oa In vivo assessment of reactive oxygen species in the brain using novel dihydroethidium-based methodology

Reactive oxygen species (ROS), e.g. superoxide, hydrogen peroxide, peroxynitrite, are important mediators for many cell signaling processes. In the central nervous system, cardiovascular function is tightly regulated by cell signaling in specific nuclei (the subfornical organ (SFO), paraventricular nucleus (PVN), and rostral venterolateral medulla (RVLM)) and an imbalance of ROS in these nuclei contributes to the development cardiovascular diseases, e.g. hypertension. There are limitations to quantify and localize ROS in brain tissue. The current state-of-the-art method to measure ROS in the brain is in situ quantification of dihydroethidium (DHE) fluorescence using confocal microscopy. DHE is a cell permeable compound, which upon reaction with ROS forms red fluorescent 2-hydroxyethidium. However, in situ DHE imaging has a number of technical problems. Recent developments with in vivo DHE methods may circumvent these problems but, this methodology has not been well established for brain, particularly cardioregulatory nuclei. The aim of this study was to test whether in vivo DHE methodology using either systemic or direct brain administration of DHE, may be an alternative to in situ DHE. Mice were injected with lipopolysaccharide (LPS) (8 micrograms/g i.p.) to elicit ROS production in the brain. In control groups we injected saline or LPS and tempol (200 microliters of 10 mM tempol, i.p.), which is a ROS scavenger. Three hours later mice were injected with DHE (150 microliters of 150 micrograms/mouse, i.p.) and brains were harvested one hour later. In a second experiment we administered DHE via an intracerebroventricular (ICV, 1 microliter of 10 micromolar DHE) cannula (including the same controls), which allows direct delivery of DHE to the brain. Our results show that LPS caused increased DHE fluorescence intensity in the SFO, RVLM and PVN but not in control areas e.g. the cortex when DHE was administrated intraperitoneally. Saline- and tempol-treated mice showed much lower DHE fluorescence. The distribution pattern was similar in both experiments. In conclusion, this study shows that DHE administered either systemically or directly into the brain leads to effective detection of LPS-induced ROS formation in brain centers important in cardiovascular homeostasis and may be an important alternative to in situ DHE.