A Standardized Protocol for Murine Endothelial Keratoplasty: From Donor Preparation to Outcome Assessment

While the first murine endothelial keratoplasty (EK) model established surgical feasibility, manual lamellar dissection and endothelial scraping introduce significant procedural variability. This study aims to develop a standardized murine EK model implementing a controlled stromal peeling technique and quadrant-based host bed preparation to minimize inconsistency.

We introduced 2 critical methodological refinements. First, donor corneas were prepared using a controlled stromal peeling approach to ensure uniform graft thickness. Second, traditional circular scraping of the recipient endothelium was replaced with quadrant-based mechanical removal to standardize the denuded area. Postoperative outcomes were quantified using AS-OCT and a novel time-resolved opacity scoring system designed to distinguish early technical failure from immunologic rejection.

Controlled stromal peeling produced posterior lamellar grafts with significantly higher thickness consistency compared with traditional needle dissection. The quadrant-based removal resulted in highly reproducible postoperative corneal edema, reflecting standardized disruption of host endothelial function. Furthermore, the time-resolved grading system allowed more granular, objective differentiation between early graft failure and delayed rejection compared with traditional binary survival assessments.

These refinements overcome the inherent variability of the initial murine EK model. By standardizing both the surgical procedure and the interpretive framework, this optimized model provides a robust, reproducible platform for investigating cellular mechanisms of graft outcomes in experimental keratoplasty.