Involvement Of Neuronal GluR2in Cerebral Ischemic Tolerance Induced By Electroacupuncture Pretreatment Through Cannabinoid CB1Receptor In Mice

Stroke is a leading cause of death and disability in adult. The major pathophysiologyfor stroke to lead to poor outcomes is ischemic cerebral injury. The accumulation ofglutamate after ischemia results in excessive stimulation of glutamate receptors leadingto neurotoxicity. Glutamate activates two major sub-families of ligand-gated postsynapticreceptors:α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) andN-methyl-D-aspartate receptor (NMDAR). AMPA receptor distribution is widely incentral nervous system, but there are obvious regional differences in its expression. AMPAreceptor was mainly expressed in the hippocampal CA1pyramidal neurons. This supportsa more pivocal role for AMPA-type glutamate receptors in the selective pattern ofneuronal loss in the hippocampus injury associated with global ischemia. AMPARs are heteromeric complexes composed of glutamate receptor subunit1–4(GluR1–GluR4). TheGluR2subunit dictates Ca2+permeability of AMPA receptor channels. The expression ofGluR2in neurons is not static and is altered after nerve system disease, especially inischemic insult. Global ischemia triggers down-regulation of GluR2protein, and enhancesAMPAR-mediated Ca2+influx in vulnerable CA1pyramidal neurons before the onset ofneuronal death.A rise in intracellular Ca2+is thought to initiate a cascade of eventsleading to the cell death. Many neuroprotective drugs are designed to inhibit excitotoxicityby acting as AMPAR antagonists. However, the clinical application of these antagonists islimted because of directly blocking AMPA receptor function which is important in normalphysiological condition. Because of the ability of GluR2subunits to adjust the action ofcalcium ion, it will be the new target which cure strok.Previous study has showed that anandamide directly inhibits AMPA receptorsubunit recombinant in Xenopus oocytes, revealing the close relationship betweenendocannabinoid and glutamate receptor. Other research showed that the endocannabinoidAEA and2-AG are activated in cerebral cells in response to excitotoxicity induced byactivition of AMPAR. The activation of the endogenous cannabinoid system plays aneuroprotective role through the cannabinoid receptors, and the inhibitor ofendocannabinoid uptake, UCM707, protected specifically against AMPA-inducedexcitotoxicity, by activating CB1Rand CB2R. The condition of excitotoxicity elicited byKA administration rapidly raised levels of endocannabinoid in hippocampus, and theseendocannabinoid induced protective mechanisms in wild-type mice. These protectivemechanisms could not be triggered in CB1knockout mice. It was observed thatWIN55.212-2inhibited the release of glutamate, which was counteracted by SR141716A.There are a series of results showing that the activation of endocannabinoids system canresist excitotoxicity induced by overly activating AMPAR, and the effect has closerelationship with CB1cannabinoid receptors.Our previous investigation has demonstrated that pretreatment withelectroacupuncture (EA) at Baihui acupoint could modulate the endocannabinoid systemby upregulating the production of the endocannabinoids, including2-arachidonylglycerol (2-AG) and anandamide (AEA), which induced ischemic tolerance through acting oncannabinoid receptors. But the mechanisms for electroacupuncturepreconditioning-induced neuroprotection are not fully understood.Therefore, we used the C57BL/6to investigated whether activation of GluR2is produced by EA pretreatment via cannabinoid CB1receptors (CB1R) aftertransient global cerebral ischemia in mice. The aim of the study was to optimize thesystem of the cerebral ischemia tolerance induced by acupuncture preconditioning, and tobring new perspective and an opportunity for AMPA treatment of stroke.Experiment1：The effect of EA pretreatment on the GluR2expression after globalcerebral ischemiaObject: Cerebral ischemia injury decrease hippocampal neurons GluR2proteinexpression, and this cause the intracellular calcium overload. This study observe whetherEA pretreatment influence the GluR2protein expression.Methods：1. The expression of GluR2subunit at different