A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

The procedures described here were approved by the National Heart, Lung, and Blood Institute Animal Care and Use Committee at the National Institutes of Health and conform to the policies outlined in The Public Health Service Policy on Humane Care and Use of Laboratory Animals, The Animal Welfare Act, and the Guide for the Care and Use of Laboratory Animals.

NOTE: The purpose of this surgical procedure is to produce an animal model of chronic myocardial ischemia that can be used to develop treatments that are clinically relevant for patients with severe CAD. This cannot be accomplished using an in vitro model.

1. Animals

1. Use male Yorkshire swine weighing between 12 and 15 kg.

2. Pre-surgical Procedures

1. For a prophylactic antibiotic, use amoxicillin and clavulanate potassium (15 mg/kg) orally twice a day beginning 24 h prior to surgery.
   NOTE: This oral antibiotic is started before surgery to make sure that the pig will swallow it and to also make sure that they will not have an allergic reaction to the medication post-operatively.
2. Withhold food and water from the pig 12 h prior to surgery.
3. On the day of surgery, anesthetize the pig with a cocktail of ketamine (33 mg/kg), midazolam (0.5 – 0.75 mg/kg), and glycopyrrolate (0.01 mg/kg) given intramuscular (IM).
4. Place a 20 G intravenous (IV) catheter in an ear vein and intubate the animal with a 7.0 Fr endotracheal tube.
   NOTE: Adjust the size of the IV catheter and the endotracheal tube as appropriate for the size of the pig.
5. Remove the hair (shave) on the left side of the pig from the shoulder to the umbilicus and from the abdominal midline to the vertebral column.
6. Administer the prophylactic antibiotics (piperacillin and tazobactam: 100 mg/kg IV) and pain medication (buprenorphine sustained release: 0.2 mg/kg subcutaneous (SC) or a fentanyl transdermal patch 25-50 µg/h).
   NOTE: The IV antibiotic is given because the animals are fasted for 12 h prior to surgery and do not receive the previously mentioned oral antibiotic during this time.
7. Transport the pig to the operating room and connect the endotracheal tube to the anesthesia machine equipped with a sidestream sensor to monitor the airway gases. Mechanically ventilate the pig using a tidal volume of 10 mL/kg as a starting point and do not exceed an airway pressure of 30 cm H₂O. Set the respiratory rate between 10-20 breaths per min (bpm) and adjust as needed to keep the end-tidal CO₂ (PetCO₂) levels between 28 and 35 mm Hg. Maintain the anesthesia with either isoflurane (1-3%) or sevoflurane (2-5%).
8. Position the pig right-side down on the operating table. Attach all necessary probes to monitor body temperature (BT), electrocardiogram (ECG), pulse oximetry (SpO₂), etc.
9. Prepare the surgical site via three alternating scrubs with 2% chlorohexidine and 70% alcohol (apply in a circular motion beginning with center and moving outward).
10. Drape the surgical site using aseptic technique²¹.

3. Surgical Procedure

1. Expose the heart through a left thoracotomy.
   1. Make a skin incision parallel to the 4^th and 5^th intercostal space about 8 – 9 cm in length using a no. 10 scalpel blade.
   2. Cut through the latissimus dorsi and serratus ventralis muscles using a pair of curved Metzenbaum scissors and Brown Adson forceps. Use electrocautery to maintain hemostasis as necessary.
   3. Enter the pleural cavity through the intercostal muscles between the 4^th and 5^th ribs with the Metzenbaum scissors cutting along the anterior aspect of the 4^th rib. To reduce the chance of accidentally damaging the lung, turn the ventilator off on the exhale just prior to entering the pleural cavity. Once the pleural cavity has been breached, turn the ventilator back on.
   4. Use a small Finochietto retractor to spread the ribs apart, exposing the heart.
2. Use Debakey forceps to grasp and lift the pericardium. Use Potts scissors to make a small hole in the pericardium, allowing air to enter the pericardial space. Continue the incision with the Potts scissors over the junction of the LAD and LCX arteries.
3. Use Babcock forceps to retract the left atrial appendage. Using Debakey and small right angle forceps, dissect the LCX artery from the surrounding tissue prior to or proximal to the 1^st obtuse marginal branch.
4. Place two vessel loops under the dissected LCX artery, one at each end. Hold a size 3.0 mm ameroid constrictor with the small right angle forceps and lift the vessel loops to gently guide the LCX artery through the opening of the constrictor. Gently rotate the constrictor so the opening is facing up. Remove the vessel loops. See Figure 1.
   NOTE: Adequate dissection of the artery is essential to prevent kinking. Select the appropriate sized constrictor for the individual animal. The appropriate sized constrictor will closely encircle the vessel without initially constricting the vessel. Domestic pigs weighing between 12 and 15 kg require ameroid constrictors sized 2.5 – 3.5 mm. The location of the ameroid constrictor on the vessel depends on the desired size of the ischemic area.
5. Re-approximate the pericardium and close with 4-0 polypropylene sutures.
6. Reestablish the negative thoracic pressure by inserting a 12 Fr chest tube or equivalent in the pleural space with the end exiting out between the layers of closure, generally two intercostal spaces posterior to thoracotomy site.
   NOTE: Observe the lung for atelectasis before closing the ribs. If any signs of underinflation are observed, re-inflate the lungs manually up to a pressure of 30 mm Hg.
7. Close the chest using 1 polypropylene sutures for the intercostal layer, 0 polypropylene sutures for the serratus and fascial layer, 2-0 polypropylene sutures for the subcutaneous layer, and 3-0 polypropylene sutures for the subcuticular layer. Staple or suture the skin.
8. Attach a three-way stop cock to the end of the chest tube and evacuate the air in the chest with a 40-60 cc syringe until a negative seal is achieved. Roll the animal onto its sternum or opposite side to facilitate removing all the air. Once the chest maintains negative pressure, remove the chest tube and suture the exit site. If animals are housed singly post-operatively, the incision can be bandaged for several days.

4. Post-operative

1. Following closure, infiltrate the incision along both sides of the thoracotomy site with 0.25% bupivacaine.
2. Wean the animal off the ventilator and turn off the anesthesia. Remove the endotracheal tube once the animal is breathing on its own and swallowing.
3. Monitor the animal until it is fully recovered (awake and sternal). There should be no abnormalities or arrhythmias on the ECG, the BT should be 38.7 – 39.8 °C, the SpO₂ should be 95 – 100%, and the respiratory rate should be 32 – 58 bpm.
4. Administer amiodarone (100 mg, orally twice a day) and clopidogrel bisulfate (75 mg, orally once a day) to prevent thrombosis and arrhythmias. Continue with amoxicillin and clavulanate potassium (15 mg/kg) orally twice a day for 10 days post-operation. Post-operative pain is controlled with buprenorphine SR  (sustained release) 0.2 mg/kg. SQ every three days.  The buprenorphine SR may be supplemented with carprofen (4.4 mg/kg) once and  then (2.2 mg/kg) PO or IM BID.
5. Keep all bedding and foraging material out of the animal's housing area until the incision is fully healed.