The Role Of P2X7 Receptor In Astrocytes Affecting Myelin Repair In Vascular Cognitive Impairment Animal

Objective: Vascular cognitive impairment(VCI)is a syndrome ranging from mild cognitive impairment to dementia caused by various cerebrovascular diseases or their risk factors,accounting for the second place in dementia.It is characterized by insidious onset,and there is a lack of efficient treatment after diagnosis,bringing heavy economic and mental burdens to society and families.It is of great significance to explore an effective treatment for VCI.We found that white matter damage associated with vascular risk factors including oligodendrocyte line or myelin damage,is one of the crucial pathogenic mechanisms involved in VCI.Astrocytes are the most abundant and widely distributed immune cells in the brain.Numerous studies have shown that astrocytes participate in the development,differentiation and maturation of white matter myelin sheath.Under ischemia or hypoxia stimulus,astrocytes could be activated into reactive astrocytes(RAS).Existing data has demonstrated that the number and function of RAS is a continuous dynamic process.P2X7 receptor is a member of purinergic receptor 2(P2Rs)family,mainly distributed on the surface of astrocytes,modulating many pathways that mediate cell signal transduction and cytotoxicity.This study was conducted to establish a VCI mouse model,exploring the effects of RAS on the repair of white matter myelin at different time points to address the role and underling mechanism of P2X7.Part one: The relationship between astrocytes activation and oligodendrocyte and myelin protein injury in VCI mouse model at different time points.Methods: VCI mouse model was established by bilateral common carotid artery ligation(BCCAO).The animals were divided into six groups: Sham group,24-hour VCI group,3-day VCI group,7-day VCI group,15-day VCI group and 30-day VCI group.The changes of learning and memory and emotion of VCI mice were observed by Morris water maze(MWM),tail suspension test(TST)and forced swimming test(FST).Immunofluorescence staining GFAP and combined with morphological changes to labeling RAS,observing its activation degree at different time points after VCI.Immunofluorescence staining of GST-pi and myelin basic protein(MBP)which observe the myelin sheath injury of white matter at different time points after VCI in striatum,corpus callosum and hippocampus.Analyzing the trends of RAS,OLs and MBP in different regions of white matter of VCI mouse at different time points.Results: The impairment of learning,memory and emotion of the five groups of VCI mice reached the highest level 15 days after BCCAO and alleviated on the 30 th days,but still worse than Sham group.The RAS in striatum and hippocampus reached the highest level in 7th day and then began to decrease,while the RAS in the corpus callosum was higher both on the 7th and 15 th day after VCI,but there was no significant difference between the two groups(p>0.05).GST-pi,MBP of striatum,corpus callosum and hippocampus reached the lowest level at 15 days and increased 30 days after operation.Key points: The changes of learning,memory and emotion of VCI mouse are consistent with the changes of myelin labeling GST-pi and MBP,the time when the expression of GST-pi and MBP was the lowest(15 days after VCI),the cognitive function of VCI mice impaired is worst as well.However,the time point of the highest degree of RAS activation was earlier than that of myelin injury and behavioral disorder.The results showed that the behavioral changes were correlated with the activation of RAS and the changes of myelin sheath injury in white matter.7-15 days after VCI may be a critical time point..Part two: study on the relationship between P2X7 receptor and reactive astrocyte function in VCI mouse at different time points.Methods: Animals and groups were the same as the first part.In the striatum,corpus callosum and hippocampus,the expressions of P2X7,iNOS and GDNF on RAS were observed by immuneofluorescence double labeling GFAP and P2X7,GFAP and iNOS,as well as GFAP and GDNF.The GLT-1expression was observed by WB.Results: Compared with the Sham group,the expression of P2X7 in the striatum,corpus callosum and hippocampus of the other five groups increased after VCI(all p < 0.05).The expression of P2X7 on RAS reached the peak at7 th day and the lowest on the 30 th days.The expression of i NO S on RAS reached the peak in all three white matter areas on the 7th day after operation.The GLT-1 in white matter was lower on 7 and 15 days after operation,but there was no significant difference(p > 0.05),but increased on the 30 th day(vs.15 d VCI group,p < 0.05).GDNF increased 24 hours after operation in the striatum and then decreased,reached the lowest on the 30 th day,GDNF reached the lowest on the 15 th days in the corpus callosum,increased on the 30 th day,and reached the lowest on the 7th day in the hippocampus,and increased significantly on the15 th day.Key points: The expression of P2X7 and iNOS on RAS,reached the peak on the 7th day after VCI.Combined with the results of the first part,the activation of RAS also reached the peak on the 7th day.It shows that the 7th day after VCI may be the most serious inflammation of RAS.GLT-1,which represents the excitotoxic glutamate uptake of RAS,was lower on 7-15 days after operation.GDNF derived from RAS varied with the time in different regions of the white matter,but it was basically in a low state from 7 th to 15 