| Background and Purpose:Ischemic cerebrovascular disease (ICVD) is a kind of significant high rate of deformity and mortality cerebral vascular disease, and its morbidity has the increasing tendency year by year, with the progressing of population aging and rising living standards in China. As we all known, ICVD has the limited method of treatment, which is the main reason for its poor prognosis. With the continuous researching of the biological characteristics of stem cell, traditional concepts of adult nerve cells do not have the ability of renewable had been broken down. In recent years, stem cell transplantation for treatment of ICVD becomes the research focus, especially researching the distribution, migration and turnover of transplanted stem cell in the host brain tissue in vivo after the transplantation of stem cell, which is essential for assessing the treatment efficacy of stem cell.Our research was divided into three parts to assessing the magnetic resonance monitoring after stem cell transplantation:1.Isolation, culture, purification and identification of rat bone marrow mesenchymal stem cells (BMSCs),2. Superparamagnetic iron oxide (SPIO) labeling of rat BMSCs and exploring the biological activity and the capability of differencing into neural-like cells after labeling,3.Magnetic resonance in vivo monitor the survival and migration of stem cells transplanted into the rat brain of middle cerebral artery occlusion (MCAO).Materials and Methods:1.Culture and identification of rat BMSCs:adherent culture and isolation method of whole bone marrow was employed to acquire BMSCs, and the cultured BMSCs were purified by continuous change of medium and pass on from generation to generation. Characteristic morphology of cultured BMSCs was observed by inverted phase contrast microscope and transmission electron microscopy, and the surface molecules of BMSCs was detected by immunocytochemistry staining, as well as the cell cycle and cell growth curve was analyzed by flow cytometry and microplate reader, respectively. Finally, the horizontal differentiation ability of BMSCs, including the osteogenic, adipogenic and neural-like differentiation,were identified. 2.SPIO labeling of rat bone marrow mesenchymal stem cells and magnetic resonance imaging of labeled cells:First, molecular surface morphology, as well as the size of particles and Zeta electric potential, between Resovist and Feridex, were measured by using scanning electron microscopy and laser particle size analyzer, respectively. Then, rat BMSCs were labeled by different concentration (14μg/ml,28μg/ml,56μg/ml and 112μg/ml) of Resovist, and intracellular iron particles were identified by using Prune's blue staining and transmission electron microscopy. The influence of Resovist labeling on rat BMSCs cell growth curve were measured by microplate reader. After the neural-like differentiation of Resovist labeled and non-labeled BMSCs, the mRNA and protein expression of neuroepithelial stem cell protein (Nestin), neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were analyzed by RT-PCR and Western blot technique. Finally, rat BMSCs labeled with different concentrations of Resovist were measured in vitro, and transplanted into normal rat brain were observed by susceptibility weighted imaging.3.MR monitoring of SPIO labeled rat BMSCs transplanting into cerebral ischemic rats:left middle cerebral artery (MCA) was occluded with the suture, and all of the rats were randomly and equally divided into 5 groups. In A group (sham group), only separated branches of the external carotid artery (ECA), and tip of the suture was not inserted within the left common carotid artery (CCA).For B group (MCAO group):only the left common carotid artery was occluded, and did not performed transplantation. As to C group (sham transplantation after MCAO), contralateral parietal cortex transplantation of PBS solution was applied after MCAO.For D group (experimental group 1 after MCAO), rat BMSCs without Resovist labeled were transplanted into MCAO model. As well as E group (experimental group 2 after MCAO), rat BMSCs labeled with Resovist were transplanted. After the success of transplantation, modified Neurological Severity Scores (mNSS) methods were performed to assess the neurological deficit at 1 day,1 week,2 weeks and 4 weeks after transplantation. The survival and migration of transplanted BMSCs were dynamically observed by Siemens Trio Tim 3.0 T superconducting high-field MRI scanner. The imaging results and brain tissue sections of HE staining together with Prussian blue staining were researched contradistinctively.Results:1.P3 generation of rat BMSCs demonstrated uniform distribution of the spindle or fiber-like cells, and showed the morphology of two different cell types under transmission electron microscope. Immunocytochemical staining results of rat CD molecules exhibited positive expression of CD44 and CD105 and negative expression of CD34 and CD45.Flow cytometry cell cycle analysis showed G0/G1 stage of rat BMSCs accounting more than 90 percent. Growth curve showed rat BMSCs had undergone halt stage, index increase stage, and plateau phase. Horizontal differentiation results revealed:ALP activity was significantly increased 14 days after osteogenic induction, and different size of droplets-like structure were detected 21 days after adipogenic induction, as well as NSE, Nestin and GFAP staining was positive after neural induction.2.Scanning electron microscope manifested that there were extremely different between Resovist and Feridex, besides, particle size and Zeta potential analysis exhibited that the diameter of the former is significantly greater than the latter, whereas the absolute value of Zeta potential of the latter is greater than former. Cell Prune's blue staining and transmission electron microscopy displayed blue iron particles or granules of high electron density, respectively. The control group and different concentrations of labeled cells group had no significant differences in optical density measured by MTT method. The mRNA of Nestin, NSE and GFAP showed high expression after neural induction detected by RT-PCR. Besides, specific positive bands were manifested in the 240 Kd position on Western blot. Susceptibility weighted imaging of in vitro EP tube revealed that the more increased concentration of labeling, the lower signal intensity were detected. Finally, lower signal density still could be demonstrated on susceptibility weighted imaging 7 weeks after transplanted into normal rat brain.3.After middle cerebral artery occlusion, HE staining showed decline of neurons number, besides, TTC staining manifest that the ischemic side of the frontal-temporal cortex and basal ganglia area had none staining. Neurological function score showed that A group vs. B and C group, C group vs. D and E group had statistically significant between each other 1 day,1 week,2 weeks and 4 weeks after transplantation. MRI results showed block oval low signal low signal with clear boundary 1 day after transplantation, along with blurred boundary 1 week later, as well as transformed to "comet-like" with a tail pointed contralateral ischemic lesions 2 weeks after transplantation, finally, tail extended to the ischemic side, and even migrate across the midline to the opposite edge of ischemic high intensity signal region 4 weeks after transplantation.Conclusion:1.The whole bone marrow adherent culture method, which had the advantage of simple, required less bone marrow, and accorded with the micro-environment of cell growth, is the ideal solution for rat BMSCs culture.2.Different concentrations labeling by using Resovist had none influence on cell proliferation of rat BMSCs, and none impact of specific neural induction.3.The more increased concentration of labeling, the lower signal intensity were detected on susceptibility weighted imaging, and lower signal density still could be demonstrated on susceptibility weighted imaging 7 weeks after transplanted into normal rat brain.4.The location and migration of Resovist labeled rat BMSCs could be detected by in vivo magnetic resonance imaging after transplanted into the ischemic rat brain as long as 4 weeks.
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