Injection Of N-methyl-D-aspartic Acid Research Articles

Role of monocarboxylate transporters in AMPK-mediated protection against excitotoxic injury in the rat retina

Activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway protects against N-methyl-D-aspartic acid (NMDA)-induced excitotoxic retinal injury. AMPK activation enhances fatty acid metabolism and ketone body synthesis. Ketone bodies are transported into neurons by monocarboxylate transporters (MCTs) and exert neuroprotective effects. In this study, we examined the distribution and expression levels of MCT1 and MCT2 in the retina and analyzed the effects of pharmacological inhibition of MCTs on the protective effects of metformin and 5-aminoimidazole-4-carboxamide (AICAR), activators of AMPK, against NMDA-induced retinal injury in rats. MCT1 was expressed in the blood vessels, processes of astrocytes and Müller cells, and inner segments of photoreceptors in the rat retina, whereas MCT2 was expressed in neuronal cells in the ganglion cell layer (GCL) and in astrocyte processes. The expression levels of MCT2, but not MCT1, decreased one day after intravitreal injection of NMDA (200 nmol). Intravitreal injection of NMDA decreased the number of cells in the GCL compared to the vehicle seven days after injection. Simultaneous injection of metformin (20 nmol) or AICAR (50 nmol) with NMDA attenuated NMDA-induced cell loss in the GCL, and these protective effects were attenuated by AR-C155858 (1 pmol), an inhibitor of MCTs. AR-C155858 alone had no significant effect on the retinal structure. These results suggest that AMPK-activating compounds protect against NMDA-induced excitotoxic retinal injury via mechanisms involving MCTs in rats. NMDA-induced neurotoxicity may be associated with retinal neurodegenerative changes in glaucoma and diabetic retinopathy. Therefore, AMPK-activating compounds may be effective in managing these retinal diseases.

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.

N-Methyl-D-Aspartic Acid Receptor-Mediated Vasodilation Is Attenuated in the Retinas of Diabetic Rats

Purpose Activation of N-methyl-d-aspartic acid (NMDA) receptors enhances nitric oxide (NO) production in retinal neuronal cells, and in turn, NO released from neuronal cells induces glial cell-mediated dilation of retinal arterioles in rats. The purpose of this study was to examine how neuronal cell-dependent, glial cell-mediated vasodilation is impacted in diabetic rat retinas. Methods Diabetes was induced in 6-week-old male Wistar rats by combining streptozotocin injection and D-glucose feeding. Two weeks later, the dilator function of retinal arterioles was assessed. Results Compared with non-diabetic rats, the dilator responses of retinal arterioles induced by intravitreal injection of NMDA and NOR3, an NO donor, were reduced in diabetic rats. Following the blockade of large-conductance Ca2+-activated K+ (BKCa) channels with iberiotoxin, no significant difference in the retinal vasodilator response to NOR3 was observed between non-diabetic and diabetic rats. Intravitreal injection of 14,15-epoxyeicosatrienoic acid, a vasodilatory factor released from glial cells, dilated retinal arterioles, and the response was diminished by diabetes. Conclusion These findings suggest that the impaired BKCa channel function in vascular cells is responsible for the diminished neuronal cell-dependent, glial cell-mediated dilation of retinal arterioles during the early stage of diabetes.

Autism-Like Behavioral Phenotypes in Mice Treated with Systemic N-Methyl-D-Aspartate.

Autism spectrum disorder (ASD) having core characteristics of social interaction problems and repetitive behaviors and interests affects individuals at varying degrees and comorbidities, making it difficult to determine the precise etiology underlying the symptoms. Given its heterogeneity, ASD is difficult to treat and the development of therapeutics is slow due to the scarcity of animal models that are easy to produce and screen with. Based on the theory of excitation/inhibition imbalance in the brain with ASD which involves glutamatergic and/or GABAergic neurotransmission, a pharmacologic agent to modulate these receptors might be a good starting point for modeling. N-methyl-D-aspartic acid (NMDA) is an amino acid derivative acting as a specific agonist at the NMDA receptor and therefore imitates the action of the neurotransmitter glutamate on that receptor. In contrast to glutamate, NMDA selectively binds to and regulates the NMDA receptor, but not other glutamate receptors such as AMPA and kainite receptors. Given this role, we aimed to determine whether NMDA administration could result in autistic-like behavior in adolescent mice. Both male and female mice were treated with saline or NMDA (50 and 75 mg/kg) and were tested on various behavior experiments. Interestingly, acute NMDA-treated mice showed social deficits and repetitive behavior similar to ASD phenotypes. These results support the excitation/inhibition imbalance theory of ASD and that NMDA injection can be used as a pharmacologic model of ASD-like behaviors.

