| Objective:Ischemic stroke is characterized by high disability rate and high mortality rate.According to the results of "The Global Burden of Disease Study-China" published by The Lancet,stroke is currently the most fatal and disabling disease in China.It is worth noting that,in addition to physical disability,about 40% of the survivors of ischemic stroke have negative emotions such as low mood,pessimism,worldweariness and irritability,which is defined as "post-stroke depression".Patients are likely to experience poor clinical outcome,which seriously affects the quality of life of the patients and brings a huge burden to the society and family.Recent studies have showed that the etiology of PSD is closely related to the inflammatory response of microglia,and the immune inflammatory process mediated by microglia can disrupt the homeostasis and synaptic plasticity of neurons,which is the biological foundation for the occurrence of post-stroke depression,but its specific molecular mechanism is not clear.Due to ischemia and hypoxia,the brain activates "damage associated molecular patterns(DAMPs)",and the release of high-mobility group box 1 protein(HMGB1),one of its key members,combined with TLR4 on microglia membrane,mediates "cascade of damage" to aggravate neuroinflammatory injury.Modulation of the inflammatory phenotype after activation of microglia cells can reduce neuroinflammation and ameliorate depressive behavior.Myocyte enhancer factor 2(MEF2D)is specifically expressed in microglial cells in the hippocampus.It has been reported that MEF2D can regulate the TLR4-mediated inflammatory response of microglia cultured in vitro.However,as a key transcription factor,there has been no study on the mechanism of MEF2D in the regulation of microglial inflammation after ischemic stroke.Based on recent advanced including our pilot studies,we proposed that the regulation of the HMGB1/TLR4 pathway by MEF2D affected the inflammatory phenotype of hippocampal microglia and thus modulated the synaptic plasticity of hippocampal neurons,thereby participating in the neuroinflammatory mechanism of post-stroke depression.By using Middle cerebral artery occlusion(MCAO)model,combined with the neurobehavioral research,RNA-Seq and bioinformatics analysis,molecular biology,in vivo AAV transfection intervention,cellular morphological analysis,et al.The experiments in this propose would explore the role of MEF2 D in regulating hippocampal microglial HMGB1/TLR4 pathway in neuroinflammation of ischemic stroke model in rats and the possible mechanism of its involvement in post-stroke depression,to provide the experimental evidence for the treatment of ischemic stroke.Materials and Methods:Adult male SD rats(body weight 180-200g)underwent embolization of middle cerebral artery(MCAO)for 90 min to simulate ischemic stroke(MCAO group).Sham group only underwent sham surgery without embolization.Laser speckle cerebral blood flow imaging,TTC staining and m NSS scoring were used to determine whether the model was successful.The depressive behavior and cognitive behavior of rats after ischemic stroke were measured by sucrose preference test(SPT),open field test(OFT),elevated plus maze test(EPM)and step-through test(STT).RNA-seq was used to analyze the enrichment pathway of differential genes.Western blot was used to detect the expression of inflammatory factors IL-1β,IL-6 and IL-10 in the hippocampus and the expression of HMGB1,TLR4 and MEF2D after 1 d,3 d,5 d,7 d and 14 d of the MCAO model.The co-localization of MEF2D and microglia cells were marked by immunofluorescence.The concentrations of CD86,CD206,i NOS and Arg1 in hippocampal microglia polarization phenotype markers were detected by immunofluorescence technique and q PCR.The expression of MEF2 D in microglia was specifically up-regulated and downregulated by stereotacically targeted injection of TLR4 pathway-specific inhibitors(TAK242 group),control solution(Vehicle group)and adeno-associated viruses(Control virus group: F4/80-NC group,MEF2 D knockdown group: F4/80-MEF2 DRNAi group,and MEF2 D overexpression group: F4/80-MEF2D-OE group).Laser confocal scanning imaging,Western blot and q PCR were used to verify the expression of specific molecules after intervention.The depressive behavior and cognitive behavior of rats with ischemic stroke after blocking TLR4 pathway,upregulated and down-regulated MEF2 D were analyzed by sucrose preference test,open field test,elevated plus maze test and step-through test.Western blot was used to detect the expressions of inflammatory cytokines IL-1β,IL-6 and IL-10 in the hippocampus,and the expressions of HMGB1,TLR4 and MEF2 D after 3 days of the MCAO model.The co-localization of MEF2 D and microglia cells was marked by immunofluorescence.The polarization phenotype markers CD86,CD206,i NOS and Arg1 in hippocampal tissues were detected by immunofluorescence technique and q PCR,and the regulation of MEF2 D on HMGB1/TLR4 pathway mediated anti-inflammatory phenotype changes of microglia cells were further detected.Morphology of microglia activation regulated by up-regulation and down-regulation of MEF2 D after ischemic stroke in rats was studied by microglia morphological analysis.Finally,Golgi staining was used to further image the dendritic spines of hippocampal neurons,and transmission electron microscopy was used to observe the synaptic ultrastructure in the hippocampal region.In vivo multi-channel recording technique was used to analyze the local field potential in hippocampal CA1 region.Immunofluorescence and Western blot were used to investigate the influence of MEF2 D on the regulation of microglial cell inflammation on the plasticity of hippocampal synapses.Results:Part I:(1)The blood flow of the cerebral cortex of rats in Sham group was unchanged compared with the pre-surgery,and there was no cerebral infarction,while the blood flow of the cerebral cortex in the rats of MCAO group was significantly decreased compared with the pre-surgery,and the brain slices showed obvious infarct area.