The authors would like to thank Janelle Ryals, Rachel Supple, and Frank Wang for technical assistance. This work was supported by NIH grants R03 DK080182 (JAC), R01 DK099611 (JAC), R01 DK103872 (JAC), Center of Biomedical Research Excellence (COBRE) grant P20 GM104936 (JAC), start-up funds and core support from the Kansas Institutional Development Award (IDeA) P20 GM103418, core support from the Kansas IDDRC P30 HD002528, and The Madison and Lila Self Fellowship Program (ANP).

All experiments described in this protocol conform to NIH guidelines in accordance with the guidelines specified by the University of Kansas Medical Center Institutional Animal Care and Use Committee.
1. Neonatal Maternal Separation (NMS)
<ol>
	<li>Monitor pregnant dams daily for litter births. 
	Note: The day the litter is born is considered P0.</li>
	<li>On P1, remove the dam from the home cage and place into a clean secondary container.</li>
	<li>Rub a handful of bedding between clean-gloved hands to maintain scent of the home cage.</li>
	<li>Add a depth of 1 - 2 cm of the home cage bedding into a clean, appropriately labeled 2 L glass beaker. Add approximately half of any additional enrichment bedding material that is present in the home cage, e.g., nestlet, crinkle paper, to the glass beaker.</li>
	<li>Gently place all of the pups from a single litter, individually, in the same beaker.</li>
	<li>Immediately place the beaker in an incubator held at 33 oC and 50% humidity for 180 min.</li>
	<li>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.</li>
	<li>Repeat this procedure for each litter undergoing NMS, using clean gloves and secondary containers for each litter/dam.</li>
	<li>At the end of the 180 min separation period, return the litters to their home cages in the same order as they were removed.</li>
	<li>Retrieve the home cage of the first litter that was separated earlier in the day. Remove the dam from the home cage and place into a clean secondary container.</li>
	<li>Remove the first beaker from the incubator and rub a handful of bedding between clean gloved hands to maintain scent of the home cage while handling the pups.</li>
	<li>Gently move the pups, individually, from the beaker to the home cage.</li>
	<li>Return any enrichment and bedding remaining in the beaker to the home cage.</li>
	<li>Return the dam from the secondary container to the home cage and return it to its appropriate housing location within the vivarium.</li>
	<li>Rinse the beaker with water and return it to the incubator. Use the same beaker for each litter throughout the separation procedure.</li>
	<li>Repeat this procedure for each litter undergoing NMS in the same order as they were placed in the incubator.</li>
	<li>Repeat this entire procedure for each litter undergoing NMS every day through P21.</li>
	<li>Wean NMS and na&#239;ve pups on P22 by placing littermates of the same sex together in clean cages complete with food and water. Na&#239;ve pups should be born and housed under the same conditions as NMS mice, but undergo no additional handling outside of normal animal husbandry tasks.</li>
</ol>
2. Perigenital Mechanical Sensitivity
<ol>
	<li>von Frey Apparatus Setup
	<ol>
		<li>Bring the mice to the testing room and allow them to acclimate for 30 min while remaining in their home cages.</li>
		<li>Prepare the testing area by placing an absorbent underpad beneath an elevated wire mesh-top table (79 cm x 28 cm) that provides enough space to allow the investigator to approach from the underside without startling the mice, approximately 55 cm in height.</li>
		<li>Place up to 12 mice, individually, under clear, perforated plastic chambers (11 cm x 5 cm x 3.5 cm) on top of the wire mesh table. Place a heavy object on top of the chambers to prevent mice from escaping.</li>
		<li>Acclimate the mice for an additional 30 min on the screen prior to withdrawal threshold assessment.</li>
	</ol>
	</li>
	<li>Perigenital Mechanical Sensitivity Assessment
	<ol>
		<li>Perform the up-down method using a standard set of graded von Frey monofilaments43. Use the following monofilaments: 1.65 g, 2.36 g, 2.83 g, 3.22 g, 3.61 g, 4.08 g, 4.31 g, and 4.74 g.
		<ol>
			<li>Hold the base of the 3.22 g monofilament and expose the retracted monofilament. 
			Note: Some models may not have a retracted monofilament.</li>
			<li>Situate the probe so that the monofilament is vertically oriented and 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.</li>
			<li>Hold the monofilament in place for 10 sec or until the animal shows a positive response. 
			Note: A positive response is considered to be a brisk jerk or jump in response to monofilament application or licking or biting behavior directed towards the monofilament. If the mouse moves during the 10 sec monofilament application without displaying these behaviors, the monofilament must be retested following a minimum 1 min-long rest period. If a mouse neither moves nor exhibits any of above listed behaviors during the 10 sec monofilament application, it is considered a negative response.</li>
			<li>Record the response, either negative or positive, in a lab notebook or laptop and repeat this procedure on all of the remaining mice using the 3.22 g monofilament.</li>
