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Cornea  |   April 2013
The ROCK Inhibitor Eye Drop Accelerates Corneal Endothelium Wound Healing
Author Affiliations & Notes
  • Naoki Okumura
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
    Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Noriko Koizumi
    Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • EunDuck P. Kay
    Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Morio Ueno
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Yuji Sakamoto
    Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Shinichiro Nakamura
    Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Japan
  • Junji Hamuro
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Shigeru Kinoshita
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, 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 April 2013, Vol.54, 2493-2502. doi:10.1167/iovs.12-11320
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      Naoki Okumura, Noriko Koizumi, EunDuck P. Kay, Morio Ueno, Yuji Sakamoto, Shinichiro Nakamura, Junji Hamuro, Shigeru Kinoshita; The ROCK Inhibitor Eye Drop Accelerates Corneal Endothelium Wound Healing. Invest. Ophthalmol. Vis. Sci. 2013;54(4):2493-2502. doi: 10.1167/iovs.12-11320.

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

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Abstract

Purpose.: To evaluate the effect of Rho kinase (ROCK)-inhibitor eye drops on a corneal endothelial dysfunction primate model and human clinical case series of corneal endothelial dysfunction.

Methods.: As a corneal-endothelial partially injured model, the corneal endothelium of seven cynomolgus monkeys was damaged by transcorneal freezing; 10 mM of ROCK inhibitor Y-27632 was then applied topically 6 times daily. The phenotype of the reconstructed corneal endothelium was evaluated by immunohistochemical analysis and noncontact specular microscopy. For clinical study, the effect of Y-27632 eye drops after transcorneal freezing was evaluated in eight corneal endothelial dysfunction patients: four central corneal edema patients and four diffuse corneal edema patients.

Results.: Slit-lamp microscopy revealed that both Y-27632–treated and –nontreated corneas became hazy after transcorneal freezing, and then recovered their transparency within 4 weeks. ROCK inhibitor Y-27632 promoted recovery of corneal endothelial cell density and wound healing in terms of both morphology and function. The percentage of ZO-1 and Na+/K+-ATPase positive cells in the regenerated area in the Y-27632 group was significantly higher than in the controls. Noncontact specular microscopy revealed that corneal endothelial cell density was significantly higher in the Y-27632 group compared with the controls at 4 weeks; cell density reached approximately 3000 cells/mm2, as opposed to 1500 cells/mm2 in the control group. In addition to the animal study findings, the clinical study findings showed that Y-27632 eye drops effectively improved corneal edema of corneal endothelial dysfunction patients with central edema.

Conclusions.: These findings show that ROCK inhibitor Y-27632 eye drops promote corneal endothelial wound healing in a primate animal model and suggest the possibility of Y-27632 as a novel therapeutic modality for certain forms of corneal endothelial dysfunction. (http://www.umin.ac.jp/ctr/ number, UMIN000003625.)

Introduction
The corneal endothelium is critical in maintaining homeostatic corneal transparency. Human corneal endothelial cells (HCECs) show severely limited proliferative ability in vivo. As a result, pathological corneal endothelial cell (CEC) loss causes a concurrent compensatory migration and enlargement of the remaining endothelial cells to achieve the functional contact-inhibited monolayer. In corneal endothelial disorders such as Fuchs' corneal dystrophy, pseudophakic bullous keratopathy, or trauma-related injuries, severe impairments of the relevant functions of CECs, namely Na+/K+-ATPase pump and barrier functions, cause irreversible corneal haziness. In the United States, 42,642 corneal transplantations were performed in 2011. 1 Since corneal endothelial dysfunction is the major indication for performing corneal transplantations, endothelial keratoplasty represented over 40% of all corneal grafts performed in both 2009 and 2010. 2 However, several severe problems can arise associated with corneal transplantation, including allograft rejection, primary graft failure, and severe loss of cell density. To the best of our knowledge, no clinically practical medical therapy has been developed to effectively treat corneal endothelial dysfunction. 
As an alternative to corneal transplantation, regenerative medical procedures might be a plausible path of therapy for treating severe corneal endothelial dysfunction. Several research groups, including ours, have reported transplantations of cultivated CECs in an animal model to establish a new clinical intervention for corneal endothelial dysfunction. 39 We recently reported the use of cell therapy to successfully achieve the recovery of corneal transparency in both rabbit and primate corneal endothelial dysfunction models. 9 However, in cases of early-stage corneal endothelial dysfunction, in which stem cells or progenitor cells are still maintained in the tissue, drug therapy may provide a less-invasive or antiprogression treatment. Our group, as well as several other groups, reported that pharmaceutical agents such as epidermal growth factor, platelet-derived growth factor, FGF-2, and small interfering RNA of connexin 43 showed the potent effect of enhancing the promotion of corneal endothelial cells, both in vitro and in vivo. 1012 However, a pharmaceutical agent has yet to be introduced into the clinical setting. 1,13 In a previous study, we demonstrated that a specific Rho kinase (ROCK)-inhibitor, Y-27632, increased the proliferative potential of cultivated primate CECs in vitro. 14 Previous studies have reported that the RhoA/ROCK pathway is involved in regulating the actin cytoskeleton, as well as cell migration, apoptosis, and proliferation. 1519 In addition, Rho GTPases reportedly suppress cell-cycle progression in several cell systems. 15,19 We also reported that the topical administration of ROCK inhibitor Y-27632 enhanced corneal endothelial wound healing in an in vivo rabbit model in which the corneal endothelium was partially damaged. 13,20,21  
In the present study, the feasibility of using topically administered ROCK inhibitor Y-27632 eye drops for clinical application was evaluated using a primate corneal endothelial dysfunction model. Our findings demonstrated that the use of ROCK inhibitor Y-27632 in eye drop form promotes corneal endothelial wound healing of the primate CECs with respect to both endothelial morphology and endothelial functions. Furthermore, CEC density, which is the most crucial indicator in the context of the clinical setting, was found to be recovered to the normal level after administration of the ROCK inhibitor Y-27632 eye drops. Of importance, we performed a pilot clinical study and demonstrated the feasibility of ROCK inhibitor Y-27632 eye-drop treatment as a therapeutic modality for corneal endothelial dysfunction. These findings provide new insights that ROCK inhibitor eye drops can be developed and employed as a new pharmaceutical agent for the treatment or antiprogression therapy of corneal endothelial dysfunction. 
Materials and Methods
Primate Corneal Endothelial Wound Model
The corneal endothelium of seven cynomolgus monkeys (3–5 years of age; estimated equivalent human age: 5–20 years) was damaged under general anesthesia by transcorneal freezing with a 7-mm–diameter probe for 15 seconds (Fig. 1A); the stainless-steel probe was immersed in liquid nitrogen for 3 minutes to stabilize its temperature at approximately −196°C. We confirmed that the transcorneal cryogenic injury damaged the central corneal endothelium in a round shape reproducibly. 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 monkey experiments were performed at the Research Center for Animal Life Science at Shiga University of Medical Science according to the protocol approved by the Animal Care and Use Committee of Shiga University of Medical Science (Approval No. 2008-10-5). 
Figure 1
 
