The Role of microRNAs Related to Apoptosis for N-Methyl-d-Aspartic Acid-Induced Neuronal Cell Death in the Murine Retina.

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.

The process of methylglyoxal-induced retinal capillary endothelial cell degeneration in rats.

Methylglyoxal, a highly reactive dicarbonyl compound, is increased and accumulated in patients with diabetic mellitus. Methylglyoxal forms advanced glycation end products (AGE), contributing to the pathogenesis of diabetic complications, including diabetic retinopathy. Recent studies have shown that methylglyoxal induces diabetic retinopathy-like abnormalities in retinal vasculature. In this study, we investigated the processes and mechanisms of methylglyoxal-induced retinal capillary endothelial cell degeneration in rats. Morphological changes in vascular components (endothelial cells, pericytes, and basement membranes) were assessed in the retinas 2, 7, and 14 days after intravitreal injection of methylglyoxal. Intravitreal methylglyoxal injection induced retinal capillary endothelial cell degeneration in a dose- and time-dependent manner. Changes in the shape and distribution of pericytes occurred before the initiation of capillary regression in the retinas of methylglyoxal-injected eyes. The receptor for AGEs (RAGEs) antagonist FPS-ZM1, and the matrix metalloproteinase (MMP) inhibitor GM6001 significantly attenuated methylglyoxal-induced capillary endothelial cell degeneration. FPS-ZM1 failed to prevent pathological changes in pericytes in methylglyoxal-injected eyes. In situ zymography revealed that MMP activity was enhanced at sites of blood vessels with reduced pericyte coverage in methylglyoxal-injected eyes. These results suggest that intravitreal methylglyoxal injection induces pathological changes in pericytes before the initiation of capillary endothelial cell degeneration via an AGE-RAGE-independent pathway. The capillary endothelial cell degeneration is mediated by activating the AGE-RAGE pathway and increasing MMP activity in endothelial cells by impairing pericyte function in the retina.

Pharmacological depletion of retinal neurons prevents vertical angiogenic sprouting without affecting the superficial vascular plexus.

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.

Impairment of endothelium-dependent vasodilator function of retinal blood vessels in adult rats with a history of retinopathy of prematurity.

Retinopathy of prematurity (ROP) is a proliferative retinal vascular disease, initiated by delayed retinal vascular growth after premature birth. In the majority of cases, ROP resolves spontaneously; however, a history of ROP may increase the risk of long-term visual problems. In this study, we evaluated the endothelial function of retinal blood vessels in adult rats with a history of ROP. ROP was induced in rats by subcutaneous injection of a vascular endothelial growth factor receptor tyrosine kinase inhibitor (KRN633) on postnatal day (P) 7 and P8. On P56, vasodilator responses to acetylcholine, GSK1016790A (an activator of transient receptor potential vanilloid 4 channels), NOR3 (a nitric oxide [NO] donor), and salbutamol (a ß2-adrenoceptor agonist) were assessed. Compared to age-matched controls, retinal vasodilator responses to acetylcholine and GSK1016790A were attenuated in P56 rats with a history of ROP. No attenuation of acetylcholine-induced retinal vasodilator response was observed under inhibition of NO synthase. Retinal vasodilator responses to NOR3 and salbutamol were unaffected. These results suggest that the production of and/or release of NO is impaired in retinal blood vessels in adult rats with a history of ROP. A history of ROP might increase the risk of impaired retinal circulation in adulthood.

Role of Epoxyeicosatrienoic Acids in Acetylcholine-Induced Dilation of Rat Retinal Arterioles in Vivo.

CYP epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to endothelium-dependent hyperpolarization (EDH)-related dilation in multiple vascular beds. The present study aimed to determine the role of EETs in the acetylcholine (ACh)-induced dilation of retinal arterioles in rats in vivo. The vasodilator responses were assessed by determining the change in diameter of the retinal arterioles on images of the ocular fundus. The intravitreal injection of 17-octadecynoic acid (1.4 nmol/eye), an inhibitor of CYP epoxygenase, and 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EE-5(Z)-E; 2 nmol/eye), an antagonist of EETs, reduced the ACh (0.3-10 µg/kg/min)-induced dilation of the retinal arterioles. The EET antagonist attenuated the vasodilator response to ACh under blockade of nitric oxide (NO) synthases and cyclooxygenases with NG-nitro-L-arginine methyl ester (30 mg/kg) plus indomethacin (5 mg/kg). Intravitreal injection of 14,15-EET (0.5 nmol/eye) dilated retinal arterioles and the response was prevented by iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (BKCa) channels (20 pmol/eye). These results suggest that ACh stimulates the production of EETs, thereby dilating the retinal arterioles via activation of BKCa channels. CYP epoxygenase-derived EETs may be involved in the EDH-related component of the ACh-induced dilation of the retinal arterioles.

