| ObjectiveTo observe the effect of acupuncture on learning and memory ability and neurobehavior of neonatal rats with hypoxic-ischemic brain damage(HIBD),and to evaluate the effect of different length of intrauterine distress(5min,10min,15min)on brain morphology and neurofunctional defect of HIBD neonatal rats,and to explore a more practical model of intrauterine distress To investigate the relationship between the different length of HIBD and autophagy of neurons,and to explore the role of autophagy of neurons in neonatal HIBD The mechanism of injury and nerve function defect.MethodsThe neonatal hypoxic-ischemic brain injury(HIBD)model was established by cesarean section with different length(5min,10min,15min).The experiment was divided into three parts:(1)to evaluate the effects of different long-term ischemia and hypoxia on neurobehavioral and learning and memory ability(open field experiment and water maze experiment)of HIBD newborn rats;(2)to observe the effects of different long-term ischemia and hypoxia on the morphology and ultrastructure of brain tissue of HIBD newborn rats by using transmission electron microscope and he staining technology;(3)to observe the differences by using Western blot technology The effects of long-term ischemia and hypoxia on the expression of autophagy protein(beclin-1,LC3II/I,p62)in hippocampal neurons of neonatal rats with HIBD were studied.Finally,a new rat model of intrauterine distress HIBD was established.A neonatal hypoxic-ischemic brain injury(HIBD)model was established by cesarean section.On the 14th day after birth(post natal day 14,pnd14),HIBD newborn rats were given acupuncture(shenting point,Benshen point,naohu point,naokong point and temporal point)intervention once a day for 14 days.After each point was injected,the handle of the needle was rotated evenly and rapidly for 10 times.After the completion of twisting,the needle came out immediately without retaining the needle.The purpose of this part of the experiment is to observe the effect of acupuncture on the expression of autophagy protein(beclin-1,LC3Ⅱ/Ⅰ,p62)and the ultrastructure of hippocampal neurons in neonatal rats with intrauterine distress HIBD,and to preliminarily reveal the mechanism of acupuncture regulating the level of autophagy of neurons and improving the brain damage and neurological function defect in neonatal rats with intrauterine distress HIBD.Neonatal hypoxic-ischemic brain disorders(HIBD)model was established by intrauterine distress cesarean section.On the 14th day after birth(PNND14),HIBD newborn rats were given acupuncture(Shenting,Bishen Shen,Naohu,Bilateral Cerebral Cavities,Bilateral Temporal Three Points)interventions,daily intervention Once,intervention was continued for 14 days.After each acupuncture point,the needle handle was twisted evenly and quickly for 10 times.After the twisting was completed,the needle was immediately released without leaving the needle.The purpose of this part(Part 4)is to observe the effects of acupuncture on autophagy protein expression(Beclin-1,LC3Ⅱ/Ⅰ,p62)and hippocampal neuron ultrastructure in neonatal rats with intrauterine distress.Reveal the mechanism of acupuncture regulating neuron autophagy and improving ischemic hypoxic brain damage and neurological deficits in neonatal rats with intrauterine distress.Results(1)The first part of the experimental study found that,compared with the normal group,there was no significant change in the average level of activity of newborn rats in the sham operation group(P＞0.05),and the average level of activity of rats in the 5min group,10min group and 15min group decreased(P＜0.05,P＜0.01,P＜0.001).Compared with the normal group,there was no significant change in the average level of activity of newborn rats in the sham operation group(P>0.05),and the average level of activity of rats in the 5min group,10min group and 15min group decreased(P＜0.05,P＜0.01,P＜0.001).Compared with the normal group,there was no significant change in the escape incubation period of rats in the sham operation group and the 5min group on each day of the navigation experiment of positioning,as well as in the number of times of crossing the platform and the total distance of swimming in the space search experiment(P＞0.05),indicating that the operation was not an interference factor of the model.Compared with the normal group,the incubation time of each day in the 10min group and the 15min group was significantly prolonged(P<0.01,P<0.001),and the number of times of crossing the platform and the total distance of swimming were reduced(P＜0.01,P＜0.001),indicating that the model group had spatial learning and memory impairment and the modeling was successful.(2)The second part of the experimental study found that the normal group and the sham group showed dense,orderly and distinct pyramidal cells with regular cell shape and clear nucleoli in the hippocampal CAI region under a 200-fold microscope.In the 15min group,the pyramidal cells reduced or even disappeared,the disordered arrangement structure was loose,the intercellular space was enlarged,the cell shape was irregular,most of the nuclei were rigid and contracted,and the nucleoli were unclear.In the 5min group,the cells were arranged neatly,the intercellular space was small,and the nucleoli were clear.In the 10min group,the arrangement of vertebral cells was looser and the number of cells was significantly reduced.In the normal group and the sham group,the perinuclear space of the prefrontal neurons was normal,the nuclear membrane was continuous and smooth,the organelles were complete,the hippocampal neurons had nuclear membrane light source,the perinuclear space was not changed,the organelles were abundant,and no obvious autophagosomes were observed.In the 5min group,the perinuclear clearance was slightly widened,the rough endoplasmic reticulum was slightly expanded,the organelle structure and morphology were clear,and autophagosomes were occasionally observed.In the 10min group,the perinuclear gap widened,the autophagosome engulfed and cleared the aging damaged organelles in many places,and the rough endoplasmic reticulum expanded significantly.In the 15min group,the cortical neurons showed increased heterochromatin,nuclear consolidation,nuclear membrane serrations,autophagosomes were rare,cell rupture contents were easy to see,the rough surface endoplasmic reticulum of hippocampal neurons expanded,mitochondria swelled and cavulated,mitochondrial cristae fell off,serrate nuclear membrane,and golgi body cavitation.