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Yasumasa Otori, Takanori Matsuoka, Miho Kumoi, Eri Tachibana, Chieko Tsujino, Satoshi Matsuda Department of Ophthalmology, National Hospital Organization, Osaka National Hospital, Osaka, JapanCorrespondence: Yasumasa Otori Email otori.yasumasa.fe@mail.hosp.go.jpPurpose: To compare the outcomes of ab interno suture trabeculotomy (AbI-TLO) and ab externo metal trabeculotomy (AbE-TLO) in adult patients with glaucoma aged over 40 years.Patients and Methods: A retrospective chart review was conducted, including adult patients with glaucoma who underwent AbI-TLO or AbE-TLO between January 2015 and June 2019. A single surgeon (YO) performed all the operations. Eighty-one patients (81 eyes) were included in this study. Surgical success was defined as a postoperative intraocular pressure (IOP) of ≤ 18 mmHg and an IOP reduction of ≥ 20% from the preoperative IOP, without requiring additional glaucoma surgery. Success rates were assessed using Kaplan–Meier survival curves and log-rank (Mantel–Cox) tests, while risk factors were analyzed using the Cox proportional hazards model.Results: Forty-nine patients who underwent AbI-TLO and 32 patients who underwent AbE-TLO were studied; the preoperative IOPs were 27.9 ± 7.3 (mean ± standard deviation) mmHg and 25.6 ± 8.1 mmHg in the AbI-TLO and AbE-TLO groups, respectively (p=0.217). The 12-month postoperative IOPs were 15.8 ± 4.0 mmHg and 16.3 ± 4.2 mmHg in the AbI-TLO and AbE-TLO groups, respectively (p=0.724). The surgical success rates at 12 months were 77.6% and 62.5% in the AbI-TLO and AbE-TLO groups, respectively (p=0.144). Postoperative hyphema with level formation and ocular hypertension over 30 mmHg were observed in 22.4% and 26.5% of patients in the AbI-TLO group and 18.8% and 12.5% of those in the AbE-TLO group, respectively. Stepwise multivariate Cox regression analysis showed that a longer axial length was a risk factor for surgical failure (hazard ratio: 2.030; p=0.042).Conclusion: AbI-TLO and AbE-TLO had similar surgical outcomes and postoperative complications. A longer axial length was associated with an insufficient IOP reduction.Keywords: axial length, gonio-assisted transluminal trabeculotomy, intraocular pressure, MIGS, outcomes, trabeculotomy

Shinichi Usui,1,2 Yasushi Ikuno,2 Sato Uematsu,1 Yuko Morimoto,1 Yoshiaki Yasuno,3 Yasumasa Otori11National Hospital Organization, Osaka National Hospital, Osaka, Japan; 2Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan; 3Computational Optics Group, University of Tsukuba, Tsukuba, JapanPurpose: To evaluate changes in axial length and choroidal thickness after trabeculectomy.Patients and methods: Fourteen patients under 80 years of age with glaucoma, were enrolled. The choroid was imaged using prototypical high-penetration optic coherence tomography (OCT) and the thickness was measured. Axial length, choroidal thickness, and intraocular pressure (IOP) were measured bilaterally at 3 pm 1 day before and 6 days after trabeculectomy. The choroidal thickness was measured at the fovea and four other locations (2 mm superior, temporal, inferior, and nasal to the center of the optic nerve head).Results: The IOP and axial length significantly decreased in eyes that underwent trabeculectomy (P < 0.0001 for the IOP; P < 0.001 for axial length comparisons). The mean choroidal thicknesses significantly increased in eyes that underwent trabeculectomy compared to preoperatively (P < 0.0001 for the fovea; P < 0.01 for four locations around the optic disc). The mean magnitude of change in IOP was correlated positively with the mean magnitude of change in axial length, but not correlated with the mean magnitude of change in choroidal thickness at the fovea that underwent trabeculectomy. The sum of the axial length and subfoveal choroidal thickness in eyes decreased significantly postoperatively (P

The type 1 ryanodine receptor (RyR1) is a Ca 2+ release channel in the sarcoplasmic reticulum that is essential for skeletal muscle contraction. RyR1 forms a channel with six transmembrane segments, in which S5 is the fifth segment and is thought to contribute to pore formation. However, its role in channel gating remains unclear. Here, we performed a functional analysis of several disease-associated mutations in S5 and interpreted the results with respect to the published RyR1 structures to identify potential interactions associated with the mutant phenotypes. We demonstrate that S5 plays a dual role in channel gating: the cytoplasmic side interacts with S6 to reduce the channel activity, whereas the luminal side forms a rigid structural base necessary for S6 displacement in channel opening. These results deepen our understanding of the molecular mechanisms of RyR1 channel gating and provide insight into the divergent disease phenotypes caused by mutations in S5., (© 2024. The Author(s).)

