Dr.Darren Freed created this ESLP device, and it uses patented negative pressure ventilation technology and software, which can't be seen anywhere else in the field of ESLP research. Our lab has previously shown that negative pressure ventilation causes less lung injury during ESLP compared to positive pressure ventilation. This is likely due to the more physiologic mechanism of negative pressure ventilation.
This device and protocol are designed to extend the safe preservation time of donor lungs and to recondition marginal lungs to acceptable transplant standards. The device itself is very simple to operate. The greatest challenge is surgically removing the juvenile pig lungs without causing any injury.
They're extremely fragile compared to adult human lungs. Begin by maintaining the expiratory tidal volume of 10 milliliters per kilogram by increasing the peak pressures for maximum alveolar recruitment. Perform a midline sternotomy.
Use electrocautery to incise the skin and subcutaneous tissue. Bluntly dissect the soft tissue attachments from the posterior table of the inferior sternum. Use a sternal saw to divide the sternum.
Insert a sternal retractor and open wide. After performing the cardiectomy, open and remove the mediastinal pleura bilaterally to expose the lungs. Then divide the pleural attachments from the diaphragm toward the left lower lung lobe, using a Deaver retractor to hold the diaphragm upwards.
Now, divide the inferior pulmonary ligament on the left and continue up toward the hilum. Next, divide the inferior vena cava and pleural attachments from the diaphragm, and then retract the diaphragm upwards using the Deaver retractor. Divide the inferior pulmonary ligament on the right side and continue up towards the hilum.
Then divide the innominate vein and arch vessels to expose the trachea. After bluntly dissecting the tissue surrounding the trachea, clamp the trachea using a tubing clamp at maximal inhalation, ensuring the expiratory tidal volume is 10 milliliters per kilogram. Transect the trachea and lift the clamp portion upwards to provide surgical traction.
Then dissect the posterior trachea from the esophagus using blunt dissection with heavy Metzenbaum scissors and a free hand. Divide any remaining pleural attachments. Transect the aorta above and below the left bronchus and remove the lungs from the chest with a segment of descending aorta.
After weighing the lungs with the clamp, quickly store them in a cooler full of ice. Begin by adding 500 milliliters of packed red blood cells to the perfusion circuit to reach a final volume of 1.5 liters of perfusate. Take photographs of the lungs for data records.
For the lung biopsy, encircle a one cubic centimeter portion with zero silk suture and tie it. Then excise this tied portion, using scissors for tissue analysis as described in the text manuscript, and divide the biopsy segment. Next, to secure the 3/8 half-inch tubing adapter to the main pulmonary artery, grasp the opposite sides of the main pulmonary artery using forceps or snaps.
Then, insert the adapter with the half-inch portion into the main pulmonary artery and hold it in place while simultaneously securing the adapter in position using zero silk or Ethibond ties. Now place the lungs in a supine position on the silicone support membrane and connect them to the ESLP device. Place a second tubing clamp on the trachea near the location of the tracheal bronchus.
After removing the more distal clamp, intubate the trachea with the endotracheal tube and secure it in position using two zip ties. Then clamp the ventilation line using a tubing clamp before releasing the proximal clamp from the trachea. Connect the pulmonary artery adapter to the pulmonary artery line.
Once the main pulmonary artery is de-aired, start the time for perfusion. On the settings page, click start heater and set the temperature to 38 degrees Celsius. Enter the pig's weight to calculate cardiac output flow.
On the main page, set the continuous positive airway pressure to 20 centimeters of water and click start continuous positive airway pressure. When ventilation begins, unclamp the ventilation line. After zeroing the arterial pressure sensor, clamp the pulmonary artery line above the pressure sensor with the tubing clamp.
Open the sensor, click zero pulmonary artery pressure and zero blood flow on the settings page and confirm the readings are zeroed on the main page. Close the pressure sensor stopcock to read the line pressure. Open the line to the pulmonary artery cannula.
Select 10%cardiac output on the main page. Click return to PA Manual and unclamp the pulmonary line. Switch the perfusate solution in the top lid before securing it to the machine to prevent condensation formation during ESLP.
Draw 10 milliliters of perfusate for centrifugal analysis and draw a time zero arterial blood gas. Place the sample on ice before centrifugation. Once the lungs have been perfused for 10 minutes, increase the flow to 20%of the cardiac output.
When the perfusate temperature reaches 32 degrees Celsius, secure the chamber lid in place with clamps to create an airtight seal. Ensure that the lungs are optimally positioned and the air leaks are repaired. With the lid secure, clamp the ventilation tubing before turning off continuous positive airway pressure.
On the settings page, select zero intrathoracic pressure, airway pressure, and airflow. Then confirm readings are zeroed on the main page. Click start CPAP before unclamping the ventilation tubing.
Set the ventilation parameters mentioned in the text manuscript and click press to start vent to activate negative pressure ventilation. Attach the sideport ventilation tubing to the chamber. Over the next few breaths, maintain the ventilation parameters as mentioned in the text manuscript.
After 10 minutes of perfusion, increase the flow to 20%and after 20 minutes of perfusion, increase the flow to 30%cardiac output. Add dextrose and insulin to the system. Evaluate lung function, using 50%cardiac output and a deoxygenating mixed sweep gas over a five minute duration, as per the manuscript instructions.
At every odd hour during preservation mode, draw a 10 milliliters sample of perfusate for future analysis. Draw one milliliter of pre-deoxygenator arterial blood gas sample every hour and from pre and post deoxygenator ports following five minutes of evaluation mode. The trends in mean pulmonary artery pressure, dynamic compliance, and pulmonary vascular resistance over 12 hours of perfusion and ventilation can be affected by the specific ESLP experimental protocol employed.
A typical trend for the ratio of partial pressure of arterial oxygen and fraction of inspired oxygen at the evaluation time points of five and 11 hours throughout negative pressure ventilation ex-situ lung perfusion is demonstrated here. Edema formation is another important index of inflammation associated with endothelial permeability, demonstrating a typical weight gain of 30%at the end of 12 hours of negative pressure ventilation ex-situ lung perfusion. The perfusate acts as a surrogate indicator of overall lung status.
Therefore, blood gas analysis of the perfusate provides extensive information on the metabolic state of the lungs. During the evaluation model of ESLP, which occurs at three, five, seven, nine, and 11 hours during a 12 hour run, an upward trend in left arterial partial pressure of oxygen is observed. It's very important to be as gentle as possible when surgically removing the lungs and to also move quickly and efficiently.
The establishment of this device, protocol, and lab has led to a successful clinical trial and several discoveries that prolong the preservation and reconditioning of donor lungs.
