Resveratrol treatment rescues neurovascular coupling in aged mice: role of improved cerebromicrovascular endothelial function and downregulation of NADPH oxidase

Abstract

Moment-to-moment adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling is essential for the maintenance of normal neuronal function. Increased oxidative stress that occurs with aging was shown to impair neurovascular coupling, which likely contributes to a significant age-related decline in higher cortical function, increasing the risk for vascular cognitive impairment. Resveratrol is a polyphenolic compound that exerts significant antiaging protective effects in large vessels, but its effects on the cerebromicrovasculature remain poorly defined. The present study was undertaken to investigate the capacity of resveratrol to improve neurovascular coupling in aging. In aged (24-mo-old) C57BL/6 mice N(ω)-nitro-l-arginine methyl ester-sensitive, nitric oxide-mediated CBF responses to whisker stimulation and to the endothelium-dependent dilator acethylcholine (ACh) were impaired compared with those in young (3-mo-old) mice. Treatment of aged mice with resveratrol rescued neurovascular coupling and ACh-induced responses, which was associated with downregulation of cortical expression of NADPH oxidase and decreased levels of biomarkers of oxidative/nitrative stress (3-nitrotyrosine, 8-isoprostanes). Resveratrol also attenuated age-related increases in reactive oxygen species (ROS) production in cultured cerebromicrovascular endothelial cells (DCF fluorescence, flow cytometry). In conclusion, treatment with resveratrol rescues cortical neurovascular coupling responses to increased neuronal activity in aged mice, likely by restoring cerebromicrovascular endothelial function via downregulation of NADPH oxidase-derived ROS production. Beneficial cerebromicrovascular effects of resveratrol may contribute to its protective effects on cognitive function in aging.

Keywords: ROS; microvascular dysfunction; oxidative stress; reactive hyperemia; senescence; vascular cognitive impairment.

Fig. 1.

Resveratrol (RSV) treatment rescues neurovascular coupling 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 (1 min, 10 Hz) in young (3 mo old), aged (24 mo old), and resveratrol-treated aged mice[0.1 arbitrary units (AU) corresponds to ∼5% increase in CBF from baseline]. B: summary data of the effect of resveratrol treatment (200 mg/kg, 10 days in food) on CBF responses to whisker stimulation and on baseline CBF (C) in young and aged mice. Data are means ± SE (n = 8 in each group). *P < 0.05 vs. young; ^#P < 0.05 vs. aged.

Fig. 2.

Resveratrol treatment rescues nitric oxide (NO) mediation of neurovascular coupling in aged mice. A: changes of CBF in response to whisker-stimulation in the absence and presence of the NO synthase inhibitor N^ω-nitro-l-arginine methyl ester (l-NAME) above the barrel cortex in control and resveratrol-treated young (3 mo old) and aged (24 mo old) mice. B: l-NAME-sensitive component of CBF responses to whisker stimulation. Data are means ± SE (n = 7–8 in each group). *P < 0.05 vs. young; ^#P < 0.05 vs. aged; ^&P < 0.05 vs. aged + resveratrol.

Fig. 3.

Resveratrol treatment rescues acetylcholine-induced, endothelial NO-mediated CBF responses in aged mice. Changes of CBF are shown in response to the endothelium-dependent dilator acethylcholine (A) and the endothelium-independent dilator adenosine (C) in the absence and presence of the NO synthase inhibitor l-NAME above the barrel cortex in control and resveratrol-treated young (3 mo old) and aged (24 mo old) mice. B: l-NAME-sensitive component of acetylcholine-induced CBF responses. Data are means ± SE (n = 7–8 in each group). *P < 0.05 vs. young; ^#P < 0.05 vs. aged; ^&P < 0.05 vs. aged + resveratrol.

Fig. 4.

Resveratrol treatment attenuates oxidative stress in aged mice. A: administration of the NADPH oxidase inhibitor apocynin improves CBF responses elicited by whisker-stimulation in aged (24 mo old) mice. CBF responses in young (3 mo old) mice are shown for comparison. Data are means ± SE (n = 6 in each group) *P < 0.05 vs. young; ^#P < 0.05 vs. aged control. B: cortical 3-nitrotyrosine content and 8-isoprostane level (C) in young, resveratrol-treated young, aged, and resveratrol-treated aged mice (n = 6 in each group). Data are means ± SE. *P < 0.05 vs. young control; ^#P < 0.05 vs. aged control.

Fig. 5.

Resveratrol decreases oxidative stress in cultured cerebromicrovascular endothelial cells and astrocytes. A: representative figures showing flow cytometric analysis of DCF fluorescence [indicating reactive oxygen species (ROS) production] in primary cerebromicrovascular endothelial cells (CMVECs) derived from young and aged F344xBN rats. Shown is the effect of resveratrol treatment (10 μmol/l, for 24 h) on ROS production by aged CMVECs. Appropriate control (unstained cells) is also shown. B: resveratrol (10 μmol/l, for 24 h) attenuates increased ROS production in aged CMVECs. Data are means ± SE (n = 8 in each group). *P < 0.05 vs. young control; ^#P < 0.05 vs. aged control. C: resveratrol (10 μmol/l, for 24 h) attenuates increased ROS production in activated astrocytes. Primary astrocytes in culture were stimulated by treatment with 10 ng/ml TNF-α, 10 ng/ml IL-1β, and 10 ng/ml IL-6 (for 4 h) with and without resveratrol pretreatment (10 μmol/l, for 24 h). Cellular ROS production was measured using the DCF fluorescence method by flow cytometry. Data are means ± SE (n = 8 in each group). *P < 0.05 vs. control; ^#P < 0.05 vs. stimulated cells (no resveratrol).

Fig. 6.

Resveratrol treatment results in downregulation of NADPH oxidase expression in the aged mouse brain. Effect of resveratrol treatment (200 mg/kg po, for 10 days) on mRNA expression of NADPH oxidase subunits Nox1 (A), Nox2 (B), Nox4 (C), and Ncf1 (p47^phox; D) in the cerebral cortex of young and aged mice. Data are means ± SE (n = 6 in each group). *P < 0.05 vs. young control; ^#P < 0.05 vs. aged control.

Fig. 7.

Resveratrol downregulates NADPH oxidase expression in aged CMVECs in culture. Shown are the effects of resveratrol treatment (10 μmol/l, for 24 h) on mRNA expression of NADPH oxidase subunits Nox2 (A) and Nox4 (B) in aged CMVECs. Data are means ± SE (n = 6 in each group). *P < 0.05 vs. young CMVECs; ^#P < 0.05 vs. untreated aged CMVECs.