Background Damage to the central nervous system(CNS)usually leads to the activation of astrocytes,followed by glial scar formation.For years,glial scar has been thought as a major obstacle for successful axon regenera...Background Damage to the central nervous system(CNS)usually leads to the activation of astrocytes,followed by glial scar formation.For years,glial scar has been thought as a major obstacle for successful axon regeneration.However,increasing evidence suggests a beneficial role for this scar tissue as part of the endogenous local immune regulation and repair process.Surprisingly,in contrast to scars in other tissues,glial scars(mainly consist of reactive astrocytes)in both rat cortex and spinal cord were recently found to be significantly softer than healthy CNS tissues.Naive astrocytes have been found to change their phenotype to reactive astrocytes and gradually into scar-forming astrocytes,upregulating the astrocyte marker glial fibrillary acidic protein(GFAP),vimentin,and inflammatory proteins in almost all known brain disorders.Such phenotype transformation process has been widely thought unidirectional or irreversible.However,recent research revealed the environment-dependent plasticity of astrocyte phenotypes,with reactive astrocytes could revert in retrograde to naive astrocytes in proper microenvironment.In consideration of the important roles of mechanical cues in CNS and the unique softening behavior of glial scars,it is of great interesting to study the effects of dynamic changes of matrix stiffness on astrocyte phenotypic switch.Materials&methods Primary astrocyes were isolated from the cortex of SpragueDawley(SD)rats at PI.After cultured for 2 weeks,astrocytes were encapsulated into a set of three-dimensional(3D)hybrid hydrogel system composed of type I collagen and alginate.Immunofluorescence and Western blot expression analysis were applied for characterizting cell responses to different and dynamically changed matrix stiffness.A molecular dynamics model was developed for simulation.Results&discussion In this work,we established an in-vitro model to study the effects of dynamic changes of matrix stiffness on astrocyte phenotypic switchings in 3D.To simulate native cellular environment,we fabricated a set of hybrid hydrogel system composed of type I collagen and alginate.The stiffness of the hybrid hydrogels was demonstrated to be dynamically changed by adding calcium chloride or sodium citrate to crosslink or decrosslink alginate,respectively.Using 3D culture models,we showed that the decrease of matrix stiffness could promote astrocyte activation,with upregulated GFAP and IL-1β.In addition,3D cultured astrocytes spread greater with decreasing matrix stiffness.Moreover,we surprisingly found that astrocyte phenotype could be switched by dynamically changing matrix stiffness.Specifically,matrix stiffening reverted the activation of astrocytes,whereas matrix softening induced astrocyte activation.We further demonstrated that matrix stiffness-induced astrocyte activation was mediated through cytoskeletal tension and YAP protein.To some extent,YAP inhibition enhanced the responses of astrocytes to matrix stiffness.These may guide researchersto re-examine the role of matrix stiffness in reactive astrogliosis in vivo,and inspire the development of novel therapeutic approach for reducing glial scar following injury,enabling axonal regrowth and improving functional recovery by exploiting the benefits of mechanobiology studies.Conclusions Taken together,our results clarify the effects of matrix stiffness and its dynamic changes on phenotypic swtich of astrocytes in three dimensions and reveal environmental factors that regulate astrocytic phenotype transformation process,which may provide potential therapeutic approach for CNS injury.展开更多
