Effects Of Electroacupuncture On Purinoceptors Mediated Neuroinflammation And The Ability Of Learning-memory In MCAO/R Rats

Objective:To investigate the effects of electroacupuncture(EA)at Baihui(DU20)and Shenting(DU24)mediated purinergic receptors of P2X7R&P2Y1R on the neuroinflammation and the ability of learning-memory in rats after cerebral ischemia and reperfusion injury.To elucidate the possible mechanism of electroacupuncture treatment on learning-memory dysfunction after cerebral ischemia and reperfusion injury.Method:1.100 Sprague-Dawley rats were randomly divided into Sham group(n=18)and Operation group(n=82).The left transient cerebral ischemia(Middle Cerebral Artery Occlusion and Reperfusion,MCAO/R)Sprague-Dawley rats were constructed.The successful model rats with neurological dysfunction evaluated by the Zea-Longa method were randomly divided into MCAO/R group(n=18),MCAO/R+EA group(n=18)and MCAO/R+Non-EA group(n=18).MCAO/R+EA group received EA intervention at DU20 and DU24 with the condensation-rarefaction wave,peak voltage of 6 V,frequency of 2/20 Hz for 30 min/day,once daily for 7 consecutive days.MCAO/R+ Non-EA group was treated with EA in the non-acupoints(located in the bilateral costal region and distal to the iliac crest),and the EA stimulation parameters were consistent with the EA treatment.2.The neurological deficits of rats was observed by neurobehavioral functional tests performed according to Zea-longa's scoring system before intervention and at days 1,3 and 7 after intervention.The cerebral ischemic infarct volume was measured by T2WI before intervention and Day 7 after intervention.The learning-memory ability was tested by Step-down avoidance test before intervention and at Day 7 after intervention and Morris water maze on days 3-7 after intervention.3.The expression of P2X7R,P2Y1R,EDI and GFAP in peri-infarct hippocampal CA1 and cortical were identified by immunohistochemical.The co-localization of P2X7R with ED1 and GFAP,P2Y1R with ED1 and GFAP were identified by immunofluorescence.The expression of inflammatory cytokines IL-1? and TNF-a in the peri-infarct hippocampus and medial prefrontal cortex were detected by ELISA.Result:1.Zea-Longa neurological deficit score Rats in the Sham group showed no behavioral impairments after the operation.There was no significant difference in neurological deficit score among the MCAO/R group,the MCAO/R+EA group and the MCAO/R+Non-EA group before intervention(P>0.05).However,these neurological scores were ameliorated in the MCAO/R+EA group at Days 7 after intervention,as compared with the MCAO/R group and the MCAO/R+Non-EA group(P<0.05).2.Outcome of learning-memory abilityMorris water maze:The escape latency of rats in the Sham group was less than MCAO/R group(P<0.01).However,escape latency was lessen in the MCAO/R+EA group at Day 7 after intervention,as compared with the MCAO/R group and the MCAO/R+Non-EA group in navigation experiments(P<0.01).The number of crossing platform of rats in the sham group was more than MCAO/R group(P<0.01),and it was increased in the MCAO/R+EA group at Days 7 after intervention,as compared with the MCAO/R group and the MCAO/R+Non-EA group(P<0.05).Step-down avoidance test:The first time-period spent prior to stepping down onto the grid(latency period)in the Sham group was more than MCAO/R group(P<0.01).There was no significant difference in latency period among the MCAO/R group,the MCAO/R+EA group and the MCAO/R+Non-EA group before intervention(P>0.05).However,it was increased in the MCAO/R+EA group at Day 7 after intervention,as compared with the MCAO/R group and the MCAO/R+Non-EA group(P<0.05).3.Volume of cerebral infarctionThe T2WI images were both intact hemispheres in the Sham group.There was no significant difference in the volume of cerebral infarction in the MCAO/R group,the MCAO/R+EA group and the MCAO/R+Non-EA group before intervention(P>0.05).However the volume of cerebral infarction was decreased in the MCAO/R+EA group as compared with the MCAO/R group and the MCAO/R+Non-EA group at Day 7 after intervention(P<0.05).4.The expression of P2X7R,P2Y1R and the activation of microglia and astrocyte Immunohistochemistry:The expression of P2X7R,P2Y1R,ED1 and GFAP in the peri-infarct hippocampal CA1 and medial prefrontal cortex was increased in the MCAO/R group compared to the Sham group(P<0.01).However,it reduced in the MCAO/R+EA group compared to the MCAO/R group and the MCAO/R+Non-EA group at Day 7 after intervention(P<0.05).Immunofluorescence:The expression of P2X7R+/ED1+,P2X7R+/GFAP+,P2Y1R+/ED1+ and P2Y1R+/GFAP+ in the peri-infarct hippocampal CA1 and medial prefrontal cortex was increased in the MCAO/R group compared to the Sham group(P<0.01).However,it reduced in the MCAO/R+EA group compared to the MCAO/R group and the MCAO/R+Non-EA group at Day 7 after intervention(P<0.05).5.The expression of inflammatory factor 1L-1? and TNF-?The level of Pro-inflammatory cytokines IL-1? and TNF-? showed elevated in the peri-infarct hippocampal and medial prefrontal cortex in the MCAO/R group compared with the Sham group(P<0.05).However,it reduced in the MCAO/R+EA group compared to the MCAO/R group and the MCAO/R+Non-EA group at Day 7 after intervention(P<0.05).ConclusionEA intervention at DU20 and DU24 could attenuate neuroinflammation in peri-infarct hippocampus and medial prefrontal cortex,including inhibition of microglia and astrocyte activation and the expression of inflammatory factor 1L-1 ?&TNF-?,and improve the ability of learning-memory in rats after cerebral ischemia and reperfusion injury via inhibitting the expresstion of purinergic receptors of P2X7R and P2Y1R.