Cell-Injection Therapy Using Cultivated Corneal Endothelial Cells Combined With a ROCK Inhibitor in a Rabbit Model

Abstract

Purpose: : To investigate the feasibility of corneal endothelial reconstruction by a cell-injection therapy using cultivated rabbit corneal endothelial cells (RCECs) combined with Y-27632.

Methods: : The rabbit corneal endothelium was intensively scraped off up to the peripheral area to make a corneal endothelial dysfunction model. Descemet’s membrane was left intact. A 2x10⁵ amount of cultivated RCECs suspended in DMEM with 100µM Y-27632 was injected into the anterior chamber of 3 eyes of 3 animals (Y-27632(+) group). The same procedure was performed without using Y-27632 in 3 eyes of 3 animals (Y-27632(-) group). In the control group, endothelial cells were scraped and RCECs were not injected (6 eyes). The eye of each animal was then kept in the face-down position for 3 hrs. We examined the in vivo celluar attachment after 3 hrs by immunohistochemistry in 1 animal per group, and performed slit-lamp examination and corneal thickness- and intraocular pressure measurement up to 7 days in 2 animals per group followed by mmunohistochemical analysis.

Results: : In the Y-27632(+) group, DiI-labeled cultivated RCECs were attached onto the Descemet’s membrane and well formed cell-cell adhesion was observed at 3 hrs after injection; corneas became clear at 48 hrs and corneal thickness was much thinner compared to the other 2 groups which demonstrated bullous keratopathy. On day 7, the Descemet’s membrane was totally covered with DiI-labeled donor endothelial cells showing Na⁺-K⁺ ATPase expression. Few Ki67-positive cells were detected in the continuous monolayer of corneal endothelial cells. No eye showed intraocular pressure elevation.

Conclusions: : This findings of this study demonstrate that ROCK inhibitor Y-27632 enhances the attachment of cultivated RCECs in vivo. We speculate that cell-injection therapy using Y-27632 might have an advantage, as it enables cultivated corneal endothelial cell transplantation without a carrier and with a less invasive procedure.

Keywords: cornea: basic science • cornea: endothelium • transplantation