Name: primary outcome assessment in a pig model of acute myocardial infarction
Uploaded: 2016-10-14T15:00:00.000+00:00
Duration: 14 min 19 s
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 anterior descending artery (LAD).
Both, three-dimensional transesophageal echocardiography (3D-TEE) and pressure-volume (PV) loop measurements can be performed at baseline, short-term and long-term follow-up. Note that these measurements are considered unreliable in the first hours after myocardial infarction due to frequent arrhythmias in this phase. Infarct size (IS) and area-at-risk (AAR) measurements are preferably assessed at short-term follow-up (24 - 72 hr)15,16, since changes in microvasculature and secondary myocardial scar thinning culminate in less reliable results. Infarct size staining is performed using 2,3,5-triphenyltetrazolium chloride (TTC) (CAUTION, irritant), which is considered highly reproducible and relatively inexpensive. TTC is a white powder that colorlessly dissolves in saline. Upon contact with various dehydrogenases, it is converted to a brick red color. Thereby, it discriminates between viable (red) and dead myocardial tissue (white). For an overview on both invasive and non-invasive infarct size determination, readers are directed to a comprehensive review on this subject17.
Figure 1 shows the timeline including anesthesia, surgical preparation and primary outcome measurements used in this study.
1. Medication and Anesthesia
<ol>
	<li>Ensure that the animal does not eat or drink for at least 5 hours prior to the procedure. Pre-treatment, anesthesia and post-operative pain treatment protocols have been described in detail elsewhere5.</li>
	<li>In short, the day prior to surgery a buprenorfine patch (5 &#181;g/hr) is applied to the skin that is active for seven days to limit post-operative pain. On the day of surgery, sedate pigs by intramuscular injection of 0.4 mg/kg midazolam, 10 mg/kg ketamine and 0.014 mg/kg atropine. Wait for approximately 10 - 15 min. Insert an 18 G cannula in one of the ear veins and administer 5 mg/kg sodium thiopental to induce anesthesia.</li>
	<li>Intubate the pig using an endotracheal tube (size 8.5 for pigs of 60 - 70 kg). If necessary, perform balloon-ventilation (frequency 12/min) and transport the pig to the operating theater.</li>
	<li>Upon arrival in the operation theater, start mechanical positive pressure ventilation with FiO2 0.50, 10 ml/kg tidal volume and a frequency of 12/min using continuous capnography recording.</li>
	<li>Start balanced anesthesia by continuous intravenous infusion of a combination of midazolam (0.5 mg/kg/hr), sufentanil (2.5 &#956;g/kg/hr) and pancuronium (0.1 mg/kg/hr).</li>
	<li>Confirm anesthesia by testing the corneal reflex and monitoring the breathing pattern (e.g., spontaneous breathing in combination with mechanical ventilation indicates incomplete anesthesia). Use vet ointment on the eyes to prevent dryness while the animal is under anesthesia.</li>
</ol>
2. 3D Transesophageal Echocardiography (TEE)
<ol>
	<li>To allow for heart rate monitoring and data acquisition, connect the animal to the 5 leads ECG on the echocardiography machine.</li>
	<li>Place the animal in the right lateral position. Make sure the probe is straight and flexible at the tip by unlocking the operating piece.</li>
	<li>Open the pig&#39;s mouth and carefully insert the echo probe in the esophagus. If necessary, use a laryngoscope for visualization. Be careful to avoid ending up in the normal anatomic pharyngeal pouch, resembling a Zenker&#39;s diverticulum18.</li>
	<li>Insert the probe for 50 - 60 cm (measure from the tip of the snout). Slowly rotate the probe and flex the head to a left anterolateral position to visualize the heart (Figure 2A - B). Make sure all walls are clearly visible.</li>
	<li>Use the &#34;3D full volume&#34; option on the display of the echocardiography machine to display two perpendicular images of the left ventricle as shown in Figure 2C - D. Then maximize the sector width that is being acquired by selecting &#34;FV Opt Volume&#34;. Pause ventilation by temporarily switching off mechanical ventilation and press &#34;Acquire&#34; to obtain full volume measurements.</li>
	<li>After echo acquisition, make sure the tip is flexible by unlocking the operating piece. Then slowly remove the probe from the animal. 
