We developed a double microdialysis fiber technique to generate hydroxyl radicals (OH) in rat spinal cord. H2O2 and FeCl2/EDTA were pumped through two parallel microdialysis fibers inserted into the spinal cord such that the reactants mix in the tissue to generate OH by the Fenton reaction. Generated OH was detected by administering phenylalanine through one fiber and measuring o-,m- and p-hydroxyphenylalanine in collected, dialysates by high pressure liquid chromatography and fluorescence detection. The hydroxyphenylalanines are produced by OH attacking the phenylalanine. OH generation was also accomplished in in vitro experiments and the results were consistent with in vivo experiments. This novel method to generate and measure OH radical in vivo overcomes difficulties in studying damage to tissue by short-lived OH. Although developed to study the role of OH in spinal cord injury, this method could be used to study other diseases involving OH damage.
- Fluorescence detection
- High pressure liquid chromatography
- Hydroxyl radical generation
- Microdialysisl Fenton reaction
- Phenylalanine hydroxylation
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