Impairment of wound healing by reactive skin decontamination lotion (RSDL®) in a Göttingen minipig® model.

Reactive Skin Decontamination Lotion (RSDL®) is an FDA-approved skin decontamination kit carried by service members for removal and neutralisation of vesicants and nerve agents. The RSDL kit, comprised of a lotion-impregnated sponge, was shown to be the superior medical decontamination device for chemical warfare agent (CWA) exposure on intact skin. In the event of a chemical exposure situation (i.e. terrorism, battlefield) physical injuries are probable, and preservation of life will outweigh the risk associated with application of RSDL to compromised skin. The purpose of this study was to quantify the rate and quality of wound healing in epidermal skin wounds treated with RSDL in a porcine model. Degree of wound healing was assessed using bioengineering methods to include ballistometry, colorimetry, evaporimetry, and high-frequency ultrasonography. Clinical observation, histopathology and immunohistochemistry were also utilised. All pigs received four bilateral superficial abdominal wounds via a pneumatic dermatome on their ventral abdomen, then were treated with the following dressings over a seven-day period: RSDL sponge, petroleum based Xeroform® gauze, 3 M™ Tegaderm™ Film, and 3 M™ Tegaderm™ Foam. Two additional non-wounded sites on the flank were used as controls. Two groups of pigs were then evaluated for a 21- or 56-day time period, representing short- and long-term wound-healing progression. Our findings indicated RSDL had a negative impact on wound-healing progression at both 21 and 56 days post-injury. Wounds receiving RSDL demonstrated a decreased skin elasticity, significant transepidermal water loss, and altered skin colouration and thickness. In addition, the rate of wound healing was delayed, and return to a functional skin barrier was altered when compared to non-RSDL-treated wounds. In conclusion, wound management care and clinical therapeutic intervention plans should be established to account for a prolonged duration of healing in patients with RSDL-contaminated wounds.

Synthesis and Molecular Properties of Nerve Agent Reactivator HLö-7 Dimethanesulfonate.

The threat of a deliberate release of chemical nerve agents has underscored the need to continually improve field effective treatments for these types of poisonings. The oxime containing HLö-7 is a potential second-generation therapeutic reactivator. A synthetic process for HLö-7 is detailed with improvements to the DIBAL reduction and ion exchange steps. HLö-7 was visualized for the first time within the active site of human acetylcholinesterase and its relative ex vivo potency confirmed against various nerve agents using a phrenic nerve hemidiaphragm assay.

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.

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: 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.

Conceptual approaches for treatment of phosgene inhalation-induced lung injury.

Toxic industrial chemicals are used throughout the world to produce everyday products such as household and commercial cleaners, disinfectants, pesticides, pharmaceuticals, plastics, paper, and fertilizers. These chemicals are produced, stored, and transported in large quantities, which poses a threat to the local civilian population in cases of accidental or intentional release. Several of these chemicals have no known medical countermeasures for their toxic effects. Phosgene is a highly toxic industrial chemical which was used as a chemical warfare agent in WWI. Exposure to phosgene causes latent, non-cardiogenic pulmonary edema which can result in respiratory failure and death. The mechanisms of phosgene-induced pulmonary injury are not fully identified, and currently there is no efficacious countermeasure. Here, we provide a proposed mechanism of phosgene-induced lung injury based on the literature and from studies conducted in our lab, as well as provide results from studies designed to evaluate survival efficacy of potential therapies following whole-body phosgene exposure in mice. Several therapies were able to significantly increase 24h survival following an LCt50-70 exposure to phosgene; however, no treatment was able to fully protect against phosgene-induced mortality. These studies provide evidence that mortality following phosgene toxicity can be mitigated by neuro- and calcium-regulators, antioxidants, phosphodiesterase and endothelin receptor antagonists, angiotensin converting enzymes, and transient receptor potential cation channel inhibitors. However, because the mechanism of phosgene toxicity is multifaceted, we conclude that a single therapeutic is unlikely to be sufficient to ameliorate the multitude of direct and secondary toxic effects caused by phosgene inhalation.

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.

Mustard Gas Inhalation Injury: Therapeutic Strategy.