	NOTE: Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	<li>Perform offline analysis with validated software as described previously19.</li>
</ol>
3. Admittance-based Pressure-volume Loop Acquisition
<ol>
	<li>Pre-soak the sensing tips of the 7 F tetra-polar admittance catheter in 0.9% saline (room temperature to 37 &#176;C) for a minimum of 20 min to ensure proper hydration and minimal baseline pressure drift during the experiment20.</li>
	<li>Administer medication and anesthesia as described in section 1.</li>
	<li>Perform surgical preparation and obtain vascular access as described previously5.
	<ol>
		<li>In short, shave and clean the neck. Disinfect the surgical area with iodine 2% and cover the non-sterile parts of the pigs with sterile surgical drapes.</li>
		<li>Make a medial incision in the neck to expose the carotid artery and internal jugular vein. Insert an 8 F sheath into the carotid artery and a 9 F sheath into the jugular vein.</li>
	</ol>
	</li>
	<li>Insert a Swan-Ganz (SG) catheter through the 9 F sheath in the jugular vein and wedge it in a small pulmonary artery by inflating the balloon at the tip of the catheter. After adequate placement in the peripheral part of the lung, deflate the balloon. Connect the SG to an external cardiac output device.</li>
	<li>Attach a 20 ml syringe containing 0.9% sterile saline to the injection port that connects to the lumen with the most proximal debouchment. Measure cardiac output by rapid infusion of 5 ml 0.9% saline (room temperature) and obtain heart rates to calculate stroke volume (SV). Repeat this procedure three times and calculate the average SV. 
	NOTE: Cardiac output is (automatically) calculated using the Stewart-Hamilton thermodilution equation and is based on temperature changes in the pulmonary artery upon infusion of room temperature saline21.</li>
	<li>Remove the SG catheter. Insert an 8 F Fogarty catheter through the 9 F sheath in the jugular vein and position it in the inferior vena cava.</li>
	<li>Calibrate the pressure signal of the PV loop catheter using the &#34;Course&#34; and &#34;Fine&#34; button, while the tip remains in 0.9% saline. Then input the measured SV into the system.</li>
	<li>Advance the PV loop catheter through the 8 F sheath in the carotid artery and center the tip in the left ventricle (LV) under fluoroscopy.</li>
	<li>Select the largest adequately placed-segment by plotting the raw conductance signal against the pressure signal. Ensure that the pressure-conductance loops are of rectangle shape. Phase signal is expected to show a sinus trace with values between 3 and 5 degrees. Pause ventilation and perform a baseline scan to convert Conductance to Volume.
	<ol>
		<li>Accept the baseline data by pressing &#34;Continue&#34; when the signals are stable (no arrhythmias), heart rate is equal to ECG or pressure derived heart rates and end-systolic (ES) / end-diastolic (ED) conductance are adequately sensed by the system20. 
		NOTE: The latter can be verified by plotting the raw conductance signal against the pressure signal and comparing ES / ED conductance values derived from the baseline scan to real-time conductance. If any of the requirements above is not met, repeat the procedure.</li>
	</ol>
	</li>
	<li>Acquire baseline pressure-volume loops by recording 10 - 12 consecutive beats during apnea by pausing ventilation.</li>
	<li>Inflate the Fogarty catheter under fluoroscopic guidance to reduce preload and record 10 - 12 consecutive beats as described above. Make sure systolic blood pressure remains &#62;60 mmHg and no arrhythmias interfere with the measurements.</li>
	<li>Remove the Fogarty and PV loop catheters. Keep recording arterial pressure before and during removal of the PV loop catheter to enable correcting for pressure drift (i.e., ex vivo pre- and post-procedural baseline pressure difference). 
	NOTE: Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	<li>Perform offline analysis of geometrical measurements and functional parameters with validated software22.</li>
</ol>
4. Area At Risk (AAR) and Infarct Size (IS) Quantification
<ol>
	<li>Dissolve 1.00 g Evans blue (CAUTION23, toxic) in 50 ml 0.9% saline, fill two 50 ml Luer lock syringes with 20 ml and 30 ml of 2% Evans blue solution respectively and keep at room temperature. 
	NOTE: Work in a fume hood and wear a dust mask to limit exposure to hazardous dusts and use gloves and protective glasses to prevent contact from skin and eyes.</li>
