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-invasive induction, as opposed to more commonly used, chemically- and immunologically-induced inflammation of the prostate34,36-39. Furthermore, NMS in mice has been shown to produce comorbid symptomology, including altered anxiety-like and anhedonic behaviors, increased micturition rates, and hindpaw hypersensitivity (16; data not shown), similar to the comorbid somatic, mood, and visceral disorders exhibited by CP/CPPS patients4-6.
The main advantage of using NMS to induce CP/CPPS in mice is that it uses a psychological intervention to initiate physiological changes. Patients with CP/CPPS have variant etiology that largely does not involve previous infection of or direct injury to the prostate1-3. Despite this fact, published models of CP/CPPS have incorporated direct or indirect inflammation of the prostate to induce hypersensitivity of the perigenital region and/or mast cell activation. A significant proportion of CP/CPPS and chronic pelvic pain patients in general, report having experienced early life adversity, which has largely gone unstudied in rodent models of urogenital pain syndromes. Previous work from our lab has shown that NMS induces vaginal hypersensitivity and associated disruption of proper functioning of the HPA axis16. Future work using this model will investigate the mechanisms that are driven by early life stress to produce the altered phenotype in adulthood, as well as to explore potential pharmacological or lifestyle interventions that could be applied in a preclinical or clinical setting.
Achieving optimal and repeatable results from this protocol depends on observing several critical steps that may need to be optimized depending on the environment and equipment used. Considering that NMS dramatically affects the functioning of the HPA axis, it is important to control for outside stimuli and environmental stress during both the neonatal and adult periods. Age-matched, non-handled na&#239;ve mice should be born and housed in concordance with each cohort of NMS mice to control for external stressors present in the environment that may affect the development of the HPA axis. It is important to determine the number of mice needed to perform the intended experiment prior to initiating breeding or ordering pregnant females into the animal facility. While performing NMS, the greatest concern is rejection of the pups by the dam, therefore it is imperative to maintain the scent of the home cage throughout the NMS protocol. To minimize the effects of diurnal rhythms, the suggested separation time for NMS is during the middle of the light cycle (from 11AM to 2PM). Perigenital sensitivity measurements should be obtained in fully mature adult male mice, approximately 8 weeks of age, and taken at the same time of day for each group, preferably early in the light cycle to coincide with the trough in diurnal rhythms. To reduce the occurrence of non-evoked movements during this procedure, assessments may be performed in a temperature-controlled, sound proof room with white noise playing (20 - 20,000 Hz). The same investigator should assess all withdrawal thresholds throughout the study to maintain consistency in the observance of positive and negative responses. As alternatives to measuring the 50% mechanical threshold, the withdrawal frequency to a single monofilament could be assessed or an electronic von Frey apparatus46 could be utilized. For mast cell visualization, all slides that are to be analyzed should be processed together to ensure equal staining of mast cells, as staining intensity can vary between experiments. A pH below 1.0 is necessary for proper contrast between the deep purple mast cells and the light blue background. Too many rinses in alcohol can wash out the stain from the mast cells and negatively affect the contrast. Finally, superimposing a grid onto the tissue can be helpful in counting cells throughout the whole tissue section.
Several considerations should be made prior to the use of NMS to induce CP/CPPS or related centralized pain disorders. First, the vast majority of publications that have incorporated early life stress have been done in rat models of NMS, using a truncated separation period of P1 - P14. Many groups have shown that this paradigm generates an IBS-like symptomology in rats and mice15; however, in our previous study a P1 - P14 separation period in C57BL/6 mice did not generate a significant increase in vaginal sensitivity16, nor did it generate colorectal hypersensitivity (data not shown). Therefore, the species and length of NMS may determine the specific outcome in adulthood, including the severity of symptomology and the specific organ(s) that is affected. Second, the behavioral and molecular changes resulting from NMS are largely due to dysregulation of the HPA axis15-18, suggesting that the effect is centrally-mediated and could have comorbid results, including changes in anxiety- and/or depression-like behaviors and increased sensitivity in other pelvic organs or more distant locations. This comorbid phenotype is indicative of what is commonly seen clinically and may be more representative of CP/CPPS patients as a whole5,28-31,42. Third, based on these changes occurring as a result of HPA axis dysregulation, the impact of stress should be of utmost concern during both the NMS procedure and later behavioral testing and in vitro analysis. Output from the HPA axis is diurnal, with greater output during the more active dark cycle and lesser output during the more quiescent light cycle, which could affect the outcome of behavioral or in vitro analysis depending on the time of the testing/sacrifice47. Likewise, exposure to stressors, including undergoing perigenital mechanical sensitivity testing, can transiently effect functioning of the HPA axis18 and potentially alter gene expression within associated brain regions and downstream peripheral tissues.

