Heiko Noack

2,7-Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation

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.

Selective upregulation of inducible nitric oxide synthase (iNOS) by lipopolysaccharide (LPS) and cytokines in microglia : In vitro and in vivo studies

A role for free radicals has been proposed in infectious brain disease, where resident microglia cells upregulate the inducible nitric oxide synthase isoform (iNOS), and thus are capable of producing nitric oxide at enhanced rates. Using the constitutively expressed NADPH oxidase, microglial cells can generate superoxide, which reacts with nitric oxide to form the powerful oxidant peroxynitrite. In a mixed cell culture system of astrocytes and microglial cells, nitrite levels, used as an indicator of nitric oxide production, were elevated after the addition of lipopolysaccharide (LPS) and cytokines. Immunohistochemistry and the NADPH diaphorase technique demonstrated selective localization of the iNOS protein in microglial cells, whereas no iNOS protein or NADPH diaphorase activity was detected in astrocytes. A similar cellular distribution was observed in vivo following injection of LPS and cytokines into the rat striatum. By contrast, LPS and interferon‐γ led to translocation of NF‐κB in microglia and in astrocytes, demonstrating that both cell types are responsive to the stimulus. Therefore, downstream control in iNOS expression is cell type‐specific. GLIA 32:51–59, 2000.

Cellular distribution of superoxide dismutases in the rat CNS

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.

Differential expression of superoxide dismutase isoforms in neuronal and glial compartments in the course of excitotoxically mediated neurodegeneration: Relation to oxidative and nitrergic stress

To examine the cellular distribution of radical scavenging enzymes in glia, in comparison to that in neurons and their behaviour during excitotoxically induced neurodegenerative processes, protein levels and the cellular localization of cytosolic and mitochondrial superoxide dismutase (Cu/Zn‐ and Mn‐SOD) were investigated in the rat brain undergoing quinolinic acid (Quin)‐induced neurodegeneration. Evidence for the specificity of the applied antibodies to detect immunocytochemically these SOD isoforms was obtained from electron microscopy and Western blotting. In control striatum Mn‐SOD was clearly confined to neurons, whereas Cu/Zn‐SOD was found, rather delicately, only in astrocytes. Microglia failed to stain with antibodies to both SOD isoforms. Quin application resulted in an initial formation of oxygen and nitrogen radicals as determined by the decline in the ratio of ascorbic to dehydroascorbic acid and by increased levels of nitrated proteins, an indicator for elevated peroxynitrite formation. Morphologically, massive neuronal damage was seen in parallel. Astroglia remained intact but showed initially decreased glutamine synthetase activities. The levels of Mn‐SOD protein increased 2‐fold 24 h after Quin injection (Western blotting) and declined only slowly over the time period considered (10 days). Cu/Zn‐SOD levels increased only 1.3‐fold. Immunocytochemical studies revealed that the increase in Mn‐SOD is confined to neurons, whereas that of Cu/Zn‐SOD was observed only in astroglial cells. Quiescent microglial cells were, as a rule, free of immunocytochemically detectable SOD, whereas in activated microglia a few Mn‐SOD immunolabeled mitochondria occurred. Our results suggest a differential protective response in the Quin lesioned striatum in that Mn‐SOD is upregulated in neurons and Cu/Zn‐SOD in astroglia. Both SOD‐isoforms are assumed to be induced to prevent oxidative and nitric oxide/peroxynitrite‐mediated damage. In the border zone of the lesion core this strategy may contribute to resist the noxious stimulus. GLIA 23:285–297, 1998.

Glutathione levels in primary glial cultures: monochlorobimane provides evidence of cell type-specific distribution.

Because glutathione (GSH) levels in glia play an important role in cellular defense against oxidative and nitrosative stress, the present study was designed to study GSH levels in the primary glial cell cultures. Here we used fluorescence microscopy and spectroscopy with monochlorobimane for measurement of intracellular glutathione content. Monochlorobimane showed high specificity for GSH with very little binding to protein sulphydryls as ascertained from the low fluorescence intensity of the protein fraction of the cells as well as from the low fluorescence of the GSH‐depleted cells. The formation of the monochlorobimane‐glutathione conjugate was observed to be enzymatically catalyzed as seen from its higher rate of formation in the presence of cell homogenate. A monochlorobimane concentration of 60 μM was used for conjugation of cellular GSH; at higher mBCl concentrations there was no appreciable increase in fluorescence. Therefore, cultures were treated with 60 μM mBCl for an incubation time of 20 min (beyond this time, export of the bimane‐glutathione adduct was significantly large) and examined by fluorescence microscopy. This adduct could be fixed with a mixture of paraformaldehyde and glutaraldehyde, and excellent fixation was observed with 4% paraformaldehyde and 0.2% glutaraldehyde. Analysis of the fluorescence images revealed differences in fluorescence intensity between astro‐ and microglial cells, which were identified by glial fibrilliary acidic protein and OX42 staining, respectively. Microglial cells isolated from primary glial cultures were found to have higher GSH content than astrocytes. Biochemical determination of GSH levels in microglia isolated from primary glial cultures corroborated this fact. From our findings it seems that owing to the greater intracellular concentration of reactive oxygen and nitrogen species to which microglia are subjected, especially under conditions of inflammation, this cell type is fortified with higher GSH levels as a means to combat oxidative and nitrosative stress. GLIA 27:152–161, 1999.

