Cees Verlaan, Joyce Visser, Merel Schurink, and Grace Croft are kindly acknowledged for their excellent technical assistance with the animal experiments.

All in vivo experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources. Experiments were approved by the local Animal Experimentation Committee.
1. Medication, Anesthesia, Venous Access, and Intubation
<ol>
	<li>Medication and Anesthesia
	<ol>
		<li>Premedication
		<ol>
			<li>Start amiodarone 150 mg/kg 10 days prior to surgery to prevent arrhythmias. Continue amiodarone in a dose of 100 mg/kg from the day of the procedure until day 28. Reduce the dosage to 50 mg/kg on day 29 and continue until the end of the study.</li>
			<li>Start anti-platelet therapy, 1&#160;mg/kg&#160;clopidogrel from day 3 prior to surgery and 4.5&#160;mg/kg acetylsalicylic acid 1 day before surgery. Continue clopidogrel 1&#160;mg/kg per day and acetylsalicylic acid 80 mg/day.</li>
			<li>Start pain medication 1 day prior to surgery by Fentanyl patch, 25 &#956;g/hr for ~70 kg pigs. Continue the use of Fentanyl patches for 24 hr&#160;post-operatively to ensure adequate pain medication. Also, daily monitor for signs of putative discomfort (i.e. behavior, breathing, gait, mobility, etc.) to increase pain medication.</li>
			<li>Fast the animal for 12&#160;hr, maintain unlimited access to water.</li>
		</ol>
		</li>
		<li>Anesthesia
		<ol>
			<li>To prevent unnecessary stress and discomfort, sedate the pig in its stable by intramuscular injection of a mixture of midazolam 0.4 mg/kg, ketamine 10 mg/kg, and atropine 0.014&#160;mg/kg.</li>
			<li>Obtain venous access by cannulating the ear vein with an 18 G i.v. cannula. Induce anesthesia by intravenous administration of 5 mg/kg sodiumthiopental, and give 1,000/100 mg amoxicillin/clavulanic acid to prevent infections. The pig will receive 1,000/100 mg amoxicillin/clavulanic acid the day after surgery as antibiotic treatment.</li>
			<li>Intubate the pig by using an endotracheal tube (size 8.5 for pigs weighing ~70 kg). If necessary perform balloon-ventilation with a frequency of 12/min and transport the pig to the operating theater.</li>
			<li>At arrival on the operation theater, immediately start mechanical positive pressure ventilation with FiO2 0.50, 10 ml/kg tidal volume, and a frequency of 12/min under continues capnography.</li>
			<li>Start balanced anesthesia by continues intravenous infusion of a combination of midazolam 0.5 mg/kg/hr, sufentanil 2.5 microg/kg/hr and pancuronium bromide 0.1 mg/kg/hr.&#160;During the entire operation, continuously monitor ECG, arterial blood pressure, temperature and capnography to measure depth of anesthesia. For example, if a sinus tachycardia is present, check if pain medication and/or anesthesia are adequate.&#160;</li>
			<li>Infuse 4.3&#160;mg/kg of amiodarone in 500 ml venofundin 6% intravenously.</li>
			<li>Monitor heart rhythm by a 5 leads ECG.</li>
			<li>Shave and clean the neck and hind limb area.</li>
			<li>Insert a Foley catheter.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Transthoracic Echocardiography
<ol>
	<li>Place the animal in the right lateral position. In landrace pigs such as Dalland pigs, only parasternal views (long and short axis) can be obtained. Due to the shape of the thorax, apical view acquisition is not feasible.</li>
	<li>Orient and obtain parasternal long axis view in 2D (B-mode). Determine the LV dimensions at end diastole and end systole in M-mode.</li>
	<li>Rotate the echo probe 90&#176; clockwise whilst maintaining its parasternal position to acquire the LV short axis views at the levels of the mitral valve, papillary muscle and apex. The short axis view of the papillary muscle and apex may require placement of the echo probe one or two intercostal spaces lower relative to the position for the mitral valve short axis view.</li>
</ol>
3. Surgical Preparation and Vascular Access
<ol>
	<li>Disinfect the surgical areas with iodine 2% and use sterile surgical drapes to cover the nonsterile parts of the pig.</li>
	<li>Make a medial incision in the neck. Pass the linea alba to minimize muscle damage and bluntly approach the carotid artery and internal jugular vein next to the trachea.</li>