(P<0.001).(2)24h after modeling,the m NSS score of rats in MCAO group was significantly higher than that in Sham group(P<0.001).(3)In the sucrose preference experiment,the percentage of sucrose water intake in MCAO group at 1,3,5 and 7days after stroke was significantly lower than that in Sham group(P<0.01).In the open field test,spontaneous activity and exploration behavior were weakened at 7days after stroke: Mean total time of rats spent at the center in MCAO group was significantly shorter than that of their counterparts in Sham group(P<0.01).Simultaneously,the change trend of time rats spent at the corner was opposite to that in the center area(P<0.01).The rearing number and grooming number in MCAO group rats were less than that of rats in Sham group(P<0.01).In the elevated plus maze experiment,anxiety behavior increased at 14 days after MCAO model in rats:percent time that rats spent in the open arms in MCAO group were significantly less than that of Sham group(P<0.01).Similar to the tendency of time spent in open arms,percent frequency of open arm entries in MCAO group were significantly less than that of Sham group(P<0.01).The Times of modifying behaviors were less than those in Sham group(P<0.01).(4)RNA-Seq analysis showed that MEF2D was a significantly down-regulated gene in ischemic stroke model rats.After enrichment analysis of differential genes,the main enrichment pathways focused on inflammatory response and immune regulation.GSEA enrichment analysis showed that TLR4pathway was significantly activated after ischemic stroke in rats.(5)The expression of pro-inflammatory factors IL-1βand IL-6 in the hippocampus of rats increased significantly on the 1st day after stroke,and reached the peak on the 3rd day,IL-1βcontinued to the 7th day,and IL-6 decreased on the 14th day after stroke,while the expression of IL-6 continued to be high on the 14th day after stroke.The expression of anti-inflammatory factor IL-10 was slightly increased on postoperative day 1,and significantly decreased on postoperative day 3,which was lower than that in the Sham group.The expression of anti-inflammatory factor IL-10 continued to be low on postoperative day 3-14.(6)The expression of HMGB1 and TLR4 increased significantly on the 1st day after stroke,and continued to the 14th day after stroke.On the contrary,the expression of MEF2D was significantly reduced on the 3rd day after stroke and remained low on the 3rd to 14th day after stroke.(7)Analysis results of microglia polarization markers:CD86 was almost not expressed in the Sham group,while CD206 was mainly expressed.In contrast,CD86 expression was significantly increased and CD206 expression was significantly decreased in the MCAO group.The q PCR results showed that the expression of i NOS m RNA in hippocampus of rats in MCAO group was significantly higher than that in Sham group,and the expression of Arg1 m RNA was significantly lower than that in Sham group.Part II :(1)After administration of TLR4 inhibitor TAK242,sucrose water intake of rats was significantly higher than that of solvent control Vehicle group in the sucrose preference test on days 1-7 after ischemic stroke(P<0.01).In the open field experiment,the movement time in the central area of the open field was significantly longer than that in the Vehicle group(P<0.01),and the movement time in the corner area was significantly less than that in the Vehicle group(P<0.01).In the elevated cross maze experiment,the time and times of entering open arms in TAK242intervention group were significantly higher than those in Vehicle group(P<0.01),and the number of modifying behaviors was significantly higher than that in Vehicle group(P<0.05).In the cognitive behavior experiment of the step-through test,there were no statistical differences in latency time,error times and travel distance between TAK242 intervention group and Vehicle group(P>0.05).(2)The TLR4 inhibitor TAK242 was given to the lateral ventricle continuously for 3 days after operation.The TLR4 pathway was significantly inhibited and TLR4 expression was significantly decreased(P<0.001).At the same time,the inhibition of MEF2D expression after stroke was reversed.Compared with the Vehicle group,the expression of MEF2D in TAK242 group was significantly up-regulated(P<0.001).The expression of IL-1βand IL-6 was significantly down-regulated in TAK242 group(P<0.001),while the expression of IL-10 was up-regulated(P<0.001).