time after global cerebralischemia-reperfusion injury.30adult male C57mice were divided randomly into two groups: Sham and I/R groups.Mice in I/R groups were subjected to global cerebral ischemia for20min. Animals werekilled at2,6,24,48,72hours after reperfusion. Western blotting was used to detect GluR2expression in hippocampal neurons at different time.2. The effect of EA pretreatment on GluR2subunit expression.20adult male C57mice were randomly divided into four groups: Sham, Con, EA+GCIand EA groups. Animals in Con and EA+GCI groups were subjected to global cerebralischemia for20min. In EA+GCI group, mice were given EA pretreatment2hours beforeglobal cerebral ischemia. Mice were killed at2hours after reperfusion. Western blottingand NeuN/GluR2double-staining immunohistochemistry were used to detect GluR2expression in hippocampal neuron.Results： 1. Two hours after reperfusion, there was a marked reduction in GluR2expression in thepyramidal neurons.Western blotting indicated that GluR2expression was significantly decreased at twohours after reperfusion in the I/R group compared with that of the Sham group,(P<0.05).But no difference was found in the content of GluR2among different time.2. EA pretreatment enhanced GluR2activation in hippocampal CA1pyramidal neuronsafter global cerebral ischemia reperfusion.Western blotting detected that Pretreatment with EA significantly increased the GluR2protein expression in the hippocampus compared with the Con group(P<0.05). But nodifference was found that the GluR2protein expression level of between EA and Shamgroup. The immunofluorescence test indicated that GluR2-positive cells co-localized withNeuN positive neurons.Conclusion: EA preconditioning can affect the whole GluR2protein expression ofhippocampal neurons after cerebral ischemia.Experiment2：Study on the involvement of GluR2in neuroprotection induced by EApreconditioningObject: Experiment1showed that EA increase GluR2protein expression after ischemia,but it is not clear that whether the GluR2protein expression affect the neuroprotection ofEA pretreatment. This research want to explore the influence of GluR2on the role of EApreconditioning.1. The verification of GluR2siRNA in the expression of GluR2in hippocampal neuron.Grouping randomly the15male C57mice into three groups: Sham, siRNA and scRNAgroups. Mice were killed at72hours after stereotaxically injection of drugs in thehippocampus. Western blotting was used to detect GluR2expression.2. Changes of EA pretreatment-induced ischemia tolerance after reduction of GluR2.Grouping randomly the20male C57mice into four groups: Sham, Con, EA+GCI andsiRNA+EA+GCI groups. Con and EA+GCI groups were subjected to global cerebralischemia for20min and mice in EA+GCI group were given EA pretreatment2hours before global cerebral ischemia. In the siRNA+EA+GCI group, mice were injectedstereotaxically in the hippocampus72hours before EA pretreatment which was describedas1. Mice were killed at24hours after reperfusion. Neurobehavioral evaluation,TUNEL and Nissl staining were used to detect the neuroprotection effect of EApretreatment.3. The influence of GluR2on apoptosis protein after global cerebral ischemia-reperfusioninjury.20adult male C57mice were randomly divided into four groups as described in3. At24hours after reperfusion, western blotting was used to detect apoptosis proteinexpression.Results：1. Knockdown of the expression of GluR2protein in the hippocampus abolishedneuroprotection which induced by EA pretreatment.Western blotting demonstrated that in the siRNA group, a marked reduction in GluR2subunit abundance in the hippocampus was visible at72hours after injectedstereotaxically in the hippocampus(P<0.05). But no difference was found in the content ofGluR2between Sham and scRNA groups.EA pretreatment improved the neurological scores at24h after reperfusion comparedwith that of the Con group(P<0.05). The neurological score of the Con group was similarto that of the siRNA+EA+GCI group.After reperfusion for24h, Nissl Staining demonstrate that pretreatment with EAsignificantly improved the percentage of healthy neurons in the hippocampus comparedwith that of the Con group(P<0.05). But the percentage of healthy neurons in the siRNA+EA+GCI group is less than