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Summary

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.

Abstract

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.

Introduction

Heart failure with reduced ejection fraction (HFrEF) accounts for about 50% of all heart failure cases, affecting an estimated 1 – 2% of people in the western world¹. Its most prevalent cause is acute myocardial infarction (AMI). As acute mortality after AMI has declined significantly due to increased awareness and improved treatment options, emphasis has shifted towards its chronic sequelae; the most prominent being HFrEF^2,3. Together with increasing health care costs⁴, the growing epidemic of heart failure stresses the need for novel diagnostics and therapies, which can be studied in a highly translational porcine model of adverse remodeling after AMI as previously described⁵.

Both, determinants (e.g., infarct size) and functional assessments (e.g., echocardiography) of adverse remodeling are often used for efficacy testing of new therapeutics, indicating the need for reliable and relatively inexpensive methods. The aim of the current paper is to address this need by introducing important and reliable outcome measures for efficacy testing in a pig model of acute myocardial infarction. These include infarct size (IS) in relation to area at risk (AAR), 3D transesophageal echocardiography (3D-TEE) and detailed admittance-based pressure-volume (PV) loop acquisition.

Infarct size is the main determinant of adverse remodeling and survival after AMI⁶. Although timely reperfusion of ischemic myocardium may salvage reversibly injured cardiomyocytes and limit infarct size, reperfusion itself causes additional damage through the generation of oxidative stress and a disproportionate inflammatory response (ischemia-reperfusion injury (IRI))⁷. Hence, IRI has been identified as a promising therapeutic target. The ability of novel therapeutics to decrease infarct size is quantified by assessing infarct size in relation to the area at risk (AAR). AAR quantification is mandatory to correct for inter-individual variability in coronary anatomy of animal models, as a larger AAR leads to a larger absolute infarct size. Since infarct size is directly related to cardiac performance and myocardial contractility, variations in AAR can influence study outcome measures irrespective of treatment modalities⁸.

Three-dimensional transesophageal echocardiography (3D-TEE) is a safe, reliable and, most importantly, clinically applicable inexpensive method to measure cardiac function non-invasively. Whereas transthoracic echocardiography (TTE) images are limited to 2D parasternal long- and short-axis views in pigs⁹, 3D-TEE can be used to obtain complete 3-dimensional images of the left ventricle. Therefore, it does not require mathematical approximations of left ventricular (LV) volumes such as the modified Simpson's rule¹⁰. The latter falls short of correctly estimating LV volumes after LV remodeling due to the lack of cylindrical geometry¹¹. Moreover, 3D-TEE is preferable over epicardial echocardiography as it does not require surgical interventions, which have been observed to exert cardioprotective effects in the present model¹². Although the use of 2D-TEE for the assessment of myocardial function has been described before^13,14, limitations regarding ventricular geometry are similar to those observed in 2D-TTE and depend on the extent of LV remodeling. Hence, the larger the infarct (and thus the higher the probability of heart failure), the more likely 2D measurements become flawed by incorrect geometrical assumptions and the higher the need for 3D techniques.

Nonetheless, most imaging modalities are limited in their ability to assess intrinsic functional properties of the myocardium. PV loops provide such relevant additional information and their acquisition is therefore described in detail below.

