| Part Ⅰ:Relationship between Neuroinflammation and Cognitive Impairment after Cerebral Ischemic StrokeObjective: To explore whether microglia-mediated neuroinflammation and cognitive impairment exist after cerebral ischemic stroke（CIS）,and to verify the correlation between them.Methods: Adult male C57BL/6J mice were used to establish middle cerebral artery occlusion（MCAO）model by using A customized wire plug with a silicone head.Triphenyltetrazolium chloride（TTC）staining was used to observe cerebral infarction,Nissel staining to detect the volume atrophy of cerebral cortex and hippocampus,and terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling（TUNEL）staining to detect cell apoptosis.Bromodeoxyuridine（Brdu）was injected intraperitoneally three times（every 8 hours）.The paraffin sections of brain tissues were prepared by cardiac perfusion of paraformaldehyde to detect the expression of Iba1,GFAP,Neu N and Brdu by immunofluorescence staining.Protein chip was used to detect the expression of inflammatory cytokines.Open-field experiment was conducted to evaluate the autonomous exploration behavior of mice;Morris water maze test was performed to assess the hippocampusdependent cognitive function of mice.In addition,the survival rate and body weight of mice were continuously observed after CIS.Besides,m NSS and Corner test were carried out to evaluate the somatosensory nerve function of mice.Results: Compared with Sham group,the survival rate of MCAO group was 56.5%,with significantly increased cerebral infarction,volume atrophy and apoptosis in right cerebral cortex and hippocampus.Meanwhile,MCAO group had significantly reduced survival of mature neurons and increased activation of microglia,as well as obviously increased expressions of inflammatory cytokines interleukin（IL）-1β,IL-6,IL-10,and tumor necrosis factor（TNF）-α in the ipsilateral cerebral infarction.Furthermore,compared with Sham group,MCAO group had significantly reduced total movement distance and residence time in the central area in Open Field.In space-learning stage of Morris water maze experiment,MCAO group showed significantly increased total escape time and total distance;while in the spatial memory stage,MCAO group revealed remarkably decreased residence time in the target quadrant and the number of times across the hidden platform.Additionally,MCAO group had significantly reduced body weight of mice,as well as evidently increased right turn rate of Corner test and the neurological function score of m NSS test after CIS.Conclusion: In the established MCAO model,microglia activated in the post-CIS recovery stage can induce the release of inflammatory cytokines to promote the death of hippocampal neurons,and then lead to hippocampal-dependent cognitive impairment.Accordingly,there may be a significant positive correlation between microglia-mediated activated neuroinflammation in the post-CIS recovery stage and cognitive impairment.Part Ⅱ:Effects of CX3CL1 and CX3CR1 Signals on Neuroinflammatory Response and Cognitive Impairment after Cerebral Ischemic StrokeObjective: To determine trend of CX3CL1 and CX3CR1 expression after stroke,and to explore whether specific regulation of CX3CL1 and CX3CR1 signal can inhibit neuroinflammatory response and improve cognitive impairment.Methods: MCAO model was constructed by using adult male wild type（WT）,CX3CR1 knockout homozygous(CX3CR1-/-)and CX3CR1 knockout heterozygous(CX3CR1+/-)C57BL/6J mice.Among them,mice in MCAO+WT group were provided with soluble recombinant CX3CL1 protein（r CX3CL1）or solvent control（phosphate buffer saline,PBS） via lateral ventricle after CIS.Genotypes were identified by gel agarose electrophoresis.The expressions of CX3CL1 and CX3CR1 were detected by Western blotting.Meanwhile,immunofluorescence to measure the co-expressions of CX3CR1-GFP and CX3CL1 with Iba1,GFAP,Neu N and Nestin proteins.The expression of Iba1,CD68,Neu N and Brdu was detected by immunofluorescence in hippocampus collected after CIS.TTC staining was used to detect cerebral infarction,Nissel staining to detect neuronal death and volume atrophy,and Tunnel staining to detect apoptosis.