Astrocytes In Oligodendrocyte Injury:Hypoxia And Inflammatory

Objective: Myelin injury are caused by ischemic,inflammatory or genetic metabolism.Oligodendrocyte precursor cells(OPCs)development are the key process of myelin regeneration after myelin injury.Astrocytes are involved in this process,but the mechanisms are still undefined.To investigate the role of astrocytes on the chronic ischemic and inflammatory mediated myelin injury,we analyzed the roles of astrocytic connexin43 and exosomes in the OPCs development respectively.To explore the mechanisms of myelin injury and the possible protective stratagems.Methods: Primary astrocytes and oligodendrocyte lineages were purified in vitro.Firstly,we performed an astrocytes-OPCs co-cultures chronic hypoxia model,and detected the proliferation,differentiation and apoptosis of OPCs by immunofluorescence and Western blot.Connexin43 inhibitors MFA and CBX were administrated to observe the effects on OPCs.Connexin43 gap junction and hemichannels activity were detected by cells scrape loading assay and dye uptake assay,respectively.The release of glutamate was detected underdifferent intervention conditions.Secondly,we collected serum from patients with Neuromyelitis optica spectrum disorders(NMOSD);sera were positive for AQP4 Ig G.We also used control sera collected from five healthy blood donors.Ig G were purified on ProteinA Sepharose 4 Fast FlowTM beads.We performed an NMO model by treated purified astrocytes with sera or NMO Ig G in vitro.The exosomes derived from astrocytes were isolated and purified by differential ultracentrifugation;then the exosomes were administrated to detect the effects on the oligodendrocytes.Results: Firstly,we successfully performed an astrocytes-OPCs co-culture chronic hypoxia model,which were verified by hypoxia inducible factor(HIF-1?)expression up-regulated and translocation from cytoplasm to nuclei.Chronic hypoxic conditions,astrocytes were activated with up-regulated expression of GFAP and connexin43 proteins,and OPCs proliferation but limited differentiation;connexin43 inhibitors MFA and CBX attenuated hypoxia-induced astrocytes activation,and significantly attenuate hypoxia-induced enhancement of GFAP and connexin43 expression at day 2 after hypoxia treatment,without affecting cells viability.Furthermore,connexin43 inhibitors rescued the limited OPCs maturation under chronic hypoxia;connexin43 inhibitors suppressed gap junction and hemichannels activity and glutamate release under chronic hypoxia.In addition,AMPA receptor inhibitor partially rescued the limited OPCs maturation under hypoxia.Secondly,sera were obtained from patients with a NMOSD or healthy donors.Prolonged exposure to seraNMO or Ig GNMO induced the functional changes of astrocytes in vitro.Exosomes derived from astrocytes were collected and using electron microscope and Western blot to detect the morphology and biomarkers of exosomes.Exosomes derived from astrocytes which treated with seraNMO or Ig GNMO regulated oligodendrocytes proteins expression detected by immunofluorescence,western blot and RT-PCR.Conclusion: Astrocytic Cx43 hemichannels could result in the increase of extracellular glutamate after chronic hypoxia,which could impair OPCs maturation and remyelination processes.Glutamate released via Cx43 hemichannels suppressed OPCs differentiationpartially mediated by AMPARs activation.Application Cx43 inhibitors could rescue hypoxia induced suppression of OPCs differentiation.Based on these findings,astrocytic Cx43 hemichannels could potentially be a therapeutic target for facilitating OPCs remyelination in hypoxia-induced white matter injury.In addition,our results confirm the pathogenic role of Ig G isolated from NMOSD patients with positive of AQP4 Ig G.Using models based on seraNMO or Ig GNMO-treated primary astrocytes,we showed that seraNMO or Ig GNMO induced alteration of astrocytes.Exosomes derived from astrocytes treated with seraNMO or Ig GNMO regulated oligodendrocytes myelin related proteins expression.To further elucidate the mechanism,which can provide new ideas for the pathogenesis of NMOSD and serve as a promising therapeutic target for NMO in clinic practice.