Mustard gas (sulfur mustard [SM], bis-[2-chloroethyl] sulfide) is a vesicating chemical warfare agent and a potential chemical terrorism agent. Exposure of SM causes debilitating skin blisters (vesication) and injury to the eyes and the respiratory tract; of these, the respiratory injury, if severe, may even be fatal. Therefore, developing an effective therapeutic strategy to protect against SM-induced respiratory injury is an urgent priority of not only the US military but also the civilian antiterrorism agencies, for example, the Homeland Security. Toward developing a respiratory medical countermeasure for SM, four different classes of therapeutic compounds have been evaluated in the past: anti-inflammatory compounds, antioxidants, protease inhibitors and antiapoptotic compounds. This review examines all of these different options; however, it suggests that preventing cell death by inhibiting apoptosis seems to be a compelling strategy but possibly dependent on adjunct therapies using the other drugs, that is, anti-inflammatory, antioxidant, and protease inhibitor compounds.

Postexposure application of Fas receptor small-interfering RNA to suppress sulfur mustard-induced apoptosis in human airway epithelial cells: implication for a therapeutic approach.

Sulfur mustard (SM) is a vesicant chemical warfare and terrorism agent. Besides skin and eye injury, respiratory damage has been mainly responsible for morbidity and mortality after SM exposure. Previously, it was shown that suppressing the death receptor (DR) response by the dominant-negative Fas-associated death domain protein prior to SM exposure blocked apoptosis and microvesication in skin. Here, we studied whether antagonizing the Fas receptor (FasR) pathway by small-interfering RNA (siRNA) applied after SM exposure would prevent apoptosis and, thus, airway injury. Normal human bronchial/tracheal epithelial (NHBE) cells were used as an in vitro model with FasR siRNA, FasR agonistic antibody CH11, and FasR antagonistic antibody ZB4 as investigative tools. In NHBE cells, both SM (300 µM) and CH11 (100 ng/ml) induced caspase-3 activation, which was inhibited by FasR siRNA and ZB4, indicating that SM-induced apoptosis was via the Fas response. FasR siRNA inhibited SM-induced caspase-3 activation when added to NHBE cultures up to 8 hours after SM. Results using annexin V/propidium iodide-stained cells showed that both apoptosis and necrosis were involved in cell death due to SM; FasR siRNA decreased both apoptotic and necrotic cell populations. Bronchoalveolar lavage fluid (BALF) of rats exposed to SM (1 mg/kg, 50 minutes) revealed a significant (P < 0.05) increase in soluble Fas ligand and active caspase-3 in BALF cells. These findings suggest an intervention of Fas-mediated apoptosis as a postexposure therapeutic strategy with a therapeutic window for SM inhalation injury and possibly other respiratory diseases involving the Fas response.

Pathological studies on the protective effect of a macrolide antibiotic, roxithromycin, against sulfur mustard inhalation toxicity in a rat model.

Macrolide antibiotics have been shown to protect airway epithelial cells and macrophages from sulfur mustard (SM)-induced cytotoxicity. In the current study, the efficacy of roxithromycin in ameliorating SM-induced respiratory injury was further evaluated in a rat model. Anesthetized rats (N = 8/group) were intratracheally exposed to SM by vapor inhalation. For the drug treatment groups, rats were orally given 10, 20, or 40 mg/kg roxithromycin one hr prior to exposure and every twenty-four hr thereafter. After one, three, or seven days of treatment, sections of the lung were examined and scored for histopathological parameters. Treatment with roxithromycin ameliorated many of the symptoms caused by SM in some animals. In particular, treatment at 40 mg/kg for three days showed significant improvements (p < .05) over the untreated group. When the evaluation was focused on trachea, treatment with roxithromycin for three days showed a trend of dose-dependent protection; moreover, the groups treated with 20 or 40 mg/kg of roxithromycin were statistically different (p < .001 and p < .05, respectively) from the untreated group. These results suggest that roxithromycin protects against some damages associated with SM injury in the lung, particularly in the upper respiratory tract.

Inflammatory effects of inhaled sulfur mustard in rat lung.

