N-methyl-D-aspartate glutamate receptors(NMDARs)play crucial roles in the pathogenesis of neuronal injuries following a stroke insult;therefore,a plethora of preclinical studies focus on better understanding functions...N-methyl-D-aspartate glutamate receptors(NMDARs)play crucial roles in the pathogenesis of neuronal injuries following a stroke insult;therefore,a plethora of preclinical studies focus on better understanding functions of NMDARs and their associated signaling pathways.Over the past decades,NMDARs have been found to exert dual effects in neuronal deaths signaling and neuronal survival signaling during cerebral ischemia.Moreover,many complex intracellular signaling pathways downstream of NMDAR activation have been elucidated,which provide novel targets for developing much-needed neuro-protectants for patients with stroke.In this review,we will discuss the recent progress in understanding the underlying mechanisms of stroke related to NMDAR activation and the potential therapeutic strategies based on these discoveries.展开更多
BACKGROUND Some isopavines can exhibit important biological activity in the treatment of neurological disorders since it is considered an antagonist of the specific Nmethyl-D-Aspartate(NMDA)receptor.Amurensinine is an...BACKGROUND Some isopavines can exhibit important biological activity in the treatment of neurological disorders since it is considered an antagonist of the specific Nmethyl-D-Aspartate(NMDA)receptor.Amurensinine is an isopavine which still has few studies.In view of the potential of isopavines as NMDA receptor antagonists,theoretical studies using bioinformatics were carried out in order to investigate whether Amurensinine binds to the NMDA receptor and to analyze the receptor/Ligand complex.This data can contribute to understanding of the onset of neurological diseases and contribute to the planning of drugs for the treatment of neurological diseases involving the NMDA receptor.AIM To investigate the interaction of the antagonist Amurensinine on the GluN1A/GluN2B isoform of the NMDA receptor using bioinformatics.METHODS The three-dimen-sional structure of the GluN1A/GluN2B NMDA receptor was selected from the Protein Data Bank(PDB)-PDB:4PE5,and the three-dimensional structure of Amurensinine(ligand)was designed and optimized using ACD/SchemsketchTM software.Prediction of the protonation state of Amurensinine at physiological pH was performed using MarvinSketch software(ChemAxon).Protonated and non-protonated Amurensin were prepared using AutoDock Tools 4 software and simulations were performed using Autodock Vina v.1.2.0.The receptor/Ligand complexes were analyzed using PyMol(Schrödinger,Inc)and BIOVIA Discovery Studio(Dassault Systemes)software.To evaluate the NMDA receptor/Amurensinine complex and validate the molecular docking,simulations using NMDA receptor and Ifenprodil antagonist were performed under the same conditions.Ifenprodil was also designed,optimized and protonated,under the same conditions as Amurensinine.RESULTS Molecular docking simulations showed that both non-protonated and protonated Amurensinine bind to the amino terminal domain(ATD)domain of the GluN1A/GluN2B NMDA receptor with significant affinity energy,-7.9 Kcal/mol and-8.1 Kcal/mol,respectively.The NMDA receptor/non-protonated Amurensinine complex was stabilized by 15 bonds,while the NMDA receptor/protonated Amurensinine complex was stabilized by less than half,6 bonds.Despite the difference in the number of bonds,the variation in bond length and the average bond length values are similar in both complexes.The complex formed by the NMDA receptor and Ifenprodil showed an affinity energy of-8.2 Kcal/mol,a value very close to that obtained for the NMDA receptor/Amurensinine complex.Molecular docking between Ifenprodil and the GluN1A/GluN2B NMDA receptor demonstrated that this antagonist interacts with the ATD of the receptor,which validates the simulations performed with Amurensinine.CONCLUSION Amurensinine binds to the NMDA receptor on ATD,similar to Ifenprodil,and the affinity energy is closer.These data suggest that Amurensinine could behave as a receptor inhibitor,indicating that this compound may have a potential biological application,which should be evaluated by in vitro and preclinical assays.展开更多
Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis is a rare disease with uncertain etiology and pathogenesis that affects young women. Its diagnosis can be delayed because of the nonspecific neuropsychiatric symp...Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis is a rare disease with uncertain etiology and pathogenesis that affects young women. Its diagnosis can be delayed because of the nonspecific neuropsychiatric symptoms in the foreground. This article describes the details of a recent complicated case of a patient with this condition which is related to an ovarian teratoma. Correct diagnostic and prompt treatment of anti-NMDA receptor encephalitis remains a serious clinical challenge due to its unspecific manifestations and varying response to treatments. The information will be of interest to clinicians working with encephalitis patients.展开更多
N-methyl-D-aspartate receptor hypofunction is the basis of pathophysiology in schizophrenia. Blocking the N-methyl-D-aspartate receptor impairs learning and memory abilities and induces pathological changes in the bra...N-methyl-D-aspartate receptor hypofunction is the basis of pathophysiology in schizophrenia. Blocking the N-methyl-D-aspartate receptor impairs learning and memory abilities and induces pathological changes in the brain. Previous studies have paid little attention to the role of the N-methyl-D-aspartate receptor subunit 1 (NR1) in neurogenesis in the hippocampus of schizophrenia. A mouse model of schizophrenia was established by intraperitoneal injection of 0.6 mg/kg MK-801, once a day, for 14 days. In N-methyl-D-aspartate-treated mice, N-methyl-D-aspartate was administered by intracerebroventricular injection in schizophrenia mice on day 15. The number of NR1-, Ki67- or BrdU-immunoreactive cells in the dentate gyrus was measured by immunofluorescence staining. Our data showed the number of NR1-immunoreactive cells increased along with the decreasing numbers of BrdU- and Ki67-immunoreactive cells in the schizophrenia groups compared with the control group. N-methyl-D-aspartate could reverse the above changes. These results indicated that NR1 can regulate neurogenesis in the hippocampal dentate gyrus of schizophrenia mice, supporting NR1 as a promising therapeutic target in the treatment of schizophrenia. This study was approved by the Experimental Animal Ethics Committee of the Ningxia Medical University, China (approval No. 2014-014) on March 6, 2014.展开更多
Objective: To observe and elucidate the neuroprotective effect of Xingnaojing (XNJ) injection on hippocampal N-methyl-D-aspartic acid (NMDA) receptors of focal cerebral ischemia in rats. Methods: Cerebral ischemia was...Objective: To observe and elucidate the neuroprotective effect of Xingnaojing (XNJ) injection on hippocampal N-methyl-D-aspartic acid (NMDA) receptors of focal cerebral ischemia in rats. Methods: Cerebral ischemia was established by occluding the middle cerebral artery with an intraluminal suture technique in rats. Neurological deficit score, infarct volume and quantity of NMDA receptors were estimated in all groups and compared. Results: After being treated with XNJ, the score decreased in the initial 6 hours and infarct volume decreased in 24 hours. And within 24 hours, the quantity of NMDA receptors obviously decreased compared with the model group (P<0. 01) It indicated that XNJ could ameliorate neurological behavior of middle cerebral artery occlusion rats and down-regulate the expression of hippocampal NMDA receptors. Conclusion: The neuroprotective effect of XNJ on focal cerebral ischemia is possibly related to down-regulating the expression of NMDA receptors in rats.展开更多
Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine.The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior,electroencephalography ...Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine.The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior,electroencephalography and 24-hour survival rate.Propofol(12.5-100 mg/kg) improved status epilepticus in a dose-dependent manner,and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection.Western blot results showed that,24 hours after induction of status epilepticus,the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus.Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels,but not the increase in N-methyl-D-aspartate receptor 2A subunit levels.The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine.This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures.展开更多
BACKGROUND:Glutamate acid ionotropic receptor N-methyl-D-aspartic acid (NMDA) takes part in long-term potentiation, thereby influencing the process of learning and memory. OBJECTIVE: To verify expression of NMDA 2...BACKGROUND:Glutamate acid ionotropic receptor N-methyl-D-aspartic acid (NMDA) takes part in long-term potentiation, thereby influencing the process of learning and memory. OBJECTIVE: To verify expression of NMDA 2A/B and 2B receptors in the anterior thalamic nucleus and subiculum complex of rats. DESIGN, TIME AND SETTING: A single-sample observation was performed at Department of Anatomy in Dalian Medical University (Dalian, Liaoning, China) from April to September in 2007. MATERIALS: Ten adult Wistar rats were used for this study, as well as rabbit anti-NMDA 2A/B and 2B antibodies. METHODS: The rats were anesthetized and perfused, followed by brain resection and coronal sectioning of the brain tissue. A 1:3 series was selected for immunohistochemistry, using antibodies specific to NMDA 2A/B and 2B receptors. Photos were taken using the Nikon image analysis system. MAIN OUTCOME MEASURES: Expression and distribution of immunohistochemistry staining of NMDA 2A/B and 2B receptor subunits. RESULTS: There were a large number of NMDA 2A/B and 2B receptor-positive neurons distributed throughout the anterior dorsal thalamic nucleus. In the anterior ventral thalamic nucleus, distribution of positive neurons was rare, staining intensity was lighter, and cell bodies were smaller compared with the anterior dorsal thalamic nucleus. In the subiculum complex, staining intensity of NMDA 2A/B and 2B-positive neurons was weakest in the molecular layer and stronger in the pyramidal layer, in particular the region with large cell bodies adjacent to the molecular layer. In the multiform layer, more positive neurons of various sizes were detected. CONCLUSION: NMDA 2A/B and 2B receptor subunits were richly distributed in the anterior thalamic nucleus, with a small difference existing between the anterior dorsal nucleus and anterior ventral nucleus. These neurons were also differentially distributed within the three layers of the subiculum complex.展开更多
AIM: Many studies have demonstrated N-methyl-D-aspartate receptor-1-subunit (NMDAR1) is associated with amblyopia. The effectiveness of levodopa in improving the visual function of the children with amblyopia has also...AIM: Many studies have demonstrated N-methyl-D-aspartate receptor-1-subunit (NMDAR1) is associated with amblyopia. The effectiveness of levodopa in improving the visual function of the children with amblyopia has also been proved. But the mechanism is undefined. Our study was to explore the possible mechanism. METHODS: Sixty 14-day-old healthy SD rats were randomly divided into 4 groups, including normal group, monocular deprivation group, levodopa group and normal saline group, 15 rats each. We sutured all the rats' unilateral eyelids except normal group to establish the monocular deprivation animal model and raise them in normal sunlight till 45-day-old. NMDAR1 was detected in the visual cortex with immunohistochemistry methods, Western Blot and Real time PCR. LD and NS groups were gavaged with levodopa (40mg/kg) and normal saline for 28 days respectively. NMDAR1 was also detected with the methods above. RESULTS: NMDAR1 in the visual cortex of MD group was less than that of normal group. NMDAR1 in the visual cortex of LD group was more than that of NS group. CONCLUSION: NMDAR1 is associated with the plasticity of visual development. Levodopa may influence the expression of NMDAR1 and improve visual function, and its target may lie in the visual cortex.展开更多
To study the effect of glutamate on the intracellular calcium signal of pure cultured rat astrocytes and the role of NMDA and AMPA receptors in the procedure, the change of calcium signal was investigated by monitorin...To study the effect of glutamate on the intracellular calcium signal of pure cultured rat astrocytes and the role of NMDA and AMPA receptors in the procedure, the change of calcium signal was investigated by monitoring the fluctuation of intracellular Ca 2+ concentration ([Ca 2+ ] i) on the basis of Fura-2 single cell fluorescent ratio (F345/F380). The changes in the effect of glutamate on the intracellular calcium signal were observed after blockage of NMDA and(or) AMPA receptors. It was found that L-glutamate could induce an increased [Ca 2+ ] i in most of the cells in concentration- and time-dependent manner. D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5, a selective antagonist of the NMDA receptor) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selective antagonist of the AMPA receptor) could abolish the effects of NMDA and AMPA respectively. The treatment of D-AP-5 and CNQX simultaneously or respectively could attenuate the effect of L-glutamate at varying degrees. All these indicated that glutamate could modulate intracellular Ca 2+ of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA receptors took part in the complex mechanisms.展开更多
Alzheimer's disease (AD) is one of the most devastating dis- eases affecting the life and health of aging population. Two hallmarks of AD are senile plaques and neurofibrillary tan- gles, and AD is well known for t...Alzheimer's disease (AD) is one of the most devastating dis- eases affecting the life and health of aging population. Two hallmarks of AD are senile plaques and neurofibrillary tan- gles, and AD is well known for the massive loss of neurons and impaired cognitive functions especially memory loss. Despite extensive search for effective treatment, available drugs have limited efficacy without affecting the course of AD. Significant efforts have been devoted to curb the pro- duction of amyloid [3 (A[3; the major component of plaques) or enhance the clearance of it, with the aim to reduce the accumulation of plaque in the brain. Antibodies that can bind A[3 to increase their removal have received a lot of at- tention although recent clinical trial results have been largely negative and disappointing (Panza et al., 2014). Targets that are not directly related to A[3 have also been pursued. One such target is N-methyl-D-aspartate (NMDA) receptors (NMDARs), a subclass of glutamate receptors. The antago- nist of NMDAR memantine has been approved for treating moderate to severe AD, although the exact mechanism un- derlying its action is still in debate (Kotermanski and John- son, 2009).展开更多
