Sex-specific effects of high-fat diet on cognitive impairment in a mouse model of VCID

Abstract

Mid-life metabolic disease (ie, obesity, diabetes, and prediabetes) causes vascular dysfunction and is a risk factor for vascular contributions to cognitive impairment and dementia (VCID), particularly in women. Using middle-aged mice, we modeled metabolic disease (obesity/prediabetes) via chronic high-fat (HF) diet and modeled VCID via unilateral common carotid artery occlusion. VCID impaired spatial memory in both sexes, but episodic-like memory in females only. HF diet caused greater weight gain and glucose intolerance in middle-aged females than males. HF diet alone impaired episodic-like memory in both sexes, but spatial memory in females only. Finally, the combination of HF diet and VCID elicited cognitive impairments in all tests, in both sexes. Sex-specific correlations were found between metabolic outcomes and memory. Notably, both visceral fat and the pro-inflammatory cytokine tumor necrosis factor alpha correlated with spatial memory deficits in middle-aged females, but not males. Overall, our data show that HF diet causes greater metabolic impairment and a wider array of cognitive deficits in middle-aged females than males. The combination of HF diet with VCID elicits deficits across multiple cognitive domains in both sexes. Our data are in line with clinical data, which shows that mid-life metabolic disease increases VCID risk, particularly in females.

Keywords: cerebral blood flow; diet-induced obesity; prediabetes; sex; vascular contributions to cognitive impairment and dementia.

Figure 1.. Timeline.

Male and female mice were placed on a high fat (HF; blue pellets) or low fat (LF; yellow pellets) diet at ~8.5 months of age. Glucose tolerance tests (GTT) were performed 2.75 and 5.75 months later. VCID (right unilateral common carotid artery occlusion) or sham surgery was performed 3 months after diet onset. Behavior testing (open field, novel object recognition test, Morris water maze, and nest building) were performed 3 months after surgery over a 2 week period. Mice were euthanized and tissue collected at ~15 months of age.

Figure 2.. Female mice on a high fat diet experience greater metabolic impairment compared to males.

Male and female mice were placed on a HF or LF diet at ~8.5 months of age. Over the course of 6 months both sexes gained weight on a HF diet (A-B), while females gained a greater percent of weight (C) than middle-aged HF males, regardless of surgery. Mice of both sexes showed impaired glucose tolerance. HF females had significantly more impairment in glucose tolerance than HF males after 6 months on the diet (D-F) (N=15-20/group). Subcutaneous fat (G) and visceral fat (H) were increased by HF diet in both sexes, but to a greater extent in females (N=10-15/group). Visceral fat TNFalpha levels (I) were increased by HF diet in both sexes (N= 4-5/group). *p<0.05 for diet effect within same sex, **p<0.01 for diet within the same sex, ***p<0.001 for diet within same sex, ****p<0.0001 for diet within same sex, #p<0.05 for sex difference within same diet group. Data are presented as mean +/− SEM.

Figure 3.. Reductions in CBF immediately post-surgery are more severe in HF compared to LF VCID females.

Cerebral blood flow (CBF) was measured via laser speckle contrast imaging immediately before and 5 minutes after unilateral carotid artery occlusion surgery. Regions of interest (ROI) were drawn for the temporal (T), parietal (P), ZOA (Z), and frontal cortical regions (F), representative images are shown (A). In the right (ischemic side) temporal region, CBF was significantly decreased in HF VCID males, LF VCID females, and HF VCID females. The decrease was greater in HF VCID vs. LF VCID females (B). In the right parietal region, CBF was significantly decreased in LF VCID and HF VCID males, and HF VCID females. The decrease was greater in HF VCID vs. LF VCID females (C). In the right ZOA region, CBF was significantly decreased in HF VCID males and HF VCID females. The decrease was greater in HF VCID vs. LF VCID females (D). In the right frontal region, CBF was significantly decreased in HF VCID males and HF VCID females. The decrease showed a trend (p=0.07) towards being grater in the HF VCID vs. LF VCID females (E). *p<0.05 for diet effect within same sex, +p<0.05 decrease in CBF. ++p<0.01 decrease in CBF, +++p<0.001 decrease in CBF. Data are presented as mean +/− SEM (N= 3-5/group).

