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.

L-Citrulline ameliorates the attenuation of acetylcholine-induced vasodilation of retinal arterioles in diabetic rats.

In our previous study, we found that the vasodilation of retinal arterioles induced by acetylcholine and BMS-191011, a large-conductance Ca2+-activated K+ (BKCa) channel opener, were diminished in diabetic rats. Currently, few agents ameliorate the impaired vasodilator responses of retinal blood vessels. Our recent finding that the intravenous infusion of L-citrulline dilated retinal arterioles, suggests that L-citrulline could be a potential therapeutic agent for circulatory disorders of the retina. In this study, we determined the effect of an oral L-citrulline treatment on impaired acetylcholine- and BMS-191011-induced vasodilation in the retinal arterioles of diabetic rats. To induce diabetes, rats were administered an intravenous dose of streptozotocin (65 mg/kg) and a 5% D-glucose solution as drinking water. The L-citrulline (2 g/kg/day) and L-arginine (2 g/kg/day) treatments commenced either 15 days before or just after the streptozotocin injection and continued throughout the experimental period. A 29-day treatment with L-citrulline, but not L-arginine, significantly ameliorated the impaired acetylcholine- and BMS-191011-induced retinal vasodilation in diabetic rats without affecting their plasma glucose levels. The 2-week L-citrulline treatment tended to ameliorate the dysfunction of the acetylcholine-induced retinal vasodilation in diabetic rats. In conclusion, these results showed that the retinal blood vessel dysfunction induced by diabetes mellitus could be prevented by the long-term administration of L-citrulline and suggest that the latter could play a potentially prophylactic role in diabetic retinopathy.

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.

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.

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.

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.

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.

Anti-cataract Effect of Resveratrol in High-Glucose-Treated Streptozotocin-Induced Diabetic Rats.

Resveratrol, which is a polyphenol found in grapes, peanuts, and other plants, has health benefits for various chronic diseases. The aim of the present study was to examine the effect of resveratrol on cataract formation in diabetic rats. Male Wistar rats (7-week-old) were treated with streptozotocin, and the streptozotocin-treated animals were administered 5% D-glucose in drinking water to promote the formation of cataracts by inducing severe hyperglycemia. Resveratrol supplementation (10 or 30 mg/kg/d) in drinking water was initiated immediately after induction of diabetes was confirmed. The full lens images of the horizontal plane were captured with the digital camera system which we developed. Cataract formation was assessed by an observer-based scoring method and by quantitative analysis of digital images of the lens. Cataracts at the peripheral region of the lens were detected 2 weeks after induction of hyperglycemia and progressed depending on the length of the diabetic period. The majority of them developed severe cataracts after 9 weeks of hyperglycemia. Resveratrol did not prevent the appearance of diabetic cataracts but significantly delayed the progression of cataracts compared with controls. The contents of sorbitol and protein carbonyls in lenses of diabetic rats were higher than those of control rats. Resveratrol suppressed the increase in protein carbonyls, but not of sorbitol, in diabetic lenses. These results suggest that resveratrol delays the progression of diabetic cataracts partially through attenuation of oxidative damage to lens proteins. Resveratrol may be beneficial in preventing the progression of diabetic cataracts.

[Selective neuronal cell death in retinal degenerative diseases].

Retinal degenerative diseases, such as glaucoma and retinitis pigmentosa (RP), are the leading causes of blindness in adults. In Japan, glaucoma is a leading cause, and RP is third major cause of acquired blindness. Specific types of neurons are injured in the patients of glaucoma and RP. Retinal ganglion cells (RGC) are specifically degenerated in glaucoma. Excitotoxicity caused by excess glutamate in the retinal extracellular space is thought to be one of the mechanisms of RGC death induced by glaucoma and retinal central artery occlusion. Retinal ischemia-reperfusion, intravitreal NMDA injection, intravitreal NO donor injection and knock out of glutamate aspartate transporter, which are used as the experimental models of glaucoma, are known to induce RGC death. RGCs are vulnerable for excess glutamate and oxidative stress related to NO, and this vulnerability may be involved in pathogenesis of glaucomatous optic neuropathy. RP, which is characterized by progressive photoreceptor-selective degeneration, is caused by mutation of the genes related to the function of photoreceptor and retinal pigment epithelium. It has not been thoroughly clarified how the mutations induce specific photoreceptor death. Tunicamycin is widely known to induce ER stress, and intravitreal tunicamycin cause photoreceptor-specific degeneration. Therefore, ER stress may cause photoreceptor-selective degeneration in RP.

