The overall goal of this experimental approach is to investigate the immune response to urinary tract infection, and to prevent direct comparison between male and female animals. This method can help answer key questions of mucosal immunology related to how males and females respond to urinary tract infection. The main advantage is that infection is established via the natural root.
We first had the idea for this method after noting how often it is stated, that the installation of the bladder of the male mice is impossible. Though this method will provide insight into urinary tract infection, it can also be applied to other disease studies, such as bladder cancer. To begin, prepare one pediatric intravenous access canula for each group of mice to be infected.
Using the inbuilt spring mechanism, divest each canula of its needle as instructed by the manufacturer. Discard the needles, preserving only the plastic intravenous canula. Sterilize the catheters in a laminar flow hood for one UV cycle of approximately 25 to 30 minutes.
After going in overnight culture of uropathogenic e coli, or UPEC, according to the text protocol, spin the bacterial suspension in a table-top microcentrifuge at 17, 000 times g for one minute, and resuspend the resulting bacterial pellet at two times 10 to the eighth CFU per milliliter in PBS. Serially dilute an aliquot of suspension, and plate on LB agar with antibiotics if appropriate, to determine the exact inoculum for each infection. Draw the bacterial inoculum into a one milliliter 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. After anesthetizing a female mouse according to an approved protocol, place the animal 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.
When the bladder is empty, 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. Next, place the tip of the catheter perpendicular to the mouse at the urethral orifice, then, 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. Once the catheter is in place, use a finger from the non-dominant hand that's resting on the mouse abdomen to very gently pull the abdominal skin towards the head of the mouse.
Note that the urethral orifice will not move. However, if the catheters is in the vagina, the tissue will move up and away from the catheter. With the hub of the catheter against the urethral orifice, slowly dispense 15 microliters of the bacterial inoculum.
A slow installation rate minimizes vesicoureteral reflux into the kidney. Slowly remove the catheter to prevent leakage. Then place the animal in its cage in a supine position.
To inoculate male mice, place two thumb forceps cranially and caudally to the mouses external genitalia. Retract the prepuce to fully expose the gland's penis. Then, once the penis is externally positioned, release the thumb forceps.
Reposition the forceps to stabilize the protruding penis perpendicular to the animal, holding the organ gently but tautly. Then through the small opening in the tip of the urethral meatus, carefully introduce the catheter and gently guide it into the penis using forceps to gently maintain tension. Once the hub of the catheters meets the tip of the penis, very slowly dispense 50 microliters of the inoculum, while maintaining the position of the penis.
Note the quality of the instillation according to a predetermined instillation quality score, such as the one shown here. Retract the catheter slowly over five seconds to prevent leakage of the inoculum. Place the mouse in its cage in a supine position.
The animal should begin to recover 30 to 45 minutes following administration of the anesthetic. After sacrificing mice according to an approved protocol, use 70%Epinal to moisten the abdomen thoroughly to minimize contamination by the fur. Using scissors, make a two centimeter incision across the lower third of the mouses abdomen, and aseptically remove the bladder and any other desired organs.
Transfer the organs into five milliliter polypropylene snap-cap tubes, containing one milliliter of sterile PBS and keep all samples on ice. With a hand-held homogenizer, homogenize the organs until almost no solid tissue remains. Discard any shiny, beige-white fat tissue that does not homogenize well.
Then, use Epinal, followed by PBS to wash the homogenizer between each experimental or organ group. After preparing serial dilutions of homogenized organ suspension, plate dilutions on LB agar plates, with antibiotics when appropriate, and incubate at 37 degrees Celsius overnight. To carry out flow cytometry on bladder tissue, after isolating the bladder as demonstrated earlier in this video, use scissors to mince the tissue well.
Add one minced bladder per tube containing digestion buffer. Then shake the tube to wash the minced tissue in the digestion buffer, and keep it on ice until all the bladders are dissected and minced. Incubate the minced bladders at 37 degrees Celsius, for 60 to 75 minutes with vigorous shaking by hand for five seconds, every 15 minutes.
When the tissue has a glassy transparent appearance resembling wet tissue paper, digestion is complete. Inactivate the digestion enzymes by adding two to three milliliters of ice-cold flow cytometry buffer, and gently mix. Then place the tubes on ice.
To ensure a single-cell suspension, and to remove any connective tissue, pass the contents of the tube through a 100 micrometer filter placed in a 15 milliliter conical tube. Then, with the end of a syringe plunger, gently press any remaining tissue through the filter. Wash the filter with an additional two milliliters of flow cytometry buffer, and keep the samples on ice.
The wash the samples by centrifugation at 200 times g and four degrees Celsius, for seven minutes. Resuspend the pellets in 100 microliters of flow cytometry buffer containing Fc block, diluted to five micrograms per milliliter, and transfer to a 96 Well round bottom plate. After 10 to 15 minutes add 100 microliters of the desired antibody cocktail to each sample.
Then incubate the samples on ice protected from light for 30 to 45 minutes. Wash the samples by centrifugation at 200 times g and four degrees Celsius for seven minutes. Finally, resuspend the cell pellets in 200 microliters of flow cytometry buffer, and pass the samples through a 40 micrometers cell strainer on a five milliliter polystyrene tube, just prior to acquisition on a flow cytometer.
This figure shows representative data from mice infected for 24 hours. Bacterial burden was equivalent between male and female mice at 24 hours post infection. To determine whether loss of inoculum at the time of infection impacted the establishment of infection each instillation was scored on a scale of one to five, with one being the most optimal.
Bacterial colonization was determined 24 hours post infection. No statistically significant differences were found among the CFU's obtained from animals with different instillation scores as assessed by a nonparametric Kruskal-Wallis test, comparing the mean rank of each column with the mean rank of every other column, and correcting for multiple comparisons using a Dunn's test. It can be concluded that suboptimal instillations, resulting in substantial leakage of bacterial inoculum during infection, do not significantly impact bacterial colonization at 24 hours.
Importantly, the frequency of suboptimal instillation will decrease with practice. Finally, while the number of CD45 positive immune cells, present in naive animals was not different between female and male mice, there was a statistically-significant increase in infiltration into the bladders of infected female mice. While attempting catheterization, it is important to use slow, gentle movements.
With its development, this technique has helped us to explore sex-based differences in immunity in the bladder of male and female animals.
