Mechanism Of Microglia CX3CR1 Pathway In Myelin Repairment On Vascular Cognitive Impairment Mice

The second most common cognitive dysfunction——vascular cognitive impairment(VCI)accounts for about 20% of all kinds of dementias.White matter lesions,or myelin or oligodendrocyte(OLs)injury,are important pathological changes in VCI.Recently,central nervous system inflammatory cell,microglia has been closely related to white matter damage.Our previous study on animal models of VCI have found myelin injury in VCI mice,with mounted microglia activation.Besides,our study also found activation of Fractalkine/CX3CR1 pathway in ischemia-induced microglia,and the expression of its downstream nuclear factor κB-p50/65(Nuclear Factor-Kappa B,NF-κB)increased,which induced more release of pro-inflammatory factors,and ultimately led to myelin damage or even cognitive impairment.Therefore,we hypothesized that regulation of CX3CR1-related signaling pathways in VCI animals might be possible to inhibit activation of microglia,reduce inflammation,promote differentiation and maturation of oligodendrocyte precursor cells(OPCs)myelin,protect and repair OLs and ultimately improve cognitive functionIn this study,the VCI model was constructed by Bilateral Common Carotid Artery Occlusion(BCCAO).We antagonized the CX3CR1 pathway and its downstream key factor NF-κB and non-receptor tyrosine protein kinase Src respectively.Tail suspension test(TST)and forced swimming test(FST)were adopted for anti-depressive function of these antagonizes.Morris water maze(MWM)was conducted for detecting hippocampal-related learning and memory function.Enzyme-linked immunosorbent Assay(ELISA)was used to detect the content of inflammatory factors and myelin-related proteins in peripheral blood.Luxol fast blue(LFB)staining and myelin basic protein(MBP)immunohistochemical staining were adopted to observe the damage and repairment of myelin in the corpus callosum(Corpus Callosum,CC)and hippocampus,ionized calcium binding adapter molecule 1(Iba-1),neuron-glial antigen 2,NG2,O4 and neuronal nuclei(Neun)were labeled to observe hippocampus activate microglia,OPCs,pre-OLs and neurons.Western blot(WB)was used to detect MBP in hippocampus.The results of our first part showed that after administration of CX3CR1 neutralizing antibody anti-FKR,VCI mice significantly shortened the time of immobility in TST and FST.On the 4th day of the MWM hidden platform test,VCI+anti-FKR mice spent less time to find the hidden platform,the number of crossing times through the target quadrant,the time spent and percentage of distance traveled in the target quadrant was significantly increased in the space exploration experiment than those of VCI group,suggesting that anti-FKR significantly improved depressive-like behavior and hippocampus related learning and memory function in VCI mice.The comparison of the corresponding IOD values of LFB and MBP staining in CC area showed that there was no statistical significant between VCI+anti-FKR and VCI+NS.The corresponding IOD value of MBP immunohistochemical staining and WB results in hippocampus was significantly increased,and peripheral MOG was induced in VCI+anti-FKR mice,indicating that inhibition of CX3CR1 protected myelin in hippocampus.The number of hippocampal Iba-1 positive cells significantly decreased,and the ELISA results showed that the levels of IL-1β and TNF-α in the VCI+anti-FKR group were significantly lower,indicating that anti-FKR can alleviate the inflammatory response in VCI mice.What’s more,CX3CR1 inhibition also slightly reduced hippocampal NG2 positive cells while significantly increased O4 and Neun positive cells,suggesting that inhibition of CX3CR1 promoted hippocampal differentiation and maturation of OPCs,protect myelin and neuron.The results of our second part showed that after the administration of NF-κB antagonist PDTC,the immobility time of VCI mice in TST and FST was significantly shorter,but the difference of immobility time was not seen in the Src antagonist PP2.In MWM,there were no significant difference between VCI+PDTC and VCI+NS to found the hidden platform,as well as swimming speed,number of crossing times,residence time and distance traveled in platform quadrant,while VCI+PP2 mice spent less time to find platform on the 4th day,increased crossing times through the target quadrant,the percentage of the total distance traveled in the target platform compared to those of the VCI group.After PDTC and PP2 intervention,the comparison of the corresponding IOD values of LFB and MBP staining in CC area to VCI+NS showed no significant difference.The corresponding IOD values of MBP immunohistochemical staining and WB results in hippocampus of PP2+NS group were significantly increased,with increased MOG content in peripheral ELISA analysis,indicating Src inhibition was capable to protect myelin from injury.Compared to VCI+NS mice,the number of Iba-1 positive cells in hippocampus and peripheral IL-6,TNF-α,HMGB1 were significantly decreased in PDTC+NS group,indicating that inhibition of NF-κB reduced microglia activation and inflammatory reaction.What’s more,the results of immunohistochemistry showed that after administration of PP2,Iba-1 positive cells were reduced while numbers of O4 positive cells were significantly increased,suggesting Src inhibition promoted differentiation and maturation of OPCs to impair injured myelin.The above experimental results have shown that the CX3CR1 neutralizing antibody anti-FKR effectively inhibits the activation of microglia and controls the inflammatory response by inhibiting the CX3CR1 pathway,which protects neurons,promoted differentiation and maturation of OPCs in the hippocampus to promotes the repair of myelin,and improves cognitive dysfunction in ischemic mice.Furthermore,the inhibitor of NF-κB,PDTC can reduce microglia activation and release of inflammatory factors induced by hypoxia,while Src inhibitor PP2 can promote OPCs maturation to reduce myelin damage and promote myelin repairment in VCI mice,thereby improving depressive-like and cognitive dysfunction in VCI mice respectively.Therefore,collective regulation of Src and NF-κB may be a potential target for the prevention and treatment of VCI in the future.