This protocol will allow researchers to perform repeat measurements of lung function in individual mice, decreasing experimental variability and reducing the number of mice needed for each experiment. The main advantage of this protocol is that it does not require visualization of the trachea through the oral cavity or traumatic dissection of the neck for intubation. This method will be of particular value to researchers studying chronic and progressive lung diseases such as asthma, COPD, emphysema, and pulmonary fibrosis.
Visual demonstration of this protocol is critical because understanding how to identify the trachea through the skin of the ventral neck is central to the method. Demonstrating the technique with Andrew Nelson will be Kate Nolan, a veterinarian from my laboratory. To create an intubation platform, fold a 15 to 20-centimeter length of 3-0 silk in half and tape the ends of the thread to the top of a three-inch three-ring binder.
Select a cannula of the appropriate size and length and cut a bevel at the distal tip of the catheter. Smooth the cut surface with abrasive paper to create a rounded tip and gently make a slight bend in the cannula approximately one centimeter from the bevel. When the materials are ready, confirm a lack of response to toe pinch in an anesthetized mouse and hook the upper incisors around the silk thread at the top of the angled surface of the platform to suspend the mouse in the supine position.
Once the mouse is squarely positioned in dorsal recumbency, gently grasp the base of the tail and retract the tail toward the table. Place a piece of tape over the base of the tail to secure the mouse and apply depilatory cream to the ventral-cervical region. After 30 to 45 seconds, use dry gauze to remove the cream and thoroughly rinse the skin with saline or distilled water.
Using forceps in the non-dominant hand, gently retract the tongue in a manner that sufficiently opens the mouth for introduction of the cannula. With the dominant hand, advance the cannula into the mouth such that the end that is distal to the slight bend is against the roof of the animal's mouth. Release the tongue and slide the flat edge of the closed forceps caudally along the ventral neck until the manubrium is reached to laterally displace the salivary glands and to flatten the muscle covering the trachea.
The trachea will appear transcutaneously as a white line. If necessary, rotate the forceps in a craniodorsal direction while maintaining tension on the skin in a caudal direction to cause the laterally displaced salivary glands to peak and to create more contrast around the trachea. Supinating the dominant hand with simultaneous flexion of the wrist, advance the cannula while simultaneously angling the distal tip of the cannula ventrally.
If the cannula has been properly placed, it will be observed within the trachea. To confirm a proper placement, connect a lung inflation bulb to the cannula and observe thoracic expansion with concurrent depression of the device. Without displacing the cannula, carefully unhook the incisors of the mouse from the intubation platform and transfer the mouse onto a horizontal platform, then attach the cannula to the adapter on the ventilator and, following a deep inflation, ventilate the mouse for 60 seconds before measuring the respiratory resistance.
Once the procedure is complete, transfer the mouse to a warmed platform and provide constant stimulation via light toe or tail pinches to encourage spontaneous respiration. Once spontaneous respiration is observed, continue to visually monitor the animal. When the respiration rate returns to normal, grasp the cannula at the level of the hub and gently pull the tube cranially and away from the mouse until the cannula is completely removed from the animal's mouth.
Once extubated, transfer the mouse to a clean recovery cage with heat support with monitoring until fully ambulatory. In this representative experiment, the respiratory resistance was measured on days 0, 3, 10, and 17, using the forced oscillation technique following a deep inflation to 25 centimeters of water held for five and 60 seconds of mechanical ventilation. No significant differences in the measured respiratory resistance were observed between any of the time points within each strain, suggesting that the absence of an increase in respiratory resistance over time indicates a lack of functionally significant intubation trauma-associated inflammation in the respiratory system over four successive time points.
This intubation procedure can be used to provide access to the trachea for the installation of experimental substances and the serial monitoring of respiratory function. This technique allows the assessment of lung function without invasive tracheostomy, facilitating the monitoring at multiple time points in chronic and progressive diseases within the same mouse.
