Dulbecco’s Modified Eagle’s Medium:F-12 nutrient mixture (D-MEM/F-12, with GlutaMAX^TM-I), B27 supplement, trypsin-EDTA solution (0.05% trypsin, 1 mM EDTA in HBSS), gentamicin, antibiotics (10,000 units/ml of penicillin, 10 mg/ml streptomycin), and trypsin (1:250) were purchased from Life Technologies (Carlsbad, CA, USA). Deoxyribonuclease 1 (DNase-1), BrdU, phenylmethylsufonyl fluoride, dithiothreitol, orthovanadate, chymostatin, leupeptin, antiparin, pepstatin A, trypan blue, LPS, and alkaline phosphatase-linked anti-rabbit secondary antibody were purchased from Sigma Chemical (St Louis, MO, USA). BCATM Protein Assay kit was obtained from Pierce (Rockford, IL, USA). EGF, bFGF, Click-iT^® EdU Alexa Fluor^® 647 HCS Assay, Hoechst 33342, anti-mouse IgG conjugated with Alexa Fluor 594 or 488, and anti-rabbit IgG conjugated with Alexa Fluor 633, 594, or 488 secondary antibodies were purchased from Life Technologies (Carlsbad, CA, USA). Cellulose acetate (0.45 μm) was obtained from Corning Inc. (Lowell, MA, USA). M-CSF and IFN-γ were purchased from Peprotech (London, UK) and NOC-18 from Alexis Biochemicals (San Diego, CA, USA). BSA and MnTBAP were obtained from Calbiochem (San Diego, CA, USA). The antibody against 3-NT conjugated with agarose beads and FeTMPyP were purchased from Cayman Chemical (Tallinn, Estonia). Rabbit anti-GFAP and DAKO fluorescent mounting medium were obtained from DakoCytomation (Glostrup, Denmark). Rat anti-mouse BrdU was obtained from Oxford Biotechnology and rat anti-mouse CD11b from Serotec (Oxford, UK). Mouse anti-nestin, rabbit anti-iNOS and mouse anti-GAPDH were purchased from BD Transduction (San Jose, CA, USA). Mouse anti-3-nitrotyrosine was purchased from Upstate Biotechnology (Charlottesville, VA, USA), while rabbit anti-EGFR, rabbit anti-phospho-Tyr1173-EGFR, rabbit anti-phospho-ERK1/2 and mouse anti-ERK1/2 were purchased from Cell Signaling (Danvers, MA, USA). Rabbit anti-nestin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and mouse anti-Sox-2 from R&D Systems (Minneapolis, MN, USA). Griess Reagent System was obtained from Promega (Madison, WI, USA). Polyvinylidene difluoride (PVDF) membranes, enhanced chemifluorescence (ECF) reagent and alkaline phosphatase-linked anti-rabbit and anti-mouse secondary antibodies were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Other reagents used in immunoblotting experiments were purchased from BioRad (Hercules, CA, USA).

C57BL/6J (iNOS^+/+) mice or B6.129P2-Nos2^tm1Lau/J (iNOS^-/-) were obtained from Charles River (Barcelona, Spain) or The Jackson Laboratory (Bar Harbor, ME, USA), respectively, and kept with food and water ad libitum in a 12 h dark:light cycle. All experiments were performed in accordance with NIH and European guidelines (86/609/EEC) for the care and use of laboratory animals. In addition, all the people working with animals have received appropriate education (FELASA course) as required by the Portuguese authorities. Furthermore, the animals were housed in our licensed animal facility (International Animal Welfare Assurance number 520.000.000.2006). This study is part of a grant approved and financed by the Foundation for Science and Technology, (FCT, Portugal), that approved the animal experimentation described (reference PTDC/SAU-NEU/102612/2008).