Purpose: To evaluate anxiety felt by glaucoma patients., Patients and Methods: In total, 472 glaucoma patients responded to a questionnaire on anxiety, subjective symptoms, and vision-related quality of life (VR-QOL) associated with glaucoma. Anxiety was evaluated using the State-Trait Anxiety Inventory (STAI), state anxiety (STAI-State) subscale along with our novel questionnaire, assessing visual function and subjective symptoms, specialized for glaucoma. VR-QOL was evaluated using 5 subitems from the 25-item National Eye Institute Visual Function Questionnaire (VFQ-25). Adherence to ophthalmic antiglaucoma agents was confirmed. As indexes of visual function, corrected visual acuity (measured by eye chart), mean deviation (MD) score (measured with static perimetry), and 4 thresholds at the center of vision were determined. Stages were classified according to the Aulhorn Classification. From the STAI-State scores, the prevalence of anxiety in glaucoma patients was evaluated. We analyzed the correlation between the STAI-State and VFQ-25, anxiety, subjective symptoms, adherence, and visual function indexes., Results: In total, 78% of glaucoma patients experienced at least an intermediate level of anxiety. The STAI-State correlated significantly with anxiety and subjective symptoms as measured by our novel questionnaire, particularly for questions "current anxiety about loss of vision" and "current anxiety in life" (r=0.468 and 0.500; both P<0.0001). However, STAI-State correlated weakly with VFQ-25, and not at all with visual function indexes and adherence., Conclusions: Many glaucoma patients feel anxiety. The STAI-State is correlated with the VR-QOL and anxiety in glaucoma patients, making it useful for understanding the anxiety present in glaucoma patients.

Purpose: We assessed vision-related quality of life (VR-QOL) in glaucoma patients and examined the relationship between VR-QOL and indices of visual function and subjective symptoms., Patients and Methods: Four hundred seventy-two glaucoma patients completed a questionnaire concerning VR-QOL and subjective symptoms of glaucoma. VR-QOL was based on 5 subscales selected from the National Eye Institute 25-item Visual Function Questionnaire (NEI VFQ-25), whereas subjective symptoms were examined using original questions that we prepared. The following were selected as visual function indices: corrected visual acuity measured with a distance visual acuity chart, mean deviation scores measured with a static perimeter, and 4 threshold levels in the innermost center of a visual field (4 pericentral test points). Disease staging was conducted according to the Aulhorn classification system. Correlations of visual function with VR-QOL were evaluated using the Spearman test., Results: Correlations were observed in almost all subscales of the NEI VFQ-25, including correlations with corrected vision, disease stage, mean deviation score, and minimum threshold values of the 4-point pericentral test on the temporal side (P<0.0001). Visual function indices affecting driving differed between daytime and night driving (P<0.0001). NEI VFQ-25 subscales were correlated with subjective symptoms felt by the patients while driving (P<0.0001)., Conclusions: Minimum threshold values of the 4 pericentral test points on the temporal side are useful for the assessment of VR-QOL. Minimum threshold values of the 4 pericentral test points on the temporal side are useful for assessing visual field abnormalities that are associated with decreased VR-QOL.

Purpose: To determine the incidence and risk factors for delayed-onset intraocular pressure (IOP) elevations after vitrectomy for Stages 4 and 5 retinopathy of prematurity and, in addition, to determine the results of treating the IOP elevations., Methods: Fifty-five consecutive eyes with successful retinal reattachment and at least 24 months of follow-up after vitrectomy were studied. The ophthalmic examinations included slit-lamp biomicroscopy, wide-field digital retinal imaging, and IOP measurements. Eyes were classified into 2 groups: eyes with a postoperative IOP elevation to ≥ 21 mmHg and eyes whose IOP was always <21 mmHg., Results: Eight eyes (14.5%) developed an IOP elevation to ≥ 21 mmHg from 2 months to 4 months (median: 2.5 months) after the vitrectomy. In two of these eyes, the IOP was controlled with antiglaucoma medications. In the other six eyes, trabeculotomy for eyes with open angles and trabeculectomy or goniosynechialysis for eyes with closed angles were performed. The IOPs were successfully controlled after an average of 1.5 glaucoma surgeries. Multivariate logistic regression analyses identified that a young gestational age (odds ratio = 1.147, 95% confidence interval: 1.024-1.346) and lensectomy (odds ratio = 8.795, 95% confidence interval: 1.262-183.3) were significantly associated with the IOP elevation., Conclusion: Delayed-onset IOP elevation after vitrectomy for retinopathy of prematurity occurs in 14.5% of the eyes, and it is significantly associated with a young gestational age and presence of lensectomy.

Purpose: To describe ocular complications in patients with human immunodeficiency virus (HIV) infection before and after highly active antiretroviral therapy(HAART)., Materials and Method: We retrospectively reviewed the medical records of 261 patients who underwent HAART and visited our clinic between April, 2007 and March, 2010, and recorded ocular complications, CD4 cell counts, visual acuity and other relevant patient information., Results: Befor HAART patients were found to have the following conditions: HIV retinopathy (41 cases), cytomegalovirus (CMV) retinitis (23 cases), and others (6 cases); and after HAART HIV retinopathy (5 cases), CMV retinitis (16 cases), Immune recovery uveitis(IRU) (4 cases), and others(9 cases). The average CD4+ T-cell counts at diagnosis of CMV retinitis were 45.2/microl before and 116.7/microl after HAART., Conclusions: Since a substantial number of patients develop CMV retinitis after the initiation of HAART, we need to examine patients to check for either the onset or reactivation of CMV retinitis and IRU even after HAART.

