In Vivo Monitoring And Assessment Of Exogenous Mesenchymal Stem Cell-derived Exosomes In Mice With Ischemic Stroke By Molecular Imaging

Part I Non-invasive monitoring of exogenous mesenchymal stem cell-derived exosomes in mice with ischemic strokeObjective: The migration ability and survival of exogenous MSC-exos in mice with remain unclear.This study aimed to extract MSC-exos and to make them visualized using a lipophilic dye.Near-infrared fluorescence imaging was performed to investigated whether MSC-exos migrate into the ischemic brain.Methods: MSC-exos were acquired by ultracentrifugation and were labeled with Di R.Then,MSC-exos labeled with Di R were injected intravenously into mice with ischemic stroke.Near-infrared fluorescence(NIRF)images in vivo were obtained on days 0,1,3,5,7,10,and 14.In vitro imaging of brain,heart,liver,spleen,lung,kidney and bowel was performed on days 3 and 14.Immunofluorescence staining of brains was performed on day 3.Results: 1)NTA and TEM showed that the MSC-exos exhibited typical morphology and size(134 nm).WB showed the expression of CD63 and CD9.2)Di R-labeled MSC-exos were detected by in vitro fluorescence imaging and the fluorescence signal intensity was highly linearly correlated with the MSC-exo concentration.3)The average radiant efficiency of the Di R-labeled MSC-exo group was significantly higher than that of the unlabeled MSC-exo group on day 1 and day 3.The average radiant efficiency peaked on day 3.4)Immunofluorescence staining showed that the Neu N signal was colocalized with the Di R-labeled MSC-exo signal in the ischemic border zone of mice treated with MSC-exos.Conclusions: MSC-exos can effectively migrate into ischemic lesions and fuse with neurons after intravenous injection.Exogenous MSC-exos are mainly excreted through the liver to the bowel,which makes it difficult for the homing of MSC-exos.Part II Effects of exogenous mesenchymal stem cell-derived exosomes in mice with ischemic strokeObjective: Mesenchymal stem cell-derived exosomes(MSC-exos)are considered an important restorative treatment for ischemic stroke.Here,we aimed to investigate the protective role of MSC-exos in mice with ischemic stroke using DTI combined with traditional methods.Methods: C57/BL mice with ischemic stroke were randomly divided into 2 groups: the control group and the MSC-exo-treated group.MSC-exos(100ug,200ul)were injected through the tail vein.Magnetic resonance(MR)images were obtained on days 1,7 and 14.On day 14,functional outcomes,angiogenesis,neurogenesis,and white matter remodeling were assessed,and western blotting assays were performed.Results: 1)Exogenous MSC-exos significantly improved neurological function recovery and decreased the infarct volume after stroke.2)Compared with the control group,angiogenesis and neurogenesis were significantly increased in the MSC-exo-treated group on day 14.3)In vivo DTI revealed that the FA value,fiber number ratio,and fiber length significantly increased in the MSC-exo-treated group.4)Exogenous MSC-exos significantly reduced the expression of pro-inflammatory cytokines IL-1βon day 14.Conclusions: With imaging methods and traditional histopathological methods,we can conclude that MSC-exos promote neurogenesis,angiogenesis and white matter recovery after stroke.DTI can be used to directly demonstrate white matter remodeling.To some extent,inflammation triggered by ischemic stroke is relieved by MSC-exo treatment.