			<li>Test all of the mice again using the next appropriate monofilament. Note: If the mouse exhibited a negative response to the 3.22 g monofilament, apply the 3.61 g monofilament in the same manner and record the response. If the mouse exhibited a positive response to the 3.22 g monofilament, apply the 2.83 g monofilament and record the response.</li>
		</ol>
		</li>
		<li>Continue testing each mouse using the next larger or smaller monofilament, as appropriate, for an additional 4 applications after the first positive response is observed.</li>
		<li>Return mice to home cages.</li>
		<li>Use the value of the final von Frey monofilament applied in the trial series in log units to calculate a 50% g withdrawal threshold as described in Chaplan et al.43.</li>
	</ol>
	</li>
</ol>
3. Acidified Toluidine Blue Mast Cell Staining
<ol>
	<li>Tissue Processing
	<ol>
		<li>Dissect prostate tissue44 from mice that have been intracardially perfused with ice-cold 4% paraformaldehyde45. Postfix the tissue in 4% paraformaldehyde for 1 hr at RT and then cryoprotect in 30% sucrose in 1x&#160;PBS at 4 &#176;C O/N.</li>
		<li>Prepare a small (30 ml) bath of heptane and place it on dry ice.</li>
		<li>Rinse the prostate tissue in 1x PBS and dry using a light duty wipe.</li>
		<li>Insert the prostate tissue into the cold heptane until it is frozen.</li>
		<li>Immediately place the frozen prostate tissue in a cryomold containing mounting media and place on dry ice until frozen.</li>
		<li>Store cryomolds at -20 &#176;C.</li>
		<li>Transversely cut the prostate tissue into 7 &#181;m-thick cryosections using a cryostat.</li>
		<li>Place 8 - 12 non-serial cryosections that span the length of the tissue, onto positively-charged microscope slides.</li>
		<li>Store finished slides at -20 &#176;C until further processing.</li>
	</ol>
	</li>
	<li>Preparing Acidified Toluidine Blue
	<ol>
		<li>Add 1 g of Toluidine Blue O to 100 ml 70% ethanol and vortex until in solution to prepare a 1% stock solution. The stock solution can be stored at RT for up to three weeks.</li>
		<li>Add 1 g NaCl to 100 ml water in a beaker placed on top of a stir plate.</li>
		<li>Adjust the pH of the NaCl solution using 12 M HCl until a pH range of 0.5 - 1.0 is achieved.</li>
		<li>Prepare the 0.25% Toluidine Blue working solution by combining 8 ml of the Toluidine Blue stock solution and 32 ml of the 1% NaCl solution. Pipette up and down to ensure total incorporation of the Toluidine Blue stock solution and mix using a vortex. The working solution must be made fresh and discarded after using.</li>
	</ol>
	</li>
	<li>Staining Prostate Cryosections
	<ol>
		<li>Remove the slides to be processed from the freezer and allow to equilibrate to RT for approximately 30 min.</li>
		<li>Wash the tissue sections by individually dipping the slides for approximately 1 sec into a 50 ml conical tube containing a sufficient volume of 1x&#160;PBS to ensure that the slide-mounted tissue sections will be completely submerged. Allow slides to air dry on a paper towel.</li>
		<li>Place slides in a glass staining jar containing enough 0.25% working Toluidine Blue solution to ensure that the slide-mounted tissue sections will be completely submerged and incubate for 10 - 15 min while on a platform rocker set at 15 rpm.</li>
		<li>Dip slides in 1x PBS for approximately 1 sec to wash out excess Toluidine Blue.</li>
		<li>Place slides in a slide box or rack in such a way that the slides are on their sides to allow for drainage.</li>
		<li>Dry the slides in an oven at 37 &#176;C for a minimum of 2 hrs or O/N at RT.</li>
		<li>Dehydrate slides quickly using alcohol, allowing the slides to completely dry between alcohol exposures. Dry slides by draining on a paper towel and wiping backs and edges with a light duty wipe.
		<ol>
			<li>Immerse the slides in 95% EtOH for approximately 1 sec and allow to completely dry. Repeat this procedure 1 - 10 times until tissue dries more blue than purple.</li>
			<li>Immerse the slides in 100% EtOH for approximately 1 sec and allow to completely dry. Repeat this procedure 1 - 10 times until tissue is dark to medium blue, but not purple.</li>
		</ol>
		</li>
		<li>Fix the stain by incubating the slides in 100% xylene for 3 min. Allow to dry.</li>
		<li>Coverslip the slides with glycerol, PBS, or xylene-based permanent mounting media. Drain excess media and store at RT.</li>
	</ol>
	</li>
	<li>Quantifying Mast Cells
	<ol>
		<li>Visualize the toluidine blue-stained tissue sections at 20X magnification using light microscopy (Figure 2).</li>
		<li>Count the total number of mast cells present within the visualized area and differentiate between non-degranulated and degranulated mast cells. 40X magnification may be necessary to confirm granulation status in some mast cells. 
		Note: Non-degranulated mast cells exhibit dense metachromasia with no or faint nuclear outline and/or no granular extrusion around the cell. Degranulated mast cells exhibit less intense metachromasia and have an obvious clear outline of the nucleus and/or free granules within the cytoplasm and outside of the cell border.</li>
		<li>Continue to assess the total number of non-degranulated and degranulated mast cells in the entirety of the current tissue section. Repeat this procedure on at least 7 additional separate sections, spanning the length each tissue.</li>
		<li>Calculate the percentage of degranulated (DG) to total mast cells for each tissue/mouse according to the following equation: (DG mast cells / Total mast cells) x 100.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

