Senescent characteristics of human corneal endothelial cells upon ultraviolet-A exposure.

PURPOSE: The objective of this study was to investigate the senescent phenotypes of human corneal endothelial cells (hCEnCs) upon treatment with ultraviolet (UV)-A. METHODS: We assessed cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation and expression of senescence markers (p16 and p21) in hCEnCs exposed to UV-A radiation, and senescent hCEnCs induced by ionizing radiation (IR) were used as positive controls. We performed RNA sequencing and proteomics analyses to compare gene and protein expression profiles between UV-A- and IR-induced senescent hCEnCs, and we also compared the results to non-senescent hCEnCs. RESULTS: Cells exposed to 5 J/cm2 of UV-A or to IR exhibited typical senescent phenotypes, including enlargement, increased SA-ß-gal activity, decreased cell proliferation and elevated expression of p16 and p21. RNA-Seq analysis revealed that 83.9% of the genes significantly upregulated and 82.6% of the genes significantly downregulated in UV-A-induced senescent hCEnCs overlapped with the genes regulated in IR-induced senescent hCEnCs. Proteomics also revealed that 93.8% of the proteins significantly upregulated in UV-A-induced senescent hCEnCs overlapped with those induced by IR. In proteomics analyses, senescent hCEnCs induced by UV-A exhibited elevated expression levels of several factors part of the senescence-associated secretory phenotype. CONCLUSIONS: In this study, where senescence was induced by UV-A, a more physiological stress for hCEnCs compared to IR, we determined that UV-A modulated the expression of many genes and proteins typically altered upon IR treatment, a more conventional method of senescence induction, even though UV-A also modulated specific pathways unrelated to IR.

Establish an In Vitro Cell Model to Explore the Impacts of UVA on Human Corneal Endothelial Wound Healing.

PURPOSE: To provide scientific data for clinical practice in making strategies for accelerating corneal endothelial wound healing, we investigated the impact of UVA on the corneal endothelial wound healing process and the underlying mechanism using an in vitro cell model. MATERIALS AND METHODS: An in vitro cell model for corneal endothelial wound healing was established by scratching the in vitro cultured human corneal endothelial cell (HCEnC) confluent layer. Then, we investigated the impacts of UVA irradiation and Ascorbic acid-2-phosphate (Asc-2p) on the wound healing process of the in vitro HCEnC model by examining wound-healing index, F-actin+ rate, Ki-67+ rate, and ROS production. RESULTS: After scratching, the Ki-67+ and F-actin+ HCEnCs occupied the scratching gap. Furthermore, the F-actin+ rates were significantly higher than Ki-67+ rates in the wound closure area. After irradiated with UVA, the wound-healing indexes, Ki-67+ rates and F-actin+ rates of the wound-healing model significantly reduced, whereas the ROS production significantly increased in a dose-dependent manner. Pretreatment with Asc-2p significantly reduced the ROS production as well as increased the wound-healing indexes, Ki-67+rates and F-actin+ rates of the UVA irradiated wound-healing model. CONCLUSION: The migration of HCEnC plays a major role in the wound healing process of the established cell model, which is like the wound healing process in vivo. UVA decreases the wound closure of the in vitro HCEnC model dose-dependently, while antioxidant Asc-2p can attenuate the damage to UVA to HCEnCs probably via reducing ROS to improve their migration.

Quantitative and Morphological Corneal Endothelial Changes After Selective Laser Trabeculoplasty and Retinal Photocoagulation.

PURPOSE: Selective laser trabeculoplasty (SLT) and retinal photocoagulation (RP) are two common laser procedures often performed at a wavelength of 532 nm, and may affect the corneal endothelium. This study used corneal specular microscopy to determine the impact of these procedures on the corneal endothelium. DESIGN: Retrospective cohort study in a private practice. METHODS: There were 249 eyes from 136 consecutive patients who underwent SLT for open-angle glaucoma and 132 eyes from 74 patients who underwent RP included. Corneal specular microscopy was performed immediately before and after each procedure and at 1-month postprocedure. Microscopy data included quantitative measures, such as cell density and central corneal thickness, and morphological measures, including percentage of hexagonal cells and coefficient of variation in cell area. RESULTS: There was a small (just over 1%) reduction in corneal endothelial cell count from pre-SLT to post-SLT (P = 0.008), and a statistically significant recovery at 1 month (P = 0.04). Central corneal thickness also transiently increased from pre-SLT to post-SLT (P = 0.03). Although polymegathism was unchanged, changes in pleomorphism were observed (P = 0.03). The only change in the RP group was an increase in polymegathism between pre-RP and post-RP (P = 0.001). CONCLUSIONS: SLT has measurable effects on both quantitative and morphological characteristics of the corneal endothelium, which seem to be transient. RP has fewer measurable effects, likely because, although the total laser energy is similar, it is delivered over a much longer time (3 ns versus 0.1 s). The changes observed in both procedures are minor and unlikely to be of clinical significance.

