New wound-healing model using cultured bovine corneal endothelial cells. 2. Role of migration and mitosis studied by immunohistochemistry.

The roles of migration and mitosis in the in vitro corneal endothelial wound-healing process were studied using an immunohistochemical method. The in vitro wound-healing model was a cultured cell sheet prepared using bovine corneal endothelium, and the wound was made at the center of the cell sheet using a rotating silicon tip. Following the wounding, specimens were incubated with bromodeoxyuridine (BrdU) for 12 hours. After the fixation with 10% phosphate-buffered formalin, the cells incorporating BrdU were stained by the avidin biotin peroxidase complex method. In 12 hours after the wounding, no stained cells were observed. In 24 hours, cell migration had started and a few cells were stained; the average number of stained cells was 14.6 +/- 4.0 cells/mm2. In 48 hours, the number of stained cells increased. The maximum number of stained cells was observed between 48 and 60 hours after the wounding. The number of stained cells decreased rapidly after 72 hours when the wound was almost closed. Stained cells were most numerous in the area within 0.8 mm of the wound edge throughout the wound-healing. The endothelial wound-healing process in this model could be divided into four phases, latent, migration, concurrence of migration and mitosis, and inhibition phases. This in vitro wound-healing model could simulate the wound-healing process in vivo and will be useful for a quantitative evaluation of drug effects on the corneal endothelial wound-healing.