[18F]-FDPA/PET was performed to detect neuroinflammatory response;and protein chip to detect the expression of inflammatory cytokines.Simultaneously,open-field experiment was conducted to evaluate the autonomous exploration behavior of mice;and Morris water maze test was performed to assess the hippocampus-dependent cognitive function of mice.Besides,the survival rate and body weight of mice were continuously observed after CIS.m NSS and Corner tests were carried out to evaluate the somatosensory nerve function of mice.Results: Compared with Sham group,there was a continuous decrease in the expression of CX3CL1 signal protein in the ipsilateral brain tissue of MCAO group;while the expression of CX3CR1 signal protein decreased first and then increased after CIS.Furthermore,compared with MCAO+CX3CR1+/-group,MCAO+CX3CR1-/-group and MCAO+r CX3CL1 group showed significant increased number of neurons and obviously inhibited phagocytic activity of microglia in the ipsilateral hippocampus.Meanwhile,following specific regulation of CX3CL1 and CX3CR1 signals,there was remarkably reduced infarct size,neuronal death and apoptosis in cerebral cortex and hippocampus,and significantly inhibited microglia-activated neuroinflammatory response and inflammatory cytokine IL-1β,IFN-γ,IL-6 and TNF-α expressions.The results of behavioral experiment showed that both MCAO+CX3CR1-/-group and MCAO+r CX3CL1 group could signaling increase survival rate and promote the recovery of somatosensory nerve function in mice.Open-field experiment revealed notably increased total distance of movement and residence time in the central area.In addition,in learning stage of Morris water maze experiment,there were significantly increased total escape time and total distance;while evidently reduced residence time in the target quadrant and the number of times across the hidden platform were found in the spatial memory stage,yet without significant difference in the average swimming speed.Conclusion: In the constructed MCAO mode,specific regulation of CX3CL1 and CX3CR1 signals can inhibit the phagocytic activity of microglia and the release of inflammatory cytokines,reduce cell apoptosis and promote the survival of hippocampal neurons,thus promoting the improvement of hippocampus-dependent cognitive function and recovery of neural function in the recovery stage.Thus,specific regulation of CX3CL1 and CX3CR1 signal axis may suppress microglia-mediated activated neuroinflammatory response and improve hippocampus-dependent cognitive impairment.Part Ⅲ:The Mechanisms of CX3CL1/CX3CR1 Axis in Mediating Neuroinflammation after Ischemic StrokeObjective: To investigate the molecular biological mechanism of CX3CL1 and CX3CR1 signals in inhibiting neuroinflammatory response after ischemic stroke.Methods:Western blotting was conducted to detect the protein expressions of IL-1β,IL-18,P65,p-P65,Caspas 1,cleave-caspas1 and GSDMD/GSDMDN-terminal in hippocampus after CIS,respectively.Meanwhile,the co-expression of Iba1 and GSDMD proteins was detected by immunofluorescence in brain tissues.The OGD/R model was established by using BV2 microglia cell line.After OGD/R modeling,cell proteins were collected for Western blotting to detect IL-1β,IL-18 and GSDMD protein expressions.ELISA was also made to measure the expression of cytokines IL-1β and IL-18 in the collected extracellular supernatant.Simultaneously,BV2 cells were intervened with r CX3CL1 or PBS in advance, followed by the detection of cell apoptosis by flow cytometry 48 hours after OGD/R modeling.Western blotting was also performed to detect the expressions of IL-1β,IL-18,P65,p-P65,Caspase 1,cleave-caspase1,GSDMD and GSDMDN-terminal following the collection of cell proteins.Additionally,the co-expression of Iba1 and GSDMD proteins was detected by immunofluorescence.Results: Compared with Sham group,MCAO+CX3CR1+/-group showed significantly increased cell count of Iba1+/GSDMD+,as well as upregulated protein expressions of IL-1β,IL-18,p-P65,cleave-caspase1 and GSDMDN-terminal.While speculated regulation of CX3CL1 and CX3CR1 signals resulted in obviously inhibited expressions of IL-1β,IL-18,p-P65,cleave-caspase1 and GSDMDN-terminal,as well as remarkably decreased cell count of Iba1+/GSDMD+.According to the in vitro experiment,in relative to OGD/R+PBS group,OGD/R+r CX3CL1 group had evidently reduced cell content of IL-1β and IL-18 in extracellular supernatant,notably suppressed intracellular protein expressions of IL-1β,IL-18,p-P65,cleave-caspase1 and GSDMDN-terminal,as well as obviously decreased cell count of Iba1+/GSDMD+ and inhibited cell apoptosis.Conclusion: CX3CL1/CX3CR1 axis may suppress the expression and release of cytokines IL-1β/IL-18 through P65/Caspase1/GSDMD signaling pathway,which may exert important regulatory effect on activated neuroinflammatory response in microglia after stroke. |
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