Inhalation of sulfur mustard (SM), a bifunctional alkylating agent that causes severe lung damage, is a significant threat to both military and civilian populations. The mechanisms mediating its cytotoxic effects are unknown and were investigated in the present studies. Male rats Crl:CD(SD) were anesthetized, and then intratracheally intubated and exposed to 0.7-1.4mg/kg SM by vapor inhalation. Animals were euthanized 6, 24, 48h or 7days post-exposure and bronchoalveolar lavage fluid (BAL) and lung tissue collected. Exposure of rats to SM resulted in rapid pulmonary toxicity, including focal ulceration and detachment of the trachea and bronchial epithelia from underlying mucosa, thickening of alveolar septal walls and increased numbers of inflammatory cells in the tissue. There was also evidence of autophagy and apoptosis in the tissue. This was correlated with increased BAL protein content, a marker of injury to the alveolar epithelial lining. SM exposure also resulted in increased expression of markers of inflammation including cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNFα), inducible nitric oxide synthase (iNOS), and matrix metalloproteinase-9 (MMP-9), each of which has been implicated in pulmonary toxicity. Whereas COX-2, TNFα and iNOS were mainly localized in alveolar regions, MMP-9 was prominent in bronchial epithelium. In contrast, expression of the anti-oxidant hemeoxygenase, and the anti-inflammatory collectin, surfactant protein-D, decreased in the lung after SM exposure. These data demonstrate that SM-induced oxidative stress and injury are associated with the generation of cytotoxic inflammatory proteins which may contribute to the pathogenic response to this vesicant.

Protective role of PI3-kinase/Akt/eNOS signaling in mechanical stress through inhibition of p38 mitogen-activated protein kinase in mouse lung.

AIM: To test the hypothesis that PI3K/Akt/eNOS signaling has a protective role in a murine model of ventilation associated lung injury (VALI) through down-regulation of p38 MAPK signaling. METHODS: Male C57BL/J6 (wild-type, WT) or eNOS knockout mice (eNOS(-/-)) were exposed to mechanical ventilation (MV) with low (LV(T), 7 mL/kg) and high tidal volume (HV(T), 20 mL/kg) for 0-4 h. A subset of WT mice was administered the specific inhibitors of PI3K (100 nmol/L Wortmannin [Wort], ip) or of p38 MAPK (SB203580, 2 mg/kg, ip) 1 h before MV. Cultured type II alveolar epithelial cells C10 were exposed to 18% cyclic stretch for 2 h with or without 20 nmol/L Wort pretreatment. At the end of the experiment, the capillary leakage in vivo was assessed by extravasation of Evans blue dye (EBD), wet/dry weight ratio and lung lavage protein concentration. The lung tissue and cell lysate were also collected for protein and histological review. RESULTS: MV decreased PI3K/Akt phosphorylation and eNOS expression but increased phospho-p38 MAPK expression along with a lung leakage of EBD. Inhibitions of phospho-Akt by Wort worsen the lung edema, whereas inhibition of p38 MAPK kinase restored activation of Akt together with alleviated capillary leakage. eNOS(-/-) mice showed an exacerbated lung edema and injury. The stretched C10 cells demonstrated that Wort diminished the activation of Akt, but potentiated phosphorylation of MAPK p38. CONCLUSION: Our results indicate that PI-3K/Akt/eNOS pathway has significant protective effects in VALI by preventing capillary leakage, and that there is a cross-talk between PI3K/Akt and p38 MAPK pathways in vascular barrier dysfunction resulting from VALI.

Evaluation of protease inhibitors and an antioxidant for treatment of sulfur mustard-induced toxic lung injury.

