Published papers

Activation of the immune system and progressive fibrosis are prominent features of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation. The main histologic findings in affected exocrine glands are marked fibrosis of the interstitium and prominent increase in the number of fibroblasts, accompanied by mild lymphocytic infiltration. Clinically, the severity of dry eye is correlated with the degree of fibrotic changes, rather than with the amount of lymphocytic infiltration, indicating that excessive extracellular matrix accumulation primarily contributes to the exocrine dysfunction. We have found that accumulated fibroblasts in the lacrimal gland of patients with cGVHD have a chimeric status. Thus, fibroblasts originating from circulating donor-derived precursors and recipient-derived fibroblasts by local epithelial mesenchymal transition may participate in the excessive fibrosis in patients with cGVHD by interacting with T cells. In this article, the immune response and pathogenic fibrosis in ocular cGVHD will be discussed. In addition, clinical findings of ocular complications after hematopoietic stem cell transplantation and currently available treatment will also be discussed. © 2010 by Lippincott Williams & Wilkins.

Purpose:- To investigate the amount of systemic oxidative stress-related DNA damage and serum total antioxidant status of patients with normal-tension glaucoma compared with healthy controls. Methods:- Forty-three patients with normal-tension glaucoma selected from 60 consecutive newly diagnosed patients with primary open-angle glaucoma were enrolled. Each patient's intraocular pressure (IOP) was measured seven times over a 24-hour period. Those whose highest IOP was over 21mmHg were excluded. Forty control subjects were recruited. The serum total antioxidant status was assessed colorimetrically by its ability to reduce the generation of the radical cation ABTS (2,2-azinobis-3-ethylbenzthiazoline sulphonate). The urinary 8-hydroxy-2′-deoxyguanosine was measured as a marker of oxidative DNA damage and normalized to creatinine. The results were compared between healthy subjects and patients with normal-tension glaucoma. Results:- The control and normal-tension glaucoma groups did now show significant differences for age, gender, serum fructosamine, cholesterol and triglyceride levels; systolic and diastolic blood pressure, body mass index; and prevalence of hypertension. The serum total antioxidant status was significantly higher (p=0.01) and the urinary 8-hydroxy-2′-deoxyguanosine/creatinine level significantly lower (p=0.03) in the patients with normal-tension glaucoma compared with the controls. Even after we adjusted the data for independent variables, the incidence of normal-tension glaucoma was significantly correlated with high serum total antioxidant levels (p=0.03; odds ratio, 1.007; 95% confidence interval, 1.001-1.013) and low urinary 8-hydroxy-2′-deoxyguanosine/creatinine (p=0.02; odds ratio 0.76; 95% confidence interval, 0.61-0.96). Conclusions:- Increased serum total antioxidant and decreased 8-hydroxy-2′- deoxyguanosine may reflect compensatory alterations in response to increased systemic oxidative stress in patients with normal-tension glaucoma. © 2010 Acta Ophthalmol.

Purpose: To investigate the applicability of tear meniscus height (TMH) measurement using Visante optical coherence tomography (OCT) in the diagnosis of dry eye disease. Design: Prospective, controlled, single-center study. Participants: Twenty-four right eyes of 24 patients (6 males, 18 females; mean age, 63.14±13.4 years) with definite dry eye according to the Japanese dry eye diagnostic criteria and 27 right eyes of 27 control subjects (12 males, 15 females; mean age, 56.04±14.22 years) were recruited. Methods: All subjects underwent slit-lamp TMH measurement, OCT upper and lower TMH measurements, tear film breakup time (BUT) measurements, vital stainings, and Schirmer test. The results were compared between the 2 groups by Mann-Whitney test. Main Outcome Measures: The correlation between the clinical findings of slit-lamp TMH, strip meniscometry examination, tear functions, vital staining scores, and the OCT upper and lower TMH parameters were tested by Spearman's correlation test. Receiver operating characteristic (ROC) curve technique was used to evaluate the sensitivity, specificity and cutoff values of OCT TMH examination in the diagnosis of dry eye. Results: The OCT upper and lower TMH values, slit-lamp TMH, strip meniscometry, tear film BUT, and vital staining scores were significantly lower in the dry eye patients compared with controls (P<0.001). A significant correlation between the OCT upper and lower TMH measurements as well as slit-lamp TMH, strip meniscometry, tear functions, vital staining scores, and the Schirmer test was found. The ROC curve technique analysis of the OCT lower TMH showed that, when the cutoff value was set at <0.30 mm, the sensitivity and specificity of the testing were 67% and 81%, respectively. Conclusions: The Visante OCT is a quick, noninvasive method for assessing the TMH, with acceptable sensitivity, specificity, and repeatability, and may have potential applications for the diagnosis and evaluation of dry eye disease. Financial Disclosure(s): The authors have no proprietary or commercial interest in any of the materials discussed in this article. © 2010 American Academy of Ophthalmology.

