Sulfur Mustard Rabbit Cornea
Sulfur Mustard Toxicity: Histology and Immunohistochemistry of Rabbit Cornea Showing Edema and Damage to the Basement Membrane. Sulfur mustard (SM) is a highly reactive bifunctional alkylating agent synthesized for chemical warfare. The eyes are particularly sensitive to SM where it causes irritation, pain, photophobia, and blepharitis, depending on the dose and duration of exposure. We hypothesize that SM-induced damage to the cornea is associated with significant alterations in the corneal stroma and the basement membrane. In these studies, the effects of SM vapor on the corneas of New Zealand white male rabbits were evaluated. The eyes of rabbits were treated with air (control) or SM vapor; after 14d and 28d animals were euthanized and histological sections of the corneas examined for damage. In control corneas, H&E staining showed a well-defined basement membrane separating columnar epithelial cells from the stroma. After 14d and 28d, areas of the basement membrane were much less well-defined. Trichrome staining showed significant areas of stromal edema where disorganization of collagen fiber bundles was evident along with prominent areas of separation of lamellar structures. This was associated with hazing of the cornea. Using immunohistochemistry in conjunction with specific antibodies, SM was found to disrupt the corneal basement membrane altering expression of cellular fibronectin, an extracellular matrix glycoprotein, and perlecan, a heparan sulfate proteoglycan. These changes were associated with increases in thickness of the basement membrane and redistribution of extracellular matrix proteins. This was also associated with an increase in basement membrane matrix metalloproteinases including ADAM17, which is important in remodeling of the basement membrane during wound healing.
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Rabbit corneal tissue was isolated and embeded in OCT. Ten micron sections were cut and stained with H&E or Masson's trichrome. For immunohistochemistry, tissue sections were incubated with citrate buffer at 37 degrees C for antigen retrieval and endogenous peroxidases inhibited with Bloxall. Tissues were incubated with primary antibodies (cellular fibronectin (1:1000, cFN, Abcam, Cambridge, UK; perlecan (1:4000, Abcam) overnight at 4 degrees C. Tissue sections were washed and incubated at room temperature with biotinylated horse anti-mouse secondary antibody (Vector Labs, Burlingame, CA). Binding was visualized with DAB Peroxidase Substrate Kit (Vector Labs). Tissue sections were counterstained with hematoxylin and cover slipped with Permount (Thermo Fisher Scientific). Images were acquired at high resolution using an Olympus BH2 microscope. For immunofluorescence, tissue sections were incubated with a mouse monoclonal antibody to ADAM17 (1:500, R&D Systems, Minneapolis, MN). After overnight incubation at 4 degree C, sections were washed with PBS and incubated at room temperature for 1 h with donkey-anti-mouse Alexafluor488 (1:1000, Invitrogen, Carlsbad, CA). 4′,6-Diamidino-2-phenylindole (DAPI) was used to visualize nuclei. Fluorescence was visualized using a Zeiss fluorescent microscope.