Name: primary outcome assessment in a pig model of acute myocardial infarction
Uploaded: 2016-10-14T15:00:00
Description: Watch this Scientific Journal Video about Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction at JoVE.com

The authors gratefully acknowledge Marlijn Jansen, Joyce Visser, Grace Croft, Martijn van Nieuwburg, Danny Elbersen and Evelyn Velema for their excellent technical support during the animal experiments.

All animal experiments were approved by the Ethical Committee on Animal Experimentation of the University Medical Center Utrecht (Utrecht, the Netherlands) and conform to the &#39;Guide for the care and use of laboratory animals&#39;.
NOTE: The protocol to perform a closed-chest balloon occlusion is not part of the current manuscript and is described in detail elsewhere5. In short, pigs (60 - 70 kg) are subjected to 75 min transluminal balloon occlusion of the midportion of the left...

<ol>
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Cardiac remodeling is largely depending on myocardial infarct size and the quality of myocardial infarct repair6,26. To assess the former in a standardized manner, the present manuscript provides an elegant method of in&#160;vivo infusion of Evans blue combined with ex&#160;vivo TTC staining, which has been validated and extensively used8,16,27,28. This method allows for quantification of the area at risk (AAR) and infarct size in relation to AAR16. The current approach ...

Heart failure with reduced ejection fraction (HFrEF) accounts for about 50% of all heart failure cases, affecting an estimated 1 - 2% of people in the western world1. Its most prevalent cause is acute myocardial infarction (AMI). As acute mortality after AMI has declined significantly due to increased awareness and improved treatment options, emphasis has shifted towards its chronic sequelae; the most prominent being HFrEF2,3. Together with increasing health care costs4, the growing epidemic of heart failure stresses the need for novel diagnostics and therapies, which can be studied in a highly translational porcine model of adverse remodeling after AMI as previously described5.
Both, determinants (e.g., infarct size) and functional assessments (e.g., echocardiography) of adverse remodeling are often used for efficacy testing of new therapeutics, indicating the need for reliable and relatively inexpensive methods. The aim of the current paper is to address this need by introducing important and reliable outcome measures for efficacy testing in a pig model of acute myocardial infarction. These include infarct size (IS) in relation to area at risk (AAR), 3D transesophageal echocardiography (3D-TEE) and detailed admittance-based pressure-volume (PV) loop acquisition.
Infarct size is the main determinant of adverse remodeling and survival after AMI6. Although timely reperfusion of ischemic myocardium may salvage reversibly injured cardiomyocytes and limit infarct size, reperfusion itself causes additional damage through the generation of oxidative stress and a disproportionate inflammatory response (ischemia-reperfusion injury (IRI))7. Hence, IRI has been identified as a promising therapeutic target. The ability of novel therapeutics to decrease infarct size is quantified by assessing infarct size in relation to the area at risk (AAR). AAR quantification is mandatory to correct for inter-individual variability in coronary anatomy of animal models, as a larger AAR leads to a larger absolute infarct size. Since infarct size is directly related to cardiac performance and myocardial contractility, variations in AAR can influence study outcome measures irrespective of treatment modalities8.
Three-dimensional transesophageal echocardiography (3D-TEE) is a safe, reliable and, most importantly, clinically applicable inexpensive method to measure cardiac function non-invasively. Whereas transthoracic echocardiography (TTE) images are limited to 2D parasternal long- and short-axis views in pigs9, 3D-TEE can be used to obtain complete 3-dimensional images of the left ventricle. Therefore, it does not require mathematical approximations of left ventricular (LV) volumes such as the modified Simpson&#39;s rule10. The latter falls short of correctly estimating LV volumes after LV remodeling due to the lack of cylindrical geometry11. Moreover, 3D-TEE is preferable over epicardial echocardiography as it does not require surgical interventions, which have been observed to exert cardioprotective effects in the present model12. Although the use of 2D-TEE for the assessment of myocardial function has been described before13,14, limitations regarding ventricular geometry are similar to those observed in 2D-TTE and depend on the extent of LV remodeling. Hence, the larger the infarct (and thus the higher the probability of heart failure), the more likely 2D measurements become flawed by incorrect geometrical assumptions and the higher the need for 3D techniques.
Nonetheless, most imaging modalities are limited in their ability to assess intrinsic functional properties of the myocardium. PV loops provide such relevant additional information and their acquisition is therefore described in detail below.

