Neonatal Maternal Separation Mice Research Articles

Exposure to early-life stress impairs weight-loss maintenance success in mice.

The impact of early-life stress on weight-loss maintenance is unknown. Mice underwent neonatal maternal separation (NMS) from 0 to 3 weeks and were weaned onto a high-fat sucrose diet (HFSD) from 3 to 20 weeks. Calorie-restricted weight loss on a low-fat sucrose diet (LFSD) occurred over 2 weeks to induce a 20% loss in body weight, which was maintained for 6 weeks. After weight loss, half of the mice received running wheels, and the other half remained sedentary. Mice were then fed ad libitum on an HFSD or LFSD for 10 weeks and were allowed to regain body weight. NMS mice had greater weight regain, total body weight, and adiposity compared with naïve mice. During the first week of refeeding, NMS mice had increased food intake and were in a greater positive energy balance than naïve mice. Female mice were more susceptible to NMS-induced effects, including increases in adiposity. NMS and naïve females were more susceptible to HFSD-induced weight regain. Exercise was beneficial in the first week of regain in male mice, but, long-term, only those on the LFSD benefited from exercise. As expected, HFSD led to greater weight regain than LFSD. Early-life stress increases weight regain in mice.

High-fat/high-sucrose diet worsens metabolic outcomes and widespread hypersensitivity following early-life stress exposure in female mice.

Exposure to stress early in life has been associated with adult-onset comorbidities such as chronic pain, metabolic dysregulation, obesity, and inactivity. We have established an early-life stress model using neonatal maternal separation (NMS) in mice, which displays evidence of increased body weight and adiposity, widespread mechanical allodynia, and hypothalamic-pituitary-adrenal axis dysregulation in male mice. Early-life stress and consumption of a Western-style diet contribute to the development of obesity; however, relatively few preclinical studies have been performed in female rodents, which are known to be protected against diet-induced obesity and metabolic dysfunction. In this study, we gave naïve and NMS female mice access to a high-fat/high-sucrose (HFS) diet beginning at 4 wk of age. Robust increases in body weight and fat were observed in HFS-fed NMS mice during the first 10 wk on the diet, driven partly by increased food intake. Female NMS mice on an HFS diet showed widespread mechanical hypersensitivity compared with either naïve mice on an HFS diet or NMS mice on a control diet. HFS diet-fed NMS mice also had impaired glucose tolerance and fasting hyperinsulinemia. Strikingly, female NMS mice on an HFS diet showed evidence of hepatic steatosis with increased triglyceride levels and altered glucocorticoid receptor levels and phosphorylation state. They also exhibited increased energy expenditure as observed via indirect calorimetry and expression of proinflammatory markers in perigonadal adipose. Altogether, our data suggest that early-life stress exposure increased the susceptibility of female mice to develop diet-induced metabolic dysfunction and pain-like behaviors.

Sex-dependent effects of postweaning exposure to an enriched environment on visceral pain and anxiety- and depression-like behaviors induced by neonatal maternal separation.

BackgroundNeonatal maternal separation (NMS) can lead to visceral pain and anxiety- and depression-like behaviors. An enriched environment (EE) can alleviate NMS-induced pain and mental disorders, but previous studies have mostly been performed in male animals. Therefore, the aim of this study was to investigate whether the effects of EE were sex dependent at different stages of development.MethodsFemale and Male C57BL/6 J mice that had been subjected to NMS alone and those subjected to both NMS and exposed to EE were used in this study. The visceral pain threshold test (PTT), open field test (OFT), sucrose preference test (SPT), and forced swimming test (FST) were conducted to evaluate visceral pain, anxiety-like behavior, and depression-like behavior in mice, respectively.ResultsCompared with the male mice in the NMS group without EE exposure, those exposed to EE from postnatal day (P)21 to 41 showed an increase of the visceral pain threshold in the PTT, an increase of the central time and central distance in the OFT, an increase of the sucrose preference rate in the SPT, and a decrease of the time of immobility in the FST. Compared with both female and male mice in the NMS group without EE exposure, those exposed to EE from P21 to P61 had an increase of the visceral pain threshold in the PTT, an increase of the central time and central distance in the OFT, an increase in the sucrose preference rate in the SPT, and a decrease of the time of immobility in the FST.ConclusionsEE is more effective in male NMS mice, while longer EE is required in female NMS mice for positive effects.

