Pulmonary Blood-less First Animal Model Of Heart Disease In Young Pigs To Establish The Pulmonary Blood To Change The Impact Of Vascular Morphology Of Immature Lung Development And Function

Part One: A Novel Hybrid Method Creating Porcine Model of Cyanotic Congenital Heart Defect with Decreased Pulmonary Blood FlowObjective. To create an experimental model of cyanotic congenital heart defect as well as the condition of decreased pulmonary blood flow with a novel hybrid method.Methods. An model of congenital heart defect with decreased pulmonary blood flow and chronically developed cyanosis was surgically produced in 8 piglets (1-2 months old) by creation of an artificial atrial septal defect through a incision of the right, third intercostal space and surgical pulmonary artery banding through a incision of the left, third intercostal space (cyanosis group). Another 8 piglets underwent a sham operation that did not include atrioseptestomy and pulmonary artery banding but did include 30 minutes of side clamp of the main pulmonary artery (PA) through a left anterolateral thoracotomy (control group). At a mean duration of 2 months following a hybrid procedure, the chest of these piglets was then reopened via a median sternotomy. Right ventricular and pulmonary artery pressures were recorded by direct puncture of a pressure-monitoring catheter. Blood samples were drawn for hematocrit, PaO2 and SaO2 determination preoperatively and 2 months postoperatively. The blood gas samples were drawn from four positions of the aortic, the right atrium, the pulmonary vein and the pulmonary artery for calculating the Qp / Qs.Results. At a mean duration of 2 months postoperatively, there were 6 long-term survivors in cyanosis group whose body weight increased to 20.6 kg. the resting gradient across the pulmonary artery band was 53.7mmHg. Qp/Qs reached 0.54:1. The arterial oxygen tension (PO2), arterial oxygen saturation (SaO₂), hematocrit value (HCT) and hemoglobin concentration(HB) were52.9 mmHg , 85.6%, 49.8%and 16.6g/dl in cyanosis group versus 118.0 mmHg, 98.0%, 37.9% and 12.2 g/dl in control group respectively (all P<0.001).Conclusions. A porcine model of cyanotic congenital heart defect with decreased pulmonary blood flow was established by a hybrid method. Application of this experimental design may enhance our understanding and possibly influence the treatment of patients having cyanotic heart disease with decreased pulmonary blood flow. Part Two: The Pulmonary Pathology of The Porcine Model of Cyanotic Congenital Heart Defect with Decreased Pulmonary Blood FlowObjective. Using quantitative morphometric techniques, we analysed the developmental characteristics of pulmonary arterial and alveolar with a porcine model of cyanotic congenital heart defect with decreased pulmonary blood flow that we had established.Methods. Lung biopsy specimens were taken from animals of control and cyanosis groups when the chests of animals were reopened at the time of 2 months postoperatively. Lung biopsy specimens were than made sections and stained with hematoxylin-eosin and elastic-van Gieson stains. The morphological characteristics of the lungs were observed with light microscope and transmission electron microscope. The method of half-quantitative morphometric technique and an image analyzer were applied to measure the following index of pulmonary microvessels: the media thickness (MT) and the percentage of media thickness (MT%), the media section area (MS) and the percentage of media section area(MS%) , the numbers of micro-arteries per square centimeter (APSC). In addition, we calculated the ratio of CMA, PMA and NMA in the same section of lung biopsy specimens.Results. A model of congenital heart defect with decreased pulmonary blood flow and chronically developed cyanosis was surgically produced in 6 piglets in D-group at a mean duration of 2 months postoperatively. There showed universally distended, irregular and thin-walled vessels in the lung biopsy specimens of group D under the light microscopy. There was an increased number of pulmonary arteries whose media were hypoplastic. The MT,MT% and MS,MS% in group D were significantly less than those of group C(all P<0.001) . the APSC was also less in group D than in group C(p< 0.01), but MAN were not significantly different between two groups(P>0.05). Electron microscopy revealed endothelial swell and irregularity, a thinned basement membrane, a disorganized or disrupted elastic fibers layer. The smooth muscle of vascular media shrinked. In addition to these, other changes included the mitochondrion vacuolization, a diminished cellular organelle and type II alveolar epithelial hyperplasia.Conclusion. The pulmonary vessels were evidently hypoplastic or degenerated in the porcine model of cyanotic congenital heart defect with decreased pulmonary blood flow, and the function was thought to be influenced by the morphological changes of the lung. To promote the pulmonary artery growth and improve this pathophysiological state, it is critical to increase and keep normal the pulmonary blood flow. Part three: Biological Changes of Pulmonary Vessels in Porcine Model of Congenital Heart Defect with Decreased Pulmonary Blood FlowObjective. There was significant structural remodelling of pulmonary extracellular matrix (ECM) in ischemic porcine lung. In this study we aimed to investigate the biological mechanisms of morphological change and ECM remodeling in the state of pulmonary ischemia through measuring the contents of structural proteins and cytokines in the porcine lungs.Methods. Sixteen healthy piglets were divided into two groups: cyanosis group (group D, n=8) and control group (group C, n=8). Cyanosis group were used to create the model of congenital heart defect with decreased pulmonary blood flow for the first step. At a mean duration of 2 months after the creation of animal model, the animals of two groups were anesthetized, lung biopsy were taken from bilateral lungs of both groups. Lung biopsy tissues were cryopreserved with liquid nitrogen. The method of enzyme linked immunosorbent assay (Elisa) was applied to measure the following biochemical indices of pulmonary parenchyma and vessels of two groups: the content of Matrix metalloproteinase-2 (MMP-2), Matrix metalloproteinase-9(MMP-9) and their tissue inhibitor of metalloproteinase-1 (TIMP-1), the content of type I and III collagens ( Col and CoIII) as well as the content of vascular endothelial growth factor (VEGF). Results. A model of congenital heart defect with decreased pulmonary blood flow and chronically developed cyanosis was surgically produced in 6 piglets in group D at a mean duration of 2 months postoperatively. The content of MMP-2 in group D and group C were 56.6±20.3 ng/ml and 77.6±20.7 ng/ml respectively, It was significantly different between two groups (P < 0.01); The content of MMP-9 in group D and group C were 4.7±1.0 ng/ml and 5.8±1.0 ng/ml respectively, there was a statistically difference between them (P < 0.01); The content of TIMP-1 in group D and group C were 14.5±3.4ng/ml and 20.2±3.8ng/ml (P < 0.001); The content of type I collagens in group D and group C were 814.6±2006.8 ng/ml and 3122.1±2865.1 ng/ml (P < 0.01); The content of type III collagens in group D and group C were 12.7±4.7 ng/ml and 8.2±4.2 ng/ml (P < 0.01); The content of VEGF in group D and group C were 13.6±3.0 ng/ml and 18.8±3.5ng/ml (P < 0.001).Conclusions. Structural remodelling of pulmonary extracellular matrix is an important feature of pulmonary ischemia which we have observed in the porcine model of cyanotic congenital heart defect with decreased pulmonary blood flow. It is just as the mechanism of flow-induced pulmonary hypertension, varies of modulating factors will be activated when the internal environment changes under the condition of pulmonary ischemia. It is inferred that the underlying mechanism of remodeling is that these modulating factors promote the convert of structural proteins and cytokines in the pulmonary extracellular matrix.