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J. V. Jester, D. Brown, M. Farid, R. F. Steinert, A. Wahlert, D. W. Kim, L. Lam, C. Nien Shy; Dose Effects of Mitomycin C (MMC) on Corneal Scarring and TGFβ Induced Myofibroblast Differentiation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2937. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
MMC treatment of photorefractive keratectomy (PRK) wounds reduces corneal scarring and haze through unknown mechanisms. The purpose of this study was to determine the concentration effects of MMC on the development of corneal haze, scarring, and the ability of keratocytes to differentiate to myofibroblasts.
Immediately following 9 diopter PRK, rabbits were treated with varying concentrations of MMC from 0.02% to 0.00002% for 10 seconds. Rabbits were then evaluated for epithelial regeneration by fluorescein staining, development of corneal haze by in vivo confocal microscopy, and stromal scarring by ex vivo 2-photon second harmonic generation (SHG). Ability of rabbit keratocytes to differentiate to corneal myofibroblasts was evaluated by treating cultured keratocytes previously exposed to MMC with 5 ng/ml of TGFβ. Keratocyte responses were then evaluated at various times by immunostaining, western blotting and RT-PCR to measure cell proliferation, cycle entry, DNA repair, expression of alpha smooth muscle actin (α-SM) and macrophage chemotactic protein 1 (MCP-1).
No effect of MMC was detected on corneal epithelial regeneration. MMC at 0.02% significantly reduced (p<0.05) peak corneal haze and showed no significant difference to baseline levels by 8 weeks after injury. MMC at 0.002% showed significantly greater haze compared to 0.02%, but returned to baseline levels by 12 weeks. Doses of MMC below 0.002% were not significantly different from vehicle treated eyes. At 12 weeks after injury, eyes treated with 0.02% MMC showed a significant decrease in corneal scarring (p < 0.05) as measured by SHG compared to other groups. Treatment of keratocytes with 0.02% MMC in culture resulted in keratocyte cell death, while lower concentrations either blocked cell proliferation in response to TGFβ or delayed cell division. Interestingly, 0.002% MMC and below significantly increased cell cycle entry in association with nuclear staining for H2AX, a recombination protein associated with DNA repair. Finally, while MMC treatment greatly enhanced TGFβ stimulated α-SM mRNA, intermediate doses blocked myofibroblast differentiation and down-regulated MCP-1 mRNA expression.
Keratocytes exposed to doses of MMC that do not inhibit corneal scarring and haze show inhibition of TGFβ induced myofibroblast differentiation, increased cell cycle entry associated with DNA repair and decreased MCP-1 expression. These findings suggest that MMC effects are not solely related to keratocyte apoptosis or inhibition of cell proliferation.
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