March 2016
Volume 57, Issue 3
Open Access
Cornea  |   March 2016
Effect of the Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) on Corneal Endothelial Wound Healing
Author Affiliations & Notes
  • Naoki Okumura
    Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Yugo Okazaki
    Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Ryota Inoue
    Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Kazuya Kakutani
    Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Shinichiro Nakano
    Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Shigeru Kinoshita
    Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Noriko Koizumi
    Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Correspondence: Noriko Koizumi, Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe 610-0321, Japan; nkoizumi@mail.doshisha.ac.jp
Investigative Ophthalmology & Visual Science March 2016, Vol.57, 1284-1292. doi:10.1167/iovs.15-18586
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Naoki Okumura, Yugo Okazaki, Ryota Inoue, Kazuya Kakutani, Shinichiro Nakano, Shigeru Kinoshita, Noriko Koizumi; Effect of the Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) on Corneal Endothelial Wound Healing. Invest. Ophthalmol. Vis. Sci. 2016;57(3):1284-1292. doi: 10.1167/iovs.15-18586.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: Ripasudil (Glanatec), a selective rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, was approved as a glaucoma and ocular hypertension treatment in Japan in 2014. The purpose of this study was to investigate the feasibility of using ripasudil eye drops to treat corneal endothelial injuries.

Methods: Cultured human corneal endothelial cells (HCECs) were treated with ripasudil, and 5-bromo-2′-deoxyuridine (BrdU) incorporation was evaluated by ELISA. A rabbit corneal endothelial damage model was also created by mechanically scraping the corneal endothelium, followed by topical ripasudil eye drop application for 2 weeks. The anterior segment was evaluated by slit-lamp microscopy, and central corneal thickness was measured by ultrasound pachymetry. Corneal specimens were evaluated by phalloidin staining and immunohistochemical analysis using antibodies against Ki67, N-cadherin, and Na+/K+-ATPase.

Results: Many more BrdU-positive cells were observed among the HCECs treated with ripasudil (0.3–30 μM) than among the control HCECs. Ripasudil-treated eyes in a rabbit model showed 91.5 ± 2.0% Ki67-positive cells after 48 hours, whereas control eyes showed 52.6 ± 1.3%. Five of six corneas became transparent in ripasudil-treated eyes, whereas zero of six corneas became transparent in the control eyes. Regenerated cell densities were higher in the eyes treated with ripasudil than in eyes treated with vehicle. Eyes treated with ripasudil expressed N-cadherin and Na+/K+-ATPase in almost all CECs, whereas this expression was decreased in control eyes.

Conclusions: Ripasudil promoted corneal endothelial wound healing, supporting its development as eye drops for treating acute corneal endothelial damage due to eye surgeries, especially cataract surgery.