Sulfur mustard (SM)-induced lung injury has been associated with protease activation, oxidative injury and inflammatory response culminating in tissue necrosis. The protease inhibitors aprotinin and ilomastat and the antioxidant trolox were evaluated for efficacy in ameliorating SM-induced lung injury. Anesthetized spontaneously breathing rats (N=6-8/group) were intratracheally intubated and exposed to 1.4 mg/kg SM (0.35 mg SM in 0.1 ml of ethanol) or ethanol alone by vapor inhalation for 50 min. At 1 min before the exposure rats were treated with one of the following: intravenous aprotinin, 4.4 mg/kg; intraperitoneal (ip) ilomastat, 25mg/kg; or ip trolox, 500 microg/kg. Aprotinin-treated animals received supplemental 2.2mg/kg doses at 1 min and 6h post-exposure (PE). A whole body plethysmograph system was used to monitor pulmonary function (PF) parameters for 1h before exposure (baseline), and from 5-6 and 23-24h post-exposure. SM inhalation caused significant increases in several PF parameters, including tidal volume, peak inspiratory flow, peak expiratory flow, end expiratory pause and enhanced pause. Consistent with the reported development of SM-induced pathology, these changes were minimal at the 5-6-h time and significant at the 23-24-h timepoint. At the later time it is known from previous work that airways are becoming obstructed with loose cellular debris, damaged cells and exudate, which contributed to the changes in PF parameters. Treatment with aprotinin or ilomastat eliminated these PF changes, yielding results comparable with controls for each of these parameters. Lung lavage fluid analysis showed that SM caused a significant increase in total protein (TP) and in the cytokines IL-1alpha and IL-13. Aprotinin treatment prevented the increases in TP and IL-1alpha production, ilomastat prevented the increased production of IL-13, and trolox treatment did not significantly prevent the SM-related increases in TP, IL-1alpha or IL-13. Histopathologic examination of lung tissue 24h post-exposure showed minimal alveolar effects caused by SM, while damage to bronchiolar regions was much more severe due to the highly reactive nature of SM. While aprotinin and ilomastat both alleviated the PF perturbations, surprisingly only aprotinin reduced the observed pathology, both grossly and histologically. These early results indicate that treatment with aprotinin and to a lesser extent ilomastat reduces some of the direct inflammatory response and damage associated with SM-induced lung injury. This research was supported by the Defense Threat Reduction Agency - Joint Science and Technology Office, Medical S&T Division.

Sulfur mustard-induced neutropenia: treatment with granulocyte colony-stimulating factor.

Although best known as a blistering agent, sulfur mustard (HD) can also induce neutropenia in exposed individuals, increasing their susceptibility to infection. Granulocyte colony-stimulating factor (G-CSF) and pegylated G-CSF (peg-G-CSF) have been approved by the U.S. Food and Drug Administration as hematopoietic growth factors to treat chemotherapy-induced neutropenia. The goal of this study was to determine the effectiveness of G-CSF and peg-G-CSF in ameliorating HD-induced neutropenia. African green monkeys (Chlorocebus aethiops) were challenged with HD and, at 1, 3, 5, or 7 days after exposure, G-CSF therapy (10 microg/kg per day for 21 days) was initiated. Peg-G-CSF (300 microg/kg, single treatment) was similarly tested, with treatment given at 3 days after exposure. Untreated HD-exposed animals recovered from neutropenia 28 days after exposure, whereas G-CSF- or peg-G-CSF-treated animals recovered 8 to 19 days after exposure (p < 0.05). These results indicate that G-CSF or peg-G-CSF may provide Food and Drug Administration-approved treatments that will reduce the duration of HD-induced neutropenia.

Monitoring sulfur mustard exposure by gas chromatography-mass spectrometry analysis of thiodiglycol cleaved from blood proteins.

A gas chromatography-mass spectrometry method for determining exposure to the chemical warfare agent 2,2'-dichlorodiethyl sulfide (sulfur mustard; HD) has been developed. The technique is based upon quantitating thiodiglycol (TDG) released from blood protein adducts that are formed upon exposure to HD. Protein was precipitated from plasma, whole blood, or packed red blood cells (RBCs) and then treated with sodium hydroxide to liberate protein-bound TDG. The TDG was derivatized with pentafluorobenzoyl chloride that enabled sensitive detection by negative-ion chemical ionization. Octadeuterothiodiglycol was used as an internal standard. Exposure of human plasma to HD (25 nM to 400 nM) resulted in a linear relationship (r2 = 0.9995) between HD concentration and released TDG levels with means ranging from 2.0 to 38 pg/mg protein. The coefficients of variation expressed as a percentage for the data points ranged from 2 to 11.5%. The application of this procedure was demonstrated in two HD animal exposure models. African green monkeys (Chlorocebus aethiops) were exposed intravenously to 1 mg/kg HD, and TDG levels in blood samples were analyzed out to 45 days post-exposure. Mean TDG levels were determined to be 220 pg/mg protein on day 1 and declined to 10 pg/mg protein on day 45. Yorkshire cross pigs (Sus scrofa) were cutaneously exposed to neat liquid HD, and TDG levels in plasma were determined out to 21 days following exposure. Mean TDG levels were found to be 60 pg/mg protein on day one and decreased to an average of 4 pg/mg protein on day 21. The data from this study indicate that the assay is sensitive and provide a relatively simple approach to assay TDG cleaved from blood proteins at relatively long time frames (21-45 days) after HD exposure. The utility of the method has been demonstrated in vivo in a non-human primate and pig HD exposure model.

