In vivo activation of N-methyl-D-aspartate receptors generates free radicals and reduces antioxidant ability in the rat hippocampus: experimental protocol of in vivo ESR spectroscopy and microdialysis for redox status evaluation.
The pathological mechanisms of various CNS diseases are closely related to glutamate neuronal excitotoxicity following NMDA receptor activation. To verify this relationship, in vivo microdialysis in the hippocampus of rats was applied to ESR spectroscopy during NMDA perfusion. Microdialysis co-perfusion of 0.1 mM NMDA dissolved in 150 mM POBN for 60 min revealed six-line carbon-centered radical ESR spectra. The hfc values were aN=15.7 G and aHbeta=2.5 G, corresponding to the values produced from the generation of lipid radicals. The antioxidant activity during the freely moving state was examined utilizing the principle that systemically applied nitroxide radicals are reduced and lose their paramagnetism by antioxidant activity in the brain. ESR analysis of sequential changes in the signal amplitude of nitroxide radicals in both the NMDA group and the control group revealed an exponential decay. The half-life of the nitroxide radical was significantly longer in the NMDA group than in the control group. The homeostasis of a steady redox balance was destroyed by acute NMDA infusion, which resulted in the generation of lipid radicals and the reduction of antioxidant ability in the hippocampus. The redox imbalance induced by the activation of NMDA-R was recovered by the inhibition of PLA2 and NOS. These results indicated that NMDA-R activation caused the shift to oxidized condition of the redox state, which subsequently leads to neuron death in the hippocampus in the model of glutamate-associated neuronal disease.
Ueda
Yuto
Y
Section of Psychiatry, Department of Clinical Neuroscience, Faculty of Medicine, University of Miyazaki 5200 Kihara, Kiyotake, Miyazaki, 889-1692, Japan. usan@med.miyazaki-u.ac.jp
Doi
Taku
T
Nagatomo
Keiko
K
Nakajima
Akira
A
D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism*; D018698:Glutamic Acid / Q000633:toxicity; D006207:Half-Life; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism*; D050356:Lipid Metabolism / Q000187:drug effects; D008297:Male; D017551:Microdialysis; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists*
D000975:Antioxidants; D005609:Free Radicals; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid