Establishment of a Mouse Model with Cough Hypersensitivity via Inhalation of Citric Acid

Chronic cough significantly impair the quality of life, causing a pressing appearance and a substantial economic burden. We focus on the pertinacity of cough, especially the mechanism of cough hypersensitivity. We establish a mouse model with cough hypersensitivity, which will be used for further study on the mechanism of chronic cough. Compared to other animal models, especially guinea pigs, easier to maintain, more cost-effective, and amenable to genetic manipulation. Our protocol is simple to operate. The model is easier to contrast. MA is more feasible. This model will be valuable too for further study on the mechanism and potential novel treatments for chronic cough.

[Narrator] To begin, place the mice in two independent chambers, each connected to an ultrasonic nebulizer. Expose the model group to 0.1 molar citric acid aerosol, and the control group to 0.9% normal saline aerosol. Set the atomization rate to the maximum value of three milliliters per minute and adjust the exposure to two hours per day over two weeks. After each session, return the mice to their respective cages. Set up the non-invasive whole body plethysmography system to measure cough sensitivity. Connect the chambers, flow transducers, bias flow, and other components. Calibrate the system by clicking the Calibrate button to ensure accurate measurements. Then, create a new cough study in the system software for mice. Configure the study parameters to allow one minute for acclimation, 10 minutes for response time, and 10 minutes for chemical solution delivery. After that, place each conscious mouse into individual chambers, ensuring only one mouse per chamber. Then, add one milliliter of the appropriate chemical solution, such as normal saline, citric acid, or capsaicin to the nebulizer to record the number of cough events once the procedure begins. Prepare the instruments for airway hyperresponsiveness measurement. Create a dose response study in the system software for mice. Configure the measurement parameters and task sequence. One minute for acclimation, 30 seconds for methacholine delivery, three minutes for response time, and one minute for recovery. Place each conscious mouse into individual chambers, ensuring only one mouse per chamber. Then, add 50 microliters of either PBS or methacholine to the nebulizer and initiate the study. Record the value of Penh, which indicates bronchoconstriction in response to increasing concentrations of methacholine. Cough sensitivity significantly increased in the model group after one week and persisted throughout the exposure period. No mortality was observed in either the model or control group during the study. After exposure, the number of spontaneous cough events was significantly higher in the model group than in the control group. Cough sensitivity induced by normal saline, citric acid, and capsaicin was also significantly higher in the model group after exposure. Airway hyperresponsiveness remained comparable between the two groups with no significant difference after modeling. To begin, collect blood from the orbital sinus of the euthanized mouse into a 1.5 milliliter microcentrifuge tube. Place the tube on ice, centrifuge the blood at 3000 G for 10 minutes at four degrees Celsius. Use a pipette to collect the supernatant and aliquot it. Then, open the chest to expose the trachea and insert a 22-gauge indwelling needle into the trachea. Lavage the trachea three times with 0.5 milliliters of pre-cooled PBS and collect the bronchoalveolar lavage fluid. Then, centrifuge the collected fluid at 500 G for 10 minutes at four degrees Celsius. Collect the supernatant, aliquot it, and store it at -80 degrees Celsius. Re-suspend the pellet in 100 microliters of PBS. Open the chest to expose the lungs and carefully remove the lung tissues. Place the collected lung tissues into cryogenic vials. No significant differences were observed between the groups in the total inflammatory and differential cell counts in bronchoalveolar lavage fluid.