We are grateful to Judith Bagott for language editing.

All procedures involving animals and their care conformed with national and international laws and policies. Approval of the study was obtained from the institutional review board of the University of Milan and governmental institution (Ministry of Health approval no. 84/2014-PR). Data that support the findings of this study are available from the corresponding author on reasonable request. The experimental model and echocardiographic protocol diagrams are detailed in Figure 1 and Figure 2.
1. Animal preparation
<ol>
	<li>Fast male domestic pigs (weight 39.8 &#177; 0.6 kg) for 8 hours overnight before the experiment. Provide free access to water.</li>
</ol>
2. Induction of anesthesia and maintenance, antibiotic prophylaxis
<ol>
	<li>Induce general anesthesia by intramuscular injection of ketamine (20 mg/kg) followed by intravenous (iv) propofol (2 mg/kg). Check for sufficient depth of anesthesia by loss of jaw tone, loss of corneal reflex with muscular relaxation and need for mechanical ventilation.</li>
	<li>Insert a catheter into the right jugular vein and advance it into the superior vena cava.</li>
	<li>Maintain anesthesia with continuous iv infusion of propofol (4-8 mg/kg/h).</li>
	<li>Inject sufentanyl (0.3 &#181;g/kg) and then ampicillin (1 g) through iv.</li>
</ol>
3. Mechanical ventilation electrocardiographic and hemodynamic monitoring
<ol>
	<li>Place a cuffed tracheal tube so the pigs are mechanically ventilated with a tidal volume of 15 mL/kg and a FiO2 of 0.21.</li>
	<li>Adjust the respiratory frequency and maintain the end-tidal partial pressure of carbon dioxide (EtCO2) between 35-40 mmHg with an infrared capnometer.</li>
	<li>Shave the pig with a mechanical razor over the whole chest and left leg (where endovascular catheters for hemodynamic measurements will be inserted surgically).</li>
	<li>Apply frontal plane electrocardiogram (ECG) plaques over the shaved feet and lower abdomen, using three ECG pads. Place two of them on front feet, and the third on the left side of the abdomen.</li>
	<li>Insert a fluid-filled catheter into the right femoral artery for mean arterial pressure measurements and for arterial blood sampling for arterial blood oxygen tension (PO2), carbon dioxide tension (PCO2), and pH.</li>
	<li>Advance a 7 F pentalumen thermodilution catheter from the right femoral vein into the pulmonary artery to measure right atrial pressure, core temperature, and cardiac output.</li>
	<li>Insert a 5 F balloon-tipped catheter from the right common carotid artery. Advance it into the aorta, and then into the left anterior descending coronary artery beyond the first diagonal branch with the aid of angiography. Confirm the correct positioning by injection of radiographic contrast medium.</li>
	<li>Advance a 5 F pacing catheter from the right subclavian vein into the right ventricle (RV) to induce ventricular fibrillation (VF).</li>
</ol>
4. Baseline transthoracic echocardiography
NOTE: On average echocardiography takes 20-30 min. For TTE, a phased-array multifrequency 2.5 to 5 MHz probe is used, while ECG is continuously recorded. Sets of frames and cine-loops consisting of at least three consecutive cardiac cycles are stored for off-line analysis.
<ol>
	<li>Take mono-dimensional (M-mode) and two-dimensional (2D) echocardiographic short- and long-axis images at the aortic level and the LV level to assess wall thickness, aortic, atrial and LV dimensions, LV function, and segmental wall motion.
	<ol>
		<li>Take a 2D short-axis view at the aortic level. This view shows the left atrium (LA, bottom center), aortic valve (center), right atrium (bottom left), tricuspid valve (left), right ventricular outflow tract (top), and pulmonary valve (right). Place the cursor in the middle of the aorta and LA to record the respective M-mode images.</li>
		<li>Take a 2D parasternal long-axis view. This view allows the visualization of the aortic root and aortic valve leaflets, interventricular septum, LV and LA. The aorta must be in the same horizontal plane and in a continuum with the interventricular septum; the aortic leaflets need to be clearly visible. Place the transducer in the third or fourth left intercostal space, with its indicator toward the right flank, making small changes in the probe angulation in order to obtain a standardized view. 
		NOTE: Parasternal short and long-axis views are used to measure the width of the aortic root and the anteroposterior dimension of the LA. The M-Mode images can be taken from either a long-axis or a short-axis at the aortic valve level (see step 4.1).</li>
		<li>Take a 2D short-axis view of the LV at the papillary level. Use a short-axis view at the papillary or chordae level for LV dimension measurements; in this way, in a ventilated animal, it is easier to obtain a standardized image, compared with that in the long-axis view. 
		NOTE: The LV must appear circular and both papillary muscles need to be clearly visible. Papillary muscles are called, by convention, anterolateral and posteromedial. If the mitral leaflets are visible and the right ventricular free wall is not a continuum, the image is not standardized.</li>
