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

Overview

This video describes a simple and reliable method of introducing liquid into the trachea for lung instillation, fixation, and lung volume measurement.

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Lung Instillation

1. Select a commercial one-inch long 20 g intravenous cannula to use for the intubation.
2. Modify the catheter tip manually bending it to generate a slight curvature at the tip as illustrated in Figure 1.
3. Anesthetize the mouse with a mixture of ketamine (100 mg/kg) and xylazine (15 mg/kg) injected IP, and confirm anesthesia by the absence of reflex motion. Apply veterinary ointment on the eyes immediately after anesthesia. Immediately after anesthesia apply veterinary ointment on the eyes and give carprofen (5-10 mg/kg SQ) for post surgical and instillation analgesia.
4. Place the mouse supine on a sloped platform. As shown in Figure 2, a large office binder with suture loops taped on works perfectly well.
5. Shave the ventral part of the neck and clean and disinfect the neck area with 70% alcohol.  With new latex- and powder-free gloves, use surgical instruments disinfected with 70% alcohol.
6. Using sharp scissors make a small surgical incision in the neck approximately 12 mm below the lower incisor.
7. With a forceps gently pull the skin in the neck caudally until the ventral wall of the trachea can be seen.
8. Gently retract the tongue and insert the cannula with the bent tip tilted toward the ventral surface of the mouse. As in 1.4, pull gently on the skin in the neck, and insert the cannula into the trachea.
   NOTE: With a little practice, the catheter will be visible moving down the trachea. If it goes in the esophagus, then there will be no visual sighting of the catheter’s movement. No incisions are made in the trachea.
9. Once the catheter is seen in the trachea in the neck, advanced it about 5 mm to be reliably passed the vocal cords but still well above the carina.
10. Prepare to instill up to 50 µl of liquid by injecting through the catheter with a gel loading pipette tip. Place the tip in the luer hub, but before injecting look carefully to observe movement of the fluid in the tip synchronous with the mouse’s breathing. Then inject the instillate.
11. With a 1 ml syringe, immediately do a relatively rapid inflation of 0.6 ml of air into the lungs through the catheter to help distribute the liquid deep in the lungs. Remove the cannula.
12. Remove the cannula.
13. Use a small amount of cyanoacrylate adhesive to close the small surgical wound as per package insert instructions for VetBond. Place mice in individual cages and visually monitor them until they wake up and behave normally without any indication of discomfort.

2. Measurement of the Fixed Lung Volume

1. Measure the lung volume using Archimedes principle as illustrated in Figure 3. Remove the fixed lung from the formaldehyde and dissect the heart and any other non-lung tissue.
2. Use a previously constructed simple homemade wire support device that is used to keep the lungs fully under water.
   NOTE: This device needs to be made compatible with whichever balance is being used. A typical device shown in Figure 4 is made from plastic pipettes and thin (20 G) wire. This system works well with the balance used in the video, but could easily be adapted to most laboratory balances.
3. Place a beaker with ≈200 ml of water on the balance and tare with the supporting cage in place in the water. See Figure 5. Remove the metal cage; place the lung on the water surface and press under water with the cage.
4. Record the weight on the balance. This number reflects the volume of water displaced and is thus a direct measure of the lung volume. Be careful to make sure the lung or suture or any part of the wire cage does not touch the sides or bottom of the beaker.
5. For accuracy, repeat this measurement. Remove the lung from the water, and dry on a tissue. Tare the beaker with cage in place again and repeat the lung volume measurement. The two volume measurements should then be averaged.
   NOTE: If the lungs are left in the formalin for more than about a week, the air in the lungs will be dissolved in the liquid. When this happens, the lung will sink, so it is no longer necessary to use any device as in Figure 4 to keep the lung submerged. In such case the volume can be measured by simply by holding the lung by one of the suture strings until it is completely submerged as illustrated in Figure 3.

Representative Results

Figure 1: Intravenous catheter with slightly curved tip used for instillation.

Figure 2: Sloped 3-ring binder used to support mice for the intubation. This binder set up to hold 3 mice.

Figure 3: Archimedes Principle. The weight of water displaced by a submerged object equals the volume of the object. Because the lung normally retains some residual air, a device as in Figure 4 is needed to keep the lung fully submerged.

Figure 4: Lab made support for keeping lungs completely underwater. Device constructed from plastic pipettes and metal wire.

Figure 5: Laboratory balance with beaker and tared submersion device ready for lung volume measurement.

Materials

Name	Company	Catalog Number	Comments
Laboratory Balance	Ohaus	Adventurer Pro Model AV 313	Other balances can be used if they have a range of 1-300 g
20 g Luer intravenous catheter	Insylte		Several other possible vendors, e.g., Jelco Optiva
500 ml laboratory bottle	Various		Several other possible vendors