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

Abstract

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.

Figure 1.

Male mice in this study underwent neonatal maternal separation (NMS) and/or pair-housing in cages equipped with running wheels. A) A schematic represents the time line of the four groups analyzed in this study, including time points for NMS and voluntary wheel running prior to behavioral and in vitro assessment at 8 weeks of age. B) NMS had a significant impact on running wheel activity, such that NMS pairs maintained a significantly lower running distance compared to naïve mice for the first three weeks of wheel running. Bracket indicates a significant effect of NMS (§, p=0.0002), two-way ANOVA, *p<0.05, Fisher’s LSD posttest. n=6 pairs (all groups).

Figure 2.

Mechanical withdrawal thresholds were measured to assess perigenital sensitivity in naïve and NMS mice. A significant impact of NMS, exercise, and an NMS/exercise interaction was observed on mechanical withdrawal thresholds. Sedentary NMS males displayed significantly lower perigenital mechanical withdrawal thresholds than naïve-Sed and NMS-Ex mice. Data were log transformed prior to analysis. Bracket indicates significant effect of NMS (§, p=0.008), exercise (ε, p<0.0001), and an NMS/exercise interaction (+, p=0.0002), two-way ANOVA, *p<0.0001 vs. naïve, ^#p<0.0001 vs. sedentary, Bonferroni posttest. n=14 (naïve-Sed, NMS-Sed), 9 (naïve-Ex), 12, (NMS-Ex).

Figure 3.

Micturition behavior was evaluated to determine changes in bladder output. A) The number of voids was significantly higher in NMS-Sed mice compared to naïve-Sed mice. No significant difference was observed between naïve-Ex and NMS-Ex mice. B) Exercise significantly decreased the total void area. The total voided volume was significantly higher in NMS-Sed mice compared to naïve-Sed and NMS-Ex. Data were rank transformed prior to statistical analysis. Bracket indicates significant effect of exercise (ε, p=0.0039), two-way ANOVA, *p<0.05 vs. naïve, ^#p<0.01 vs. sedentary, Fisher’s LSD posttest, n=19 (naïve-Sed), 28 (NMS-Sed), 6 (naïve-Ex, NMS-Ex).

Figure 4.

Sucrose preference and elevated plus maze testing was performed to evaluate depression- and anxiety-like behaviors, respectively. A) No significant impact of NMS or exercise was observed on the percent of sucrose consumed. B) Exercise significantly increased the volume of water (p=0.0346) and sucrose (p=0.0017) that was consumed, across naïve and NMS mice. NMS-Ex mice consumed significantly more water than NMS-Sed mice. An NMS/exercise interaction (p=0.0405) significantly impacted the volume of sucrose consumed, with naïve-Ex mice consuming significantly more than naïve-Sed and NMS-Ex mice. C) Exercise significantly decreased the amount of time spent in the open arms of an EPM, across both groups (p=0.0110), and particularly in NMS-Ex compared to NMS-Sed. D) The number of entries into the open arms was also significantly lower in exercised groups (p=0.0021), with naïve-Ex and NMS-Ex showing significantly fewer entries compared to their sedentary counterparts. Brackets indicate significant effect of exercise (ε) or an NMS/exercise interaction (+), two-way ANOVA, *p<0.05 vs. naïve, ^#p<0.05 vs. sedentary, Fisher’s LSD posttest. A-B: n=23 (naïve-Sed), 25 (NMS-Sed), 8 (naïve-Ex), 7 (NMS-Ex); C-D: n=8 (naïve-Sed, NMS-Sed, NMS-Ex), 7 (naïve-Ex).

Figure 5.

Serum corticosterone and mast cell degranulation were evaluated to determine downstream activation of the hypothalamic-pituitary-adrenal axis. A) NMS (p=0.0403) and exercise (p=0.0149) both significantly decreased serum CORT levels. Sedentary naïve mice had significantly higher serum corticosterone levels compared to NMS-Sed and naïve-Ex mice. Photomicrographs are shown of acidified toluidine blue-stained prostate sections from naïve (B, B’) and NMS (C, C’) mice to demonstrate intact (arrow) and degranulated (arrowhead) mast cells. Higher magnification images illustrate dense metachromasia (red arrows, B’ and C’) and faint staining with extruded granules (red arrowheads, C’) of intact and degranulated mast cells, respectively. D) A significant impact of exercise (p=0.0481) was observed on mast cell degranulation in the bladder. A trend toward increased mast cell degranulation was observed in bladders from NMS-Sed mice compared to naïve-Sed (p=0.0805). NMS-Ex mice exhibited significantly lower percentages of degranulated mast cells compared to NMS-Sed mice. E) A significant impact of NMS (p=0.0004) and an NMS/exercise interaction (p=0.0002) was also observed on mast cell degranulation in the prostate. NMS-Sed mice exhibited significantly higher percentages of degranulated mast cells compared to both naïve-Sed and NMS-Ex mice. Brackets indicate significant effect of NMS (00A7), exercise (ε), or an NMS/exercise interaction (+), two-way ANOVA; *p<0.05 vs. naïve, ^#p<0.05 vs. sedentary, Fisher’s LSD posttest. Scale bars equal 100μm. A: n=10 (all groups); D-E: n=3–4 (all groups).

Figure 6.

Neurogenesis was evaluated in the hippocampus using BrdU. Examples of BrdU-labeled neurons (red) in the subgranular zone of the dentate gyrus are shown from naïve-Sed (A), NMS-Sed (B), naïve-Ex (C), and NMS-Ex (D) mice. NeuN (green) labeled mature neurons in the granular zone, along with Hoechst stain (blue). E) A significant impact of NMS and exercise was observed on BrdU labeled neurons. NMS-Sed mice had significantly fewer BrdU-positive neurons per section, compared to naïve-Sed. Exercise significantly increased the number of BrdU-positive neurons in both naïve and NMS mice compared to their sedentary counterparts. Brackets indicate an effect of NMS (§, p=0.0014) or exercise (ε, p=0.0118); two-way ANOVA, *p<0.05 vs. naïve, ^#p<0.05 vs. sedentary, Fisher’s LSD posttest. n=3 (naïve-Sed, naïve-Ex), 4 (NMS-Sed, NMS-Ex).