Cell-injection Therapy Using Rho Kinase Inhibitor in a Corneal Endothelial Dysfunction Rabbit Model

Abstract

Purpose: To investigate feasibility of corneal endothelial reconstruction by a cell-injection therapy using cultivated rabbit corneal endothelial cells (RCECs) in a corneal endothelial dysfunction rabbit model and to determine the optimum cell numbers for the cell-injection therapy.

Methods: Rabbit corneal endothelium was denuded by intensive mechanical scraping. Cultivated RCECs in the presence of 100μM of Rho kinase (ROCK) inhibitor, Y-27632, were injected into the anterior chamber of the host animals at three concentrations (2x10⁵ cells, 5.0x10⁵cells, or 1.0x10⁶cells). The eyes of each animal were kept in the face-down position for 3 hours. Slit-lamp examinations, corneal thickness- and intraocular pressure- measurements, and immunohistochemical analysis were performed for up to 14 days.

Results: All eyes received cell-injection therapy showed improved corneal clarity; corneal clarity and corneal thickness recovered the fastest rate in the host animals that received cultivated RCECs at 1.0x10⁶cell numbers. In all animal groups, corneal endothelium demonstrated the characteristic contact-inhibited monolayer with polygonal cells that express the functional endothelial phenotypic proteins, ZO-1 and Na+/K+-ATPase. When the endothelial cell density of the host animals was measured, the animal that received a cell injection at 1.0x10⁶ cells demonstrated significant high cell density with 3296.6±365.1 cells/mm², while the other two animal groups showed 1432.0±200.8 cells/mm² (injected cell numbers: 2.0x10⁵) and 2252.2±204.6 cells/mm² (injected cell numbers: 5.0x10⁵ cells). None of the eyes of the experimental animals showed elevated intraocular pressure or immunological rejection.

Conclusions: The findings provide evidence that the cell-injection therapy using appropriate cell numbers with ROCK inhibitor enables corneal endothelial regeneration by tissue engineering technique and may be a useful clinical alternative for corneal transplantation.

Keywords: 481 cornea: endothelium • 480 cornea: basic science • 687 regeneration