The corneal endothelium regulates corneal transparency by balancing corneal hydration through pump and barrier functions. Hexagonal corneal endothelial cells (CECs) form a monolayer sheet at a cell density of approximately 2500 cells/mm2 in late adulthood, but corneal endothelial damage—due to Fuchs' endothelial corneal dystrophy, cataract surgery, and corneal trauma—induces compensatory migration and spreading, which results in a cell density drop. When corneal endothelial cell density reaches a critical level, typically 500 cells/mm2, the corneal endothelial function is not compensated, and the cornea then exhibits haziness, together with edema. The only therapy is corneal transplantation, as no pharmaceutical intervention has yet been introduced into clinical settings.1 
In 2009, we reported that the rho-associated kinase (ROCK) inhibitor, Y-27632, promoted in vitro cell proliferation of cultured CECs.2 Subsequently, we demonstrated that topical application of Y-27632 in the form of eye drops enhances in vivo corneal endothelial wound healing in rabbit and monkey models.3,4 Following the animal experiments, we performed clinical research on corneal endothelial dysfunction patients who were treated with Y-27632 eye drops following a 2-mm-diameter transcorneal freezing procedure. Rho-associated kinase inhibitor eye drops showed effectiveness in reducing the central corneal thickness in patients with early stage Fuchs' endothelial corneal dystrophy (these patients still had residual healthy corneal endothelium at the peripheral cornea), whereas the ROCK inhibitor did not reduce central corneal thickness in patients with an advanced stage of corneal endothelial dysfunction with diffuse edema.4,5 These results from our clinical research suggest that ROCK inhibitor eye drops enhance corneal endothelial wound healing by promoting residual CEC proliferation, as long as some relatively healthy CECs remain. However, despite accumulating evidence, no commercially available ROCK inhibitor has been developed for the treatment of corneal endothelial injury. 
In the field of ophthalmology, ROCK inhibitors have attracted much interest among investigators as potent drugs for glaucoma treatment, because ROCK inhibitors reduce the intraocular pressure by increasing the outflow facility of the aqueous humor.610 Ripasudil hydrochloride hydrate, a selective ROCK inhibitor, has been developed as a glaucoma drug and has been shown in phase 1 clinical trials to reduce IOP in healthy adult volunteers.11 Randomized clinical studies have demonstrated that 0.4% ripasudil eye drops reduced intraocular pressure in patients with primary open-angle glaucoma and ocular hypertension.12,13 Subsequently, in 2014, a commercial preparation of ripasudil hydrochloride hydrate eye drops (Glanatec ophthalmic solution 0.4%, Kowa Company Ltd., Nagoya, Japan) was approved in Japan for the treatment of glaucoma and ocular hypertension.14 The 50% inhibitory concentration (IC50) of ripasudil is 0.051 μM for ROCK 1 and 0.019 μM for ROCK 2, whereas the IC50 for PKACα, PKC, and CaMKIIα are 2.1, 27, and 0.37 μM, respectively.15 This high selectivity for ROCK is considered to contribute to the safety of ripasudil, which shows minimal off-target effects through inhibition or activation of other signaling pathways. 
In the present study, we tested the feasibility of repositioning ripasudil as a treatment for corneal endothelial injuries. Drug repositioning (i.e., the repurposing of an existing drug for new indications1618) is one possible strategy for drug development that could have a faster approval time and lower investment cost than traditional drug discovery. Here we showed that ripasudil treatment promotes in vitro CEC proliferation in cultured human CECs (HCECs) in a similar fashion to that shown by the conventional ROCK inhibitors Y-27632 and fasudil. We also demonstrated that ripasudil eye drops promoted wound healing and facilitated regeneration of the corneal endothelium in a rabbit wound model. 
Methods
Animal Experiment Approval
In all experiments, animals were housed and treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rabbit experiments were performed at Doshisha University (Kyoto, Japan) according to the protocol approved by the University's Animal Care and Use Committee (Approval A15012). Human donor corneas were obtained from SightLife (Seattle, WA, USA) for research purposes. 
Cell Culture
A total of four human donor corneas (from persons >40 years of age) were used for cultivation of HCECs by the protocol described previously.19 Briefly, Descemet's membranes containing the HCECs were stripped from donor corneas, and the membranes were digested with 1 mg/mL collagenase A (Roche Applied Science, Penzberg, Germany) at 37°C for 12 hours. The HCECs were seeded in 1 well of a 48-well plate coated with laminin E8 fragments (iMatrix-511; Nippi, Inc., Tokyo, Japan) (0.5 μg/cm2). The culture medium was prepared according to published protocols. 
First, bone marrow mesenchymal stem cells (BM-MSCs) were cultured according to previously reported protocols.20 Briefly, basal medium for HCECs was prepared (OptiMEM-I [Life Technologies Corp., Carlsbad, CA, USA] containing 8% fetal bovine serum, 5 ng/mL epidermal growth factor [Sigma-Aldrich Corp., St. Louis, MO, USA], 20 μg/mL ascorbic acid [Sigma-Aldrich Corp.], 200 mg/L calcium chloride, 0.08% chondroitin sulfate [Wako Pure Chemical Industries, Ltd., Osaka, Japan], 50 μg/mL gentamicin, and 10 μM SB431542 [Merck Millipore, Billerica, MA, USA]) and conditioned by culturing BM-MSCs for 24 hours. The basal medium conditioned with BM-MSCs was then collected for use as the culture medium for HCECs. The HCECs were cultured with Y-27632 (Wako Pure Chemical Industries, Ltd.), fasudil (Wako Pure Chemical Industries, Ltd.), or ripasudil (Kowa Company, Ltd., Nagoya, Japan) to evaluate the effect of each ROCK inhibitor on HCEC proliferation. 
Immunohistochemistry
Cultured HCECs or rabbit corneal specimens were fixed in 4% formaldehyde and incubated in 1% BSA to block nonspecific binding. The samples were investigated by conducting immunohistochemical analyses of Ki67 (Sigma-Aldrich Corp.), N-cadherin (1:300; BD Biosciences, San Jose, CA, USA), and Na+/K+-ATPase (1:300; Upstate Biotechnology, Lake Placid, NY, USA) antibodies. Alexa Fluor 488–conjugated goat anti-mouse (Life Technologies Corp.) was used as a secondary antibody at a 1:1000 dilution. Cell morphology was evaluated after actin staining with a 1:400 dilution of Alexa Fluor 594–conjugated phalloidin (Life Technologies Corp.). Proliferative cells were evaluated by 5-ethynyl-2 Click-iT 5-ethynyl-2′-deoxyuridine (EdU) imaging kits (Life Technologies Corp.) according to the manufacturer's instructions. Briefly, the HCECs (1 × 104 cells per well) were cultured in a 96-well plate and incubated with 10 μM EdU for 6 hours at 37°C. Following fixation with 4% paraformaldehyde and permeabilization with 0.3% Triton X-100 (Nacalai Tesque, Kyoto, Japan), the HCECs were incubated with a reaction cocktail. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). The specimens were viewed with a fluorescence microscope (TCS SP2 AOBS; Leica Microsystems, Wetzlar, Germany). 
Cell Proliferation Assay
Human CECs were cultured at a density of 5000 cells per well in a 96-well plate. Cell proliferation was determined by evaluating incorporation of 5-bromo-2′-deoxyuridine (BrdU) into DNA by use of the Cell Proliferation Biotrak ELISA system, version 2 (GE Healthcare Life Sciences, Buckinghamshire, United Kingdom) according to the manufacturer's instructions. Briefly, HCECs were incubated with 10 μM BrdU for 24 hours. Cultured cells were incubated with fixation solution and incubated with 100 μL monoclonal antibody against BrdU for 30 minutes. The BrdU absorbance was measured by a spectrophotometric microplate reader. 
Rabbit Corneal Endothelial Damage Model by Corneal Freezing
As an in vivo wound model, the corneal endothelium of 48 eyes of 24 Japanese white rabbits was damaged in a modified protocol, as described previously.3,21,22 Briefly, a stainless steel 7-mm-diameter probe was immersed in liquid nitrogen for 3 minutes, and the center of the rabbit cornea was cryofrozen with the probe for 15 seconds under general anesthesia. This procedure was carefully confirmed not to induce complete blindness or any severe general adverse effect. One 0.4% ripasudil eye drop (four times daily) or 0.8% ripasudil eye drop (two times daily) was topically instilled (50 μL) in one eye of each of six rabbits, whereas vehicle was instilled in the fellow eye of each rabbit as a control (n = 6). Ripasudil eye drops and vehicles were provided by the Kowa Company. After 48 hours of treatment, the rabbits were euthanized, and the corneal endothelium wound area was evaluated by Alizarin red staining and examined with a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). The Ki67-positive cells located at the leading edge (3.5 mm distant from the center of the cornea), middle area (4.5 mm distant from the center of the cornea), and periphery (5.5 mm distant from the center of the cornea) were also evaluated in the same specimens. These experiments were performed in duplicate. 
Rabbit Corneal Endothelial Damage Model by Mechanical Scraping
A rabbit corneal endothelial damage model was created to mimic surgical trauma. Half the area of the corneal endothelium of 24 eyes of 12 Japanese white rabbits was mechanically scraped from the Descemet's membrane with a 20-gauge silicone needle (Soft Tapered Needle; Inami & Co., Ltd., Tokyo, Japan) while the animals were under general anesthesia. The scraped area was confirmed as damaged by 0.04% trypan blue staining. The experimenter who created the endothelial damage model was blinded to subsequent treatment with ripasudil or vehicle. Before this procedure, the lenses were removed with an Alcon Series 20000 Legacy Surgical System (Alcon, Inc., Fort Worth, TX, USA) to deepen the anterior chamber. One eye of each rabbit was used for 0.4% ripasudil treatment and the contralateral eye served as the control. The corneal endothelium of both eyes was used in the experiment to reduce the number of rabbits required for the study and to provide a correct evaluation of the effect, because wound healing varied with the individual rabbits. This procedure was confirmed not to induce complete blindness or any severe general adverse effects. 
Rabbit Corneal Assessment
Anterior segments were evaluated by slit-lamp microscopy for 2 weeks. Scheimpflug images and a corneal thickness map were obtained with a Pentacam instrument (OCULUS; Optikgeräte GmbH, Wetzlar, Germany). Corneal endothelium was evaluated by contact specular microscopy (Konan scanning slit specular microscope; Konan Medical, Nishinomiya, Japan). Corneal thickness was determined with an ultrasound pachymeter (SP-2000; Tomey, Nagoya, Japan), and the mean of 10 measured values was then calculated (up to a maximum thickness of 1200 μm, the instrument's maximum reading). 
Statistical Analysis
The statistical significance (P value) of differences in the mean values of the two-sample comparison was determined with the Student's t-test. Values shown on the graphs represent the mean ± SEM. 
Results
Effect of Ripasudil on HCECs Proliferation In Vitro
Cultured HCECs were seeded on a culture plate and cultured for 24 hours, and then HCECs were treated with the following ROCK inhibitors for 48 hours: Y-27632 (10 μM), fasudil (10 μM), and ripasudil (0.3–100 μM). Representative phase contrast images showed that higher numbers of cells were observed when cells were treated with ROCK inhibitors (Fig. 1A). The proliferative potential was assessed by BrdU incorporation into the newly synthesized DNA. An ELISA showed that concentrations of ripasudil from 0.3 to 30 μM significantly enhanced BrdU incorporation (Fig. 1B). The numbers of cells staining for the proliferative EdU and Ki67 markers were greater for the ripasudil (10 μM) treatment than for the control treatment (Figs. 1C, 1D). 
Figure 1
 