The Impact of Long-term High-fat Diet on Co-morbid Disorders in an Early Life Stress Mouse Model

Exposure to stress early in life has been associated with adult-onset co-morbidities such as chronic pain, obesity, and metabolic disorders. We have established a mouse model of early life stress using neonatal maternal separation (NMS) that results in urogenital hypersensitivity and increased body weight and adiposity compared to naive (non-separated) mice. In this study we tested the hypothesis that consumption of a high fat/high sucrose (HFS) diet will worsen NMS-related outcomes. Male and female naive and NMS mice were pair-housed with a sex matched littermate. At 4-weeks of age mice were assigned to either control diet or HFS diet and weight gain and food intake were measured weekly. Mechanical sensitivity and body composition were measured at 22-weeks of age. At 25-29 weeks of age mice were subject to a glucose tolerance test. Mice were then sacrificed, and tissues were fixed and frozen. Adipose tissue was analyzed via RT-PCR and fixed liver tissue was stained and analyzed for steatosis. NMS mice on a HFS diet were found to have increased body weight and percent body fat compared to naive mice on a HFS diet and to NMS mice on a control diet. Additionally, both NMS and the HFS diet resulted in significantly lower hind paw mechanical withdrawal thresholds indicative of hypersensitivity. Both NMS and HFS diet consumption were found to result in impaired glucose homeostasis. Upon histological examination of the liver, HFS diet fed NMS mice were observed to have increased steatosis and hepatocyte ballooning. Our results suggest that HFS diet exacerbates the outcomes of NMS in both males and females, resulting in widespread hypersensitivity, weight gain, glucose intolerance, and steatosis. Future work will explore possible underlying mechanisms for the increased weight gain and sensitivity associated with stress and diet. Grant support from RO1DK099611 (JAC), RO1DK103872 (JAC), T32HD057850 (OCE), COBRE grant P20GM104936, IDeA grant P20GM103418, core support from IDDRC grant P30HD002528.

Exercise Attenuates Hindpaw Hypersensitivity, Depression-like Behaviors, and Morphological Changes in the Hippocampus of Mice Exposed to Early Life Stress

Early life stress (ELS) is associated with the development of chronic pain and mood disorders. Exposure to ELS has been correlated with suppressed hippocampal neurogenesis across the lifespan. Conversely, exercise promotes hippocampal neurogenesis, as well as prevents pain-like behaviors in ELS mice. Here, we are using an established mouse model of ELS, neonatal maternal separation (NMS), to test the hypothesis that increasing physical activity can attenuate ELS-induced pain- and depression-like behaviors and increase hippocampal neurogenesis. NMS was performed by separating whole litters from the dam for 3h/day from postnatal day 1 (P1) to P21. Mice were weaned and pair-housed with same-sex littermates on P22 and remained in sedentary caging (-Sed) or were given running wheels (-Ex) at 4 weeks of age. Mice underwent monthly hindpaw von Frey and Hargreaves testing for 4 months and were subsequently tested for sucrose preference and nest building. Ethynyldeoxyuridine (EdU) was used to quantify hippocampal neurogenesis at 5 months of age. A significant impact of NMS and exercise was observed on mechanical sensitivity in female mice, such that NMS-Ex mice had higher thresholds compared to NMS-Sed and naive-Ex mice. Male NMS-Sed mice had significantly lower withdrawal thresholds compared to naive-Sed mice, only at the end of the study. A significant impact of exercise and an NMS/Ex interaction was observed on female thermal latency with naive-Ex mice having lower withdrawal latencies. Male NMS-Ex mice had significantly lower sucrose preference compared to NMS-Sed and naive-Ex mice. Exercise significantly increased nest construction quality in female mice, with NMS-Sed mice having lower scores than naive-Sed or NMS-Ex mice. Finally, exercise increased the number of EdU-immuno-positive cells in the hippocampus of NMS mice. Taken together, our results suggest that increased voluntary physical activity can prevent the development of ELS-associated outcomes, including hypersensitivity, depression-like behaviors, and decreased hippocampal neurogenesis. This work was supported by RO1DK099611 (JAC), RO1DK103872 (JAC), T32HD057850 (BML), COBRE grant P20GM104936, IDeA grant P20GM103418, core support from IDDRC grant P30HD002528.

