In vivo fluorometric measurement of cerebral oxidative stress using 2'-7'-dichlorofluorescein (DCF)

The time course of oxidative stress following cerebral ischemia and reperfusion has been obscure, although oxygen-derived free radicals have been postulated to play an important role in the progression of reperfusion injury. We have examined the time profile of hydrogen peroxide (H2O2) generation in the rat cortex following incomplete forebrain ischemia and reperfusion. We used 20 male Sprague-Dawley rats anesthetized with alpha-chloralose and urethane. A closed cranial window was created in the temporoparietal skull, and 2'-7'-dichlorofluorescein (DCF), a sensitive fluorescent probe for H2O2, was loaded intracellularly by the superfusion technique. We simultaneously monitored DCF fluorescence and reflectance from the cortex with an in vivo fluoromicroscope having two photomultiplier tubes, and subtracted the hemodynamic artifact from DCF fluorescence. Incomplete forebrain ischemia was induced by temporal ligation of both common carotid arteries combined with hypotension. The results showed that corrected DCF fluorescence remained unchanged during ischemia, but increased following reperfusion, indicating enhanced H2O2 generation. Pretreatment by intraperitoneal injection of catalase attenuated H2O2 generation significantly. that H2O2 generation is mainly enhanced following reperfusion.