Ex Situ Heart Perfusion: A Technique to Preserve Isolated Porcine Heart Function

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Pre-surgical Preparations

1. Place the organ chamber properly on the apparatus cart and install the silicon support membrane inside the organ chamber. The aorta (Ao), pulmonary artery (PA) and left atrium (LA) connection points can be seen in Figure 1.
2. Install the ex situ heart perfusion (ESHP) tubing network (represented in Figure 2A, B) oxygenator and filter. Attach the heat exchanger water lines and the sweep gas tubing to the oxygenator.
3. Place the flow probes for measuring coronary sinus/PA and LA flow on the corresponding tubing.
4. Connect the Ao and LA pressure transducers to the representative lines on the circuit.
5. Ensure that all the tubing connections are firmly attached and all the stopcocks and luer locks are properly closed on the unattached sites.
6. Prime the circuit with 750 mL of modified Krebs-Henseleit buffer (NaCl, 85; KCl, 4.6; NaHCO₃, 25; KH₂PO, 1.2; MgSO₄, 1.2; glucose, 11; and CaCl₂, 1.25 mmol/L) containing 8% albumin. De-air the Ao and LA pumps by positioning the pump outlet above the inlet so that the air leaves the pump chamber (Figure 3). The solution typically does not need to be oxygenized before the start of the perfusion.
7. Initiate the software after the Ao and LA pumps are de-aired and the circuit is primed.

2. ESHP Software Initialization and Adjustments

NOTE: The ESHP apparatus used here is equipped with a custom software program to allow control of pump speed in order to achieve and maintain desired LA and Ao pressures. The software also analyzes functional parameters and provides a visual evaluation of pressure waveforms (Figure 4).

1. To start the ESHP program, click on the program shortcut on the monitor.
2. In the "setting" page, click "initialize". The initializing message will appear on the board (Figure 5).
3. On the same page, zero the flow sensors clicking the "zero LA flow" and "zero PA flow". The message will appear on the board.
4. Adjust the height of the pressure transducers to the height of the silicon support. To zero the pressure transducers, open the Ao and LA pressure transducers (and any other transducers set to check the pressure) to the atmosphere, then click "zero all pressures" button. The message will appear on the board.
5. In the "main" page, increase the Ao pump speed gradually up to the point where flow from the Ao cannula appears in the organ chamber. In the present system, this is achieved with 900–1000 revolutions per minute (RPM).
6. Add 750 mL of blood to the perfusate solution to bring the total perfusate volume to 1.5 L (as described in the "Surgery, harvesting blood, and heart procurement" section) and then increase the LA pump PRM (800-900 RPM) so that no air remains in the LA cannula or the LA tubing beneath the silicone support membrane.
7. After initializing the controlling software and de-airing of the ESHP apparatus, donor heart procurement may proceed.

3. Preparations and Anesthesia

1. Administer 20 mg/kg of ketamine and 0.05 mg/kg of atropine intramuscularly for premedication.
2. Transfer the pig to the surgical suite and place the pig on the operating table with tabletop heating to maintain normothermia.
3. Titrate oxygen flow rate for mask induction according to animal weight and the anesthetic system. For the closed-circle anesthetic circuits the oxygen flow should be 20 – 40 mL/kg.
4. Turn on isoflurane to 4–5%; after one or two minutes this may be reduced to 3%.
5. Evaluate the depth of anesthesia. The pig is in the surgical plane if there is no withdrawal reflex in response to noxious stimulus.
6. After confirmation of the appropriate depth of anesthesia, proceed to intubation.
7. Place the pulse oximeter probe on the tongue (preferred) or ear. The oxygen saturation measured by pulse oximetry should remain above 90%.
8. Shave patches of hair off on the left and right elbow regions, and left stifle. Wash off skin oils with soap and water, rinse with rubbing alcohol and dry completely. Place the ECG contacts. Avoid lead wire interference with the surgical site. Connect the leads to the correct locations.
9. To maintain the anesthesia, adjust oxygen flow (20–40 mL/kg) and inhalant gas rate (1–3%). The heart rate should be 80–130 beats/min. Respiration rate should be 12–30 breaths/min.
10. Shave, wash and aseptically prepare the incision site.

