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

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

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

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

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

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

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