Effect And Mechanism Of Microglia In Glial Scar Formation After Spinal Cord Injury

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Fascin-1 is highly expressed specifically in microglia after spinal cord injury and regulates microglial migrationBackground:Recent research indicates that after spinal cord injury(SCI),microglia accumulate at the borders of lesion between astrocytic and fibrotic scars and perform inflammation-limiting and neuroprotective functions;however,the mechanism of microglial migration remains unclear.Fascin-1 is a key actin-bundling protein that regulates cell migration,invasion and adhesion,but its role during SCI has not been reported.Methods:A mouse model of thoracic(T10)spinal cord compression injury was used.Western blot was used to detect the expression level and changes of Fascin-1 protein before and 3,7 and 14 days after spinal cord injury.Immunofluorescence was used to make Fascin-1 and the main cellular markers of spinal cord double labeled respectively,and the cellular localization of Fascin-1 was analyzed.Microglia were cleared in vivo and the changes of Fascin-1 were observed.Scratch assay and Transwell were used to observe the effect of Fascin-1 on microglia migration in vitro.Polarization of microglia was induced in vitro,and the expression of Fascin-1 was observed.Results:We found that at 7-14 days after SCI in mice,Fascin-1 is significantly upregulated(P<0.001),mainly distributed around the lesion,and specifically expressed in CX3CR1-positive microglia.The percentage of Fascin-1~+CX3CR1~+costained cells relative to the total number of Fascin-1~+or CX3CR1~+cells in the injured spinal cord reached 94.06±0.82%or 87.65±1.08%,respectively.However,Fascin-1 is not expressed in GFAP-positive astrocytes,Neu N-positive neurons,NG2-positive cells,PDGFR?-positive cells,or blood-derived Mac2-positive macrophages infiltrating into the lesion core.The expression of Fascin-1 is correspondingly decreased after microglia are specifically depleted in the injured spinal cord by the colony-stimulating factor 1receptor(CSF1R)inhibitor PLX5622(P<0.0001).The upregulation of Fascin-1expression is observed when microglia are activated by myelin debris in vitro,and microglial migration is prominently increased(P<0.05).The inhibition of Fascin-1expression using small interfering RNA(si RNA)markedly suppresses the migration of microglia,but this effect can be reversed by treatment with myelin(P<0.05).The M1/M2 polarization of microglia does not affect the expression of Fascin-1.Conclusions:Our results suggest that Fascin-1 is highly expressed specifically in microglia after SCI and can play an important role in the migration of microglia and the formation of microglial scars.Hence,the elucidation of this mechanism will provide novel therapeutic targets for the treatment of SCI.Part ? Microglia M1 polarization can promote astrocytes to produce CSPG through TGF? 1/SOX9 pathway after spinal cord injuryBackground: After spinal cord injury,astrocytes and microglia gradually migrate around the lesion core,astrocytes proliferate and hypertrophy to form astrocytic scar,and produce CSPG,to limit the spread of inflammation,but also hinder the growth of axons.Microglia form microglial scar and can also show pro-inflammatory M1 polarization and anti-inflammatory M2 polarization according to different microenvironment.However,the effect of microglia polarization on astrocytes is not clear.Methods: A mouse model of thoracic spinal cord compression injury(T10)was used.Tissue immunofluorescence was used to observe the polarization of microglia M1(i NOS)and M2(Arg1)in pre-operation and 3,7 and 14 days after spinal cord injury,and the location relationship between microglia and astrocytes on 14 days.Mouse microglia and astrocyte cell lines were cultured in vitro.Western blot and immunofluorescence were used to verify the polarization induction of microglia,and the expression of TGF?1 in microglia was detected.Tissue immunofluorescence was used to observe the expression of TGF?1 in microglia before and 3,7 and 14 days after spinal cord injury.Microglia were polarized and cultured astrocytes in conditioned medium to detect the expression of SOX9 and CS56 in vitro.A rescue experiment was designed to verify that microglia M1 polarization promotes astrocytes to produce CSPG through TGF?1 / SOX9 pathway.Results: At 14 days after SCI,both microglia(CX3CR1+)and astrocytes(GFAP+)formed dense scars,and the spatial positions of the two cells were close to each other.The polarization of microglia was different before and 3,7 and 14 days after SCI.M1 polarization(i NOS+CX3CR1+)was mainly observed at 3 and 7 days after injury,accounting for 54.3 ± 4.4% and 76.7 ± 3.6% of CX3CR1+ microglia,respectively,while M2 polarized microglia(Arg1+CX3CR1+)were slightly expressed at 7 and 14 days after injury,accounting for 16.6 ± 3.5% and 24.9 ± 2.8% of CX3CR1+ microglia,respectively.Polarization of microglia was successfully induced in vitro,and high expression of TGF?1 was found in M1 polarized microglia(P<0.0001).Tissue immunofluorescence also confirmed that M1 microglia showed high expression of TGF?1 at 3 and 7 days.The polarized conditioned medium of microglia M1 could promote the astrocytes secret CSPG(P<0.01),by and the effect disappeared after SOX9 was knocked down,but the addition of TGF?1 cytokines could not be reversed.Conclusion: Microglia are polarized in M1 and highly express TGF?1 at 3 and 7 days after SCI.M1 polarized microglia promote astrocytes to produce CSPG through TGF?1 / SOX9 pathway.