| Objectives: Excessive activation of NMDA receptors(NMDARs)is implicated in pathological form of plasticity named post-ischemic long-term potentiation(i-LTP)which was observed in glutamate mediated excitotoxity after stroke.In the past decades,many efforts focused on the NMDARs inhibitors for the therapeutic intervention of stroke.However,most of these compounds designed to directly inhibit NMDARs caused severe psychotomimetic side effects and failed in clinical investigations in unexcepted way.Recently,in light of the researches which reported the hypofunction of NMDARs in stroke and other neurodegenerative diseases,our study focus on enhancing NMDAR fuctions and improving synaptic plasticity after reducing the excitotoxicity.GLYX-13 is a NMDA receptor modulator with glycine site partial agonist properties.It was reported that GLYX-13 appears promising in reducing ischemic neuronal death in previous study.However,the underlying molecular mechanism of GLYX-13 attenuating the ischemic neuronal damage remains elusive.Bases on vivo MCAO and vitro OGD model,our study aims to investigate the effects of GLYX-13 on NMDARs and synaptic plasticity in stroke and characterizes the underlying molecular mechanisms.Methods: To study of the neuroprotective effects of GLYX-13 on MCAO model(1)Animals and groups: 2-month-old male C57BL/6 mice were randomly divided into 3 groups.They were named Sham,Vehicle,and GLYX-13 respectively.(2)Established the transient middle Cerebral Artery Occlusion(MCAO)model in vivo: the silicon coated monofilament was inserted in the right MCA of mice.The occlusion was maintained 60 min.(3)Cerebral blood flow was measured by Laser Doppler Flowmetry: it is used to evaluate the success of transient MCAO and only those mice which ipsilateral regional cerebral blood flow(after MCAO)?20% of baseline were used for further study.(4)Application of GLYX-13: GLYX-13 were administrated in mice by intraperitoneal injection(10mg/kg).(5)Neurological score also contributed to evaluate the success of transient MCAO and to estimate the degree of neurological deficits and severity of the injury.0: Normal;1: mild circling and <50% attempts to rotate to contralateral side;2: mild circling with >50% attempts to rotate to contralateral side;3: consistent circling with a rotation position more than 1-2sec and the nose of mice almost reaches its tail;4: severe rotation and lose the reflex;5: coma or moribund.(6)TTC staining was performed to measure the infarct volume in each group.(7)HE staining and Fluoro Jade C staining were performed to assess the extent of neuronal injury in each group.To explore the protective mechanism of GLYX-13 on ischemic neuronal injury(1)Western blot analysis was performed to determine the expression of NMDAR subunit(NR2A,NR2 B,p-NR2 A,p-NR2B)4h or 24 h after reperfusion.(2)Electrophysiological study(whole cell patch clamp):(1)Groups: Con,OGD,OGD+10?mol/L GLYX-13,OGD+10?mol/L GLYX-13+100?mol/L D-serine.The above drugs were administrated when OGD began,and they were added into the ACSF during examination.(2)Established the Oxygen glucose deprivation model in vitro: OGD model used to mimic the situation of lack the energy supply in ischemic brain tissue.OGD was induced by replacing 95%O2+5%CO2 with 95%N2+5%CO2 and switching to an ACSF containing 10mmol/L sucrose instead of glucose for 5min.(3)Whole cell patch clamp recording: synaptic responses were evoked by stimulating the Schaffer collateral pathway.EPSC,NMDA-EPSC and NR2 A or NR2 B –EPSC were recorded.(3)TTC staining was performed to measure the infarct volume in vivo MCAO model in following 5 groups: Vehicle,GLYX-13,GLYX-13+D-serine,GLYX-13+ NVP-AAM077,GLYX-13+Ro256981.Results: 1.GLYX-13 exerts potential neuroprotective effects on MCAO model mice in vivo.(1)GLYX-13 treatment reduced Neurological severity scores in MCAO mice 24 h after reperfusion.(2)GLYX-13 decreased the infarct volume in MCAO mice 24 h after reperfusion..(3)GLYX-13 treatment reduced injury of neurons in hippocampus caused by MCAO 24 h after reperfusion.2.GLYX-13 attenuates cerebral ischemia injury in vivo and vitro ischemic model by differential modulations of NMDAR subunit components.(1)Results of Western blot analysis : GLYX-13 down-regulated the phosphorylated level of NR2 B at site Tyr 1472 and up-regulated the phosphorylation of NR2 A at site Tyr 1325 in vivo MCAO mice 4hrs after reperfusion.Besides GLYX-13 reduced the loss of NR2 A 24hrs after reperfusion.(2)Results of Electrophysiological study:(1)GLYX-13 is against pathological synaptic plasticity by targeting the NMDAR glycine coagonist site.(2)GLYX-13 regulated the NMDAR subunit components which have been already changed by OGD.(3)GLYX-13 exerts neuroprotective effects via elevating NR2 A components with reducing NR2 B components in vivo MCAO model.Conclusion: 1.GLYX-13 exerts potential neuroprotective effects on MCAO model mice in vivo.(1)GLYX-13 treatment reduced Neurological severity scores in MCAO mice 24 h after reperfusion.(2)GLYX-13 decreased the infarct volume in MCAO mice 24 h after reperfusion.(3)GLYX-13 treatment reduced injury of neurons in hippocampus caused by MCAO 24 h after reperfusion.2.GLYX-13 attenuates cerebral ischemia injury in vivo and vitro ischemic model by differential modulations of NMDAR subunit components.(1)GLYX-13 down-regulated the phosphorylated level of NR2 B at site Tyr 1472 and up-regulated the phosphorylation of NR2 A at site Tyr 1325 in vivo MCAO mice 4hrs after reperfusion.Besides GLYX-13 reduced the loss of NR2 A 24hrs after reperfusion.(2)GLYX-13 is against pathological synaptic plasticity by targeting the NMDAR glycine coagonist site in vitro OGD model.(3)GLYX-13 regulated the NMDAR subunit components which have been already changed by OGD.(4)GLYX-13 exerts neuroprotective effects via elevating NR2 A components with reducing NR2 B components in vivo MCAO model. |
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