Abstract

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

Highlights

  • Preservation of corneal transparency is essential for vision

  • The human corneal endothelial cells (HCEC) apoptosis status was examined by activated caspase3 immunostaining

  • Corneal grafts are retrieved and evaluated by eye bank before to be use in clinics and the main criteria for clinical eligibility of cornea is the endothelial cell density (ECD). It is representative of the functional capacity of the endothelium and a minimal density of 2000 cells/mm2 is required for corneal transplantation

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Summary

Introduction

Preservation of corneal transparency is essential for vision. This requires integrity of the three layers of the cornea, the stratified squamous epithelium, the stroma and the inner surface endothelium. A recent study demonstrated that a few proliferating cells were found exclusively in extreme periphery of endothelium on human corneas with a short postmortem time and that a very slow and continuous centripetal cell migration might exist to partially compensate the physiological cell loss in vivo [5]. This mechanism cannot immediately compensate neither acute nor chronic important CEC losses which are replaced by enlargement and migration of neighboring cells resulting in shape modification and increase of cell size [6,7]. There is a worldwide shortage of donor corneas available for transplantation

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