| Objective:In childhood,the unilateral common carotid artery ligation(Vannucci)combined with 8%hypoxic environment was used to establish the HIBD model.After adulthood,the chronic unpredictable Mild Stress(CUMS)was used to establish a depression model.The susceptibility to depression and the immunological mechanism involved in adulthood after hemorrhagic hypoxic brain injury in neonatal period provide a certain reference for the mechanism research and clinical pharmacology research of post-stroke depression.Methods:Male C57BL/6J mice weighing 4-6 g on day 9(P9)were randomly divided into sham operation group(Sham)and ischemia-anoxia group(HIBD),established by left common carotid artery ligation(Vannucci).The HIBD model was then exposed to a mixture of ambient temperature of 34?-34.5? and 8%O 2+92%N 2 for 45 min.After the establishment of the HIBD animal model,surviving mice and sham-operated(Sham)mice were continuously placed in adulthood at 21%02 in a temperature of 28?.From week 6(P42 days),mice randomly selected from HIBD model mice weighing 18-23 g were randomly divided into ischemia-anoxia group(HIBD)+ blank control group(control)and ischemia-anoxic group(HIBD).+chronic stress group(CUMS);also randomly divided into sham operation group(Sham)+blank control group(sham)+sham operation group(Sham)+chronic stress group(CUMS)from sham operation group(Sham)),the experimental group and the control group of mice C57BL/6J every 4 cages,normal feeding.A chronic unpredictable mild stress was administered to the Sham+CUMS group and the HIBD+CUMS group daily to establish a mouse model of depression,while the Sham+control and HIBD+control mice were not stressed,but were reared.The conditions are the same.Whether the HIBD model was successfully established by The grip test,Hanging test,weight and laser speckle blood flow imaging;whether the mice were depressed by a series of behavioral experiments,including tail suspension experiment,forced swimming test,and sugar water preference test Behavior;black-and-white box experiment and three-box experiment were used to observe whether there was anxiety-like behavior and social function impairment in mice;finally,flow cytometry was used to detect monocytes,microglia,B lymphocytes and The content of T lymphocytes.Results:Part I Establishment and evaluation of a hypoxic-ischemic brain injury model(HIBD)in neonatal mice1.In laser speckle blood flow imaging,cerebral blood flow was significantly decreased in the left injury area of the mild injury group(HIM)group and the severe injury group(HIS)compared with the Sham group(HIM vs Sham,F=27.44,p=0.01;HIS vs Sham,F=27.44,p<0.01);blood flow in the left lesion of the HIS group was significantly lower than that in the HIM group(HIS vs HIM,F=27.44,p=0.04),while the right uninjured area There was no difference in cerebral blood flow between the groups.2.In the neurological deficit score,the grip test,P10-P12,HIM group and HIS group were significantly lower than the Sham group(HIM vs Sham,p<0.05;HIS vs Sham,p<0.05).P11-P12,HIS group grasp score was significantly lower than HIM group(HIS vs HIM,p<0.05).In the Hanging test,the suspension time of the P10-P11 and HIS groups was less than that of the Sham group(HIS vs Sham,p<0.05),while the HIM group had no difference with Sham;P12,compared with the Sham group,the HIM group.Suspension time decreased significantly in the HIS group(HIM vs Sham,F=10.38,p=0.048;HIS vs Sham,F=10.38,p<0.01).3.In terms of body weight,the weight gain of the mice in the HIM group and the HIS group was slower than that in the Sham group(p<0.001),while the weight gain in the HIS group was slower(p<0.001).From the first day after surgery(P10-P16),the body weight of HIM group and HIS group were lower than that of Sham group(HIM vs Sham,p<0.05;HIS vs Sham,p<0.05);-P16,the weight of the HIS group was significantly lower than that of the HIM group(HIS vs HIM,p<0.05).Part ? Establishment and evaluation of depression model1.The TST results showed that the HIBD group had a low immobility time(Sham vs HIBD,t=2.35,p=0.024)compared with the Sham group,and the Sham+CUMS group began with the onset of chronic stress.Compared with the HIBD+CUMS group,the stationary time was gradually extended(F=5.623,p=0.001).From the first week to the third week,the stationary time of the Sham+CUMS group was longer than that of the HIBD+CUMS group(Sham+CUMS).Vs HIBD+CUMS,p<0.05),by week 4,there was no difference between the two groups.2.The FST results showed that before the chronic stress,the HIBD group was stationary for a shorter period of time than the Sham group(HIBD vs Sham,t=2.46,p=0.019).With the onset of chronic stress,the stationary time of the Sham+CUMS group and the HIBD+CUMS group gradually prolonged(F=11.731,p<0.001).From the 3rd week,the Sham+CUMS group and the HIBD+CUMS group were still.The immobility time was longer than that of the Sham+control group and the HIBD+control group(Sham+CUMS vs Sham+control,p<0.05;HIBD+CUMS vs HIBD+control,p<0.05),but the Sham+CUMS group and HIBD+CUMS There is no difference between the groups.3.The results of the saccharification preference experiment showed that the sucrose preference rate of