AN ORGAN CULTURE MODEL FOR STUDYING CORNEAL WOUND HEALING

Abstract

Wound healing at the epithelial‐stromal junction was studied in rabbit corneal air‐lifted organ cultures. Dissected corneas with scleral rims, filled with agarose, were placed epithelial side up in culture dishes, with medium 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 for 10 days. Mild injury was induced by adding 20 μl of 10mM 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. Plus or minus Dox washes were repeated 3 times in 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 good nuclei staining. By electron microscopy, hemidesmosomes, the basement membrane and the anterior stroma ECM were partially degraded after CEES exposure. Corneas treated with Dox after CEES had a normal epithelial BM‐stromal interface. This data suggests that even mild mustard injuries cause some DNA damage to the basal epithelial cells, and some ECM degradation. These adverse affects are reversed by doxycycline.