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D. Roh, S. Rhee, A. Cook, A. Joshi, R. Kowolski, D. Dhaliwal, J. Funderburgh; A Single Application of Mitomycin-C Induces DNA Cross-Linking in Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3959.
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A single intraoperative application of the DNA cross-linking agent mitomycin-C (MMC) is common for prophylactic haze reduction during corneal refractive surgery. The extent of DNA crosslinking induced in corneal cells by such treatment is unknown. Such crosslinking could affect function of the corneal endothelium (CE) a nondividing layer vital to the transparency of the cornea. This study aims to determine the presence of MMC and MMC-induced DNA crosslinking in CE in both intact globes and in culture.
MMC concentration in CE was examined after a 2 min application of a 0.02% solution to intact goat globes. Inhibition of E. coli growth by sliding microtome sections of treated corneas was compared to that of MMC standards. DNA cross-linking was examined in cultured CE treated with MMC at varying times and concentrations and also in intact globes under conditions emulating photorefractive keratectomy: after epithelial debridement, after debridement and laser ablation, and after debridement followed by 24 hour incubation. CE cells were embedded in agarose, spread on slides, lysed, irradiated to induce dsDNA breaks, and electrophoresed under denaturing conditions. DNA from individual cells was stained and photographed. Quantitative image analysis determined cross-linked DNA with reduced electrophoretic mobility, classified as %DNA-in-Head.
After epithelial exposure to MMC the concentration in the CE determined from a standard curve reached 0.37 microg/mL. In both ocular and cultured CE, all MMC treatments generated more %DNA-in-Head when compared to untreated controls (ANOVA p<0.0001, Dunnett’s p<0.01). In intact globes, increased length of treatment at 0.02% resulted in increased DNA crosslinking. In cultured CE, MMC treatment at concentrations present in the CE also resulted in significant increases in DNA crosslinking.
A single topical MMC application results in its presence in the corneal endothelium. This exposure leads to significant DNA crosslinking in a time-dependent and concentration-dependent manner in both intact globes and in culture.
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