Methyl isocyanate inhalation induces tissue factor-dependent activation of coagulation in rats.

Methyl isocyanate (MIC) is a highly toxic industrial chemical causing acute lethality after inhalation. The objective of this study was to determine whether alterations in hemostasis also occur in the immediate hours after exposure. Male rats were exposed to MIC (125-500 ppm) by nose-only vapor inhalation for 30 min. Arterial O2 saturation was monitored prior to exposure, and hourly thereafter. Rats were euthanized at 1, 2, 4, and 8 hr and plasma analyzed for recalcification clotting time, tissue factor (TF) activity, and protein levels. Hypoxemia, as assessed by pulse oximetry, was an early feature of MIC inhalation. In contrast to sham or low (125 ppm) concentrations, 250 and 500 ppm MIC caused significant declines in blood oxygen saturation (% SpO2) at 1 hr, which remained at deficit during the postexposure period. Commensurate with hypoxemia, plasma clotting time was significantly accelerated 1 hr after MIC inhalation (sham treatment: 955 ± 62.8 s; 125 ppm MIC: 790 ± 62 s; 250 ppm: 676 ± 28.0 s; 500 ppm: 581 ± 175 s). This procoagulant effect was transient, with no difference observed between sham and all MIC groups by 8 hr. Similarly, elevated TF activity and protein were detected in plasma 1 hr after MIC inhalation, each of which showed a progressive decline back to control levels at later timepoints. This study demonstrates that MIC inhalation resulted in hypoxemia and transient hypercoagulability of blood. Accelerated clotting occurred rapidly and was likely due to intravascular TF, which initiates the extrinsic coagulation pathway.

Bronchiolitis Obliterans and Pulmonary Fibrosis after Sulfur Mustard Inhalation in Rats.

Inhalation of powerful chemical agents, such as sulfur mustard (SM), can have debilitating pulmonary consequences, such as bronchiolitis obliterans (BO) and parenchymal fibrosis (PF). The underlying pathogenesis of disorders after SM inhalation is not clearly understood, resulting in a paucity of effective therapies. In this study, we evaluated the role of profibrotic pathways involving transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF) in the development of BO and PF after SM inhalation injury using a rat model. Adult Sprague-Dawley rats were intubated and exposed to SM (1.0 mg/kg), then monitored daily for respiratory distress, oxygen saturation changes, and weight loss. Rats were killed at 7, 14, 21, or 28 days, and markers of injury were determined by histopathology; pulmonary function testing; and assessment of TGF-ß, PDGF, and PAI-1 concentrations. Respiratory distress developed over time after SM inhalation, with progressive hypoxemia, respiratory distress, and weight loss. Histopathology confirmed the presence of both BO and PF, and both gradually worsened with time. Pulmonary function testing demonstrated a time-dependent increase in lung resistance, as well as a decrease in lung compliance. Concentrations of TGF-ß, PDGF, and PAI-1 were elevated at 28 days in lung, BAL fluid, and/or plasma. Time-dependent development of BO and PF occurs in lungs of rats exposed to SM inhalation, and the elevated concentrations of TGF-ß, PDGF, and PAI-1 suggest involvement of these profibrotic pathways in the aberrant remodeling after injury.

Oxygen Administration Improves Survival but Worsens Cardiopulmonary Functions in Chlorine-exposed Rats.

Chlorine is a highly reactive gas that can cause significant injury when inhaled. Unfortunately, its use as a chemical weapon has increased in recent years. Massive chlorine inhalation can cause death within 4 hours of exposure. Survivors usually require hospitalization after massive exposure. No countermeasures are available for massive chlorine exposure and supportive-care measures lack controlled trials. In this work, adult rats were exposed to chlorine gas (LD58-67) in a whole-body exposure chamber, and given oxygen (0.8 FiO2) or air (0.21 FiO2) for 6 hours after baseline measurements were obtained. Oxygen saturation, vital signs, respiratory distress and neuromuscular scores, arterial blood gases, and hemodynamic measurements were obtained hourly. Massive chlorine inhalation caused severe acute respiratory failure, hypoxemia, decreased cardiac output, neuromuscular abnormalities (ataxia and hypotonia), and seizures resulting in early death. Oxygen improved survival to 6 hours (87% versus 42%) and prevented observed seizure-related deaths. However, oxygen administration worsened the severity of acute respiratory failure in chlorine-exposed rats compared with controls, with increased respiratory acidosis (pH 6.91 ± 0.04 versus 7.06 ± 0.01 at 2 h) and increased hypercapnia (180.0 ± 19.8 versus 103.2 ± 3.9 mm Hg at 2 h). In addition, oxygen did not improve neuromuscular abnormalities, cardiac output, or respiratory distress associated with chlorine exposure. Massive chlorine inhalation causes severe acute respiratory failure and multiorgan damage. Oxygen administration can improve short-term survival but appears to worsen respiratory failure, with no improvement in cardiac output or neuromuscular dysfunction. Oxygen should be used with caution after massive chlorine inhalation, and the need for early assisted ventilation should be assessed in victims.

