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Tsukasa Mochizuki, Kazuyuki Hirooka, Naoki Okada, Hiromitsu Onoe, Kana Tokumo, Hideaki Okumichi, Yoshiaki Kiuchi Department of Ophthalmology and Visual Science, Hiroshima University, Hiroshima, 734-8551, JapanCorrespondence: Kazuyuki Hirooka, Department of Ophthalmology and Visual Science, Hiroshima University, 1-2-3 Kasumi, Minami-Ku, Hiroshima, 734-8551, Japan, Tel +81-82-257-5247, Fax +81-82-257-5249, Email khirooka9@gmail.comPurpose: The aim of this study was to evaluate ab interno trabeculotomy outcomes without phacoemulsification.Methods: This retrospective study evaluated 118 eyes of patients aged 18 and above who underwent ab interno trabeculotomy between December 2017 and August 2022. When surgeries were performed in both eyes, only the eye undergoing the initial surgery was evaluated. Prior to and after surgery, the intraocular pressure (IOP) and mean number of IOP-lowering medications were compared. An IOP of ≤ 21 mmHg (A) and ≤ 18 mmHg (B) along with a ≥ 20% reduction in the preoperative IOP was defined as survival. Cases that required reoperation for glaucoma were defined as surgical failure. The Kaplan–Meier method was used to evaluate the survival rates. A Cox proportional hazards model was used to analyze the preoperative factors that influenced survival rates.Results: At 36 months postoperatively, the 13.4 ± 2.8 mmHg average IOP was significantly decreased from the preoperative 23.5 ± 9.8 mmHg value (P < 0.0001). Moreover, a significant decrease in the mean number of the IOP-lowering medications to 2.3 ± 1.4 at 36 months was found versus the initial 3.9 ± 1.1 preoperative value (P < 0.0001). The survival rates for criteria A and B at 36 months postoperatively were 28% and 25%, respectively. Only the preoperative IOP was identified by multivariate analysis as a factor influencing survival rates (P < 0.0001). Hyphema in 36 eyes (30.5%) and an IOP spike in 20 eyes (16.9%) were the only observed complications. Additional glaucoma surgery was required in 27 eyes (22.9%) during the follow-up period.Conclusion: Utilization of ab interno trabeculotomy effectively lowered the IOP and reduced the number of IOP-lowering medications. Patients with higher preoperative IOP exhibited better postoperative outcomes.Keywords: microhook, Kahook Dual Blade, intraocular pressure, glaucoma

Naoki Okada,1,2 Kazuyuki Hirooka,1,2 Hiromitsu Onoe,1,2 Kana Tokumo,1,2 Hideaki Okumichi,1,2 Yoshiaki Kiuchi1,2 1Department of Ophthalmology and Visual Science, Hiroshima University, Hiroshima, Japan; 2Hiroshima Eye Clinic, Hiroshima, JapanCorrespondence: Kazuyuki Hirooka, Department of Ophthalmology and Visual Science, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan, Tel +81-82-257-5247, Fax +81-82-257-5249, Email khirooka9@gmail.comPurpose: To examine the potential risk factors affecting the long-term outcomes following a combination of phacoemulsification with ab interno trabeculotomy with the microhook (μLOT-Phaco) and goniotomy with the Kahook Dual Blade (KDB-Phaco).Methods: For 12 months, we retrospectively examined a total of 100 eyes of 100 patients with primary open-angle glaucoma (POAG) and exfoliation glaucoma (EG) who had previously undergone surgery between December 2016 and December 2020. Patients with a preoperative intraocular pressure (IOP) ˂12 mmHg were excluded. Probability of success was calculated using the Kaplan–Meier method, with surgical failure defined as an IOP > 18 mmHg, < 20% IOP reduction or additional glaucoma surgery. The Cox proportional hazards model was used to examine the potential risk factors for failure, which included age, gender, type of glaucoma, surgical techniques, preoperative IOP, number and type of preoperative IOP-lowering medications, preoperative visual field mean deviation (MD) value, and axial length.Results: For the 51 males and 49 females, mean preoperative age was 74.4 ± 9.0 years, with μLOT-Phaco performed in 44 and KDB-Phaco in 56 subjects. The type of glaucoma was POAG in 68 and EG in 32 eyes. Preoperative IOP was 20.5 ± 6.7 mmHg, while postoperative IOPs were 14.4 ± 4.2 mmHg, 13.7 ± 2.8 mmHg, and 14.6 ± 3.9 mmHg, respectively (P < 0.001). Significant decreases from the preoperative number of IOP-lowering medications (3.1 ± 1.2) were observed at 12, 24, and 36 months, respectively (1.2 ± 1.3, 1.6 ± 1.3, and 2.1 ± 1.4 (P < 0.001)). Probability of success at 12, 24, and 36 months postoperatively was 52.0%, 49.6%, and 47.7%, respectively. Lower preoperative IOP was shown to be a potential risk factor for surgical failure.Conclusion: Long-term follow-ups showed IOP decreased in μLOT-Phaco and KDB-Phaco patients. Results suggest that patients with higher preoperative IOP may have better postoperative outcomes.Keywords: MIGS, Kahook Dual Blade, intraocular pressure, glaucoma

