Effect of cAMP signaling in ethanol mediated oligoprotection following injury

Lydia E. Cortes BetancourtUniversity of MiamiLeadership Alliance

Introduction•Ethanol improves motor function in injured animals

•cAMP increases axon growth in cell implants and improve function of the cells•cAMP signaling cAMP/PKA/CREB•PKA is a tetrameric protein with two subunits.•Research has proven that etoh increases cAMP and increases PKA

ethanol

Increases cAMP

Increases PKA

Purpose

• 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 and

hydrogen peroxide

neitherhydrogen peroxide

only

Methods

Injury Thoracic spinal cord contusion In T8

Tissue obtaining Spinal cord and brain’s cells were

obtained from rats

Cell Culture Method derived from Chen Y et al

•Cleaned under the dissection microscope

•Brain and spinal cord’s cells were diced to 1.5 mm3 chunks

•Put the cells in collagenase/dispase enzyme mix for two hours

•Triturate the cells with the pipette

•Run them in the strainer

•Spin cells in the centrifuge

•Red Blood cell wash

•Let sit in ice for 10 minutes

•Spin cells and add 20 ml DMEM20S

Staining To see if the cells we are interested in

grow

Fix •4% paraformaldehyde- 15 min•Wash with DPBS

Permeabilizing•Add 0.1% Triton -100 in PBS•Leave for no more than 10 minutes•Wash two times with DPBS

Blocking•10%NGS in DPBS (normal goat serum)•For one hour•One wash

1ry antibody•GFAP monoclonal (1:1000)•RIP polyclonal (1:200)•overnight

2ry antibody

•GAM 594•GAR 488 •Leave one hour•Clean with DPBS

Adding ethanol and hydrogen peroxide

•Added 1 micromolar H2O2 for half an hour

•Put Ethanol 5% or 15% for three hours or twenty four hours

•Take cells from the dish

•Centrifuge

•Take out supernatant

•Resuspend with lysis buffer

•Keep product for analysis in western blot

Western Blot Analysis

The gel for Western Blot was prepared

Proteins were transferred to nitrocellulose membranes and probed using antiPKA and antiCREB.

Each tissue was incubated with the antibody, washed and then exposed to autoradiographic film.

Staining of slides to see apoptosis

•Treated the cells with PI for 10 min

•Repeated the staining procedure, but with the secondary Hoechst

Results

Cell death assay

cAMP signaling changes

PKA

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CREB

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Conclusion

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.

References

Baud O., Greene A., Li J., Wang H. , Volpe J, and Rosenberg P. (2004) Glutathione Peroxidase-Catalase Cooperativity Is Required for Resistance to Hydrogen Peroxide by Mature Rat Oligodendrocytes. The Journal of Neuroscience. 24(7). 1531-1540.

Belayev L, Khoutorova L, Zhang Y, Belayev A, Zhao W, Busto R and Ginsberg M. (2004) Caffeinol confers cortical but not subcortical neuroprotection after transient focal cerebral ischemia in rats. Brain Research. 1008. 278-283.

Bracken MB. (2001) Methylprednisolone and acute spinal cord injury: an update of the randomized evidence. Spine. 26 (24). 47-54.

Chen Y, Balasubramaniyan V, Tallquist M, Li J and Richard Lu Q. (2007) Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nature Protocols 2. 1044 – 1051.

Chiarugi P. (2003) Reactive oxygen species as mediators of cell adhesion. Ital J Biochem. 52. 28–32.

Dohrman D, Diamond I and Gordon A. (1996) Ethanol causes translocation of cAMP- dependent protein kinase catalytic subunit to the nucleus. The Proceedings of the National Academy of Sciences Online (U.S.). 93. 10217- 10221.

Halliwell B, Gutteridge JMC (1999) Antioxidant defence enzymes: the glutathione peroxidase family. In: Free radicals in biology and medicine, Ed 3, pp 140-146, 170-172. Oxford, UK: Oxford UP.

Haynes RL, Folkerth RD, Keefe RJ, Sung I, Swzeda LI, Rosenberg PA, Volpe JJ, Kinney HC (2003) Nitrosative and oxidative injury to premyelinating OLs in periventricular leukomalacia. J Neuropathol Exp Neurol .62. 441-450

Hoek JB and Pastorino JG. (2002) Ethanol, oxidative stress, and cytokine-induced liver cell injury. Alcohol. 27 (1). 63-8

Pearse D, Pereira F, Marcillo A, Bates M, Berrocal Y, Filbin M and Bartlett M. (2004) cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Nature Medicine. 1-7

Qun W, Sun A, Simonyi A, Kalogeris T, Miller D, Sun G and Korthuis R. (2007) Ethanol preconditioning protects against ischemia/reperfusion-induced brain damage: Role of NADPH oxidase-derived ROS. Free Radic Biol Med. 43 (7). 1048- 1060.

Ryu H., Lee J.,Impey S., Ratan R., and Ferrante R. (2005) Antioxidants modulate mitochondrial PKA and increase CREB binding to D-loop DNA of the mitochondrial genome in neurons. Proc Natl Acad Sci U S A. 102(39). 13915–13920.

Wang L, Renault G., Garreau H and Jacquet M. (2004) Stress induces depletion of Cdc25p and decreases the cAMP producing capability in Saccharomyces cerevisiae. Microbiology. 150. 3383-3391.

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