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

Abstract

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.

Keywords: calcitonin gene-related peptide; hypothalamic-pituitary-adrenal axis; mast cell; migraine; mouse grimace scale; photophobia; voluntary wheel running.

Figure 1

Representative timeline of evoked migraine experiments and outcome measures. All groups consisted of mice that underwent neonatal maternal separation (NMS) from postnatal day (P) 1 to 21 (depicted as the red shaded period) and non-separated, naïve mice. Mice in Cohort A were singly-housed with running wheels at 8 weeks of age and sacrificed at 12 weeks of age. Dura mater was collected to evaluate mast cell (MC) characteristics and measure protein level using Western Blot. Mice in Cohort B were pair-housed with running wheels at 4 weeks of age. At 8 weeks of age, they received either inflammatory soup (IS) or saline applied to the dura mater and, 1 h later, mouse grimace score (MGS) was measured over 1 h. Forepaw mechanical withdrawal threshold was measured immediately after cessation of MGS. At 40 weeks of age, Cohort B mice received a single intraperitoneal (IP) injection of nitroglycerin (NTG) and 30 min later were assessed for MGS over 1 h, immediately followed by hindpaw mechanical withdrawal threshold. Mice in Cohort C were also pair-housed with running wheels at 4 weeks of age and assessed for MGS, without dural stimulation of NTG, at 12 weeks of age. The mice were given an IP NTG injection at 40 weeks, followed 30 min later by photophobia testing and sacrifice. The level of corticosterone was measured in the serum and the level of calcitonin gene-related peptide (CGRP) was measured in the serum, dura, and trigeminal ganglia (TG).

Figure 2

Dura mater was stained with toluidine blue (TB) to visualize mast cells and the total number and percent of degranulated mast cells were quantified in sedentary (Sed) or exercised (Ex) naïve and NMS mice. (A) NMS-Sed mice had a non-significant increase in the number of mast cells in the dura compared to naïve-Sed and NMS-Ex mice. (B) There was a significant NMS/exercise interaction on the percent of degranulated dural mast cells. NMS-Sed mice had a significantly greater percent of degranulated dura mast cells compared to naïve-Sed mice. This effect was attenuated by exercise, as NMS-Ex mice had a significantly lower percent of degranulated mast cells compared to NMS-Sed mice. (C) Representative image of dura mater from an NMS-Sed mouse stained with TB. Arrow heads indicate intact mast cells and arrows indicate degranulated or activated mast cells. Scale bar in C equals 25 μm. Bracket indicates a significant NMS/exercise interaction (⁺p < 0.05), two-way ANOVA; **p < 0.01 vs. naïve, ##p < 0.01 vs. sedentary, Fisher’s LSD posttest. n = 3–6.

Figure 3

Protein levels of protease-activated receptor 2 (PAR2) and transient receptor potential ankyrin 1 (TRPA1) were measured in the dura of Sed and Ex naïve and NMS mice using Western blot. (A) There was a significant impact of NMS on dural PAR2 protein levels. (B) A significant interaction effect of NMS and exercise was observed on dural TRPA1 protein levels. NMS-Ex mice had significantly lower TRPA1 protein levels compared to naïve-Ex mice. Bracket indicates a significant effect of NMS (§p < 0.05) or a NMS/exercise interaction (+p < 0.05), two-way ANOVA; **p < 0.01 vs. naïve, Fisher’s LSD posttest. n = 5–7.

Figure 4

Inflammatory soup or saline was applied to the dura through a modified injection cannula of Sed or Ex naïve and NMS mice. (A) MGS was measured every 5 min for 1 h beginning 1 h after dural injection. Forepaw mechanical thresholds were measured immediately after. (B) An overall significant effect of exercise was observed on MGS, such that naïve-Ex-Saline, NMS-Ex-Saline, and NMS-Ex-IS mice all had significantly higher MGS compared to their sedentary counterparts. (C) A significant overall effect of IS was observed on forepaw mechanical withdrawal threshold. Bracket indicates a significant effect of exercise (εεεεp < 0.0001) or IS (δp < 0.05), three-way ANOVA; #p<0.05 vs. sedentary, Fisher’s LSD posttest. n = 4–6.

