Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

The overall goal of the following experiment is to determine the impact of early life stress on the urogenital system by measuring peri genital mechanical sensitivity and mast cell activation within the prostate in male C 57 black six mice that underwent neonatal maternal separation. This is achieved by first performing neonatal maternal separation on litters of C 57 black six mouse pups to induce early life stress for 20 consecutive days. The second step is to measure peri genital mechanical sensitivity by application of a series of Von Fre monofilaments to the peri genital region.

Next acidified toluidine blue staining is performed on cryostat sections of the mouse prostates. In order to quantify mast cell activation. The results show that neonatal maternal separation increases peri genital mechanical sensitivity and prosthetic mast cell activation based on our behavioral and histological analyses.

The main advantage of this technique over existing methods such as experimental autoimmune prostatitis, is that it incorporates stress exposure during early development, which precipitates symptomology later in life, similar to what is observed in patients with chronic prostatitis. This method can help answer key questions in the chronic pelvic pain field, such as the impact of early life stress on behavior, and provide histological evidence of altered pain states. Though this method can provide insight into chronic prostatitis, it can also be applied to other functional pain syndromes such as interstitial cystitis, painful bladder syndrome, vulvodynia, fibromyalgia, or migraines.

It can even be applied to other common psychological comorbidities such as anxiety and depression The day after birth, remove the dam from the home cage and place her into a clean secondary container. Rub a handful of bedding between clean gloved hands to maintain the scent of the home cage, and then transfer a depth of one to two centimeters of the home cage bedding into a clean, appropriately labeled two liter glass speaker. Additionally, at approximately half of any additional enrichment bedding material that is present in the home cage such as nestlets or crinkle paper to the glass speaker next individually, place all of the pups from a single litter into the prepared beaker and immediately place the beaker at 33 degrees Celsius and 50%humidity for 180 minutes.

During the separated incubation period, remove the dam from the secondary container and return her to the home cage. Return the home cage to its appropriate housing location within the vivarium. At the end of the separation period, retrieve the home cage and remove the dam, placing her into a clean secondary container.

Then remove the beaker from the incubator and drop a handful of bedding between clean gloved hands to maintain the scent of the home cage while handling the pups. Gently move the pups individually from the beaker to the home cage and return any enrichment and bedding remaining in the beaker to the home cage. Once all the pups have been returned, move the dam from the secondary container to the home cage and return it to its appropriate housing location within the vivarium.

Repeat this entire procedure for each litter undergoing neonatal maternal separation every day through postnatal day 21. Then wean the pups and house them in the same conditions as the control mice throughout the duration of the experiment. Prepare the testing area by placing an absorbent underpad beneath an elevated wire mesh top table 55 centimeters in height.

The table should provide enough space to allow the investigator to approach from the underside without startling the mice. Place up to 12 eight week old mice individually under clear perforated plastic chambers on top of the wire mesh table. Place a heavy object on top of the chambers to prevent the mice from escaping.

Hold the base of a three point 22 gram von frame monofilament and expose the retracted monofilament. Situate the probe so that the monofilament is vertically oriented. When the mouse is alert, immobile and its body weight is distributed evenly between the hind paws.

Apply it to either the left or right side of the scrotum, avoiding the midline until a slight bend is observed in the filament. Hold the monofilament in place for 10 seconds or until the animal shows a positive response such as a brisk jerk or jump or licking or biting behavior directed towards the monofilament. If a mouse neither moves, nor exhibits any positive responses during the ten second monofilament application, record a negative response test all of the mice using the same size filament, and then switch to the next appropriate monofilament.

Continue testing each mouse using the next larger or smaller monofilament as appropriate for an additional four applications. After the first positive response is observed, dissect and then cryo section, fixed prostate tissue from nine week old mice into seven micron thick cryo sections. Next, prepare a 1%toluidine blue stock solution by adding one gram of toluidine blue O to 100 milliliters of 70%ethanol and vortex the mixture until it goes into solution.

Store the stock solution at room temperature for up to three weeks. In another beaker, add one gram of sodium chloride to 100 milliliters of water placed on top of a stir plate. Adjust the pH of the salt solution using 12 molar hydrochloric acid until a pH range of 0.5 to 1.0 is achieved.

Then prepare a 0.25%acidified toine blue working solution by combining eight milliliters of the Udine blue stock Solution and 32 milliliters of the acidic 1%sodium chloride solution. Pipe it up and down to ensure total incorporation of the odine blue Stock Solution and mix Using a vortex, prepare the working solution fresh for each batch of slides. Wash the tissue sections by individually dipping the slides for approximately one second into a 50 milliliter conical tube containing a sufficient volume of PBS to completely submerge the tissue.

Allow the slides to air dry on a paper towel. Next place slides in a glass standing jar containing enough of the 0.25%toluidine blue working solution to ensure the tissue sections will be completely submerged. Incubate the slides for 10 to 15 minutes while on a platform rocker set at 15 RPMs.

Then dip slides in PBS for approximately one second to wash out any excess tall udine blue and place the slides in a slide box so that the slides are on their sides. To allow for drainage. Dry the slides in an oven at 37 degrees Celsius for a minimum of two hours or overnight at room temperature.

Once dry immerse the slides in 95%ethanol for approximately one second, and then allow the section to completely dry. Repeat this procedure one to 10 times until the tissue dries more blue than purple. Next, immerse the slides in 100%ethanol for approximately one second and allow the section to completely dry.

Repeat this procedure one to 10 times until the tissue is dark to medium blue but not purple. Finally, fix the stain by incubating the slides in 100%xylene for three minutes. Allow them to dry and then cover, slip the slides with glycerol PPS or a xylene based permanent mounting media.

Drain any excess media and store the completed slides at room temperature. When tested with a graded series of Von Frame Monofilaments, eight week old neonatal maternal separation, mice displayed peri genital mechanical allodynia when compared to naive counterparts. This is evidenced as a significant reduction in the mechanical withdrawal threshold that elicited a positive behavioral response.

Additional evidence of chronic prostatitis chronic pelvic pain syndrome can be seen histologically shown here are cryostat sections of prostate tissues that were stained with acidified o toluidine blue to observe tryptase granules housed within mast cells. A significantly higher percentage of de granulated mast cells were observed in prostate sections from neonatal separation mice. When compared to naive mice While performing neonatal maternal separation, it's important to remember to maintain the scent of the home cage to avoid rejection of the pups by the dam Following this procedure.

Other methods like open field testing or a two choice liquid preference can be used to assess behavioral evidence for comorbid mood disorders. After watching this video, you should have a good understanding of how to assess peri genital mechanical sensitivity and prostatic mast cell activation as behavioral and histological evidence of chronic prostatitis and mice following neonatal maternal separation.