Central transcorneal freezing alters the proliferation pattern of endothelial cells in the corneal periphery

Abstract

Purpose : Studies on corneal endothelial cell (CEC) proliferation related to endothelial injury, can lead to a better understanding of the lack of sufficient regeneration in corneal endothelial dysfunction. We used an in vivo rabbit model of transcorneal freezing to describe CEC proliferation in response to injury and to describe the localization of slow-cycling endothelial cells.

Methods : A central endothelial lesion was induced in 5-weeks old rabbits (n=4) by transcorneal freezing, followed by intraperitoneal injection of 5-Ethynyl-2’-deoxyuridine (EdU). EdU injection was repeated at T_24hours and T_{48 hours} to label cells in s-phase during wound healing. Control group underwent the same EdU loading protocol without transcorneal freezing. Animals were euthanized after 2, 5 or 40 days and corneoscleral buttons were flat-mounted. Fluorescence ex vivo confocal microscopy (LSM 710, ZEISS, Oberkochen, Germany) was performed to assess the expression of proliferation marker Ki-67 and EdU-labeling.

Results : Central transcorneal freezing induced denudation of endothelial cells and Descemet's membrane (4 mm Ø). In the central cornea, the CECs adjacent to the denuded area showed extensive proliferation (Ki-67⁺ and EdU⁺), covering the lesion within 5 days. No central proliferation (Ki-67^- and EdU^- ) was detected 40 days after transcorneal freezing in any of the groups. In the periphery, proliferating CECs were present at all time points in both groups, yet during the first 5 days after injury the proliferating CECs were arranged more clustered, compared with the control group, where they continuously showed a scattered pattern. CECs retaining the EdU-label at day 40 were present only in the periphery in both groups.

Conclusions : Proliferation and migration of adjacent CECs seems to be the main mechanism for endothelial repopulation after injury. During central wound healing, alterations in peripheral CEC proliferation towards a clustered cell pattern, indicate central-peripheral communication and possible presence of slow-cycling progenitor cells in the periphery. These data will be usefull for many animal experiments using rabbit, and might explain differences between the rabbit model and human, in which the proliferation of CECs remains much more discreet.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.