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><tbody><tr><td>Pregnant C57BL/6 female mice&amp;nbsp;</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>2 L 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&amp;deg;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.&amp;nbsp;</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&amp;deg;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&amp;nbsp;</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&amp;nbsp;</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.&amp;nbsp;&amp;nbsp;</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 application or licking or biting behavior directed towards the monofilament.
Mice that were exposed to NMS also displayed histological evidence of CP/CPPS. Cryostat sections of prostate tissue were stained with acidified o-Toluidine Blue to observe tryptase granules housed within mast cells (Figure 3A-D). The percentage of mast cells that showed evidence of activation, e.g., diffuse metachromasia, granules present outside of the cell border (Figure 3D), was significantly increased in prostate tissue from 8 week-old NMS mice, compared to na&#239;ve (p &#60; 0.0001, Student&#8217;s t-test, Figure 3E). The total number of infiltrated mast cells, regardless of granulation status, was not significantly different between na&#239;ve (99.5 &#177;&#160;15.2 cells/section) and NMS mice (108.2 &#177;&#160;22.5 cells/section).
<img alt="Figure 1" src="/files/ftp_upload/53181/53181fig1.jpg" /> 
Figure 1. Procedural time line. The schematic outlines a recommended time line for performing the described methodology. Neonatal maternal separation (NMS) is performed daily from postnatal day (P) 1 until P21 and mice are weaned on P22. Mice remain undisturbed outside of normal animal husbandry until 8 weeks of age when they are tested for perigenital mechanical sensitivity. If the same mice are going to be assessed for mast cell staining, one week should be allowed to elapse following behavioral testing to allow for any residual stress effects to resolve.
<img alt="Figure 2" src="/files/ftp_upload/53181/53181fig2.jpg" /> 
Figure 2. Perigenital mechanical sensitivity. Perigenital mechanical sensitivity was measured using von Frey monofilament application. Male mice that underwent neonatal maternal separation (NMS) displayed a significant reduction in withdrawal threshold, compared to na&#239;ve mice, indicative of mechanical allodynia. Data represents mean SEM, n = 4 for each group. *p &#60; 0.05, Student&#8217;s t-test.
<img alt="Figure 3" src="/files/ftp_upload/53181/53181fig3.jpg" /> 
Figure 3.&#160;Mast cell activation. Acidified toluidine blue was used to visualize tryptase granules and calculate the percentage of activated/degranulated mast cells in cryostat sections of prostate tissue. Representative photomicrographs are shown of toluidine blue-stained sections from na&#239;ve (A) and NMS (B) prostate with arrows indicating intact (non-degranulated) and arrowheads indicating activated (degranulated) mast cells. Higher magnification images from na&#239;ve (C) and NMS (D) bladder are shown to illustrate histological differences from non-degranulated (C) and degranulated (D) mast cells. A significantly higher percentage of degranulated mast cells were observed in prostate sections from NMS mice when compared to na&#239;ve mice (E). Data represents mean SEM, n = 4 for each group. ****p &#60; 0.0001, Student&#8217;s t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53181/53181fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

assessment-perigenital-sensitivity-prostatic-mast-cell-activation

Nanyang Technological University

Research

JoVE Journal

Medicine

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.

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

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

Neuroscience

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

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 pain8-10. 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.

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.

