Regeneration of corneal endothelium following complete endothelial cell loss in rat keratoplasty.

PURPOSE

Corneal endothelial cells (EC) are crucial for maintaining corneal clarity before and after keratoplasty. Since it is thought that corneal graft rejection leads to irreversible EC loss and transplant failure, we quantified immune mediated EC loss in the rat keratoplasty model and analyzed whether the EC layer would then regenerate.

METHODS

Rats were subjected to orthotopic penetrating keratoplasty. We compared endothelial responses to immunological EC loss following allogeneic transplantations between Fisher and Lewis rats (group R) to those following mechanical EC removal in a syngeneic setting between Lewis rats (group S). Animals were followed clinically for corneal opacity for up to one year. Bulbi were excised and prepared for histological examination at different time points: ECs were defined and characterized using Alicarin red S/ DAPI staining on corneal flatmounts. Ki-67/ DAPI staining on flatmount preparations served to detect cell proliferation. Immunohistochemical staining of corneal cryosections was used to characterize infiltrating immune cells.

RESULTS

GROUP R: After about two weeks the allografts were completely opaque, which was accompanied by a massive leukocyte infiltration in conjunction with EC destruction, signifying rejection. EC loss without an immune reaction (group S) resulted only in medium opacity levels. In both groups, all grafts regained clarity in the following weeks to months, and a newly-formed endothelial cell layer with irregular and enlarged ECs became apparent on the formerly EC free grafts. Scattered Ki-67 positive cells within the endothelial cell layer were observed during re-endothelialization. In addition to re-endothelialization, the immunological infiltration seen in the allografts at the time of rejection had subsided after one year.

CONCLUSIONS

Re-endothelialization following keratoplasty takes place in the rat in vivo and restores graft clarity, following both immunological or surgical destruction of ECs. Following rejection, EC replacement is accompanied by a reduction of immune infiltrates. Peripheral recipient ECs are a sufficient source for graft re-endothelialization, as seen in rats following EC removal. Our results suggest that ECs both proliferate and enlarge during re-endothelialization in the rat keratoplasty model.