Lydia Cortes, University of Miami Dr. Damien Pearse, Dr. Fenghan Yu and Daniella Garcia, Miller School of Medicine Effect of cAMP signaling in ethanol mediated protection following injury Introduction: •Ethanol improves motor function in injured animals •cAMP increases axon growth in cell implants and improve function of the cells •PKA is released from the regulatory subunit, phosphorylates nearby proteins, then translocates to the nucleus, where it regulates gene expression Objective • Prove if ethanol’s protective capacity works against oxidative stress present in injured organisms or if it has to do with another pathogenesis of the injury •Find the concentration and duration necessary for the ethanol treatment to be functional. •Prove if treatment with ethanol increases cAMP signaling and prevents cell apoptosis. Hypothesis : ethanol Increases cAMP Increases PKA Ethanol and hydroge n peroxid e neither hydroge n peroxid e only 24 hours ethanol and hydrogen peroxide Three hours ethanol and hydrogen peroxide Half an hour hydrogen peroxide Three hours hydrogen peroxide Twenty four hours hydrogen peroxide Methods: •Tissue obtaining •Cell Culture Cell culture was prepared according to a method derived from Chen Y et al. •Adding ethanol and hydrogen peroxide Added 1 micromolar concentration of hydrogen peroxide to each slide. Then added 15 % or 5% ethanol. •Staining of slides PI and Hoechst •Western Blot Analysis PKA and PCREB Results: Fig2. cAMP signaling changes Fig 1. Cell death assay Fig3. Cells stained with PI and Hoechst Hoechst staining of 5% ethanol PI staining of 5% ethanol PI staining of 15% ethanol for twenty four hours for twenty four hours for twenty four hours Hoechst staining of 15% ethanol for twenty four hours Conclusions: A functional treatment to cure paralysis in injured organisms is the combination of 5% ethanol for twenty four hours with another pharmacological agent. This is because, according to our results, administration of 5% ethanol to cells presenting oxidative stress increases PKA and CREB production, and proved to create a low percentage of cell death. Treatment with 15% ethanol causes cell death and low production of PKA and CREB. PKA U ntreated H 2O 2 3 hours H 2O 2 24 hours 5% ethanol3 hours 5% ethanol24 hours 15% ethanol3 hours 15% ethanol24 hours 0 5 10 15 fold change CREB U ntreated H 2O 2 3hours H 2O 2 24 hours 5% ethanol3 hours 5% ethanol24 hours 15% ethanol3 hours 15% ethanol24 hours 0 1 2 3 4 5 6 7 8 9 10 11 12 fold change
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Lydia Cortes, University of MiamiDr. Damien Pearse, Dr. Fenghan Yu and Daniella Garcia, Miller School of Medicine
Effect of cAMP signaling in ethanol mediated protection following injury
Introduction:
•Ethanol improves motor function in injured animals
•cAMP increases axon growth in cell implants and improve function of the cells
•PKA is released from the regulatory subunit, phosphorylates nearby proteins, then translocates to the nucleus, where it regulates gene expression
Objective• Prove if ethanol’s protective capacity works against oxidative stress present in injured organisms or if it has to do with another pathogenesis of the injury •Find the concentration and duration necessary for the ethanol treatment to be functional. •Prove if treatment with ethanol increases cAMP signaling and prevents cell apoptosis.
Hypothesis
:
ethanol
Increases cAMP
Increases PKA
Ethanol and
hydrogen peroxide
neitherhydrogen peroxide
only
24 hours ethanol and hydrogen peroxide
Three hours ethanol and hydrogen peroxide
Half an hour
hydrogen peroxide
Three hours
hydrogen peroxide
Twenty four hours
hydrogen peroxide
Methods:
•Tissue obtaining
•Cell CultureCell culture was prepared according to a method derived from Chen Y et al.
•Adding ethanol and hydrogen peroxideAdded 1 micromolar concentration of hydrogen peroxide to each slide. Then added 15 % or 5% ethanol.
•Staining of slides PI and Hoechst
•Western Blot Analysis PKA and PCREB
Results: Fig2. cAMP signaling changes Fig 1. Cell death assay
Fig3. Cells stained with PI and Hoechst
Hoechst staining of 5% ethanol PI staining of 5% ethanol PI staining of 15% ethanol for twenty four hours for twenty four hours for twenty four hours
Hoechst staining of 15% ethanol for twenty four hours
Conclusions: A functional treatment to cure paralysis in injured organisms is the combination of
5% ethanol for twenty four hours with another pharmacological agent. This is because,
according to our results, administration of 5% ethanol to cells presenting oxidative
stress increases PKA and CREB production, and proved to create a low percentage of cell
death. Treatment with 15% ethanol causes cell death and low production of PKA and
CREB.
PKA
Un
trea
ted
H2O
2 3
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urs
H2O
2 24
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5% e
than
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5% e
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rs
15%
eth
ano
l 3 h
ou
rs
15%
eth
ano
l 24
ho
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0
5
10
15
fold
ch
ang
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CREB
Un
trea
ted
H2O
2 3h
ou
rs
H2O
2 24
ho
urs
5% e
than
ol 3
ho
urs
5% e
than
ol 2
4 h
ou
rs
15%
eth
ano
l 3 h
ou
rs
15%
eth
ano
l 24
ho
urs
0123456789
101112
fold
ch
ang
e
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