Reactive oxygen species (ROS) have been implicated as an important causative factor in cell damage, including apoptosis and necrosis. Their proposed actions comprise lipid peroxidation, DNA damage, destruction of the mitochondrial respiratory chain and protein modifications. Recent experiments underline the importance of peroxynitrite, the reaction product of the two potent reactive species nitric oxide and Superoxide. Several fluorogenic compounds have been used in order to determine ROS formation in living cells. Besides dihydrorhodamine-123 (DHR-123), at present mostly applied to monitor peroxynitrite, 2,7-dihydrodichlorofluorescein (DCF-H) is used for detection of hydrogen peroxide and nitric oxide. We employed a cell free approach to evaluate the specificity and sensitivity of DCF-H to various oxidizing compounds. Our studies imply that DCF-H is much more sensitive to peroxynitrite oxidation than any other compound tested. In order to study peroxynitrite generation within individual cells, primary glial cultures loaded with DCF-H were monitored with a laser scanning microscope. Microglia, stimulated to simultaneously produce the peroxynitrite precursors nitric oxide and Superoxide, displayed the greatest increase in DCF fluorescence, whereas microglia producing either nitric oxide or Superoxide alone showed a relatively small increase in DCF fluorescence. In conclusion, DCF-H was demonstrated to be an excellent peroxynitrite marker with the potential to detect peroxynitrite formation in living cells. © 1997 Federation of European Biochemical Societies.

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Superoxide dismutase (SOD) is considered to be a major factor in protection of nervous tissue against excitotoxic and ischemic/hypoxic lesion. Controversial reports about the localization of SOD after such an insult prompted us to re‐investigate immunocytochemically the localization of the enzyme in the brain and spinal cord using specific antibodies against the manganese (Mn‐SOD) and copper/zinc (Cu/Zn‐SOD) containing isoenzyme in combination with cell type specific markers. CNS tissue sections were analyzed by confocal laser scanning microscopy and digital photo imaging. Cu/Zn‐SOD immunoreactivity was found to be located predominantly in astrocytes throughout the CNS. The staining was found in the cytoplasm, in cellular processes and, less intensive, in the nucleus sparing the nucleolus. At a lower level the enzyme was also detectable in neuronal perikarya and in structures of the neuropil. Motoneurons of the spinal cord displayed an enhanced Cu/Zn‐SOD staining intensity, when compared to brain neurons. In contrast the Mn‐containing isoenzyme was predominantly localized to neurons and their processes throughout the brain and the spinal cord. Confirming the mitochondrial localization of the enzyme, a granular staining pattern sparing the nucleus was observed. Mn‐SOD stained mitochondria were also seen in astroglial cells but the staining intensity was, on the whole, much lower compared to neurons, and often hardly detectable. It seems reasonable to conclude that differences in the basal content of SOD‐isoenzymes may contribute to different cellular susceptibilities in neurodegenerative processes that are accompanied by oxidative stress. GLIA 29:25–34, 2000. © 2000 Wiley‐Liss, Inc.

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