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James V. Jester, Chyong Jy Nien, Donald J. Brown; Mitomycin C (MMC) Blocks Keratocyte DNA Repair Leading to Prolonged Inhibition of Myofibroblast Differentiation and Wound Repair. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2211.
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While MMC is currently used as a corneal anti-fibrotic to reduce the incidence of corneal haze following refractive surgery, the cellular mechanism and long-term safety remain unclear. The purpose of this study was to evaluate the long-term effects of MMC on rabbit corneal keratocytes in vitro and in vivo.
Keratocytes were isolated from rabbit eyes and cultured under serum-free conditions. Cells were then treated with MMC (0.02 % to 0.002%) for 10 minutes and allowed to recover. Cells were then evaluated for: 1) DNA repair by staining for H2AX, a phosphorylated nuclear histone that marks DNA intrastrand crosslinks (ICLs), and comet assay to assess DNA repair, and 2) TGFβ-induced cell proliferation following 7 day recovery from MMC treatment. The epithelium from live rabbit corneas was also removed and treated with 0.02% and 0.002% MMC applied for 15 and 60 seconds and then evaluated for, 1) H2AX staining immediately after MMC treatment, and 2) 6 week recovery followed by wound healing response to lamellar keratectomy injury as evaluated by in vivo confocal.
MMC treated keratocytes showed increased H2AX staining that peaked 2 days after treatment that was coupled with decreasing tail moment measured by comet assay, suggesting MMC induced DNA was not repaired. Recovery of keratocytes for 7 days after 0.02% and 0.002% MMC treatment failed to restore a normal TGFβ response, suggesting prolonged or permanent keratocyte damage. Staining of rabbit corneas for H2AX after MMC treatment showed nuclear keratocyte and corneal endothelial cell staining that was marked with 0.02% and greatly reduced at 0.002%, suggesting that the antifibrotic effect of 0.02% over 0.002% MMC is due to DNA damage. Corneas treated with MMC and allowed to recover for 6 weeks and then wounded showed no fibrotic response indicating that MMC has prolonged effects in vivo.
This data suggests that the anti-fibrotic effects of MMC in the cornea are related to DNA damage and the formation of ICLs that are not repaired. Furthermore, normal wound healing responses years after surgery may be significantly impaired in eyes treated with MMC for refractive surgery.
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