Asami Mori

Retinal blood vessels are damaged in a rat model of NMDA-induced retinal degeneration.

Retinal ischemia-reperfusion causes capillary degeneration but the mechanisms of damage are not well understood. The NMDA receptor plays an important role in neuronal damage after ischemia-reperfusion. Therefore, we determined whether retinal blood vessels are damaged structurally and functionally in a rat model of retinal degeneration induced by NMDA. At 7 days after a single intravitreal injection of NMDA (200nmol) into the eye, loss of retinal ganglion cells and thinning of the inner plexiform layer were observed. Endothelial cells disappeared in some regressing vessels and empty basement membrane sleeves were left as remnants of the vessels. The number of basement membrane sleeves was increased in the NMDA-treated retina and non-perfused vessels were found in the injured retina. These results indicate that retinal blood vessels are damaged in the NMDA-induced retinal degeneration model. Neuronal cells may play a role in maintaining normal structure and function of the vasculature in the retina.

Protective effects of TGF-β inhibitors in a rat model of NMDA-induced retinal degeneration

Recent studies have shown that the retinal blood vessels are damaged in experimental models of retinal degeneration, but the mechanisms underlying their damage are not fully understood. In this study, we examined the possible role of transforming growth factor (TGF)-β in retinal neuron loss and capillary degeneration induced in rats by an intravitreal injection of N-methyl-d-aspartate (NMDA). The number of cells in the ganglion cell layer was significantly decreased 2 days after NMDA treatment, and a further decrease was observed at 7 days. Enhanced capillary degeneration was detected 7 days after NMDA treatment. Simultaneous injection of NMDA and the TGF-β inhibitor (SB431542 or LY364947) slightly but significantly attenuated the reduction in number of cells in the ganglion cell layer and almost completely prevented the enhancement of capillary degeneration. These results suggest that activation of the TGF-β signaling pathway induces neuronal and vascular cell damage in rat retina.

The prostanoid EP2 receptor agonist ONO-AE1-259-01 protects against glutamate-induced neurotoxicity in rat retina

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.

Activation of the TRPV1 channel attenuates N-methyl-d-aspartic acid-induced neuronal injury in the rat retina

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; 200 nmol/eye) in rats in vivo. Under ketamine/xylazine anesthesia, male Sprague-Dawley rats were subjected to intravitreal NMDA injection. Capsaicin (5.0 nmol/eye) was intravitreally admianeously with NMDA injection. SA13353 (10mg/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. Capsaicin and SA13353 almost completely prevented this damage. Treatment with capsazepine (TRPV1 antagonist, 0.5 nmol/eye), CGRP (8-37) (calcitonin gene-related peptide (CGRP) receptor antagonist, 0.5 pmol/eye), or RP67580 (tachykinin NK1 receptor antagonist, 0.5 nmol/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.

Role of calcium-activated potassium channels in acetylcholine-induced vasodilation of rat retinal arterioles in vivo

The vascular endothelium plays an important role in regulating retinal blood flow via actions of several vasodilators, including nitric oxide (NO), prostaglandin I2, and an endothelium-derived hyperpolarizing factor (EDHF). Our previous in vivo studies demonstrated that acetylcholine (ACh) dilates the rat retinal arteriole partly through NO- and prostaglandin-independent pathway, possibly the EDHF-mediated pathway, but the underlying mechanism(s) remains to be elucidated. It has been suggested that activation of Ca2+-activated K+ (KCa) channels contributes to the EDHF-mediated responses; therefore, the roles of KCa channels in ACh-induced vasodilation of retinal arterioles were examined in rats. The retinal vascular responses were assessed by determining changes in diameters of retinal arterioles in ocular fundus images that were captured with an original fundus camera system. Intravitreal injection of charybdotoxin, an inhibitor of intermediate- and large-conductance KCa (I/BKCa) channels, or iberiotoxin, an inhibitor of large-conductance KCa (BKCa) channels, significantly reduced the ACh-induced vasodilation of retinal arterioles, whereas neither apamin, an inhibitor of small-conductance KCa (SKCa) channels, nor TRAM-34, an inhibitor of intermediate-conductance KCa (IKCa) channels, altered the response. The vasodilator response to ACh observed under the combined blockade of NO synthase and cyclooxygenase with NG-nitro-L-arginine methyl ester plus indomethacin was also diminished by iberiotoxin. Iberiotoxin did not affect the NO donor NOR3-induced vasodilation of retinal arterioles, whereas it significantly reduced the BKCa channel opener BMS-191011-induced responses. These results suggest that activation of BKCa channels is involved in the EDHF-mediated component of the vasodilator response to ACh in the rat retinal arterioles in vivo.

