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Background: In mice, a tri-layered (superficial, intermediate, and deep) vascular structure is formed in the retina during the third postnatal week. Short-term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor inhibitors delays the formation of superficial vascular plexus and this allows us to investigate the developmental process of superficial and deep vascular plexuses at the same time. Using this model, we examined the effect of pharmacological depletion of retinal neurons on the formation of superficial and deep vascular plexuses., Results: Neuronal cell loss induced by an intravitreal injection of N-methyl-d-aspartic acid on postnatal day (P) 8 delayed vascular development in the deep layer but not in the superficial layer in mice treated with KRN633, a VEGF receptor inhibitor, on P0 and P1. In KRN633-treated mice, neuronal cell loss decreased the number of vertical sprouts originating from the superficial plexus without affecting the number of angiogenic sprouts growing in front. Neuronal cell loss did not impair networks of fibronectin and astrocytes in the superficial layer., Conclusions: Our results suggest that inner retinal neurons play a crucial role in forming the deep vascular plexus by directing the sprouts from the superficial blood vessels to the deep layer., (© 2020 American Association of Anatomists.)

An electrical communication between the endothelial and smooth muscle cells via gap junctions, which provides the signaling pathway known as endothelium-dependent hyperpolarization (EDH), plays a crucial role in controlling the vascular tone. In this study, we investigated the role of gap junctions in the acetylcholine (ACh)-induced EDH-type dilation of rat retinal arterioles in vivo. The dilator response was evaluated by measuring the diameter of retinal arterioles. Intravitreal injection of gap junction blockers (18β-glycyrrhetinic acid and carbenoxolone) reduced the ACh-induced dilation of retinal arterioles. Moreover, the retinal arteriolar response to ACh was attenuated by 18β-glycyrrhetinic acid under treatment with a combination of N G -nitro-L-arginine methyl ester (a nitric oxide (NO) synthase inhibitor; 30 mg/kg) and indomethacin (a cyclooxygenase inhibitor; 5 mg/kg). The NO- and prostaglandin-independent, EDH-related component of ACh-induced dilation of retinal arterioles was prevented by intravitreal injection of iberiotoxin, which inhibits large-conductance Ca 2+ -activated K + channels. Furthermore, the combination of 18β-glycyrrhetinic acid and iberiotoxin produced greater attenuation in the EDH-related response than that by the individual agent. Treatment with 18β-glycyrrhetinic acid revealed no significant effect on NOR3 (an NO donor)-induced retinal vasodilator response. These results suggest that gap junctions contribute to the ACh-induced, EDH-type dilation of rat retinal arterioles in vivo.

Misdirected vascular growth frequently occurs in the neovascular diseases in the retina. However, the mechanisms are still not fully understood. In the present study, we created capillary-free zones in the central and peripheral retinas in neonatal mice by pharmacological blockade of vascular endothelial growth factor (VEGF) signaling. Using this model, we investigated the process and mechanisms of revascularization in the central and peripheral avascular areas. After the completion of a 2-day treatment with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5, revascularization started on P8 in the central avascular area where capillaries had been dropped out. The expression levels of VEGF were higher in the peripheral than in the central avascular area. However, the expansion of the vasculature in the peripheral avascular retina remained suppressed until revascularization had been completed in the central avascular area. Additionally, we found disorganized endothelial cell division, misdirected blood vessels with irregular diameters, and abnormal fibronectin networks at the border of the vascular front and the avascular retina. In the central avascular area, a slight amount of fibronectin as non-vascular component re-formed to provide a scaffold for revascularization. Mechanistic analysis revealed that higher levels of VEGF attenuated the migratory response of endothelial cells without decreasing the proliferative activity. These results suggest that the presence of concentration range of VEGF, which enhances both migration and proliferation of the endothelial cells, and the structurally normal fibronectin network contribute to determine the proper direction of angiogenesis.

An impairment of cellular interactions between the elements of the neurovascular unit contributes to the onset and/or progression of retinal diseases. The present study aims to examine how elements of the neurovascular unit are altered in a rat model of retinopathy of prematurity (ROP). Neonatal rats were treated subcutaneously with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg) on postnatal day (P) 7 and P8 to induce ROP. Morphological assessments were performed of blood vessels, astrocytes and neuronal cells in the retina. Aggressive angiogenesis, tortuous arteries and enlarged veins were observed in the retinal vasculature of KRN633-treated (ROP) rats from P14 to P28, compared to age-matched control (vehicle-treated) animals. Morphological abnormalities in the retinal vasculature showed a tendency toward spontaneous recovery from P28 to P35 in ROP rats. Immunofluorescence staining for glial fibrillary acidic protein and Pax2 (astrocyte markers) revealed that morphological changes to and a reduction in the number of astrocytes occurred in ROP rats. The developmental cell death was slightly accelerated in ROP rats; however, no visible changes in the morphology of retinal layers were observed on P35. The abnormalities in astrocytes might contribute, at least in part, to the formation of abnormal retinal blood vessels and the pathogenesis of ROP.

Because visual information accounts for 80-90% of sensory information that we get from our circumstance, loss of vision seriously diminishes our quality of life. According to a recent epidemiological study, glaucoma is the first, and retinitis pigmentosa (RP) is the second leading causes of acquired blindness in Japan. Degeneration of the retinal ganglion cells (RGC) and photoreceptor cells causes glaucoma and RP, respectively. Intraocular pressure-lowering therapy is an only effective treatment for glaucoma, and the agents that protect RGC directly against glaucomatous injury have not been available yet. In addition, there is no effective treatment for RP at present. microRNAs are a class of small, endogenous, non-coding RNAs comprised of approximately 20 nucleotides. It has been clarified that microRNAs reduces the stability of the target mRNAs and/or repress the translation of the target genes. A single microRNA can affect the transcription of multiple mRNAs, and almost 30% of human genes are thought to be regulated by microRNAs. Therefore, it has been considered that the expression changes of microRNAs are possible to cause various diseases, such as cancer and neurodegenerative diseases. Recently, the expression changes in microRNAs have been reported in the retina of experimental model animals for glaucoma and RP. The expressional changes of microRNAs are suggested to be related with development and progression of glaucoma and RP. Here, we will discuss about the relationship between the expressional changes of microRNAs and neuronal cell death in glaucoma and RP.