Ultraviolet A light induces DNA damage and estrogen-DNA adducts in Fuchs endothelial corneal dystrophy causing females to be more affected.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.

Essential role of ultraviolet radiation in the decrease of corneal endothelial cell density caused by pterygium.

PURPOSE: To investigate the essential role of ultraviolet (UV) radiation in the decrease of corneal endothelial cell density (ECD) caused by pterygium. METHODS: In this retrospective controlled study, 76 consecutive patients with unilateral primary pterygium were enrolled from January 2016 to August 2016. The Central corneal ECD was measured in both eyes. Divide the subjects into two subgroups according to the UV exposure time, and the difference of corneal ECD between both eyes was analyzed using independent samples t-test. The relationship between the percentage of difference of corneal ECD and the disease duration, the percentage of pterygium to the cornea and the astigmatism value were analyzed. RESULTS: For subjects with longer UV exposure, the average of ECD in pterygium group and control group were, respectively, 2264.83 ± 349.78 cells/mm2 and 2477.16 ± 300.36 cells/mm2 (P = 0.004). For subjects with short UV exposure, the average of ECD in pterygium group and control group were, respectively, 2596.46 ± 373.24 cells/mm2 and 2690.66 ± 321.34 cells/mm2 (P = 0.262). The decrease of corneal ECD was positively correlated with the period of the disease (P = 0.035). There was no statistical correlation between a decrease in the corneal ECD and the percentage of pterygium to cornea (R = -0.055, P = 0.635) and the astigmatism value in the pterygium eye (R = -0.135, P = 0.246). CONCLUSIONS: UV radiation exposure played a significant role in the effect of pterygium decreasing the corneal ECD.

The protective effect of simvastatin against ultraviolet B-induced corneal endothelial cell death.

Purpose: Excessive ultraviolet B (UVB) exposure causing corneal endothelium injury, including apoptosis, is a serious condition. Therefore, drugs that can inhibit apoptosis in corneal endothelial cells represent an effective strategy. Simvastatin is widely used as a specific inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase, can reduce levels of low density lipoprotein (LDL) cholesterol, and exerts anti-inflammatory effects. However, the protective effect of simvastatin on corneal endothelial cells remains unclear. Therefore, the aim of this study was to elucidate whether UVB promotes the initiation of apoptosis in corneal endothelial cells and injury reversible by simvastatin treatment. Methods: We detected the cell viability, subG1 population, and caspase-3 activity. Results: Results showed that simvastatin alleviates UVB-induced cell death, cell apoptosis, and caspase-3 activity. Conclusion: Our findings indicated that simvastatin alleviated UVB-induced corneal endothelial cell apoptosis via caspase-3 activity.

Elucidating the molecular basis of PPCD: Effects of decreased ZEB1 expression on corneal endothelial cell function.

