Kinetics of keratocyte proliferation in response to epithelial debridement

Over the past 40 years, several groups have shown that epithelial debridement results in the death of keratocytes subjacent to the wound area. More recently this cell death has been shown to involve apoptosis. The purpose of this project was to examine the proliferative response of the normally quiescent keratocytes to repopulate the apoptotic area. Three mm wounds were made in the central cornea of adult rats and allowed to heal 4 hr to 14 days. Cryostat sections were stained with propidium iodide to mark the nuclei of all cells. Actively proliferating cells were identified with anti-Ki67, a marker of the late G1-M phase of the cell cycle. Anti-alpha-smooth muscle actin was used to determine if myofibroblasts were present. In unwounded corneas, keratocytes were uniformly spread throughout the stroma, and less than one proliferating cell per mm was observed. By 4 hr after wounding, the anterior one-half to three-fourths of the stroma subjacent to the wound was devoid of cells. No increase in Ki67-expressing cells was observed in the stroma until 24 hr after wounding (3.9 +/- 0.5 and 6. 3 +/- 0.5 mm(-1)in the wound center and edge, respectively). The number of Ki67-expressing cells steadily increased, peaking 44 hr after debridement (41.2 +/- 1.7 and 39.6 +/- 1.0). These cells were confined to a narrow zone adjacent to the area of cell death. No change in the number of cells expressing Ki67 was observed in the keratocytes distal to the original debridement. Ki67 levels did not return to control levels until 7 days after wounding. No alpha-smooth muscle actin was detected at any time point. This study indicates that epithelial debridement stimulates a synchronous increase in keratocyte proliferation. This stimulation is specific for cells immediately adjacent to the area of cell death. This activation does not involve the transformation of the stromal cells to a myofibroblast phenotype.