th day.It is suggested that the effect of RAS is mainly pro-inflammatory during this time,and P2X7 receptor,which is mainly expressed on RAS,may play an important role.Inte rvention of P2X7 in 7th day after operation may reduce inflammatory reaction and protect white matter myelin sheath.Part Ⅲ: Effects of antagonizing P2X7 receptor on functional status and myelin repair of reactive astrocytes in VCI mouse.Methods: Animals are the same as the first part.The animals were divided into four groups: Sham group,VCI group,VCI+24h P2X7 antagonist group and VCI+7d P2X7 antagonist group.A-804598,a specific antagonist of P2X7 receptor,was selected to block the effe ct of P2X7 recepter.The changes of cognition and emotion of VCI mouse at different time points were observed by MWM,TST and FST.The GLT-1 expression of RAS was observed by WB.In striatum,corpus callosum and hippocampus,immunofluorescence double labeling GFAP and iNOS,GFAP and GDNF were used to observe the expression of inflammatory factor iNOS and neurotrophic factor GDNF on RAS at different time points,immunofluorescence staining for GST-pi and MBP to observe the injury of myelin sheath in white ma tter at different time points.Results: Compared with VCI group,learning,memory and emotional behavior were improved in VCI+24h P2X7 antagonist group and VCI+7d P2X7 antagonist group.The expression of P2X7 in white matter was significantly decreased,th e expression of GLT-1 was increased.The expression of iNOS in striatum,corpus callosum and hippocampus was significantly decreased,while the expression of GST-pi and MBP was increased.The expression of GDNF in striatum and corpus callosum in both antag onistic groups was not significantly different from that in VCI group,but the expression in hippocampal VCI+7d P2X7 antagonist group was higher than that in VCI group(p<0.05).Compared with VCI+24h P2X7 antagonist group,GLT-1 expression increased,iNOS expression decreased and GST-pi increased in white matter of VCI+7d P2X7 antagonism group;MBP increased in striatum and corpus callosum,but not statistically significant in hippocampus;GDNF increas ed in three white matter regions but had no statistical significance.Key points: P2X7 is one of the important receptors of RAS in the process of myelin repair in VCI mouse.Blocking it promotes myelin repair after VCI.The mechanism may be related to the inhibition of iNOS and other inflammatory pathways on RAS and the promotion of excitotoxic amino acid transport pathway such as GLT-1.The anti-inflammatory effect of antagonizing P2X7 receptor in 7th day after operation was better than that of 24 hours af ter operation.7th day after surgery may be a appropriate time for VCI to interfere with P2X7 after operation.Part Ⅳ Effects of antagonizing P2X7 receptor on the functional status of reactive astrocytes and the maturation of oligodendrocyte precursors i n vitro.Methods: Primary astrocytes were extracted from newborn 24 h ICR mouse,then subcultured.Establish oxygen and glucose deprivation(OGD)model of astrocytes in vitro.The specific antagonist of P2X7,A-804598 was added to block P2X7 receptor on RAS.According to different treatment methods,astrocytes were divided into 5 groups: Sham group,1h OGD group,12 h OGD group,1h P2X7 antagonist group and 12 h P2X7 antagonist group.After the primary OPCs medium was extracted from newborn 24 h SD rats for proliferation,then divided into 5 groups: Sham group,1h OGD group,OGD12 h group,1h P2X7 antagonist group and 12 h P2X7 antagoni st group.Five groups of astrocyte supernatants collected at the corresponding time point were added to each group,and the normal OPCs differentiation medium was added to the differentiation OPCs at the same time.The medium was changed every day,and the cells were collected for WB detection after 5 days.The levels of P2X7 and GLT-1 in the lysate of astrocytes were detected by WB,and the concentrations of iNOS and GDNF were detected by enzyme-linked immunosorbent assay(ELISA).The expression of MBP in OPCs of 5 groups was detected by WB.Results: Compared with the non-antagonist group,the expression of P2X7 and iNOS decreased,the expression of GLT-1 increased and the expression of MBP increased in the antagonist group.Compared with the 1h P2X7 antagonist group,the expression of P2X7 and iNOS decreased and the expression of MBP increased in the 12 h P2X7 antagonist g roup(p < 0.001).There was no significant difference in the expression of GLT-1(p > 0.001).Compared with Sham group,the expression of GDNF in the other four groups increased,but there was no significant difference among the four groups.Key points: The antagonism of P2X7 receptor on RAS immediately and12 h after OGD is beneficial to myelin repair.Under the condition of ischemia and hypoxia,P2X7 receptors on RAS play an important role in myelin repair,which may play a protective role in pro moting myelin maturation by directly reducing the expression of inflammatory factors and promoting the transport of excitotoxic amino acids.Conclusion: P2X7 is one of the important receptors of white matter damage caused by RAS after ischemia and hypoxia.Blocking it at an appropriate time is helpful to improve myelin sheath repair and improve the cognitive impairment of VCI mouse.The mechanism may be related to the inhibition of iNOS and other inflammatory pathways on RAS and the promotion of excitotoxi c amino acid transport pathway such as GLT-1.This pathway can be used as a potentially important therapeutic target for VCI therapy.