Objective To study the effect of salicylate on the expression and function of NMDA receptors in spiral ganglion neurons (SGNs). Methods The mRNA of NR1 subunit of NMDA receptor in modiolus tissues were detected by R...Objective To study the effect of salicylate on the expression and function of NMDA receptors in spiral ganglion neurons (SGNs). Methods The mRNA of NR1 subunit of NMDA receptor in modiolus tissues were detected by Real time fluorescence quantitative PCR (FQ-PCR). NMDA receptor whole-cell currents were recorded using patch clamp in acute isolated SGNs. Results Compared with the control group, salicylate significantly increased the mRNA level of NR1 subunit in SGNs. NMDA of concentrations ranging from 0.1 mM to 10 mM evoked no current in SGNs. NMDA (0. 1mM and 0.5 mM) applied with salicylate (5 mM), however, induced inward currents (212.6±15.2pA, n=5; 607.9±44.3pA, n=5) in a dose-dependent manner, which could be inhibited by APV. Salicylate alone did not produce any current in SGNs. Conclusion Salicylate increases the expression of NMDA receptors and facilitates the currents mediated by NMDA receptors in SGNs.展开更多
The N-methyl-d-aspartate(NMDA)receptors,which belong to the ionotropic Glutamate receptors,constitute a family of ligand-gated ion channels.Within the various subtypes of NMDA receptors,the GluN1/2A subtype plays a si...The N-methyl-d-aspartate(NMDA)receptors,which belong to the ionotropic Glutamate receptors,constitute a family of ligand-gated ion channels.Within the various subtypes of NMDA receptors,the GluN1/2A subtype plays a significant role in central nervous system(CNS)disorders.The present article aims to provide a comprehensive review of ligands targeting GluN2A-containing NMDA receptors,encompassing negative allosteric modulators(NAMs),positive allosteric modulators(PAMs)and competitive antagonists.Moreover,the ligands’structure–activity relationships(SARs)and the binding models of representative ligands are also discussed,providing valuable insights for the clinical rational design of effective drugs targeting CNS diseases.展开更多
Summary: Adult-bom neurons undergo a transient period of plasticity during their integration into the neural circuit. This transient plasticity may involve NMDA receptors containing NR2B, the major sub unit expressed...Summary: Adult-bom neurons undergo a transient period of plasticity during their integration into the neural circuit. This transient plasticity may involve NMDA receptors containing NR2B, the major sub unit expressed at early developmental stages. The main objective of the present study was to investigate the effects of NR2B gene knockdown on the functional integration of the adult-born granule cells gen- erated from the subgranule zone (SGZ) in the hippocampus. The small interfering RNA (siRNA) was used to knock down the NR2B gene in the adult-born hippocampal neurons. In the functional integration test, the mice were exposed to a novel environment (open field arena), and the expression of c-fos was immunohistochemically detected in the hippocampus. After exposure to the novel environment, siRNA-NR2B mice were significantly different from control mice in either the number of squares or the number of rears they crossed, showing decreased horizontal and vertical activity (P〈0.05). Moreover, the c-fos expression was increased in both control and siRNA-NR2B mice after open field test. But, it was significantly lower in siRNA-NR2B neurons than in control neurons. It was concluded that the neu- ral activity of newborn neurons is regulated by their own NR2B-containing NMDA glutamate receptors during a short, critical period after neuronal birth.展开更多
Previous reports have shown that N-methyl-D-aspartate (NMDA) receptors are extensively involved in epilepsy genesis and recurrence. Recent studies have shown that synaptic and extrasynaptic NMDA receptors play diffe...Previous reports have shown that N-methyl-D-aspartate (NMDA) receptors are extensively involved in epilepsy genesis and recurrence. Recent studies have shown that synaptic and extrasynaptic NMDA receptors play different, or even opposing, roles in various signaling pathways, including synaptic plasticity and neuronal death. The present study analyzed changes in synaptic and extrasynaptic NMDA receptor-mediated currents during epilepsy onset. Mouse models of lithium chloride pilocarpLne-induced epilepsy were established, and hippocampal slices were prepared at 24 hours after the onset of status epilepticus. Synaptic and extrasynaptic NMDA receptor-mediated excitatory post-synaptic currents (NMDA-EPSCs) were recorded in CA1 pyramidal neurons by whole-cell patch clamp technique. Results demonstrated no significant difference in rise and delay time of synaptic NMDA-EPSCs compared with normal neurons. Peak amplitude, area-to-peak ratio, and rising time of extrasynaptic NMDA-EPSCs remained unchanged, but decay of extrasynaptic NMDA-EPSCs was faster than that of normal neurons, These results suggest that extrasynaptic NMDA receptors play a role in epileptogenesis.展开更多
The latencies of motor- and somatosensory-evoked potentials were prolonged to different degrees, and wave amplitude was obviously decreased, after injection of dynorphin into the rat subarachnoid cavity. The wave ampl...The latencies of motor- and somatosensory-evoked potentials were prolonged to different degrees, and wave amplitude was obviously decreased, after injection of dynorphin into the rat subarachnoid cavity. The wave amplitude and latencies of motor- and somatosensory-evoked potentials were significantly recovered at 7 and 14 days after combined injection of dynorphin and either the kappa opioid receptor antagonist nor-binaltorphimine or the N-methyl-D-aspartate receptor antagonist MK-801. The wave amplitude and latency were similar in rats after combined injection of dynorphin and nor-binaltorphimine or MK-801. These results suggest that intrathecal injection of dynorphin causes damage to spinal cord function. Prevention of N-methyl-D-aspartate receptor or kappa receptor activation lessened the injury to spinal cord function induced by dynorphin.展开更多
This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine (L-NA) on the survival of retinal gangl...This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine (L-NA) on the survival of retinal ganglion cells (RGCs) at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 (1.0, 3.0 and 4.5 mg/kg) and L-NA (1.5, 3.0, 4.5 and 6.0 mg/kg) increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.展开更多
In the present study, 7 day postnatal C57/BL6 wild-type mice (hyperoxia group) and 7 day postnatal N-methyI-D-aspartate receptor subtype 3A knockout mice (NR3A KO group) were exposed to 75% oxygen and 15% nitrogen...In the present study, 7 day postnatal C57/BL6 wild-type mice (hyperoxia group) and 7 day postnatal N-methyI-D-aspartate receptor subtype 3A knockout mice (NR3A KO group) were exposed to 75% oxygen and 15% nitrogen in a closed container for 5 days. Wild-type mice raised in normoxia served as controls. TdT-mediated dUTP nick end labeling (TUNEL)/neuron-specific nuclear protein (NeuN) and 5-bromo-2'-deoxyuridine (BrdU)/NeuN immunofluorescence staining showed that the number of apoptotic cells and the number of proliferative cells in the dentate subgranular zone significantly increased in the hyperoxia group compared with the control group. However, in the same hyperoxia environment, the number of apoptotic cells and the number of proliferative cells significantly decreased in the NR3A KO group compared with hyperoxia group. TUNEL+/NeuN+ and BrdU+/NeuN~ cells were observed in the NR3A KO and the hyperoxia groups. These results demonstrated that the NR3A gene can promote cell apoptosis and mediate the potential damage in the developing brain induced by exposure to non-physiologically high concentrations of oxygen.展开更多