Kana Tokumo,1 Naoki Okada,1 Hiromitsu Onoe,1 Kaori Komatsu,1 Shun Masuda,1 Hideaki Okumichi,1 Kazuyuki Hirooka,1 Ryo Asaoka,2 Yoshiaki Kiuchi1 1Department of Ophthalmology and Visual Science, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan; 2Seirei Hamamatsu General Hospital Department of Ophthalmology, Shizuoka, JapanCorrespondence: Kana Tokumo, Department of Ophthalmology and Visual Science, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan, Tel +81-822575247, Fax +81-822575249, Email kanatokumo@hiroshima-u.ac.jpPurpose: To compare the efficacy of Ex-PRESS implantation (EXP) with that of trabeculectomy (TLE) with mitomycin C for maintaining low target intraocular pressure (IOP) in patients with open-angle glaucoma.Patients and Methods: Patients were randomly assigned to receive EXP or TLE. Surgical success was defined according to three target mean IOP ranges (5 mmHg ≤ IOP ≤ 18 mmHg [criterion A], 5 mmHg ≤ IOP ≤ 15 mmHg [criterion B], and 5 mmHg ≤ IOP ≤ 12 mmHg [criterion C]) representing reductions of at least 20% below the baseline on two consecutive follow-up visits 3 months post-surgery, with or without antiglaucoma medication and without further glaucoma surgery. Participants were divided into three subgroups based on baseline mean deviation (MD) values: early (MD ≥ − 6 dB), moderate (− 6 dB > MD ≥ − 12 dB), and advanced (− 12 dB > MD). Survival rates were calculated by subgroup.Results: A total of 73 patients, including 30 in the EXP group and 43 in the TLE group, were included in the study. No significant differences in baseline ocular or demographic characteristics were found between the two groups. No significant difference in IOP was noted every 6 months. After the 3-year follow-up, success rates were A) 60.0% and 60.2%, B) 45.7% and 58.1%, and C) 31.5% and 40.5% for the EXP and TLE groups, respectively. Moreover, there was no difference in success rate based on glaucoma level. Many glaucoma medications administered before surgery were associated with a higher failure rate in the TLE group but not in the EXP group.Conclusion: Both procedures resulted in similar IOP reductions and success rates for a low target IOP. The number of preoperative glaucoma medications was a risk factor for TLE failure.Keywords: filtration surgery, glaucoma, randomized controlled clinical trial, visual field

Hiroshi Saito,1 Yuya Nakamura,2,3 Masahiro Inagaki,2,4 Shiho Yamadera,5 Hideo Misawa,2,3 Naoki Sato,2,6 Tatsunori Oguchi,2 Takae Inagaki,7 Yuya Tsuji,2 Mayumi Tsuji,2 Isao Ohsawa,3 Hiromichi Gotoh,3 Yuji Kiuchi2 1JA Hiroshima Genneral Hospital, Hatsukaichi City, Hiroshima, Japan; 2Department of Pharmacology, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan; 3Saiyu Soka Hospital, Soka City, Saitama, Japan; 4Faculty of Arts and Sciences at Fujiyoshida, Showa University, Fujiyoshida City, Yamanashi, Japan; 5Department of Hospital Pharmaceutics, Showa University School of Pharmacy, Shinagawa-ku, Tokyo, Japan; 6Department of Research Center, Tanabe Pharmacy Inc., Chuo-ku, Tokyo, Japan; 7Graduate School of Nursing and Rehabilitation Science, Yokohama City, Kanagawa, JapanCorrespondence: Yuya Nakamura Email y.nakamura@med.showa-u.ac.jpPurpose: Lipopolysaccharides (LPS) induce inflammation by binding to the Toll-like receptor (TLR) 4 complex, including LPS-binding protein (LBP). The anti-inflammatory effects of linagliptin in LPS-induced inflammation in the TLR4-independent pathway have not been examined before. We examined the anti-inflammatory effects of linagliptin in the TLR4- and the LBP-independent pathway.Methods: U937 cells were cultured in the medium supplemented with 10% fetal bovine serum (FBS) and treated with 100 nM phorbol myristate acetate for 48 h. Cells were then left untreated or were treated with 10 μg/mL anti-TLR4 antibodies alone or in combination with linagliptin for 1 h in media supplemented with or without 10% FBS. The cells were divided into 5 groups: a) control cells (untreated) b) cells treated with LPS c) cells treated with 10 μg/mL anti-TLR4 antibodies d) cells treated with LPS and 10 μg/mL anti-TLR4 antibodies and e) cells treated with LPS, 10 μg/mL anti-TLR4 antibodies, and linagliptin. The LPS concentrations used were 50 pg/mL or 100 pg/mL for cells treated in the presence of 10% FBS and 100 pg/mL or 1 μg/mL for cells treated in the absence of FBS. Linagliptin concentrations of 1 nM, 10 nM, and 100 nM were used for treatment. The supernatants were analyzed for interleukin (IL)-6 production after 24 h of various treatments.Results: LPS increased IL-6 production compared to the untreated control cells, and anti-TLR4 antibody suppressed LPS-induced increased IL-6 levels. Linagliptin suppressed LPS-induced IL-6 production in a concentration-dependent manner in the presence of FBS. However, only 100 nM linagliptin could suppress LPS-induced IL-6 production in the absence of FBS.Conclusion: Concentration-dependent and -independent inflammatory suppression was observed following linagliptin treatment after LPS induction in an experimental model of TLR4 inhibition by anti-TLR4 antibodies. Our results showed that linagliptin may inhibit inflammation through multiple mechanisms centered around the TLR-4-mediated pathway.Keywords: lipopolysaccharide, U937 cells, fetal bovine serum, anti- toll-like receptor 4 antibody, interleukin-6

