Extensive misalignment of plate-haptic rotationally asymmetric multifocal toric intraocular lens.

PURPOSE: Plate-haptic rotationally asymmetric multifocal toric intraocular lenses (IOL) (Lentis Comfort Toric) occasionally rotate extensively after surgery. We conducted the current study to investigate the incidence of extensive misalignment of this IOL and its association with clinical parameters. STUDY DESIGN: Retrospective case series. SUBJECTS AND METHODS: Data were collected from patients who had undergone phacoemulsification and implantation of a plate-haptic multifocal toric IOL. RESULTS: Among 332 eyes, extensive misalignment of toric IOLs ≥ 45º occurred in 3.3% (11 eyes). The amount of misalignment in eyes with extensive misalignment was 81.6 ± 22.9º, while in those without extensive misalignment, it was 3.0 ± 2.7º. The eyes with extensive misalignment showed significantly greater axial length (p < 0.001), larger corneal diameter (p = 0.034), and flatter corneas (p = 0.044) than those without extensive misalignment. Repositioning surgery to correct toric IOL misorientation was conducted in 9 eyes between 7 and 28 days after cataract surgery. In 2 eyes, repositioning surgery was carried out twice. CONCLUSIONS: In majority of cases plate-haptic multifocal toric IOLs showed satisfactory rotational stability, but extensive misalignment ≥ 45º occurred in 3.3% of cases.

Long-term outcomes of cataract surgery with toric intraocular lens implantation by the type of preoperative astigmatism.

Surgical outcomes of toric intraocular lens (IOL) implantation for 8 years after surgery were analyzed. Data were retrospectively collected in 176 eyes of 176 patients before and 1 month, 1, 3, 5, and 8 years after phacoemulsification and implantation of a toric IOL. Preoperative corneal and postoperative manifest astigmatism was analyzed by converting to power vector notations; horizontal/vertical (J0) and oblique (J45) astigmatism components. Toric IOL implantation significantly reduced pre-existing astigmatism by decreasing J0 in eyes with preoperative with-the-rule (WTR) astigmatism, increasing J0 in eyes with against-the-rule (ATR) astigmatism, and correcting J45 in eyes with oblique astigmatism. After surgery, the eyes with preoperative ATR astigmatism showed a significant ATR astigmatic shift, and J0 at 5 and 8 years was significantly smaller than that at 1 month postoperatively. Uncorrected distance visual acuity was also significantly worse at 5 and 8 years than at 1 month postoperatively. In eyes with WTR and oblique astigmatism, the effects of toric IOLs on astigmatism and visual acuity were sustained for 8 years. The long-term astigmatism-correcting effects did not differ among the models of toric IOL used in this study, SN6AT3-8 (Alcon Laboratories). In eyes with preoperative ATR astigmatism, astigmatism-correcting effects of toric IOLs decreased at 5 years and later postoperatively, indicating that overcorrection may be considered at the time of cataract surgery. In eyes with WTR and oblique astigmatism, the effects of toric IOLs were maintained throughout the 8-year follow-up period.

Influence of Dry Eye Disease on the Measurement Repeatability of Corneal Curvature Radius and Axial Length in Patients with Cataract.

The influence of dry eye disease (DED) on ocular biometric measurements is unclear. We aimed to investigate the effect of DED on the repeatability of ocular biometric measurements in cataract patients. Overall, 114 eyes scheduled for cataract surgery were enrolled. Before surgery, DED parameters including tear film break-up time (BUT), corneal and conjunctival staining scores, and subjective symptoms were examined. Corneal curvature radius and axial length were assessed twice on the same day using IOLMaster-500 (Carl Zeiss Meditec), and the absolute difference between the two measurements was calculated and used as an index of measurement repeatability. The measurement repeatability of the steep meridian of corneal curvature radius was significantly worse in eyes with DED than in those without DED (p = 0.044) and was significantly associated with BUT (r = -0.206, p = 0.031). The measurement repeatability of axial length was negatively correlated with BUT (r = -0.199, p = 0.041) and positively correlated with the corneal staining score (r = 0.253, p = 0.009). In conclusion, the measurement repeatability of corneal curvature radius declined in eyes with DED. Shortened BUTs were associated with a deterioration in the measurement repeatability of corneal curvature radius and axial length.