	<li>Taking similar precautions, dissolve 1% 2,3,5-triphenyl-tetrazoliumchloride (TTC) (CAUTION, irritant) in 37 &#176;C 0.9% saline and keep at 37 &#176;C.</li>
	<li>Surgically prepare the animal to obtain vascular access to both carotid arteries. Perform a sternotomy to allow for direct visualization of the effect of in vivo Evans blue infusion5.</li>
	<li>Insert a 7 F and an 8 F introducer sheath in the respective carotid artery. Alternatively, insert both introducer sheaths in one single carotid artery or use one of the femoral arteries for one of both guiding catheters.</li>
	<li>Connect two standard Y-connectors to a 7 F JL4 and an 8 F JL4 guiding catheter respectively. For a femoral approach, use a JR4 for the right coronary artery (RCA) and a JL4 for the left main coronary artery (LCMA). Connect an additional three-way tap with 10 cm extension to both Y-connectors.</li>
	<li>Administer 100 IU/kg heparin. Position the 8 F JL4 guiding catheter in the ostium of the LMCA via one of two introducer sheaths.</li>
	<li>Using a 0.014&#34; guidewire, advance a coronary dilatation catheter through the LCMA catheter and position the balloon at the site where coronary occlusion was performed during MI induction. Do not inflate yet.</li>
	<li>Position the second 8 F JL4 guiding catheter in the ostium of the RCA via the second introducer sheath.</li>
	<li>Perform a coronary angiography (CAG) by infusing contrast agent under fluoroscopy to confirm correct positioning of both guiding catheters and the balloon in the coronary arteries, using anteroposterior and LAO 30&#176; views.</li>
	<li>Attach the two 50 ml syringes containing 30 ml (LCMA) and 20 ml (RCA) 2% Evans blue to the respective three-way taps attached to the Y-connectors on the guiding catheters.</li>
	<li>Inflate the balloon and confirm occlusion of the coronary artery by CAG. Only when the balloon completely blocks the passage of any contrast agent, inject Evans blue dye through both guiding catheters (5 ml/s) while the balloon is inflated.</li>
	<li>Directly after the completion of Evans blue infusion, induce ventricular fibrillation by placing a 9 V battery on the non-infarcted part of the heart.</li>
	<li>Incise the caval vein to release pressure and make sure a suction unit is available to allow for drainage of blood.</li>
	<li>Deflate the balloon, retract it together with both guiding catheters and explant the heart by dissecting surrounding membranes. A transverse cut through the large vessels (i.e., aorta, pulmonary artery/veins) allows for complete explantation. Rapidly wash off blood and superfluous dye on the exterior surface and in the cardiac cavities using 0.9% saline.</li>
	<li>Carefully dissect the left ventricle and make cuts in 5 equal 10 mm thick sections from apex to base, in a plane parallel to the atrioventricular (AV) groove.</li>
	<li>Photograph both sides of all five slices separately under ambient light conditions, as a possible Evans blue washout may occur in the subsequent step. For calibration, make sure a ruler is present in the image.</li>
	<li>Incubate for 10 min in 1% TTC solution at 37 &#176;C, turning the sections around after 5 min for equal staining.</li>
	<li>Again, photograph both sides of all five slices separately under ambient light conditions and make sure a ruler is visualized in the image for calibration.</li>
	<li>Weigh all slices. Use software suitable for the analyses5. When using ImageJ (version 1.47), click the &#34;Straight line&#34; button. Now, draw a straight line with a known distance using the ruler in the image (e.g., 5 cm). Click &#34;Analyze&#34; -&#62; &#34;Set scale&#34; and enter the distance in the box &#34;Known distance&#34;. This procedure allows for calibration of distance in pixels to SI units of length.</li>
	<li>Using the &#34;Polygon selections&#34;button, select the total area that corresponds to the LV myocardium in the present image, click &#34;Analyze&#34; -&#62; &#34;Measure&#34; to acquire measurements. Perform this procedure for both sides of each slice of myocardium, and average per slice.
	<ol>
		<li>Multiply by the weight of the slice proportional to the total weight of all five slices and average these measurements for all slices.</li>
	</ol>
	</li>
	<li>Perform similar measurements for area at risk (AAR) and infarct size (IS). Divide IS/AAR, AAR/LV and IS/LV and multiply by 100% to obtain respective outcome measurements5.</li>
</ol>