Primary glial cultures were prepared from the brains of 0 to 3-day-old C57BL/6J (iNOS^+/+) or B6.129P2-Nos2^tm1Lau/J (iNOS^-/-) mice according to the method of Giulian and Baker (1986). Briefly, the brains were removed from the skull, following decapitation, and placed in dissection medium composed of Ca²⁺- and Mg²⁺-free HBSS (137 mM NaCl, 5.36 mM KCl, 0.44 mM KH₂PO₄, 0.34 mM Na₂PO₄ . 2H₂O, 4.16 mM NaHCO₃, 5 mM glucose, 1 mM sodium pyruvate, 10 mM HEPES, pH 7.4), supplemented with 0.25% gentamicin. The enveloping meninges and the cerebellum were discarded and the cortex tissue was mechanically dissociated and digested with trypsin (0.1%) and DNase 1 (0.001%) in Ca²⁺- and Mg²⁺- free HBSS for 20 min, at 37°C. Cells were seeded in 75 cm² flasks coated with poly-L-lysine, at a density of 0.2 × 10⁶ cells/cm² and cultured in D-MEM/F-12 with GlutaMAX^TM-I supplemented with 10% FBS, 0.25% gentamicin and 0.25 ng/ml M-CSF, at 37°C and 95% air-5% CO₂ in a humidified incubator. Culture medium was changed every 3–4 days and confluency was achieved after 10–14 DIV. Microglia were detached from the mixed glial cultures 3–10 days after reaching confluency, by shaking at 200 rpm for 2 h, and collected from the supernatant by centrifuging at 1500 rpm, for 5 min. Cells were then seeded for 3 days at a density of 0.035 × 10⁶ cells/cm² onto 16-mm diameter glass coverslips, for immunocytochemistry assays, or on 12-well plates, for preparation of lysates, both coated with poly-L-lysine, in serum-free medium, without M-CSF. Next, cultures were treated with an acute inflammatory stimulus (except the controls): 100 ng/ml LPS plus 0.5 ng/ml IFN-γ, for 24 h (Saura et al., 2003).

Neural stem cell cultures were obtained from the SVZ of postnatal day 0–3 wild-type or transgenic C57BL/6J mice expressing enhanced green fluorescent protein (GFP) under the control of the actin promoter, as previously described by Carreira et al. (2010). The SVZ-derived NSCs were allowed to develop as primary neurospheres in a 95% air-5% CO₂ humidified atmosphere at 37°C, during 7 days. Next, neurospheres were collected, dissociated and plated for 5 days on 16-mm diameter glass coverslips, for immunocytochemistry assays, or on 12-well plates, coated with poly-L-lysine, in the same medium as above, without growth factors, for preparation of lysates.

Green fluorescent protein-positive SVZ neurospheres were collected, dissociated and plated together with iNOS^+/+ or iNOS^-/- microglial cell cultures (from now on denominated as iNOS^+/+ or iNOS^-/- mixed cultures, respectively) on 16-mm diameter glass coverslips, for immunocytochemistry assays, or on 12-well plates, for preparation of lysates, both coated with poly-L-lysine, and kept in fresh D-MEM/F-12 with GlutaMAX^TM-I medium, supplemented with 1% B27, 0.25% gentamicin, 10 ng/ml EGF and 10 ng/ml bFGF, at 37°C and 95% air-5% CO₂ in a humidified incubator, for 3 days. GFP-positive SVZ neurospheres were also dissociated and seeded alone, for 3 days, on 16-mm diameter coverslips or on 12-well plates, coated with poly-L-lysine, and cultured in the same medium as above, for control experiments. Cultures were treated with 100 ng/ml LPS plus 0.5 ng/ml IFN-γ, for 24 h. Control cultures were left untreated. The cell-permeable superoxide dismutase mimetic and ONOO^- scavenger MnTBAP (100 μM; Szabo et al., 1996) or the ONOO^- decomposition catalyst FeTMPyP (50 μM; Misko et al., 1998), when used, were added 30 min before LPS plus IFN-γ and kept throughout the incubation period.

Subventricular zone-derived NSC were exposed to the NO donor NOC-18 (100 μM) for 48 h. MnTBAP (100 μM) and FeTMPyP (50 μM), were added 30 min before NOC-18 and kept throughout the incubation period.

To analyze proliferation of SVZ-derived NSCs, 10 μM BrdU was added to the cultures 16 h prior to fixation (Alvaro et al., 2008; Carreira et al., 2010). Nuclei that incorporated BrdU in this time-window were detected by immunofluorescence, as detailed next. Following 20 min fixation with 4% paraformaldehyde/4% sucrose in PBS (0.1 M), the cells were permeabilized with 1% Triton X-100 for 5 min, and DNA was denaturated by treatment with 1 M HCl for 30 min, at 37°C. Non-specific binding was blocked with 3% BSA in 0.2% Tween-20 in PBS (PBS-T) for 1 h, and then BrdU-positive cells were labeled with a rat anti-BrdU antibody (1:50) for 90 min, at room temperature. The cells were then incubated with a secondary antibody goat anti-rat IgG conjugated with Alexa Fluor 594 (1:200), for 1 h, at room temperature. Nuclei were stained with Hoechst 33342 (1 μg/ml) for 5 min. Coverslips were mounted on glass slides using DAKO fluorescence mounting medium. The images were acquired in a laser-scanning microscope LSM 510 META (Zeiss, Jena, Germany) or in a fluorescence microscope (Axioskop 2 Plus, Zeiss, Jena, Germany). The number of BrdU-positive nuclei was counted in 8–10 randomly selected fields for each coverslip (in a total of ∼900–1,200 cells per coverslip), and the data were expressed as percentage of the total number of live cells. A minimum of three independent experiments, from NSC cultures prepared from different animals, was analyzed for each condition.

