of hippocampus in AD brains. In conclusion, up-regulated expression of GMF in the hippocampus, and the co-localization of GMF and thioflavin-S stained NFTs and APs suggest that GMF may play important role in the pathogenesis of AD.

Glia maturation factor expression in entorhinal cortex of Alzheimer's disease brain.

Alzheimer's disease (AD) is characterized by the presence of neuropathological lesions containing amyloid plaques (APs) and hyperphosphorylated Tau containing neurofibrillary tangles (NFTs) and is associated with neuroinflammation and neurodegeneration. Entorhinal cortex (Brodmann's area 28) is involved in memory associated functions and is one of the first brain areas targeted to form the neuropathological lesions and also severely affected cortical region in AD. Glia maturation factor (GMF), a central nervous system protein and a proinflammatory molecule is known to be up-regulated in the specific areas of AD brain. Our previous immunohistochemical studies using temporal cortex showed that GMF is expressed in the vicinity of APs and NFTs in AD brains. In the present study, we have analyzed the expression of GMF and its association with APs and NFTs in the entorhinal cortex of AD brains by using immunohistochemistry combined with thioflavin-S fluorescence labeling methods. Results showed that GMF immunoreactive glial cells, glial fibrillary acidic protein labeled reactive astrocytes and ionized calcium binding adaptor molecule-1 labeled activated microglia were increased in the entorhinal cortical layers especially at the sites of 6E10 labeled APs and Tau containing NFTs. In conclusion, increased expression of GMF by the glial cells in the entorhinal cortex region, and the co-localization of GMF with APs and NFTs suggest that GMF may play important proinflammatory roles in the pathogenesis of AD.