Effect of ripasudil on in vitro corneal endothelial cell proliferation. (A) Cultured HCECs were seeded onto the culture plate and incubated for 24 hours, followed by treatment with the ROCK inhibitors Y-27632 (10 μM), fasudil (10 μM), and ripasudil (0.3–100 μM) for 48 hours. Representative phase contrast images are shown. Scale bar: 50 μm. (B) The HCECs were cultured at the density of 5000 cells per well in a 96-well plate for 24 hours, and DNA synthesis was detected by ELISA as incorporation of BrdU into DNA. BrdU incorporation was significantly enhanced at the ripasudil concentrations from 0.3 to 30 μM. **P < 0.01. (C, D) The effect of ripasudil on proliferation of corneal endothelium was evaluated by EdU and Ki67 staining after 48 hours of treatment. The numbers of EdU- and Ki67-positive cells were significantly higher following treatment of HCECs with ripasudil. **P < 0.01.
Figure 1
 
Effect of ripasudil on in vitro corneal endothelial cell proliferation. (A) Cultured HCECs were seeded onto the culture plate and incubated for 24 hours, followed by treatment with the ROCK inhibitors Y-27632 (10 μM), fasudil (10 μM), and ripasudil (0.3–100 μM) for 48 hours. Representative phase contrast images are shown. Scale bar: 50 μm. (B) The HCECs were cultured at the density of 5000 cells per well in a 96-well plate for 24 hours, and DNA synthesis was detected by ELISA as incorporation of BrdU into DNA. BrdU incorporation was significantly enhanced at the ripasudil concentrations from 0.3 to 30 μM. **P < 0.01. (C, D) The effect of ripasudil on proliferation of corneal endothelium was evaluated by EdU and Ki67 staining after 48 hours of treatment. The numbers of EdU- and Ki67-positive cells were significantly higher following treatment of HCECs with ripasudil. **P < 0.01.
Effect of Ripasudil Eye Drops on Corneal Endothelial Proliferation in a Transcorneal Freezing Rabbit Model
We tested the effect of 0.4% ripasudil eye drop (four times daily) and 0.8% ripasudil (two times daily) in the rabbit partial corneal endothelial wound model, because the topical and systemic safety of doses lower than these was already confirmed in commercially available ripasudil eye drops (Glanatec ophthalmic solution 0.4%; Kowa Company, Ltd.) in clinical trials.11 Slit-lamp microscopy showed that control rabbits with 7-mm-diameter corneal endothelial wounds created by transcorneal freezing exhibited hazy corneas after 48 hours. On the other hand, rabbits treated with 0.4% or 0.8% ripasudil eye drops exhibited less haze in their corneas, and no other adverse effects, such as delay of corneal epithelial wound healing, severe conjunctival injection, and corneal opacity, were observed (Fig. 2A). Coincidently, representative Alizarin red staining images showed that the wound area tended to be smaller in eyes treated with 0.4% or 0.8% ripasudil eye drops than in control eyes (Fig. 2B). The Ki67 staining demonstrated that 0.4% or 0.8% ripasudil eye drops promoted cell proliferation throughout the wound edge to the peripheral area of the residual corneal endothelium (Fig. 2C). The percentage of Ki67-positive cells in the leading edge was 72.8 ± 4.3% in the control, but it was 84.4 ± 2.5% and 90.5 ± 1.7% in the eyes treated with 0.4% and 0.8% ripasudil, respectively. In the peripheral area, Ki67-positive cells accounted for 33.5 ± 3.7% of the cells in the control and 40.4 ± 2.7% and 47.1 ± 3.1% of the cells in the eyes treated with 0.4% and 0.8% ripasudil, respectively (Fig. 2D). 
Figure 2
 
Effect of ripasudil eye drops on corneal endothelial proliferation in a rabbit corneal freezing model. (A) A stainless-steel 7-mm-diameter probe was immersed in liquid nitrogen for 3 minutes and then placed onto the rabbit cornea for 15 seconds with the animal under general anesthesia. Then, one 0.4% ripasudil (four times daily) or 0.8% ripasudil (two times daily) eye drop was topically instilled, whereas vehicle was instilled in the fellow eye of each rabbit as a control (n = 6). Slit-lamp microscopy showed that control eyes exhibited hazy corneas after 48 hours, but eyes treated with 0.4% or 0.8% ripasudil eye drops exhibit less hazy corneas. No other adverse effects, such as the delay of corneal epithelial wound healing, severe conjunctival injection, and corneal opacity were observed. (B) The wound area of the corneal endothelium was evaluated by Alizarin red staining after 48 hours of treatment. Alizarin red staining images showed that the wound area tended to be smaller in eyes treated with 0.4% or 0.8% ripasudil eye drops than in control eyes. Scale bar: 1 mm. (C, D) Ki67+ cells located at the leading edge (3.5 mm distant from the center of the cornea), middle area (4.5 mm distant from the center of the cornea), and periphery (5.5 mm distant from the center of the cornea) were evaluated. Ki67 staining confirmed that 0.4% or 0.8% ripasudil eye drops promoted cell proliferation throughout the wound edge to the peripheral area. Administration of 0.8% ripasudil (two times per day) enhanced Ki67 expression to a higher level than was observed with 0.4% ripasudil eye drops (four times daily). Morphology was evaluated using actin staining performed with Alexa Fluor 594–conjugated phalloidin. Nuclei were stained with DAPI. Representative images of pupil centers are shown. Scale bar: 50 μm. These experiments were performed in duplicate.
Figure 2
 