PURPOSE: To investigate the functional role that the zinc e-box binding homeobox 1 (ZEB1) gene, which underlies the genetic basis of posterior polymorphous corneal dystrophy 3 (PPCD3), plays in corneal endothelial cell proliferation, apoptosis, migration, and barrier function. METHODS: A human corneal endothelial cell line (HCEnC-21T) was transfected with siRNA targeting ZEB1 mRNA. Cell proliferation, apoptosis, migration, and barrier assays were performed: Cell proliferation was assessed with cell counting using a hemocytometer; cell apoptosis, induced by either ultraviolet C (UVC) radiation or doxorubicin treatment, was quantified by measuring cleaved caspase 3 (cCASP3) protein levels; and cell migration and barrier function were monitored with electric cell-substrate impedance sensing (ECIS). RESULTS: ZEB1 knockdown in HCEnC-21T cells transfected with siRNA targeting ZEB1 did not result in a significant difference in cell proliferation when compared with control. Although knockdown of ZEB1 in HCEnC-21T cells sensitized the cells to UV-induced apoptosis, ZEB1 knockdown did not alter the cells' susceptibility to doxorubicin-induced apoptosis, as measured with cCASP3 protein levels, compared with controls. Similarly, no difference was observed in cell migration following ZEB1 knockdown. However, cell barrier function increased significantly following ZEB1 knockdown. CONCLUSIONS: The corneal endothelium in PPCD3 is characterized by morphologic, anatomic, and molecular features that are more consistent with an epithelial-like rather than an endothelial-like phenotype. Although these characteristics have been well documented, we demonstrate for the first time that susceptibility to UV-induced apoptosis and cell barrier function are significantly altered in the setting of reduced ZEB1. The significance of an altered cellular response to apoptotic stimuli and increased cell barrier function in the pathobiology of PPCD remains to be fully elucidated.

UV-A Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3-Dependent Apoptosis in Corneal Endothelial Cells.

PURPOSE: To examine whether Nrf2-regulated antioxidant defense and p53 are activated in human corneal endothelial cells (CEnCs) by environmental levels of ultraviolet A (UV-A), a known stimulator of oxidative stress. METHODS: Immortalized human CEnCs (HCEnCi) were exposed to UV-A fluences of 2.5, 5, 10, or 25 J/cm2, then allowed to recover for 3 to 24 hours. Control HCEnCi did not receive UV-A. Reactive oxygen species (ROS) were measured using H2DCFDA. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. Levels of Nrf2, HO-1, NQO-1, p53, and caspase3 were detected by immunnoblotting or real-time PCR. Activated caspase3 was measured by immunoblotting and a fluorescence assay. RESULTS: Exposure of HCEnCi to 5, 10, and 25 J/cm2 UV-A increased ROS levels compared with controls. Nrf2, HO-1, and NQO-1 mRNA increased 1.7- to 3.2-fold at 3 and 6 hours after irradiation with 2.5 and 5 J/cm2 UV-A. At 6 hours post irradiation, UV-A (5 J/cm2) enhanced nuclear Nrf2 translocation. At 24 hours post treatment, UV-A (5, 10, and 25 J/cm2) produced a 1.8- to 2.8-fold increase in phospho-p53 and a 2.6- to 6.0-fold increase in activated caspase3 compared with controls, resulting in 20% to 42% cell death. CONCLUSIONS: Lower fluences of UV-A induce Nrf2-regulated antioxidant defense and higher fluences activate p53 and caspase3, indicating that even near-environmental levels of UV-A may affect normal CEnCs. This data suggest that UV-A may especially damage cells deficient in antioxidant defense, and thus may be involved in the etiology of Fuchs' endothelial corneal dystrophy (FECD).

Evaluation of UVA Cytotoxicity for Human Endothelium in an Ex Vivo Corneal Cross-linking Experimental Setting.

PURPOSE: To evaluate endothelial cytotoxicity after exposure of human corneas to ultraviolet-A (UVA) (λ = 365 nm; 5.4 J/cm(2)) in an experimental ex vivo corneal cross-linking setting. METHODS: Sixteen pairs of human donor corneas were cut into two pieces. One piece of each cornea was treated with 0.025% riboflavin solution prior to UVA irradiation (5 minutes; 18 mW/cm(2)), whereas the other piece was not irradiated but treated with riboflavin (right eye) or preservation medium (left eye). By irradiating from the endothelial side, the UVA dosage applied to endothelial cells exceeded at least eight times the cytotoxic threshold established in animal models (0.65 J/cm(2)). Endothelial cell counts were performed by two independent investigators after storage (4 to 5 days at 31 °C) and staining (trypan blue, alizarin red). Normality (Q-Q plot; Shapiro-Wilk test) and equivalence (mixed-effects modeling with a 10% equivalence threshold) of the endothelial cell counts of the different groups were evaluated. RESULTS: Equivalence of mean endothelial cell density between both groups was observed: 2,237 ± 208 cells/mm(2) in UVA-irradiated pieces and 2,290 ± 281 cells/mm(2) in control pieces (mean difference of 53 ± 240 cells/mm(2) between both groups). CONCLUSIONS: Despite direct irradiation of human donor endothelium using the clinical dosage for cross-linking, equivalence in endothelial cell counts was observed between irradiated tissues and controls. Ex vivo human corneal endothelial cells seem to be far more resistant to riboflavin-enhanced UVA irradiation than previously estimated by animal experiments.

