Preparation of a Murine Oropharyngeal Aspiration Model to Establish Bacterial Lung Infections

Begin with a secured anesthetized mouse. Gently pull its tongue to expose the oropharynx, the region of the throat located behind the mouth.

Deliver a small volume of bacterial suspension into the oropharynx.

The liquid remains in the oropharynx and triggers a breathing reflex.

This reflex causes oropharyngeal aspiration, pulling the bacteria down the trachea into the lungs.

The bacteria reach the small airways and alveoli, where they multiply and attach to the epithelial lining of the lung tissue.

This activates resident macrophages and leads to the release of signaling molecules that recruit neutrophils. These neutrophils release cytokines, leading to inflammation.

Return the mouse to its cage and monitor it.

After the mouse has been euthanized, cut the trachea and nearby blood vessels to remove the lungs.

Embed the lungs in tissue-freezing medium and freeze them for further analysis of bacteria-infected lung tissue.

To inoculate the mice, hang the first anesthetized animal by its top incisors on a strong, thin string. Secure it to a fixed object above the operating surface, at a height approximately twice the mouse's body length. Use sterile blunt-ended forceps to gently pull out the tongue, and transfer the tongue to sterile, gloved fingers to allow access to the oropharynx. Depress the plunger of a micropipette to the first stop to place 50 microliters of the inoculum into the oropharynx, until the mouse inhales the bacterial suspension, by reflexive aspiration.

The mouse will stop breathing for a few seconds when the inoculum is placed in the oropharynx, and eventually, reflexive aspiration will cause the bacterial suspension to be inhaled, indicated by the distinctive crackling noise of liquid entering the lungs.

Then return the animal to its cage, with monitoring, until full recumbency. At the appropriate experimental endpoint, cut the trachea, pulmonary artery, and pulmonary vein to allow removal of the lung tissue.

For histopathological analysis of the disease progression, place the lungs into a specimen mold, and fill the mold with optimal cutting temperature compound, until the tissue is completely submerged. Then freeze the samples at minus 80 degrees Celsius until their sectioning and analysis.