The Effects Of Modulating Of Autophagy Flux In Astrocytes On Neuron Survival During Ischemic Stroke

Stroke contributes to the most of disability and death in the world.It has surpassed cancer and become the first cause of death in China.As we know,neurons in central nervous system are quite difficult to repair and regeneration suffering from ischemia and denaturation injury.The neuronal death resulted from injury is the basic reason for the high disability rate of stroke and neurodegenerative diseases.However,there are not effective treatments or interventions against neuronal damage or necrosis.The role of autophagy in different type of acute brain injuries varies.Generally speaking,autophagy mediates a clear neuroprotective activity in intoxication by various psychoactive agents,subarachnoid haemorrhage and spinal cord injury(SCI).In other scenarios,autophagic cell death actively contributes to neuronal demise in neonatal hypoxic–ischaemic encephalopathy.However,it still remains to be determined whether autophagy has a cytoprotective or a cytotoxic role in stroke.Previous studies mostly focused on the role of neurons in brain injury rather than astrocytes.Astrocytes comprise the largest class of glial cells in the mammalian central nervous system(CNS).They play key roles in maintaining the blood–brain barrier,regulating regional blood flow,providing trophic,antioxidant and metabolic support to neurons,neurotransmitter recycling,and regulating synaptogenesis and synaptic transmission.Neurons release and transfer damaged mitochondria to astrocytes for disposal and recycling.Recent study shows that astrocytes can release functional mitochondria that enter neurons,suggesting the neuroglia materials transformation may be a critical endogenous neuroprotection mechanism.So,whether modulating autophagy flux of astrocytes contributes to improving neuron survival after stroke remains unknown.The current study aims to investigate the effects of the survival of neurons from ischemic injury by regulating autophagy flux of astrocytes.Experiment 1: To investigate the time course of autophagy flux change in cultured neurons and astrocytes after OGDObjective: To investigate the time course of autophagy flux in neurons and astrocytes exposed to OGD and re-oxygenation.Methods: 1.In this study,we cultured primary neurons and astrocytes respectively and established oxygen glucose deprivation / re-oxygenation(OGD / R)model.The primary neurons and astrocytes were randomly divided into 6 groups: Sham group and OGD 1 h / re-oxygenation(0 h,6 h,12 h,24 h,48 h)groups.Besides,primary astrocytes were randomly divided into 6 groups: Sham group and OGD 4 h / re-oxygenation(0 h,6 h,12 h,24 h,48 h)groups.Western Blot detected the expression of autophagy flux marker proteins LC3 and p62.In addition,transmission electron microscope(TEM)was used to monitor the number of autophagosomes and autolysosomes in neurons in each group.Moreover,neurons and astrocytes were divided into 2 groups: rapamycin and control groups.Western blot was used to detect the expression of autophagy flux in neurons in each group.Besides,neurons and astrocytes were infected with lentivirus encoding m RFP-GFP-LC3.After virus infection was stable,fluorescence was used to detect the autophagy flux in neurons.Results: 1.In normal condition,autophagy flux of neurons remained low level and was activated immediately when exposed to OGD(P<0.05).Autophagy flux of neurons was inhibited after oxygenation 12 h,decreasing to the baseline level.However,it was induced again after oxygenation 24 h as well as 48 h(P<0.05).After rapamycin treatment,autophagy flux of neurons maintained a high level(P<0.01).2.In normal condition,autophagy flux of astrocytes remained a high level.OGD 1 h and re-oxygenation didn’t change autophagy flux level of astrocytes while OGD 4 h changed autophagy flux level of astrocytes.Autophagy flux of astrocytes was inhibited immediately when exposed to OGD and decreased to the lowest level after oxygenation 12 h(P<0.05).While it was induced again after oxygenation 24 h as well as 48 h consistent with the basic condition.After treatment with rapamycin,autophagy flux of astrocytes was at a high level(P<0.01).Conclusions: 1.Autophagy flux of neurons is induced immediately when exposed to OGD and inhibited after re-oxygenation 6 h and 12 h,decreasing to the baseline level,and activated again after oxygenation 24 h.2.Autophagy flux of astrocytes is inhibited immediately when exposed to OGD and decreased to the lowest level after re-oxygenation 12 h,and activated again after re-oxygenation 24 h consistent with the basic condition.Experiment 2 To investigate the influence of astrocytes on the ability of neurons against OGD injury.Objectives: To observe the influence of astrocytes on the ability of neurons against OGD injury when neurons were cultured alone or neuron-astrocytes were co-cultured.Methods: Primary neurons were cultured alone and