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Effects of Electroacupuncture on Learning and Memory Dysfunction in Rats Following Cerebral Hypoperfusion and ItsMechanisms with PKA/CREB Signaling PathwayObjective:To investigate the effects of electroacupuncture(EA)on spatial learning and memory function and long-term potentiation(LTP)in rats with cerebral hypoperfusion,and to explore whether the cAMP-dependent protein kinase-cyclic adenosine monophosphate response element binding protein signaling pathway(PKA/CREB)was involved in these effects.Methods:50 Sprague-Dawley(SD)rats were randomly divided into five groups:a sham-operated control group(Con),a model group(Mod),an EA group(EA),an EA combined with intracerebroventricular(ICV)injection of PKA blocker H89 group(EA+H89)and an EA combined with ICV injection of normal saline(NS)group(EA +NS).Cerebral hypoperfusion was induced by permanent,bilateral common carotid artery occlusion(two-vessel occlusion,2VO).EA was given on GV20 and GV14.Morris water maze(MWM)analysis system was used to evaluate the spatial learning and memory ability of rats.The long-term potentiation(LTP)of the Schaffer collateral-CA1 were recorded.The expression of phosphoCREB(pCREB)protein of hippocampus were evaluated by western blot.Results:1.In the MWM test:Compared with the control group,the model group rats exhibited significantly longer escape latencies from the 2nd to the 5th time during the training period(P<0.05 respectively).Compared with the model group,the EA group rats shortened the atencies from the 3rd to the 5th time during the training period(P<0.05 respectively).The EA? H89 group rats demonstrated significantly prolonged escape latencies than the EA ? NS group rats from the 2nd to the 5th time during the training period(P<0.05 respectively).The time spent in the target quadrant was significantly decreased in the model group(P<0.01 vs.control group),while it was significantly increased in the EA group(P<0.01 vs.model group).The EA ? H89 group rats exhibited significantly shorter time than that of the EA ? NS group(P<0.01).2.HFS of the Schaffer collateral inputs to CA1 pyramidal cells induced a stable LTP in the slope of fEPSP in control rats.Contrastingly,in the model group,fEPSP slope was significantly reduced(P<0.01 vs.the control group).EA reversed the 2VO-induced LTP impairment(P<0.01 vs.the model group).The normalized fEPSP slope was significantly decreased in EA ? H89 group than that in the EA ? NS group(P<0.01 vs.EA ? NS group).3.The expression of the pCREB protein was decreased 7 days after the 2VO operation(P<0.01 vs.the control group).Treatment of EA impeded the reduction of pCREB protein in 2VO rats(P<0.05 vs.the model group).Moreover,this protective effect of EA was partially reversed in EA ? H89 group rats,when compared with the rats in EA ? NS group(P<0.05,vs.the EA ? NS group).Conclusions:1.EA could ameliorate the spatial learning and memory deficits and LTP impairment induced by 2VO.2.EA could reverse the decrease of pCREB protein expression of 2VO rats,suggesting that EA might exert effects through activation of the PKA/CREB signaling pathway.3.The effects of EA could be inhibited by PKA blocker H89,further verified that the activation of the PKA/CREB signaling pathway might be a potential mechanism of EA.Part ? Effects of Electroacupuncture on Neuronal Apoptosis and Its Potential Mechanism with the PKA/CREB Signaling Pathway in Rats with Cerebral HypoperfusionObjective:To investigate the effects of electroacupuncture(EA)on hippocampal neurons apoptosis and its probable mechanism with the PKA/CREB signaling pathway in rats following cerebral hypoperfusion.Methods:120 Sprague-Dawley(SD)rats were randomly divided into five groups:a sham-operated control group(Con),a model group(Mod),an EA group(EA),an EA combined with intracerebroventricular(ICV)injection of PKA blocker H89 group(EA+H89)and an EA combined with ICV injection of normal saline(NS)group(EA +NS).Each group included three time courses:7d,14d and 21d.Cerebral hypoperfusion was induced by permanent,bilateral common carotid artery occlusion(two-vessel occlusion,2VO).EA was given on GV20 and GV14.TUNEL and western blot were employed to observe apoptosis of neurons and expression changes of Bax and Bcl-2 protein of hippocampus.Results:1.In TUNEL stain:? As the observation time is prolonged,the apoptosis rates were significantly higher on day 14 and 21 than on day7 in the Mod groups(P<0.05);The apoptotic rate of hippocampal neurons in EA groups also increased gradually,but there was no significant difference(P>0.05).? As compared between the groups at the 3 time points respectively,on day7,14 and 21,percentages of apoptotic neurons were higher in the Mod groups compared with the Con groups(P<0.05),while it was lower in the EA groups compared with the Mod groups(P<0.05);In the EA +H89 groups,the apoptosis rates were significantly higher when compared with the EA +NS groups.2.Western blotting suggested that:? As the observation time is prolonged,the