| Object:We injected BMSCs into rats after transient middle cerebral artery occlusion (MCAO) model through intraarterial, intravenous, and intracerebral routes, then to investigate the therapeutic effect and the detailed mechanisms which exerted in the improvment of neurological function after ischemia.Methods:BMSCs were isolated, purified and amplified with the adherent culture method. Surface antigens of CD90, CD29, CD 106, CD34, CD45, CD11b in BMSCs were identified by flow cytometry. BMSCs were labeled with 10μmol/L Brdu. MCAO model was established with suture emboli method. In this study, we injected 3×106BMSCs into rats at day 7 after MCAO model via intraarterial, intravenous, and intracerebral routes. The effects on neurologic functional outcome after BMSCs treatment were assessed at post-ischemic days (PIDs) 1,7,14,21,28,35 using the behavioral tests (mNSS test and adhesive test) and body weight. The rats were humanely killed at PID 35, then the brain tissues were processed for histochemical and immunohistochemical staining:Bielshowsky's-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 immunofluorescence; The expressions of NSE,Nogo-A,SYN,Ki-67,GFAP and VEGF in brain were analyzed with immunohistochemical staining.ResuIts:The morphology of BMSCs in vitro was the thin spindle shape, which was adherent growth. After sub-culturing with 10μm/L Brdu for 48h, the percentage of Brdu+-BMSCs was found to be 95.0±8.40%. Flow cytometry indicated that the positive rates of high expression of CD90, CD29, CD 106 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%, There was a significant recovery of behavioral tests (mNSS test and adhesiv test) in intraarterial, intravenous, and intracerebral group, as compared to PBS group respectively (p<0.05), furthermore, such therapeutic effects in intraarterial group were more obvious than intravenous and intracerebral groups(p<0.05). BMSCs treatment enhanced the axon-myelin remodeling in the area of corpus callosum and the corpus striatum in the ipsilateral hemisphere, as compared to PBS group(p<0.05). the expressions of SYN, Ki-67, GFAP, VEGF in brain were significantly increased(p<0.05), the expression of Nogo-A in brain was significantly decreased (P<0.05), nevertheless, the number of NSE-positive cells in brai,the infarct volume and the thickness of glial scar were no significant difference (P>0.05), As BMSCs treated groups which were intraarterial, intravenous, and intracerebral group compared with PBS group at day 35 after MCAO, respectively. Moreover, intraarterially transplanted BMSCs significantly upregulated the expression of SYN (P<0.05), Ki-67 (P<0..05), and reduced the expression of Nogo-A (P<0.05) as compared to intracerebral or intravenous injection. Ki-67 expression significantly positively correlated with GFAP expression (r=0.742, p<0.05), the expression of GFAP also significantly positively correlated with the expression of VEGF (r=0.725, p<0.05), and Nogo-A expression significantly negatively correlated with the expression of SYN (r=-0.842, p<0.05).Conclusions Our results demonstrated that the treatment of cerebral ischemia transplanted with BMSCs via intravenous, intraarterial and intracerebral routes significantly improved neurologic function, and the degree and speed of neurologic functional recovery in intraarterial way were the best compared to those in intravenous and intracerebral way at the same time point after infarction. Treated-BMSCs increased endogenous cells proliferation, angiogenesis, synaptogenesis, enhanced the axons regeneration and the protective function of astrocytes, all of which may contribute to neurological functional recovery. |
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