Promotion of wound healing by ROCK inhibitor Y-27632 in a primate model. (AC) Central corneal endothelium was partially damaged by transcorneal cryogenic injury. (D) A 10-mM amount of Y-27632 (50 μL) was topically applied in one eye of each animal six times daily for 2 days. (E) DAPI staining shows the corneal endothelial wound. The wound area of the Y-27632–treated eye was smaller than that of the control eye after 48 hours. The white dotted line indicates the wound area. Scale bar: 500 μm. (F) Flatmount examination of the posterior side of the corneal tissue 4 weeks after Y-27632 treatment. Green fluorescein indicates F-actin staining (phalloidin) and blue indicates nuclear staining (DAPI). In the central area of the corneal endothelium, the actin cytoskeleton is organized at the cortex of the hexagonal cells in the Y-27632–treated eyes and exhibits a monolayer cell shape, while the actin cytoskeleton is disrupted in the control eyes. In the peripheral area, actin filaments are organized at the cortex, both in the Y-27632–treated eyes and the control eyes. Scale bar: 100 μm. (G) Immunohistochemical staining for Ki67, a cell proliferation marker, in the regenerated CECs. Both the Y-27632–treated eyes and the control eyes showed no Ki67-positive cells 4 weeks after cryoinjury. Scale bar: 100 μm. (H) Morphological scanning electron microscopy (SEM) analysis of the regenerated corneal endothelium of the central area revealed that ROCK inhibitor Y-27632 promotes morphological recovery. There is a large variation in cell size with some giant endothelial cells in the control eye, while the Y-27632–treated cornea shows fairly normal endothelial morphology without a large variation in cell size (left). CECs in the control eyes exhibited poorly formed cell junctions, while those in the Y-27632-treated eye exhibited fairly normal junctions with a hexagonal cell shape (right).
Figure 1
 
Promotion of wound healing by ROCK inhibitor Y-27632 in a primate model. (AC) Central corneal endothelium was partially damaged by transcorneal cryogenic injury. (D) A 10-mM amount of Y-27632 (50 μL) was topically applied in one eye of each animal six times daily for 2 days. (E) DAPI staining shows the corneal endothelial wound. The wound area of the Y-27632–treated eye was smaller than that of the control eye after 48 hours. The white dotted line indicates the wound area. Scale bar: 500 μm. (F) Flatmount examination of the posterior side of the corneal tissue 4 weeks after Y-27632 treatment. Green fluorescein indicates F-actin staining (phalloidin) and blue indicates nuclear staining (DAPI). In the central area of the corneal endothelium, the actin cytoskeleton is organized at the cortex of the hexagonal cells in the Y-27632–treated eyes and exhibits a monolayer cell shape, while the actin cytoskeleton is disrupted in the control eyes. In the peripheral area, actin filaments are organized at the cortex, both in the Y-27632–treated eyes and the control eyes. Scale bar: 100 μm. (G) Immunohistochemical staining for Ki67, a cell proliferation marker, in the regenerated CECs. Both the Y-27632–treated eyes and the control eyes showed no Ki67-positive cells 4 weeks after cryoinjury. Scale bar: 100 μm. (H) Morphological scanning electron microscopy (SEM) analysis of the regenerated corneal endothelium of the central area revealed that ROCK inhibitor Y-27632 promotes morphological recovery. There is a large variation in cell size with some giant endothelial cells in the control eye, while the Y-27632–treated cornea shows fairly normal endothelial morphology without a large variation in cell size (left). CECs in the control eyes exhibited poorly formed cell junctions, while those in the Y-27632-treated eye exhibited fairly normal junctions with a hexagonal cell shape (right).
Histological Examination
To evaluate the effect of ROCK inhibitor Y-27632 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) on the morphological wound healing process, corneas obtained from one monkey euthanized 48 hours after the transcorneal freeze injury were examined by DAPI (Vector Laboratories, Burlingame, CA) staining. A 10-mM amount of Y-27632 was topically applied to one eye of each animal 6 times daily for the first 48 hours. PBS was applied to the fellow eye of each animal as a control. The wound area was then evaluated and calculated by use of Java-based image processing software (Image J; National Institutes of Health, Bethesda, MD). 
To investigate the phenotype of reconstructed corneal endothelium obtained from four monkeys euthanized 4 weeks after transcorneal freezing in the presence or absence of Y-27632 treatment, immunohistochemical analyses of actin, Ki67, ZO-1, and Na+/K+-ATPase were performed. Actin staining was performed with 1:400 diluted AlexaFluor 488-conjugated phalloidin (Life Technologies Corporation, Carlsbad, CA). ZO-1 or Na+/K+-ATPase staining was performed with 1:200 diluted ZO-1 polyclonal antibody (Zymed Laboratories, South San Francisco, CA), or Na+/K+-ATPase monoclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY). Ki67 staining was performed using 1:400-dilution antimouse Ki67 antibody (Sigma-Aldrich Co., St. Louis, MO). AlexaFluor 488-conjugated goat antimouse IgG (1:2000; Life Technologies Corporation) was used as a secondary antibody. Each nucleus was stained with DAPI and examined under a confocal microscope (Leica TCS SPE; Leica Microsystems, Wetzlar, Germany). 
Scanning Electron Microscopy
To evaluate the effect of Y-27632 on functional wound healing, corneal specimens were obtained from four monkeys at 4 weeks after the treatment. Excised corneas were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M Sörensen buffer (pH 7.2 to 7.4) for at least 3 hours at room temperature. Following several washes in the buffer and postfixation with 1% aqueous osmium tetroxide, the corneas were dehydrated through an ascending ethanol series and transferred to hexamethyldisilazane (Agar Scientific, Stansted, UK), which was allowed to sublimate off. The samples were then mounted on stubs and sputter-coated with gold before being examined under a scanning electron microscope (model 5600; JEOL Ltd., Tokyo, Japan). 
Evaluation of Corneal Appearance and Examination by Specular Microscopy
The corneal endothelium of seven cynomolgus monkeys was injured by transcorneal freezing as described above. Next, 10 mM of ROCK inhibitor Y-27632 diluted in PBS (50 μL) was topically applied in one eye of each animal 6 times daily for 2 days, while PBS was applied in the animal's fellow eye as a control. One animal was euthanized at 48 hours after cryoinjury to determine the morphological wound healing process, while the in vivo corneal endothelium of the surviving animals was examined by use of a noncontact specular microscope (Noncon ROBO SP-8800; Konan Medical, Nishinomiya, Japan). Four weeks after transcorneal injury, four animals were euthanized and processed for immunohistological analysis and electron microscopy. The other two animals were kept alive for long-term observation. 
Clinical Trial of ROCK Inhibitor Eye Drops
The clinical trial performed in this study was conducted in accordance with the tenets set forth in the Declaration of Helsinki. This study was performed according to a protocol approved by the Institutional Review Board of Kyoto Prefectural University of Medicine (approval number: C-626-2). Prior to this, a phase 1 clinical study of ROCK inhibitor Y-27632 eye drops involving 10 healthy volunteers was conducted (approval number: C-626-1), which confirmed that 10 mM of Y27632 applied 6 times daily for 7 days caused no systemic or local side effects. Clinical trial registration was obtained from the Japanese Infrastructure for Academic Activities, University hospital Medical Information Network (UMIN) (No. UMIN000003625; in the public domain at http://www.umin.ac.jp/english/). After proper informed consent was obtained from all subjects, eight eyes of 8 patients scheduled for Descemet's Stripping Automated Endothelial Keratoplasty (DSAEK) surgery were enrolled in this study, which ran from May to August 2010. 
The patients were divided into two groups consisting of four patients per group according to the severity and extent of their corneal edema. In the first group, categorized as “diffuse corneal edema,” patients presented with widespread corneal edema throughout the central and peripheral cornea. In the other group of patients, categorized as “central corneal edema,” more peripheral corneal regions were clear. All of those four patients were clinically diagnosed as “late-onset Fuchs corneal dystrophy” in our cornea clinic based on the typical findings of multiple guttae and corneal edema that progressed after 40 years of age. One of these Fuchs' dystrophy cases (case 1) was recently reported in another manuscript as a case report. 22 The demographic data and pretreatment clinical manifestations of the patients are summarized in the Table
Table
 