Apoptosis in Rat Cornea After In Vivo Exposure to Ultraviolet Radiation at 300 nm.

PURPOSE: Peak toxicity for in vivo ultraviolet radiation (UVR) exposure to the lens is in the 300-nm wavelength region. However, little is known about corneal cell damage at 300 nm. The purpose of the study was to determine the time evolution of apoptosis in the cornea after in vivo exposure to 300-nm UVR. METHODS: Altogether, 16 Sprague Dawley rats were divided into 4 groups and unilaterally exposed to 5 kJ/m UVR (λmax: 300 nm; λ0.5: 10 nm) for 15 minutes. After a predetermined latency period of 1, 5, 24, and 120 hours, depending on the group, the animals were killed and eyes were enucleated. Eye globes were further cryosectioned in 10-µm thick midsagittal sections. For the detection of apoptosis, the TUNEL method was applied. RESULTS: TUNEL-positive signals were observed in the superficial epithelial cells in the exposed and control eyes at all latency periods. At 5 hours, TUNEL staining was detected in the exposed corneas in epithelial cells, keratocytes, and endothelial cells with a maximum signal at 24 hours. At 120 hours, no TUNEL staining was found in endothelial cells and only occasionally in keratocytes in exposed corneas. Signs of ulceration and stromal thinning were observed at 120 hours. CONCLUSIONS: UVR in the 300-nm wavelength region induces TUNEL staining in all 3 corneal layers. TUNEL staining of all 3 corneal layers is an early postexposure event observed after a 5-hour latency period. Corneal sterile keratolysis occurs in the time window of 24 to 120 hours probably induced by neutrophils.

Comparative study of corneal endothelial cell damage after femtosecond laser assisted deep stromal dissection.

PURPOSE: To find a relatively safe designed stromal bed thickness to avoid endothelial damage for lamellar keratoplasty with an Allegretto Wavelight FS200 femtosecond laser. METHODS: Twelve rabbits were randomly divided into 50 µm and 150 µm groups according to the anticipated residue stromal bed thickness preparation with a femtosecond laser. Six rabbits without laser cutting were used as a control group. Central endothelial images were analyzed with in vivo confocal microscopy and scanning electron microscopy. The apoptosis of endothelium was evaluated with Hoechst 33342 staining and a TUNEL assay. RESULTS: The endothelium of the 50 µm group had extensive injuries upon in vivo confocal and scanning electron microscopic observation, and minor injuries were observed in the 150 µm group. Moreover, more apoptotic cells were observed in the 50 µm group. CONCLUSIONS: When using a FS200 femtosecond laser assisted anterior lamellar keratoplasty, there was minor endothelium damage with a 150 µm stromal bed, and a more than 150 µm thickness stromal bed design may prevent the damage of corneal endothelium.

Decreased DJ-1 leads to impaired Nrf2-regulated antioxidant defense and increased UV-A-induced apoptosis in corneal endothelial cells.

PURPOSE: To investigate the role of DJ-1 in Nrf2-regulated antioxidant defense in corneal endothelial cells (CECs) at baseline and in response to ultraviolet A (UV-A)-induced oxidative stress. METHODS: DJ-1-deficient CECs were obtained by transfection of an immortalized normal human corneal endothelial cell line (HCECi) with DJ-1 small interfering RNA (siRNA) or by isolation of CECs from ex vivo corneas of DJ-1 knockout mice. Levels of reactive oxygen species (ROS), protein carbonyls, Nrf2 subcellular localization, Nrf2 target genes, and protein interaction between Keap1/Nrf2 and Cul3/Nrf2 were compared between normal and DJ-1-deficient CECs. Oxidative stress was induced by irradiating HCECi cells with UV-A, and cell death and levels of activated caspase3 and phospho-p53 were determined. RESULTS: DJ-1 siRNA-treated cells exhibited increased levels of ROS production and protein carbonyls as well as a 2.2-fold decrease in nuclear Nrf2 protein when compared to controls. DJ-1 downregulation led to attenuated gene expression of Nrf2 and its target genes HO-1 and NQO1. Similar levels of Nrf2 inhibitor, Keap1, and Cul3/Nrf2 and Keap1/Nrf2 were observed in DJ-1 siRNA-treated cells as compared to controls. Ultraviolet A irradiation resulted in a 3.0-fold increase in cell death and elevated levels of activated caspase3 and phospho-p53 in DJ-1 siRNA-treated cells compared to controls. CONCLUSIONS: Downregulation of DJ-1 impairs nuclear translocation of Nrf2, causing decreased antioxidant gene expression and increased oxidative damage. The decline in DJ-1 levels leads to heightened CEC susceptibility to UV-A light by activating p53-dependent apoptosis. Targeting the DJ-1-Nrf2 axis may provide a potential therapeutic approach for enhancing antioxidant defense in corneal endothelial disorders.

