Details of a Researcher - Tsubota, Kazuo

Papers - Tsubota, Kazuo

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Neurodegenerative influence of oxidative stress in the retina of a murine model of diabetes

Sasaki M., Ozawa Y., Kurihara T., Kubota S., Yuki K., Noda K., Kobayashi S., Ishida S., Tsubota K.

Diabetologia 53 ( 5 ) 971 - 979 2010.05

ISSN 0012-186X

Aims/hypothesis: Diabetic retinopathy is a progressive neurodegenerative disease, but the underlying mechanism is still obscure. Here, we focused on oxidative stress in the retina, and analysed its influence on retinal neurodegeneration, using an antioxidant, lutein. Methods: C57BL/6 mice with streptozotocin-induced diabetes were constantly fed either a lutein-supplemented diet or a control diet from the onset of diabetes, and their metabolic data were recorded. In 1-month-diabetic mice, reactive oxygen species (ROS) in the retina were measured using dihydroethidium and visual function was evaluated by electroretinograms. Levels of activated extracellular signal-regulated kinase (ERK), synaptophysin and brain-derived neurotrophic factor (BDNF) were also measured by immunoblotting in the retina of 1-month-diabetic mice. In the retinal sections of 4-month-diabetic mice, histological changes, cleaved caspase-3 and TUNEL staining were analysed. Results: Lutein did not affect the metabolic status of the diabetic mice, but it prevented ROS generation in the retina and the visual impairment induced by diabetes. ERK activation, the subsequent synaptophysin reduction, and the BDNF depletion in the diabetic retina were all prevented by lutein. Later, in 4-month-diabetic mice, a decrease in the thickness of the inner plexiform and nuclear layers, and ganglion cell number, together with increase in cleaved caspase-3- and TUNEL-positive cells, were avoided in the retina of lutein-fed mice. Conclusions/interpretation: The results indicated that local oxidative stress that has a neurodegenerative influence in the diabetic retina is prevented by constant intake of a lutein-supplemented diet. The antioxidant, lutein may be a potential therapeutic approach to protect visual function in diabetes. © 2010 Springer-Verlag.

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The role of oxidative stress and inflammation in conjunctivochalasis

Ward Samantha K., Wakamatsu Tais Hitomi, Dogru Murat, Ibrahim Osama M A, Kaido Minako, Ogawa Yoko, Matsumoto Yukihiro, Igarashi Ayako, Ishida Reiko, Shimazaki Jun, Schnider Cristina, Negishi Kazuno, Katakami Chikako, Tsubota Kazuo

Investigative Ophthalmology and Visual Science 51 ( 4 ) 1994 - 2002 2010.04

ISSN 0146-0404

PURPOSE. To investigate the status of oxidative stress and histopathologic alterations in patients with conjunctivochalasis and compare the findings with those in healthy control subjects. METHODS. Eleven patients (n = 20 eyes) with Yokoi grade 3 conjunctivochalasis and 11 health control subjects (n = 22 eyes) were prospectively recruited. ELISA for tear hexanoyllysine (HEL) and inflammatory cytokines, tear film break-up time tests, Schirmer test measurements, and fluorescein and rose bengal vital staining were performed. Conjunctival specimens obtained during surgery for conjunctivochalasis and cataract underwent immunohistochemical staining for HEL+8- OHdG, MMP-3, and MMP-9, and positively stained cells were counted. Transmission electron microscopy was also performed, with staining for elastic fibers in the conjunctival stroma. RESULTS. The mean tear stability and vital staining scores were significantly worse in the conjunctivochalasis patients than in the control subjects. The tear HEL and tear cytokine levels showed significantly higher values in eyes with conjunctivochalasis. IL-1β and IL-6 levels showed a significant correlation with corneal epithelial damage. IL-1β and TNFα showed a significant correlation with 8-OHdG-stained cell counts. Specimens from patients with conjunctivochalasis revealed a significantly higher number of cells positively stained for HEL, 8-OHdG, MMP-3, and MMP-9 than did specimens from age- and sex-matched control subjects. Transmission electron microscopy showed decreased intercellular cohesiveness, with the conjunctival stroma showing an accumulation of elastic fibers. CONCLUSIONS. Lipid and DNA oxidative stress were present in the conjunctiva. Increased tear inflammation seemed to coexist with loss of conjunctival epithelial cohesiveness and increased collagenolytic activity, which may explain the conjunctival laxity observed in patients with conjunctivochalasis. © Association for Research in Vision and Ophthalmology.

