To begin, place the autoclaved surgical instruments on the surgical table. Shave the abdomen of the anesthetized rat and place the rat in the supine position on the surgical board. Clean the abdomen with povidone iodine followed by 70%ethanol.
Place ophthalmic ointment under the rat's eyes to prevent dryness. Secure the rat in place on the surgical board. Start the data acquisition software and begin recording.
Turn on the ventilator at four milliliters per kilogram and ensure positive end expiratory pressure is around two centimeters of water. Using scissors, perform a midline laparotomy from the xiphoid process to the pubic symphysis. Then with the help of a blunt instrument, perform a medial-lateral visceral rotation and visualize the infrahepatic inferior vena cava.
Inject heparin into the inferior vena cava with a 20-gauge needle. Using a pair of scissors, cut the skin from the sternal notch to just below the angle of the mandible and begin to dissect toward the trachea. Then, bluntly dissect away necessary strap muscles to expose the trachea.
Make a transverse incision on the anterior trachea between the cartilaginous rings, ensuring not to cut through the posterior portion of the trachea. Place a 5-0 silk suture around the trachea. Insert the endotracheal tube into the cartilaginous rings, and secure it with the 5-0 silk suture.
Connect the endotracheal tube to the ventilator and ensure proper chest rising. Using scissors, perform a median sternotomy and enter the thoracic cavity. Place chest wall retractors to expose the heart and lungs, avoiding any inadvertent manipulation of the lungs.
With the combination of sharp and blunt dissection, remove the thymus from the anterior mediastinum. Identify the pulmonary artery and place a 5-0 silk suture around it to prepare for cannulation. Make a two to three millimeter incision in the right ventricular outflow tract using scissors to place the arterial cannula within the pulmonary artery and secure it with the 5-0 suture.
After euthanizing the rat, quickly connect the de-aired lung preservation fluid to the arterial cannula to gravity flush the lungs. Connect the arterial cannula to the EVLP circuit. Turn on the roller pump and allow a small amount of perfusate to flow through the lung and out of the left ventricle into the thoracic cavity.
Once perfusate begins to flow out of the left atrium, turn off the roller pump. Next, place a small forceps in the left ventricle and gently stretch the mitral valve annulus. Place a 5-0 silk tie around the heart and loosely tie.
Insert the left atrium cannula into the left ventricle and advance the cannula until it can be seen within the atrium. Secure the left atrium with the pre-tied 5-0 suture. Identify the esophagus and clamp it with a hemostat as close to the diaphragm as possible.
Then, cut the esophagus below the hemostat. Using the spine as a guide, cut all ligamentous attachments connecting the heart-lung block to the surrounding structures with scissors. Then dissect the trachea from the neck and cut the trachea above the endotracheal tube to free the heart-lung block.
Move the heart-lung block to the thoracic jacket within the EVLP circuit and attach the left atrium cannula to the circuit. Turn the roller pump on and connect the ventilator monitor. Check the bubble trap to ensure no air emboli are being introduced into the system.
Slowly change ventilation and perfusion settings to the desired experimental levels during the initial 15 minutes. Additionally, increase the perfusion flow rate to the desired rate and pressure. At designated time points, check perfusate gas levels and pulmonary function tests.
All tested perfusates showed a slight decrease in left atrium partial pressure of oxygen with the RBC-based perfusate significantly decreasing at one hour. For the next several hours, both polyhHB and control perfusates had stable left atrium partial pressure of oxygen. The delta partial pressure of oxygen significantly decreased at one hour in the RBC perfusate group while it remained stable in the polyhHB and control perfusates with a non-significant trend.
Left atrium partial pressure of carbon dioxide was significantly lower in the RBC and control perfusate compared to polyhHB after the first hour, and this trend continues over the following hours. The delta partial pressure of carbon dioxide was significantly increased in the RBC perfusate after one hour and after that remained stable in both the polyhHB and control perfusate. Real-time lung physiological data demonstrate that the RBC perfusate significantly increased pulmonary vascular resistance within the first hour.
While both polyhHB and control perfusates maintained low and stable pulmonary vascular resistance over the period, the change in lung weight was significant in the RBC perfusate initially, with a continued increase in all perfusates, slightly more in polyhHB. Compliance decreased significantly in the RBC perfusate within the first hour, while it decreased non-significantly in other perfusates. PolyhHB had the highest compliance after four hours.