Does beta-ray emitting therapy of ciliary body tumors decrease central corneal endothelial cell density.

PURPOSE: To evaluate the effect of plaque radiation therapy using beta-radioactive isotope for anterior segment tumors on the density and morphology of endothelial cells. 
 METHODS: Endothelial cell density (ECD) and morphometry in 15 eyes of 15 patients with ciliary body tumor was examined prior to and 6 months after radiation therapy. After irradiation, central ECD values were also compared with peripheral (2.0 mm from limbus) ECD values measured around the plaque. The ECD, average cell area, coefficient of variation of cell area, and pachymetry measurements were conducted with contact specular microscopy.
 RESULTS: The mean corrected ECD values prior to irradiation were 2147 ± 128 cells/mm2 and 2050 ± 108 cells/mm2 after the radiation therapy. After irradiation, the mean peripheral ECD values were 2056 ± 101 cells/mm2. A significant decrease in ECD values was observed after radiation (p = 0.007). Peripheral ECD values measured around the plaque showed no significant difference (p = 0.86) as compared to central ECD values.
 CONCLUSIONS: According to our measurements, plaque therapy for tumors in the anterior segment decreases ECD significantly, but not highly, even in case of plaques containing beta-radiation isotope, and the plaques are not placed direct on the cornea surface. The decreased ECD causes no changes in corneal thickness or transparency, but it may have an influence on a subsequent cataract surgery, which generates further endothelial loss.

Comparison of central corneal thickness using ultrasound pachymetry during corneal collagen cross-linking.

PURPOSE: To study variation in central corneal thickness (CCT) during corneal collagen cross-linking(CXL) using ultrasound pachymetry. METHODS: Twenty patients (26 eyes) with progressing keratoconus undergoing riboflavin-UVA-induced CXL were involved in this study. Intraoperative CCT measurement using ultrasonic pachymetry was performed during the procedure. Measurements were obtained before operation, after epithelial removal, after riboflavin drop instillation, and after UVA irradiation. RESULTS: Mean CCT was 495 +/- 56 and 450 +/- 52 microm before and after epithelial removal, respectively. Mean CCT was 443 +/- 42 and 411 +/- 39 microm after riboflavin drop instillation and after UVA irradiation, respectively. Statistically significant decreases in CCT occurred between the preoperative period and after epithelial removal, after riboflavin drop instillation and after UVA irradiation. Twenty-six eyes from 20 patients undergoing CXL were divided into 2 groups (I with CCT > or = 400 microm after UVA irradiation and II with CCT < 400 microm after UVA irradiation). No statistically significant difference was noted between I and II in preoperative endothelial cell count, but a statistically greater postoperative endothelial cell count was noted in I compared to II. A statistically significant difference was evident between preoperative and postoperative endothelial cell counts in Group II (P < 0.05). CONCLUSION: Performing CXL with the use of riboflavin and UVA irradiation resulted in a statistically significant decrease in CCT, to a level where the corneal endothelium may be damaged.

Stiffening of rabbit corneas by the bacteriochlorophyll derivative WST11 using near infrared light.

