Assessment of graft rejection and failure in a murine model of corneal endothelial keratoplasty

Abstract

Purpose : Endothelial keratoplasty (EK) is a widely adopted transplantation procedure performed for corneal endothelial disorders with high (~90%) survival rate. However, the mechanisms causing graft rejection post-EK are yet to be elucidated. Herein, we define the thresholds for classification of graft rejection and failure post-EK in a murine model of EK.

Methods : EK was performed using C57BL/6 as donors and BALB/c mice as recipients. The central endothelium and Descemet’s membrane were removed from the recipient cornea and replaced by a 1.5-mm graft (including the posterior stroma and Descemet's membrane) from the donor. Mice were followed and evaluated using slit lamp biomicroscope and AS-OCT for 16 weeks to evaluate opacity (0-5) and central corneal thickness (CCT). Subsequently, the grafted corneas were harvested, and endothelial cell density (anti-ZO-1 staining) was assessed by confocal microscopy. Lymph nodes were collected, and CD4⁺ T cells were MACS-sorted and co-cultured with syngeneic or allogeneic antigen-presenting cells (APC) to assess the IFN-γ expression levels by alloreactive Th1 cells (ELISPOT) in response to the direct (donor APC) or indirect (host APC) pathways of sensitization.

Results : Animals with irreversible opacity, graft detachment, or anterior synechia at 2-weeks post-transplantation were considered graft failure and excluded. The remaining animals (N=4) were divided into two groups (group 1: 0-1 and group 2: 2-5) based on corneal opacity scores at 4 weeks post-EK. CCT was significantly higher for grafts in group 2 compared to group 1 (p=0.0095). The frequencies of IFN-γ⁺ T-cells were significantly higher in group 2 grafted corneas compared to group 1. Additionally, the total IFN-γ expression were higher in DLNs of mice with group 2 compared to group 1 (p=0.0012). The immune response post-EK was predominantly attributed to donor APC sensitization and partly to recipient APC sensitization (direct pathway: p=0.0056, indirect pathway: p=0.0092). The endothelial cell density was significantly higher in the corneas with low compared to high grade opacity (p<0.0001).

Conclusions : These results demonstrate that immune-mediated allograft rejection in the host post-EK is initiated primarily by donor-derived APCs. We observed distinct differences in the post-procedure morphology of the corneas with low- versus high-grade opacity.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.