Involvement of Gap Junctions in Acetylcholine-Induced Endothelium-Derived Hyperpolarization-Type Dilation of Retinal Arterioles in Rats.

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 (18ß-glycyrrhetinic acid and carbenoxolone) reduced the ACh-induced dilation of retinal arterioles. Moreover, the retinal arteriolar response to ACh was attenuated by 18ß-glycyrrhetinic acid under treatment with a combination of NG-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 Ca2+-activated K+ channels. Furthermore, the combination of 18ß-glycyrrhetinic acid and iberiotoxin produced greater attenuation in the EDH-related response than that by the individual agent. Treatment with 18ß-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.

Metformin Protects against NMDA-Induced Retinal Injury through the MEK/ERK Signaling Pathway in Rats.

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.

The process of revascularization in the neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

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.

4-Aminopyridine, a Voltage-Gated K+ Channel Inhibitor, Attenuates Nitric Oxide-Mediated Vasodilation of Retinal Arterioles in Rats.

Nitric oxide (NO) is an important regulator of the retinal blood flow. The present study aimed to determine the role of voltage-gated K+ (KV) channels and ATP-sensitive K+ (KATP) channels in NO-mediated vasodilation of retinal arterioles in rats. In vivo, the retinal vasodilator responses were assessed by measuring changes in the diameter of retinal arterioles from ocular fundus images. Intravitreal injection of 4-aminopyridine (a KV channel inhibitor), but not glibenclamide (a KATP channel blocker), significantly attenuated the retinal vasodilator response to the NO donor (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3). Intravitreal injection of indomethacin (a non-selective cyclooxygenase inhibitor) also reduced the NOR3-induced retinal vasodilator response. The combination of 4-aminopyridine and indomethacin produced a greater reduction in the NOR3-induced response than either agent alone. 4-Aminopyridine had no significant effect on pinacidil (a KATP channel opener)-induced response. These results suggest that the vasodilatory effects of NO are mediated, at least in part, through the activation of 4-aminopyridine-sensitive KV channels in the retinal arterioles of rats. NO exerts its dilatory effect on the retinal vasculature of rats through at least two mechanisms, activation of the KV channels and enhancement of prostaglandin production.

Abnormal Vascular Phenotypes Associated with the Timing of Interruption of Retinal Vascular Development in Rats.

Pathological angiogenesis is a leading cause of blindness in several retinal diseases. The key driving factor inducing pathological angiogenesis is the pronounced hypoxia leading to a marked, increased production of vascular endothelial growth factor (VEGF). The aim of this study was to determine whether the abnormal vascular growth occurs in a manner dependent on the degree of the vascular defects. Vascular defects of two different degrees were created in the retina by subcutaneously treating neonatal rats with the VEGF receptor (VEGFR) tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5 (P4/5) or P7 and P8 (P7/8). The structure of the retinal vasculature changes was examined immunohistochemically. Prevention of vascular growth and regression of some preformed capillaries were observed on the next day, after completion of each treatment (i.e., P6 and P9). The vascular regrowth occurred as a result of eliminating the inhibitory effect on the VEGFR signaling pathway. KRN633 (P4/5)-treated rats exhibited a retinal vasculature with aggressive intravitreal neovascularization on P21. On the other hand, the appearance of tortuous arteries is a representative vascular pathological feature in retinas of KRN633 (P7/8)-treated groups. These results suggest that an interruption of the retinal vascular development at different time points induces different vascular pathological features in the retina. Pharmacological agents targeting the VEGF signaling pathway are useful for creating an abnormal retinal vasculature with various pathological features in order to evaluate the efficacy of anti-angiogenic compounds.

Involvement of matrix metalloproteinases in capillary degeneration following NMDA-induced neurotoxicity in the neonatal rat retina.

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.

Probucol Slows the Progression of Cataracts in Streptozotocin-Induced Hyperglycemic Rats.

