Precision Induction and Distinction of Coughing and Sneezing Reflexes in Mice

This protocol focuses on respiratory reflex in mice. We aim to stimulate the trachea and the nasal cavity to induce coughing and sneezing, and it will differentiate between these two types of events based on the audio features. Previous studies often used nebulized capsaicin or citric acid to stimulate the respiratory check and coughing and sneezing behaviors in mice.

However, it remains unclear whether respiratory check stimulation by nebulization induces coughing, sneezing, or both, as there's currently no reliable method for distinguishing these two responses. For this project, we established a method to induce coughing and sneezing by selectively stimulating the trachea or the nasal cavity. We also used acoustic analysis to identify the distinctive features between cough and sneeze.

Our research en masse provides a powerful tool that will directly support the development of future drugs for coughing and sneezing disorders. To begin, cut a 28-gauge needle into a cannula of approximately five millimeters in length. Using forceps and abrasive paper, smooth both its ends, and attach one end to silicone tubing.

Connect the tube-attached cannula to a one-milliliter syringe, and flush the cannula with water to verify patency. Next, bend the cannula at a 90-degree angle with a two-to three arm ratio. Insert the longer arm into the five-centimeter-long silicone tube.

Randomly assign mice to three groups, with six mice each. Now, secure the anesthetized mouse in a supine position on the table. After shaving the cranial and cervical hair, disinfect the area with iodophor and alcohol.

Under a microscope, incise the cervical skin for approximately one centimeter, and bluntly dissect through tissues and muscles to expose the trachea. Puncture the soft tissue between the cricoid and tracheal cartilages using a 32-gauge needle. Then, create a minimal tracheal opening with ophthalmic scissors.

Insert the silicone-sheathed metal cannula approximately one millimeter into the tracheal lumen, and secure the cannula-trachea junction with a 7-0 suture. Now, incise the scalp for approximately one centimeter, and bluntly tunnel subcutaneously from the neck up to the skull. Route the silicone tube through the subcutaneous tunnel to the cranium, and fix it onto the skull using dental cement, and then stitch the skin.

Suture the cervical incision and let the animal recover for five days. Fit the ultrasonic probe with a port-compatible sealing ring and insert the probe into the whole body plethysmography chamber port to ensure airtight contact. Connect the whole-body plethysmography chamber to the flow sensor and bias flow system.

Attach a skull-fixed silicone tube to a 30-centimeter extension tube, and place the mouse into the whole-body plethysmography chamber. Extend the tube through the top port of the chamber, and seal the port using modeling clay around the tubing. Verify air tightness by confirming that the respiratory waveform peaks fall within four, plus or minus two.

Connect a one-milliliter syringe loaded with five milliliters of capsaicin solution to the extension tube, and mount it onto the microinjection pump. Position a high-speed camera facing the chamber. Adjust the camera distance and focus for a clear view, and set the sampling rate to 160 frames per second.

Now, simultaneously start the whole-body plethysmography, ultrasound, and camera software. Trigger the pump to begin capsaicin infusion, and record cough counts for 10 minutes. Prepare the animals and all the materials required for the procedure.

After anesthetizing and preparing the mouse, secure it in a stereotaxic frame. Disinfect the surgical area with iodophor and alcohol. Incise the skin over the skull and nasal bone, using a scalpel to expose the nasal bone.

Using a microdrill, create a 0.5-millimeter hole at the junction between the nasal bone and the lateral process of the nasal septal cartilage. Now, expose the nasal mucosa through the burr hole. Gently lift the nasal mucosa using a 32-gauge needle to expose the nasal cavity.

Then, mount the polyethylene tube into the stereotaxic arm. Align the tip of the tube flush with the burr hole opening, and secure the tube onto the nasal bone using dental cement, and then stitch the skin. Place the mouse on a heating pad and allow it to recover until consciousness returns.

Connect the whole-body plethysmography chamber to the flow sensor and bias flow system according to the manufacturer's manual. Next, fit the ultrasonic probe with a port-adapted gasket. Insert the probe into the plethysmography chamber and verify an airtight seal.

Transfer the mouse into the plethysmography chamber, and extend the polyethylene tube through the top port. Seal the port using modeling clay to secure the tubing. Verify chamber air tightness and set up the camera as demonstrated earlier.

Finally, advance a 0.25-millimeter-diameter nylon filament through a soft tube into the nasal cavity to a depth of zero to three millimeters. Keep five centimeters of the filament exposed beyond the handheld tube. During each stimulation, press the free end of the filament until it bends to approximately 45 degrees, delivering around 0.6 grams of force to the nasal mucosa for one second.

Repeat the stimulation once every 30 seconds while recording sneezing responses for 10 minutes. Mice subjected to tracheal or nasal surgery with mechanical nasal stimulation and capsaicin tracheal challenge significantly increased sneeze and cough events, respectively. Whole-body plethysmography recordings showed both single and double-peak respiratory traces for capsaicin-induced coughing and mechanically-induced sneezing.

Sound oscillograms revealed that cough audio had an abrupt-onset peak intensity at the start, and was concentrated in the zero-to-30 kilohertz range. Whereas sneeze audio built progressively in intensity, spanned a zero-to-80 kilohertz range, and had a longer duration. The compression phase of single-peak coughs averaged around 32.39 milliseconds, significantly shorter than double-peak coughs, at around 53.17 milliseconds.

The average compression phase of single-peak sneezes was around 44.03 milliseconds, while double-peak sneezes averaged at around 74.24 milliseconds and were significantly longer. Single-peak patterns occurred in 77.55%of coughs, whereas only 32.42%of sneezes exhibited single peaks, with double-peak patterns dominating at 67.58%Sneezing audio durations were significantly longer than coughing durations, though with overlapping ranges. Transection of nasal sensory afferents significantly reduced capsaicin-induced sneezing, while coughing remained unaffected.