	<li>Carefully isolate the carotid artery and internal jugular vein. Make sure the vagal nerve is undamaged. Place Vicryl 2-0 sutures around both vessels to gain vessel control. Achieve arterial access by cannulating the internal carotid artery with an 8F sheath using the Seldinger technique. Fix the sheath to the artery, make sure the artery is not fully occluded by the suture. Venous access can be acquired by cannulating the jugular vein with a 9F sheath also using the Seldinger technique. Before securing the sheath make sure the vein is ligated. Alternatively, the femoral artery can also be used for arterial access.</li>
	<li>Administer 100 IE/kg heparin immediately after inserting the sheaths to inhibit thrombus formation.</li>
	<li>For a stable and constant arterial pressure measurement, cannulate one of the smaller arteries in one of the hind limbs by making a small incision above the artery. The artery is found just under the skin, pulsations can be felt through the skin. Isolate the artery from its surrounding tissue. Place 2 Vicryl 2-0 sutures around the vessel, 1 proximal and 1 distal. Ligate the distal side and insert an 18 G i.v. cannula and secure tightly connect the pressure.</li>
</ol>
4. Invasive Pressure Volume Loop Analysis
<ol>
	<li>Insert a Swann-Ganz catheter (SG) via the previously placed sheath in the internal jugular vein.</li>
	<li>Connect a cardiac output device to the part of the SG that culminates in the proximal lumen.</li>
	<li>Inject 5 ml of 0.9% saline into the proximal lumen of the SG and measure cardiac output; repeat this three times and average the indices.</li>
	<li>Calibrate the PV system by using the previously determined cardiac output.</li>
	<li>Insert the 7F conductance catheter via the carotid artery into the left ventricle under fluoroscopic guidance.</li>
	<li>Select the largest segment present in the LV for volume measurements and perform a baseline scan under apnea.</li>
	<li>After volume calibration is completed, record 10-15 beats under apnea.</li>
	<li>Remove the SG and place a balloon catheter in the inferior caval vein at the level of the diaphragm under fluoroscopic guidance.</li>
	<li>Perform preload reduction by inflating the balloon under apnea and record the corresponding PV-Loops.</li>
</ol>
5. Intracoronary Pressure and Flow Measurement
<ol>
	<li>Dilute nitroglycerin in a concentration of 100 &#956;g/ml and dilute adenosine in a concentration of 30 &#956;g/ml.</li>
	<li>Position an 8F guiding catheter in the ostium of the left coronary artery.</li>
	<li>Place the combined pressure/flow wire in the proximal part of the left coronary artery.</li>
	<li>Administer 200 &#956;g of nitroglycerin intracoronary and normalize the distal pressure (Pd), measured by the wire, with the arterial pressure.</li>
	<li>Place the wire in the mid part of the left anterior descending artery (LAD).</li>
	<li>Start measuring baseline pressure and flow. Induce hyperemia by administering 60 &#956;g of adenosine intracoronary, flush with 2 ml of saline and measure hyperemic pressure and flow. Wait for the flow to restore to baseline values. Repeat the measurement twice.</li>
	<li>Infuse another 200 &#956;g nitroglycerin intracoronary and repeat steps 1.6 and 1.7 for the left circumflex coronary artery.</li>
</ol>
6. Induction of MI
<ol>
	<li>Place the intracardiac defibrillation catheter in the right ventricle using the venous sheath. The distal electrodes should be in the apex of the ventricle, the proximal electrodes in the atrium and/or superior caval vein. Connect the catheter to the defibrillator and set it to 50 J.</li>
	<li>Measure the diameter of the LAD distal from the second diagonal (D2) in AP and LAO 30&#176; view.</li>
	<li>Choose an angioplasty balloon with a diameter according the diameter of the LAD distal from the D2 (Figure 1).</li>
	<li>Position a guidewire through the guiding catheter distally in the LAD.</li>
	<li>Advance the balloon catheter over the guidewire. Place the balloon distal from the D2.</li>
	<li>Administer 30 IE/kg heparin.</li>