<table border="0" cellpadding="0" cellspacing="0" width="875">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">3-dimensional transesophageal echocardiography</td>
			<td style="width:180px;"></td>
			<td style="width:108px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">iE33 ultrasound device</td>
			<td style="width:180px;">Philips</td>
			<td style="width:108px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">X7-2t transducer</td>
			<td style="width:180px;">Philips</td>
			<td style="width:108px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Aquasonic&#174; 100 ultrasound transmission gel</td>
			<td style="width:180px;">Parker Laboratories Inc.</td>
			<td style="width:108px;">01-34</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Battery handle type C (laryngoscope handle)</td>
			<td style="width:180px;">Riester</td>
			<td style="width:108px;">12303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Ri-Standard Miller blade MIL 4 (laryngoscope blade)</td>
			<td style="width:180px;">Riester</td>
			<td style="width:108px;">12225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Qlab 10.0 (3DQ Advanced) analysis software</td>
			<td style="width:180px;">Philips</td>
			<td style="width:108px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Pressure-volume loop acquisition</td>
			<td style="width:180px;"></td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Cardiac defibrillator</td>
			<td style="width:180px;">Philips</td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.9% saline</td>
			<td style="width:180px;">Braun</td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">8F Percutaneous Sheath Introducer Set</td>
			<td style="width:180px;">Arrow</td>
			<td style="width:108px;">CP-08803</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9F Radifocus&#174; Introducer II Standard Kit&#160;</td>
			<td style="width:180px;">Terumo</td>
			<td>RS*A90K10SQ</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">8F Fogarty catheter</td>
			<td style="width:180px;">Edward Life Sciences</td>
			<td>62080814F</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7F Criticath&#8482; SP5107H TD catheter (Swan-Ganz)</td>
			<td style="width:180px;">Becton Dickinson (BD)</td>
			<td>680078</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:397px;">Ultraview SL Patient Monitor and Invasive Command Module (external cardiac output device)</td>
			<td>Spacelabs Healthcare</td>
			<td>91387</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ADVantage system&#8482;</td>
			<td>Transonic SciSense</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7F tetra-polar admittance catheter (7.0 VSL Pigtail / no lumen)</td>
			<td>Transonic SciSense</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-channel acquisition system (Iworx 404)</td>
			<td>Iworx</td>
			<td style="width:108px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Labscribe V2.0 analysis software</td>
			<td style="width:180px;">Iworx</td>
			<td style="width:108px;">-</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Infarct size / area-at-risk quantification</td>
			<td style="width:180px;"></td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Diathermy</td>
			<td style="width:180px;">-</td>
			<td style="width:108px;"></td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Lebsch knife</td>
			<td style="width:180px;">-</td>
			<td style="width:108px;"></td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Hammer</td>
			<td style="width:180px;">-</td>
			<td style="width:108px;"></td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Bone marrow wax</td>
			<td style="width:180px;">Syneture</td>
			<td style="width:108px;"></td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Klinkenberg scissors</td>
			<td style="width:180px;">-</td>
			<td style="width:108px;"></td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Retractor</td>
			<td style="width:180px;">-</td>
			<td style="width:108px;"></td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Surgical scissors</td>
			<td style="width:180px;">-</td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7F Percutaneous Sheath Introducer Set&#160;</td>
			<td style="width:180px;">Arrow</td>
			<td>CP-08703</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8F Percutaneous Sheath Introducer Set&#160;</td>
			<td style="width:180px;">Arrow</td>
			<td style="width:108px;">CP-08803</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">7F JL4 guiding catheter&#160;</td>
			<td style="width:180px;">Boston Scientific</td>
			<td style="width:108px;">H749 34357-662</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8F JL4 guiding catheter&#160;</td>
			<td style="width:180px;">Boston Scientific</td>
			<td>H749 34358-662&#160;</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">COPILOT Bleedback Control Valves&#160;</td>
			<td>Abbott Vascular</td>
			<td>1003331</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Connecta&#8482;&#160;</td>
			<td style="width:180px;">Franklin Lakes</td>
			<td>394995</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Contrast agent</td>
			<td style="width:180px;">Telebrix</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:397px;">Persuader 9 Steerable Guidewire 9 (0.014&#34;, 180 cm, straight tip), hydrophilic coating</td>
			<td style="width:180px;">Medtronic Inc.</td>
			<td>9PSDR180HS</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:397px;">SAPPHIRE&#8482; Coronary Dilatation Catheter (PTCA balloon suitable for the size of the particular coronary artery (2.75 - 3.25 mm))</td>
			<td style="width:180px;">OrbusNeich</td>
			<td style="width:108px;">103-3015</td>
			<td>Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evans Blue&#160;</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td>E2129-100G</td>
			<td>Toxic. Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,3,5-triphenyl-tetrazolium chloride (TTC)</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td>T8877-100G</td>
			<td>Irritant. Alternative product can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9V battery</td>
			<td style="width:180px;">-</td>
			<td style="width:108px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Ruler</td>
			<td style="width:180px;">-</td>
			<td style="width:108px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Photocamera</td>
			<td style="width:180px;">Sony</td>
			<td style="width:108px;">-</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:397px;">ImageJ</td>
			<td style="width:180px;">National Institutes of Health</td>
			<td style="width:108px;">-</td>
			<td>Alternative product can be used</td>
		</tr>
	</tbody>
</table>