		<li>Place the cursor in the middle of the LV and record an M-mode image of the LV at the papillary level.</li>
		<li>Repeat steps 4.1.3 and 4.1.4 looking for the sub-papillary and apical level of the LV.</li>
	</ol>
	</li>
	<li>Take a 2D apical 4-chamber view (AP4CH). The LV, the LA, the right ventricle (RV), and the right atrium (RA) are visible together with the mitral and tricuspid valves and the interatrial and interventricular septum. Position the probe at the level of the cardiac apex (fourth intercostal space; the marker on the probe needs to be oriented to the left). The structure that helps standardize the view is the interventricular septum, which should be displayed parallel to the ultrasound beam. This is possible by moving the transducer either medially or laterally. 
	NOTE: Foreshortening occurs when the imaging plane does not pass through the true LV apex, resulting in an oblique view of the LV cavity. Foreshortening underestimates LV volumes and overestimates LVEF. Foreshortening is avoided by changing the probe positions and/or moving it to a lower intercostal space and laterally. The LV long-axis needs to be larger than 4.8 cm in pigs with body weight 33-35 kg.</li>
	<li>Take an apical two-chamber view (AP2CH). From AP4CH, rotate the transducer 45-60&#176; counterclockwise direction; only the LA and LV must be visible, so avoid the interventricular septum and verify that the cursor passes in the middle of the LA and LV.</li>
	<li>Take an apical three-chamber (AP3CH) view or apical long-axis. From AP4CH, rotate the transducer 45-60&#176; counterclockwise. In AP3CH, the LV apex is visible, together with the anterior septum and posterolateral LV segments. The other visible structures are LVOT, LA, and the aortic valve.</li>
	<li>Take an apical five-chamber view (AP5CH). Start from the AP4CH view and angle the probe ventrally, and then laterally in order to visualize an oblique septum, the aorta with LVOT, LV, RV, and both atria.</li>
	<li>Pulsed Doppler (PW) echocardiography 
	NOTE: This method allows: (1) measurement of transvalvular flow velocities, cardiac output and stroke volume; (2) measurement of intervals, e.g., pulmonary artery acceleration time, and (3) evaluation of LV diastolic function. 
	NOTE: The aliasing phenomenon is avoided by lowering the baseline pulse repetition frequency or increasing it when new disturbing frequencies appear.
	<ol>
		<li>To obtain a standardized AP4CH view, use color Doppler and record a cine-loop.</li>
		<li>Place the PW sample volume at the cusp of mitral leaflets, and use color Doppler to place the cursor orthogonally to the mitral flow and aligned to the LV long-axis. Then, switch to PW and record at least three cardiac cycles.</li>
		<li>To obtain a standardized AP5CH view, use color Doppler and record a cine-loop with at least three cardiac cycles.</li>
		<li>Use color Doppler to place the cursor orthogonally to the aortic flow. Move the sample volume toward the aortic valve until the flow velocity accelerates. Record at least three cardiac cycles.</li>
	</ol>
	</li>
	<li>Use tissue Doppler imaging (TDI): from a 2D standardized AP4CH, PW TDI measures peak longitudinal myocardial velocity from a single segment. 
	NOTE: The principal limitation of TDI is its angle dependence. If the angle of incidence exceeds 15&#176;, there is about 4% underestimation of velocity.</li>
</ol>
5. Induction of myocardial infarction
<ol>
	<li>Inflate the balloon of the catheter in the left anterior descending coronary artery with 0.7 mL of air. Confirm the occlusion by the rapid progressive ECG ST-segment elevation5.</li>
</ol>
6. Cardiac arrest
<ol>
	<li>Cardiac arrest is defined as soon as ventricular fibrillation occurs. After 10 min of occlusion, ventricular fibrillation may occur spontaneously. Otherwise induce it through a pacing catheter with&#160;1 to 2 mA alternate current (AC) delivered to the right ventricular endocardium.</li>
	<li>Discontinue ventilation after the onset of ventricular fibrillation and deflate the balloon-tipped catheter5.</li>
</ol>
7. Cardiopulmonary resuscitation
<ol>
	<li>After 12 min of untreated ventricular fibrillation, start cardiopulmonary resuscitation (CPR) maneuvers. These include chest compression with the mechanical chest compressor and mechanical ventilation with oxygen (tidal volume 500 mL, 10 breaths per minute).</li>
	<li>After 2 min and every 5 min of CPR, inject epinephrine (30 &#181;g/kg) through the catheter positioned in the right atrium.</li>
	<li>After 5 min of CPR, attempt defibrillation with&#160;150 joule shock, using a defibrillator. 
	&#8203;NOTE: Successful resuscitation is defined as restoration of organized cardiac rhythm with mean arterial pressure &#62;60 mmHg5.</li>
</ol>
8. Post-cardiac arrest supportive care
<ol>
	<li>After successful resuscitation, maintain anesthesia and inflate the balloon in the left anterior descending coronary artery.</li>
	<li>Forty-five min after resuscitation, deflate the balloon and withdraw the left anterior descending coronary artery catheter5&#160;(Figure 1).</li>