	<li>Inflate the balloon until the pressure matches the right diameter of the LAD.</li>
	<li>Check total occlusion of the LAD by angiography (Figure 1).</li>
	<li>Cover the sterile working field and the wound in the neck with sterile drapes cloths. Free the chest from any coverage to make it available for chest compressions or transthoracic defibrillation.</li>
	<li>Check the pressure in the balloon during the next 90 min and restore the pressure if necessary.</li>
	<li>In case of ventricular fibrillation:
	<ol>
		<li>Immediately start chest compressions with a frequency of 100/min.</li>
		<li>Administer 300 mg amiodarone intravenously as a fast bolus (~1 min).</li>
		<li>Start intracardiac defibrillation, give shocks of 50 J.</li>
		<li>After 5 unsuccessful shocks, restart chest compressions. Change intracardiac defibrillation to transthoracic defibrillation and shock with 150 J. In case of unsuccessful shock, change to 200 J.</li>
		<li>If necessary, administer another dose of 150 mg amiodaron and/or 1 mg adrenalin. Repeat adrenalin twice with intervals of 3-5 min, when necessary.</li>
		<li>Continue chest compressions, interspersed with transthoracic defibrillation</li>
	</ol>
	</li>
</ol>
Finishing the Surgical Procedure (for Long Term Follow Up)
<ol>
	<li>After 90 min check by angiography if the LAD is still fully occluded.</li>
	<li>Administer another 30 IE/kg heparin and deflate the balloon. Check for reperfusion. Remove the deflated balloon with the guiding catheter from the carotid sheath.</li>
	<li>Carefully remove the arterial sheath and clamp the carotid artery immediately with an anastomosis clamp (Figure 1). Use continues stitches (6-0 prolene) to close the carotid artery. Remove the clamp and check for leakages.</li>
	<li>Remove the internal defibrillation catheter and remove the sheath from the internal jugular vein. Ligate proximal of the sheath entry.</li>
	<li>Close the subcutis and skin of the neck in two layers using 2-0 Vicryl.</li>
</ol>
7. Cardiac Magnetic Resonance Imaging
<ol>
	<li>Place the animal on the MRI table head first in the supine position under continuous anesthesia.</li>
	<li>Place a dedicated phased-array cardiac coil over the chest of the animal.</li>
	<li>For image planning obtain scout images in short axis and two-chamber long axis views.</li>
	<li>Acquire ECG-gated steady-state free precession (SSFP) cine of short axis (from apex to base of LV) and two chamber long-axis views.</li>
	<li>Late gadolinium enhancement (LGE) can be acquired using an inversion recovery 3D-turbo-gradient-echo-technique 15 min after double-dose i.v. bolus injection of a gadolinium based contrast agent.</li>
	<li>Perform offline analysis with validated software of functional parameters. Assess left-ventricular ejection fraction (LVEF), LV mass, end diastolic volume, end systolic volume, stroke volume, cardiac output, and scar mass.</li>
</ol>
8. End of Study and Infarct Size
<ol>
	<li>At the end of the study, follow Protocols 1-5 and 7 to acquire follow up measurements.</li>
	<li>Make a median 30-40 cm incision from just below the suprasternal notch to a point just below the xiphoid process. Advance through the linea alba down to the sternum. Split the xiphoid and use Klinkenberg scissors to separate the posterior sternum from the pericardium with caution. After using the scissors bluntly continue further separation. Perform a sternotomy by e.g. using a hammer and Lebsch knife. Bone marrow bleeding is minimized by rubbing bone wax on the marrow. Open the thorax with a sternum retractor.</li>
	<li>Enter the 3rd pleural space and locate the inferior caval vein in the mediastinum.</li>
	<li>Humanely euthanize the animal by cutting the inferior caval vein under deep anesthesia. Remove blood with a suction device. Place a 9 V battery on the apex to induce ventricular fibrillation.</li>
	<li>After excision of the heart, cut the right and left ventricle into five slices from base to apex and incubated in 1% triphenyl-tetrazolium chloride dissolved in 0.9% saline at 37 &#176;C for 15 min. Next, wash the slices in 0.9% saline and photograph the slices from both sides.</li>
</ol>

