Yugo Okazaki

Effect of the Rho Kinase Inhibitor Y-27632 on Corneal Endothelial Wound Healing.

PURPOSE The purpose of this study was to investigate the feasibility of using Rho-associated kinase (ROCK) inhibitor eye drops for treating severe corneal endothelial damage due to surgical invasion. METHODS A rabbit corneal endothelial damage model was created by mechanically scraping half the area of the corneal endothelium of eighteen eyes of Japanese white rabbits. A selective ROCK inhibitor, Y-27632 (10 mM), was applied topically for 2 weeks, and then the anterior segment was evaluated by slitlamp microscopy. The corneal endothelium was evaluated by phalloidin staining and immunohistochemical analysis. We then conducted pilot clinical research and applied Y-27632 eye drops topically to three patients who exhibited severe corneal edema due to corneal endothelial damage. RESULTS In the corneal endothelial damage rabbit model, more Ki67-positive cells were detected in Y-27632-treated eyes than in control eyes. Five of six corneas became transparent in Y-27632-treated eyes, whereas zero of six corneas became transparent in the control eyes (P < 0.01). Actin fibers were distributed at the cell cortex in the eyes treated with Y-27632, whereas actin distribution was partially disrupted, and stress fibers were observed in control eyes. N-cadherin and Na+/K+-ATPase were expressed in almost all cells in Y-27632-treated eyes, but expression decreased in control eyes. Preliminary human cases confirmed that ROCK inhibitor eye drops were considerably effective for treatment of corneal edema associated with cataract surgery. CONCLUSIONS ROCK inhibitor may be developed as an eye drop for treating acute corneal endothelial damage to prevent progression of bullous keratopathy. (University Hospital Medical Information Network Clinical Trial Registry no. UMIN000003625; www.umin.ac.jp/ctr).

Density-gradient centrifugation enables the purification of cultured corneal endothelial cells for cell therapy by eliminating senescent cells

The corneal endothelium is essential for maintaining corneal transparency; therefore, corneal endothelial dysfunction causes serious vision loss. Tissue engineering-based therapy is potentially a less invasive and more effective therapeutic modality. We recently started a first-in-man clinical trial of cell-based therapy for treating corneal endothelial dysfunction in Japan. However, the senescence of corneal endothelial cells (CECs) during the serial passage culture needed to obtain massive quantities of cells for clinical use is a serious technical obstacle preventing the push of this regenerative therapy to clinical settings. Here, we show evidence from an animal model confirming that senescent cells are less effective in cell therapy. In addition, we propose that density-gradient centrifugation can eliminate the senescent cells and purify high potency CECs for clinical use. This simple technique might be applicable for other types of cells in the settings of regenerative medicine.

Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) Transiently Alters the Morphology of Corneal Endothelial Cells

PURPOSE Ripasudil (Glanatec), a selective Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, was approved in Japan in September 2014 for the treatment of glaucoma and ocular hypertension. The purpose of this study was to investigate the effect of ripasudil eye drops on corneal endothelial morphology, as ROCK signaling is known to modulate the actin cytoskeleton. METHODS Morphological changes in the corneal endothelium were evaluated in human subjects by specular and slit-lamp microscopy, following topical administration of ripasudil. We also used a rabbit model to evaluate the effect of ripasudil on clinical parameters of the corneal endothelium. Twenty-four hours after ripasudil application, corneal specimens were evaluated by phalloidin staining, immunohistochemical analysis, and electron microscopy. RESULTS Specular microscopy revealed morphological changes in human eyes, and slit-lamp microscopy showed guttae-like findings. The rabbit model showed morphological changes similar to those seen in human eyes after ripasudil administration. Electron microscopy demonstrated that these alterations are due to the formation of protrusions along the cell-cell borders, but this formation is transient. Expression of corneal endothelial function-related markers was not disrupted; corneal thickness and corneal volume were not changed; and no cell death was observed following ripasudil administration. CONCLUSIONS Ripasudil induces transient guttae-like findings in humans, most likely due to protrusion formation along intracellular borders caused by the reduction in actomyosin contractility of the corneal endothelial cells. No severe adverse effects were observed. Physicians should be aware that ROCK inhibitors can cause these guttae-like findings, to avoid misdiagnosing patients as having Fuchs endothelial corneal dystrophy. (www.umin.ac.jp/ctr number, UMIN000018340.).

Immune Cells on the Corneal Endothelium of an Allogeneic Corneal Transplantation Rabbit Model

Purpose Corneal endothelial cell density undergoes a progressive decrease for many years after transplantation, eventually threatening patients with late endothelial failure. The purpose of this study was to investigate the possibility of an immunologic response in successfully grafted corneal endothelium. Methods The corneal endothelium of patients who had undergone corneal transplantation was evaluated by specular microscopy. Rabbit models were subjected to penetrating keratoplasty (PK) with either syngeneic or allogeneic corneal transplants and Descemets stripping endothelial keratoplasty (DSEK) with allogeneic corneal transplants. The presence of immune cells and expression of proinflammatory cytokines were determined by immunostaining. The corneal endothelium and immune cells were also evaluated by scanning electron microscopy. Results Scanning slit contact specular microscopy of patients with no features of graft rejection revealed cell-like white dots on the grafted corneal endothelium. The corneal endothelium of the allogeneic PK and DSEK rabbit models displayed the presence of immune cells, including CD4+ T-helper cells, CD8+ cytotoxic T cells, CD20+ B lymphocytes, CD68+ macrophages, and neutrophils, but these immune cells were rarely observed in the syngeneic PK model. These immune cells also produced proinflammatory cytokines. Notably, some of the corneal endothelial cells situated near these immune cells exhibited features of apoptosis. Conclusions T lymphocytes, B lymphocytes, macrophages, and neutrophils are present on the grafted corneal endothelium in both PK and DSEK allogeneic rabbit models. The potential involvement of immune cells as an underlying pathophysiology for late endothelial failure deserves further examination.