Mesenchymal Stem Cells Attenuate Blood-Brain Barrier Leakage After Cerebral Ischemia In Mice

Background:Ischemic stroke induced the increase of matrix metalloproteinase-9(MMP-9),which increased the permeability of blood-brain barrier(BBB)by extracellular matrix degradation.As a popular cell type in stem cell therapy,bone marrow derived mesenchymal stem cells(MSCs)have a great potential for the cerebral ischemia therapy.Studies demonstrated that transplantation of bone marrow mesenchymal stem cells reduced the blood-brain barrier disruption and improve the neurological function in acute phase of cerebral ischemic injury;however,the molecular mechanism is largely unknown.In this study,we examined whether mesenchymal stem cells played a positive role in maintaining blood-brain barrier function through directly affecting the release of matrix metalloproteinase-9 after ischemic brain injury,and further explored underlying molecular mechanism.We hypothesized that mesenchymal stem cells inhibited intercellular adhesion molecule-1(ICAM-1)expression,reduced neutrophil adhesion,infiltration,and migration;thereby reducing neutrophil-induced matrix metalloproteinase-9 release,which attenuated the blood-brain barrier disruption.Methods:Adult ICR male mice(n=118)underwent 90-minute transmiet middle cerebral artery occlusion(t MCAO)and received 2X105 mesenchymal stem cell stereotactic transplantation in the ischemic penumbra region within 20 minutes.Experiment was divided into four groups: sham group,ischemia + PBS treated group,ischemia + stem cell treated group,ischemic +SB-3CT treated(positive control)group.Neurobehavioral outcomes,infarct volume,blood-brain barrier permeability were determined at 1 and 3 days after middle cerebral artery occlusion.Then matrix metalloproteinase-9 expression and activity,neutrophil adhesion,infiltration and migration,myeloperoxidase activity were further measured.We also studied the changes in the intercellular adhesion molecule-1.In in vitro experiments,we used mesenchymal stem cells and their condition medium to cocultured with b End.3 cells after glucose oxygen deprivation,which simulated ischemia and detected the intercellular adhesion molecule-1 expression and its possible pathways.Results:Compared with the PBS group,the behavioral function,infarct volume and the leakage of Ig G were significantly decreased in the stem cell treated group(p<0.05).Stem cell therapy increases the gap junction of occludin,ZO-1,claudin-5,and m RNA levels of inflammatory factors IL-1β,IL-6,TNF-α(p<0.05).Stem cell therapy also reduced the number of neutrophil infiltration and myeloperoxidase activity(p<0.05).In addition,we also demonstrated that stem cell therapy effectively inhibited the upregulation and activity of matrix metalloproteinase-9 after ischemia.Stem cell therapy decreased the expression of intercellular adhesion molecule-1.Furthermore,matrix metalloproteinase-9 was downregulated in the SB-3CT treated mice(inhibition of matrix metalloproteinase-9),which was used as positive control.The co-culture of stem cells and/or their conditioned medium with b End.3 cells after glucose and oxygen deprivation(OGD)in vitro showed that stem cell therapy reduced intercellular adhesion molecule-1 expression and AMPK phosphorylation(p<0.05).The compound C,the inhibitor of AMPK,effectively inhibited the AMPK(p<0.05).Conclusion:Mesenchymal stem cell therapy attenuated blood-brain barrier disruption in mice after middle cerebral artory occlusion.It attenuated the upward trend of matrixmetallo-proteinase-9 which could be via downregulating the intracellular adhetion molecule-1 in endothelial cells potentially,suggesting that stem cells influenced matrixmetalloproteinase-9 from neutrophils and resident cells which might be via AMPK pathway and intracellular adhetion molecule-1 acted as a key factor in the paracrine actions of mesenchymal stem cell.Our study suggests a new target of matrix metalloproteinase-9 in mesenchymal stem cell therapy for cerebral ishemia.