Purpose: To investigate the relationship between relative lens position (RLP) and appositional closure in eyes with narrow angles., Methods: Ultrasound biomicroscopy (UBM) was used to measure anterior chamber depth (ACD) and lens thickness (LT), and the IOLMaster to measure axial length (AL). The number of quadrants with appositional closure was assessed by UBM under dark conditions. The RLP was calculated thus: RLP = 10 × (ACD + 0.5 LT) /AL., Results: This study comprised 30 consecutive patients (30 eyes) with narrow-angle eyes defined as Shaffer grade 2 or lower and without peripheral anterior synechiae (24 women, 6 men; mean age ± SD, 67.3 ± 10.4 years; range, 42-87 years). Under dark conditions, 66.7% of the eyes with narrow angles showed appositional closure in at least one quadrant. Of the various ocular biometric parameters, only the RLP significantly decreased with appositional closure in at least one quadrant (P = 0.005)., Conclusion: A decrease in the RLP can be predictive of appositional closure for narrow-angle eyes under dark conditions.

Purpose: To assess the usefulness of anterior segment optical coherence tomography (AS-OCT) in evaluating postoperative filtering bleb function., Methods: Forty-eight eyes of 39 patients who had earlier undergone trabeculectomy were enrolled. Blebs were imaged with a commercially available AS-OCT system. The correlation between intraocular pressure (IOP) and 4 quantitative parameters, bleb height, bleb wall thickness, and the height and extent of internal cavity, and 2 qualitative parameters, bleb wall reflectivity, and the presence or absence of posterior episcleral fluid, were analyzed., Results: Of the 39 participants, 25 (64.1%) were males (mean age, 65.3±10.0 y). The height and extent of the internal cavity and the bleb height were not correlated with the IOP. The bleb wall thickness was negatively (P=0.004) correlated with the IOP. The mean IOP in eyes with a low-reflectivity bleb wall was significantly (P=0.001) lower than in eyes with a high-reflectivity wall. The IOP levels were significantly (P=0.008) lower in eyes with posterior episcleral fluid than in eyes without it., Conclusions: AS-OCT is a useful tool to evaluate filtering bleb function. The height and extent of the total bleb and the internal cavity were not correlated with IOP control. A low-reflectivity wall and the presence of episcleral fluid were associated with lower IOP.

Background and Objective: To assess and explore the effectiveness of phacoemulsification and trabeculotomy combined with goniosynechialysis in lowering intraocular pressure in eyes with uncontrollable chronic angle-closure glaucoma., Patients and Methods: The medical charts of 24 eyes of 22 patients with chronic angle-closure glaucoma were reviewed retrospectively. The probability of successful treatment in 12 eyes that had earlier glaucoma attacks and 12 that did not have an attack were evaluated with Kaplan-Meier life-table analysis with the target pressure set at 15 mm Hg., Results: Mean preoperative intraocular pressure for all eyes was 33.3 +/- 12.0 mm Hg and decreased to 11.8 +/- 2.3 mm Hg. The success rate was 86.6% at 1 year after surgery. Life-table analysis showed that there was no significant difference in the two groups., Conclusion: Phacoemulsification and trabeculotomy combined with goniosynechialysis is an effective surgical treatment for chronic angle-closure glaucoma in eyes with or without a previous glaucoma attack., (Copyright 2010, SLACK Incorporated.)

Purpose: To evaluate the biologic efficacy of intravitreal bevacizumab (IVB) for iris neovascularization (INV) or neovascular glaucoma (NVG) in patients with ischemic retinal disorders., Design: Retrospective, consecutive, interventional case series., Participants: Thirty patients (41 eyes) with INV or NVG secondary to ischemic retinal disorders., Methods: Patients received IVB (1 mg) as the initial treatment for INV or NVG and were followed up for at least 6 months. Ophthalmic evaluations included measurement of visual acuity and intraocular pressure (IOP), a complete ophthalmic examination, and fluorescein angiography. Patients were divided into 3 subgroups: INV without elevated IOP (INV group), NVG with an open angle (O-NVG group), and NVG with angle closure (C-NVG group) for outcomes analysis., Main Outcome Measures: The controllability of IOP by IVB, incidence of recurrence, and requirement for surgery to treat NVG., Results: No significant ocular or systemic adverse events developed during follow-up (range, 6-22 months; mean, 13.3 months). The mean IOP levels were 14.7, 31.2, and 44.9 mmHg at baseline in the INV, O-NVG, and C-NVG groups, respectively. In the INV group (9 eyes), the INV regressed or resolved after 1 injection. Iris neovascularization recurred in 4 eyes by 6 months and stabilized after repeated injections without IOP elevation. In the O-NVG group (17 eyes), rapid neovascular regression with successful IOP normalization (

