Recurrent urinary tract infections are a serious health concern. This protocol will help us understand how short-lived exposures to urogenital bacteria result in damage to the bladder and trigger E.Coli-recurrent UTI. An advantage of this model is that bladder exfoliation and recurrent UTI is triggered by the urogenital bacterium Gardnerella vaginalis, which is often abundant in women who experience recurrent UTI.
Begin by cycling the G.vaginalis strain from a minus 80-degree-Celsius freezer stock into an anaerobic chamber. Streak the bacteria onto an NYCIII plate without antibiotics. Incubate the plate at 37 degrees Celsius anaerobically for 24 hours.
In the anaerobic chamber, inoculate five milliliters of anaerobic NYCIII media with a one-microliter loop full of cells from the NYCIII plate and incubate the culture statically at 37 Celsius under anaerobic conditions for 18 hours. Do not include antibiotics in the growth medium. Determine the OD600 of the culture using a spectrophotometer.
Then, centrifuge it at 9, 600 times G for one minute and aspirate the media. Resuspend the bacterial pellet in PBS to the desired concentration and serially dilute and plate the inoculum. Add 10 microliters of urine to 90 microliters of PBS in a cytofunnel cassette with an attached filter.
Place the cassettes in the cytocentrifuge with a slide and spin them at 600 to 800 times G for six minutes with high acceleration. After removing the slides and allowing them to dry overnight, stain the slides with a hematology staining kit and analyze them by light microscopy for the presence of PMNs and epithelial cells. Once tuberculin slip-tip syringe with fixative, affix a catheter to the end, bevel facing opposite the syringe markings.
Snip off the excess tubing one to two millimeters from the end of the needle, taking care not to expose the needle tip. Flick the syringe to remove bubbles and push the plunger to void air. Then fill the catheter with fixative over a microcentrifuge tube After anesthetizing and sacrificing the mouse, once the legs are secured, open the mouse's pelvic area with forceps and a pair of surgical scissors to expose the bladder.
Carefully push aside the adjacent fat, but leave the bladder in place. Hold the syringe with the dominant hand with the needle pointing down. Dip the catheter tip into sterile lubricant and position the catheter tip at the urethral opening, holding the syringe barrel away at a 30 to 45 degree angle over the mouse body.
Apply downward pressure with slight clockwise motion and gently insert the catheter into the urethra As the catheter tip enters, hinge the syringe toward the tail of the mouse while continuing to slide the catheter further into the urethra until the syringe barrel is parallel to the working surface. The entire catheter needle shaft should enter the mouse, positioning the catheter tip within the bladder lumen. Slowly deliver 50 to 80 microliters of fixative, causing the bladder to inflate like a balloon.
Keep the catheter in place and raise the syringe slightly, tilting the tip up. With the other hand, open a hemostat and slide one prong under the catheter needle at the intersection of the urethra. Partially close the hemostat until it just makes contact with the needle.
Gently slide the catheter needle out of the bladder while simultaneously clamping down and locking the hemostat completely to prevent loss of the fixative. Grip the hemostat so that it is parallel to the working surface with the bladder resting on top. Lift up gently and carefully cut under the hemostat to remove the bladder with the hemostat still attached.
Place the bladder and attached hemostat into a Falcon tube containing warmed fixative. Ensure that the bladder is fully submerged in the fluid and not pressed against the walls of the tube. In order to image the bladder by scanning electron microscopy, sagittally bisect with a clean, double-sided razor blade or scalpel and make a second cut tangential to the hemostat to release the bladder.
This results in two half-bladder cups. If any remaining fat pads exist on the exterior of the bladder, gently remove them. Rinse the bladder halves three times in sodium cacodylate buffer.
Stain the tissue with 1%osmium tetroxide in 0.15-molar cacodylate buffer for one hour at room temperature. Perform this step with the staining vessel wrapped in foil to maintain a dark environment. After staining, rinse the bladder halves three times in ultra-pure water.
If osmicated oil is seen on the surface of the water, aspirate or wick it off to prevent contamination during the drying steps. After dehydrating the tissue, bisesct the bladder halves with a clean, double-sided razor to generate four total pieces. Following inoculation, UPEC titers are detectable in urine.
The level of UPEC bacteriuria is high on day one and may increase during the first week before decreasing at later time points. Approximately 65 to 80%of mice will have no detectable UPEC in the urine by 28 days post-inoculation. These mice can be used in the subsequent steps of the model.
Mice that remain bacteriuric should be eliminated from the experiment. Two sequential G.vaginalis exposures, given 12 hours or one week apart, result in the emergence of UPEC from intracellular reservoirs to cause recurrent bacteriuria. Urine cytology analysis can be used to detect polymorphonuclear cells in urine from G.Vaginalis-exposed mice with displayed UPEC emergence.
In the model with two exposures given one week apart, UPEC titers in bladder tissue are lower in G.vaginalis-exposed mice compared to PBS, presumably due to emergence of UPEC from reservoirs and subsequent clearance. Visualization of in situ fixed-bladder tissue by SEM reveals large, superficial umbrella urothelial cells lining the bladder surface in control mice exposed only to PBS. Urothelial exfoliation is evidenced by a loss of superficial umbrella cells, revealing smaller underlying transitional epithelium in mice exposed to G.vaginalis.
Early after UPEC inoculation, during the establishment of intracellular reservoirs, bacterial cells are visible on the urothelium and filamenting out of exfoliating cells. The methods described here pave the way for future studies to investigate the effect of additional Gardnerella strains or other urogenital species on E.coli rUTI.
