Visualization of Urinary Tract Infection in a Mouse Model Using Bioluminescence

Samenvatting

Source: Luyts, N., et al. Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging. J. Vis. Exp. (2021).

This video demonstrates the induction and tracking of urinary tract infection in a mouse model using bioluminescent E. coli injected into the bladder, where bacterial adhesion, invasion, and intracellular replication occur. The emitted light from the bioluminescent operon serves as a real-time marker of bacterial load during infection progression.

Protocol

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

1. Inoculation of the animals

1. Preparation of the animals
   1. Choose the desired mouse strain(s) depending on the research question and availability of knock-out lines, experimental details, and differences in UTI susceptibility. Keep in mind that transurethral catheterization is easier in female mice. Do not use animals younger than 8 weeks, as they are immunologically immature. Here, 12-week-old female C57Bl/6J mice were used.
   2. Order animals well in advance and let them acclimate, ideally for 7 days. Group house animals in individually ventilated cages, under standard 12 h light/dark conditions.
   3. Shave the abdominal region of the animals to limit the loss of signal. Do not use hair removal cream, as it can burn the skin of the animals quickly. Restrain the animal by tightly holding the scruff and hind limbs with the non-dominant hand while shaving with the dominant hand. Alternatively, shave under isoflurane anesthesia.
      NOTE: Animals were shaved 2 days prior to imaging, considering the animals will groom the shaved area even further.
   4. Provide water and standard food ad libitum throughout the experiment. However, deprive animals of water 2 h prior to instillation to minimize the bladder volume during instillation.
   5. Mount a sterile 24 G angiocatheter tip on a 100 µL syringe and fill the syringe with the bacterial solution.
      NOTE: To determine the background luminescence, obtain a baseline image prior to the instillation (see step 2, below).
2. Instillation of the animals
   1. Place the animals in an induction chamber and anesthetize them using inhalation of isoflurane with pure oxygen as a carrier gas (induction at 3% and maintenance at 1.5%).
   2. Place one animal on a working surface in the supine position and maintain a stable isoflurane anesthesia using a nose cone during the instillation. Apply the eye ointment.
   3. Expel the residual urine by applying gentle compression and making circular movements on the suprapubic region. Clean the lower abdomen with 70% ethanol prior to instillation.
   4. Lubricate the catheter tip with normal saline. Put the index finger of the non-dominant hand on the abdomen and push it gently upwards. Start the catheterization of the urethra at a 90° angle (vertically) and once resistance is encountered, tilt it horizontally before inserting it further (0.5 cm).
      NOTE: Never forcefully push the catheter, as this will cause harm to the urethra. Gentle turning motions can be helpful in catheter insertion. On the other hand, a lack of resistance usually indicates erroneous insertion into the vagina.
   5. Perform a slow (5 µL/s) instillation of 50 µL of the bacterial inoculum (2 x 10⁷ CFU).
      NOTE: Higher volumes or faster instillation might cause reflux to the kidneys. During practice of the technique, blue ink can be used to evaluate reflux.
   6. After the instillation, keep the syringe and catheter in place for a few more seconds and then slowly retract to prevent leakage. Record any irregularities such as a high amount of leakage or a bloody meatus and exclude animals, if necessary.
   7. Position the animal in the supine position at the nose cone of the imaging chamber and repeat the preceding steps 1.1.1-1.1.6 for all the remaining animals. Use one catheter per experimental group. Ensure anesthesia is continued and minimize the time between the first and the last animal.
   8. If necessary, administer antibiotics or experimental drugs prior to or after the imaging (step 2). For example, to administer enrofloxacin, add 40 µL of the enrofloxacin (100 mg/mL) solution to 3.96 mL of physiological saline to obtain a 1/100 dilution. Inject 100 µL/10 g subcutaneously at 9 am and 5 pm to administer 10 mg/kg of enrofloxacin twice daily for 3 days.

2. Bioluminescence imaging

1. Preparation and selection of imaging parameters
   1. Open the BLI acquisition software (see Table of Materials) and click on Initialize in the imaging device (see Table of Materials) to test the camera and stage controller system and to cool the CCD camera to -90 °C.​
      NOTE: During this process, the door is locked, and the progress of the initialization can be followed on the control panel. A green light indicates that the temperature of -90 °C has been reached. A warning will appear if imaging is attempted before completion of the initialization.
   2. Ensure data is saved automatically: Click on acquisition Auto-save and select the correct folder.
   3. Select Luminescence and Photograph. Check the default luminescence settings: Set excitation filter to Block and emission filter to Open.
   4. Set the exposure time to Auto when taking the first image, especially when expecting a dim signal, to ensure an adequate number of photon counts. For in vivo measurements and bright signals, set the Exposure Time to ~30 s. If the image is saturated, a warning will appear. If this happens, reduce the exposure time.
   5. Select the medium Binning, F/stop 1, and choose the correct field of view (FOV) (D for 5 animals).
   6. Set the subject height to 1 cm when imaging mice.
   7. Click on Add three times in the Acquisition Control Panel to obtain a sequence of three images, as technical replicates.
2. Imaging
   1. Place mice in the imaging chamber in the supine position and use the manifold nose cone to maintain anesthesia (isoflurane 1.5%) throughout the experiment. Image up to 5 mice simultaneously and separate the animals using the light baffle to prevent reflection.
   2. Close the door and click on Acquire to start the imaging sequence.
   3. Fill in detailed information about the experiment (wild type and knockout animals, treatments, day of imaging, etc.). The imaging settings, such as exposure time, are saved automatically.
   4. Remove mice from the imaging chamber and return them to their cage. Check for the full recovery after anesthesia. Within minutes, the animals should be fully awake and explorative. Do not provide analgesia as this might interfere with the UTI course.
   5. Return the cages to the ventilated racks until the next imaging cycle. After completion of the experiment, euthanize the animals by CO₂ asphyxiation or cervical dislocation. Do this before the recovery of isoflurane anesthesia to minimize distress

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Anesthesia vaporizer	Harvard apparatus limited	N/A	https://www.harvardapparatus.com/harvard-apparatus-anesthetic-vaporizers.html
Baytril 100 mg/mL	Bayer	N/A	Enrofloxacin
BD Insyte Autoguard 24 GA	BD	382912	Yellow angiocatheter, use sterile plastic tip for instillation
C57Bl/6J mice	Janvier	N/A
Centrifuge 5804R	Eppendorf	EP022628146
Dropsense 16	Unchained Labs	Trinean	to measure OD 600nm
Dulbecco's Phosphate Buffered Saline, Gibco	ThermoFisher Scientific	REF 14040-083
Ethanol 70% denaturated 5L	VWR international	85825360
Falcon 14ml Round Bottom Polystyrene Tube, Snap-Cap	Corning	352057
Falcon 50ml cellstart	Greiner	227285
Hamilton GASTIGHT syringe, PTFE luer lock, 100 µL	Sigma-Aldrich	26203	to ensure slow bacterial instillation of 50 µL
Inoculation loop	Roth	6174.1	holder: Art. No. 6189.1
Iso-Vet 1000mg/g	Dechra Veterinary products	N/A	Isoflurane
IVIS Spectrum In Vivo Imaging System	PerkinElmer	REF 124262	imaging device