Reseach The Role Of CB2R/EAAT2in Electroacupuncture Pretreatment-induced Cerebral Ischemic Tolerance

During cerebral ischemic injury, the brain will release a large number of freeexcitatory amino acids, among which the most important is glutamate. Glutamate acts onits receptors, N-methyl-d-aspartate (NMDA) receptors located in the postsynapticmembrane, and result in receptor-dependention channels opening causes fatal calciumoverload, lead to neuronal necrosis or apoptosis, and increase secondary brain injury.Owing to the lack of biodegradable enzymes for glutamate in the neurons, glutamateclearance mainly relays on the glutamate transporter system located on the cellularmembrane of neurons and glial glutamate transporters. The excitatory glutamatetransporters (EAATs) on the cellular membrane of neurons and glial transport glutamate into the cells where glutamate is converted into glutamine by glutamine synthetase and thelatter is in turn transpored back to neurons[1]. EAATs include EAAT1-5types, amongwhich the EAAT2is the richest in the cortex and striatum and only expressed in glial cells,EAAT2undertakes90%glutamate transportation and is the most important glutamatetransporter during cerebral ischemic injury[2]. Rats after stroke given electroacupuncturetreatment can play the neuroprotective effects by significantly increasing the expression ofEAAT2and enhancing glutamate reuptake function[3]. Our previous study found thatelectroacupuncture (EA) pretreatment can activate CB2receptors in rat brain ischemictissue and play a neuroprotective role[4]. As previously reported, CB2receptor mainlyexpressed in microglia and astrocytes[5]. Meanwhile, the research group using cell culturetechniques found that pretreatment of the CB2receptor agonist AM1241reducedlipopolysaccharide (LPS)-gamma-interferon (IFN-gamma)-induced microglial activationand injury[6], reduced microglial activation and injury caused by glutamate-mediated[7].EA pretreatment generated neuroprotective effect has been clearly associated with theCB2receptor. At the same time, the role of electroacupuncture treatment is concerned withthe regulation of the glutamate uptake and degradation. However, electroacupuncturepretreatment how to play a role in the activation of CB2receptors is currently not veryclear. Therefore, we hypothesize that electroacupuncture preconditioning activates CB2receptors and acts the EAAT2to play the neuroprotective effection. In this study, usingthe C57BL/6mice global cerebral ischemic injured model to verify this hypothesis.Experiment1: The study of EAAT2involved in EA pretreatmentinduced-neuroprotectionObjective:To reconfirm the effect of EA pretreatment and to abserve EAAT2whether involve inthe neuroprotective effect induced-electroacupucture pretreatment.Methods64adult male C57BL/6mice, were randomly divided into8groups (n=8):un-operated group (Sham group), control group (Con group), ceftriaxone group (Ceftriaxone Sodium, CXT groups), solvent group (V group), electroacupuncturepreconditioning group (EA group), Dihydrokainate+EA group (DHK+EA group)，DHKsolvent+EA group (V+EA group) and DHK group.The animals in the CXT group and Vgroup accepted intraperitoneally injection of CXT600mg/kg or equal volume normalsaline0.1mL1time/day, for5consecutive days. DHK group wasintracerebroventricularly injected DHK200ug/kg before1h ischemia. Anesthetizedanimals in the EA, DHK+EA and V+EA groups were accepted electroacupunctureat"Baihui Point". The parameters were density wave, frequency2/15Hz, the1mA currentintensity,30min/day, for5consecutive days. At24h after the last management, all animalsaccepted bilateral common carotid artery occlusion for20min in order to produce theglobal cerebral ischemia. The animals in the DHK+EA group and V+EA group wereintracerebroventricularly injected DHK200ug/kg or the same volume of solvent saline2uL1h before ischemia. The mice were sacrificed to evaluate the score of neurologicalbehavior after24h of reperfusion, and the brain tissues were collected. By using HEstaining, cell morphology was observed and the injured neurons in hippocampal CA1region were calculated under the light microscope.Results1． EAAT2agonist CXT pretreatment can reduce the ischemic brain injury ofC57BL/6miceThe mouse neurological behavioral scores and histopathological results in thehippocampal CA1region: the mouse neurobehavioral scores were significant reduced(P<0.05) and the numbers of injured neurons were increased (P<0.05) in the Con groupcompared to that in Sham group; the mouse neurobehavioral scores were significantincreased (P<0.05) and the numbers of damaged neurons were decreased in the CXTgroup in comparison to Con group (P<0.05), but there was no significant diversificationbetween V+EA group and EA group.2．