Table 1.
The oligonucleotide sequences for each primer sequence were obtained from Affymetrix database using the probe set IDs and Primer3 software. The primers were custom prepared and used as described in the Methods.
Fig 1.
Diet and genotype effects on cognitive endpoints from three different behavioral tests.
The performance of C57BL/6 (WT) or LDLr-/- mice in the Y-maze alternation was determined after they were fed a control (CD) or western (WD) diet (A). The mean percentage of triplicates (number of alternations, number of times 3 adjacent arms, divided by the total number of arms entered) is shown (n = 8/grp). These same animals were also exposed to the Morris Water Maze (MWM, B) where % time spent in the platform quadrant during the probe trial is shown. In separate but identically treated groups of WT and LDLr -/- animals, mice were exposed to the Radial Arm Water Maze (RAWM, C) and the number of trial 4 errors in day’s 1–4 vs 5–9 of testing is shown (n = at least 9/grp). Values are mean ±SEM and analyzed by two-way ANOVA with Tukey’s multiple comparisons post hoc test.
Fig 2.
Western diet increases BBB transfer coefficient (Ki).
Wild type (WT) and LDLr -/- animals on either a control (CD) or western diet (WD) are plotted against their mean Ki (min-1) ± SEM (A). Representative images of the Ki maps of WT CD, LDLr -/- CD, WT WD and LDLr -/- WD are shown (B) where brain is circled in red. Two-way ANOVA showed a significant effect of diet on the BBB transfer coefficient Ki where WD increased Ki when WT and LDLr-/- data were pooled denoted by *, p<0.05 (n = at least 6/grp).
Fig 3.
Factor VIII immunostaining density increases in hippocampus and thalamus of LDLr -/- while IBA1 increases in the cortex and hippocampus of C57BL/6 (WT) fed a western diet.
Area density of factor VIII and IBA1 immunostaining in coronal section of C57BL/6 (WT) or LDLr -/- mice fed control (CD) or western diet (WD) as determined by image analysis (A and D). Representative images comparing factor VIII immunostaining in thalamus of LDLr -/- mice fed CD (B) or WD (C) (scale bar = 50μm). WD fed animals had more granules of factor VIII positive material in capillaries (arrows). Microglia in control fed mice (E) have most IBA-1 positivity surrounding the nucleus while those from western diet (F) fed animals have denser staining that extends through elongate strands of cytoplasm (scale bar = 20μm). Representative images comparing IBA1 immunostaining in cortex (red), hippocampus (blue) and thalamus (green) of WT mice fed CD (G) or WD (H) (scale bar = ~0.5mm). Statistical differences from WT (*) or LDLr -/- (#) CD fed mice or LDLr-/-WD fed ($), as determined by ANOVA with Tukey’s multiple comparisons post hoc test p<0.05, n = at least 5/grp).
Table 2.
Shift in gene expression with western diet and/or Ldlr -/-.
C57BL/6 (WT) or LDLr-/- mice were fed a control (CD) or western (WD) diet for 12 weeks and their brain gene expression was measured. Values are fold of WT on a CD ±SEM. Means in a row without a common subscript letter differ (p≤ 0.05) as analyzed by two-way ANOVA and Tukey test. GxD = Genotype x Diet interaction effect (n = at least 5/grp).
Fig 4.
Genotype but not diet alters brain glucose uptake.
18FDG uptake measured by mean standardized uptake value (SUVglu) corrected for plasma glucose from a summed static image from 20–30 min post injection of C57BL/6 (WT) or LDLr -/- mice fed control (CD) or western diet (WD). Statistical differences by two-way ANOVA between WT and LDLr -/- mice (*) p<0.05, n = at least 4/grp.
Fig 5.
In vivo brain metabolite content determined by 1H-MRS.
Localized 1H spectra were obtained from an 18.75 μl volume (white box) positioned within the left or right hemisphere of the brain. Metabolite content was determined by spectral fitting with LC model using a basis set of 17 metabolites plus components due to macromolecules and intracellular lipids.
Table 3.
Shift in brain metabolites assessed by 1H-MRS by western diet and/or LDLr -/-.