Pathological retinal angiogenesis contributes to the pathogenesis of several ocular diseases. Valproic acid, a widely used antiepileptic drug, exerts anti-angiogenic effects by inhibiting histone deacetylase (HDAC). Herein, we investigated the effects of valproic acid and vorinostat, a HDAC inhibitor, on pathological retinal angiogenesis in mice with oxygen-induced retinopathy (OIR). OIR was induced in neonatal mice by exposure to 80% oxygen from postnatal day (P) 7 to P10 and to atmospheric oxygen from P10 to P15. Mice were subcutaneously injected with valproic acid, vorinostat, or vehicle once a day from P10 to P14. At P15, retinal neovascular tufts and vascular growth in the central avascular zone were observed in mice with OIR. Additionally, immunoreactivity for phosphorylated ribosomal protein S6 (pS6), an indicator of mammalian target of rapamycin (mTOR) activity, was detected in the neovascular tufts. Both valproic acid and vorinostat reduced the formation of retinal neovascular tuft without affecting vascular growth in the central avascular zone. Valproic acid reduced the pS6 immunoreactivity in neovascular tufts. Given that vascular endothelial growth factor (VEGF) activates mTOR-dependent pathways in proliferating endothelial cells of the neonatal mouse retina, these results suggest that valproic acid suppresses pathological retinal angiogenesis by interrupting VEGF-mTOR pathways., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background: A short-term interruption of vascular development causes structural abnormalities in retinal vasculature. However, the detailed changes in vascular components (endothelial cells, pericytes, and basement membranes) remain to be fully determined. The present study aimed to provide a detailed description of morphological changes in vascular components following a short-term interruption of retinal vascular development in mice., Results: Two-day treatment of neonatal mice with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg, subcutaneously) on postnatal day (P)0 and P1 (P0/1) and P4 and P5 (P4/5) induced different degrees and patterns of impairment of retinal vascular development. Three days after completion of the treatment, the delayed radial vascular growth occurred in P0/1 group mice, whereas in P4/5 group mice, revascularization preferentially occurred in the central avascular area, and radial vascular growth remained suppressed by P10. Differences in α-smooth muscle actin expression in pericytes were noted in the processes between normal vascular formation and vascular regrowth. The changes in vascular cells were associated with the hypoxia-induced enhancement of VEGF expression in the superficial retinal layer., Conclusions: These findings suggest that the phenotype of vascular cells is altered following a short-term interruption of vascular development in the retina. Developmental Dynamics 247:699-711, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Abnormalities in retinal blood vessels and neuronal function persist in eyes undergoing retinopathy of prematurity. In this study, we examined morphological and functional changes in retinal blood vessels and neurons in mice that had undergone short-term interruption of retinal vascular development through inhibition of vascular endothelial growth factor (VEGF) signaling. In mice treated with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 0 and 1, the vascular density in the retinal surface increased by P12, but development of deep retinal vascular plexus and choroidal vasculature was delayed until P14. Overall retinal morphology was mostly normal in KRN633-treated mice during the observation period (∼P28), with the exception of P8 and P14. On P28, abnormalities in retinal vascular patterns were evident, but electroretinogram and retinal blood perfusion were within the normal range. Abnormal architecture of retinal vasculature disturbs retinal hemodynamics; therefore, mice treated postnatally with VEGF receptor inhibitors could serve as an animal model for studying the regulatory mechanism of local retinal blood flow and the effect of persistent abnormal retinal vascular patterns on the risk of onset of retinal ischemia., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Methylglyoxal, a highly reactive dicarbonyl compound, is formed as a by-product of glycolysis and plays an important role in the pathogenesis of diabetic complications, including diabetic retinopathy. However, it remains to be determined how methylglyoxal affects the regulatory mechanisms of retinal blood flow. In this study, we examined the effects of methylglyoxal on β 2 -adrenoceptor-mediated vasodilatory mechanisms in rat retinal arterioles. The retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in the fundus images. Intravitreal injection of methylglyoxal significantly diminished the vasodilation of retinal arterioles induced by the β 2 -adrenoceptor agonist salbutamol. The vasodilator effect of BMS-191011, a large-conductance Ca 2+ -activated K + (BK Ca ) channel opener, on retinal arterioles was also attenuated by methylglyoxal. In contrast, methylglyoxal had no significant effect on retinal vasodilator response to forskolin. Methylglyoxal attenuated retinal vasodilator response to salbutamol under blockade of BK Ca channels with iberiotoxin, an inhibitor of the channels. These results suggest that methylglyoxal attenuates β 2 -adrenoceptor-mediated retinal vasodilation by impairing the coupling of the β 2 -adrenoceptor to the guanine nucleotide-binding protein (Gs protein) and the function of the BK Ca channel. Increased methylglyoxal in the eyes may contribute to the impairment of regulatory mechanisms of retinal blood flow in patients with diabetic retinopathy.

Recently, we reported that capsaicin, a transient receptor potential vanilloid type1 (TRPV1) agonist, protected against excitotoxicity induced by intravitreal N-methyl-D-aspartic acid (NMDA) in the rats in vivo. It has been reported that morphine, an opioid receptor agonist, ameliorated excitotoxicity induced by ischemia-reperfusion in the retina, and that capsaicin-induced neuroprotection was reduced by naloxone, an opioid receptor antagonist in the brain. The aim of the present study is to clarify whether activation of opioid receptors is involved in the capsaicin-induced neuroprotection in the retina. Under ketamine/xylazine anesthesia, male Sprague-Dawley rats were subjected to intravitreal NMDA injection (200nmol/eye). Capsaicin (5.0nmol/eye), calcitonin gene-related peptide (CGRP; 0.05pmol/eye), β-endorphin (0.5 pmol/eye), substance P (5nmol/eye), and naloxone (0.5nmol/eye) were intravitreally administered simultaneously with NMDA. Morphometric evaluation 7 days after NMDA injection showed that intravitreal NMDA injection resulted in ganglion cell loss. Capsaicin, CGRP, β-endorphin, and substance P prevented this damage. Treatment with naloxone (0.5nmol/eye) almost completely negated the protective effects of capsaicin, CGRP, β-endorphin, and substance P in the NMDA-injected rats. These results suggested that activation of opioid receptors is possibly involved in the protective effect of capsaicin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Astrocytes migrate into the retina through the optic nerve head by means of the axons of retinal ganglion cells, and spread radially toward the peripheral retina. Endothelial cells migrate along the astrocyte cellular network to form the retinal surface vasculature. Here, we examined the effects of a delay in retinal vascularization on the migration and proliferation status of astrocytes in mice., Results: A dose-dependent delay in retinal vascularization was observed in mice that had been treated with KRN633 (1-10 mg/kg), a VEGF receptor inhibitor, on the day of birth and on the following day. Delayed vascularization resulted in a delay in the astrocyte network formation, and an increase in astrocyte number in the optic nerve head and the vascular front. The increase in the number of astrocytes may be attributed to increased proliferation and delayed migration. These abnormalities in astrocyte behavior correlated with the degree of delay in retinal vascularization. The vascularization delay also led to retinal hypoxia, which subsequently stimulated VEGF leading to an increase in vascular density., Conclusions: These findings suggest that a delay in normal vascularization leads to abnormal astrocyte behavior, which results in the formation of abnormal astrocyte and endothelial cell networks in the mouse retina. Developmental Dynamics 246:186-200, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

High-mobility group Box-1 (HMGB1) is known to be released from injured cells and to induce an inflammatory response. Although HMGB1 was reported to mediate ischemia-reperfusion injury of the brain, its role in glutamate excitotoxicity of the retina remains controversial. Here, the authors demonstrated the evidence that HMGB1 is involved in the retinal damage induced by NMDA. Under ketamine/xylazine anesthesia, male Sprague-Dawley rats were subjected to intravitreal injection of NMDA (200 nmol/eye) or HMGB1 protein derived from bovines (5-15 μg/eye). Intravitreal anti-HMGB1 IgY (5 μg/eye) was simultaneously administered with NMDA or HMGB1. Seven days later, animals were killed and 5-μm retinal sections through the optic nerve head were obtained. These specimens were subjected to morphometry. Intravitreal NMDA and HMGB1 protein evoked cell loss in the ganglion cell layer 7 days later. Intravitreal anti-HMGB1 IgY reduced these damages. Anti-HMGB1 IgY reduced the number of 8-hydroxy-deoxyguanosine (8-OHdG)-positive cells induced by intravitreal NMDA. Toll-like receptor 2/4 antagonist peptide, receptor for advanced glycation end-products (RAGE) antagonist peptide, and FPS-ZM1 significantly reduced the retinal damage induced by HMGB1 protein. The results in the present study suggest that HMGB1 is at least in part involved in NMDA-induced retinal injury, and probably induces cell death of retinal ganglion cells with increase of oxidative stress, via activation of toll-like receptor 2/4 and RAGE in the rat retina., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

L-Ornithine is a non-proteinogenic amino acid, abundant in freshwater clams and commercially available as an oral nutritional supplement. L-Ornithine is metabolized by ornithine-δ-aminotransferase. Deficiency of this enzyme causes gyrate atrophy of the choroid and retina, an autosomal recessive hereditary disease characterized by the triad of progressive chorioretinal degeneration, early cataract formation, and type II muscle fiber atrophy, with hyperornithinemia. However, it is unknown whether long-term L-ornithine supplementation affects visual function and retinal histology. The aim of the present study is to determine the effect of long-term supplementation of excess amounts of L-ornithine on visual function and retinal histology in rats. Male Brown Norway rats at six weeks of age were allowed free access to chow containing 4% (w/w) L-ornithine (the high ornithine diet) or that containing 4% (w/w) casein (the control diet) for 49 weeks. The dose of L-ornithine calculated from the food intake was approximately 0.8 g/d/animal, which was 100 times higher than the recommended dose for healthy humans. The amplitude of the a-wave of the scotopic rod-cone electroretinogram and the number of cells in the ganglion cell layer in the L-ornithine-treated group were larger than those in the control group 49 weeks after initiating the test diet. No functional or histological damage to the retina was seen up to 49 weeks after the start of the high-ornithine diet. The present study demonstrated that long-term supplementation of very high doses of L-ornithine for at least 49 weeks did not induce retinal damage.