NMDA receptor channels play a significant role in learning and memory and their dysfunction can cause neuronal cell death leading to dementia. Research had shown that lipids change the risk for dementia, especially so...NMDA receptor channels play a significant role in learning and memory and their dysfunction can cause neuronal cell death leading to dementia. Research had shown that lipids change the risk for dementia, especially some omega-3 lipids appear to lower Alz-heimer’s risk, yet only limited research exists on the modulation of NMDA receptor channels by lipids. Here we review recent literature concerning molecular determinants that influence the NMDA receptor channel gating via membrane lipids and fatty acids with profound significance for understanding how altered NMDA signalling leads to neuronal cell death linked to age-related dementia’s. Future discovery of lipid-like modulators of NMDA receptor function offer the potential for the development of new bioceu-ticals and affordable nutritional supplements to combat neuronal degeneration as well as to promote well being and healthy aging.展开更多
BACKGROUND: Activation of N-methyl-D-aspartate receptor (NMDAR) is a key link of exitotoxicity at the phase of cerebral ischemic injury. Because NMDAR is a main way to mediate internal flow of Ca2+ among glutamic acid...BACKGROUND: Activation of N-methyl-D-aspartate receptor (NMDAR) is a key link of exitotoxicity at the phase of cerebral ischemic injury. Because NMDAR is a main way to mediate internal flow of Ca2+ among glutamic acid receptors, over-excitation can cause neuronal apoptosis. Calcitonin gene related peptide has a strongly biological activity. On one hand, it can protect ischemic neurons through inhibiting the expression of NMDAR1 mRNA; on the other hand, it can play the protective effect through down-regulating the expression of NMDAR1 mRNA by exogenous calcitonin gene related peptide. OBJECTIVE: To observe the expression of NMDAR1 and the regulatory effect of calcitonin gene related peptide on the expression of NMDAR1 mRNA and protein in the cerebral cortex of rats with focal cerebral ischemia/reperfusion (I/R). DESIGN: Randomized controlled animal study. SETTING: China Medical University. MATERIALS: A total of 216 healthy male Wistar rats, general grade, weighing 250-280 g, were selected in this study. Twelve rats were randomly selected to regard as control group; meanwhile, other 204 rats were used to establish middle cerebral artery occlusion/reperfusion (MACO) models. The main reagents were detailed as follows: calcitonin gene related peptide (Sigma Company); calcitonin gene related peptide kit (Boster Company); antibody Ⅰ, Ⅱ and antibody β-actin Ⅰ, Ⅱ of NMDAR1 mRNA and chemiluminescence reagent (Santa Cruz Company, USA). METHODS: The experiment was carried out in the Laboratory of Neurobiology of China Medical University from August 2005 to June 2006. ① Right MCAO models of rats were established to cause focal ischemia and scored based on Zea Longa five-grade scale. If the scores were 1, 2 and 3 after wakefulness, the MACO models were established successfully and involved in the experiment. A total of 120 rats with successful modeling were randomly divided into I/R group and administration group with 60 in each group. All rats in the both groups were observed at five time points, including 6, 12, 24, 48 and 72 hours after reperfusion and after 2-hour ischemia, with 12 experimental animals at each time point. Six rats were prepared for detection of hybridization in situ, and the other 6 were used for Western blotting histochemical detection. Rats in the control group were opened their skin to separate common carotid artery and not treated with line and drugs. In addition, rats in the I/R group were treated with 1 mL saline at 2 hours after focal cerebral ischemia, and then, rats in the administration group were treated with 1 mL (1 g/L) calcitonin gene related peptide at 2 hours after focal cerebral ischemia. ② The expression of NMDAR1 mRNA was detected with hybridization in situ at various time points; moreover, the expression of NMDAR1 protein was measured with Western blotting method at various time points. The results were analyzed with Metamoph imaging analytical system. MAIN OUTCOME MEASURES: The expression of NMDAR1 mRNA and its protein in cortical neurons of rats at various time points. RESULTS: A total of 84 rats were excluded because of non-symptoms, exanimation or death; and then, 132 rats were involved in the final analysis. The expression of NMDAR1 mRNA and its protein in cortical neurons of rats in the control group was 0.205±0.001 and 0.184±0.001, respectively; after I/R, expression of NMDAR1 mRNA and its protein was up-regulated, especially, expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.245±0.003, 0.287±0.004, 0.354±0.008, 0.284±0.002 and 0.217±0.006, respectively; moreover, expression of protein at 6, 12, 24, 48 and 72 hours was 0.222±0.003, 0.261±0.028, 0.311±0.004, 0.259±0.013 and 0.210±0.008, respectively. There was significant difference between the two groups (0.205±0.001, P < 0.01). The expression was up-related in the former 24 hours, reached peak at 24 hours, down-regulated, and decreased to the level of control group at 72 hours. Except 72 hours, the expression of NMDAR1 mRNA and its protein was lower in administration group than that in I/R group at other four time points. In addition, the expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.223±0.005, 0.243±0.001, 0.292±0.002, 0.250±0.003 and 0.213±0.003, respectively; moreover, the expression of protein at 6, 12, 24, 48 and 72 hours was 0.216±0.006, 0.245±0.025, 0.276±0.003, 0.241±0.045 and 0.202±0.013, respectively. There was significant difference at various time points (P < 0.05). CONCLUSION: The expressions of NMDAR1 mRNA and its protein of peripheral cortical neurons are up-related in ischemic area after focal cerebral I/R. Meanwhile, exogenous calcitonin gene related peptide can protect cortical neurons through inhibiting expression of NMDAR1 mRNA and its protein after focal cerebral I/R.展开更多
文摘N-methyl-D-aspartate glutamate receptors(NMDARs)play crucial roles in the pathogenesis of neuronal injuries following a stroke insult;therefore,a plethora of preclinical studies focus on better understanding functions of NMDARs and their associated signaling pathways.Over the past decades,NMDARs have been found to exert dual effects in neuronal deaths signaling and neuronal survival signaling during cerebral ischemia.Moreover,many complex intracellular signaling pathways downstream of NMDAR activation have been elucidated,which provide novel targets for developing much-needed neuro-protectants for patients with stroke.In this review,we will discuss the recent progress in understanding the underlying mechanisms of stroke related to NMDAR activation and the potential therapeutic strategies based on these discoveries.