Reduced Hippocampal Volume and Neurochemical Response to Adult Stress Exposure in a Female Mouse Model of Urogenital Hypersensitivity.

Early life stress exposure significantly increases the risk of developing chronic pain syndromes and comorbid mood and metabolic disorders later in life. Structural and functional changes within the hippocampus have been shown to contribute to many early life stress-related outcomes. We have previously reported that adult mice that underwent neonatal maternal separation (NMS) exhibit urogenital hypersensitivity, altered anxiety- and depression-like behaviors, increased adiposity, and decreased gene expression and neurogenesis in the hippocampus. Here, we are using magnetic resonance imaging and spectroscopy (MRI and MRS) to further investigate both NMS- and acute stress-induced changes in the hippocampus of female mice. Volumetric analysis of the whole brain revealed that the left hippocampus of NMS mice was 0.038 mm3 smaller compared to naïve mice. MRS was performed only on the right hippocampus and both total choline (tCho) and total N-acetylaspartate (tNAA) levels were significantly decreased due to NMS, particularly after WAS. Phosphoethanolamine (PE) levels were decreased in naïve mice after WAS, but not in NMS mice, and WAS increased ascorbate levels in both groups. The NMS mice showed a trend toward increased body weight and body fat percentage compared to naïve mice. A significant negative correlation was observed between body weight and phosphocreatine levels post-WAS in NMS mice, as well as a positive correlation between body weight and glutamine for NMS mice and a negative correlation for naïve mice. Together, these data suggest that NMS in mice reduces left hippocampal volume and may result in mitochondrial dysfunction and reduced neuronal integrity of the right hippocampus in adulthood. Hippocampal changes also appear to be related to whole body metabolic outcomes.

Voluntary Wheel Running Partially Attenuates Early Life Stress-Induced Neuroimmune Measures in the Dura and Evoked Migraine-Like Behaviors in Female Mice.

Migraine is a complex neurological disorder that affects three times more women than men and can be triggered by endogenous and exogenous factors. Stress is a common migraine trigger and exposure to early life stress increases the likelihood of developing chronic pain disorders later in life. Here, we used our neonatal maternal separation (NMS) model of early life stress to investigate whether female NMS mice have an increased susceptibility to evoked migraine-like behaviors and the potential therapeutic effect of voluntary wheel running. NMS was performed for 3 h/day during the first 3 weeks of life and initial observations were made at 12 weeks of age after voluntary wheel running (Exercise, -Ex) or sedentary behavior (-Sed) for 4 weeks. Mast cell degranulation rates were significantly higher in dura mater from NMS-Sed mice, compared to either naïve-Sed or NMS-Ex mice. Protease activated receptor 2 (PAR2) protein levels in the dura were significantly increased in NMS mice and a significant interaction of NMS and exercise was observed for transient receptor potential ankyrin 1 (TRPA1) protein levels in the dura. Behavioral assessments were performed on adult (>8 weeks of age) naïve and NMS mice that received free access to a running wheel beginning at 4 weeks of age. Facial grimace, paw mechanical withdrawal threshold, and light aversion were measured following direct application of inflammatory soup (IS) onto the dura or intraperitoneal (IP) nitroglycerin (NTG) injection. Dural IS resulted in a significant decrease in forepaw withdrawal threshold in all groups of mice, while exercise significantly increased grimace score across all groups. NTG significantly increased grimace score, particularly in exercised mice. A significant effect of NMS and a significant interaction effect of exercise and NMS were observed on hindpaw sensitivity following NTG injection. Significant light aversion was observed in NMS mice, regardless of exercise, following NTG. Finally, exercise significantly reduced calcitonin gene-related peptide (CGRP) protein level in the dura of NMS and naïve mice. Taken together, these findings suggest that while voluntary wheel running improved some measures in NMS mice that have been associated with increased migraine susceptibility, behavioral outcomes were not impacted or even worsened by exercise.

