Effects of Ripasudil on Mouse Subconjunctival Immune Cell Motility Activated by Monocyte Chemoattractant Protein-1

Purpose: The postoperative wound-healing response significantly affects the outcome of glaucoma filtering surgery. Understanding this response at the cellular level is crucial. This study investigated the effects of the Rho kinase inhibitor ripasudil on immune cells activated by monocyte chemoattractant protein-1 (MCP-1), a known risk factor for glaucoma filtering surgery failure. Methods: Single-cell RNA sequencing and immunocytochemical analyses were performed to identify the type of lysozyme M (LysM)-positive cells. LysM-eGFP knock-in mice were observed using multiphoton microscopy after MCP-1 loading. Raw image data were analyzed with Imaris software to quantify the number and velocity of LysM-positive cells. In the Boyden chamber assay, THP-1 and RAW264.7 cells were loaded into the upper chambers, with MCP-1 in the lower wells acting as a chemoattractant. The effect of ripasudil was examined in each experiment. Results: LysM-positive cells were identified as macrophages in mouse subconjunctival tissue. MCP-1 significantly increased both the velocity and number of LysM-positive cells compared with the intact state. Pretreatment with ripasudil significantly reduced cell velocity from 6.48 ± 3.87 to 2.97 ± 1.59 µm/min (P < 0.0001, Welch's t-test), although the change in cell number was not significant. The Boyden chamber assay showed that MCP-1 induced THP-1 and RAW264.7 cell migration, which was almost completely inhibited by 10 µM ripasudil. Conclusions: Ripasudil attenuated MCP-1-activated macrophage behavior in both intravital imaging and in vitro experiments. These findings suggest that ripasudil may have therapeutic potential for preventing excessive wound healing after glaucoma filtering surgery.