Naoki Sato,1,2 Yuya Nakamura,1,3 Shiho Yamadera,4 Masahiro Inagaki,1,5 Sachiyo Kenmotsu,5 Hiroshi Saito,4,6 Tatsunori Oguchi,1 Mayumi Tsuji,1 Hirokazu Chokki,1 Isao Ohsawa,3 Hiromichi Gotoh,3 Shinichi Iwai,6 Yuji Kiuchi1 1Department of Pharmacology, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan; 2Department of Research Center, Tanabe Pharmacy Inc., Tokyo, Japan; 3Department of Nephrology, Saiyu Soka Hospital, Soka City, Saitama-ken, Japan; 4Department of Hospital Pharmaceutics, Showa University School of Pharmacy, Shinagawa-ku, Tokyo, Japan; 5Fuculty of Arts and Sciences at Fujiyoshida, Showa University, Fujiyoshida City, Yamanashi-ken, Japan; 6Department of Healthcare and Regulatory Sciences, Showa University School of Pharmacy, Shinagawa-ku, Tokyo, JapanCorrespondence: Yuya NakamuraDepartment of Pharmacology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, JapanTel +81-3-3784-8125Fax +81-3-3787-4790Email y.nakamura@med.showa-u.ac.jpPurpose: Dipeptidyl peptidase-4 inhibitors, including linagliptin, prevent inflammation. However, the in vitro effects of linagliptin are unclear. Moreover, although linagliptin inhibits lipopolysaccharide (LPS)-induced inflammation, the anti-inflammatory effects of linagliptin in this context are not concentration-dependent. In the absence of LPS-binding protein (LBP), the pro-inflammatory effects of LPS involve pathways other than the Toll-like receptor (TLR) 4 pathway. Here, we aimed to determine the anti-inflammatory mechanisms of linagliptin in an experimental model in which LBP was added to the medium.Methods: Human U937 monocytes were cultured at 1 × 106 cells/mL in Roswell Park Memorial Institute medium and differentiated into macrophages using phorbol myristate acetate. All processes were carried out in medium containing 10% fetal bovine serum (FBS). After 48 hrs of culture, we replaced the medium and pretreated the cells with 100, 250, 500, or 2500 nM linagliptin for 1 hr. We exchanged the medium again, and the cells were treated with 1 ng/mL LPS with or without 100, 250, 500, or 2500 nM linagliptin. Interleukin (IL)-6 and LBP in the supernatant, nuclear factor (NF)-κB/p65 in the nucleus, and reactive oxygen species (ROS) in the cells, as important markers of the mechanism of inflammation induction by LPS, were measured using enzyme-linked immunosorbent assay kits.Results: Linagliptin significantly prevented LPS-stimulated IL-6 production and intranuclear NF-κB/p65 levels in a concentration-dependent manner. LPS-induced intracellular ROS levels were significantly decreased by linagliptin at all concentrations. LBP levels were markedly higher in FBS-containing medium than in medium without FBS. However, LBP levels did not change following administration of linagliptin and/or LPS.Conclusion: Concentration-dependent and -independent inflammatory suppression was observed following linagliptin treatment in the context of LPS-induced pro-inflammatory responses. Thus, our findings suggested that linagliptin induced two different mechanisms to repress inflammation, i.e., TLR4-dependent and -independent mechanisms.Keywords: linagliptin, lipopolysaccharide-binding protein, interleukin-6, intranuclear subunit of nuclear factor-κB/p65, reactive oxygen species

Tomofusa Yamauchi,1 Hitoshi Tabuchi,1 Kosuke Takase,1 Zaigen Ohara,1 Hitoshi Imamura,1 Yoshiaki Kiuchi2 1Department of Ophthalmology, Tsukazaki Hospital, Himeji, Japan; 2Department of Ophthalmology, Hiroshima University, Hiroshima, Japan Aim: We compared the visual performance of toric intraocular lenses (IOLs) and non-toric IOLs made of the same material.Patients and materials: The subjects included patients implanted with either Acrysof IQ® toric IOLs (SN6AT3-9) or Acrysof IQ® IOLs (SN60WF) bilaterally. The toric group included 103 patients who were implanted with Acrysof IQ toric IOLs bilaterally. The non-toric group was a corneal astigmatism-matched control group and included 103 patients who were implanted with Acrysof IQ IOLs bilaterally.Results: The uncorrected distance visual acuity was significantly better in the toric group, whereas the uncorrected 50 cm visual acuity was better in the non-toric group. There was no significant difference in contrast sensitivity (with and without glare) between both the groups. The rate of spectacle dependency for distance vision was significantly lower in the toric group. There were no significant differences between the two groups in all items of the postoperative quality-of-vision questionnaire (25-item Visual Function Questionnaire).Conclusion: The toric IOLs used in this study reduced spectacle dependency more than the non-toric IOLs and did not compromise the subjective visual function, but the uncorrected 50 cm vision was worse in toric IOL implanted eyes. Keywords: cataract, cataract surgery, astigmatism, visual function

