2008 Annual Report
1a.Objectives (from AD-416)
Aim 1: Continue to determine the effects of BB supplementation on behavioral aging using paradigms sensitive to: motor (probes sensitive to balance, strength, coordination) and cognitive (short- and long-term memories) behaviors.
Aim 2: Determine the effects of BB supplementation on signaling and neurogenesis and correlate these with alterations in behavioral parameters determined in SA1.
Aim 3: Determine the mechanisms involved in the anti-inflammatory/antioxidant activity of BB in cells.
1b.Approach (from AD-416)
After 8 weeks supplementation of 2% (of the blueberry diet), performance will be examined in the rats using age-sensitive motor (e.g., accelerating rotarod) and cognitive (Morris water maze) behaviors. The effects of BB dietary supplementation on neurogenesis proliferation and differentiation will be examined using immunocytochemistry, i.e., bromodeoxyuridine (BrDU) incorporation in hippocampus obtained from the supplemented behaviorally-assessed animals. We will also assess the effects of BB on kainic acid and lipopolysaccharide (LPS) treated BV-2 microglial cells and COS-7 cells will be examined by assessing cell death and calcium clearance. Recently, we have found that several markers of inflammation, including the cytokine, Il-1 beta, the transcription factor, NfkappaB, are both suppressed when the BV-2 cells are pre-treated with BB before being exposed to LPS or kainic acid. We will continue these experiments under the present proposal to determine if the antioxidant/anti-inflammatory properties of BB involve reductions in inflammatory/oxidant signals. We will use imaging, Western blot, and gene array with real-time PCR analyses for these experiments.
Previously we assessed three different BB-derived diets, all equated on phenolic levels, to determine whether the effects observed with the whole, crude BB extract are due to polyphenolics or if the other compounds were contributing to the age-related improvements in behavior. Old (19 mo) F344 rats were fed a control diet or one with 5.4% crude BB extract (as before), a 2% pre-C18 column BB extract, or a 0.1% post-C18 column semi-purified BB extract (a mixture of only BB phenolics with the sugars and organic acids removed) for 8 weeks prior to motor and cognitive testing. Results showed that only the crude BB extract diet improved rotarod performance, while all three BB-derived diets improved working memory in the Morris water maze. In work recently completed, we carried out analyses of new neuronal growth (neurogenesis) in these groups by using cell counts of bromodeoxyuridine (BrdU) incorporation in dentate gyrus of the hippocampus. This is an important memory control area that shows considerable decline in function, as well as reductions in neurogenesis in aging. The results of these analyses indicated that there was a trend for the animals fed the crude BB extract to show increases in BrdU incorporation into the dentate gyrus, while the animals fed the pre or post C-18 BB extract diets showed significantly more neurogenesis than either the control or crude BB extract fed diets. Therefore, one mechanism involved in this enhanced increased in cognitive performance may be related to increased neurogenesis by BB polyphenols. These experiments were continued this year with analyses of the putative alterations in various neuronal signals induced by the BB-derived diets. We assessed changes in cyclic AMP response element binding protein (CREB), protein kinase C gamma (PKC gamma), and PKCa which are activated by oxidative stress and extracellular signal regulated kinase-1 (ERK) which is protective against oxidative stress. The results indicated that CREB was reduced, while mixed effects were seen with respect to the other signals. This year we have been utilizing neuronal and glial markers to assess neurogenesis in the dentate gyrus of these animals (as we have described for the strawberry project, Projects #51000-063-10T and 17T) using doublecortin to stain neurons and GFAP to stain glia. Additional details can be found under Project Nos: 51000-063-10T and 51000-063-17T, and for the parent CRIS 1950-51000-063-00D Nutritional Modulation of Brain Aging and Cognitive Decline.
This work is aligned with NP 107 – Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Models.