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

An ex vivo lung perfusion system allows the recreation of blood circulation and ventilation in isolated human and animal lungs to assess their function. Begin with isolated rabbit lungs with an attached heart on sterile gauze. Insert a tracheal cannula and ventilate the lungs via positive pressure for continuous lung activity.

Cut off the heart ventricles and insert cannulae into the pulmonary artery and the left atrium. Secure the cannulae with the neighboring tissues to avoid distention during perfusion. Inject isotonic saline through the arterial cannulae to flush out residual blood. Assemble the isolated lungs into a lung chamber by attaching the tracheal cannula to the ventilation system and the arterial cannulae to the perfusion system. Both systems are connected to a data acquisition system that measures different physiological parameters.

Now, switch the ventilation from positive to negative pressure to ensure optimal oxygenation and perfusion in the lungs. Then, slowly perfuse the lungs with artificial perfusate and gradually step up to a maximum flow rate. Modify the arterial and venous pressure to achieve zone three ventilation, a physiological state when both the arterial and venous pressures are higher than the alveolar pressure, causing maximum pulmonary vessels to open. This allows the proper distribution of perfusate into the pulmonary capillaries.

Collect real-time data for the desired parameters to evaluate lung function. 

Lift the isolated lungs out of the thorax, and carefully place the lungs over a sterile gauze on a Petri dish. To prevent atelectasis, ventilate the lungs using positive-pressure ventilation, with positive end-expiratory pressure set at 2 centimeters of water column. Cut the ventricles off the heart at the level of the atrioventricular groove.

After opening the two ventricles, introduce the pulmonary artery cannula through the right pulmonary artery, and the left atrium cannula through the mitral valve, into the left atrium. Fix the cannulae with a 4-0 silk suture, including the surrounding tissues in the ligatures of the pulmonary artery and left atrium, to avoid structural distension. Inject 250 milliliters of saline isotonic solution through the arterial cannulae to flush the remaining blood from the vascular bed.

For the perfusion setup, place the isolated lungs carefully into the lung chamber. Attach the trachea to the transductor on the chamber cover, and connect the cannulated vessels to the perfusion system. Then, close and secure the chamber with the rotary lock. Next, attach the chamber lid and switch over the stopcock to shift from positive to negative pressure ventilation. Perfuse the lungs with 200 milliliters of artificial, blood-free perfusate, starting with the flow at 3 mL/min/kg.

Then, slowly, step up the flow over 5 minutes to 5 mL/min/kg, and for the next 5 minutes, allow the flow to reach 8 mL/min/kg, followed by a maximum flux of 10 mL/min/kg for 5 minutes. Ventilate the lungs with humidified air at a frequency of 30 beats per minute, a tidal volume of 10 mL//kg and an end-expiratory pressure of 2 centimeters of water column.

To achieve zone three conditions, wait for 10 to 15 minutes to obtain an equilibrium characterized by an isogravimetric state, ensuring that the venous pressure is stable throughout the registry. Ensure that the weight of the lung remains constant and the arterial and left atrial pressures are stable to achieve zone three conditions to open up a maximum number of pulmonary vessels and maintain the microvascular bed content during the experiment.

For the 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.