Histochemical demonstration of endothelial superoxide and hydrogen peroxide generation in ischaemic and reoxygenated rat tissues

Objective: The aims were to test and evaluate two novel and independent histochemical methods for detecting the initial postischaemic burst of superoxide and hydrogen peroxide in buffer perfused rat tissues during reflow after 60 min warm ischaemia. Methods: The first is a high manganese/diaminobenzidine technique, in which superoxide oxidises Mn²⁺ to Mn³⁺, which in turn oxidises diaminobenzidine to form amber coloured polymers, observable by light microscopy. The second is a high iron/diaminobenzidine technique, in which hydrogen peroxide oxidises diethylenetriaminepenta-acetate chelated Fe²⁺ to form intermediate species, which in turn oxidise diaminobenzidine similarly to Mn³⁺. Various isolated organs of the rat were rendered ischaemic for 60 min, and reperfused with oxygen or air equilibrated buffers containing diaminobenzidine and either Mn²⁺ or Fe²⁺. Tissues were fixed by perfusion with Trump's solution and processed for light microscopy. Results: Both manganese and iron methods consistently showed the appearance of reaction product on the luminal surfaces of arterial, capillary, and venular endothelial cells in lung, heart, and intestine of the rat during the first 2 to 3 min of reoxygenation after ischaemia. The histochemical reactions were nearly absent in non-manganese-treated and non-iron-treated controls. Superoxide dismutase strongly inhibited Mn²⁺/diaminobenzidine reaction product formation and catalase strongly inhibited Fe²⁺/diaminobenzidine reaction product formation, when tested in specially perfused lung preparations in which these specific antioxidant enzymes were concentrated. Conclusions: These histochemical techniques provide direct, visual evidence that a burst of reactive oxygen species is generated in postischaemic rat tissues. The Mn²⁺/diaminobenzidine and Fe²⁺/diaminobenzidine techniques permit investigation of the endothelium derived reactive oxygen by simple laboratory procedures available to almost any investigator at low marginal cost. The endothelial oxidants so revealed may be of pathophysiological significance in a variety of cardiovascular disorders.