This method can help answer key questions in the field of antibiotic resistance about novel therapeutics with a treatment of ventilator-associated pneumonia. The main advantage of this technique is that it accurately replicates clinical infection in a quick and easily reproducible manner. 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-handed forceps to gently pull out the tongue, and transfer the tongue to sterile, gloved fingers to allow access to the oral pharynx. Depress the plunger of a micro pipette to the first stop to place 50 microliters of the inoculum into the oral pharynx, 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 oral pharynx, and eventually reflexive aspiration will cause the bacterial suspension to be inhaled, indicative 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.
To estimate the bacterial burden, transfer the harvested lung tissue to a 50 milliliter conical vial, and determine its mass with an analytical balance. Then transfer five milliliters of sterile PBS to the vial. Homogenize the lung tissue with a tissue homogenizer.
And perform serial dilutions in the homogenate and sterile PBS, to achieve approximately 1000 colony forming units, or CFU, per millimeter, based on the expected bacterial burden in the infected lungs. Plate 100 microliters of each dilution onto individual tryptic soy agar plates. And use glass beads to evenly distribute the homogenates.
Then store the plates in a humidified, 37 degree Celsius incubator over night. Before calculating the number of colony forming units per dilution. It is necessary to infect the mice with various inoculum concentrations, to determine the LD 100.
Under these representative experimental conditions, two times 10 to the eighth CFU per mouse was too high. Five times 10 to the seventh and two times 10 to the seventh, were too low. And one times 10 to the eighth was just right Hematoxylin and ee-ah-zin staining of lung sections before and 24 hours after oral pharyngeal aspiration of the bacterial species commonly encountered in ventilator-associated pneumonia in humans, typically results in substantial alveolar inflammation.
While attempting this procedure, it's important to remember the inoculum must be prepared and atomy surd identically from experiment to experiment to obtain reproducible results. After watching this video, you should have a good understanding of how to recapitulate ventilator-associated pneumonia using a mouse model.
