| ObjectiveIn this study,we used randomized controlled experimental method of acupuncture intervention in the rat model of hypoxic-ischemic brain damage(HIBD).To observe the influence mechanism of acupuncture for autophagy and energy metabolism and recovery of neurons,we took detection with expression of ROS and NIX protein and the change of ATP/ADP in the hippocampus.Our goal is to provide an accurate theoretical basis for clinical treatment of HIE.Part oneMethodsThe experimental conditions and methods:20 rats(type of SD)with half of female rats were provided by the animal center of medical laboratory with a SPF degree.The license number was[Guangdong:SYXK(Guangdong)2013-0002].Weight of rats were about 230-270g for female and 400-450g for male.Rats were given a routine quarantine for 3 days and fed in a cage in couples to breed for 12h.rats in the morning check to observe vaginal suppository,vaginal suppository for pregnancy zeroth days,3 days of continuous breeding.Finally,we got 10 pregnant rats:2 rats with normal delivery,cesarean section for 4 rats,2 rats for raising neonatal rats and 2 spare rats.Pregnant rats which were going to give birth to their babies were given an isoflurane inhalation anesthesia to receive a delay caesarean delivery with clipping bilateraluterine artery for 10 minute to create neonatal rat models withhypoxic-ischemic brain damage(HIBD).Experimental groups dividing:the successful model of the rats to nurse were breastfeeding to 14 days of age were the successful model of the rats were divided into model acupuncture group and model group;natural childbirth neonatal rats were randomly divided into normal group and normal acupuncture group and acupuncture group,8 rats in each group,4 groups in total.Intervention:Rats in acupuncture intervention groups were given daily Jin' s three needle acupuncture,with acupuncture once a day,each point twist manipulating needle after 10 times of the needle and took out the needle.A total intervention of 14 days.The rats in model group and normal group were caught every day at the same time to reduce anthropogenic factors.Detection:Took out the whole brain tissue under the ice on a low temperature and divided the brain into various parts with a blunt separation,marking and weighing.The average fluorescence intensity expression of ROS was detected by flow cytometry with the hippocampus,and the expression of NIX was detected by the method of Western blot.Results1.Weight:The experimental results showed that the normal group with acupuncture had no significant difference in body weight in Day13-Day35 compared with normal group(P>0.05).The weight of rats in model group was significantly different in Day13-Day35(P<0.01),the weight of rats in model group with acupuncture in Dayl3-Day21 was statistically significant different(P<0.01).Compared with the model group,the weight of rats in acupuncture group was different but not statistically significant(P>0.05).The weight of rats in the model acupuncture group was increased in Day21-Day35 and there was no statistically significant difference(P>0.05).2.behavioral testing:1)Field trials:Comparing the frequency of activities between the normal group,normal with acupuncture group,the model group and the model with acupuncture group,the normal group and the normal with acupuncture group had a higher frequency of activities and had no significant difference between the two groups(P>0.05),activities of model with acupuncture group and model group were lower than normal group,which was statistically significant(P<0.05);Compared with the model group,the frequency of activities in model with acupuncture group was better than the model group and the difference was significant.2)Water maze Test(space exploration):Comparing the residence time of the target quadrant(the first quadrant)in each group of rats,It was no significant difference between normal group and normal group with acupuncture(P>0.05),Residence time of the first quadrant in model group with acupuncture and model group were shorter than normal group,which was significant(P<0.05);Compared with model group,the residence time of the target quadrant of the model group with acupuncture was longer than the model group,and the difference was statistically significant(P<0.05).3.The average fluorescence intensity of ROS expression test results:Comparing with normal group,the difference between normal acupuncture group and normal group is not obvious(P>0.05),model group and model acupuncture group ROS mean fluorescence intensity expression was significantly higher than normal group,the difference was statistically significant(P<0.05);Compared with model group,the average fluorescence expression of ROS in rat model acupuncture group decreased markedly,and the difference was significant(P<0.05).4.Test results of NIX protein content:Compared with normal group,the expression of NIX in hippocampus of normal acupuncture group and normal group of rats was not significant(P>0.05);model group and model acupuncture group were lower than normal group,the difference was remarkable(P<0.05),compared with the model group,the acupuncture group expressed higher than the model group,the difference was remarkable(P<0.05).Part twoMethodsThe experimental conditions and methods were the same as experiment one Experimental groups:the successful model of the rats to nurse were breastfeeding to 14 days of age were the successful model of the rats were divided into model acupuncture group and model group;rats were randomly divided into normal group of natural childbirth,10 rats in each group,a total of 3 groups.Experimental methods:the same as experiment part 1.Detection:Transmission electron microscopy detection of autophagy in the hippocampus CAl area was detected on the 1th day of birth in Aberdeen mice,14 days(acupuncture before),21 days(acupuncture),28 days(after acupuncture).The ATP/ADP ratio in the brain tissues was detected by HPLC in the 1th Day,28 days(after acupuncture),respectively.Results1.The weight of brain tissue of rats in each group:On the first day,there was no significant difference in brain weight between normal group and model group(P>0.05).On the 28th day,compared with normal group,the weight of left cortex of rats in acupuncture group was not significantly increased(P>0.05),but the weight of left cortex in model group decreased significantly(P<0.05).Compared with the model group,the left cortex weight of the acupuncture group increased significantly,and the difference was significant(P<0.05).2.Transmission electron microscope:On the first day,the normal group and model group did not discover the complete neuron;14th day,before acupuncture,the normal group and model group are visible neurons,but the autophagy expression is not obvious;On the 21st day,the acupuncture intervention 7 days after,the expression of autophagy in normal group was not obvious,although there were few autophagy in the model group,the number of autophagy was more than the other two groups in the Acupuncture group.On day 28th,the expression of autophagy in each group was not obvious after 14 days of acupuncture intervention.3.Detected the ATP/ADP ratio in brain tissues with high performance liquid chromatography:the ATP/ADP ratio of normal group is higher than that of model group at the first day,the difference is statistically significant;At day 28th,acupuncture group is lower than normal group,but compared with the model group,the difference is statistically significant.Conclusions1.Acupuncture could improve the weight of brain tissue in HIBD rats.2.Acupuncture could improve the learning and memory ability of HIBD rats.3.The acupuncture of Jin' s three needle can increase the autophagy which has been reduced to low expression,but it is only in the early stage of acupuncture intervention.4.The ratio of ROS to ATP/ADP in acupuncture group was expressed as negative correlation trend.Suggesting that acupuncture of Jin three needles needle acupoint to promote the brain function repair mechanism of HIBD model rats with hypoxic-ischemic brain injury may be able to effectively reduce oxidative stress injury in the brain tissues of HIBD model rats with acupuncture of Jin three needles needle acupoint group,it can improve mitochondrial energy metabolism of brain tissues of HIBD model rats. |
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