Myocardial Infarction and Functional Outcome Assessment in Pigs

Todos in vivo experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources. Experiments were approved by the local Animal Experimentation Committee.

1. Medication, Anesthesia, Venous Access, and Intubation

1. Medication and Anesthesia
   1. Premedication
      1. Start amiodarone 150 mg/kg 10 days prior to surgery to prevent arrhythmias. Continue amiodarone in a dose of 100 mg/kg from the day of the procedure until day 28. Reduce the dosage to 50 mg/kg on day 29 and continue until the end of the study.
      2. Start anti-platelet therapy, 1 mg/kg clopidogrel from day 3 prior to surgery and 4.5 mg/kg acetylsalicylic acid 1 day before surgery. Continue clopidogrel 1 mg/kg per day and acetylsalicylic acid 80 mg/day.
      3. Start pain medication 1 day prior to surgery by Fentanyl patch, 25 μg/hr for ~70 kg pigs. Continue the use of Fentanyl patches for 24 hr post-operatively to ensure adequate pain medication. Also, daily monitor for signs of putative discomfort (i.e. behavior, breathing, gait, mobility, etc.) to increase pain medication.
      4. Fast the animal for 12 hr, maintain unlimited access to water.
   2. Anesthesia
      1. To prevent unnecessary stress and discomfort, sedate the pig in its stable by intramuscular injection of a mixture of midazolam 0.4 mg/kg, ketamine 10 mg/kg, and atropine 0.014 mg/kg.
      2. Obtain venous access by cannulating the ear vein with an 18 G i.v. cannula. Induce anesthesia by intravenous administration of 5 mg/kg sodiumthiopental, and give 1,000/100 mg amoxicillin/clavulanic acid to prevent infections. The pig will receive 1,000/100 mg amoxicillin/clavulanic acid the day after surgery as antibiotic treatment.
      3. Intubate the pig by using an endotracheal tube (size 8.5 for pigs weighing ~70 kg). If necessary perform balloon-ventilation with a frequency of 12/min and transport the pig to the operating theater.
      4. At arrival on the operation theater, immediately start mechanical positive pressure ventilation with FiO₂ 0.50, 10 ml/kg tidal volume, and a frequency of 12/min under continues capnography.
      5. Start balanced anesthesia by continues intravenous infusion of a combination of midazolam 0.5 mg/kg/hr, sufentanil 2.5 microg/kg/hr and pancuronium bromide 0.1 mg/kg/hr. During the entire operation, continuously monitor ECG, arterial blood pressure, temperature and capnography to measure depth of anesthesia. For example, if a sinus tachycardia is present, check if pain medication and/or anesthesia are adequate. 
      6. Infuse 4.3 mg/kg of amiodarone in 500 ml venofundin 6% intravenously.
      7. Monitor heart rhythm by a 5 leads ECG.
      8. Shave and clean the neck and hind limb area.
      9. Insert a Foley catheter.

2. Transthoracic Echocardiography

1. Place the animal in the right lateral position. In landrace pigs such as Dalland pigs, only parasternal views (long and short axis) can be obtained. Due to the shape of the thorax, apical view acquisition is not feasible.
2. Orient and obtain parasternal long axis view in 2D (B-mode). Determine the LV dimensions at end diastole and end systole in M-mode.
3. Rotate the echo probe 90° clockwise whilst maintaining its parasternal position to acquire the LV short axis views at the levels of the mitral valve, papillary muscle and apex. The short axis view of the papillary muscle and apex may require placement of the echo probe one or two intercostal spaces lower relative to the position for the mitral valve short axis view.

3. Surgical Preparation and Vascular Access

1. Disinfect the surgical areas with iodine 2% and use sterile surgical drapes to cover the nonsterile parts of the pig.
2. Make a medial incision in the neck. Pass the linea alba to minimize muscle damage and bluntly approach the carotid artery and internal jugular vein next to the trachea.
3. Carefully isolate the carotid artery and internal jugular vein. Make sure the vagal nerve is undamaged. Place Vicryl 2-0 sutures around both vessels to gain vessel control. Achieve arterial access by cannulating the internal carotid artery with an 8F sheath using the Seldinger technique. Fix the sheath to the artery, make sure the artery is not fully occluded by the suture. Venous access can be acquired by cannulating the jugular vein with a 9F sheath also using the Seldinger technique. Before securing the sheath make sure the vein is ligated. Alternatively, the femoral artery can also be used for arterial access.
4. Administer 100 IE/kg heparin immediately after inserting the sheaths to inhibit thrombus formation.
5. For a stable and constant arterial pressure measurement, cannulate one of the smaller arteries in one of the hind limbs by making a small incision above the artery. The artery is found just under the skin, pulsations can be felt through the skin. Isolate the artery from its surrounding tissue. Place 2 Vicryl 2-0 sutures around the vessel, 1 proximal and 1 distal. Ligate the distal side and insert an 18 G i.v. cannula and secure tightly connect the pressure.

4. Invasive Pressure Volume Loop Analysis

1. Insert a Swann-Ganz catheter (SG) via the previously placed sheath in the internal jugular vein.
2. Connect a cardiac output device to the part of the SG that culminates in the proximal lumen.
3. Inject 5 ml of 0.9% saline into the proximal lumen of the SG and measure cardiac output; repeat this three times and average the indices.
4. Calibrate the PV system by using the previously determined cardiac output.
5. Insert the 7F conductance catheter via the carotid artery into the left ventricle under fluoroscopic guidance.
6. Select the largest segment present in the LV for volume measurements and perform a baseline scan under apnea.
7. After volume calibration is completed, record 10-15 beats under apnea.
8. Remove the SG and place a balloon catheter in the inferior caval vein at the level of the diaphragm under fluoroscopic guidance.
9. Perform preload reduction by inflating the balloon under apnea and record the corresponding PV-Loops.

