A ROCK Inhibitor Enhances Corneal Endothelial Wound Healing in Both an in vitro and in vivo Model

Abstract

Purpose: : Corneal endothelial cells (CECs) show poor regenerative ability in humans, and non-compensatory damage of CECs causes irreversible corneal haziness in cases of bullous keratopathy. We previously demonstrated that the Rho kinase (ROCK) inhibitor Y-27632 enhances cell proliferation in primate CECs in vitro (ARVO, 2008). The current study was conducted to demonstrate the usefulness of Y-27632 for corneal endothelial wound healing using in vitro and in vivo models.

Methods: : CECs of cynomolgus monkeys were cultured and grown to confluence in culture dishes. As an in vitro 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 Y-27632. Culture medium without Y-27632 was used for the control. The length of the wound was determined by ImageJ after a 24-hour incubation. As an in vivo model, corneal endothelium of 3 Japanese white rabbits were damaged by transcorneal freezing with a 7-mm-diameter probe. Ten mM of Y-27632 was applied topically in 1 eye of each animal 4 times daily, while PBS was applied in the other eye as a control. Corneal transparency and thickness were assessed by slit-lamp microscope and ultrasound pachymeter. The rabbits were euthanized after 48 hours of treatment, and the wound area of the corneal endothelium was evaluated by Alizarin red staining after enucleation.

Results: : The mean lengths of the wound in the monkey CECs in vitro model were significantly shorter in the Y-27632 group than in the control group after 24 hours (37.9±5.7% and 64.9±11.4% as a ratio of the initial length of the damaged area, respectively; p<0.01). In the in vivo model, slit-lamp microscopy examination revealed that the corneal transparency was higher and corneal thickness was thinner in the Y-27632 group compared to the control group. The mean wound area of the Y-27632 group was smaller than that of control group after 48 hours (2.2±2.0% and 10.5±9.6% as a ratio of the initial damaged area, respectively).

Conclusions: : We have demonstrated that ROCK inhibitor Y-27632 enhances corneal endothelial wound healing both in in vitro and in vivo models. We speculate that our results will be applicable for humans who are in a corneal endothelial deficient condition, such as Fuchs’ corneal endothelial dystrophy, post keratoplasty, or trauma.

Keywords: cornea: endothelium • wound healing • cornea: basic science