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Biconvex posterior chamber accommodating intraocular lens implantation after cataract surgery

Saiki Megumi, Negishi Kazuno, Dogru Murat, Yamaguchi Takefumi, Tsubota Kazuo

Journal of Cataract and Refractive Surgery 36 ( 4 ) 603 - 608 2010.04

ISSN 0886-3350

Purpose: To evaluate the long-term efficacy of a biconvex accommodating intraocular lens (IOL) to restore near visual performance. Setting: Department of Ophthalmology, Keio University Hospital, Tokyo, Japan. Methods: This 4-year study comprised eyes that had cataract surgery with implantation of a 1CU accommodating IOL. At all postoperative visits, the following were assessed: subjective spherical equivalent (SE) refraction; uncorrected (UDVA) and corrected (CDVA) distance visual acuities; uncorrected (UNVA), distance-corrected (DCNVA), and corrected (CNVA) near visual acuities; change in minimum additional (add) power (diopters) to attain CNVA; and accommodation amplitude. A questionnaire was administered at the last visit. Results: Twelve eyes of 8 cataract patients (mean age 59.0 years ± 18.4 [SD]) were evaluated. Postoperatively, the mean SE at 1 year and 4 years was significantly more hyperopic than at 1 month (P<.05). The mean UDVA increased significantly at 4 years compared with 1 month (P<.05). There were no significant changes in CDVA, UNVA, CNVA, and DCNVA throughout the follow-up period. The change in the minimum add power to attain CNVA and in the subjective and objective accommodation amplitudes also did not change significantly over time. Eighty-three percent of patients reported being satisfied with the results. Conclusion: Four years after implantation of an accommodating IOL, most patients had good vision and were satisfied, although the accommodation amplitude for near vision was not sufficient throughout the follow-up. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned. © 2010 ASCRS and ESCRS.

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The Efficacy, Sensitivity, and Specificity of In Vivo Laser Confocal Microscopy in the Diagnosis of Meibomian Gland Dysfunction

Ibrahim Osama M A, Matsumoto Yukihiro, Dogru Murat, Adan Enrique Sato, Wakamatsu Tais Hitomi, Goto Tateki, Negishi Kazuno, Tsubota Kazuo

Ophthalmology 117 ( 4 ) 665 - 672 2010.04

ISSN 0161-6420

Purpose: To evaluate the efficacy, sensitivity and specificity of confocal microscopy (CM) parameters: meibomian gland (MG) acinar longest diameter (MGALD), MG acinar shortest diameter (MGASD), inflammatory cell density (ICD), and MG acinar unit density (MGAUD) in the diagnosis of MG dysfunction (MGD). Design: Prospective, controlled, single-center study. Participants: Twenty MGD patients (9 males, 11 females; mean age, 63.5±16.5 years) and 26 age- and gender-matched control subjects (13 males, 13 females; mean age, 53.2±15.7 years) were recruited. Methods: All subjects underwent slit-lamp examinations, tear film break-up time (BUT) measurements, assessment of tear evaporation rate from the ocular surface (TEROS), vital stainings, Schirmer test, meibography, MG expressibility, and CM of the MG. Data were compared between the 2 groups using the Mann-Whitney and chi-square tests. Main Outcome Measures: The correlation between the clinical findings of tear functions, vital staining scores, and the 4 CM parameters were tested by Spearman's correlation coefficient by rank test. Receiver operating characteristic curve technique was used to evaluate the sensitivity, specificity, and cutoff values of CM parameters. Results: The mean tear film BUT, vital staining scores, TEROS values, MG expressibility, and MG dropout grades by meibography were significantly worse in MGD patients compared with controls (P<0.001). The mean values of the MGALD, MGASD, ICD, and MGAUD in MGD patients were significantly worse than those observed in the controls with CM. All CM parameters showed a strong, significant correlation with tear functions, ocular surface vital stainings, MG expressibility, and MG dropout grades. The cutoff values for MGALD, MGASD, ICD, and MGAUD in the diagnosis of MGD were 65 μm, 25 μm, 300 cells/mm², and 70 glands/mm², respectively. The sensitivity and specificity values of these parameters under these cutoff values were 90% and 81% for MGALD, 86% and 96% for MGASD, 100% and 100% for ICD, 81% and 81% for MGAUD. Conclusions: Confocal microscopy has the potential to diagnose the simple MGD with high sensitivity and specificity. The CM-based diagnostic parameters correlated significantly and strongly with the status of the ocular surface 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.

