Real-time monitoring of superoxide accumulation and antioxidant activity in a brain slice model using an electrochemical cytochrome c biosensor.

The overproduction of reactive oxygen species and the resulting damage are central to the pathology of many diseases. The study of the temporal and spatial accumulation of reactive oxygen species has been limited because of the lack of specific probes and techniques capable of continuous measurement. We demonstrate the use of a miniaturized electrochemical cytochrome c (Cyt c) biosensor for real-time measurements and quantitative assessment of superoxide production and inactivation by natural and engineered antioxidants in acutely prepared brain slices from mice. Under control conditions, superoxide radicals produced from the hippocampal region of the brain in 400-μm-thick sections were well within the range of detection of the electrode. Exposure of the slices to ischemic conditions increased the superoxide production twofold and measurements from the slices were stable over a 3- to 4-h period. The stilbene derivative and anion channel inhibitor 4,4'-diisothiocyano-2,2'-disulfonic stilbene markedly reduced the extracellular superoxide signal under control conditions, suggesting that a transmembrane flux of superoxide into the extracellular space may occur as part of normal redox signaling. The specificity of the electrode for superoxide released by cells in the hippocampus was verified by the exogenous addition of superoxide dismutase (SOD), which decreased the superoxide signal in a dose-dependent manner. Similar results were seen with the addition of the SOD mimetic cerium oxide nanoparticles (nanoceria), in that the superoxide anion radical scavenging activity of nanoceria with an average diameter of 15 nm was equivalent to 527 U of SOD for each 1 μg/ml of nanoceria added. This study demonstrates the potential of electrochemical biosensors for studying real-time dynamics of reactive oxygen species in a biological model and the utility of these measurements in defining the relative contribution of superoxide to oxidative injury.