The Study Of Brain-derived Microparticles Activated Microglia Leading To The Secondary Inflammatory After Traumatic Brain Injury

Purposes: Traumatic brain injury(TBI)is a series of complex pathophysiological processes.The combined effect of primary injury and secondary injury leads to the destruction of brain.Secondary neuroinflammation is an important pathophysiological reaction after TBI,and plays an important role in secondary brain injury.A better understanding of the regulation mechanism of microglia phenotypic transformation can improve our understanding of nerve injury and repair after TBI,However,the molecular regulation mechanism of microglia activation and differentiation is extremely complex,the specific mechanism is still unclear.Microparticles(MPs)are small cell vesicles ranging in size from 0.1 to 1um.They contain a variety of biologically active molecules,including proteins,lipids and nucleic acids.They shuttle between cells to act as transmitters,and are important mediators for the body to produce related inflammatory reactions.However,the molecular biological characteristics of Brain-derived Microparticles(BDMPs)are still unclear,and there are no reports on the role of BDMPs in the secondary inflammation after TBI.Therefore,we used subcortical injection model of mice and microglia in vitro to observe the effect of BDMPs on microglia activation and related inflammatory response,and to explore the role and mechanism of BDMPs in the secondary inflammation of TBI by mediating microglia.Method: 1.Establish a mouse model of hydraulic impact brain injury(TBI).BDMPs were extracted from the brain injury area after TBI by enzyme digestion gradient centrifugation.The phenotype and component analysis of BDMPs were performed by flow cytometry.2.The activation,proliferation and differentiation of microglia were observed by immunofluorescence staining and immunohistochemical staining.The RNA content of inflammatory factors TNF-a,IL-6,IL-l0 and monocyte chemokine-1(MCP-1)were detected by RT-PCR.3.BDMPs were co-cultured with microglia(BV2).The activation and transformation of microglia were observed by ion conductivity microscopy.Western-blot and ELISA were used to detect the changes of inflammatory factors TNF-α,IL-1β,IL-6,IL-l,MCP-1,CCL-2.4.The damage of blood-brain barrier(BBB)was observed by Evans blue injection,and the degree of brain edema was observed.Human umbilical vein endothelial cells(HUVEC)were cultured in vitro to simulate the BBB.The permeability of HUVEC barrier was observed by adding BDMPs.Result: 1.BDMPs have a complete membrane structure with various biomarkers(NSE and GFAP)on the surface.And BDMPs are composed of multicellular MPs,mainly neuronal and glial cell-derived MPs,and also contain MPs from blood cells and endothelial cells.2.Microglia can be activated by BDMPs.The morphology of microglia transforms from branching to spherical,and the phenotype of M1/M2 changes dynamically.The results of PCR showed that the expressions of IL-1beta,TNF-a,IL-6,CCL2 and NOS2 in brain tissue increased.3.In vitro,microglia and BDMPs began to deform after 52 minutes of co-culture,and fully activated after 104 minutes.After 3 hours,the levels of IL-6,TNF-α and MCP-1 in cell lysate increased significantly,while the levels of TNF-α and MCP-1 in supernatant increased by ELISA,but the levels of IL-10 did not change significantly.4.After BDMPs injection,obvious edema and Evans blue leakage were observed on the surface of brain tissue in mice.When BDMPs was added in vitro,the leakage of FITC-Dextran by HUVEC barrier increased significantly.Conclusion: 1.BDMPs can combine and mediate the activation,deformation and differentiation of microglia,promote the expression of TNF-α,IL-1β,IL-6 and MCP-1/CCL-2,and play an inflammatory role in the secondary inflammation after TBI.2.BDMPs can increase BBB permeability and promote local inflammatory response.