C57BL/6 (WT) or LDLr-/ mice were fed a control (CD) or western (WD) diet for 12 weeks and their brain fatty acid and TCA cycle intermediates were measured. Values are mean of abundance relative to Cr+PCr ±SEM. Means in a row without a common subscript letter differ (p≤ 0.05) as analyzed by two-way ANOVA and Tukey test. GxD = Genotype x Diet interaction effect (p≤ 0.05, n = at least 7/grp).
Table 4.
Shift in brain fatty acid and TCA cycle intermediate abundance with western diet and/or LDLr -/-.
C57BL/6 (WT) or LDLr-/- mice were fed a control (CD) or western (WD) diet for 12 weeks and their brain fatty acid and TCA cycle intermediates were measured. Values are mean in μmol/g tissue ±SEM. Means in a row without a common subscript letter differ (p≤ 0.05) as analyzed by two-way ANOVA and Tukey test. GxD = Genotype x Diet interaction effect (n = 4/grp).
Table 5.
Shift in negatively charged complex lipids with western diet and/or LDLr -/-.
C57BL/6 (WT) or LDLr-/- mice were fed a control (CD) or western (WD) diet for 12 weeks and their brain complex lipids were measured by LC CSH-(+)ESI QTOF MS. Values are mean in μmol/g tissue ±SEM. Means in a row without a common subscript letter differ (p≤ 0.05) as analyzed by two-way ANOVA and Tukey test. GxD = Genotype x Diet interaction effect (n = 4/grp).
Table 6.
Shift in positively charged complex lipids with western diet and/or LDLr -/-.
C57BL/6 (WT) or LDLr-/- mice were fed a control (CD) or western (WD) diet for 12 weeks and their complex lipids were measured by LC CSH- (-) ESI QTOF MS. Values are mean in μmol/g tissue ±SEM. Means in a row without a common subscript letter differ (p≤ 0.05) as analyzed by two-way ANOVA and Tukey test. GxD = Genotype x Diet interaction effect (n = 4/grp).
Fig 6.
Shift in brain free oxylipins, endocannabinoids and polyunsaturated fatty acids (PUFA) to a proinflammatory profile by diet.
Partial least squares analysis (A) discriminate between C57BL/6 (WT) or LDLr-/ mice, fed a control (CD) or western (WD) diet due to differences in brain metabolite pattern seen in associated loading plot (B). Hierarchical cluster analysis categorized measured variables into clusters and ANOVA analysis tested for effect of the diet, genotype, and the diet x genotype interaction. Clusters identified as having either a significant change are shown (C). Loading plot: variables with VIP scores >1 are shown. The variables are colored according to their clusters affiliations.
Table 7.
Non-fasting physiological parameters at 20 weeks.
Data are represented as mean ± SEM for wild type (WT) LDLr -/- on either a control (CD) or western (WD) diet, n = 20 for body weight and 4 for all other parameters. a-c Means in a row without a common superscript letter differ (P < 0.05) as analyzed by two-way ANOVA and the TUKEY test. G × D1 = Genotype × Diet interaction effect. n = 20 for body weight and 4 for all other parameters.
Fig 7.
Cardiac and liver histopathology.
In heart, CD (A) and WD-fed (B) wild type mice had normal aortic sinus structures, but the aortic sinus of LDLr -/- mice on CD had subendothelial foam cell plaques and medial accumulations of extracellular cholesterol (C), both were more extensive in LDLr -/- mice fed a WD (D). In liver, compared with CD fed (A), WT mice given a WD (B) had marked microvesicular and macrovesicular hepatic lipidosis that was most marked in the periacinar regions. CD fed LDLr -/-mice (C) had more hepatocellular glycogen than WT mice but were otherwise histologically normal. LDLr -/- mice given a WD (D) had generalized macrovesicular lipidosis, enlarged Kupffer cells with foamy cytoplasm (Arrowheads), periportal mononuclear inflammation, and individual hepatocyte necrosis accompanied by mixed inflammatory cell infiltrates (Arrow). Scale bar: heart = 100μM and liver = 50μM.
Fig 8.
Model of physiological pathway in which diet or genotype induced hyperlipidemia alters cognitive function.
Table 8.
Functional changes by western diet (WD) or in a genetic (G) model of hyperlipidemia.
Table 9.
Cardiac and liver histopathology with western diet and/or LDLr -/-.