Alpha-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in
ischemia-reperfusion injury of brain by forming adducts of
oxygen free radicals including .
OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of
oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of
methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked
cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent alpha-(4-pyridyl-1-oxide)-N-tert butylnitrone (
POBN 100 mM) for 20 min. In addition 50 mg/kg BW of
POBN was administered intraperitoneally (
IP) 20 min prior to
ischemia in order to improve our ability to detect
free radical adducts. Cup fluid was subsequently replaced every 15 min during
ischemia and every 10 min during reperfusion with fresh
POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection,
cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. .
OH radical adducts (characterized by six line EPR spectra) were detected during
ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-
acetic acid (100 microM;
DETAPAC) a
chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress .
OH radical adduct formation during
ischemia as well as reperfusion. PBN
free radical adducts were detected in EPR spectra of the
lipid extracts of PBN treated rat brains subjected to
ischemia/reperfusion. Thus this study suggests that PBN's protective action in
cerebral ischemia/
reperfusion injury is related to its ability to prevent a cascade of
free radical generation by forming spin adducts.