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.
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.
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% 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’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, 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.
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)
2. Perigenital Mechanical Sensitivity
3. Acidified Toluidine Blue Mast Cell Staining
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 naïve counterparts (n = 5, Figure 2). This is evidenced as a significant reduction (p < 0.01; Student’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 naïve (p < 0.0001, Student’s t-test, Figure 3E). The total number of infiltrated mast cells, regardless of granulation status, was not significantly different between naïve (99.5 ± 15.2 cells/section) and NMS mice (108.2 ± 22.5 cells/section).
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.
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 naïve mice, indicative of mechanical allodynia. Data represents mean SEM, n = 4 for each group. *p < 0.05, Student’s t-test.
Figure 3. 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 naïve (A) and NMS (B) prostate with arrows indicating intact (non-degranulated) and arrowheads indicating activated (degranulated) mast cells. Higher magnification images from naïve (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 naïve mice (E). Data represents mean SEM, n = 4 for each group. ****p < 0.0001, Student’s t-test. Please click here to view a larger version of this figure.
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 naïve 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.
The authors have nothing to disclose.
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).
Pregnant C57BL/6 female mice | Charles River | 027 | Timed or untimed pregnant females should be monitored daily for in-house birth. |
2L glass beaker(s) | Sigma-Aldrich | CLS10002L | Each NMS litter will be held in the same beaker throughout the 21 day separation period. |
VWR Forced Air Incubator, Basic | VWR International | 414005-124 | The incubator should be held at 33°C and 50% humidity. |
Touch Test Sensory Evaluator, Kit of 20 (Semmes-Weinstein Von Frey Aesthesiometer for touch assessment) | Stoelting | 58011 | 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. |
Animal Enclosure (12 mice 6 rats) | IITC | 435 | Be sure to place a heavy object on top of the individual, acrylic animal enclosures to prevent mice from escaping. |
Mesh Stand for mice and rats (12 mice 6 rats) | IITC | 410 | Place stand on a stable table top for comfortable access. |
Phosphate buffered saline | Sigma-Aldrich | P5493 | Dilute the 10x PBS stock to 1x PBS. |
Paraformaldehyde | Sigma-Aldrich | P6148 | A 4% paraformaldehyde solution is needed to intracardially perfuse mice and postfix tissue in preparation for mast cell staining. |
Sucrose | Sigma-Aldrich | S5016 | Cryoprotect fixed tissue in 30% sucrose solution at 4°C overnight. |
Heptane | Sigma-Aldrich | 246654 | Chill heptane on dry ice to freeze tissue. |
Standard Cryomold | VWR International | 4557 | To assist in freezing prostate tissue prior to cryostat sectioning |
Tissue-Tek OCT Compound | Sakura | 4583 | 12 x 125 mL |
VWR Superfrost Plus Micro Slide | VWR International | 48311-703 | 75 x 25 x 1 mm |
Toluidine Blue O | Sigma-Aldrich | 198161 | Certified by the Biological Stain Commission. Suitable for use as a metachromatic stain for mast cells. |
Ethanol, Absolute (200 Proof) | Fisher Scientific | BP2818-4 | Molecular Biology Grade, Fisher Bioreagents; 95% and 100% solutions will be needed to dehydrate stained cryosections before mast cell visualization. |
Sodium Chloride | Sigma-Aldrich | S9888 | Acidified NaCl solution should be made fresh before staining cryosections. |
GeneMate Sterile 50 mL Centrifuge Tubes | BioExpress | C-3394 | Disposable tubes used to mix toulidine blue elements or dip slides into 1xPBS. |
Coplin staining dish for 10 slides, with ground glass cover | Fisher Scientific | 08-815 | 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. |
Xylenes (Histological) | Fisher Scientific | X3P | Once dehydrated, tissue is cleared with xylene. |
Microscope Slide Boxes, 100-Place | VWR International | 82003 | Boxes can be used for storage and transportation of slides. |
Fisherbrand Microscope Slide Box | Fisher Scientific | 22-363-400 | These slide boxes are typically small enough to fit inside a cryostate. |
Glycerol | Sigma-Aldrich | G5516 | Glycerol is a traditional mounting medium. |
Mounting Medium, Richard-Allan Scientific | VWR International | 4111 | Mounting medium firmly bonds the coverslip to the slide. |
Micro Cover Glasses, Rectangular, No. 1 | VWR International | 48393-106 | A coverslip is placed over the dehydrated and cleared tissue to protect the sample. |
Light Microscope | Nikon | The Advanced Automated Research Microscope Eclipse 90i, used by our lab, has been discontinued and replaced Eclipse Ni-E. |