PURPOSE: We evaluated the efficacy and safety of photochemical corneal stiffening by palladium bacteriochlorin 13'-(2-sulfoethyl)amide dipotassium salt (WST11) and near infrared (NIR) illumination, using ex vivo and in vivo rabbit eye models. METHODS: Corneas of post mortem rabbits and living rabbits were pretreated topically with 2.5 mg/mL WST11 in saline or in 20% dextran T-500 (WST-D), washed and illuminated with an NIR diode laser (755 nm, 10 mW/cm(2). Studies with corneas of untreated fellow eyes served as controls. Tensile strength measurements, histopathology, electron spin resonance, and optical spectroscopy and fluorescence microscopy were used to assess treatment effects. Comparative studies were performed with standard riboflavin/ultraviolet-A light (UVA) treatment. RESULTS: WST11/NIR treatment significantly increased corneal stiffness following ex vivo or in vivo treatment, compared to untreated contralateral eyes. The incremental ultimate stress and Young's modulus of treated corneas increased by 45, 113, 115%, and 10, 79, and 174% following 10, 20, and 30 minutes of incubation with WST11, respectively. WST-D/NIR had a similar stiffening effect, but markedly reduced post-treatment edema and shorter time of epithelial healing. WST11/NIR and WST-D/NIR generate hydroxyl and superoxide radicals, but no singlet oxygen in the cornea. Histology demonstrated a reduction in the keratocyte population in the anterior half of the corneal stroma, without damage to the endothelium. CONCLUSIONS: Treatment of rabbit corneas, with either WST11/NIR or WST-D/NIR, increases their biomechanical strength through a mechanism that does not involve singlet oxygen. The WST-D/NIR treatment showed less adverse effects, demonstrating a new potential for clinical use in keratoconus and corneal ectasia after refractive surgery.

Is there an endothelial cell toxicity of light-adjustable lens UVA irradiation on the human corneal endothelium?

PURPOSE: In cataract and refractive surgery, implantation of a light-adjustable lens (LAL) represents a promising method to noninvasively correct deviation from target refraction. In this method, UVA irradiation is used to induce controlled changes in the form and refraction of the lens. UVA irradiation is known to have a damaging effect on corneal tissue. So far, no studies about the toxicity of LAL treatment on the human corneal endothelium exist. We tested the effect of LAL treatment on human donor endothelium under organ culture conditions. METHODS: Two pairs of human donor corneas not suitable for transplantation but with good endothelial quality were exposed either to LAL treatment with a target refraction of the maximum adjustable profile for hyperopia with astigmatism (right corneas) or to the white light of the same slit lamp for identical time frames (left corneas). Development of endothelial cell number, morphology and percentage of necrosis was observed. After 6 weeks, histologic analysis was performed. RESULTS: Comparing morphology, cell count, percentage of necrosis, and histologic examination in vitro, no significant differences between right and left corneas could be detected. CONCLUSIONS: The maximum dose of UVA irradiation used in LAL treatment was not detrimental for human corneal endothelium in vitro. Further in vivo studies and longer observation periods should be performed for ensuring the safety of LAL treatment as a routine procedure.

SOD2 contributes to anti-oxidative capacity in rabbit corneal endothelial cells.

PURPOSE: Corneal endothelial cells are rich in mitochondria, a potential source of reactive oxygen species (ROS). ROS have been implicated in endothelial cell loss during aging or in endothelial dystrophies. In this study we examined the anti-oxidative role of mitochondrial superoxide dismutase (SOD2) in corneal endothelial cells. METHODS: SOD2 expression was examined by RT-PCR and western blot analysis in fresh rabbit corneal endothelium (RCE) and cell cultures. SOD2 activity, total reactive oxygen species (ROS), mitochondrial ROS, mitochondrial membrane potential (MMP), and apoptotic levels were examined in untreated, SOD2 siRNA and viral vector shRNA treated RCE cells. Scrambled siRNA and shRNA sequence targeting non-mammalian genes were used as controls. RESULTS: SOD2 is expressed in both fresh and cultured rabbit corneal endothelium. SOD2 expression was reduced by ~80%-90% in cultured RCE using either siRNA or shRNA approaches. SOD2 activity was decreased by ~70%-80% for both approaches. Total cell ROS was significantly increased in shSOD2 lentivirus treated cells (9%±6%) relative to control transduction (0.4%±0.1%). MitoSOX™ staining for mitochondrial ROS in siSOD2 treated RCE cells was dramatically increased. Two minutes of UV irradiation increased total ROS levels by 15%, whereas in shSOD2 treated cells UV induced ROS was increased 29%±5% (p<0.05). MMP was reduced in shSOD2 viral treated cells by 66%±3%, significantly greater than in control transduced cells (15%±8%, p<0.05). Apoptosis increased by 1.5 fold in shSOD2 virus treated samples compared with scrambled virus and untreated cells. CONCLUSIONS: SOD2 is expressed in both fresh and cultured rabbit corneal endothelium. siRNA and shRNA approaches are able to efficiently knockdown SOD2 expression and reduce enzyme activity in RCE cells. Decreased SOD2 activity causes elevated ROS production, mitochondrial membrane potential loss and early cell apoptosis. These results indicate that SOD2 is a significant anti-oxidative enzyme in RCE cells.

