The Role And Mechanism Of Microglia Polarization In White Matter Lesions Induced By Chronic Cerebral Hypoperfusion

ObjectiveTo explore the effect of microglia polarization on white matter lesions and its mechanism under chronic cerebral hypoperfusion.MethodPart Ⅰ: The selection and construction of white matter lesions model.Adult male SD rats were randomly divided into sham operation group(SG),classic bilateral common carotid artery occlusion group(BG)and modified bilateral staged common carotid artery occlusion group(GG).The Chronic Cerebral Hypoperfusion rats model was established by classical or modified bilateral carotid artery occlusion.The mortality of each group was observed at 3 days and 4weeks after operation.The disappearance of pupil reflection to light in each group was observed 7 days after operation.The pathological changes of white matter were observed by Kluver-Barrera staining at 4 weeks.We used Morris water maze to evaluate the cognitive impairment of rats at 4 weeks.Part Ⅱ: Phenotypic changes of microglia in white matter lesions induced by chronic cerebral hypoperfusion.Adult male SD rats were randomly divided into sham operation group(SG)and hypoperfusion group(CG).Chronic cerebral hypoperfusion rat model was established by modified staging bilateral carotid artery occlusion.Iba-1 immunohistochemical staining was used to observe the activation of microglia in corpus callosum.The lesions of oligodendrocytes and myelin sheath were observed by MBP immunohistochemical staining.The polarization of M1/M2-type microglia in the white matter region of brain was observed in vivo model by double labeling of Iba-1 and CD16 cells and Iba-1 and CD206 cells.The expressions of inflammatory factors IL-1 β,IL-6 and IL-10 in the corpus callosum were detected by Western-blot.White matter pathological changes were observed by Kluver-Barrera staining.Morris water maze was used to evaluate the cognitive impairment of rats.Part Ⅲ: The mechanism of phenotypic changes of microglia in chronic cerebral hypoperfusion white matter lesions.(1)Relationship between Adenosine 2a Receptor(A2AR)and phenotype changes of microglia under chronic cerebral hypoperfusion.Adult male SD rats were randomly divided into sham operation group(SG),hypoperfusion group(CG),A2 AR inhibitor group(IG),and A2 AR agonist group(AG).Chronic cerebral hypoperfusion rat model was established by bilateral carotid artery staging occlusion.The pathological changes of white matter were observed by Kluver-Barrera staining.Morris water maze was used to observe the cognitive impairment of rats.The expressions of IL-1 β,IL-6,IL-10 in the corpus callosum were detected by Western-blot assay,and the expressions of i NOS and Arg1,two polarization markers of microglia in the corpus callosum were detected by Western-blot.The lesions and demyelination of oligodendrocytes in white matter were observed by using single fluorescent immunohistochemical staining to label MBP.The polarization of M1/ M2-type microglia in the white matter region of brain was observed in vivo model by double labeling of Iba-1 and CD16 double positive cells and Iba-1 and CD206 double positive cells.(2)Pathways of adenosine 2a Receptor(A2AR)regulates phenotypic changes of microglia cells under chronic cerebral hypoperfusion.The oligodendrocyte-microglia co-culture line was established.Chronic cerebral hypoperfusion cell model was established by oligoendrocytes and microglia co-culture using hypoxia and hypoglycemia method,and A2 AR agonist and PKA inhibitor were respectively used for intervention.Control group(SG),hypoperfusion group(CG),A2 AR agonist group(AG),and A2 AR agonist combined with PKA group inhibitor(AP)were set up.The expression of three inflammatory factors,IL-1 β,IL-6 and IL-10,was detected by EILSA assay,and PKA activity was detected by ELISA.LDH was detected by ELISA for oligodendrocyte injury index.The expression of i NOS and Arg1 polarization markers in microglia was detected by Western-blot method,and the related molecules p-CREB and CREB in PKA-CREB signaling pathway were detected.To investigate the regulation of microglial polarization and white matter lesions induced by chronic cerebral hypoperfusion by A2 AR through PKA-CREB signaling in an in vitro model.ResultPart Ⅰ: The selection and construction of brain white matter lesions model.1.The mortality rates at 3 days after operation were 0%,30% and 5%,respectively,and at 4 weeks after operation were 0%,35% and 5%.2.The disappearance rate of pupil reflectance was 0% before surgery,however 7 days after surgery were 0%,41.6% and 0%.3.Kluver-Barrera staining showed that compared with SG group,the white matter area of brain of rats in BG group and GG group showed sparse,disordered arrangement of nerve fibers and inclusion of vacuolation.There was no significant difference between BG and GG