Neonatal maternal separation increases the number of p27-positive Sertoli cells in prepuberty

Neonatal maternal separation (NMS) is an experimental model for early life stress, which affects the growth and development of various organs, resulting in adverse health effects in humans and animals. In our previous study, we demonstrated that NMS [(0.5-, 1-, 2-h/day NMS, from postnatal day (PND) 1–10] induced morphological changes to the male reproductive system, including decreased Sertoli cell numbers in mouse testes at PND 70. To clarify the mechanism by which NMS decreases Sertoli cell numbers, we evaluated the effects of NMS on mouse testes at PNDs 10 and 16. At PND 10, the Sertoli cell number was not significantly different among experimental groups; however, it decreased in 0.5- and 2-h/day NMS mice at PND 16. The termination of Sertoli cell proliferation in prepuberty can be induced by p27, a cyclin-dependent kinase inhibitor. At PND 10, we observed an increase in the number of p27-positive Sertoli cells in 2-h/day NMS mice. The seminiferous tubule diameters decreased significantly in 1- and 2-h/day NMS mice, and the relative interstitial area increased in 2-h/day NMS mice. Serum corticosterone level significantly increased, and serum testosterone level significantly decreased in the 2-h/day NMS mice. At PND 16, the tubule diameters and height of seminiferous epithelium were significantly higher in 0.5- and 2-h/day NMS mice. Our results suggest that NMS disturbs serum corticosterone and testosterone levels and increases the number of p27-positive Sertoli cells at PND 10, resulting in a decrease in the number of Sertoli cells at PND 16.

Voluntary wheel running improves outcomes in an early life stress-induced model of urologic chronic pelvic pain syndrome in male mice.

Patients with a history of early life stress (ELS) exposure have an increased risk of developing chronic pain and mood disorders later in life. The severity of ELS in patients with urologic chronic pelvic pain syndrome (UCPPS) is directly correlated with symptom severity and increased comorbidity, and is inversely related to likelihood of improvement. Voluntary exercise improves chronic pain symptoms, and our group and others have shown that voluntary wheel running can improve outcomes in stress-induced UCPPS models, suggesting that exercise may negate some of the outcomes associated with ELS. Here, we provide further evidence that voluntary wheel running can attenuate increased perigenital mechanical sensitivity, bladder output, and mast cell degranulation in the bladder and prostate in male mice that underwent neonatal maternal separation (NMS). Sedentary male NMS mice had reduced serum corticosterone, which was not impacted by voluntary wheel running, although stress-related regulatory gene expression in the hypothalamus and hippocampus was significantly increased after exercise. Neurogenesis in the dentate gyrus of the hippocampus was diminished in sedentary NMS mice and significantly increased in both exercised naïve and NMS mice. Sucrose consumption increased in exercised naïve but not NMS mice, and anxiety behaviors measured on an elevated plus maze were increased after exercise. Together these data suggest that voluntary wheel running is sufficient to normalize many of the UCPPS-related outcomes resulting from NMS. Exercise also increased hippocampal neurogenesis and stress-related gene expression within the hypothalamic-pituitary-adrenal axis, further supporting exercise as a nonpharmacological intervention for attenuating outcomes related to ELS exposure.

Neonatal maternal separation causes decreased numbers of sertoli cell, spermatogenic cells, and sperm in mice

Neonatal maternal separation is an experimental model used to evaluate the effects of toxic stress in neonates, or early life stress. Although various physiological and psychological stresses during childhood have been reported, the effects of neonatal maternal separation on the male reproductive system remain unclear. Therefore, the present study evaluated the effects of neonatal maternal separation on the male reproductive system. In neonatal male ICR mice, maternal separation was performed for 0.5, 1, 2, and 4 hours/day, from postnatal day 1 to 10. At 10 weeks of age, the neonatal maternal separation mice exhibited decreases in both testicular weight and epididymal sperm number, along with various testicular morphological changes involving germ cells, Sertoli cells, and interstitial cells. Notably, neonatal maternal separation mice showed decreased numbers of Sertoli cells. Animals subjected to 0.5-, 1-, and 2-h/day neonatal maternal separation exhibited decreases in serum levels of testosterone but not in those of gonadotropin (luteinizing hormone and follicle-stimulating hormone). Together, these data showed that neonatal maternal separation in male mice causes decreased Sertoli cell numbers following puberty, resulting in subsequent decreased spermatogenic activity.

