Ex vivo Lung Perfusion: A Technique for Pulmonary Research in a Rabbit Model

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

1. Cannulation:

1. Place the freshly isolated rabbit's lungs along with heart over a sterile gauze on a petri dish.
2. To prevent atelectasis, ventilate the lungs using positive-pressure ventilation with positive end-expiratory pressure (PEEP) set at 2 cmH₂O.
3. Remove the ventricles by cutting them off the heart at the level of the atrioventricular groove.
4. After opening the two ventricles, introduce the OD 4.6 mm pulmonary artery cannula for the rabbit with a basket through the pulmonary artery and introduce the OD 5.9 mm left atrium cannula for the rabbit with the basket through the mitral valve into the left atrium.
5. Use a 4-0 silk suture in the pulmonary artery and left atrium to fix the cannulae. Include the surrounding tissues in the ligatures of the pulmonary artery and left atrium to avoid the distension of these structures.
6. Inject 250 mL of saline isotonic solution through the arterial cannulae to flush the remaining blood from the vascular bed.

2. Perfusion technique:

1. Setup:
   1. Place the isolated lungs carefully into the lung chamber.
   2. Attach the trachea to the transductor on the cover of the chamber.
   3. Connect the cannulated vessels to the perfusion system.
   4. Close the chamber and secure it with the rotary lock.
      NOTE: The recirculating perfusion circuit consists of an open venous reservoir, a peristaltic pump, a heat exchanger, and a bubble trap.
   5. At this point, attach the chamber lid and switch over a stopcock to switch from positive to negative pressure ventilation. To check the negative pressure ventilation of the lungs and airtight closure of the chamber, inspect the respiratory excursion of the lung and chamber pressure on the pressure gauge.
   6. Perfuse the lungs with 200 mL of artificial blood-free perfusate (a Krebs-Ringer bicarbonate buffer containing 2.5% of bovine albumin).
   7. Start the perfusate flow at 3 mL/min/kg, then slowly step up the flow over a 5-min period to 5 mL/min/kg. Reach a flow of 8 mL/min/kg over the next 5 min and then after another 5-min period reach a maximum flux of 10 mL/min/kg. Take care of avoiding air from getting into the circuit.
      NOTE: Maintain the pH and the temperature of the perfusate within physiological ranges (pH 7.4-7.5; temperature, 37 °C-38 °C). To adjust the pH, add NaHCO₃ (1N) or increase the flow of carbon dioxide. Alternatively, use HCl (0.1N) to acidify.
2. Parameters:
   1. Check whether the predetermined perfusion and ventilation parameters are set as required.
   2. Ventilate the lungs with humidified air at a frequency of 30 bpm, a tidal volume of 10 mL/kg, and an end-expiratory pressure (Pe) of 2 cmH₂O.
      NOTE: The pulmonary arterial pressure (0-20 mmHg) corresponds to the height of the liquid level in the oxygenator or reservoir in centimeters above the pulmonary trunk, while the pulmonary venous pressure corresponds to the height of the pressure equilibration chamber above the left atrium. Both values can be modified. Note that left atrium pressure is also 0-20 mmHg.
3. Achieving zone 3 conditions:
   1. Use the two catheters connected to side ports of the cannulae secured in the pulmonary artery, left atrium, and pressure transducers to measure the arterial (Pa) and venous (Pv) pressures.
   2. Set the baseline pressures at the level of the lung hilum (Zero-reference).
   3. Conduct the experiments under zone 3 ventilation conditions. To achieve this, wait for 10-15 min to obtain an equilibrium characterized by an isogravimetric state.
   4. Ensure that the venous pressure is higher than the alveolar pressure (Palv) and the arterial pressure remains higher than both (Pa > Pv > Palv) for Zone 3 conditions to occur.
   5. Ensure that the lungs' weight remains constant and arterial and left atrial pressures are stable to achieve zone 3 conditions to open up a maximum number of pulmonary vessels and maintain the microvascular bed content during the experiment. 
      NOTE: The measurement of Kfc as an indicator of pulmonary edema has no variation between a manual and an automatic perfusion system.
4. Electronic control and signal processing: Ensure that the respiratory flow, weight changes, microvascular pressure, tidal volume, vascular resistance, among others, are registered on a multiple central electronics unit that integrates signals coming from the transducers and displays them on the evaluation system.