RESUMEN
Objective To investigate the therapeutic effect and the detailed mechanisms of intraarterially delivery of bone marrow mesenchymal stem cells ( BMSCs) for treatment of middle cerebral artery occlusion (MCAO) in rats.Methods BMSCs were isolated,purified and amplified with the adherence culture method.BMSCs were labeled with 5-bromo-2-deoxyuridine ( BrdU ) (10 μmol/L) for 48 h before transplation.Surface antigens of CD90,CD29,CD106,CD34,CD45,CD11b were identified by flow cytometry.The MCAO model was established with suture emboli method.In this study,3×106 BMSCs were injected into rats with MCAO through intraarterial route at day 7 after stroke.The effects on functional and physical recovery were assessed with the behavioral tests (mNSS test and adhesive test) and body weight.Bielshowsky-Luxol Fast Blue double staining was used to demonstrate the reconstruction of axon and myelin.The Brdu-labeled BMSCs in vitro and in vivo were detected with direct immunofluorescent staining.The expression of neuron specific enolase ( NSE),neurite outgrowth inhibitor-A ( Nogo-A),synaptophysin (SYN),ki-67 nuclear antigen (Ki-67),glial fibrillary acid protein( GFAP),vascular endothelial growth factor ( VEGF) in brain were analyzed with immunohistochemical staining.Results Flow cytometry indicated that the positive rates of high expression of CD90,CD29,CD106 in BMSCs were respectively 91.70%,88.40% and 52.20%.Meanwhile,the positive rates of low expression of CD34,CD45,CD11b in BMSCs were 2.70%,5.65% and 7.82%,respectively.There was a significant difference in behavioral tests ( mNSS test and adhesive test) between BMSCs group and PBS group at day 21,28,35 after MCAO (mNSS:4.89 ±1.36,7.00 ±1.67,3.78 ±1.30 and 6.33 ±1.21,2.44 ±1.13,5.67 ± 1.51;t =2.69,3.83,4.75;adhesive test:54.00 ± 10.48,68.17 ± 11.09,36.89 ±9.80 and 59.33 ± 12.40,23.44 ± 9.04,46.50 ±9.38;t =2.51,3.92,4.77;P ＜0.05).Meanwhile,a significant difference in body weight was discovered between them at day 28,35 after MCAO.In BMSCs group,the area of corpus callosum in the ipsilateral hemisphere was significantly enlarged,the positive number of Brdu,SYN,Ki-67,GFAP,VEGF in brain was significantly increased,the expression of Nogo-A in brain was significantly decreased,nevertheless,the number of NSE-positive cells in brain and the infarct volume were not significant different from PBS group at day 35 after MCAO.Conclusions These results suggest that intra-arterial transplantation of BMSCs is an efficient treatment protocol for stroke.Treatment with BMSCs increases endogenous cells proliferation,angiogenesis,synaptogenesis,enhances axonal regeneration and the protective function of astrocytes,all of which may contribute to neurological functional recovery.