Early life stress reduces voluntary exercise and its prevention of diet-induced obesity and metabolic dysfunction in mice.

The development of obesity-related metabolic syndrome (MetS) involves a complex interaction of genetic and environmental factors. One environmental factor found to be significantly associated with MetS is early life stress (ELS). We have previously reported on our mouse model of ELS, induced by neonatal maternal separation (NMS), that displays altered regulation of the hypothalamic-pituitary-adrenal (HPA) axis and increased sensitivity in the urogenital organs, which was attenuated by voluntary wheel running. Here, we are using our NMS model to determine if ELS-induced changes in the HPA axis also influence weight gain and MetS. Naïve (non-stressed) and NMS male mice were given free access to a running wheel and a low-fat control diet at 4-weeks of age. At 16-weeks of age, half of the mice were transitioned to a high fat/sucrose (HFS) diet to investigate if NMS influences the effectiveness of voluntary exercise to prevent diet-induced obesity and MetS. Overall, we observed a greater impact of voluntary exercise on prevention of HFS diet-induced outcomes in naïve mice, compared to NMS mice. Although body weight and fat mass were still significantly higher, exercise attenuated fasting insulin levels and mRNA levels of inflammatory markers in epididymal adipose tissue in HFS diet-fed naïve mice. Only moderate changes were observed in exercised NMS mice on a HFS diet, although this could partially be explained by reduced running distance within this group. Interestingly, sedentary NMS mice on a control diet displayed impaired glucose homeostasis and moderately increased pro-inflammatory mRNA levels in epididymal adipose, suggesting that early life stress alone impairs metabolic function and negatively impacts the therapeutic effect of voluntary exercise.

Early life stress disrupts intestinal homeostasis via NGF-TrkA signaling

Early childhood is a critical period for development, and early life stress may increase the risk of gastrointestinal diseases including irritable bowel syndrome (IBS). In rodents, neonatal maternal separation (NMS) induces bowel dysfunctions that resemble IBS. However, the underlying mechanisms remain unclear. Here we show that NMS induces expansion of intestinal stem cells (ISCs) and their differentiation toward secretory lineages including enterochromaffin (EC) and Paneth cells, leading to EC hyperplasia, increased serotonin production, and visceral hyperalgesia. This is reversed by inhibition of nerve growth factor (NGF)-mediated tropomyosin receptor kinase A (TrkA) signalling, and treatment with NGF recapitulates the intestinal phenotype of NMS mice in vivo and in mouse intestinal organoids in vitro. Mechanistically, NGF transactivates Wnt/β-catenin signalling. NGF and serotonin are positively correlated in the sera of diarrhea-predominant IBS patients. Together, our findings provide mechanistic insights into early life stress-induced intestinal changes that may translate into treatments for gastrointestinal diseases.

(267) The Influence of Long-Term Western Diet and Voluntary Exercise in an Early Life Stress Mouse Model of Co-Morbid Disorders

Early life stress is associated with the development of chronic pain, mood, and obesity-related metabolic disorders in adulthood. Although the underlying mechanisms of these syndromes are unknown, altered regulation of the hypothalamic-pituitary-adrenal (HPA) axis has been implicated. It is hypothesized that early life stress induces HPA axis dysfunction leading to the development of overlapping disorders. We have established a mouse model of early life stress, neonatal maternal separation (NMS), that displays molecular evidence of altered HPA axis regulation and urogenital hypersensitivity and dysfunction, which can be mitigated by voluntary exercise. Adult NMS mice also weigh more and have greater body fat percentage than naive mice. Therefore, the present study is investigating whether outcomes of NMS are exacerbated by a Western diet and what effect voluntary exercise has on these outcomes. NMS was performed by separating whole litters from the dam for 3h/day starting on postnatal day 1 (P1) to P21. At 4 weeks of age, NMS and naive (non-separated) male mice either received access to a running wheel in their home cage (exercised) or remained in sedentary housing. At 16 weeks of age, a subset of mice received a high-fat/high-sucrose diet (20% kcal protein, 35% kcal carbohydrate (15% sucrose), 45% kcal fat) for 11 weeks. Perigenital mechanical sensitivity, weight, and body composition were measured throughout the study, and glucose tolerance was measured before sacrifice. Our results suggest that NMS mice were more susceptible to diet-induced weight gain and glucose intolerance compared to naive mice. Additionally, exercise had a stronger protective effect in naive mice, although both NMS and diet had a significant negative impact on running distance. We also observed a significant impact of NMS and diet on perigenital sensitivity. Future work will investigate a potential underlying mechanism for susceptibility to weight gain associated with stress and diet.