The effects of light regimes and hormones on corneal growth in vivo and in organ culture.

When chicks are exposed to constant light (CL) during growth, their corneas become flatter and lighter in weight, and their anterior segments become shallower than those of chicks exposed to cyclical periods of light and dark. These effects have been correlated with CL suppression of cyclical changes in melatonin levels. The question of whether light directly influences corneal growth (e.g. via cryptochromes in the cornea) or acts remotely via the suppression of the melatonin rhythm has not yet been answered. Retinoic acid (RA), an ubiquitous morphogen, also causes non-functional flattening during corneal growth, but its effect in vivo has not been correlated with light regimes. We wished to characterize and distinguish between hormonal and light effects on corneal growth. We used organ culture to study the direct effects of light regimes, melatonin, and RA, and compared these results with those of parallel in vivo experiments. In this study, eye drops containing melatonin or RA were applied to corneas exposed to CL in vivo or in organ culture, and effects on corneal mass and hydration were measured. We applied a melatonin blocker, luzindole, to chick corneas in normal light/dark conditions to confirm that the observed melatonin effects are mediated at the cell membrane. Anterior chamber depth and refraction in vivo were measured. We found that, during CL exposure, combined application of melatonin and RA eye drops increased the depth of the anterior segment in vivo, (P = 0.003) and interestingly, both also reduced the hyperopia of CL exposure after 2 weeks (P = 0.002), thus partially reversing the effects of CL. RA increased corneal hydration in vivo (P = 0.030) but not in organ culture. Melatonin had no effect on corneal hydration in vivo, but in organ culture, melatonin significantly decreased hydration (P < 0.001). We found no evidence for a direct effect of light on corneal hydration in growing chick corneas in culture. Melatonin is required for normal corneal growth in vivo, and together melatonin and RA, or RA alone, affects the regulation of water content within the chick cornea. Melatonin also affects corneal hydration in vitro, but RA does not.

Corneal endothelial safety after ultraviolet light treatment of the light-adjustable intraocular lens.

PURPOSE: To assess the safety to the corneal endothelium of ultraviolet (UV) light treatment to a light-adjustable intraocular lens (IOL). SETTING: Codet Vision Institute, Tijuana, Mexico. DESIGN: Cohort study. METHODS: The study evaluated consecutive patients scheduled for phacoemulsification cataract surgery with implantation of a light-adjustable IOL. Endothelial cell density (ECD), variation in the size of endothelial cells, and the percentage of hexagonal cells were determined centrally with a specular microscope preoperatively, 1 week postoperatively (before UV treatment), and 6 months postoperatively. Two adjustments and 2 lock-in procedures were performed with a light-delivery system between 2 weeks and 4 weeks postoperatively. RESULTS: The study enrolled 10 patients. Specular microscopy showed a mean ECD of 2344 cells/mm(2) preoperatively. The mean overall central ECD was 2047 cells/mm(2) 1 week postoperatively and 2130 cells/mm(2) at 6 months, representing an endothelial cell loss of 12.6% and 9.1%, respectively, from preoperative values. There was no statistically significant difference in the coefficient of variation or percentage of hexagonal cells at either postoperative evaluation. CONCLUSIONS: The endothelial cell loss at 6 months correlated well with previous reports of endothelial damage after phacoemulsification with IOL implantation. The light-adjustable IOL irradiation protocol for adjustment and lock-in procedures did not seem to add to the endothelial damage caused by the cataract surgery, indicating the protocol is safe to the corneal endothelium.