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/pubmed?term=((Out-of-hospital+cardiac+arrest+does+not+affect+post-discharge+survival+in+patients+with+acute+myocardial+infarction%5BTitle%5D)+AND+%22Circulation+Reports%22%5BJournal%5D)">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/pubmed?term=((Immediate+coronary+angiography+in+survivors+of+out-of-hospital+cardiac+arrest%5BTitle%5D)+AND+%22The+New+England+Journal+of+Medicine%22%5BJournal%5D)">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/pubmed?term=((European+Resuscitation+Council+and+European+Society+of+Intensive+Care+Medicine+guidelines+2021%3A+post-resuscitation+care%5BTitle%5D)+AND+%22Intensive+Care+Medicine%22%5BJournal%5D)">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/pubmed?term=((Postresuscitation+myocardial+diastolic+dysfunction+following+prolonged+ventricular+fibrillation+and+cardiopulmonary+resuscitation%5BTitle%5D)+AND+%22Critical+Care+Medicine%22%5BJournal%5D)">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/pubmed?term=((Ventilation+with+argon+improves+survival+with+good+neurological+recovery+after+prolonged+untreated+cardiac+arrest+in+pigs%5BTitle%5D)+AND+%22Journal+of+the+American+Heart+Association%22%5BJournal%5D)">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/pubmed?term=((et+al+Recommendations+for+the+evaluation+of+left+ventricular+diastolic+function+by+echocardiography%3A+An+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging%5BTitle%5D)+AND+%22Journal+of+the+American+Society+of+Echocardiography%22%5BJournal%5D)">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/pubmed?term=((et+al+Recommendations+for+cardiac+chamber+quantification+by+echocardiography+in+adults%3A+an+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging%5BTitle%5D)+AND+%22Journal+of+the+American+Society+of+Echocardiography%22%5BJournal%5D)">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/pubmed?term=((Investigation+of+myocardial+stunning+after+cardiopulmonary+resuscitation+in+pigs%5BTitle%5D)+AND+%22Biomed+Environ+Sci%22%5BJournal%5D)">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/pubmed?term=((Cardiac+arrest+in+pigs+with+48+hours+of+post-resuscitation+care+induced+by+2+methods+of+myocardial+infarction%3A+A+methodological+description%5BTitle%5D)+AND+%22Journal+of+the+American+Heart+Association%22%5BJournal%5D)">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/pubmed?term=((Postresuscitation+treatment+with+argon+improves+early+neurological+recovery+in+a+porcine+model+of+cardiac+arrest%5BTitle%5D)+AND+%22Shock%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Resuscitation%22%5BJournal%5D)">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><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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2.8K 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 ...

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

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 ...

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, antiarrhythmic drugs administered intravenously can produce drops in systemic pressure in the already compromised cardiac patient. While performing open-heart procedures, surgeons will often either create a small port or form a pericardial cradle to create suitable fields for operation. This access yields opportunities for target pharmacological delivery (antiarrhythmic or ischemic preconditioning agents) directly to the myocardial tissue without undesired side effects.
We have developed a swine model for testing pharmacological agents for target delivery within the pericardial fluid. While fully anesthetized, each animal was instrumented with a Swan-Ganz catheter as well as left and right ventricle pressure catheters, and pacing leads were placed in the right atrial appendage and the right ventricle. A medial sternotomy was then performed and a pericardial access cradle was created; a plunge pacing lead was placed in the left atrial appendage and a bipolar pacing lead was placed in the left ventricle. Utilizing a programmer and a cardiac mapping system, the refractory period of the atrioventricular node (AVN), atria and ventricles was determined. In addition, atrial fibrillation (AF) induction was produced utilizing a Grass stimulator and time in AF was observed. These measurements were performed prior to treatment, as well as 30 min&#160;and 60 min&#160;after pericardial treatment. Additional time points were added for selected studies. The heart was then cardiopleged and reanimated in a four chamber working mode. Pressure measurements and function were recorded for 1 hr after reanimation. This treatment strategy model allowed us to observe the effects of pharmacological agents that may decrease the incidence of cardiac arrhythmias and/or ischemic damage, during and after open-heart surgery.

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

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 ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

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 ...

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic dobutamine stress echocardiography in humans, a reductionist investigation approach to unmask subtle changes in cardiac function has become possible. Here, we provide a protocol for using this technique in mice to facilitate expanded analysis of LV architecture and function in physiology and pathology enabling the observation of alterations in models of cardiac disease hidden in unstressed hearts. This investigation can be performed in one and the same animal and allows both, basal and pharmacologically stress-induced measurements. We outline detailed criteria for appropriate anesthesia, imaging-based LV analysis, consideration of intra- and interobserver variability, and obtaining positive inotrope response that can be attained in mice after intraperitoneal injection of dobutamine under near physiological conditions. To recapitulate the characteristics of human physiology and disease in small animal models, we highlight critical pitfalls in evaluation, e.g., a pronounced Bowditch effect in mice. To further meet translational objectives, we compare stress-induced effects in humans and mice. When used in translational studies, attention must be paid to physiological differences between mice and human. Experimental rigor dictates that some parameters assessed in patients can only be used with caution due to restrictions in spatial and temporal resolution in mouse models.

Left ventricular dysfunction constitutes the final common pathway for a host of cardiac disorders. We here provide a detailed protocol of transthoracic dobutamine stress echocardiography approach for comprehensive evaluation of the left ventricular function of mouse models of cardiac disease as well as cardiac phenotyping.

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

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Transthoracic Echocardiography in Mice

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

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

Myocardial Infarction and Functional Outcome Assessment in Pigs

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in Göttingen Minipigs and Landrace Pigs

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice