The long term objective of this project is to reveal mechanisms whereby free radicals damage neurons following spinal cord injury - a major health care issue. The initial trauma is worsened by secondary destructive processes, including the release of toxic substances into the extracellular space by the injured cells. Better characterization of the mechanisms whereby secondary damage agents cause neuronal degeneration should pave the way to improving pharmacological methods of disrupting the secondary damage cascade. The PI is funded by NIH (R29) to study the correlation between free radical production and membrane lipid oxidation. The present project is a complementary study, to test the hypothesis that oxidation of major cell components is the final pathway whereby free radicals cause neuronal degeneration and death in spinal cord injury.
The specific aims are: 1. To verify that oxidative damage to proteins and DNA is associated with spinal cord injury. The time courses of production of 8- hydroxy-2-deoxyguanosine (8-OHdG)- a marker of DNA oxidation, protein carbonyl content (PCC)- an indicator of protein oxidation, and nitrotyrosine (NTyr)- an indicator of protein nitration, will be determined, protein degradation examined by Western blots. Apoptosis will be examined by in situ immunohistochemical staining, enzyme-linked immuno-sorbent assay (ELISA) for quantitation of DNA fragmentation, and laddering of DNA fragments by electrophoresis. Neuronal death will be quantitated by counting neurons by specific immunohistochemical staining of neurofilament protein in spinal cord tissue following impact spinal cord injury. 2. To apply free radical suppressors (superoxide dismutase and glutathione peroxidase), a nitric oxide synthase inhibitor (nitro-L-arginine), and an iron chelator (desferrioxamine) into the intrathecal space of the rat spinal cord by a microdialysis or a microcannula loop. The time course of hydrogen peroxide and hydroxyl radical in the extracellular space will be measured by microdialysis sampling; 8-OHdG, PCC and NTyr are measured, protein degradation examined, apoptosis characterized, and neuron death quantitated as in Aim 1 in spinal cord tissue following impact injury. This will further correlate free radical production to the oxidative neuronal damage. 3. To test whether free radicals at the levels induced by trauma cause oxidation of protein and DNA and trigger apoptotic neuron death in the rat spinal cord. The free radicals will be generated in the spinal cord at the levels measured upon trauma in the PI's previous studies by applying precursors and enzymes through the microcannula or microdialysis loop implanted in the intrathecal space of the spinal cord. 8-OHdG, PCC and NTyr will be measured, protein degradation examined, apoptosis characterized, and neuron death quantitated in spinal cord tissue as in Aim 1. This will directly test whether free radicals damage neurons by inducing oxidation of major cell components and will provide strategies for therapy.
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