Figure 5

Mouse grimace score and hindpaw mechanical withdrawal threshold was measured in naïve and NMS mice that were Sed or Ex following an intraperitoneal injection of saline or NTG. (A) MGS was measured every 5 min over 1 h, beginning 30 min after NTG injection. Hindpaw mechanical thresholds were measured immediately afterward. (B) A significant overall effect of NTG was observed on MGS. Naïve-Ex-NTG mice had a significantly higher MGS then naïve-Sed-NTG and naïve-Sed-Saline mice. NMS-Ex-NTG mice had a significantly lower MGS compared to naïve-Ex-NTG mice and a significantly higher MGS than NMS-Ex-Saline mice. (C) A significant overall effect of NMS and a NMS/exercise interaction was observed on hind paw mechanical withdrawal threshold. NMS-Sed-Saline mice and naïve-Ex-Saline had significantly lower hind paw withdrawal thresholds compared to naïve-Sed-Saline mice. A trend toward a decreased withdrawal threshold was observed in NMS-Sed-NTG mice compared to naïve-Sed-NTG (p = 0.067) and NMS-Ex-NTG mice (p = 0.071). Bracket indicates a significant effect of NTG (δδδδ p < 0.0001), NMS (§ p < 0.05), or a NMS/exercise interaction (+p < 0.05), three-way ANOVA; #p < 0.05 vs. sedentary, &, &&&&p < 0.05, 0.0001 vs. saline, *p < 0.05 vs. naïve, Fisher’s LSD posttest. n = 3–5.

Figure 6

Mouse grimace score was measured in Sed and Ex naïve and NMS mice that were briefly anesthetized and allowed to recover for 1 h. There was a significant overall effect of exercise (εεε p < 0.001; two-way ANOVA). Naïve-Ex had a significantly greater MGS compared to naïve-Sed mice (## p < 0.01; Fisher’s LSD posttest). n = 4–5.

Figure 7

Photophobia-like behavior was measured in Sed and Ex naïve and NMS mice. (A) Light aversion behavior was measured 6 [baseline day 1 (BL1)] and 3 [baseline day 2 (BL2)] days prior to testing day and 30 min after an intraperitoneal NTG injection. (B) The percent of time spent on the light side of a light/dark box was quantified at each time point. A significant overall effect of time (p < 0.0001) and a NMS/time interaction (p < 0.05) was observed on the time spent in the light. Between BL1 and the treatment day, naïve-Sed, NMS-Sed, and NMS-Ex mice significantly decreased their time spent in the light side. Both NMS-Sed and NMS-Ex mice significantly decreased their time spent in the light between BL2 and NTG. (C) Total distance traveled was measured during the testing period and there was a significant overall effect of time across all groups (p < 0.0001). Naïve-Sed and NMS-Ex mice displayed significant changes in their distance traveled from BL1 to BL2 and from BL2 to NTG. On testing day, naïve-Ex mice traveled a significantly farther distance compared to either naïve-Sed or NMS-Ex mice. (D) Comparisons between groups revealed a significant impact of NMS on time in the light side following NTG treatment. NMS-Sed mice spent significantly less time in the light compared to naïve-Sed mice. (E) Likewise, when calculated as a percent of BL2 time spent in the light, there was a significant overall effect of NMS. Brackets indicate significant within-group differences between time points (three-way RM ANOVA, α naïve-Sed, β NMS-Sed, γ naïve-Ex, p < 0.05, 0.01, 0.0001, Fisher’s LSD posttest, A,B) or NMS (§p < 0.05) two-way ANOVA. *p < 0.05 vs. naïve, #p < 0.05 vs. sedentary, Fisher’s LSD posttest. n = 10.

Figure 8

Calcitonin gene-related peptide and corticosterone levels were measured in Sed and Ex naïve and NMS mice 2 h after nitroglycerin treatment. (A) CGRP content in the dura was significantly decreased by exercise, such that naïve-Ex and NMS-Ex mice had significantly lower CGRP protein levels compared to their sedentary counterparts. CGRP content in the TG (B) and serum (C) was not different between groups. (D) Serum corticosterone levels were also not significantly different between groups. Bracket indicates a significant effect of exercise (εp < 0.05) two-way ANOVA; #, ###p < 0.05, 0.001 vs. sedentary, Fisher’s LSD posttest. n = 5–6.