Dose-dependent emergence of acute and recurrent corneal lesions in sulfur mustard-exposed rabbit eyes.

Sulfur mustard (SM) is a lipid soluble alkylating agent that causes genotoxic injury. The eye is highly sensitive to SM toxicity and exposures exceeding 400 mg min/m3 can elicit irreversible corneal pathophysiologies. Development of medical countermeasures for ocular SM exposure has been hindered by a limited understanding of dose-dependent effects of SM on corneal injury. Here, clinical, histological and ultrastructural analyses were used to characterize the effects of SM dose on corneal injury progression. Corneas were evaluated for up to 20 wk following exposure to saturated SM vapor for 30-150 s, which corresponds to 300-1,500 mg min/m3. In acute studies, a ceiling effect on corneal edema developed at doses associated with full-thickness corneal lesions, implicating endothelial toxicity in corneal swelling. Recurrent edematous lesions (RELs) transiently emerged after 2 wk in a dose-dependent fashion, followed by the development of secondary corneal pathophysiologies such as neovascularization, stromal scarring and endothelial abnormalities. RELs appeared in 96 % of corneas exposed for ≥ 90 s, 52 % of corneas exposed for 60 s and 0 % of corneas exposed for 30 s. While REL latency was variable in corneas exposed for 60 s, REL emergence was synchronized at exposures ≥ 90 s. Corneas did not exhibit more than one REL, suggesting RELs are part of a programmed pathophysiological response to severe alkylating lesions. In post-mortem studies at 12 wk, corneal edema was positively correlated to severity of endothelial pathologies, consistent with previous findings that endothelial toxicity influences long-term outcomes. These results provide novel insight into long-term corneal pathophysiological responses to acute toxicity and identify exposure conditions suitable for therapeutic testing.

Corneal Endothelial Cell Toxicity Determines Long-Term Outcome After Ocular Exposure to Sulfur Mustard Vapor.

PURPOSE: Ocular exposure to sulfur mustard (SM) vapor causes acute loss of corneal endothelial cells (CECs). Persistent corneal endothelial pathologies are observed in eyes that do not recover from SM exposure, suggesting that endothelial toxicity contributes to mustard gas keratopathy (MGK). Here, we evaluated the contributions of endothelial loss to acute and chronic corneal injuries in SM-exposed eyes. METHODS: Rabbit eyes were exposed in vivo to equivalent doses of SM using 9-, 11-, or 14-mm vapor caps. The effects of exposure area on corneal injury progression were longitudinally evaluated over 12 weeks using clinical evaluations. The effects of exposure area on CEC morphology, endothelial and epithelial ultrastructure, and endothelial barrier function were determined from 1 day to 12 weeks. RESULTS: SM exposure caused loss of CECs and failure of endothelial barrier integrity at 1 day, independent of exposure cap size. By 3 weeks, eyes exposed with the 14-mm vapor cap exhibited increased corneal permeability, repopulation of the endothelium by cells with fibroblastic morphology, and abnormal deposition of extracellular matrix. Eyes exposed with 9- or 11-mm vapor caps exhibited transient symptoms of injury that fully resolved, with the rate of recovery correlated with cap size. CONCLUSIONS: The nonlinear correlation between endothelial lesion size and probability of developing MGK suggests that the CEC loss is a determinative factor for emergence of MGK. These studies illustrate the importance of endothelial repair in preventing MGK. Furthermore, they exclude chemical modification of basement membrane as a mechanistic cause of recurrent epithelial erosions in MGK eyes.

Apoptotic cell death in rat lung following mustard gas inhalation.

To investigate apoptosis as a mechanism of sulfur mustard (SM) inhalation injury in animals, we studied different caspases (caspase-8, -9, -3, and -6) in the lungs from a ventilated rat SM aerosol inhalation model. SM activated all four caspases in cells obtained from bronchoalveolar lavage fluid (BALF) as early as 6 h after exposure. Caspase-8, which is known to initiate the extrinsic Fas-mediated pathway of apoptosis, was increased fivefold between 6 and 24 h, decreasing to the unexposed-control level at 48 h. The initiator, caspase-9, in the intrinsic mitochondrial pathway of apoptosis as well as the executioner caspases, caspase-3 and -6, all peaked (P < 0.01) at 24 h; caspase-3 and -6 remained elevated, but caspase-9 decreased to unexposed-control level at 48 h. To study further the Fas pathway, we examined soluble as well as membrane-bound Fas ligand (sFas-L and mFas-L, respectively) and Fas receptor (Fas-R) in both BALF cells and BALF. At 24 h after SM exposure, sFas-L increased significantly in both BALF cells (P < 0.01) and BALF (P < 0.05). However, mFas-L increased only in BALF cells between 24 and 48 h (P < 0.1 and P < 0.001, respectively). Fas-R increased only in BALF cells by 6 h (P < 0.01) after SM exposure. Apoptosis in SM-inhaled rat lung specimens was also confirmed by both immunohistochemical staining using cleaved caspase-3 and -9 antibodies and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining as early as 6 h in the proximal trachea and bronchi, but not before 48 h in distal airways. These findings suggest pathogenic mechanisms at the cellular and molecular levels and logical therapeutic target(s) for SM inhalation injury in animals.

Gene expression profiling of rat hippocampus following exposure to the acetylcholinesterase inhibitor soman.

Soman (O-pinacolyl methylphosphonofluoridate) is a potent neurotoxicant. Acute exposure to soman causes acetylcholinesterase inhibition, resulting in excessive levels of acetylcholine. Excessive acetylcholine levels cause convulsions, seizures, and respiratory distress. The initial cholinergic crisis can be overcome by rapid anticholinergic therapeutic intervention, resulting in increased survival. However, conventional treatments do not protect the brain from seizure-related damage, and thus, neurodegeneration of soman-sensitive brain areas is a potential postexposure outcome. We performed gene expression profiling of the rat hippocampus following soman exposure to gain greater insight into the molecular pathogenesis of soman-induced neurodegeneration. Male Sprague-Dawley rats were pretreated with the oxime HI-6 (l-(((4-aminocarbonyl)pyridinio)methoxyl)methyl)-2-((hydroxyimino)methyl)-pyridinium dichloride; 125 mg/kg, ip) 30 min prior to challenge with soman (180 microg/kg, sc). One minute after soman challenge, animals were treated with atropine methyl nitrate (2.0 mg/kg, im). Hippocampi were harvested 1, 3, 6, 12, 24, 48, 72, 96, and 168 h after soman exposure and RNA extracted to generate microarray probes for gene expression profiling. Principal component analysis of the microarray data revealed a progressive alteration in gene expression profiles beginning 1 h postexposure and continuing through 24 h postexposure. At 48 h to 168 h postexposure, the gene expression profiles clustered nearer to controls but did not completely return to control profiles. On the basis of the principal component analysis, analysis of variance was used to identify the genes most significantly changed as a result of soman at each postexposure time point. To gain insight into the biological relevance of these gene expression changes, genes were rank ordered by p-value and categorized using gene ontology-based algorithms into biological functions, canonical pathways, and gene networks significantly affected by soman. Numerous signaling and inflammatory pathways were identified as perturbed by soman. These data provide important insights into the molecular pathways involved in soman-induced neuropathology and a basis for generating hypotheses about the mechanism of soman-induced neurodegeneration.