Editor's Highlight: Pulmonary Vascular Thrombosis in Rats Exposed to Inhaled Sulfur Mustard.

Sulfur mustard (SM) is a chemical warfare agent. When inhaled, SM causes significant injury to the respiratory tract. Although the mechanism involved in acute airway injury after SM inhalation has been well described previously, the mechanism of SM's contribution to distal lung vascular injury is not well understood. We hypothesized that acute inhalation of vaporized SM causes activated systemic coagulation with subsequent pulmonary vascular thrombi formation after SM inhalation exposure. Sprague Dawley rats inhaled SM ethanolic vapor (3.8 mg/kg). Barium/gelatin CT pulmonary angiograms were performed to assess for pulmonary vascular thrombi burden. Lung immunohistochemistry was performed for common procoagulant markers including fibrin(ogen), von Willebrand factor, and CD42d in control and SM-exposed lungs. Additionally, systemic levels of d-dimer and platelet aggregometry after adenosine diphosphate- and thrombin-stimulation were measured in plasma after SM exposure. In SM-exposed lungs, chest CT angiography demonstrated a significant decrease in the distal pulmonary vessel density assessed at 6 h postexposure. Immunohistochemistry also demonstrated increased intravascular fibrin(ogen), vascular von Willebrand factor, and platelet CD42d in the distal pulmonary vessels (<200 µm diameter). Circulating d-dimer levels were significantly increased (p < .001) at 6, 9, and 12 h after SM inhalation versus controls. Platelet aggregation was also increased in both adenosine diphosphate - (p < .01) and thrombin- (p < .001) stimulated platelet-rich plasma after SM inhalation. Significant pulmonary vascular thrombi formation was evident in distal pulmonary arterioles following SM inhalation in rats assessed by CT angiography and immunohistochemistry. Enhanced systemic platelet aggregation and activated systemic coagulation with subsequent thrombi formation likely contributed to pulmonary vessel occlusion.

From the Cover: ImpairedProliferation and Differentiation of the Conducting Airway Epithelium Associated With Bronchiolitis Obliterans After Sulfur Mustard Inhalation Injury in Rats.

Sulfur mustard (SM) is a chemical warfare agent that causes chronic airway remodeling. This study's objective was to assess for changes to the bronchiolar epithelium after SM exposure to explain its contribution to chronic airway remodeling. Materials and methods: Adult male rats were exposed to a sublethal dose of SM inhalation (1.0-1.2 mg/kg) for 50 min. Histological sections of the bronchiolar epithelium were analyzed for changes using hematoxylin and eosin, trichrome, and immunofluorescent staining for acetylated tubulin (AT) and club cell secretory protein (CCSP). CCSP in bronchoalveolar lavage fluid was assessed using western blot. A bromodeoxyuridine (BRDU) assay was used to assess for epithelial proliferation, and real-time PCR measured changes in Notch mRNA expression. Results: SM caused significant proximal bronchiolar epithelial injury with epithelial denudation, loss of acetylated tubulin and CCSP staining, and reduced bronchoalveolar lavage fluid CCSP levels. bromodeoxyuridine (BRDU) + staining of proximal bronchiolar epithelial cells was not increased, but staining was increased in the distal bronchiolar epithelium. One month after injury, the proximal bronchiolar epithelium was not fully repaired. Significant collagen deposition surrounded proximal bronchioles with luminal obstruction, consistent with bronchiolitis obliterans. These changes corresponded with a downregulation of Notch1, Notch3, and Hes1 mRNA expressions. Conclusions: This study demonstrates that SM exposure resulted in severe proximal airway epithelial injury, persistent morphological changes, impaired epithelial proliferation and, ultimately, bronchiolitis obliterans. These changes occurred at the same time that the Notch signaling genes were downregulated. Thus, the lung epithelium and the Notch signaling pathway may be worthy targets for the prevention of chronic airway remodeling after SM inhalation injury.

From the Cover: Catalytic Antioxidant Rescue of Inhaled Sulfur Mustard Toxicity.