randomly divided into 4 groups: Sham group and OGD(15 min,30 min,60 min)/ re-oxygenation 24 h groups.Primary astrocytes were cultured alone and were divided into 2 groups: Sham group and OGD 1 h / oxygenation 24 h groups.Cell Count Kits were used to analyze cell viability in each group of neurons and astrocytes.In addition,neurons and astrocytes were co-cultured and were divided into 2 groups: Sham group and OGD 1 h / re-oxygenation 24 h groups.Likewise,neuron viability was detected.Results: Neuron viability was reduced significantly to 65% when exposed to OGD 60 min compared with OGD 0 min(P<0.001).In addition,Astrocytes which were cultured alone were insulted by OGD 60 min and 24 h re-oxygenation showed no significant difference compared with OGD 0 min.While addition of astrocytes to neuronal cells culture indirectly displayed higher neuron viability(80%)(P<0.01)compared with OGD 0 min,which is higher than neuron viability that cultured alone.Conclusions: Neurons are vulnerable to OGD injury while astrocytes are resistant to OGD injury.Astrocytes enhance the viability of neurons against OGD injury and play a cytoprotective role.Experiment 3 To investigate the effect of modulating of autophagy flux of astrocytes on themselves against OGD injuryObjectives: Astrocytes autophagy flux were up-regulated or down-regulated via agents or lentivirus.Then the astrocytes were subjected to OGD 4 h / oxygenation 24 h and cell viability of astrocytes was detected to investigate the effect of modulating autophagy flux of astrocytes on themselves against OGD injury.Methods: Primary astrocytes were cultured alone and were divided into 5 groups: Sham group,Ctrl group,administration of RAPA,3-MA and small reference RNA(si RNA)groups,so as to up-regulate or down-regulate autophagy flux of astrocytes.OGD 4 h / R 24 h model was established in astrocytes except for Sham group and cell viability of astrocytes in each group was detected.Results: Down-regulation of autophagy flux of astrocytes with interfering with autophagy related gene 7 genetically weakened the ability of astrocytes after OGD injury(P<0.001).Conclusions: Inhibition of autophagy flux of astrocytes weakens the ability of astrocytes against OGD injury,meaning that the autophagy flux of astrocytes plays a critical role in maintaining the function of astrocytes.Experiment 4 To investigate the effects of modulating of autophagy flux of astrocytes on neuron survival under normal condition or OGD / re-oxygenationObjectives: The modulated astrocytes were co-cultured with neurons indirectly and viability of neurons was detected under normal condition or exposed to OGD / R.Methods: 1.Astrocytes which were modulated by si Atg5,3-MA or RAPA as well as control groups and neurons were co-cultured indirectly then followed OGD 0 min or 60 min,after 24 h re-oxygenation LDH release,cell viability assay as well as TUNEL staining were used to evaluate the effects of regulating autophagy flux in astrocytes on the co-cultured system cytotoxicity as well as neurons viability.Results: No significant difference was observed in each group under normal condition.Up-regulation of autophagy flux of astrocytes with RAPA decreased LDH level(P<0.05)and apoptosis of neurons in the co-cultured system(P<0.01)and enhanced neurons viability(P<0.01)whereas down-regulation of autophagy flux in astrocytes with 3-MA or si Atg5 increased LDH level(P<0.05)and apoptosis of neurons in the co-cultured system(P<0.05,P<0.01)and decreased neurons viability(P<0.05,P<0.01)following OGD 60 min / R 24 h.Conclusions: Modulation of autophagy flux in astrocytes does not affect neuron survival under the normal condition.Up-regulation of autophagy flux of astrocytes plays a cytoprotective role.Down-regulation of autophagy flux of astrocytes plays a cytotoxic role.Experiment 5 To investigate the effects of modulating of autophagy flux in astrocytes on brain infarct volume and neuron survival against ischemia / reperfusion in vivo.Objectives: To clear the effects of modulating of autophagy flux of astrocytes on brain infarct volume and neuron survival subjected to MCAO 1 h / reperfusion 24 h.Methods: Wild type C57BL6 mice were divided into 2 groups: AAV-GFAP-GFP and AAV-GFAP-ATG7 groups.Two types of adeno-associated viruses(AAV)mentioned above that were used to up-regulate autophagy flux of astrocytes were injected into penumbra tissue of each mouse.Three weeks later,ischemia / reperfusion model was established and TTC staining were used to measure the brain infarct volume of each mouse and IF were used to measure the survival of neurons in penumbra tissue.Results: Up-regulation of autophagy flux in astrocytes with AAV-GFAP-ATG7 decreased the brain infarct volume compared with AAV-GFAP-GFP(P<0.05)and increased the survival of neurons subjected to MCAO / reperfusion injury in penumbra tissue(P<0.05).Conclusions: Up-regulation of autophagy flux in astrocytes plays a neuroprotective role in ischemic stroke.