Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Summary

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

Abstract

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis¹. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain², a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)^3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity⁵.

A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method⁶ to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum⁷ as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.

CP/CPPS is primarily characterized by the presence of tactile allodynia⁴. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain^8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Protocol

1. Bacteria Preparation for Mouse Inoculation

The following must be done under aseptic conditions.

1. Take one 17 x 100 mm polypropylene tubes with caps and add 3 ml of fresh Luria broth (LB) media. Using autoclaved tips, take some frozen bacteria glycerol stock of strain CP1² and transfer a small quantity of the culture into the tube containing the LB media. Replace the tube cap so that oxygen is still able to enter the tube - the culture needs to grow under aerobic conditions. Place tube at 37 °C in a shaking incubator at 220 rpm overnight.
2. The next day, transfer 5 μl of overnight culture to a fresh tube with 3 ml LB media and grow under static conditions in an incubator overnight at 37 °C.
3. On day 3 transfer 40 μl of culture into a a 50 ml tube containing 40 ml LB media each. Place in a 37 °C static incubator overnight.
4. Turn on and set centrifuge to 4 °C. Once centrifuge has reached final temperature, transfer each 40 ml culture into a 40 ml Nalgene centrifuge tube.
5. Weigh tubes to balance centrifuge - adjust volume with LB media as needed. Spin tubes at 6,000 rpm for 20 min.
6. Carefully remove supernatant and gently suspend bacterial pellet in 40 ml sterile PBS.
7. Repeat step 1.5 with PBS.
8. Aspirate off supernatant and suspend bacteria in 500 μl sterile PBS. Transfer the solution into a 1.5 ml microfuge tube. Take 10 μl of suspension and dilute in 990 μl of ice-cold PBS. Use a spectrophotometer to read the OD_{420 nm} to determine the volume needed for inoculation. To determine the appropriate volume of ice-cold PBS to suspend pellet in: Take the OD_{420 nm} number (in ml) and subtract the estimated volume of the bacterial pellet [ex. OD_{420 nm} = 0.545, pellet approximately 0.075 ml. 0.545 - 0.075 = 0.470].
9. Remove 10 μl of bacterial suspension and dilute in 990 μl ice-cold PBS. Read the OD_{420 nm}. The goal is to achieve an OD_{420 nm} value for the diluted suspension of 1.000 ± .010. If the number of your dilution is above this target, add more volume to the suspension and take another reading. If the number is below the .990 then spin down the suspension and repeat OD_{420 nm} reading until obtaining desired reading.

2. Animal Infection

Mice (5 to 7 weeks old, ten per group) were purchased from Jackson Laboratory (Bar Harbor, ME).

1. Mice are anesthetized by inhalation of 1-4% isoflurane in a Plexiglas chamber and are monitored until recumbent.
2. One mouse at a time is taken for instillation of bacteria by catheterization with a polyethylene (PE10) catheter attached to a modified 30 G needle of a glass Hamilton syringe (length of 1.5-2.0 cm). The catheter is inserted to the hub of the needle. The mouse is placed on its dorsal surface under anesthesia maintained using a nose cone.
3. The penis of the mouse is extruded by gentle pressure and liberal amounts of lubricant on cotton swabs is used for lubrication of the entrance to the penile urethra.
4. 10 μl of phosphate-buffered saline containing 1 x 10⁸ bacteria is introduced into the urethra of anesthetized mice following catheterization.
5. Mice are maintained in an anesthetized state for 15 min in the Plexiglas chamber after bacterial introduction to allow bacterial attachment and to prevent immediate urination.
6. Mice are placed back in their cages and monitored for the next 24 hr.

3. Behavior Testing

Mice were tested prior to infection (baseline) and on days 3, 7, 14, 21 and 28 after infection. Referred hyperalgesia and tactile allodynia was tested using von Frey filaments applied to the abdomen^11,12 and the plantar region of the hind paw¹³. Testing was perfomed at a fixed time-of-day, standard methodology and single experimenter testing of all animals were employed. Blinded testing of groups was utilized to combat the limitations of behavior-based pain testing in animal models. Five individual von Frey filaments with forces of 0.04, 0.16, 0.4, 1.0 and 4.0 g (Stoelting, USA) were applied to the abdomen and frequency of withdrawal responses was calculated.

1. After a 30 min period of acclimatization, mice are placed in individual Plexiglas chambers (6 x 10 x12 cm) made in-house with a stainless steel wire grid floor.
2. Referred hyperalgesia and tactile allodynia were tested using the five von Frey filaments. Each filament was applied for 1-2 sec with an inter-stimulus interval of 5 sec for a total 10 times, and the hairs were tested in ascending order of force.
3. Each filament is applied to the lower abdominal area in the general vicinity of the prostate and care was taken to stimulate different areas within this region to avoid desensitization or "wind up" effects. Filaments were applied in increasing order of force for 1-2 sec with at least 5 sec intervals between stimulations for a total of 10 times.
4. Three types of behavior are considered a positive response to filament stimulation: 1) sharp retraction of the abdomen; 2) immediate licking or scratching of the area of filament stimulation; 3) jumping.
5. Response frequency was calculated as the percentage of positive response (out of 10, e.g. 5 responses of 10 = 50%) and data was reported as the mean percentage of response frequency ± SE
6. Animals with more than 25 positive total baseline responses are excluded from the study.
7. Tactile allodynia was tested on the plantar region of the hind paw using von Frey filaments with forces of 0.04, 0.16, 0.4, 1.0 and 4.0 g. The median 50% withdrawal threshold (5) was assessed using the up-down method where testing was started with 0.04 g filament applied perpendicularly to the plantar surface of the hind paw until the filament bent slightly. Filaments were tested in ascending order until a positive response was observed. A positive response to the filament was defined as either a sharp withdrawal of the paw or licking of the test paw. When a positive response was recorded the next weaker filament was applied, and if a negative response was observed, then the next stronger filament was applied.
8. The experiments and methodology described have been reviewed and approved by the Northwestern University animal care and use committee.

Results

We examined NOD/ShiLtj and C57BL/6J male mice for the presence of chronic pain upon bacterial infection. Male mice (5 to 7 weeks old) were instilled with saline or bacteria into the urethra (Figure 1). Mechanical stimulation of the pelvic area with von Frey filaments of saline-instilled C57BL/6J mice and UPEC-infected C57BL/6J mice resulted in a response frequency that did not change during the 28-day course of the experiment (Figure 1). In contrast, UPEC-infected NOD mice exhibited resp...

Discussion

Infection of the mouse prostate with UPEC allows for the in vivo modeling of events that may be involved in the pathogenesis of bacterial prostatitis, CP/CPPS or as a predisposing event in chronic inflammation. The methods described for bacterial preparation and instillation draw upon a large body of literature on UPEC models in female urinary tract infection^7,14. The model has wide applicability for studying pathogenesis, testing potential vaccine candidates and mechanisms of immune modulation. The a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Culture tubes	BD Falcon	352059
LB Broth Miller	EMD	EM1.10285.0500
Nalgene Centrifuge Tubes	Thermo	3118-0050
Isoflurane	Butler Schein	NDC 11695-6776-1
Catheter needle 30G	Hamilton	91030
PE10 tubing	BD intramedic	427400
Von Frey Filaments	Stoelting	58025-31