文摘BACKGROUND Some isopavines can exhibit important biological activity in the treatment of neurological disorders since it is considered an antagonist of the specific Nmethyl-D-Aspartate(NMDA)receptor.Amurensinine is an isopavine which still has few studies.In view of the potential of isopavines as NMDA receptor antagonists,theoretical studies using bioinformatics were carried out in order to investigate whether Amurensinine binds to the NMDA receptor and to analyze the receptor/Ligand complex.This data can contribute to understanding of the onset of neurological diseases and contribute to the planning of drugs for the treatment of neurological diseases involving the NMDA receptor.AIM To investigate the interaction of the antagonist Amurensinine on the GluN1A/GluN2B isoform of the NMDA receptor using bioinformatics.METHODS The three-dimen-sional structure of the GluN1A/GluN2B NMDA receptor was selected from the Protein Data Bank(PDB)-PDB:4PE5,and the three-dimensional structure of Amurensinine(ligand)was designed and optimized using ACD/SchemsketchTM software.Prediction of the protonation state of Amurensinine at physiological pH was performed using MarvinSketch software(ChemAxon).Protonated and non-protonated Amurensin were prepared using AutoDock Tools 4 software and simulations were performed using Autodock Vina v.1.2.0.The receptor/Ligand complexes were analyzed using PyMol(Schrödinger,Inc)and BIOVIA Discovery Studio(Dassault Systemes)software.To evaluate the NMDA receptor/Amurensinine complex and validate the molecular docking,simulations using NMDA receptor and Ifenprodil antagonist were performed under the same conditions.Ifenprodil was also designed,optimized and protonated,under the same conditions as Amurensinine.RESULTS Molecular docking simulations showed that both non-protonated and protonated Amurensinine bind to the amino terminal domain(ATD)domain of the GluN1A/GluN2B NMDA receptor with significant affinity energy,-7.9 Kcal/mol and-8.1 Kcal/mol,respectively.The NMDA receptor/non-protonated Amurensinine complex was stabilized by 15 bonds,while the NMDA receptor/protonated Amurensinine complex was stabilized by less than half,6 bonds.Despite the difference in the number of bonds,the variation in bond length and the average bond length values are similar in both complexes.The complex formed by the NMDA receptor and Ifenprodil showed an affinity energy of-8.2 Kcal/mol,a value very close to that obtained for the NMDA receptor/Amurensinine complex.Molecular docking between Ifenprodil and the GluN1A/GluN2B NMDA receptor demonstrated that this antagonist interacts with the ATD of the receptor,which validates the simulations performed with Amurensinine.CONCLUSION Amurensinine binds to the NMDA receptor on ATD,similar to Ifenprodil,and the affinity energy is closer.These data suggest that Amurensinine could behave as a receptor inhibitor,indicating that this compound may have a potential biological application,which should be evaluated by in vitro and preclinical assays.
文摘Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis is a rare disease with uncertain etiology and pathogenesis that affects young women. Its diagnosis can be delayed because of the nonspecific neuropsychiatric symptoms in the foreground. This article describes the details of a recent complicated case of a patient with this condition which is related to an ovarian teratoma. Correct diagnostic and prompt treatment of anti-NMDA receptor encephalitis remains a serious clinical challenge due to its unspecific manifestations and varying response to treatments. The information will be of interest to clinicians working with encephalitis patients.
基金supported by the National Natural Science Foundation of China,No.81160169(to JL),81460214(to JL),31660270(to JD),31460255(to JD)the Natural Science Foundation of Ningxia Hui Autonomous Region of China,No.2018AAC02005(to JL)
文摘N-methyl-D-aspartate receptor hypofunction is the basis of pathophysiology in schizophrenia. Blocking the N-methyl-D-aspartate receptor impairs learning and memory abilities and induces pathological changes in the brain. Previous studies have paid little attention to the role of the N-methyl-D-aspartate receptor subunit 1 (NR1) in neurogenesis in the hippocampus of schizophrenia. A mouse model of schizophrenia was established by intraperitoneal injection of 0.6 mg/kg MK-801, once a day, for 14 days. In N-methyl-D-aspartate-treated mice, N-methyl-D-aspartate was administered by intracerebroventricular injection in schizophrenia mice on day 15. The number of NR1-, Ki67- or BrdU-immunoreactive cells in the dentate gyrus was measured by immunofluorescence staining. Our data showed the number of NR1-immunoreactive cells increased along with the decreasing numbers of BrdU- and Ki67-immunoreactive cells in the schizophrenia groups compared with the control group. N-methyl-D-aspartate could reverse the above changes. These results indicated that NR1 can regulate neurogenesis in the hippocampal dentate gyrus of schizophrenia mice, supporting NR1 as a promising therapeutic target in the treatment of schizophrenia. This study was approved by the Experimental Animal Ethics Committee of the Ningxia Medical University, China (approval No. 2014-014) on March 6, 2014.