Hideaki Okumichi,1 Yoshiaki Kiuchi,1 Tetsuya Baba,2 Takashi Kanamoto,3 Tomoko Naito,4,5 Shunsuke Nakakura,6 Hitoshi Tabuchi,6 Hiroki Nii,7 Chie Sueoka,7 Yosuke Sugimoto1,8 1Department of Ophthalmology and Visual Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan; 2Shirai Eye Hospital, Mitoyo, Japan; 3Department of Ophthalmology, Hiroshima Memorial Hospital, Hiroshima, Japan; 4Department of Ophthalmology, Okayama University Graduate School of Medicine, Okayama, Japan; 5Department of Ophthalmology, Konko Hospital, Asakuchi, Japan; 6Department of Ophthalmology, Saneikai Tsukazaki Hospital, Himeji, Japan; 7Department of Ophthalmology, Hiroshima General Hospital, Hiroshima, Japan; 8Department of Ophthalmology, Hiroshima Prefectural Hospital, Hiroshima, Japan 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. Keywords: tafluprost/timolol fixed combination, latanoprost, glaucoma, ocular-surface safety, intraocular pressure

Muhammad Irfan Kamaruddin,1,2 Momoko Nakamura-Shibasaki,1 Yu Mizuno,1 Yoshiaki Kiuchi1 1Department of Ophthalmology and Visual Sciences, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; 2Department of Ophthalmology, Hasanuddin University, Makassar, South Sulawesi, Indonesia Purpose: Ripasudil is a novel Rho-associated protein kinase inhibitor that is used to treat ocular hypertension. However, the comparison of the intraocular pressure (IOP)-lowering effects between ripasudil alone and other ocular hypotensive drugs has not been studied thoroughly. The purpose of this study is to examine the ocular hypotensive effects of 0.4% ripasudil, 2% pilocarpine, 0.5% timolol and 0.1% dorzolamide in rabbits. We also studied the IOP changes when 0.4% ripasudil was combined with 2% pilocarpine, 0.5% timolol or 0.1% dorzolamide. Methods: One drop of saline solution, 0.4% ripasudil, 0.5% timolol, 2% pilocarpine or 1% dorzolamide or a combination of these agents was applied topically to the left eyes of eight healthy albino rabbits. Posttreatment changes in the IOP of albino rabbits were monitored using a rebound tonometer over a 5-h time course. Changes in IOP after application of saline served as the control. One-way analysis of variance and Dunnett’s post hoc tests were used for statistical analyses. Results: After topical instillation, 0.4% ripasudil resulted in significant decreases in IOP at 0.5 and 1 h compared with the control group. Treatment with timolol, pilocarpine or dorzolamide had no significant effect on IOP. Treatment with timolol, pilocarpine or dorzolamide in combination with ripasudil resulted in significant reductions in IOP at 1 h. However, none of these agents enhanced the IOP-lowering effects of ripasudil. Conclusion: Ripasudil has stronger IOP-lowering effects than timolol, pilocarpine or dorzolamide hypotensive agents in our rabbit model. Addition of timolol, pilocarpine or dorzolamide did not enhance the IOP-lowering effects of ripasudil alone. Keywords: ripasudil, timolol, pilocarpine, dorzolamide, intraocular pressure, rabbit

Yosuke Harada,1 Tomoyuki Akita,2 Joji Takenaka,1 Yuko Nakamura-Kadohiro,1 Junko Tanaka,2 Yoshiaki Kiuchi1 1Department of Ophthalmology and Visual Science, Graduate School of Biomedical Sciences, 2Department of Epidemiology, Infectious Disease Control and Prevention, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan Aim: The aim of this study was to determine the reproducibility and reliability of optic disk evaluation in supine subjects with a Heidelberg Retina Tomograph II (HRT II).Methods: One eye was randomly selected for evaluation by HRT II in each of eleven healthy subjects. Five images were obtained by each of two experienced operators at two separate visits with the subjects in both sitting and supine positions. A stand was constructed to allow stable, handheld operation of the HRT II head for imaging of the supine subjects. Measurements of optic disk parameters obtained in the supine position were compared with those obtained in the sitting position. The reliability of measurements in the supine position was evaluated by calculating both the coefficients of variation for measurements made by one observer on the first visit and the interobserver and intervisit intraclass correlation coefficients (ICCs); the values obtained were compared with those obtained for the sitting position. Results: Measurements of all parameters made in the supine position were highly similar to those made in the sitting position. The coefficients of variation obtained for each parameter in the sitting and supine positions ranged from 5.67% to 14.16% and from 2.18% to 16.08%, respectively. The interobserver ICC values in the sitting and supine positions were ≥0.978 and ≥0.989, respectively. The intervisit ICC values in the sitting and supine positions were ≥0.958 and ≥0.983, respectively.Conclusion: Handheld operation of the HRT II in a custom-designed stand is feasible for optic disk evaluation in the supine subjects who were not able to maintain the sitting position. Keywords: glaucoma, optic disk evaluation, supine position, Heidelberg retina tomograph

