[62] Histochemical methods for localization of endothelial superoxide and hydrogen peroxide generation in perfused organs

Publisher Summary

This chapter describes two histochemical techniques capable of revealing discrete sources of biological oxidants in perfused organs at the light or electron microscopic levels. The first is a high manganese/diaminobenzidine technique, in which superoxide oxidizes Mn²⁺ to Mn³⁺, which in turn oxidizes diaminobenzidine (DAB) to form amber-colored, osmiophilic polymers, observable by light or electron microscopy. The second is a high iron/diaminobenzidine technique, in which hydrogen peroxide oxidizes diethylenetriaminepentaacetate-chelated Fe³⁺ to form intermediate species, which in turn oxidize DAB similarly to Mn³⁺. Both the manganese and iron methods can readily demonstrate the appearance of reaction product on the luminal surfaces of arterial, capillary, and venular endothelial cells during the first 2 to 3 rain of reoxygenation after ischemia. The histochemical reactions are nearly absent in non-manganese- and noniron-treated controls. Superoxide dismutase strongly inhibits the Mn²⁺/DAB reaction, and catalase strongly inhibits the Fe²⁺/DAB reaction, when tested in specially perfused lung preparations in which these specific antioxidant enzymes are concentrated. These histochemical techniques can provide direct, visual evidence that a burst of reactive oxygen species is generated by isolated or cultured cells or by perfused tissues, using simple laboratory procedures available to almost any investigator at marginal cost.