The Effects Of Rho-Associated Kinase (Rock) Inhibitor Supplementation In Corneal Preservation Media For Donor Tissue Used In Descemet’S Membrane Endothelial Keratoplasty (Dmek)
Published 2026 - 44th Congress of the ESCRS
Reference: FP14.13 | Type: Free paper | DOI: 10.82333/wsam-n144
Authors: Dr Hon Shing Ong* 1 , Dr Deepika Malhotra 2 , Ms Dawn Neo 3 , Ms Xiao Yu Ng 3 , Dr Gary Peh 3 , Prof. Jodhbir Mehta 1
1Corneal and Refractive Surgery Department,Singapore National Eye Centre,Singapore,Singapore;Regenerative Therapy Research Group,Singapore Eye Research Institute,Singapore,Singapore;Singhealth Duke-NUS Ophthalmology & Visual Sciences Academic Clinical Programme,Duke-NUS Graduate Medical School,Singapore,Singapore, 2Corneal and Refractive Surgery Department,Singapore National Eye Centre,Singapore,Singapore, 3Regenerative Therapy Research Group,Singapore Eye Research Institute,Singapore,Singapore
Purpose
To evaluate the effects of pre-treating donor corneal tissue with a Rho-associated kinase inhibitor (ROCKi), Netarsudil (AR-13324), on corneal endothelial cell (CEC) viability during Descemet’s membrane endothelial keratoplasty (DMEK) graft preparation and insertion, using endothelium-in and endothelium-out techniques.
Setting
Ex-vivo comparative study of human donor corneas.
Methods
This study utilized 22 pairs of research grade human donor corneas preserved in Optisol-GS. One cornea from each pair was incubated in 10 µM AR-13324 (ROCKi) for 2 hours, while the contralateral cornea served as the control. DMEK tissues were prepared and pulled-through using either endothelium-in (Endoglide) or endothelium-out (Geuder glass injector) technique. 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 analysis. Correlations between donor age, preservation duration, and cell viability were also analyzed.
Results
For the endothelium-out model, live cell percentage increased from 89.60 ± 5.35% in controls to 95.14 ± 3.74% in ROCKi treated corneas (p = 0.0001), while percentage dead cells decreased from 7.13 ± 3.71% to 2.16 ± 1.39% (p = 0.0017). For the endothelium-in model, live cell percentage increased from 86.75 ± 4.81% to 94.39 ± 3.26% (p = 0.0001), with corresponding decrease in percentage fallout (8.21 ± 5.02% to 4.05 ± 2.82%; p = 0.0036) and 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 survivability between the two DMEK techniques after ROCKi treatment.
Conclusions
Pre-incubation with ROCKi 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 during tissue preparation may help improve endothelial preservation, particularly in preloaded DMEK grafts, thereby supporting broader clinical application and improved graft survival outcomes.