This protocol provides methodology for using neonatal stress, in the form of NMS, to induce symptomology indicative of CP/CPPS in adult male mice. As adults, the NMS mice display significant perigenital mechanical allodynia, as well as evidence of mast cell degranulation in prostate tissue. The use of NMS is a novel approach to developing a preclinical model of CP/CPPS in that it replicates the early life stress that is often reported by patients with chronic pelvic pain12-14, as well as incorporating a non-in...

Chronic pelvic pain is not in itself a disease, but rather a term associated with the ongoing spontaneous and/or evoked pain experienced by patients diagnosed with irritable bowel syndrome (IBS), interstitial cystitis/painful bladder syndrome (IC/PBS), vulvodynia, or chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). These syndromes are often comorbid and share many characteristics in that they have no associated pathology or identified underlying etiology, although dysfunction within the immune system, central nervous system, and peripheral nervous system has been shown to contribute towards the maintenance and progression of these disorders1-3. Patients with chronic pelvic pain are more likely to present with symptoms of additional, non-pelvic-related functional pain disorders and mood disorders, including anxiety, depression, and panic disorder4-6, which has been associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA) axis7-10. Exposure to early life stress or trauma is a significant risk factor for developing HPA abnormalities and associated chronic pain syndromes10,11 and, as such, a significant subset of patients with functional pelvic pain disorders report having experienced adverse childhood events such as abuse or neglect12-14.
Rodent models of neonatal maternal separation (NMS), which involves removing the pups from their dam for a set period of time during the preweaning period, have been used for the past two decades to study the outcomes of early life stress. In general, NMS has been shown to increase HPA axis activation, and resultant anxiety-like behaviors, by directly affecting gene expression within the hypothalamus, as well as disrupting downstream regulation from limbic structures15-18. Disruption of proper HPA axis functioning has been shown to contribute towards increased colorectal19-22 and vaginal16 sensitivity displayed by rodent NMS models, but despite extensive characterization of the long-term effect of postnatal bladder inflammation23-25, the impact of early life stress has largely gone unstudied in the urogenital organs. Therefore, the following study will describe how to perform NMS in mice and later evaluate perigenital mechanical sensitivity and mast cell infiltration/activation in the prostate to validate the use of male NMS mice as a preclinical model for CP/CPPS.
Of all of the diagnosed chronic pelvic pain disorders, CP/CPPS is perhaps the least well-recognized and characterized syndrome, despite having a lifetime prevalence of approximately 14%&#160;26 and annual patient costs estimated at $4,400 (twice that of low back pain or rheumatoid arthritis27). Patients with CP/CPPS report pain in the perineum, rectum, prostate, penis, testicles, and/or abdomen28, experience a higher degree of psychological stress than control patients29, and commonly present with symptoms of or are diagnosed with comorbid chronic pelvic pain or mood disorders5,29-31. Recurrent infection, leaky epithelium, neurogenic inflammation, and autoimmunity have all been surmised as potential underlying causes of CP/CPPS2,32,33, as well as mast cell activation and degranulation34. Expressed prostatic secretions from men with CP/CPPS had increased mast cell tryptase and nerve growth factor (NGF) levels34, and a later study confirmed that tryptase and carboxypeptidase A (CPA3), a marker of mast cell activation, were also increased