	<li>If resuscitation is not immediately achieved, resume CPR and continue it for 1 min before subsequent defibrillation.</li>
	<li>If ventricular fibrillation recurs, treat it by immediate defibrillation.</li>
	<li>Use no supportive measures other than epinephrine.</li>
</ol>
9. Four-hour (h) observation
<ol>
	<li>After successful resuscitation, maintain anesthesia.</li>
	<li>Monitor the animals hemodynamically during the 4 h (short-term) observation period.</li>
	<li>Keep the animals&#39; temperature at 38 &#177; 0.5 &#176;C.</li>
	<li>At 2 h and 4 h post-resuscitation, repeat a complete echocardiographic examination, following the steps described in section 4. 
	NOTE: Broken ribs may be a consequence of chest compression. In this case, it is important to move the probe pressing it gently at the intercostal spaces.</li>
	<li>After a 4 h observation, extubate the pigs and return them to their cage.</li>
	<li>Give analgesia with butorphanol (0.1 mg/kg) by intramuscular injection (IM) or&#160;as recommended by the institutional animal care guidelines.&#160;</li>
	<li>Then, inject ampicillin (1 g) by IM.</li>
</ol>
10. 96-hours observation and euthanasia
<ol>
	<li>At the end of the 96 h post-AMI-cardiac arrest-ROSC (mid-term), re-anesthetize the animals (step 2) for echocardiographic examination (step 4). Monitor the ECG continuously as previously described (step 3).</li>
</ol>
11. Echocardiographic measurements
NOTE: Take all recordings and measurements according to the recommendations of the American and European Societies of Echocardiography Guidelines6,7. Send all echocardiographic recordings by a remote desktop connection to be stored in a local database for analysis. A cardiologist blinded to the study groups averages at least three measurements for each variable.
<ol>
	<li>For the aortic and LA diameter, measure from M-mode of the short-axis views at the level of the aortic sinuses using the leading-edge to leading-edge method.</li>
	<li>For LV outflow tract (LVOT) diameter, measure it 0.5-1 cm below the aortic cusp (proximal) from a parasternal long-axis view.</li>
	<li>For end-diastolic anteroseptal and posterior diastolic wall thickness at the papillary level, measure at end-diastole from the border between the myocardial wall and the cavity and the border between the myocardium wall and the pericardium.</li>
	<li>For the LV ejection fraction (LVEF), calculate it as: (LV end-diastolic volume (EDV)-LV end-systolic volume (ESV)) / (LVEDV) * 100. Define end-diastole as the first frame after mitral valve closure or the frame in which LV dimension is most frequently the largest. Define end-systole as the frame after the aortic valve closure or the frame where cardiac dimensions are smallest. Follow the tracings of LV area measurements at the boundary between the myocardium and the LV cavity. Measure LV areas and calculate LV volumes by modified Simpson&#39;s single plane rule from the AP4CH view.</li>
	<li>Repeat step 11.4 in AP2CH view for the biplane Simpson Method that uses the end-diastolic and end-systolic AP4CH and AP2CH views to calculate LV volumes and LVEF.</li>
	<li>For PW peak mitral inflow velocity (E vel) (cm/s), A velocities (A vel) and E-wave deceleration time (DT), measure these from the mitral flow spectrum (Figure 6).</li>
	<li>For TDI systolic s&#39; velocities and diastolic e&#39; and a&#39; velocities, measure these from the TDI spectrum images at the AP4CH view from the septal or lateral annulus and calculate averages at baseline and 96 h after coronary occlusion. 
	NOTE: The E vel to TDI-derived e&#39; velocity ratio (cm/sec) (E/e&#39;) is an indicator of diastolic function. The normal E/e&#8242; ratio should be 9 or less or more than 15; values between 8-14 present a not defined significance.</li>
	<li>Calculate stroke volume (SV) as the volume of blood pumped out of the left ventricle with each systole. The SV formula is: SV =&#160;&#960; * [LVOT diameter/2]2 * LVOT VTI.</li>
	<li>Calculate cardiac output (CO, mL/min) as the blood flow passing across the outflow tract every minute. It is calculated using the formula: CO = SV * HR.</li>
	<li>For analysis of LV regional motility, divide LV in 16 segments&#160;(visualized in the short axis views and/or the apical 2, 3, 4 chamber views). Score each segment using the following criteria: normo-kinesia (1 point) for normal wall thickening and excursion; hypokinesia (2 points) for reduced wall thickening and reduced wall excursion; akinesia (3 points) no wall thickening or wall excursion; dyskinesia (4 points); systolic outward or LV wall thinning includes aneurysmal wall motion, with eccentric bulges during both systole and diastole. Calculate the wall motion score index (WMSI) using the formula: total score/16. In a normo-kinetic ventricle, the WMSI is 1.</li>
</ol>
12. Statistical analysis
<ol>
	<li>Express data as mean &#177; SEM. Use one-way ANOVA, for repeated measurements and Tukey&#39;s post-hoc test. *p &#60; 0.05 vs baseline (BL); &#167; p &#60; 0.05 2 h post AMI-cardiac arrest-ROSC vs 96 h post AMI-cardiac arrest-ROSC; # p &#60; 0.05 4 h post AMI-cardiac arrest-ROSC vs 96 h post AMI-cardiac arrest-ROSC.</li>
</ol>

