Published papers

The lacrimal gland has a multifaceted role in maintaining a homeostatic microenvironment for a healthy ocular surface via tear secretion. Dry-eye disease, which is caused by lacrimal gland dysfunction, is one of the most prevalent eye diseases that cause corneal epithelial damage and results in significant loss of vision and a reduction in the quality of life. Here we demonstrate orthotopic transplantation of bioengineered lacrimal gland germs into adult mice with an extra-orbital lacrimal gland defect, a mouse model that mimics the corneal epithelial damage caused by lacrimal gland dysfunction. The bioengineered lacrimal gland germs and harderian gland germs both develop in vivo and achieve sufficient physiological functionality, including tear production in response to nervous stimulation and ocular surface protection. This study demonstrates the potential for bioengineered organ replacement to functionally restore the lacrimal gland. © 2013 Macmillan Publishers Limited. All rights reserved.

Purpose. We applied a recently reported next-generation sequencing (NGS) strategy for screening the ABCA4 gene in a British cohort with ABCA4-associated disease and report novel mutations. Methods. We identified 79 patients with a clinical diagnosis of ABCA4-associated disease who had a single variant identified by the ABCA4 microarray. Comprehensive phenotypic data were obtained, and the NGS strategy was applied to identify the second allele by means of sequencing the entire coding region and adjacent intronic sequences of the ABCA4 gene. Identified variants were confirmed by Sanger sequencing and assessed for pathogenicity by in silico analysis. Results. Of the 42 variants detected by prescreening with the microarray, in silico analysis suggested that 34, found in 66 subjects, were disease-causing and 8, found in 13 subjects, were benign variants. We detected 42 variants by NGS, of which 39 were classified as disease-causing. Of these 39 variants, 31 were novel, including 16 missense, 7 splice-site-altering, 4 nonsense, 1 in-frame deletion, and 3 frameshift variants. Two or more disease-causing variants were confirmed in 37 (47%) of 79 patients, one disease-causing variant in 36 (46%) subjects, and no disease-causing variant in 6 (7%) individuals. Conclusions. Application of the NGS platform for ABCA4 screening enabled detection of the second disease-associated allele in approximately half of the patients in a British cohort where one mutation had been detected with the arrayed primer extension (APEX) array. The time- and cost-efficient NGS strategy is useful in screening large cohorts, which will be increasingly valuable with the advent of ABCA4-directed therapies. © 2013 The Association for Research in Vision and Ophthalmology, Inc.

Age-related macular degeneration (AMD) is a vision-threatening disease, and its prevalence is increasing worldwide. Two approaches are commonly used to suppress the pathological choroidal neovascularization (CNV) that characterizes 'wet' AMD. One is photodynamic therapy (PDT), which involves using a systemic photosensitizer in combination with low-power laser irradiation of CNV through the neural retina and retinal pigment epithelium (RPE); the other involves the intravitreous injection of drugs targeting vascular endothelial growth factor (VEGF). In some cases, both methods are used simultaneously. However, recent results from animal experiments indicate that transient increase in VEGF in the tissue following PDT is required for retinal cell survival in overcoming the cellular stress caused by PDT. To maximize the visual outcome and retinal protection, it will be necessary to optimize the combination therapy, which requires the elucidation of the molecular and cellular changes that occur in the retinal tissue during the course of treatment. © 2013 - IOS Press and the authors. All rights reserved.

Aging is commonly defined as the accumulation of diverse deleterious changes in cells and tissues with advancing age. To investigate whether aging changes are involved in the lacrimal glands of chronic graft-versus-host disease (cGVHD) model mice, we obtained the specimens from cGVHD model mice, untreated aged and young mice, and examined by histopathology, and immunoblotting. Oxidative stress markers, 8-OHdG, 4-HNE, and hexonoyl lesion (HEL), and other aging markers, p16 and p38, were used to assess the samples. The infiltrating mononuclear cells and endothelia of capillaries in the cGVHD and aged mice expressed the oxidative stress markers and other aging markers, but not in the young mice. Histological changes and the expression of aging markers in the samples from cGVHD mice exhibited similar features to those in aging mice. These results suggest that changes that typically appear with advanced age occur earlier in the lives of mice with lacrimal gland cGVHD.