Influence of frosted haptics on rotational stability of toric intraocular lenses.

We investigated the unfolding property and rotational stability of a new toric intraocular lens (IOL); TECNIS toric II (toric-II, ZCW, Johnson & Johnson) that is an improved version of TECNIS toric IOL (toric-I, ZCV). Both IOLs are based on an identical platform, except for the frosted haptics with toric-II IOL. The study consisted of two parts; experimental study and clinical, retrospective, case series. Experimental study indicated that the overall time from IOL ejection to unfolding to 11 mm was significantly shorter with toricII than toric-I IOLs (p = 0.032), due to the earlier separation of the haptics from the optic with toric-II IOL. Clinical study included 131 eyes of 99 patients who had undergone phacoemulsification and toric IOL implantation. At 3 months postoperatively, toric-II IOL showed significantly better rotational stability than toric-I IOL, including smaller residual manifest astigmatism (p = 0.018), less amount of axis misalignment from the intended axis (p = 0.04), lower incidence of misalignment > 10º (p = 0.0044), and less degree of prediction errors (p = 0.043). Postoperative uncorrected distance visual acuity tended to be better in the toric-II than in the toric-I groups, with marginal statistical difference (p = 0.057). TECNIS toric II IOL with the frosted haptics showed significantly better rotational stability than its predecessor, probably due to quicker unfolding and greater friction with the capsular bag.

Influence of posterior corneal astigmatism on the outcomes of toric intraocular lens implantation in eyes with oblique astigmatism.

PURPOSE: To assess whether the outcomes of toric intraocular lens (IOL) implantation in eyes with oblique astigmatism can be improved by direct measurements of posterior corneal astigmatism using anterior segment optical coherence tomography (AS-OCT) instead of by using anterior corneal measurements alone. STUDY DESIGN: Retrospective case series. METHODS: Two toric IOL power calculation methods were compared: anterior corneal astigmatism was used in the keratometry group, whilst total corneal astigmatism determined by ray tracing through the measured anterior and posterior corneal surfaces was used in the AS-OCT group. In a total of 279 eyes of 232 patients, subgroup analysis was conducted for with-the-rule (WTR) (85 eyes in the keratometry group and 34 eyes in the AS-OCT group), against-the-rule (ATR) (73/29 eyes), and oblique (26/32 eyes) astigmatism. RESULTS: In the WTR and ATR astigmatism groups, the uncorrected distance visual acuity (UDVA) was significantly better in the AS-OCT group than in the keratometry group (P = 0.012 and P < 0.001, Mann-Whitney test), and the residual astigmatism was significantly smaller in the AS-OCT group than in the keratometry group (P = 0.037 and P < 0.001). In eyes with oblique astigmatism, the UDVA (P = 0.299) and residual astigmatism (P = 0.373) of the keratometry and AS-OCT groups did not differ. CONCLUSION: Incorporation of posterior corneal astigmatism measured with AS-OCT can significantly improve the outcomes of toric IOL implantation in eyes with WTR and ATR astigmatism, but not in eyes with oblique astigmatism.

Comparison of incidence of repositioning surgery to correct misalignment with three toric intraocular lenses.