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Cardiol. 59 (13), E1564 (2012).</li><li>Keith Jones, ., W, , 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=Peripheral+nociception+associated+with+surgical+incision+elicits+remote+nonischemic+cardioprotection+via+neurogenic+activation+of+protein+kinase+C+signaling.">Peripheral nociception associated with surgical incision elicits remote nonischemic cardioprotection via neurogenic activation of protein kinase C signaling.</a> Circulation. 120, S1-S9 (2009).</li><li>Gross, G. J., Baker, J. E., Moore, J., Falck, J. R., Nithipatikom, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+Surgical+Incision+Induces+Remote+Preconditioning+of+Trauma+(RPCT)+via+Activation+of+Bradykinin+Receptors+(BK2R)+and+the+Cytochrome+P450+Epoxygenase+Pathway+in+Canine+Hearts.">Abdominal Surgical Incision Induces Remote Preconditioning of Trauma (RPCT) via Activation of Bradykinin Receptors (BK2R) and the Cytochrome P450 Epoxygenase Pathway in Canine Hearts.</a> Cardiovasc. Drugs Ther. 25 (6), 517-522 (2011).</li><li>Van Hout, G. P. J., de Jong, R., Vrijenhoek, J. E. P., Timmers, L., Duckers, H. J., Hoefer, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Admittance-based+pressure-volume+loop+measurements+in+a+porcine+model+of+chronic+myocardial+infarction.">Admittance-based pressure-volume loop measurements in a porcine model of chronic myocardial infarction.</a> Exp. Physiol. 98 (11), 1565-1575 (2013).</li><li>Sunagawa, K., Maughan, W. L., Burkhoff, D., Sagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+interaction+with+arterial+load+studied+in+isolated+canine+ventricle.">Left ventricular interaction with arterial load studied in isolated canine ventricle.</a> Am. J. Physiol. 245 (5 Pt 1), H773-H780 (1983).</li><li>Steendijk, P., Baan, J., Der Velde, E. T. V. a. n., Baan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+critical+coronary+stenosis+on+global+systolic+left+ventricular+function+quantified+by+pressure-volume+relations+during+dobutamine+stress+in+the+canine+heart.">Effects of critical coronary stenosis on global systolic left ventricular function quantified by pressure-volume relations during dobutamine stress in the canine heart.</a> J. Am. Coll. Cardiol. 32 (3), 816-826 (1998).</li></ol>

