| PART ONE:Optimization of open-chest myocardial infarction model of swineObjectiveDifferent occlusion sites of left anterior descending artery(LAD) have been used for establishing model of myocardial infarction(MI) in pigs.However,little is known about the correlation between the LAD ligating positions and the quality of MI model. The aims of this study are:①to compare the characteristics,stability in particular,of acute myocardial infarction swine model by two different procedures:ligation just below D2 branch,or ligation at one-third distal section of LAD.And to explore the correlation among site of LAD ligation,infarct size,and left ventricular function.②to explore the characteristics and risk factors of ventricular fibrillation(Vf) in open-chest acute myocardial infarction models of swine.Methods①Forty-seven miniswines were divided into group A(ligation just below D2 branch,n=24) and group B(ligation at one-third distal section of LAD,n=23).Left ventricular ejection fraction(LVEF) was measured by echocardiography before and after MI.Coronary angiography was used for confirming the ligation position after MI and the histological ischemic area was examined at 4 weeks after MI.The variations of each parameter were compared between the two groups.Linear regression analysis was used to analyze the correlation between LAD ligation position and infarct size as well as LVEF.②Forty-seven miniswines mentioned before,along with another 10 miniswines underwent same surgery were divided into Vf group and Vf-free group based on the occurrence of Vf during procedure.The weight,gender,heart rate(HR) before and after MI,LVEF,thoracotomy route(intercostal or parasternal),operation duration, percentage point of LAD ligation,arrhythmia after MI were recorded and compared between Vf group and Vf-free group.The risk factors of Vf were determined by logistic regression analysis.ResultsThirty-nine pigs survived the operation.Ischemic area was significantly smaller, post MI LVEF was significantly higher and the variations of these parameters were significantly smaller in group B(n=20) than that in group A(n=19).The difficulty in identifying ventricular branches,along with great variation of branching pattern, encumbers the precise ligation in group A.Eighteen of the 57 pigs suffered from ventricular fibrillation within 35 min after LAD ligation and 11 cases were died.Compared with Vf-free group,Vf group featured a higher position of LAD ligation,a higher incidence of ventricular tachycardia and maximal HR＞160 bpm post MI.Logistic regression analysis showed the position of LAD ligation was the sole risk factors of Vf after MI with statistical significance.Pigs with HR＞160 bpm or＜60 bpm post MI was susceptible to Vf.Conclusion①Ligating LAD with its whole length rather than ventricular branch as coordinate may be more practical and advisable in establishing reproducible MI models,allowing for standardization the location of occlusion and infarct size.②The position of LAD ligation is the vital determinant of Vf in establishing acute MI model of swine.Attentions should be paid to the first 30 min after LAD ligation, HR,and ventricular tachycardia.PART TWO:Labeling of swine bone marrow mesenchymal stem cells with Resovist and MR imaging In vitroObjectiveTo label swine bone marrow mesenchymal stem cells(MSCs) with Resovist combined with poly-1-lysine(PLL),and to determine the characteristics of magnetically-labeled stem cells with MR imaging in vitro.MethodsSwine mesenchymal stem cells were isolated,culture-expanded 3 passages,and identified by Fluorescence-activated Cell Sorting(FACS) in vitro.MSCs were magnetically labeled with different concentration of Resovist in culture medium for different incubation duration,so as to determine the appropriate labeling methods according to both cellular iron incorporation and cell toxity.Accumulation of particles of SPIO in MSC was assessed by Prussian Blue staining,atomic absorption spectrometry and electron microscopy.Cell toxity were determined by trypan-blue exclusion test,CCK-8 test and electron microscopy.Magnetically-labeled stem cells suspensions underwent MR imaging with T1-weighted spin-echo,T2-weighted fast field-echo and T2-weighted flash sequences at a clinical 1.5 T MR system to determine the characteristics of MR imaging in vitro.The attenuation of Resovist within the labeled cells was assessed by Prussian Blue staining,atomic absorption spectrometry and MR imaging.ResultsLabeling MSCs with 25μg/ml or 50μg/ml of Resovist in culture medium for 24 hours is appropriate.Intracytoplasmic particles stained with Prussian blue stain were detected for all cells with mean cellular iron content of(13.13±2.30) pg per cell for 25μg/ml Resovist in medium,and(23.77±2.06) pg per cell for 50μg/ml Resovist in medium.In vitro MR images showed signal intensity changed most obviouly in T2-weighted flash sequence.The percentage change of signal intensity increased with increasing cell numbers.As few as 5×104-1×105 cells could be detected using this approach.With division of stem cells,the plasma iron decreased gradually,and approached the prelabeling baseline level 12 to 16 days after labeling.ConclusionSwine bone marrow mesenchymal stem cells can be successfully and efficiently labeled with Resovist