The overall goal of this procedure is to reliably intubate mice orally. This is accomplished by first carefully positioning the anesthetized animal in the proper orientation. On the otoscope speculum, the vocal cords are then visualized and the endotracheal tube is passed into the throat using a stylet.
Next, the intubated mouse is placed on a ventilator to verify proper placement of the tube. Alternatively, the otoscope can be used to deliver test substances to the lung. Ultimately, accurate pressure volume relationships in the lung can be measured.
The main advantages of this technique over existing methods like tracheostomy are that it is fast, bloodless causes no trauma and can be repeated. Visual demonstration of this technique is critical as the intubation steps are difficult to learn and written instructions have little meaning without simultaneous visualization of the anatomy and the mechanics of intubation. To perform an endotracheal intubation using an otoscope first scruff, an anesthetized eight-week old mouse, and then use cushioned fine forceps to gently extend the tongue from the mouth.
Maintain the extension by holding the tongue between forceps and applying gentle force, and then pull the mouse up onto the speculum of the otoscope with a vertical motion. Look through the ocular of the otoscope while continuing to scruff the animal tightly and gently pull the mouse onto the full length of the speculum of the otoscope by the tongue and the scruff. Look carefully for the vocal cords, which should be easily visible and move with each breath the structure of the laryngeal opening.
The laryngeal ATUs should look relatively white. If the cords are not moving or are poorly visualized, rotate the animal slightly and gently hyperextend the neck. Then with the dominant hand, pick up a 20 gauge, one inch catheter equipped with a one centimeter length of PE 10 tubing extending through the tip of the catheter and hold it like a pencil.
Then insert the catheter into the side of the speculum. Direct the PE 10 tubing through the vocal cords and advance the catheter over the tubing until the hub is at the level of the lower incisor. Then remove the tubing quickly.
Next, gently transfer the animal from the speculum to a mechanical ventilator, and verify the tube location by visualizing the expired air bubbles going through the peep trap. Observe an airway pressure tracing for negative deflections to confirm the proper placement of the tube. In the case of esophageal intubation, the pressure tracing will reveal significantly higher pressures and no negative deflections.
Alternatively, place a small amount of water into a piece of IV tubing and connect the tubing to the intubation tube, verifying the movement to and from the mouse with respiration. An alternative use of the otoscope method is to deliver test substances to the mouse. Use the speculum to insert a gel loading pipette up to the vocal cords and instill up to 50 microliters of the treatment of interest.
The mouse will aspirate the liquid through the cords verified by the ous. Breathing sounds heard until the fluid is distributed fully in the lungs. After administering the treatment, gently remove the pipette and place the mouse on a heating pad.
Alternatively, place the animal inside of a cage that is on a temperature controlled warming table. When the animal has fully recovered, place it back into its cage. Intubation through the otoscope is reliable and fast.
In this representative airway pressure tracing the blue line reflects the airway pressure, and the red line reflects the tidal volume tracing in the mouse immediately after intubation. The negative downward deflections of the pressure curve indicate the generation of negative intrathoracic pressure in the mouse by spontaneous respiratory effort and proper placement of the endotracheal tube. For this representative pressure volume curve, the mouse was intubated with a 20 gauge intravenous catheter as just demonstrated paralyzed with pantonium and mechanically ventilated.
The curves illustrate that the catheter does not leak up to 30 centimeters of water pressure and can therefore be used to make accurate measurements of pressure volume relationships in the mouse. In this figure, a representative dose response curve is shown. In this experiment, the control animal had a minimal response to methylcholine up to 25 milligrams per milliliter, while the albumin, immunized and challenged mouse demonstrated hyperresponsiveness to the treatment.
The measurements were made with mice intubated with a 20 gauge catheter as just demonstrated, and were indistinguishable from those made in a tracheostomies mouse by using direct laryngoscopy. Quantitative delivery to the lung is both more reliable and more uniform. For example, as illustrated in this image, the direct installation technique can be used to accurately deliver antigen to the lower respiratory tract in a model of allergic inflammation.
After watching this video, you should have a good understanding of how to use an otoscope to perform a direct laryngoscopic intubation in a mouse.