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Replacement of multifocal by monofocal intraocular lens in two cases for asthenopia and photophobia

Torii Hidemasa, Negishi Kazuno, Dogru Murato, Tsubota Kazuo

Japanese Journal of Clinical Ophthalmology 64 ( 4 ) 459 - 463 2010.04

ISSN 0370-5579

Purpose : To report two cases who received replacement of multifocal intraocular lens (IOL) by monofocal IOL due to postoperative dissatisfaction. Cases : A 75-year-old male received bilateral cataract surgery with implantation of diffractive multifocal IOL. Asthenopia and photophobia became worse after surgery. In accordance to his wish, the multifocal IOL was replaced by monofocal IOL 4 months later. His subjective complaints persisted but improved after treatment for essential blepharospasm. Another 52-year-old female received similar bilateral cataract surgery with implantation of refractive multifocal IOL. The lens was replaced by monofocal one due to photophobia 4 months later. Her subjective complaints persisted. Conclusion : These cases illustrate that replacement of multifocal by monofocal IOL due to dissatisfaction of patients need careful considerations.

Induction of corneal epithelium-like cells from cynomolgus monkey embryonic stem cells and their experimental transplantation to damaged cornea

Kumagai Yuta, Kurokawa Manae S., Ueno Hiroki, Kayama Maki, Tsubota Kazuo, Nakatsuji Norio, Kondo Yasushi, Ueno Satoki, Suzuki Noboru

Cornea 29 ( 4 ) 432 - 438 2010.04

ISSN 0277-3740

Purpose: We previously reported the successful transplantation of corneal epithelium-like cells derived from mouse embryonic stem (ES) cells onto injured mouse cornea. Here, we tested whether nonhuman primate ES cells have ability to differentiate into corneal epithelial cells and whether monkey ES cell-derived corneal epithelium-like cells were applicable for the experimental transplantation to damaged cornea. Methods: Monkey ES cells were cultivated on type IV collagen-coated dishes for various days to induce differentiation into corneal epithelium-like cells. The differentiation was evaluated by reverse transcription-polymerase chain reaction and immunostaining. The corneal epithelium-like cells were transplanted to the injured mouse cornea. Reconstitution of the corneal epithelium was evaluated by immunostaining. Results: The cells cultured on type IV collagen showed cobblestone like appearance resembling epithelial cells. They expressed messenger RNA of pax6, p63, E-cadherin, CD44, proliferating cell nuclear antigen, keratin 3, and keratin 12. Protein expressions of pax6, keratin 3/12, p63, proliferating cell nuclear antigen, E-cadherin, and CD44 were confirmed by immunostaining. When the corneal epithelium-like cells were transplanted, they adhered to the corneal stroma, leading to formation of multiple cell layers. The grafted cells were stained with anti-human nuclear protein antibody, which crossreacted with nuclei of monkey cells but not with those of mouse cells. They retained the expressions of keratin 3/12, E-cadherin, and CD44. Conclusions: We induced corneal epithelium-like cells from monkey ES cells with moderate efficiency. The cells were successfully transplanted onto the injured mouse cornea. This is the first demonstration that nonhuman primate ES cells were induced to differentiate into corneal epithelium-like cells, which were applicable for transplantation to an animal model of corneal injury. © 2010 Lippincott Williams & Wilkins.

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Erratum

Kawakita T., Kawashima M., Murat D., Tsubota K., Shimazaki J.