5. Intracoronary Pressure and Flow Measurement

1. Dilute nitroglycerin in a concentration of 100 μg/ml and dilute adenosine in a concentration of 30 μg/ml.
2. Position an 8F guiding catheter in the ostium of the left coronary artery.
3. Place the combined pressure/flow wire in the proximal part of the left coronary artery.
4. Administer 200 μg of nitroglycerin intracoronary and normalize the distal pressure (Pd), measured by the wire, with the arterial pressure.
5. Place the wire in the mid part of the left anterior descending artery (LAD).
6. Start measuring baseline pressure and flow. Induce hyperemia by administering 60 μg of adenosine intracoronary, flush with 2 ml of saline and measure hyperemic pressure and flow. Wait for the flow to restore to baseline values. Repeat the measurement twice.
7. Infuse another 200 μg nitroglycerin intracoronary and repeat steps 1.6 and 1.7 for the left circumflex coronary artery.

6. Induction of MI

1. Place the intracardiac defibrillation catheter in the right ventricle using the venous sheath. The distal electrodes should be in the apex of the ventricle, the proximal electrodes in the atrium and/or superior caval vein. Connect the catheter to the defibrillator and set it to 50 J.
2. Measure the diameter of the LAD distal from the second diagonal (D2) in AP and LAO 30° view.
3. Choose an angioplasty balloon with a diameter according the diameter of the LAD distal from the D2 (Figure 1).
4. Position a guidewire through the guiding catheter distally in the LAD.
5. Advance the balloon catheter over the guidewire. Place the balloon distal from the D2.
6. Administer 30 IE/kg heparin.
7. Inflate the balloon until the pressure matches the right diameter of the LAD.
8. Check total occlusion of the LAD by angiography (Figure 1).
9. Cover the sterile working field and the wound in the neck with sterile drapes cloths. Free the chest from any coverage to make it available for chest compressions or transthoracic defibrillation.
10. Check the pressure in the balloon during the next 90 min and restore the pressure if necessary.
11. In case of ventricular fibrillation:
    1. Immediately start chest compressions with a frequency of 100/min.
    2. Administer 300 mg amiodarone intravenously as a fast bolus (~1 min).
    3. Start intracardiac defibrillation, give shocks of 50 J.
    4. After 5 unsuccessful shocks, restart chest compressions. Change intracardiac defibrillation to transthoracic defibrillation and shock with 150 J. In case of unsuccessful shock, change to 200 J.
    5. If necessary, administer another dose of 150 mg amiodaron and/or 1 mg adrenalin. Repeat adrenalin twice with intervals of 3-5 min, when necessary.
    6. Continue chest compressions, interspersed with transthoracic defibrillation

Finishing the Surgical Procedure (for Long Term Follow Up)

1. After 90 min check by angiography if the LAD is still fully occluded.
2. Administer another 30 IE/kg heparin and deflate the balloon. Check for reperfusion. Remove the deflated balloon with the guiding catheter from the carotid sheath.
3. Carefully remove the arterial sheath and clamp the carotid artery immediately with an anastomosis clamp (Figure 1). Use continues stitches (6-0 prolene) to close the carotid artery. Remove the clamp and check for leakages.
4. Remove the internal defibrillation catheter and remove the sheath from the internal jugular vein. Ligate proximal of the sheath entry.
5. Close the subcutis and skin of the neck in two layers using 2-0 Vicryl.

7. Cardiac Magnetic Resonance Imaging

1. Place the animal on the MRI table head first in the supine position under continuous anesthesia.
2. Place a dedicated phased-array cardiac coil over the chest of the animal.
3. For image planning obtain scout images in short axis and two-chamber long axis views.
4. Acquire ECG-gated steady-state free precession (SSFP) cine of short axis (from apex to base of LV) and two chamber long-axis views.
5. Late gadolinium enhancement (LGE) can be acquired using an inversion recovery 3D-turbo-gradient-echo-technique 15 min after double-dose i.v. bolus injection of a gadolinium based contrast agent.
6. Perform offline analysis with validated software of functional parameters. Assess left-ventricular ejection fraction (LVEF), LV mass, end diastolic volume, end systolic volume, stroke volume, cardiac output, and scar mass.

8. End of Study and Infarct Size

1. At the end of the study, follow Protocols 1-5 and 7 to acquire follow up measurements.
2. Make a median 30-40 cm incision from just below the suprasternal notch to a point just below the xiphoid process. Advance through the linea alba down to the sternum. Split the xiphoid and use Klinkenberg scissors to separate the posterior sternum from the pericardium with caution. After using the scissors bluntly continue further separation. Perform a sternotomy by e.g. using a hammer and Lebsch knife. Bone marrow bleeding is minimized by rubbing bone wax on the marrow. Open the thorax with a sternum retractor.
3. Enter the 3rd pleural space and locate the inferior caval vein in the mediastinum.
4. Humanely euthanize the animal by cutting the inferior caval vein under deep anesthesia. Remove blood with a suction device. Place a 9 V battery on the apex to induce ventricular fibrillation.
5. After excision of the heart, cut the right and left ventricle into five slices from base to apex and incubated in 1% triphenyl-tetrazolium chloride dissolved in 0.9% saline at 37 °C for 15 min. Next, wash the slices in 0.9% saline and photograph the slices from both sides.