Protocol

All animal experiments were approved by the Ethical Committee on Animal Experimentation of the University Medical Center Utrecht (Utrecht, the Netherlands) and conform to the 'Guide for the care and use of laboratory animals'. NOTE: The protocol to perform a closed-chest balloon occlusion is not part of the current manuscript and is described in detail elsewhere5. In short, pigs (60 – 70 kg) are subjected to 75 min transluminal balloon occlusion of the midportion of the left anterior descending artery (LAD). Both, three-dimensional transesophageal echocardiography (3D-TEE) and pressure-volume (PV) loop measurements can be performed at baseline, short-term and long-term follow-up. Note that these measurements are considered unreliable in the first hours after myocardial infarction due to frequent arrhythmias in this phase. Infarct size (IS) and area-at-risk (AAR) measurements are preferably assessed at short-term follow-up (24 – 72 hr)15,16, since changes in microvasculature and secondary myocardial scar thinning culminate in less reliable results. Infarct size staining is performed using 2,3,5-triphenyltetrazolium chloride (TTC) (CAUTION, irritant), which is considered highly reproducible and relatively inexpensive. TTC is a white powder that colorlessly dissolves in saline. Upon contact with various dehydrogenases, it is converted to a brick red color. Thereby, it discriminates between viable (red) and dead myocardial tissue (white). For an overview on both invasive and non-invasive infarct size determination, readers are directed to a comprehensive review on this subject17. Figure 1 shows the timeline including anesthesia, surgical preparation and primary outcome measurements used in this study. 1. Medication and Anesthesia Ensure that the animal does not eat or drink for at least 5 hours prior to the procedure. Pre-treatment, anesthesia and post-operative pain treatment protocols have been described in detail elsewhere5. In short, the day prior to surgery a buprenorfine patch (5 µg/hr) is applied to the skin that is active for seven days to limit post-operative pain. On the day of surgery, sedate pigs by intramuscular injection of 0.4 mg/kg midazolam, 10 mg/kg ketamine and 0.014 mg/kg atropine. Wait for approximately 10 – 15 min. Insert an 18 G cannula in one of the ear veins and administer 5 mg/kg sodium thiopental to induce anesthesia. Intubate the pig using an endotracheal tube (size 8.5 for pigs of 60 – 70 kg). If necessary, perform balloon-ventilation (frequency 12/min) and transport the pig to the operating theater. Upon arrival in the operation theater, start mechanical positive pressure ventilation with FiO2 0.50, 10 ml/kg tidal volume and a frequency of 12/min using continuous capnography recording. Start balanced anesthesia by continuous intravenous infusion of a combination of midazolam (0.5 mg/kg/hr), sufentanil (2.5 μg/kg/hr) and pancuronium (0.1 mg/kg/hr). Confirm anesthesia by testing the corneal reflex and monitoring the breathing pattern (e.g., spontaneous breathing in combination with mechanical ventilation indicates incomplete anesthesia). Use vet ointment on the eyes to prevent dryness while the animal is under anesthesia. 2. 3D Transesophageal Echocardiography (TEE) To allow for heart rate monitoring and data acquisition, connect the animal to the 5 leads ECG on the echocardiography machine. Place the animal in the right lateral position. Make sure the probe is straight and flexible at the tip by unlocking the operating piece. Open the pig's mouth and carefully insert the echo probe in the esophagus. If necessary, use a laryngoscope for visualization. Be careful to avoid ending up in the normal anatomic pharyngeal pouch, resembling a Zenker's diverticulum18. Insert the probe for 50 – 60 cm (measure from the tip of the snout). Slowly rotate the probe and flex the head to a left anterolateral position to visualize the heart (Figure 2A – B). Make sure all walls are clearly visible. Use the "3D full volume" option on the display of the echocardiography machine to display two perpendicular images of the left ventricle as shown in Figure 2C – D. Then maximize the sector width that is being acquired by selecting "FV Opt Volume". Pause ventilation by temporarily switching off mechanical ventilation and press "Acquire" to obtain full volume measurements. After echo acquisition, make sure the tip is flexible by unlocking the operating piece. Then slowly remove the probe from the animal. NOTE: Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered. Perform offline analysis with validated software as described previously19. 3. Admittance-based Pressure-volume Loop Acquisition Pre-soak the sensing tips of the 7 F tetra-polar admittance catheter in 0.9% saline (room temperature to 37 °C) for a minimum of 20 min to ensure proper hydration and minimal baseline pressure drift during the experiment20. Administer medication and anesthesia as described in section 1. Perform surgical preparation and obtain vascular access as described previously5. In short, shave and clean the neck. Disinfect the surgical area with iodine 2% and cover the non-sterile parts of the pigs with sterile surgical drapes. Make a medial incision in the neck to expose the carotid artery and internal jugular vein. Insert an 8 F sheath into the carotid artery and a 9 F sheath into the jugular vein. Insert a Swan-Ganz (SG) catheter through the 9 F sheath in the jugular vein and wedge it in a small pulmonary artery by inflating the balloon at the tip of the catheter. After adequate placement in the peripheral part of the lung, deflate the balloon. Connect the SG to an external cardiac output device. Attach a 20 ml syringe containing 0.9% sterile saline to the injection port that connects to the lumen with the most proximal debouchment. Measure cardiac output by rapid infusion of 5 ml 0.9% saline (room temperature) and obtain heart rates to calculate stroke volume (SV). Repeat this procedure three times and calculate the average SV. NOTE: Cardiac output is (automatically) calculated using the Stewart-Hamilton thermodilution equation and is based on temperature changes in the pulmonary artery upon infusion of room temperature saline21. Remove the SG catheter. Insert an 8 F Fogarty catheter through the 9 F sheath in the jugular vein and position it in the inferior vena cava. Calibrate the pressure signal of the PV loop catheter using the "Course" and "Fine" button, while the tip remains in 0.9% saline. Then input the measured SV into the system. Advance the PV loop catheter through the 8 F sheath in the carotid artery and center the tip in the left ventricle (LV) under fluoroscopy. Select the largest adequately placed-segment