Eye 24 ( 4 ) 746 2010.04

ISSN 0950-222X

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Mechanism of visual sensations experienced during pars plana vitrectomy under retrobulbar anesthesia

Sugisaka Eiko, Shinoda Kei, Sano Ronaldo Yuiti, Ishida Susumu, Imamura Yutaka, Ozawa Yoko, Shinoda Hajime, Suzuki Kotaro, Tsubota Kazuo, Inoue Makoto

Ophthalmologica 224 ( 2 ) 103 - 108 2010.02

ISSN 0030-3755

Purpose: To investigate the visual sensations experienced by patients during vitrectomy under retrobulbar anesthesia. Methods: 30 men and 45 women with a mean age of 65.3 ± 10.6 years underwent vitrectomy under retrobulbar anesthesia for macular disease. 28 eyes had an idiopathic epiretinal membrane, 13 had an idiopathic macular hole, 32 had macular edema (17 diabetic retinopathy and 15 retinal vein occlusion), and 2 had submacular hemorrhage. 49 patients with nonmacular disease underwent similar vitrectomy procedures and were used for comparison. An interview was conducted with the patient about his/her visual sensations during and within 3 h of the vitrectomy. Results: 70 (93.3%) of the patients reported seeing lights, 53 (70.7%) reported seeing colors, and 48 (64.0%) reported seeing movements or moving objects. Of the patients who reported seeing movements or moving objects, 44 (58.7%) reported seeing surgical instruments, and 5 (6.7%) saw the surgeon's fingers or hands. Patients with macular diseases tended to report more visual sensations than patients with nonmacular diseases. The patients' description and drawings appeared to arise mainly from the shadows cast by the intravitreal objects, and some patients perceived highly accurate details including the movements and color of the objects. Conclusions: Visual sensations are experienced by approximately 90% of the patients, and there may be a common mechanism by which patients perceive the intravitreal objects that are not focused on by the retina through the eye's optical system. © 2009 S. Karger AG, Basel.

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Conjunctival in vivo confocal scanning laser microscopy in patients with sjögren syndrome

Wakamatsu Tais Hitomi, Sato Enrique Adan, Matsumoto Yukihiro, Ibrahim Osama M A, Dogru Murat, Kaido Minako, Ishida Reiko, Tsubota Kazuo

Investigative Ophthalmology and Visual Science 51 ( 1 ) 144 - 150 2010.01

ISSN 0146-0404

PURPOSE. To demonstrate the conjunctival alterations in patients with Sjögren's (SSDE) and non-Sjögren's syndrome dry eye (NSSDE) using a new generation confocal microscope (HRTII/ RCM; Heidelberg Engineering, Heidelberg, Germany), in a prospective controlled study. METHODS. Twenty-eight right eyes of 28 patients with SSDE (28 women; mean age, 58.2 ± 14.3 years), 7 right eyes of patients with NSSDE (7 women; mean age, 66.1 ± 14.4 years), and 14 right eyes of 14 age- and sex-matched control subjects were studied. All subjects underwent the Schirmer test, tear film breakup time (BUT), vital staining, and confocal microscopy of the temporal bulbar conjunctiva. The density of conjunctival epithelial cells, epithelial microcysts, and conjunctival and corneal inflammatory infiltrates were also assessed. RESULTS. The tear quantity, stability, and vital staining scores were significantly worse in patients with SSDE or NSSDE than in control subjects (P < 0.001 and P < 0.05, respectively). Eyes of the patients with SSDE or NSSDE had a significantly higher density of conjunctival and corneal inflammatory infiltrates than did the control eyes (P < 0.001). Conjunctival inflammatory cell densities showed a negative correlation with tear stability and tear quantity and a positive correlation with the vital staining scores. Conjunctival epithelial cell densities were significantly lower in SSDE and NSSDE compared with control subjects (P < 0.05). The density of epithelial cysts was significantly higher in SS than in healthy control eyes (P < 0.001). CONCLUSIONS. Confocal scanning laser microscopy was an efficient and a noninvasive tool for the quantitative assessment of the conjunctival inflammation and epithelial cell densities as well as evaluation of conjunctival morphologic alterations, such as microcysts in patients with SSDE and NSSDE dry eye disease. © Association for Research in Vision and Ophthalmology.

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Subepithelial corneal fibrosis partially due to epithelial-mesenchymal transition of ocular surface epithelium

Kawashima Motoko, Kawakita Tetsuya, Higa Kazunari, Satake Yoshiyuki, Omoto Masahiro, Tsubota Kazuo, Shimmura Shigeto, Shimazaki Jun