Purpose: To determine whether alpha-amino-3-hydroxy-5-methylisoxazole-4-propioate (AMPA) receptor (AMPAR) subunit proteins are expressed in cultured retinal ganglion cells (RGCs)., Methods: RGCs were purified from dissociated rat retinal cells (postnatal days 6-8), using a modified two-step panning method and cultured in serum-free medium containing neurotrophic factors and forskolin. Immunohistochemistry was performed on cultured RGCs on days 1, 3, and 7 in vitro (1 DIV, 3 DIV, and 7 DIV) using specific antibodies against AMPAR subunits GluR1 to 4 and microtubule-associated protein (MAP) 2, which is a neuronal marker. Glutamate-induced Ca(2+) influx was measured with fura-2 acetoxymethyl ester fluorescence., Results: GluR1 to 4 proteins were expressed in the cell body of RGCs on 1 DIV. RGCs showed strong GluR1 to 4 immunoreactivity in both cell bodies and processes on 3 DIV and 7 DIV, with the gradual spreading of expression and the growth of processes. At all time points examined, GluR2 immunoreactivity was equal to that of the other subunits. Accumulation of intracellular Ca(2+) levels in RGCs induced by glutamate occurred equally on both 3 DIV and 7 DIV., Conclusion: All AMPAR subunits are almost equally expressed in cultured RGCs.

Purpose: The anterior ocular segment topography in angle-closure glaucoma was studied using the non-contact three-dimensional anterior segment analyzer Pentacam., Subjects and Methods: The central anterior chamber (AC) depth, mid-peripheral AC depth, peripheral AC depth, AC volume, central AC depth/ocular axial length ratio and angles of a narrow angle group (47 eyes of 29 patients), a post laser iridotomy (LI) group (43 eyes of 30 patients), and an open-angle glaucoma (OAG) group (37 eyes of 21 patients) were measured and compared. In addition, changes in the parameters before and after LI (13 eyes of 9 patients) were investigated., Results: The AC volume for the narrow angle group (74.5 +/- 21.1 microl) was significantly smaller than for the other groups (post LI group: 96.4 +/- 21.4 microl; OAG group: 144.2 +/- 31.6 microl, p<0.001). The most significant association was detected between AC volume and the peripheral AC depth. Only two parameters, AC volume and peripheral AC depth, increased significantly after LI (p< 0.001)., Discussion: Measurement of the AC volume and the peripheral AC depth using Pentacam is useful for evaluating the anterior ocular segment topography in narrow angle eyes.

Purpose: To determine whether repeated intravitreal triamcinolone (IVTA) injections in patients with Behçet disease that is resistant to conventional therapy will prevent recurrences of uveitis., Design: Prospective preliminary clinical trial., Methods: Nine eyes of five patients with Behçet disease with sight-threatening uveitis that was resistant to conventional therapy received repeated IVTA injections. Each patient was examined monthly, and IVTA was given when the TA particles that remained in the vitreous were judged to be gone before the next visit., Results: Repeated IVTA completely prevented the recurrence of uveitis in eight eyes, and the one eye that had an attack experienced the uveitis when the TA particles disappeared quicker than expected. It was possible to stop systemic corticosteroid and/or immunosuppressive agents within 1 year after IVTA in three patients., Conclusion: These results indicate that repeated IVTA will prevent the recurrence of uveitis in patients with Behçet disease with frequent ocular attacks that is resistant to conventional therapy.

The neuroprotective effect of donepezil, an acetylcholinesterase inhibitor on retinal ganglion cells (RGCs), was examined. The survival of purified RGCs after exposure to glutamate with or without donepezil (10(- 7) M to 10(- 5) M) was measured after 3 days in culture. In vivo, the neuroprotective effect of donepezil was examined by quantifying the number of viable RGCs 7 days after axotomy in adult rats. In vitro, donepezil dose-dependently reduced RGC death caused by glutamate toxicity. Oral administration of donepezil (10 mg kg(-1) day(-1)) significantly reduced RGC death after axotomy. In conclusion, donepezil exerts a protective effect on RGCs both in vitro and in vivo.

Purpose: To evaluate the effect of substituting latanoprost(LAT) 0.005% for unoprostone(UNO) 0.12% after a trial of unilateral treatment., Methods: We treated 30 patients with primary open-angle glaucoma(n = 8), ocular hypertension (n = 1), or normal-tension glaucoma(n = 21) with UNO for 4 weeks in one eye and then substituted LAT for UNO. Four weeks later we measured the intraocular pressure(IOP) in the ipsilateral eye., Results: The mean baseline IOP level was 18.6 +/- 3.8(mean +/- standard deviation) mmHg. The mean IOP levels(reduction rates) after UNO and LAT therapy were 16.7 +/- 3.1 mmHg (16.6%) and 14.1 +/- 3.2 mmHg (28.9%), respectively(p < 0.001). All patients who responded to UNO also responded to LAT; however, 55% of those who did not respond to UNO responded to LAT., Conclusions: If LAT is substituted for UNO, it can be predicted that 63.3% of the patients will respond.

