| Objective:Observing electricity on morphine withdrawal rats brain tissue spatial learning and memory ability,and different brain regions pathomorphism,single amine neurotransmitter,Ca2+-CaM-CaMK-after CREB signaling pathway and the effect of study will reduce the mechanism of action of morphine withdrawal symptoms,for morphine withdrawal provide experimental foundation and theoretical basis for clinical treatment.Method:Using Morris water maze select learning and memory ability of normal SD rat 56,only will be randomly divided into control group 14,module of 42.Dose escalation method is used to the back of the neck subcutaneous injection of morphine,5 in a row,2 times a day;Three hours after the last morphine injection and naloxone on acute urged withdrawal.Building successful 42 rats were randomly divided into model group,acupuncture group,the electric acupuncture group,each group of 14 only.The control group and model group and acupuncture group the same time,the same degree of catch catch a stimulus,will not be any treatment intervention.Acupuncture group rats "god court","the will","shen shu" point on both sides,regular disinfection,with filiform needle Pierce the acupoints,respectively,into the needle 2-3mm,fast turning 1 min,15min,retaining needle 1/d,6 d continuous intervention.Electric acupuncture group with acupuncture group and find out in the acupuncture group operation will be the basis of "god court" and "best" connect to lead is negative;Bilateral"shen shu" point connected to wires are negative,turn on the electric acupuncture therapeutic apparatus,selection of dilatational wave(frequency for 2/100hz),intervention in 15 min,1/d,6 d continuous intervention.Using Morris water maze test rats learning and memory ability;Under light microscopy to observe the VTA and NAc brain tissue pathology change;Using the ELISA method for determination of blood listing amine neurotransmitter(DA and 5-HT,NE)content;Flow cytometry to detect the VTA and NAc nerve cells[Ca2+]i and CaM activity;Immunohistochemical staining detection CaMK ? and after CREB positive expression,and CaMK?and after CREB phosphorylation;Real-time fluorescent quantitative PCR detection VTA and NAc neurons CaMK II alpha subunit mRNA expression and CaMK ? alpha subunit phosphorylation level;Western Blot detection VTA and NAc neurons CaMK ? alpha protein expression and CaMK ? alpha phosphorylation level.SPSS18.0 software package was used to statistical analysis,measurement data to plus or minus S said,mean comparison between groups using single factor analysis of variance,covariance together with LSD method when compared between two groups,the variance is used when Tamhane'S T2 test,P<0.05,the difference was statistically significant.Result:1.The body quality change:after acupuncture intervention,compared with control group,model group rats significantly lower body quality,statistically significant difference(P<0.01).Compared with model group,body acupuncture group and the curative group rats quality specific significant difference(P<0.05).Compared with acupuncture group,the curative group of rats body quality there was no significant difference(P>0.05).2.Morris water maze test results:in the directional navigation experiments,the control group,model group,acupuncture group,the curative group rats with the increase of training test times,each rat escape latent period shorten.At the same time point compared with control group,model group rats escape significantly prolong the incubation period(P<0.01).At the same time point compared with model group,acupuncture group and the curative group significantly shorten the escape latency of rats(P<0.01).At the same time point compared with acupuncture group,rat escape latent period shorten electric acupuncture group(P<0.05).In the search space experiments,compared with control group,model group rats significantly extend across platform(P<0.05).Compared with model group,acupuncture group and the curative group rats crossing platform number increased significantly(P<0.05,P<0.01).Compared with acupuncture group,the curative group rats crossing platform number increased significantly(P<0.05).3.HE staining results:normal group:the brain tissue staining is clear,neat,nerve cells in the form of cytoplasm nucleus boundaries clear,no tissue edema,scattered in a small amount of inflammatory cells,blood vessels without the congestion.Model group:brain tissue structure is loose,owe neat,nerve cells form part of the nucleus pycnosis neurons and glial cells increased obviously,increased number of inflammatory cells,capillary swelling.Acupuncture group and the curative group:brain tissue pathologic conditions have obvious change,main show is loose significantly reduce brain tissue,reduced Numbers of nucleus pycnosis neurons,glial cells and inflammatory cells number also reduce,capillary swelling reduce.4.Blood listing amine neurotransmitter content:compared with control group,model group rats serum DA,5-HT,NE content increased significantly,statistically significant difference(P<0.01).Acupuncture group and the curative group of morphine withdrawal rats serum DA,5-HT,NE content is significantly reduced,compared with model group,the difference statistically significant(P<0.01).Compared with acupuncture group,serum DA electric acupuncture group,5-HT,NE content reduced,statistically significant difference(P<0.05).5.Flow cytometry results:acupuncture group and the curative group rat VTA and NAc nerve cells[Ca2+]i and CaM activity than model group significantly reduced(P<0.05,P<0.01).Inhibition compared with acupuncture group,electric acupuncture intervention of chronic morphine dependence naloxone in nerve cells caused by acute urged withdrawal[Ca2+]i and CaM activity dramatically increases more significantly(P<0.05).6.Immunohistochemical results:compared with model group,acupuncture and cupping intervention can significantly reduce morphine withdrawal rats VTA and NAc neurons CaMK? positive cells expression(P<0.05,P<0.01),decrease after CREB positive cells expression(P<0.05,P<0.01),inhibit after CREB phosphorylation(P<0.05,P<0.01),and electric acupuncture grpup is more significant than acupuncture group(P<0.05).-7.A real-time fluorescent quantitative PCR results:acupuncture and cupping intervention can significantly lower CaMK ? alpha mRNA expression,inhibition of CaMK? alpha subunit phosphorylation,with significant difference compared with model group(P<0.05,P<0.01),and the curative group is more significant than acupuncture group(P<0.05).8.Western Blot results:acupuncture and cupping intervention can significantly lower CaMK ? alpha mRNA,protein expression,inhibition of CaMK? alpha subunit phosphorylation,with significant difference compared with model group(P<0.05,P<0.01),and electric acupuncture group is more significant than acupuncture group(P<0.05).Conclusion:1.Electric acupuncture can reduce acute chronic morphine dependence naloxone urged withdrawal rats lost body quality,could shorten the morphine withdrawal rats platform to escape the incubation period,increase the cross platform,to improve the spatial learning and memory ability.2.Electric acupuncture can improve pathomorphism changes of morphine withdrawal rats neurons.3.Electric acupuncture can reduce morphine withdrawal rats blood listing amine neurotransmitter DA,5-HT,NE content.4.Electric acupuncture can inhibit chronic morphine dependence naloxone in acute urged withdrawal caused by intracellular[Ca2+]i and CaM activity rise sharply,cut the VTA and NAc neurons CaMK ? positive expression,cut the VTA and NAc neurons CaMK ? alpha subunit mRNA expression and protein expression,inhibition of CaMK ? alpha subunit phosphorylation,cut after CREB nuclear transcription factor protein expression,and suppress after CREB phosphorylation.5.Will inhibit morphine withdrawal reaction,the ability of learning and memory in the rat model of morphine withdrawal by Ca2+-CaM-CaMK ?-after CREB signaling pathways play its biological effect. |
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