Molecular Vision 16 2727 - 2732 2010

ISSN 1090-0535

Purpose: To determine whether epithelial-mesenchymal transition is involved in the development of corneal subepithelial fibrosis (pannus). Methods: Frozen samples of pannus tissue removed from human corneas with a diagnosis of total limbal stem cell deficiency were characterized by immunostaining for both epithelial and mesenchymal markers. We selected transformation-related protein 63 (p63) and pancytokeratin as epithelial markers and vimentin and α-smooth muscle actin (α-SMA) as mesenchymal markers. Immunostaining for β-catenin and E-cadherin was performed to determine wingless- Int (Wnt)-pathway activation. RT-PCR analysis was also performed on epithelial tissue obtained from pannus samples after dispase digestion. Results: Immunohistochemistry revealed strong nuclear expression of p63 and weak intercellular expression of E-cadherin in epithelial basal cells of pannus tissue. Furthermore, translocation of β-catenin from intercellular junctions to the nucleus and cytoplasm was also observed. Double-positive cells for both p63 and α-SMA were observed in the subepithelial stroma of pannus tissue, which was supported by RT-PCR and cytospin analysis. Conclusions: Epithelial-mesenchymal transition may be partially involved in the development of subepithelial corneal fibrosis due to total limbal stem cell deficiency. © 2010 Molecular Vision.

Topical tranilast for treatment of the early stage of mild dry eye associated with chronic GVHD

Ogawa Y., Dogru M., Uchino M., Tatematsu Y., Kamoi M., Yamamoto Y., Ogawa J., Ishida R., Kaido M., Hara S., Matsumoto Y., Kawakita T., Okamoto S., Tsubota K.

Bone Marrow Transplantation 45 ( 3 ) 565 - 569 2010

ISSN 0268-3369

Fibrosis and excessive extracellular matrix production are characteristic features of lacrimal gland chronic GVHD (cGVHD). Tranilast (n-3,4-anthoranilic acid), used for fibrotic skin disease, inhibits transforming growth factor-Β-induced matrix production. We conducted a non-randomized study comparing 8 patients (five men, three women; median age, 47 years) given topical tranilast with 10 patients (three men, seven women; median age, 37 years) receiving therapy with topical artificial tears, sodium hyaluronate and vitamin A for mild ocular cGVHD. The tranilast group instilled topical tranilast and artificial tears q.i.d., beginning the day of dry eye diagnosis. The ocular surface and tear dynamics of each patient were evaluated before hematopoietic stem cell transplant, at the onset of dry eye and after 3 months of treatment. At 3 months, the scores of the Rose Bengal test and Schirmer test with nasal stimulation had significantly improved in the tranilast group compared with that in the control group (P<0.05). Furthermore, although five control patients (50) developed severe dry eye within the treatment period, only one tranilast-treated patient (12.5) did; the rest still had mild dry eye (P<0.05). These results suggest the hypothesis that topical tranilast may effectively retard the progression of mild dry eye associated with cGVHD. © 2010 Macmillan Publishers Limited.

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Lacrimal hypofunction as a new mechanism of dry eye in visual display terminal users

Nakamura Shigeru, Kinoshita Shigeru, Yokoi Norihiko, Ogawa Yoko, Shibuya Michiko, Nakashima Hideo, Hisamura Ryuji, Imada Toshihiro, Imagawa Tomohiro, Uehara Masato, Shibuya Izumi, Dogru Murat, Ward Samantha, Tsubota Kazuo

PLoS ONE 5 ( 6 ) 2010

Background: Dry eye has shown a marked increase due to visual display terminal (VDT) use. It remains unclear whether reduced blinking while focusing can have a direct deleterious impact on the lacrimal gland function. To address this issue that potentially affects the life quality, we conducted a large-scale epidemiological study of VDT users and an animal study. Methodology/Principal Findings: Cross sectional survey carried out in Japan. A total of 1025 office workers who use VDT were enrolled. The association between VDT work duration and changes in tear film status, precorneal tear stability, lipid layer status and tear secretion were analyzed. For the animal model study, the rat VDT user model, placing rats onto a balance swing in combination with exposure to an evaporative environment was used to analyze lacrimal gland function. There was no positive relationship between VDT working duration and change in tear film stability and lipid layer status. The odds ratio for decrease in Schirmer score, index of tear secretion, were significantly increased with VDT working year (P = 0.012) and time (P = 0.005). The rat VDT user model, showed chronic reduction of tear secretion and was accompanied by an impairment of the lacrimal gland function and morphology. This dysfunction was recovered when rats were moved to resting conditions without the swing. Conclusions/Significance: These data suggest that lacrimal gland hypofunction is associated with VDT use and may be a critical mechanism for VDT-associated dry eye. We believe this to be the first mechanistic link to the pathogenesis of dry eye in office workers. © 2010 Nakamura et al.