We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N-methyl-D-aspartic acid (NMDA)-induced retinal neurotoxicity, but the mechanism underlying this protection is not fully understood. The present study aimed to examine the effects of everolimus, another inhibitor of mTOR, on neuronal cell loss and inflammation in a rat model of NMDA-induced retinal neurotoxicity, and to determine whether the extracellular signal-regulated kinase (ERK) pathway contributes to the protective effect of everolimus. Intravitreal injection of NMDA (200 nmol) resulted in (1) cell loss in the ganglion cell layer, (2) increase in the numbers of CD45-positive leukocytes and Iba1-positive microglia, and (3) phosphorylation of ribosomal protein S6 (pS6), a downstream indicator of mTOR activity. Simultaneous injection of everolimus with NMDA significantly attenuated these NMDA-induced responses. The neuroprotective effect of everolimus was almost completely prevented by the mitogen-activated protein kinase/ERK kinase inhibitor U0126 (1 nmol). NMDA increased the level of phosphorylated ERK (pERK) in Müller cells, and increase in pERK levels was also observed after co-injection of NMDA and everolimus. These results suggest that everolimus has a neuroprotective effect against NMDA-induced retinal neurotoxicity, an effect that seems to be mediated partly by activation of the ERK pathway in Müller cells.

Recent studies have demonstrated that inhibition of the mammalian target of rapamycin (mTOR) protects against neuronal injury, but the mechanisms underlying this protection are not fully understood. The present study investigates whether rapamycin, an inhibitor of the mTOR pathway, protects against N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity and whether the extracellular signal-regulated kinase (ERK) pathway contributes to this protective effect in rats. Significant cell loss in the ganglion cell layer and a reduction in thickness of the inner plexiform layer were observed 7 days after a single intravitreal injection of NMDA (200 nmol/eye). These NMDA-induced morphological changes were significantly reduced by rapamycin (20 nmol/eye). The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells had increased 6 hr after NMDA injection, an effect that was significantly attenuated by rapamycin. The ERK inhibitor U0126 (1 nmol/eye) almost completely abolished rapamycin's inhibition of NMDA-induced apoptosis. Immunohistochemical studies showed that NMDA caused a time-dependent increase in levels of the phosphorylated form of the ribosomal protein S6 (pS6), a downstream indicator of mTOR activity. The increased pS6 levels were markedly decreased by rapamycin. Both NMDA and rapamycin increased the level of phosphorylated ERK (pERK) in Müller cells, and coinjection of both agents further increased pERK levels. These results suggest that rapamycin has a neuroprotective effect against NMDA-induced retinal neurotoxicity and that this effect could be patially mediated by activation of the ERK pathway in retinal Müller cells., (Copyright © 2014 Wiley Periodicals, Inc.)

In the present study, we histologically examined the effects of nilvadipine on neuronal injury induced by intravitreal (i.v.) N-methyl-D-aspartate (NMDA) (200 nmol/eye) and intravitreal NOC12 (400 nmol/eye), a nitric oxide donor, in the rat retina. Morphometric evaluation at 7 d after injection of NMDA or NOC12 showed that treatment with nilvadipine (1 mg/kg, i.v.) 15 min prior to injection of NMDA or NOC12 dramatically reduced the retinal damage. These results suggest that nilvadipine protects neurons against excitotoxic injury in the rat retina in vivo at least in part via an antioxidative effect.

Increasing evidence suggests that the complex interactions among multiple cell types including neuronal, glial, and vascular cells, are critical for maintaining adequate cerebral blood flow that is necessary for normal brain function and survival. The disturbance of these interactions contributes to the pathogenesis of central nervous system disorders such as stroke and Alzheimer's disease. The retina is part of the central nervous system, and the properties of vasculature in the retina are similar to those in the brain. The interactions among multiple cell types in the retina also play an important role in the maintenance of tissue homeostasis, and the impairment of interactions can contribute to the onset and/or progression of retinal diseases. In this review, we describe the neurovascular interactions in the retina and alternations of interactions in pathological conditions such as diabetic retinopathy and glaucoma.

We examined the effects of the serine protease inhibitor nafamostat mesilate on neuronal and vascular injury in rat retinas treated with N-methyl-D-aspartate (NMDA). The degree of neuronal degeneration was assessed by measuring the number of cells in the ganglion cell layer and the thickness of the inner plexiform layer. The degree of capillary degeneration was assessed by measuring the number of empty basement membrane sleeves that were left as remnants of the vessels. Significant neuronal and capillary degeneration was observed 7 d after a single intravitreal injection of NMDA into the eye. Both forms of degeneration were significantly prevented by simultaneous injection of nafamostat mesilate with NMDA. These results indicate that nafamostat mesilate affords protection against the neuro/vascular injury seen in NMDA-treated retinas. Nafamostat mesilate may be considered as a candidate for neuro/vascular protective interventions in retinal diseases associated with glutamate-induced excitotoxicity, such as glaucoma and diabetic retinopathy.

Purpose: Recently, cell cycle reentry has been reported in the experimental brain ischemia model and Alzheimer's disease brain. Cyclin-dependent kinases (CDKs) are known to be key molecules that regulate the cell cycle. The aims of the present study were to determine the change in expression of the proteins involved in regulation of G1 phase, and to test the effect of small molecule CDK inhibitors on neuronal injury in the ischemic-reperfused rat retina., Methods: Male Sprague-Dawley rats were anesthetized and subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mmHg. One day after this procedure, the retinal tissues were homogenized and immunoblot analyses were done to check the expression levels of cell cycle regulatory proteins. Additional groups of rats received 3 mg/kg roscovitine, a CDK inhibitor, and 1 mg/kg CDK4 inhibitor intravenously (i.v.) 15 min before ischemia and underwent 60 min of ischemia. On the 7th day of the retinal ischemia-reperfusion, eyes were subjected to morphometry., Results: Immunoblot analysis revealed that ischemia-reperfusion significantly increased expression levels of cyclin D1 and CDK4, and decreased those of p16(INK4) and p27(KIP1). Treatment with a CDK4 inhibitor (1 mg/kg, i.v.) 15 min before ischemia significantly reduced death of the retinal ganglion cells., Conclusion: Upregulation of cell cycle regulatory proteins and activation of nonmitotic CDK5 are shown to be associated with neuronal cell death. Although the CDK4 inhibitor used in the present study is known to reduce the activity of mitotic CDK4, there is no information about its blocking effect on other CDKs, such as nonmitotic CDK5. The results in the present study suggest that abnormal progression of the cell cycle and/or activation of nonmitotic CDK are involved in the neuronal cell death induced by retinal ischemia-reperfusion. Furthermore, as shown with a CDK4 inhibitor, agents that alter the activity of CDK may be good candidates for inhibitors of neuronal cell death induced by ischemia-reperfusion injury.

The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate vascular smooth muscle tone but the role of BK(Ca) channels in regulation of retinal circulation remains unclear. In the present study, we examined the effects of BMS-191011 and NS 1619, openers of BK(Ca) channels, on rat retinal blood vessels in vivo. Male Wistar rats (8- to 10-week-old) were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally (i.p.)) and treated with tetrodotoxin (50 µg/kg, intravenously (i.v.)) to eliminate any nerve activity and prevent movement of the eye under artificial ventilation. A mixture solution of adrenaline and noradrenaline (9:1) was infused to maintain adequate systemic circulation. BMS-191011 (10-100 µg/kg, i.v.) and NS 1619 (0.1-1.0 µg/kg, i.v.) increased the diameter of retinal arterioles without altering systemic blood pressure and heart rate significantly. The vasodilator responses to BMS-191011, but not to NS 1619, were significantly diminished by intravitreal injection of iberiotoxin (an inhibitor of BK(Ca) channels, 20 pmol/eye). These results suggest that BMS-191011 dilates rat retinal arterioles through activation of iberiotoxin-sensitive BK(Ca) channels in vivo. The BK(Ca) channel opener could be considered as a candidate for improving retinal circulation without severe cardiovascular side-effects.

We histologically examined the effects of all-trans retinoic acid (ATRA) on neuronal injury induced by intravitreous injection of N-methyl-d-aspartic acid (NMDA) (200nmol/eye). Treatment with ATRA for 7 days (15mg/kg for the first two days and 10mg/kg for the following five days, p.o.) reduced the decrease of cell number in the ganglion cell layer and the inner nuclear layer 7 days after NMDA injection. TUNEL staining 6h after NMDA injection showed that treatment with ATRA (15mg/kg, p.o.) 1h prior to NMDA injection reduced the number of apoptotic cells in the ganglion cell layer and inner nuclear layer. The anti-apoptotic effect of ATRA was vanished by intravitreous injection of U0126, an extracellular signal-regulated kinase/mitogen-activated protein kinase kinase inhibitor (1nmol/eye). These results suggest that ATRA has a protective effect, which is medicated by extracellular signal-regulated kinase pathway, on NMDA-induced apoptosis in the rat retina. ATRA may be useful as a therapeutic drug against retinal diseases that cause glutamate neurotoxicity.