Early life voiding dysfunction leads to lower urinary tract dysfunction through alteration of muscarinic and purinergic signaling in the bladder

Lower urinary tract dysfunction (LUTD) is a common problem in children and constitutes up to 40% of pediatric urology clinic visits. Improved diagnosis and interventions have been leading to better outcomes in many patients, whereas some children are left untreated or do not respond to the treatment successfully. In addition, many of these patients are lost by the pediatric urologists during their teenage years, and the outcome in later life largely remains unidentified. Studies suggest childhood LUTD is associated with subsequent adult urinary tract symptoms. However, whether and how early life LUTD attributes to urinary symptoms in those patients later in life remains to be elucidated. In the current study, we investigated the effects of early life voiding perturbation on bladder function using a neonatal maternal separation (NMS) protocol in mice. The NMS group displayed a delayed development of voluntary voiding behavior, a significant reduction of functional bladder capacity, and bladder overactivity compared with control mice later in life. In vitro evaluation of detrusor smooth muscle and molecular study showed a decrease in muscarinic contribution alongside an increase in purinergic contribution in detrusor contractility in NMS mice compared with control group. These results suggest that early life bladder dysfunction interfered with the normal maturation of the voluntary micturition control and facilitated LUTD in a later stage, which is at least partly attributed to an alteration of muscarinic and purinergic signaling in the urinary bladder.

Voluntary wheel running attenuates urinary bladder hypersensitivity and dysfunction following neonatal maternal separation in female mice.

Patients with interstitial cystitis/painful bladder syndrome (IC/PBS) commonly suffer from widespread pain and mood disorder, which has been attributed to improper functioning of the hypothalamic-pituitary-adrenal (HPA) axis. Voluntary exercise has been shown to improve HPA axis function, therefore we are determining whether voluntary wheel running can attenuate urological pain and dysfunction following neonatal maternal separation (NMS) in female mice. Mice underwent NMS for 3 h/day from postnatal Day 1-21, were caged with free access to running wheels at 4 weeks of age, and assessed 4 weeks later for bladder sensitivity, micturition, reward behavior, mast cell degranulation, and HPA axis-related in vitro analysis. Increased bladder sensitivity, void frequency, and mast cell degranulation was observed in adult sedentary (-Sed) NMS mice, compared to naïve-Sed controls. Sucrose preference was increased in NMS-Sed mice and corticotropin-releasing factor receptor 1 (CRF1 ) and glucocorticoid receptor mRNA levels were significantly reduced in the hippocampus. Exercise normalized bladder sensitivity, micturition output, and increased brain-derived neurotrophic factor (BDNF) mRNA levels in the hippocampus of NMS mice. Mast cell degranulation was also normalized in NMS bladders following exercise. Voluntary exercise normalized behavioral outcomes resulting from NMS in female mice, increased hippocampal BDNF mRNA levels, and decreased mast cell degranulation in the bladder. Together these results provide novel insight into the efficacy of voluntary exercise to attenuate comorbid outcomes resulting from exposure to early life stress.

(108) - The role of corticotropin-releasing factor receptors in mediating stress-induced bladder sensitivity in female mice

