Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The overall goal of the procedure shown here is to demonstrate simple methods that will greatly aid in the setup and analysis of mice with lung cancer or other lung pathologies. First is a method to directly instill cancer cells or chemical treatments to the lungs. Then postmortem fixation and lung volume measurements are described.

This is accomplished by first intubating the mouse with a slightly modified intravenous cannula. Next, the fluid of interest is instilled through the cannula into the lung. Then after all in vivo experiments are done and the mouse is dead.

The lungs are connected to a reservoir for fixation. In the final step, the lungs are dissected and the lung volume is measured. Ultimately, the quantitative morphology of the fixed lung tissue can be compared between experimental lung samples.

The main advantage of this new installation technique over existing methods is that with our method, there is almost no chance of incorrectly placing the cannula into the trachea. The procedure to fix the lungs under constant pressure is simple and leads to a consistent fixation in different lungs. Finally, the method to measure the fixed mouse lung volume allows for proper normalization for quantifying lung cells and structural elements.

Use up this method. Ensure both a direct delivery of the drug or other agent of interest to the lung, and an accurate and precise measurement of the lung. Will you Before beginning the procedure manually bend the tip of a commercial one inch long, 20 gauge intravenous cannula into a slight curve.

Then after confirming anesthesia by lack of response to toe pinch, apply ointment to the eyes of an adult mouse and secure the mouse in the supine position on a sloped platform with a large office binder and suture loops. Shave the ventral part of the neck and disinfect the skin with 70%alcohol. Then using sharp scissors, make a small surgical incision in the neck approximately 12 millimeters below the lower incisor.

Next, use forceps to gently pull the skin in the neck coddly until the ventral wall of the trachea can be seen. Then gently retract the tongue and insert the cannula with a bent tip tilted toward the ventral surface of the mouse. Pulling gently on the neck skin again, as just demonstrated.

Insert the cannula into the trachea when the catheter can be observed in the trachea, advance the catheter about five millimeters to pass the vocal cords. Now, place the tip of a gel loading pipette loaded with 50 microliters of the installate into the lure hub of the catheter. Confirm movement of the fluid in the tip synchronous with the mouse's breathing.

Then inject the installate using a one milliliter syringe. Immediately inflate the lungs with 0.6 milliliters of air through the catheter to help distribute the liquid, deepen the lungs. Then remove the cannula.

Close the surgical wound with a small amount of cyanoacrylate adhesive, and place the mouse in an individual cage with monitoring until it is fully recovered. After the final experimental time point, make a small cut in the neck of the animal to expose the ventral side of the trachea. Then insert an 18 gauge stub needle tip into the trachea.

Using thread, tie the needle into the trachea and carefully open the thorax with a midline incision. Then cut away the diaphragm and remove the lateral chest walls to expose the lungs. Next, make sure there is no air in the fixation tubing by running formaldehyde out the end of the stopcock.

Then connect the formal and reservoir and filled tubing directly to the lure end of the tracheostomy needle. Set the top surface of the formaldehyde 25 centimeters above the level of the mouth and open the stopcock to inflate the lungs with a fixative. After leaving the lungs under pressure for at least 20 minutes, slowly pull back on the needle to expose more of the un cannulated trachea while tying off the trachea beyond the end of the stub needle.

When the trachea is securely tied, remove the stop cock and carefully dissect out the lungs. Then place the lungs in formaldehyde overnight. Here, a typical device for holding the lungs in place during the fixed lung volume made from plastic pipettes and thin 20 gauge wire is shown to measure the fixed lung volume first, dissect the heart and any other non lung tissue from the lungs.

Next place a beaker containing approximately 200 milliliters of water equipped with the wire support device onto a balance and tear the equipment. Then remove the device. Place the lung on the water surface and use the device to press the tissue under the water.

Taking care that the lung suture or any part of the device does not touch the sides or the bottom of the beaker, record the weight on the balance. This number reflects the volume of water displaced and is thus a direct measure of the lung volume. Then dry the lung on a wiper, tear the beaker with a device again, and repeat the lung volume measurement.

The two volume measurements should then be averaged here. An example of a lung into which trian blue was instilled as just demonstrated is shown. Note, the widespread distribution of the dye, similar to what has been observed for other dyer tra tracers administered directly into the mouse trachea While attempting this procedure, it's important to remember to be careful with the insertion of the cannula and to keep the tip ventral side up.

The techniques we have described here are useful in studies of lung cancer, but they're also applicable to most other lung disease models in the mouse.