Prostaglandin E(2) (PGE(2)) plays an important role in promoting inflammation and neurological disorders. The actions of PGE(2) are mediated by four different G-protein-coupled receptors (EP(1), EP(2), EP(3), and EP(4)). The purpose of this study was to determine whether stimulation of prostanoid EP(2) receptors has the potential to prevent the excitotoxic injuries in the retina. For this purpose, we examined the effect of 11,15-O-dimethyl prostaglandin E(2) (ONO-AE1-259-01), a selective prostanoid EP(2) receptor agonist, on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in the rat retina. ONO-AE1-259-01 (2 or 20 nmol) together with NMDA (200 nmol) was given intravitreally, and histological evaluation was performed at 1 week after the injection. ONO-AE1-259-01 concentration-dependently prevented NMDA-induced cell loss in ganglion cell layer and reduction in thickness of inner plexiform layer. These results indicate that ONO-AE1-259-01 protects the excitotoxic injuries in the rat retina, and that the prostanoid EP(2) receptor may be a target for neuroprotective intervention in the retinal diseases associated with glutamate-induced excitotoxicity, such as glaucoma and diabetic retinopathy.

We examined the effects of vasodilatory prostaglandins (prostacyclin and prostaglandin E(2)) and selective agonists for prostanoid EP(2) and EP(4) receptor on the diameters of retinal blood vessels and fundus (retinal/choroidal) blood flow in rats. Male Wistar rats (8- to 10-week-old) were treated with tetrodotoxin (50 microg/kg, i.v.) to eliminate any nerve activity and prevent movement of the eye and infused with a mixture solution of norepinephrine and epinephrine (1:9) to maintain adequate systemic circulation under artificial ventilation. Fundus images were captured with a digital camera that was equipped with the special objective lens for small animals, and the diameters of retinal arterioles and venules were measured on a personal computer. Fundus blood flow was estimated using a laser Doppler flowmetry. Intravenous infusions of prostacyclin and prostaglandin E(2) dilated retinal blood vessels, increased fundus blood flow and decreased systemic blood pressure in a dose-dependent manner. The effects of vasodilatory prostaglandins on retinal arterioles were greater than those on retinal venules. Similarly, a prostanoid EP(2) receptor agonist (ONO-AE1-259-01) dilated retinal blood vessels, and increased fundus blood flow and decreased systemic blood pressure. However, a prostanoid EP(4) receptor agonist (ONO-AE1-329) failed to increase fundus blood flow, despite its comparable depressor response with those to vasodilatory prostaglandins and the prostanoid EP(2) receptor agonist. The responses to forskolin, an activator of adenylyl cyclase, were very similar to those to prostacyclin and the prostanoid EP(2) receptor agonist. These results suggest that prostacyclin and prostaglandin E(2) act as vasodilators in retinal and choroidal circulation, and prostanoid IP and EP(2) receptors play an important role in the regulation of ocular hemodynamics in rats.

We established an experimental system for measuring blood flow in the rat fundus and examined whether intravenously administered vasodilatory prostaglandins (PGE(1), PGE(2), and PGI(2)), 8-(4-chlorophenylthio)-cAMP (a cAMP analogue), and nicardipine (a Ca(2+)-channel blocker) increase fundus blood flow (FBF). Under artificial ventilation, rats were injected with tetrodotoxin (50 microg/kg, i.v.) to eliminate any nerve activity and prevent movement of the eye. After tetrodotoxin, the rats were infused with norepinephrine (0.3 - 0.5 microg . kg(-1) . min(-1)) and epinephrine (2.7 - 4.5 microg . kg(-1) . min(-1)) simultaneously to maintain adequate systemic circulation. We found that intravenous infusion of PGE(1) (2 - 10 microg . kg(-1) . min(-1)), PGE(2) (3 - 30 microg . kg(-1) . min(-1)), and PGI(2) (1 - 10 microg . kg(-1) . min(-1)) increased the FBF in a dose-dependent manner. The vasodilatory PGs decreased arterial pressure, whereas they did not affect heart rate. Like vasodilatory PGs, 8-(4-chlorophenylthio)-cAMP (30 micromol/kg, i.v.) increased FBF and decreased arterial pressure. While infusion of nicardipine (0.3 - 3 microg . kg(-1) . min(-1)) produced comparable depressor responses with those to vasodilatory PGs and the cAMP analogue, it did not increase FBF. These results suggest that vasodilatory PGs and cAMP act more selectively than Ca(2+)-channel blockers on retinal/choroidal blood vessels. Therefore, the vasodilatory PGs might be considered to be possible candidates for the therapeutics to treat disorders of retinal/choroidal circulation.

Brief ischemia was reported to protect retinal cells against injury induced by subsequent ischemia-reperfusion with de novo protein synthesis, and this phenomenon is known as late ischemic preconditioning. The aims of the present study were to determine whether nitric oxide synthase (NOS) was involved in the mechanism of late ischemic preconditioning in rat retina using pharmacological tools. Under anesthesia with pentobarbital sodium, male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 24 hrs before 60 min of ischemia. Retinal sections sliced into 5 microm thick were examined 7 days after ischemia. Additional groups of rats received NG-nitro-L-arginine and NG-monomethyl-L-arginin, non-selective NO synthase inhibitors, 7-nitroindazole, a neuronal NOS inhibitor, and aminoguanidine and L-N6-(1-iminoethyl) lysine, inducible NO synthase (iNOS) inhibitors before preconditioning, and were subjected to 60 min of ischemia. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Ischemic preconditioning for 5 min completely protected against the histological damage induced by 60 min of ischemia applied 24 hrs thereafter. Treatment of rats with aminoguanidine and L-N6-(1-iminoethyl) lysine, but not NG-nitro-L-arginine, NG-monomethyl-L-arginine or 7-nitroindazole, wiped off the protective effect of ischemic preconditioning. The inhibitory effect of aminoguanidine was abolished by L-arginine, but not D-arginine. The results in the present study suggest that NO synthesized by iNOS is involved in the histological protection by late ischemic preconditioning in rat retina.

Brief ischemia was reported to protect various cells against injury induced by subsequent ischemia-reperfusion, and this phenomenon is known as ischemic preconditioning. The aims of the present study were to clarify whether early ischemic preconditioning could be observed in the rat retina by histological examination. Male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 5-60 min before 60 min of ischemia. Additional groups of rats received 10 mg/kg 8-phenyltheophiline and 4.5 mg/kg 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A1 receptor antagonists, 5 mg/kg 5-hydroxydecanoate and 1 mg/kg glibenclamide, ATP-sensitive K+ channel blockers, or 2.5 mg/kg chelerythrine and 0.1 mg/kg bisindolylmaleimide I, protein kinase C inhibitors, 15 or 30 min before preconditioning. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Five minutes of preconditioning ischemia 20-40 min before 60 min of sustained ischemia completely prevented the retinal tissue damage induced by the sustained ischemia. Treatment with 8-phenyltheophylline, DPCPX, 5-hydroxydecanoate, glibenclamide, chelerythrine and bisindolylmaleimide I almost completely reduced the protective effect of early ischemic preconditioning. The results in the present study indicated that early ischemic preconditioning was demonstrated in the rat retina. Stimulation of adenosine receptors, opening of ATP-sensitive K+ channels and activation of protein kinase C might be involved in the underlying protective mechanisms.

