| IntroductionVascular cognitive impairment is a kind of cognitive dysfunction caused by cerebral vascular disorders,the diagnostic criteria of American heart association(AHA)and American stroke association(ASA)defined in 2011 consists of three parts,firstly,cognitive dysfunction exists,secondly,there is evidence of cerebrovascular disease,thirdly,there is correlation between cognitive dysfunction and cerebrovascular disease.Besides,the AHA/ASA defines VCI as the whole process from mild cognitive function impairment to dementia,so we can presume that VCI possesses characteristics of persistent and progressive.Once VCI occurs,we have no effective treatment so it brings heavy economic and mental burden to the family and society.However,the mechanisms of cognitive dysfunction caused by cerebral vascular disorders are not fully understood.Large numbers of clinical studies in the past 30 years indicate that cognitive dysfunction correlate strongly with the white matter hyperintensities on MRI T2 series,which often prompts cerebral white matter lesions.Animal studies by Shibata et al proclaim that chronic cerebral hypoperfusion successfully damaged white matter,and on the thirtieth day they observed that animals’ working memory impairment and white matter damage occur on the same time.Therefore,white matter lesions induced by cerebral ischemia,namely myelin loss or oligodendrocyte injury is an important reason of VCI.Thus,what factors make important influence on myelin damage and repairment?Being the sentinel immune cell of the brain,microglia are tasked as producer of inflammatory factors and initiator of cerebral white matter lesions.But in addition to inflammation,there is also some other underlying mechanism in downstream of microglia activation induce cognitive dysfunction.Our previous study indicate that microglia not only mediate the myelin loss in VCI,but also are essential with the generation of myelin,this is seemingly contradictory.To consider of the the presence of multiple activation phenotypes for microglia detected in recent years,namely M1 phenotype cells producing pro-inflammatory cytokines and M2 phenotype cells which express anti-inflammatory cytokines,namely the polarization of microglia,we suppose that destruction of homeostasis caused by cerebral ischemia activate microglia,and push activatied microglia much easier switching to M1 phenotype through a certain molecular pathway,and then cause the release of inflammatory factors,finally induce the occurrence of VCI.So,what is the mechanism of the regulation of the polarization direction of microglia? Researchs have shown that the Fractalkine/CX3CR1 pathway may play an important role in this process,and our study aims to discovery mechanism of this.Therefore,we hypothesized that microglia polarization mediated by Fractalkine/CX3CR1 pathway plays an important role in the process of VCI occurs.The present study will establish VCI mice models different on ischemia duration by bilateral carotid artery occlusion method,and observed animal models’ cognitive behavior variance by Morris water maze,and then we select a kind of mice model which possess the most stable cognitive and behavioral impairment,use immunohistochemistry,western blotting(WB),immunofluorescence technique to detect circumstance of myelin loss,activation of microglia,polarization direction of microglia,investigating the mechanism of the polarization of microglia involved in the occurrence of VCI,to provide new ideas and targets for the treatment of VCI.Methods Part Ⅰ: The Study of establishment and myelin damage of vascular cognitive impairment mice modelsHealthy adult male ICR mice were randomly divided into two groups,model group(VCI)and sham operation group(Sham),24 mice were in VCI group and 8 in Sham group.VCI group was randomly divided into three groups: ischemia duration of 10 minutes group(VCI-10min),ischemia duration of 30 minutes group(VCI-30min),ischemia duration of 60 minutes group(VCI-60min).0.3% pentobarbital was used in anesthesia of the model group before the surgery of bilateral carotid artery occlusion to establish VCI mice models,time of blocking cerebral blood perfusion varied in different VCI groups.Mice in Sham operation group were conducted the same surgery except occluding bilateral carotid artery.Morris water maze test was used to assess cognitive function after surgery on post operation day(POD)7 to POD 10.On POD 10,mice in each groups were anesthetized and transcardially perfused with normal saline and 4% paraformaldehyde respectively after cognitive function test,paraffin sections were prepared for Luxol fast blue(LFB)staining and myelin basic protein(MBP)and Glutathione S-transferase(GST-pi)immunohistochemistry to observe status of demyelination,oligodendrocytes in hippocampus and white matter lesions in corpus callosum.Part Ⅱ: the study of microglia polarization in hippocampus of vascular cognitive impairment mice modelAdult healthy male ICR mice were randomly divided into model group(VCI)and sham operation group(Sham);mice in VCI group were randomly divided into post operation day(POD)1 subgroup(VCI-24h),POD 7 subgroup(VCI-7d)and POD 15 subgroup(VCI-15d)with 16 mice in each subgroup.There are 48 mice in sham group,16 mice were randomly selected on POD 1,POD 7,POD 15 to gather tissues.0.3% pentobarbital