OBJECTIVE Astrocytes activa⁃tion and glial scar formation are the important causes that hinder the recovery of motor function after cerebral ischemia.However,its precise mechanism has not been clarified.Peroxisome pro...OBJECTIVE Astrocytes activa⁃tion and glial scar formation are the important causes that hinder the recovery of motor function after cerebral ischemia.However,its precise mechanism has not been clarified.Peroxisome proliferator-activated receptorα(PPARα)is a ligand-activated nuclear transcriptional factor.This study aims to further clarify the role of PPARαin astrocyte activation after cerebral isch⁃emia and explore the underlying mechanism.METHODS Astrocyte activation in vivo model was induced by transient middle cerebral artery occlusion(tMCAO)in mice and in vitro model was induced by oxygen-glucose deprivation/reox⁃ygenation(OGD/R)in primary culture of mouse astrocyte.The effects of PPARαon astrocyte ac⁃tivation and autophagy flux were observed in the condition of PPARαdysfunction(PPARαnull mice)or PPARαactivation by oleoylethanol⁃amide(OEA).RESULTS PPARαmainly ex⁃pressed in activated astrocytes during the chron⁃ic phase of brain ischemia and PPARαdysfunc⁃tion promoted astrocytes activation after brain ischemia in vivo and in vitro.After cerebral isch⁃emia,the expressions of LC3-Ⅱ/Ⅰand P62 both increased in the brain tissue near the infarct core.Autophagic vesicles accumulation was ob⁃served by electron microscopy in astrocytes,and mRFP-GFP-LC3 adenovirus infection assay indi⁃cated the block of autophagy flux.PPARαdys⁃function aggravated autophagy flux block,while PPARαactivation preserved the lysosome func⁃tion and restored autophagy flux in astrocytes after OGD/R.Autophagy flux blocker bafilomycin A1 and chloroquine antagonized the effect of OEA on inhibiting astrocyte activation.CONCLU⁃SION PPARαactivation inhibites the over-activa⁃tion of astrocytes by restoring the autophagy flux after cerebral ischemia.展开更多
OBJECTIVE To investigate the effect of icariin Ⅱ(ICS Ⅱ) on lipopolysaccharide(LPS)-induced inflammation and amyloid production in astrocytes.METHODS The cerebral cortex of newborn SD rats was isolated in vitro,and t...OBJECTIVE To investigate the effect of icariin Ⅱ(ICS Ⅱ) on lipopolysaccharide(LPS)-induced inflammation and amyloid production in astrocytes.METHODS The cerebral cortex of newborn SD rats was isolated in vitro,and the primary astrocytes were extracted and cultured.Astrocytes were pre-treated with ICSⅡ(5,10 and20 μmol·L^(-1)) or dexamethasone(1 μmol·L^(-1)) for1 h.Cell inflammation models were established with LPS and treated with ICS Ⅱ or dexamethasone for another 24 h.The anti-neuroinflammation and anti-amyloid effects of ICS Ⅱ in astrocytes were detected by ELISA and Western blotting respectively.RESULTS ICS Ⅱ decreased the levels of beta secretase 1(BACE1),Aβ1-40 and Aβ1-42 in astrocytes in a concentration-dependent manner.Moreover,the levels of tumor necrosis factor-alpha,interleukin-1β,reactive oxygen species,inducible nitric oxide synthase,cyclooxygenase-2 and transforming growth factor-β1 in astrocytes were significantly inhibited by ICS II(5,10 and 20 μmol·L^(-1)).In addition,ICSⅡhas a significant inhibitory effect on LPS-induced IκB-α degradation and NF-κB activation.CONCLUSION ICS Ⅱ exerts neuroprotective effects on LPS-induced inflammation in astrocytes,through regulating IKK/IκB/NF-κB signaling pathway.展开更多
OBJECTIVE To establish a method for primary cultured and iden-tified Sprague-Dawley(SD) rats cerebral cortical astrocytes. METHODS Cerebral co-rtex of SD neonatal rats within 24 h was taken with stereo microscope and ...OBJECTIVE To establish a method for primary cultured and iden-tified Sprague-Dawley(SD) rats cerebral cortical astrocytes. METHODS Cerebral co-rtex of SD neonatal rats within 24 h was taken with stereo microscope and was cut topieces(1 mm^3),digested by Accutase and 0.1% DNAase(37℃,15 min),anddispersed cell suspension was made by mechanical method and filtered. The fibroblast cells and microglia were removed through differential adhesion and sha-ke. Passaged cells were identified by immunofluorescent with anti-Glial fibrillaryacidic protein(GFAP) antibody. RESULTS The astrocytes of rats cerebral cortex were cultured in this method,which had a large number of cells,good activity,high purity,abundant and elongated cell processes,and were interwoven into a network,showing a typical and good growth state. The third generation of the cel s comprised >95% astrocytes. CONCLUSION This simple and reliable cultivation method of astrocyte from rat cerebral tissue is established with high purity,and in a good growth condition.展开更多