We examined the effect of probucol, an antihyperlipidemic drug with potent antioxidant properties, on cataract formation in streptozotocin (STZ)-induced hyperglycemic rats that were given 5% D-glucose as drinking water. Probucol treatment was initiated immediately after the induction of hyperglycemia was confirmed. Using full horizontal-plane lens images captured with an original digital camera system, the opacity of central region of lens was assessed by measuring the opaque area in the region. Central opacities were detected after 3 weeks of hyperglycemia, and progressed in a time-dependent manner. The majority of STZ-induced hyperglycemic rats developed severe cataracts after 9 weeks of hyperglycemia. Probucol slowed the progression of cataracts in a dose-dependent manner. Levels of sorbitol and protein carbonyls in lenses of STZ-induced hyperglycemic rats were higher than those of control rats. Probucol suppressed the increase in protein carbonyls, but not of sorbitol, in lenses of STZ-induced hyperglycemic rats. Probucol had no significant effect on increases in plasma concentrations of glucose, total cholesterol, and triglyceride observed in STZ-induced hyperglycemic rats. These results suggest that probucol slows the progression of sugar cataracts, independent of its lipid-lowering effects. The beneficial effect of probucol on cataracts is partially attributable to the attenuation of oxidative damage to lens proteins.

Role of Neuron⁻Glia Signaling in Regulation of Retinal Vascular Tone in Rats.

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.

Transient phenotypic changes in endothelial cells and pericytes in neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

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.

Brilliant Blue G protects against photoreceptor injury in a murine endotoxin-induced uveitis model.

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.

Retinal neuronal cell loss prevents abnormal retinal vascular growth in a rat model of retinopathy of prematurity.

A short-term blockade of the vascular endothelial growth factor (VEGF)-mediated pathway in neonatal rats results in formation of severe retinopathy of prematurity (ROP)-like retinal blood vessels. The present study aimed to examine the role of retinal neurons in the formation of abnormal retinal blood vessels. Newborn rats were treated subcutaneously with the VEGF receptor tyrosine kinase inhibitor, KRN633 (10 mg/kg), or its vehicle (0.5% methylcellulose in water) on postnatal day (P) 7 and P8. To induce excitotoxic loss of retinal neurons, N-methyl-D-aspartic acid (NMDA) was injected into the vitreous chamber of the eye on P9. Changes in retinal morphology, blood vessels, and proliferative status of vascular cells were evaluated on P11 and P14. The number of cells in the ganglion cell layer and the thickness of the inner plexiform layer and inner nuclear layer were significantly decreased 2 days (P11) after NMDA treatment. The pattern and degree of NMDA-induced changes in retinal morphology were similar between vehicle-treated (control) and KRN633-treated (ROP) rats. In ROP rats, increases in the density of capillaries, the tortuosity index of arteries, and the proliferating vascular cells were observed on P14. The expansion of the endothelial cell network was prevented, and the capillary density and the number of proliferating cells were reduced in NMDA-treated retinas of both control and ROP rats. Following NMDA-induced neuronal cell loss, no ROP-like blood vessels were observed in the retinas. These results suggest that retinal neurons play an important role in the formation of normal and ROP-like retinal blood vessels.

Iron-chelating agents attenuate NMDA-Induced neuronal injury via reduction of oxidative stress in the rat retina.

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 Fe2+, 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 Fe2+ using SiRhoNox-1, a fluorescent probe of labile Fe2+ 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 Fe2+ 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), ZnCl2 (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 ZnCl2, 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.

Establishment of an abnormal vascular patterning model in the mouse retina.

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.

Anti-angiogenic effects of valproic acid in a mouse model of oxygen-induced retinopathy.

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.

Methylglyoxal Impairs ß2-Adrenoceptor-Mediated Vasodilatory Mechanisms in Rat Retinal Arterioles.

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 ß2-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 ß2-adrenoceptor agonist salbutamol. The vasodilator effect of BMS-191011, a large-conductance Ca2+-activated K+ (BKCa) 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 BKCa channels with iberiotoxin, an inhibitor of the channels. These results suggest that methylglyoxal attenuates ß2-adrenoceptor-mediated retinal vasodilation by impairing the coupling of the ß2-adrenoceptor to the guanine nucleotide-binding protein (Gs protein) and the function of the BKCa channel. Increased methylglyoxal in the eyes may contribute to the impairment of regulatory mechanisms of retinal blood flow in patients with diabetic retinopathy.