Demographic Data of the Patients Involved in the ROCK Inhibitor Eye Drop Clinical Trial
Table
 
Demographic Data of the Patients Involved in the ROCK Inhibitor Eye Drop Clinical Trial
Eye Sex Age Type of Edema Cause of Endothelial Decompensation Central Corneal Thickness, μm BCVA, logMar Ocular Complications
Pre 6M Pre 6M
1 L M 52 Central Fuchs' dystrophy 703 568 0.7 −0.18 None
2 R F 75 Central Fuchs' dystrophy 809 (722:DSAEK) 1 0.7 Cataract
3 L F 57 Central Fuchs' dystrophy 682 663 0.52 0.52 None
4 L F 62 Central Fuchs' dystrophy 759 687 0.7 0.52 Myopic CRA
5 L F 76 Diffuse Laser iridotomy 683 506 1 1.7 Cataract
6 L F 70 Diffuse Laser iridotomy 920 920 0.7 1.52 Cataract
7 L F 72 Diffuse Laser iridotomy 827 827 0.7 0.7 Cataract
8 R F 72 Diffuse Pseudoexfoliation syndrome 721 757 0.4 1 Cataract
Treatment Procedures
Prior to the administration of ROCK inhibitor Y-27632 eye drops, corneal endothelial cells were removed from the central part of cornea by transcorneal freezing according to the previous reports, yet with some modifications. 13 In brief, a 2-mm–diameter stainless steel rod was immersed in liquid nitrogen for 3 minutes to stabilize its temperature at approximately −196°C. Under topical anesthesia using oxybuprocaine hydrochloride (Santen Pharmaceutical Co., Ltd., Osaka, Japan), the frozen rod was pressed gently onto the central cornea for 15 seconds in order to damage diseased corneal endothelial cells. After the frozen rod was removed and the cornea had thawed, 10 mM of Y-27632 dissolved in 50 μL of PBS was topically applied in eye drop form 6 times daily for 7 days. Gatifloxacin hydrate eye drops (0.3%; Senju Pharmaceutical Company, Ltd., Osaka, Japan) were also applied 4 times daily to prevent ocular surface infection. Slit-lamp examination, noncontact specular microscopy, anterior segment optical coherence tomography (OCT; Visante; Carl Zeiss Meditec, Tokyo, Japan), and intraocular pressure measurements were performed daily for the first 7 days. Thereafter, the eyes were examined every week up to 1 month, and every 4 weeks up to 6 months. Prior to treatment, and at 3 and 6 months following treatment, the eyes were examined to elucidate any systemic side effects related to the ROCK inhibitor eye drop application. 
Statistical Analysis
Statistical analysis was performed by use of a commercial spreadsheet program (Excel; Microsoft Corporation, Redmond, WA). The statistical significance (P value) in mean values of the two-sample comparison was determined with the Student's t-test. A P value of <0.05 was considered statistically significant. Values shown represent the mean ± SEM. 
Results
ROCK Inhibitor Enhanced Morphological Recovery of Corneal Endothelium
In a previous report, we demonstrated that ROCK inhibitor Y-27632 facilitated wound healing in a rabbit animal model. 13 However, rabbit corneal endothelium has the ability to proliferate in vivo. Thus, we attempted to confirm that Y-27632 eye drops promote wound healing of primate corneal endothelium, in which cell proliferation is barely observed in vivo. In order to test this, we created partial corneal endothelial wounds by transcorneal freezing in the eyes of cynomolgus monkeys. Following cryoinjury, 10 mM of Y-27632 was topically applied to the eyes in eye drop form 6 times daily (Figs. 1A–D). Consistent with the rabbit findings, 13 the wounded area of the corneal endothelium following Y-27632 treatment was significantly reduced in comparison with that of the control eye (Fig. 1E). After treating the monkey eyes with ROCK inhibitor for 4 weeks, an actin cytoskeleton was observed at the cortex in the peripheral undamaged area, both in the control eyes and the Y-27632–treated eyes. However, in the control eyes, organization of the actin cytoskeleton of the corneal endothelium in the damaged central region was greatly disturbed and the cell shapes were severely altered (Fig. 1F). On the other hand, the Y-27632–treated eyes showed actin cytoskeleton at the cell cortex of hexagonal cells in the central area (Fig. 1F). Ki67-positive cells were not observed, in both the control eyes and the Y-27632–treated eyes, regardless of the region (Fig. 1G). Thus, it is likely that the proliferation of the CECs after administration of Y-27632 was blocked when CECs covered the wound area and formed contact-inhibited phenotypes. Analysis by scanning electron microscopy further confirmed the effect of Y-27632 on morphological recovery after 4 weeks of treatment (Fig. 1H). CECs in the Y-27632–treated eyes showed the characteristic hexagonal morphology of CECs, which have similar cell sizes. On the other hand, cells in the control eyes showed a large variation of cell sizes and the presence of enlarged cells. In addition, the cell junctions in the control eyes were poorly formed, whereas the cell junctions in the Y-27632–treated eyes were fairly normal among the hexagonal-shape cells. These findings demonstrate that the topical administration of ROCK inhibitor Y-27632 eye drops greatly enhances morphological recovery during the corneal endothelial wound repair process. 
ROCK Inhibitor Enhanced Functional Recovery of Corneal Endothelium