Metformin, a biguanide antihyperglycemic drug, can exert various beneficial effects in addition to its glucose-lowering effect. The effects of metformin are mainly mediated by AMP-activated protein kinase (AMPK)-dependent pathway. AMPK activation interferes with the action of the mammalian target of rapamycin complex 1 (mTORC1), and blockade of mTORC1 pathway suppresses pathological retinal angiogenesis. Therefore, in this study, we examined the effects of metformin on pathological angiogenesis and mTORC1 activity in the retinas of mice with oxygen-induced retinopathy (OIR). OIR was induced by exposing the mice to 80% oxygen from postnatal day (P) 7 to P10. The OIR mice were treated with metformin, rapamycin (an inhibitor of mTORC1), or the vehicle from P10 to P12 or P14. The formation of neovascular tufts, revascularization in the central avascular areas, expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) 2, and phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, were evaluated at P10, P13, or P15. Neovascular tufts and vascular growth in the central avascular areas were observed in the retinas of P15 OIR mice. The formation of neovascular tufts, but not the revascularization in the central avascular areas, was attenuated by metformin administration from P10 to P14. Metformin had no significant inhibitory effect on the expression of VEGF and VEGFR2, but it reduced the pS6 immunoreactivity in vascular cells at the sites of angiogenesis. Rapamycin completely blocked the phosphorylation of ribosomal protein S6 and markedly reduced the formation of neovascular tufts. These results suggest that metformin partially suppresses the formation of neovascular tufts on the retinal surface by blocking the mTORC1 signaling pathway. Metformin may exert beneficial effects against the progression of ocular diseases in which abnormal angiogenesis is associated with the pathogenesis. [ABSTRACT FROM AUTHOR]

Glaucoma is one of the leading causes of acquired blindness and characterized by retinal ganglion cell (RGC) death. MicroRNAs are small noncoding RNAs that degrade their target mRNAs. Apoptosis is one of the common mechanisms leading to neuronal death in many neurodegenerative diseases, including glaucoma. In the present study, we identified microRNAs that modulate RGC death caused by the intravitreal injection of N-methyl-d-aspartic acid (NMDA). We found an upregulation of miR-29b and downregulation of miR-124 in the retina of the NMDA-injected eyes. The intravitreal injection of an miR-29b inhibitor 18 h before NMDA injection reduced RGC death and the downregulation of myeloid cell leukemia 1 (MCL-1), an anti-apoptotic factor, induced by intravitreal NMDA. The intravitreal injection of an miR-124 mimic 18 h before NMDA injection also reduced RGC death and the upregulation of B-cell/chronic lymphocytic leukemia lymphoma 2 (bcl-2)-associated X protein (Bax) and bcl-2 interacting protein (Bim), pro-apoptotic factors, induced by intravitreal NMDA. These data suggest that expressional changes in microRNA are involved in the excitotoxicity of RGCs, and that complement and/or inhibition of microRNA may be a potential therapeutic approach for the diseases related to the excitotoxicity of RGCs, such as glaucoma and retinal central artery occlusion. [ABSTRACT FROM AUTHOR]

We previously identified that NO derived from neuronal cells acts on glial cells and causes vasodilation in the healthy rat retina via the release of epoxyeicosatrienoic acids (EETs) and prostaglandins (PGs) by activation of the arachidonic acid cascade. However, it is not clear which PG types are involved in these responses. The aim of the present study was to identify prostanoid receptors involved in glial cell-derived vasodilation induced by NO in rat retina. Male Wistar rats were used to examine the effects of intravitreal pretreatment with indomethacin, a cyclooxygenase inhibitor; PF-04418948, a prostanoid EP2 receptor antagonist; and CAY10441, a prostanoid IP receptor antagonist, on the changes in the retinal arteriolar diameter induced by intravitreal administration of NOR3, an NO donor. Retinal arteriolar diameters were measured using ocular fundus images captured with a high-resolution digital camera in vivo. The increase in the retinal arteriolar diameter induced by intravitreal injection of NOR3 was significantly suppressed by intravitreal pretreatment with indomethacin and PF-04418948, but not by CAY10441. The dose of PF-04418948 and CAY10441 injected intravitreally in the present study significantly reduced the increase in the retinal arteriolar diameter induced by prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2), respectively. These results suggest that activation of the arachidonic acid cascade and subsequent stimulation of prostanoid EP2 receptors are involved in rat retinal vasodilatory responses evoked by NO-induced glial cell stimulation. Therefore, glial cell-derived PGE2, similar to EETs, may play an important role in retinal vasodilatory mechanisms. [ABSTRACT FROM AUTHOR]

Circulating catecholamines contribute to the regulation of retinal vascular tone. Our previous studies have demonstrated that the activation of large-conductance Ca2+-activated K+ (BKCa) channels is involved in the β2-adrenoceptor-mediated dilation of retinal arterioles in rats. The present study aimed to examine the role of Gi protein in the β2-adrenoceptor-mediated activation of BKCa channels in the retinal arterioles. Images of in vivo rat ocular fundi were captured, and the diameters of retinal arterioles were measured. Systemic blood pressure and heart rate were recorded continuously. Intravenous infusion of formoterol (0.01–0.3 μg/kg/min), a β2-adrenoceptor agonist, increased the diameter of retinal arterioles but decreased mean arterial pressure in a dose-dependent manner. Intravitreal injection of iberiotoxin (20 pmol/eye), an inhibitor of BKCa channels, significantly attenuated the formoterol-induced dilation of retinal arterioles. Similar results were obtained when salbutamol (0.03–3 μg/kg/min), another β2-adrenoceptor agonist, was used instead of formoterol. However, iberiotoxin had no significant effect on retinal vasodilator responses to intravenous infusion of denopamine (1–30 μg/kg/min; a β1-adrenoceptor agonist), CL316243 (0.3–10 μg/kg/min; a β3-adrenoceptor agonist), prostaglandin I2 (0.03–10 μg/kg/min; a prostanoid IP receptor agonist), and forskolin (1–10 μg/kg/min; an adenylyl cyclase activator). Intravitreal injection of pertussis toxin (66 ng/eye; a Gi protein inhibitor) significantly attenuated the dilation of retinal arterioles induced by formoterol but not by denopamine and CL316243. In the presence of pertussis toxin, iberiotoxin had no inhibitory effect on formoterol-induced dilation of retinal arterioles. These results suggest that stimulation of β2-adrenoceptors dilates retinal arterioles through pertussis toxin–sensitive Gi protein–dependent activation of BKCa channels in rats in vivo. [ABSTRACT FROM AUTHOR]

Abstract We previously reported that P2X7 receptor antagonists prevented the retinal injury caused by N-methyl- d -aspartic acid. It has been reported that activation of P2X7 receptor is involved in the secretion of proinflammatory cytokines by macrophages, monocytes, and microglia. Although retinal inflammation is known to cause photoreceptor cell death, it is unclear whether a noncompetitive antagonist of P2X7 receptor can protect photoreceptor cells against inflammation-induced injury. We examined whether Brilliant Blue G (BBG), a potent non-competitive antagonist of P2X7 receptor, had neuroprotective effects on photoreceptor cell injury in a murine endotoxin-induced uveitis (EIU) model. EIU was evoked by lipopolysaccharides (LPS; 10 mg/kg/day) administered intraperitoneally once a day for 4 days. BBG (50 mg/kg/day) and indomethacin (10 mg/kg) were also injected intraperitoneally just before LPS injection. BBG significantly prevented photoreceptor cell loss and reduction of the amplitudes of dark-adapted and light-adapted flush electroretinograms induced by LPS, whereas indomethacin did not show such protective effects. These results indicated that BBG is protective against photoreceptor cell injury in EIU in the mice in vivo , suggesting that P2X7 receptor antagonists may be good candidates for preventing photoreceptor degeneration induced by inflammation. Highlights • Activation of P2X7 receptor causes the injury of the outer retina in a murine endotoxin-induced uveitis (EIU) model. • Brilliant blue G, a P2X7 receptor antagonist, had neuroprotective effects on photoreceptor cell injury in a murine EIU model. • Blockade of P2X7 receptor is a good candidate for protection against outer retinal degeneration induced by inflammation. • BBG may be superior to other dyes in inner limiting membrane staining for macular surgery. [ABSTRACT FROM AUTHOR]