Introduction: Dry eye is a visually disabling disease encountered in many countries with a wide variation of treatment practices all over the world. On that front, the 2007 Report of the International Dry Eye WorkShop (DEWS) reviewed the current knowledge on all aspects of dry-eye disease (DED), in an evidence-based manner, and outlined the trends and recommendations in the treatment of DED on the basis of disease severity. Areas covered: This review mainly focuses on treatments for DED based on severity as recommended in the DEWS report, particularly artificial eye drops, hyaluronate sodium eye drops, autologous serum, anti-inflammatory eye drops including cyclosporine and steroids, and mucin secretagogues. New dry-eye treatment modalities in current trials outlined on the clinicaltrial.gov site are also outlined. Expert opinion: Further investigations into the mechanism of action of the new mucin and tear secretagogues which have been suggested to have anti-inflammatory properties will enrich our understanding in relation to relevant ocular surface responses after treatment with these new agents. © 2013 Informa UK, Ltd. © 2013 Informa UK, Ltd.

We characterized subtypes of fundus autofluorescence (AF) and the progression of retinal atrophy, and correlated these findings with genotype in Stargardt disease. Full clinical examination and AF imaging was undertaken in 68 patients with Stargardt disease. The baseline data were compared to those at follow-up. Patients were classified into three AF subtypes: type 1 had a localized low signal at the fovea surrounded by a homogeneous background, type 2 had a localized low signal at the macula surrounded by a heterogeneous background with numerous foci of abnormal signal, and type 3 had multiple low signal areas at the posterior pole with a heterogeneous background. At baseline, there were 19 patients with type 1, 41 with type 2, and 8 with type 3 disease. The areas of reduced AF signal were measured and rate of atrophy enlargement (RAE) was calculated as the difference of the atrophy size over time (mm2) divided by the follow-up interval (years). Molecular screening of ABCA4 was undertaken. The mean follow-up interval was 9.1 years. A total of 42% cases with type 1 disease progressed to type 2, and 12% with type 2 progressed to type 3. The RAE (mm2/y) based upon baseline AF subtypes was significantly different; 0.06 in type 1, 0.67 in type 2, and 4.37 in type 3. ABCA4 variants were identified in 57 patients. There was a significant association between AF subtype and genotype. The AF pattern at baseline influences the enlargement of atrophy over time and has genetic correlates. These data are likely to assist in the provision of counseling on prognosis in Stargardt disease and be valuable for future clinical trials.

Aim: To evaluate the association between the magnitude of relative afferent pupillary defect (RAPD) and the laterality of visual fields in glaucoma patients using pupillography. Methods: Evaluations were made with an RAPDx (Konan Medical USA, Irvine, California, USA), which is designed to analyse pupil responses to multiple colour stimuli (white, red, green, blue, and yellow) at multiple controlled stimulus intensities. Humphrey automated perimetry (SITA standard 30-2) was performed to determine mean deviations (MDs), which were used to evaluate associations between MD differences (better eye MD - worse eye MD) and RAPDx amplitude and latency measurements in pupil response to multiple colour stimuli. Results: 58 glaucoma patients (35 men, 23 women; mean age: 62.6±12.8 years; range 28-88 years) were enrolled. The MD values were -2.00±3.1 (range -9.64 to 2.33) dB in the better eyes and -8.17±7.1 (range -26.33 to 1.07) dB in the worse eyes. The differences in MD values (better eye MD - worse eye MD) were 6.17±6.1 (range 0.05-26.41) dB. The results of linear regression analysis between the log-scaled RAPD amplitudes and differences in MD values were as follows: white: R2=0.45 (p<0.001), red: R2=0.18 (p=0.002), green: R2=0.30 (p<0.001), blue: R2=0.29 (p<0.001), and yellow: R2=0.32 (p<0.001). The log-scaled RAPD latencies and the differences in MD values were: white: R2=0.17 (p=0.001), red: R2=0.03 (p=0.22), green: R2 =0.01 (p=0.52), blue: R2=0.1 (p=0.02), and yellow: R2=0.05 (p=0.11). Conclusions: The log-scaled RAPD amplitudes correlated moderately with the differences in MD values, but the log-scaled RAPD latencies showed a weaker correlation. Stimulation with white light produced the strongest correlation with differences in MD values.