Objective:To explore the effects of dopamine receptor D2(DRD2)on astrocytic dedifferentiation based on SOX2-regulated genes in neural stem cells(NSCs)and astrocytes.Methods:Immunofluorescence staining and SOX2-GFP mic...Objective:To explore the effects of dopamine receptor D2(DRD2)on astrocytic dedifferentiation based on SOX2-regulated genes in neural stem cells(NSCs)and astrocytes.Methods:Immunofluorescence staining and SOX2-GFP mice were used to examine the lineage differentiation of SOX2-positive cells during the development of cerebral cortex.Primary NSCs/astrocytes culture,ChIP-seq and Western Blot were adopted to analyze and verify the expression of candidate genes.Pharmacological manipulation,neurosphere formation,photochemical ischemia,immunofluorescence staining and behavior tests were adopted to evaluate the effects of activating DRD2 signaling on astrocytic dedifferentiation.Results:Immunofluorescence staining demonstrated the NSC-astrocyte switch of SOX2-expression in the normal development of cerebral cortex.ChIP-seq revealed enrichment of DRD2 signaling by SOX2-bound enhancers in NSCs and SOX2-bound promoters in astrocytes.Western Blot and immunofluorescence staining verified the expression of DRD2 in NSCs and reactive astrocytes.Application of quinagolide hydrocholoride(QH),an agonist of DRD2,significantly promoted astrocytic dedifferentiation both in vitro and in vivo following ischemia.In addition,quinagolide hydrocholoride treatment improved locomotion recovery.Conclusion:Activating DRD2 signaling facilitates astrocytic dedifferentiation and may be used to treat ischemic stroke.展开更多
Aquaporin-4 （AQP-4）, a water-channel protein,is highly expressed in the brain, which is important ele- ments in the formation of brain edema and plays an important role in the rapid transmembrane transport. AQP-4 ex...Aquaporin-4 （AQP-4）, a water-channel protein,is highly expressed in the brain, which is important ele- ments in the formation of brain edema and plays an important role in the rapid transmembrane transport. AQP-4 ex- pression up-regulates after ischemia-reperfusion injury in rats, making the astrocytic endfeet swelling, with the con- sequence of the injury of blood-brain barrier（BBB） , increasing the permeability of BBB, render too much water in the blood flow to the brain parenchyma, which results in cytotoxic edema, disordering the stability of the central nervous system. In addintion, the increased permeability of BBB is one of the important reasons for the cerebral stroke, therefore, it is essential that research the relationship between AQP-4 with BBB further and restore the blood-brain barrier injury be a new strategy for the prevention and treatment of stroke, worthy of further research.展开更多
基金financially supported by the National Natural Science Foundation of China ( 11872298, 11602191,1161101223,11532009)the China Postdoctoral Science Foundation ( 2018M631141)the Shaanxi Postdoctoral Science Foundation,and the Fundamental Research Funds for the Central Universities ( Z201811336)
文摘Background Damage to the central nervous system(CNS)usually leads to the activation of astrocytes,followed by glial scar formation.For years,glial scar has been thought as a major obstacle for successful axon regeneration.However,increasing evidence suggests a beneficial role for this scar tissue as part of the endogenous local immune regulation and repair process.Surprisingly,in contrast to scars in other tissues,glial scars(mainly consist of reactive astrocytes)in both rat cortex and spinal cord were recently found to be significantly softer than healthy CNS tissues.Naive astrocytes have been found to change their phenotype to reactive astrocytes and gradually into scar-forming astrocytes,upregulating the astrocyte marker glial fibrillary acidic protein(GFAP),vimentin,and inflammatory proteins in almost all known brain disorders.Such phenotype transformation process has been widely thought unidirectional or irreversible.However,recent research revealed the environment-dependent plasticity of astrocyte phenotypes,with reactive astrocytes could revert in retrograde to naive astrocytes in proper microenvironment.In consideration of the important roles of mechanical cues in CNS and the unique softening behavior of glial scars,it is of great interesting to study the effects of