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N. Okumura, N. Koizumi, M. Ueno, Y. Sakamoto, H. Takahashi, K. Hirata, J. Hamuro, S. Kinoshita; The Effect of a ROCK Inhibitor on Corneal Endothelial Cell Migration in an in vitro Wound-Healing Model. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2830.
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Corneal endothelial cells (CECs) show poor regenerative ability in humans, and non-compensatory damage of CECs causes irreversible corneal haziness. We previously demonstrated that the Rho-kinase (ROCK) inhibitor Y-27632 promotes cell proliferation of cultivated CECs and enhances corneal endothelial wound healing in an animal model (ARVO, 2009). Since wound healing is an integrated outcome of cell proliferation and cell migration, this current study was conducted to demonstrate the effect of Y-27632 on the cell migration of CECs by using an in vitro wound-healing model.
CECs of cynomolgus monkeys were cultured and grown to confluence in culture dishes with culture medium containing 10% fetal bovine serum (FBS). The cells were then cultured with culture medium containing 0.1% FBS for 5 days to arrest the cell cycle by serum starvation. As an in vitro wound-healing model, the cultivated CECs were scraped with a plastic pipette tip to create 6 linear defect sites in each dish. The culture medium was then replaced with fresh medium containing 10 µM of Y-27632. Culture medium without Y-27632 was used for the control. The length of the wound was determined by ImageJ software after 12 and 24 hours of incubation.
The mean lengths of the wound in the monkey CECs in the Y-27632 group were 99.9±5.8% and 96.8±7.5%, while those in the control group were 75.5±6.0% and 56.7±8.7% as a ratio of the initial length of the damaged area after 12 and 24 hours, respectively. The mean lengths of the wound were significantly wider in the Y-27632 group than in the control group at both 12 and 24 hours after incubation (p<0.01).
We have demonstrated that ROCK inhibitor Y-27632 suppresses corneal endothelial cell migration in an in vitro cell-cycle-arrested wound-healing model. We speculate that enhancement of corneal endothelial wound healing by Y-27632 is mainly caused by the promotion of cell proliferation by Y-27632 as we recently reported.
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