PURPOSE: To compare the incidence of re-orientating surgery to improve misalignment of three models of acrylic toric intraocular lenses: AcrySof toric intraocular lens (Alcon Laboratories, Inc.), TECNIS toric intraocular lens (Johnson & Johnson Vision, Inc.) and HOYA 355 toric intraocular lens (HOYA). METHODS: In this retrospective, multicenter case series, medical charts were reviewed for collecting data on realignment surgery of toric intraocular lenses at 10 ophthalmic surgical sites in Japan. RESULTS: Over all, intraocular lens repositioning surgery was conducted in 89 of 9430 eyes (0.944%) at an average of 10.5 ± 9.7 days after the initial cataract surgery. The incidence was 0.213% (11/5155), 1.797% (62/3451) and 1.942% (16/824) with AcrySof, TECNIS and HOYA toric intraocular lenses, respectively. The incidence was significantly lower with AcrySof than with other two brands of toric intraocular lenses (p < 0.0001). In those eyes which underwent reorientation surgery, the amount of misalignment was 26.4 ± 21.9°, 29.7 ± 15.4° and 28.1 ± 20.7° with AcrySof, TECNIS and HOYA toric intraocular lenses, respectively; there was no significant difference among groups (p = 0.821). The repositioning surgery significantly reduced misalignment in all three groups. CONCLUSION: The rotational stability was considerably different among toric intraocular lenses of different manufacturers. The incidence of repositioning surgery was significantly lower with AcrySof than with TECNIS and HOYA toric intraocular lenses.

Blue light-filtering and violet light-filtering hydrophobic acrylic foldable intraocular lenses: Intraindividual comparison.

PURPOSE: To compare the clinical outcomes after cataract surgery and implantation of a blue light-filtering IOL (AcrySof IQ SN60WF) or a violet light-filtering intraocular lens (IOL) (OptiBlue ZCB00V). SETTING: Four surgical sites in Japan. DESIGN: Prospective case series. METHODS: One eye of patients with bilateral cataract was randomly allocated to the blue light-filtering IOL and the fellow eye to the violet light-filtering IOL. Visual acuity and contrast sensitivity were assessed over 3 months. The incidence of cyanopsia was evaluated using the neutralization method. RESULTS: The study enrolled 110 eyes of 55 patients. There was no significant difference in visual acuity between the two IOLs. Based on the neutralization results 1 week postoperatively, 15 cases (27.8%) with the light-filtering IOL and 8 cases (14.8%) with the violent light-filtering IOL had cyanopsia; the difference reached statistical significance (P = .049). After 2 weeks, the difference in the incidence of cyanopsia was not significant. Postoperative contrast sensitivity under photopic condition at 1 week and 3 months and contrast sensitivity under mesopic conditions at 3 months were significantly better with the violet light-filtering IOL than with the blue light-filtering IOL (P < .05). CONCLUSIONS: The violet light-filtering IOL yielded highly satisfactory clinical outcomes, including reduction of cyanopsia and a potential improvement in contrast sensitivity. The different chromophores of the IOL and its different material and design might have contributed.

Effects of astigmatic defocus on binocular contrast sensitivity.

PURPOSE: To determine the effects of astigmatism on contrast sensitivity (CS). METHODS: Eighteen normal volunteers (30.5 ± 6.0 [mean ± SD] years) were recruited. After correcting each refractive error by spectacles, against-the-rule (ATR) or with-the-rule (WTR) astigmatism of +1.00, +2.00 and +3.00 D was intentionally produced in both eyes, and then binocular CS was measured. The cylindrical addition of different powers (+1.00-+3.00 D) was compensated with spherical lenses so that the spherical equivalent refraction became zero in each eye. Subsequently, the above cylindrical addition was monocularly induced, and binocular CS was measured again. The relation between CS and astigmatic power, axis, and monocular or binocular astigmatism was investigated. RESULTS: With binocular ATR and WTR astigmatism, increases in astigmatic power significantly correlated with decreases in the area under the log contrast sensitivity function (AULCSF). With monocular astigmatic defocus, astigmatic power addition did not affect AULCSF. With binocular astigmatic defocus of high-power (+2.00 and +3.00 D), ATR astigmatism deteriorated AULCSF more than WTR astigmatism. In a comparison between binocular and monocular astigmatic defocus, CS was significantly worse with binocular astigmatic defocus than with monocular astigmatic defocus at higher spatial frequencies regardless of astigmatic power. CONCLUSIONS: Binocular astigmatic defocus deteriorates CS depending on the amount of astigmatic power. ATR astigmatism reduces CS more than WTR astigmatism dose. In addition, binocular astigmatic defocus affects CS more severely than monocular astigmatic defocus especially at high spatial frequencies.

Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Corneal Endothelial Cell Proliferation.