4. Blood Collection and Heart Procurement

1. Evaluate the anesthesia level every minimum every 5 min to confirm the surgical plane (no pedal reflex and no blink reflex, no response to painful stimuli).
2. Perform a median sternotomy.
   1. Identify jugulum and xiphoid as landmarks.
   2. Using electrocautery, develop the midline between the landmarks by dividing the subcutaneous tissue and the fascia between the fibers of the pectoralis major muscle.
   3. Mark the midline along the sternal bone with the cautery. Perform sternal osteotomy with an electric or air-powered saw. To prevent creating injuries to the underlying structures (e.g., pericardium and brachiocephalic vein, and innominate artery), proceed gradually with the saw.
   4. Retract the sternum gradually, using a sternal retractor. To avoid excessive tension and vascular injury, do not place the retractor too far cranially.
   5. Free the sternopericardial ligaments from the posterior surface of the sternum using cautery.
   6. Open the pericardium with a Metzenbaum scissor and fix the pericardial edges to the sternum using 1-0 silk suture.
3. Extend the midline incision cranially by 2–3 cm and expose the right common carotid artery and internal jugular vein.
4. Obtain proximal and distal control of the vessels by encircling the vessels with silk ties (2-0).
5. Tie the cranial encircling ties on each vessel.
6. Open the anterior 1/3 of each vessel with an 11-blade and then insert a 5–6 F sheath into each vessel. Tie the caudal encircling tie around each vessel to secure the respective sheathes.
7. Monitor the arterial and central venous pressures by connecting each sheath to a pressure transducer.
8. Deliver 1,000 U/kg heparin intravenously.
9. Place a 3-0 polypropylene purse-string suture around the right atrial appendage and secure it with a snare.
10. Inside the purse-string suture, create a 1 cm incision on the appendage using an 11 blade. Insert a two-stage venous cannula (28/36 FR) inside the incision and position the distal tip in the IVC. Secure the cannula by tying snare to the venous cannula. Control the outlet of the cannula with a tubing clamp.
11. From the two-stage venous cannula placed in the right atrium, collect 750 mL of whole blood from the pig gradually over a period of 15 min into an autoclaved glass container and simultaneously replace the volume with 1 L of an isotonic crystalloid solution such as Plasmalyte A.
12. Add the blood to the perfusion circuit (which has been previously primed with 750 mL Krebs-Henseleit buffer containing 8% albumin) to reach a final volume of 1.5 L of perfusate. The perfusate is a 1:1 combination of Krebs-Henseleit containing 8% albumin solution and whole blood from the donor animal.
13. Place a cardioplegia needle (14–16 F) in the ascending Ao and secure it with a snare.
14. Connect the cardioplegia cannula to the cardioplegia bag and add 100 mL of blood to 400 mL of cardioplegia (St. Thomas Hospital Solution) to reach a final volume of 500 mL blood cardioplegia.
15. Euthanize the pig by exsanguination. If intending to add more blood to the perfusate after starting of the perfusion (according to the aims of the study), collect the blood and add 10–30 U/mL of heparin to it and store it in a glass container or a plastic bag at 4 °C for short durations (hours).
16. Cross-clamp the ascending Ao with an Ao clamp and deliver the cardioplegic solution into the Ao root.
17. After delivery of the cardioplegic solution is completed, remove the cross-clamp and perform the cardiectomy.
    1. For ease of attaching the Ao and PA to their representative cannula, partially dissect the ascending Ao from the PA using a Metzenbaum scissors.
    2. Transect the superior and inferior vena cava, leaving roughly 1 cm of length on each.
    3. Separate the heart from the posterior mediastinum by transecting the pulmonary veins.
    4. Excise the heart ensuring all of the Ao arch vessels are procured along with a segment of descending Ao. Preserve up to the PA bifurcation.
18. Weigh the empty heart. The amount of weight gain over the ex situ preservation interval can be used as a metric for organ edema.

