Study On The Mechanism Of Myeloid Cells Phagocytosis-Inflammatory Imbalance Participates In Synapse Damage After Hypoxia-ischemia Brain Injury

Objective:Hypoxia-ischemia brain injury(HIBD)is the leading cause of death and disability in newborns.30%of surviving children will have neurological sequelae,but the pathogenesis is unclear.Synapses are the core of information transmission and synaptic damage is the key mechanism of abnormal behavior in children with HIBD.Myeloid cells refer to all myeloid cells differentiated from immature cells in the pre-myeloid lineage,including granulocytes,erythroid cells,megakaryocytes and monocytes in a broad sense,in the brain,they are mainly microglia cells(MGs)and monocyte-derived macrophages(MDMs).Neuroinflammation caused by active myeloid cells is one of the core mechanisms of HIBD.Phagocytosis and inflammation are the main functions of myeloid cells(MGs and MDMs),which regulate synaptic plasticity.Under physiological conditions,phagocytosis and inflammation in the developing brain are maintained at a certain level,and the "phagocytosis-inflammation" balance will help to form stability neural circuit.Under pathological conditions,"phagocytosis" and/or "inflammation" functions will lose its original steady state and will activate or inhibite,and no longer maintain a balanced state.This is the "phagocytosis-inflammation" imbalance.In addition to phagocytosis and inflammation,current research believes that MGs and MDMs,as the most important immune cells in the brain.Under physiological conditions,it can participate in the regulation of synaptic pruning through phagocytosis and the release of certain inflammatory mediators,thereby participating in the formation of neural circuits.Under pathological conditions,they initiate brain’ inflammatory response in the brain,and can also play a role in the damage and repair of the homeostasis of the microenvironment through phagocytosis.After HIBD,the "phagocytosis-inflammation" of MGs and MDMs in the brain is out of balance.The pathogenesis of HIBD-induced synaptic damage,and its molecular mechanism,especially possible regulation,need to be further studied.Previous studies in our laboratory have confirmed that in addition to the activation of MGs after HIBD,MDMs also invaded into the brain parenchyma,and the inflammation and phagocytosis of MGs were active,but the relationship between the two and the repair of synaptic damage is still unclear.Based on this,this study will focus on the "phagocytosis-inflammation"imbalance caused by myeloid cells(MGs and MDMs)in HIBD,and focus on their function to synaptic damage and repair,try to reveal the mechanism and possible regulation of the "phagocytosis-inflammation" imbalance of the two myeloid cells in the synapse damage after HIBD.Methods:1.CX3CR1GFP/+CCR2RFP/+ double transgenic mice and C57BL/6J mice were used to establish a neonatal mouse HIBD model through the modified Rice-Vannucci method to further confirm the functions of the two different myeloid cells.After that,the animals were randomly divided into sham operation group(Sham)and hypoxia-ischemia group(HI).The HI group was further divided into hypoxia-ischemia mild injury group(HIM)and hypoxia-ischemia severe injury group(HIS)based on previous studies.2.Behavioral experiments were used to confirm the behavioral changes of mice after HIBD 6 w.3.TTC staining was used to confirm the cerebral infarction area of mice brain after HIBD 3 d.4.HE staining was used to reveal the morphological changes of brain tissue injury after HIBD 3 d and 6 w.5.Nissl staining was performed to evaluate the damage of brain tissue neurons after HIBD 3 d and 6 w.6.Tunel staining was used to assess the damage of brain tissue cells after HIBD 3 d.7.FJB staining was used to evaluate the damage of brain tissue neurons after HIBD 1 d and 3 d.8.Western Blotting was used to detect the expression of synapse-related proteins(PSD95 and SYP)and TREM2 after HIBD 3 d,as well as the changes in the expression of postsynaptic membrane protein-PSD95 in the MGs cell line BV2 before and after OGD.9.Immunofluorescence technology was used to detect the expression of two kinds of myeloid cells in the cortex and hippocampus of the brain after HIBD 3 d,and the relationship between them and synapse-associated proteins.10.Immunofluorescence technology was used to detect the expression of LAMP 1 and PSD95 in CD11b+cells in the brain after HIBD 3 d.11.Two myeloid cell lines(MGs cell line-BV2,mononuclear macrophage cell line-Raw264.7)were used for phagocytic stimulation experiments to detect the changes in the phagocytic ability of the two myeloid cells before and after OGD.12.Flow cytometry experiment combined with magnetic bead sorting to detect the expression of two myeloid cells TREM2 and CD200R in the brain after HIBD 3 d.13.Immunofluorescence technology was used to detect the expression of two myeloid cells TREM2 and its adaptor DAP 12 and pro-inflammatory factors IL-1(3 and IL-6 in the brain after HIBD 3 d.14.Immunofluorescence technology was used to detect the secretion of PSD95 protein in primary MGs after stimulation by inflammatory factors(IL-1(3,IL-6).Results:1.Verify the brain damage after HIBD(1)Behavioral changes after HIBDa.The results of the open field experiment confirmed that the motor function of the mice in each group at 6 w after HIBD was not significantly impaired,but the mice in the HIM group were detected in the open field test within 10-15 min and 20-25 min,the staying time in the middle area was longer than that in the Sham group.b.The results of the shuttle box experiment confirmed that after HIBD 6 w,in the HIM and HIS groups,the number of through between the black box and the white box were decreased.