Purpose: We have performed clinical research on cell-based therapy for corneal endothelial decompensation since 2013. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for promoting proliferation of human corneal endothelial cells (HCECs). Methods: HCECs were cultured in media supplemented with various low-molecular-weight compounds to screen for the effect of those compounds on cell proliferation. Activation of substrates of p38 MAPK and cell cycle regulatory proteins were evaluated by western blotting. Corneal endothelial wounds were created in a rabbit model, and p38 MAPK was applied in eye drop form, followed by evaluation of cell proliferation in the corneal endothelium by Ki67-immunostaining. Results: HCECs cultured with SB203580 exhibited hexagonal morphology and similar size and morphology, whereas control HCECs cultured without inhibitor exhibited monolayer morphology and varied in size and morphology. Flow cytometry demonstrated that cell proliferation was significantly increased by SB203580. Western blotting showed activation of ATF2 and HSP27 (substrates of p38 MAPK), and upregulation of cyclin D and downregulation of p27 were induced by inhibiting p38 MAPK. In the rabbit model, promotion of wound healing of the corneal endothelium was associated with significant upregulation of Ki67-positive proliferating cells following topical administration of SB203580 when compared with untreated endothelium (50.9% and 36.1%, respectively). Conclusions: Activation of p38 MAPK signaling due to culture stress might suppress the proliferation of HCECs, whereas a p38 MAPK inhibitor can counteract this activation and enable efficient in vitro HCEC expansion.

Immune Cells on the Donor Corneal Endothelium After Corneal Transplantation.

PURPOSE: The aim of this study was to investigate the existence of presumed immune cells observed by contact specular microscopy in patients who underwent penetrating keratoplasty (PK) and Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: This cross-sectional study was conducted on consecutive patients who underwent follow-up visits between January and March 2015 for previously performed PK or DSAEK. Presumed immune cell-suspected "cell-like white dots" were evaluated by scanning slit contact specular microscopy. The association between the grading of presumed immune cells with clinical parameters, such as corneal endothelial cell density, time after surgery, and the titer of steroid administration, was also investigated. RESULTS: A total of 54 eyes of 54 patients who underwent PK (32 eyes/32 patients) and DSAEK (22 eyes/22 patients) were evaluated, and suspected immune cells were observed in all patients. In the PK and DSAEK groups, the number of patients in the presumed immune cell grades 1, 2, 3, and 4 were 19, 10, 2, and 1 and 10, 8, 2, and 2, respectively (P = 0.663). No statistically significant association was found between the immune cell grades and the clinical parameters. CONCLUSIONS: Immune cells were observed on the corneal endothelial grafts in all 54 patients who underwent PK or DSAEK. Although the number of immune cells varied between patients and showed no correlation with clinical parameters, it would be beneficial to conduct a future prospective study to analyze the effect of immune cells on postoperative corneal endothelial cell loss.

Incidence and Outcomes of Repositioning Surgery to Correct Misalignment of Toric Intraocular Lenses.

PURPOSE: To analyze the incidence and appropriate timing of repositioning surgery to correct misalignment of acrylic foldable toric intraocular lenses (IOLs). DESIGN: Retrospective, multicenter case series. PARTICIPANTS: Patients who had undergone phacoemulsification and implantation of toric IOL at 8 surgical sites. METHODS: Patient charts were reviewed to collect data on repositioning surgery of toric IOLs. MAIN OUTCOME MEASURES: Incidence, timing, and outcomes of repositioning surgery. RESULTS: Among 6431 eyes implanted with toric IOLs, 42 eyes (0.653%) of 42 patients underwent repositioning surgery at an average of 9.9±7.5 days (range, 0-30 days) after IOL implantation. The repositioning surgery significantly reduced misalignment from 32.9°±15.7° to 8.8°±9.7° (P < 0.001), which was measured at 7.6±5.0 weeks postoperatively. Refractive cylinder was significantly reduced from 2.4±1.1 diopters (D) to 1.1±0.8 D (P < 0.001). There was a significant negative correlation between the interval from cataract surgery to repositioning procedure and the degree of residual misalignment (r = -0.439, P < 0.001). The residual misalignment was 13.1°±13.5° when the repositioning surgery was performed within 6 days after cataract surgery, whereas the residual misalignment was 6.3°±5.9° when the IOL was repositioned 7 days or later (P < 0.001). In 2 eyes that were treated within 24 hours after cataract surgery, the IOL re-rotated significantly, and additional surgical intervention was required. CONCLUSIONS: Toric IOLs were repositioned in 0.653% of cases. A relationship was found between the timing of repositioning surgery and surgical outcome. These data suggest that repositioning surgery should be performed 1 week after IOL implantation.