We evaluated whether factors secreted by human amniotic epithelial cells (HAECs) have the neuroprotective effect on rat retinal ganglion cells (RGCs) purified by immunopanning. After culture in B27 complete medium containing B27 supplement, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and forskolin, the medium was changed to: (1). medium containing N2 supplement and forskolin (N2 basal medium); (2). medium conditioned by HAECs containing N2 supplement and forskolin (HAEC-CM); and (3). N2 basal medium containing several neurotrophic factors. HAEC-CM promoted the RGC survival compared to N2 basal medium. The effect of HAEC-CM was significantly higher than that of BDNF, neurotrophin-3 and CNTF. These results suggest that HAECs may produce unknown neuroprotective factors, suggesting its potential for the treatment of RGC degenerative diseases.

We determined the effect of nilvadipine, a dihydropyridine-type calcium channel blocker, in preventing glutamate neurotoxicity in purified retinal ganglion cells (RGCs). RGCs were purified from dissociated rat retinal cells (postnatal days 6-8), using a modified two-step panning method, and cultured in serum-free medium containing neurotrophic factors and forskolin. RGC survival after exposure to glutamate (25 microM) with nilvadipine or other calcium channel blockers was measured by calcein-acetoxymethyl ester staining after 3 days in culture. Changes in the level of intracellular Ca(2+) ([Ca(2+)](i)) were measured with fura-2 fluorescence. Induction of apoptosis was evaluated using the TDT-dUTP terminal nick-end labeling technique. The neurotoxic effects of low doses of glutamate were blocked by a specific alpha-amino-3-dihydro-5-methylisoxazole-4-propionate-kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (20 microM). Simultaneous application of nilvadipine (1-100 nM) with glutamate protected against glutamate neurotoxicity in a dose-dependent manner. Calcium-imaging experiments showed that the glutamate-evoked [Ca(2+)](i) increase was significantly blocked by nilvadipine (P<0.001), but not nifedipine and diltiazem, in about 50% of RGCs. In addition, the application of nilvadipine significantly reduced glutamate-induced apoptosis (P<0.001). These findings suggest that nilvadipine may partly inhibit glutamate-induced apoptotic cell death by blocking calcium influx via voltage-dependent calcium channels in purified RGCs.

The purpose of this study was to determine whether timolol, an ocular hypotensive drug, has retinal neuroprotective effects in experimental in vitro and in vivo models. For in vitro studies, we used retinal neuron cultures from rat embryos and purified retinal ganglion cells (RGCs) from newborn rats. In the former, neurotoxicity was induced using 1 mM glutamate and cell viability was assessed. In RGCs, neurotoxicity was induced using 25 microM glutamate for 3 days and cell viability was assessed. For the in vivo study, we used a rat model of retinal ischemic injury induced by elevating intraocular pressure (IOP) by raising the hydrostatic pressure. The retinal damage was evaluated by counting the number of cells in the ganglion cell layer (GCL) and by examining the a- and b-waves in the electroretinogram (ERG). For the intraocular distribution study, 0.5% [3H]timolol was topically applied to rat eyes, and these were enucleated after various intervals and divided into parts. Each part was combusted and the radioactivity measured. Timolol (0.1 and 1 microM) markedly reduced the glutamate-induced neuronal cells in retinal neuron cultures and in RGCs. After ischemic-reperfusion, both the number of cells in the GCL and a- and b-waves in the ERG decreased; however, topically applied 0.5% timolol reduced these effects. Topically applied 0.5% timolol was detected at a concentration of approximately 1 microg/g wet tissue in retina-choroid at 30 min after its application. In conclusion, timolol was effective against retinal neuron damage both in vitro and in vivo. Furthermore, topically applied timolol reached the retina-choroid. These findings suggest that timolol has a direct neuroprotective effect against retinal damage., (Copyright 2002 Elsevier Science B.V.)

Purpose: Low concentrations of excitotoxic agents such as glutamate decrease survival of retinal ganglion cells (RGCs) and may be an important cause of RGC death in a variety of retinal diseases. Arachidonic acid (AA), an intercellular messenger in the central nervous system, has been reported to have multiple effects on glutamate receptors, including an inhibitory effect on non-N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to test the hypothesis that AA could protect RGCs from glutamate neurotoxicity., Methods: RGCs were purified from the rat retina on postnatal days 7 and 8 by a modified two-step panning method. Survival of RGCs after exposure to glutamate, with or without AA treatment, was measured after 3 days in culture. To visualize calcium signals, RGCs were loaded with a calcium indicator dye, fluo-3 acetoxymethyl ester, and the fluorescence was measured by laser scanning confocal microscopy. Electrophysiological effects of AA on non-NMDA ionotropic receptors were examined by using whole-cell patch clamp configurations., Results: Incubation of RGCs with 25 microM glutamate caused 60% loss of RGCs. This glutamate neurotoxicity was significantly ameliorated by low concentrations of AA. Concentrations of AA above 10 microM were toxic to RGCs. Calcium imaging showed that glutamate-, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA) and kainate-induced intracellular calcium accumulation in these cells was reduced by AA. Electrophysiological recordings revealed that currents mediated by non-NMDA ionotropic receptors were inhibited by AA in a dose-dependent manner., Conclusions: Low concentrations of AA can reduce glutamate neurotoxicity to RGCs by the inhibition of non-NMDA ionotropic receptors. These results suggest that endogenous or exogenous AA may be used to protect RGCs from glutamate neurotoxicity and that AA may be one potential treatment for RGC loss in a variety of eye diseases, including glaucoma.

