| The morbidity of ischemic cerebral vascular diseases is increasing gradually, and this kind of diseases attracts more and more attention because of its threat against the health and lives of human beings. After acute cerebral ischemia, the brain cells in the ischemic tissues will die in a short period of time and lead to deficit of neurological function. Until now, there is no effective therapy to it. how to improve the injured neurological functions of the patients suffering from cerebral ischemia is still the key problem of the present time, the isolation of neural stem cells (NSCs) in the embryo in the early years of 1990s, especially NSCs were discovered in the brains of adult mammalian throw new light on the treatment of CNS diseases. The discovery of NSCs suggests the existence of self-repair of the brain. After cerebral injuries, the activation of endogenous neural stem cells contributes to the recovery of injured neurological functions. But, this effect was limited because there are not enough new neural cells derived from limited endogenous NSCs to substitute injured neural cells. Although the transplantation of exogenous NSCs can be used to improve the neurological functions , the application of exogenous NSCs is confined due to their immunologic rejection, ethical issues, limited resources, difficult isolation and expansion. Bone Marrow-derived Mesenchymal Stromal Cells (MSCs) have multipotential differentiation capability and can be easily obtained from the bone marrow . There is no moral problem and immunological rejection in autogenous MSCs transplantation . If the transplanted MSCs can be largely trans-differentiated into neural cells in brain and greatly improve functional recovery following CNS injury, MSCs will become the ideal cells to the recovery of CNS injury. The aim of our experiment is to explore the detail relationship between proliferation, migration and differentiation of MSCs transplanted intravenously to the rats following Focal Cerebral Ischemia in the brain. We also explore the effect and mechanism of MSCs repairing the injured neurological function.Materials and Method:1. Mesenchymal Stromal Cells were obtained from two-months male SD rats' long bones. MSCs were isolated by first taking advantage of their preferential adherence to the plastic surface of the culture bottle. Then, MSCs were proliferated, purified and passaged. Cells harvested from the third to fifth passage were used in the experiment. We check the third passage of MSCs with CD90/CD45 antibody by using Fluorescence Activated Cell Sorting (FRCS), and make further identification by inducing MSCs differentiated into adipogenic cells in vitro.2. Health male SD rats were divided into MSCs group (n=20) and control group (n=20) by random. They all were subjected to permanent middle cerebral artery occlusion (MCAO) with suture embolic method and were generally assessed by the scores of neurological deficits. HE and TTC staining were employed to observe the position and area of cerebral ischemia. At 5 days after MCAO, MSCs group were transplanted intravenously with 1ml monoplast suspension(containing 4×10~6 MSCs), and control group were infused intravenously with 1ml serum-free medium. Then, they were performed following process:①Neurological Severity Score (NSS) were detected before and at 1, 7, 14, and 21 days after MCAO;②At 21 days after MCAO, five rats selected by random from each group were sacrificed and made cerebral TTC staining to survey volume of infarction by using SimplePCI image analysis system;③Five rats selected by random from each group were sacrificed at 7,14, and 21 days after MCAO, BrdU was injected intraperitoneally to label dividing cells of all of ischemic rats at 6,13, and 20days after MCAO, and the five rats were killed at 1 day after the last injection. Their brain tissues were gotten through poly-methanal-fixed, paraffin-embedded, and then were made into paraffin sections. To evaluate the relationship between proliferation, migration and differentiation, bromodeoxyuridine (BrdU), neuronspecific enolase (NSE) and glial flbrillary acidic protein (GFAP) were choosed as the markers of proliferation and differentiation. BrdU-labeled cells, BrdU-labeled cells with NSE expression (BrdU/NSE -labeled cells) and BrdU-labeled cells with GFAP expression (BrdU/GFAP-labeled cells) were identified by immunohistochemistry staining.Results:1. MSCs got purified through isolation and passage. MSCs expressed various markers of different lineage cells including mesenchymal cells, but not hemopoietic stem cells. By using Fluorescence Activated Cell Sorting (FRCS), the result showed that the third passage of MSCs expressed the specific cell surface antigen of mesenchymal stem cells: CD90,but not expressed the specific marker of hemopoietic stem cells: CD45. In vitro differentiation study showed that MSCs cound differentiate into adipogenic cells which are one of the different types of differentiated mesenchymal lineage progeny (adipogenic cells, osteogenic cells, and chondrogenic cells, et al). Therefore, these results confirmed that the cells we cultured were MSCs.2. Right hemiplegia and left Horror's sign were observed after left MCAO with a nylon suture. TTC and HE staining showed that ischemic regions were located in left striatum and temporoparietal cortex. The position and area were stable at each time.3. Significant recovery of Neurological Severity Score was found in MSCs at 21 days after MCAO compared with control group (p<0.05). There was no significant difference in infarction volume between MSCs group and control group.4. The numbers of BrdU/GFAP double-positive cells, BrdU/NSE double-positive cells and BrdU-positive cells that were detected in SVZ of the lateral ventricle, the ischemic zone and the ischemic boundary zone at the same fixed time points showed that there was a significant difference between MSCs group and control group. Immunostaining showed that MSCs had migrated to the ischemic and boundary zone. A few MSCs differentiated into neurons-like cells and astrocyte-like cells, these cells were mainly present in the ischemic boundary zone.5. The number of GFAP-positive cells in the ischemic boundary zone of MSCs group were apparently less than those of control group at 14, 21 days after MCAO.Conclusion:1. Our experiment isolated and cultured MSCs successfully from SD rats' long bones and got purified MSCs with the potentiality of proliferation and differentiation.2. The MCAO model can be used as an ideal animal model for the study of self-repair and activation of neural stem cells after focal ischemia. Cerebral ischemia promotes the increases of endogenous neural stem cells in SVZ of the lateral ventricle. These cells are proved to be in a state of proliferation by BrdU-labeled staining and migrate to the injuries zone. Some of those can differentiate into mature neurons and astrocytes gradually.3. MSCs transplanted intravenously to rats after MCAO can migrate to the ischemic and boundary zone, proliferate and differentiate to substitute the injured neural cells probably. This is one cause that MSCs can ameliorate neurological deficits after transplanted to the rats. Other causes include the increased expression of trophic factors, the activation of neural stem cells in SVZ of the lateral ventricle, the inhibition of formation of glial scar in the boundary zone of infarction, and so on.
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