The Role And Mechanism Of Microglia Depletion Impairs Proliferation Of Astrocyte In Spinal Cord Injury By Regulating The STAT3 Signaling Pathway

Objective:Astrocytes and microglia play orchestrated roles in response to spinal cord injury(SCI);however,the molecular mechanisms through which microglia regulate astrocytes after SCI are not yet fully understood.Herein,pharmacological treatments were used to specifically deplete microglia and thereby investigate the effects of microglia on the astrocytic response,and the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway were explored.Methods:In vivo,female C57BL/6 mice were subjected to contusion SCI.Microglial depletion was induced with the colony-stimulating factor 1 receptor(CSF1R)inhibitor PLX3397 starting 2 weeks before injury and was sustained until sacrifice.The thymidine analog 5-ethynyl-2-deoxyuridine(EdU)was intraperitoneally injected to examine the proliferating cells.The proinflammatory cytokines TNF-α,IL-1β and IL-6 were detected using a specific Bio-Plex ProTM Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3)levels were compared between groups.In vitro,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to examine the specific role of STAT3 signaling in mediating astrocyte proliferation induced by microglia.Results:Specific depletion of microglia by PLX3397 resulted in disrupted glial scar formation,increased inflammatory spillover,diffuse tissue damage and impaired functional recovery after SCI.Microglial depletion markedly decreased the number of EdU-positive proliferating cells,especially GFAP-positive/Edu-positive astrocytes,in the spinal cord at 7 days after SCI.RNA-seq analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased the expression of pSTAT3 in astrocytes.Importantly,in vitro coculture of astrocytes and microglia further revealed that astrocyte proliferation induced by activated microglia was abolished by STA21 administration.Conclusion:Microglial depletion impaired astrocyte proliferation and astrocytic scar formation and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following SCI.