Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The overall goal of this procedure is to set up a consistent rat, ex vivo lung perfusion model that will yield reproducible results. The first step is to cannulate the trachea and begin mechanical ventilation. Next, the pulmonary artery and left atrium are cannulated.

The final step is to remove the heart lung block from the chest cavity and place it in the ex vivo lung perfusion circuit. Ultimately, pressure sensors, weight transducers, and a blood gas analyzer are used to show the condition of the lung in real time by measuring pulmonary arterial pressure and other parameters. Ex vivo lung profusion is a remarkable technology that has allowed us to expand the donor pool for lung transplantation.

We anticipate that through this technology to serve as a platform for organ evaluation, we can improve the quality of organs being brought to transplant and overall transplant outcomes. Here at the Copper laboratory, we have coined the phrase arming the organ, so assessing, remodeling, or repairing, and then modifying the organ to improve the quality and thus improve transplant outcomes. We have developed a robust and reproducible small animal rat model of ex vivo lung perfusion.

Through this video, we'll demonstrate the key and critical steps for you to develop this ex vivo lung perfusion model, which then can serve as a platform for perfusion and transplantation Research. Begin by setting up the EVLP circuit and circulating warm perfuse eight throughout the system. Set the warm water bath, heat exchanger, and artificial thorax to 37 degrees Celsius.

Run a deoxy oxygenation gas through the PERFUSE eight in the gas filter to ensure that it has approximately 6%dissolved oxygen for the experiment. After opening the data acquisition program, connect the pulmonary artery pressure transducer, tracheal, differential pressure transducer, respiratory flow, differential pressure transducer, lung weight transducer, and pump speed transducer to the analog to digital converter box. Finally, set up the operating table and prepare all operating tools and supplies needed for the procedure.

After verifying sedation by toe, pinch, place the anesthetized animal in the supine position on the operating table. Begin recording data using the data acquisition program. When ready, enter the abdominal cavity and inject heparin into the inferior vena cava.

Following standard procedures, cannulate the trachea and connect the cannula to a ventilation circuit. Turn on the mechanical ventilator to start mechanically ventilating the lungs. Next, retract the thoracic cavity to expose the lungs taking care to avoid touching them.

Remove the thymus and shift the abdominal contents to one side to expose either the inferior vena cava or the mesenteric vein. After euthanasia, place a silk suture behind the pulmonary artery and aorta. In preparation for securing the pulmonary artery cannula, make a two to three millimeter incision on the anterior surface of the right ventricular outflow tract and insert the cannula into the main pulmonary artery.

Secure with silk suture, remove clots from the pulmonary vasculature by perfusing approximately 15 milliliters of a low potassium positive electrolyte solution through the pulmonary artery and out through the apex of the heart. Next position a silk suture behind the heart and around the ventricles. To dilate the mitral valve and facilitate cannulation, insert a small pair of surgical forceps into the apex through the mitral valve and into the left atrium.

When ready, insert a cannula into the apex through the mitral valve and into the left atrium. Secure the left atrium cannula with the silk suture. Next, connect the pulmonary artery cannula to the EVLP circuit.

Ensure that the inflow line coming from the circuit is primed with perfuse eight. To prevent air from entering the heart and lungs, turn on the main peristaltic pump and set it to a low speed. This will allow perfuse eight to run through the pulmonary artery and out the left ventricle into the chest cavity.

Monitor the pressure of the pulmonary artery to make sure that it does not spike indicating either a blockage or poor cannulation. When ready, turn off the peristaltic pump. Connect the pulmonary artery cannula to the ex vivo lung perfusion circuit.

Do not connect the left atrium cannula to the circuit until the heart lung block has been removed from the body. Remove the heart lung block by clamping the esophagus with a hemostat and cutting below the clamp so that the esophagus can be used to raise the cardiopulmonary structures bluntly, dissect away the surrounding tissue and cut the descending aorta and auxiliary vessels to free the heart lung block while raising it via the esophagus. Transect the trachea to free the heart lung block and place it into the designated location on the EVLP circuit.

Then connect the left atrium cannula to the outflow line and start the peristaltic pump. To record ventilation data and monitor pressure quickly remove the ventilation line from the top of the EVLP apparatus and attach the housing with the pressure sensors. Next, reinsert the ventilation line on top of the EVLP apparatus.

Make sure that the bubble trap is adequately filled with perfuse eight. To avoid introducing air bubbles into the lungs during the initial 15 minutes, slowly change the ventilation and perfusion settings to the desired experimental levels. Increase the perfusion flow rate to the desired rate and pressure during this initial ramp up phase as well.

It is recommended to program the ventilator to produce intermittent side breaths to facilitate movement of fluid out of the lung space and delay the onset of edema. Define time zero as the time when ventilation parameters are at a tidal volume of four milliliters per kilogram positive. End expiratory pressure at two centimeters.

H2O and perfusion parameters are at their expected levels and remaining constant. If desired, take Perfuse eight samples from the sample port flash freeze in liquid nitrogen, and note the time the samples were taken. When the experiment is complete, isolate any necessary anatomical pieces for collection and either flash freeze them in liquid nitrogen or place into fixing solution for further studies.

The lung weight of male Sprague dolly rats did not significantly change throughout 60 minutes of ex vivo lung perfusion after the first 10 minutes. There was also no change in pulmonary arterial pressure. Similarly, pulmonary vascular resistance was not altered throughout the duration of the study.

After one hour of ventilation at high tidal volumes and high peep pro-inflammatory cytokine concentrations were increased in the PERFUSE eight, while anti-inflammatory cytokine concentrations remained unchanged. After watching this video, you should have a good understanding of the key steps necessary to establish a small animal ex Viva lung profusion model in rats in your laboratory.