During the regenerative wound healing process, there was a compensatory migration and enlargement of the neighboring corneal endothelial cells that migrated into the injury site to restore the contact-inhibited monolayer. After full recovery of the contact-inhibited monolayer of the corneal endothelium, the CECs were then able to exert their characteristic barrier and pump functions. We examined the fully recovered wound sites as divided into three areas as follows: the recovered central area that was previously damaged, the peripheral undamaged area, and the transition zone between the two areas (Figs. 2A, 2B). When cells were stained with anti-ZO-1 (the barrier-function-associated protein marker) antibody, the subcellular localization of ZO-1 in the control eyes was greatly disturbed in the central area and in the transition zone (Figs. 2A, 2B). On the other hand, the characteristic plasma-membrane staining pattern was observed in the Y-27632–treated eyes in all three of the examined areas (Figs. 2A, 2B). To confirm these findings, the histological phenotypes were further determined using confocal microscopy. The regenerated corneal endothelial cells of the Y-27632–treated eyes demonstrated plasma-membrane staining of ZO-1 in both the central and peripheral areas, while the staining pattern of ZO-1 was greatly disturbed in the central area of the control eye as opposed to the normal phenotypes of the peripheral region of the same cornea (Fig. 2C). Similar findings were observed when corneal endothelium was stained for Na+/K+-ATPase (the pump-function-associated protein marker). Y-27632 promoted the authentic subcellular localization of the of Na+/K+-ATPase at the plasma membrane of the regenerated cells in the central area, whereas the distribution of Na+/K+-ATPase was greatly disturbed in the cells of the central area in the absence of Y-27632 (Fig. 2D). The percentages of ZO-1- and Na+/K+-ATPase–positive cells in the central area were significantly higher in the Y-27632–treated eyes than those in the control eyes (Figs. 2E, 2F), suggesting that Y-27632 rapidly enhances functional recovery as well as morphological recovery. 
Figure 2
 
ROCK inhibitor Y-27632 promoted the functional recovery of regenerated corneal endothelium. (A, B) Subcellular localization of ZO-1 was disturbed in the central area of the control eyes, while the subcellular localization of ZO-1 in the Y-27632–treated eye was demonstrated at the plasma membrane, the physiological location. Scale bar: 500 μm. (C, D) In the Y-27632-treated eye, all regenerated cells in the wounded central area expressed ZO-1 and Na+/K+-ATPase. On the other hand, the expression of ZO-1 and Na+/K+-ATPase was decreased and their subcellular location was greatly disturbed in the control eyes. Scale bar: 100 μm. (E, F) The percentages of ZO-1 and Na+/K+-ATPase–positive cells in the wounded area are significantly higher in the Y-27632–treated eye than in the control eye. *P < 0.01, **P < 0.05.
Figure 2
 
ROCK inhibitor Y-27632 promoted the functional recovery of regenerated corneal endothelium. (A, B) Subcellular localization of ZO-1 was disturbed in the central area of the control eyes, while the subcellular localization of ZO-1 in the Y-27632–treated eye was demonstrated at the plasma membrane, the physiological location. Scale bar: 500 μm. (C, D) In the Y-27632-treated eye, all regenerated cells in the wounded central area expressed ZO-1 and Na+/K+-ATPase. On the other hand, the expression of ZO-1 and Na+/K+-ATPase was decreased and their subcellular location was greatly disturbed in the control eyes. Scale bar: 100 μm. (E, F) The percentages of ZO-1 and Na+/K+-ATPase–positive cells in the wounded area are significantly higher in the Y-27632–treated eye than in the control eye. *P < 0.01, **P < 0.05.
ROCK Inhibitor Eye Drop Enabled Recovery of CEC Density in a Primate Model
Slit-lamp microscopy examination revealed that both Y-27632–treated and –nontreated corneas became hazy immediately after the corneal endothelial damage induced by transcorneal freezing; their transparency was recovered within 1 month (Fig. 3A). No severe side effects such as irreversible corneal haze and persistent corneal epithelial defect were observed during the wound healing process following cryoinjury. To test whether or not Y-27632 actually promoted the recovery of endothelial cell density, which is the most important clinical indicator, endothelial cell density in the monkey eyes was determined by use of noncontact specular microscopy. Since monkey CECs (MCECs) barely proliferate in vivo, corneal endothelial damage induced a compensatory migration and enlargement of the remaining neighboring endothelial cells. Consistent with this finding, in the control group, noncontact specular microscopy showed enlarged CECs, with a cell density of approximately 1500 cells/mm2, 1 week after the injury (Figs. 3B, 3C). However, the corneal endothelium of the Y-27632–treated group was reconstructed without the compensatory enlargement of cell size, and the cell density reached approximately 3000 cells/mm2, which is in the high range of endothelial cell density (Figs. 3B, 3C). The fact that the CEC density was significantly higher in the Y-27632–treated group compared with that of the controls suggests that ROCK inhibitor eye drops may stimulate the peripheral undamaged cells to proliferate, subsequently resulting in the recovery of functional corneal endothelium with a normal high cell density. 
Figure 3
 
