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Forty-seven donor corneas were preserved in McCarey-Kaufman (M-K) medium at 4 degrees C for 1 day, then in organ culture at 34 degrees C for approximately 1 month, then in M-K medium at 4 degrees C for an additional two days before transplantation. The central donor endothelium was examined by specular microscopy before and after organ culture and 2 months after keratoplasty. No significant change in central endothelial cell density occurred during organ culture. The 47 transplants were compared with 47 grafts preserved only in M-K medium at 4 degrees C for approximately 36 hours. All transplants were performed by the same surgeon over the same period, and the two groups contained similar types of surgical procedures. The organ-cultured grafts were thicker on the first post-operative day and took longer to epithelialize . Two months after keratoplasty all of the 94 grafts were clear and thin, but the mean central endothelial cell loss was 28% in the 47 organ-cultured transplants and 10% in the 47 transplants preserved only in M-K medium (P less than 0.0001). These results indicate that the endothelium of corneas preserved by organ culture at 34 degrees C and then placed in M-K medium at 4 degrees C for 2 days may be more susceptible to surgical trauma than those preserved only in M-K medium at 4 degrees C.

We investigated the combined fluorescent dyes acridine orange (AO) and ethidium bromide (EB) for corneal endothelial evaluation. Both dyes intercalate with DNA and RNA and are mutagenic at high concentrations. Optimum differential staining was obtained after 5 minutes exposure to 1 microgram/ml of each dye. Electron microscopy confirmed that the dye combination simultaneously identified both viable and nonviable cells. Swelling of perfused dog corneas for 3 hours after exposure to both dyes for 5 minutes was not increased. Exposure to visible light from a fluorescence microscope for 2-5 minutes caused increased corneal swelling and abnormal intercellular junctions by electron microscopy in paired dog corneas stained for 5 minutes with either AO or EB. The addition of 10% calf serum to the staining and observation media decreased the toxic effect of AO/EB and light seen by specular microscopy and by electron microscopy. Human corneas exposed to AO and EB were not mutagenic in the Salmonella/microsome assay. These results indicate that AO and EB, 1 microgram/ml, provide a rapid identification of both viable and nonviable corneal endothelial cells. When the corneas are exposed to light, however, the dyes are toxic; this toxicity is decreased by the presence of 10% calf serum in the staining and observation media.