AREA AND DEPTH OF SURFACTANT-INDUCED CORNEAL INJURY CORRELATES WITH CELL-DEATH

PURPOSE. in previous studies in which in vivo confocal microscopy (CM) was used, quantifiable differences mere identified in the corneal epi thelium and stroma for surfactants producing different degrees of ocul ar irritation. In the present study, in vive confocal microscopy was u sed to determine area and depth of the initial corneal changes, and th e correlation of the data to cell death was characterized by ex vive L ive-dead assay. METHODS. In four groups of rabbits (12 animals each), 10 mu l surfactants known to produce slight, mild, moderate, or severe irritation was applied to the central cornea of one eye; 4 untreated rabbits served as Controls. Measurements of group total mean epithelia l thickness: epithelial cell area, and depth of keratocyte loss in fou r corneal regions were made by in vive CM in G rabbits of each group a nd in 4 control animals at 3 hours and in the remaining rabbits at 3 h ours and 1 day. Corneas were then removed and fixed for conventional h istologic examination (two eyes/treatment/group), or regions were exci sed and placed in culture media containing 2 mu M calcein-acetoxymethy l ester (calcein-AM) and 4 mu M ethidium homodimer. Using laser scanni ng CM, the number of dead epithelial or stromal cells in a 300 x 300 x 170 mu m (in the x, y, and z axes, respectively) volume of the cornea was determined. RESULTS. Confocal microscopy showed that application of the slight irritant resulted in decreased epithelial thickness at 3 hours (41.2 +/- 2.6 mu m in treated eyes versus 43.6 +/- 3 mu m in co ntrol eyes n = 6 and 4, respectively) and a significant decrease (P < 0.001) in epithelial cell size (630 +/- 203 mu m(2) versus 1427.2 +/- 90.7 mu m(2)). On day 1, mild, moderate, and severe irritants caused c omplete loss of epithelium and disappearance of keratocytes to a depth of 30.8 +/- 10.7 mu m, 47.2 +/- 10.4 mu m, and 764.6 +/- 159.6 mu m ( n = 6, 5, and 6), respectively. At 3 hours, live-dead assay detected m ore dead epithelial cells as a percentage of total surface cells (49.2 . +/- 4.5% in slightly irritated eyes versus 20.9 +/- 3.2% in control eyes): significantly correlating with the measurement by in vivo CM of average epithelial cell size in each eye (r = -0.96; P < 0.005). On d ay 1, mild and moderate irritants showed increasing stromal cell death from 9.8 +/- 16.2 cells to 36.4 +/- 17.7 cells, which significantly c orrelated with the depth of stromal injury determined by in vive CM (r = 0.75); P < 0.00001). No surviving keratocytes were detected in seve rely irritated eyes. CONCLUSIONS. The data support the hypothesis that differences in surfactant-induced ocular irritation are directly rela ted to area and depth of acute corneal injury.