Factor XII protects neurons from apoptosis by epidermal and hepatocyte growth factor receptor‐dependent mechanisms

BackgroundFactor XII (FXII) is a serine protease that participates in the intrinsic coagulation pathway. Several studies have shown that plasma FXII exerts a deleterious role in cerebral ischemia and traumatic brain injury by promoting thrombo‐inflammation. Nevertheless, the impact of FXII on neuronal cell fate remains unknown. ObjectivesWe investigated the role of FXII and FXIIa in neuronal injury and apoptotic cell death. MethodsWe tested the neuroprotective roles of FXII and FXIIa in an experimental model of neuronal injury induced by stereotaxic intracerebral injection of N‐methyl‐D‐aspartic acid (NMDA) in vivo and in a model of apoptotic death of murine primary neuronal cultures through serum deprivation in vitro. ResultsHere, we found that exogenous FXII and FXIIa reduce brain lesions induced by NMDA injection in vivo. Furthermore, FXII protects cultured neurons from apoptosis through a growth factor‐‐like effect. This mechanism was triggered by direct interaction with epidermal growth factor (EGF) receptor and subsequent activation of this receptor. Interestingly, the “proteolytically” active and two‐chain form of FXII, FXIIa, exerts its protective effects by an alternative signaling pathway. FXIIa activates the pro‐form of hepatocyte growth factor (HGF) into HGF, which in turn activated the HGF receptor (HGFR) pathway. ConclusionThis study describes two novel mechanisms of action of FXII and identifies neurons as target cells for the protective effects of single and two‐chain forms of FXII. Therefore, inhibition of FXII in neurological disorders may have deleterious effects by preventing its beneficial effects on neuronal survival.

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.

Glutamatergic neurons from medial prefrontal cortex to dorsal raphe nucleus regulates maternal aggression in mice

Lactating female mice nurture their pups and attack intruders in their territory. These behaviors are the representatives of maternal behavior. The neuronal mechanisms underlying each distinct behavior have yet to be clarified. The dorsal raphe nucleus (DRN) regulates both maternal care and aggression. In the present study, we investigated whether the glutamatergic signals from the medial prefrontal cortex (mPFC) to the DRN is involved in regulating maternal care and aggression in lactating female mice. Injections of N-methyl-D-aspartic acid (NMDA) receptor antagonists or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonists into the DRN decreased the frequency of biting behavior, while injection of NMDA or AMPA inhibited maternal care. In dams that were injected with a retrograde tracer Fluorogold into the DRN, the number of c-Fos- and Fluorogold-positive neurons in the mPFC increased in the dams that displayed biting behavior in response to an intruder. Furthermore, optogenetic activation or inhibition of glutamatergic neurons projecting from the mPFC to the DRN increased or decreased the frequency of biting behavior, respectively. On the other hand, the optogenetic manipulations did not affect the display of maternal care. These results suggest that glutamatergic neurons from the mPFC to the DRN regulate maternal aggression, but not maternal care, in lactating female mice.

Induction of analgesia using atorvastatin in experimental diabetic neuropathy through NMDA receptor and inflammatory cytokines inhibition