Purpose: To evaluate the efficacy and safety of new moist cool air device (MCAD) for ocular symptoms, tear film stability and ocular surface status in office workers with dry eye disease (DED). Methods: In this prospective single-centre clinical trial, 40 eyes of 20 patients with DED were recruited and randomly divided into two groups (group with MCAD exposure and group without MCAD). All subjects are visual display terminals (VDTs) workers spending at least 4 h/day in front of VDTs. Patients using MCAD underwent moist air applications for 4 h/day for a total of five working weekdays during VDT works at their offices. The other group of patients performed their VDT work without moist cool air device exposure. The change in symptoms of ocular surface (OS) dryness, fatigue and discomfort was evaluated using visual analogue scale (VAS) scores. Changes in visual function, tear functions and ocular surface status were evaluated using best-corrected visual acuity (BCVA), the functional visual acuity (FVA) test, blink rate, BUT measurements, strip meniscometry (SM), tear evaporation rate, fluorescein staining and rose bengal staining scores. Tear film lipid layer interferometry was also performed to assess the status of the lipid layer over the tear film. In addition, adverse events were recorded. Results: In group with MCAD, symptoms of OS dryness during VDTs work, and FVA and BUT were significantly improved. SM and tear evaporation rate were significantly improved. There were no statistically significant differences on lipid layer stability and corneal staining scores in both groups. Blink rate was significantly increased in group without MCAD. No adverse events were reported during this trial. Conclusion: Moist cool air device use provided symptomatic relief of ocular dryness and improvement on tear stability in office workers with DED. This new device seems to be a safe and promising alternative in the treatment of DED. © 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.

The International Chronic Ocular GVHD Consensus Group held 4 working meetings to define new diagnostic metrics for chronic ocular graft-versus-host disease (GVHD). After considering the factors currently used to diagnose chronic ocular GVHD, the Consensus Group identified 4 subjective and objective variables to measure in patients following allogeneic hematopoietic stem cell transplantation (HSCT): OSDI, Schirmer's score without anesthesia, corneal staining, and conjunctival injection. Each variable was scored 0-2 or 0-3, with a maximum composite score of 11. Consideration was also given to the presence or the absence of systemic GVHD. On the basis of their composite score and the presence or absence of systemic GVHD, patients were assigned to one of three diagnostic categories: NO, PROBABLE, or DEFINITE ocular GVHD. New diagnostic criteria for chronic ocular GVHD are presented by the Consensus Group. Validation studies are needed to identify the best combination of the proposed metrics to maximize diagnostic sensitivity and specificity.

Tissue-engineering approaches to cultivate corneal endothelial cells (CECs) or induce CECs from stem cells are under investigation for the treatment of endothelial dysfunction. Before clinical application, a validation method to determine the quality of these cells is required. In this study, we quantified the endothelial pump function required for maintaining the corneal thickness using rabbit CECs (RCECs) and a human CEC line (B4G12). The potential difference of RCECs cultured on a permeable polyester membrane (Snapwell), B4G12 cells on Snapwell, or B4G12 cells on a collagen membrane (CM6) was measured by an Ussing chamber system, and the effect of different concentrations of ouabain (Na,K-ATPase specific inhibitor) was obtained. A mathematical equation derived from the concentration curve revealed that 2 mM ouabain decreases pump function of RCECs to 1.0 mV, and 0.6 mM ouabain decreases pump function of B4G12 on CM6 to 1.0 mV. Ouabain injection into the anterior chamber of rabbit eyes at a concentration of <2 mM maintained the corneal thickness, while those over 3 mM significantly increased the corneal thickness. B4G12 cell sheets transplanted into rabbit eyes treated with 0.6 mM ouabain maintained the corneal thickness, while 3.5 mM ouabain significantly increased the corneal thickness. Taken together, pump function >1.0 mV is required to maintain the corneal thickness. These results can be used for standardization of CEC pump function and validation of tissue-engineered CEC sheets for clinical use. © Copyright 2013, Mary Ann Liebert, Inc. 2013.