Figure 4.. Reductions in CBF 4 months post-surgery persist mainly in the temporal cortical region.

CBF was measured via laser speckle contrast imaging 4 months after unilateral carotid artery occlusion surgery. Representative images are shown of ROIs in a sham and a VCID mouse (A). In right temporal region, relative CBF was significantly decreased in all VCID mice (B). In the right parietal region, there was no relative decrease in CBF in any group (B). In the right ZOA region, CBF was significantly decreased in LF VCID mice of both sexes (D). In the right frontal region, there was no decrease in CBF in any group (E). +p<0.05 decrease in CBF. ++p<0.01 decrease in CBF. Data are presented as mean +/− SEM (N=5-8/group).

Figure 5.. Effects of diet, sex, and VCID on anxiety-like behavior, activities of daily living, and episodic-like memory.

At the end of the study behavior was assessed. In the open field, track length (locomotor activity) was decreased in HF diet mice (A) and in females there were an overall effect of diet to increase anxiety-like behavior (less time in center), however post-hoc comparisons between groups did not reach significance (B). Nest building (24hrs), an assessment of activities of daily living), was poor in all middle aged mice (C), with the median score being a two which indicates less than half of the nestlet was torn. Episodic-like memory was tested in the novel object recognition test (NORT) test. Only 3 groups showed recognition of the novel object (intact memory): LF sham males, LF VCID males, and LF sham females. All other groups showed no recognition of the novel object (D). A-B) *p<0.05 for diet effect within same sex, **p<0.01 for diet within the same sex, ****p<0.0001 for diet within same sex. D) ⁺⁺p<0.01 vs. chance (50%), ⁺⁺⁺p<0.001 vs. chance (50%), ⁺⁺⁺⁺p<0.0001 vs. chance (50%). Red line = chance (50% of time). Data are presented as mean +/− SEM for A, C, D (N=13-19/group) and median +/− interquartile range for B (N=8-14/group).

Figure 6.. Spatial memory is impaired by VCID in both sexes, and HF diet in females only.

At the end of the study spatial memory was assessed via the Morris water maze (MWM). During the training trials, the males (A) and females (B) improved performance across both visible and hidden trials (3 way repeated measures ANOVA, main effect of trial). Representative heat maps are shown for the probe trial, with target quadrant in lower right (C). During the probe trial, LF sham and HF sham males and females and LF VCID females showed a preference for the target quadrant, while all other groups showed no preference, indicating a memory impairment (D). In females, the HF VCID mice also showed a lower preference than LF sham females. ⁺p<0.05 vs. chance, ⁺⁺p<0.01 vs. chance, ⁺⁺⁺p<0.001 vs. chance, ⁺⁺⁺⁺p<0.0001 vs. chance,*p<0.05 LF sham female vs. LF VCID female, **p<0.01 LF sham female vs. HF VCID female. Red line = chance (25% of time). Data are presented as mean +/− SEM (N=13-19/group).

Figure 7.. Sex-Specific Correlations Between Metabolic and Cognitive Measures.

Using a correlation matrix, we compared the relationship between percent of body weight gained during the study (Weight; N=70/sex), visceral fat pad weight as a percentage of body weight (% Vis fat; N=51-52/sex), visceral fat TNFalpha levels (TNFɑ; N= 18-19 sex), the area under the curve for the glucose tolerance test performed after 6 months on the diet (GTT; N= 69-70/sex), percent time spent with the novel object during the novel object recognition test (NORT; N=66/sex), and the amount of time spent in the target quadrant region during the probe trial of the Morris water maze (MWM; N=63-68/sex). Pearson r values are presented. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Green = positive correlation, blue = negative correlation.