Histochemical method for localization of hydrogen peroxide and oxygen radicals in the intact neonatal brain

The generation of reactive oxygen species has been implicated in the pathog enesis of a wide variety of diseases of the central nervous system. Often t hese pathological conditions involve damage to specific cell types within s elected areas of the brain. Thus, there is a marked need for a method which allows microscopic visualization/detection of these oxygen radicals in dis crete brain areas. We are reporting a method to histochemically localize, w ith single cell resolution,hydrogen peroxide (H2O2) and oxygen radicals in the neonatal brain in vivo. This method expands on the technique developed to visualize H2O2 and the superoxide anion radical (O-2-) in isolated perfu sed organs (e.g., lung, heart) (Babbs, 1994). With our technique, the intac t brain is perfused inrtracardially with warm oxygenated saline to remove b lood, followed by perfusion with buffers containing either iron and diethyl enetriaminepentaacetate for the detection of H2O2 or manganese for the dete ction of oxygen radicals. The free radical oxidizes its respective metal, w hich in turn oxidizes diaminobenzidine (DAB) to form a brown reaction produ ct which can be visualized using light microscopy. (C) 1998 Prous Science. All rights reserved.