Materials

Dulbecco's modified Eagle medium and fetal calf serum were from Gibco; poly-L-lysine, penicillin G, steptomycin and bovine serum albumin were from Sigma. A Mn SOD rabbit polyclonal antibody was prepared in our laboratory by injection of an oligopeptide NH ₂-Tyr-Val-Ser-Gln-Arg-Tyr-Ile-Val-Cyst-Lys-Lys-COOH of rat Mn SOD (Pinteaux et al.1996). Guinea pig polyclonal 2',3' cyclic nucleotide 3'-phophodiesterase (CNPase) antiserum was prepared in our laboratory according to Drummond et al.(1978). Mouse monoclonal glial fibrillary acid protein (GFAP) antibody was from Euromedex. Hybridoma cells were from ATCC Rockville USA and mouse monoclonal OX-42 antibody from Cerdic, France. CY3-conjugated donkey anti-mouse IgG and CY3-conjugated donkey antiguinea pig IgG were from Jackson Immuno Research Lab. CY 2-conjugated goat antirabbit IgG was from Amersham Like Science, Inc. Rabbit polyclonal nitrotyrosine antiserum was from Upstate Biotechnology and rabbit polyclonal inducible nitric oxide synthetase antiserum was from Transduction Laboratories.

Cell cultures

Cultures of glial cells were prepared from cerebral hemispheres of 1-day-old newborn rats accordong to Weibel et al. 1984, with modifications. Cells were plated at a density of 10⁶ cells/ml on poly-L-lysine treated glass coverslips in 35 mm diameter Falcon Petri dishes and cultured in Dulbecco'smodified Eagle's medium supplemented with 10% fetal calf serum, 50U/ml penicillin and 50 microg/ml streptomycine. The cells were grown to confluency at 37°C in a 5% CO_2> humidified atmosphere. The culture medium was changed after 5 days and then twice a week, for a period of two weeks. To obtain secondary cultures of oligodendroglial cells, O-2A progenitors present in the primary glial cell culture were, according to Deloulme et al. 1993, detached from the astroglial underlayer by gently syringing culture medium onto the cell layer. The detached cells were sedimented by centrifugation and replated onto poly-L-lysine coated glass coverslips in DMEM medium supplemented with 10% calf serum. After 1 h. this medium was replaced by a serum-free medium consisting of DMEM plus 5 microg/ml insulin, 50 microg/ml transferrin and 0.5 mg/ml bovine serum albumin. Oligodendroglial cells were grown in this medium for 4 days before analysis. In parallel, some secondary cultures were maintained for the same period of time in DMEM supplemented with 10% fetal calf serum.

Immunocytochemical procedures

Double immunofluorescence staining with surface markers for O-2A precursors (A2B5) or for microglial cells (OX-42) or an oligodendrocyte maturation marker (CNPase) and with MnSOD antibodies was performed on cell cultures. Detection of surface antigens was performed on living cells. Cells were incubated with mouse monoclonal A2B5 (hybridoma supernatant, 1:1 dilution) or OX-42 (hybridoma supernatant, 1:2 dilution) antibodies in DMEM for 35 min at room temperature. After one rinse with DMEM and then with with PBS, cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and then washed with PBS. For detection of intracellular MnSOD or CNPase, cultures were then permeabilized with methanol for 5 min at room temperature. After washing with PBS, cells were incubated with blocking buffer (3% BSA in PBS) for 15 min. After 3 rinses with PBS, cells were incubated for 2 h with Mn SOD or CNPase polyclonal antibodies (1:100 dilution) in blocking buffer. After washing with PBS, cells were incubated for 35 min at room temperature with a mixture of CY3-conjugated donkey anti-mouse IgG (1:200 dilution) or CY3-conjugated donkey anti-guinea pig IgG (1:800) and CY2-conjugated goat anti-rabbit IgG(1:400 dilution). To carry out double immunofluorescence staining with intracellular marker GFAP (an astrocyte marker) OX-42 (a microglial marker) and Mn SOD, iNOs and nitrotyrosine antibodies, the cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with methanol for 15 min. After washing with PBS, cultures were incubated for 2 h at room temperature with a mixture of mouse monoclonal GFAP antibody (1:800 dilution) and rabbit polyclonal MnSOD antibody (1:100 dilution) in blocking buffer. Cells were rinsed with PBS and incubated for 35 min at room temperature with the same mixture of CY2- and CY3-conjugated antibodies, under the same conditions as mentioned above. In all immunofluorescence staining procedures, controls were performed by omitting the primary antibody. All preparations were mounted with aqua polymount and examined using a Leitz DMRD fluorescence microscope.

Primary glial cell cultures obtained by setting up dissociated cerebral cortices of 1-day-old rat pups and grown in a serum supplemented medium were analyzed. The majority of the cells presented a flat epithelium shape GFAP positive, corresponding to type 1 astrocytes. Double immunostaining analysis revealed only a faint MNSOD immunoreactivity of the perinuclear cytoplasm and cell expansions present in almost all the GFAP positive astroglial cells. Exposure of these cells to 0.1 micro g/ml LPS added to the culture medium caused an increase in SOD activity which affects specifically the Mn form of the enzyme whereas the CuZn enzyme remains unaffected.