Purpose: To determine if Chlamydia pneumoniae infection is associated with primary open-angle glaucoma (POAG). Patients and Methods: Consecutive patients with newly diagnosed primary POAG attending the Glaucoma clinic of Keio University Hospital between June 2007 and January 2008 were considered for inclusion in this prospective case-control study. Forty consecutive POAG patients and 41 normal healthy individuals as a control population met the inclusion criteria. The exclusion criteria for both groups were; taking steroids or immunosuppressive agents, smoking, and history of any acute or chronic systemic disease including stroke, heart attack, diabetes mellitus, hypertension, asthma, and autoimmune diseases. The serum was analyzed for C. pneumoniae and C. trachomatis immunoglobulin G antibody titers by enzyme-linked immunosorbent assay. Seroactivity to each antigen between case and control groups was evaluated by Mann-Whitney U test. Results: The age, male/female ratio, and intraocular pressure of the cases and control groups were not significantly different. Immunoglobulin G titers for C. pneuemoniae was significantly higher in patients with POAG than in controls (P=0.009). The titers to C. trachomatis were not significantly different between the 2 groups (P=0.99). Conclusion: The results suggest that higher C. pneumoniae titers are associated with POAG. If confirmed, this may indicate either a common factor that causes susceptibilities to both glaucoma and C. pneumoniae infection or that C. pneumoniae may be a causal factor for developing POAG. Copyright © 2010 by Lippincott Williams & Wilkins.

Light damage to the retina accelerates retinal degeneration in human diseases and rodent models. Recently, the polyphenolic phytoalexin resveratrol has been shown to exert various bioactivities in addition to its classical antioxidant property. In the present study, we investigated the effect of resveratrol on light-induced retinal degeneration together with its underlying molecular mechanisms. BALB/c mice with light exposure (5000-lux white light for 3 hours) were orally pretreated with resveratrol at a dose of 50 mg/kg for 5 days. Retinal damage was evaluated by TdT-mediated dUTP nick-end labeling, outer nuclear layer morphometry, and electroretinography. Administration of resveratrol to mice with light exposure led to a significant suppression of light-induced pathological parameters, including TdT-mediated dUTP nick-end labeling-positive retinal cells, outer nuclear layer thinning, and electroretinography changes. To clarify the underlying molecular mechanisms, the nuclear translocation of activator protein -1 subunit c-fos was evaluated by enzyme-linked immunosorbent assay, and the retinal activity of sirtuin 1 was measured by deacetylase fluorometric assay. Retinal activator protein-1 activation, up-regulated following light exposure, was significantly reduced by application of resveratrol. In parallel, retinal sirtuin 1 activity, reduced in animals with light damage, was significantly augmented by resveratrol treatment. Our data suggest the potential use of resveratrol as a therapeutic agent to prevent retinal degeneration related to light damage. Copyright © American Society for Investigative Pathology.

Purpose: To evaluate the use of topography-guided conductive keratoplasty in eyes with keratoconus. Design: Interventional case series. Methods: We examined 21 eyes in 21 patients with advanced keratoconus. Topography-guided conductive keratoplasty was performed with intraoperative monitoring of corneal astigmatism using a surgical keratometer. Uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), corneal topography, manifest refraction, intraocular pressure (IOP), corneal endothelial cell counts, complications, and eventual outcomes were evaluated. Results: UCVA (logarithm of the minimal angle of resolution [logMAR]), which was 1.65 ± 0.49 preoperatively, improved to 1.04 ± 0.64 at 1 week (P < .001) and 1.12 ± 0.61 at 1 month after surgery (P < .001). BSCVA, which was 1.02 ± 0.56 preoperatively, improved to 0.76 ± 0.65 at 1 week (P = .026) and 0.76 ± 0.60 at 1 month after surgery (P = .003). Manifest refraction, which was -15.13 ± 6.66 diopters (D) before surgery, declined to -9.97 ± 6.71 D at 1 month after surgery (P = .002). Although corneal topography reverted to the preoperative pattern and UCVA and BSCVA also regressed toward preoperative values, 12 of 21 eyes were better able to tolerate and conduct normal daily activities using contact lenses. Five subjects have undergone or are considering corneal transplantation after unsatisfactory postoperative results. No serious perioperative complication was observed. Conclusions: Topography-guided conductive keratoplasty may be effective in reshaping corneal configuration in eyes with keratoconus, without serious complications, and possibly contributed to avoiding or delaying corneal transplantation. © 2010 Elsevier Inc. All Rights Reserved.

