Research Component 4 - Crews Molecular Pathology of Ethanol Induced Brain Damage. Our recentstudies indicate that neuroinflammation contributes to alcoholic neurodegeneration. Using an in vivo modelof binge induced brain damage we found that corticolimbic damage has genetic and adolescentsusceptibility, is associated with proinflammatory gene induction and causes long lasting perseverativechanges disrupting relearning behavior. Both in vivo binge and in vitro cultured brain slice studies suggestthat ethanol-induced neurotoxicity involves activation of the oxidation sensitive transcription factor NFicB, aswell as cytokine and oxidative enzyme induction. The anti-oxidant BHT blocks ethanol neurotoxicity andNFicB-DNA binding. These studies lead to the overall hypothesis that ethanol induces long lasting changesin CNS cvtokines and other components of neuroinflammation that contribute to oxidative stress.neurodegeneration and alcoholic dysfunction. Our discoveries indicate that systemic cytokines induce braincytokines and neurotoxicity prompting experiments on the effects of ethanol on induction of brain, liver andsystemic cytokines and oxidative enzymes. The TLR agonist, LPS, is potentiated by ethanol in brain, liverand serum. To understand the role of systemic cytokines in neurotoxicity, mice will be treated for varioustimes with ethanol alone and in combination with cytokine inducing TLR agonists. Multiple cytokines,oxidative enzymes and NFicB transcription factors will be investigated. Gene expression and protein will bedetermined in brain as well as serum and liver (mRNA by RTPCR, protein by ELISA). Brains will besectioned and neurotoxicity related to brain region and cell specific gene induction. To investigatemechanisms of ethanol induction of cytokines and other gene transcription, neurotoxicity and oxidative stresswill be studied in brain slice cultures. In slice culture ethanol increases NFicB-DNA binding and synthesis ofcytokines and oxidative enzymes. To explore the direct actions of ethanol and cytokines on brain, slices willbe treated with ethanol and/or cytokines to follow oxidative stress, NFicB-DNA binding (EMSA, reportermice), gene induction (mRNA-RTPCR), protein (ELISA-histochemistry) as well as neurotoxicity andneurogenesis. Mechanisms will be determined using time courses and dose response curves in combinationwith siRNA, antibodies, and transgenic animals. These studies will contribute to our understanding ofalcoholic pathology.
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