Takashi Kanamoto

Purpose: The purpose of this study was to investigate the physiological changes of amino acids in the rat retina caused by ocular hypertension. Methods: Adult Wistar rats were used as an experimental model of ocular hypertension. Retinas were hydrolyzed with HCl at 108°C to isolate amino acids. Residual amino acids were measured by reverse phase high-performance liquid chromatography and the total volume of residual amino acids and the ratio of D- and L-amino acids were analyzed. Free D- and L-alanine levels were also measured using two-dimensional HPLC. Results: The amount of retinal alanine decreased in ocular hypertension compared with the control (p < 0.05, Student’s t-test); the amounts of other amino acids did not differ between the two conditions. The D/L ratio of alanine was higher than that of other amino acids. Ocular hypertension reduced the D/L ratio of retinal alanine, while that of other amino acids was unchanged. Ocular hypertension increased the D/L ratio of free alanine. Conclusions: Ocular hypertension reduced the D/L ratio of retinal alanine, presumably due in large part to alanine peptides.

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins.Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA.Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed ofα-enolase, S-arrestin, and ATP synthase subunitsαandβ, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats.Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

Purpose: The purpose of this study is to investigate the role of free D-serine in the death of retinal cells caused by ocular hypertension.Methods: Adult Wistar rats were used as an experimental model of ocular hypertension. Immunohistochemistry was used to identify the retinal sites and expression patterns of D-serine and serine racemase in the rat retina. The concentrations of free D-serine and L-serine in the retina were measured by two-dimensional high-performance liquid chromatography. Retinal cell death was investigated by Immunohistochemistry.Results: D-serine was expressed on the retinal ganglion cell layer in the retinas of rats with ocular hypertension. A serine racemase was specifically expressed in the retinal ganglion cells. The ratio of free D-/L-serine in the retinas with ocular hypertension was higher than that in the retinas with normal tension. Annexing-V-positive cells were observed in the retinal ganglion cell layer in the retinas of the rats with ocular hypertension, and these cells were also co-localized with D-serine expression.Conclusions: We suspect that the up-regulation of serine racemase expression induced by ocular hypertensionleads to an increase in free D-serine converted from free L-serine in retinal ganglion cells and that retinal cell death is associated with D-serine expression.

Purpose: To evaluate the ocular-surface safety of a 0.001% benzalkonium chloride-containing tafluprost/timolol fixed combination (TTFC) in patients with primary open-angle glaucoma (POAG) or ocular hypertension who have inadequate intraocular pressure (IOP) control with latanoprost monotherapy. Methods: This study is a multicenter, prospective, single-arm, open-label clinical study. Patients with POAG or ocular hypertension who have inadequate IOP control with latanoprost monotherapy were considered eligible. After providing informed consent, patients continued latanoprost monotherapy for 12 weeks, followed by a switch to TTFC. We evaluated the extent of ocular-surface damage using superficial punctate keratopathy (SPK) score, tear breakup time (TBUT), hyperemia score, IOP, systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate at 0, 4, and 12 weeks after switching. Results: A total of 68 patients were enrolled, of whom, 64 patients were included in the final analysis. No significant changes in SPK score, TBUT, or hyperemia score were observed at 4 and 12 weeks compared with week 0. IOP decreased significantly at 4 (13.9±2.5 mmHg) and 12 (14.1±2.5 mmHg) weeks, relative to week 0 (15.3±2.7 mmHg). No significant changes in either SBP or DBP were observed during the study, although patients’ mean heart rate decreased significantly after switching to TTFC. Adverse drug reactions to TTFC occurred in seven patients including two incidences of asthma and one of arrhythmia, and no events were serious. Conclusion: The ocular-surface safety of TTFC is not significantly different to that of latanoprost. Furthermore, switching from latanoprost to TTFC in patients with insufficient IOP control has additive IOP-lowering effects. TTFC is an effective approach for patients receiving latanoprost monotherapy who require more intensive IOP reduction.

Background. To investigate how transcorneal electric stimulation (TES) affects the retina, by identifying those proteins up- and downregulated by transcorneal electric stimulation (TES) in the retina of rats.Methods. Adult Wistar rats received TES on the left eyes at different electrical currents while the right eyes received no treatment and served as controls. After TES, the eye was enucleated and the retina was isolated. The retinas were analyzed by proteomics.Results. Proteomics showed that twenty-five proteins were upregulated by TES. The identified proteins included cellular signaling proteins, proteins associated with neuronal transmission, metabolic proteins, immunological factors, and structural proteins.Conclusions. TES induced changes in expression of various functional proteins in the retina.