ROCK inhibitor Y-27632 eye drops promoted the recovery of cell density in a corneal-endothelial partially damaged primate model. (A) Slit-lamp microscopy examination revealed that both Y-27632–treated and –nontreated corneas recovered their transparency 1 month after cryoinjury. (B) In the control group, noncontact specular microscopy shows enlarged corneal endothelium migrating into the damaged area at the density of approximately 1500 cells/mm2 1 week after the injury. However, corneal endothelium of the Y-27632–treated group was reconstructed without compensatory enlargement with a normal cell density of approximately 3000 cells/mm2. (C) Noncontact specular microscopy analysis revealed that the CEC density was significantly higher in the Y-27632–treated group than in the control group throughout the 4-week observation period (*P < 0.01).
Figure 3
 
ROCK inhibitor Y-27632 eye drops promoted the recovery of cell density in a corneal-endothelial partially damaged primate model. (A) Slit-lamp microscopy examination revealed that both Y-27632–treated and –nontreated corneas recovered their transparency 1 month after cryoinjury. (B) In the control group, noncontact specular microscopy shows enlarged corneal endothelium migrating into the damaged area at the density of approximately 1500 cells/mm2 1 week after the injury. However, corneal endothelium of the Y-27632–treated group was reconstructed without compensatory enlargement with a normal cell density of approximately 3000 cells/mm2. (C) Noncontact specular microscopy analysis revealed that the CEC density was significantly higher in the Y-27632–treated group than in the control group throughout the 4-week observation period (*P < 0.01).
ROCK Inhibitor Eye Drops Restored the Corneal Thickness of Human Corneal Endothelial Dysfunction
Finally, we tested whether or not ROCK inhibitor eye drops enhance the proliferation of HCECs in vivo. This clinical trial involved four patients with central corneal edema and four patients with diffuse corneal edema. The patients were treated with the ROCK inhibitor eye drops 6 times daily for 7 days. Corneal thickness and best corrected visual acuity (BCVA) data of these 8 patients before and 6 months after treatment are shown in the Table
A representative case of a central corneal edema patient is shown in Figures 4A to 4D. Before treatment, central corneal edema accompanied by the epithelial bulla was detected in patient No. 1. (Figs. 4A, 4B). The corneal edema was significantly reduced and BCVA recovered from logMAR 0.7 to −0.18 (Figs. 4C, 4D) at 6 months after treatment. A representative case of a diffuse corneal edema patient is shown in Figures 4E to 4H. Before treatment, diffuse corneal edema due to argon laser iridotomy-induced bullous keratopathy (ALI-BK) was observed (Figs. 4E, 4F). The corneal edema persisted and recovery of visual acuity was not obtained at 6 months after treatment (Figs. 4G, 4H). Specular microscopic examination was found to be difficult to perform on most of these patients due to corneal edema. We found some portion of relatively healthy corneal endothelial cells in the paracentral area before treatment in patient no. 1, though no analyzable image was obtained from the center cornea. Moreover, we confirmed the remodeling of corneal endothelial cells both of the center and paracentral area in this patient after treatment (Fig. 4I). In the central corneal edema patients, central corneal thickness was reduced 6 months after treatment compared to pretreatment levels (Fig. 4J). In contrast, there was no reduction of central corneal thickness in the eyes with diffuse corneal edema (Fig. 4J). Although no statistical significance was shown from this small cohort, reduction of corneal thickness may indicate recovery of corneal endothelial function in patients with central edema. In terms of the visual acuity, not much improvement was seen even in the patients with central edema, except in patient No. 1, due to the presence of preexisting senile cataract or macular degeneration. However, vision was kept at the same level or was slightly improved during the 6-month observation period. In all patients, no complications such as intraocular pressure elevation or systemic complications were detected in relation to the transcorneal freezing or the ROCK inhibitor eye drop application. 
Figure 4
 
Clinical trial of ROCK inhibitor Y-27632 eye drops for treating patients with central corneal edema and diffuse corneal edema. (A, B) Representative case of a central corneal edema patient is shown. Before treatment, central corneal edema was detected in patient 1. (C, D) Six months after treatment, the corneal edema was significantly reduced and visual acuity recovered from logMAR 0.70 to −0.18. (E, F) Representative case of a diffuse corneal edema patient is shown. Before treatment, diffuse corneal edema due to argon laser iridotomy-induced bullous keratopathy (ALI-BK) was observed. (G, H) Six months after treatment, the corneal edema persisted and recovery of visual acuity was not obtained. (I) The corneal endothelium of case 1 observed by noncontact-specular microscopy before (A, B) and 6 months after treatment (C, D). Before treatment, we could not obtain clear image of corneal endothelium from the center part of cornea due to corneal edema (A). In contrast, some endothelial cells with guttae were observed para-central area of the same eye (B). Six months after treatment, specular microscopic images were obtained from both of center (C) and peripheral cornea (D). Approximate cell density after treatment was 1200 to 1500 cells/mm2 in both areas. (J) In the central corneal edema patients, central corneal thickness was reduced 6 months after treatment compared to pretreatment levels. In contrast, the central corneal thickness did not reduce in eyes with diffuse corneal edema.
Figure 4
 
