An Omega-3-rich Anti-inflammatory Diet Improved Widespread Allodynia and Worsened Metabolic Outcomes in Adult Mice Exposed to Neonatal Maternal Separation

Abstract

Inflammation plays a key role in the progression and maintenance of chronic pain, which impacts the lives of millions of Americans. Despite growing evidence that chronic pain can be improved by treating underlying inflammation, successful treatments are lacking and pharmaceutical interventions are limited due to drug side effects. Here we are testing whether a 'healthy human' diet (HHD), with or without anti-inflammatory components (HHAID), improves pain-like behaviors in a preclinical model of chronic widespread hypersensitivity induced by neonatal maternal separation (NMS). The HHD and HHAID are isocaloric and macronutrient-matched, have a low glycemic index, and fat content (35 kcal%) that is high in omega-3 fatty acids, while only the HHAID includes a combination of key anti-inflammatory compounds, at clinically relevant doses. Mice on these diets were compared to mice on a control diet with a macronutrient composition commonly used in rodents (20% protein, 70% carbohydrate, 10% fat). Our results demonstrate a benefit of the HHAID on pain-like behaviors in both male and female mice, despite increased caloric intake, adiposity, and weight gain. In female mice, HHAID specifically increased measures of metabolic syndrome and inflammation compared to the HHD and control diet groups. Male mice were susceptible to worsening metabolic measures on both the HHAID and HHD. This work highlights important sexual dimorphic outcomes related to early life stress exposure and dietary interventions, as well as a potential disconnect between improvements in pain-like behaviors and metabolic measures.

Keywords: Chronic pain; Early life stress; Inflammation; Nutrition; Obesity.

Fig. 1.

Impact of NMS and diet on perigenital mechanical withdrawal threshold. Perigenital withdrawal threshold was measured every 4 weeks in female (A) and male (C) mice and the area under the curve (AUC) was calculated to produce a cumulative threshold (B, D). (A) In female mice, there was a significant effect of diet over the 16 weeks (p < 0.0001). (B) AUC measurements also revealed a significant effect of diet (p = 0.0005) with HHAID-fed mice having significantly higher thresholds than HHD- or control-fed mice, regardless of NMS status. (C) In males, there was a significant effect of diet (p < 0.0001) over the 16 weeks. (D) AUC measurements also revealed a significant effect of diet (p < 0.0001) with HHAID-fed mice having significantly lower withdrawal thresholds compared to HHD- and control-fed for both naïve and NMS mice. Additionally, NMS-HHD mice had significantly greater withdrawal thresholds than both NMS-Control and Naïve-HHD mice. δ denotes a significant effect of diet, three-way RM ANOVA (A, C) or two-way ANOVA (B, D). ††p < 0.01 HHD vs. control, &, &&, &&&&p < 0.05, 0.01, 0.0001 HHAID vs. control, #, ####p < 0.05, 0.0001 HHAID vs. HHD, Fisher’s LSD. n = 6–16 per group.

Fig. 2.

Impact of NMS and diet on hindpaw mechanical withdrawal threshold. Hindpaw mechanical withdrawal threshold was measured every 4 weeks (A, C) and the area under the curve (AUC, (B, D)) was calculated to produce a cumulative threshold. In female mice, there was an overall significant effect of NMS (p = 0.001) and time (p < 0.0001) across all experimental time points (A), and of NMS for the cumulative measurement ((B), p = 0.001). (B) Both NMS-Control and NMS-HHD female mice had significantly lower withdrawal thresholds compared to their naïve counterparts. (C, D) In males, there was a significant effect of time (p = 0.0069) and a trend toward an NMS effect (p = 0.081), but no effect of diet. § and τ denote a significant impact of NMS or time, respectively, three-way RM ANOVA (A) or two-way ANOVA (B). *, ***p < 0.05, 0.001 vs. same-diet naïve, Fisher’s LSD posttest. n = 6–16 per group.

Fig. 3.

Impact of NMS and diet on nest building behavior. (A) In female mice, there was a significant overall effect of diet (p = 0.048) on nest quality. NMS-HHAID mice had a significantly lower nest score compared to naïve-control mice. (B) Nest quality was not affected by diet or NMS in male mice. (C) Examples of nests with scores ranging from 5 (left) to 1.5 (right) are shown. δ denotes significant effect of diet, two-way ANOVA. *p < 0.05 vs. naïve-control mice, Fisher’s LSD posttest. n = 6–16 per group.

