Nitro blue tetrazolium staining: a morphological demonstration of superoxide in the rat retina

Nitro blue tetrazolium (NBT) is an artificial electron acceptor which has been suggested to be a useful tool in studies of free radicals. To establish whether NBT could be useful in morphological demonstration of free radicals in the retina, the staining pattern was examined in normal rat retina and after incubation with the free radical scavenger superoxide dismutase or with inhibitors of free radical protective enzymes, such as potassium cyanide, dicoumarol, and aminotriazole. After 60 min incubation in 1 mM NBT, staining was observed in photoreceptors, in some cells in the inner nuclear and ganglion cell layers, and in glial cells. Staining also appeared in the plexiform layers and occasionally in small blood vessels. Addition of superoxide dismutase caused a decreased staining of the inner parts of the retina. The inhibitors of free radical scavengers caused a dose-dependent increase in staining of the inner parts of the retina and, at high concentrations of potassium cyanide, of the photoreceptors. Thus, NBT seems indeed to be useful in morphological studies of free radicals in the retina.