Clinical trial of ROCK inhibitor Y-27632 eye drops for treating patients with central corneal edema and diffuse corneal edema. (A, B) Representative case of a central corneal edema patient is shown. Before treatment, central corneal edema was detected in patient 1. (C, D) Six months after treatment, the corneal edema was significantly reduced and visual acuity recovered from logMAR 0.70 to −0.18. (E, F) Representative case of a diffuse corneal edema patient is shown. Before treatment, diffuse corneal edema due to argon laser iridotomy-induced bullous keratopathy (ALI-BK) was observed. (G, H) Six months after treatment, the corneal edema persisted and recovery of visual acuity was not obtained. (I) The corneal endothelium of case 1 observed by noncontact-specular microscopy before (A, B) and 6 months after treatment (C, D). Before treatment, we could not obtain clear image of corneal endothelium from the center part of cornea due to corneal edema (A). In contrast, some endothelial cells with guttae were observed para-central area of the same eye (B). Six months after treatment, specular microscopic images were obtained from both of center (C) and peripheral cornea (D). Approximate cell density after treatment was 1200 to 1500 cells/mm2 in both areas. (J) In the central corneal edema patients, central corneal thickness was reduced 6 months after treatment compared to pretreatment levels. In contrast, the central corneal thickness did not reduce in eyes with diffuse corneal edema.
Discussion
In most tissues, the wound repair process consists of cell migration and cell proliferation. Unlike such a generalized mechanism of wound healing, the regenerative wound repair observed in human corneal endothelium is accomplished by cell migration and attenuation of neighboring cells adjacent to the injury site, with limited involvement of cell proliferation. Of interest, species-specific differences exist in regard to cell proliferation ability during wound healing (e.g., rabbit, mouse, and bovine CECs exhibit proliferative ability, while the proliferative ability of human, monkey, and cat CECs is severely limited). 2327  
The corneal endothelium is critical for maintaining homeostatic-corneal transparency; corneal endothelium has to retain sufficient cell density to maintain the contact-inhibited monolayer, which is crucial to perform the ionic pump and barrier functions. If the corneal endothelium fails to retain sufficient cell density due to either the aging process or severe injury, the result of this abnormality is an increase of overall cell size and an alteration of the cell shape to a pleomorphic shape. Enlarged corneal endothelial cells, as well as cells with abnormal morphology, are closely associated with endothelial dysfunction. Therefore, it has been widely studied to trigger the proliferation of CECs in vivo in the absence of pathological complications leading to another ocular dysfunction. Of importance, HCECs are arrested at the G1 phase of the cell cycle, 27 suggesting that they are not terminally differentiated but do possess proliferative potential. 
In our search for a biological tool that could proliferate CECs, we investigated the efficacy of the selective ROCK inhibitor Y-27632. We recently published that Y-27632 inhibited dissociation-induced apoptosis and promoted the adhesion and proliferation of MCECs. 14 Moreover, we reported that the topical application of Y-27632 promoted corneal endothelial wound healing in a rabbit model. 20,21 In this present study, we attempted to establish a new pharmacological intervention in the form of Y-27632 eye drops that would promote corneal endothelial wound healing. Using a primate corneal endothelial dysfunction model, we demonstrated that: the topical application of Y-27632 eye drops greatly enhanced wound healing of corneal endothelium; that the regenerated corneal endothelium of the central damaged region demonstrated physiological hexagonal cell morphology and resumed the characteristic adhesion profiles (ZO-1 and Na+/K+-ATPase) and actin cytoskeleton when treated with Y-27632 eye drops; and that Y-27632 eye drops greatly enhanced cell density to the normal level. These important findings suggest that the administration of ROCK inhibitor Y-27632 eye drops enhances both the functional and morphological recovery. In addition, we demonstrated that Y-27632 eye drops proved effective for the recovery of corneal transparency and the gradual reduction of corneal thickness for up to 6 months in human patients who had central edema due to endothelial dysfunction. Those findings suggest that the use of Y-27632 eye drops may be clinically beneficial to a certain group of patients with central edema caused by endothelial dysfunction (i.e., Fuchs' corneal dystrophy patients). Moreover, it has been reported that spontaneous remodeling may take place in Y-27632–treated patients with central edema, similar to that observed in HCECs after Descemet's stripping procedures, 28,29 likely due to the existence of corneal endothelial precursors with higher proliferative ability in the peripheral cornea. However, our data (Figs. 3B, 3C) using primate CECs, whose proliferative behavior is similar to that of HCECs, supports the finding that ROCK inhibitor greatly stimulates the proliferation of MCECs. 
Rho GTPases members (RhoA, Rac1, and Cdc42) reportedly play an important role at many aspects of the cell cycle. 19 Earlier studies have shown that Rho contributes to cell cycle progression and that inactivation of Rho by C3 blocks G1/S progression in Swiss 3T3 fibroblast. 15,30 Unlike these findings, we revealed that inhibition of Rho/ROCK signaling by selective ROCK inhibitor Y-27632 promotes the proliferation of cultured CECs. 14 Therefore, Rho/ROCK activity on the cell cycle may be cell-type dependent. 31 However, the mechanism by which ROCK inhibitor promotes corneal endothelial cell proliferation has yet to be elucidated. In regard to the clinical application, Fasudil, one of the ROCK inhibitors, has already been approved for clinical use and has been administered in over 124,000 cases in Japan. 32 Moreover, ROCK inhibitors have been developed for a wide range of diseases such as cardiovascular disease, pulmonary disease, and cancer. 32 In the field of ophthalmology, another ROCK inhibitor (Y-39983) has been developed for treating glaucoma and is currently undergoing clinical trials. 33 It is likely that the application of ROCK inhibitor might also be of clinical benefit for the treatment of corneal endothelial dysfunction. Thus, we hypothesize that the combined treatment regimen consisting of partial denudation of diseased CECs via transcorneal freezing and the topical application of Y-27632 in eye drop form may be useful to promote the proliferation of corneal endothelium in patients with central cornea edema. However, there are several concerns to be addressed before advancing the current findings toward clinical application; they include which endothelial diseases, stage of disease, method of performing the transcorneal freezing, and the duration and dosage of the ROCK inhibitor eye drops. Such issues will influence the healing pattern. Nevertheless, the current pilot study offers encouragement that some patients with endothelial dysfunction, especially those with Fuchs' corneal dystrophy, might be good candidates for transcorneal freezing/ROCK inhibitor eye drop treatment as an alternative to graft surgery. 
In summary, our results demonstrate that ROCK inhibitor Y-27632 promotes corneal endothelium wound healing in a primate animal model. Furthermore, this is the first report of a case series demonstrating a pharmaceutical agent being successfully used to treat corneal endothelial dysfunction in human eyes in the absence of other ocular complications, and our findings encourage us to further develop ROCK inhibitor eye drops as a novel therapy for certain forms of corneal endothelial dysfunction. 
Acknowledgments
The authors thank Yoshiki Sasai and Masatoshi Ohgushi for their assistance and advice about ROCK inhibitors, Hideaki Tsuchiya and Kenta Yamasaki for technical support, Tsutomu Inatomi, Takahiro Nakamura, and Hiroko Nakagawa for assistance with the clinical trial, and John Bush for reviewing the manuscript. 
Supported by the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (AS2314212G: SK, NO), the Funding Program for Next Generation World-Leading Researchers from the Cabinet Office in Japan (LS117: NK), and the Highway Program for realization of regenerative medicine (SK, NO). 
Disclosure: N. Okumura, None; N. Koizumi, None; E.P. Kay, None; M. Ueno, None; Y. Sakamoto, None; S. Nakamura, None; J. Hamuro, None; S. Kinoshita, None 
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Figure 1
 
