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A. S. DeSantis, R. A. Hahn, J. Beloni, D. R. Gerecke, K. K. H. Svoboda, M. K. Gordon; Doxycycline Promotes Healing of Corneal Injuries Induced by Half Mustard (CEES). Invest. Ophthalmol. Vis. Sci. 2008;49(13):3386. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In the cornea, exposure to mustard compounds has a severe effect on the basal epithelial cells and on the extracellular matrix where the basement membrane abuts the stroma. Our goal was to characterize mild mustard injury in a rabbit corneal organ culture model system, and determine whether, 2 hr after injury, the application of the matrix metalloproteinase inhibitor doxycycline would lessen the injury.
Rabbit corneas were dissected with scleral rims and prepared for air-lifted organ culture as follows: Corneas were placed epithelial side down in a dish, and the endothelial side of the concave structure was filled with agarose. Corneas were then flipped, placing epithelial side up in culture dishes. Medium was added up to the corneal-scleral junction. Every 4-8 hr, 200 ul of medium was applied dropwise onto the central cornea. Corneas were viable and transparent in organ culture for a minimum of 7 days. Mild injury was induced in 1 day old cultures by adding 20 µl of 10 mM half mustard (CEES) dropwise onto the central cornea. After a 2 hr 37oC incubation, to half of the samples 1.5 ml medium containing 200mM doxycycline (Dox) was added, also dropwise. Medium alone was added to the remaining half. Medium washes plus or minus Dox were repeated 3 times more over the course of 22 hr. Corneas were then fixed, embedded, and sectioned.
22 hr after CEES treatment, suprabasal epithelial cell nuclei stained well, but basal cell nuclei were pale, staining poorly with DAPI or toluidine blue, suggesting DNA damage. Dox induced basal cell proliferation and resulted in strong staining of nuclei. By electron microscopy, hemidesmosomes, the basement membrane and the anterior stroma ECM showed distinct signs of degradation after CEES exposure. Corneas treated with Dox after CEES had a nearly normal epithelial BM-stromal interface.
Our data suggests that even mild mustard injuries cause some degree of DNA damage to the basal epithelial cells, and detectible ECM degradation. These adverse affects are ameliorated by doxycycline.
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