Kinetics of keratocyte proliferation in response to epithelial debridement

Over the past 40 years, several groups have shown that epithelial debrideme nt results in the death of keratocytes subjacent to the wound area. More re cently this cell death has been shown to involve apoptosis. The purpose of this project was to examine the proliferative response of the normally quie scent keratocytes to repopulate the apoptotic area. Three mm wounds were ma de in the central cornea of adult rats and allowed to hear 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-cl-smooth muscle acti n was used to determine if myofibroblasts were present. In unwounded cornea s, keratocytes were uniformly spread throughout the stroma. and less than o ne proliferating cell per mm was observed. By 4 hr after wounding, the ante rior one-half to three-fourths of the stroma subjacent to the wound was dev oid of cells. No increase in Ki67-expressing cells was observed in the stro ma until 24 hr after wounding (3.9 +/- 0.5 and 6.3 +/- 0.5 mm(-1) in the wo und center and edge, respectively). The number of Ki67-expressing cells ste adily 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 c ell death. No change in the number of cells expressing Ki67 was observed in the keratocytes distal to the original debridement. Ki67 levels did not re turn to control levels until 7 days after wounding. No a-smooth muscle acti n was detected at any time point. This study indicates that epithelial debr idement 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. (C) 2000 Academic Press.