Promotion of wound healing by ROCK inhibitor Y-27632 in a primate model. (AC) Central corneal endothelium was partially damaged by transcorneal cryogenic injury. (D) A 10-mM amount of Y-27632 (50 μL) was topically applied in one eye of each animal six times daily for 2 days. (E) DAPI staining shows the corneal endothelial wound. The wound area of the Y-27632–treated eye was smaller than that of the control eye after 48 hours. The white dotted line indicates the wound area. Scale bar: 500 μm. (F) Flatmount examination of the posterior side of the corneal tissue 4 weeks after Y-27632 treatment. Green fluorescein indicates F-actin staining (phalloidin) and blue indicates nuclear staining (DAPI). In the central area of the corneal endothelium, the actin cytoskeleton is organized at the cortex of the hexagonal cells in the Y-27632–treated eyes and exhibits a monolayer cell shape, while the actin cytoskeleton is disrupted in the control eyes. In the peripheral area, actin filaments are organized at the cortex, both in the Y-27632–treated eyes and the control eyes. Scale bar: 100 μm. (G) Immunohistochemical staining for Ki67, a cell proliferation marker, in the regenerated CECs. Both the Y-27632–treated eyes and the control eyes showed no Ki67-positive cells 4 weeks after cryoinjury. Scale bar: 100 μm. (H) Morphological scanning electron microscopy (SEM) analysis of the regenerated corneal endothelium of the central area revealed that ROCK inhibitor Y-27632 promotes morphological recovery. There is a large variation in cell size with some giant endothelial cells in the control eye, while the Y-27632–treated cornea shows fairly normal endothelial morphology without a large variation in cell size (left). CECs in the control eyes exhibited poorly formed cell junctions, while those in the Y-27632-treated eye exhibited fairly normal junctions with a hexagonal cell shape (right).
Figure 1
 
Promotion of wound healing by ROCK inhibitor Y-27632 in a primate model. (AC) Central corneal endothelium was partially damaged by transcorneal cryogenic injury. (D) A 10-mM amount of Y-27632 (50 μL) was topically applied in one eye of each animal six times daily for 2 days. (E) DAPI staining shows the corneal endothelial wound. The wound area of the Y-27632–treated eye was smaller than that of the control eye after 48 hours. The white dotted line indicates the wound area. Scale bar: 500 μm. (F) Flatmount examination of the posterior side of the corneal tissue 4 weeks after Y-27632 treatment. Green fluorescein indicates F-actin staining (phalloidin) and blue indicates nuclear staining (DAPI). In the central area of the corneal endothelium, the actin cytoskeleton is organized at the cortex of the hexagonal cells in the Y-27632–treated eyes and exhibits a monolayer cell shape, while the actin cytoskeleton is disrupted in the control eyes. In the peripheral area, actin filaments are organized at the cortex, both in the Y-27632–treated eyes and the control eyes. Scale bar: 100 μm. (G) Immunohistochemical staining for Ki67, a cell proliferation marker, in the regenerated CECs. Both the Y-27632–treated eyes and the control eyes showed no Ki67-positive cells 4 weeks after cryoinjury. Scale bar: 100 μm. (H) Morphological scanning electron microscopy (SEM) analysis of the regenerated corneal endothelium of the central area revealed that ROCK inhibitor Y-27632 promotes morphological recovery. There is a large variation in cell size with some giant endothelial cells in the control eye, while the Y-27632–treated cornea shows fairly normal endothelial morphology without a large variation in cell size (left). CECs in the control eyes exhibited poorly formed cell junctions, while those in the Y-27632-treated eye exhibited fairly normal junctions with a hexagonal cell shape (right).
Figure 2
 
ROCK inhibitor Y-27632 promoted the functional recovery of regenerated corneal endothelium. (A, B) Subcellular localization of ZO-1 was disturbed in the central area of the control eyes, while the subcellular localization of ZO-1 in the Y-27632–treated eye was demonstrated at the plasma membrane, the physiological location. Scale bar: 500 μm. (C, D) In the Y-27632-treated eye, all regenerated cells in the wounded central area expressed ZO-1 and Na+/K+-ATPase. On the other hand, the expression of ZO-1 and Na+/K+-ATPase was decreased and their subcellular location was greatly disturbed in the control eyes. Scale bar: 100 μm. (E, F) The percentages of ZO-1 and Na+/K+-ATPase–positive cells in the wounded area are significantly higher in the Y-27632–treated eye than in the control eye. *P < 0.01, **P < 0.05.
Figure 2
 
