Abstracts

RATIONALE: Injury to hippocampal structures during epileptogenesis is associated with the excitotoxic effects of glutamate especially through activation of NMDA receptors (NMDA-R). Such NMDA-R activation results in phospholipase A2 (PLA2) mediated generation of superoxide (O2-). Both enzymatic dismutation and spontaneous disproportion rapidly convert superoxide into hydrogen peroxide (H2O2). Although H2O2 is an important reactive oxygen species related to initiation of lipid peroxidation, there is no direct evidence that NMDA-R activation actually mediates H2O2 generation.
METHODS: After placement of a hippocampal probe in rats designed to allow in vivo microdialysis, hydrogen peroxide was generated by co-perfusion of 10 mM NMDA for 10 minutes. Electron spin resonance (ESR) spectroscopy was used to measure formation of stable nitroxide radicals in the perfusate. Spin traps included p-acetamidophenol (p-AP) and 4-hydrazonomethyl-1-hydroxy-2,2,5,5,-tetramethyl-3-imidazoline-3-0xide (HHTIO). To confirm whether H2O2 generation was derived from PLA2 activation, an experimental group was given 250 mM quinacrine (QA,PLA2 inhibitor).
RESULTS: During perfusion with 10 mM NMDA measurement of the appearance of stable nitroxide radicals with ESR showed a rapid increase in the generation of H2O2. Following QA co-perfusion NMDA failed to induce the appearance of H2O2 in the perfusate. Lipid peroxidation is initiated by hydrogen abstraction from polyunsaturated lipid acids by hydroxyl radicals that are formed by the transformation of H2O2 and O2- and catalyzed by transition metals. Neural injury during seizures generates lipid peroxidation products. This study of in vivo changes demonstrated that NMDA-R activation induces H2O2 generation. Further PLA2 activation appears to be an important pathway involved in seizure-related neurotoxicity through lipid peroxidation.
CONCLUSIONS: Kindling induced epileptogeneisis depends upon changes modurated through NMDA receptors. While calcium-mediated neurotoxic effects involve glutamate effects on NMDA-R, we have shown that in freely moving rats that NMDA-mediated phospholipase A2 activation will generate H2O2, leading ultimately to lipid peroxidative injury as well.
[Supported by: This study was supported by a Grant-in-Aid for Encouragement of Young Scientists (12770537) from the Ministry of Education, Science, Sport and Culture, Japan (to Y.U.).]