Morphometric assessment of normal human ciliary body using ultrasound biomicroscopy.

PURPOSE: To quantitatively assess the biometry of the ciliary body in normal human eyes using ultrasound biomicroscopy. METHODS: We evaluated 85 eyes of 85 normal subjects (35 men and 50 women), whose age ranged from 11 to 86 years (mean ± SD, 56.8 ± 20.4 years). The eyes were assessed along the 3-, 6-, 9-, and 12-o'clock meridians relative to the center of the cornea. Clinical data were collected, including age, axial length, ciliary body length (CBL), ciliary body thickness (CBT), anterior chamber depth, iris root thickness, trabecular-iris angle, and scleral-ciliary process angle. Axial length was measured using A-scan ultrasonography. RESULTS: CBL and CBT tended to be larger in the superior than in the inferior quadrant, but the differences among the four quadrants were not statistically significant. The average CBL showed a significant positive correlation with the average CBT (r = 0.40, P < 0.001). Average CBL and CBT were significantly correlated with axial length (r = 0.33, P = 0.031; r = 0.46, P < 0.01 respectively). In addition, the average CBL was significantly correlated with anterior chamber depth (r = 0.23, P < 0.05), trabecular-iris angle (r = 0.29, P = 0.01), and scleral-ciliary process angle (r = 0.40, P < 0.001). CONCLUSIONS: Ultrasound biomicroscopic imaging demonstrated that the ciliary body is similar in size in all circumferences, and eyes with longer axial length have an elongated and thicker ciliary body. The values obtained in the present study may serve as standard clinical references.

A Surgical Cryoprobe for Targeted Transcorneal Freezing and Endothelial Cell Removal.

PURPOSE: To examine the effects of transcorneal freezing using a new cryoprobe designed for corneal endothelial surgery. METHODS: A freezing console employing nitrous oxide as a cryogen was used to cool a series of different cryoprobe tip designs made of silver for high thermal conductivity. In vitro studies were conducted on 426 porcine corneas, followed by preliminary in vivo investigations on three rabbit corneas. RESULTS: The corneal epithelium was destroyed by transcorneal freezing, as expected; however, the epithelial basement membrane remained intact. Reproducible endothelial damage was optimally achieved using a 3.4 mm diameter cryoprobe with a concave tip profile. Stromal edema was seen in the pre-Descemet's area 24 hrs postfreeze injury, but this had been resolved by 10 days postfreeze. A normal collagen fibril structure was seen 1 month postfreeze, concurrent with endothelial cell repopulation. CONCLUSIONS: Transcorneal freezing induces transient posterior stromal edema and some residual deep stromal haze but leaves the epithelial basement membrane intact, which is likely to be important for corneal re-epithelialization. Localized destruction of the endothelial monolayer was achieved in a consistent manner with a 3.4 mm diameter/concave profile cryoprobe and represents a potentially useful approach to remove dysfunctional corneal endothelial cells from corneas with endothelial dysfunction.