Joji Takenaka,1 Eriko Kunihara,1 Ulfah Rimayanti,2 Junko Tanaka,3 Makoto Kaneko,4 Yoshiaki Kiuchi1 1Department of Ophthalmology and Visual Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; 2Faculty of Medicine and Health Sciences, UIN Alauddin Makassar, South Sulawesi, Indonesia; 3Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; 4Graduate School of Engineering, Osaka University, Osaka, Japan Purpose: To compare the reliability and accuracy of intraocular pressure (IOP) measured while wearing soft contact lenses (SCLs) using a non-contact tonometer (NCT), Goldmann applanation tonometer (GAT), iCare rebound tonometer (RBT) and the Tono-Pen XL.Methods: Twenty-six healthy subjects were examined. The IOP was measured using NCT, GAT, RBT, and the Tono-Pen XL, while the subjects wore SCLs -5.00 D, -0.50 D and +5.00 D. Bland–Altman plots and a regression analysis were used to compare the IOPs obtained with those instruments and the IOPs of the naked eyes measured using GAT (the standard IOPs in this study).Results: The IOPs obtained by the Tono-Pen XL while the subjects were wearing -5.00 D, -0.50 D, and +5.00 D SCLs were significantly higher than those of the naked eyes obtained using GAT. RBT showed that the IOPs were similar to the GAT standard IOPs under all conditions. The IOPs measured with NCT and GAT while the subjects were wearing -5.00 D and -0.50 D SCLs were similar to the GAT standard IOPs. The IOPs obtained with RBT and NCT while the subjects were wearing -5.00 D and -0.50 D SCLs exhibited a good correlation with the standard IOPs.Conclusion: The NCT and RBT are best when measuring IOP through hydrogel SCLs. Keywords: soft contact lens, intraocular pressure, rebound tonometer, non-contact tonometer

Yoshie Shimizu,1 Shunsuke Nakakura,1 Makiko Nishiyama,1 Hitoshi Tabuchi,1 Yoshiaki Kiuchi2 1Department of Ophthalmology, Saneikai Tsukazaki Hospital, Himeji, 2Department of Ophthalmology and Visual Sciences, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Background: We investigated the efficiency, safety and patient preference of switching from dorzolamide 1%/timolol 0.5% to brinzolamide 1%/timolol 0.5% while maintaining the prostaglandin F2α analog.Methods: We initially enrolled 44 eyes from 44 primary open angle glaucoma patients, and a total of 42 patients completed the study. All patients were under treatment with various prostaglandin F2α analogs and dorzolamide 1%/timolol 0.5%. While maintaining the prostaglandin F2α analog, dorzolamide 1%/timolol 0.5% was switched to brinzolamide 1%/timolol 0.5%. Conjunctival hyperemia, superficial punctate keratopathy, and intraocular pressure (IOP) were evaluated at baseline and at 4, 12, and 24 weeks. Adverse events and patient preferences, measured using a questionnaire at study initiation and at 24 weeks, were also noted.Results: The IOP was 17.7±1.7, 16.8±2.6, 16.7±2.2, and 16.7±2.4 mmHg at baseline and at 4, 12, and 24 weeks, respectively, with no significant differences in IOP values at any time point (P=0.117, one-way analysis of variance). In addition, no significant differences were found in the incidence of conjunctival hyperemia or SPK score at any time point (all P>0.5, by Kruskal–Wallis test). Based on the evaluation of side effects using the questionnaire, stinging/burning was less common (P=0.042), while blurred vision was more common (P=0.003), after switching to brinzolamide 1%/timolol 0.5%. Regarding patient preferences, 13 patients (31%) preferred dorzolamide 1%/timolol 0.5%, 12 patients (29%) preferred brinzolamide 1%/timolol 0.5%, and 17 patients (40%) preferred neither.Conclusion: When switching from dorzolamide 1%/timolol 0.5% to brinzolamide 1%/timolol 0.5%, the IOP values and incidence of superficial punctate keratopathy and conjunctival hyperemia were sustained throughout the 24-week observation period, and the patient preferences were similar for the two regimens. However, differences were observed in the ocular sensations of stinging/burning with dorzolamide 1%/timolol 0.5% and blurred vision with brinzolamide 1%/timolol 0.5%. Keywords: glaucoma, prostaglandin F2α, brinzolamide, dorzolamide, fixed combination, timolol

Shunsuke Nakakura,1 Minamai Yamamoto,1 Etsuko Terao,1 Nozomi Nagatomi,1 Naoko Matsuo,1 Yausko Fujisawa,1 Yuki Fujio,1 Hitoshi Tabuchi,1 Yoshiaki Kiuchi2 1Department of Ophthalmology, Saneikai Tsukazaki Hospital, Himeji, Japan; 2Department of Ophthalmology and Visual Sciences, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Purpose: We investigated the incidence of prostaglandin-associated periorbitopathy (PAP) in subjects with glaucoma treated with latanoprost ophthalmic solution.Subjects and methods: One eye and the forehead in 22 subjects were evaluated. All patients had used latanoprost for more than 1 year (range, 12 to 45 months; mean, 26.0 months) and were prostaglandin F2α analogue treatment-naïve. Digital photographs of the subjects obtained before latanoprost therapy and at the last examination were compared retrospectively. Four signs of PAP (deepening of the upper eyelid sulcus (DUES), upper eyelid ptosis, flattening of the lower eyelid bags, and inferior scleral show) and supplemental side effects around the eyelids (eyelash growth, poliosis, and eyelid pigmentation) were judged to be negative or positive by three independent observers. If the observers unanimously rated a sign as positive, the result was defined as positive.Results: Twelve subjects (54.5%) had no apparent signs. Three subjects were judged to have DUES (13.6%), and two subjects each were judged to have flattening of the lower eyelid bags and eyelid pigmentation (9.0%). The other signs were judged as positive in only one subject each, respectively (4.5%). A univariate logistic regression analysis showed no significant associations between any of the signs and age, sex, or the duration of therapy.Conclusion: Latanoprost induced DUES, upper eyelid ptosis, flattening of the lower eyelid bags, inferior scleral show, and supplemental side effects around the eyelids; however, the rates of such occurrence might be relatively low. Keywords: glaucoma, prostaglandin F2α, deepening of the upper eyelid sulcus