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.

3D Transesophageal Echocardiography
3D transesophageal echocardiography (3D-TEE) can be used for the assessment of global cardiac function. After AMI, global cardiac function differs from healthy baseline values. In particular, left ventricular ejection fraction (LVEF) decreases from 59 &#177; 4% to 37 &#177; 6% after one week of reperfusion (n = 10) (GPJ van Hout, 2015). An increase in end-systolic volume (51 &...

Watch this Scientific Journal Video about Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction at JoVE.com

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

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 ...

The overall goal of this methodology is to acquire reliable and reproducible outcome measurements for the evaluation of new therapies in a pig model of acute myocardial infarction. This method can help answer key questions in the cardiovascular field, such as the effect of therapeutic interventions on the schema of a fusion injury and for modeling also an acute myocardial infarction. The main advantage of these techniques is that they allow for an accurate and a clinically relevant assessment of ischemia reperfusion injury and cardiac remodeling.Through this method, can provide insight into myocardial ischemia reperfusion injury. It can also be applied to other studies involving the evaluation of cardiac function. The current protocol allows for the acquisition of primary outcome measurements prior to, or after an acute myocardial infarction.The infarction is made using a 75-minute transluminal balloon occlusion of the left anterior descending artery, which is detailed by the cited video. This video demonstrates how to perform three dimensional transesophageal echocardiography, and the pressure of volume loop measurements for baseline, short-term, and long-term follow up measurements. The video also demonstrates how to measure infarct size and area at risk staining after termination.Prepare the pig with anesthesia as described in the text protocol, and connect the animal to the echocardiography machine using the EKG leads. Now, prepare the echo probe. Put some ultrasound gel on the tip and unlock it so that it is flexible.Adjust the imaging angle to 90 degrees using the handheld operating piece. With the pig in a right lateral position, open the mouth and carefully insert the probe into the esophagus. Be careful not to apply too much pressure, and to avoid the normal anatomic pharyngeal pouch, which resembles a Zenker's diverticulum.Then, insert the probe 50 to 60 centimeters, measure from the tip of the snout, and slowly rotate and flex the head to a left anterolateral position to visualize the heart. Make sure all of the heart's walls are clearly visible. If necessary, lock the echo probe tip to stabilize the image acquisitions.On the display, toggle the 3D Full Volume option to produce to perpendicular images of the left ventricle. Then, maximize the sector width that is being acquired by selecting FV Opt Volume. Now, pause the ventilation and press Acquire to obtain full volume measurements.After obtaining a complete measurement, switch on the mechanical ventilation. After the echo acquisition, unlock the probe and slowly remove it from the animal. Then, monitor the pig until it has recovered from the surgery.In preparation, soak the sensing tips of the catheter in 0.9%saline for at least 20 minutes. After preparing the pig for surgery, infuse 100 international units of heparin per kilogram, and place an 8F introducer sheath in the carotid, and a 9F sheath in the jugular vain. Next, insert a Swan Ganz catheter thought the 9F sheath, inflate the balloon at the tip of the catheter, and wedge it in a small pulmonary artery.Once adequately placed in there peripheral part of the lung, deflate the balloon and connect the Swan Ganz catheter to an external cardiac output device. Upon connection, view the cardiac output menu on the external cardiac output device. Enter the weight of the animal into the system and calculate the length so the BMI is 22.5.Proceed by loading a 20 milliliter syringe with room temperate 0.9%saline and attaching it to the injection port connected to the lumin with the most proximal debouchment. Switch off the ventilation and wait for five heartbeats. First, press