Alcoholics suffer from neuroticdeneration and loss of CNS function, which could occur due to hinge drinking. Studies in vivo using a 4-day animal binge model found neurodegeneration in corticolimbic areas, i.e. Binge-Induced Brain Damage (BIBD). BIBD is characterized by significant neuronal damage, long-lasting expression of markers of inflammation, and changes in behavior. Although in vitro studies have found glutamate-NMDA excitotoxicity following chronic ethanol treatment, BIBD is not blocked by glutamate-NMDA antagonists, hut BIBD is blocked by antioxidants. This proposal will investigate the role of oxidative stress in BIBD and how it alters signaling pathways involved in neuronal survival. We will also determine the contribution of multiple ethanol exposures and ethanol-induced plasma toxins to alcoholic neurodegeneration as both of these pathological states can lead to oxidative stress. Ethanol-induced plasma toxins may contribute to oxidative stress and multiple ethanol exposures may lead to cumulative stress prompting experiments to determine the contributions of these factors to alcoholic neurodegeneration. The following hypotheses will be tested.
Aim 1. BIBD is related to oxidative stress. This hypothesis will be tested by measuring nitro-tyrosine, 4-hydroxynenenol and 8-hydroxyguanosine, indicators of protein, lipid and nucleic acid oxidation respectively. In addition, genes related to oxidative stress, e.g., NADPH oxidase, cytochrome P(450)2E1,TNFalpha and NFkappaB, will be studied as potential components of BIBD. Oxidative stress and TNFalpha can activate NFkappaB, which is known to induce COX-2, iNOS and MnSOD expression. We will also investigate prosurvival signaling molecules such as MAPK/ERK and CREB, which provide resistance to neurotoxic events such as oxidative stress. Preliminary studies comparing the P and NP rat strains indicate that ethanol increases MAPK/ERK activation, atrophic signaling pathway that causes prosurvival gene induction, likely as a protective response to ethanol-induced oxidative stress. MAPK/ERK activation is inversely related to BIBD, that is, P rats have greater BIBD and reduced MAPK/ERK activation compared to NP rats. Thus, a second component of this aim will be to investigate aspects of P-MAPK/ERK and P-CREB. The antioxidant butylated hydroxytoluene (BHT) pre-vents BIBD and these studies will be extended using other antioxidants. The effect of antioxidants on induction of oxidative stress markers, enzymes, and the signaling cascades mentioned above will also be determined using immunohistochemistry, EMSA, western and blot analyses. Experiments using transgenic mice strains including NFkappaB reporter gene mice, TNFalpha knockouts, CYP2E1 knockouts and PKC knockouts will test the role of these specific genes in B1BD.
Aim 2. Peripheral tissue damage is involved in BIBD. Our 4-day binge elevates plasma ammonia and enzyme levels. Further, intragastric models of liver fibrosis induce CNS inflammation. Thus, models that are known to induce hepatotoxicity will be investigated for brain gene activation and neuropathology as described above.
Aim 3. Repeated binge exposure will increase BIBD. Clinical studies have shown that multiple withdrawals lead to a higher incidence of seizures and correlates with memory and learning impairments. Thus, the effects of multiple episodes of alcohol exposure on BIBD, gene induction and oxidative stress will be determined. These in vivo studies will greatly increase our understanding of the factors that cause neurodegeneration in alcoholics.
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