Effect of ripasudil eye drops on corneal endothelial proliferation in a rabbit corneal freezing model. (A) A stainless-steel 7-mm-diameter probe was immersed in liquid nitrogen for 3 minutes and then placed onto the rabbit cornea for 15 seconds with the animal under general anesthesia. Then, one 0.4% ripasudil (four times daily) or 0.8% ripasudil (two times daily) eye drop was topically instilled, whereas vehicle was instilled in the fellow eye of each rabbit as a control (n = 6). Slit-lamp microscopy showed that control eyes exhibited hazy corneas after 48 hours, but eyes treated with 0.4% or 0.8% ripasudil eye drops exhibit less hazy corneas. No other adverse effects, such as the delay of corneal epithelial wound healing, severe conjunctival injection, and corneal opacity were observed. (B) The wound area of the corneal endothelium was evaluated by Alizarin red staining after 48 hours of treatment. Alizarin red staining images showed that the wound area tended to be smaller in eyes treated with 0.4% or 0.8% ripasudil eye drops than in control eyes. Scale bar: 1 mm. (C, D) Ki67+ cells located at the leading edge (3.5 mm distant from the center of the cornea), middle area (4.5 mm distant from the center of the cornea), and periphery (5.5 mm distant from the center of the cornea) were evaluated. Ki67 staining confirmed that 0.4% or 0.8% ripasudil eye drops promoted cell proliferation throughout the wound edge to the peripheral area. Administration of 0.8% ripasudil (two times per day) enhanced Ki67 expression to a higher level than was observed with 0.4% ripasudil eye drops (four times daily). Morphology was evaluated using actin staining performed with Alexa Fluor 594–conjugated phalloidin. Nuclei were stained with DAPI. Representative images of pupil centers are shown. Scale bar: 50 μm. These experiments were performed in duplicate.
Effect of Ripasudil Eye Drops on Corneal Endothelial Wounds in a Mechanical Scraping Rabbit Model
Next, we evaluated whether ripasudil eye drops are an effective treatment for severe corneal endothelial damage; for example, the damage occurring during ocular surgeries such as cataract surgery, which is one of the leading causes of bullous keratopathy. We mechanically scraped half the area of the rabbit corneal endothelium and applied 0.4% ripasudil eye drops (three times daily). Slit-lamp microscopic images showed that eyes treated with vehicle as a control exhibited corneal edema, whereas eyes treated with ripasudil exhibited almost clear corneas (Fig. 3A). Five of six eyes treated with ripasudil recovered corneal transparency in the pupil center, whereas six of six control eyes did not. Scheimpflug images and corneal thickness maps obtained with a Pentacam HR instrument also demonstrated that control eyes exhibited corneal edema throughout the whole area. By contrast, ripasudil-treated eyes showed less edema in the cornea, including the pupillary area, although edema was still evident in the peripheral damaged area (Fig. 3B). The central corneal thickness of eyes treated with ripasudil was 392.8 ± 12.8 μm, but that of eyes treated with vehicle was 1200 μm (more than the maximum readings of ultrasound pachymetry in all eyes; Fig. 3C). The Ki67-positive cells in the wounded area at the center of cornea were evaluated after 48 hours of ripasudil eye drops. The control eye showed 52.6 ± 1.3% of the cell population as Ki67 positive, whereas the ripasudil-treated eyes showed 91.5 ± 2.0% Ki67-positive cells (Figs. 3D, 3E). 
Figure 3
 
Effect of ripasudil eye drops on wound healing in a mechanical damage corneal endothelial rabbit model. (A) Half the area of the corneal endothelium was mechanically scraped in six rabbits, and 0.4% ripasudil eye drops were applied (three times daily) for 2 weeks. Vehicle was applied to the fellow eyes as a control. The damage to the scraped area was confirmed by 0.04% trypan blue staining, and the experimenter who created the endothelial damage model was blinded to the subsequent treatment with ripasudil or vehicle. Representative slit-lamp microscope images are shown. (B) Representative Scheimpflug images and corneal thickness maps obtained by Pentacam HR are shown. Values for the corneal thickness map are indicated in micrometers. (C) The central corneal thickness was evaluated by ultrasound pachymetry. The central corneal thickness of eyes treated with ripasudil was 392.8 ± 12.6 μm, but that of eyes treated with vehicle was 1200 μm. **P < 0.01. (D, E) After 48 hours of ripasudil eye drops, Ki67-positive cells in the wounded area at the center of cornea were evaluated. The control eye showed 52.6 ± 1.32% of the cell population as Ki67 positive, whereas the ripasudil-treated eyes showed 91.5 ± 1.97% Ki67-positive cells. Scale bar: 50 μm. **P < 0.01.
Figure 3
 
Effect of ripasudil eye drops on wound healing in a mechanical damage corneal endothelial rabbit model. (A) Half the area of the corneal endothelium was mechanically scraped in six rabbits, and 0.4% ripasudil eye drops were applied (three times daily) for 2 weeks. Vehicle was applied to the fellow eyes as a control. The damage to the scraped area was confirmed by 0.04% trypan blue staining, and the experimenter who created the endothelial damage model was blinded to the subsequent treatment with ripasudil or vehicle. Representative slit-lamp microscope images are shown. (B) Representative Scheimpflug images and corneal thickness maps obtained by Pentacam HR are shown. Values for the corneal thickness map are indicated in micrometers. (C) The central corneal thickness was evaluated by ultrasound pachymetry. The central corneal thickness of eyes treated with ripasudil was 392.8 ± 12.6 μm, but that of eyes treated with vehicle was 1200 μm. **P < 0.01. (D, E) After 48 hours of ripasudil eye drops, Ki67-positive cells in the wounded area at the center of cornea were evaluated. The control eye showed 52.6 ± 1.32% of the cell population as Ki67 positive, whereas the ripasudil-treated eyes showed 91.5 ± 1.97% Ki67-positive cells. Scale bar: 50 μm. **P < 0.01.
Effect of Ripasudil Eye Drops on Regenerated Corneal Endothelium
Contact specular microscopy showed that control eyes exhibited blurred corneal endothelial images from the border (center of the cornea) to the damaged area. On the other hand, regenerated corneal endothelium was observed throughout whole area of the cornea with hexagonal and monolayer morphology (Fig. 4A). Cell density was significantly higher in the eyes treated with ripasudil than in eyes treated with vehicle, although the corneal endothelial cell density tended to be lower in the damaged area than in nondamaged areas in both treated eyes and control eyes (Fig. 4B). 
Figure 4
 