<ol>
	<li>Roger, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics-2012+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2012 update: a report from the American Heart Association.</a> Circulation. 125 (1), (2012).</li><li>Mosterd, A., Hoes, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+epidemiology+of+heart+failure.">Clinical epidemiology of heart failure.</a> Heart. 93 (9), 1137-1146 (1136).</li><li>vander Spoel, T. I. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+relevance+of+pre-clinical+studies+in+stem+cell+therapy:+systematic+review+and+meta-analysis+of+large+animal+models+of+ischaemic+heart+disease.">Human relevance of pre-clinical studies in stem cell therapy: systematic review and meta-analysis of large animal models of ischaemic heart disease.</a> Cardiovasc. Res. 91 (4), 649-658 (2011).</li><li>Crick, S., Sheppard, M., Ho, S., Gebstein, L., Anderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+pig+heart+:+comparisons+with+normal+human+cardiac+structure.">Anatomy of the pig heart : comparisons with normal human cardiac structure.</a> J. Anat. 193 (1), 105-119 (1998).</li><li>Stubhan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cardiovascular+and+ECG+parameters+in+the+normal,+freely+moving+G&#246;ttingen+Minipig.">Evaluation of cardiovascular and ECG parameters in the normal, freely moving G&#246;ttingen Minipig.</a> J. Pharmacol. Toxicol. Methods. 57 (3), 202-211 (2008).</li><li>Heusch, G., Skyschally, A., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in-situ+pig+heart+with+regional+ischemia/reperfusion+-+ready+for+translation.">The in-situ pig heart with regional ischemia/reperfusion - ready for translation.</a> J. Mol. Cell Cardiol. 50 (6), 951-963 (2011).</li><li>Skyschally, A., van Caster, P., Iliodromitis, E. K., Schulz, R., Kremastinos, D. T., Heusch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+postconditioning:+experimental+models+and+protocol+algorithms.">Ischemic postconditioning: experimental models and protocol algorithms.</a> Basic Res Cardiol. 104 (5), 469-483 (2009).</li><li>Iekushi, K., Seeger, F., Assmus, B., Zeiher, A. M., Dimmeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cardiac+microRNAs+by+bone+marrow+mononuclear+cell+therapy+in+myocardial+infarction.">Regulation of cardiac microRNAs by bone marrow mononuclear cell therapy in myocardial infarction.</a> Circulation. 125 (14), 1765-1773 (2012).</li><li>Sijbesma, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+polymers+formed+from+self-complementary+monomers+using+quadruple+hydrogen+bonding.">Reversible polymers formed from self-complementary monomers using quadruple hydrogen bonding.</a> Science. 278 (5343), 1601-1604 (1997).</li><li>van Slochteren, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+measurement+techniques+of+regional+myocardial+function+to+assess+the+effects+of+cardiac+regenerative+therapy+in+different+models+of+ischaemic+cardiomyopathy.">Advanced measurement techniques of regional myocardial function to assess the effects of cardiac regenerative therapy in different models of ischaemic cardiomyopathy.</a> Eur. Heart J. Cardiovasc. Imaging. 13 (10), 808-818 (2012).</li><li>McCall, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarction+and+intramyocardial+injection+models+in+swine.">Myocardial infarction and intramyocardial injection models in swine.</a> Nature Protoc. 8 (7), 1479-1496 (2012).</li><li>Roussi, J., Andre, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+functions+and+haemostasis+parameters+in+pigs:+absence+of+side+effects+of+a+procedure+of+general+anaesthesia.">Platelet functions and haemostasis parameters in pigs: absence of side effects of a procedure of general anaesthesia.</a> Thromb Res. 81 (3), 297-305 (1996).</li></ol>

