The overall aim of this procedure is to measure tactile allodynia in a mouse resulting from a bacterial infection of the prostate. This is accomplished by first preparing a bacterial suspension for installation into a male mouse that is placed under anesthesia. The second step is to gently extrude the penis of the mouse, followed by intraurethral catheterization.
Next bacteria are instilled and initiate an ascending infection of the mouse prostate. The final step is measurement of tactile allodynia following infection. Ultimately, increased response frequency to vray fibers applied to the pelvic area is used to show enhanced tactile elo DIA of the pelvic region in response to bacterial prostatitis.
The main advantage of this technique over existing methods such as hormone induced path prostatitis or autoimmune prostatitis, if that it uses clinically irrelevant bacteria and a natural ascending infection methodology that are really important for prostate disease pathogenesis, Generally, individuals that are new to this method will struggle because of an inability to consistently register positive responses to vray fiber stimulation. All procedures in this protocol must be performed according to institution and government guidelines. Testing for referred hyperalgesia and tactile allodynia is performed at a fixed time of day using standard methodology with a single experimenter testing all of the animals.
Blind testing of groups should be utilized to combat the limitations of behavior-based pain testing in animal models. To obtain a baseline measurement for all animals in the experiment, first acclimate five to seven week old male mice to the testing room for 30 minutes, and then place each mouse in an individual plexiglass chamber with a stainless steel wire grid floor to test for tactile allodynia. Apply individual von Fray filaments with forces of 0.04, 0.16, 0.4, 1.0, and 4.0 grams to the lower abdominal area in the general vicinity of the prostate.
Apply each filament in increasing order of force for one to two seconds with at least five second intervals between stimulations for a total of 10 times. Take care to stimulate different areas within this region to avoid desensitization or wind up effects. Three types of behavior are considered a positive response to filament stimulation.
One sharp retraction of the abdomen, two, immediate licking or scratching of the area of filament stimulation.Three. Jumping calculate response frequency as the percentage of positive response and report data as the mean percentage of response. Frequency plus minus standard error.
Animals with more than 25 positive baseline responses are excluded from the study to examine for specificity of the allodynia response. The tactile allodynia response of the pore region is tested immediately following the pelvic area testing by applying the von Frey filaments to the planter region of the hind pore until the filament bends slightly. Test the filaments in ascending order until a positive response defined as either a sharp withdrawal or licking of the test pore is observed.
After a positive response is observed, apply the weaker filament to identify the fiber with the greatest amount of force that evokes no response. First, prepare the catheter by inserting a polyethylene catheter into the hub of a 30 gauge needle attached to a glass 1.5 to two centimeter Hamilton syringe loaded with the bacterial suspension after anesthetization with one to 4%isof fluorine in a plexiglass chamber. Transfer amounts to a nose cone dorsal side down for maintenance of anesthesia, extrude the penis by applying gentle pressure.
Then using a cotton swab, apply a liberal amount of artificial tears as lubricant to the entrance of the penile urethra. Carefully insert the catheter into the urethra. Then introduce 10 microliters of PBS containing one times 10 to the eight CP one bacteria into the urethra by slowly decompressing the plunger of the syringe.
Once the bacterial suspension has been administered carefully remove the catheter. Return the mouse to the anesthesia chamber for 15 minutes to allow bacterial attachment and prevent immediate urination. Once the 15 minutes have elapsed, place the mouse back into the home cage and monitor for the next 24 hours.
Repeat testing for referred hyperalgesia and tactile allodynia responses at suitable intervals. Testing on days 3, 7, 14, 21 and 28 after infection is recommended. Tactile EALs measured its responses to mechanical stimulation of the pelvic region using Von Fray filaments of five calibrated forces.
Data are reported as the mean percentages of positive responses. Plus minus standard error before installation of bacteria and at post-infection days 7, 14, 21 and 28 and over indicated a significant increase in response frequency at post infection days seven to 28 compared with that at baseline for all filaments tested in UPEC infected not mice. The percent response at each PID was calculated as total responses to all fibers relative to baseline responses.
After watching this video, you should have a good understanding of how to initiate bacterial prostatitis in male mice, followed by behavior testing to measure pelvic tactile nia in these mice.
