| How to prevent and treat cerebral and spinal ischemic injuries has been one of themost important issues in the medical field. Ischemic preconditioning has broken newground for ischemic organ protection. To develop novel and rational measures to reducecerebral and spinal ischemia, ischemic tolerance has been a hotspot in medical research.Electroacupunture (EA) is a novel therapy based on traditional acupuncture combinedwith modern electrotherapy. Our laboratory constantly strives to conduct research on theeffect and mechanism of EA-induced ischemic tolerance. Previous investigationsconducted in our laboratory have demonstrated that electroacupuncture can induce the ischemic tolerance both in the brain and spinal cord. However, the underlying mechanismfor EA neuroprotection was not been fully elucidated.Multiple studies have shown that excitatory amino acid transporters (EAATs) thathave been shown to be neuroprotective, especially EAAT2and EAAT3, in the brainagainst ischemia. However, the expression and potential role of EAATs in the ischemictolerance induced by EA preconditioning remains unclear. First of all, we investigated theEAAT expression in the presence and absence of electroacupuncture. Secondly, weexplored the different role of EAAT2and EAAT3in cerebral ischemic tolerance inducedby repeated EA preconditioning via using EAAT2inhibitor DHK and EAAT3-/-mice.Our previous studies have demonstrated that repeated electroacupuncture can inducethe ischemic tolerance in the spinal cord. However, the observation time of our previousinvestigations did not exceed72hours after reperfusion. It was suspected that EApreconditioning didn’t permanently prevent cell death after ischemic/reperfusion injury,but only postponed the evolution of the progress of cell death. Hence, the initialinvestigation was to elucidate if electroacupuncture preconditioning induced long-standingischemic tolerance. HMGB1is a nonhistone nuclear protein with dual function. Inside thecells, HMGB1binds DNA and plays a role in transcriptional regulation. Outside the cell,HMGB1can activate infammatory pathways and serves as a key late cytokine-likemediator of systemic infammation. Furthermore, HMGB1is massively releasedextracellular and amplifcation of plural infammatory responses in the ischemic region inbrain. These studies suggested HMGB1served as a key late cytokine-like mediator ofsystemic infammation after I/R in the central nervous system. Nonetheless, the role ofHMGB1in the induced neuroprotection by EA preconditioning in the spinal cord remainunclear. In present study, we investigated the role and mechanism of HMGB1in the spinalischemic tolerance induced by repeated EA preconditioning. Part I The Neuroprotective Effect of ElectroacupuncturePreconditioning is Mediated by EAATsExperiment I. The expression of EAATs in cerebral cortex and hippocampus afterrepeated electroacupunctureObjective To investigate the expression of EAATs in the cerebral cortex andhippocampus after repeated electroacupuncture without MCAO.Methods Male SD rats were randomly allocated into4groups as follows (5in eachgroup):(1) Control group (Con);(2) Pentobarital group (PB): sodium pentobarbital group(40mg/kg, ip,5d);(3) Para-electroacupuncture group (PEA): received electroacupunctureat the point1.0cm away from “Baihui” mA,2/15Hz,30min/d,5d) under sodiumpentobarbital anesthesia; and (4) Electroacupuncture group (EA): receivedelectroacupuncture at the “Baihui” acupoint (1mA,2/15Hz,30min/d,5d) under sodiumpentobarbital anesthesia. Brain cortices and hippocampus of rats were harvested at24hafter the last electroacupuncture. The expressions of EAAT1, EAAT2and EAAT3weredetected by Weston Blot.Results EAAT1expression in the cerebral cortex and hippocampus was not significantlydifferent among the control, pentobarbital, para-electroacupuncture andelectroacupuncture groups (P>0.05). EAAT2expression in the cerebral cortex of ratswith electroacupuncture was significantly higher than that in the control rats (P<0.05).This difference was not observed in the hippocampus. Electroacupuncture tended toincrease EAAT3expression in the hippocampus (P=0.158) when compared with thecontrol condition.Conclusion: Repeated electroacupuncture preconditioning at Baihui acupiont significantlyincreased the level of EAAT2protein in the cerebral cortex.Experiment II. The role of EAAT2in cerebral ischemic tolerance induced by repeatedEA preconditioning Objective To investigate the role of EAAT2in cerebral ischemic tolerance induced byrepeated EA preconditioning.Methods Male SD rats were randomly divided into6groups as follow (8in each group):(1) Control group (Con);(2) Pentobarital