Purpose: To evaluate the irregularity of the anterior and posterior cornea after Descemet stripping automated endothelial keratoplasty (DSAEK) and penetrating keratoplasty (PK). Methods: This clinical study comprised 39 eyes: 13 consecutive eyes after DSAEK, 13 consecutive eyes after PK, and 13 age-matched normal eyes. Corneal elevation data were acquired using a rotating Scheimpflug camera 1 and 3 months after DSAEK and PK. Anterior and posterior corneal elevation data were decomposed into a set of Zernike polynomials up to eighth order. Total higher-order root mean square (RMS) and RMS from third to eighth order were calculated. The astigmatism and irregularity of the anterior and posterior surfaces were compared between DSAEK and PK. Results: The regular astigmatism and tilt components of the anterior surface were significantly lower after DSAEK than after PK at 1 and 3 months (P < 0.001), whereas there was no difference in astigmatism of the posterior surface between the groups (P = 0.07, 0.22). The higher-order RMS and RMSs of third- to eighth-order components of the anterior surface were significantly larger after PK than those after DSAEK at 1 and 3 months (P < 0.01), whereas there were no significant differences between DSAEK and PK in higher-order aberration RMS and RMSs of third- to eighth-order components of the posterior surface. Conclusions: Postoperative corneal irregularity of the anterior surface was greater after PK than after DSAEK, whereas there was no significant difference in posterior surface irregularity. DSAEK is superior to PK in terms of the higher-order irregularity of the anterior surface. Copyright © 2010 by Lippincott Williams & Wilkins.

The process of aging involves the accumulating changes in the microenvironment that lead to cell senescence or apoptosis, and subsequent tissue or organ dysfunction. Multiple extrinsic and intrinsic events that cause DNA instability are associated with aging. Cells containing unstable DNA are biologically vulnerable, and if the DNA damage is too great for the cell to repair, it becomes senescent or dies by apoptosis. Thus, the cell's capacity to repair its DNA determines the progress of aging, at least in part. Here, we focus on the sirtuins, the mammalian homologs of the yeast life-span-extending molecule, Sir2. Among the sirtuin family proteins in mammals, the one most similar to yeast Sir2 is SIRT1, which is involved in multiple pathways, including the repair of DNA double-strand breaks. Although the role of SIRT1 in mammalian longevity is not clear, it is expressed throughout the retina, where it may suppress aging. In fact, a mutant mouse model of retinal degeneration shows an abnormal subcellular localization of SIRT1 protein and accelerated retinal cell apoptosis. Further analyses are required to elucidate the mechanism of DNA damage and repair, including the contributions of the sirtuins, in the aged or diseased retinas, which will help us understand the mechanisms of retinal aging. © 2010 S. Karger AG, Basel.

The involvement of reactive oxygen species (ROS) in the pathophysiology of Sjögren's syndrome (SS), an autoimmune disorder, and irradiation-induced impairments in salivary secretion has been reported. Meanwhile, the strong antioxidant astaxanthin (Ast) has been suggested to have therapeutic effects on various diseases. In the present study, we examined the ROS scavenging capacity of Ast using a human salivary gland epithelial cell line (HSY) and investigated the effects of Ast on salivary secretion in a mouse model of irradiation-induced salivary gland dysfunction. Furthermore, we performed a clinical study of Ast in six SS patients and six normal individuals, quantifying the volume of saliva secretion and the level of oxidative stress markers in the saliva. Ast partially suppressed hydrogen peroxide-induced ROS in HSY cells. The mouse model demonstrated that the preadministration of Ast resulted in the suppression of irradiation-induced hyposalivation. Furthermore, the administration of Ast appeared to increase salivary output in both the SS and normal groups. The level of oxidative stress marker, hexanoyl-lysine, in the saliva was reduced after Ast intake. These results suggest that Ast might act as an ROS scavenger, providing benefits to SS patients with impaired salivary secretion.

