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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
Visualization of the Infection with Bioluminescence
- To visualize the progress of the infection, insert a chromosomal bioluminescent operon (luxCDABE) into the Pseudomonas aeruginosa PGN5 and VE2 strains tested. The plasmids and protocol used to label these strains were developed in the Schweizer lab and may not be compatible with all species/strains of bacteria. Importantly, visualization of the infection is optional; thus, genomic insertion of this operon is not necessary to perform the mortality study.
- Prepare and validate strains. Additionally, check for bioluminescence in labeled strains at each validation step.
- After strains are prepared, inject the animals in groups of 10 with the bioluminescent strains following the steps above.
- Image the animals every 3 h for 24 h using an animal imaging system capable of bioluminescence.
- First, prepare the imager by setting the camera parameters and heating the stage for the animals. Then, set the oxygen flow to 1.5 L per min (or follow the manufacturer's recommendations).
- After the imager and stage are stabilized, place one mouse into the anesthesia chamber immediately following injection and administer 3.5% isoflurane into the chamber with O2 flow for about 4 min. The anesthesia methods may vary depending upon the chamber and/or anesthetic agent used; follow the manufacturer's recommendations. Determine proper anesthesia via the withdrawal reflex test.
- Move the mouse to the temperature-stabilized stage. Position the mouse on its back with arms outstretched and fit the mouse with a nose cone for administration of 2.5% isoflurane throughout the imaging procedure.
- Close the door and take bioluminescent images and X-rays of the mouse.
- When imaging is complete, return the mouse to its cage and monitor it. The mouse should regain consciousness within 3-5 min.
- Continue to image mice every 3 h for 24 h, each time using a different mouse from each group. Do not re-image a mouse within 24 h due to the possibility of adverse effects from re-exposure to anesthesia. A single mouse should only receive one dose of anesthesia every 24-36 h. Clean the imaging platform after each mouse is imaged. Turn off the imager between imaging time points.
NOTE: Bioluminescence will fade, regardless of the strain injected. The intensity and longevity of bioluminescence will vary depending on many factors, including the number of bacteria injected, the mouse strain, the bacterial strain, etc.