group (PB): sodium pentobarbital (40mg/kg,ip,5d);(3) Para-electroacupuncture group (PEA): received electroacupuncture at the point1.0cm away from “Baihui”(1mA,2/15Hz,30min/d,5d) under sodium pentobarbitalanesthesia;(4) Electroacupuncture group (EA): received electroacupuncture at the“Baihui” acupoint (1mA,2/15Hz,30min/d,5d) under sodium pentobarbital anesthesia;(5) DHK group: administered with dihydrokainate (DHK,10mg/kg, ip) before the onset ofbrain ischemia; and (6) DHK+EA group: administered DHK (10mg/kg, ip) before theonset of brain ischemia after EA pretreatment at Baihui acupoint (1mA,2/15Hz,30min/d,5d). At24h after the last treatment, the right middle cerebral artery occulsionmodel (MCAO) was induced for120mins in all animals. Their brain infarct volumes,neurologic functions, and the levels of EAAT2in the cerebral cortex were evaluated.Results Compared to the control group, repeated EA preconditioning remarkedlyimproved the neurological deficit scores and reduced the cerebral infarct fraction(P<0.05). When dihydrokainate (DHK) was injected30min before the MCAOsignificantly worsened the neurological deficit scores when compared to the controlgroup(P<0.05). Dihydrokainate also significantly worsened the neurological deficitscores in rats with electroacupuncture pretreatment when compared to electroacupuncturealone (P <0.05). Rats with electroacupuncture preconditioning had higher EAAT2expression in the cerebral cortex at2h after the right MCAO than control rats (P<0.05).Conclusion: EAAT2inhibitor DHK abolished the cerebral ischemic tolerance induced byrepeated EA pretreatment. It suggested that EAAT2was involved in the cerebral ischemictolerance induced by repeated EA pretreatment in rats.Experiment III. The role of EAAT3in cerebral ischemic tolerance induced by repeatedEA preconditioningObjective To explore the effect of EAAT3in cerebral ischemic tolerance induced by repeated EA preconditioningMethods: Male EAAT3-/-mice were randomly divided into2groups (8in each group):control and electroacupuncture (1mA,2/15Hz,30min/d,5d) groups. All EAAT3-/-micewere subjected to right middle cerebral artery occlusion for90minutes. Their brain infarctvolumes, neurologic functions, were evaluated.Male EAAT3-/-mice were randomly divided into2groups (5in each group): control andelectroacupuncture (1mA,2/15Hz,30min/d,5d) group. The levels of EAAT1, EAAT2inthe cerebral cortex and hippocampus were evaluated.Result: Electroacupuncture pretreatment at the Baihui acupoint significantly reduced theinfarct volumes and improved the neurological deficit scores in the EAAT3knockout mice.Electroacupunture also increased EAAT2expression in the cerebral cortex andhippocampus. Electroacupunture did not affect the level of EAAT1in the cerebral cortexor hippocampus.Conclusion: EAAT3-/-mice could not abolish the cerebral ischemic tolerance induced byrepeated EA pretreatment. It indicated that EAAT3was not involved in the cerebralischemic tolerance induced by repeated EA pretreatment.Part II The Role of HMGB1in long-standing Ischemic Tolerance Inducedby EA preconditioning in Spinal CordExperiment I: EA preconditioning induced long-standing ischemic tolerance viareducing HMGB1expression after reperfusion in the spinal cord of ratsObjective To investigate if EA preconditioning induced long-standing ischemic tolerancevia reducing HMGB1expression after reperfusion in spinal cord of ratsMethods Experiment1: Male SD rats were randomly assigned to5groups (8in eachgroup)：(1) Sham group；(2) r HMGB1group (r HMGB1200μg, intrathecal injection,1/d,5d)；(3) I/R group；(4) EA group: received electroacupuncture at the “Zusanli”acupoint (1mA,2/15Hz,30min/d,5d)；and (5) EA+rHMGB1group：received rHMGB1 (rHMGB1200μg, intrathecal injection) daily at30min prior to EA for5days. At24hafter the last treatment, all animals were subjected to spinal cord ischemia induced byballoon inflation of a2F Fogarty catheter in the thoracic aorta, and the proximal meanarterial blood pressure was maintained at45mm Hg for12minutes. At2,5,7and14daysafter reperfusion, the neurologic function was scored by the Tarlov criteria. Another fouranimals in each group were sacrificed2and21days for histopathologic examination andTUNEL staining. Experiment2: Male SD rats were randomly assigned to3groups asfollows (16in each group): Sham, I/R and EA. The rats in the I/R and EA groups weresubjected to spinal cord ischemia for12minutes. Four animals in each group weresacrificed at2,5,7and14days after reperfusion and the spinal cords (L5-7) wereremoved immediately for RT-PCR and Western Blot.Results Experiment1： The neurologic function scores in the I/R group