Sex differences in the effects of high fat diet on underlying neuropathology in a mouse model of VCID

Abstract

Background: Damage to the cerebral vasculature can lead to vascular contributions to cognitive impairment and dementia (VCID). A reduction in blood flow to the brain leads to neuropathology, including neuroinflammation and white matter lesions that are a hallmark of VCID. Mid-life metabolic disease (obesity, prediabetes, or diabetes) is a risk factor for VCID which may be sex-dependent (female bias).

Methods: We compared the effects of mid-life metabolic disease between males and females in a chronic cerebral hypoperfusion mouse model of VCID. C57BL/6J mice were fed a control or high fat (HF) diet starting at ~ 8.5 months of age. Three months after diet initiation, sham or unilateral carotid artery occlusion surgery (VCID model) was performed. Three months later, mice underwent behavior testing and brains were collected to assess pathology.

Results: We have previously shown that in this VCID model, HF diet causes greater metabolic impairment and a wider array of cognitive deficits in females compared to males. Here, we report on sex differences in the underlying neuropathology, specifically white matter changes and neuroinflammation in several areas of the brain. White matter was negatively impacted by VCID in males and HF diet in females, with greater metabolic impairment correlating with less myelin markers in females only. High fat diet led to an increase in microglia activation in males but not in females. Further, HF diet led to a decrease in proinflammatory cytokines and pro-resolving mediator mRNA expression in females but not males.

Conclusions: The current study adds to our understanding of sex differences in underlying neuropathology of VCID in the presence of a common risk factor (obesity/prediabetes). This information is crucial for the development of effective, sex-specific therapeutic interventions for VCID.

Keywords: Diet-induced obesity; High fat diet; Neuroinflammation; Prediabetes; Sex; Vascular contributions to cognitive impairment and dementia; White matter.

Fig. 1

Experimental design. Experimental timeline. Ctrl control diet (10% fat), HF high fat diet (60% fat), VCID vascular contributions to cognitive impairment and dementia, RT-qPCR real time quantitative polymerase chain reaction, IF immunofluorescent labeling. *Results of the behavioral testing can be found in Salinero et al. [30]

Fig. 2

White matter changes are driven by VCID in males and HF diet in females. Representative images of myelin basic protein (MBP) labeling (top panel) and CC1 labeling (mid panel) are shown in A for the corpus callosum (CC) and F for the hippocampus. In the merged image shown in the bottom panel, MBP is shown in magenta, CC1 in cyan and DAPI in blue. Scale bar 100 µm. Labeling density quantification in the CC (B, C) and the hippocampus (G, H). Data are represented as mean + SEM. 2-way ANOVA with Tukey’s post hoc test ^#p < 0.05, ^##p < 0.01 effect of VCID; *p < 0.05, **p < 0.01 effect of diet. 3-way ANOVA results are reported under the graph *p < 0.05. Correlations of glucose intolerance (GTT-AUC) with MBP (D, I) and CC1 (E, J) densities were analyzed using simple linear regression for males and females combined and for each sex separately with r² and p values noted for each. n = 3–6 mice/group. Ctrl control diet, HFD high fat diet

Fig. 3

High fat diet induces microglia activation in males but suppresses it in females. Microglia were labeled using Iba1 (cyan) and CD68 (magenta). Cells were classified into 4 groups depending on morphology and CD68 immunoreactivity: ramified cells with or without CD68 and ameboid cells with or without CD68 (A, i–iv). Scale bar 20 µm. The % area occupied by the Iba1 labeling was quantified in the CC (B) and the hippocampus (H). Cell densities were quantified in the corpus callosum (C–E) and the CA1 region of the hippocampus (I–K). Data are represented as mean + SEM. 2-way ANOVA with Tukey’s post hoc test. ^#p < 0.05 effect of VCID; *p < 0.05, **p < 0.01 effect of diet. 3-way ANOVA results are reported under the graph *p < 0.05, **p < 0.01, ***p < 0.001. Correlation of Iba1+ ameboid cell density with body weight change expressed as % of body weight at the start of the experiment in the CC (F) and in the CA1 of the hippocampus (L). Correlation of ameboid Iba1+CD68+ cell density with the novel object recognition index (NORI) as a measure of episodic-like memory in the CC (G) and in the CA1 of the hippocampus (M). Correlation analyzed using simple linear regression for each sex with r² and p values noted for each. *p < 0.05, ***p < 0.001, n = 4–6 mice/group. Ctrl  control diet, HFD high fat diet

Fig. 4

Diet effects on inflammation-related gene changes in the hippocampus are sex-specific. Relative expression of mRNA of Iba1 (A), inflammatory cytokines IL-1β (B) and TNF-α (C), anti-inflammatory factor Arg1 (D) and pro-resolution factors: AnxA1 (E) and its receptor FPR2 (F). mRNA levels were quantified using the ΔΔCq method and reported as fold change relative to the males on a Ctrl diet with a sham surgery. Data are represented as mean + SEM. 2-way ANOVA with Tukey’s post hoc test *p < 0.05 effect of diet. 3-way ANOVA results are reported under the graph *p < 0.05, **p < 0.01. Correlation of IL-1β expression with the % time spent in the target quadrant during the probe trial of the Morris water maze as a readout for spatial memory (G). Correlation analyzed using simple linear regression for each sex with r² and p values noted for each. n = 4–8 mice/group. Ctrl control diet, HFD high fat diet