Sulfur mustard (bis 2-chloroethyl ethyl sulfide, SM) is a powerful bi-functional vesicating chemical warfare agent. SM tissue injury is partially mediated by the overproduction of reactive oxygen species resulting in oxidative stress. We hypothesized that using a catalytic antioxidant (AEOL 10150) to alleviate oxidative stress and secondary inflammation following exposure to SM would attenuate the toxic effects of SM inhalation. Adult male rats were intubated and exposed to SM (1.4 mg/kg), a dose that produces an LD50 at approximately 24 h. Rats were randomized and treated via subcutaneous injection with either sterile PBS or AEOL 10150 (5 mg/kg, sc, every 4 h) beginning 1 h post-SM exposure. Rats were euthanized between 6 and 48 h after exposure to SM and survival and markers of injury were determined. Catalytic antioxidant treatment improved survival after SM inhalation in a dose-dependent manner, up to 52% over SM PBS at 48 h post-exposure. This improvement was sustained for at least 72 h after SM exposure when treatments were stopped after 48 h. Non-invasive monitoring throughout the duration of the studies also revealed blood oxygen saturations were improved by 10% and clinical scores were reduced by 57% after SM exposure in the catalytic antioxidant treatment group. Tissue analysis showed catalytic antioxidant therapy was able to decrease airway cast formation by 69% at 48 h post-exposure. To investigate antioxidant induced changes at the peak of injury, several biomarkers of oxidative stress and inflammation were evaluated at 24 h post-exposure. AEOL 10150 attenuated SM-mediated lung lipid oxidation, nitrosative stress and many proinflammatory cytokines. The findings indicate that catalytic antioxidants may be useful medical countermeasure against inhaled SM exposure.

Airway tissue plasminogen activator prevents acute mortality due to lethal sulfur mustard inhalation.

RATIONALE: Sulfur mustard (SM) is a chemical weapon stockpiled today in volatile regions of the world. SM inhalation causes a life-threatening airway injury characterized by airway obstruction from fibrin casts, which can lead to respiratory failure and death. Mortality in those requiring intubation is more than 80%. No therapy exists to prevent mortality after SM exposure. Our previous work using the less toxic analog of SM, 2-chloroethyl ethyl sulfide, identified tissue plasminogen activator (tPA) an effective rescue therapy for airway cast obstruction (Veress, L. A., Hendry-Hofer, T. B., Loader, J. E., Rioux, J. S., Garlick, R. B., and White, C. W. (2013). Tissue plasminogen activator prevents mortality from sulfur mustard analog-induced airway obstruction. Am. J. Respir. Cell Mol. Biol. 48, 439-447). It is not known if exposure to neat SM vapor, the primary agent used in chemical warfare, will also cause death due to airway casts, and if tPA could be used to improve outcome. METHODS: Adult rats were exposed to SM, and when oxygen saturation reached less than 85% (median: 6.5 h), intratracheal tPA or placebo was given under isoflurane anesthesia every 4 h for 48 h. Oxygen saturation, clinical distress, and arterial blood gases were assessed. Microdissection was done to assess airway obstruction by casts. RESULTS: Intratracheal tPA treatment eliminated mortality (0% at 48 h) and greatly improved morbidity after lethal SM inhalation (100% death in controls). tPA normalized SM-associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress. Moreover, tPA treatment resulted in greatly diminished airway casts, preventing respiratory failure from airway obstruction. CONCLUSIONS: tPA given via airway more than 6 h after exposure prevented death from lethal SM inhalation, and normalized oxygenation and ventilation defects, thereby rescuing from respiratory distress and failure. Intra-airway tPA should be considered as a life-saving rescue therapy after a significant SM inhalation exposure incident.

Intratracheal heparin improves plastic bronchitis due to sulfur mustard analog.

BACKGROUND: Inhalation of sulfur mustard (SM) and SM analog, 2-chloroethyl ethyl sulfide (CEES), cause fibrinous cast formation that occludes the conducting airways, similar to children with Fontan physiology-induced plastic bronchitis. These airway casts cause significant mortality and morbidity, including hypoxemia and respiratory distress. Our hypothesis was that intratracheal heparin, a highly cost effective and easily preserved rescue therapy, could reverse morbidity and mortality induced by bronchial cast formation. METHODS: Sprague-Dawley rats were exposed to 7.5% CEES via nose-only aerosol inhalation to produce extensive cast formation and mortality. The rats were distributed into three groups: non-treated, phosphate-buffered saline (PBS)-treated, and heparin-treated groups. Morbidity was assessed with oxygen saturations and clinical distress. Blood and bronchoalveolar lavage fluid (BALF) were obtained for analysis, and lungs were fixed for airway microdissection to quantify the extent of airway cast formation. RESULTS: Heparin, given intratracheally, improved survival (100%) when compared to non-treated (75%) and PBS-treated (90%) controls. Heparin-treated rats also had improved oxygen saturations, clinical distress and airway cast scores. Heparin-treated rats had increased thrombin clotting times, factor Xa inhibition and activated partial thromboplastin times, indicating systemic absorption of heparin. There were also increased red blood cells (RBCs) in the BALF in 2/6 heparin-treated rats compared to PBS-treated control rats. CONCLUSIONS: Intratracheal heparin 1 hr after CEES inhalation improved survival, oxygenation, airway obstruction, and clinical distress. There was systemic absorption of heparin in rats treated intratracheally. Some rats had increased RBCs in BALF, suggesting a potential for intrapulmonary bleeding if used chronically after SM inhalation.