weresignificantly less than that in the EA group at2,5,7and14days after reperfusion(P<0.05). The numbers of normal neurons in the anterior spinal cord in EA groups werealso significantly greater than in I/R groups at2and21days (P<0.05). Compared with I/Rgroup, the numbers of TUNEL positive neurons in the EA groups were significantlyreduced at2and21days (P<0.05); Experiment2：The expression of HMGB1mRNA andprotein significantly increased in the I/R group at2,5,7and14days after reperfusion(P<0.05, Sham vs I/R). Compared with the I/R group, the expression of HMGB1mRNAsignificantly decreased at2,5,7and14days after reperfusion and the level of HMGB1protein only reduced at5and21days (P<0.05, EA+IR vs I/R).Conclusion: Repeated EA preconditioning induced long-standing ischemic tolerance viareducing the expression of HMGB1and apoptosis after I/R injuries in the spinal cordof rats.Experiment II: HMGB1mediated neurons apoptosis via increasing the secretion ofTNF-α after stimulation of RAW264.7macrophageObjective To investigate if HMGB1mediated neurons apoptosis via increasing thesecretion of TNF-after stimulation RAW264.7macrophage Methods Experiment1: Primary cultured neurons were assigned to8groups：(1) PBSgroup；(2) HMGB1-10ng/mL group；(3) HMGB1-50ng/mL group；(4) HMGB1-100ng/mLgroup；(5) PBS+RAW264.7macrophage；(6)Neuron treated with10μl conditionedmedium of HMGB1-treated RAW264.7group；(7)Neuron treated with50μl conditionedmedium of HMGB1-treated RAW264.7group；and (8) Neuron treated with100μlconditioned medium of HMGB1-treated RAW264.7group.The cellular viabilities ofspinal neurons was measured at1,3,5,7and9days by MTT assay after treatment. Flowcytometry was carried out to assess neuron apoptosis at7days after treatment.Experiment2: RAW264.7macrophage were divided into2groups: Control group andHMGB1group. The level of TNF-, IL-1β, IL-6, IL-8were determined by Elisa.Experiment3: Primary cultured neurons were divided into4groups:(1) controlmedium+non-immune lgG；(2) control medium+TNF-neutralizing antibody；(3)conditioned medium of HMGB1-treated RAW264.7+non-immune lgG； and (4)conditioned medium of HMGB1-treated RAW264.7+TNF-neutralizing antibody. Thecellular viabilities of neurons was meassured at1,3,5,7and9days by MTT assay aftertreatment. Flow cytometry was carried out to assess neuron apoptosis at7days aftertreatment.Results Experiment1: Compared with the PBS group, HMGB1directly treated withneuron had no effect on the apoptosis and cellular viabilities. However, spinal cordneurons treated with3different concentration of conditioned medium of HMGB1-treatedRAW264macrophage, occurred abundant apoptosis accompanied by suppressed cellactivity (P<0.05). Experiment2: Compared with the control group, the level of TNF-,IL-1β, IL-6, IL-8remarkably increased in HMGB1-treated RAW264.7macrophagenon-immune. Experiment3: HMGB1mediated neurons apoptosis via increasing thesecretion of TNF-after stimulation RAW264.7macrophage by HMGB1. TNF-neutralizing antibody can inhibit the apoptosis of neurons and improved neurons (P<0.05),which induced by conditioned medium of HMGB1-treated RAW264macrophage.Conclusion: Spinal cord neurons apoptosis can be induced by conditioned medium ofHMGB1-treated RAW264macrophage, not by HMGB1treatment directly. TNF- neutralizing antibody can reversed the apoptosis of neurons mediated by conditionedmedium of HMGB1-treated RAW264macrophage.SUMMARY1. Repeated EA pretreatment at Baihui acupiont up-regulated the expression ofEAAT2in the cerebral cortex. The EAAT2antagonist DHK reversed theneuroprotective effect of EA pretreatment. These results indicateelectroacupuncture at Baihui induces brain ischemic tolerance in rats, which maybe mediated by EAAT2.2. EAAT3-/-mice could not abolish the cerebral ischemic tolerance induced byrepeated EA pretreatment. It suggest that EAAT3is not involved in the cerebralischemic tolerance induced by repeated EA pretreatment.3. Repeated EA pretreatment at Zusanli acupiont induces ischemic tolerance in thespinal cord of rats and the effect was long-standing via reducing HMGB1expression and apoptosis after I/R injury in the spinal cord. Recombinant HMGB1(r HMGB1) reversed the neuroprotective effect of EA pretreatment. These resultsindicate electroacupuncture at Zusanli induces spinal cord ischemic tolerance inrats, which may be mediated by HMGB1expression after reperfusion in the spinalcord.4. HMGB1could not directly induce neurons apoptosis. HMGB1mediated neuronsapoptosis via increasing the secretion of TNF-after stimulation RAW264.7macrophage by HMGB1, which can be reversed by TNF-neutralizing antibody. |
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