Histochemical studies of mitochondrial activities of cultured corneal endothelial cells of cat during wound-healing.

The corneal endothelial cells of the cat were cultured, and in 3 weeks the cells became confluent and formed a cell sheet. The cells were stained with rhodamine 123 and nitroblue tetrazolium (NBT). The frequencies of cells stained by these two methods changed during the 3-week culture, but at the end of the culture the frequencies were stabilized at 58 +/- 3.9 (mean +/- SD) % for the rhodamine 123 staining method and 35 +/- 3.7% for the NBT method. An oval wound of 600 X 400 microns was made in the center of the cultured endothelial cell sheet, and the cell migration was observed by phase contrast microscopy. Six hours after the wounding, the cells began to migrate toward the center of the wounded area, and in 24 hours the denuded area was almost completely covered by migrated cells. In 48 hours the wound was tightly covered by the migrated cells. One hour after the wounding, the cells in the wound margin were strongly stained by rhodamine 123, but not by NBT. The cultured cell sheet was divided into 4 concentric areas, ie, S1 was the center of the wound, S2 was the 100-microns-wide zone inside the original wound edge, S3 was the 100-microns-wide zone outside the original wound edge, and S4 was the 100-microns-wide zone outside the S3 zone. Topographical changes in rhodamine- and NBT-stained cells were observed over a 48-hour period. Rhodamine-positive cells increased in the S1 area, while a decrease occurred in the S2 and S3 areas. NBT-positive cells peaked at 12 hours after the wounding and decreased thereafter. It was concluded that the endothelial cells migrating to cover the wounded area showed a significant enhancement of mitochondrial activity, as indicated by the rhodamine 123 staining, but the succinic dehydrogenase activity revealed by NBT staining was rather suppressed.