in the urine of CP/CPPS patients35. The potential role for mast cells in the onset and maintenance of CP/CPPS has been a major focus of animal research on this syndrome thus far. The most commonly employed rodent model used to study CP/CPPS is an experimental autoimmune prostatitis (EAP) model generated by subcutaneous injection of prostate antigen in Complete Freund&#8217;s adjuvant, which results in varied degrees of prostatic inflammation depending on species and strain used34,36-39. Mast cell infiltration and activation/degranulation has been shown to increase following induction of EAP34,35,40. Transgenic mice deficient in either mast cells34 or the tryptase receptor PAR235 do not develop prostatic tactic sensitivity following EAP, unlike wildtype EAP mice. While this preclinical model replicates many of the characteristics of human CP/CPPS, the induction protocol is not indicative of the human condition, which has a diverse etiology and often does not involve direct inflammation, infection, or injury of the prostate.
The influence of early life stress on the development of CP/CPPS in humans has largely gone uninvestigated; however, a study by Hu, et al.41,&#160;demonstrated that men who reported a history of childhood physical, emotional, and/or sexual abuse were significantly more likely to experience symptoms suggestive of CP/CPPS. Furthermore, they showed that both pain and urinary scores were increased in patients with a history of physical and emotional abuse. We have previously demonstrated that the same NMS paradigm in female C57BL/6 mice produces vaginal hypersensitivity and abnormal gene expression in both the vagina and bladder suggestive of dysfunctional HPA axis output16. This evidence combined with the high prevalence of CP/CPPS patients presenting with other comorbidities42, including IC/PBS and mood disorders, that have more clearly been shown to be linked with early life stress exposure12-14, provides the rationale for using an NMS model to investigate CP/CPPS in mice.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pregnant C57BL/6 female mice&#160;</td>
			<td>Charles River</td>
			<td>027</td>
			<td>Timed or untimed pregnant females should be monitored daily for in-house birth.</td>
		</tr>
		<tr>
			<td>2L glass beaker(s)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS10002L</td>
			<td>Each NMS litter will be held in the same beaker throughout the 21 day separation period.</td>
		</tr>
		<tr>
			<td>VWR Forced Air Incubator, Basic</td>
			<td>VWR International</td>
			<td>414005-124</td>
			<td>The incubator should be held at 33&#176;C and 50% humidity.</td>
		</tr>
		<tr>
			<td>Touch Test Sensory Evaluator, Kit of 20 (Semmes-Weinstein Von Frey Aesthesiometer for touch assessment)</td>
			<td>Stoelting</td>
			<td>58011</td>
			<td>The following monofilaments should be used for perigenital sensitivity assessment: 1.65 g, 2.36 g, 2.83 g, 3.22 g, 3.61 g, 4.08 g, 4.31 g, and 4.74 g.</td>
		</tr>
		<tr>
			<td>Animal Enclosure (12 mice 6 rats)</td>
			<td>IITC</td>
			<td>435</td>
			<td>Be sure to place a heavy object on top of the individual, acrylic animal enclosures to prevent mice from escaping.&#160;</td>
		</tr>
		<tr>
			<td>Mesh Stand for mice and rats (12 mice 6 rats)</td>
			<td>IITC</td>
			<td>410</td>
			<td>Place stand on a stable table top for comfortable access.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>Dilute the 10x PBS stock to 1x PBS.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>A 4% paraformaldehyde solution is needed to intracardially perfuse mice and postfix tissue in preparation for mast cell staining.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S5016</td>
			<td>Cryoprotect fixed tissue in 30% sucrose solution at 4&#176;C overnight.</td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Sigma-Aldrich</td>
			<td>246654</td>
			<td>Chill heptane on dry ice to freeze tissue.</td>