Excitoneurotoxicity is regarded as one of the mechanisms of the death of retinal ganglion cells induced by retinal central artery occlusion and glaucoma. Oxidative stress is at least in part involved in excitoneurotoxicity. Fenton reaction, which is catalyzed by Fe 2+ , is known to cause formation of hydroxyl radical, one of reactive oxygen species, suggesting that chelation of iron may be protective against excitoneurotoxicity. In the present study, we histologically evaluated whether zinc-deferoxamine (Zn-DFO) and deferasirox (DFX), common iron-chelating agents, were protective against N-methyl-D-aspartate (NMDA)-induced retinal injury in the rat in vivo . Male Sprague–Dawley rats were subjected to intravitreal NMDA injection (200 nmol/eye). Zn-DFO (1, 3, 10, and 30 mg/kg), Zn (0.1, 0.2 and 0.6 mg/kg) and DFX (20 mg/kg) were intraperitoneally administered. Morphometric evaluations using paraffin-embedded retinal sections, and detection of Fe 2+ using SiRhoNox-1, a fluorescent probe of labile Fe 2+ in the retinal frozen sections were carried out. Intravitreal NMDA resulted in strong positive signals of SiRhoNox-1 in the ganglion cell layer 24 h after NMDA injection, suggesting that intravitreal NMDA caused Fe 2+ accumulation in the retinal ganglion cells. Intravitreal NMDA induced retinal ganglion cell loss 7 days after NMDA injection. Zn-DFO (1, 3, 10, and 30 mg/kg), ZnCl 2 (0.2 mg/kg, a corresponding dose of 1 mg/kg Zn-DFO) and DFX (20 mg/kg) prevented the damage of retinal ganglion cells, whereas 0.6 mg/kg ZnCl 2 , which is a corresponding dose of 3 mg/kg Zn-DFO, did not show any protective effects. Zn-DFO (30 mg/kg) significantly decreased the intensity of the fluorescence of SiRhoNox-1 and the transferrin immunofluorescence 24 h after NMDA injection, the number of TUNEL-positive cells 24 h after NMDA injection, that of 8-OHdG-positive cells, and that of 4-hydroxy-2-nonenal-positive cells 12 and 24 h after NMDA injection. These data suggest that iron-chelating agents protected retinal neurons against excitoneurotoxicity via reduction of iron content and oxidative stress in the rats in vivo . We proposed that treatment with iron-chelating agents would be a new strategy for the retinal diseases caused by excitoneurotoxicity. [ABSTRACT FROM AUTHOR]

Purpose: Our recent study demonstrated that herkinorin, a non-opioid μ-receptor agonist derived from salvinorin A, dilates retinal arterioles through stimulation of μ-opioid receptors in rats. Activation of neuronal nitric oxide (NO) synthase and the presence of ganglion cells in the retina appear to be crucial for inducing μ-opioid receptor-mediated retinal vasodilation. In the present study, we examined the role of the interaction between neurons and glia in the retinal vasodilator mechanism involving μ-opioid receptors in rats. Materials and methods: The localization of μ-opioid receptors and neuronal NO synthase (nNOS) in the rat retina was examined using immunohistochemistry. The retinal vascular responses were evaluated by measuring the diameter of retinal arterioles inin vivofundus images. Both systemic blood pressure and heart rate were continuously recorded. Results: Immunoreactivity of μ-opioid receptors was found in ganglion cells and astrocytes, while that of nNOS was detected in ganglion cells and amacrine cells. Herkinorin increased retinal arteriolar diameter without significantly changing mean blood pressure and heart rate. The retinal vasodilator response to herkinorin was significantly attenuated by treatment with glial toxins (fluorocitrate and disialoganglioside-GD1b). The glial toxins markedly prevented vasodilation induced by intravitreal injection, but not by intravenous infusion, of NOR3, an NO donor. Conclusion: These results suggest that retinal glial cells play an important role in the μ-opioid receptor-mediated retinal vasodilation in rats. Stimulation of μ-opioid receptors on retinal ganglion cells may affect the activity of glial cells, thereby changing retinal vascular tone. [ABSTRACT FROM PUBLISHER]

The interruption of vascular development could cause structural and functional abnormalities in tissues. We have previously reported that short-term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors induces abnormal retinal vascular growth and patterns. An exposure of neonatal mice to high-concentration oxygen disturbs normal retinal vascular development. The present study aimed to determine (1) whether vascular abnormalities are observed in the retina of newborn mice exposed to high concentrations of oxygen, and (2) how astrocyte network formation is affected following the exposure to hyperoxia. Newborn (postnatal day 0) mice were exposed to 75% oxygen for 48 or 96 hr. During hyperoxia exposure, VEGF expression decreased, and the onset of retinal vascularization was completely suppressed. After completion of the hyperoxic period, retinal vascularization occurred, but it was delayed in a hyperoxic exposure duration-dependent manner. In retinas of hyperoxia-exposed mice, dense capillary plexuses were found, and the number of arteries and veins decreased. The astrocyte network formation was slightly delayed under hyperoxic conditions, and the network became denser in retinas of mice with an episode of hyperoxia. Expression of VEGF levels in the avascular retina of mice that were exposed to hyperoxia was higher than that of control mice. These results suggest that short-term interruption of the onset of vascular development resulting from the reduction in VEGF signals induces abnormal vascular patterns in the mouse retina. The abnormalities in retinal astrocyte behavior might contribute to the formation of an abnormal retinal vascular growth. [ABSTRACT FROM AUTHOR]

Retinal ganglion cell death in glaucoma is caused at least in part by a large Ca 2+ influx through N-methyl-D-aspartic acid (NMDA) receptors. Apelin is a peptide originally found in the tissue extracts of bovine stomach. Recent studies have been shown that apelin protects against the ischemic-reperfused injury in the brain. We examined whether apelin had protective effects on the NMDA-induced retinal ganglion cell (RGC) death using B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice, which express the enhanced cyan fluorescent protein in RGCs in the retina, in vivo . The mice were anesthetized by ketamine and xylazine, and NMDA (40 nmol/eye) was intravitreally injected. We evaluated the effects of apelin-13, [Glp 1 ]-apelin-13, a potent agonist of apelin receptor, and apelin-36 on the NMDA-induced retinal ganglion cell death. NMDA-induced retinal ganglion cell loss was clearly seen 7 days after NMDA injection. Intravitreal apelin-36 (0.33 nmol/eye), but not apelin-13 (1 nmol/eye) nor [Glp 1 ]-apelin-13 (1 nmol/eye), simultaneously injected with NMDA significantly reduced the cell loss. The protective effect of apelin-36 was not reduced by ML221 (0.1 nmol/eye; 5-[(4-Nitrobenzoyl)oxy]-2-[(2-pyrimidinylthio)methyl]-4 H -pyran-4-one), an apelin receptor antagonist, GF109203X (0.03 nmol/eye), a protein kinase C inhibitor, U0126 (0.2 nmol/eye), a MAPK/ERK kinase inhibitor, LY294002 (0.1 nmol/eye), a phosphoinositide 3-kinase inhibitor, Akti 1/2 (0.05 nmol/eye), an Akt inhibitor, or 4,5,6,7-tetrabromobenzotriazole (0.2 nmol/eye), a casein kinase-2 inhibitor. In addition, human apelin-36 did not affect the kainic-acid (20 nmol/eye)-induced ganglion cell death. The present study suggests that apelin-36 protects against the NMDA-induced ganglion cell death independently of the activation of apelin receptor in the murine retina in vivo . [ABSTRACT FROM AUTHOR]

Activation of N-methyl- d -aspartic acid (NMDA) receptors followed by a large Ca 2+ influx is thought to be a mechanism of glaucoma-induced neuronal cell death. It is possible that damage-associated molecular patterns leak from injured cells, such as adenosine triphosphate, causing retinal ganglion cell death in glaucoma. In the present study, we histologically investigated whether antagonists of the P2X7 receptor protected against NMDA-induced retinal injury in the rat in vivo . Under ketamine/xylazine anesthesia, male Sprague–Dawley rats were subjected to intravitreal injection of NMDA. We used A438079 (3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine) and brilliant blue G as P2X7 receptor antagonists. Upon morphometric evaluation 7 days after an intravitreal injection (200 nmol/eye), NMDA-induced cell loss was apparent in the ganglion cell layer. Intravitreal A438079 (50 pmol/eye) simultaneously injected with NMDA and intraperitoneal brilliant blue G (50 mg/kg) administered just before the NMDA injection as well as 24 and 48 h after significantly reduced cell loss. In addition, A438079 decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells 12 h after NMDA injection. P2X7 receptors were immunolocalized in the ganglion cell layer and the inner and outer plexiform layers, whereas the immunopositive P2X7 receptor signal was not detected on the Iba1-positive microglial cells that infiltrated the retina 12 h after NMDA injection. The present study shows that stimulation of the P2X7 receptor is involved in NMDA-induced histological damage in the rat retina in vivo . P2X7 receptor antagonists may be effective in preventing retinal diseases caused by glutamate excitotoxicity, such as glaucoma and retinal artery occlusion. [ABSTRACT FROM AUTHOR]

