In situ detection of reactive oxygen species and nitric oxide production in normal and pathological tissues: improvement by differential interference contrast

Abstract

Reactive oxygen species (ROS), among which nitric oxide (NO) is currently included, play a plethora of (patho)physiological roles. Harman's free radical theory of aging put forth over 40 years ago received full support since then. A nitric oxide hypothesis of aging recently proposed by McCann, is very likely to be the object of widespread investigation in the near future. Therefore, the possibility of localizing at the (sub)cellular level under the light microscope the sites of ROS and NO production with simple and reliable methods appears as a powerful tool for analytic cytology and pathology. Various histochemical methods were developed for visualizing ROS production; a recently improved version to localize superoxide (and possibly also singlet oxygen), based on a DAB–Mn2+–Co2+ reaction, appears very promising. Since the direct detection of NO is still very difficult, the action sites of NO are currently localized by the identification of NO synthase (NOS). The most widespread method to reveal the catalytic activity of NOS is that of demonstrating the fixation-resistant NADPH diaphorase activity with the tetrazolium salt method. We have improved this method by using a tetrazolium salt whose formazan particles are very thin and lipid insoluble (tetranitroblue tetrazolium, TNBT) and by including a tissue protectant, polyvinyl alcohol, in the incubation medium. Here significant examples of application of the DAB–Mn2+–Co2+ technique for ROS and the TNBT–PVA method for NOS to normal liver and brain and to solid tumors are presented. We further document the usefulness of Nomarkis's differential interference contrast (DIC) to analyze wide tissue areas where ROS production or NOS activity is low or even nil. The improved version for NOS allowed for the first time to demonstrate NOS activity in liver fat-storing cells and in astrocyte-like cells in the brain.