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-gamma led to translocation of NF-kappaB 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.

Nitrosative stress in primary glial cultures after induction of the inducible isoform of nitric oxide synthase (i-NOS).

Primary glial cultures are able to express the inducible isoform of nitric oxide synthase (i-NOS) upon stimulation by bacterial lipopolysaccharides (LPS) and gamma-interferon (gamma-IfN). Immunocytochemical studies revealed, that under our experimental conditions i-NOS is expressed exclusively by the microglial cells and not in the astrocytes. Nitric oxide (NO) formation represents an oxidative load for the microglial cells, as observed by the oxidation rate of the ROS- and peroxynitrite indicator dichloro-dihydrofluorescein (DCF-H) in these cells. However, cell viability was not affected by the nitric oxide formation, indicating some form of protection against the higher oxidative load. Upregulation of Mn-SOD in the mitochondria in the course of the induction of i-NOS and, compared to the astrocytes, higher GSH levels in the microglial cells probably explain the resistance of the cultures against nitrosative stress. Increased SOD-activities in the mitochondria could lower the superoxide concentration in this organelle and may prevent an oxidative and/or nitrosative damage via a decreased peroxynitrite formation. The higher GSH-levels in the microglial cells of unstimulated cultures represents a buffer which, under the conditions of i-NOS catalyzed NO-formation, prevents a decline of the microglial GSH-levels below that of the astrocytes.

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.

Induction of nitric oxide synthesis lowers intracellular glutathione in microglia of primary glial cultures.

Glutathione (GSH) is one of the most important antioxidants involved in detoxification of reactive oxygen and nitrogen species. We investigated the changes in intracellular GSH in primary glial cultures stimulated to produce inducible nitric oxide synthase and subsequently nitric oxide by bacterial lipopolysaccharide and gamma interferon treatment. Intracellular GSH content was measured by both the monochlorobimane fluorescence microscopy method and the GSH reductase recycling assay (Tietze. Anal Biochem 27:502-522, 1969.). Our results show that irrespective of the assay used the GSH content in stimulated cultures decreased to almost half that of control cultures. This decrease in GSH content was accompanied by an increase in S-nitrosoglutathione in the stimulated cultures. Analysis of the GSH related fluorescence images showed that the fluorescence intensity was lowered exclusively in microglial cells whereas that of astrocytes remained almost unchanged. The present study in conjunction with our previous investigation (Chatterjee et al. Glia 27:152-161, 1999) can be interpreted to imply that the higher GSH levels in untreated microglia are a mechanism to withstand nitric oxide synthase induced oxidative and nitrosative stress and therefore the GSH levels in microglia drop to astrocyte levels after induction.

Photodynamic effects induced by meso-tetrakis[4-(carboxymethyleneoxy)phenyl] porphyrin on isolated Sarcoma 180 ascites mitochondria.

Using mitochondria isolated from Sarcoma 180 ascites tumour in Swiss mice as a model system, we have evaluated the ability of a novel porphyrin, meso-tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin (H2T4CPP), to induce damage on photosensitization. Oxidative damage to mitochondria, one of the primary and crucial targets of the photodynamic effect, is assessed by measuring products of lipid peroxidation such as thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH), besides the loss of activity of the mitochondrial marker enzyme succinate dehydrogenase (SDH). Analysis of product formation, the effect of deuteration and selective inhibition by scavengers of reactive oxygen species (ROS) show that the damage observed is due mainly to singlet oxygen (1O2) and to a minor extent to hydroxyl radicals (.OH). The 1O2 generation and triplet lifetime of this porphyrin have also been estimated. Fluorescence spectroscopy, used to ascertain the binding of this porphyrin to the mitochondrial proteins, shows a rapid association within 0-2 h and a decline thereafter. Confocal microscopy reveals intracellular localisation of this porphyrin in cells in vitro. Our overall results suggest that the porphyrin H2T4CPP, due to its ability to bind to mitochondrial protein components and to generate ROS upon photoexcitation, may have potential applications in photodynamic therapy.

