Methods for Detecting Cough and Airway Inflammation in Mice

My research focuses on chronic cough. To understand the pathophysiology of cough, my studies required test samples to verify changes in key factor levels. I aim to establish standardized procedures for harvesting tissue samples from mice.

Cough hypersensitivity is often triggered by a viral infection. For the first time, we found that viral infection might trigger cough hypersensitivity while increasing the release of interferon-gamma from T lymphocytes in the lung. Most patients with chronic cough have cough hypersensitivity, characterized by increased neural responsivity to a range of stimuli that affects airways and the lungs.

In the future, we will focus on peripheral and the central processes contributing to cough hypersensitivity. To begin, take the deeply anesthetized H1N1-infected mouse after cough detection, and collect blood from the eye orbits. Shake the blood collection tube to fully mix the blood and anticoagulant to prevent blood coagulation.

Then, centrifuge the collected blood at 800 G for five minutes at four degrees Celsius. Collect the supernatant and store it. Resuspend the pellet in one milliliter of D-Hank solution.

Spread 10 microliters of the blood cell suspension on a glass slide to determine cell profiles. To harvest the spleen, open the mouse's chest, remove the left auricle, and then perfuse the pulmonary and systemic circulation with five milliliters of normal saline. Using surgical forceps, remove the whole spleen from the mouse.

Now, measure the weight of the spleen. Cut the spleen into two halves. Fix the first half with 4%paraformaldehyde at room temperature for histopathological analysis.

Store the second half at minus 80 degrees Celsius for cytokine measurement. For bronchoalveolar lavage fluid collection, separate the right lung by ligating at the right main stem bronchus. Collect the bronchoalveolar lavage fluid from the left lungs by lavaging three times with 0.5 milliliters of PBS.

Centrifuge collected bronchoalveolar lavage fluid at 800 G for five minutes at four degrees Celsius. Then, collect the supernatant. Resuspend the pellet in 200 microliters of PBS.

Using a counting slide, count the leukocytes in the bronchoalveolar lavage fluid. To determine cell profiles by differential counts, smear 50 microliters of the cell suspension onto glass slides and allow it to dry. Fix the slide with 4%paraformaldehyde overnight before staining with hematoxylin eosin.

After bronchoalveolar lavage fluid collection, remove and fix half of the right lung and trachea with 4%paraformaldehyde for histopathological analysis. Store the other half at minus 80 degrees Celsius for western blotting, RT-qPCR, and enzyme-linked immunosorbent assay. H1N1 virus infection resulted in inflammatory changes in mouse lungs, including edema and many lymphocytes and neutrophil infiltration.

H1N1 virus infection induced inflammatory changes in mouse tracheae, including cilia shedding and inflammatory cell infiltrations. H1N1 virus infection significantly increased the ratio of the white pulp area to the whole spleen area in mice, with lymphocytes accumulating in the white pulp of the spleen.