		</tr>
		<tr>
			<td>Standard Cryomold&#160;</td>
			<td>VWR International</td>
			<td>4557</td>
			<td>To assist in freezing prostate tissue prior to cryostat sectioning</td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT Compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>12 x 125 mL</td>
		</tr>
		<tr>
			<td>VWR Superfrost Plus Micro Slide</td>
			<td>VWR International</td>
			<td>48311-703</td>
			<td>75 x 25 x 1 mm</td>
		</tr>
		<tr>
			<td>Toluidine Blue O</td>
			<td>Sigma-Aldrich</td>
			<td>198161</td>
			<td>Certified by the Biological Stain Commission. Suitable for use as a metachromatic stain for mast cells.</td>
		</tr>
		<tr>
			<td>Ethanol, Absolute (200 Proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>Molecular Biology Grade, Fisher Bioreagents; 95% and 100% solutions will be needed to dehydrate stained cryosections before mast cell visualization.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>Acidified NaCl solution should be made fresh before staining cryosections.</td>
		</tr>
		<tr>
			<td>GeneMate Sterile 50 mL Centrifuge Tubes&#160;</td>
			<td>BioExpress</td>
			<td>C-3394</td>
			<td>Disposable tubes used to mix toulidine blue elements or dip slides into 1xPBS.</td>
		</tr>
		<tr>
			<td>Coplin staining dish for 10 slides, with ground glass cover</td>
			<td>Fisher Scientific</td>
			<td>08-815</td>
			<td>Hold up to 10 standard 3 x 1 in. (75 x 25mm) slides back-to-back. Use separate dishes for Toluidine Blue working solution, 95% EtOH, 100% EtOH, and xylene.</td>
		</tr>
		<tr>
			<td>Xylenes (Histological)</td>
			<td>Fisher Scientific</td>
			<td>X3P</td>
			<td>Once dehydrated, tissue is cleared with xylene.</td>
		</tr>
		<tr>
			<td>Microscope Slide Boxes, 100-Place</td>
			<td>VWR International</td>
			<td>82003</td>
			<td>Boxes can be used for storage and transportation of slides.</td>
		</tr>
		<tr>
			<td>Fisherbrand Microscope Slide Box</td>
			<td>Fisher Scientific</td>
			<td>22-363-400</td>
			<td>These slide boxes are typically small enough to fit inside a cryostate.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Glycerol is a traditional mounting medium.</td>
		</tr>
		<tr>
			<td>Mounting Medium, Richard-Allan Scientific</td>
			<td>VWR International</td>
			<td>4111</td>
			<td>Mounting medium firmly bonds the coverslip to the slide.</td>
		</tr>
		<tr>
			<td>Micro Cover Glasses, Rectangular, No. 1</td>
			<td>VWR International</td>
			<td>48393-106</td>
			<td>A coverslip is placed over the dehydrated and cleared tissue to protect the sample.</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>The Advanced Automated Research Microscope Eclipse 90i, used by our lab, has been discontinued and replaced Eclipse Ni-E.&#160;&#160;</td>
		</tr>
	</tbody>
</table>

assessment of perigenital sensitivity and prostatic mast cell activation in a mouse model of neonatal maternal separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

Mice that have undergone NMS showed behavioral evidence indicative of CP/CPPS. When tested with a graded series of von Frey monofilaments, 8 week-old NMS mice (n = 4) displayed perigenital mechanical allodynia when compared to na&#239;ve counterparts (n = 5, Figure 2). This is evidenced as a significant reduction (p &#60; 0.01; Student&#8217;s t-test) in the mechanical withdrawal threshold that elicited a positive behavioral response, recorded as a brisk jerk or jump in response to monofilament ...

Watch this Scientific Journal Video about Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation at JoVE.com

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

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

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9.2K Views.  University of Kansas Medical Center. We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm – neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.