Establishment of a Urinary Tract Infection Model in Female Mice

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

1. Preparation of Catheters

1. Prepare one pediatric intravenous-access cannula for each group of mice to be infected. Using the inbuilt spring mechanism, divest each cannula of its needle, as instructed by the manufacturer. Discard the needles, preserving only the plastic intravenous cannula.
2. Sterilize catheters in a laminar flow hood for one ultraviolet (U.V.) cycle, typically 25 – 30 min.
   Note: Catheters can be used for more than one mouse, however, a new catheter should be used between experimental groups, sexes, or bacterial strains.

2. Preparation of Uropathogenic Escherichia coli for Infection

1. At least two days prior to infection, using a sterile inoculation loop, streak uropathogenic Escherichia coli (UPEC) from a frozen bacterial stock onto a Luria Broth (LB) agar plate, containing antibiotics if appropriate. Incubate at 37 °C overnight. Plates can be stored at 4 °C for up to one week.
2. In a 100 ml sterile Erlenmeyer flask, inoculate 10 ml LB, containing antibiotics if appropriate, with a single colony from the LB plate and incubate standing at 37 °C overnight (approximately 16 – 18 hr).
   Note: Standing cultures permit the expression of type 1 pili, which are necessary for infection. Cultures grown shaking will have less efficient pili expression, and therefore, inconsistent rates of infection.
3. Measure the optical density (OD)₆₀₀ of the overnight culture and calculate the number of bacteria per ml using a pre-determined growth curve. As an example, for UPEC strain UTI89, OD₆₀₀ = 0.35 is equivalent to 2 x 10⁸ colony forming unit (CFU) per ml, thus, an overnight culture with an OD₆₀₀ = 2.4 would be equivalent to 13.7 x 10⁸ CFU per ml. Empirically determine the relationship between the OD₆₀₀ and viable bacteria with every new strain employed for infection.
4. Determine the number of bacteria needed by considering the number of animals to be infected and that each mouse should receive approximately 10⁷ colony-forming units (CFU) in 50 μl phosphate-buffered saline (PBS). Include in the calculation the ~130 µl dead volume of the catheter and syringe nub and 100 µl needed to determine the inoculum.
   1. Spin the bacterial suspension in a tabletop microcentrifuge at 17,000 x g for 1 min and resuspend the resulting bacterial pellet at 2 x 10⁸ CFU per ml in PBS. Serially dilute an aliquot of this suspension and plate it on LB agar, with antibiotics if appropriate, to determine the exact inoculum for each infection.
5. Draw the bacterial inoculum into a 1 ml syringe and attach the catheter to the end of the syringe. Tap the syringe to remove any air and depress the plunger to fill the dead air space in the catheter before beginning the instillation.

3. Preparation of Mice

1. Anesthetize mice via intraperitoneal injection of 100 mg/kg ketamine and 5 mg/kg xylazine. Supplemental heat can be provided. 
2. Ensure that each mouse is fully sedated by squeezing the footpad with medium pressure. Mice are fully sedated when they do not react and require 3 – 5 min to reach this state.
3. Place mice supine and apply medium pressure to the lower abdomen to empty the bladder of urine. Full bladders feel like a pea under the skin between the iliac crests.
   Note: Inhaled isoflurane has been used to catheterize female mice, however, it has not been tested whether male mice are sufficiently anesthetized by inhaled isoflurane for this procedure.

4. Transurethral Instillation of Female Mice

1. Using the non-dominant hand, place the thumb on the tail and a finger of the same hand on the abdomen of the mouse and apply gentle pressure in opposing directions to hold the mouse firmly in place.
2. Place the tip of the catheter perpendicular to the mouse at the urethral orifice. With gentle pressure, slide the catheter into the urethra, until the hub meets the urethral orifice, while simultaneously lowering the syringe so that it is parallel to the working surface. The catheter requires no lubrication. Do not push or force the catheter into the urethra. The catheter should slide smoothly and easily into the urethra, with little to no resistance.
   1. Before instilling the inoculum (step 4.3), once the catheter is in place, very gently pull the abdominal skin towards the head of the mouse with the finger from the non-dominant hand that is already on the abdomen of the mouse (step 4.1). If the catheter is in the urethra, the tissue composing the urethra orifice will not move, whereas if the catheter is incorrectly placed in the vagina, the tissue will move up and away from the catheter. This rapid test should be performed on each mouse.
3. When the hub of the catheter meets the urethral orifice, slowly dispense 50 µl of the bacterial inoculum. A slow instillation rate minimizes vesicoureteral reflux into the kidney. Slowly remove the catheter to prevent leakage, over a count of 5. Place mice in their cages in a supine position.