Long-term omega-3 supplementation modulates behavior, hippocampal fatty acid concentration, neuronal progenitor proliferation and central TNF-α expression in 7 month old unchallenged mice

Abstract

Dietary polyunsaturated fatty acid (PUFA) manipulation is being investigated as a potential therapeutic supplement to reduce the risk of developing age-related cognitive decline (ARCD). Animal studies suggest that high omega (Ω)-3 and low Ω-6 dietary content reduces cognitive decline by decreasing central nervous system (CNS) inflammation and modifying neuroimmune activity. However, no previous studies have investigated the long term effects of Ω-3 and Ω-6 dietary levels in healthy aging mice leaving the important question about the preventive effects of Ω-3 and Ω-6 on behavior and underlying molecular pathways unaddressed. We aimed to investigate the efficacy of long-term Ω-3 and Ω-6 PUFA dietary supplementation in mature adult C57BL/6 mice. We measured the effect of low, medium, and high Ω-3:Ω-6 dietary ratio, given from the age of 3-7 months, on anxiety and cognition-like behavior, hippocampal tissue expression of TNF-α, markers of neuronal progenitor proliferation and gliogenesis and serum cytokine concentration. Our results show that a higher Ω-3:Ω-6 PUFA diet ratio increased hippocampal PUFA, increased anxiety, improved hippocampal dependent spatial memory and reduced hippocampal TNF-α levels compared to a low Ω-3:Ω-6 diet. Furthermore, serum TNF-α concentration was reduced in the higher Ω-3:Ω-6 PUFA ratio supplementation group while expression of the neuronal progenitor proliferation markers KI67 and doublecortin (DCX) was increased in the dentate gyrus as opposed to the low Ω-3:Ω-6 group. Conversely, Ω-3:Ω-6 dietary PUFA ratio had no significant effect on astrocyte or microglia number or cell death in the dentate gyrus. These results suggest that supplementation of PUFAs may delay aging effects on cognitive function in unchallenged mature adult C57BL/6 mice. This effect is possibly induced by increasing neuronal progenitor proliferation and reducing TNF-α.

Keywords: TNF-α; cognition; neurogenesis; omega 3; polyunsaturated fatty acid.

Figure 1

Locomotor activity and anxiety in the open field. (A) Locomotor activity of mice in the open field measured by distance traveled and (B) anxiety-like behavior in mice measured by time spent in the center of the open field. Data analyzed by One-Way ANOVA within strains with Tukey's post-hoc test, ^*p < 0.05, as indicated on figure. All data presented as mean ± SEM (n = 10/group).

Figure 2

Learning behavior in the Barnes Maze. Learning ability of mice on the Barnes Maze measured by latency to the escape location over 4 days of training. All data represent mean ± SEM (n = 10/group).

Figure 3

Total number of TNF-α positive cells in the hippocampus. The total number of TNF-α positive cells in (A) the hippocampus (including the dentate gyrus) and (B) the dentate gyrus only. Data analyzed by One-Way ANOVA within strains with Tukey's post-hoc test, ^*p < 0.05, ^***p < 0.001, as indicated on figure. All data presented as mean ± SEM (n = 5/group).

Figure 4

Representative images of TNF-α expression in the dentate gyrus. Representative images of the hippocampus centered on the dentate gyrus to demonstrate expression of TNF-α positive cells at 20× magnification in (A) High Ω-3:Ω-6 mice, (B) medium Ω-3:Ω-6 mice, (C) low Ω-3:Ω-6 mice and at 40× magnification in (D–F), respectively. Arrows signify relevant stained cells.

Figure 5

Effect of Ω-3:Ω-6 diet on neurogenesis in the dentate gyrus. Number of KI67 positive cells (A) and DCX positive cells (B) in the dentate gyrus. Data analyzed by One-Way ANOVA within strains with Tukey's post-hoc test, ^*p < 0.05 as indicated on figure. All data represented as mean ± SEM (n = 5/group).

Figure 6

Representative images of DCX expression in the dentate gyrus. Representative images of the hippocampus centered on the dentate gyrus to demonstrate expression of DCX positive cells at 20× magnification in (A) high Ω-3:Ω-6 mice, (B) medium Ω-3:Ω-6 mice, (C) low Ω-3:Ω-6 mice and at 40× magnification in (D–F), respectively. Arrows signify relevant stained cells.

Figure 7

Representative images of Ki67 expression in the dentate gyrus. Representative images of the hippocampus centered on the dentate gyrus to demonstrate expression of Ki67 positive cells at 20× magnification in (A) high Ω-3:Ω-6 mice, (B) medium Ω-3:Ω-6 mice, (C) low Ω-3:Ω-6 mice and at 40× magnification in (D–F), respectively. Arrows signify relevant stained cells.

Figure 8

Effect of Ω-3:Ω-6 diet on oxidative stress in the dentate gyrus. Representative images of oxo8dG/oxo8G expression in the dentate gyrus at 20× magnification in (A) high Ω-3:Ω-6 mice, (B) medium Ω-3:Ω-6 mice, (C) low Ω-3:Ω-6 mice, with representation of the median intensity of staining in each group (D).