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

Abstract

Source: Nielsen, T. B., et al., Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia. J. Vis. Exp. (2018)

This video demonstrates the procedure of delivering bacteria through the oropharynx into the lungs of an anesthetized mouse to induce lung infection and trigger an immune response.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Inoculating/Infecting Mice

1. Suspend an anesthetized mouse, hanging it by its top incisors on a strong, thin string (e.g., dental floss) secured to a fixed object at a height approximately twice the mouse’s body length (e.g., 20 cm) above the operating surface (e.g., laboratory bench).
2. Gently pull the mouse's tongue out of its mouth using sterile, blunt-ended forceps. Transfer the tongue to a sterile, gloved finger to prevent accidental crushing of the mouse’s tongue. Hold the tongue outside of the mouth, allowing access to the oropharynx and preventing swallowing of the inoculum to the side.
3. While holding the mouse’s tongue, place the 50-μL inoculum (bacterial suspension) into the oropharynx using a micropipette; the oropharynx is located at the junction of the oral cavity and the pharynx toward the back of the mouth.
   NOTE: It is very important to only deliver liquid by pipetting to the first stop on the micropipette. The mouse will stop breathing for a few seconds when the inoculum is placed in the oropharynx; eventually, reflexive aspiration will cause the mouse to inhale the bacterial suspension, indicated by the distinctive crackling noise of liquid entering the lungs, which results in pneumonia infection.
4. If the inoculum is not placed far back enough to block normal respiration, pinch the nares with forceps to force reflexive aspiration through the mouth and subsequent inhalation of the inoculum.
5. After inoculation, release the tongue, remove the mouse from the suspension string, place it in a cage, and monitor until it wakes up from anesthesia.

2. Monitoring Disease Progression

NOTE: Due to animal suffering, various indicators should be used to indicate when mice become moribund; euthanasia should be performed following this determination, in accordance with a previously approved IACUC protocol; various markers of moribundity include body temperature, weight loss, appearance, gait, and other biomarkers that can be obtained from blood (e.g., via iSTAT).

1. Euthanize the mice according to previously approved IACUC protocol (e.g., CO₂ followed by cervical dislocation).
2. Remove the lungs from a euthanized mouse by cutting the trachea, pulmonary artery, and pulmonary vein close to the lungs.
   1. Microscopy
      1. Place the lungs (or portion) in a specimen mold.
      2. Fill the specimen mold with optimal cutting temperature (O.C.T.) compound, completely submerging the tissue.
      3. Freeze the sample at -80 °C.
      4. Send the sample to a pathology lab to section and mount on slides for microscopy.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chamber for Anesthesia	Kent Scientific Corporation	VetFlo-0720	Accommodates up to 5 mice
Cryomold, Intermediate Size	Sakura Tissue-Tek	4566	Disposable vinyl specimen molds, 15×15×5 mm
Dental Floss	Oral-B	37000469537	Tie to stable post approx. 6" above table height
Forceps	VWR	82027-440	Used to gently pull tongue out of mouse's mouth
Isoflurane for Anesthesia	Abbott	10015516	Alternative drug can be used; modify procedure accordingly
iSTAT Cartridge	Abbott	03P79-25	Various cartridges are available to suit your needs
Ketamine, 100 mg/mL	Western Medical Supply	4165	Dilute 1:10 in PBS to 1 mg/mL and combine with Xylazine at 1 mg/mL
Ointment for Eyes	Akorn	Tears Renewed	Avoid touching eye with tip of dispenser
Phosphate-Buffered Saline (PBS)	Corning	21-031-CM	Dulbecco's PBS without calcium and magnesium
Pipette Tips, 200-μL	VWR	10017-044	Autoclave before use
Pipetter, 200-μL	Gilson	Pipetman P200	Autoclave and calibrate before use
Spreader, Bacterial Cell	Bel-Art	F377360006	Sterilize by baking or autoclaving before each use
Vial, Cryogenic, 2.0 mL	Corning	430659	Used for cryogenic storage of concentrated bacterial inocula