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 gamma-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 lactate 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.

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 microM was used for conjugation of cellular GSH; at higher mBCl concentrations there was no appreciable increase in fluorescence. Therefore, cultures were treated with 60 microM 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.

Collateral branch formation related to cellular structures in the axon tract during corticopontine target recognition.

The corticopontine projection develops exclusively by collateral branches that form along the length of corticospinal axons days after they have passed their hindbrain target, the basilar pons. In vitro evidence suggests that the basilar pons releases a diffusible activity that initiates and directs the growth of collateral branches. This study investigates whether contact-dependent mechanisms may also influence the formation of collateral branches. By using immunocytochemistry, electron microscopy, and neuronal tracing techniques, we examined the region of the axon tract, the cerebral peduncle, overlying the basilar pons for cellular structures that correlate spatially and temporally with collateral branch formation. We found that radial glia are excluded from the tract. Oligodendrocyte precursors are found only at low density. Although mature astrocytes are absent, immature astrocytes are present throughout the tract. However, our evidence does not suggest a direct role for glial cell types in collateral branch formation. In contrast, dendrites of basilar pontine neurons are transiently present in the tract during the time of collateral branch formation. Although collateral branches are observed in regions of the tract devoid of dendrites, the orientation and location of most collateral branches correlates at the light microscopic level with dendrites. Electron microscopy reveals sites of increased collateral branch formation near neuronal cell bodies or dendrites. However, cell processes, whether dendritic or otherwise, are rarely found in direct contact with collateral branch points. A common and unexpected feature is the bundles of corticopontine collateral branches, oriented transversely to their parent corticospinal axons and directed across the tract to the basilar pons. Dendrites were often apposed to or embedded within the transverse bundles. These findings suggest that dendrites are not essential for collateral branch formation but that they may enhance this process and define discrete preferred locations for collateral branch initiation and elongation within the cerebral peduncle.

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.

American football injuries in Germany. First results from Bundesliga football.

This study investigated the incidence of injury in German Bundesliga football based on the evaluation of two American football teams before and during the season 1995. Certified team physicians and team physiotherapists were the initial medical professionals providing on-site diagnosis, injury documentation and first aid for all injuries. An injury was defined as minor (group I) causing the player to miss practice or game sessions up to 1 week without requiring surgery; as severe (group II) causing the player to miss practice or game sessions for more than 1 week up to 3 months or requiring surgery; and as catastrophic (group III) if the incident led to treatment for more than 3 months, treatment in an intensive care unit or persistent neurological or orthopaedic disability or death. From the data collected it was possible to calculate the risk of injury per time of exposure per athlete. The athlete's function, influence of the weather and mechanisms of the injuries were registered. In total, 242 injuries were documented. The rate of injury was calculated as 16 per 1000 hours of practice and game per athlete. Severe injuries (group II) were found in 94 cases. Catastrophic injuries were not seen during the study. The knee was found to be the most common site of injury, while the ankle ranked second. Our study showed that the risk of injury in American football in German Bundesliga is comparable with soccer or handball.

[Value of small bowel double contrast enema in clinical interventions]. / Die Wertigkeit des Dünndarmdoppelkontrasteinlaufes im klinischen Einsatz.

The double contrast enema is the most effective morphological screening method for the evaluation of the whole small bowel. Its sensitivity is 85%, its specifity 96.7%. In specific clinical problems the number of pathological roentgen findings rises: from 34.4% when all indications are taken into consideration to 58% in indications specific to the small intestine such as Morbus Crohn or the malabsorption syndrome. Search for tumours and the double contrast of the small bowel in unclear gastro-intestinal bleeding are unproductive. The weak point of this screening method is the lower part of the small intestine. Therefore, the selective peroral or retrograde analysis of the terminal ileum supplement the contrast method. A precondition for good results is an adequate technical standard. Besides the clinical results some technical results are therefore discussed such as contrast medium quantities, examination and X-ray time, radiation exposure and influences on the image quality.