Fig. 4.

Impact of NMS and diet on body weight and composition. (A) In female mice, there was a significant overall effect of NMS (p = 0.007), diet (p = 0.006), and time (p < 0.0001) on body weight across 18 weeks. NMS-HHAID mice weighed significantly more than NMS-control (weeks 7, 9, and 14–18) and NMS-HHD (weeks 14–18) mice. (B) Body composition in females was significantly impacted by diet (p < 0.0001) and time (p < 0.0001). NMS-HHAID mice had significantly higher body fat percentage compared to NMS-control mice (every timepoint) and NMS-HHD mice (all but 12 weeks). At 18 weeks on the diet, naïve-HHAID mice had significantly higher body fat percentage than naïve-control mice. (C) In male mice, there was a significant overall effect of diet (p < 0.0001) and time (p < 0.0001)on body weight. NMS-HHAID (weeks 5–18) and-HHD (weeks 10–18) mice weighed more than NMS-control mice. Similarly, naïve-HHAID (weeks 14–18) and -HHD (weeks 10–18) mice weighed more than naïve-control mice. (D) Body composition analyses in male mice found a significant overall effect of diet (p < 0.0001) and time (p < 0.0001) on body fat percentage. NMS-HHAID mice had significantly higher body fat percentage compared to NMS-control mice (weeks 8–18). At week 16, naïve-HHAID mice had significantly higher body fat percentage compared to naïve-control mice and naïve-HHD had significantly higher body fat percentage compared to naïve-control (weeks 12–18). §, δ, and τ denote significant effects of NMS, diet, and time, respectively, three-way RM ANOVA. *p < 0.05 vs. same-diet naïve, †p < 0.05 HHD vs. control, &p < 0.05 HHAID vs. control, #p < 0.05 HHAID vs. HHD, Fisher’s LSD posttest. n = 6–16 per group.

Fig. 5.

The impact of NMS and diet on end-of-study body weight, body fat percentage, and gonadal fat weight. (A) Final body weight in female mice was significantly impacted by NMS (p = 0.044) and diet (p < 0.0001). HHD- and HHAID-fed mice were significantly heavier than control-fed mice, regardless of NMS status. NMS-HHAID female mice were also significantly heavier than naïve-HHAID mice. (B) Final body fat percentage was significantly impacted by diet (p < 0.0001), specifically in HHAID-fed mice. (C) Periovarian fat weight was significantly impacted by diet (p < 0.0001) with NMS-HHAID mice having significantly heavier fat pads than NMS-control and NMS-HHD mice. In male mice, final body weight (D), body fat percentage (E), and epididymal fat pad weight (F) were all significantly impacted by diet (p < 0.0001) with HHD- and HHAID-fed mice having significantly higher values compared to control-fed mice, regardless of NMS status. § and δ denote significant effects of NMS and diet, respectively, two-way ANOVA. *p < 0.05 vs. same-diet naïve, †, ††, †††, †††† < 0.05, 0.01, 0.001, 0.0001 HHD vs. control, &, &&, &&&, &&&&p < 0.05, 0.01, 0.001, 0.0001 HHAID vs. control, #p < 0.05 AID vs. HHD, Fisher’s LSD posttest. n = 6–16 per group.

Fig.6.

Effect of diet and early life stress on food intake and feed efficiency. (A) In female mice, there was a significant effect of diet (p = 0.042) on caloric intake. NMS and naïve mice fed either HHAID or HHD consumed more calories than their control-fed counterparts throughout the experiment. (B) Feed efficiency in female mice was significantly impacted by diet (p = 0.0004) with naïve-HHAID and NMS-HHAID mice having significantly higher feed efficiencies compared to their HHD-fed counterparts, despite being calorically identical. (C) In male mice, there was a significant overall effect of diet (p = 0.007) on caloric intake with HHAID- and HHD-fed mice consuming more calories than control-fed mice at every time point of the study. (D) There was also a significant effect of diet (p = 0.046) on feed efficiency in male mice, but no statistical difference between HHD- and HHAID-fed mice. δ denotes a significant effect of diet, three-way RM ANOVA (A, C) or two-way ANOVA (B, C). †p < 0.05 HHD vs. control, &p < 0.05 HHAID vs. control, #, ##p < 0.05, 0.01 HHAID vs. HHD, Fisher’s LSD posttest. n = 3–8 pairs per group.