This work was supported by the HGG Group BV (SK), the &#8220;Wijnand M. Pon Stichting&#8221; (SC, SK). This research forms part of the Project P1.04 SMARTCARE of the BioMedical Materials institute, cofunded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation (SJL, JG). All authors have reported that they have no relationships to disclose.&#160;

Intracoronary balloon occlusion of the LAD provides a reproducible and consistent preclinical MI model in pigs that can be used to investigate safety and the efficacy of new cardiovascular therapies that closely mimics the human situation. As shown in Figure 5, the presented ischemia/reperfusion infarction model provides the platform that can be further tailored to investigate different phases of MI and post-MI remodeling whilst the initial ischemia/reperfusion injury is identical for both.
The success of the described protocol outlined here is dependent on the myocardial ischemia as the most critical phase of the protocol. Correct placement of the balloon distal to the second diagonal branch of the LAD is crucial for reaching adequate infarct size whilst ensuring a high survival rate. Based on this MI model, a ~15% mortality rate was observed, while extensive mid and apical segments of the anterior, septal and inferior walls were infarcted as seen on CMR and TTC staining (Figures 2A and 2B). The duration of ischemia can be tailored according to the desired infarct size. Although we have used Landrace pigs in this protocol, minipigs (i.e. G&#246;ttingen minipigs) usually require longer durations of myocardial ischemia (e.g. 150 min occlusion).
Outcome analysis in preclinical and clinical MI studies is often based on LVEF. Although lower LVEF has been firmly associated with increased risk for cardiovascular mortality, it remains dependent on hemodynamical parameters such as preload9. Arguably, given that on average only 10-15% of the LV is infarcted, several conceptual and practical limitations are related to LVEF being a global measure of LV systolic function rather than reflecting local improvement10. Therefore, the proposed measures of outcome used in this model shed light on different aspects of MI and post-MI remodeling thereby providing the investigators the means to accurately assess the efficacy of new therapies on multiple levels.
To optimize translation from preclinical models to clinical practice, we choose using large pigs instead of minipigs. Hemodynamic measurements, medication dosages and surgical devices can easily be exchanged with clinical practice. Compared to minipigs, large pigs gain relatively much weight. This may cause a problem in long-term follow up, with regard to comparability of serial results. Female Dalland Landrace pigs weigh approximately 70 kg at an age of 6 months. To prevent abundant weight gain during the follow up period, animals are kept on a restricted diet. Pigs receive 750 g of custom made low calorie food (containing: proteins 15.6%, fat 2.0%, fibers 14.8%, ashes 8.8%, calcium 0.9%, phosphorous 0.57%, magnesium 0.29%, and potassium 0.18%) twice a day and gain about 10 kg of weight in 4 weeks.
McCall and coworkers have previously published a similar protocol for myocardial infarction in pigs11. Considerable overlap exists between this protocol and theirs, emphasizing the preference for the LAD rather than the left circumflex artery (LCX) or the right coronary artery (RCA). In our experience, there is a lesser extent of infarct size of the total left ventricle using the LCX while the RCA infarction is accompanied with higher chance of unwanted conduction disturbances (i.e. sinus node dysfunction, AV-node dysfunction). One difference between the two protocols pertains to the use of increased pharmacological platelet inhibition in this protocol, as we have observed higher rates of no-reflow based on thrombus formation as the result of 90 min of hemostasis in the occluded coronary artery. This observation is in line with known hypercoagulability observed in pigs12. Although McCall proposed using a single, high-dose, bolus of heparin, this protocol relies on the use of heparin in multiple lower doses spread throughout the surgery to minimize thrombotic complications.
In summary, we present a porcine MI model that enables researchers to make use of an effective, reproducible and above all practical large animal model of human disease to study new therapeutics as an essential step towards a first-in-man clinical trial.

