Neuroprotective Role of Dietary Supplementation with Omega-3 Fatty Acids in the Presence of Basal Forebrain Cholinergic Neurons Degeneration in Aged Mice

Abstract

As major components of neuronal membranes, omega-3 polyunsaturated fatty acids (n-3 PUFA) exhibit a wide range of regulatory functions. Recent human and animal studies indicate that n-3 PUFA may exert beneficial effects on aging processes. Here we analyzed the neuroprotective influence of n-3 PUFA supplementation on behavioral deficits, hippocampal neurogenesis, volume loss, and astrogliosis in aged mice that underwent a selective depletion of basal forebrain cholinergic neurons. Such a lesion represents a valid model to mimic a key component of the cognitive deficits associated with dementia. Aged mice were supplemented with n-3 PUFA or olive oil (as isocaloric control) for 8 weeks and then cholinergically depleted with mu-p75-saporin immunotoxin. Two weeks after lesioning, mice were behaviorally tested to assess anxious, motivational, social, mnesic, and depressive-like behaviors. Subsequently, morphological and biochemical analyses were performed. In lesioned aged mice the n-3 PUFA pre-treatment preserved explorative skills and associative retention memory, enhanced neurogenesis in the dentate gyrus, and reduced volume and VAChT levels loss as well as astrogliosis in hippocampus. The present findings demonstrating that n-3 PUFA supplementation before cholinergic depletion can counteract behavioral deficits and hippocampal neurodegeneration in aged mice advance a low-cost, non-invasive preventive tool to enhance life quality during aging.

Keywords: aging; cholinergic system; cognitive deficits; neuroprotection; omega-3 fatty acids; prevention.

Figure 1

Experimental procedures. After 8-week oral supplementation with n-3 PUFA, 21-month old aged mice have been subjected to intracerebroventricular (i.c.v.) injections of mu-p75-saporin or saline (sham lesion) to selectively deplete the forebrain cholinergic system. Two weeks after the lesion, the animals were behaviorally tested by means of validated tasks (Elevated Plus Maze, EPM; Splash Test, ST; Social Interactions, SI; Hidden Food Test, HFT; Predator Odor Fear Conditioning, POFC; Porsolt Test, PT). At the end of testing battery, mice were sacrificed, and brains collected for morphological and biochemical analyses.

Figure 2

Elevated Plus Maze data. Duration and frequency of exploration of the closed and open arms in the four experimental groups. Data are reported as mean and SEM (oil sham, n = 12; n-3 PUFA sham, n = 10; oil sap, n = 10; n-3 PUFA sap, n = 10). Arm effect: *** p < 0.000001; Diet effect: § p < 0.05; Diet x lesion effect: # p < 0.05.

Figure 3

Predator Odor Fear Conditioning. Duration of freezing in the four experimental groups during habituation and context test sessions were used to calculate the retention index. Data are reported as mean and SEM (oil sham, n = 11; n-3 PUFA sham, n = 10; oil sap, n = 10; n-3 PUFA sap, n = 10). Asterisks indicate the level of statistical significance of the post-hoc comparisons between oil sap and the remaining groups: * p < 0.05, ** p < 0.01.

Figure 4

Porsolt Test. Duration and frequency of active and passive coping strategies displayed by the four experimental groups. Data are reported as mean and SEM (oil sham, n = 11; n-3 PUFA sham, n = 10; oil sap, n = 10; n-3 PUFA sap, n = 10). Strategy effect: *** p < 0.00001.

Figure 5

Morphological results. (A–C). Diagrams showing the variations of volume in the whole hippocampus, Ammon’s Horn (CA1 + CA3) and Dentate Gyrus (DG) in the four experimental groups (n = 5 mice/group). (D). Graph showing the large increase of Ki67-positive neuroblasts in the n-3 PUFA sap group in respect to the other groups. (E). Graph representing the unchanged values detected in the SOX2⁺ sub-populations. (F). Diagram showing the enhancement of DCX-positive neuroblasts in the n-3 PUFA sap group in respect to the other groups. (G). Histogram indicating the GFAP⁺ cell number in the DG. (H). Representative images showing the increase in the DCX⁺ cells (red, arrows) in the DG of the n-3 PUFA sap mice, when compared with oil sham group. (I). Representative fluorescence images of GFAP⁺ cells (red), showing the increased astrogliosis in the oil sap group, counteracted by the pre-treatment with n-3 PUFA. Scale bar 100 µm. * p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t test.

Figure 6

Densitometric hippocampal VAChT expression. (A–D). Confocal images from the hippocampus, namely CA3 region, of oil sham, n-3 PUFA sham, oil sap and n-3 PUFA sap mice stained with Neuro-trace (blue) and VAChT (red) showing the expression of VAChT immunostaining. (E–G). Representative densitometric graph of the expression levels of VAChT in CA1 (E), CA3 (F) and dentate gyrus (DG; G) of oil sham, n-3 PUFA sham, oil sap and n-3 PUFA sap mice. The F/A ratio defines mean fluorescence of individual samples (F) normalized to total Area (A). Data of the four experimental groups are depicted as mean and SEM (n = 5/group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar: 50 µm.

Figure 7

Hippocampal VAChT and GFAP immunoblotting results. (A) Immunoblots of total hippocampal proteins from 24-month-old mice. (B) The histogram shows densitometric quantification of changes in gray values, expressed as % of oil sham group. Data of the four experimental groups are depicted as mean and SEM. GAPDH was used as loading control. (VAChT analysis: oil sham, n = 11; n-3 PUFA sham, n = 10; oil sap, n = 8; n-3 PUFA sap, n = 7; GFAP analysis: oil sham, n = 11; n-3 PUFA sham, n = 9; oil sap, n = 9; n-3 PUFA sap, n = 7). Statistical significance of the post-hoc comparisons between oil sap and the remaining groups: *** p < 0.001; **** p < 0.00001.