The Effects Of TPO On Apoptosis And Brain Edema In Traumatic Brain Injury Of Mice

Objective:Overall objective: To clarify the neuroprotective effect of TPO in mice.Specific objectives:(1)TPO in mouse brain injury model;(2)To explore the effect of TPO on nerve cell apoptosis in craniocerebral trauma in mice;(3)Explore the influence of TPO on brain edema in mice;(4)Explore the effect of TPO on nerve function in mice,and provide new ideas for clinical treatment of brain edema.Methods:ELISA,Nestin immunohistochemical method,biobehavioral method,HE staining method,dry and wet weight method,western blotting,quantitative PCR were used to observe the changes of TPO;study the exogenous TPO to reduce brain edema,effectively reduce the resulting brain dysfunction,and further demonstrate that TPO has protective effect in mice.The specific contents are as follows:One.TPO was differentially changed in normal brain tissue and at 12 h,1d,3d,7d,14 d,and 21 d after the cranbrain injury1.Using CCI(controlled cortical impact),the mice were killed at12 h,1d,3d,7d,14 d,and 21 d and brain tissue was collected2.Compare the change of TPO content in the trauma group and the normal brain tissue,and select the intervention time point;3.Formulate the drug concentration gradient according to the change,evaluate the concentration interval with neuroprotection effect according to the neurological defect score,and select the best TPO dose for intervention.Two.The efficacy of sham,TPO,inhibition(LY294002),TPO control(double steaming),TPO + LY294002,and LY294002 control(DMSO)on posttraumatic nerve cells and brain edema in mice.1.At the intervention time point,rh TPO was injected intraperitoneally for TPO group;the same dose of inhibitor,LY294002 group;TPO + LY294002group;the same dose of double steaming as the control of TPO treatment group;the same dose of DMSO as LY294002 control and sham group,including six experimental mice;2.Compare the results of water maze and neurological deficit score method in each group;3.On the fifth day after grouping,the mice were killed and the brain tissue was collected to compare the number and morphology of nerve cells in each group by Nestin immunohistochemistry and HE staining in each group;4.In the same step 3,brain tissue was obtained,and the groups were compared to determine cerebral edema by dry-wet specific gravity method,RT-PCR method and western blotting method.Three.statistical method:The experimental data were expressed using mean(x)or mean ± standard deviation(x±s),and were analyzed by Analysis of variance(Analysis of variance,anova)or multi-way analysis of variance(two-way anova)using the SPSS.Statistics.27 statistical analysis software,followed by Tukey’s HSD post hoc test,t-test,and Holm-Bonferroni correctionResults:1.Brain tissue was obtained at 12 h,1d,3d,5d,7d,14 d and 21 d after modeling.By ELISA,TPO content of brain tissue in mice doubled in 12h-1days,plummeted in 1-5 days,and gradually recovered similar to that of normal mouse brain tissue in 5-21 days.The best dose of rh TPO was designed based on the change of TPO concentration.2.After successful modeling,modeling mice developed different degrees of behavioral abnormalities such as motor function,alertness and physiological behavior.The experimental results showed that the NSS index score of mice gradually decreased with time,and the neurological function gradually recovered.However,during the experiment,the NSS score of TPO group was significantly lower than that of no-load group and TPO + LY294002 group,indicating that the TPO group showed obvious neurological recovery.3.Water maze experiment results: TPO group and false surgery group in positioning navigation test,show better learning ability,and in the space search experiment,TPO group and false surgery group has a good space environment memory ability,TPO group relative to the empty group,better space search skills,after five days of learning,the sixth day TPO group in the platform quadrant search swimming time is closer to the false surgery group.4.By HE staining method in the TPO group,the morphology of the glial cells and the no-load and TPO + LY294002 groups.The mice in the LY294002 group were disturbed,with focal infarction,edema in the surrounding tissues,hyperplasia,necrosis and hypertrophic degeneration appeared in glial cells,and abundant cytoplasm,which was slightly more severe than that of the DMSO group.5.Nestin Immunohistochemistry showed that the TPO group had more positive Nestin particles than the empty group and TPO + LY294002 group,and slightly more in the DMSO group than the LY294002 group,while the number of positive Nestin particles in TPO + LY294002 group,DMSO group and no load group was not significantly different,between TPO group and LY294002 group.6.Dry and wet weight method of mice: the brain water content of LY294002 group was significantly higher than that of DMSO group,that of TPO group was significantly lower than that of empty load group and TPO +LY294002 group,and the brain water content of sham operation group was the lowest.7.RT-PCR showed that the expression level of MMP-9 in TPO group was significantly lower than that of null and TPO + LY294002 groups;the TIMP-1m RNA in TPO group was significantly higher than that of null and TPO +LY294002 group,and the expression level of TIMP-1 m RNA in LY294002 group was significantly lower than that of DMSO group.8.western blotting The results showed that MMP-9 protein expression was significantly higher than TPO group and TPO group,and TIMP-1 protein expression was significantly lower than TPO group;MMP-9 in LY294002 group was significantly higher than that of DMSO group,and TIMP-1 protein expression was significantly lower than that of DMSO group.Conclusion:1.In early TBI occurs,TPO stress increased,and then plunge,gradually tend to normal,the experimental results found that the TPO increased in the short term,can in the TBI complications after the development of neuroprotection,and effectively reduce the resulting brain dysfunction,its mechanism may be related to reduce TBI of brain tissue neuronal degeneration and necrosis.2.Timely application of TPO in the early stage of TBI can reduce brain edema in mouse brain trauma,and the mechanism of action may interact with the possible activation of TPO through the PI3 K / AKT signaling pathway.The mechanism of action may be related to the role of TPO to inhibit MMP-9(metalloproteinase-9)expression of MMP-9 and regulate the content of MMP-9 and TIMP-1(tissue inhibitor-1)to maintain the integrity of blood-brain barrier to avoid secondary brain edema and brain injury.