Patients with interstitial cystitis/painful bladder syndrome (IC/PBS) often report that their symptoms worsen during periods of heightened stress. Exposure to stress early in life can also increase susceptibility to developing IC/PBS, which has been linked to altered regulation of the hypothalamic-pituitary adrenal (HPA) axis. The HPA axis responds to a stressor by releasing corticotropin-releasing factor (CRF), driving downstream release of glucocorticoids. CRF also has peripheral actions on immune cells, bladder urothelium, and enteric neurons. Two CRF receptors have been described, CRF1 and CRF2, that act in opposition to one other to increase and decrease output from the HPA axis, respectively. Antagonism of either receptor has been shown to stabilize mast cells, thereby preventing stress-induced visceral hypersensitivity. Here, we are investigating the potential role of CRF receptors in preventing stress-induced bladder hypersensitivity in a mouse model of early life stress. Mouse pups underwent neonatal maternal separation (NMS) for 3 hours per day from postnatal day (P) 1 to 21 and were weaned on P22. At 10 weeks of age, the visceromotor response (VMR) to urinary bladder distension (UBD) was performed on female naïve and NMS mice. One week later, mice were treated with saline or a CRF1 antagonist (NBI 35965, 20 mg/kg, intraperitoneal) twenty minutes prior to a one hour exposure to water avoidance stress (WAS). Mice were retested for VMR during UBD at 24 hours and 7 days after WAS exposure. Saline-treated NMS mice had a significant increase in VMR at 1d post-WAS compared to baseline measurements and saline-treated naïve-mice. Treatment with CRF1 antagonist significantly decreased VMR in both naïve and NMS mice, at 1d-, but not 7d-post WAS. Our results suggest that antagonizing CRF1 may prevent stress-induced exacerbation of bladder hypersensitivity. Future studies will include testing with CRF2 antagonist and additional behavioral tests. Supported by NIH grant DK099611.

(111) - Investigating the influence of exercise and diet on early life stress-induced pain and obesity in mice

Individuals who experience adverse childhood events show a susceptibility to developing chronic pain disorders. These disorders frequently present co-morbidly and with both mood and obesity-related metabolic disorders. Although the underlying mechanisms of these syndromes are yet to be established, it is recognized that the hypothalamic-pituitary-adrenal (HPA) axis plays a role in the regulation of the stress, nociceptive, and metabolic systems. Therefore, it is hypothesized that HPA axis dysfunction influences their pathogenesis. The present study is investigating the effect of exercise and diet interventions in a mouse model of early life stress that displays molecular evidence of altered HPA axis regulation as well as behavioral evidence of urogenital pain disorders. Neonatal maternal separation (NMS) was performed by separating entire litters from the dam for 3h/day starting on postnatal day 1 (P1) to P21. Naive pups remained undisturbed and all pups were weaned on P22. At 4 weeks of age NMS and naive mice either received free access to a running wheel in their home cage (exercised [-Ex]) or remained in sedentary housing (-Sed). Body composition analysis was assessed in adult mice using EchoMRI. Both male and female NMS-Sed mice weighed significantly more and had greater percent body fat compared to naive or Ex mice. At 16 weeks of age, male mice were placed on a high-fat/sucrose diet for one week. All groups displayed a significant increase in weight and percent body fat. Interestingly, a diet-induced increase in perigenital mechanical sensitivity was observed in all mice except for NMS-Sed, which had an increased withdrawal threshold compared to their baseline measurements. Our findings suggest that exercise can attenuate early life stress-induced development of obesity and potential metabolic syndrome. Future work will investigate the efficacy of an anti-inflammatory diet and long-term consequences of high-fat/sucrose diet consumption. Supported by NIH grants DK099611, DK103872, and HD057850.

Differential Influence of Early Life and Adult Stress on Urogenital Sensitivity and Function in Male Mice.

