Aging exacerbates obesity-induced cerebromicrovascular rarefaction, neurovascular uncoupling, and cognitive decline in mice

Abstract

Epidemiological studies show that obesity has deleterious effects on the brain and cognitive function in the elderly population. However, the specific mechanisms through which aging and obesity interact to promote cognitive decline remain unclear. To test the hypothesis that aging exacerbates obesity-induced cerebromicrovascular impairment, we compared young (7 months) and aged (24 months) high-fat diet-fed obese C57BL/6 mice. We found that aging exacerbates the obesity-induced decline in microvascular density both in the hippocampus and in the cortex. The extent of hippocampal microvascular rarefaction and the extent of impairment of hippocampal-dependent cognitive function positively correlate. Aging exacerbates obesity-induced loss of pericyte coverage on cerebral microvessels and alters hippocampal angiogenic gene expression signature, which likely contributes to microvascular rarefaction. Aging also exacerbates obesity-induced oxidative stress and induction of NADPH oxidase and impairs cerebral blood flow responses to whisker stimulation. Collectively, obesity exerts deleterious cerebrovascular effects in aged mice, promoting cerebromicrovascular rarefaction and neurovascular uncoupling. The morphological and functional impairment of the cerebral microvasculature in association with increased blood-brain barrier disruption and neuroinflammation (Tucsek Z, Toth P, Sosnowsk D, et al. Obesity in aging exacerbates blood-brain barrier disruption, neuroinflammation and oxidative stress in the mouse hippocampus: effects on expression of genes involved in beta-amyloid generation and Alzheimer's disease. J Gerontol Biol Med Sci. 2013. In press, PMID: 24269929) likely contribute to obesity-induced cognitive decline in aging.

Keywords: Endothelial dysfunction; Learning and memory.; MCI; Vascular cognitive impairment.

Figure 1.

Obesity-related changes in hippocampal function in aging. (A) Body mass of young and aged mice fed a high-fat diet (HFD) or standard diet (SD) at the time of sacrifice. Data are means ± SEM. *p < .05 vs young mice (SD), ^# p < .05 vs aged mice (SD), ^$ p < .05 vs young mice (HFD). (B) Aging exacerbates obesity-induced cognitive impairment (elevated plus maze-based learning protocol; see Methods section for details). For obese aged mice, transfer latency was similar on Days 1 and 2 (corresponding to a learning index: ~0), indicating that these mice had significantly impaired learning ability. Data are given as mean ± SEM. *p < .05 vs young mice (SD), ^# p < .05 vs aged mice (SD), ^$ p < .05 vs young mice (HFD).

Figure 2.

Obesity-induced changes in cerebral capillary density in aging. (A–D) Representative confocal images showing CD31 positive capillary endothelial cells (red) in the hippocampi of young and aged mice fed an HFD or SD. Hoechst 33342 was used for nuclear counterstaining (original magnification: ×5). (E) Representative confocal microscopic analysis of CD31 positive capillaries (green) in the CA1 region of the hippocampus of an obese aged mouse (original magnification: ×20). (F and G) Summary data for obesity-induced changes of capillary length density in the CA1 region of the hippocampus (F), retrosplenial cortex and corpus callosum (G) of young and aged mice. Data are given as mean ± SEM. *p < .05 vs young mice (SD), ^# p < .05 vs aged mice (SD), ^$ p < .05 vs young mice (HFD). (H) Relationship between capillary length density in the CA1 region of the hippocampus of young and aged mice fed an HFD or SD and the learning index obtained in these group of animals.

Figure 3.

Obesity-induced changes in pericyte coverage of hippocampal capillaries in aging. (A) Representative confocal image showing perivascular localization of a platelet-derived growth factor receptor β (PDGFRβ) expressing pericyte (red) surrounding CD31 positive capillary endothelial cells (green) in the CA1 region of the mouse hippocampus. Hoechst 33342 was used for nuclear counterstaining. (B–E) Representative confocal microscopy analysis of PDGFRβ expressing pericyte coverage (red) of CD31 positive capillaries (green) in the CA1 region of the hippocampi of young SD-fed (B), young HFD-fed (C), aged SD-fed (D) and aged HFD-fed (E) animals. (F) Summary data showing obesity- and aging-dependent loss of pericyte coverage in the hippocampus (see Methods section for details). *p < .05 vs young mice (SD), ^# p < .05 vs aged mice (SD), ^$ p < .05 vs young mice (HFD).

Figure 4.

Panel A: Mean t statistic for positive and negative angiogenesis regulators and the signed aggregate of both groups. Panel B: Gene set enrichment analysis (GSEA) scores for positive and negative regulators of angiogenesis and the signed aggregate of both groups. HFD appears to increase the angiogenesis signature, age and the age–diet interaction appear to have mixed effects, whereas the age–diet interaction shows a trend toward decreased angiogenic signature. “Age Effect” refers to old vs young, “Diet Effect” refers to HFD vs control, and “Age–Diet Interaction” refers to the changes in gene expression that occur with the combination of age and HFD that cannot be accounted for by age or diet alone.

Figure 5.

Effect of factors present in the circulation of young and aged obese and lean mice on endothelial angiogenic capacity. Treatment with sera collected from young mice fed an HFD does not affect formation of capillary-like structures by cerebromicrovascular endothelial cells (CMVECs). In contrast, treatment with sera collected from aged mice fed an SD or an HFD significantly increases endothelial angiogenic capacity. CMVECs were plated on Geltrex-coated wells, and tube formation was induced by treating cells with vascular endothelial growth factor (100ng/mL, for 24h). Representative examples of capillary-like structures are shown on Panels A–D. Summary data, expressed as total tube length per total area scanned (µm tube/mm²), are shown in Panel E. Data are given as means ± SEM (n = 5 in each group). *p < .05 vs control.

Figure 6.

Obesity-induced neurovascular uncoupling and upregulation of NADPH oxidase in aged mice. (A) Representative traces of cerebral blood flow (CBF) measured with a laser Doppler probe above the whisker barrel cortex during contralateral whisker stimulation (1min, 10 Hz) in young and aged mice fed an HFD or SD (0.1 AU corresponds to ~5% increase in CBF from baseline). Panel B depicts the summary data of the effect of HFD-induced obesity on CBF responses to whisker stimulation in young and aged mice. Data are given as mean ± SEM (n = 8 in each group). *p < .05 vs young mice (SD), ^# p < .05 vs aged mice (SD), ^$ p < .05 vs young mice (HFD). (C) Summary data of the effect of apocynin on CBF responses to whisker stimulation in HFD-fed young and aged mice. p < .05 vs young mice (HFD), ^$ p < .05 vs aged mice (HFD). (D) Summary data showing flow cytometric analysis of dihydroethidium fluorescence (indicating reactive oxygen species production) in primary CMVECs treated with sera collected from young and aged mice fed an SD or an HFD. Data are given as means ± SEM (n = 6 in each group). *p < .05 vs young mice (SD), ^# p < .05 vs aged mice (SD), ^$ p < .05 vs young mice (HFD). (E) Effects of obesity on mRNA expression of NADPH oxidase subunit Nox2 in the hippocampi and cerebral cortex of young and aged mice fed an SD or an HFD. Data are given as mean ± SEM (n = 5 in each group). *p < .05 vs young mice (SD), ^# p < .05 vs aged mice (SD), ^$ p < .05 vs young mice (HFD).