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JoVE Encyclopedia of Experiments
Encyclopedia of Experiments: Cancer Research

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Lung Instillation and Fixation


Lung Instillation and Fixation: Introducing Liquid into the Trachea and Fixing Lungs to Measure the Fixed Lung Volume



- Lung instillation is used to introduce substance directly into the trachea for analyzing mouse lungs with cancer or other pathologies. First, place an anesthetized mouse on a slope intubation platform and shave the hair on the neck. Make a small incision with a scissor below the neck and gently pull the skin using forceps to reveal the trachea. Insert a catheter into the trachea through the mouth and observe it through an incision. Inject example liquid into the trachea and rapidly inflate the lungs by introducing air with an empty syringe to distribute the liquid properly.

Close the wound and place the mouse in a recovery chamber. After the desired period, sacrifice the mouse and expose the lungs. Inflate it with fixative to quantify total lung capacity. Place the lungs in a tube with a fixative. Measure the fixed lung volume by using the Archimedes' principle. Place the lungs into the water and press it with a wired device. Note the weight of water displaced by submerged lungs. The fraction of the lung volume in the fixed left lung is usually smaller than the one measured in vivo.

- Before beginning the procedure, manually bend the tip of a commercial 1-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 caudally until the ventral wall of the trachea can be seen. Then gently retract the tongue and insert the cannula with the 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 5 millimeters to pass the vocal chords.

Now place the tip of a gel loading pipette loaded with 50 microliters of the instillate into the lower hub of the catheter. Confirm movement of the fluid in the tip synchronous with the mouse's breathing, then inject the instillate. Using a 1-milliliter syringe, immediately inflate the lungs with 0.6 milliliters of air through the catheter to help distribute the liquid deep in 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 lung. Next make sure there is no air in the fixation tubing by running formaldehyde at the end of the stopcock. Then connect the formalin reservoir and filled tubing directly to the lower 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 uncannulated trachea while tying off the trachea beyond the end of the stub needle. When the trachea is securely tied, remove the stopcock 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 a wire support device onto a balance and tare 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, tare the beaker with the device again, and repeat the lung volume measurement. The two volume measurements should then be averaged.

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