Abstract
Purpose :
Fuchs endothelial corneal dystrophy (FECD) is a progressive loss of corneal endothelial cells (CECs) that are post-mitotically arrested with limited proliferative capacity. Therefore, wound healing is mainly achieved through cell enlargement and migration. Inhibition of Rho-kinase, a key regulator of cytoskeletal reorganization has been shown to promote cellular migration. The purpose of this study was therefore to investigate the effect of a novel Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (K-115) in promoting CEC cell migration on Descemet’s membrane in FECD ex vivo.
Methods :
Normal and FECD Descemet’s membrane-CEC tissues were obtained from cadaveric corneas or patients undergoing Descemet’s membrane endothelial keratoplasty (DMEK) surgeries. The tissues were preserved in Optisol-GS and after washing in PBS were stained with Hoechst 33342 for 30 sec and attached to the plastic plates by air drying for 3 min with the endothelial cell side facing up. The tissues were treated with 1uM of K-115 drug and monitored using live cell imaging microscope (Leica DMi8) for 5 hours. Controls were treated with Chen’s media without the drug. The images from three biological and three technical replicates were collected per group, and the velocity and displacement of the cells were analyzed using the TrackMate plugin in ImageJ. Mann-Whitney and one-way Anova tests were used for statistical analysis with p<0.05 being statistically significantly different.
Results :
Mean velocity (pixels/hour±SD) of CECs without the drug was 0.45±0.11 in normal and 0.64±0.21 in FECD tissues; and increased to 0.65±0.20 (p<0.05) and 0.82±0.39 (p<0.001) with K-115, respectively. Mean displacement (pixels/hour±SD) of the cells from the normal and FECD tissues without the drug was 4.33±2.19 and 6.63±5.8, which increased to 13.49±10.32 (p<0.001) and 15.02±13.10 (p<0.001) respectively with K-115. Although the fold change in mean displacement between normal and FECD cells did not change significantly following the treatment with K-115, a significantly higher mean velocity (p<0.01) was observed in FECD compared to normal cells.
Conclusions :
FECD cells migrate faster on the Descemet’s membrane following the treatment with K-115 compared to the normal cells, which further provides a promising therapeutic approach for the treatment of FECD using ripasudil after Descemetorhexis without endothelial keratoplasty (DWEK).
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.