(2)Abnormal brain histopathology after HIBDa.After HIBD 3 d,there were obvious infarcts in the brain tissue of the mice in the HIS group,but no obvious infarcts were seen in the Sham group and HIM group.b.After HIBD 3 d,the cerebral cortex and hippocampus of the HIS group mice were loose,stained lightly,and phagocytic neuron phenomenon were seen.And focal macrophages,MGs and a small amount of neutrophil infiltrate into brain,and some cells showed typical necrosis.There was no obvious change in the morphology of the brain tissue of the HIM group.After HIBD 6 w,the hippocampal-CA1,CA2,and CAS areas of the HIS group showed tissue structure destruction,cell number decreased,and neuron loss,HIM group hippocampal-CA1 area cells were decreased,and some neurons were lost.(3)Neuron’ damage after HIBDa.After HIBD 3 d,the number of Nissl bodies in the cerebral cortex and hippocampus of the HIS group were decreased and the staining became lighter,while it did not change significantly in HIM group,after HIBD 6 w,the number of Nissl bodies in the hippocampus-CA1,CA2,and CA3 of the HIS group were reduced and the staining became lighter,the number of Nissl bodies in the hippocampal-CA1 area of the HIM group were reduced and the staining became lighter.b.After HIBD 3 d,a large number of apoptotic cells appeared in the cerebral cortex and hippocampus-CA1,CA2,CA3 of the HIS group,and the cortex and CA2 were the most serious.No apoptotic cells were seen in the hippocampal-dentate gyrus.c.There were a large number of acute necrotic neurons in the cerebral cortex and hippocampus-CA2 area after HIBD 1 and 3 d.2.Verify synapse damage after HIBD(1)Synapse loss and clearance:Synapse loss increases after HIBD,and MGs are the myeloid cells that phagocytize synapsesa.After HIBD 3 d,PSD95 in the Sham group and HIM group were scattered around the MGs,MDMs were not observed in the brain parenchyma.MGs had smaller cell bodies,longer protrusions,and more branches.PSD95 could be seen in the branches and cell bodies.PSD95 in the cortex and hippocampus of HIS group increased,the cell body of MGs became rounded,and the branches were shortened.There were a large number of PSD95 in the cell body,some of which could be distributed along the cell membrane.MDMs mainly existed in the central area of infarction,but did not contact the postsynaptic membrane protein PSD95.b.After HIBD 3 d,the SYP of the Sham group and the HIM group showed a thick layer,spread on one end of the MGs cell body.MDMs were not observed in the brain parenchyma.The MGs cell body were smaller,with longer protrusions and branches and cells.SYP could be seen in the branches and cell bodies.SYP in the cortex and hippocampus of HIS group were still thick,but not on one end of the MGs cell body,but acrossed the middle of the MGs cell body.The MGs cell body becomed rounded and the branches were shortened.The protrusions of some cells were almost invisible,It was a small amount of SYP in the cell body.MDMs mainly existed in the central area of the infarcts,but they hardly uncontacted with the SYP.c.After HIBD 3 d,the expression of synaptophysin protein-SYP in the cerebral cortex and hippocampus-CA2 area of the HIS group were decreased in the central area of the infarcts,and were increased in the edge area of the infarcts,and mainly co-localized with MGs.The expression of synaptophysin protein SYP in the cerebral cortex and hippocampus CA2 area of mice were increased in HIM group.(2)MGs express synaptic proteina.After HIBD 3 d,the postsynaptic membrane protein PSD95 were increased in the cerebral cortex area of the HIS group mice,and it distributed around the MGs,parallel to the MGs cell bodies in spatial distribution,the cell bodies of the MGs became rounded,and the branches were shortened.Some cells had almost no branches.There were a large number of postsynaptic membrane protein PSD95 in the cell body,some of which can be distributed along the cell membrane.MDMs mainly existed in the central area of the infarcts,but hardly did not contact the PSD95,in the Sham and HIM groups,the PSD95 protein in the cerebral cortex and hippocampus-CA2 area were scattered around MGs,and is parallel to the MGs in spatial distribution.MGs had smaller cell bodies,longer protrusions,and more branches.PSD95 protein can be seen in the MGs’ branches and cells and no MDMs were observed in the brain parenchyma.b.After HIBD 3 d,in the cerebral