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Serum free fatty acids levels not associated with normal tension glaucoma

Yuki Kenya, Kimura Itaru, Tsubota Kazuo

Clinical Ophthalmology 4 ( 1 ) 91 - 94 2010

ISSN 1177-5467

Purpose: To determine the free fatty acid levels in the sera of patients with normal-tension glaucoma and compare it with that of normal controls. Methods: Forty-four consecutive patients with newly diagnosed normal-tension glaucoma and forty-four age and gender matched controls were evaluated. The type and level of fatty acids in the sera were measured by gas chromatography (Model GC17A; Shimazu, Kyoto, Japan). Twenty-four fatty acids were identified from 12:0 to 24:1. The values were compared between the normal-tension glaucoma and control groups by Mann-Whitney U tests. Results: No statistically significant difference was found in the levels of any free fatty acids between the normal-tension glaucoma group and control group. Conclusion: No significant association was found in the serum free fatty acids levels including docosahexaenoic acid and eicosapentaenoic acid between normal-tension glaucoma patients and controls. © 2010 Yuki et al, publisher and licensee Dove Medical Press Ltd.

Reduced-serum vitamin C and increased uric acid levels in normal-tension glaucoma

Yuki Kenya, Murat Dogru, Kimura Itaru, Ohtake Yuichiro, Tsubota Kazuo

Graefe's Archive for Clinical and Experimental Ophthalmology 248 ( 2 ) 243 - 248 2010

ISSN 0721-832X

Background: Vitamin A, B₉, C, E, and uric acid are well-known antioxidants and may prevent age-related eye disorders. The aim of the present study was to investigate the levels of antioxidant vitamins, A, B₉, C, E, and antioxidative substance, uric acid in the serum of Japanese patients with normal-tension glaucoma and compare the results with normal controls. Methods: All subjects with suspicion of primary open-angle glaucoma who came to the glaucoma subspeciality clinic of Keio University Hospital were enrolled in this study. Sixty patients (28 males, 32 females; mean age ± standard deviation: 59.9 ± 9.8 years) with newly diagnosed primary open-angle glaucoma patients were consecutively enrolled in this study. After the diagnosis of primary open-angle glaucoma, the patients underwent 24-h IOP measurements. Forty-seven newly diagnosed consecutive normal-tension glaucoma patients (18 males, 29 females; mean age ± standard deviation: 59.5 ± 10.2 years) were enrolled in this study. The control subjects were recruited from subjects who came to the clinic for annual refractive check-up. The 44 consecutive control subjects of the current study, (16 males, 28 females; 62.7 ± 14.8 years) did not have any ocular diseases. The serum levels of vitamins A, B₉, C, E, and uric acid were measured. The values were compared between the normal-tension glaucoma and control groups by the Mann-Whitney U test. Results: Serum levels of vitamin C were significantly lower in normal-tension glaucoma patients than in normal healthy controls (P = 0.04; normal-tension glaucoma; 4.6 ± 4.0 μg/ml control; 6.3 ± 3.9 μg/ml). Uric acid level was significantly higher in normal-tension glaucoma patients than in controls (P = 0.01; normal-tension glaucoma; 5.8 ± 1.5 mg/dl control; 4.9 ± 1.4 mg/dl). No statistically significant difference was seen in vitamin A (P = 0.41; normal-tension glaucoma; 82.1 ± 26.7 μg/dl control; 77.1 ± 30.1 μg/dl), B₉ (P = 0.37; normal-tension glaucoma; 8.7 ± 4.3 ng/ml control; 8.0 ± 3.1 ng/ml)and E (P = 0.83; normal-tension glaucoma; 1.5 ± 0.6 control; 1.5 ± 0.6) levels between normal-tension glaucoma and control groups. Conclusion: Normal-tension glaucoma patients had lower serum levels of vitamin C and increased levels of uric acid. These observations may pave the way for possible alternative treatment for normal-tension glaucoma. © 2009 Springer-Verlag.