A correction was made to <a href="/video/51269">Myocardial Infarction and Functional Outcome Assessment in Pigs</a>. An author's middle initial was omitted at publication.
The author's name was updated from:
Sanne Jansen of Lorkeers
to:
Sanne J. Jansen of Lorkeers

A correction was made to <a href="/video/51269">Myocardial Infarction and Functional Outcome Assessment in Pigs</a>.
The middle initial J. has been added to author Sanne Jansen of Lorkeers.

Erratum: Myocardial Infarction and Functional Outcome Assessment in Pigs

Acute myocardial infarction (AMI) and its long-term sequelae such as chronic heart failure (CHF) profoundly impact patient prognosis and quality of life, let alone the high cost restraints imposed on our available healthcare resources1. The prevalence of CHF in the western world is estimated at 1-2%, of which ~60% of cases are the consequence of AMI as primary cause2. In the USA alone, about 5.7 million patients suffer from CHF accounting for approximately $30 billion in annual healthcare costs in 2008, with a predicted triplicate in costs rising to $97 billion annually in 20301. Taken together, these numbers make a strong argument for the development of new cardioregenerative treatments that, for swift translation, rely on a reproducible and reliable large animal myocardial infarction model that accurately mimics the human scenario.
Pigs (Sus scrofa) are increasingly being used in cardiovascular research for pharmacological and toxicological testing3. One of the traits responsible for this success as a translational research tool is their similarity in cardiac function and anatomy with the human heart4,5. For instance, pig heart-to-body weight ratio, cardiac size and coronary artery anatomy distribution have all shown to be remarkably similar to man4. Moreover, cardiomyocyte metabolism, electrophysiological properties and response to an ischemic insult such as AMI have been reported to show high levels of agreement with the human situation6,7. Ultimately, to fulfill the above described criteria, a standardized MI-protocol that produces robust and sustainable MI for testing of investigational new drugs (IND) is needed. Here, we present such a standardized model that uses a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD) followed by reperfusion, thereby creating reproducible myocardial infarction covering the anteroapical, septal and inferoseptal walls of the left ventricle.