was used in anesthesia of the model group before the surgery of bilateral carotid artery occlusion to establish VCI mice models,time of blocking cerebral blood perfusion is 60 minutes.Mice in Sham operation group were conducted the same surgery except occluding bilateral carotid artery.On POD 1,POD 7,POD 15,mice in appropriate subgroup and sham operation group were anesthetized and transcardially perfused with normal saline and 4% paraformaldehyde respectively,paraffin sections and hippocampas tissue homogenate protein were prepared.CD11b/iNOS(inducible nitric oxide synthase)and Arginase-1(Arg-1)immunofluorescence and Western blot were used to observe status of microglia activation,direction of microgia polarization to M1 phenotype or M2 phenotype and the status of Fractalkine /CX3CR1 signal pathway.Results Part Ⅰ: The Study of establishment and myelin damage of vascular cognitive impairment mice modelsThe latency of finding platform on POD 9 was significantly longer in mice of VCI-60 min group(18.07 ± 5.86 s vs 41.90 ± 4.52 s,t =-9.11,P=0.000).Among the four groups,the latency on POD 9 was significantly different(18.07 ± 5.86 s vs 26.64 ± 9.37 s vs 33.44 ± 6.65 vs 41.90 ± 4.52 s,F = 17.52,P=0.000);time of probe test was significantly different(26.15 ± 6.02 s vs 22.08 ± 3.49 s vs 20.67 ± 3.53 s vs 12.22 ± 2.26,F = 16.63,P=0.000),compared with the sham group,VCI-10 min group didn’t decrease significantly(P> 0.05),VCI-30 min group and VCI-60 min group decreased significantly(P <0.05).MBP immunohistochemical staining of brain slice: among the four groups,the average IOD ratios were significantly different(1.00 ± 0.07 vs 0.71 ± 0.08 vs 0.53 ± 0.08 vs 0.29 ± 0.13,F = 82.28,P=0.000),compared with the sham group,VCI-10 min group,VCI-30 min group and VCI-60 min group reduced significantly(P <0.05).LFB staining of brain slice: among the four groups,the average IOD ratios were significantly different(1.00 ± 0.32 vs 0.82 ± 0.16 vs 0.62 ± 0.12 vs 0.40 ± 0.19,F = 12.16,P=0.000),compared with the sham group,VCI-10 min group didn’t decrease significantly(P> 0.05),VCI-30 min group and VCI-60 min group decreased significantly(P <0.05).GST-pi immunohistochemical staining of brain slice: among the four groups,the numbers of GST-pi labeled mature oligodendrocyte were significantly different(23.02 ± 4.83/mm~2 vs 21.01 ± 2.14/mm~2 vs 18.08 ± 2.01/mm~2 vs 15.03 ± 1.66/mm~2,F = 11.22,P=0.000),compared with the sham group,VCI-10 min group didn’t change significantly(P> 0.05),VCI-30 min group and VCI-60 min group reduced significantly(P <0.05).Part II: the study of microglia polarization in hippocampus of vascular cognitive impairment mice modelCompared with sham group,CD11 b IOD ratio of VCI-15 d group increased significantly(0.21 ± 0.11 vs 0.61 ± 0.17,t =-5.66,P=0.000).Among four groups,iNOS IOD ratios were significantly different(1.04 ± 0.13 vs 0.99 ± 0.09 vs 1.13 ± 0.08 vs 1.17 ± 0.17,F = 3.37,P =0.032,compared with sham group,iNOS IOD ratios of VCI-24 h group didn’t reduce significantly(P> 0.05),VCI-7d group and VCI-15 d group didn’t raise significantly(P >0.05),but compared with VCI-24 h group,VCI-15 d group raised significantly(P <0.05).Among four groups,Arg-1 IOD ratios were significantly different(0.82±0.08 vs 0.96±0.14 vs 0.94±0.11 vs 0.64±0.26,F = 6.35,P =0.002),compared with sham group,Arg-1 IOD ratios of VCI-24 h group and VCI-7d group didn’t increase significantly(P>0.05),VCI-15 d group increased significantly(P <0.05).Immunofluorescence staining: compared with sham group,the numbers of both CD11 b and iNOS positive cells in VCI-15 d group raised significantly(15.66 ± 0.78/mm~2 vs 22.11 ± 2.27/mm~2,t =-7.62,P=0.000),the numbers of Arg-1 positive cells in VCI-15 d group decreased significantly(19.18 ± 2.45/mm~2 vs 12.18 ± 2.16/mm~2,t = 6.06,P=0.000).Results of protein about Fractalkine / CX3CR1 signal pathway western blotting: compared with sham group,the CX3CL1 IOD ratios in VCI-15 d group increased significantly(0.23 ± 0.12 vs 0.47 ± 0.22,t =-2.83,P=0.013,the CX3CR1 IOD ratios in VCI-15 d group increased significantly(0.32±0.11 vs 0.45±0.10,t=﹣2.60,P=0.021),the Nuclear Factor-Kappa B(NF-κB)P50 IOD ratios in VCI-15 d group increased significantly(0.46 ± 0.14 vs 0.78 ± 0.12,t =-5.01,P=0.000),the cAMP Response Element-Binding Protein(CREB)IOD ratios in VCI-15 d group didn’t raised significantly(0.11 ± 0.05 vs 0.13 ± 0.09,t =-0.60,P=0.56).ConclusionWe established and compared different groups of vascular cognitive impairment mice models with varied carotid artery occlusion time,proved that transient ischemia can reduce oligodendrocytes impairment and myelin lesions,and cognitive impairment.In addition,mice of 60 minutes of carotid artery occlusion time behave the most stable on cognitive impairment and pathology lesions,hence it is appropriate model to study the polarization of microglia.Observing the status of microglia activation of VCI model,status of microglia polarization to M1 phenotype or M2 phenotype,and the status of Fractalkine /CX3CR1 signaling pathway,we indicated that in VCI models activated microglia mainly polarize to M1 phenotype,Fractalkine/CX3CR1 signal pathway activated follow the direction of NF-κB inducing M1 phenotype activation thus resulting in pro-inflammatory response causing myelin lesions. |
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