Diets enriched in foods with high antioxidant activity reverse age-induced decreases in cerebellar beta-adrenergic function and increases in proinflammatory cytokines

Abstract

Antioxidants and diets supplemented with foods high in oxygen radical absorbance capacity (ORAC) reverse age-related decreases in cerebellar beta-adrenergic receptor function. We examined whether this effect was related to the antioxidant capacity of the food supplement and whether an antioxidant-rich diet reduced the levels of proinflammatory cytokines in the cerebellum. Aged male Fischer 344 rats were given apple (5 mg dry weight), spirulina (5 mg), or cucumber (5 mg) either in 0.5 ml water by oral gavage or supplied in the rat chow daily for 14 d. Electrophysiologic techniques revealed a significant decrease in beta-adrenergic receptor function in aged control rats. Spirulina reversed this effect. Apple (a food with intermediate ORAC) had an intermediate effect on cerebellar beta-adrenergic receptor physiology, and cucumber (low ORAC) had no effect, indicating that the reversal of beta-adrenergic receptor function decreases might be related to the ORAC dose. The mRNA of the proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha) and TNFbeta was also examined. RNase protection assays revealed increased levels of these cytokines in the aged cerebellum. Spirulina and apple significantly downregulated this age-related increase in proinflammatory cytokines, whereas cucumber had no effect, suggesting that one mechanism by which these diets work is by modulation of an age-related increase in inflammatory responses. Malondialdehyde (MDA) was measured as a marker of oxidative damage. Apple and spirulina but not cucumber decreased MDA levels in the aged rats. In summary, the improved beta-adrenergic receptor function in aged rats induced by diets rich in antioxidants is related to the ORAC dose, and these diets reduce proinflammatory cytokine levels.

Fig. 1.

Peri-event time histograms showing the response to iontophoretically applied GABA onto single cerebellar Purkinje neurons from an aged rat on the control diet (left) or an aged rat fed a spirulina-enriched diet for 2 weeks (right). The top panels (A, D) show baseline responses to a 5 sec application of GABA. The middle panels (B, E) show responses to GABA during concurrent application of ISO. The bottom panels(C, F) represent recovery after termination of the ISO. Histograms represent averages of four drug responses. Thex-axis represents time in seconds and they-axis represents action potentials per 0.5 sec time bin.

Fig. 2.

The ability of ISO to augment GABAergic inhibition of cerebellar Purkinje neurons was examined in young rats (n = 7 rats, 42 neurons for gavage;n = 7 rats, 57 neurons for control diet) and aged rats fed apple (n = 6 rats, 38 neurons, fed via oral gavage), spirulina (n = 6 rats, 38 neurons, fed via oral gavage), vehicle (n = 5 rats, 40 neurons, fed via gavage; n = 6 rats, 33 neurons, normal diet), or cucumber (n = 6 rats, 35 neurons, fed via diet supplement) for 2 weeks. There were no differences between young or aged controls on the diet versus gavage treatment; therefore the samples were pooled. The ability of ISO to augment GABAergic inhibition of Purkinje cell spontaneous firing was the dependent measure. The spirulina diet significantly reversed the age-induced decrease in the β-adrenergic receptor response (p < 0.05; Fisher’s exact test). The apple diet had an intermediate effect (not significantly different from either control or spirulina), and the cucumber diet did not produce any change.

Fig. 3.

Representative image of a 5% denaturing polyacrylamide gel obtained by a phosphorimager on which double-stranded RNase-protected fragments were resolved. Total mRNA was extracted from the cerebellum of rats aged 4 and 20 months. The template consisted of radiolabeled antisense RNA probes for IL-1α (protected probe size, 403 nt), IL-1β (361 nt), TNFβ (322 nt), IL-3 (286 nt), IL-4 (256 nt), IL-5 (226 nt), IL-6 (202 nt), IL-10 (181 nt), TNFα (160 nt), IL-2 (142 nt), and IFNγ (129 nt).Tem, Template.

Fig. 4.

Age-related mRNA expression of TNFα and TNFβ in cerebellar tissue of male Fischer 344 rats. TNFα and TNFβ mRNA expression was significantly increased in cerebellar tissues dissected from aged rats (20 months; n = 6) compared with young rats (4 months; n = 6) (one-way ANOVA: TNFα, F = 49, p < 0.0001; TNFβ, F = 152, p < 0.0001). An internal standard control gene, ribosomal protein L32, was included in the template, and the OD of each band for cytokine mRNA was normalized relative to the OD of the L32 band by the following expression: (OD of the cytokine mRNA band/OD of the L32) × 100.

Fig. 5.

The age-related increase in TNFα and TNFβ mRNA expression in rat cerebellar tissue is prevented by diets rich in antioxidants. The TNFα and TNFβ mRNA expression was significantly decreased in cerebellar tissue obtained from aged rats (18 months) fed for 2 weeks with either spirulina (n = 6) (0.33% w/w dry; spirulina vs aged controls; p < 0.005 andp < 0.001 for TNFα and TNFβ, respectively) or apple (n = 6) (5 mg/d dry weight; apple vs aged controls; p < 0.005 and p < 0.001 for TNFα and TNFβ, respectively) compared with aged rats fed on control diet. The cucumber diet did not produce any significant change in TNFα and TNFβ.

Fig. 6.

MDA values are increased in control aged rats (n = 6) compared with young rats (n = 6), and apple (n = 6) and spirulina (n = 6) diets bring those values back toward those observed in young rats (p < 0.01; one-way ANOVA). Lipid peroxidation by MDA was measured by a TBA reaction where the MDA–TBA adduct end-product was chromatographically separated. A single MDA–TBA peak is detected by spectrophotometry with absorbance at 532 nm and emission at 553 nm. Values are expressed in picomoles of MDA per milligram wet weight of tissue.