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

Immunology and Infection

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and offspring, including the development of neuropsychiatric disorders. Risk factors linked to the emergence of affective dysregulation in the maternal-infant dyad have been extensively studied. Exposure to psychosocial stress during pregnancy has consistently emerged as one of the strongest predictors. Several rodent models have been created to explore this association; however, these models rely on the use of physical stressors or a limited number of psychosocial stressors presented in a repetitive fashion, which do not accurately capture the type, intensity, and frequency of stressors experienced by women. To overcome these limitations, a chronic psychosocial stress (CGS) paradigm was generated that employs various psychosocial insults of different intensity presented in an unpredictable fashion. The manuscript describes this novel CGS paradigm where pregnant female mice, from gestational day 6.5 to 17.5, are exposed to various stressors during the day and overnight. Day stressors, two per day separated by a 2 h break, range from exposure to foreign objects or predator odor to frequent changes in bedding, removal of bedding, and cage tilting. Overnight stressors include continuous light exposure, changing cage mates, or wetting bedding. We have previously shown that exposure to CGS results in the development of maternal neuroendocrine and behavioral abnormalities, including increased stress reactivity, the emergence of fragmented maternal care patterns, anhedonia, and anxiety-related behaviors, core features of women suffering from perinatal mood and anxiety disorders. This CGS model, therefore, becomes a unique tool that can be used to elucidate molecular defects underlying maternal affective dysregulation, as well as trans-placental mechanisms that impact fetal neurodevelopment and result in negative long-term behavioral consequences in the offspring.

The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and ...

Behavior

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in offspring. Animal models of MIA are used to test causality, investigate mechanisms, and develop diagnostics and treatments for these disorders. Despite their widespread use, many MIA models suffer from a lack of reproducibility and almost all ignore two important aspects of this risk factor: (i) many offspring are resilient to MIA, and (ii) susceptible offspring can exhibit distinct combinations of phenotypes. To increase reproducibility and model both susceptibility and resilience to MIA, the baseline immunoreactivity (BIR) of female mice before pregnancy is used to predict which pregnancies will result in either resilient offspring or offspring with defined behavioral and molecular abnormalities after exposure to MIA. Here, a detailed method of inducing MIA via intraperitoneal (i.p.) injection of the double stranded RNA (dsRNA) viral mimic poly(I:C) at 12.5 days of gestation is provided. This method induces an acute inflammatory response in the dam, which results in perturbations in brain development in mice that map onto similarly impacted domains in human psychiatric and neurodevelopmental disorders (NDDs).

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring

Maternal infection is a risk factor for neurodevelopmental disorders. Mouse models of maternal immune activation (MIA) may elucidate infection's impact on brain development and function. Here, general guidelines and a procedure are provided to produce reliably resilient and susceptible offspring exposed to MIA.

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in ...

Creating a Preterm Rat Model for Early Postnatal Pain Research

A Preterm Rat Model for Pain Studies

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain sensitivity. The primary objective of this protocol was to investigate the impact of neonatal pinprick stimuli on the pain threshold in the later stages of life using a preterm rat model. By establishing this model, we aim to advance the research on understanding and managing early postnatal pain associated with prematurity. The findings of this study indicate that while the baseline thresholds to mechanical stimuli remained unaffected, there was a notable increase in mechanical hypersensitivity following complete Freund's adjuvant (CFA) injection in adult rats. Interestingly, compared with male rats, female rats demonstrated heightened inflammatory hypersensitivity. Notably, maternal behavior, the weight of the litters, and the growth trajectory of the offspring remained unchanged by the stimulation. The manifestation of altered nociceptive responses in adulthood after neonatal painful stimuli could be indicative of changes in sensory processing and the functioning of glucocorticoid receptors. However, further research is needed to understand the underlying mechanisms involved and to develop interventions for the consequences of prematurity and neonatal pain in adults.

Here, we present a concise protocol for creating a preterm rat model, facilitating research on early postnatal pain management. The method involves performing a cesarean section three days before the expected birth, extracting preterm rat pups via hysterectomy, and integrating them with the biological offspring of a surrogate dam.

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain ...