Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

To study pulsatile pulmonary pressure flow relationships. This protocol explains how to isolate, ventilate and perfuse mouse lungs. The trachea is surgically exposed for ventilation.

Then the heart and lungs are isolated so that the pulmonary artery and left atrium can be cannulated. The pulmonary system is then perfused. Experiments are performed by altering pulmonary artery flow, magnitude, waveform and or frequency.

The resulting pulmonary artery and left atrial pressure wave forms are recorded and demonstrate the pulsatile pulmonary pressure flow relationships. The main advantage of this technique over existing methods, such as in vivo preparations, is that changes in pressure flow relationships. Pulsatile pressure flow relationships can be obtained in the absence of changes in volume status, sympathetic nervous system tone, activity, and effects of anesthesia.

Also, most isolated lung preparations measure only steady pressure flow relationships, whereas our technique permits pulsatile pressure flow relationships to be determined. This method can help answer key questions in the field of pulmonary physiology, such as how does pulmonary vascular disease affect pulsatile pulmonary hemodynamics, or how does large pulmonary artery stiffening affect right ventricular afterload? Visual demonstration of this method is critical as exposing the heart and lungs and securing the cannula in the pulmonary artery is difficult to learn.

Excess pressure from instrumentation can cause edema in the lungs and improper cannula placement can puncture the pulmonary artery. Before beginning the experiment, it is necessary to prepare the IL one system used to ventilate and perfuse the isolated lung. The tubing should be connected to the pressure transducers.

O solitary pump and flow meter completely Flush the system with distilled water warmed to 37 degrees Celsius and remove any bubbles in the tubing. Then prime the Endur tech pump tubing with 1%PBS. Now zero.

The pressure for each P 75 transducer by first closing its valve to the IL one system and opening its valve to the atmosphere. Then use the plug SIS amplifier to automatically zero the pressure before preparing the animal humidify the IO one setup by soaking the ceramic porous piece in distilled water. After anesthetizing the mouse stabilize the mouse for surgery by pinning its front paws to corkboard.

Now spray the chest with 95%alcohol. Use straight forceps to grab the skin at the neck and cut a one centimeter opening with the straight scissors. Once the inside of the neck is exposed, cut away white glandular tissue in superficial muscles while looking for the esophagus and trachea.

Isolate the esophagus and trachea from tissue on either side and posteriorly and tie a loose suture around the trachea. To cannulate the trachea carefully cut a small V into one side of it with the small scissors. Now take the mouse to the IL one system using blunt forceps.

Grab the tracheal cannula and slide it into the trachea. Secure the cannula with a suture. Begin ventilation with room air first, spray the chest with alcohol and remove the skin using straight forceps and straight scissors.

Cut the skin upward along the sternum. Lift the skin on each side and cut along the line of the lower ribs. Now grasp the xiphoid process with tweezers and use ball tip scissors to cut a hole in the diaphragm where it meets the bottom of the sternum.

Grab the diaphragm and cut it away from the ribs. Using ball tipped scissors, grasp the xiphoid process again and use the ball tipped scissors to carefully cut up the sternum through the ribs without damaging the lungs, heart, or blood vessels. To expose the heart, grab the left ribs and cut off their tips.

Now slowly inject the right ventricle with 0.1 milliliters of heparin After injection, cut away the rest of the ribs. Use the back or rounded end of the forceps to gently push the lungs away from the rib while cutting the ribs under a microscope. Remove glandular tissue in the fat on top of the heart.

Use the tweezers to pull away the tissues and trim them off. With the spring scissors to place a suture around the aorta and pulmonary artery, gently spoon the heart with blunted tweezers. There should be little resistance.

Now tie a loose suture with the double throw. Next, cut away the lower abdomen. Use angled scissors to cut down through ribs and spine.

This will cause a significant amount of blood to flow. Use a Q-tip to staunch the flow. Once the blood flow is stopped, prime the IL system with four milliliters of RPMI from a syringe filled with 10 milliliters of RPMI.

It's good to double check that the tubing contains no bubbles. Now cannulate the right ventricle by cutting a small notch near the apex and inserting the cannula Aim down into the right to reach the pulmonary artery. The tip of the probe should be visible through the nearly transparent wall of the pulmonary artery.

Then tighten the suture around the cannula, aorta and pulmonary artery to cannulate the left ventricle. Cut a notch in the apex and insert the cannula aiming upwards into the left atrium. Slight pressure may be needed to find the valve, but once found, the cannula will slip through and be secure without a suture.

Manually infuse RPMI from the 10 milliliter syringe at 0.3 milliliters per minute until you see flow in the outflow tubing. If there is no flow in the outflow tubing, reposition the left ventricle cannula. However, if there's no flow in the lungs, abort the experiment because the pulmonary artery likely has a leak.

Now that the heart is fully cannulated, connect the 60 milliliter syringe to the IL one system. Using the steady flow pump and infuse the RPMI Perfuse eight at a rate of one milliliter per minute. Check for leakage during the initial perfusion.

Gradually perfuse the lungs for another two minutes during which time the lungs will turn white. For pulsatile flow studies. First set the solitary pump piston displacement at the desired levels.

Then set the steady flow rate. Now open the valve to the osli pump and start recording data right before running the osli flow profile. Once you are done, open the data output file and plot the experimental flow based on the experimental flows from initial trial.

Adjust the oscillatory pump piston displacement at each frequency so that Q max is as desired. Hold ventilation at a constant pressure During data collection for steady flow trials, close the valve to the solitary pump and set the steady flow rate to the desired level. Start recording data and step through the steady flow protocol.

Collecting data for at least 10 seconds At each flow rate between trials. Reset the flow rate to 0.5 milliliters per minute. Be sure to hold ventilation at a constant pressure during data collection.

If zero left atrial pressure trials are desired, remove outflow tubing while conducting trials. Vigilantly watch for RPMI in the airway tubing. RPMI must not be allowed to reach the airway pressure transducers or they will be damaged.

Here are results of the steady flow perfusion with flow Q in the top row and resulting pulmonary artery and left atrial pressures in the second row under a variety of conditions. In the first two panels, left atrial pressure increased and decreased with Q because the outflow tubing was in place, the first panel shows air pressure at end, inspiratory pressure, and the second shows air pressure at end expiratory pressure. Next, the tubing was removed so that the left atrial pressure was constant and independent of Q.Then the height of the outlet tubing was adjusted manually so that the left atrial pressure was higher.

But independent of Q again, the first panel shows air pressure at end, inspiratory pressure, and the second shows air pressure at end. Expiratory pressure. The resulting steady pulmonary artery pressure flow relationships for each case can be obtained by plotting pressure versus flow.

Here are the results of the pulsatile flow perfusion with flow Q in the top panel and resulting pulmonary artery, left atrial and air pressures. In the bottom panel. From these pulsatile pulmonary pressure flow relationships, pulmonary vascular impedance can be calculated, which reflects the total right ventricular afterload.

Once mastered, this technique can be done in 20 to 30 minutes. While attempting this procedure, it is important to take your time and relax so that your hands are steady. Once the lungs are adequately perfused, the hard part is over.

Following this procedure, other techniques can be performed such as histology or biochemical assays on the lung tissue to answer additional questions such as what molecular mechanisms are responsible for the observed changes in pulmonary pulsatile pressure flow relationships. This technique paves the way for researchers in the field of pulmonary physiology to explore the role of specific genes and proteins in the development and progression of pulmonary vascular disease using genetically modified mice.