Architectural and Biochemical Expressions of Mustard Gas Keratopathy: Preclinical Indicators and Pathogenic Mechanisms

<div><p>A subset of victims of ocular sulfur mustard (SM) exposure develops an irreversible, idiotypic keratitis with associated secondary pathologies, collectively referred to as mustard gas keratopathy (MGK). MGK involves a progressive corneal degeneration resulting in chronic ocular discomfort and impaired vision for which clinical interventions have typically had poor outcomes. Using a rabbit corneal vapor exposure model, we previously demonstrated a clinical progression with acute and chronic sequelae similar to that observed in human casualties. However, a better understanding of the temporal changes that occur during the biphasic SM injury is crucial to mechanistic understanding and therapeutic development. Here we evaluate the histopathologic, biochemical and ultrastructural expressions of pathogenesis of the chronic SM injury over eight weeks. We confirm that MGK onset exhibits a biphasic trajectory involving corneal surface regeneration over the first two weeks, followed by the rapid development and progressive degeneration of corneal structure. Preclinical markers of corneal dysfunction were identified, including destabilization of the basal corneal epithelium, basement membrane zone abnormalities and stromal deformation. Clinical sequelae of MGK appeared abruptly three weeks after exposure, and included profound anterior edema, recurring corneal erosions, basement membrane disorganization, basal cell necrosis and stromal degeneration. Unlike resolved corneas, MGK corneas exhibited frustrated corneal wound repair, with significantly elevated histopathology scores. Increased lacrimation, disruption of the basement membrane and accumulation of pro-inflammatory mediators in the aqueous humor provide several mechanisms for corneal degeneration. These data suggest that the chronic injury is fundamentally distinct from the acute lesion, involving injury mechanisms that operate on different time scales and in different corneal tissues. Corneal edema appears to be the principal pathology of MGK, in part resulting from persistent necrosis of the basal corneal epithelium and deterioration of the basement membrane. The findings also provide a potential explanation as to why administration of anti-inflammatories transiently delays, but does not prevent, the development of MGK sequelae.</p> </div

Histopathology scores over an 8 week period following a 2.5-min corneal exposure to SM vapor.

<p>The degree and extent of corneal sequelae from 1–8 weeks (n = 6 for all) and resolved cornea at 8 weeks (8r; n = 4; black bars) were scored. Qualitative descriptors corresponding to numerical scores are presented on the right axis. Data shown represent mean plus standard error and asterisks indicate values that are significantly different from 2-week scores (<i>p</i><0.05; cross-hatched bars).</p

Representative light microscopy of H&E-stained sections demonstrating cellular aspects of wound repair in MGK and resolving corneas.

<p>Panels A–D were imaged at the lesion margin, with the limbus to the right, and panels E–F were imaged at the central cornea. Scale bar is 100 µm for all panels except 1 week, where it is 20 µm.</p

Changes in the ultrastructure of the BMZ from 1–5 weeks during the onset of MGK.

<p>(A–F) TEM images from control and 1–5 weeks. Scale bar  = 2 µm. Thin panels represent high magnification images of the BMZ. Basal cell (bc); hemidesmosomes (arrows); nucleus (n); stroma (st); Bowman’s like-layer (bll); necrotic basal cell (nec); proteinaceous void (*); lamina densa (ld); epithelial process penetrating into stroma (c); and breaks in the lamina densa (arrowheads).</p

Light microscopy of H&E-stained sections demonstrating representative changes in corneal histology from 1–8 weeks after a 2.5-min SM vapor exposure.

<p>Arrows in panel B represent boundaries of acute epithelial lesion.</p

MGK corneas are exposed to keratoactive factors at the anterior and posterior boundaries.

<p>(A) Lacrimation increases over time in MGK corneas. (B) Cytokine levels in the AH increase during the acute and the chronic SM injury. For A and B: * represents <i>p</i><0.05. (C, D) Measurement of (C) MMP-2 and (D) MMP-9 activity in the AH. All data are presented as mean plus standard error.</p

Intra-corneal deposition of epithelial cells during MGK.

<p>(A, B) Light microscopy demonstrating stromal residency of vacuolated (A) and compact (A, B) epithelial cell clusters. Scale bar in (B) is 40 µm. (C) Transmission electron micrograph of mid-stromal island demonstrating characteristic epithelial cell features. Scale bar is 2 µm. (D, E) Regions marked in (C) are shown at higher magnification to demonstrate desmosomal plaques.</p

The physiology and toxicology of acute inhalation phosphine poisoning in conscious male rats

<p>Phosphine (PH₃) is a toxidrome-spanning chemical that is widely used as an insecticide and rodenticide. Exposure to PH₃ causes a host of target organ and systemic effects, including oxidative stress, cardiopulmonary toxicity, seizure-like activity and overall metabolic disturbance. A custom dynamic inhalation gas exposure system was designed for the whole-body exposure of conscious male Sprague-Dawley rats (250–350 g) to PH₃. An integrated plethysmography system was used to collect respiratory parameters in real-time before, during and after PH₃ exposure. At several time points post-exposure, rats were euthanized, and various organs were removed and analyzed to assess organ and systemic effects. The 24 h post-exposure LCt₅₀, determined by probit analysis, was 23,270 ppm × min (32,345 mg × min/m³). PH₃ exposure affects both pulmonary and cardiac function. Unlike typical pulmonary toxicants, PH₃ induced net increases in respiration during exposure. Gross observations of the heart and lungs of exposed rats suggested pulmonary and cardiac tissue damage, but histopathological examination showed little to no observable pathologic changes in those organs. Gene expression studies indicated alterations in inflammatory processes, metabolic function and cell signaling, with particular focus in cardiac tissue. Transmission electron microscopy examination of cardiac tissue revealed ultrastructural damage to both tissue and mitochondria. Altogether, these data reveal that in untreated, un-anesthetized rats, PH₃ inhalation induces acute cardiorespiratory toxicity and injury, leading to death and that it is characterized by a steep dose-response curve. Continued use of our interdisciplinary approach will permit more effective identification of therapeutic windows and development of rational medical countermeasures and countermeasure strategies.</p