基金Provided financial assistance by"Hundred Talented Projects"of Shanghai Health Bureau (No. 97BR016)
文摘Objective: To observe and elucidate the neuroprotective effect of Xingnaojing (XNJ) injection on hippocampal N-methyl-D-aspartic acid (NMDA) receptors of focal cerebral ischemia in rats. Methods: Cerebral ischemia was established by occluding the middle cerebral artery with an intraluminal suture technique in rats. Neurological deficit score, infarct volume and quantity of NMDA receptors were estimated in all groups and compared. Results: After being treated with XNJ, the score decreased in the initial 6 hours and infarct volume decreased in 24 hours. And within 24 hours, the quantity of NMDA receptors obviously decreased compared with the model group (P<0. 01) It indicated that XNJ could ameliorate neurological behavior of middle cerebral artery occlusion rats and down-regulate the expression of hippocampal NMDA receptors. Conclusion: The neuroprotective effect of XNJ on focal cerebral ischemia is possibly related to down-regulating the expression of NMDA receptors in rats.
基金supported by National Natural Science Foundation of China,No. 30500482
文摘Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine.The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior,electroencephalography and 24-hour survival rate.Propofol(12.5-100 mg/kg) improved status epilepticus in a dose-dependent manner,and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection.Western blot results showed that,24 hours after induction of status epilepticus,the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus.Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels,but not the increase in N-methyl-D-aspartate receptor 2A subunit levels.The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine.This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures.
基金the National Natural Science Foundation of China, No. 30470551a Category Project of the Education Department of Liaoning Province, No. 05L1021
文摘BACKGROUND:Glutamate acid ionotropic receptor N-methyl-D-aspartic acid (NMDA) takes part in long-term potentiation, thereby influencing the process of learning and memory. OBJECTIVE: To verify expression of NMDA 2A/B and 2B receptors in the anterior thalamic nucleus and subiculum complex of rats. DESIGN, TIME AND SETTING: A single-sample observation was performed at Department of Anatomy in Dalian Medical University (Dalian, Liaoning, China) from April to September in 2007. MATERIALS: Ten adult Wistar rats were used for this study, as well as rabbit anti-NMDA 2A/B and 2B antibodies. METHODS: The rats were anesthetized and perfused, followed by brain resection and coronal sectioning of the brain tissue. A 1:3 series was selected for immunohistochemistry, using antibodies specific to NMDA 2A/B and 2B receptors. Photos were taken using the Nikon image analysis system. MAIN OUTCOME MEASURES: Expression and distribution of immunohistochemistry staining of NMDA 2A/B and 2B receptor subunits. RESULTS: There were a large number of NMDA 2A/B and 2B receptor-positive neurons distributed throughout the anterior dorsal thalamic nucleus. In the anterior ventral thalamic nucleus, distribution of positive neurons was rare, staining intensity was lighter, and cell bodies were smaller compared with the anterior dorsal thalamic nucleus. In the subiculum complex, staining intensity of NMDA 2A/B and 2B-positive neurons was weakest in the molecular layer and stronger in the pyramidal layer, in particular the region with large cell bodies adjacent to the molecular layer. In the multiform layer, more positive neurons of various sizes were detected. CONCLUSION: NMDA 2A/B and 2B receptor subunits were richly distributed in the anterior thalamic nucleus, with a small difference existing between the anterior dorsal nucleus and anterior ventral nucleus. These neurons were also differentially distributed within the three layers of the subiculum complex.
文摘AIM: Many studies have demonstrated N-methyl-D-aspartate receptor-1-subunit (NMDAR1) is associated with amblyopia. The effectiveness of levodopa in improving the visual function of the children with amblyopia has also been proved. But the mechanism is undefined. Our study was to explore the possible mechanism. METHODS: Sixty 14-day-old healthy SD rats were randomly divided into 4 groups, including normal group, monocular deprivation group, levodopa group and normal saline group, 15 rats each. We sutured all the rats' unilateral eyelids except normal group to establish the monocular deprivation animal model and raise them in normal sunlight till 45-day-old. NMDAR1 was detected in the visual cortex with immunohistochemistry methods, Western Blot and Real time PCR. LD and NS groups were gavaged with levodopa (40mg/kg) and normal saline for 28 days respectively. NMDAR1 was also detected with the methods above. RESULTS: NMDAR1 in the visual cortex of MD group was less than that of normal group. NMDAR1 in the visual cortex of LD group was more than that of NS group. CONCLUSION: NMDAR1 is associated with the plasticity of visual development. Levodopa may influence the expression of NMDAR1 and improve visual function, and its target may lie in the visual cortex.
基金ThisprojectwassupportedbyagrantfromNationalNaturalSciencesFoundationofChina (No .30 0 4 0 0 37)
文摘To study the effect of glutamate on the intracellular calcium signal of pure cultured rat astrocytes and the role of NMDA and AMPA receptors in the procedure, the change of calcium signal was investigated by monitoring the fluctuation of intracellular Ca 2+ concentration ([Ca 2+ ] i) on the basis of Fura-2 single cell fluorescent ratio (F345/F380). The changes in the effect of glutamate on the intracellular calcium signal were observed after blockage of NMDA and(or) AMPA receptors. It was found that L-glutamate could induce an increased [Ca 2+ ] i in most of the cells in concentration- and time-dependent manner. D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5, a selective antagonist of the NMDA receptor) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selective antagonist of the AMPA receptor) could abolish the effects of NMDA and AMPA respectively. The treatment of D-AP-5 and CNQX simultaneously or respectively could attenuate the effect of L-glutamate at varying degrees. All these indicated that glutamate could modulate intracellular Ca 2+ of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA receptors took part in the complex mechanisms.
文摘Alzheimer's disease (AD) is one of the most devastating dis- eases affecting the life and health of aging population. Two hallmarks of AD are senile plaques and neurofibrillary tan- gles, and AD is well known for the massive loss of neurons and impaired cognitive functions especially memory loss. Despite extensive search for effective treatment, available drugs have limited efficacy without affecting the course of AD. Significant efforts have been devoted to curb the pro- duction of amyloid [3 (A[3; the major component of plaques) or enhance the clearance of it, with the aim to reduce the accumulation of plaque in the brain. Antibodies that can bind A[3 to increase their removal have received a lot of at- tention although recent clinical trial results have been largely negative and disappointing (Panza et al., 2014). Targets that are not directly related to A[3 have also been pursued. One such target is N-methyl-D-aspartate (NMDA) receptors (NMDARs), a subclass of glutamate receptors. The antago- nist of NMDAR memantine has been approved for treating moderate to severe AD, although the exact mechanism un- derlying its action is still in debate (Kotermanski and John- son, 2009).
