This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory function. Isolated lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. This method could provide insight into the characterization of the non-respiratory capabilities of the pulmonary tissues, such as metabolic activity and the activities of various components like alveolar macrophage and endothelial tissue.
To begin with, assemble the working apparatus, ensuring that it contains the main steel column mounted on a base plate holding the artificial thorax with the pneumotachometer and weight transducer located above the column and behind the preheating coil with a bubble trap. Connect all the transducers to the central electronics unit and prepare the ventilation system besides the apparatus. After shaving the surgical site of the anesthetized rabbit, make a ventral median line incision of three to five centimeters from the sternum manubrium up to the neck.
Use the operating scissors to cut the anterior 2/3 of the trachea between the cartilage rings. Insert a five millimeter tracheal cannula through the tracheal fibrous membrane and fix the cannula carefully with a 4-0 silk suture. Place the forceps or tweezers underneath the trachea to ensure the cannula does not bend against the trachea.
Connect the respiration pump to the tracheal cannula, set the tidal volume at 10 milliliters per kilogram and initiate the ventilation quickly after the tracheotomy and before the thorax is opened to maintain positive pressure in the lungs for preventing lung collapse during the surgery. To access the thoracic cavity, use a scalpel or scissors to open the thorax wall and perform a medial sternotomy up to the upper third of the thorax. Hold the thorax halves open with two retractors.
Localize the superior and inferior vena cava and tag them with threads. Before the exsanguination of the animal, identify the right ventricle and inject 1, 000 international units per kilogram of heparin. Immediately after the injection, ligate the superior and inferior vena cava with the pre-looped thread.
Cut through the manubrium sterni to extend the medial sternotomy towards the tracheal cannula, releasing the trachea on both sides from connecting tissue. Now resect the trachea above the tracheal cannula and gently pull up the cannula in a cranial/caudal axis. After exsanguination for harvesting the cardiopulmonary block, use direct digital dissection or spring scissors to separate the connective tissue and remove the lungs from the thorax.
Next, dissect the vasculature and esophagus. 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 two 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 cannula 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 three milliliters per minute per kilogram, then slowly step up the flow over five minutes to five milliliters per minute per kilogram. And for the next five minutes, allow the flow to reach eight milliliters per minute per kilogram, followed by a maximum flux of 10 milliliters per minute per kilogram for five minutes.
Ventilate the lungs with humidified air at a frequency of 30 beats per minute, a tidal volume of 10 milliliters per kilogram and an end expiratory pressure of two centimeters of water column. To achieve zone three conditions, wait for 10 to 15 minutes to obtain an equilibrium characterized by an ISO gravimetric 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. The concentration of 5-HT and monoamine oxidase involved in the lung metabolism and vascular permeability were assessed. The concentration of 5-HT and monoamine oxidase peaked after 15 minutes of preservation and then decreased during the next six hours.
Afterward, perfusion levels showed a non-statistically significant increase up to 24 hours. The release rates of 5-HT and monoamine oxidase indicated that during the first hour of the preservation, 5-HT levels rose higher than monoamine oxidase and decreased within six hours after being recaptured by endothelial cells and platelets, as well as monoamine oxidase-mediated catabolism. Neutral endopeptidase activity increased at nine hours and then decreased and remained stable during 12 to 24 hours.
Angiotensin-converting enzyme activity decreased after four hours, then increased and remained stable through time til 24 hours. The effect of pulmonary preservation in capillary permeability was assessed for 24 hours, indicating that the perfused lung suffered a progressive increase in capillary filtration coefficient. The effect of different additives in the capillary permeability of the isolated lung perfusion system under diverse conditions was checked and a maximum increase in the permeability was observed with atropin.
The most important maneuver is to correctly place the lungs into a lung chamber and connect the cannulated vessels to the perfusion system. The implications of this technique extend toward interactions between circulatory substances and the effects of inhaled or perfused substances as in drug testing and various lung pathologies.