by plotting the raw conductance signal against the pressure signal. Ensure that the pressure-conductance loops are of rectangle shape. Phase signal is expected to show a sinus trace with values between 3 and 5 degrees. Pause ventilation and perform a baseline scan to convert Conductance to Volume. Accept the baseline data by pressing "Continue" when the signals are stable (no arrhythmias), heart rate is equal to ECG or pressure derived heart rates and end-systolic (ES) / end-diastolic (ED) conductance are adequately sensed by the system20. NOTE: The latter can be verified by plotting the raw conductance signal against the pressure signal and comparing ES / ED conductance values derived from the baseline scan to real-time conductance. If any of the requirements above is not met, repeat the procedure. Acquire baseline pressure-volume loops by recording 10 – 12 consecutive beats during apnea by pausing ventilation. Inflate the Fogarty catheter under fluoroscopic guidance to reduce preload and record 10 – 12 consecutive beats as described above. Make sure systolic blood pressure remains >60 mmHg and no arrhythmias interfere with the measurements. Remove the Fogarty and PV loop catheters. Keep recording arterial pressure before and during removal of the PV loop catheter to enable correcting for pressure drift (i.e., ex vivo pre- and post-procedural baseline pressure difference). NOTE: Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered. Perform offline analysis of geometrical measurements and functional parameters with validated software22. 4. Area At Risk (AAR) and Infarct Size (IS) Quantification Dissolve 1.00 g Evans blue (CAUTION23, toxic) in 50 ml 0.9% saline, fill two 50 ml Luer lock syringes with 20 ml and 30 ml of 2% Evans blue solution respectively and keep at room temperature. NOTE: Work in a fume hood and wear a dust mask to limit exposure to hazardous dusts and use gloves and protective glasses to prevent contact from skin and eyes. Taking similar precautions, dissolve 1% 2,3,5-triphenyl-tetrazoliumchloride (TTC) (CAUTION, irritant) in 37 °C 0.9% saline and keep at 37 °C. Surgically prepare the animal to obtain vascular access to both carotid arteries. Perform a sternotomy to allow for direct visualization of the effect of in vivo Evans blue infusion5. Insert a 7 F and an 8 F introducer sheath in the respective carotid artery. Alternatively, insert both introducer sheaths in one single carotid artery or use one of the femoral arteries for one of both guiding catheters. Connect two standard Y-connectors to a 7 F JL4 and an 8 F JL4 guiding catheter respectively. For a femoral approach, use a JR4 for the right coronary artery (RCA) and a JL4 for the left main coronary artery (LCMA). Connect an additional three-way tap with 10 cm extension to both Y-connectors. Administer 100 IU/kg heparin. Position the 8 F JL4 guiding catheter in the ostium of the LMCA via one of two introducer sheaths. Using a 0.014" guidewire, advance a coronary dilatation catheter through the LCMA catheter and position the balloon at the site where coronary occlusion was performed during MI induction. Do not inflate yet. Position the second 8 F JL4 guiding catheter in the ostium of the RCA via the second introducer sheath. Perform a coronary angiography (CAG) by infusing contrast agent under fluoroscopy to confirm correct positioning of both guiding catheters and the balloon in the coronary arteries, using anteroposterior and LAO 30° views. Attach the two 50 ml syringes containing 30 ml (LCMA) and 20 ml (RCA) 2% Evans blue to the respective three-way taps attached to the Y-connectors on the guiding catheters. Inflate the balloon and confirm occlusion of the coronary artery by CAG. Only when the balloon completely blocks the passage of any contrast agent, inject Evans blue dye through both guiding catheters (5 ml/s) while the balloon is inflated. Directly after the completion of Evans blue infusion, induce ventricular fibrillation by placing a 9 V battery on the non-infarcted part of the heart. Incise the caval vein to release pressure and make sure a suction unit is available to allow for drainage of blood. Deflate the balloon, retract it together with both guiding catheters and explant the heart by dissecting surrounding membranes. A transverse cut through the large vessels (i.e., aorta, pulmonary artery/veins) allows for complete explantation. Rapidly wash off blood and superfluous dye on the exterior surface and in the cardiac cavities using 0.9% saline. Carefully dissect the left ventricle and make cuts in 5 equal 10 mm thick sections from apex to base, in a plane parallel to the atrioventricular (AV) groove. Photograph both sides of all five slices separately under ambient light conditions, as a possible Evans blue washout may occur in the subsequent step. For calibration, make sure a ruler is present in the image. Incubate for 10 min in 1% TTC solution at 37 °C, turning the sections around after 5 min for equal staining. Again, photograph both sides of all five slices separately under ambient light conditions and make sure a ruler is visualized in the image for calibration. Weigh all slices. Use software suitable for the analyses5. When using ImageJ (version 1.47), click the "Straight line" button. Now, draw a straight line with a known distance using the ruler in the image (e.g., 5 cm). Click "Analyze" -> "Set scale" and enter the distance in the box "Known distance". This procedure allows for calibration of distance in pixels to SI units of length. Using the "Polygon selections"button, select the total area that corresponds to the LV myocardium in the present image, click "Analyze" -> "Measure" to acquire measurements. Perform this procedure for both sides of each slice of myocardium, and average per slice. Multiply by the weight of the slice proportional to the total weight of all five slices and average these measurements for all slices. Perform similar measurements for area at risk (AAR) and infarct size (IS). Divide IS/AAR, AAR/LV and IS/LV and multiply by 100% to obtain respective outcome measurements5.

Representative Results

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

Discussion

Cardiac remodeling is largely depending on myocardial infarct size and the quality of myocardial infarct repair^6,26. To assess the former in a standardized manner, the present manuscript provides an elegant method of in vivo infusion of Evans blue combined with ex vivo TTC staining, which has been validated and extensively used^8,16,27,28. This method allows for quantification of the area at risk (AAR) and infarct size in relation to AAR¹⁶. The current approach …

Materials

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