Effect of ripasudil eye drops on regenerated corneal endothelium in a mechanical damage corneal endothelial rabbit model. (A) Regenerated corneal endothelium was evaluated by contact specular microscopy after 14 days. Nondamaged areas, borders (pupil center), and damaged areas were evaluated. (B) The corneal endothelial cell density of the eyes treated with ripasudil was significantly higher in all areas (nondamaged areas, border, and damaged areas) than in the eyes treated with vehicle. (C) Regenerated corneal endothelium was evaluated histologically after 14 days. Morphology was evaluated using actin staining performed with Alexa Fluor 594–conjugated phalloidin. (D, E) Functional recovery was analyzed by immunostaining for N-cadherin (D) and Na+/K+-ATPase (E). (F) Cell proliferative status was analyzed by Ki67 immunostaining. No Ki67-positive cells were observed in either the ripasudil treated eyes or the control eyes after 14 days. Nuclei were stained with DAPI. Representative images of pupil centers are shown. Scale bar: 50 μm.
Figure 4
 
Effect of ripasudil eye drops on regenerated corneal endothelium in a mechanical damage corneal endothelial rabbit model. (A) Regenerated corneal endothelium was evaluated by contact specular microscopy after 14 days. Nondamaged areas, borders (pupil center), and damaged areas were evaluated. (B) The corneal endothelial cell density of the eyes treated with ripasudil was significantly higher in all areas (nondamaged areas, border, and damaged areas) than in the eyes treated with vehicle. (C) Regenerated corneal endothelium was evaluated histologically after 14 days. Morphology was evaluated using actin staining performed with Alexa Fluor 594–conjugated phalloidin. (D, E) Functional recovery was analyzed by immunostaining for N-cadherin (D) and Na+/K+-ATPase (E). (F) Cell proliferative status was analyzed by Ki67 immunostaining. No Ki67-positive cells were observed in either the ripasudil treated eyes or the control eyes after 14 days. Nuclei were stained with DAPI. Representative images of pupil centers are shown. Scale bar: 50 μm.
Regenerated corneal endothelium in the damaged area of the control eye exhibited a fibroblastic phenotype with the formation of stress fibers, but that of the treated eye exhibited a hexagonal monolayer phenotype and actin fibers were distributed at the cell cortex (Fig. 4C). Eyes treated with ripasudil expressed N-cadherin (a marker of adherence junctions) and Na+/K+-ATPase (a marker of pump function) in almost all the CECs, but this expression was lower in the control eyes (Figs. 4D, 4E). The Ki67 expression was not observed in either the control or the ripasudil-treated eyes (Fig. 4F). This indicates that corneal endothelial cell proliferation was no longer observed once the remaining CECs had covered the damaged area and cell–cell contact was reestablished, even following treatment with ripasudil eye drops. 
Discussion
In many countries, bullous keratopathy caused by cataract surgery is one of the leading causes of corneal transplantation due to corneal endothelial dysfunction.2325 In the United States, 12.2% of the corneas are used for post–cataract surgery corneal edema.26 As in western countries, 20–40% of corneal transplantation in Asian countries is performed to treat bullous keratopathy caused by cataract surgery.2729 The World Health Organization reported that 51% of blindness is caused by cataracts on a global basis30; therefore, it is reasonable to estimate that numerous patients will undergo cataract surgery in the near future, and a certain percentage of those patients will unfortunately experience severe corneal endothelial damage during their surgeries. The development of pharmaceutical treatments is urgently needed. 
Wound healing is a combined effect of cell migration, spreading, and proliferation in various tissues. However, wound healing of the corneal endothelium is mainly caused by migration and spreading due to the severely limited proliferation ability of corneal cells.31 Consequently, critical damage induces impairment of wound healing and loss of function.32 For that reason, researchers, including our group, have devoted much effort in the search for pharmaceutical agents (e.g., epidermal growth factor,33 platelet-derived growth factor (PDGF),34 FGF-2,35 and ROCK inhibitors3) that can promote proliferation of CECs for the treatment of corneal endothelial disease. 
We recently reported a preliminary successful result for pilot clinical research of using the ROCK inhibitor, Y-27632, as a form of eye drops for treating corneal endothelial injury.36 In that clinical research, patients had experienced severe corneal edema due to cataract surgery, and they recovered their corneal transparency following treatment with Y-27632 eye drops.36 We proposed that the ROCK inhibitor promotes cell proliferation of residual undamaged healthy CECs and ultimately reduces the risk of development of bullous keratopathy; therefore, ROCK inhibitors might be developed as eye drops for treating corneal endothelial injury. However, Y-27632 is currently only available for research use, not for clinical use, so the development of ROCK inhibitors other than Y-27632 represents a bottleneck for treating corneal endothelial injury in the clinical setting. 
Injury to the corneal endothelium decreases the corneal endothelial cell density, but severe damage due to various corneal endothelial diseases induces fibroblastic transformation37,38 that is thought to represent an endothelial–mesenchymal transformation.39 Fibroblastic transformation of CECs causes the cells to lose not only their morphologic character but also the activity of the corneal endothelial pump and barrier functions.40 Similarly, we showed here that the CECs of a rabbit wound model exhibited a fibroblastic morphology with corneal edema. Although this current study showed that ROCK inhibitor treatment regenerated a higher density of CECs and also suppressed fibroblastic formation, further investigation is needed to determine whether suppression of fibroblastic changes is a consequence of faster wound healing or a direct anti fibroblastic effect of the ROCK inhibitor.41,42 
The Ki67-positive proliferating cells were observed 48 hours after corneal endothelial damage in the current rabbit model, but were not observed when the damaged area was fully covered by CECs in both the control and ROCK inhibitor–treated eyes. This finding suggests that ROCK inhibitors do not induce cell proliferation once CECs form cell–cell contacts between adjacent cells. In other words, if a pharmaceutical agent that promotes cell proliferation is available in the clinical settings, the “golden time” begins immediately following the injury until just before the injured area is covered by compensatory migration and spreading of the remaining CECs. 
ROCK has been researched as a potential therapeutic target for vascular disease, cancer, neuronal degenerative disease, asthma, and glaucoma, because ROCK signaling activation is involved in numerous diseases.41,42 In contrast to the accumulation of evidence showing that ROCK inhibitors have potency against various diseases, only two drugs have been approved for clinical use: (1) fasudil for the treatment of cerebral vasospasm43 and (2) ripasudil for the treatment of glaucoma and ocular hypertension.14 Ripasudil clinical trials revealed slight to mild conjunctival hyperemia in more than half of the participants, whereas no other severe adverse effect was observed.11,12 In terms of the corneal endothelium, we reported that ripasudil induces guttae-like findings in humans, but these are transient, and no corneal endothelial damage was observed.44 We also reported that the guttae-like findings arise due to the formation of protrusions along cell–cell junctions as a consequence of the reduction in actomyosin contractility caused by inhibition of ROCK signaling.45 In the present in vitro evaluation, ripasudil enhanced cell proliferation to almost the same level seen with Y-27632. In the rabbit wound model, ripasudil eye drops restored transparency in five of six corneas, and Y-27632 eye drops showed the same effect in a previous report.36 The effects of long-term use of ripasudil eye drops on the corneal endothelium in clinical settings still remain in question. Therefore, further study to confirm its safety and efficacy is necessary for the development of ripasudil eye drops as a therapeutic modality for corneal endothelial diseases. 