基金supported by a grant from National Nature Science Fund of China(No.81060082,30860098)Nature Science Fund of Guangxi(No.2011jjA40056)to Jiping Su
文摘Objective To study the effect of salicylate on the expression and function of NMDA receptors in spiral ganglion neurons (SGNs). Methods The mRNA of NR1 subunit of NMDA receptor in modiolus tissues were detected by Real time fluorescence quantitative PCR (FQ-PCR). NMDA receptor whole-cell currents were recorded using patch clamp in acute isolated SGNs. Results Compared with the control group, salicylate significantly increased the mRNA level of NR1 subunit in SGNs. NMDA of concentrations ranging from 0.1 mM to 10 mM evoked no current in SGNs. NMDA (0. 1mM and 0.5 mM) applied with salicylate (5 mM), however, induced inward currents (212.6±15.2pA, n=5; 607.9±44.3pA, n=5) in a dose-dependent manner, which could be inhibited by APV. Salicylate alone did not produce any current in SGNs. Conclusion Salicylate increases the expression of NMDA receptors and facilitates the currents mediated by NMDA receptors in SGNs.
基金The work was supported by the National Natural Science Foundation of China(82204200)the International Postdoctoral Exchange Fellowship Program(Talent-Introduction Program,YJ20210279,China)+1 种基金the China Postdoctoral Science Foundation(2022M711939)to Fabao Zhao,and the Natural Science Foundation of Shandong Province(ZR2022QH287 to Fabao Zhao and ZR2022QH312 to Na Liu,China).
文摘The N-methyl-d-aspartate(NMDA)receptors,which belong to the ionotropic Glutamate receptors,constitute a family of ligand-gated ion channels.Within the various subtypes of NMDA receptors,the GluN1/2A subtype plays a significant role in central nervous system(CNS)disorders.The present article aims to provide a comprehensive review of ligands targeting GluN2A-containing NMDA receptors,encompassing negative allosteric modulators(NAMs),positive allosteric modulators(PAMs)and competitive antagonists.Moreover,the ligands’structure–activity relationships(SARs)and the binding models of representative ligands are also discussed,providing valuable insights for the clinical rational design of effective drugs targeting CNS diseases.
基金supported by the National Natural Science Foundation of China(No.81000537)
文摘Summary: Adult-bom neurons undergo a transient period of plasticity during their integration into the neural circuit. This transient plasticity may involve NMDA receptors containing NR2B, the major sub unit expressed at early developmental stages. The main objective of the present study was to investigate the effects of NR2B gene knockdown on the functional integration of the adult-born granule cells gen- erated from the subgranule zone (SGZ) in the hippocampus. The small interfering RNA (siRNA) was used to knock down the NR2B gene in the adult-born hippocampal neurons. In the functional integration test, the mice were exposed to a novel environment (open field arena), and the expression of c-fos was immunohistochemically detected in the hippocampus. After exposure to the novel environment, siRNA-NR2B mice were significantly different from control mice in either the number of squares or the number of rears they crossed, showing decreased horizontal and vertical activity (P〈0.05). Moreover, the c-fos expression was increased in both control and siRNA-NR2B mice after open field test. But, it was significantly lower in siRNA-NR2B neurons than in control neurons. It was concluded that the neu- ral activity of newborn neurons is regulated by their own NR2B-containing NMDA glutamate receptors during a short, critical period after neuronal birth.
基金Shanghai Pujiang Program,No. 06PJ14053the National Natural Science Foundation of China,No. 30600177+2 种基金the Scientific Research Founda-tion for the Returned Over-seas Chinese Scholars,State Education MinistryDoctoral Fund of Ministry of Education of China,No. 20070248083Shanghai Leading Aca-demic Discipline Project,No. B205
文摘Previous reports have shown that N-methyl-D-aspartate (NMDA) receptors are extensively involved in epilepsy genesis and recurrence. Recent studies have shown that synaptic and extrasynaptic NMDA receptors play different, or even opposing, roles in various signaling pathways, including synaptic plasticity and neuronal death. The present study analyzed changes in synaptic and extrasynaptic NMDA receptor-mediated currents during epilepsy onset. Mouse models of lithium chloride pilocarpLne-induced epilepsy were established, and hippocampal slices were prepared at 24 hours after the onset of status epilepticus. Synaptic and extrasynaptic NMDA receptor-mediated excitatory post-synaptic currents (NMDA-EPSCs) were recorded in CA1 pyramidal neurons by whole-cell patch clamp technique. Results demonstrated no significant difference in rise and delay time of synaptic NMDA-EPSCs compared with normal neurons. Peak amplitude, area-to-peak ratio, and rising time of extrasynaptic NMDA-EPSCs remained unchanged, but decay of extrasynaptic NMDA-EPSCs was faster than that of normal neurons, These results suggest that extrasynaptic NMDA receptors play a role in epileptogenesis.
基金Key Science and Technology Research and Development Program of Liaoning Province, China, No. 20112250021, 20112250041.
文摘The latencies of motor- and somatosensory-evoked potentials were prolonged to different degrees, and wave amplitude was obviously decreased, after injection of dynorphin into the rat subarachnoid cavity. The wave amplitude and latencies of motor- and somatosensory-evoked potentials were significantly recovered at 7 and 14 days after combined injection of dynorphin and either the kappa opioid receptor antagonist nor-binaltorphimine or the N-methyl-D-aspartate receptor antagonist MK-801. The wave amplitude and latency were similar in rats after combined injection of dynorphin and nor-binaltorphimine or MK-801. These results suggest that intrathecal injection of dynorphin causes damage to spinal cord function. Prevention of N-methyl-D-aspartate receptor or kappa receptor activation lessened the injury to spinal cord function induced by dynorphin.
基金supported by research grants from Chinese National Key Project for Basic Research,No. 2011CB504402the National Natural Science Foundation of China, No. 30901649 and 30872829
文摘This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine (L-NA) on the survival of retinal ganglion cells (RGCs) at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 (1.0, 3.0 and 4.5 mg/kg) and L-NA (1.5, 3.0, 4.5 and 6.0 mg/kg) increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.