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

Purpose: Corneal endothelial cell density undergoes a progressive decrease for many years after transplantation, eventually threatening patients with late endothelial failure. The purpose of this study was to investigate the possibility of an immunologic response in successfully grafted corneal endothelium. Methods: The corneal endothelium of patients who had undergone corneal transplantation was evaluated by specular microscopy. Rabbit models were subjected to penetrating keratoplasty (PK) with either syngeneic or allogeneic corneal transplants and Descemet's stripping endothelial keratoplasty (DSEK) with allogeneic corneal transplants. The presence of immune cells and expression of proinflammatory cytokines were determined by immunostaining. The corneal endothelium and immune cells were also evaluated by scanning electron microscopy. Results: Scanning slit contact specular microscopy of patients with no features of graft rejection revealed cell-like white dots on the grafted corneal endothelium. The corneal endothelium of the allogeneic PK and DSEK rabbit models displayed the presence of immune cells, including CD4+ T-helper cells, CD8+ cytotoxic T cells, CD20+ B lymphocytes, CD68+ macrophages, and neutrophils, but these immune cells were rarely observed in the syngeneic PK model. These immune cells also produced proinflammatory cytokines. Notably, some of the corneal endothelial cells situated near these immune cells exhibited features of apoptosis. Conclusions: T lymphocytes, B lymphocytes, macrophages, and neutrophils are present on the grafted corneal endothelium in both PK and DSEK allogeneic rabbit models. The potential involvement of immune cells as an underlying pathophysiology for late endothelial failure deserves further examination.

Rho kinase inhibitor enables cell-based therapy for corneal endothelial dysfunction.

The corneal endothelium maintains corneal transparency; consequently, its dysfunction causes severe vision loss. Tissue engineering-based therapy, as an alternative to conventional donor corneal transplantation, is anticipated to provide a less invasive and more effective therapeutic modality. We conducted a preclinical study for cell-based therapy in a primate model and demonstrated regeneration of the corneal endothelium following injection of cultured monkey corneal endothelial cells (MCECs) or human CECs (HCECs), in combination with a Rho kinase (ROCK) inhibitor, Y-27632, into the anterior chamber. We also evaluated the safety and efficacy of Good Manufacturing Practice (GMP)-grade HCECs, similar to those planned for use as transplant material for human patients in a clinical trial, and we showed that the corneal endothelium was regenerated without adverse effect. We also showed that CEC engraftment is impaired by limited substrate adhesion, which is due to actomyosin contraction induced by dissociation-induced activation of ROCK/MLC signaling. Inclusion of a ROCK inhibitor improves efficiency of engraftment of CECs and enables cell-based therapy for treating corneal endothelial dysfunction as a clinically relevant therapy.

Effect of the Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) on Corneal Endothelial Wound Healing.

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.

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

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

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

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

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

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

Involvement of cyclin D and p27 in cell proliferation mediated by ROCK inhibitors Y-27632 and Y-39983 during corneal endothelium wound healing.

PURPOSE: To investigate the molecular mechanism of Rho-associated kinase (ROCK) inhibitors Y-27632 and Y-39983 on corneal endothelial cell (CEC) proliferation and their wound-healing effect. METHODS: The expression of G1 proteins of the cell cycle and expression of phosphorylated Akt in monkey CECs (MCECs) treated with Y-27632 were determined by Western blotting. The effect of Y-39983 on the proliferation of MCECs and human CECs (HCECs) was evaluated by both Ki67 staining and incorporation of BrdU. As an in vivo study, Y-39983 was topically instilled in a corneal-endothelial partially injured rabbit model, and CEC proliferation was then evaluated. RESULTS: Investigation of the molecular mechanism of Y-27632 on CEC proliferation revealed that Y-27632 facilitated degradation of p27Kip1 (p27), and promoted the expression of cyclin D. When CECs were stimulated with Y-27632, a 1.7-fold increase in the activation of Akt was seen in comparison to the control after 1 hour. The presence of LY294002, the PI 3-kinase inhibitor, sustained the level of p27. When the efficacy of Y-39983 on cell proliferation was measured in a rabbit model, Y-39983 eye-drop instillation demonstrated rapid wound healing in a concentration range of 0.095 to 0.95 mM, whereas Y-27632 demonstrated rapid wound healing in a concentration range of 3 to 10 mM. CONCLUSIONS: These findings show that ROCK inhibitors employ both cyclin D and p27 via PI 3-kinase signaling to promote CEC proliferation, and that Y-39983 may be a more potent agent than Y-27632 for facilitating corneal endothelium wound healing.