Perfusion and Inflation of the Mouse Lung for Tumor Histology

Demonstrating the procedure will be Mackenzie Davenport, a graduate student from the Edmonds Laboratory. After euthanizing the mouse, use surgical scissors to make a 3.5 to five millimeter horizontal incision in the middle of the lower abdomen. Next, insert surgical scissors into the incision and cut vertically up the center midline to just below the neck of the mouse.

Pull the skin back with fingers and inspect the axillary lymph nodes. Use surgical scissors to make a 3.5 millimeter lateral incision to open the abdominal cavity. Then cut in the interior direction up to the bottom of the thorax.

Inspect the organs in the abdominal cavity such as the liver, spleen, and kidneys. With the flat of the surgical scissors move the liver to expose the diaphragm. Inspect the diaphragm for tumor growth or metastases, then gently snip the diaphragm on the operator's right side, allowing it to expand.

Cut the diaphragm from right to left to expose the thoracic cavity and lungs, taking care not to cut the lungs. Cut up through the lateral extreme of the left rib cage to inspect the left lobe of the lungs. Gently move the right lobes of the lung out of the way.

Then cut up the lateral extreme of the right rib cage and remove the rib cage. If fresh or frozen lung tissue is required, use hemostat forceps to clamp the bronchus of the left lobe and resect the left lung with surgical scissors prior to perfusion. Use the forceps to lift the tissue covering the trachea and cut away any excess tissue, then gently cut the thin tissue lining the trachea to expose the airway.

Cut through the renal artery with surgical scissors. To perfuse the lungs, use a three milliliter syringe with a 22 gauge needle to inject PBS with 10 units per milliliter heparin into the right ventricle of the heart. Slowly profuse the lungs at approximately 300 microliters per second causing the lungs to turn white.

For lung inflation, use a three milliliter syringe with a 22 gauge needle and hold it parallel to the trachea. Insert the needle into the trachea and inject 10%formalin with a flow rate no greater than 200 microliters per second until the lungs have fully inflated. Once the lungs are inflated, formalin will backflow out of the trachea.

Hold the needle in place for a few more seconds and then withdraw. Use forceps to lift the heart, insert surgical scissors directly behind the lungs and cut the connective tissue to resect to the lungs. Then cut the to remove it from the lungs.

Place the lungs in a cassette labeled with the mouse ID or study ID, then place the cassette in 10%buffered formalin for 24 to 48 hours. Lungs can be left in fixative for over a year if desired. When finished, transfer the cassette containing the lungs to 70%ethanol and proceed with histology.

This protocol allows for quick perfusion, inflation, and fixation of mouse lungs. Tumor histology in lungs which have been perfused and inflated using this technique is demonstrated here. When the profusion step is skipped, or the lungs fail to perfuse correctly, excess blood in the lungs creates less than ideal histology and can make it challenging to fully observe lung architecture.

Inflation is an important step in this protocol. It is more difficult to identify areas of hyperplasia in uninflated lungs due to the compression and close proximity of the alveoli. However, in overinflated lungs, many of the alveolar walls have been broken which could be mistaken for emphysema.