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">0.9% Saline</td>
			<td style="width:224px;">Braun</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">6-0 Prolene</td>
			<td style="width:224px;">Ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Acetylsalicylic acid</td>
			<td style="width:224px;">80 mg (Ratiopharm)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Adenosine</td>
			<td style="width:224px;">3mg/ml apotheek UMCU</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">AdVantage PV loop system</td>
			<td style="width:224px;">Transonic Scisence</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:216px;">Amiodarone</td>
			<td style="width:224px;">Cordarone I.V. (Sanofi)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:224px;">Amiodaron HCl (PCH)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amoxicilline/ clavulaanzuur</td>
			<td style="width:224px;">500/50mg (Sandoz)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Atropinesulfaat</td>
			<td style="width:224px;">0,5 mg/ml (PCH)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Bone Marrow Wax</td>
			<td style="width:224px;">Syneture</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Cardiac Defibrillator</td>
			<td style="width:224px;">Philips</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Clopidogrel</td>
			<td style="width:224px;">Clopidrogel 75A (Apothecon B.V.)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Contrast agent</td>
			<td style="width:224px;">Telebrix</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Endotracheal tube</td>
			<td style="width:224px;">Covidien</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fentanyl patch</td>
			<td style="width:224px;">Durogesic&#174; 25 mcg/h (Janssen-Cilag)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Fogarty catheter</td>
			<td style="width:224px;">Edwards Lifesciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Gadolinium</td>
			<td style="width:224px;">Gadovist</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Guidewire</td>
			<td style="width:224px;">Abbott</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Heparin</td>
			<td style="width:224px;">Leo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">I.V. cannula</td>
			<td style="width:224px;">Abbocath&#174; (Hospira venisystems)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Iodine</td>
			<td style="width:224px;">Jodiumtictuur 2% (Eurovet)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Ketamine</td>
			<td style="width:224px;">Narketan&#174; 10 V&#233;toquinol</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Midazolam</td>
			<td style="width:224px;">5mg/ml (Actavis)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Nitroglycerin</td>
			<td style="width:224px;">1mg/ml Pohl Boskamp</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Pancuronium bromide</td>
			<td style="width:224px;">2mg/ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Seldinger vascular sheath 8F</td>
			<td style="width:224px;">Arrow</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sufentanil&#160;</td>
			<td style="width:224px;">50 mcg/ml (Sufentanil-hameln)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Swann-Ganz catheter</td>
			<td style="width:224px;">Criticath ref 680078 (Argon)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Synolux</td>
			<td style="width:224px;">250 mg (Pfizer)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Tetra-polar catheter</td>
			<td style="width:224px;">Transonic Scisence</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Thiopental</td>
			<td style="width:224px;">0,5 g (Inresa)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Triphenyl-tetrazolium chloride</td>
			<td style="width:224px;">Merck</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Venofundin</td>
			<td style="width:224px;">Braun</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Vicryl 2-0</td>
			<td style="width:224px;">Ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Volcano ComboMap system</td>
			<td style="width:224px;">Volcano</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

myocardial infarction and functional outcome assessment in pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