Table I: Time effect of LPS treatment on Mn- and CuZn-SOD activities of glial cell cultures.
Duration of LPS treatment (hours)	0	5	10	15	20	30
CuZn SOD activity	100	61	96	113*	126*	139*
Mn SOD activity	100	79	365*	483*	596*	635*

LPS (final concentration 0.1 micro g/ml) was directly added to the culture medium of 2-week-old cultures. SOD activities were determined as indicated in Materials and Methods (mean+/- S.E.M, n=4; * value significantly different from co,trol; p <.05 )

Until 5 h after LPS addition, there was neither a change nor a small decrease in the specific activity and the concentration of Mn SOD. Between 5 and 30 h of treatment, an approximately fivefold increase in activity and enzyme protein level (estimated by densitometry of the western blot, not shown) was observed. Beside the astroglial cells, some GFAP negative cells, with amoeboide shape and very well positive to OX-42 immunostaining express strong MN SOD immunoreactivity in the perinuclear area and as punctate labeling along the cell processes. Regarding their OX-42 immunoreactivity, these Mn SOD positive cells correspond to microglia. An increase in Mn SOD immunoreactivity is also observed in this cell population after LPS treatment. Careful examination of the primary glial cell cultures derived from newborn rat cerebrum reveals some smaller phase dark cells with stellate appearance, dispersed on top of the underlayer formed by the polygonal shaped astroglial cells. These cells characterized by the ganglioside marker A2B5 on their surface may correspond to bipotential O2-A precursors. We observed that A2B5- positive cells did not express any immunostaining for Mn SOD. Oligodendroglial cells derived from O-2A precursors, after 4 days in secondary culture and strongly immunoreactive for CNPase did not reveal any Mn SOD immunostaining.LPS treatment of these cells did not induce any MN SOD immunostaining, even after LPS treatment.

Concerning inducible NOs, its presence is exclusively observed immunocytochemically in a fraction of the OX-42 positive microglial cell population, and only after LPS treatment. In our conditions of growth, astroglial nor oligodendroglial cells never express any iNOs immunoreactivity, even in response to LPS treatment. This specifical iNOs expression by LPS treated microglia was confirmed by western blotting analysis of total extracts of OX-42 positive cells (not shown). Immunostaining analysis for nitrotyrosine reveals a significant titrotyrosine immunostaining within OX-42 positive microglial cells exposed to LPS. A faint nitrotyrosine immunostaining is also observed in astroglia surrounding OX-42 nitrotyrosine positive microglia. In the absence of any LPS treatment, no nitrotyrosine immunostaining may be observed either in microglia nor in astroglia.Similarly there is no observable nitrotyrosine immunoreactivity in response to LPS treatment lower than 5 hours.

It is well admitted that astrocytes are among the most functionally diverse group of cells in the brain. They are particularly active during the development of its structure and the regulation of the brain cell environment. Astrocytes have a great potential to remove superoxide radicals resulting from cell respiratory activity (Copin et al. 1992). Mn SOD, an inducible superoxide dismutase, seems to play an important role in the antioxidant defense system occuring in the brain: total SOD activity of the astrocytes is significantly higher than that of neurons (Geremia et of al. 1990). Immunocytochemical analysis of MnSOD distribution among the various glial cell types present in a mixed primary cell culture has revealed its presence in GFAP positive type 1 astrocytes and also in OX-42 immunoreactive microglial cells. Exposure of astroglia and microglia to LPS significantly reinforces their Mn SOD immunoreactivity.By contrast, O-2A precursors of oligodendroglial and type 2, A2B5 positive astroglial cells never present any significant MN SOD immunoreactivity, even after LPS exposure. It is well known that microglial cells grown in primary culture may secrete superoxide (Tanaka et al. 1994). Our demonstration of the presence of Mn SOD in microglia suggest that these cells may convert superoxide radicals to hydrogen peroxide. Superoxide and peroxide radicals may react by the iron catalyzed Haber-Weiss reaction which may lead to the synthesis of hydroxyl radicals and hydroxyl ions (Halliwell and Gutteridge, 1986). All these radicals are well known to damage various surrounding biomolecules and membrane structures. Moreover, since hydrogen peroxyde is freely diffusible across membranes, it could serve as second messenger system able to modulate various fondamental cellular functions. For example it is known that hydrogen peroxide mediates beta amyloid protein toxicity (Behl et al.1994).

Concerning iNOs, we observed its presence exclusively in some microglial cells in response to LPS treatment. Astroglial nor oligodendroglial cells never expressed any iNOs immunoreactivity in our conditions of culture, even after LPS treatment. NO releases under these conditions by microglia may diffuse to the surrounding cells or generate through its reaction with oxygen superoxide the subsequent formation of peroxinitrite (Becman, 1994). As with hydrogen peroxide (other biological oxidant) it seems likely that peroxynitrite may modulate various specific signalling systems, contributing to cell regulating processes. For example, a modulation of tyrosine phosphorylation by peroxynitrite has recently been reported (Li et al. 1998).

Under our culture conditions, A2B5 positive glial cells, corresponding to O2-A bipotential glial progenitors (Raff, 1983) and differentiated oligodendrocytes do not express any observable level of Mn SOD in secondary cultures, even after LPS treatment. The absence of observable Mn SOD in oligodendrocytes could be a characteristic of this cell type. One may suggest that mitochondria of O-2A precursors, oligodendroglia ant type 2 astrocytes present a much poorer ability to scavenge ROS than other types of glial cells. This is in accordance with the observation by Noble et al.(1994) pointing out that toxicity of catecholamine metabolism toward oligodendrocytes and resulting from the generation of reactive free radical species was completely prevented by co-culture with astrocytes. One may imagine that the great sensitivity of oligodendrocytes to ROS damage (Husain and Juurlink, 1995) could be efficiently antagonized by co-culture with gkial cells expressing a high ROS scavenging potential. This also suggest that oligoendroglial vulnerability resulting from a very low Mn SOD content could play an important role in the development of dismyelinating disorders.

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