5. Placement of the Heart onto the ESHP Apparatus and Initiation of Perfusion

1. Trim excess tissue around the LA with a Metzenbaum scissor and cut between the pulmonary veins to create a common orifice.
2. Place a purse-string suture around the LA orifice using a 3-0 polypropylene suture.
3. Suture and close the inferior vena cava with a 3-0 polypropylene suture. Leave the superior vena cava open at the beginning of the perfusion to ensure the right ventricle (RV) remains decompressed until the perfusate is warmed and an organized rhythm is achieved.
4. Place the LA cannula into the LA orifice and secure it with a snare (Figure 6).
5. Gently squeeze the ventricles to de-air the heart” from 5.5. and add it to 5.7. as Increase the Ao pump speed to 1600 RPM as gently squeezing the heart. The remaining air in Ao root will be ejected through the innominate and subclavian branches.
6. Attach the Ao to the Ao cannula embedded in the silicone membrane. Secure the Ao around the cannula with a silk tie. Trim the Ao to achieve a proper lie without tension or kinking.
7. Increase the Ao pump speed to 1600 RPM. The remaining air in Ao root will be ejected through the innominate and subclavian branches.
8. Connect the Ao purge line to the innominate artery. Secure the connection with a silk tie.
9. Snare the left subclavian artery orifice with a silk tie. Secure the closure with a snare and snap. Through the orifice of the subclavian artery, place an introducer sheath (5f). Ensure that the length of the catheter and its orientation is properly adjusted so that it does not interfere with Ao valve function.
10. Connect the Ao pressure transducer to the introducer sheath side port.
11. Read the Ao pressure on the monitor. Adjust the Ao pump speed to reach a mean pressure of 30 mm Hg. At this point (Time 0), the perfusion will have started in the non-working mode (Langendorff mode) and appearance of a dark deoxygenated perfusate in the PA line is a reflector of reestablishment of coronary flow. Set a timer to follow duration of the perfusion if needed.
12. Turn on the heat exchanger and set the temperature to 38 °C. The perfusate will warm up to 37–38 °C in approximately 10 min. For normothermic perfusion of a porcine heart, keep the temperature at 38 °C throughout the perfusion.
13. Maintain the perfusion in the non-working mode for the first hour of the perfusion. Adjust the LA pump speed to maintain the LA pressure at 0 mmHg.
14. Once the perfusate temperature is >34 °C, evaluate the heart rhythm and pace and defibrillate as required (5–20 joules). Ensure the heart is completely decompressed before attempting cardioversion.
15. Check the dissolved gas status using a blood gas analyzer. Adjust the gas mixture to maintain a pH: 7.35–7.45, arterial partial pressure of carbon dioxide (PaCO2): 35–45 mmHg, arterial partial pressure of oxygen (PaO2): of 100–150 mmHg, and oxygen saturation (sO2) ≥95%.
16. Once the heart is normothermic and in a stable rhythm, ligate the superior vena cava.
17. Attach temporary pacemaker leads to the right atrial wall and pace the heart in an AAI mode at 100 beats/min.
18. Attach the epicardial electrocardiography electrodes to the surface of the heart.
19. Switch to working mode after 1 h of perfusion in Langendorff mode. For this purpose, enter the desire LA pressure (typically 6–8 mmHg) on the left side of the main page, in the "desired LAP" section of the software, and click on the button to initiate the feedback loop. The activated working mode will appear as a green button, and the LA pump speed will automatically increase and decrease to reach and maintain the desired LA pressure.
20. As the heart begins to work, coronary vascular resistance will drop resulting in a low diastolic pressure. Adjust the Ao pump speed to maintain the Ao diastolic pressure of 40 mmHg as afterload during perfusion in the working mode.