Background: Diabetic neuropathy is a complication of diabetes causing damage to the nerves. Objectives: Considering the neuroprotective anti-inflammatory antioxidant characteristics of statins, the current study aimed at determining the effects of atorvastatin on diabetic neuropathy through assessing the involvement of N-methyl-D-aspartic acid (NMDA) receptor, factors of oxidative stress, and inflammatory cytokines in rats with diabetes. Methods: Male rats were randomly assigned into six groups of the saline- and atorvastatin-treated controls, and streptozotocin (STZ)-induced diabetic animals treated with vehicle, diabetic animals treated with morphine (5 mg/kg), and rats treated with atorvastatin (10 mg/kg/day for 10 weeks) alone or in combination with NMDA receptor agonist. The hot plate and formalin tests were carried out on the rats. Moreover, malondialdehyde level, catalase and superoxide dismutase activities, levels of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in the dorsal root ganglia (DRG) of the animals were measured. Finally, the expression of the NMDA receptor in DRG was investigated in the current study. Results: Diabetes resulted in analgesia in all tests, and pretreatment with atorvastatin exacerbated diabetes-induced analgesic effects in the hot plate and early phase of the formalin test (P≤0.01 and P≤0.05 in comparison to those reported for the diabetic vehicle-treated group, respectively). The injection of NMDA could reverse the atorvastatin-induced analgesia in the hot plate test (P≤0.05). Diabetes caused to increase the levels of IL-1β, IL-6, and TNF-α in DRG in comparison to those reported for the control group (P≤0.05). Furthermore, the pretreatment of rats using atorvastatin could significantly reverse the increase in TNF-α level (P≤0.05). Conclusions: Atorvastatin showed analgesic properties, which might be acting through NMDA receptors and reduction of inflammatory cytokines.

Comparison of three administration modes for establishing a zebrafish seizure model induced by N-Methyl-D-aspartic acid.

BACKGROUNDEpilepsy is a complex neurological disorder characterized by recurrent, unprovoked seizures resulting from the sudden abnormal discharge of brain neurons. It leads to transient brain dysfunction, manifested by abnormal physical movements and consciousness. It can occur at any age, affecting approximately 65 million worldwide, one third of which are still estimated to suffer from refractory seizures. There is an urgent need for further establishment of seizure models in animals, which provides an approach to model epilepsy and could be used to identify novel anti-epileptic therapeutics in the future.AIMTo compare three administration modes for establishing a seizure model caused by N-Methyl-D-aspartic acid (NMDA) in zebrafish.METHODSThree administration routes of NMDA, including immersion, intravitreal injection and intraperitoneal injection, were compared with regard to their effects on inducing seizure-like behaviors in adult zebrafish. We evaluated neurotoxicity by observing behavioral changes in zebrafish and graded those behaviors with a seizure score. In addition, the protective effects of MK-801 (Dizocilpine) and natural active constituent resveratrol against NMDA-induced alterations were studied.RESULTSThe three NMDA-administration methods triggered different patterns of the epileptic process in adult zebrafish. Seizure scores were increased after increasing NMDA concentration regardless of the mode of administration. However, the curve of immersion continuously rose to a high plateau (after 50 min), while the curves of intravitreal injection and intraperitoneal injection showed a spike in the early stage (10-20 min) followed by a steady decrease in seizure scores. Furthermore, pretreatment with resveratrol and MK-801 significantly delayed seizure onset time and lowered seizure scores.CONCLUSIONBy comparing the three methods of administration, intravitreal injection of NMDA was the most suitable for establishing an acute epileptic model in zebrafish. Thus, intraperitoneal injection in zebrafish can be applied to simulate diseases such as epilepsy. In addition, NMDA immersion may be an appropriate method to induce persistent seizures. Moreover, MK-801 and resveratrol showed strong anti-epileptic effects; thus, both of them may be clinically valuable treatments for epilepsy.

Establishment of an adult zebrafish model of retinal neurodegeneration induced by NMDA.

To establish a model of retinal neurodegeneration induced by N-Methyl-D-aspartic acid (NMDA) in adult zebrafish. We compared the effects of three different NMDA delivery methods on retinal neurodegeneration in adult zebrafish: immersion (I.M.), intravitreal injection (I.V.), and intraperitoneal injection (I.P.), and examined retinal pathology and degeneration by hematoxylin and eosin and TUNEL staining in the treated zebrafish. Effects of the NMDA receptor antagonist MK-801 and the natural product resveratrol on NMDA-induced retinal neurodegeneration were also assessed. The thickened inner retina was seen in histology with 100 µmol/L NMDA by I.M. administration. Significant apoptosis in the retinal ganglion cell layer and retinal thickness reduction occurred in 0.5 mol/L NMDA I.P. administration group.Seizure-like behavioral changes, but no retinal histological alteration occurred in 16 mg/kg NMDA I.P. administration group. Resveratrol and MK-801 prevented NMDA-induced retinal neurodegeneration in the zebrafish. Among the three drug administration methods, I.V. injection of NMDA is the most suitable for establishment of an acute retinal damage model in zebrafish. I.M. with NMDA is likely the best for use as a chronic retinal damage model. I.P. treatment with NMDA causes brain damage. Resveratrol and MK801 may be a clinically valuable treatment for retinal neurodegeneration.

