Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing

Our protocol can be used to examine the respiratory effects of leptin on the carotid body in the peripheral chemoreflex breathing control, and physiological responses to hypoxia. We can also assess the effects of loss and gain of carotid body function using surgical and chemical denervation and adenovirus transfection respectively. Before beginning the procedure, measure the temperature inside a custom made whole body plasthemagrophy chamber, and measure the relative humidity of the lab.

Next, measure the body weight and rectal temperature of the experimental mouse. Place the mouse inside the chamber and place an oximeter collar around the shaved neck of the animal. And be sure to completely seal the chamber to avoid air leakages.

Wait approximately 30 minutes until the mouse is quiet and the chamber is at a constant volume. Before beginning to record the respiratory signals and peripheral oxygen saturation at normoxia. After 20 minutes of normoxia, expose the animal to a constant mixed gas flow of 10%oxygen and 3%carbon dioxide.

And begin recording the first cycle of acute hypoxia in respiratory signals and peripheral oxygen saturation. During the first 30 seconds of hypoxia, open the two small side ports at the base of the chamber to allow in-plugs of the mix gases. After the initial 30 seconds, close one of the small side ports of the chamber and continue to record at a constant hypoxia at 10%of the fraction of inspired oxygen for five minutes.

At the end of the hypoxia treatment, switch the in-flow source to re-expose the mouse to room air and allow the animal to recover at normoxia for at least 30 minutes before beginning the next analysis. At the end of the experiment, plug a syringe into one of the small side ports at the base of the chamber and inject one milliliter of room air into the whole blood plethysmography chamber three times to calibrate the chamber. After the third flush, measure the temperature in the chamber with the animal still inside and the chamber still closed.

Then, open the chamber and measure the rectal temperature of the mouse before returning the animal to its home cage. For surgical denervation of the carotid sinus nerve, administer analgesia to the anesthetize mouse, and remove the hair at the ventral region of the neck. Disinfect the exposed skin with Betadine and alcohol and apply ointment to the animal's eyes.

After making a midline incision, remove the connective and adipose tissues to expose the bifurcation of the common carotid arteries and identify the hypoglossal nerve. Lift up the hypoglossal nerve to expose the glossopharyngeal nerve immediately below and use micro-spring scissors to bilaterally dissect the carotid sinus nerves. When the carotid bodies have been exposed, gently suspend one microliter of the appropriate adenovirus in four microliters of liquid Matrigel matrix on ice, per side, and apply five microliters of the viral suspension to the carotid body area bilaterally.

Wait two to three minutes until the liquid Matrigel matrix has solidified before closing the incision with 6-0 silk suture and administering one milliliter of normal saline subcutaneously. Then house the mouse in a recovery chamber with monitoring until sternal recumbency before returning the animal to its home cage. Continuous infusion of leptin significantly increases the hypoxic ventilatory response in lean C57BL/6 mice while carotid sinus nerve dissection abolishes this leptin induced increase.

As expected, no attenuating effects of carotid sinus nerve dissection on the hypoxic ventilatory response are observed in the sham surgery group after leptin infusion. The induction of leptin receptor long isoform expression in the carotid bodies of leptin receptor long isoform deficient obese db/db mice results in a significant increase in the hypoxic ventilatory response. Whereas after transfection with control LacZ adenovirus, no change is observed.

These techniques allow analysis of the role of carotid bodies in the peripheral chemoreflex and the effects of leptin on carotid bodies in the control of breathings.