Assessment of Corneal Graft Outcomes in a Murine Model of Endothelial Keratoplasty

Abstract

Objectives: In this study, we establish a protocol for evaluating the outcomes of endothelial keratoplasty, including graft survival, rejection, or failure. Additionally, we also evaluate the alloimmune response in graft recipients. Methods: We performed EK using C57BL/6 (allogeneic) and BALB/c (syngeneic) as donors and BALB/c mice as recipients. Slit-lamp examination and optical coherence tomography were performed for clinical evaluations for 16 weeks post-procedure. Criteria for the assessment of corneal opacity were established and the animals were graded weekly. Additionally, we assessed corneal endothelial cell density by harvesting the corneas and staining with zonula occludens-1 (ZO-1). Lastly, lymph nodes were collected, and CD4⁺ T cells were MACS-sorted and co-cultured with syngeneic or allogeneic antigen-presenting cells (APCs) to assess the IFN-γ expression levels by alloreactive Th1 cells (ELISPOT) in response to the direct (donor) or indirect (host) pathways of sensitization. Results: We observed graft failure in four animals, including irreversible corneal opacity, graft detachment, and anterior synechiae in the first four weeks. The remaining animals were graded between 0 and 5 as per the established criteria. The total and graft corneal thickness and endothelial cell density progressively worsened with a higher grade of corneal opacity. The direct allosensitization of Th1 cells was significantly higher in mice with a higher grade of corneal opacity. At 16 weeks follow-up, the grafts remained stable with low opacity scores in syngeneic EK recipients; however, the opacity scores were higher and variable in allogeneic EK recipients. Conclusions: These findings establish a standardized protocol to assess the graft outcomes in a murine model of EK. Furthermore, we delineate the underlying immunological pathway that contributes to the immune-mediated rejection of grafts in this model.

Keywords: endothelial keratoplasty; graft failure; graft rejection; murine model.

Figure 1

Algorithm for the clinical assessment of graft outcomes in a murine model of endothelial keratoplasty.

Figure 2

(A) Scoring system and (B) corresponding representative images for grading corneal opacity in a murine model of endothelial keratoplasty.

Figure 3

Representative slit-lamp examination and optical coherence tomography images of (A) irreversible opacity, (B) graft detachment, and (C) anterior synechiae, which are classified as graft failure in mice following endothelial keratoplasty.

Figure 4

(A) Optical coherence tomography and corresponding zonula occludens-1 confocal images assessing the (B) total and graft corneal thickness, and (C) corneal endothelial cell density (CEnC), respectively. (D) Linear regression showed an inverse correlation between endothelial cell density and thickness in tissues with different grades of corneal opacity. The intergroup comparison was performed using a one-way analysis of variance test (* p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001). Scale = 100 μm (for confocal imaging).

Figure 5

(A) The enzyme-linked immunospot (ELISPOT) assay showed a significant association between increased (B) direct allosensitization of Th1 cells (indicated by higher IFN-γ⁺ spots at four weeks) with a higher grade of corneal opacity in recipients following allogeneic EK. (C) A moderately higher indirect Th1 allosensitization was observed in animals with a higher grade of corneal opacity following allogeneic EK. (D) Linear regression analysis showed an inverse correlation between IFN-γ⁺ spots (direct pathway) on ELISPOT assay and CEnC density in tissues derived from mice with various grades of corneal opacity. Intergroup comparison was performed using a one-way analysis of variance test (*** p < 0.001).

Figure 6

Corneal opacity grading in (A) syngeneic and (B) allogeneic EK over 16-week post procedure. At four weeks post-procedure, recipients with syngeneic EK remained consistently low corneal opacity scores, (C) whereas the animals with allogeneic EK had higher scores which were variable during the remaining 12 weeks of follow-up. (D) The Kaplan–Meier curve shows that all the recipients with syngeneic EK showed graft survival at the end of 16 weeks, whereas the graft survival rate was 75% in allogeneic EK recipients.