Short-Term Long Chain Omega3 Diet Protects from Neuroinflammatory Processes and Memory Impairment in Aged Mice

Regular consumption of food enriched in omega3 polyunsaturated fatty acids (ω3 PUFAs) has been shown to reduce risk of cognitive decline in elderly, and possibly development of Alzheimer's disease. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the most likely active components of ω3-rich PUFAs diets in the brain. We therefore hypothesized that exposing mice to a DHA and EPA enriched diet may reduce neuroinflammation and protect against memory impairment in aged mice. For this purpose, mice were exposed to a control diet throughout life and were further submitted to a diet enriched in EPA and DHA during 2 additional months. Cytokine expression together with a thorough analysis of astrocytes morphology assessed by a 3D reconstruction was measured in the hippocampus of young (3-month-old) and aged (22-month-old) mice. In addition, the effects of EPA and DHA on spatial memory and associated Fos activation in the hippocampus were assessed. We showed that a 2-month EPA/DHA treatment increased these long-chain ω3 PUFAs in the brain, prevented cytokines expression and astrocytes morphology changes in the hippocampus and restored spatial memory deficits and Fos-associated activation in the hippocampus of aged mice. Collectively, these data indicated that diet-induced accumulation of EPA and DHA in the brain protects against neuroinflammation and cognitive impairment linked to aging, further reinforcing the idea that increased EPA and DHA intake may provide protection to the brain of aged subjects

Impaired Interleukin-1β and c-Fos Expression in the Hippocampus Is Associated with a Spatial Memory Deficit in P2X7 Receptor-Deficient Mice

Recent evidence suggests that interleukin-1β (IL-1β), which was originally identified as a proinflammatory cytokine, is also required in the brain for memory processes. We have previously shown that IL-1β synthesis in the hippocampus is dependent on P2X7 receptor (P2X7R), which is an ionotropic receptor of ATP. To substantiate the role of P2X7R in both brain IL-1β expression and memory processes, we examined the induction of IL-1β mRNA expression in the hippocampus of wild-type (WT) and homozygous P2X7 receptor knockout mice (P2X7R−/−) following a spatial memory task. The spatial recognition task induced both IL-1β mRNA expression and c-Fos protein activation in the hippocampus of WT but not of P2X7R−/− mice. Remarkably, P2X7R−/− mice displayed spatial memory impairment in a hippocampal-dependant task, while their performances in an object recognition task were unaltered. Taken together, our results show that P2X7R plays a critical role in spatial memory processes and the associated hippocampal IL-1β mRNA synthesis and c-Fos activation

Preference for novelty was measured using a 2-min ITI in WT and P2X7R^−/− mice.

<p>Both strain exhibited a clear preferential exploration of the novelty (Arm effect, ***p<0.001).</p

IL-1β mRNA expression after the exposure to the Y maze in P2X₇R^−/− mice.

<p>IL-1β mRNA was measured by real-time PCR on WT and P2X₇R^−/− mice sacrificed either after free exploration (control group) or completion of behavioral testing (Y-maze group). All data are expressed as mean relative fold change±SEM (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006006#s2" target="_blank">Materials and Methods</a> for explanation). (A) IL-1β mRNA on hippocampus extracts from the 2-min ITI Y-maze group and their controls (left-hand panel) and the 30-min ITI Y-maze group and their controls (right-hand panel). (B) IL-1β mRNA on hypothalamus extracts from the 30-min ITI Y-maze group and their controls. Y maze exposure significantly increased the IL-1β mRNA expression in the hippocampus of WT mice but not of P2X₇R^−/− mice. Whatever the group considered (WT or P2X₇R^−/−), no induction of IL-1β mRNA expression was detected in hypothalamus after the Y maze experience. ***p<0.001, **p<0.01.</p

Recognition memory performance after 30 minutes of retention in P2X₇R^−/− mice.

<p>(A) Time spent (sec) in the novel or the familiar arm after a 30-min ITI. (B) Time spent (sec) exploring the novel or the familiar object after a 30-min ITI. Spatial recognition memory, but not object recognition memory, was impaired in P2X₇R^−/− mice after a 30-min ITI. ***p<0.001, **p<0.01,*p<0.05.</p

Brain fatty acid composition.

<p>dGLA: dihomo-gamma-linolenic acid (20:3 ω6); AA: arachidonic acid (20:4 ω6); EPA: eicosapentaenoic acid (20:5 ω3); DHA: docosahexaenoic acid; DMA: dimethyl acetal; LC ω6: long chain ω6 (20:2 ω6+20:3 ω6+20:4 ω6+22:4 ω6+22:5 ω6); LC ω3: long chain ω3 (20:5 ω3+22:5 ω3+22:6 ω3); NS: not significant.</p>***<p>p<0.001 as compared to aged control diet;</p>**<p>p<0.01 as compared to aged control diet;</p>*<p>p<0.05 as compared to aged control diet.</p

Spatial and object recognition memory.

<p>(A) Time spent (in sec) in the novel or the familiar arm after a 5-min ITI in 3-month-old (young) and 22-month-old (aged) mice fed with the control diet or the LCω3 diet for 2 months. (B) Time spent (in sec) in the novel or the familiar object after a 1-hr ITI in 3-month-old (young) and 22-month-old (aged) mice fed with the control diet or the LCω3 diet for 2 months. *** p<0.001, ** p<0.01, * p<0.05.</p

c-Fos expression in the DG, CA1 and CA3 regions of the hippocampus.

<p>c-Fos immunohistochemical analysis was performed in the hippocampus of 3-month-old (young) and 22-month-old (aged) mice fed with the control diet or the LCω3 diet for 2 months and sacrificed 90 min after the spatial recognition acquisition session. (A) Representative images of c-Fos immunohistochemistry in the DG (left panel), the CA1 (central panel) and the CA3 region (right panel) of the hippocampus. Scale bar = 100 µm. (B) Quantification of c-Fos-positive cells was performed in the DG, the CA1 and the CA3 regions of the hippocampus. Data are presented as mean ± SEM. ** p<0.01. (C) Correlation between the number of c-Fos positive cells induced by the Y-maze task in the DG (left panel), the CA1 (central panel) and the CA3 (right panel) and the spatial recognition score. Pearson's correlation coefficients (r) and corresponding significance (p) are displayed within each correlation window. The number of c-Fos positive cells in the DG and CA1, but not in the CA3 regions of the hippocampus was positively correlated to the spatial recognition score.</p

Morphometry of astrocytic processes in the hippocampus.

<p>(A) Confocal analysis of GFAP immunofluorescence was performed in the DG, the CA1 and the CA3 regions of the hippocampus of 3-month old (young) and 22-month old (aged) mice fed with the control diet or the LCω3 diet for 2 months using a 63X oil-immersion lens with a 3.10X zoom and IMARIS software. Images are representative of GFAP 3D immunofluorescence in the DG (upper panel), CA1 (central panel) and CA3 (lower panel). Scale bar = 8.5 µm. (A) Morphometric analysis of astrocytes in the DG, CA1 and CA3 regions of the hippocampus was performed using the “filament tracer” program/function. Data present means of primary and secondary processes lengths expressed in µm ± SEM. *** p<0.001, ** p<0.01, * p<0.05.</p