Purpose: N-syndecan is a transmembrane heparan sulfate proteoglycan, that is highly expressed in neural tissues. In the current study, changes in N-syndecan expression during retinal development were examined., Methods: Localization of N-syndecan in developing rat retina was examined by immunohistochemistry and in situ hybridization. The amount of the core protein was evaluated by immunoblot analysis, using retinal homogenates at various developmental stages. In addition, mRNA expression was semiquantified by reverse transcription-polymerase chain reaction (RT-PCR). To understand better the localization of N-syndecan in retinal neuronal cells, we performed immunocytochemistry using retinal ganglion cells in culture., Results: N-syndecan is highly expressed in nerve fiber-rich layers of the retina at early postnatal stages (between postnatal day [P]0 and P14). In contrast, immunoreactivity was faint during embryonic stages and late postnatal stages. In addition, in retinal flatmounted sections, N-syndecan immunoreactivity was observed on the axons of retinal ganglion cells. Intense signals were observed in the ganglion cell layer during in situ hybridization. Immunoblot analyses demonstrated that the amount of N-syndecan core protein reached a peak at approximately P14. The RT-PCR analyses using N-syndecan primers showed that an intense amplified band was observed in the cDNA derived from P14 retinas, whereas only faint bands were detected in the embryonic day (E)16 and P42 retinas. In retinal ganglion cells in culture, N-syndecan was located on the long, extended neurites., Conclusions: The data show that N-syndecan is transiently expressed, primarily in retinal neural fibers, during retinal development, indicating that it may be involved in formation of the retinal neural network.

Purpose: Neurocan and phosphacan are nervous tissue-specific chondroitin sulfate proteoglycans (CSPGs) that are highly expressed in postnatal rat retina. To elucidate potential roles of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells (RGCs), in vitro experiments were conducted with purified RGCs., Methods: Neurocan and phosphacan were purified from postnatal rat brain by DEAE-column chromatography and subsequent gel chromatography. RGCs were obtained from postnatal rat retinas by a two-step immunopanning procedure using an anti-Thy 1,1 antibody and an anti-macrophage antibody. Neurite outgrowth from RGCs was examined on poly-L-lysine (PLL)-conditioned plates, and PLL-conditioned plates treated with neurocan or phosphacan., Results: Compared with PLL-conditioned plates, neurocan and phosphacan inhibited neurite outgrowth from RGCs at 48 and 72 hours after seeding. When chondroitin sulfate side chains linked to the core proteins were digested by chondroitinase ABC, the inhibitory effect remained, indicating that the core proteins are related to the effect. Furthermore, the digestion of chondroitin sulfate side chains linked to phosphacan core protein significantly promoted the inhibitory effect of phosphacan on neurite outgrowth from RGCs., Conclusions: Neurocan and phosphacan, which are highly expressed in postnatal rat retina, inhibit neurite outgrowth from postnatal rat RGCs, indicating that these proteoglycans may be inhibitory factors against neurite outgrowth from RGCs during retinal development.

Purpose: Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan present exclusively in central nervous system tissues. In the current study the expression pattern and characterization of NGC during the development of the retina were investigated., Methods: Expressional changes of NGC mRNAs during rat retinal development were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The localization and characterization of NGC core proteins were investigated by immunoblot analysis and immunohistochemistry using an anti-NGC antibody., Results: Immunohistochemical analysis revealed that NGC was highly expressed in the nerve fiber layer (NFL) and inner plexiform layer (IPL) in rat postnatal developing retina. At embryonal stages, NGC immunoreactivities were faint. In contrast, at postnatal developmental stages (approximately postnatal day [P]7), intense immunoreactivity was observed in the NFL and IPL, where active dendrite branching was observed, and conventional synapses began to be formed. As retinal layer differentiation proceeded (from P14 to P42), immunoreactivities in the inner retinal layers gradually became fainter. Immunoblot and semiquantitative RT-PCR analyses showed that the peak level of NGC expression occurred on approximately P7 and P14. Glycosylation of the NGC core protein changed as the retinal layers matured. In immunoelectron microscopic analysis, NGC immunoreactivity was located on the axonal membranes of neuronal cells in the postnatal retina, whereas immunoreactivity was reduced on membranes at the adult stage. In retinal ganglion cells in vitro, NGC was highly localized in their spiny budding neurites., Conclusions: The results show spatiotemporal expression patterns of NGC, and suggest that it plays a role in the formation of neural networks in retinal development.

