Use of H2O2 to Cause Oxidative Stress, the Catalase Issue

Addition of hydrogen peroxide (H2O2) is a method commonly used to trigger cellular oxidative stress. However, the doses used (often hundreds of micromolar) are disproportionally high with regard to physiological oxygen concentration (low micromolar). In this study using polarographic measurement of oxygen concentration in cellular suspensions we show that H2O2 addition results in O2 release as expected from catalase reaction. This reaction is fast enough to, within seconds, decrease drastically H2O2 concentration and to annihilate it within a few minutes. Firstly, this is likely to explain why recording of oxidative damage requires the high concentrations found in the literature. Secondly, it illustrates the potency of intracellular antioxidant (H2O2) defense. Thirdly, it complicates the interpretation of experiments as subsequent observations might result from high/transient H2O2 exposure and/or from the diverse possible consequences of the O2 release.

Oxygen and seizure dynamics: I. Experiments

We utilized a novel ratiometric nanoquantum dot fluorescence resonance energy transfer (NQD-FRET) optical sensor to quantitatively measure oxygen dynamics from single cell microdomains during hypoxic episodes as well as during 4-aminopyridine (4-AP)-induced spontaneous seizure-like events in rat hippocampal slices. Coupling oxygen sensing with electrical recordings, we found the greatest reduction in the O2 concentration ([O2]) in the densely packed cell body stratum (st.) pyramidale layer of the CA1 and differential layer-specific O2 dynamics between the st. pyramidale and st. oriens layers. These hypoxic decrements occurred up to several seconds before seizure onset could be electrically measured extracellularly. Without 4-AP, we quantified a narrow range of [O2], similar to the endogenous hypoxia found before epileptiform activity, which permits a quiescent network to enter into a seizure-like state. We demonstrated layer-specific patterns of O2 utilization accompanying layer-specific neuronal interplay in seizure. None of the oxygen overshoot artifacts seen with polarographic measurement techniques were observed. We therefore conclude that endogenously generated hypoxia may be more than just a consequence of increased cellular excitability but an influential and critical factor for orchestrating network dynamics associated with epileptiform activity.