Shunsuke Nakakura1, Hitoshi Tabuchi1, Yukio Baba2, Futoshi Maruiwa2, Nobuko Ando2, Takashi Kanamoto3, Yoshiaki Kiuchi31Department of Ophthalmology, Saneikai Tsukazaki Hospital, Himeji, Hyogo Prefecture, Japan; 2Department of Ophthalmology, Baba Eye Clinic, Hiroshima, Japan, 3Department of Ophthalmology and Visual Sciences, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima City, JapanObjective: To compare the safety and effectiveness of fixed-combination regimes (latanoprost–timolol and brinzolamide 1% compared to dorzolamide 1%/timolol and latanoprost) in open-angle glaucoma patients after switching from a combination of three topical antiglaucoma eye drops.Methods: We conducted an open, randomized 12-week multicenter prospective study. We randomly allocated 39 patients who had been treated with three antiglaucoma eye drops (prostaglandin F2α analogues plus beta-blockers and carbonic anhydrase inhibitors) into two groups. Group A (n = 20) were treated with latanoprost–timolol and brinzolamide 1% therapy and Group B (n = 16) were treated with dorzolamide 1%/timolol and latanoprost. Thirty-six patients completed all 12 weeks of this study. The major clinical parameters measured were intraocular pressure (IOP), conjunctive hyperemia, superficial punctate keratopathy and hyperpigmentation of eyelid at baseline, 4, and 12 weeks. Additionally noted were adverse events and patient preferences, measured using a questionnaire at study initiation and at 12 weeks.Results: At baseline, IOPs were (Group A: 14.1 ± 2.9 mmHg, B: 14.5 ± 2.9 mmHg; P = 0.658), (Group A: 13.8 ± 2.6 mmHg, B: 14.3 ± 2.8 mmHg; P = 0.715) at 4 weeks, and (Group A: 14.1 ± 2.7 mmHg, B: 14.2 ± 2.7 mmHg; P = 0.538) at 12 weeks. Among the groups, there was no significant difference at any time point after baseline (P = 0.923, 0.951, respectively). All adverse events were not remarkably different after therapy. In regards to patient preference before and after switching therapy, 10 patients (50%) in Group A and 10 patients (63%) in Group B preferred using fixed-combination eye drop therapy.Conclusions: Effectiveness and safety were maintained in both groups after switching therapy. Overall, patients generally preferred using a fixed-combination therapy.Keywords: glaucoma, latanoprost–timolol, dorzolamide–timolol, brinzolamide, fixed combination, intraocular pressure

Tetrahydrobiopterin (BH4) content in the pancreas from GK (Goto-Kakizaki) rats as a type II diabetic model was markedly decreased compared to Wistar rats as a healthy control. Moreover, insulin stimulated BH4 synthesis in a hamster pancreatic β-cell line, HIT-T15 cells. Therefore, in diabetic condition, the reduction of insulin action is likely to lead to the low levels of BH4 content in pancreatic β-cells. In this study, we examined the effects of the decrease in BH4 content on insulin secretion by L-arginine (L-Arg) or glucose using HIT-T15 cells. Cytosolic calcium concentration ([Ca2+]i) in HIT-T15 cells was increased by L-Arg. Under the low BH4 content using 2,4- diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of BH4 synthesis, L-Arg-induced both Ca2+ response and insulin secretion were stimulated. These stimulating effects were recovered by BH4 content recovering, using sepiapterin, a precursor of BH4 biosynthesis. By contrast, glucose-induced Ca2+ response was not affected by the BH4 content. These results suggest that the decrease of BH4 content in β-cells may stimulate insulin secretion by L-Arg, but not by glucose, under diabetic conditions.

We examined the role of endogenous tetrahydrobiopterin (BH4) on L-tyrosine-induced increase in cytosolic Ca: concentration ([Ca2+],) in the ß-cell line, HIT-T15. Addition of L-tyrosine to the cells increased [Ca2+], in a conccntration- depcndent manner. Moreover, the L-tyrosine-induced [Ca2+], elevation was blocked by L-3,4-dihydroxyphenylalanine (L-DOPA). Under low levels of BH4 by treatment with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of BH4 biosynthesis, elevation of [Ca2+], by L-tyrosine was stimulated. Moreover, co-prctreatment with sepiapterin, a precursor of BH4 biosynthesis, reduced the decrease of BH4 content by DAHP, and suppressed L-tyrosine-induccd [Ca2+ ]¡ elevation in DAHP-pretreated cells. These findings suggest that BH4 negatively regulates L-tyrosine-induced [Ca2+] elevation via the production of L-DOPA in ß-cells.