Sulfur mustard-induced apoptosis in hairless guinea pig skin.

The present study was aimed to examine whether apoptosis is involved in the pathogenesis of sulfur mustard (SM)-induced basal cell death. Skin sites of the hairless guinea pig exposed to SM vapor for 8 minutes were harvested at 3, 6, 12, 24, and 48 hours postexposure. Immunohistochemical detection of basal cell apoptosis was performed using the ApopTag in situ apoptosis labeling kit. Only occasional apoptotic basal cells (BC)were observed in nonexposed and perilesional control sites. At lesional sites, apoptosis of BC was not detected at 3 hours postexposure. However, at 6 hours and 12 hours postexposure, 18% and 59% of BC were apoptotic, respectively. At 24 and 48 hours postexposure, individual apoptotic basal cells were not clearly recognizable due to necrosis. At the ultrastructural level, degenerating BC exhibited typical apoptotic morphology including nuclear condensation and chromatin margination. The results suggest that apoptotic cell death is a cytotoxic mechanism with the number of BC undergoing apoptosis significantly increasing from 6 to 12 hours postexposure. In addition, because necrosis is preferential at 24 hours postexposure, we believe that SM-induced cell death involves early apoptosis and late necrosis, which temporally overlap to produce a single cell death pathway along an apoptotic-necrotic continuum.

A vapor exposure model for neonatal mice.

Sulfur mustard (HD) is a vesicant compound that was first used as a chemical warfare agent in World War I. (Papirmeister et al. 1991). Numerous animal models have been used to study HD-induced vesication. In this article, we describe modifications of the vapor cup model of Mershon and colleagues (1990) to establish a new vapor cup model for use in neonatal mice. The need to develop this model resulted from the development of gene-targeted knockout mice that can be used to evaluate the function of specific genes and their contribution to HD-induced pathology. However, the knockouts are haired mice; therefore, it is necessary to perform vapor exposures on the pups prior to their growing hair. Neonatal mice were anesthetized with isofluorane inhalation and placed in sternal recumbency on a 37 degrees C isothermal pad to maintain body heat during exposure. The vapor cup consisted of a 1.5-mL microfuge tube cap (8 mm inside diameter) modified using a Dremel tool to contour its rim to better fit the curve of a mouse pups back. The inside of the cap was fitted with an 8-mm disk of Whatman #2 filter paper, and the rim of the cap was coated with a thin bead of Thomas Lubriseal grease. Ten muL of neat HD was placed on the filter paper disk, and the cup was immediately inverted and placed onto the back of an anesthetized mouse pup. Exposure times varied from 10 to 30 min. At 24 h postexposure, the mice were euthanized; the HD-exposed skin was removed and fixed in 10% neutral buffered formalin. Following a minimum of 24 h of formalin fixation, the skin sections were bisected across the exposed area. The sections were embedded in paraffin with the central straight-cut surfaces being the focus of histological evaluation. The amount of damage associated with the HD vapor cup exposure varied with time in a dose response fashion. Typical damage consisted of varying amounts of epidermal necrosis at the basal cell level, with occasional separation of epidermis from dermis (microvesication). In severe cases there was complete coagulation of the epidermis and no microvesication. This model should prove useful in identifying the biochemical mechanism of action of HD and ultimately aid in the evaluation of treatment compounds. It may also provide a relevant exposure model for other compounds for which the assessment of vapor-induced damage is necessitated.