Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

Improving the quality of donor lungs is the key to improving the success rate of lung transplantation. We hope to establish a simple and reproducible animal model to study the effect of ex vivo lung profusion, improving the quality of donor lungs. EVLP has been proven to be an effective technique measure to improve the quality of donor lungs, but there are still many problems in EVLP that need to be studied and clarified.

The perfusion conditions still need to be explored, and the ability of EVLP to attenuate ischemia-reperfusion injury needs to be further verified. Compared with other technique and models. our rat EVLP platform is most simple, operable, reproducible, inexpensive, and much more cost-effective, which provides an efficient research platform for EVLP-related research.

Attention should be made to the therapeutic impact of ex vivo lung profusion technology on marginal donor lungs, which is similar to DCD in China. At the same time, this technology can provide a platform for future research. To begin, place the anesthetized rat supine on an operating table and secure it with sutures.

Using rodent surgical scissors, make a two to three centimeter longitudinal incision along the midline of the neck in front of the trachea. Cut the skin and dissect the muscle tissue in front of the trachea to fully expose the trachea. Then, using a syringe, inject 1, 000 units per kilogram of heparin into the tail vein and wait for five minutes to ensure adequate heparinization of the blood.

Free the space behind the trachea. Pass a 3-0 suture through the trachea and tie a loose knot for later use. Make a V-shaped incision of 0.5 centimeters above the knot in the trachea.

Insert a 14 gauge tracheal tube into the trachea. Tighten the suture knot to secure the tube. Connect the tracheal tube to the ventilator.

Turn on the ventilator to begin ventilation of the lungs. Monitor the vital signs of the rat every five minutes and stop the ventilation. After confirming rat death following cardiac arrest using the following parameters, restart mechanical ventilation in pressure-controlled mode and ventilate for five minutes.

Clamp the trachea and rest the donor lung at room temperature for one hour of warm ischemic time. Next, for harvesting the lung, adjust the operating table to a 45 degree head high and feet low inclined position. Using a hair shaver, remove the hair from the middle of the rat's chest and abdomen.

Disinfect the surgical area with iodine three times and drape it with a surgical towel. Make a six to seven centimeter longitudinal incision along the abdomen's midline with rodent surgical scissors. Cut the skin, open the abdominal wall to the abdominal cavity, and expose the organs.

Using a syringe, inject 1, 000 units per kilogram of heparin into the inferior vena cava and wait for five minutes to ensure adequate heparinization of the blood. Then, use scissors to cut the rat's inferior vena cava and start mechanical ventilation of the lungs. After ventilation, use forceps to lift the xiphoid process and make a longitudinal incision along the sternum from bottom to top.

Use a rib spreader to expose the chest cavity, then, remove the thymus tissue to expose the heart and the major blood vessels below it. Free the rat's aorta and the space behind the pulmonary artery. Pass a 3-0 suture through the pulmonary artery and tie a loose knot for later use.

Make a two to three millimeter V-shaped incision on the anterior surface of the right ventricle outflow tract. Insert a pulmonary artery cannula into the pulmonary artery through the incision and tighten the pretied suture to secure the cannula. Cut the rat heart tip and insert hemostatic forceps into the left ventricle.

Disrupt the valves between the left ventricle and atrium to ensure the left atrial outflow tract is clear. Next, connect the pulmonary artery cannula to the perfusion circuit and use 15 milliliters of low potassium solution to flush the residual blood at a flow rate of 0.6 to one milliliter per minute. Place a suture behind the heart and encircle the ventricle.

Insert the cannula through the incision in the left ventricle and tighten the prettied suture to secure the cannula. Now, cut off the trachea above the tracheal tube. Lift the trachea and use scissors to separate the connective tissue behind the trachea downward to the diaphragm.

Then, cut off the inferior vena cava and the main pulmonary artery above the diaphragm. Separate the heart and lungs. To keep the lungs inflated, immediately clamp the lower third of the trachea at the end of inhalation.

Collect the heart and lungs and place them in a low potassium solution for preservation. Place the heart and lungs in the ex vivo lung perfusion, or EVLP circuit's, designated position and connect the left ventricle cannula to the perfusion circuit. After assembling the EVLP setup, fill the bubble trap with a sufficient amount of lung perfusion repair solution to prevent bubbles from entering the lungs.

Place the cardiopulmonary unit in the organ chamber and connect it to the EVLP device. Then, turn on the ventilator and peristaltic pump. Start the lung perfusion at 20%of the target flow rate.

After calculating the target flow rate, gradually increase the flow rate to the target flow rate within one hour. Set the heat exchanger to 40 degrees Celsius to maintain a lung temperature of 37.5 degrees Celsius. After 20 minutes of perfusion, remove the endotracheal clamp, and using the following parameters, start mechanical ventilation.

Then, start the flow of hypoxic gas. To maintain the perfusion solution, partial pressure of carbon dioxide entering the pulmonary artery between 35 and 45 millimeters simultaneously during ventilation. During perfusion, constantly monitor the perfusion solution flow rate, arteriovenous pressure, airway peak pressure, and lung function parameters.

Pulmonary graft oxygenation levels and vascular resistance in DCD donor lungs remained stable, with no significant differences over the four-hour perfusion period. Dynamic compliance of DCD donor lungs gradually decreased during the four-hour perfusion period. Glucose levels in the perfusate of DCD donor lungs steadily declined throughout the four-hour perfusion.

Electrolyte levels, including sodium and potassium, remained consistent across the groups during the four-hour perfusion. A significantly higher number of apoptotic cells were detected in the DCD donor group compared to other groups, with the cold preservation group showing more positive cells than the EVLP group. The lung injury score was significantly lower in the EVLP group compared to the cold static preservation and control groups.

Alveolar wall thickening and alveolar hemorrhage were prominent in the four-hour cold static preservation group, while normal alveolar structure was preserved in the EVLP group.