cortex area and hippocampus of the HIS group,the synaptophysin protein-SYP,compared with the postsynaptic membrane protein PSD95,showed a thick layer that traverses the MGs cell bodies and were perpendicular to the MGs cell bodies.The cell bodies of the MGs became rounded,and the branches were shortened.Some cells had almost no branches.And there was a small amount of synaptophysin protein-SYP in the cell body.MDMs mainly existed in the central area of the infarcts,but hardly did not contact with the S YP protein.In the Sham and HIM group,the synaptophysin protein SYP in the cortex presented a thick layer and was concentrated at one end of the MGs cell bodies and which was perpendicular to it.The cell body of MGs is smaller,with longer protrusions,and more branches.The synaptophysin protein SYP can be seen in the branches and cell bodies,no MDMs were observed in the brain parenchyma.c.After HIBD 3 d,in the cerebral cortex area and hippocampus of HIS group,some postsynaptic membrane protein PSD95 and lysosomal protein 1 LAMP1 co-localized in CD11b+cells,and some did not co-localize with LAMP1.d.After OGD 0 h,the expression of PSD95 in the MGs cell line were increased significantly,and returned to the pre-OGD level at 24 h after OGD.e.After OGD 0 h,the expression of PSD95 in primary MGs increased.After stimulation by IL-6,the MGs secreted more PSD95 than the group without inflammatory factors under normal culture condition.After OGD,the level of PSD95 protein secreted by MGs was further increased,which was higher than that of the normal culture group after inflammation stimulation.3.The "phagocytosis-inflammation" imbalance in the brain after HIBD(1)Phagocytosis was active after HIBD,and MGs mainly performed the function of phagocytosis of synapsesa.After HIBD 3 d,the total protein expression of TREM2 in the cerebral cortex and hippocampus of the HIS group were increased.b.After HIBD 3 d,MDMs infiltrated into brain in the cerebral cortex and hippocampus of the HIS group.c.Under normal culture conditions and after glucose and oxygen deprivation(OGD),the phagocytic capacity of mouse MGs cell line-BV2 was always stronger than that of mononuclear macrophage cell line-Raw264.7,BV2 cells remained strong phagocytic ability after OGD,but the phagocytic ability of Raw264.7 cell line were decreased after OGD.(2)The inflammatory response is active after HIBD.Both MGs and MDMs express inflammatory mediators,but only MDMs express IL-6a.After HIBD 3 d,the levels of pro-inflammatory factor IL-1β of MGs and MDMs in the edge area of cerebral cortex infarction in the HIS group were increased,the levels of IL-1β in the cerebral cortex of the HIM group did not change significantly,the IL-1β of MGs and MDMs in the hippocampus-CA2 central and edge area were increased.In the hippocampal-CA2,the IL-1β of MGs and MDMs were decreased in the HIM group.b.After HIBD 3 d,the level of pro-inflammatory factor IL-6 of MDMs were increased in the cerebral cortex and hippocampus-CA2 central area of the infarcts in the HIS group,while were decreased in the edge area of the infarcts.In HIM group,there was no significant change in IL-6 of MDMs in brain cortex,but it was decreased in the hippocampal.No MGs expressed IL-6 in each group.4.Possible regulatory mechanism of "phagocytosis-inflammation" imbalance in the brain after HIBDa.After HIBD 3 d,the expression of TREM2 and CD200R of MGs in the cerebral cortex of the HIS group were increased,but the expression of MDMs did not change significantly,in the HIM group,only MDMs expressed CD200R was increased.b.After HIBD 3 d,the expression of TREM2 of MGs in the center and edge areas of infarcts in the cerebral cortex and hippocampus-CA2 area of HIS group were increased,while there were no significant changes in the HIM group,and MDMs did not express TREM2 in each group.The expression of TREM2 adaptor DAP 12 in MGs and MDMs in the center and edge areas of infarcts in the cortex and hippocampus-CA2 area of HIS group was increased,while there was no significant change in HIM group.c.After HIBD 3 d,the expression of PSD95,TREM2,and DAP 12 were increased in the central area of the infarcts in the cerebral cortex and hippocampus-CA2 area of the HIS group.After zooming to 600X,we found that some cells can co-express TREM2,PSD95 and DAP 12.But the specific cell types need to be further studied.Conclusions:1.Neurons and synapses in the brain are damaged after HIBD.MGs are myeloid cells that perform synaptic phagocytosis in the brain and can express the phagocytic protein TREM2.TREM2 may be involved in regulating the phagocytosis of MGs and MDMs after HIBD.2.MG can swallow and secrete the postsynaptic membrane protein PSD95,and inflammatory factors(IL-6 and IL-1β)can stimulate the expression of PSD95 in MGs.3.Both MGs and MDMs can release the inflammatory factor IL-1β,but MDMs have a stronger inflammatory response and mainly express IL-6,and the IL-6 released by them may have a regulatory effect on the phagocytic function of MGs.CD200R is involved in regulating the inflammatory release of MGs and MDMs.