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- Home - Abstract - Introduction - Materials - Methods - Detailed Methods - Results - Calculations - Discussion and Relevance - Conclusion - References - Acknowledgements - Project Info |
Neurodegenerative diseases are caused by cell death (either necrosis or apoptosis), when the number of cells which are dying either increases due to disease, physical insult or tissue assault, or the human aging processes. The brain requires large amounts of Oxygen, and evidence now indicates that the levels oxidative stress, caused by free radicals as byproducts of chemical reactions in the mitochondria, increase as humans age. Polyphenolic compounds have been known to decrease levels of oxidative stress, resulting in increased cell apoptosis. Blueberries, bilberries, and other such vegetation are known carriers of polyphenolic compounds, and as such are perfect organic materials for treatment and prevention of brain diseases caused by oxidative stress. To test the neuroprotective (anti oxidative) properties, of blueberry extract, several experiments were conducted, as outlined within this website. Originally, PC12 cells were selected as the in vetro test cells. However, PC12 cells were unavailable, and Shy-Y cells were used instead. First, ShyY cells underwent the zapoglobin assay, so the amount of hydrogen peroxide required for 50% cell death could be accurately calculated. They were plated from stock cells grown in media (MEM, HS, FBS, P/S). Cells plated at 10^5 cells/ml in dishes or wells coated with collagen substrate were incubated in fresh media with 100ng/ml 7S NGF for 6 (six) days. The cells were pre-counted, and resuspended with 200 ul zapoglobin cell lysis solution (HBSS). Intact nuclei were counted with a hemocytometer and microscope. Next, cells underwent the same process, but Hoechst stain was used, and living cells were counted. It was determined that 500ul at 15 minutes hydrogen peroxide exposure caused 50% cell death. Finally, the Roche MTT Cell proliferation assay was instigated. Different combinations of hydrogen peroxide, blueberry extract and control were used at 15 minute and 30 minute intervals. Ratios of dead VS living cells were calculated using a spectrophotometer. It was found that 0.1% blueberry extract not only provided neuroprotection to cells after hydrogen peroxide exposure but also seemed to cause a significant survival in the number of neurites per cell, and that, most importantly, wells containing blueberry extract had less cell death than those with hydrogen peroxide, or even exclusively control. |
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Copyright 2004 Gabriel Rigg, Michael
Armson, David Dickinson. |
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