<ol>
	<li>Kubota, T., 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=Out-of-hospital+cardiac+arrest+does+not+affect+post-discharge+survival+in+patients+with+acute+myocardial+infarction.">Out-of-hospital cardiac arrest does not affect post-discharge survival in patients with acute myocardial infarction.</a> Circulation Reports. 3 (4), 249-255 (2021).</li><li>Spaulding, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+coronary+angiography+in+survivors+of+out-of-hospital+cardiac+arrest.">Immediate coronary angiography in survivors of out-of-hospital cardiac arrest.</a> The New England Journal of Medicine. 336 (23), 1629-1633 (1997).</li><li>Nolan, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=European+Resuscitation+Council+and+European+Society+of+Intensive+Care+Medicine+guidelines+2021:+post-resuscitation+care.">European Resuscitation Council and European Society of Intensive Care Medicine guidelines 2021: post-resuscitation care.</a> Intensive Care Medicine. 47 (4), 369-421 (2021).</li><li>Xu, T., 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=Postresuscitation+myocardial+diastolic+dysfunction+following+prolonged+ventricular+fibrillation+and+cardiopulmonary+resuscitation.">Postresuscitation myocardial diastolic dysfunction following prolonged ventricular fibrillation and cardiopulmonary resuscitation.</a> Critical Care Medicine. 36 (1), 188-192 (2008).</li><li>Fumagalli, F., 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=Ventilation+with+argon+improves+survival+with+good+neurological+recovery+after+prolonged+untreated+cardiac+arrest+in+pigs.">Ventilation with argon improves survival with good neurological recovery after prolonged untreated cardiac arrest in pigs.</a> Journal of the American Heart Association. 9 (24), 016494 (2020).</li><li>Nagueh, S. F., 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=et+al+Recommendations+for+the+evaluation+of+left+ventricular+diastolic+function+by+echocardiography:+An+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">et al Recommendations for the evaluation of left ventricular diastolic function by echocardiography: An update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> Journal of the American Society of Echocardiography. 29 (4), 277-314 (2016).</li><li>Lang, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al+Recommendations+for+cardiac+chamber+quantification+by+echocardiography+in+adults:+an+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">et al Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> Journal of the American Society of Echocardiography. 28 (1), 1-39 (2015).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+myocardial+stunning+after+cardiopulmonary+resuscitation+in+pigs.">Investigation of myocardial stunning after cardiopulmonary resuscitation in pigs.</a> Biomed Environ Sci. 24 (2), 155-162 (2011).</li><li>Vammen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+arrest+in+pigs+with+48+hours+of+post-resuscitation+care+induced+by+2+methods+of+myocardial+infarction:+A+methodological+description.">Cardiac arrest in pigs with 48 hours of post-resuscitation care induced by 2 methods of myocardial infarction: A methodological description.</a> Journal of the American Heart Association. 10 (23), 022679 (2021).</li><li>Ristagno, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postresuscitation+treatment+with+argon+improves+early+neurological+recovery+in+a+porcine+model+of+cardiac+arrest.">Postresuscitation treatment with argon improves early neurological recovery in a porcine model of cardiac arrest.</a> Shock. 41 (1), 72-78 (2014).</li><li>Rysz, S., 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=et+al+The+effect+of+levosimendan+on+survival+and+cardiac+performance+in+an+ischemic+cardiac+arrest+model+-+A+blinded+randomized+placebo-controlled+study+in+swine.">et al The effect of levosimendan on survival and cardiac performance in an ischemic cardiac arrest model - A blinded randomized placebo-controlled study in swine.</a> Resuscitation. 150, 113-120 (2020).</li></ol>

