Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

This is an inexpensive method that combines the advantages of air inflation and vascular perfusion fixation for preserving lungs for structural and functional analyses. The main advantage of this technique is the preservation of both cell morphology and location within the airspaces of the lung. Care should be taken while placing the lure stub adapter into the trachea and the perfusion needle into the right ventricle to ensure adequate inflation and fixation.

To set up the air-inflation apparatus, place a syringe for the water column into a ring holder and measure a vertical height of 25 centimeters from the animal platform to 25 centimeters on the water column. Attach to the end of the water column tube to the stopcock on the air chamber and attach a tube from the female lure of the air chamber to the stopcock on the animal processing container. Confirm that the cap to the air chamber and the stopcock on the outside of the animal processing container are closed, and that the stopcock on the tubing leading from the water column to the air inflation chamber is open.

Then fill the syringe with water to the 25 centimeter mark. Water will flow through the syringe and tubing into the air chamber. Once the pressure is equalized, the water will stop flowing.

To prepare the lungs for inflation make one lateral incision in the abdominal wall below the rib cage of the euthanized mouse, and a second lateral abdominal wall incision above the hips. Cut along the midline from the interior incision toward the superior incision, and to use blunt scissors to carefully make an incision into the lateral side of the diaphragm. The lungs should collapse as soon as the diaphragm is punctured.

Cut transversely along the diaphragm to open the thoracic cavity and cut superiorly along the sternum from the xiphoid process to the juggler notch, and laterally above the rib cage to fully expose the heart and lungs. Pin down the sides of the rib cage and make a midline incision in the neck, above the trachea. Remove the skin, muscle, thyroid gland, and connective tissue surrounding the trachea and use curved forceps to slide two pieces of suture under the posterior trachea.

Use one piece of suture to hold the inflation lure stub adapter in place and use an 18 gauge by one inch needle to make a small hole in the trachea. Insert a 20 gauge lure stub adapter into the hole and tie the suture around the trachea immediately distal to where the lure stub adopter enters the hole. Then transfer the animal to the animal processing container and attach the lure stub adapter to the female lure on the inside of the animal processing container.

To inflate the lungs, place the 25 gauge by 5/8th inch needle attached to the perfusion apparatus tubing into the right ventricle of the heart and cut the abdominal aorta to allow blood to drain from the heart and to promote the flow of perfusate through the lungs. Open the stopcock on the outside of the animal processing container and inflate the lungs at 25 centimeters of water for five minutes, while taking care that the water level in the syringe does not decrease too quickly. During the last minute of the lung inflation, turn on the peristaltic pump to a flow rate of 10 milliliters per minute.

Heparin solution should flow from the bottle through the tubing into the animal. After inflating the lungs for five minutes, turn off the pump and switch the perfusate from heparin to fixative. Lower the water column syringe to 20 centimeters.

It's normal for air bubbles to move within the water column as the pressure changes. Check the water level in the syringe. It should be at the 25 centimeter mark.

Then wait one minute to allow the lungs to deflate from 25 to 20 centimeters of water pressure before restarting the pump at a 6.5 milliliter per minute flow rate for 10 to 15 minutes, vascular fixative perfusion. For extraction of the inflated and fixed lung tissue, tightly tie the second piece of thread around the trachea, distal to the lure stub adapter, and remove the lure stub adapter from the trachea. Remove the needle from the heart and use blunt scissors to cut the connective tissue, posterior to the mediastinum, to free the lungs and heart from the thoracic cavity taking care to avoid puncturing the lungs.

Carefully remove the heart from the lungs and place the lungs into 20 to 25 milliliters of fixative in a 50 milliliter conical tube. Pull the suture holding the trachea through the opening in the conical tube and secure the suture by the threads of the cap. Then invert the tube to ensure that the buoyant, air-inflated lungs remain fully submerged in fixative and process the lungs for histologic studies according to standard protocols.

When the diaphragm is entered during dissection, the integrity of the pleural space will be abolished and the lungs should collapse. Upon the application of 25 centimeters of water pressure, air will enter the lungs via the trachea and inflation should be easily observed. Once the lungs have fully expanded, the inflation pressure can be decreased to 20 centimeters to keep the lungs inflated without overdistension.

The lungs should remain inflated after tracheal ligation and removal from the thorax. Deflation can occur if the lungs are punctured during the animal preparation or lung extraction. Histological analysis of inflated lung sections reveals that very few immune cells are present in the airway lumen of tissues fixed using traditional liquid-based inflation.

In contrast, inflammatory cells are preserved within the airspaces of tissues fixed via vascular perfusion with air inflation. Following air inflation with profusion fixation, lung tissue may be embedded in formalin for tissue sectioning and subsequent analysis via histology techniques, including staining, immunohistochemistry, and immunofluorescent imaging.