and can be depicted with a standard 1.5-T MR imager in vitro. The incorporated Resovist particles could retain within cells for 2 weeks.PART THREE:In vivo cardiac magnetic resonance Imaging of Resovist-labeled mesenchymal stem cells in swinesObjectiveTo explore the feasibility and value of magnetic resonance imaging(MRI) of transplanted Resovist-labeled MSCs in swine heart with acute MI.MethodsMSCs were isolated from miniswines.After 24-hour incubation with nanometer Resovist,the third-passage MSCs was further labeled with DNA dye 4'-6-diamidino-2-phenylindole(DAPI) and aliphatic red fluorescent dye PKH26-GL. Approximately 1 hour following the ligation of left anterior descending coronary artery in 20 swines,SPIO-labeled MSCs(5×105 per 150μl injection,n=3;1×106 per 150μl injection,n=3;and 2×106 per 150μl injection,n=3),MSCs without SPIO (1×106 per 150μl injection,n=3),and SPIO without MSCs(2μl Resovist per 150μl injection,n=3) were transextracardially implanted into the freshly infarcted myocardium.Each swine received 3 injections.All 20 miniswines were performed MR imaging with T1-weighted spin-echo,T2-weighted fast field-echo,T2-weighted flash,T2-MAP,and T2-weighted flash sequence after the intravenous injection of magnevist at a clinical 1.5 T MR system 20 to 24 hours post infarction.All 20 miniswines subsequently sacrificed for histology just atl hour post MRI.ResultsNo areas of low signal were identified in the SPIO-free cell transplanted hearts.All injected sites containing SPIO-MSCs,as well as SPIO without MSCs were detected in vivo in a clinical 1.5T scanner,presenting low signal intensity spots with the scanning T2-weighted flash sequence,while obscure or even invisible with T1-weighted spin-echo and T2-weighted fast field-echo sequences.The quality of MR image with T2-MAP or T2-weighted flash sequence after the intravenous injection of magnevist was superior to T2-weighted flash.The area of low signal with T2-MAP is linearly correlated to echo time and cell number transplanted.As few as 5×105 cells could be detected in a clinical 1.5T scanner.Prussian blue positive,as well as DAPI and PKH26 fluorescently labeled cells were detected on histology in sections corresponding to lesions shown on MRI in animals transplanted with SPIO-labeled MSCs.ConclusionMagnetically labeled MSCs transplanted in swine myocardial ischemia can be visualized in vivo with MRI at clinical 1.5-T field strength,suggesting the feasibility of tracking SPIO-MSC clinically.PART FOUR:Dose-effect relationship of magnetically-labeled mesenchymal stem cells in swines with acute myocardial infarction: MRI assessmentObjectiveThere is a paucity of information correlating the number of transplanted stem cell after myocardial infarction(MI) with the beneficial effect on infracted heart. Therefore,we assessed the dose-effect relationship of transplanted magnetically-labeled mesenchymal stem cells(MSCs) in swines with acute MI using magnetic resonance imaging(MRI).MethodsAfter 24-hour incubation with nanometer Resovist and poly-1-lysine(PLL),MSCs were transextracardially implanted into the freshly infarcted myocardium approximately 1 hour following the ligation of LAD in 15 swines.Swines were randomized into 3 groups according to the number of MSCs:low dose group(n=5), 5×106;medium dose group(n=5),1×106;high dose group(n=5),2×107.Another 5 swines were received DMEM medium only as control group.The dynamic changes of transplanted cells,infarct area and ventricular function were monitored by 1.5-T clinical MRI at 24 hours,1,3 and 6 months post infarction.All 20 miniswines subsequently sacrificed for histology at months post infarction.Immunohistochemical staining with antibodies against CD68 was performed to show the distribution of macrophage.The number of blood vessels was identified by the expression of yon Willebrand factor.The apoptosis was assessed by Tunel immunohistochemical staining.HE staining,Mallory staining and Prussian Blue staining were also performed.ResultsThe improvement of LVEF was obviously related to the number of transplanted MSCs 3 months after transplantation[0.12%in control group,1.78%in low dose group,3.72%in medium dose group(P＜0.05,compared with control),6.68%in high dose group(P＜0.05,compared with control),respectively],as well as 6 months after transplantation[-1.08%in control group,1.82%in low dose group,4.10%in medium dose group(P＜0.05,compared with control),7.24%in high dose group(P＜0.05, compared with control),respectively].The changes of infarct area,the number of blood vessels and the apoptosis index are also related to the number of transplanted MSCs.SPIO-labeled MSCs presenting low signal intensity spots with the scanning T2*WI-Flash2d sequence.The duration of discernable spots and the migration extent of the sports were related to the dose of injected cells.Histology showed the majority of iron particles were extracellularly distributed.ConclusionsLocally injected MSCs in infracted myocardium can improve ventricular function in a dose-dependent manner.The persistence of significant iron-dependent MRI signal derived from extracellular Resovist indicates that MRI of Resovist-labeled cells does not reliably report long-term stem cell engraftment in the heart.
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