Purpose: Previous studies have shown that injury to the retinal vasculature, including capillary degeneration, occurs following N-methyl- d-aspartate (NMDA)-induced neuronal cell loss, but it is unclear whether there are age-dependent differences in the severity of vascular damage. The purpose of the present study was to determine age-related changes in retinal capillary degeneration in NMDA-induced retinal damage rat model. Materials and Methods: A single intravitreal injection of NMDA (200 nmol/eye) was performed in 1-, 2-, 3-, 7- and 15-week-old male Sprague-Dawley rats. Seven days after NMDA injection, damage to retinal neurons and blood vessels were assessed by measuring the number of cells in the ganglion cell layer and the length of empty basement membrane sleeves, left behind by regressing endothelial cells, respectively. Results: At all ages examined, the NMDA-induced neurotoxicity exhibited similar levels of ganglion cell death. However, the degeneration of capillaries in NMDA-treated retina was severe during the early stages of retinal vascular development, and the degree of capillary degeneration decreased with age. Conclusion: The degree of retinal capillary degeneration in NMDA-induced retinal damage rat model decreases in an age-dependent manner. Retinal capillaries may be resistant to retinal neuronal damage in young adults. [ABSTRACT FROM AUTHOR]

We previously reported that treatment of mid-pregnant mice with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, caused fetal growth restriction resulting from diminished vascularization in the placenta and fetal organs. In this study, we examined how the treatment of mid-pregnant mice with KRN633 affects the development and morphology of vascular components (endothelial cells, pericytes, and basement membrane) in the retinas of their newborn pups. Pregnant mice were treated with KRN633 (5 mg/kg) once daily from embryonic day 13.5 until the day of delivery. Vascular components were examined using immunohistochemistry with specific markers for each component. Radial vascular growth in the retina was slightly delayed until postnatal day 4 (P4) in the newborn pups of KRN633-treated mothers. On P8, compared with the pups of control mothers, the pups of KRN633-treated mothers exhibited decreased numbers of central arteries and veins and abnormal branching of the central arteries. No apparent differences in pericytes or basement membrane were observed between the pups of control and KRN633-treated mothers. These results suggest that a critical period for determining retinal vascular patterning is present at the earliest stages of retinal vascular development, and that the impaired vascular endothelial growth factor signaling during this period induces abnormal architecture in the retinal vascular network [ABSTRACT FROM AUTHOR]

Abstract: Capsaicin, a transient receptor potential vanilloid type1 (TRPV1) agonist, has been reported to protect against ischemia-reperfusion injury in various organs, including the brain, heart, and kidney, whereas activation of TRPV1 was also reported to contribute to neurodegeneration, including pressure-induced retinal ganglion cell death in vitro. We histologically investigated the effects of capsaicin and SA13353, TRPV1 agonists, on retinal injury induced by intravitreal N-methyl-d-aspartic acid (NMDA; 200nmol/eye) in rats in vivo. Under ketamine/xylazine anesthesia, male Sprague–Dawley rats were subjected to intravitreal NMDA injection. Capsaicin (5.0nmol/eye) was intravitreally admianeously with NMDA injection. SA13353 (10mg/kg) was intraperitoneally administered 15min before NMDA injection. Morphometric evaluation at 7 days after NMDA injection showed that intravitreal NMDA injection resulted in ganglion cell loss. Capsaicin and SA13353 almost completely prevented this damage. Treatment with capsazepine (TRPV1 antagonist, 0.5nmol/eye), CGRP (8–37) (calcitonin gene-related peptide (CGRP) receptor antagonist, 0.5pmol/eye), or RP67580 (tachykinin NK1 receptor antagonist, 0.5nmol/eye) almost completely negated the protective effect of capsaicin in the NMDA-injected rats. Seven days after intravitreal NMDA injection, the cell number of retinal ganglion cell was significantly smaller than in the eye that had received capsaicin in B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice that express the enhanced cyan fluorescent protein in retinal ganglion cells in the retina. These results suggested that activation of TRPV1 protects retinal neurons from the injury induced by intravitreal NMDA in rats in vivo. Activation of CGRP and tachykinin NK1 receptors is possibly involved in underlying protective mechanisms. [Copyright &y& Elsevier]

Abstract: Although hydrogen sulfide (H2S) is generally thought to be a toxic gas, it has been reported to protect various tissues against ischemia-reperfusion injury. In the present study, we histologically investigated whether H2S, using sodium hydrosulfide (NaHS) as its donor, had a protective effect on N-methyl-d-aspartate (NMDA)-induced retinal injury in the rat in vivo. Under ketamine/xylazine anesthesia, male Sprague–Dawley rats were subjected to intravitreal NMDA injection. NaHS (0.163–120 μmol/kg) was intraperitoneally administered 15 min before NMDA injection. Morphometric evaluation at 7 days after NMDA injection showed that intravitreal NMDA injection resulted in ganglion cell loss. NaHS dose-dependently prevented this damage. NaHS (120 μmol/kg) significantly decreased the numbers of TUNEL-positive, 4-hydroxy-2-nonenal-positive, and 8-OHdG-positive cells 12 h after NMDA injection. In another experimental series, we demonstrated that NaHS (120 μmol/kg) significantly reduced the retinal injury induced by intravitreal NOC12 (400 nmol/eye), which was a nitric oxide donor and reported to induce oxidative stress, in the retina, 7 days after intravitreal injection. These results suggested that H2S protects retinal neurons against the injury induced by intravitreal NMDA in rats in vivo. Anti-oxidative activity of H2S are possibly involved in underlying protective mechanisms. [Copyright &y& Elsevier]

Purpose: The aim of this study was to examine the vasodilator effect of fasudil, a Rho-kinase inhibitor, on retinal arterioles in stroke-prone spontaneously hypertensive rats (SHRSPs) and in age-matched normotensive Wistar-Kyoto rats (WKYs).Methods: Rats (12-14 weeks-old) were anesthetized with thiobutabarbital (120 mg/kg, intraperitoneal). Fundus images were captured with a digital camera that was equipped with a special objective lens. The vasodilator responses of retinal arterioles were assessed by measuring changes in the diameters of the vessels.Results: The baseline diameter of the retinal arteriole was significantly smaller in SHRSPs than in WKYs. Fasudil (50-200 microg/kg/min, intravenous) dose-dependently increased the diameter of the retinal arteriole and decreased the systemic blood pressure in both groups. The vasodilator effect of fasudil on the retinal arteriole in SHRSPs was greater than in WKYs.Conclusions: These results suggest that fasudil has beneficial effects on retinal vascular complications associated with chronic hypertension. [ABSTRACT FROM AUTHOR]

Metformin, an anti-hyperglycemic drug of the biguanide class, exerts positive effects in several non-diabetes-related diseases. In this study, we aimed to examine the protective effects of metformin against N-methyl-D-aspartic acid (NMDA)-induced excitotoxic retinal damage in rats and determine the mechanisms of its protective effects. Male Sprague–Dawley rats (7 to 9 weeks old) were used in this study. Following intravitreal injection of NMDA (200 nmol/eye), the number of neuronal cells in the ganglion cell layer and parvalbumin-positive amacrine cells decreased, whereas the number of CD45-positive leukocytes and Iba1-positive microglia increased. Metformin attenuated these NMDA-induced responses. The neuroprotective effect of metformin was abolished by compound C, an inhibitor of AMP-activated protein kinase (AMPK). The AMPK activator, AICAR, exerted a neuroprotective effect in NMDA-induced retinal injury. The MEK1/2 inhibitor, U0126, reduced the neuroprotective effect of metformin. These results suggest that metformin protects against NMDA-induced retinal neurotoxicity through activation of the AMPK and MEK/extracellular signal-regulated kinase (ERK) signaling pathways. This neuroprotective effect could be partially attributable to the inhibitory effects on inflammatory responses. [ABSTRACT FROM AUTHOR]

The interactions between neuronal, glial, and vascular cells play a key role in regulating blood flow in the retina. In the present study, we examined the role of the interactions between neuronal and glial cells in regulating the retinal vascular tone in rats upon stimulation of retinal neuronal cells by intravitreal injection of N-methyl-d-aspartic acid (NMDA). The retinal vascular response was assessed by measuring the diameter of the retinal arterioles in the in vivo fundus images. Intravitreal injection of NMDA produced retinal vasodilation that was significantly diminished following the pharmacological inhibition of nitric oxide (NO) synthase (nNOS), loss of inner retinal neurons, or intravitreal injection of glial toxins. Immunohistochemistry revealed the expression of nNOS in ganglion and calretinin-positive amacrine cells. Moreover, glial toxins significantly prevented the retinal vasodilator response induced by intravitreal injection of NOR3, an NO donor. Mechanistic analysis revealed that NO enhanced the production of vasodilatory prostanoids and epoxyeicosatrienoic acids in glial cells in a ryanodine receptor type 1-dependent manner, subsequently inducing the retinal vasodilator response. These results suggest that the NO released from stimulated neuronal cells acts as a key messenger in neuron–glia signaling, thereby causing neuronal activity-dependent and glial cell-mediated vasodilation in the retina. [ABSTRACT FROM AUTHOR]

