| Objective:To investigate the effects of electroacupuncture on learning and memory ability andα7 nAChR expression and microglia subtype conversion brain inflammatory response in hippocampal of mice after internal fixation of tibial fracture.Methods:In this study,a total of 95 SPF 6-month-old C57BL/6 male mice were selected as experimental subjects.According to the number table method,they were randomly divided into control group,simple tibial fracture model group(surgery group),acupoint electric stimulation + tibial fracture model group(electroacupuncture group),α7nAChR agonist PNU282987 + tibial fracture model group(PNU group)and acupoint electric stimulation +α7nAChR antagonist MLA + tibial fracture model group(MLA+electroacupuncture group),with 19 rats in each group.The control group was not subjected to other treatments except the daily water maze positioning cruise test.The other groups were treated with internal fixation for tibial fracture of right hind limb after5 days of water maze positioning cruise.Electroacupuncture group received 7 days of acupuncture point electric stimulation.PNU group was given α7nAChR agonist for 7 days postoperatively.MLA+ electroacupuncture group was given 7 days after the operation,and α7nAChR antagonist was also given.In the whole experiment,positioning cruise experiment and space exploration experiment were used to evaluate the cognitive function of mice.Mice in each group were randomly removed from the neck 1 day before surgery,1 day after surgery,3 days after surgery and 7 days after surgery,and 4 mice in each group were sacrificed at each time point and 7 days after surgery.The levels of tumor necrosis factor-α(TNF-α),interleukin-1 β(IL-1β),interleukin-4(IL-4),interleukin-10(IL-10)and other cytokines in the hippocampus of mice were detected by enzyme-linked immunosorbent assay.The microglial cell typing in the hippocampus was detected by flow cytometry 7 days after the operation.The expression of α7nAChR in hippocampus of mice was detected by immunofluorescence assay(ELISA)on the 7th day after operation.Results:1.General situation:1)Conditions of mice during the whole experiment cycle: 12 hours of alternating light and dark environment,suitable temperature and humidity,good development of mice in each group,no significant change in body weight,no fighting and biting during feeding.2)The internal fixation model of tibial fracture was established,the anesthesia induction of intraoperative mice was stable,the wound was small,the blood loss was small,the intraoperative heat preservation measures were taken,the postoperative mice recovered well,the activity was not restricted,and the overall condition was good.2.Morris water maze test results:1)Adaptive training: 95 mice were put into the water maze one by one,and the mice without swimming ability,defective vision and suspended animation in water were removed,and the qualified standby mice were replaced.2)Positioning cruise test: modeling after five days of orientation cruise before experiment,each group of mice swimming route by the original attached to purposeful swam straight lines in the direction of the platform,with the increasing number of days training,the type has been able to find more quickly in the water maze of platform,to escape the incubation period shorten,gradually in the training to the fourth day,there was no significant difference in escape latency of each group after statistical comparison and analysis.These results indicated that mice in each group had a better memory of platform position in water maze after adaptive training.After modeling,the escape latency of mice in each group was observed at three time points of 1 day,3 days and 7 days after operation.The results showed that compared with the control group,the escape latency of mice in other groups after internal fixation of tibial fracture was significantly prolonged,with statistical significance.3)Space exploration test: After modeling,the platform was removed at each time point to measure the times of crossing the platform of mice in each group.Space exploration experiment was conducted 2 hours before the start of positioning cruise experiment.The results showed that compared with the control group,The Times of crossing the platform in the other groups were significantly reduced,and the aimless wall adhesion movement was more common,with statistical significance.3.ELISA results:In this study,the contents of cytokines including IL-1β,IL-4,IL-10 and TNF-α in the hippocampus of mice were determined 1 day before operation,1day after operation,3 days after operation and 7 days after operation.Results of each index: There was no statistical significance in the contents of four cytokines measured in the hippocampus of each group before operation(P > 0.05).Compared with the control group,the contents of IL-1β and TNF-α in other groups were significantly increased 1day after operation(P < 0.05).3 days after surgery,the contents of IL-1β and TNF-α in MLA+ electroacupuncture group had no significant change(P < 0.05),while the contents of IL-1β,TNF-α,IL-4 and IL-10 in electroacupuncture group and PNU group increased(P < 0.05).7 days after surgery,the contents of IL-1β and TNF-α in the operation group were still higher than those in the control group,with statistical significance(P<0.05),while the contents of IL-1β and TNF-α in the electroacupuncture group,PNU group and MLA+ electroacupuncture group were decreased(P>0.05).Compared with the operation group,the contents of IL-1β and TNF-α in the PNU group were significantly increased 1day after surgery(P<0.05),while the contents of IL-1β and TNF-α in the MLA+electroacupuncture group were not significantly increased(P>0.05).The contents of IL-1β and TNF-α in PNU group had no significant change(P<0.05),while the contents of IL-4 and IL-10 increased(P<0.05).The contents of IL-1β and TNF-α in MLA+electroacupuncture group had no significant change(P>0.05),while the contents of IL-4and IL-10 increased(P<0.05).The contents of IL-1β and TNF-α in PNU group were decreased(P<0.05),while the contents of IL-4 and IL-10 were increased(P<0.05).The contents of IL-1β and TNF-α in MLA+ electroacupuncture group were decreased,but there was no statistical significance(P>0.05),while the contents of IL-4 and IL-10 were increased(P<0.05).4.Flow cytometry results:In this study,CD206 and CD86 were measured by flow cytometry 7 days after the operation to determine the content of M1 and M2 subtypes of microglia in the hippocampus of mice.The microglial cells in the hippocampus of the control group were mostly in a resting state due to the absence of internal fixation of tibial fracture.The contents of M1 and M2 microglia in the hippocampus of mice in the operation group and MLA+ electroacupuncture group were increased,and M1 was much higher than M2.The content of M1 and M2 microglia in hippocampus of electroacupuncture group and PNU group was increased,and M2 was much higher than M1.5.Immunofluorescence staining results:The content of α7nAChR on the surface of microglia in the hippocampus of mice was determined by immunofluorescence staining 7days after operation.Through statistical analysis of IOD and surface density,the results showed that:Compared with the control group,the content of α7nAChR on the surface of microglia in the hippocampus of mice in the surgery group was significantly decreased,and that in the electroacupuncture group,PNU group and MLA+ electroacupuncture group was significantly increased,and the content of α7nAChR in the electroacupuncture group and PNU group was significantly increased.Compared with MLA+electroacupuncture group,it was statistically significant.The content of α7nAChR on the surface of microglia in the hippocampus of mice in the PNU group was higher than that in the electroacupuncture group,and there was statistical significance between the two groups.Conclusion:1.After internal fixation of tibial fracture,the learning and memory ability of mice was decreased,and the decreased learning and memory ability was positively correlated with the decrease of α7nAChR content and anti-inflammatory factors(IL-4 and IL-10)in the hippocampus,and negatively correlated with the decrease of pro-inflammatory factors(IL-1β and TNF-α).2.Electroacupuncture can improve learning and memory in tibial fracture model mice by decreasing inflammatory factors in the hippocampus.3.Electroacupuncture stimulation can change the M1/M2 ratio of microglia in the hippocampus of model mice,which may be a key link in regulating the content of anti-inflammatory factors and pro-inflammatory factors in the hippocampus.4.The change of M1/M2 ratio of microglia may be related to the activation ofα7nAChR.The effect of electroacupuncture stimulation was similar to that of α7nAChR agonist. |
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