Recent advances in the understanding of aging have paved a new way of thinking about intervening with the aging process. There is a global agreement in the scientific community that calorie restriction (CR) can actually extend the life span of various kinds of animals so that this has become a real intervention in aging. In addition to the CR theory, the free radical theory is another important hypothesis, which is believed to be involved in aging. According to this theory, we can manage the aging process by controlling calories or reactive oxygen species. In this paper, these two important aging theories, CR and free radical aging, are reviewed, and it is discussed how to apply these theories to the prevention and treatment of eye diseases. Finally, we share the preliminary results of our animal study on dry eye, and I report my personal experience as a dry eye patient, which has been alleviated by the antiaging approach. © 2010 S. Karger AG, Basel.

PURPOSE. To investigate the cytologic findings of superior bulbar conjunctiva in superior limbic keratoconjunctivitis (SLK) using laser scanning confocal microscopy and impression cytology in a prospective controlled study. METHODS. Twenty-one eyes of 11 SLK patients (9 women, 2 men; mean age, 49.3 ± 17.9 years) and 18 eyes of 9 control subjects (6 women, 3 men; mean age, 46.4 ± 8.7 years) underwent tear function tests including vital stainings, Schirmer test, tear clearance test, digital confocal laser scanning microscopy, and conjunctival impression cytology. After confocal microscopy and impression cytology images were obtained, the mean individual epithelial cell area (MIECA), nucleocytoplasmic (N/C) ratio, and inflammatory cell density were analyzed. The correlation between confocal microscopy and impression cytology parameters was investigated. RESULTS. The MIECA of SLK patients and control subjects in confocal microscopy was 786.54 ± 463.88 μm2 and 311.50 ± 78.30 μm2, respectively. The mean N/C ratio was 0.356 ± 0.090 and 0.490 ± 0.038, respectively. The MIECA and N/C ratio in impression cytology showed significant correlation with the corresponding confocal microscopy parameters (MIECA, P ± 0.0028; N/C, P= 0.0051). The inflammatory cell density in confocal microscopy significantly correlated with superior bulbar conjunctival Rose-Bengal scores (P= 0.0264). CONCLUSIONS. Laser scanning confocal microscopy seems to be an efficient noninvasive tool in the evaluation of phenotypic alterations of the conjunctival epithelium in SLK and may serve as an alternative for impression cytology. N/C ratio and inflammatory cell density appear to be two new promising parameters of in vivo confocal microscopy in the assessment of ocular surface disease in SLK. © Association for Research in Vision and Ophthalmology.

PURPOSE. The Na+-/K+-dependent ATPase (Na, K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. METHODS. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na, K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na, K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. RESULTS. Insulin increased the Na, K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na, KATPase α1-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na, K-ATPase α1-subunit. CONCLUSIONS. These results suggest that insulin increases the Na, K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by α1-subunit dephosphorylation. © Association for Research in Vision and Ophthalmology.

Purpose. To investigate the changes in functional visual acuity (VA) and higher order aberrations in dry eye patients. Methods. In this prospective comparative case series, 22 right eyes were classified into those with or without superficial punctate keratopathy (SPK) in the central cornea of 22 patients with Sjögren syndrome; 10 right eyes of 10 normal subjects served as the control. Serial measurements of VAs using a functional VA measurement system and higher order aberrations using a wavefront sensor were performed under blink-free conditions without topical anesthesia over a 10-second period. The parameters for each measurement were compared among the SPK-positive and -negative and normal groups. The correlation between those parameters was also analyzed. Results. Dry eye with SPK showed significant deterioration of visual function and optical quality compared with dry eye without SPK and in normal eyes, as detected by both the visual maintenance ratio (VMR; P < 0.05) and the variation of VA (P < 0.05) and by comalike and total higher order aberrations (P < 0.05). Moreover, the severity of epithelial damage at the central cornea correlated significantly with VMR (P < 0.01) and variation of VA (P < 0.01) as well as comalike (P < 0.05) and total higher order aberrations (P < 0.05). The dry eye group without SPK showed minor visual deterioration compared with normal eyes, as detected only by VMR (P < 0.05). Conclusions. Optical disturbances at the central optical zone of the cornea in dry eye disease may affect visual performance. Functional VA measurement may be an applicable method of evaluating visual performance in dry eyes that is as efficient as wavefront aberration measurements. © 2011 The Association for Research in Vision and Ophthalmology, Inc.

