Name: assessment of perigenital sensitivity and prostatic mast cell activation in a mouse model of neonatal maternal separation
Uploaded: 2015-08-13T14:00:00
Description: 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

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 clea...

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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