Location: Boston, Massachusetts2011 Annual Report
1a. Objectives (from AD-416)
To determine the dose and duration effects of the wild blueberry powder (WBP) on age-related differences in resistance to oxidative stress/inflammation (OS/INF) by exposing young and old animals to lipopolysaccharide (LPS). Brain regional differences in stress signaling (e.g,, nuclear factor kappa B, Nf'B), cytokine activation (e.g., TNFa), and OX-6, a marker of microglial activation, as well as differences in microglial morphology, will be assessed. Tissue levels of the polyphenols will be assessed following the various doses and durations of WBP to examine bioavailability.
1b. Approach (from AD-416)
AIM 1 - The effects of the age-related differences in resistance to inflammation will be assessed by supplementing (for 2, 4, or 8, weeks) young (4 mo) and old (19 mo) animals with a control diet or a diet containing the equivalent of 0, ½, or 1.5, 4.5 cups/day (in human terms) of WBP. One half of each group of animals will then be administered a vehicle (saline) or LPS (5 mg/kg i.p.) just before they euthanized and stress (e.g., Nf'B, cytokines) and protective (ERK) signals assessed (Figure 1). We will also determine levels of OX-6, a marker of microglial activation, as well as differences in microglial morphology. A subset of animals will be euthanized at each time-point for assessment of stress signaling and tissue levels of the polyphenols. AIM 2 - We will also compare and correlate the degree of alterations in the various signals to the rats’ behavioral (e.g., radial arm water maze, and various motor tests e.g., rotorod) performance. Additionally, using bromodeoxyuridine (BrdU) to identify areas of the dentate gyrus showing increased DNA incorporation (an indicator of cell proliferation) and neuronal (Ngn2) and glial (GFAP) markers, we will determine differences in neurogenesis among the various groups. Dr. Kalt will measure tissue levels of polyphenols to assess bioavailability of the various WBP doses.
3. Progress Report
Previously, we demonstrated that blueberry (BB) can attenuate declines in cognitive and motor behavior in aging. However, it is not known if different berries act synergistically when incorporated into a diet or if they have individual effects in the same or different brain regions. This year we elucidated this by examining the effects of diets supplemented with 2% BB, 2% strawberry (SB), or 2% BB + 2% SB on age-related cognitive and motor dysfunction. Compared to young rats, aged control-diet fed rats displayed impaired cognitive performance in a water maze that was indicative of impaired function of different memory areas of the brain. However, perhaps due to the difficulty of the task, which may have obscured any behavioral enhancements, the aged, berry-supplemented rats were not improved compared to control rats. Analyses are currently underway to measure levels of inflammatory cytokines and activation of microglia in different brain areas. If the different polyphenolic compounds in these berry fruits are acting in different brain regions, then it becomes important to eat a variety of fruits and vegetables to maintain cognitive function in old age. We have also previously determined that some of the major effects of wild and cultivated BB application to cells may be to alter oxidative stress/inflammatory signaling in brain cells, possibly mediating some of the beneficial effects of BB on age-related cognitive decline and loss of cell function. This year, we examined various treatment durations and doses for the BB and SB, individually and in combination, on inflammatory markers in cell models to elucidate individual vs. synergistic effects. The data indicate longer and higher doses of the berries were more effective in reducing inflammation. Under most treatment conditions, the BB was more effective than the SB treatment. Additionally, the combination of BB+SB (at full strength) was not more effective than the BB alone. . Therefore, it may be that the individual polyphenols in the different berries might exert their effects through different and/or independent mechanisms. We also conducted a study to examine the active components in BB that may be responsible for the protection provided by BB in cell models of aging. Stilbenes, the most well known of which is resveratrol, are a group of compounds in BB that may be responsible for their beneficial effects, possibly by decreasing inflammation and oxidative stress. One of these stilbenes, pterostilbene, reversed the deleterious effects of aging on cognitive performance in aged rats similar to what was shown in blueberries. We also determined that pterostilbene was effective in preventing stress-induced calcium imbalance in cells. We are currently assessing if pterostilbene, resveratrol, and two anthocyanins from blueberries, delphinidin and malvidin, are efficacious in protecting microglia from inflammatory-induced stress signaling. We are also examining the protective effects of the stilbene and anthocyanin compounds on hippocampal cell calcium buffering in comparison to whole BB extract.