RELATIONS BETWEEN TOCOPHEROL DEPLETION AND COENZYME Q DURING LIPID PEROXIDATION IN RAT LIVER MITOCHONDRIA

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe++/ascorbate and NADPH/ADP/Fe++ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.

Enhanced cellular glutathione peroxidase immunoreactivity in activated astrocytes and in microglia during excitotoxin induced neurodegeneration

The cellular distribution pattern of cellular glutathione peroxidase (GPx) was analyzed immunocytochemically in the normal rat central nervous system (CNS) and following exposure to the excitotoxin quinolinic acid (Quin). In the normal CNS, GPx was localized predominantly to microglia, identified by staining with isolectin B4 or the monoclonal antibody OX‐42. Three days after intrastriatal administration of 90 μmoles Quin, GPx immunoreactivity was increased in activated microglia and also in astrocytes costained for glial fibrillary acidic protein (GFAP). Whereas GPx‐positive astrocytes were seen predominantly in the environment of the lesion, most intensive immunoreactivity was located in globular‐shaped microglia in the lesion core. GPx staining was generally low in neurons and failed to increase its intensity after lesion. In the case of excitotoxin‐induced generation of oxygen‐derived free radicals, the elevation of GPx levels in microglia, and likewise in activated astroglia, may provide an important mechanism to withstand oxidative stress. GLIA 24:252–256, 1998.

Comparison of protein oxidation and aldehyde formation during oxidative stress in isolated mitochondria.

Oxidative stress is known to cause oxidative protein modification and the generation of reactive aldehydes derived from lipid peroxidation. Extent and kinetics of both processes were investigated during oxidative damage of isolated rat liver mitochondria treated with iron/ascorbate. The monofunctional aldehydes 4-hydroxynonenal (4-HNE), n-hexanal, n-pentanal, n-nonanal, n-heptanal, 2-octenal, 4-hydroxydecenal as well as thiobarbituric acid reactive substances (TBARS) were detected. The kinetics of aldehyde generation showed a lag-phase preceding an exponential increase. In contrast, oxidative protein modification, assessed as 2,4-dinitrophenylhydrazine (DNPH) reactive protein-bound carbonyls, continuously increased without detectable lag-phase. Western blot analysis confirmed these findings but did not allow the identification of individual proteins preferentially oxidized. Protein modification by 4-HNE, determined by immunoblotting, was in parallel to the formation of this aldehyde determined by HPLC. These results suggest that protein oxidation occurs during the time of functional decline of mitochondria, i.e. in the lag-phase of lipid peroxidation. This protein modification seems not to be caused by 4-HNE.

Role of endogenous and exogenous antioxidants in the defence against functional damage and lipid peroxidation in rat liver mitochondria

Mitochondria are cellular organelles where the generation of reactive oxygen species may be high. They are, however, effectively protected by their high capacities of antioxidative systems, as enzymes and either water or lipid soluble low molecular weight antioxidants.

Peroxynitrite mediated damage and lowered superoxide tolerance in primary cortical glial cultures after induction of the inducible isoform of NOS

The effect of the induction of i‐NOS in primary glial cultures was studied with respect to the protein levels of reactive oxygen species (ROS) scavenging enzymes and the cytotoxicity of nitric oxide (⋅NO) formation at different levels of artificially generated superoxide. Stimulation of the cultures by bacterial lipopolysaccharides and γ‐interferon resulted in an induction of i‐NOS exclusively in microglial cells. Among the ROS scavenging enzymes superoxide dismutase (Cu/Zn‐ and Mn‐isoform), glutathione peroxidase and catalase only mitochondrial Mn‐SOD was found to be upregulated in the course of i‐NOS induction (Western blots). Although ⋅NO formation did not affect cell viability at physiological levels of superoxide over a time period of 4 days, it caused an oxidative load particularly in microglial cells as observed by monitoring the oxidation of dichloro‐dihydrofluorescein, an indicator for the formation of peroxynitrite and ROS. Elevated levels of superoxide, generated either intracellularly by paraquat or extracellularly via xanthine oxidase and hypoxanthine, resulted dose‐dependently in a larger decline of cell viability in the ⋅NO forming cultures compared to controls (release of lac‐tate dehydrogenase, citrate synthase, stainability by propidium iodide, and tetramethylrhodamine). NOS‐inhibitors reduced the degree of cell damage to that seen for control cultures, indicating an ONOO−‐/⋅NO mediated mechanism of cell damage. Our data support the concept that i‐NOS catalyzed ⋅NO‐formation leads to an ONOO−‐mediated increased oxidative load. At physiological levels of superoxide and within a wide range of higher superoxide levels this nitrosative stress is well balanced in cultured glial cells by protective mechanisms. GLIA 28:13–24, 1999.