基金supported by the National Institutes of Health, USA, No. NS 045810, NS 057255the BasicClinical Scientific Research Foundation Program of the Capital Medical University, China, No. 2006JL19
文摘In the present study, 7 day postnatal C57/BL6 wild-type mice (hyperoxia group) and 7 day postnatal N-methyI-D-aspartate receptor subtype 3A knockout mice (NR3A KO group) were exposed to 75% oxygen and 15% nitrogen in a closed container for 5 days. Wild-type mice raised in normoxia served as controls. TdT-mediated dUTP nick end labeling (TUNEL)/neuron-specific nuclear protein (NeuN) and 5-bromo-2'-deoxyuridine (BrdU)/NeuN immunofluorescence staining showed that the number of apoptotic cells and the number of proliferative cells in the dentate subgranular zone significantly increased in the hyperoxia group compared with the control group. However, in the same hyperoxia environment, the number of apoptotic cells and the number of proliferative cells significantly decreased in the NR3A KO group compared with hyperoxia group. TUNEL+/NeuN+ and BrdU+/NeuN~ cells were observed in the NR3A KO and the hyperoxia groups. These results demonstrated that the NR3A gene can promote cell apoptosis and mediate the potential damage in the developing brain induced by exposure to non-physiologically high concentrations of oxygen.
文摘NMDA receptor channels play a significant role in learning and memory and their dysfunction can cause neuronal cell death leading to dementia. Research had shown that lipids change the risk for dementia, especially some omega-3 lipids appear to lower Alz-heimer’s risk, yet only limited research exists on the modulation of NMDA receptor channels by lipids. Here we review recent literature concerning molecular determinants that influence the NMDA receptor channel gating via membrane lipids and fatty acids with profound significance for understanding how altered NMDA signalling leads to neuronal cell death linked to age-related dementia’s. Future discovery of lipid-like modulators of NMDA receptor function offer the potential for the development of new bioceu-ticals and affordable nutritional supplements to combat neuronal degeneration as well as to promote well being and healthy aging.
文摘BACKGROUND: Activation of N-methyl-D-aspartate receptor (NMDAR) is a key link of exitotoxicity at the phase of cerebral ischemic injury. Because NMDAR is a main way to mediate internal flow of Ca2+ among glutamic acid receptors, over-excitation can cause neuronal apoptosis. Calcitonin gene related peptide has a strongly biological activity. On one hand, it can protect ischemic neurons through inhibiting the expression of NMDAR1 mRNA; on the other hand, it can play the protective effect through down-regulating the expression of NMDAR1 mRNA by exogenous calcitonin gene related peptide. OBJECTIVE: To observe the expression of NMDAR1 and the regulatory effect of calcitonin gene related peptide on the expression of NMDAR1 mRNA and protein in the cerebral cortex of rats with focal cerebral ischemia/reperfusion (I/R). DESIGN: Randomized controlled animal study. SETTING: China Medical University. MATERIALS: A total of 216 healthy male Wistar rats, general grade, weighing 250-280 g, were selected in this study. Twelve rats were randomly selected to regard as control group; meanwhile, other 204 rats were used to establish middle cerebral artery occlusion/reperfusion (MACO) models. The main reagents were detailed as follows: calcitonin gene related peptide (Sigma Company); calcitonin gene related peptide kit (Boster Company); antibody Ⅰ, Ⅱ and antibody β-actin Ⅰ, Ⅱ of NMDAR1 mRNA and chemiluminescence reagent (Santa Cruz Company, USA). METHODS: The experiment was carried out in the Laboratory of Neurobiology of China Medical University from August 2005 to June 2006. ① Right MCAO models of rats were established to cause focal ischemia and scored based on Zea Longa five-grade scale. If the scores were 1, 2 and 3 after wakefulness, the MACO models were established successfully and involved in the experiment. A total of 120 rats with successful modeling were randomly divided into I/R group and administration group with 60 in each group. All rats in the both groups were observed at five time points, including 6, 12, 24, 48 and 72 hours after reperfusion and after 2-hour ischemia, with 12 experimental animals at each time point. Six rats were prepared for detection of hybridization in situ, and the other 6 were used for Western blotting histochemical detection. Rats in the control group were opened their skin to separate common carotid artery and not treated with line and drugs. In addition, rats in the I/R group were treated with 1 mL saline at 2 hours after focal cerebral ischemia, and then, rats in the administration group were treated with 1 mL (1 g/L) calcitonin gene related peptide at 2 hours after focal cerebral ischemia. ② The expression of NMDAR1 mRNA was detected with hybridization in situ at various time points; moreover, the expression of NMDAR1 protein was measured with Western blotting method at various time points. The results were analyzed with Metamoph imaging analytical system. MAIN OUTCOME MEASURES: The expression of NMDAR1 mRNA and its protein in cortical neurons of rats at various time points. RESULTS: A total of 84 rats were excluded because of non-symptoms, exanimation or death; and then, 132 rats were involved in the final analysis. The expression of NMDAR1 mRNA and its protein in cortical neurons of rats in the control group was 0.205±0.001 and 0.184±0.001, respectively; after I/R, expression of NMDAR1 mRNA and its protein was up-regulated, especially, expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.245±0.003, 0.287±0.004, 0.354±0.008, 0.284±0.002 and 0.217±0.006, respectively; moreover, expression of protein at 6, 12, 24, 48 and 72 hours was 0.222±0.003, 0.261±0.028, 0.311±0.004, 0.259±0.013 and 0.210±0.008, respectively. There was significant difference between the two groups (0.205±0.001, P < 0.01). The expression was up-related in the former 24 hours, reached peak at 24 hours, down-regulated, and decreased to the level of control group at 72 hours. Except 72 hours, the expression of NMDAR1 mRNA and its protein was lower in administration group than that in I/R group at other four time points. In addition, the expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.223±0.005, 0.243±0.001, 0.292±0.002, 0.250±0.003 and 0.213±0.003, respectively; moreover, the expression of protein at 6, 12, 24, 48 and 72 hours was 0.216±0.006, 0.245±0.025, 0.276±0.003, 0.241±0.045 and 0.202±0.013, respectively. There was significant difference at various time points (P < 0.05). CONCLUSION: The expressions of NMDAR1 mRNA and its protein of peripheral cortical neurons are up-related in ischemic area after focal cerebral I/R. Meanwhile, exogenous calcitonin gene related peptide can protect cortical neurons through inhibiting expression of NMDAR1 mRNA and its protein after focal cerebral I/R.