Vasodilation of retinal arterioles induced by activation of BKCa channels is attenuated in diabetic rats

The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate the retinal vascular tone, but question of whether the impairment of the channel function contributes to abnormalities of retinal circulation has not yet been completely elucidated. The purpose of this study was to examine effects of diabetes on the vasodilation induced by activation of BK(Ca) channels. Male Wistar rats were treated with streptozotocin and experiments were performed 2 weeks later. The streptozotocin-treated animals were given drinking water containing 5% d-glucose to shorten the term in the development of retinal vascular dysfunction. The retinal vascular responses were assessed by measuring diameter of retinal arterioles in the fundus images that were captured with an original fundus camera system. In non-diabetic rats, vasodilator effects of acetylcholine on retinal arterioles were significantly reduced by iberiotoxin, an inhibitor of BK(Ca) channels. However, the inhibitory effect of iberiotoxin was not observed in diabetic rats, and the responses to the BK(Ca) channel opener BMS-191011 were almost completely abolished. The retinal vasodilator response to acetylcholine, possibly an endothelium-derived hyperpolarizing factor-mediated response, observed after treatment with N(G)-nitro-l-arginine methyl ester and indomethacin was markedly reduced in diabetic rats. The responses to pinacidil, an opener of ATP-sensitive K(+) channels, were unchanged. These results suggest that the retinal vasodilator response mediated through mechanisms involving activation of BK(Ca) channels is diminished at the early stage of diabetes in rats. The impairment of BK(Ca) channel function may contribute to abnormal retinal hemodynamics in diabetes and consequently play an important role in the pathogenesis of diabetic retinopathy.

β-Adrenoceptor-mediated vasodilation of retinal blood vessels is reduced in streptozotocin-induced diabetic rats

We investigated the effects of epinephrine and dopamine on retinal blood vessels in streptozotocin (STZ, 80 mg/kg, i.p.)-treated rats and age-matched control rats to determine whether diabetes mellitus alters the retinal vascular responses to circulating catecholamines. Experiments were performed 6-8 weeks after treatment with STZ or the vehicle. The fundus images were captured with the digital fundus camera system for small animals we developed and diameters of retinal blood vessels contained in the digital images were measured. Epinephrine increased the diameters of retinal blood vessels, but the vasodilator responses were reduced in diabetic rats. Dopamine produced a biphasic retinal vascular response with an initial vasoconstriction followed by a vasodilation. The vasoconstrictor effects of dopamine on retinal arterioles were enhanced in diabetic rats, whereas the difference between the two groups was abolished by treatment with propranolol. The vasodilator effect of isoproterenol, but not of the activator of adenylyl cyclase colforsin, on retinal blood vessels was reduced in diabetic rats. No difference in vasoconstriction of retinal blood vessels to phenylephrine between non-diabetic and diabetic rats was observed. The vasodilator responses of retinal blood vessels to 1,1-dimethyl-4-phenylpiperazinium, a ganglionic nicotinic receptor agonist, were also attenuated in diabetic rats. These results suggest that diabetes mellitus alters the retinal vascular responses to circulating catecholamines and the impairment of vasodilator responses mediated by beta-adrenoceptors contributes to the alteration.

Role of vascular endothelial growth factor in maintenance of pregnancy in mice.

It is well known that withdrawal of progesterone from the maternal circulation is a critical stimulus to parturition in rodents, such as rats and mice. However, mechanisms that determine the timing of progesterone withdrawal are not completely understood. In the present study, we examined whether the vascular endothelial growth factor (VEGF) system in the corpus luteum (CL) contributes to the regulation of circulating progesterone levels and acts as a determinant of the timing of parturition in mice. We found that reduction in the expression levels of VEGF and VEGF receptor-2 in the CL precedes the impairment of luteal circulation and a series of events leading to parturition (i.e., reduction of plasma progesterone, enhancement of myometrium contractility, and onset of parturition). Blocking of VEGF signaling by using the inhibitor of VEGFR tyrosine kinase KRN633 at mid-pregnancy caused a similar sequence of events and induced preterm birth. These results suggest that the VEGF system in the CL plays a critical role in maintaining a high level of circulating progesterone, and determining the timing of parturition in mice.

Structural and functional changes in retinal vasculature induced by retinal ischemia-reperfusion in rats.

Recent studies have shown retinal blood vessel damage in experimental models of retinal degeneration. The present study aimed to provide a detailed description of the structural and functional changes in retinal vasculature induced by retinal ischemia-reperfusion (I/R) in rats. Retinal ischemia was induced for 60 min by raising the intraocular pressure to 130 mmHg. Morphological changes in vascular components (endothelial cells, pericytes, and basement membranes), the patency and perfusion of blood vessels, and expression of vascular endothelial growth factor (VEGF) were assessed in the retinas at 2, 7, and 14 days after I/R. Significant reductions in vascular densities were observed at 7 and 14 days after I/R. Pericyte loss occurred after the appearance of endothelial cell degeneration, whereas the vascular basement membranes remained unchanged. Some vessels showed no perfusion in damaged retina. A decrease in the immunoreactivity of VEGF in the region extending from the ganglion cell layer to the outer plexiform layer was evident 2 days after I/R. In retinal I/R model, retinal ganglion cells are rapidly (<2 day) damaged following reperfusion, therefore, the current results suggest that neuronal cell damage precedes capillary degeneration, and neuronal cells may play an important role in maintaining vascular structure and function through the production and release of endothelial cell survival factors, including VEGF. Neuronal cell damage could be an additional cause of progression of ischemic retinal damage by reducing blood supply to the retinal neurons due to the destruction of the blood vessel network.

Rapamycin prevents N-methyl-D-aspartate-induced retinal damage through an ERK-dependent mechanism in rats

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 rapamycins 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.