Here, we showed that ripasudil eye drops are potent enhancers of corneal endothelial wound healing in a rabbit model that mimics severe corneal endothelial injury. Thus, we propose that drug repositioning of ripasudil for treating corneal endothelial diseases would be a judicious strategy for delivering ROCK inhibitor eye drops in the clinical setting. 
Acknowledgments
The authors thank Kowa Company for providing ripasudil eye drops. 
Supported by Program for the Strategic Research Foundation at Private Universities from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (NK, NO). 
SK holds patent no. 5657252, regarding the application of ROCK inhibitor for corneal endothelium. NO and NK are listed as inventors on the patent. 
Disclosure: N. Okumura, P; Y. Okazaki, None; R. Inoue, None; K. Kakutani, None; S. Nakano, None; S. Kinoshita, P; N. Koizumi, P 
References
Tan DT, Dart JK, Holland EJ, Kinoshita S. Corneal transplantation. Lancet. 2012; 379: 1749–1761.
Okumura N, Ueno M, Koizumi N, et al. Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor. Invest Ophthalmol Vis Sci. 2009; 50: 3680–3687.
Okumura N, Koizumi N, Ueno M, et al. Enhancement of corneal endothelium wound healing by Rho-associated kinase (ROCK) inhibitor eye drops. Br J Ophthalmol. 2011; 95: 1006–1009.
Okumura N, Koizumi N, Kay EP, et al. The ROCK inhibitor eye drop accelerates corneal endothelium wound healing. Invest Ophthalmol Vis Sci. 2013; 54: 2493–2502.
Koizumi N, Okumura N, Ueno M, Nakagawa H, Hamuro J, Kinoshita S. Rho-associated kinase inhibitor eye drop treatment as a possible medical treatment for fuchs corneal dystrophy. Cornea. 2013; 32: 1167–1170.
Honjo M, Tanihara H, Inatani M, et al. Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility. Invest Ophthalmol Vis Sci. 2001; 42: 137–144.
Tokushige H, Inatani M, Nemoto S, et al. Effects of topical administration of y-39983, a selective rho-associated protein kinase inhibitor, on ocular tissues in rabbits and monkeys. Invest Ophthalmol Vis Sci. 2007; 48: 3216–3222.
Williams RD, Novack GD, van Haarlem T, Kopczynski C. Group ARPAS. Ocular hypotensive effect of the Rho kinase inhibitor AR-12286 in patients with glaucoma and ocular hypertension. Am J Ophthalmol. 2011; 152: 834–841.
Inoue T, Tanihara H. Rho-associated kinase inhibitors: a novel glaucoma therapy. Prog Retin Eye Res. 2013; 37: 1–12.
Van de Velde S, Van Bergen T, Sijnave D, et al. AMA0076, a novel, locally acting Rho kinase inhibitor, potently lowers intraocular pressure in New Zealand white rabbits with minimal hyperemia. Invest Ophthalmol Vis Sci. 2014; 55: 1006–1016.
Tanihara H, Inoue T, Yamamoto T, et al. Phase 1 clinical trials of a selective Rho kinase inhibitor, K-115. JAMA Ophthalmol. 2013; 131: 1288–1295.
Tanihara H, Inoue T, Yamamoto T, et al. Phase 2 randomized clinical study of a Rho kinase inhibitor, K-115, in primary open-angle glaucoma and ocular hypertension. Am J Ophthalmol. 2013; 156: 731–736.
Tanihara H, Inoue T, Yamamoto T, et al. Intra-ocular pressure-lowering effects of a Rho kinase inhibitor, ripasudil (K-115), over 24 hours in primary open-angle glaucoma and ocular hypertension: a randomized, open-label, crossover study. Acta Ophthalmol. 2015; 93: e254–260.
Garnock-Jones KP. Ripasudil: first global approval. Drugs. 2014; 74: 2211–2215.
Isobe T, Mizuno K, Kaneko Y, Ohta M, Koide T, Tanabe S. Effects of K-115, a rho-kinase inhibitor, on aqueous humor dynamics in rabbits. Curr Eye Res. 2014; 39: 813–822.
Jin G, Wong ST. Toward better drug repositioning: prioritizing and integrating existing methods into efficient pipelines. Drug Discov Today. 2014; 19: 637–644.
Langedijk J, Mantel-Teeuwisse AK, Slijkerman DS, Schutjens MH. Drug repositioning and repurposing: terminology and definitions in literature. Drug Discov Today. 2015; 20: 1027–1034.
Wurth R, Thellung S, Bajetto A, Mazzanti M, Florio T, Barbieri F. Drug-repositioning opportunities for cancer therapy: novel molecular targets for known compounds. Drug Discov Today. 2016; 21: 190–199.
Okumura N, Kusakabe A, Hirano H, et al. Density-gradient centrifugation enables the purification of cultured corneal endothelial cells for cell therapy by eliminating senescent cells. Sci Rep. 2015; 5: 15005.
Nakahara M, Okumura N, Kay EP, et al. Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium. PLoS One. 2013; 8: e69009.
Matsubara M, Tanishima T. Wound-healing of the corneal endothelium in the monkey: a morphometric study. Jpn J Ophthalmol. 1982; 26: 264–273.
Matsubara M, Tanishima T. Wound-healing of corneal endothelium in monkey: an autoradiographic study. Jpn J Ophthalmol. 1983; 27: 444–450.
Al-Yousuf N, Mavrikakis I, Mavrikakis E, Daya SM. Penetrating keratoplasty: indications over a 10 year period. Br J Ophthalmol. 2004; 88: 998–1001.
Ghosheh FR, Cremona FA, Rapuano CJ, et al. Trends in penetrating keratoplasty in the United States 1980-2005. Int Ophthalmol. 2008; 28: 147–153.
Keenan TD, Jones MN, Rushton S, et al. Trends in the indications for corneal graft surgery in the United Kingdom: 1999 through 2009. Arch Ophthalmol. 2012; 130: 621–628.
Eye Bank Association of America. Eye Banking Statistical Report. Washington, DC; 2013.
Dandona L, Naduvilath TJ, Janarthanan M, Ragu K, Rao GN. Survival analysis and visual outcome in a large series of corneal transplants in India. Br J Ophthalmol. 1997; 81: 726–731.
Shimazaki J, Amano S, Uno T, Maeda N, Yokoi N. National survey on bullous keratopathy in Japan. Cornea. 2007; 26: 274–278.
Tan DT, Janardhanan P, Zhou H, et al. Penetrating keratoplasty in Asian eyes: the Singapore Corneal Transplant Study. Ophthalmology. 2008; 115: 975–982.
Pascolini D, Mariotti SP. Global estimates of visual impairment: 2010. Br J Ophthalmol. 2012; 96: 614–618.
Joyce NC. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res. 2003; 22: 359–389.
Bourne WM. Clinical estimation of corneal endothelial pump function. Trans Am Ophthalmol Soc. 1998; 96: 229–239. discussion 239–242.
Hoppenreijs VP, Pels E, Vrensen GF, Oosting J, Treffers WF. Effects of human epidermal growth factor on endothelial wound healing of human corneas. Invest Ophthalmol Vis Sci. 1992; 33: 1946–1957.
Hoppenreijs VP, Pels E, Vrensen GF, Treffers WF. Effects of platelet-derived growth factor on endothelial wound healing of human corneas. Invest Ophthalmol Vis Sci. 1994; 35: 150–161.
Lu J, Lu Z, Reinach P, et al. TGF-beta2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation. Exp Cell Res. 2006; 312: 3631–3640.
Okumura N, Inoue R, Okazaki Y, et al. Effect of the Rho kinase inhibitor Y-27632 on corneal endothelial wound healing. Invest Ophthalmol Vis Sci. 2015; 56: 6067–6074.
Naumann GO, Schlotzer-Schrehardt U. Keratopathy in pseudoexfoliation syndrome as a cause of corneal endothelial decompensation: a clinicopathologic study. Ophthalmology. 2000; 107: 1111–1124.
Kawaguchi R, Saika S, Wakayama M, Ooshima A, Ohnishi Y, Yabe H. Extracellular matrix components in a case of retrocorneal membrane associated with syphilitic interstitial keratitis. Cornea. 2001; 20: 100–103.
Lee HT, Lee JG, Na M, Kay EP. FGF-2 induced by interleukin-1 beta through the action of phosphatidylinositol 3-kinase mediates endothelial mesenchymal transformation in corneal endothelial cells. J Biol Chem. 2004; 279: 32325–32332.
Okumura N, Kay EP, Nakahara M, Hamuro J, Kinoshita S, Koizumi N. Inhibition of TGF-beta signaling enables human corneal endothelial cell expansion in vitro for use in regenerative medicine. PLoS One. 2013; 8: e58000.
Liao JK, Seto M, Noma K. Rho kinase (ROCK) inhibitors. J Cardiovasc Pharmacol. 2007; 50: 17–24.
Olson MF. Applications for ROCK kinase inhibition. Curr Opin Cell Biol. 2008; 20: 242–248.
Hu E, Lee D. Rho kinase as potential therapeutic target for cardiovascular diseases: opportunities and challenges. Expert Opin Ther Targets. 2005; 9: 715–736.
Nakagawa H, Koizumi N, Okumura N, Suganami H, Kinoshita S. Morphological changes of human corneal endothelial cells after rho-associated kinase inhibitor eye drop (ripasudil) administration: a prospective open-label clinical study. PLoS One. 2015; 10: e0136802.
Okumura N, Okazaki Y, Inoue R, et al. Rho-associated kinase inhibitor eye drop (ripasudil) transiently alters the morphology of corneal endothelial cells. Invest Ophthalmol Vis Sci. 2015; 56: 7560–7567.
Figure 1
 