Purpose: Low concentrations of excitotoxic agents such as glutamate and nitric oxide decrease survival rates of purified retinal ganglion cells (RGCs). In the retina, RGCs are ensheathed by retinal Müller glial (RMG) cell processes. The purpose of this study was to determine whether RMG cells could protect RGCs from these excitotoxic injuries., Methods: RGCs were purified from 7- or 8-day-old Long Evans rats and cultured on polylysine/laminin-coated coverslips in serum-free medium for 2 days. The coverslips were then moved to dishes containing either confluent RMG monolayers or no glial cells in glutamate-free medium. Some dishes with confluent RMG cells were exposed to D,L-threo-beta-hydroxyaspartate (THA), a blocker of glutamate uptake. Three days after exposure to various concentrations of glutamate or the NO donor, 2, 2'-(hydroxynitroso-hydrazino)bisethanamine, survival rates of RGCs were measured by calcein-acetoxymethyl ester staining. Glutamate concentrations in the medium were measured using amino acid analysis., Results: Without RMG cells, the application of increasing concentrations (5-500 microM) of glutamate caused a dose-dependent increase in RGC death after 3 days. The neurotoxic effects of glutamate were blocked in the RMG cell cocultures, even when there was no direct contact between the cell types. The protective effect of RMG cells was weakened by THA treatment. NO also had toxic effects on RGC. RMG cells prevented this toxicity but only when in direct contact with the RGCs., Conclusions: RMG cells can protect RGCs from glutamate and NO neurotoxicity. We suggest that functional disorders of glutamate uptake in RMGs might be one of the etiologies of glaucoma.

Purpose: To determine whether low concentrations of glutamate induce cell death in purified rat retinal ganglion cells (RGCs)., Methods: Rat retinal ganglion cells were purified from dissociated retinal cells by a modified two-step panning method and were cultured in serum-free medium containing neurotrophic factors and forskolin. Survival of RGCs after exposure to glutamate, with or without glutamate receptor antagonists, was measured by calcein-acetoxymethyl ester staining after 3 days in culture. To visualize calcium signals, RGCs were loaded with the calcium indicator dye, fluo-3 acetoxymethyl ester, and fluorescence was measured by laser scanning confocal microscope. Electrophysiological properties of RGCs were examined by using the whole-cell, patch-clamp technique., Results: The application of increasing concentrations (5-500 microM) of glutamate caused a dose-dependent increase in RGC death after 3 days in culture. Neurotoxic effects of low doses of glutamate were totally blocked by a specific alpha-amino-3-dihydro-5-methyl-isoxazol-4-propionic acid-kainate (AMPA-KA) receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), but not by a specific N-methyl-D-aspartate receptor antagonist, 2-amino-5-phosphonovalerate (APV). In addition, calcium imaging and patch-clamp recordings showed that intracellular calcium accumulation and glutamate-evoked inward currents were completely blocked by DNQX but not by APV., Conclusions: Low doses of glutamate can activate AMPA-KA receptors in RGCs, which causes increases in intracellular calcium and decreases in cell survival. This is the first report to show the functional role of calcium-permeable AMPA-KA receptors in cultured RGCs.

The expression of the proto-oncogenes c-fos and c-jun was examined by in situ hybridization at various timepoints following transient retinal ischemia by means of ligation of the retinal central artery in the rat. Ischemia of 90-minute duration resulted in the degeneration of neurons in both the ganglion cell layer and the inner nuclear layer at 48 hours after reperfusion. The expression of c-fos and c-jun messenger RNA throughout the entire inner nuclear layer was transiently coinduced following 90-minute retinal ischemia with a peak at 1 hour after reperfusion. This expression was also found in the ganglion cell layer at 3 hours after reperfusion. Weak signals for c-fos and c-jun mRNA were observed at 24 hours after reperfusion and returned to near control levels by 48 hours. c-jun protein expression was detected in the ganglion cell layer, the middle of the inner nuclear layer, and optic nerve head at 3 hours, but not 1 hour, after lethal ischemia/reperfusion; however, c-fos protein expression was not detected after reperfusion. Whereas no neuronal degenerative changes were found at 7 days after 30-minute ischemic retina, c-fos and c-jun messenger RNA were also induced at 1 hour postreperfusion. To our knowledge, this study is the first report to show expression patterns of immediate-early genes after retinal ischemia/reperfusion. These results suggest that changes in expression of c-fos and c-jun after transient retinal ischemia are similar to those after transient brain ischemia, and the selective occlusion of the central retinal artery will provide a useful model for studying ischemic neuronal degeneration in vivo in the rat retina.

We studied the cellular localization and osmotic regulation of taurine transporter (TauT) mRNA in the rat eyes using in situ hybridization. TauT mRNA signals were expressed in the ciliary body, and the outer part of the inner nuclear layer (INL), the outer nuclear layer (ONL) and the inner segment (IS) of the adult rat retina. Chronic hypernatrema, induced by gavaging with 1 ml/100 g body weight of 5% NaCl every other day for 7 days, markedly increased in TauT mRNA in the retina compared with control rats. However, there was little change in TauT mRNA in the eyes in acute hypernatremic state that is induced by single injection of high concentration of NaCl. On the contrary, acute hypernatremic rats displayed markedly elevated Na+/myo-inositol cotransporter (SMIT) mRNA in the retina and the iris-ciliary body and the lens epithelium. Under chronic hypernatremic conditions, there was no significant increase in SMIT mRNA in rat eyes. These findings suggest that TauT mRNA is osmotically regulated in vivo to protect retinal neuronal function, especially against chronic hypernatremic conditions, in contrast to rapid up-regulation of SMIT mRNA in acute hypernatremic rats.

