We have focused on the role of oxidative mechanisms in ischemia/reperfusion induced brain damage in the Mongolian gerbil model. We have found that: A) In vivo salicylate hydroxylation is increased implicating an increased flux of hydroxyl free radicals, B) In vivo spin-trapping techniques show formation of spin-trapped free radicals, C) Certain spin-traps (PBN, alpha- phenyl-t-butyl nitrone) administered to gerbils offer protection, D) Lesioned brain tissue homogenate is more prone to peroxidation and E) Proteins are oxidatively damaged in lesioned brain. Other related leads we have recently made indicate: A) Oxidative damage to RNA and DNA in peroxidized brain, B) Mediated loss of enzymes which may be responsible for the build up of the excitatory toxic amino acid glutamate in lesioned brain, C) An enhanced expression of specific genes in lesioned brains, and D) Older gerbils have an increased susceptibility to ischemia/reperfusion injury. Focus will be directed toward investigating the mechanism of action of spin-traps in the context of rigorously assessing brain regional localization of oxidative damage to nuclei acids and proteins and on the time-course and the nature of the free radicals produced in the ischemia/reperfusion insult (IRI)-lesioned gerbil brain. We will use the Mongolian gerbil brain ischemia/reperfusion model and: A) Determine using spin-trapping techniques combined with HLPC and mass spectroscopy the nature of the spin-trapped free radicals produced in lesioned brain. A novel approach of utilizing a combination of alpha-carbon isotopes of the spin-trap PBN (alpha-12CPBN, alpha-13CPBN) administered at specific time points in the development of the IRI-induced injury will be used to determine the time-course of the free radicals produced. B) Determine if oxygen free radical damage to RNA and DNA, namely the presence of increased levels of 8-hydroxyguanosine (8-OHG) and 8-hydroxydeoxyguanosine (8-OHdG), respectively, are formed in lesioned brains. C) Determine the extent of oxidative damage to proteins and characterize the regional localization and time-course of oxidative damage. D) 31P-NMR in vivo spectroscopy will be used to standardize the extent of cortical ischemia for subsequent determination of oxidative damage to brain tissue.
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