Effect of ripasudil on in vitro corneal endothelial cell proliferation. (A) Cultured HCECs were seeded onto the culture plate and incubated for 24 hours, followed by treatment with the ROCK inhibitors Y-27632 (10 μM), fasudil (10 μM), and ripasudil (0.3–100 μM) for 48 hours. Representative phase contrast images are shown. Scale bar: 50 μm. (B) The HCECs were cultured at the density of 5000 cells per well in a 96-well plate for 24 hours, and DNA synthesis was detected by ELISA as incorporation of BrdU into DNA. BrdU incorporation was significantly enhanced at the ripasudil concentrations from 0.3 to 30 μM. **P < 0.01. (C, D) The effect of ripasudil on proliferation of corneal endothelium was evaluated by EdU and Ki67 staining after 48 hours of treatment. The numbers of EdU- and Ki67-positive cells were significantly higher following treatment of HCECs with ripasudil. **P < 0.01.
Figure 1
 
Effect of ripasudil on in vitro corneal endothelial cell proliferation. (A) Cultured HCECs were seeded onto the culture plate and incubated for 24 hours, followed by treatment with the ROCK inhibitors Y-27632 (10 μM), fasudil (10 μM), and ripasudil (0.3–100 μM) for 48 hours. Representative phase contrast images are shown. Scale bar: 50 μm. (B) The HCECs were cultured at the density of 5000 cells per well in a 96-well plate for 24 hours, and DNA synthesis was detected by ELISA as incorporation of BrdU into DNA. BrdU incorporation was significantly enhanced at the ripasudil concentrations from 0.3 to 30 μM. **P < 0.01. (C, D) The effect of ripasudil on proliferation of corneal endothelium was evaluated by EdU and Ki67 staining after 48 hours of treatment. The numbers of EdU- and Ki67-positive cells were significantly higher following treatment of HCECs with ripasudil. **P < 0.01.
Figure 2
 
Effect of ripasudil eye drops on corneal endothelial proliferation in a rabbit corneal freezing model. (A) A stainless-steel 7-mm-diameter probe was immersed in liquid nitrogen for 3 minutes and then placed onto the rabbit cornea for 15 seconds with the animal under general anesthesia. Then, one 0.4% ripasudil (four times daily) or 0.8% ripasudil (two times daily) eye drop was topically instilled, whereas vehicle was instilled in the fellow eye of each rabbit as a control (n = 6). Slit-lamp microscopy showed that control eyes exhibited hazy corneas after 48 hours, but eyes treated with 0.4% or 0.8% ripasudil eye drops exhibit less hazy corneas. No other adverse effects, such as the delay of corneal epithelial wound healing, severe conjunctival injection, and corneal opacity were observed. (B) The wound area of the corneal endothelium was evaluated by Alizarin red staining after 48 hours of treatment. Alizarin red staining images showed that the wound area tended to be smaller in eyes treated with 0.4% or 0.8% ripasudil eye drops than in control eyes. Scale bar: 1 mm. (C, D) Ki67+ cells located at the leading edge (3.5 mm distant from the center of the cornea), middle area (4.5 mm distant from the center of the cornea), and periphery (5.5 mm distant from the center of the cornea) were evaluated. Ki67 staining confirmed that 0.4% or 0.8% ripasudil eye drops promoted cell proliferation throughout the wound edge to the peripheral area. Administration of 0.8% ripasudil (two times per day) enhanced Ki67 expression to a higher level than was observed with 0.4% ripasudil eye drops (four times daily). Morphology was evaluated using actin staining performed with Alexa Fluor 594–conjugated phalloidin. Nuclei were stained with DAPI. Representative images of pupil centers are shown. Scale bar: 50 μm. These experiments were performed in duplicate.
Figure 2
 