(3)Compared with the F4/80-NC group,the percentage of sucrose water intake in the F4/80-MEF2D-RNAi group was significantly decreased on day 1,3 and 7 after surgery(P<0.001),and the sucrose water intake in the F4/80-MEF2D-OE group was significantly increased(P<0.001).(4)In the open field experiment,the upregulation of MEF2D significantly improved the spontaneous activity and exploration behavior of rats at 7 days after stroke:the movement time of central region was significantly decreased(P<0.05),and the activity time of peripheral corner region was significantly increased(P<0.05);The central area movement time of rats in F4/80-MEF2D-OE group was significantly longer than that in F4/80-NC group and F4/80-MEF2D-RNAi group(P<0.001),and the peripheral corner area movement time was significantly shorter than that in F4/80-NC group and F4/80-MEF2D-RNAi group(P<0.01),and the upright frequency and grooming frequency were increased(P<0.05).In the elevated cross maze experiment,the up-regulation of MEF2D significantly improved the anxiety behavior of rats 14days after stroke:the time and times of entering open arms were significantly decreased in F4/80-MEF2D-RNAi group(P<0.05);F4/80-MEF2D-OE overexpression group significantly increased the time and times of entering open arms(P<0.001).(5)Upregulation of MEF2D improved the inflammatory response in the ischemic hippocampus of rats after stroke:the expression of inflammatory cytokines in the hippocampus showed that the expressions of IL-1βand IL-6 were significantly down-regulated in F4/80-MEF2D-OE group(P<0.05),and the expression of IL-10was significantly up-regulated(P<0.001).(6)The percentage of CD86-positive cells in F4/80-MEF2D-RNAi group was significantly higher than that in F4/80-NC group(P<0.01);On the contrary,the percentage of CD86 positive cells in F4/80-MEF2DOE group was significantly lower than that in F4/80-NC group(P<0.001),and the percentage of CD206 positive cells was significantly higher than that in F4/80-NC group(P<0.001).The q PCR results showed that the m RNA expression of i NOS in F4/80-MEF2D-OE group was significantly lower than that in F4/80-NC group(P<0.001),and the m RNA expression of Arg1 was significantly higher than that in F4/80-NC group(P<0.01).(7)After ischemic stroke,the number of synapses and concentric circles crossing and the length of total synapses of hippocampal microglia in the F4/80-MEF2D-OE group were more than those in F4/80-NC group,and the activation index was lower than that in the F4/80-NC group.The number of intersections of microglia and concentric circles and the length of total synapses in the down-regulated MEF2D group were decreased compared with the F4/80-NC group.Microglia were significantly activated,with enlargement of cell body,shortening and thickening of synapses,increasing in number and shape index compared with the F4/80-NC group.Part III:(1)Upregulation of MEF2 D improved dendritic branching complexity and dendritic spine density of pyramidal neurons in hippocampal CA1 region of ischemic stroke rats: Dendritic complexity and dendritic spine density of pyramidal neurons were significantly decreased in MEF2 D down-regulation group,but the above indexes were significantly improved by MEF2 D overexpression.Compared with F4/80-MEF2D-RNAi group,the number of dendritic branches of pyramidal neurons in F4/80-MEF2D-OE group was significantly increased at 40 μm,60 μm,70μm,and 80 μm from the neuronal body.The density of secondary dendritic spines of apical dendrites increased significantly per 10μm length.(2)The results of transmission electron microscopy showed that the number of synapses,intrasynaptic vesicles and postsynaptic membrane density in the hippocampus of rats in the F4/80-MEF2D-OE group were significantly increased,while that in the F4/80-MEF2 DRNAi group was significantly decreased.(3)In vivo multi-channel electrophysiological recording results showed that the phase amplitude coupling of LFPs θ,high and low frequency γ waves in hippocampal CA1 region of rats in F4/80-NC group decreased,and γ energy was inhibited.In F4/80-MEF2D-RNAi group,the phase amplitude coupling of θ,high and low frequency γ waves decreased more obviously,and γ energy inhibition was more significant.However,for the rats in the F4/80-MEF2D-OE group,the corresponding frequency peaks of LFPs in the hippocampal CA1 region were observed,and the energy in this region was increased.Meanwhile,the phase amplitude coupling of θ wave,high γ and low γ wave was also enhanced.(4)The results of immunofluorescence and Western blot showed that the expression levels of presynaptic membrane protein SYN1 and postsynaptic membrane protein PSD95 in hippocampus of rats in F4/80-MEF2D-OE group were significantly increased.F4/80-MEF2D-RNAi group was significantly decreased.Conclusion:1.Activation of HMGB1/TLR4/MEF2 D pathway in hippocampal microglia mediates neuroinflammation-induced post-stroke depression in ischemic stroke model rats.2.MEF2 D regulates the HMGB1/TLR4 inflammatory pathway to promote the transformation of microglia into anti-inflammatory phenotype to reduce neuroinflammation and ameliorate post-stroke depression.3.The mechanism by which MEF2 D regulates microglial neuroinflammation and alleviates post-stroke depression is related to its improvement of hippocampal synaptic plasticity. |
PDF Full Text Request