The authors have nothing to disclose.

A complete echocardiographic examination in a pig experimental model of AMI, cardiac arrest and resuscitation may give different information on the evolution of LV function and LV structural changes, although some amount of data are available in the literature5,8. In &#34;pure&#34; models of experimental cardiac arrest (restricted to induced ventricular fibrillation), myocardial function impairment reverses in the first days after ROSC, but little is known of wha...

Cardiac arrest frequently happens minutes after the onset of typical chest pain, and in some cases it is the first manifestation of coronary artery disease1. In fact, 48% of survivors of out-of-hospital cardiac arrest present occlusion of a coronary artery on angiography2. In addition, for patients who return to spontaneous circulation (ROSC) after cardiac arrest, cardiac dysfunction is one of the most important determinants of morbidity and mortality3.
Transthoracic echocardiography (TTE) is a non-invasive diagnostic and prognostic tool used in patients to assess post-resuscitation myocardial dysfunction, structural changes and/or AMI extension after ROSC and in the days that follow. In experimental ischemic and non-ischemic cardiac arrest models in pigs, TTE is frequently used to noninvasively serially assess cardiac systolic&#160;function, hemodynamics, and the response to therapy. In 2008, changes in diastolic dysfunction were described in terms of increase in mitral E velocity and tissue Doppler (TDI) e&#39; velocity ratio (E/e&#39;) and decrease in mitral E velocity and A velocity ratio (E/A) shortly after resuscitation in a non-ischemic pig model of cardiac arrest4.
The present study describes the different methodologic steps followed to assess left ventricular (LV) structure and LV systolic and diastolic function by TTE at different time-points in an ischemic pig model of cardiac arrest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aquasonic</td>
			<td>Parker</td>
			<td>-</td>
			<td>ultrasound gel</td>
		</tr>
		<tr>
			<td>Adult foam ECG disposable monitoring and stress testing, wet gel, non-invasive patien</td>
			<td>Philips</td>
			<td>40493E</td>
			<td>ECG electrode</td>
		</tr>
		<tr>
			<td>Bellavista 1000</td>
			<td>Bellavista</td>
			<td>MB230000</td>
			<td>ventilator with infrared capnometer</td>
		</tr>
		<tr>
			<td>ComPACS</td>
			<td>Medimatic SRL</td>
			<td>-</td>
			<td>local database and software</td>
		</tr>
		<tr>
			<td>CX50</td>
			<td>Philips</td>
			<td>-</td>
			<td>Echocardiographic machine</td>
		</tr>
		<tr>
			<td>InTube Tracheal tube</td>
			<td>Intersurgical Ltd</td>
			<td>8040080</td>
			<td>cuffed tracheal tube</td>
		</tr>
		<tr>
			<td>LUCAS2</td>
			<td>Phisio-Control Inc</td>
			<td>-</td>
			<td>mechanical chest compressor</td>
		</tr>
		<tr>
			<td>MRx defibrillator</td>
			<td>Philips</td>
			<td>-</td>
			<td>defibrillator</td>
		</tr>
		<tr>
			<td>S5-1</td>
			<td>Philips</td>
			<td>-</td>
			<td>Phased array probe</td>
		</tr>
		<tr>
			<td>Swan-Ganz catheter 2 lumen 5fr</td>
			<td>Edwards</td>
			<td>110F5</td>
			<td>for the coronary artery occlusion</td>
		</tr>
		<tr>
			<td>Swan-Ganz catheter 2 lumen 7fr</td>
			<td>Edwards</td>
			<td>111F7</td>
			<td>for mean arterial pressure measurement</td>
		</tr>
		<tr>
			<td>Swan-Ganz catheter for thermodiluition 7fr</td>
			<td>Edwards</td>
			<td>131F7</td>
			<td>to measure right atrial pressure, core temperature and cardiac output</td>
		</tr>
	</tbody>
</table>