Purpose: To describe the phenotypic presentation of a cohort of individuals with homozygous disease-associated ABCA4 variants. Design: Retrospective case series. Participants: Eighteen affected individuals from 13 families ascertained from a total cohort of 214 families with ABCA4-related retinal disease presenting to a single center. Methods: A detailed history was obtained, and color fundus photography, autofluorescence (AF) imaging, optical coherence tomography (OCT), and electrophysiologic assessment were performed. Phenotypes based on ophthalmoscopy, AF, and electrophysiology were assigned using previously reported characteristics. ABCA4 mutation detection was performed using the ABCR400 microarray (Asper Biotech, Tartu, Estonia) and high-throughput DNA sequencing, with direct sequencing used to assess segregation. Main Outcome Measures: Detailed clinical, electrophysiologic, and molecular genetic findings. Results: Eleven disease-associated homozygous ABCA4 alleles were identified, including 1 frame shift, 2 stops, 1 intronic variant causing splice-site alteration, 2 complex missense variants, and 5 missense variants: p.Glu905fsX916, p.Arg1300X, p.Gln2220X, c.4253+4 C>T, p.Leu541Pro and p.Ala1038Val (homozygosity for complex allele), p.Val931Met and p.Arg1705Gln (complex allele), p.Arg212Cys, p.Cys1488Arg, p.Arg1640Trp, p.Gly1961Glu, and p.Leu2027Phe. Eight of these 11 homozygous alleles have not been reported previously. Six of 7 patients with homozygous null alleles had early-onset (<10 years) disease, with all 7 having a severe phenotype. Two patients with homozygous missense variants (p.Leu541Pro and p.Ala1038Val [complex], and p.Arg1640Trp) presented with a severe phenotype. Three patients with homozygous p.Gly1961Glu had adult-onset disease and a mild phenotype. One patient with homozygous p.Leu2027Phe showed a spared fovea and preserved visual acuity. Conclusions: The phenotypes represented in patients identified as homozygous for presumed disease-associated ABCA4 variants gives insight into the effect of individual alleles. Null alleles have severe functional effects, and certain missense variants are similar to nulls, suggesting complete abrogation of protein function. The common alleles identified, p.Gly1961Glu and p. Leu2027Phe, both have a mild structural and functional effect on the adult retina; the latter is associated with relatively retained photoreceptor architecture and function at the fovea. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. © 2013 by the American Academy of Ophthalmology.