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Evaluation of lipid oxidative stress status and inflammation in atopic ocular surface disease

Wakamatsu Tais H., Dogru Murat, Ayako Igarashi, Takano Yoji, Matsumoto Yukihiro, Ibrahim Osama M A, Okada Naoko, Satake Yoshiyuki, Fukagawa Kazumi, Shimazaki Jun, Tsubota Kazuo, Fujishima Hiroshi

Molecular Vision 16 2465 - 2475 2010

ISSN 1090-0535

Background: Although the oxidative stress status in atopic skin disease has been reported to be elevated, there are still no studies related to the status of oxidative stress in atopic ocular surface disease. The purpose of this study was to evaluate the ocular surface lipid oxidative stress status and inflammation in atopic keratoconjunctivitis (AKC) patients and normal subjects. Methods: Twenty eight eyes of 14 patients (9 males, 5 females) with AKC and 18 eyes of 9 age and sex matched (4 males and 5 females) normal healthy controls were examined in this prospective study. The severity of atopic dermatitis (AD) was scored by the SCORing Atopic Dermatitis (SCORAD) index. All subjects underwent Schirmer test, tear film break up time (BUT), fluorescein/Rose Bengal stainings, tear collection, and brush cytology from the upper palpebral conjunctiva. The brush cytology samples were stained with Diff-Quik for differentiation of inflammatory cells and immunohistochemistry (IHC) staining with HEL (hexanoyl-lysine) and 4-HNE (4-hydroxy-2-nonenal) to study lipid oxidation. HEL and cytokine (interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), tumor necrosis factoralpha (TNF-α), interferon-gamma (IFN-γ)) levels were measured by enzyme-linked immunosorbent assay (ELISA) from tear samples of AKC patients and control subjects. Toluidine Blue and IHC staining with HEL, 4-HNE and cluster of differentiation 45 (CD45) were performed on papillary samples of AKC patients. This study was conducted in compliance with the "Declaration of Helsinki." Results: The tear stability and vital staining scores were significantly worse in eyes of AKC patients (p<0.05) compared to the controls. Inflammatory cells and positively stained conjunctival epithelial cells for HEL and 4-HNE showed a significant elevation in brush cytology samples of AKC patients. Significantly higher levels of HEL and cytokines were detected in tears of AKC patients compared to controls. Papillary specimens also revealed many CD45 inflammatory cells as well as many cells positively stained with HEL and 4-HNE in IHC. A strong significant linear positive correlation between conjunctival inflammation and epithelial lipid oxidative stress status was observed. Conjunctival lipid oxidative stress also correlated strongly with tear HEL levels and epithelial damage scores. Conclusions: The ocular surface disease in AKC was characterized by marked tear instability, ocular surface epithelial damage, increase in inflammatory infiltrates and presence of increased lipid oxidation. © 2010 Molecular Vision.

The effect of topical amiloride eye drops on tear quantity in rabbits

Hara Shuya, Hazama Akihiro, Miyake Masao, Kojima Takashi, Sasaki Yasumasa, Shimazaki Jun, Dogru Murat, Tsubota Kazuo

Molecular Vision 16 2279 - 2285 2010

ISSN 1090-0535

Purpose: To investigate the presence of epithelial sodium channels (ENaC) in rabbit and human conjunctival epithelium and to test the effects of topical amiloride, a potassium-sparing diuretic that blocks the ENaC, on tear quantity in rabbits. Methods: Both healthy normal human and rabbit conjunctival tissues underwent immunohistochemistry staining for ENaC-α and γ subunits as well as for reverse transcription polymerase chain reaction (RT-PCR) for detection of ENaC- α and ENaC-γ subunit mRNA expression. Rabbits were instilled topical amiloride eye drops and tear function tests were performed before and after instillations. Results: Immunohistochemical staining for ENaC-α subunit in all rabbit eyes showed positive staining in apical and basal conjunctival epithelial cells. Human conjunctival epithelia revealed positive staining with ENaC-α antibody especially in the apical and basal layers. Immunohistochemistry staining with ENaC- γ antibody also revealed positive staining of the conjnunctival epithelial cells especially in the basal layers. The ENaC-α mRNA was detected in samples from healthy white rabbit conjunctival epithelia, and ENaC-α and ENaC- γ mRNAs were detected in samples from healthy human conjunctival epithelia. The mean tear quantity showed a significant increase at 15 and 30 min compared to the pre-instillation value in eyes assigned to amiloride eye drops. The mean tear quantity at 15 and 30 min was significantly higher in the amiloride group compared to the control eyes. Conclusions: Topical amiloride application appears to increase the quantity of preocular tears owing to inhibition of conjunctival epithelial sodium channels. © 2010 Molecular Vision.

