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

Protocollo

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.

Materiali

Name	Company	Catalog Number	Comments
2-Stop Tygon E-Lab Tubing, 3.17 mm ID, 12/pack, Black/White	Hugo Sachs Elektronik (HSE)	73-1864
Adapter for Positive Pressure Ventilation on IPL-4	Hugo Sachs Elektronik (HSE)	73-4312
Adapter for Positive Pressure Ventilation on IPL-4	Hugo Sachs Elektronik (HSE)	73-4312
Alternative Pressure-Free Gas Supply for IPL-4: To supply the trachea with gas mixture different from room air during negative ventilation	Hugo Sachs Elektronik (HSE)	73-4309
Base Unit for the Rabbit to Fetal Pig Isolated Perfused Lung	Hugo Sachs Elektronik (HSE)	73-4138
Bovine serum A2:D41albumin lyophilized powder	sigma	3912	500 g
Calcium chloride, CaCl2·2H2O.	JT Baker	10035-04-8
Cryogenic vials	Corning	430659	2 mL
D-glucose, C6H12O6.	sigma	G5767
Differential Low Pressure Transducer DLP2.5, Range +- 2.5 cmH2O, HSE Connector	Hugo Sachs Elektronik (HSE)	73-3882
Differential Pressure Transducer MPX, Range +- 100 cmH2O, HSE Connector	Hugo Sachs Elektronik (HSE)	73-0064
Eppendorf tubes
Ethanol absolute HPLC grade	Caledon
Falcon tubes			14 mL
Harvard Peristaltic Pump P-230 (Complete with Control Box and P-230 Motor Drive)	Hugo Sachs Elektronik (HSE)	70-7001
Heated Linear Pneumotachometer 0 to 10 L/min flow range	Hugo Sachs Elektronik (HSE)	59-9349
Heater Controller for Single Pneumotachometer 230 VAC, 50 Hz	Hugo Sachs Elektronik (HSE)	59-9703
Heparin	PISA		5000 UI
HPLC Column (C18 100A 5U)	Alltech	98121213	150 mm x 4.6 mm
Hydrophilic Syringe Filter	Millex	SLLGR04NL	4 mm
IPL-4 Core System for Isolated Rabbit to Fetal Pig Lung, 230	Hugo Sachs Elektronik (HSE)	73-4296
IPL-4 Core System for Isolated Rabbit to Fetal Pig Lung, 230 V	Hugo Sachs Elektronik (HSE)	73-4296
Jacketed Glass Reservoir for Buffer Solution, with Frit and Tubing, 6.0 L	Hugo Sachs Elektronik (HSE)	73-0322
Lauda Thermostatic Circulator, Type E-103, 230 V/50 Hz, 3 L Bath Volume, Temperature Range 20 to 150°C	Hugo Sachs Elektronik (HSE)	73-0125
Left Atrium Cannula for Rabbit with Basket, OD 5.9 mm	Hugo Sachs Elektronik (HSE)	73-4162
Low Range Blood Pressure Transducer P75 for PLUGSYS Module	Hugo Sachs Elektronik (HSE)	73-0020
Magnesium sulfate heptahydrate, MgSO4·7H2O	JT Baker	10034-99-8
Microcentrifuge Tube	Corning	430909
Negative Pressure Ventilation Control Option with Pressure Regulator for IPL-4	Hugo Sachs Elektronik (HSE)	73-4298
New Zeland rabbits
PISABENTAL (Pentobarbital sodium)	PISA	Q-7833-215
PLUGSYS Case, Type 603* 7	Hugo Sachs Elektronik (HSE)	73-0045
PLUGSYS TCM Time Counter Module	Hugo Sachs Elektronik (HSE)	73-1750
PLUGSYS Transducer Amplifier Module (TAM-A)	Hugo Sachs Elektronik (HSE)	73-0065
PLUGSYS Transducer Amplifier Module (TAM-D)	Hugo Sachs Elektronik (HSE)	73-1793
PLUGSYS VCM-4R Ventilation Control Module with Pressure Regulator	Hugo Sachs Elektronik (HSE)	73-1755
Potassium chloride, KCl.	JT Baker	3040-01
Potassium dihydrogen phosphate, KH2PO4	JT Baker	7778-77-0
Pulmonary Artery Cannula for Rabbit with Basket, OD 4.6 mm	Hugo Sachs Elektronik (HSE)	73-4161
Servo Controller for Perfusion (SCP	Hugo Sachs Elektronik (HSE)	73-2806
Snap Cap Microcentrifuge Tube	Costar	3620	1.7 mL
Sodium bicarbonate, NaHCO3	sigma	S6014
Sodium chloride, NaCl.	sigma	S9888
Surgical gloves No. 7 1/2
Surgical gloves No. 8
Taygon tubes	Masterflex
Tracheal Cannula for Rabbit, OD 5.0 mm	Hugo Sachs Elektronik (HSE)	73-4163