Maho Shibata

Involvement of P2X7 receptors in the hypoxia-induced death of rat retinal neurons.

PURPOSE To investigate the hypoxia-induced death of rat retinal neurons and to determine whether P2X(7) activation is involved in this type of neuronal death. METHODS Cultured retinal neurons from fetal rats were used. The effects and time course of various degrees of hypoxia (1%-5% O(2)) in the death of retinal neurons, were examined. The effects of P2X(7) antagonists, oxidized adenosine triphosphate (oxidized ATP; 30-100 microM), and brilliant blue G (BBG; 100 nM-10 microM) on hypoxia-induced neuronal death, including apoptosis, were assessed by using trypan blue exclusion, TUNEL assays, and cleaved caspase-3 immunoreactivity. Immunocytochemical analysis was performed to determine whether these neurons express P2X(7) receptors. The effects of P2X(7) receptor stimulation, induced by the P2X(7) agonist benzoyl- benzoyl-ATP (BzATP), on neuronal viability and intracellular Ca(2+) levels ([Ca(2+)](i)) were examined. RESULTS Retinal neuronal death increased according to the degree of hypoxia and became more severe after 12 hours. Both oxidized ATP and BBG significantly decreased hypoxia-induced neuronal death. Immunocytochemistry demonstrated that P2X(7) receptors were expressed by the cultured retinal neurons. ATP and BzATP caused P2X(7) receptor-dependent neuronal death in a dose-dependent manner and led to a sustained increase in [Ca(2+)](i), with BzATP being more effective than ATP. These effects were hypoxia-induced factor-1alpha- independent and were prevented by oxidized ATP. CONCLUSIONS The results suggest that the death of retinal neurons can be triggered by hypoxia and that P2X(7) activation is involved in the hypoxia-induced death of retinal neurons. P2X(7) antagonists can prevent hypoxia-induced damage in retinal neurons.

The first report on intermediate-term outcome of Ex-PRESS glaucoma filtration device implanted under scleral flap in Japanese patients.

Purpose: This paper compares the outcomes of the Ex-PRESS® Glaucoma Filtration Device (Alcon, Fort Worth, TX) implant observed in Japanese patients for 1 year with those of patients undergoing trabeculectomy. Patients and methods: The subjects comprised ten eyes of ten cases with open-angle glaucoma for which filtration surgery using Ex-PRESS (P-50) was performed by one operator from February 2008 and observed for at least 1 year (Ex-PRESS Group), and eleven eyes of eleven cases for which trabeculectomy was performed by the same operator (TE Group). For both groups, mitomycin C was used and a scleral flap was created after a fornix-based incision of the conjunctiva. Results: Hypotony and choroidal detachment were observed as early postoperative complications during a 1-week period in one-third of the cases in the TE Group, and failing vision in about 45%, while these were seen in fewer cases in the Ex-PRESS Group. No significant difference in intraocular pressure (IOP) was observed during the period, but IOP variations on the day following the surgery were obviously narrower in the Ex-PRESS Group than in the TE Group. Visual acuity was significantly poorer from 1 week to 3 months in the TE Group while it was stable in the Ex-PRESS Group. The Ex-PRESS Group had fewer cases of laser suture lysis and fewer administrations of glaucoma eyedrop, and no cases of progression in the stage of visual field defect. Conclusion: Filtration surgery using the Ex-PRESS is unlikely to cause early complications in Japanese patients. Similarly to the trabeculectomy, the intermediate-term control of IOP showed favorable results.

Clinical results of selective laser trabeculoplasty in open-angle glaucoma in Japanese eyes: comparison of 180 degree with 360 degree SLT.

PurposeTo evaluate the efficacy of selective laser trabeculoplasty (SLT) in the adjunctive treatment of medically diagnosed open-angle glaucoma and to compare the difference in intraocular pressure (IOP) lowering effects between 180-degree and 360-degree SLT. MethodsThis study is a retrospective consecutive chart review of open-angle glaucoma patients who had undergone first-time SLT from January of 2005 to July of 2007. All the patients had primary open-angle glaucoma or pseudoexfoliation glaucoma under medical treatment and followed for at least 3 months after the procedure. The IOP reduction and treatment success were compared with the 2 treatment types. ResultsTwenty-nine patients underwent 180-degree SLT (35 eyes) and 25 patients underwent 360-degree SLT (34 eyes). The average follow-up was 19.5 months (range 3 to 36) for 180-degree group and 17.9 months (range 3 to 36) for 360-degree group. During the follow-up period, the 360-degree SLT group showed significantly lower posttreatment IOP at each follow-up point relative to pretreatment IOP, and its IOP reduction rate stayed statistically higher than the 180-degree group. We found a positive correlation between the pretreatment IOP and the IOP reduction rate for 360-degree SLT. The lower the pretreatment IOP was, the lower IOP reduction rate became. A Kaplan-Meier survival analysis showed higher success rates after 360-degree SLT than after 180-degree SLT. ConclusionsThe 360-degree SLT was shown to be more effective than180-degree SLT for intermediateterm reduction in IOP of Japanese patients with open-angle glaucoma as an adjunctive treatment protocol.