dynamic changes of matrix stiffness on astrocyte phenotypic switch.Materials&methods Primary astrocyes were isolated from the cortex of SpragueDawley(SD)rats at PI.After cultured for 2 weeks,astrocytes were encapsulated into a set of three-dimensional(3D)hybrid hydrogel system composed of type I collagen and alginate.Immunofluorescence and Western blot expression analysis were applied for characterizting cell responses to different and dynamically changed matrix stiffness.A molecular dynamics model was developed for simulation.Results&discussion In this work,we established an in-vitro model to study the effects of dynamic changes of matrix stiffness on astrocyte phenotypic switchings in 3D.To simulate native cellular environment,we fabricated a set of hybrid hydrogel system composed of type I collagen and alginate.The stiffness of the hybrid hydrogels was demonstrated to be dynamically changed by adding calcium chloride or sodium citrate to crosslink or decrosslink alginate,respectively.Using 3D culture models,we showed that the decrease of matrix stiffness could promote astrocyte activation,with upregulated GFAP and IL-1β.In addition,3D cultured astrocytes spread greater with decreasing matrix stiffness.Moreover,we surprisingly found that astrocyte phenotype could be switched by dynamically changing matrix stiffness.Specifically,matrix stiffening reverted the activation of astrocytes,whereas matrix softening induced astrocyte activation.We further demonstrated that matrix stiffness-induced astrocyte activation was mediated through cytoskeletal tension and YAP protein.To some extent,YAP inhibition enhanced the responses of astrocytes to matrix stiffness.These may guide researchersto re-examine the role of matrix stiffness in reactive astrogliosis in vivo,and inspire the development of novel therapeutic approach for reducing glial scar following injury,enabling axonal regrowth and improving functional recovery by exploiting the benefits of mechanobiology studies.Conclusions Taken together,our results clarify the effects of matrix stiffness and its dynamic changes on phenotypic swtich of astrocytes in three dimensions and reveal environmental factors that regulate astrocytic phenotype transformation process,which may provide potential therapeutic approach for CNS injury.
基金National Natural Science Foundation of China(81603093)and the Open Research Fund of State Key Laboratory of Cellu⁃lar Stress Biology,Xiamen University(SKLC⁃SB2019KF016)。
文摘OBJECTIVE Astrocytes activa⁃tion and glial scar formation are the important causes that hinder the recovery of motor function after cerebral ischemia.However,its precise mechanism has not been clarified.Peroxisome proliferator-activated receptorα(PPARα)is a ligand-activated nuclear transcriptional factor.This study aims to further clarify the role of PPARαin astrocyte activation after cerebral isch⁃emia and explore the underlying mechanism.METHODS Astrocyte activation in vivo model was induced by transient middle cerebral artery occlusion(tMCAO)in mice and in vitro model was induced by oxygen-glucose deprivation/reox⁃ygenation(OGD/R)in primary culture of mouse astrocyte.The effects of PPARαon astrocyte ac⁃tivation and autophagy flux were observed in the condition of PPARαdysfunction(PPARαnull mice)or PPARαactivation by oleoylethanol⁃amide(OEA).RESULTS PPARαmainly ex⁃pressed in activated astrocytes during the chron⁃ic phase of brain ischemia and PPARαdysfunc⁃tion promoted astrocytes activation after brain ischemia in vivo and in vitro.After cerebral isch⁃emia,the expressions of LC3-Ⅱ/Ⅰand P62 both increased in the brain tissue near the infarct core.Autophagic vesicles accumulation was ob⁃served by electron microscopy in astrocytes,and mRFP-GFP-LC3 adenovirus infection assay indi⁃cated the block of autophagy flux.PPARαdys⁃function aggravated autophagy flux block,while PPARαactivation preserved the lysosome func⁃tion and restored autophagy flux in astrocytes after OGD/R.Autophagy flux blocker bafilomycin A1 and chloroquine antagonized the effect of OEA on inhibiting astrocyte activation.CONCLU⁃SION PPARαactivation inhibites the over-activa⁃tion of astrocytes by restoring the autophagy flux after cerebral ischemia.
基金National Natural Science Foundation of China (81560585).