Glutamate transporters play an important role in maintaining the extracellular glutamate concentration below the neurotoxic level. We investigated the expression of glutamate/aspartate transporter (GLAST) mRNA in the periventricular region of rats with kaolin-induced hydrocephalus by in situ hybridization (ISH). The density of GLAST mRNA-positive cells and the level of hybridization signals per positive cell significantly increased in the acute stage of hydrocephalus. We also demonstrated co-localization of GLAST mRNA and GFAP immunoreactivity in a single cell using the combined methods of ISH and immunohistochemistry. These findings suggest that GLAST is expressed in the reactive astrocytes of the periventricular area and regulates extracellular glutamate concentration after hydrocephalic brain injury.

We studied the localization of Na+/myo-inositol cotransporter (SMIT) mRNA in normal and hypertonic stress rat eyes by in situ hybridization histochemistry using cRNA probes. SMIT mRNA signals were observed in the iris-ciliary body, the lens epithelial cells, and the ganglion cell layer and the inner nuclear layer of the retina. There was a rapid increase on SMIT mRNA in the retina of hypertonic stress rats compared with control rats. These findings suggest that Na+/myo-inositol cotransporter gene expression is osmotically regulated in vivo to protect retinal neuronal function against hypertonic stress.

We have demonstrated the cellular localization of glutamate-aspartate transporter (GLAST/GluT-1) mRNA in the rat retina and its induction after ischemia by in situ hybridization. GLAST mRNA was expressed in the inner two-thirds of the inner nuclear layer (INL) and in sparse small cells in the inner portion of the ganglion cell layer (GCL) of the adult rat retina. GLAST mRNA was also found in about 90% of cells in the optic nerve head where more than 90% of cells express glial fibrillary acidic protein (GFAP) mRNA. Moreover, experimental occlusion of the central retinal artery followed by reperfusion for 48 h resulted in degeneration of neurons and a marked increase in GLAST mRNA expression in the INL. These findings suggest that GLAST may be expressed in Müller cells and astrocytes in the retina, and may play an important role in regulation of extracellular glutamate concentration especially under ischemic conditions.

Day-night variation of the preprosomatostatin mRNA level in the suprachiasmatic nucleus (SCN), the site of the circadian pacemaker, was determined in rats maintained under light-dark cycles by semiquantitative Northern blot hybridization of micropunched tissues. Preprosomatostatin mRNA abundance in the SCN showed significant variation during a day with a peak level at around light on and a trough at around 4 h after light off. This time course was essentially identical with that previously found in blinded rats, indicating that environmental light did not produce an immediate effect on the preprosomatostatin mRNA level in the SCN. Moreover, this rhythm in the preprosomatostatin mRNA appeared specific to the SCN, since levels in the cortex and anterior hypothalamus did not oscillate even under light-dark conditions.

Temporal changes of serotonin (5-HT) content in the median (MRN) and dorsal (DRN) raphe nuclei were measured in rats kept under various lighting conditions. Serotonin content in the MRN and DRN under light-dark (LD) condition showed diurnal rhythmicity, with a peak during early light phase and a trough during the dark phase. In constant dark (DD) condition, a single peak was observed and was out of phase to the 5-HT peak found under LD condition. Animals exposed to constant light (LL) after 2 days in DD showed marked increase in 5-HT after lights on. These results suggest that changes in 5-HT in the MRN and DRN are regulated by an endogenous pacemaker and by light.

Neuropeptide Y (NPY)-like immunoreactivity (LI) in the iris-ciliary body of rats kept under constant darkness (DD) and in 12 h light-12 h dark cycle (LD) was determined by enzyme immunoassay. NPY-LI contents in the iris-ciliary body were found to oscillate in circadian fashion under DD and LD conditions, with a peak at about circadian time 12 (CT 12) and a trough at around CT 0. Unilateral superior cervical ganglionectomy caused a significant decrease in NPY-LI levels in the sympathectomized eye compare to the contralateral intact eye, independent of lighting phase. These results suggest the presence of an endogenous circadian rhythm in NPY-LI content in the rat iris-ciliary body, and the possible involvement of a sympathetic input from the superior cervical ganglion.

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a circadian pacemaker in mammals. We determined the circadian profiles of arginine-vasopressin (AVP), a major peptide in the dorsomedial SCN, in rats under light-dark (LD), constant dark (DD) and constant light (LL) conditions. Under LD conditions, AVP levels in the SCN showed circadian rhythmicity with a peak at early light phase and a broad trough during the dark phase. This rhythm in the AVP contents was maintained even after 14 days of free-running under DD conditions and 3 days under LL conditions. These circadian patterns of AVP are similar to those of somatostatin, another peptide in the dorsomedial SCN. This indicates a common mode of regulation for peptides in this subfield of the SCN.