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.

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.

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.

Activation inhibitors of nuclear factor kappa B protect neurons against the NMDA-induced damage in the rat retina.

We reported that high-mobility group Box-1 (HMGB1) was involved in excitoneurotoxicity in the retina. HMGB1 is known to activate nuclear factor kappa B (NF-κB). However, the role of NF-κB in excitotoxicity is still controversial. Here, we demonstrated that NF-κB activation induced by NMDA led to the retinal neurotoxicity. Male Sprague-Dawley rats were used, and NMDA (200 nmol/eye) and bovine HMGB1 (15 µg/eye) were intravitreally injected. Triptolide (500 pmol/eye), BAY 11-7082 (500 pmol/eye), and IMD-0354 (7.5 nmol/eye), NF-κB inhibitors, were co-injected with NMDA or HMGB1. Retinal sections were obtained seven days after intravitreal injection. Cell loss in the ganglion cell layer was observed in the HMGB1- and the NMDA-treated retina. All of the NF-κB inhibitors used in this study reduced the damage. BAY 11-7082 reduced the expression of phosphorylated NF-κB 12 h after NMDA injection, upregulation of GFAP immunoreactivity induced by NMDA 12 and 48 h after NMDA injection, and the number of TUNEL-positive cells 48 h after NMDA injection. The results suggest that NF-κB activation is one of the mechanisms of the retinal neuronal death that occurs 48 h after NMDA injection or later. Prevention of NF-kB activation is a candidate for the treatment of retinal neurodegeneration associated with excitotoxicity.

Probucol prevents the attenuation of ß2-adrenoceptor-mediated vasodilation of retinal arterioles in diabetic rats.

Probucol is an antihyperlipidemic drug with potent antioxidant properties. Oxidative stress plays an important role in the pathogenesis of diabetic retinopathy. In this study, we aimed to investigate the protective effects of probucol against diabetes-induced retinal vascular dysfunction in a rat model of diabetes. Diabetes was induced by a combination of streptozotocin treatment and D-glucose feeding, and retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles. The vasodilator effect of salbutamol, a ß2-adrenoceptor agonist, on retinal arterioles was significantly diminished 2 weeks after the induction of diabetes. In non-diabetic rats, vasodilator responses to salbutamol were significantly reduced after an intravitreal injection of iberiotoxin, a blocker of large-conductance KCa (BKCa) channels. However, this effect was not observed in diabetic rats. Probucol had no significant effect on salbutamol-induced changes in diameter of retinal arterioles in non-diabetic rats, whereas it could prevent the attenuation of retinal vasodilator response to salbutamol in diabetic rats. These results suggest that the reduced function of BKCa channels is involved in the attenuation of ß2-adrenoceptor-mediated retinal vasodilation in diabetic rats. Probucol preserves the BKCa channel function in retinal arterioles under diabetic conditions; therefore, it may show beneficial effects on diabetic retinopathy by preventing or slowing the impairment of the retinal circulation in patients with diabetes mellitus.

Opioid receptor activation is involved in neuroprotection induced by TRPV1 channel activation against excitotoxicity in the rat retina.

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), ß-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, ß-endorphin, and substance P prevented this damage. Treatment with naloxone (0.5nmol/eye) almost completely negated the protective effects of capsaicin, CGRP, ß-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.