groups.4.The water maze experiment showed that compared with the SG group,the latency time of reaching the platform in CG group was longer(P < 0.01),and the number of crossing the platform was decreased(P<0.01).Compared with CG group,GG group had no significant difference in the latency time to reach the platform and the number of crossing the platform(P>0.05).Part Ⅱ: Phenotypic changes of microglia in chronic cerebral hypoperfusion white matter lesions.1.Kluver-Barrera staining results showed that,compared with the SG group,sparse,disordered and vacuolated nerve fibers were observed in the white matter of rats in the CG group at 2,4 and 8 weeks after surgery.2.Water maze test showed cognitive impairment in chronic cerebral hypoperfusion rats:compared with SG group,the latency time of CG group to reach the platform was significantly prolonged at 4 and 8weeks after surgery(P<0.05,P<0.01),but there was no conspicuousness difference at 2 weeks;Compared with the sham SG group,the number of CG crossing the platform was significantly decreased at 2,4 and 8 weeks postoperatively(P<0.01).3.Immunohistochemical staining showed the changes of glial cells in the white matter area of rats with Chronic Cerebral Hypoperfusion: microglia cells were labeled with Iba-1antibody,and Iba-1 immunohistochemical staining showed statistically significant differences in number of positive cells between the sham operation group and the chronic cerebral hypoperfusion group at 2,4 and 8 weeks(P < 0.01).The results of SNK test showed that compared with the sham operation group,the number of positive cells of the chronic cerebral hypoperfusion group of 2,4 and 8 weeks increased,and the increase was most obvious in the chronic cerebral hypoperfusion group of 4w.MBP antibody labeled oligoendrocyte involved in myelination.MBP immunohistochemistry showed that there was a statistically significant difference in IOD value between the sham operation group and the chronic cerebral hypoperfusion group at 2,4 and 8 weeks(P<0.05).The results of SNK test showed that compared with the sham operation group,IOD values in the chronic cerebral hypoperfusion group of 2,4 and 8 weeks all decreased,and the decrease was most obvious in the chronic cerebral hypoperfusion group of 4 and 8 weeks.4.The polarization of glial cells in the white matter region of rats with chronic cerebral hypoperfusion was shown by fluorescence immunohistochemistry: CD16 and Iba-1 double label showed that compared with the SG group,the double positive cells in the 2,4 and 8weeks in CG group were obviously increased(P<0.01),while no significant different point in the double positive cells was found between the 2,4 and 8weeks in CG group.CD206 and Iba-1 double label showed that compared with the SG group,the double positive cells in the 2 CG group were increased(P<0.05),while no significant different point in the double positive cells was found in the 4 and 8weeks in CG group.5.We use western blotting to detect the expression of inflammatory factors IL-1β,IL-6and IL-10 in white matter.In comparison with sham operation group,IL-1 β in chronic cerebral hypoperfusion group was significantly increased at 2 and 4 weeks postoperatively(P<0.01),but the difference was not statistically significant at 8w(P>0.05).Compared with the sham operation group,IL-6 in the chronic cerebral hypoperfusion group was increased at 2,4 and 8 weeks(P<0.05),and significantly increased at 4 weeks postoperatively(P<0.01),while IL-10 in the chronic cerebral hypoperfusion group decreased at 4 and 8 weeks after surgery(P<0.05),but increased at 2 weeks(P>0.05).Part Ⅲ: The role and mechanism of phenotypic changes of microglia in white matter lesions induced by chronic cerebral hypoperfusion.1.Kluver-Barrera staining results showed that compared with SG group,nerve fibers in CG group became loose,some fibers were disordered,and local vacuolation appeared,and the changes were more obvious with the increase of slow perfusion time.Compared with CG group,nerve fibers in IG group became more loose and disordered after treatment with A2 AR inhibitor,and vacuolation increased.After treatment with A2 AR agonist,the nerve fibers in AG group became compact,the degree of disturbance was reduced,and the vacuoles were reduced compared with those in CG group.The results indicate that chronic cerebral hypoperfusion causes cerebral white matter lesionsin.A2 AR agonists can alleviate the injury after chronic cerebral hypoperfusion,while A2 AR inhibitors can aggravate the lesions after chronic cerebral hypoperfusion.2.Morris water maze experiment showed,in terms of latency time to reach the platform,there were statistically significant differences among groups(P<0.01),SNK test showed that,compared with SG group,latency time of CG group increased(P<0.01);Compared with CG group,latency time of AG group was