ROCK inhibitor Y-27632 promoted the functional recovery of regenerated corneal endothelium. (A, B) Subcellular localization of ZO-1 was disturbed in the central area of the control eyes, while the subcellular localization of ZO-1 in the Y-27632–treated eye was demonstrated at the plasma membrane, the physiological location. Scale bar: 500 μm. (C, D) In the Y-27632-treated eye, all regenerated cells in the wounded central area expressed ZO-1 and Na+/K+-ATPase. On the other hand, the expression of ZO-1 and Na+/K+-ATPase was decreased and their subcellular location was greatly disturbed in the control eyes. Scale bar: 100 μm. (E, F) The percentages of ZO-1 and Na+/K+-ATPase–positive cells in the wounded area are significantly higher in the Y-27632–treated eye than in the control eye. *P < 0.01, **P < 0.05.
Figure 3
 
ROCK inhibitor Y-27632 eye drops promoted the recovery of cell density in a corneal-endothelial partially damaged primate model. (A) Slit-lamp microscopy examination revealed that both Y-27632–treated and –nontreated corneas recovered their transparency 1 month after cryoinjury. (B) In the control group, noncontact specular microscopy shows enlarged corneal endothelium migrating into the damaged area at the density of approximately 1500 cells/mm2 1 week after the injury. However, corneal endothelium of the Y-27632–treated group was reconstructed without compensatory enlargement with a normal cell density of approximately 3000 cells/mm2. (C) Noncontact specular microscopy analysis revealed that the CEC density was significantly higher in the Y-27632–treated group than in the control group throughout the 4-week observation period (*P < 0.01).
Figure 3
 
ROCK inhibitor Y-27632 eye drops promoted the recovery of cell density in a corneal-endothelial partially damaged primate model. (A) Slit-lamp microscopy examination revealed that both Y-27632–treated and –nontreated corneas recovered their transparency 1 month after cryoinjury. (B) In the control group, noncontact specular microscopy shows enlarged corneal endothelium migrating into the damaged area at the density of approximately 1500 cells/mm2 1 week after the injury. However, corneal endothelium of the Y-27632–treated group was reconstructed without compensatory enlargement with a normal cell density of approximately 3000 cells/mm2. (C) Noncontact specular microscopy analysis revealed that the CEC density was significantly higher in the Y-27632–treated group than in the control group throughout the 4-week observation period (*P < 0.01).
Figure 4
 
Clinical trial of ROCK inhibitor Y-27632 eye drops for treating patients with central corneal edema and diffuse corneal edema. (A, B) Representative case of a central corneal edema patient is shown. Before treatment, central corneal edema was detected in patient 1. (C, D) Six months after treatment, the corneal edema was significantly reduced and visual acuity recovered from logMAR 0.70 to −0.18. (E, F) Representative case of a diffuse corneal edema patient is shown. Before treatment, diffuse corneal edema due to argon laser iridotomy-induced bullous keratopathy (ALI-BK) was observed. (G, H) Six months after treatment, the corneal edema persisted and recovery of visual acuity was not obtained. (I) The corneal endothelium of case 1 observed by noncontact-specular microscopy before (A, B) and 6 months after treatment (C, D). Before treatment, we could not obtain clear image of corneal endothelium from the center part of cornea due to corneal edema (A). In contrast, some endothelial cells with guttae were observed para-central area of the same eye (B). Six months after treatment, specular microscopic images were obtained from both of center (C) and peripheral cornea (D). Approximate cell density after treatment was 1200 to 1500 cells/mm2 in both areas. (J) In the central corneal edema patients, central corneal thickness was reduced 6 months after treatment compared to pretreatment levels. In contrast, the central corneal thickness did not reduce in eyes with diffuse corneal edema.
Figure 4
 
Clinical trial of ROCK inhibitor Y-27632 eye drops for treating patients with central corneal edema and diffuse corneal edema. (A, B) Representative case of a central corneal edema patient is shown. Before treatment, central corneal edema was detected in patient 1. (C, D) Six months after treatment, the corneal edema was significantly reduced and visual acuity recovered from logMAR 0.70 to −0.18. (E, F) Representative case of a diffuse corneal edema patient is shown. Before treatment, diffuse corneal edema due to argon laser iridotomy-induced bullous keratopathy (ALI-BK) was observed. (G, H) Six months after treatment, the corneal edema persisted and recovery of visual acuity was not obtained. (I) The corneal endothelium of case 1 observed by noncontact-specular microscopy before (A, B) and 6 months after treatment (C, D). Before treatment, we could not obtain clear image of corneal endothelium from the center part of cornea due to corneal edema (A). In contrast, some endothelial cells with guttae were observed para-central area of the same eye (B). Six months after treatment, specular microscopic images were obtained from both of center (C) and peripheral cornea (D). Approximate cell density after treatment was 1200 to 1500 cells/mm2 in both areas. (J) In the central corneal edema patients, central corneal thickness was reduced 6 months after treatment compared to pretreatment levels. In contrast, the central corneal thickness did not reduce in eyes with diffuse corneal edema.
Table
 
Demographic Data of the Patients Involved in the ROCK Inhibitor Eye Drop Clinical Trial
Table
 
Demographic Data of the Patients Involved in the ROCK Inhibitor Eye Drop Clinical Trial
Eye Sex Age Type of Edema Cause of Endothelial Decompensation Central Corneal Thickness, μm BCVA, logMar Ocular Complications
Pre 6M Pre 6M
1 L M 52 Central Fuchs' dystrophy 703 568 0.7 −0.18 None
2 R F 75 Central Fuchs' dystrophy 809 (722:DSAEK) 1 0.7 Cataract
3 L F 57 Central Fuchs' dystrophy 682 663 0.52 0.52 None
4 L F 62 Central Fuchs' dystrophy 759 687 0.7 0.52 Myopic CRA
5 L F 76 Diffuse Laser iridotomy 683 506 1 1.7 Cataract
6 L F 70 Diffuse Laser iridotomy 920 920 0.7 1.52 Cataract
7 L F 72 Diffuse Laser iridotomy 827 827 0.7 0.7 Cataract
8 R F 72 Diffuse Pseudoexfoliation syndrome 721 757 0.4 1 Cataract
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