In Vivo Labeling and Tracking of Proliferating Corneal Endothelial Cells by 5-Ethynyl-2′-Deoxyuridine in Rabbits

PURPOSE: To develop a method to label proliferating corneal endothelial cells (ECs) in rabbits in vivo and track their migration over time. METHODS: We compared intraperitoneal (IP) and intracameral (IC) administration of 5-ethynyl-2′-deoxyuridine (EdU) in two experiments: (1) six rabbits received IP or IC EdU. Blood and aqueous humor (AH) samples were incubated with HL-60 cells. Flow cytometry detected the EdU incorporation, representing the bioavailability of EdU. (2) In vivo EdU labeling was investigated in pulse-chase study: 48 rabbits received EdU IP or IC. The corneas were flat-mounted after 1, 2, 5, or 40 days and imaged using fluorescence microscopy. EdU(+) and Ki67(+) ECs were quantified and their distance from the peripheral endothelial edge was measured. RESULTS: EdU was bioavailable in the AH up to 4 hours after IC injection. No EdU was detected in the blood or the AH after IP injection. High quality EdU labeling of EC was obtained only after IC injection, achieving 2047 ± 702 labeled ECs. Proliferating ECs were located exclusively in the periphery within 1458 ± 146 µm from the endothelial edge. After 40 days, 1490 ± 397 label-retaining ECs (LRCs) were detected, reaching 2219 ± 141 µm from the edge, indicating that LRCs migrated centripetally. CONCLUSIONS: IC EdU injection enables the labeling and tracking of proliferating ECs. LRCs seem to be involved in endothelial homeostasis, yet it remains to be investigated whether they represent endothelial progenitor cells. TRANSLATIONAL RELEVANCE: EdU labeling in animal models can aid the search for progenitor cells and the development of cell therapy for corneal endothelial dysfunction.