Disruption of Gap Junctions May Be Involved in Impairment of Autoregulation in Optic Nerve Head Blood Flow of Diabetic Rabbits

PURPOSE To determine whether an impairment of the autoregulatory mechanism of blood flow in the optic nerve head (ONH) is present in diabetic rabbits and whether the impairment results from the uncoupling of gap junctions. METHODS Experiments were performed on six alloxan-induced diabetic rabbits and six healthy control animals. In a test of the integrity of the autoregulatory mechanism, the intraocular pressure (IOP) was elevated from the 20-mm Hg baseline to 50 and then to 70 mm Hg. The capillary blood flow in the ONH was measured every 10 minutes by the laser speckle method, with simultaneous measurements of blood pressure. Ocular perfusion pressure (OPP) was calculated at each step, and the relationship between blood flow and OPP was analyzed. In addition, octanol, gap27 (gap junction uncouplers), or balanced saline solution was injected into the vitreous of healthy rabbits, with the balanced saline solution-injected eyes serving as the control. Changes in the ONH blood flow in response to the IOP elevation were determined in the same way. RESULTS Diabetic rabbits had a significant decrease in ONH blood flow when the OPP was reduced by an elevation of the IOP to 50 or to 70 mm Hg, whereas the ONH blood flow was well maintained in healthy rabbits. After injection of octanol (10.0 mM) or gap27 (10 μM), a reduction of OPP resulted in a significant decrease in ONH blood flow in the healthy rabbits. CONCLUSIONS These results indicate that autoregulation is disrupted in diabetic animals, and uncoupling the gap junctions in healthy rabbits also disrupts the autoregulation.

Magnetic Resonance Imaging Findings of Terson's Syndrome Suggesting a Possible Vitreous Hemorrhage Mechanism

BackgroundThe mechanism of Terson’s syndrome is controversial. Here we report magnetic resonance (MR) imaging findings of Terson’s syndrome that may help clarify the mechanism responsible for vitreous hemorrhage.CaseA 49-year-old man suffered from subarachnoid hemorrhage with bilateral intraocular hemorrhage. Fundus examination revealed sub-internal limiting membrane and retinal hemorrhages in both eyes. MR images were recorded after the neurosurgery.ConclusionThe vitreous hemorrhage may be caused by a large amount of blood, originally formed by Terson’s syndrome, entering the subarachnoid space around the optic nerve and from there infiltrating the intraocular space through the perivascular space around the central retinal vessels within the optic nerve.

Involvement of Glial Cells in the Autoregulation of Optic Nerve Head Blood Flow in Rabbits

PURPOSE To investigate the involvement of glial cells in the autoregulation of optic nerve head (ONH) blood flow in response to elevated intraocular pressure (IOP). METHODS Rabbit eyes were treated with an intravitreal injection of l-2-aminoadipic acid (LAA), a gliotoxic compound. Twenty-four hours after the injection IOP was artificially elevated from a baseline of 20 to 50 or 70 mm Hg and maintained at each IOP level for 30 minutes. ONH blood flow was measured by laser speckle flowgraphy every 10 minutes. Ocular perfusion pressure (OPP) was calculated to investigate the relationship between ONH blood flow and OPP. To evaluate the effects of LAA on the function and morphology of retinal neurons and glial cells, electroretinogram (ERG) was monitored after injections of LAA (2.0 and 6.0 mM) or saline as a control. Histologic and immunohistochemical examinations were then performed. RESULTS In the LAA-treated eyes, histologic changes selectively occurred in the retinal Müller cells and ONH astrocytes. There was not any significant reduction of amplitude or elongation of implicit time of each parameter in the ERG after LAA injection compared with control. ONH blood flow in LAA-treated eyes was significantly decreased with a reduction of OPP during IOP elevation to 50 and 70 mm Hg, whereas blood flow was maintained in control eyes during IOP elevation to 50 mm Hg. CONCLUSIONS These results indicate the involvement of glial ells in the autoregulation of ONH blood flow during IOP elevation.

Changes in optic nerve head blood flow, visual function, and retinal histology in hypercholesterolemic rabbits

We investigated the effects of hypercholesterolemia on optic nerve head (ONH) blood flow, visual function, and retinal histology in a rabbit model. Hypercholesterolemia was induced in rabbits by feeding them a high cholesterol (1%) diet for 12 weeks. Changes in blood pressure, intraocular pressure (IOP), and ONH blood flow were monitored at 6 and 12 weeks after treatment. The autoregulation of ONH blood flow as detected by laser speckle flowgraphy was verified by an artificial elevation of IOP at 12 weeks. Visually evoked potentials (VEPs) were also recorded and analyzed at 6 and 12 weeks. Finally, a histological examination as well as immunohistochemistry to endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) was performed. In the hypercholesterolemic rabbits, blood pressure, IOP, and ONH blood flow did not alter significantly throughout this study. The autoregulation of ONH blood flow against IOP elevation was found to be impaired at 12 weeks. The amplitudes of the first negative peak of VEPs were diminished. Both the density of the retinal ganglion cells and the thickness of the inner nuclear layer and photoreceptor cell layer were reduced. Immunoreactivity to eNOS was reduced and that to iNOS was enhanced in the hypercholesterolemic rabbits compared to those in the normal control rabbits. The results of this study show that hypercholesterolemia induces impairment in the autoregulation of ONH blood flow and deterioration in visual function and histology. Downregulation of eNOS activity might be one of the causes for impairment of the autoregulation. Enhanced activity of iNOS might be involved in the impaired visual function and histology.