Fig. 7.

Impact of diet and early life stress on glucose tolerance, fasting insulin, and HOMA-IR. (A) In female mice, there was a significant effect of diet (p < 0.001) on glucose tolerance with female NMS-HHAID mice having significantly higher serum glucose compared to NMS-Control mice at 15, 30, and 60 min and higher than NMS-HHD mice at 30 min. Additionally, Naïve-HHAID mice had a higher serum glucose level at 30 min compared to Naïve-Control mice. (B) Area under the curve (AUC) measurements revealed a significant diet effect (p = 0.0003) with both naïve-HHAID and NMS-HHAID mice having significantly higher serum glucose compared to their control counterparts. NMS-HHD mice were also significantly higher than NMS-Control. There was no significant effect of diet or NMS on fasting serum insulin levels (C) or calculated HOMA-RI (D). (E) In male mice, a significant effect of diet (p = 0.004) was observed on glucose tolerance in male mice with naïve-HHD mice having significantly higher serum glucose levels at 30 and 60 minutes compared to naïve-control mice. (F) AUC measurements were also significantly impacted by diet (p = 0.005) with naïve-HHD mice having higher serum glucose levels than naïve-control mice. A significant impact of diet was observed on fasting serum insulin levels (p = 0.040) and HOMA-IR (p = 0.032) in male mice with naïve-HHAID and naïve-HHD mice having significantly higher levels than naïve-control mice. δ denotes a significant effect of diet, three-way RM ANOVA (A, E) or two-way ANOVA (B-D, F-H). †, ††p < 0.05, 0.01 HHD vs. control, &, &&&p < 0.05, 0.001 HHAID vs. control, #p < 0.05 HHAID vs. HHD, Fisher’s LSD posttest. n = 6–16 per group.

Fig. 8.

Effects of diet and early life stress on serum corticosterone concentrations. (A) In female mice, there was a significant effect of diet (p = 0.008) on serum corticosterone level. NMS-HHAID had significantly lower serum corticosterone compared to NMS-control mice. (B) In male mice, there was also a significant effect of diet (p = 0.021) on serum corticosterone levels. NMS-HHD mice had significantly lower serum corticosterone compared to naïve-HHD and NMS-HHAID mice and NMS-HHAID mice also had significantly higher corticosterone compared to NMS-control. δ denotes a significant effect of diet, two-way ANOVA. *p < 0.05 vs. same diet-fed naïve, &, &&&p < 0.05, 0.001 HHAID vs. control, ##p < 0.01 HHAID vs. HHD, Fisher’s LSD posttest. n = 6–16 per group.

Fig. 9.

The effect of diet and early life stress on gene expression of inflammatory markers in gonadal adipose tissue. (A) In females, there was a significant effect of diet on F4/80 (p < 0.0001), CD68 (p < 0.0001), CD11b (p = 0.001), CD11c (p = 0.024), and TNFα (p = 0.016). HHAID-fed mice had significantly higher mRNA levels compared to both control-fed and HHD-fed for most genes analyzed, regardless of NMS status. (B) In males, there was a significant effect of diet on CD68 (p = 0.003), CD11c (p < 0.0001), and IL-10 (p = 0.021) and additional effects of NMS (p < 0.0001) and an interaction effect of NMS and diet (p = 0.012) on CD11c. F4/80 mRNA levels were significantly higher in NMS-HHAID mice compared to NMS-control or NMS-HHD mice. CD11c was significantly higher in NMS-HHAID and -HHD mice compared to NMS-control mice and to naïve mice fed the same diet. Il-10 was elevated in naïve-HHAID mice compared to naïve-Control mice. δ denotes a significant effect of diet, two-way ANOVA. *p < 0.05 vs. same diet-fed naïve, †p < 0.05 HHD vs. control, &p < 0.05 HHAID vs. control, #p < 0.05 HHAID vs. HHD, Fisher’s LSD posttest. n = 5 per group.