The Effects of Rho-Associated Kinase (ROCK) Inhibitor Supplementation in Corneal Preservation Media for Donor Tissue Used in Descemet’s Membrane Endothelial Keratoplasty (DMEK)

Purpose: This study seeks to evaluate the effects of pre-treating donor corneal tissue with a Rho-associated kinase inhibitor (ROCKi), Netarsudil (AR-13324), on the viability of corneal endothelial cells (CECs) during Descemet’s membrane endothelial keratoplasty (DMEK) graft preparation and insertion using endothelium-in and endothelium-out techniques. Methods: This ex vivo comparative study utilized 24 pairs of research-grade human donor corneas preserved in Optisol-GS. One cornea from each pair was incubated in 1 µM AR-13324 for 2 h, while the contralateral cornea served as a control. DMEK tissues were prepared and simulated insertion was performed using either the endothelium-in (Endoglide) or endothelium-out (Geuder glass injector) technique. The surgeon performing the tissue preparation and insertion was masked to the ROCKi treatment allocation. Post-manipulation, tissues underwent live/dead cytotoxicity assay using Calcein-AM and Ethidium homodimer-1 staining. Percentages of live, fallout, and dead CECs were quantified via fluorescence microscopy and ImageJ (version 1.54p) analysis. Correlations between donor age, preservation duration, and cell viability were also analyzed. Results: For the endothelium-out model, live cell percentage was lower in controls compared to ROCKi-treated corneas (89.6 ± 5.35% vs. 95.1 ± 3.74%, p = 0.0001), while the percentage of dead cells was higher in controls (7.13 ± 3.71% vs. 2.16 ± 1.39%, p = 0.0017). For the endothelium-in model, live cell percentage was lower in controls compared to ROCKi-treated corneas (86.8 ± 4.81% vs. 94.4 ± 3.26%, p = 0.0001), with corresponding higher percentage fallout (8.21 ± 5.02% vs. 4.05 ± 2.82%; p = 0.0036) and higher percentage dead cells (5.04 ± 3.52% to 1.56 ± 1.13%; p = 0.0070). No significant correlations were found between donor age or preservation time and cell viability outcomes. Comparative analysis showed no significant difference in cell survivability between the two DMEK techniques with ROCKi treatment. Conclusions: Pre-incubation with the ROCKi AR-13324 significantly enhances corneal endothelial cell viability and reduces cell death during DMEK graft preparation and manipulation, regardless of the graft insertion technique used. Incorporating ROCKi treatment into tissue preparation protocols may enhance endothelial preservation, potentially expanding its clinical utility in eye banking and improving corneal graft survival.