2008 Annual Report
1a.Objectives (from AD-416)
1: Determine the effects of SB supplementation on behavioral aging using paradigms sensitive to cognitive (short and long-term memories) behaviors.
2: Determine the effects of SB supplementation on calcium dependent and neuronal signaling (2) and neurogenesis (2b) correlate these with alterations in behavioral parameters determined in SA 1.
3: Determine whether the efficacies of these supplementations in the behavioral assays are associated with enhanced resistance to oxidative stress or inflammation.
1b.Approach (from AD-416)
Aim 1. The effects of SB dietary supplementation (2% SB extract in the diet for 8 weeks. After 8 weeks performance will be examined in the rats using age-sensitive cognitive behaviors that are selective for reference and working memories. The latter behaviors will be assessed using the Morris water maze and the radial arm water maze, as well as novelty tests. All of these tests have been validated as being age sensitive (e.g. demonstrated a significant decline as a function of age).
Aim 2. The effects of SB dietary supplementation on neurogenesis and differentiation will be examined using immunocytochemistry, bromodeoxyuridine (BrDU) incorporation in hippocampus and olfactory bulb obtained from the supplemented behaviorally-assessed animals Calcium signaling will be assessed by examining Ca45 clearance in tissues (as above) taken from the brain.
Aim 3. For the basal assessments we will use immunohistochemistry to assess various markers of oxidative stress (e.g., HO-1) and inflammation (e.g., cytokines) as well as immunoblotting to detect HO-1 and bcl 2 expression. The responses of the tissue (muscarinic receptor sensitivity, HSP-70 activation) to oxidant (hydrogen peroxide, 10 uM) or inflammatory (LPS) stressors will be assessed by exposing cross-cut slices of the various brain regions obtained from the supplemented animals.
Previously, we have shown that whole, crude berry extracts are able to reverse several parameters of brain aging, as well as age-related motor and cognitive deficits when fed to rats from 19-21 months of age. These effects may be the result of direct effects on brain signaling or indirect effects through antioxidant and anti-inflammatory properties of the polyphenols. Two different berryfruit diets (blueberry, BB and strawberry, SB) were employed to determine whether the effects observed are indeed the result of differential effects of the polyphenols on the brain. Old (19 mo) F344 rats were fed a control, 2% BB, or 2% SB diet for 8 weeks prior to motor and cognitive testing. Results showed that SB-fed rats had improved performance compared to the BB-fed rats on the large plank. BB rats were better than SB rats on rod walking, while both diets improved motor function on the rotarod compared to control. Both berryfruit diets enhanced working memory in the Morris water maze. We are currently assessing regional localization of the BB and SB polyphenols and their putative differential effects on signaling parameters in these rats, with a view toward determining selective bioavailability and mechanism(s) of action. This year we quantified cells in the dentate gyrus (an area of the hippocampus important for memory control) expressing the protein doublecortin, a marker for neurons that are newly generated from the supplemented rats. We have shown that the number of proliferating precursor cells and surviving cells in the dentate gyrus of the SB group to be higher than the control group, with the BB group in between these two groups. In double-labeling experiments, it was confirmed that the bromodeoxyuridine (BrdU)-positive cells did not co-localize with the glial fibrillary acidic protein (GFAP)-positive cells, showing that new neuronal cells and not glial cells were increased. We are now counting more slides per rat to reduce the variability. This work is aligned with NP 107 – Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Models.