the Sham+CUMS group began to decrease from the first week(F=33.218,p<0.001),which was lower than that of the Sham+control group(Sham+CUMS vs Sham+control,p<0.05),while the preference rate of mice in the HIBD+CUMS group was lower than that of the control group from the second week(HIBD+CUMS vs HIBD+control,p<0.05).4.In terms of body weight,before the chronic stress,the weight of the mice in the HIBD group was lower than that in the Sham group(HIBD vs Sham,t=5.83,p<0.01).After chronic stress,the weight of the HIBD+CUMS group and the Sham+CUMS group were lower than those of the HIBD+control group and the Sham+control group(HIBD+CUMS vs HIBD+control,p<0.05;Sham+CUMS vs Sham+control,p<0.05),but there was no difference between the HIBD+CUMS and the Sham+CUMS groups.5.The experiment results of black and white box showed that from the first week,the accumulation time of the clear box of HIBD+CUMS group and Sham+CUMS group gradually decreased(F=5.321,p=0.004),and the HIBD+CUMS group decreased compared with the control group(HIBD+CUMS vs Sham+CUMS,p<0.01),the third week of chronic stress,the accumulation time of the clear box in the Sham+CUMS group was lower than that of the control group(Sham+CUMS vs Sham+control,p<0.05),and the fourth stage of chronic stress Week,the accumulation time of the clear box in the HIBD+CUMS group was lower than that in the Sham+CUMS group(HIBD+CUMS vs Sham+CUMS,F=14.38,p=0.029).6.Three boxes of experimental results showed that from the second week,the sniff time of the HIBD+CUMS group was lower than that of the HIBD+control group(HIBD+CUMS vs HIBD+control,p<0.05),while Sham+CUMS The scent time of the mice in the group gradually decreased from the third week(F=10.759,p<0.001).At the fourth week of chronic stress,the smear time of the HIBD+CUMS group was lower than that of the Sham+CUMS group(HIBD+CUMS).Vs Sham+CUMS,F=30.89,p=0.015).In the social initiative stage(Socialbility),the social initiative coefficient(S1/(S1+E))of the Sham+CUMS group and the HIBD+CUMS group gradually decreased from the first week(F=4.686,p=0.004).In the middle of the week,the social initiative coefficient of the HIBD+CUMS group was significantly lower than that of the Sham+CUMS group(HIBD+CUMS vs Sham+CUMS,F=8.00,p=0.012).In the social memory stage,the social memory coefficient(S2/(S1+S2))of the HIBD+CUMS group was lower than that of the control group from the first week(HIBD+CUMS vs HIBD+control,p<0.05).The social memory coefficient of the Sham+CUMS group was lower than that of the control group at the 4th week(Sham+CUMS vs Sham+control,F=24.24,p=0.031).The social memory coefficient of the HIBD+CUMS group was higher than that of Sham.+CUMS reduction was evident at weeks 1,3,and 4(HIBD+CUMS vs Sham+CUMS,p<0.05).Part III Expression of hippocampal immune cells in mice with post-stroke depressionIn flow cytometry,the percentage of MDMs(CD45high/CD11b+)in the Sham+CUMS group was higher than that in the control group(Sham+CUMS vs Sham+control,F=7.08,p=0.049),and HIBD+CUMS was decreased(HIBD+CUMS vs HIBD+control,F=7.08,p=0.014);Sham+CUMS group MC(CD451ow/CD11b+)percentage reduction(Sham+CUMS vs Sham+control,F=7.09,p=0.049),HIBD+CUMS The percentage of group MC(CD451ow/CD 11 b+)increased(HIBD+CUMS vs HIBD+control,F=7.09,p=0.014);the percentage of B lymphocytes(CD45+CD19+)in Sham+CUMS group increased(Sham+CUMS vs Sham+control,F=19.39,p=0.002),while the HIBD+CUMS group decreased(HIBD+CUMS vs HIBD+control,F=13.39,p<0.01);the percentage of T lymphocytes(CD4+/CD45+)in the Sham+CUMS group increased(Sham+CUMS vs Sham+control,F=6.55,p=0.004),percentage reduction of T lymphocytes(CD4+/CD45+)in HIBD+CUMS group(HIBD+CUMS vs HIBD+control,F=6.55,p=0.05);HIBD+The percentage of T lymphocytes(CD8+/CD45+)was reduced in the CUMS group(HIBD+CUMS vs HIBD+control,F=2.89,p=0.041).Conclusion:1.On the 9th day after birth,the ischemic stroke mice were prepared by Vannucci method.The results of laser speckle blood flow imaging,neurological function assessment and body weight change proved that the model of hypoxic-ischemic brain damage(HIBD)was successfully established..2.In adulthood(6w),after chronic unpredictable mild stress(CUMS),the FST,TST immobility time gradually increased,the syrup preference decreased gradually,and the weight gain was slow.The FST and TST results suggest that hypoxic-ischemic brain damage increases the susceptibility of depression in adult mice,and the basal level of depression-like behavior in HIBD mice is lower.3.The anxiety-like behavior and social function of the HIBD model group were not significantly different from those of the sham-operated group,but with the progress of chronic stress,the anxiety-like behavior and social function of the HIBD group were more significantly impaired.4.After neonatal rats undergo negative stress events such as ischemia and hypoxia,the pathogenesis of depression in adulthood may involve the involvement of immune cells in the brain,which may be manifested by down-regulation of monocyte expression and B lymphocytes(Down-regulation of CD 19+)and T lymphocytes(CD4+,CD8+)expression and upregulation of microglia expression. |
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