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 pathogenesis of a wide variety of diseases of the central nervous system. Often these pathological conditions involve damage to specific cell types within selected areas of the brain. Thus, there is a marked need for a method which allows microscopic visualization/detection of these oxygen radicals in discrete brain areas. We are reporting a method to histochemically localize, with 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 (O₂-) in isolated perfused organs (e.g., lung, heart) (Babbs, 1994). With our technique, the intact brain is perfused intracardially with warm oxygenated saline to remove blood, followed by perfusion with buffers containing either iron and diethylenetriaminepentaacetate for the detection of H₂O₂ or manganese for the detection of oxygen radicals. The free radical oxidizes its respective metal, which in turn oxidizes diaminobenzidine (DAB) to form a brown reaction product which can be visualized using light microscopy.