Experiences of adverse childhood events have been associated with improper output of the hypothalamic-pituitary-adrenal (HPA) axis in adulthood, as well as development of comorbid functional pain disorders. Symptoms of chronic prostatitis/chronic pelvic pain syndrome frequently overlap with those of interstitial cystitis/painful bladder syndrome and symptom severity is often triggered by stress. The objective of this study was to investigate the influence early life stress and acute adult stress on (1) perigenital sensitivity, (2) micturition, (3) anhedonia, and (4) HPA axis regulation and output in male C56Bl/6 mice. Neonatal maternal separation (NMS) was performed for 3 h a day from postnatal day 1 to 21 and naïve pups remained unhandled during this time. As adults, male mice were tested for referred prostate sensitivity and micturition patterning prior to and 1 and 8 days after exposure to 1 h of water avoidance stress (WAS). Following testing, prostate and bladder tissues were used for mast cell and Western blot analysis and RT-PCR was performed on mRNA from hypothalamus, amygdala, and hippocampus. Serum corticosterone (CORT) was also measured by enzyme-linked immunosorbent assay (ELISA). A significant increase in perigenital sensitivity and micturition frequency was observed in NMS mice and these measures were exacerbated by WAS exposure. Exposure to NMS significantly increased mast cell degranulation in both the bladder and prostate. Mast cell degranulation was also increased in naïve prostate tissue following WAS exposure. Cytokine mRNA levels were influenced by both NMS and WAS exposure, though WAS had a larger impact on central gene expression. Protein levels of CRF1 were differentially regulated by NMS and WAS in the bladder and prostate and serum CORT levels were significantly diminished following stress exposure. Taken together, these data suggest that NMS results in neurogenic inflammation and hypersensitivity within the urogenital organs, coupled with diminished gene expression and output from the HPA axis. Future studies of NMS in male mice may provide a useful tool as a preclinical model of male chronic urological pain syndromes for investigating potential pharmacological and interventional therapies.

Neonatal maternal separation increases susceptibility to experimental colitis and acute stress exposure in male mice

Experiencing early life stress can result in maladjusted stress response via dysregulation of the hypothalamic-pituitary-adrenal axis and serves as a risk factor for developing chronic pelvic pain disorders. We investigated whether neonatal maternal separation (NMS) would increase susceptibility to experimental colitis or exposure to acute or chronic stress. Male mice underwent NMS from postnatal day 1–21 and as adults were assessed for open field behavior, hindpaw sensitivity, and visceromotor response (VMR) to colorectal distension (CRD). VMR was also measured before and after treatment with intracolonic trinitrobenzene sulfonic acid (TNBS) or exposure to acute or chronic water avoidance stress (WAS). Myeloperoxidase (MPO) activity, proinflammatory gene and corticotropin-releasing factor (CRF) receptor expression were measured in distal colon. Baseline VMR was not affected by NMS, but undergoing CRD increased anxiety-like behaviors and mechanical hindpaw sensitivity of NMS mice. Treatment with TNBS dose-dependently decreased body weight and survival only in NMS mice. Following TNBS treatment, IL-6 and artemin mRNA levels were decreased in the distal colon of NMS mice, despite increased MPO activity. A single WAS exposure increased VMR during CRD in NMS mice and increased IL-6 mRNA and CRF2 protein levels in the distal colon of naïve mice, whereas CRF2 protein levels were heightened in NMS colon both at baseline and post-WAS exposure. Taken together, these results suggest that NMS in mice disrupts inflammatory- and stress-induced gene expression in the colon, potentially contributing towards an exaggerated response to specific stressors later in life.

Urinary bladder hypersensitivity and dysfunction in female mice following early life and adult stress

Early adverse events have been shown to increase the incidence of interstitial cystitis/painful bladder syndrome in adulthood. Despite high clinical relevance and reports of stress-related symptom exacerbation, animal models investigating the contribution of early life stress to female urological pain are lacking. We examined the impact of neonatal maternal separation (NMS) on bladder sensitivity and visceral neuroimmune status both prior-to, and following, water avoidance stress (WAS) in adult female mice. The visceromotor response to urinary bladder distension was increased at baseline and 8d post-WAS in NMS mice, while colorectal sensitivity was transiently increased 1d post-WAS only in naïve mice. Bladder micturition rate and output, but not fecal output, were also significantly increased following WAS in NMS mice. Changes in gene expression involved in regulating the stress response system were observed at baseline and following WAS in NMS mice, and WAS reduced serum corticosterone levels. Cytokine and growth factor mRNA levels in the bladder, and to a lesser extent in the colon, were significantly impacted by NMS and WAS. Peripheral mRNA levels of stress-responsive receptors were differentially influenced by early life and adult stress in bladder, but not colon, of naïve and NMS mice. Histological evidence of mast cell degranulation was increased in NMS bladder, while protein levels of protease activated receptor 2 (PAR2) and transient receptor potential ankyrin 1 (TRPA1) were increased by WAS. Together, this study provides new insight into mechanisms contributing to stress associated symptom onset or exacerbation in patients exposed to early life stress.