MEK/ERK- and calcineurin/NFAT-mediated mechanism of cerebral hyperemia and brain injury following NMDA receptor activation.

N-methyl-d-aspartate (NMDA) receptor activation increases regional cerebral blood flow (rCBF) and induces neuronal injury, but similarities between these processes are poorly understood. In this study, by measuring rCBF in vivo, we identified a clear correlation between cerebral hyperemia and brain injury. NMDA receptor activation induced brain injury as a result of rCBF increase, which was attenuated by an inhibitor of mitogen-activated protein kinase or calcineurin. Moreover, NMDA induced phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear translocation of nuclear factor of activated T-cell (NFAT) in neurons. Therefore, a MEK/ERK- and calcineurin/NFAT-mediated mechanism of neurovascular coupling underlies the pathophysiology of neurovascular disorders.

l-Citrulline ameliorates cerebral blood flow during cortical spreading depression in rats: Involvement of nitric oxide- and prostanoids-mediated pathway.

l-Citrulline is a potent precursor of l-arginine, and exerts beneficial effect on cardiovascular system via nitric oxide (NO) production. Migraine is one of the most popular neurovascular disorder, and imbalance of cerebral blood flow (CBF) observed in cortical spreading depression (CSD) contributes to the mechanism of migraine aura. Here, we investigated the effect of l-citrulline on cardiovascular changes to KCl-induced CSD. in rats. Intravenous injection of l-citrulline prevented the decrease in CBF, monitored by laser Doppler flowmetry, without affecting mean arterial pressure and heart rate during CSD. Moreover, l-citrulline attenuated propagation velocity of CSD induced by KCl. The effect of l-citrulline on CBF change was prevented by l-NAME, an inhibitor of NO synthase, but not by indomethacin, an inhibitor of cyclooxygenase. On the other hand, attenuation effect of l-citrulline on CSD propagation velocity was prevented not only by l-NAME but also by indomethacin. In addition, propagation velocity of CSD was attenuated by intravenous injection of NOR3, a NO donor, which was diminished by ODQ, an inhibitor of soluble guanylyl cyclase. These results suggest that NO/cyclic GMP- and prostanoids-mediated pathway differently contribute to the effect of l-citrulline on the maintenance of CBF.

Stimulation of µ-opioid receptors dilates retinal arterioles by neuronal nitric oxide synthase-derived nitric oxide in rats.

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-NH2 (CTOP), a selective µ-opioid receptor antagonist. Nω-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, Nω-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.

Stimulation of ß1- and ß2-adrenoceptors dilates retinal blood vessels in rats.

Our previous studies have demonstrated that adrenaline dilates rat retinal arterioles by stimulating propranolol-sensitive ß-adrenoceptors and ß3-adrenoceptors, and selective stimulation of ß2- or ß3-adrenoceptors causes retinal vasodilator responses. In the present study, we compared the effects of ß1- and ß2-adrenoceptor stimulation on rat retinal arterioles in vivo. Rat ocular fundus images were captured using an original high-resolution digital fundus camera. Diameters of retinal arterioles contained in the images were measured. Systemic blood pressure and heart rate were recorded continuously. Denopamine, a ß1-adrenoceptor agonist, increased the diameter of retinal arterioles and heart rate, and produced a small but statistically insignificant decrease in mean arterial pressure. CGP20712A, a ß1-adrenoceptor antagonist, but not ICI118551, a ß2-adrenoceptor antagonist, significantly prevented denopamine-induced retinal vasodilator and heart rate responses. Salbutamol, a ß2-adrenoceptor agonist, increased the diameter of retinal arterioles and decreased mean arterial pressure without significantly changing heart rate. The effects of salbutamol were significantly prevented by ICI118551, but not by CGP20712A. These results suggest that stimulation of ß1- and ß2-adrenoceptors dilates retinal blood vessels and indicate that all three ß-adrenoceptor subtypes (ß1, ß2, and ß3) may be involved in the retinal vasodilator response to adrenaline in rats.