The authors acknowledge Alfreda and Kung Tak Chung for their excellent technical support during the animal experiments.

All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and regulations of the University of Hong Kong, and the protocol was approved by the Committee on the Use of Live Animals in Teaching and Research (CULTAR) at the University of Hong Kong.
NOTE: Female farm pigs weighing 35-40 kg (9-12 months old) were used for this study. The flowchart of this experiment is shown in <strong class="xfig"...

<ol>
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Standard animal models are of paramount importance to understand the pathophysiology and mechanisms of diseases and test novel therapeutics. Our protocol establishes a porcine model of HF induced by left circumflex artery blockage and rapid pacing. Eight weeks after the induction of MI, the animals developed significant impairment of LVEF, LVEDD, LVESD, +dP/dt, and ESPVR. This protocol also tests the administration method of stem cell therapy for heart regeneration by intramyocardial injection. The infarct size, and card...

Cardiovascular diseases, coronary artery disease (CAD) in particular, remain the major cause of morbidity and mortality in Hong Kong and worldwide1. In Hong Kong, a 26% increase from 2012 to 2017 of the number of CAD patients treated under the Hospital Authority was projected2. Among all CADs, acute myocardial infarction (MI) is a leading cause of death and subsequent complications, such as heart failure (HF). These contribute to significant medical, social, and financial burdens. In patients with MI, thrombolytic therapy or primary percutaneous coronary intervention (PCI) is an effective therapy in preserving life, but these therapies can only reduce cardiomyocyte (CM) loss during MI. The treatments available are unable to replenish the permanent loss of CMs, which leads to cardiac fibrosis, myocardial remodeling, cardiac arrhythmia, and eventually heart failure. The mortality rate at 1-year post-MI is around 7% with more than 20% patients developing HF3. In end-stage HF patients, heart transplantation is the only available effective therapy, but it is limited by a shortage of available organs. Novel therapies are necessary to reverse the development of post-MI HF. As a result, cell-based therapy is considered an attractive approach to repair the impaired CMs and ameliorate left ventricular (LV) function in HF following MI. Our previous studies found stem cell transplantation to be beneficial for heart function improvement after direct intramyocardial transplantation in small animal models of MI4,5. Standardized preclinical large animal HF protocols are thus needed to further test the efficacy and safety of stem cell transplantation before clinical use.
Recent decades have witnessed the widespread use of pigs in cardiovascular research for stem cell therapy. HF pigs are a promising model of translational research due to their similarity to humans in terms of cardiac size, weight, rhythm, function, and coronary artery anatomy. Moreover, porcine HF models can mimic post-MI HF patients in terms of CM metabolism, electrophysiological properties, and neuroendocrine changes under ischemic conditions6. The protocol presented here uses such a standardized pig HF model, employing a closed-chest coronary balloon occlusion of the left circumflex artery (LCX) followed by rapid pacing induced by pacemaker implantation. The study also optimizes the route of intramyocardial administration of stem cells for the treatment of post-MI HF. The purpose is to produce a porcine animal model of chronic myocardial infarction that can be used to develop treatments that are clinically relevant for patients with severe CAD.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amiodarone</td>
			<td>Mylan</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Anaesthetic machines and respirator</td>
			<td>Drager</td>
			<td>Fabius plus XL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Angiocath</td>
			<td>Becton Dickinson</td>