EAAT2antagonist DHK can reverse EA pretreatment-induced ischemic toleranceeffectThe mouse neurological function scores and histopathological results in the hippocampal CA1region: the mouse neurobehavioral scores were significant reduced(P<0.05) and the numbers of damaged neurons were increased (P<0.05) in the Con groupcompared to Sham group; the mouse neurobehavioral scores were significant increased(P<0.05) and the numbers of injured neurons were decreased (P<0.05) in the EA groupand V+EA group in comparison to Con group (P<0.05; the mouse neurobehavioral scoreswere lower (P<0.05) and the numbers of injured neurons were increased (P<0.05) in theDHK+EA group compared to EA group, there was no difference in the neurologicalfunctional scores and the number of injured neurons between the V+EA group and EAgroup.Conclusion:Electroacupuncture pretreatment improved the injury effect of cerebral ischemic,increased the mouse neurological behavior score and reduced the number of injuryneurons. The EAAT2antagonist DHK was injected before ischemia, significantly reversedthe neuroprotective effect induced-electroacupucture pretreatment. But EAAT2agonistCXT pretreatment increased the mouse neurological behavior score and reduced thenumber of the injured neurons. Suggesting that EAAT2takes part in the neuroprotectiveeffect of electroacupuncture pretreatment.Experiment2: The study on the role of CB2R/EAAT2pathway in the ischemictolerance protective effect induced by electroacupucture preconditioningObjective:To confirm CB2receptor involving in the neuroprotective effectinduced-electroacupucture pretreatment, and further investigate the activation CB2recptorafter EA pretreatment to adjust the EAAT2of glial cell membranes play a role of cerebralischemia tolerance.Methods136adult male C57BL/6mice were randomly divided into11groups: Sham group,Con group, EA group, AM630+EA group, solvent+EA group (V+EA group), AM1241group, solvent group, ACEA group, AM251+EA group, AM630group and AM251group. The animals in the AM1241group, ACEA group, V group, AM630group and AM251group were accepted intraperitoneally injection of AM1241600μ g/kg, ACEA2.5mg/kg,equal volume normal solvents, AM6303mg/kg and AM2511mg/kg. The anesthetizedanimals in the EA group, AM630+EA, AM251+EA group and V+EA group wereaccepted electroacupuncture "Baihui Point", the parameters were the same experiment1,at the same time intraperitoneal injection AM1241, AM251and solvents beforeelectroacupuncture3h and30min. At24h after the last management, all animals wereaccepted bilateral common carotid artery occlusion for20min in order to produce theglobal cerebral ischemia. Using the Western blotting to detect EAAT2protein expressionfor6each group. Among Sham group, Con group, EA group, AM630+EA group, V+EAgroup, AM630group and AM251group，8of each group were sacrificed to evaluate thescore of neurological behavior after24h of reperfusion, and the brain tissues werecollected. By using HE staining, cell morphology was observed and the injured neurons inhippocampal CA1region were calculated under the light microscope.8of each groupusing double immunofluorescence staining to detect the number of the cellularcolocalization of EAAT2with the Glial Fibrillary Acidic Protein (a specific marker forastroglia cells, GFAP）after24h of reperfusion.Results1. CB2receptor antagonist AM630can reduce EA pretreatmentinduced-nueroprotectionThe mouse neurological behavior scores and histopathological results in thehippocampal CA1region show: the mouse neurological behavior scores weresignificantly reduced (P<0.05) and the numbers of injured neurons were increased(P<0.05) in the Con group compared to that in Sham group; the mouse neurobehavioralscores were significantly increased (P <0.05) and the numbers of injured neurons weredecreased (P<0.05) in the EA group and V+EA group in comparison to Con group(P<0.05;the mouse neurobehavioral scores were lower (P<0.05) and the numbers ofinjured neurons were increased (P<0.05) in the AM630+EA group compared to EA group,but there was no effect between the neurological behavioral scores and the number of injured neurons in V+EA group.2. CB2receptor agonist AM1241pretreatment can increase the EAAT2proteinexpression of brain tissue, but CB2receptor antagonist AM630reversed theup-regulation of the EAAT2protein expression induced EA pretreatmentWestern blotting results: the EAAT2expression level increased in Con group, but wasnot statistically significant compared to Sham group (P>0.05), the EAAT2proteinexpression was significantly increased in the EA and V+EA groups compared to Congroup (P<0.05); the EAAT2protein expression decreased in AM630+EA group (P<0.05),and V+EA group was not statistically significant in comparison to EA group (P>0.05).The EAAT2protein expression level was significantly increased in AM1241groupcompared to Con group (P<0.05), but there was not statistically significant between the Vgroup and Con group (P>0.05).3. EA pretreatment increased the astrocyte-specific marker GFAP and EAAT2protein expression levels in the brain tissue and this effect can be reversed by theCB2receptor antagonist AM630Results of double immunofluorescence staining and Western blotting were consistent:after ischemia reperfusion for24h, there was no significant difference in the EAAT2expression between Sham and Con groups. The EAAT2expression induced-EApretreatment was significantly more than Con group, the AM630+EA group was weakercompared to the EA group. The strength of GFAP did not differ between Sham group andCon group. The expression strength of GFAP was significantly stronger in EA group incomparison to Con group and AM630+EA group. EAAT2-GFAP localization positive cellswere significantly stronger in the EA group compared with Con group, but theAM630+EA group was weaker.4. CB1receptor agonist ACEA pretreatment had no effect on the EAAT2proteinexpression, CB1receptor antagonist AM251did not also reverse the up-regulation ofthe EAAT2protein expression induced-EA pretreatmentWestern blotting results: the EAAT2expression level increased in Con group, but wasnot statistically significant compared to Sham group (P>0.05). The EAAT2protein expression in the EA group and V+EA group was significantly increased compared withCon group (P<0.05), the EAAT2protein levels decreased in AM251+EA group, but therewas no statistically effect in EA group and V+EA group (P>0.05). The EAAT2proteinexpression was not statistically significant in the ACEA group and V group compared withCon group (P>0.05).Conclusion:1. The neurological behavior scores and histopathological results prove that: CB2receptor antagonist AM630can reverse the protective effect induced electroacupuncturepretreatment induced-neuroprotective. Western blotting results and immunofluorescentstaining results showed that the electroacupuncture pretreatment increased the number ofthe EAAT2–GFAP location cells, AM630antagonistic this effect of electroacupuncturepretreatment. CB2receptor agonist AM1241pretreatment also improved the EAAT2protein expression. Suggesting that the electroacupuncture pretreatment plays aneuroprotective effect through the CB2R-EAAT2pathway in the brain ischemia model.2． Suggesting that CB1receptors do not participate in the up-regulation EAAT2protein expression induced-electroacupuncture pretreatment.SummaryThe experiment results show EAAT2participates in the cerebral ischemic toleranceinduced by EA pretreatment. Further experimental results show that the activaton CB2receptors by EA pretreatment can up-regulation EAAT2on membrane of Glial cells topaly the cerebral ischemic tolerance.Therefore this experiment presents EA pretreatmentinduces cerebral ischemia tolerance mechanism for a new pathways-CB2R/EAAT2.