Immunosuppressive therapy is the main postoperative treatment for keratoplasty, but there are considerable differences in protocols for the use of steroids and other immunosuppressants. Therefore, we conducted 2 prospective randomized clinical trials and 1 prospective nonrandomized clinical trial on keratoplasty postoperative treatment. One study evaluated the efficacy and safety of long-term topical corticosteroids after a penetrating keratoplasty was performed. Patients who underwent keratoplasty and maintained graft clarity for >1 year were randomly assigned to either a steroid or a no-steroid group. At the 12-month follow-up, the no-steroid group developed significantly more endothelial rejection than did the steroid group. A second study elucidated the effectiveness and safety of systemic cyclosporine in high-risk corneal transplantation. The patients were assigned to a systemic cyclosporine or control group. At a mean follow-up of 42.7 months, no difference was observed in the endothelial rejection rates and graft clarity loss between the 2 groups. A third study elucidated the effectiveness and the safety of systemic tacrolimus in high-risk corneal transplantation. Of 11 consecutive eyes decompensated despite systemic cyclosporine treatment, there was no irreversible rejection in eyes treated with tacrolimus, which was significantly better than in previous penetrating keratoplasty with systemic cyclosporine treatment. Prognosis after keratoplasty in patients with keratoconus is relatively good, but special attention is required for patients with atopic dermatitis. Postkeratoplasty atopic sclerokeratitis (PKAS) is a severe form of sclerokeratitis after keratoplasty in atopic patients. Our retrospective study showed that 35 eyes of 29 patients from a total of 247 keratoconus eyes undergoing keratoplasty were associated with atopic dermatitis, of which 6 eyes of 5 patients developed PKAS. Eyes with PKAS had a significantly higher incidence of atopic blepharitis and preoperative corneal neovascularization, and therefore, we suggest systemic corticosteroids or cyclosporine to prevent PKAS in such high-risk cases. © 2013 by Lippincott Williams & Wilkins.

Sirtuins (Sirt1-7) are nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylases/ADP-ribosyltransferases that modulate many metabolic responses affecting aging. Sirtuins expressed in tissues and organs involved in systemic metabolism have been extensively studied. However, the characteristics of sirtuins in the retina, where local energy expenditure changes dynamically in response to light stimuli, are largely unknown. Here we analyzed sirtuin mRNA levels by real-time PCR, and found that all seven sirtuins are highly expressed in the retina compared with other tissues, such as liver. We then analyzed the sirtuin mRNA profiles in the retina over time, under a 12-h light/12-h dark cycle (LD condition) and in constant darkness (DD condition). All seven sirtuins showed significant daily variation under the LD condition, with all except Sirt6 being increased in the dark phase. The expression patterns were different under the DD condition, suggesting that sirtuin mRNA levels except Sirt6 are affected by light-dark condition. These findings were not obtained in the brain and liver. In addition, the mRNA expression patterns of Nicotinamide phosphoribosyltransferase (Nampt), peroxisome proliferator-activated receptor gamma coactivator (PGC1α), and transcription factor A, mitochondrial (Tfam) in the retina, were similar to those of the sirtuins except Sirt6. Our observations provide new insights into the metabolic mechanisms of the retina and the sirtuins' regulatory systems. © 2013 The Authors.

Purpose: To investigate the long-term effects of postoperative flat anterior chamber (FAC) development on outcomes following trabeculectomy with mitomycin C. Methods: This was a retrospective cohort study. Data on 383 consecutive patients (383 eyes) who underwent trabeculectomy at our institution between 1999 and 2009 were followed up. Patients who developed FAC after trabeculectomy and patients with maintained anterior chamber were evaluated. The primary outcome variable was the success of the initial trabeculectomy, which was defined at 3 different levels by the achievement of the following intraocular pressure (IOP)-related criteria without secondary IOP-lowering surgery: (a) IOP ≤ 12 mmHg and ≤30 % reduction in IOP from the preoperative level; (b) IOP ≤ 16 mmHg and ≤20 % reduction in IOP; and (c) IOP ≤ 21 mmHg. The hazard ratios (HRs) for the failure of trabeculectomy caused by FAC within 5 years of surgery were examined in conjunction with the Cox proportional hazards regression model. Results: FAC was observed in 90 of the 383 eyes examined (23.4 %). Postoperative mild FAC was associated with the long-term success of trabeculectomy when evaluated according to our strictest success criterion, Criterion-A [HR = 0.72 (95 % CI 0.53-0.98); P = 0.04]. In contrast, severe FAC was inversely associated with the long-term success of the surgery when evaluated according to our most lenient criterion, Criterion-C [HR = 1.93 (95 % CI 1.16-3.22); P = 0.01]. Conclusion: Mild postoperative FAC after trabeculectomy is associated with a favorable long-term outcome, whereas severe postoperative FAC leads to an unfavorable prognosis. © 2013 Japanese Ophthalmological Society.