			<td>381147</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Anti-human nuclear antigen</td>
			<td>abcam</td>
			<td>ab19118</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Axio Plus image capturing system</td>
			<td>Zeiss</td>
			<td>Axioskop 2 PLUS</td>
			<td>Axioskop 2 plus</td>
		</tr>
		<tr>
			<td>AxioVision Rel. 4.5 software</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Baytril</td>
			<td>Bayer</td>
			<td>-</td>
			<td>enrofloxacin</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Mundipharma</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>CardioLab Electrophysiology Recording Systems</td>
			<td>GE Healthcare</td>
			<td>G220f</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Culture media</td>
			<td>MesenCult</td>
			<td>05420</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cyclosporine</td>
			<td>Novartis</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Defibrillator</td>
			<td>GE Healthcare</td>
			<td>CardioServ</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dorminal</td>
			<td>TEVA</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Echocardiographic system</td>
			<td>GE Vingmed</td>
			<td>Vivid i</td>
			<td>-</td>
		</tr>
		<tr>
			<td>EchoPac software</td>
			<td>GE Vingmed</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Electrophysiological catheter</td>
			<td>Cordis Corp</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Embozene Microsphere</td>
			<td>Boston Scientific</td>
			<td>17020-S1</td>
			<td>700 &#956;m</td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>Vet Care</td>
			<td>VCPET70PCW</td>
			<td>Size 7</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR chemicals</td>
			<td>20821.33</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Sigma</td>
			<td>HT501320</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>IVC balloon Dilatation Catheter</td>
			<td>Boston Scientific</td>
			<td>3917112041</td>
			<td>Mustang</td>
		</tr>
		<tr>
			<td>JR4 guiding catheter</td>
			<td>Cordis Corp</td>
			<td>67208200</td>
			<td>6F</td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Quala</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Mersilk</td>
			<td>Ethicon</td>
			<td>W584</td>
			<td>2-0</td>
		</tr>
		<tr>
			<td>Metoprolol succinate</td>
			<td>Wockhardt</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>RM2125RT</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Mobile C arm fluoroscopy equipment</td>
			<td>GE Healthcare</td>
			<td>OEC 9900 Elite</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pacemaker</td>
			<td>St Jude Medical</td>
			<td>PM1272</td>
			<td>Assurity MRI pacemaker</td>
		</tr>
		<tr>
			<td>Pacemaker generator</td>
			<td>St Jude Medical</td>
			<td>Merlln model 3330</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pressure-volume catheter</td>
			<td>CD Leycom</td>
			<td>CA-71103-PL</td>
			<td>7F</td>
		</tr>
		<tr>
			<td>Pressure&#8211;volume signal processor</td>
			<td>CD Leycom</td>
			<td>SIGMA-M</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Programmable Stimulator</td>
			<td>Medtronic Inc</td>
			<td>5328</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PTCA Dilatation balloon Catheter</td>
			<td>Boston Scientific</td>
			<td>H7493919120250</td>
			<td>MAVERICK over the wire</td>
		</tr>
		<tr>
			<td>Ramipril</td>
			<td>TEVA</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sheath introducer</td>
			<td>Cordis Corp</td>
			<td>504608X</td>
			<td>8F, 9F, 12F</td>
		</tr>
		<tr>
			<td>Steroid</td>
			<td>Versus Arthritis</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Temgesic</td>
			<td>Nindivior</td>
			<td>-</td>
			<td>buprenorphine</td>
		</tr>
		<tr>
			<td>Venous indwelling needle</td>
			<td>TERUMO</td>
			<td>SR+OX2225C</td>
			<td>22G</td>
		</tr>
		<tr>
			<td>Vicryl</td>
			<td>Ethicon</td>
			<td>VCP320H</td>
			<td>2-0</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Alfasan International B.V.</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac New Zealand Limited</td>
			<td>-</td>
			<td>tiletamine+zolezepam</td>
		</tr>
	</tbody>
</table>

