| Backround and ObjectiveCerebral ischemia is a major cause of death and disability worldwide. Thrombolytic therapy is an effective therapy when administered within 4.5 h after stroke. However, only a few patients are able to receive treatment due to the limited therapeutic window. Further, thrombolytic treatment may increase the risk of intracranial hemorrhage. Therefore, development of alternative therapies for ischemic stroke is important.Mesenchymal stem cells (MSCs), a heterogeneous population of plastic-adherent cells, have been successfully used for the treatment of experimental cerebral ischemia. Enhanced functional recovery was observed even at one year after administration in ischemic rats. Patients with ischemic stroke treated with autologous bone marrow-derived MSCs (BMSCs) also showed functional improvement, with no adverse effects. Transplantation of BMSCs after stroke was recently shown to induce angiogenesis in the ischemic boundary zone (IBZ) in rats. However, the neurorestorative mechanisms by which BMSCs improve functional recovery remain largely unknown.Generally, cerebral ischemia stimulates endogenous neurogenesis in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) within the dentate gyrus of the hippocampus, and promotes neural stem/progenitor cells (NSPCs) migration toward the ischemic lesion to reconstruct the neural network. However, the majority of these cells promptly undergo apoptosis and have a limited affect on functional recovery. This rapid cell death may be attributed to a deleterious microenvironment after ischemia, which lacks adequate trophic support. As such, we examined whether BMSCs-induced angiogenesis enhances cerebral tissue perfusion and creates a suitable microenvironment within the ischemic brain, which in turn accelerates endogenous neurogenesis and leads to improved functional recovery. MethodsBone marrow was collected from iliac aspirates from healthy adult male volunteers after informed consent was obtained. At the fifth passage, human BMSCs (hBMSCs) were collected, counted, and analyzed by flow cytometry and karyotype analysis. Adult female rats subjected to 2 h middle cerebral artery occlusion (MCAO) were transplanted with 5X105 hBMSCs in 10μL saline or an equal volume of saline into the ipsilateral brain parenchymal at 3 d after MCAO. Bromodeoxyuridine (BrdU,50 mg/kg, i.p.) was injected every two days immediately after transplantation for a total of 14 d. (1) The modified neurological severity scores (mNSS) test and the adhesive-removal somatosensory test were performed to valuate the recovery of neurological function; (2) Hematoxylin and eosin staining was used to detect relative infarct volume; (3) 18F-FDG micro-PET scanning was used to assess the glucose metabolic activity of the brain lesion; (4) Cells co-stained for both BrdU and vWF were considered proliferating endothelial cells and CD31 immunofluorescence was considered neovasculature; (5) Transcranial laser Doppler flowmetry was used to monitor relative cerebral blood flow (CBF); (6) The staining of BrdU, nestin, and musashil were used to detect NSPCs in the SVZ of the lateral ventricle and SGZ within the dentate gyrus of the hippocampus; (7) Double-immunofluorescence staining was used to detect the migration, differentiation, and apoptosis of NSPCs; (8) Western blot analysis was used to detect the levels of VEGF, nestin, musashil, ki-67 and DCX in the ipsilateral hemisphere; (9) Fluorescence in situ hybridization was used to track donor male hBMSCs in female recipients.ResultshBMSCs-treated rats exhibited significant behavioral recovery beginning at 2 w after cerebral ischemia compared with controls. Moreover, rats treated with hBMSCs showed increased glucose metabolic activity and reduced infarct volume. hBMSCs treatment significantly increased the expression of vascular endothelial growth factor, promoted angiogenesis, and facilitated cerebral blood flow in the ischemic boundary zone. Further, Flk-1+hBMSCs treatment enhanced proliferation of NSPCs in the SVZ and SGZ. Finally, more NSPCs migrated toward the ischemic lesion and differentiated to mature neurons or glial cells with less apoptosis in hBMSCs-treated rats. hBMSCs survived after transplantation, the majority of which were distributed close to the injured tissue at 12 w after transplantation.ConclusionThese data indicate that angiogenesis induced by hBMSCs promotes endogenous neurogenesis, which may cause functional recovery after cerebral ischemia.
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