Manual, then press Start on the output device.Then, rapidly infuse five milliliters of saline to measure the cardiac output. Within 15 seconds, the device measurements will be shown. Then, switch on the ventilation.Repeat this procedure three times and calculate the average stroke volume. Between infusions, wait a minute or two to allow the end-tidal carbon dioxide to return to preexisting values. Now, remove the Swan Ganz catheter and insert an 8F Fogarty catheter through the 9F sheath in the jugular vein, and position it in the inferior vena cava.While the tip remains in 0.9%saline, calibrate the pressure signal of the PV loop catheter as close to 0 as possible, using the course and fine buttons. Then, input the measured stroke volume into the system. The next step is to advance the PV loop catheter through the 8F sheath in the carotid artery, and, while using fluoroscopy, advance and center the tip into the left ventricle.Now, plot the raw conductance signal against the pressure signal, which should make a rectangular loop. Using this graph, select the largest adequately placed segment. Then, pause the mechanical ventilation and press enter on the pressure volume loop module to take a baseline scan to convert conductance to volume.When the module shows the heart rate, systolic and diastolic conductance, turn the respiration back on. Before proceeding, the measured heart rate must be equal to the EKG, or pressure derived heart rate. There cannot be any arrhythmia, and the systolic and diastolic conductance must be adequately sensed.If the baseline data is determined to be adequate, accept it by pressing enter and write down the heart rate and systolic and diastolic conductance shown on the PV loop module. Then, plot the volume signal against the pressure signal. The loops must still be rectangular.Now, pause the ventilation and acquire baseline pressure volume by recording 10 to 12 consecutive heartbeats. Then, switch on the ventilation again and allow the end-tidal carbon dioxide level to return to preexisting values. Next, under fluoroscopic guidance, inflate the Fogarty catheter to reduce the preload.Then, record 10 to 12 consecutive beats without ventilation as before. The systolic blood pressure must remain above 60 millimeters of mercury, and no arrhythmia can interfere with the measurements. After acquiring the data, deflate the Fogarty catheter, switch on the ventilation, and wait for the end-tidal carbon dioxide to return to preexisting levels.Now, remove the Fogarty and PV loop catheters while continuing to record arterial pressure to correct for pressure drift. Finally, monitor the animal until it has recovered before returning it to the other animals. After preparing the animal for surgery, access both carotid arteries using a surgical exploration of the neck.Then, insert a 7F and an 8F introducer sheath in the respective carotid artery. Now, administer heparin at 100 units per kilo. In order to perform a sternotomy, make a medium 30 to 40 centimeter incision from just below the suprasternal notch to a point just below the xiphoid process.Then, advance through the linea alba down to the sternum. Use diathermy to carefully split the xiphoid and use Klinkenberg scissors to separate the posterior sternum from the pericardium. Continue the separation bluntly, moving cranially.Make sure to point the scissor tips upwards towards the sternum to avoid cardiac damage. Perform a sternotomy by using a hammer and Lebsche knife. Bone marrow bleeding can be minimized by rubbing bone marrow wax on the marrow.Open the thorax with a retractor. Now, open the pericardium using surgical scissors. Connect standard Y catheters to the guiding catheters.Then, connect a three-way tap with a 10 centimeter extension to both Y connectors. Then, position one of the guiding catheters in the ostium of the left main coronary artery via one of the sheaths. Next, using a guide wire inserted into the left anterior descending artery, advance a coronary dilatation catheter through the left main coronary artery catheter, and position the balloon where the coronary occlusion was performed during the myocardial infarction induction.Do not inflate yet. Continue by positioning the other guiding catheter in the ostium of the right coronary artery using the other sheath. Now, perform