Anti-aging in ophthalmology.

Tsubota Kazuo

Ophthalmic research 44 ( 3 ) 145 2010

Pattern-reversal visual-evoked potential in patients with occult macular dystrophy

Hanazono Gen, Ohde Hisao, Shinoda Kei, Tsunoda Kazushige, Tsubota Kazuo, Miyake Yozo

Clinical Ophthalmology 4 ( 1 ) 1515 - 1520 2010

ISSN 1177-5467

Purpose: Occult macular dystrophy (OMD) is a hereditary retinal disease characterized by a normal fundus, normal full-field electroretinograms (ERGs), progressive decrease of visual acuity, and abnormal focal macular ERGs. The purpose of this study was to report pattern-reversal visual-evoked potential (pVEPs) findings in OMD patients. Patients and method: The pVEPs recorded from four patients with OMD (aged 42-61 years; 2 men and 2 women) were reviewed. The visual acuities ranged from 20/200 to 20/30. The amplitudes of the N-75 and P-100 (P2 amplitude) and the latency of the N-75 components (N1 latency) were analyzed. Results: The mean (±SD) P2 amplitude was 2.7 ± 1.9 μV for the 5′, 4.8 ± 2.9 μV for the 10′, 3.2 ± 2.1 μV for the 20′, and 4.4 ± 3.5 μV for the 40′ checkerboard stimuli. The N1 latency was 122.2 ± 6.4 ms for the 5′, 105.0 ± 11.5 ms for the 10′, 97.7 ± 10.0 ms for the 20′, and 91.0 ± 13.7 ms for the 40′ checkerboard stimuli. The mean P2 amplitude was reduced and the N1 latency was delayed in comparison with the laboratory standard for the Keio University Hospital. Conclusions: The delayed latency and reduced amplitude suggest a major contribution of the central cone pathway to the pVEPs. © 2010 Hanazono et al.

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Selenoprotein P controls oxidative stress in cornea

Higuchi Akihiro, Takahashi Kazuhiko, Hirashima Masaki, Kawakita Tetsuya, Tsubota Kazuo

PLoS ONE 5 ( 3 ) 2010

The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress. © 2010 Higuchi et al.

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Eicosapentaenoic acid suppresses ocular inflammation in endotoxin-induced uveitis

Suzuki Misa, Noda Kousuke, Kubota Shunsuke, Hirasawa Manabu, Ozawa Yoko, Tsubota Kazuo, Mizuki Nobuhisa, Ishida Susumu

Molecular Vision 16 1382 - 1388 2010

ISSN 1090-0535

Purpose: To investigate the effect of eicosapentaenoic acid (EPA) on acute ocular inflammation in an animal model of endotoxin-induced uveitis (EIU). Methods: C57Bl/6 mice (6-week-old males) were orally treated with EPA at a dose of 50 mg/kg/day for 5 days. EIU was then induced in the animals by intraperitoneal injection of 160 μg lipopolysaccharide (LPS). Twenty-four hours after LPS injection, leukocyte adhesion to the retinal vasculature was evaluated by the concanavalin A lectin perfusion-labeling technique, and leukocyte infiltration into the vitreous cavity was quantified. Furthermore, the protein levels of monocyte chemotactic protein (MCP)-1, interleukin (IL)-6, intercellular adhesion molecule-1 and phospholyrated nuclear factor (NF)-κB p65 in the retina and retinal pigment epithelium (RPE)-choroid complex were examined by enzyme-linked immunosorbent assay (ELISA). Results: At 24 h after LPS injection, the EIU animals treated with oral EPA administration showed a significant decrease in leukocyte adhesion to the retinal vessels by 43.4% (p<0.01) and leukocyte infiltration into the vitreous cavity by 49.2% (p<0.05). In addition, EPA significantly reduced the protein levels of MCP-1 and IL-6 in the retina and the RPE-choroid complex. Furthermore, phosphorylation of NF-κB was suppressed by EPA treatment. Conclusions: Our data suggest that EPA inhibits multiple inflammatory molecules in vivo. EPA may become a novel strategy in the prevention and/or treatment of ocular inflammatory diseases. © 2010 Molecular Vision.

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