expression of Bcl-2 protein was significantly higher in the 21d subgroups than in the 14d subgroups in the EA groups(P<0.05),while there were no significant differences in other groups.? As compared between the groups at the 3 time points respectively,on day7,14 and 21,the expression of Bax protein were significantly up-regulated(P<0.05)and the expression of Bcl-2 protein were significantly down-regulated(P<0.05)when comparing the Mod groups with the Con groups,while the Bax protein significantly reduced(P<0.05)and the Bcl-2 protein significantly improved(P<0.05)when comparing the EA groups with the Mod groups;In the EA+H89 groups,the Bax protein significantly increased(P<0.05)while the Bcl-2 protein significantly decreased(P<0.05)when compared with the EA+NS groups(but the expression of Bcl-2 protein was no significant difference on day 14).Conclusions:1.EA could significantly alleviate 2VO-induced apoptosis of hippocampal neurons on day7,day 14 and day21.2.EA may play an anti-neuronal apoptosis by regulating the expression of Bax protein and Bcl-2 protein.3.The effects of EA could be inhibited by PKA blocker H89,further verified the underlying mechanism of the PKA/CREB signaling pathway in effects of EA.Part ? Effect of Electroacupuncture on Synaptic Plasticity of Hippocampal Neurons and Its Potential Mechanism with the PKA/CREB Signaling Pathway in Rats with Cerebral HypoperfusionObjective:To investigate the effects of electroacupuncture(EA)on dendritic spine density and its probable mechanism with the PKA/CREB signaling pathway in rats following cerebral hypoperfusion.Methods:Firstly,30 Sprague-Dawley(SD)rats were randomly divided into five groups:a sham-operated control group(Con),a model group(Mod),an EA group(EA),an EA combined with intracerebroventricular(ICV)injection of PKA blocker H89 group(EA+H89)and an EA combined with ICV injection of normal saline(NS)group(EA +NS),n=6.On day7,Golgi silver stain was employed to observe the dendritic spine density.Secondly,75 Sprague-Dawley(SD)rats were randomly divided into five groups as described above,but each group up included three subgroups based on time courses:7d,14d and 21d,n=5.These rats sacrificed for quantitative real-time PCR(RT-PCR)to detect the expression changes of microRNA132(miR132),and for western blot to detect the expression changes of phosphoCREB(pCREB)protein,brain derived neurotrophic factor(BDNF)and p250GAP protein-a GTPase activating protein,in hippocampus.Cerebral hypoperfusion was induced by permanent,bilateral common carotid artery occlusion(two-vessel occlusion,2VO)and EA was delivered the day after the operation at Baihui(GV20)and Dazhui(GV14)acupoints.Results:1.On day7,the dendritic spine density was markedly decreased in model group rats(P<0.01 vs.the control group),and it was significantly improved in EA group rats(P<0.01 vs.the model group).The EA ? H89 group rats had significant fewer dendritic spines than that of the EA ? NS group rats(P<0.01 vs.the EA ? NS group).2.On day7,14 and 21,the expression of pCREB protein in the model groups were significantly lower than that in the control groups(P<0.05),and it were significantly higher in the EA groups than that of the model groups(P<0.05).On day7 and 14,the expression of pCREB protein in EA + H89 groups were significantly lower than that in EA+NS groups(P<0.05),but there was no significant difference on day21(P>0.05).3.On day7,14 and 21,the expression of BDNF protein in the model groups were significantly lower than that in the control groups(P<0.05).On day7 and 21,the expression of BDNF protein were significantly higher in the EA groups than that of the model groups(P<0.05).On day7,14 and 21,the expression of BDNF protein in EA + H89 groups were significantly lower than that in EA+NS groups(P<0.05).4.On day7,14 and 21,the expression of p250GAP protein were significantly increased when compared the model groups with the control groups(P<0.05),while it were significantly decreased when compared the EA groups with the model groups(P<0.05).On day7,the expression of p250GAP protein in EA + H89 groups were significantly higher than that in EA+NS groups(P<0.05),but there was no significant difference on day14 and 21(P>0.05).5.On day7,14 and 21,the expression of miR132 were significantly decreased when compared the model groups with the control groups(P<0.05),while it were significantly increased when compared the EA groups with the model groups(P<0.05).Meanwhile,it were significantly decreased when compared the EA+H89 groups with the EA+NS groups(P<0.05).Conclusions:1.EA could promote synaptic plasticity by alleviating the reduction of dendritic spine density in 2VO rats.2.EA could regulate the expressions of the pCREB protein,BDNF protein,miR132 and p250GAP protein through activating the PKA/CREB signaling pathway.3.The effects of EA could be partially inhibited by PKA blocker H89,further verified the underlying mechanism of the PKA/CREB signaling pathway in effects of EA. |
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