a coronary angiography.Infuse contrast agent under fluoroscopy. First, confirm correct positioning of the guiding catheter in the left main coronary artery with an anteroposterior view. Thereafter, visualize the right coronary artery.The contrast infusion may propel the balloon further into the coronary artery. Reposition it if necessary. Then, attach syringes with freshly made 2%Evans Blue dye to the three-way taps.Next, using a coronary angiography, inflate the balloon and confirm that it completely occludes part of the coronary artery. The occlusion is critical. While the balloon is inflated, inject the dye through both guiding catheters at 5 milliliters per second.The heart should not turn blue, except for the part of the left ventricle that is deprived of blood flow. If the heart does not go into ventricular fibrillation spontaneously, touch a nine volt battery to the non-infarcted part of the heart five beats after dye infusion. Then, incise the cable vein to release pressure and use suction to drain out the blood.Now, deflate the balloon. Retract it along with both guiding catheters. Then, explant the heart by dissecting the surrounding membranes, and making a transverse cut through the large vessels.Make sure to wash off all the blood and dye with 0.9%saline, both on the outside and on the inside. Then, carefully dissect the left ventricle. Then, make five equal 10 millimeter thick sections from apex to base, in a plane parallel to the atrial ventricular groove.Photograph both sides of all five slices under ambient light conditions using a ruler in the image for calibration. Then, stain the slices in TTC and photograph them again. 3D transesophageal echocardiography was used for the assessment of global cardiac function.One week after an acute myocardial infarction, global cardiac function deteriorated when compared to healthy baseline values, based on results from 10 animals. This was accounted for by reduced contraction of the anteroseptal wall. Pressure volume loops were also used, both to assess global cardiac function, and to calculate intrinsic myocardial muscle properties.Outcome measurements of the former were easily calculated, and include EDV int he lower right corner, and ESV in the upper left corner. These values provide important information on left ventricular geometry. From these measures, the left ventricular rejection fraction, an important measure of left ventricular pump function, was calculated, along with several other parameters.For intrinsic cardiac performance, different measurements were derived from the PV loops, such as the end systolic and end diastolic pressure volume relationship. After myocardial infarction, the end systolic volume pressure relationship slope may decrease, indicating decreased contractility. Ultimately, stains were used to identify the remote myocardium from the area at risk, and the infarcted myocardium.About 22%of the left ventricle made up the area at risk, most of which was infarcted. After watching this video, you should have a good understanding of how to perform 3D transesophageal echocardiography, pressure volume loop data acquisition, and infarct size to area at risk staining. Once mastered, transesophageal echocardiography can be performed in 20 minutes.Pressure volume loop data acquisition in 30 minutes. And infarct size and area at risk staining in 60 minutes. While attempting PV loops and infarct size area at risk staining, it is important to remember to be cautious not to induce and arrhythmias.The techniques shown in this video can help researchers in the field of cardiovascular disease to explore ischemia and reperfusion injury in large animal models.

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Research

JoVE Journal

Medicine

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 ...

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 ...

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.

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 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 ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

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 ...

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.

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 ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...