establishing a swine model of post-myocardial infarction heart failure for stem cell treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

Mortality 
A total of 24 pigs were used in this study. Three of them died during MI induction because of sustained VT. One animal died in the open-heart surgery for cell injection because of wound bleeding. Two animals died because of severe infection. Two animals were excluded because of slight EF reduction (LVEF reduction &#62; 40% of baseline). As a result, 16 animals completed the whole study protocol.
...

Watch this Scientific Journal Video about Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment at JoVE.com

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

This swine heart failure model, induced by left circumflex artery blockage and rapid pacing, can be used to assess the effects of direct intramyocardial stem cell injection on heart regeneration. Our swine colony heart failure model is quite stable. The new infarct size of this model is around 16%of left ventricle and left ventricle injection infarction reduced by at least 40%from baseline.With the experimental pig fixed in the supine position, locate the right carotid artery and juggler vein in the carotid triangle, and use hemostatic forceps and aseptic technique to isolate the right carotid artery and jugular vein. Ligate the distal end of the right carotid artery and juggler vein and use an angiocath to cannulate the right juggler vein. After inserting a pacemaker lead into the right ventricle under x-ray guidance use forceps to isolate the sternocleidomastoid and anterior scalene muscles and connect the pacemaker to the lead.Then implant a pacemaker between the two muscles. Then close the two muscles with 2-0 silk. To assess changes in left ventricle function, isolate the right femoral artery and femoral vein in the femoral triangle, and calculate the right femoral artery with an angiocath.Place a guidewire into the femoral vein via the angiocath and remove the angiocath. Cannulate a 12 French sheath into the femoral vein under the guidance of the guidewire and remove the guidewire. Cannulate the right femoral artery with a nine French sheath as just demonstrated.And insert a balloon catheter through the 12 French sheath into the interior vena cava under x-ray guidance. Calibrate a seven French pressure volume catheter in isotonic saline with a pressure volume signal processor and insert the catheter into the left ventricle apex through the nine French sheath under x-ray guidance. Suspend the ventilation and use the pressure volume signal processor to measure the left ventricle maximal and negative pressure derivative, and systolic pressure and end diastolic pressures.Then restart the ventilation. For myocardial infarction induction, administer five milligrams per kilogram amiodarone and 1.5 milligrams per kilogram lidocaine bolus intravenously over one hour to prevent ventricular arrhythmias before calculating the right carotid artery with an eight French sheath as demonstrated. Perform the coronary angiography through a six French sheath over-the-wire guiding catheter via the placed sheath guided by standard C arm fluoroscopy.Occlude to the left circumflex coronary artery distal to the first obtuse marginal branch with percutaneous transluminal coronary angioplasty dilation balloon catheter inflation under x-ray guidance. Load one milliliter of 700 micrometers sponge microspheres mixed with seven milliliters of iohexol in a 10 milliliter syringe and inject the mixture through the balloon catheter to block the left circumflex coronary artery. Then deflate the balloon and perform an angiogram to confirm the occlusion.Eight weeks after infarction induction load two times 10 to the eighth human-induced pluripotent stem cell-derived mesenchymal stem cells in two milliliters of normal saline into a five milliliter syringe and sterilize 10 centimeters around the apex beat area of the anesthetized experimental animal. Use a retractor to perform a left thoracotomy at the four or five intercostal space and perform a pericardiotomy to expose the infarcted lateral wall. When the infarcted areas visible, perform five to eight intra myocardial injections of approximately 300 microliters of cells per injection before closing the intercostal space with iron wire and closing the muscle layer with 2-0 silk sutures and the subcutaneous tissues and skin layers with 2-0 vicryl sutures.To assess the inducibility of ventricular tachyarrhythmia after cell therapy, insert a six French electrophysiological catheter into the right ventricular apex via the femoral vein and display the intracardiac recordings with the surface electrocardiogram leas I, II, and III on the electrophysiological recording system at a speed of 200 millimeters per second. Use a stimulator to deliver a two millisecond pulse width at two times the diastolic threshold and deliver a pacing train of eight stimuli at one 200 millisecond and one 300 millisecond drive cycle length followed by one or two premature extra stimuli. Then sequentially shortened the coupling intervals until the ventricular effective refractory period of arrhythmia is induced, noting the presence of an inducible sustained ventricular tachyarrhythmia.In this representative experiment, serial echocardiographic examination showed that the left ventricle ejection fraction significantly decreased from baseline while the left ventricle end-diastolic dimension and end systolic dimension significantly increased at eight weeks after myocardial infarction induction. In contrast, the left ventricle ejection fraction significantly increased and the end systolic dimension remarkably decreased in the cell transplantation group eight weeks after administration. The negative pressure derivative and end systolic pressure volume relationship significantly decreased from baseline at eight weeks after induction of myocardial infarction while intramyocardial administration of mesenchymal stem cells resulted in an increase in these functions.Measurement of the left ventricle infarct wall thickness in five to seven serial one centimeter thick sections from each animal revealed a percentage of left ventricle infarction of approximately 16%There was no cell survival around the injection site in the infarct area eight weeks after transplantation. But a small number of the surviving injected stem cells were visible in the periinfarct area. The incidents of inducible sustained ventricular tachyarrhythmias could be easily increased in animals with heart failure.Of note, stem cell transplantation did not significantly modify the underlying myocardial substrate to reduce susceptibility to ventricular tachyarrhythmia. As left circumflex coronary artery blockage may induce arrhythmia. Be sure to monitor the ECG and to immediately use external biphasic defibrillator to re-establish sinuses as necessary.This method provide a stable and reproducible clinically relevant large animal model of heart failure for testing the efficacy of stem cell-based therapies.

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Research

JoVE Journal

Medicine

6.5K visualizações.  University of Hong Kong. Este modelo de insuficiência cardíaca suína, induzido pelo bloqueio da artéria circunflexo esquerda e ritmo rápido, pode ser usado para avaliar os efeitos da injeção direta de células-tronco intramiocárdia na regeneração cardíaca. Nosso modelo de insuficiência cardíaca da colônia suína é estável. O novo tamanho do infarto deste modelo é de cerca de 16% do ventrículo esquerdo e o infarto de injeção de ventrículo esquerdo reduzido em pelo menos 40% da linha de base.