The authors have nothing to disclose.

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

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11.4K Views.  University Medical Center Utrecht.  Betrouwbare en nauwkeurige uitkomst evaluatie is de sleutel voor de vertaling van preklinische therapieën in de klinische behandeling. De huidige paper beschrijft hoe drie klinisch relevante primaire uitkomstmaten van cardiale prestaties en de schade op te nemen in een varken acuut hartinfarct model. 

Video: Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

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 ...

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 ...

Histological Quantification of Chronic Myocardial Infarct in Rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

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 ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

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 ...

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in G&#246;ttingen Minipigs and Landrace Pigs

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an unmet clinical need for prevention and therapy of post-myocardial infarction heart failure (post-MI HF). Clinically relevant pig models of post-MI HF are prerequisites for final proof-of-concept studies before entering into clinical trials in drug and medical device development.
Here we aimed to characterize a closed-chest porcine model of post-MI HF in adult G&#246;ttingen minipigs with long-term follow-up including serial cardiac magnetic resonance imaging (CMRI) and to compare it with the commonly used Landrace pig model.
MI was induced by intraluminal balloon occlusion of the left anterior descending coronary artery for 120 min in G&#246;ttingen minipigs and for 90 min in Landrace pigs, followed by reperfusion. CMRI was performed to assess cardiac morphology and function at baseline in both breeds and at 3 and 6 months in G&#246;ttingen minipigs and at 2 months in Landrace pigs, respectively.
Scar sizes were comparable in the two breeds, but MI resulted in a significant decrease of left ventricular ejection fraction (LVEF) only in G&#246;ttingen minipigs, while Landrace pigs did not show a reduction of LVEF. Right ventricular (RV) ejection fraction increased in both breeds despite the negligible RV scar sizes. In contrast to the significant increase of left ventricular end-diastolic (LVED) mass in Landrace pigs at 2 months, G&#246;ttingen minipigs showed a slight increase in LVED mass only at 6 months.
In summary, this is the first characterization of post-MI HF in G&#246;ttingen minipigs in comparison to Landrace pigs, showing that the G&#246;ttingen minipig model reflects post-MI HF parameters comparable to the human pathology. We conclude that the G&#246;ttingen minipig model is superior to the Landrace pig model to study the development of post-MI HF.

The overall goal of the current study is to present the techniques of induction of myocardial infarction (MI) and post-myocardial infarction heart failure (post-MI HF) in closed-chest, adult G&#246;ttingen minipigs and the characterization of post-MI HF model in G&#246;ttingen minipigs as compared to Landrace pigs.

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...

Transthoracic Echocardiography to Assess Post-Resuscitation Left Ventricular Dysfunction After Acute Myocardial Infarction and Cardiac Arrest in Pigs

One of the main causes of out-of-hospital cardiac arrest is acute myocardial infarction (AMI). After successful resuscitation from cardiac arrest, approximately 70% of patients die before hospital discharge due to post-resuscitation myocardial and cerebral dysfunction. In experimental models, myocardial dysfunction after cardiac arrest, characterized by an impairment in both left ventricular (LV) systolic and diastolic function, has been described as reversible but very little data are available in cardiac arrest models associated with AMI in pigs. Transthoracic echocardiography is the first-line diagnostic test for the assessment of myocardial dysfunction, structural changes and/or AMI extension. In this pig model of ischemic cardiac arrest, echocardiography was done at baseline and 2-4 and 96 hours after resuscitation. In the acute phase, the examinations are done in anesthetized, mechanically ventilated pigs (weight 39.8 &#177; 0.6 kg) and ECG is recorded continuously. Mono- and bi-dimensional, Doppler and tissue Doppler recordings are acquired. Aortic and left atrium diameter, end-systolic and end-diastolic left ventricular wall thicknesses, end-diastolic and end-systolic diameters and shortening fraction (SF) are measured. Apical 2-, 3-, 4-, and 5-chamber views are acquired, LV volumes and ejection fraction are calculated. Segmental wall motion analysis is done to detect the localization and estimate the extent of myocardial infarction. Pulsed Wave Doppler echocardiography is used to record trans-mitral flow velocities from a 4-apical chamber view and trans-aortic flow from a 5-chamber view to calculate LV cardiac output (CO) and stroke volume (SV). Tissue Doppler Imaging (TDI) of LV lateral and septal mitral anulus is recorded (TDI septal and lateral s&#39;, e&#39;, a&#39; velocities). All the recordings and measurements are done according to the recommendations of the American and European Societies of Echocardiography Guidelines.

Transthoracic echocardiography is the first-line diagnostic test for post-resuscitation left ventricular dysfunction and structural changes in a pig model of cardiac arrest.

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

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