Dorsal raphe nucleus 5-Hydroxytryptamine 2A receptors are critical for the organisation of panic attack-like defensive behaviour and unconditioned fear-induced antinociception elicited by the chemical stimulation of superior colliculus neurons

Microinjections of N-methyl-d-aspartic acid (NMDA) in the midbrain tectum structures produce panic attack-like defensive behaviours, followed by an antinociceptive response. It has been suggested that fear-related defensive responses organised by brainstem neurons can be modulated by 5-hydroxytryptamine (5-HT). However, there is a shortage of studies showing the role of dorsal raphe nucleus (DRN) 5-HT2A receptors in the modulation of panic-like behaviour and fear-induced antinociception organised by the superior colliculus (SC). The purpose of this study was to investigate the participation of DRN 5-HT2A receptors in the modulation of panic attack-like behaviour and antinociception evoked by intra-SC injections of NMDA. In experiment I, the animals received microinjections of physiological saline or NMDA (6, 9 and 12 nmol) in the deep layers of the SC (dlSC). In experiment II, the most effective dose of NMDA (12 nmol) or vehicle was preceded by microinjections of vehicle or the 5-HT2A receptor selective antagonist R-96544 at different concentrations (0.5, 5 and 10 nM) in the DRN. Both proaversive and antinociceptive effects elicited by intra-dlSC injections of NMDA were attenuated by DRN pretreatment with R-96544. In addition, a morphological analysis showed that 5-HT2A receptors are present in GABAergic interneurons in the DRN. Taken together, these findings suggest that DRN 5-HT2A receptors are critical for the modulation of both panic attack-like defensive behaviour organised by SC neurons and unconditioned fear-induced antinociception. A possible interaction between serotonergic inputs, GABAergic interneurons and serotonergic outputs from the DRN was also considered.

Regional Differences Within the Anterior Cingulate Cortex in the Generation Versus Suppression of Pain Affect in Rats

The anterior cingulate cortex (ACC) modulates emotional responses to pain. Whereas, the caudal ACC (cACC) promotes expression of pain affect, the rostral ACC (rACC) contributes to its suppression. Both subdivisions receive glutamatergic innervation, and the present study evaluated the contribution of N-methyl-d-aspartic acid (NMDA) receptors within these subdivisions to rats’ expression of pain affect. Vocalizations that follow a brief noxious tail shock (vocalization afterdischarges, VAD) are a validated rodent model of pain affect. The threshold current for eliciting VAD was increased in a dose-dependent manner by injecting NMDA into the rACC, but performance (latency, amplitude, and duration) at threshold was not altered. Alternately, the threshold current for eliciting VAD was not altered following injection of NMDA into the cACC, but its amplitude and duration at threshold were increased in a dose-dependent manner. These effects were limited to Cg1 of the rACC and cACC, and blocked by pretreatment of the ACC with the NMDA receptor antagonist d-2-amino-5-phosphonovalerate. These findings demonstrate that NMDA receptor agonism within the cACC and rACC either increases or decreases emotional responses to noxious stimulation, respectively. PerspectiveNMDA receptor activation of the rostral and caudal ACC respectively inhibited or enhanced rats’ emotional response to pain. These findings mirror those obtained from human neuroimaging studies; thereby, supporting the use of this model system in evaluating the contribution of ACC to pain affect.

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.

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.

