| In this study, bone marrow mesenchymal stem cells (MSCs) were labeled by superparamagnetic iron oxide(SPIO), and transplanted into the rat model of myocardial infarction. The aim was to explore the feasibility of in vivo tracking the long-term fate of SPIO-labeled MSCs using a7.0T superconducting small animal magnetic resonance imaging. Overall, The study was composed of the following two parts.Part1Development of the rat model of myocardial infarction and cine MR imaging using an ultra-high field strength (7.0T) small animalmagnetic resonance scanner[Objective]To establish an optimized method of coronary ligation to develop the rat model of acute myocardial infarction and investigate the feasibility of evaluate the cardiac function of rat model of myocardial infarction with cine sequence using an ultra-high field strength (7.0T) small animal magnetic resonance imaging system.[Methods]Myocardial infarction was experimentally induced in15adult female Lewis rats by permanent ligation of the left anterior descending coronary artery. In vivo serial cine MRI was performed to assess the cardiac function using a7.0T horizontal-bore animal scanner at24hours before the surgery and2weeks after MI, respectively. After the last MR imaging, the animals were sacrificed for the histopathological test to confirm the myocardial necrosis and fibrosis by HE staining.[Results] The MI incidence and the mortality by permanent ligation of the left anterior descending coronary artery was66.7%and33.3%, respectively. After obstruction of the left anterior descending artery, the myocardium below the ligation and apex became pale or bruises. Cine MR imaging was carried out before MI and two weeks after MI, which showed that the anterior wall of left ventricle became thinner, the motion of the anterior and apical wall decreased or disappeared, and aneurysm appeared after myocardial infarction. The heart function decreased significantly at2weeks after MI than before MI (LVEF:56.45±7.46vs79.84±9.34, P<0.05; EDV:3.43±0.39vs3.02±0.32, P<0.05; ESV:1.54±0.33vs1.29±0.23, P<0.05). The myocardium mass also decreased (3.25±0.43vs3.45±0.33, P>0.05), but there was no statistical diffrences. HE staining confirmed the myocardial infarction after ligation of coronary.[Conclusion]Ligation of the left anterior descending artery is a reliable method of developing a rat model of MI, which can meet the experimental requirements. The changes of regional myocardium can be used as preliminary indicators of myocardial infarction. Cine MR imaging can be used for the assessment of cardiac functional parameters in rat models of myocardial infarction. Part2Magnetic Resonance Imaging with SPIO to Track the Long-term Fate of Mesenchymal Stem Cells After Transplantation in the Heart[Objective]To evaluate the outcomes of allogeneic bone marrow mesenchymal stem cells (MSCs) transplantation in rat models of myocardial infarction, and to test the feasibility and accuracy of tracking the long-term fate of the SPIO labelled MSCs following intramyocardially injection in the experimental acute myocardial infarction in rats using a7.0T magnetic resonance imaging.[Methods]According to the different adherent characteristics, MSCs were isolated and cultured from bone marrow of male Lewis rats, and doubly labeled with SPIO and4,-6-diamidino-2-phenylindole dihydrochloride (DAPI). Myocardial infarction was induced by left coronary artery ligation in female Lewis rats. MRI was performed to screening the qualified animals2weeks after myocardial infarction (baseline), which were allocated randomizedly to2groups. MSCs (1x106) were injected into the peri-infarct region as MSCs transplantation group, while the animals in control group only received equal volume of phosphate buffered saline. In vivo serial MRI was performed using a7.0T horizontal-bore animal scanner at3days,1,2, and4weeks after cell delivery, respectively. Electrocardiography-gated T2*WI gradient echo sequence and cine MRI were performed for in vivo cell tracking and assessing cardiac function. After each MRI scan,5rats from each group were sacrificed, The survival, migration and apoptosis of grafted MSCs were assessed by polymerase chain reaction analysis for the rat Y-chromosome-specific SRY gene, histopathological examination and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively.[Results]Serial follow-up MRI demonstrated large persistent intramyocardial signal-voids as large black spots representing SPIO during the follow-up of4weeks, and MSCs moderate the left ventricular dysfunction compared with controls and baseline at3days,1and2weeks after cell transplantation (LVEF:53.27±9.44vs51.42±7.41, P>0.05;55.57±12.52vs51.42±7.41, P>0.05;54.43±10.99vs51.42±7.41, P>0.05;53.27±9.44vs49.67±7.03, P>0.05;55.57±12.52vs47.66±6.76, P>0.05;54.43±10.99vs48.30±8.61, P>0.05, and ESV:1.37±0.57vs1.46±0.33, P>0.05;1.29±0.66vs1.46±0.33, P>0.05;1.33±0.58vs1.46±0.33, P>0.05;1.37±0.57vsl.51±0.38, P>0.05;1.29±0.66vs1.39±0.11, P>0.05;1.33±0.58vs1.49±0.30, P>0.05), respectively. But there were no statistical differences. The signal intensity of the spots increased, the contrast of the spots decreased and the size of the spots diminished during the follow-up. The signal intensity (257.05±56.43vs227.56±87.62), the contrast (79.74±4.41vs85.27±6.04)and the size (4.98±0.22vs5.34±0.45) of the spots showed no statistical difference between3days and1week after injection. The signal intensity increased statistically at2w and4w than3d after cell transplantation (554.76±95.25vs227.56±87.62, P<0.05;798.74±121.89vs227.56±87.62, P<0.05). Also, the contrast (67.14±2.67vs85.27±6.04,P<0.05;59.73±3.58vs85.27±6.04,P<0.05) and the size (2.43±0.34vs5.34±0.45,P<0.05;1.62±0.23vs5.34±0.45,P<0.05) of the spots decreased rapidly at2w and4w than3d after cell transplantation. The TUNEL analysis confirmed that MSCs engrafted in infarcted hearts underwent apoptosis. The presence of engrafted cells was confirmed by real-time polymerase chain reaction on postmortem specimens, which showed that the expression of Y-chromosome-specific SRY gene of MSCs from male donors in infarcted hearts of female recipients was consistent with the results of the the TUNEL assessment. The histopathological studies revealed that the site of cell injection was infiltrated by inflammatory cells and the iron-positive cells were macrophages identified by CD68staining, but very few or no DAPI-positive stem cells in the animals at4weeks after cells transplantation.[Conclusion]MRI enables in vivo evaluation of the long-term therapeutic potential of MSCs for myocardial infarction, while does not reliably track the long-term fate of SPIO-labeled MSCs engraftment in the heart. |
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