The effect of dichlorotriazinyl aminofluorescein on human keratocytes in vitro

Dichlorotriazinyl aminofluorescein (DTAF) has been used to stain corneal stromal collagen as part of in vivo animal experiments for many years. Toxicity of this drug, if present, might alter the observed wound healing. To determine if this drug has any deleterious effect on keratocytes, we evaluated it in vitro. Human keratocytes prepared in 24-well plates were exposed to varying concentrations of DTAF (10(-4), 10(-3), 10(-2), 1, 10, 10(2) microgram/ml). Exposure times of 1 hour and 24 hours at each concentration of DTAF were evaluated. The cell number was measured 1 and 3 days after initiation of exposure to DTAF using a Coulter counter. Keratocyte proliferation was not affected by 1-hour exposure to DTAF, but keratocyte proliferation measured 3 days after initiation of exposure to DTAF for 24 hours was inhibited in a dose-dependent manner (p = 0.02) and was significantly inhibited at concentrations of 10 and 100 microgram/ml (p < 0.05). Fluorescent microscopy showed binding of DTAF to keratocytes. We have demonstrated that prolonged exposure to DTAF inhibits proliferation of cultured keratocytes. These results suggest that DTAF-induced cytotoxicity may alter net production of collagen in the corneal stroma in animal models.

Antiproliferative effect of basic fibroblast growth factor-saporin mitotoxin on keratocytes in culture

We evaluated the effect of the conjugate of basic fibroblast growth factor (FGF2) and saporin (FGF2-SAP) on proliferation of cultured keratocytes. Cultured rabbit and human keratocytes were incubated in medium containing 0.01 to 100 nM of chemical conjugate of EGF2 conjugated by disulfide bond to saporin (CCFS1), FGF2 genetically fused to saporin (rFGF2-SAP), FGF2, or saporin for three hours or four days and cell proliferation was quantified four days after the drug treatment. Proliferation of rabbit and human keratocytes was effectively inhibited by three hour and by four day exposure to CCFS1 and rFGF2-SAP in a dose-dependent manner, whereas it was affected minimally by four day exposure to saporin. Their inhibitory effects were detected at concentrations above 0.1 or 1 nM, and were most prominent in serum-stimulated rabbit keratocytes. These results suggest a potential role for FGF2-SAP in limiting proliferation of keratocytes during corneal wound healing.