(3)The third part of the experimental study found that the relative beclin-1 protein expression in the normal group,sham group,5min group,10min group and 15min group followed a normal distribution(kolmogorov-smirnov=0.193,0.221,0.146,0.215,0.251,P=0.200,0.200,0.200,0.200,0.148).Homogeneity of variance was tested and homogeneity of variance(Levene’s=0.944,P=0.473)was performed.LSD test was used for comparison between groups.The relative expression of p62 protein in each group followed a normal distribution(kolmogorov-smirnov=0.184,0.231,0.096,0.194,0.196,P=0.200,0.200,0.200,0.200,0.200).After homogeneity test of variance,variance was not homogeneous(Levene’s=5.729,P=0.000),and Dunnett T3 test was used for comparison between groups.The relative expression of LC3II/I protein in each group followed a normal distribution(kolmogorov-smirnov=0.106,0.152,0.146,0.249,0.141,P=0.200,0.200,0.200,0.154,0.200).After homogeneity test of variance,variance was not homogeneous(Levene’s=3.497,P=0.006).Compared with the normal group,the expressions of beclin-1,p62 and LC3II/I in the hippocampus of rats in the sham operation group showed no significant changes(P=0.108,P=1.000,P=0.826).Compared with the normal group,beclin-1,p62 and LC3II/I expressions in the hippocampus of rats in the 5min group were increased(P=0.016,P=0.036,P=0.027),beclin-1 and LC3II/I expressions in the hippocampus of rats in the 10min group were increased(P=0.000,P=0.000),and p62 expression was increased(P=0.001),while beclin-1,p62 and LC3II/I expressions in the hippocampus of rats in the 15min group were increased(P=0.001,P=0.000,P=0.000).(4)The fourth part of the experimental study showed that after the modeling of intrauterine distress HIBD,the rats in the model group walked out of the central area for a longer time(P＜0.01),the central area distance and the total distance were significantly shortened(P<0.01,P＜0.05),and the number of activities was significantly reduced(P＜0.01).In the acupuncture group,the time to walk out of the central area was significantly shortened(P＜0.05),the distance to the central area and the total distance were significantly increased(P<0.01,P<0.05),and the number of activities was significantly increased(P＜0.01).In the positioning navigation experiment,with the increase of training days,the escape incubation period of each group showed a trend of gradually shortening,and the difference between the groups was statistically significant(P<0.001).On day 1-5,the escape latency period of the model group was significantly longer than that of the normal group(P＜0.05).In the space exploration experiment,the time of crossing the platform quadrant for the first time was significantly longer(P<0.01),the residence time of the target quadrant was significantly shorter(P＜0.01),and the number of crossing the platform was significantly reduced(P<0.01).However,in the positioning navigation experiment from day 1 to 5,the escape latency period of the acupuncture group was significantly shorter than that of the model group(P<0.05),the time of crossing the platform quadrant for the first time in the space exploration experiment was significantly shorter(P<0.05),the residence time of the target quadrant was significantly longer(P＜0.01),and The Times of crossing the platform were significantly increased(P＜0.05).Compared with the normal group,LC3II/I,beclin-1 and p62 in the hippocampus of newborn rats in the HIBD model group increased significantly(P＜0.001,P＜0.001,P<0.001).Compared with the HIBD model group,the expression of LC3II/I and beclin-1 in the hippocampus of newborn rats in the acupuncture group increased(P＜0.001,P<0.001),and the expression of p62 decreased(P<0.001).In the sham operation group,the prefrontal neurons had normal perinuclear space,continuous smooth nuclear membrane,complete organelles,hippocampal neurons had nuclear membrane light source,no changes in perinuclear space,abundant organelles,and occasionally autophagosomes.In the HIBD model group,the cortical neurons increased heterochromatin,nuclear consolidation,nuclear membrane serrations,autophagosomes increased,cell rupture contents were easy to see,hippocampal neurons expanded rough endoplasmic reticulum,mitochondria swelled and cavitated,mitochondrial cristae fell off,serrate nuclear membrane,and golgi body cavitation.The nuclear membrane of cortical neurons in the acupuncture group was smooth,organelles were abundant,the perinuclear gap was narrower than that in the model group,autophagosomes were increased,cell necrosis was not observed,the rough endoplasmic reticulum of hippocampal neurons was slightly expanded,and autophagosomes were increased.ConclusionHIBD neonatal rat hippocampal neurons have autophagy and neuronal morphological damage and other manifestations,confirming that ischemia and hypoxia can induce autophagy,and at the same time cause neuronal damage and even necrosis.Acupuncture can repair neurological deficits caused by HIBD.Acupuncture intervention activates and promotes autophagy levels,to a certain extent,promotes the repair of damaged neurons.Acupuncture activates autophagy,promotes autophagy levels,and repairs damaged neurons.It was revealed that the level of autophagy induced by ischemic and hypoxic environment is not enough to repair neuronal damage,and the promotion of further autophagy level is beneficial to the repair of neuronal damage. |
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