The Applied Basic Research Of Bone Marrow Mononuclear Cells Autologous Transplantation Into Infarcted Myocardium In Dogs

Objectives: 1. To establish myocardial infarction (MI) model in dog and noninvasive evaluation the size of MI by myocardial contrast echocardiography (MCE) and contrast-enhanced magnetic resonance imaging (MRI) then to compare with histological standards. 2. To study the role of improvement of post-MI LV function and myocardial blood flow by autologous bone marrow mononuclear cells (BM-MNCs) transplantation in canine models. 3. To explore the method of tracking magnetically labeled BM-MNCs in vivo in a canine model.Methods: 1. To induce a MI, twenty male adult dogs were intubated and ventilated. After left sided thoracotomy, left anterior descending coronary artery were occluded by a permanent ligation. Two weeks later, the size of MI was measured by MCE and enhanced-MRI among four MI model dogs, which was compared with postmortem size of fibrosing scar. 2. Fourteen canine MI models were randomized into three groups. After two weeks post-MI, groupl received 10~7 BM-MNCs via epicardium, group2 received 10~5 BM-MNCs by the same way and group3 as controls was injected identical quantitative phosphate buffered saline (PBS). Four weeks after BM-MNCs or PBS injection, all the animals underwent transthoracic echocardiography and MCE. Finally, the hearts were excised and then calculated microvascular density by HE staining. 3. Feridex is one FDA-approved SPIO nanoparticle and shows hypointense T2 signal on MRI. Autologous BM-MNCs were labeled by Feridex in vitro, and then were injected into MI regions on four models. To assess Feridex labeled BM-MNCs, animals were imaged at one, two and four weeks after cell injection by 1.5T MRI. Scan parameters for ECG gated, free-breathing navigator and three dimensional steady state free precession (3DSSFP ) were optimized to detect the cells. After humane euthanasia, the hearts were excised and sliced along short-axis plane. Histology corresponding to MRI slices was performed.Results: 1. The MI incidence and the mortality was 90% and 10% respectively. The MI size measured by MCE, enhanced-MRI and postmortem analysis was 2. 29±0. 50cm~2,1. 73±0. 72 cm~2 and 1. 70±0. 85 cm~2 respectively. There were significant differences (p<0.05). Viable myocardium around MI was all detected accurately by MCE and enhanced-MRI. 2. After BM-MNCs injection 4 weeks, LVEF (62%, 58% vs 48%) and E/A value (0.78, 0.89 vs 0.42) both improved significantly (p<0.05). The size of MI decreased from2.74cm~2 to 1.91 cm~2 in group 1 and from 2.49cm~2 to 2.03 cm~2 in group2 and there were significant differences (p<0.05). Mi-thickness showed no differences among the three groups (p>0.05). The A,K and A×K values measured by MCE were significantly higher than those in pre-injection of BM-MNCs (p<0.05), but no changes in control group (p>0.05). 3. Prussian blue staining revealed more than 98% of BM-MNCs were labeled by Feridex and BM-MNCs appeared to be unaffected by magnetically labeling. Three dogs injected Feridex- BM-MNCs were visualized at one week by MRI, but none of unlabeled BM-MNCs were seen. All lesions that were detected within one week could not be visualized over four week. The dot injected Feridex-cells which fixated by 4 % methanal also was seen at one week by MRI, but the signal disappeared at two week. DE-MRI confirmed the presence of Feridex-BM-MNCs injection within the infarcted myocardium. 4. A large amount of neovascularization appeared in BM-MNCs injection dogs under light microscope. The mean microvascular density in three groups was 79.8±4.8/mm2, 74.8±6.5/mm~2 and 28.6±5.2/ mm~2 respectively (p<0.05). By Prussian blue staining, differentiation into vascular endothelial cell and cardiocyte-like cells could be found under light microscope and Feridex could be englobed by macrophages.Conclusions: 1. The method of establishing canine MI model by ligating LAD and branches after thoracotomy is reliable and preferred. MCE and enhanced-MRI can accurately detect and assess MI and viable myocardium. 2. Autologous BM-MNCs engrafts via epicardium improve cardiac function and myocardial blood flow of post-MI on canine models. 3. The sites of injection of Feridex-BM-MNCs can be detected by clinical 1.5T MR. Pathological microscopic pictures show that BM-MNCs can differentiate into cardiocyte-like cells and vascular endothelial cell. 4. MCE and MRI technology can be applied to clinical researches about stem cells as noninvasive methods.