decreased(P<0.01).In terms of the times of crossing the platform,there were statistically significant differences among all groups(P<0.01),SNK test showed that,compared with SG group,CG group was reduced(P<0.01);Compared with the CG group,the times of AG group increased(P<0.05).3.The expression of inflammatory factors IL-1β,IL-6 and IL-10 in white matter was detected by western blot.Compared with SG group,IL-1 β and IL-6 in CG group were significantly increased(P<0.01),and IL-10 was significantly decreased(P<0.01).Compared with CG group,IL-1β and IL-6 in IG group were significantly increased(P<0.01),but IL-10 had no significant difference(P>0.05);Compared with CG group,IL-1 β and IL-6 in AG group were significantly decreased(P<0.01),and IL-10 was significantly increased(P<0.01).4.Western blot was used to test the expression of polarization markers in microglia.In comparison with SG group,i NOS in CG group was increased markedly(P<0.01),but for Arg-1,no significant difference was found(P>0.05).Compared with CG group,i NOS in IG group was significantly increased(P<0.01),but Arg-1 no significant difference was found(P>0.05).Compared with CG group,i NOS in AG group was obviously decreased(P<0.01),while Arg-1 was significantly increased(P<0.01).5.Fluorescent immunohistochemical staining shows glial cell changes in white matter:fluorescence immunohistochemical single label MBP showed that compared with SG group,IOD value in CG group was decreased obviously(P<0.01).Compared with CG group,IOD value in IG group was reduced(P<0.05);Compared with CG group,IOD value in AG group was increased markedly(P<0.01).Compared with SG group,CD16 and Iba-1 double positive cells were hugely increased in CG group(P<0.01).Compared with CG group,double positive cells in IG group were increased(P<0.05);Compared with CG group,the number of double positive cells in AG group was significantly decreased(P<0.01).Compared with SG group,there was no significant difference in CD206 and Iba-1double positive cells in CG group(P>0.05).Compared with CG group,there was no significant difference in double positive cells in IG group(P>0.05).Compared with CG group,the number of double positive cells in AG group was increased significantly(P<0.01).(2)Pathways that A2 AR regulates phenotypic changes of microglia cells under chronic cerebral hypoperfusion1.In the oligodendrocyte-microglia co-culture system,microglia were activated and oligodendrocytes were damaged after hypoxia and hypoglycemia treatment.2.IL-1 β and IL-6 in CG group were significantly increased compared with SG group(P<0.01),while IL-10 was decreased(P<0.01).Compared with CG group,IL-1β and IL-6 in AG group were decreased obviously(P<0.01),and IL-10 was significantly increased(P<0.01).Compared with AG group,IL-1βand IL-6 in AP group were significantly increased(P<0.01),and IL-10 was significantly decreased(P<0.01).3.PKA activity in CG group was higher than that in SG group by ELISA(P<0.05);Compared with CG group,PKA activity in AG group was significantly increased(P<0.01).Compared with AG group,PKA activity in AP group was significantly decreased(P<0.01).4.Indices of oligodendrocyte injury detected by ELISA: Compared with SG group,LDH of CG group was significantly increased(P < 0.01);Compared with CG group,LDH of AG group was significantly decreased(P < 0.01);LDH of Compared with AG group,AP group was significantly increased(P < 0.01).5.Western-blot method was used to detect the expression of polarization markers in microglia cells.Compared with SG group,i NOS in CG group was significantly increased(P<0.01).Compared with CG group,i NOS in AG group was significantly decreased(P<0.01),while Arg-1 was significantly increased(P<0.01).Compared with AG group,i NOS in AP group was significantly increased(P<0.01),and Arg-1 was significantly decreased(P<0.01).6.The expression of PKA-CREB signaling pathway related molecules was detected by Western-blot: Compared with SG group,p-CREB/CREB in CG group was increased(P<0.05).Compared with CG group,p-CREB/CREB in AG group was significantly increased(P<0.01);Compared with AG group,p-CREB/CREB in AP group was significantly decreased(P<0.01).Conclusion1.The rat model of modified staged bilateral carotid artery occlusion is an ideal model of white matter lesions induced by chronic cerebral hypoperfusion.2.Under chronic cerebral hypoperfusion,microglia in the white matter region of rats were activated and polarized to the M1 phenotype,mediating the inflammatory injury of white matter and the impairment of cognitive function.3.Activation of A2 AR can polarize microglia to the M2 phenotype,and play a protective role in the white matter lesions.Inhibition of A2 AR had the opposite effect.4.A2 AR regulates microglial polarization through the PKA-CREB signaling pathway,thereby regulating white matter lesions in chronic cerebral hypoperfusion.