Generating Urinary Tract Infection Model: A Technique to Administer Uropathogenic Bacteria in Mouse to Develop Urinary Tract Infection Model

Protocolo

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 from 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 a bacterial solution.
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 in 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 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 3). 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.

Materiais

Name	Company	Catalog Number	Comments
Aesculap ISIS	Aesculap	GT421	hair trimmer, with GT608 cap
Anesthesia vaporizer	Harvard apparatus limited	N/A	https://www.harvardapparatus.com/ harvard-apparatus-anestheticvaporizers.html
BD Insyte Autoguard 24 GA	BD	382912	Yellow angiocatheter, use sterile plastic tip for instillation
C57Bl/6J mice	Janvier	N/A
Dulbecco's Phosphate Buffered Saline, Gibco	ThermoFisher Scientific	REF 14040-083
Ethanol 70% denaturated 5L	VWR international	85825360
Hamilton GASTIGHT syringe, PTFE luer lock, 100 µL	Sigma-Aldrich	26203	to ensure slow bacterial instillation of 50 µL
Iso-Vet 1000mg/g	Dechra Veterinary products	N/A	Isoflurane