Effects of Gelatin Hydrogel Containing Chymase Inhibitor on Scarring in a Canine Filtration Surgery Model

PURPOSE To investigate the effects of gelatin hydrogel (GH) containing a chymase inhibitor (CI) on intraocular pressure (IOP) and conjunctival scarring in a canine model of glaucoma surgery. METHODS Glaucoma surgery models were made in beagles. As the first experiment, GH was implanted. IOP was measured for 4 weeks, followed by histologic evaluation. As the second experiment, GH containing a CI or GH alone was implanted. IOP and bleb features were evaluated for 12 weeks. The densities of proliferative cell nuclear antigen (PCNA)-positive cells, fibroblasts, and mast cells were quantified. The mRNA levels of transforming growth factor-β (TGF-β) and chymase were determined by real-time PCR. RESULTS In the first experiment, IOP was significantly lower in the eyes treated with GH than that in the control eyes. The conjunctival area normalized by the scleral area was reduced in the treated eyes. In the second experiment, IOP reduction was maintained for 12 weeks in the eyes treated with GH containing a CI, but not in the eyes treated with GH alone. In addition, the bleb score was larger, whereas the adhesion score and densities of PCNA-positive cells, fibroblasts, mast cells, and chymase-positive cells were lower in the eyes treated with GH containing a CI. The mRNA levels of TGF-β and chymase were significantly decreased in the eyes treated with GH containing a CI. CONCLUSIONS Implanting GH alone maintained IOP reduction, whereas GH containing a CI enhanced the IOP-reducing effect by suppressing cell proliferation. This drug delivery system might be useful for maintaining filtering blebs for a longer duration after glaucoma surgery.

Purinergic Vasotoxicity: Role of the Pore/Oxidant/KATP Channel/Ca2+ Pathway in P2X7-Induced Cell Death in Retinal Capillaries

P2X7 receptor/channels in the retinal microvasculature not only regulate vasomotor activity, but can also trigger cells in the capillaries to die. While it is known that this purinergic vasotoxicity is dependent on the transmembrane pores that form during P2X7 activation, events linking pore formation with cell death remain uncertain. To better understand this pathophysiological process, we used YO-PRO-1 uptake, dichlorofluorescein fluorescence, perforated-patch recordings, fura-2 imaging and trypan blue dye exclusion to assess the effects of the P2X7 agonist, benzoylbenzoyl-ATP (BzATP), on pore formation, oxidant production, ion channel activation, [Ca2+]i and cell viability. Experiments demonstrated that exposure of retinal microvessels to BzATP increases capillary cell oxidants via a mechanism dependent on pore formation and the enzyme, NADPH oxidase. Indicative that oxidation plays a key role in purinergic vasotoxicity, an inhibitor of this enzyme completely prevented BzATP-induced death. We further discovered that vasotoxicity was boosted 4-fold by a pathway involving the oxidation-driven activation of hyperpolarizing KATP channels and the resulting increase in calcium influx. Our findings revealed that the previously unappreciated pore/oxidant/KATP channel/Ca2+ pathway accounts for 75% of the capillary cell death triggered by sustained activation of P2X7 receptor/channels. Elucidation of this pathway is of potential therapeutic importance since purinergic vasotoxicity may play a role in sight-threatening disorders such as diabetic retinopathy.

Expression Sites of Neural Stem Cell-Related Genes in the Monkey Retina

Objectives: Based on the hypothesis that undifferentiated retinal stem cell (RSC)-like cells exist in the fovea (the light-stressed, concave, avascular center of the retina where light is focused), we investigated the expression sites of neural stem cell (NSC)-related genes in the monkey retina. Methods: Cynomolgus monkeys were euthanized, and both eyes were then enucleated. Each eye was hemisected near the limbus, and flat-mounted retina samples were then prepared. Using a stereomicroscope, 1-mm x 1-mm blocks of the retina at the fovea, mid-periphery, and extreme periphery were then excised. These samples were used for real-time polymerase chain reaction analysis of the NSC-related gene (nestin, PAX6, and SOX2) expression at each site. Results: Nestin expression was high in the fovea, with a lower expression in the mid-periphery and extreme periphery. No differences in PAX6 gene expression were found in the fovea, mid-periphery, and extreme periphery. SOX2 expression was highest in the extreme periphery, with decreased expression in the mid-periphery and fovea. Conclusions: Our finding that nestin expression was highest in the fovea suggests that foveal retinal cells may have more undifferentiated characteristics that are different from retinal cells at other sites.