Abstract.: Intracellular ADP-ribose is an activator of TRPM2, which is a Ca2+-permeable channel and mediates H2O2-induced cell death, in the TRPM2-expressing rat β-cell line RIN-5F. We examined the effect of extracellular-added ADP-ribose on intracellular Ca2+ concentration in RIN-5F cells. ADP-ribose induced Ca2+ release from the thapsigargin-sensitive Ca2+ store, but not Ca2+ entry across the plasma membrane. A phospholipase C (PLC) inhibitor and a non-specific IP3 receptor inhibitor blocked its Ca2+ release. H2O2-induced Ca2+ entry through TRPM2 was not affected by extracellular ADP-ribose. These findings suggest that extracellular-added ADP-ribose induces Ca2+ release through the PLC-IP3 pathway and does not act as a TRPM2 activator. Keywords:: ADP-ribose, hydrogen peroxide, TRPM2

The purpose of this study was to examine whether basic fibroblast growth factor (bFGF) stimulates tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells. BH4 content was determined by oxidation under acidic conditions as biopterin and analysed with reversed-phase high Performance liquid chromatography. Measurement of the mRNA level of QTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of the de novo pathway of BH4 synthesis. The addition of bFGF to endothelial cells increased the BH4 content and GTPCH mRNA levels in an incubation period- and a concentration-dependent manner. 2,4-Diamino-6- hydroxypyrimidine, an inhibitor of GTPCH, strongly reduced the bFGF-induced increase in BH4 content. These findings suggest that bFGF stimulates BH4 synthesis via a de novo pathway with the induction of GTPCH.

We examined the effect of immunosuppressant cyclosporin A (CsA) on the synthesis of tetrahydrobiopterin (BH4), which is a cofactor for nitric oxide synthase (NOS), during treatment with lipopolysaccharide (LPS) in mouse brain microvascular endothelial cells. Addition of LPS to the endothelial cells increased the BH4 content and the mRNA level of GTP-cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH4 synthesis, and the LPSínduced increases in both the BH4 content and expression of GTPCH mRNA were further stimulated by the cotreatment with CsA. 2,4-Diamino-6-hydroxypyrimidine, an inhibitor of GTPCH, blocked the increase in BH4 content induced by CsA during the LPS treatment. Moreover. CsA stimulated the expression of inducible NOS (iNOS) mRNA during the LPS treatment. These findings suggest that CsA stimulates LPS-induced BH4 synthesis through the induction of GTPCH, and iNOS expression. CsA may increase NO production during LPS treatment in brain microvascular endothelial cells.

Tetrahydrobiopterin (BH4) is one of the cofactors for nitric oxide synthase (NOS), and also has a protective effect against reactive oxygen species (ROS)- and/or nitric oxide (NO)-induced cytotoxicities. Lipopolysaccharide (LPS) induces NO production and BH4 synthesis, and also ROS release. This study examined whether hydrogen peroxide, one of the ROS, has a regulatory role in BH4 synthesis in vascular endothelial cells. Addition of LPS to the endothelial cells increased the BH4 content. Although the LPS-induced increase in BH4 content was not affected by the treatment with superoxide dismutase and catalase, co-treatment with hydrogen peroxide further stimulated the LPS-induced increase in BH4 content. Moreover, the hydrogen peroxide-induced increase in BH4 content in the presence of LPS was completely inhibited by the treatment with 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP-cyclohydrolase I (GTPCH). These findings suggest that hydrogen peroxide may stimulate LPS-induced BH4 synthesis through the induction and/or activation of GTPCH. Under inflammation, hydrogen peroxide seems to stimulate BH4 synthesis.

We examined the role ofprotein kinase C (PKC) in the insulin-induced biosynthesis oftetrahydrobiopterin (BH4)' which is one of the co factors of nitric oxide (NO) synthase (NOS), in vascular endothelial cells. The BH. level was determined as biopterin by reversed-phase high performance liquid chromatography with fluorometric detection. Measurement of the level ofrnRNA for GTP cyclohydrolase I (GTPCH), which is the fust and the rate-limiting enzyme for the novo pathway for BH4 synthesis, was performed by reverse transcription-polymerase chain reaction (RT-PCR). Treatment with insulin increased the BH4 level and the GTPCH rnRNA level in endothelial cells. The insulin-induced increases of both GTPCH rnRNA and BH4 levels were enhanced by co-treatment with bisindolylmaleimide I or Ro 31-8220, inhibitors of PKC. On the other hand, the insulin-induced increases of BH4 synthesis and expression of GTPCH rnRNA were also enhanced by co-incubation with a PKC activator, phorbol 12-myristate 13-acetate (PMA) for 24 h. Long-term treatment with PMA is known to down-regulate PKC. Addition of insulin after pretreatment with PMA for 24 h markedly enhanced BH4 synthesis and expression of .:JTPCH rnRNA. These findings suggest that PKC negatively regulates the induction of BH4 synthesis by insulin -:ndothelial cells. Insulin-induced BH4 synthesis may be suppressed in diabetes mellitus, since PKC has been shown to be up-regulated in diabetes mellitus.

The purpose of this study was to examine whether 17β-estradiol stimulates the synthesis of tetrahydrobiopterin : BH4), which is one of the cofactors of nitric oxide (NO) synthase, in mouse brain microvascular endothelial cells. Addition of 17()-estradiol to endothelial cells time- and concentration-dependently increased intracellular BH4 level. 17β-Estradiol also stimulated the mRNA level of GTP-cyclohydrolase I (GTPCH), which is a rate-limiting enzyme of the de novo BH4 synthetic pathway. In addition, the 17β-estradiol-induced expression of GTPCH mRNA was strongly attenuated by treatment with an inhibitor of 17β-estradiol receptor 4-hydroxy-tamoxlfen. These results suggest that 17β-estradiol stimulates BH4 synthesis through the induction of GTPCH by tamoxifensensitive receptor in vascular endothelial cells. The 17β-estradiol-induced increase in BH4 level might be implicated in not only NO production, but also protective effects of 17β-estradiol against ischemic brain damage and atherosclerosis, since BH4 is an intracellular antioxidant.