Purpose. To determine if glaucomatous visual field defect severity is associated with fear of falling. Methods. This is a cross-sectional study. A total of 387 consecutive subjects with POAG were enrolled in this study along with 293-ocular disease-free control subjects, who were screened at the same institutions. We defined mild POAG as MD of -6 dB or better, moderate POAG as MD of -6 to -12 dB, and severe POAG as MD of -12 dB or worse in the better eye. All participants were requested to answer a questionnaire on fear of falling. Associations between POAG severity and the prevalence of fear of falling were evaluated with the Cochran-Armitage trend test. Multivariable factors including age-adjusted odds ratios (ORs) for the prevalence of fear of falling and 95% confidence intervals (CIs) were evaluated with logistic regression models. Results. The p revalence of fear of falling was 35/293 (11.9%) in the control group, 38/313 (12.1%) in the mild POAG group, 12/48 (25.0%) in the moderate POAG group, and 6/26 (23.1%) in the severe POAG group, and the trend was statistically significant (P =0.028 Cochran-Armitage trend test). The adjusted ORs for prevalence in the mild, moderate, and severe POAG groups compared with that in the control group were 1.44 (95% CI: 0.83-2.51), 2.33 (95% CI: 1.00-5.44), and 4.06 (95% CI: 1.39-11.90), respectively. Conclusions. Among patients with POAG, the severity of visual field defects is associated with fear of falling. (http://www.umin.ac.jp/ctr/index.htm number, UMIN000005574.). © 2013 The Association for Research in Vision and Ophthalmology, Inc.

Purpose. To investigate the efficacy of 2% rebamipide ophthalmic solution on the tear functions and ocular surface status of the superoxide dismutase-1(Sod1-/-) mice. Methods. Two percent Rebamipide ophthalmic solution was applied to 40-week-old male Sod1-/- and wild-type (WT) mice four times a day for 2 weeks. We examined the cytokine concentrations in the tear fluid (by CytoBead assay), tear film break-up time, amount of tear production, and expressions of mucins 1, 4, and 5AC, by RT-PCR. We also performed vital staining of the ocular surface, PAS staining for muc5AC, and immunohistochemical stainings for 4-hydroxy-2-nonenal (4-HNE), 8-hydroxy-2′-deoxyguanosine (8-OHdG), in the conjunctiva to compare the Results before and after rebamipide instillations. Results. The tear functions and ocular surface epithelial damage scores were significantly worse in the Sod1-/- than in the WT mice. Application of 2% rebamipide for 2 weeks significantly improved the tear film break-up time, the amount of tear production, and the corneal epithelial damage scores, which also significantly increased the conjunctival goblet cell density and muc5 mRNA expression, in the Sod1-/- mice. The mean IL-6, IL-17, TNF-α, and IFN-γ levels in the tear fluid were reduced significantly along with a significant decrease in the density of cells positive for 4-HNE and 8-OHdG in the conjunctiva. Conclusions. Two percent Rebamipide ophthalmic solution significantly improved the tear stability and corneal epithelial damage, and enhanced the expression of muc5 mRNA on the ocular surface. We also observed anti-inflammatory effects in the tear film together with antioxidative effects in the conjunctiva, suggesting the efficacy of rebamipide in age-related dry eye disease attributable to SOD1 knockout. © 2013 The Association for Research in Vision and Ophthalmology, Inc.