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.

Twelve pigs underwent coronary artery occlusion followed by 12 min of ventricular fibrillation and 5 min of CPR. Eight pigs were successfully resuscitated, and seven survived at 96 h post AMI-cardiac arrest-ROSC. All echocardiographic variables at different time-points during the study are summarized in Table 1.
Changes in heart rate (HR) and systolic echocardiographic parameters 
HR increased significantly at 2 h and 4 h post-AMI-cardiac arrest-ROSC comp...

Watch this Scientific Journal Video about Transthoracic Echocardiography to Assess Post-Resuscitation Left Ventricular Dysfunction After Acute Myocardial Infarction and Cardiac Arrest in Pigs at JoVE.

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

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

One of the main causes of out-of-hospital cardiac arrest is acute myocardial infarction (AMI). After successful resuscitation from cardiac arrest, ...

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2.7K Views.  Istituto di Ricerche Farmacologiche Mario Negri IRCCS. Transthoracic echocardiography is the first-line diagnostic test for post-resuscitation left ventricular dysfunction and structural changes in a pig model of cardiac arrest.

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

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

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 ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

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 ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

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

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

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

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

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

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

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

Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the examiners&#39; expertise. Speckle tracking Echocardiography (STE) is a promising but technically challenging method that can be used to quantitatively assess regional and global systolic and diastolic myocardial performance. Myocardial strain and strain rate can be measured in all three dimensions&#160;&#8212; radial, circumferential, longitudinal &#8212; of myocardial deformation. Standard cross-sectional two-dimensional B-mode images are recorded and subsequently postprocessed by automated continuous frame-by-frame tracking and motion analysis of speckles within the myocardium. Images are recorded as digital loops and synchronized to a 3-lead EKG for timing purposes. Longitudinal deformation is assessed in the apical 4-, 3-, and 2-chamber views. Circumferential and radial deformation are measured in the parasternal short axis plane.
Optimal image quality and accurate tissue tracking are paramount for the correct determination of myocardial performance parameters. Utilizing transthoracic STE in a healthy volunteer, the present article is a detailed outline of the essential steps and potential pitfalls of quantitative echocardiographic myocardial deformation analysis.

Speckle tracking echocardiography is an emerging diagnostic imaging technique for the quantitative assessment of global and regional myocardial performance. Standard view echocardiographic motion images are recorded and deformation parameters are subsequently measured by automated continuous frame-by-frame tracking and motion analysis of speckles within the B-mode images of the myocardium.

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the ...

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 ...

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 ...