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Anna Rizza and Rahel Meisels Effects of creatine and green tea extract on bloodinduced damages in retinal and neuronal cells. Supervision: Prof. Dr. Hans Rudolf Widmer and PD Dr. Volker Enzmann Tutors: Dr. Stefano Di Santo and Stephanie Lötscher, Cluster for Regenerative Neurosciences, DKF & Inselspital Bern Introduction Intracerebral hemorrhage (ICH) is a subtype of stroke which is characterized by a high morbidity and mortality. Age-related macular degeneration (AMD) is the leading cause of blindness in the industrialized world. In the wet form of the disease, leaky blood vessel lead to retinal cell damage. So far, no effective treatment option is available for these devastating disease states. In the present study we investigated the protective potential of creatine and green tea extract administration on blood-damaged neuronal and retinal cells. Methods Required substances Creatine: This is a natural substance and primarily found in red meat. It is synthesized from three different amino acids: Arginine, glycine and methionine. Many sportsmen eat it as a food supplement to build up muscles. It was, however, also shown that creatine Anna Rizza and Rahel Meisels supports survival of neurons. In our study, we tried to prove that creatine has a positive influence on cells exposed to blood-induced damage. Green tea extract (Epigallocatechin gallate, EGCG): This natural substance is found in many vegetal products such as green, white and black tea, various vegetables, red wine and nuts. In our study, we tried to prove that EGCG has a positive influence on cells exposed to blood-induced damage. Blood: Cells are damaged by blood if the latter gets in direct contact with them, mainly because of the iron in its erythrocytes. Blood reaches the brain/retinal cells if a blood vessel breaks. Cells: We performed the research using mouse cell lines: o 661W retinal cells (cones) o C17.2 neuronal stem cells Procedures C17.2 neural stem cells and 661W retinal cells were grown in culture dishes containing medium in a humified atmosphere of 5% CO2 at 37°C. Photograph of the incubator. With the help of a microscope the number of cells per volume was counted. This way, we did know the approximate number of cells we put into wells. Like that, we were able to follow the development (growth, viability and proliferation) of those cells. Blood was collected in heparin-coated tubes under sterile conditions. The blood was sonicated to lysate the erythrocytes and the blood lysate sterile-filtered. Cells Anna Rizza and Rahel Meisels were exposed to the blood lysates in concentrations of 2.5, 5, 20, and 50 %. Cell cultures were exposed to the blood lysates in absence or presence of creatine [10 mM] or green tea extracts (EGCG) [5 µM for C17.2 cells; 50 µM for 661W cells]. Cultures without blood lysates served as controls. We also prepared some control wells that were treated with NaIO3, a substance that is toxic to the cells. Eventually, three different methods of measurement were applied to indicate the development of our samples. Measurement XTT: As this substance is added to living cells, it becomes orange due to cell activity. Therefore, the number of cells is directly proportional to the intensity of the orange color. So, we can find out the ratios between dead and living cells in each sample. PrestoBlue: PrestoBlue functions in a way very similar to XTT. However, here the blue color turns red and it works outside of the cell. Therefore, the cells are not killed as with the XTT method. Immunocytochemistry: Cells are treated with paraformaldehyde in order to fix the protein structures. Then, primary and secondary antibodies are added. The primary antibodies attach to the cell surface and the secondary ones attach to the primary antibodies. Since we used secondary antibodies with an attached fluorochrome labeled cells become fluorescent after excitation under the microscope. C17.2 cells were immunocytochemically stained for the neuronal marker β-III-tubulin and the glial marker GFAP. Fixed 661W cells were stained for the cone marker G-alpha transducin 2 (GαT2). Afterwards, pictures were taken using an epifluorescence microscope equipped with a digital Anna Rizza and Rahel Meisels camera. Thus, we could see different parts of the cells in different colors, depending on which antibodies were bound to a particular part of the cell. Results Phase contrast C17.2 Phase contrast 661W Anna Rizza and Rahel Meisels Cell viability in presence of creatine (XTT)................................. …………… 661W Cell number (% rel to Blood 0%) 120.0 No Crea + Crea [10 mM] 100.0 80.0 60.0 40.0 20.0 0.0 Blood 0% Blood 2.5% Blood 5% Blood 20% Blood 50% Positive Ctr (6 mM NaIO3) Cell viability in presence of blood lysate and creatine (PrestoBlue) C17.2 Cell number (% rel to Blood 0% no creatine) 140.0 No crea +crea [10 mM] 120.0 100.0 80.0 60.0 40.0 20.0 0.0 Blood 0% Blood 2.5% Blood 5% Blood 20% Blood 50% Positive Ctr (6 mM NaIO3) Anna Rizza and Rahel Meisels Cell viability in presence of blood lysate (PrestoBlue) 661W Cell number (% rel to blood 0%) 120 100 80 60 40 20 0 Blood 0% Blood 2.5% Blood 5% Blood 20% Blood 50% Cell viability in presence of blood lysate and EGCG (PrestoBlue) Cell number (% rel to Blood 0% no EGCG) C17.2 160.0 No EGCG 140.0 +EGCG [5 uM] 120.0 100.0 80.0 60.0 40.0 20.0 0.0 Blood 0% Blood 2.5% Blood 5% Blood 20% Blood 50% Positive Ctr (6 mM NaIO3) Anna Rizza and Rahel Meisels Schematic summary of the effect of creatine or EGCG on cell viability Creatine EGCG 661W C17.2 XTT PrestoBlue Toxic effect of blood only Little blood toxicity even at highest concentration at high concentrations Negative effect of creatine Inconsistent effect of at the highest blood creatine concentration blood only PrestoBlue (Odd) increase of cell PrestoBlue viability by blood Blood toxicity increasing Thus, no cyto-protective with concentration effect of EGCG ICC 661W: DAPI, GαT2 ICC C17.2: DAPI, GFAP, β-III-tubulin Anna Rizza and Rahel Meisels Conclusions and discussion • In these experiments blood lysate did show toxicity on retinal and neuronal cells at high concentrations only. • Green tea extract and creatine appeare to have a negative effect on challenged retinal and neuronal cell viability. However, these results need to be repeated, since the study was based on a very limited number of replicates. Furthermore, a lot of our cells died. Many of them dried out while we were changing the medium as we did not refill the wells quickly enough. Finally, we cannot rule out that our cells have been infected by bacteria because they were not treated in a sterile way. Acknowledgments Even though our results did not match our expectations, we have made many interesting new experiences and gotten to know many lab procedures. We would like to thank Swiss Youth in Science for the wonderful opportunity of participating in this research week. To the members lab we are thankful for their patience and willingness to share their knowledge and passion with us.