Download Effects of creatine and green tea extract on blood

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.