Effect of ripasudil eye drops on corneal endothelial proliferation in a rabbit corneal freezing model. (A) A stainless-steel 7-mm-diameter probe was immersed in liquid nitrogen for 3 minutes and then placed onto the rabbit cornea for 15 seconds with the animal under general anesthesia. Then, one 0.4% ripasudil (four times daily) or 0.8% ripasudil (two times daily) eye drop was topically instilled, whereas vehicle was instilled in the fellow eye of each rabbit as a control (n = 6). Slit-lamp microscopy showed that control eyes exhibited hazy corneas after 48 hours, but eyes treated with 0.4% or 0.8% ripasudil eye drops exhibit less hazy corneas. No other adverse effects, such as the delay of corneal epithelial wound healing, severe conjunctival injection, and corneal opacity were observed. (B) The wound area of the corneal endothelium was evaluated by Alizarin red staining after 48 hours of treatment. Alizarin red staining images showed that the wound area tended to be smaller in eyes treated with 0.4% or 0.8% ripasudil eye drops than in control eyes. Scale bar: 1 mm. (C, D) Ki67+ cells located at the leading edge (3.5 mm distant from the center of the cornea), middle area (4.5 mm distant from the center of the cornea), and periphery (5.5 mm distant from the center of the cornea) were evaluated. Ki67 staining confirmed that 0.4% or 0.8% ripasudil eye drops promoted cell proliferation throughout the wound edge to the peripheral area. Administration of 0.8% ripasudil (two times per day) enhanced Ki67 expression to a higher level than was observed with 0.4% ripasudil eye drops (four times daily). Morphology was evaluated using actin staining performed with Alexa Fluor 594–conjugated phalloidin. Nuclei were stained with DAPI. Representative images of pupil centers are shown. Scale bar: 50 μm. These experiments were performed in duplicate.
Figure 3
 
Effect of ripasudil eye drops on wound healing in a mechanical damage corneal endothelial rabbit model. (A) Half the area of the corneal endothelium was mechanically scraped in six rabbits, and 0.4% ripasudil eye drops were applied (three times daily) for 2 weeks. Vehicle was applied to the fellow eyes as a control. The damage to the scraped area was confirmed by 0.04% trypan blue staining, and the experimenter who created the endothelial damage model was blinded to the subsequent treatment with ripasudil or vehicle. Representative slit-lamp microscope images are shown. (B) Representative Scheimpflug images and corneal thickness maps obtained by Pentacam HR are shown. Values for the corneal thickness map are indicated in micrometers. (C) The central corneal thickness was evaluated by ultrasound pachymetry. The central corneal thickness of eyes treated with ripasudil was 392.8 ± 12.6 μm, but that of eyes treated with vehicle was 1200 μm. **P < 0.01. (D, E) After 48 hours of ripasudil eye drops, Ki67-positive cells in the wounded area at the center of cornea were evaluated. The control eye showed 52.6 ± 1.32% of the cell population as Ki67 positive, whereas the ripasudil-treated eyes showed 91.5 ± 1.97% Ki67-positive cells. Scale bar: 50 μm. **P < 0.01.
Figure 3
 
Effect of ripasudil eye drops on wound healing in a mechanical damage corneal endothelial rabbit model. (A) Half the area of the corneal endothelium was mechanically scraped in six rabbits, and 0.4% ripasudil eye drops were applied (three times daily) for 2 weeks. Vehicle was applied to the fellow eyes as a control. The damage to the scraped area was confirmed by 0.04% trypan blue staining, and the experimenter who created the endothelial damage model was blinded to the subsequent treatment with ripasudil or vehicle. Representative slit-lamp microscope images are shown. (B) Representative Scheimpflug images and corneal thickness maps obtained by Pentacam HR are shown. Values for the corneal thickness map are indicated in micrometers. (C) The central corneal thickness was evaluated by ultrasound pachymetry. The central corneal thickness of eyes treated with ripasudil was 392.8 ± 12.6 μm, but that of eyes treated with vehicle was 1200 μm. **P < 0.01. (D, E) After 48 hours of ripasudil eye drops, Ki67-positive cells in the wounded area at the center of cornea were evaluated. The control eye showed 52.6 ± 1.32% of the cell population as Ki67 positive, whereas the ripasudil-treated eyes showed 91.5 ± 1.97% Ki67-positive cells. Scale bar: 50 μm. **P < 0.01.
Figure 4
 
Effect of ripasudil eye drops on regenerated corneal endothelium in a mechanical damage corneal endothelial rabbit model. (A) Regenerated corneal endothelium was evaluated by contact specular microscopy after 14 days. Nondamaged areas, borders (pupil center), and damaged areas were evaluated. (B) The corneal endothelial cell density of the eyes treated with ripasudil was significantly higher in all areas (nondamaged areas, border, and damaged areas) than in the eyes treated with vehicle. (C) Regenerated corneal endothelium was evaluated histologically after 14 days. Morphology was evaluated using actin staining performed with Alexa Fluor 594–conjugated phalloidin. (D, E) Functional recovery was analyzed by immunostaining for N-cadherin (D) and Na+/K+-ATPase (E). (F) Cell proliferative status was analyzed by Ki67 immunostaining. No Ki67-positive cells were observed in either the ripasudil treated eyes or the control eyes after 14 days. Nuclei were stained with DAPI. Representative images of pupil centers are shown. Scale bar: 50 μm.
Figure 4
 
Effect of ripasudil eye drops on regenerated corneal endothelium in a mechanical damage corneal endothelial rabbit model. (A) Regenerated corneal endothelium was evaluated by contact specular microscopy after 14 days. Nondamaged areas, borders (pupil center), and damaged areas were evaluated. (B) The corneal endothelial cell density of the eyes treated with ripasudil was significantly higher in all areas (nondamaged areas, border, and damaged areas) than in the eyes treated with vehicle. (C) Regenerated corneal endothelium was evaluated histologically after 14 days. Morphology was evaluated using actin staining performed with Alexa Fluor 594–conjugated phalloidin. (D, E) Functional recovery was analyzed by immunostaining for N-cadherin (D) and Na+/K+-ATPase (E). (F) Cell proliferative status was analyzed by Ki67 immunostaining. No Ki67-positive cells were observed in either the ripasudil treated eyes or the control eyes after 14 days. Nuclei were stained with DAPI. Representative images of pupil centers are shown. Scale bar: 50 μm.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×