Mortality and Infarct Size
In our center, out of 32 pigs (Female Dalland Landrace, 6 months old, ~70 kg) that were subjected to this MI protocol, five (15.6%) died due to refractory ventricular fibrillation during ischemia. This protocol creates an infarct covering approximately 10-15% of the left ventricle, located in the anteroseptal, septal and inferoseptal walls (Figure 2A). If serial noninvasive assessment of infarct size is warranted, late gadolinium enhancement (LGE) on CMR can be used to follow the nonviable infarct area over time (Figure 2B).
Cardiac Function and Remodeling
Four weeks after MI, global and regional parameters reflecting cardiac function should be decreased compared to healthy baseline values. Specifically, LV ejection fraction (LVEF) should decrease to approximately ~35-45% four weeks post-MI. Besides global systolic function, several parameters reflecting post-MI adverse remodeling can also be measured, such as LV morphology and diameters using CMR and echocardiography (Figures 3A and 3B). Four weeks after MI, an increase in end diastolic volume (EDV) as a sign of adverse remodeling can be expected (Figures 3A and 3B).
Coronary flow and pressure parameters
Angiogenesis and formation of new capillaries are often regarded as important treatment goals in ischemic heart disease8. Assessment of microvascular resistance can be indirectly based on the combined measurement of intracoronary pressure and flow velocity. Representative pressure and flow velocity measurement under normal conditions and maximal hyperemia is shown in Figure 4. Four weeks after MI, the hyperemic microvascular resistance should be increased in the infarct related coronary artery (LAD) compared to the baseline situation8.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51269/51269fig1highres.jpg" src="/files/ftp_upload/51269/51269fig1.jpg" /> 
Figure 1. MI model based on LAD balloon occlusion. (A) Standard surgical equipment with: 1) towel clamps; 2) mosquitos; 3) dissecting forceps; 4) round container; 5) needle holders (fine and rough); 6) Klinkenberg scissor; 7) dissecting scissors (straight and curved); 9) forceps (De-Bakey, fine and rough); 10) hose clamp; 11) anastomosis clamp; 12) gauzes; 13) electrosurgical pencil; 14) scalpel holder; 15) Dreesman (suction); 16) retractor; 17) lamp holders. (B) Left anterior oblique fluoroscopic view of the LAD and the LCX. (C) After visualizing the second diagonal branch, position the two radiopaque markers (see inset, black arrowheads) of the balloon just distally of the D2. Inflate and ensure that coronary blood flow is successfully blocked by contrast injection (see asterisk). Intracardiac defibrillator lead can be seen in the right ventricle (see white arrowhead). LAD denotes Left anterior descending artery; LCX denotes left circumflex artery; LAO denotes left anterior oblique view; AP denotes anterior posterior view; D1 denotes first diagonal branch; D2 denotes second diagonal branch. <a href="https://www.jove.com/files/ftp_upload/51269/51269fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51269/51269fig2highres.jpg" src="/files/ftp_upload/51269/51269fig2.jpg" /> 
Figure 2. Infarct size after MI. (A) The 90&#160;min&#160;balloon occlusion of the LAD leads to extensive myocardial damage and scar formation (white color), visualized by TTC staining at 1 month follow up. (B) Schematic infarct distribution shows that the infarction is located in the anterior, anteroseptal and inferoseptal segments of the heart. (C,D) Short and long axis late gadolinium enhanced CMR images show the extensive infarct scar (white signal, see black arrowheads) localized in the anterior, anteroseptal and inferoseptal segments of the heart. LGE-CMR denotes late gadolinium enhanced cardiac magnetic resonance. Scale bar denotes 3 cm. <a href="https://www.jove.com/files/ftp_upload/51269/51269fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="4in" src="/files/ftp_upload/51269/51269fig3highres.jpg" width="400" /> 
Figure 3. Assessment of cardiac function in ischemic MI models. (A) Representative CMR cine-loop images at end diastole and end systole showing functional impairment of the infarct scar segments. (B) M-Mode image of 2D parasternal long axis by echocardiography, showing LV dilatation (increase in LVIDd) 1 month after MI, as well as functional impairment (absence of septal thickening). EDV denotes end diastolic volume; ESV denotes end systolic volume; LVIDd denotes left ventricular internal diameter at diastole and LVIDs denotes left ventricular internal diameter at systole. <a href="https://www.jove.com/files/ftp_upload/51269/51269fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51269/51269fig4highres.jpg" src="/files/ftp_upload/51269/51269fig4.jpg" /> 
Figure 4. Intracoronary pressure and flow velocity derived parameters. Intracoronary pressure and flow velocity recordings using the Combowire showing (A) reference values prior to MI with high response to hyperemia (black arrowhead). (B) 1 month after MI, the infarct related artery (LAD) has a decreased hyperemic response in coronary flow velocity (black arrowheads). As a result, pressure and flow velocity derived parameters (HMR) or flow velocity reserve (CFR) are decreased compared to the baseline. bAPV denotes basal average peak velocity; pAPV denotes peak average peak velocity; CFR denotes coronary flow reserve; HMR denotes hyperemic microvascular resistance. <a href="https://www.jove.com/files/ftp_upload/51269/51269fig4highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51269/51269fig5highres.jpg" src="/files/ftp_upload/51269/51269fig5.jpg" /> 
Figure 5. Overview of different study designs. (A) Schematic of multiple possible study designs to validate investigational new drugs (INDs) in various stages of MI using this LAD MI pig model. Dependent on the chosen phase of MI that is under investigation, functional analysis can be performed just prior to the treatment allocation as the baseline value and assessment of the area at risk. <a href="https://www.jove.com/files/ftp_upload/51269/51269fig5highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Myocardial Infarction and Functional Outcome Assessment in Pigs at JoVE.com

Myocardial Infarction and Functional Outcome Assessment in Pigs

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

myocardial-infarction-and-functional-outcome-assessment-in-pigs

Nanyang Technological University

Research

JoVE Journal

Medicine

27.7K Views.  University Medical Center Utrecht. This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Video: Myocardial Infarction and Functional Outcome Assessment in Pigs

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...