HARVARD DEPARTMENT OF OPHTHALMOLOGY
Category : Basic and Translational Research Candidate : Mohit Parekh Poster #: B18
Effect of ROCK Inhibitor on Cell Migration in Fuchs Endothelial Corneal Dystrophy
Mohit Parekh , Annie Miall , Neha Deshpande , Ula V Jurkunas
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 ).