Opioids contribute to the regulation of cerebral vascular tone. The purpose of this study was to examine the effects of herkinorin, a non-opioid μ-opioid receptor agonist derived from salvinorin A, on blood vessels in the rat retina and to investigate the mechanism underlying the herkinorin-induced retinal vasodilatory response. Ocular fundus images were captured using an original high-resolution digital fundus camera in vivo . The retinal vascular responses were evaluated by measuring the diameter of retinal arterioles in the fundus images. Both systemic blood pressure and heart rate were continuously recorded. Herkinorin increased the retinal arteriolar diameter without significantly changing mean blood pressure and heart rate. The retinal vasodilator response to herkinorin was almost completely prevented following treatment with naloxone, a nonselective opioid receptor antagonist and H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH 2 (CTOP), a selective μ-opioid receptor antagonist. N ω -nitro-L-arginine methyl ester, a nonselective nitric oxide (NO) synthase inhibitor, or indomethacin, a cyclooxygenase inhibitor, significantly attenuated the herkinorin-induced retinal vasodilator responses. In addition, N ω -propyl-L-arginine, an inhibitor of neuronal NO synthase, diminished the herkinorin-induced retinal vasodilator responses. Seven days after an intravitreal injection of N -methyl-D-aspartic acid, loss of inner retinal neurons and abolishment of the retinal vasodilator response to herkinorin were observed. These results suggest that herkinorin dilates rat retinal arterioles through stimulation of retinal μ-opioid receptors. The μ-opioid receptor-mediated retinal vasodilator response is likely mediated by NO generated by neuronal NO synthase. Retinal neurons play an important role in the retinal vasodilator mechanism involving μ-opioid receptors in rats. [ABSTRACT FROM AUTHOR]

Interactions between neuronal cells and vascular cells in the retina are critical for maintaining retinal tissue homeostasis. Impairment of cellular interactions contributes to development and progression of retinal diseases. Previous studies demonstrated that neuronal cell damage leads to capillary degeneration in an N -methyl-D-aspartic acid (NMDA)-induced retinal degeneration model. However, the mechanisms underlying this phenomenon are not fully understood. In this study, we examined the possible role of matrix metalloproteinase (MMP)-9 in neuronal cell loss and capillary degeneration in NMDA-treated retinas of neonatal rats. Intravitreal injection of NMDA (50 or 200 nmol) was performed on postnatal day (P) 7 and morphological changes in retinal neurons and vasculature were examined on P14. The MMP inhibitor CP101537 (100 nmol) or vehicle (dimethyl sulfoxide) was intravitreally injected simultaneously with, or 2 days after, NMDA injection. CP101537 protected against neurovascular degeneration in a time-dependent manner as follows: 1) simultaneous injection of CP101537 with NMDA prevented morphological changes in retinal neurons induced by NMDA (50 nmol); and 2) reduction in capillary density and number of vertical sprouts induced by NMDA (200 nmol) was prevented when CP101537 was injected 2 days after NMDA injection. Gelatin zymography and western blot analyses indicated that activity and protein levels of MMP-9 were enhanced from 4 h to 2 days after NMDA injection. Increased activity and protein levels of MMP-9 were suppressed by MMP inhibitors (CP101537 and GM6001). In situ zymography revealed that MMP activity was enhanced throughout the retinal vasculature in NMDA-treated retinas. These results indicate that MMP-9 plays an important role in neurovascular degeneration in the injured retina. Inhibition of MMP-9 may be an effective strategy for preventing and reducing neurovascular degeneration. • Excessive dose of NMDA induces retinal neurovascular degeneration. • Neuronal cell loss precedes capillary degeneration following NMDA treatment. • MMP inhibitors protect neuronal and vascular cells in NMDA-treated retinas. • MMP-9 plays an important role in neurovascular degeneration in injured retinas. • Inhibition of MMP-9 may be an effective strategy for neurovascular degeneration. [ABSTRACT FROM AUTHOR]

• Intravenous injection of resveratrol induces dilation of retinal arterioles. • Intravitreal injection of resveratrol protects against NMDA-induced retinal injury. • These effects of resveratrol are mediated via the AMPK-dependent pathway. Resveratrol, a natural polyphenolic compound, reportedly possesses numerous biological activities, including anti-inflammatory and antioxidant effects. In the current study, we examined (1) the dilator effects of resveratrol on retinal arterioles, (2) the protective effects of resveratrol against excitotoxic retinal injury, and (3) whether these effects are mediated by the AMP-activated kinase (AMPK)-dependent pathway in rats. Male Wistar rats (7 to 10 weeks old) were used in this study. The diameters of the retinal arterioles, mean arterial pressure, and heart rate were measured in vivo. The retinal injury was assessed by histological examination. Intravenous injection of resveratrol (3 mg/kg) increased the diameter of the retinal arterioles without affecting the mean arterial pressure and heart rate. The AMPK inhibitor, compound C (5 mg/kg, intravenously), significantly attenuated the retinal vasodilator response to resveratrol. Seven days after intravitreal injection of N -methyl- d -aspartic acid (NMDA; 25, 50, and 100 nmol/eye), the number of cells located in the ganglion cell layer (GCL) was reduced, along with thinning of the inner plexiform layer. Intravitreal resveratrol injection (100 nmol/eye) reduced the NMDA (25 and 50 nmol/eye)-induced cell loss in the GCL. The neuroprotective effect of resveratrol was significantly but not completely reversed by compound C (10 nmol/eye). These results suggest that resveratrol dilates retinal arterioles and protects against NMDA-induced retinal neurodegeneration via an AMPK-dependent pathway in rats. Resveratrol may have the potential to slow the onset and progression of diseases associated with retinal ischemia by improving impaired retinal circulation and protecting retinal neuronal cells. [ABSTRACT FROM AUTHOR]

Na+/K+-ATPase (NKA) plays an important role in ion homeostasis and neurotransmitter uptake. In the retina, multidirectional communications among neurons, glia, and blood vessels (that is, neuro-glio-vascular interaction) are crucial for maintaining tissue homeostasis. We investigated the role of NKA in the elements of neuro-glio-vascular unit in neonatal and adult rat retinas. Male Sprague-Dawley rats (1- and 8-week-old) were injected intravitreally with ouabain (20 nmol/eye), an inhibitor of NKA. Morphological changes in retinal neurons, glia, and blood vessels were examined. The intravitreal injection of ouabain decreased the number of cells in the ganglion cell layer, as well as the thicknesses of the inner plexiform and inner nuclear layers in neonatal and adult rats compared to age-matched controls. The ouabain-induced neuronal cell damage was partially prevented by D-(−)-2-amino-5-phosphonopentanoic acid, an antagonist of N -methyl-D-aspartic acid receptors. In the deep retinal vascular plexus of the ouabain-injected eyes, angiogenesis was delayed in neonatal rats, whereas capillary degeneration occurred in adult rats. The immunoreactivity of glutamine synthetase and vascular endothelial growth factor (VEGF) decreased in the retinas of neonatal and adult rats injected intravitreally with ouabain. The immunoreactivity of glial fibrillary acidic protein was enhanced in the retinas of ouabain-injected adult eyes. After the ouabain injection, CD45-positive leukocytes and Iba1-positive microglia increased in the inner retinal layer of neonatal rats, whereas they increased in the middle retinal layer of adult rats. These results suggest that the inhibition of NKA induces the degeneration of neuronal and vascular cells and alteration of glial cells in both neonatal and adult retinas. In addition to the direct effects of NKA inhibition, the disturbance of retinal glutamate metabolism and decreased VEGF expression may contribute to neurovascular degeneration. The activity of NKA is crucial for maintaining elements of neuro-glio-vascular unit in the retina. • Ouabain induces morphological changes in retinal neurons, glia, and blood vessels. • Ouabain-induced retinal changes are altered in an age-dependent manner. • The role of Na+/K+-ATPase in the retinal homeostasis changes during development. • Na+/K+-ATPase is crucial for maintaining elements of neuro-glio-vascular unit. [ABSTRACT FROM AUTHOR]