Glutamatergic Signals in the Dorsal Raphe Nucleus Regulate Maternal Aggression and Care in an Opposing Manner in Mice

Lactating female mice nurture their pups and attack intruders in their territory. When an intruder invades a dam’s territory, she needs to switch her behavior from care to aggression to protect her pups and territory. Although the neuronal mechanisms underlying each distinct behavior have been studied, it is unclear how these behaviors are displayed alternatively. The dorsal raphe nucleus (DRN) regulates both nurturing and aggressive behaviors. In the present study, we examined whether the DRN is involved in regulating alternative display of maternal care and aggression. We first examined neuronal activity in the medial prefrontal cortex (mPFC) and lateral habenula (LHb), which send glutamatergic input to the DRN, in dams by injecting Fluorogold, a retrograde tracer, into the DRN. The number of c-Fos- and Fluorogold-positive neurons in the mPFC and LHb increased in the dams that displayed biting behavior in response to an intruder, but remained unchanged in the dams that displayed nurturing behavior. Injections of N-methyl-d-aspartic acid (NMDA) receptor antagonists or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonists into the DRN inhibited biting behavior but not nurturing behavior. In contrast, injections of NMDA or AMPA into the DRN inhibited nurturing behavior. These results suggest that glutamatergic signals in the DRN, which may originate from the mPFC and/or LHb, regulate the preferential display of biting behavior over nurturing behavior in dams.

GYY4137, an Extended-Release Hydrogen Sulfide Donor, Reduces NMDA-Induced Neuronal Injury in the Murine Retina.

We previously reported that systemic administration with sodium hydrogen sulfide, a rapid-release donor compound of hydrogen sulfide (H2S), protected retinal neurons against N-methyl-D-aspartic acid (NMDA)-induced injury. For clinical application of H2S donors for retinal neurodegeneration, topical administration with an extended-release donor compound will be better. In the present study, we histologically investigated whether GYY4137, an extended-release hydrogen sulfide donor, had a protective effect on NMDA-induced retinal injury in the mice in vivo. Male and female B6.Cg-Tg(Thy1-CFP)23Jrs/J and C57BL/6J mice anesthetized with a mixture of ketamine and xylazine were subjected to intravitreal NMDA injection (80 nmol/eye). GYY4137 was intravitreally administered with NMDA simultaneously. Morphometric evaluation was carried out seven days after NMDA injection. Intravitreal NMDA induced retinal ganglion cell loss. GYY4137 (1, 10 and 100 nmol/eye) significantly reduced retinal ganglion cell loss seven days after NMDA injection. GYY4137 (10 nmol/eye) decreased the numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells 12 h after NMDA injection. These results suggest that extended release donor compounds of H2S protect retinal neurons against excitotoxicity induced by intravitreal NMDA in the mice in vivo through its anti-oxidative activity.

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), 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.

Apelin-36 is protective against N-methyl-D-aspartic-acid-induced retinal ganglion cell death in the mice

Retinal ganglion cell death in glaucoma is caused at least in part by a large Ca2+ 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, [Glp1]-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 [Glp1]-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]-4H-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.

Protective effects of PF-4708671 against N-methyl-d-aspartic acid-induced retinal damage in rats.

We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N-methyl-d-aspartic acid (NMDA)-induced retinal damage in rats. Rapamycin inhibits mTOR activity, thereby preventing the phosphorylation of ribosomal protein S6, which is a downstream target of S6 kinase. Therefore, we aimed to determine whether PF-4708671, an inhibitor of S6 kinase, protects against NMDA-induced retinal injury. Intravitreal injection of NMDA (200 nmol/eye) caused cell loss in the ganglion cell layer and neuroinflammatory responses, such as an increase in the number of CD45-positive leukocytes and Iba1-positive microglia. Surprisingly, simultaneous injection of PF-4708671 (50 nmol/eye) with NMDA significantly attenuated these responses without affecting phosphorylated S6 levels. These results suggest that PF-4708671 and rapamycin likely protect against NMDA-induced retinal damage via distinct pathways. The neuroprotective effect of PF-4708671 is unlikely to be associated with inhibition of the S6 kinase, even though PF-4708671 is reported to be a S6 kinase inhibitor.

P2X7 receptor antagonists protect against N-methyl-d-aspartic acid-induced neuronal injury in the rat retina

Activation of N-methyl-d-aspartic acid (NMDA) receptors followed by a large Ca2+ 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 (200nmol/eye), NMDA-induced cell loss was apparent in the ganglion cell layer. Intravitreal A438079 (50pmol/eye) simultaneously injected with NMDA and intraperitoneal brilliant blue G (50mg/kg) administered just before the NMDA injection as well as 24 and 48h after significantly reduced cell loss. In addition, A438079 decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells 12h 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 12h 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.