To investigate the clinical effects of travoprost-timolol fixed combination on ocular hypotensive agents, ocular surface agents and adherence in Japanese glaucoma patients. 28 Japanese glaucoma patients, who had used topical prostaglandin F2alpha analogue (28 eyes; PG group), were assigned to treatment with travoprost-timolol fixed combination. Reduction of intraocular pressure (IOP), grades of conjuctival follicle, conjuctival injection and keratoepitheliopathy, as well as interviews for topical administration, were evaluated. 38 patients, who switched from the topical prostaglandin F2alpha analogue and a beta-blocker to travoprost-timolol fixed combination (38eyes: PG+BB group) were also evaluated in the same manner. While IOP was significantly reduced in the PG group, IOP was not changed in the PG+BB group. Both groups showed no significant changes in scores for conjuctival follicle, keratoepitheliopathy, or interview. These results suggest that travoprost-timolol fixed combination reduces IOP and produces low toxicity on the ocular surface.

Aim: To report on the effectiveness of systemic steroid-pulse therapy in treating idiopathic choroidal detachment. Case Presentation:Our patient developed idiopathic choroidal detachment after cataract surgery for pseudoexfoliation glaucoma and glaucoma filtration surgery. Systemic steroid-pulse therapy was performed, and the choroidal detachment resolved completely. Conclusion: We suggest systemic steroid-pulse therapy as a treatment option for idiopathic choroidal detachment with low intraocular pressure.

The purpose of this study was to investigate the role played by platelet-derived growth factor-α (PDGFRα) in oxidative stress-induced retinal cell death. A previous proteomic study from our laboratory showed that expression of PDGFRα is elevated considerably in the retinas of an animal model of glaucoma-the excitatory amino acid carrier (EAAC) 1-deficient (EAAC1-/-) mouse. Retinal sites and expression patterns of PDGFRα were determined by immunohistochemistry in the retinas of EAAC1-/- and control CRL:CD1(ICR) mice. A retinal cell line was exposed to hydrogen peroxide, and expression PDGFRα determined by Western blot analysis. Effects of PDGF-AA and PDGFRα-siRNA on hydrogen peroxide-induced retinal cell death were examined. PDGFRα was detected in the retinal ganglion cell layer (RGL) of both EAAC1-/- and ICR mice, and was also localized in the internal nuclear layer (INL) of EAAC1-/- mice. While treatment with excess PDGF-AA had no additional effect on retinal cell death, expression of PDGFRα increased with exposure to hydrogen peroxide. Hydrogen peroxide-induced retinal cell death was inhibited by exposure to PDGF-AA via phosphatidylinositol 3 kinase (PI3K); cell death was promoted by PDGFRα-siRNA. PDGFRα is expressed in mouse retina, where it is essential for retinal cell survival under conditions of oxidative stress.

Purpose: To investigate the expression in the mouse retina and the function in a retinal cell of cullin5 that is known as a protein that is associated with ubiquitilation. Methods: The retinal sites of expressing cullin5 were determined by immunohistochemistry with a specific antibody for cullin5. Retinal cells were transfected with HA-tagged cullin5 using an adenovirus system, and retinal cells were exposed to L-glutamate with or without an over-expression of cullin5. Results: Cullin5 was expressed in the retina of C57BL/6N mice and the retinal ganglion cell layer (RGL) of retina was strongly immunostained. Further, cullin5 was localized in the retinal ganglion cell (RGC). An over-expression of cullin5 reduced the percentage of retinal cell deaths induced by L-glutamate. Conclusions: The expression of cullin5 on retinal cells and reduction in the percentage of dead retinal cells induced by L-glutamate suggest that cullin5 has neuroprotective properties in retinal cells.

The purpose of this study is to evaluate the intraocular pressure (IOP) -lowering effect and safety of topical travoprost with sofzia and without benzalkonium chloride on Japanese patients with glaucoma. Topical travoprost (0.04%) was used on 39 glaucoma patients with no prior use of topical prostaglandin F2alpha analogues (Beginning group). The IOP, number of conjunctival follicules, degrees of conjunctival hyperemia, and degrees of superficial punctate keratitis (SPK, AD-classification) were determined at the beginning of the treatment and after 1 month and 3 months. 37 other patients who were using 0.005% topical latanoprost were switched to 0.04% topical travoprost and analyzed in the same way (Switched group). For the Beginning group, the IOP was significantly decreased after 3 months (p < 0.0001). The conjunctival follicule score was decreased significantly (p = 0.033). Both the SPK area score and density score for the cases with SPK at the baseline decreased significantly (p = 0.034 and p = 0.024). In the switched group, the IOP was not changed significantly at 3 months after the switch (p = 0.118). Both the conjunctival follicule and hyperemia score were significantly decreased at 3 month (p = 0.0074 and p = 0.0047). The SPK area score for the cases with SPK at the time of switch decreased significantly (p = 0.013). Travoprost with sofzia preservative had an equal effect in reducing the IOP as latanoprost. It had low toxicity on the ocular surface of Japanese glaucoma patients.