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Abstract
Formation of excess free radical causes cellular oxidative stress, which has been shown to be associated with a variety of pathologic conditions. While electron spin resonance (ESR) spectroscopy has been the only method to demonstrate the presence of free radicals, its application to tissue samples has been challenging. We report here the successful ESR detection in thin-sliced fresh tissues or frozen sections in a rat model. Ferric nitrilotriacetate (Fe-NTA) induces oxidative renal tubular damage that ultimately leads to high incidence of renal carcinoma in rodents. Twenty minutes after administration of 5 mg iron/kg Fe-NTA to rats, a thin-slice of the kidney was mounted on a tissue-type cell and analyzed by ESR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An ESR signal from alkylperoxyl radical adduct was obtained, and the signal was inversely proportional to renal α-tocopherol content which was modulated through diet. Furthermore, we undertook ex vivo study using frozen sections. Fe-NTA (1 mM) was added to a rat kidney frozen section for 10 min. After washing the specimen was mounted on a tissue-type cell and analyzed with ESR spin trapping using DMPO. Alkylperoxyl radical signal was dependent on thickness, incubation time and renal tissue levels of α-tocopherol, and was reduced by preincubation with catalase or dimethyl sulfoxide but not with α-tocopherol outside tissue. This versatile method facilitates identification of free radicals in pathologic conditions, and may be useful for selection of antioxidants.