that in the EA+GCI group(P<0.05).2. Involvement of GluR2activation in neuroprotection by electroacupuncture pretreatmentthrough inhibiting neuron opoptosis.TUNEL staining showed that there were less TUNEL-positive cells in the EA+GCIgroup compared with that of the Con group(P<0.05). But a large number ofTUNEL-positive cells in the in the hippocampus were seen in the siRNA+EA+GCI groups(P<0.05).After reperfusion for24h, Western blotting indicated that the ratio of Bax to Bcl-2inthe Con group was higher than that in the Sham group, then EA pretreatment significantlyreduce the ratio compared with that of the Con group(P<0.05). In the siRNA+EA+GCIgroup, the ratio of Bax to Bcl-2was markedly upregulated compared with that of theEA+GCI group(P<0.05).Conclusion: GluR2protein in the hippocampal neurons take part in neuroprotectioninduced by EA preconditioning through inhibiting apoptosis of neurons after ischemia.Experiment3：The effect of endocannabinoids pretreatment on GluR2expressionafter cerebral ischemia insultObject: To investigate if hippocampal neurons GluR2protein expression was affected byendocannabinoids pretreatment.Methods：1. The influence of endocannabinoids2-arachidonoylglycerol pretreatment on GluR2expression in hippocampal neurons and potential mechanism.The20male C57mice were divided into four groups: Sham, Con,2-AG, AM251+2-AG and V groups. Mice in Con groups were subjected to global cerebral ischemia for20min. Animals were killed at2hours after reperfusion. Western blotting was used todetect GluR2expression in hippocampal neurons.Results:1.2-AG pretreatment increases the expression of GluR2after two hours after reperfusion.AM251abolish this effect of2-AG.Western blotting showed that in2-AG group, hippocampal neuronal GluR2expressionwas significantly up-regulated compared with that in Con group (P<0.05); Compared withthe2-AG group, the GluR2expression was lower in the AM251+2-AG group (P<0.05).But no difference was found between V and Sham groups.Conclusion: Endocannabinoids2-AG can raise GluR2protein expression of hippocampus,and this effect relate to cannabinoid receptors CB1R. Experiment4：The influence of CB1R on the effect of EA preconditioning on theGluR2expression after global cerebral ischemia-reperfusion injuryObject: To explore whether CB1R involved in the effect of EA pretreatment on GluR2protein expression of hippocampus after ischemia.Methods：1. The effect of CB1receptor agonists ACEA and WIN55212-2on the expreesion ofGluR2.Grouping randomly the25male C57mice into five groups: Con, EA+GCI,ACEA+GCI, WIN+GCI and V+GCI groups. At2hours after reperfusion, western blottingwas used to detect GluR2expression.2. The effect of CB1receptor antagonists AM251and SR141716A on the GluR2expreesion.Grouping randomly the25adult male C57mice into five groups: Con, EA+GCI,AM251+EA+GCI, SR141716A+EA+GCI and V+EA+GCI groups. Mice were killed at2hours after reperfusion. Western blotting was used to detect GluR2expression.Results：1. CB1receptor agonist can simulate the effect of EA preconditioning on the GluR2molecules expreesion in the hippocampal neurons after cerebral ischemia injury.Western blotting showed that2h after reperfusion, the expression of GluR2in theACEA+GCI and WIN+GCI groups were increased compared with that of the Con andV+GCI groups(P<0.05). But no difference was found in the content of GluR2betweenCon and V+GCI groups.2. CB1receptor antagonist can reverse the effect of EA preconditioning on the expreesionof GluR2molecules in the hippocampal neurons after cerebral ischemia injury.Western blotting detected that After reperfusion for2h, the expression of GluR2in theAM251+EA+GCI and SR141716A+EA+GCI groups were significantly decreasedcompared with that of the EA+GCI and V+EA+GCI groups(P<0.05), but no significantdifference was found compared with that of the EA+GCI and V+EA+GCI groups. Conclusion: CB1R involve in the up regulation of EA preconditioning on GluR2proteinexpression.SummaryThis research reveals that EA pretreatment enhances GluR2activation and in turninhibits neuron opoptosis via CB1R to protect against global cerebral ischemia. Theresults suggest that EA pretreatment is a new therapy for cerebral ischemia-reperfusionlesion which aim at GluR2subunit of AMPAR.