Antifibrinolytic mechanisms in acute airway injury after sulfur mustard analog inhalation.

Acute lung injury in response to mustard gas (sulfur mustard [SM]) inhalation results in formation of fibrin casts, which obstruct the airway. The objective of this study was to identify fibrinolytic pathways that could be contributing to the persistence of airway casts after SM exposure. Rats were exposed to the SM analog, 2-chloroethyl ethyl sulfide, via nose-only aerosol inhalation. At 4 and 18 hours after exposure, animals were killed and airway-capillary leak estimated by measuring bronchoalveolar lavage fluid (BALF) protein and IgM content. The fibrin clot-degrading and plasminogen-activating capabilities of BALF were also assessed by activity assays, whereas Western blotting was used to determine the presence and activities of plasminogen activator inhibitor-1, thrombin activatable fibrinolytic inhibitor and α2-antiplasmin. Measurement of tissue-specific steady-state mRNA levels was also conducted for each fibrinolytic inhibitor to assess whether its synthesis occurs in lung or at extrapulmonary sites. The results of this study demonstrate that fibrin-degrading and plasminogen-activating capabilities of the airways become impaired during the onset of 2-chloroethyl ethyl sulfide-induced vascular leak. Findings of functionally active reservoirs of plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor, and α2-antiplasmin in BALF indicate that airway fibrinolysis is inhibited at multiple levels in response to SM.

Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation.

UNLABELLED: Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence. METHODS: Rats were exposed to the SM analog 2-chloroethyl ethyl sulfide (CEES) via nose-only aerosol inhalation. One hour later, TFPI (1.5mg/kg) in vehicle, or vehicle alone, was instilled into the trachea. Arterial O2 saturation was monitored using pulse oximetry. Twelve hours after exposure, animals were euthanized and bronchoalveolar lavage fluid (BALF) and plasma were analyzed for prothrombin, thrombin-antithrombin complex (TAT), active plasminogen activator inhibitor-1 (PAI-1) levels, and fluid fibrinolytic capacity. Lung steady-state PAI-1 mRNA was measured by RT-PCR analysis. Airway-capillary leak was estimated by BALF protein and IgM, and by pleural fluid measurement. In additional animals, airway cast formation was assessed by microdissection and immunohistochemical detection of airway fibrin. RESULTS: Airway obstruction in the form of fibrin-containing casts was evident in central conducting airways of rats receiving CEES. TFPI decreased cast formation, and limited severe hypoxemia. Findings of reduced prothrombin consumption, and lower TAT complexes in BALF, demonstrated that TFPI acted to limit thrombin activation in airways. TFPI, however, did not appreciably affect CEES-induced airway protein leak, PAI-1 mRNA induction, or inhibition of the fibrinolytic activity present in airway surface liquid. CONCLUSIONS: Intratracheal administration of TFPI limits airway obstruction, improves gas exchange, and prevents mortality in rats with sulfur mustard-analog-induced acute lung injury.

Tissue plasminogen activator prevents mortality from sulfur mustard analog-induced airway obstruction.

Sulfur mustard (SM) inhalation causes the rare but life-threatening disorder of plastic bronchitis, characterized by bronchial cast formation, resulting in severe airway obstruction that can lead to respiratory failure and death. Mortality in those requiring intubation is greater than 80%. To date, no antidote exists for SM toxicity. In addition, therapies for plastic bronchitis are solely anecdotal, due to lack of systematic research available to assess drug efficacy in improving mortality and/or morbidity. Adult rats exposed to SM analog were treated with intratracheal tissue plasminogen activator (tPA) (0.15-0.7 mg/kg, 5.5 and 6.5 h), compared with controls (no treatment, isoflurane, and placebo). Respiratory distress and pulse oximetry were assessed (for 12 or 48 h), and arterial blood gases were obtained at study termination (12 h). Microdissection of fixed lungs was done to assess airway obstruction by casts. Optimal intratracheal tPA treatment (0.7 mg/kg) completely eliminated mortality (0% at 48 h), and greatly improved morbidity in this nearly uniformly fatal disease model (90-100% mortality at 48 h). tPA normalized plastic bronchitis-associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress (i.e., clinical scores) while decreasing airway fibrin casts. Intratracheal tPA diminished airway-obstructive fibrin-containing casts while improving clinical respiratory distress, pulmonary gas exchange, tissue oxygenation, and oxygen utilization in our model of severe chemically induced plastic bronchitis. Most importantly, mortality, which was associated with hypoxemia and clinical respiratory distress, was eliminated.