Purpose: To report a case who received implantation of piggyback intraocular lens (IOL) for residual refractive error after cataract surgery. Case: A 70-year-old man presented to our clinic complaining of decreased visual acuity in his both eyes. Both eyes had received radial keratotomy 23 years before and photorefractive keratectomy 12 years before. Findings: Visual acuity was 0.5 right and 0.4 left without correction, and 0.9 right and 1.0 left with correction. Under diagnosis of age-related cataract, both eyes received phacoemulsification-aspiration with IOL implantation. Visual acuity was 0.5 in either eye without correction and 1.2 right and 1.0 left with correction one year later. The left eye had refraction of +3.00D()cyl-2.00D 65°. By request of the patient, monofocal piggyback IOL was implanted into the left eye. Six months after surgery, left visual acuity was 0.63 without and 1.0 with correction. There has been no complication throughout. Conclusion: Implantation of piggyback IOL can be a therapeutic option for residual refractive error after cataract surgery.

Corneal epithelial stem cells are located in the limbus, the junction between the cornea and the conjunctiva. A limbal epithelium model in vitro would be useful for the study of epithelial stem cells, as well as improving the quality of cultivated epithelial sheets for the treatment of limbal stem cell deficiency. In this study, we succeeded in constructing a limbal epithelium-like structure that could be maintained for at least 5 months in vitro. We modified conventional medium by replacing epidermal growth factor with keratinocyte growth factor (KGF) and adding Y-27632, a rho kinase inhibitor. Using this medium, epithelial cells freshly isolated from human limbus were cocultured with human mesenchymal stem cell-derived feeder cells. Cells formed a stratified layer without air exposure, and both basal and suprabasal layers maintained their unique morphologies for up to 5 months. Basal layers expressed the progenitor marker p63 uniformly and K15 heterogeneously. Expressions of PAX6, K3, and K12 indicated that cell sheets underwent normal differentiation in the corneal epithelium lineage. Although medium was changed daily after day 7, cell debris was observed every day, suggesting that cell sheets underwent turnover. Furthermore, secondary colonies were observed from cells dissociated from 1-month and 3-month cultured sheets. In conclusion, human limbal epithelial cell sheet cultures with KGF and Y-27632 maintained stratification, high expression of both stem/progenitor markers and differentiation markers, and colony-forming cells long-term. This protocol may be useful as an in vitro limbal epithelial model for basic studies. © AlphaMed Press 2013.

The N-methyl- d-aspartate (NMDA) receptor-induced apoptosis is implicated in the pathological mechanisms of neural tissues, increasing the release of reactive oxygen species (ROS), resulting in a type of apoptotic cell death called excitotoxicity. Although intrinsic mechanisms to remove ROS, such as antioxidant enzymes, are provided by the tissue, the association between NMDA-induced excitotoxicity and antioxidative enzymes is not well understood. In this study, we focused on superoxide dismutase 1 (SOD1), an antioxidant enzyme, and investigated the role of SOD1 in the NMDA-induced neuronal cell death in the retina. NMDA was intravitreally injected into wild-type (WT) and SOD1 total knock-out (SOD1-deficient) mice. The number of TUNEL-positive cells in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) counted in the retinal sections and flatmount retinas were significantly higher in the SOD1-deficient mice than the WT mice after NMDA injection. Visual function assessed by dark-adapted electroretinogram (ERG) showed that the amplitudes of a-wave, b-wave, and oscillatory potential 2 were significantly reduced in the NMDA-injected SOD1-deficient mice. The level of ROS in the GCL and INL, measured using dihydroethidium, and the number of positive cells for γ-H2AX, a marker for DNA double strand breaks, and 8-OHdG, a marker for DNA oxidation, in the GCL were significantly increased in the SOD1-deficient mice after NMDA injection. We also measured mRNA and protein levels of SOD1 and SOD2 in the retina of WT mice, to find that mRNA and protein levels of SOD1, but not SOD2, were significantly reduced after NMDA injection. SOD1 deficiency exacerbated NMDA-induced damage to the inner retinal neurons, and NMDA reduced SOD1 levels in the retina of WT mice. Therefore, SOD1 protected retinal neurons against NMDA-induced retinal neurotoxicity, and NMDA-induced SOD1 reduction may be involved in neuronal vulnerability to excitotoxicity. © 2013 Elsevier Ltd.