We investigated the effects of troglitazone, which is a ligand of nuclear transcription factor peroxisome proliferator- activated receptor-γ (PPAR-γ), on insulin-induced tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells (MBMECs). BH4 content was determined as biopterin, by reversed-phase high performance liquid chromatography using fluorometric detection. Measurement of mRNA level of GTP cyclohydrolase I (GTPCH), which is a rate-limiting enzyme for de novo BH4 synthesis, was performed by reverse transcription- polymerase chain reaction (RT -PCR). Addition of insulin (10µg/ml) to endothelial cells increased intracellular BH4 content and GTPCH mRNA level, and the insulin-induced increase in BH4 content and GTPCH mRNA level was further stimulated by co-treatment with troglitazone (0.3-3 µM). However, the expression of PPAR-γ mRNA in untreated and insulin-treated endothelial cells was not detected. Moreover, bezafibrate (1-10 µM), a ligand of PPAR-δ, did not affect BH4 synthesis in insulin-treated endothehal cells. These findings suggest that troglitazone stimulates insulin-induced BH4 synthesis in endothelial cells with the induction of GTPCH through PPAR-γ- and PPAR-δ-independent mechanism

We examined the effects of insulin on tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells (MBMECs). Treatment of MBMECs with insulin increased the intracellular BH4 content in a time- and concentration-dependent manner. The insulin-induced increase in BH4 content was inhibited by treatment with 2,4-diamino-6-hydroxypyrimidine, a selective inhibitor of GTP cyclohydrolase I, and Nacetylserotonin, a selective inhibitor of sepiapterin reductase. These findings indicate that insulin stimulates BH4 synthesis in MBMECs through a de novo synthetic pathway of BH4.

The purpose of this study was to characterize the protective effect of tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, against NO-induced endothelial cell injury. The addition of S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, to endothelial cells induced the release of lactate dehydrogenase (LDH), a marker for cell injury. The SNAP-induced endothelial cell injury was markedly reduced by pretreatment with sepiapterin, a precursor of BH4 synthesis. On the other hand, exogenous BH4 had little effect on the SNAP-induced endothelial cell injury. We recently found that NO-induced endothelial cell injury involves a part of H202 production, since the injury was blocked by the treatment with catalase. Although BH4 released reactive oxygen species (ROS) in cell-free conditions, the increase in intracellular BH4 by pretreatment with sepiapterin strongly reduced H202-induced intracellular oxidative stress. These findings suggest that the increase in intracellular BH4 content but not extracellular BH4, strongly attenuates NO-induced endothelial cell injury by at least one of the mechanisms by which BH4 reduces H202-induced oxidative stress. Intracellular BH4 seems mainly to play a role as an antioxidant or as a ROS-scavenger.

Abstract Background Little is known regarding the molecular pathways that underlie the process of retinal development. The purpose of this study was to identify proteins which may be involved in development of retina. We used a proteomics-based approach to identify proteins that are up- or down-regulated during the development of the embryo chick retina. Results Two-dimensional gel electrophoresis was performed with the retina of embryo chicken, which was obtained from embryos of day 7 (ED7) and of day 11 (ED11). The protein spots showing significant differences were selected for identification by MALDI mass spectrometry. Thirteen proteins were differentially expressed; seven proteins were up-regulated in embryo retina of chicken at ED 11 and six proteins were down-regulated. Significant proteins were also evaluated in embryo day 15 (ED15). Some of identified proteins were known to regulate cell proliferation, cell death, transport, metabolism, organization and extracellular matrix, and others also included novel proteins. Conclusion We identified thirteen proteins which differentially expressed in embryonal retina of chicken at day 7, as compared to the retina of embryo of day 11. They were various regulatory proteins for cellular signaling.

Abstract Introduction To report the effect of topical latanoprost on the position of a subluxated lens. Case presentation After 0.005% latanoprost was administered topically to a patient with ocular hypertension due to a pseudoexfoliation syndrome and a subluxated lens, the position of the lens was examined by slit-lamp biomicroscopy, and the ciliary body thickness by ultrasound biomicroscopy. The lens had moved posteriorly, and the thickness of the ciliary body had decreased after the latanoprost. Conclusion We suggest that the decrease in the thickness of the ciliary body resulted in an increase in the tension of the zonule of Zinn fibers, thus pulling the subluxated lens posteriorly.

Abstract Background Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter found in neuronal tissues and is extensively expressed in the retina. EAAC1 plays a role in a variety of neural functions, but its biological functions in the retina has not been fully determined. The purpose of this study was to identify proteins regulated by EAAC1 in the retina of mice. To accomplish this, we used a proteomics-based approach to identify proteins that are up- or down-regulated in EAAC1-deficient (EAAC1-/-) mice. Results Proteomic analyses and two-dimensional gel electorphoresis were performed on the retina of EAAC1-/- mice, and the results were compared to that of wild type mice. The protein spots showing significant differences were selected for identification by mass spectrometric analyses. Thirteen proteins were differentially expressed; nine proteins were up-regulated and five proteins were down-regulated in EAAC1-/- retina. Functional clustering showed that identified proteins are involved in various cellular process, e.g. cell cycle, cell death, transport and metabolism. Conclusion We identified thirteen proteins whose expression is changed in EAAC-/- mice retinas. These proteins are known to regulate cell proliferation, death, transport, metabolism, cell organization and extracellular matrix.