Background: Anti-vascular endothelial growth factor (VEGF) drugs and/or photodynamic therapy (PDT) constitute current treatments targeting pathological vascular tissues in tumors and age-related macular degeneration. Concern that PDT might induce VEGF and exacerbate the disease has led us to current practice of using anti-VEGF drugs with PDT simultaneously. However, the underlying molecular mechanisms of these therapies are not well understood.Methods: We assessed VEGF levels after PDT of normal mouse retinal tissue, using a laser duration that did not cause obvious tissue damage. To determine the role of PDT-induced VEGF and its downstream signaling, we intravitreally injected a VEGF inhibitor, VEGFR1 Fc, or a PI3K/Akt inhibitor, LY294002, immediately after PDT. Then, histological and biochemical changes of the retinal tissue were analyzed by immunohistochemistry and immunoblot analyses, respectively.Results: At both the mRNA and protein levels, VEGF was upregulated immediately and transiently after PDT. VEGF suppression after PDT resulted in apoptotic destruction of the photoreceptor cell layer in only the irradiated area during PDT. Under these conditions, activation of the anti-apoptotic molecule Akt was suppressed in the irradiated area, and levels of the pro-apoptotic protein BAX were increased. Intravitreal injection of a PI3K/Akt inhibitor immediately after PDT increased BAX levels and photoreceptor cell apoptosis.Conclusion: Cytotoxic stress caused by PDT, at levels that do not cause overt tissue damage, induces VEGF and activates Akt to rescue the neural tissue, suppressing BAX. Thus, the immediate and transient induction of VEGF after PDT is neuroprotective. © 2011 Suzuki et al; licensee BioMed Central Ltd.

Smoking is a serious public health problem around the world and causes many diseases such as chronic obstructive pulmonary disease, lung cancer, and some eye diseases. Cytochrome P450s (CYPs) are xenobiotic-metabolizing enzymes and are distributed in the corneas, protecting the ocular surface against chemical compounds in the environment. Although CYPs are principally detoxification enzymes, CYP1A1 and CYP2A6 are known to participate in the induction of lung cancer by smoking. We studied the participation of CYPs in corneal dysfunction caused by exposure to mainstream cigarette smoke (MCS) in a smoking rat model. Six-week-old male Sprague-Dawley rats were exposed to MCS. Exposure to MCS caused corneal damage and lacrimal gland dysfunction. Immunohistochemical analysis revealed that CYP1A1 expression was upregulated in the corneal epithelium and ducts of the lacrimal glands, accompanied by an increase in production of reactive oxygen species (ROS). An increase in 8-hydroxy-2′- deoxyguanosine, which is a marker of oxidative DNA damage, was detected only in areas where CYP1A1 was expressed, whereas the level of hexanoyl-lysine adduct, which is an initial marker of oxidative damage of phospholipids, did not increase. Exposure to MCS damaged the corneas and lacrimal glands probably through DNA oxidation by ROS produced by CYP1A1. Although the influence of other components in MCS remains unclear, CYPs, especially CYP1A1, probably participate in corneal damage and lacrimal gland dysfunction induced by smoking. © 2011 Elsevier Inc. All rights reserved.

Background: Application of induced pluripotent stem (iPS) cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required. Methodology/Principal Findings: We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method) with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo. Conclusions/Significance: These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets. © 2011 Yoshida et al.

Background: The purpose of this study was to evaluate the correlation between contrast sensitivity and calculated higher-order aberrations based on individual natural pupil diameter after cataract surgery. Methods: This prospective study included 120 eyes from 92 patients who were randomized to receive one of four lenses, including three aspheric lenses (Acrysof SN60WF, Tecnis ZA9000, and Hoya Py60AD) and one spherical lens (Acrysof SN60AT). Contrast sensitivity, higher-order aberrations of the whole eye, and pupil diameter under photopic and mesopic conditions were measured 1 month postoperatively. Higher-order aberrations were decomposed into Zernike coefficients, calculated according to individual pupil diameter. The correlation between higher-order aberrations and contrast sensitivity was evaluated. Results: There were no significant differences in contrast sensitivity function between the four types of lenses under photopic conditions. However, the contrast sensitivity function and area under log contrast sensitivity function in the aspheric lenses were significantly better than in the spherical lens under mesopic conditions. Under mesopic conditions, spherical aberration in eyes with aspheric lenses was significantly lower than in eyes with spherical lenses (P,<0.05). Under photopic conditions, coma aberration had a significant negative correlation with contrast sensitivity at 12 cycles/degree. Under mesopic conditions, spherical aberration had a significant negative correlation with contrast sensitivity at 3, 6, and 12 cycles/degree with glare, and with contrast sensitivity at 6 and 18 cycles/degree without glare. Conclusion: In terms of influence on visual function, coma aberration may be more significant under photopic conditions and spherical aberration under mesopic conditions. © 2011 Yamaguchi et al, publisher and licensee Dove Medical Press Ltd.