文摘OBJECTIVE To investigate the effect of icariin Ⅱ(ICS Ⅱ) on lipopolysaccharide(LPS)-induced inflammation and amyloid production in astrocytes.METHODS The cerebral cortex of newborn SD rats was isolated in vitro,and the primary astrocytes were extracted and cultured.Astrocytes were pre-treated with ICSⅡ(5,10 and20 μmol·L^(-1)) or dexamethasone(1 μmol·L^(-1)) for1 h.Cell inflammation models were established with LPS and treated with ICS Ⅱ or dexamethasone for another 24 h.The anti-neuroinflammation and anti-amyloid effects of ICS Ⅱ in astrocytes were detected by ELISA and Western blotting respectively.RESULTS ICS Ⅱ decreased the levels of beta secretase 1(BACE1),Aβ1-40 and Aβ1-42 in astrocytes in a concentration-dependent manner.Moreover,the levels of tumor necrosis factor-alpha,interleukin-1β,reactive oxygen species,inducible nitric oxide synthase,cyclooxygenase-2 and transforming growth factor-β1 in astrocytes were significantly inhibited by ICS II(5,10 and 20 μmol·L^(-1)).In addition,ICSⅡhas a significant inhibitory effect on LPS-induced IκB-α degradation and NF-κB activation.CONCLUSION ICS Ⅱ exerts neuroprotective effects on LPS-induced inflammation in astrocytes,through regulating IKK/IκB/NF-κB signaling pathway.
文摘OBJECTIVE To establish a method for primary cultured and iden-tified Sprague-Dawley(SD) rats cerebral cortical astrocytes. METHODS Cerebral co-rtex of SD neonatal rats within 24 h was taken with stereo microscope and was cut topieces(1 mm^3),digested by Accutase and 0.1% DNAase(37℃,15 min),anddispersed cell suspension was made by mechanical method and filtered. The fibroblast cells and microglia were removed through differential adhesion and sha-ke. Passaged cells were identified by immunofluorescent with anti-Glial fibrillaryacidic protein(GFAP) antibody. RESULTS The astrocytes of rats cerebral cortex were cultured in this method,which had a large number of cells,good activity,high purity,abundant and elongated cell processes,and were interwoven into a network,showing a typical and good growth state. The third generation of the cel s comprised >95% astrocytes. CONCLUSION This simple and reliable cultivation method of astrocyte from rat cerebral tissue is established with high purity,and in a good growth condition.
文摘Objective:To explore the effects of dopamine receptor D2(DRD2)on astrocytic dedifferentiation based on SOX2-regulated genes in neural stem cells(NSCs)and astrocytes.Methods:Immunofluorescence staining and SOX2-GFP mice were used to examine the lineage differentiation of SOX2-positive cells during the development of cerebral cortex.Primary NSCs/astrocytes culture,ChIP-seq and Western Blot were adopted to analyze and verify the expression of candidate genes.Pharmacological manipulation,neurosphere formation,photochemical ischemia,immunofluorescence staining and behavior tests were adopted to evaluate the effects of activating DRD2 signaling on astrocytic dedifferentiation.Results:Immunofluorescence staining demonstrated the NSC-astrocyte switch of SOX2-expression in the normal development of cerebral cortex.ChIP-seq revealed enrichment of DRD2 signaling by SOX2-bound enhancers in NSCs and SOX2-bound promoters in astrocytes.Western Blot and immunofluorescence staining verified the expression of DRD2 in NSCs and reactive astrocytes.Application of quinagolide hydrocholoride(QH),an agonist of DRD2,significantly promoted astrocytic dedifferentiation both in vitro and in vivo following ischemia.In addition,quinagolide hydrocholoride treatment improved locomotion recovery.Conclusion:Activating DRD2 signaling facilitates astrocytic dedifferentiation and may be used to treat ischemic stroke.
文摘Aquaporin-4 （AQP-4）, a water-channel protein,is highly expressed in the brain, which is important ele- ments in the formation of brain edema and plays an important role in the rapid transmembrane transport. AQP-4 ex- pression up-regulates after ischemia-reperfusion injury in rats, making the astrocytic endfeet swelling, with the con- sequence of the injury of blood-brain barrier（BBB） , increasing the permeability of BBB, render too much water in the blood flow to the brain parenchyma, which results in cytotoxic edema, disordering the stability of the central nervous system. In addintion, the increased permeability of BBB is one of the important reasons for the cerebral stroke, therefore, it is essential that research the relationship between AQP-4 with BBB further and restore the blood-brain barrier injury be a new strategy for the prevention and treatment of stroke, worthy of further research.