Neural stem cell proliferation was also assessed by incorporation of the thymidine analog EdU, which is incorporated into DNA of dividing cells during S phase (Buck et al., 2008; Cappella et al., 2008; Chehrehasa et al., 2009). EdU (10 μM) was added to the cultures 4 h prior to fixation. Nuclei that incorporated EdU in this time-window were detected by immunofluorescence, as follows. Following 20 min fixation with 4% paraformaldehyde/4% sucrose in PBS (0.1 M), the cells were washed with 3% BSA/PBS and then permeabilized with 0.5% Triton X-100/PBS for 15 min, at room temperature. The cells were then incubated with the Click-iT reaction cocktail [1x at 87.5% (v/v) Click-iT Reaction Buffer, 2% (v/v) CuSO₄, 0.05% (v/v) fluorescent azide (Alexa Fluor 647), and 1x at 10% (v/v) Reaction Buffer Additive], protected from light. Cells were then washed twice in 3% BSA/PBS and an immunocytochemistry was performed as detailed next.

The number of EdU-positive nuclei was counted in 8–10 randomly selected fields for each coverslip (in a total of ∼900–1,200 cells per coverslip), and the data were expressed as percentage of the total number of live cells. A minimum of three independent experiments was analyzed for each condition.

Following fixation and permeabilization, non-specific binding was blocked with 3% BSA. Cells were incubated with the primary antibodies for 90 min, at room temperature. After rinsing with PBS, the cells were incubated with the appropriate secondary antibodies for 1 h (1:200, anti-mouse, anti-rabbit or anti-rat IgGs conjugated with Alexa Fluor 488, 594, or 633), at room temperature. All antibodies were prepared in blocking solution. Nuclei were labeled with Hoechst 33342 (1 μg/ml) for 5 min, after incubation with the secondary antibodies. Coverslips were mounted on glass slides, the cells were visualized using a fluorescence microscope (Axioskop 2 Plus, Zeiss, Jena, Germany) and the images were acquired with the Axiovision software (release 4.7) or in a laser scanning fluorescence microscope LSM 510 META (Zeiss, Jena, Germany). The primary antibodies and the concentrations used were: mouse anti-Sox-2, 1:100; rabbit anti-nestin, 1:100; rabbit anti-GFAP, 1:400; mouse anti-nestin, 1:500; rat anti-mouse-CD11b, 1:200; rabbit anti-iNOS, 1:200.

Nitric oxide production was indirectly assessed by measuring the concentration of nitrites in the culture medium (Green et al., 1982), in primary microglia cultures, SVZ-derived NSC cultures or mixed cell cultures. A commercial kit from Promega was used, and the standard protocol provided by the supplier was followed. The concentration of nitrite for each sample was calculated from a standard curve using a sodium nitrite solution and data were expressed in μM.

Cells were lysed in 50 mM Tris-HCl, 10 mM EGTA, 1% Triton X-100 and 2 mM MgCl₂, supplemented with 100 μM phenylmethylsufonyl fluoride, 1 mM dithiothreitol, 1 μg/ml chymostatin, 1 μg/ml leupeptin, 1 μg/ml antiparin, 5 μg/ml pepstatin A, 1 mM sodium orthovanadate, 50 mM NaF, pH 7.4, at 4°C. Protein concentration was determined by the bicinchoninic acid (BCA) method, and the samples were used for Western blot analysis, after adding 6x concentrated sample buffer [0.5 M Tris, 30% glycerol, 10% sodium dodecyl sulfate (SDS), 0.6 M dithiothreitol, 0.012% bromophenol blue] and heating, for 5 min, at 95°C. Equal amounts of protein were separated by electrophoresis on SDS-polyacrylamide gels, and transferred electrophoretically to PVDF membranes. These were then blocked for 1 h at room temperature, in Tris-buffered saline (137 mM NaCl, 20 mM Tris-HCl, pH 7.6) containing 0.1% Tween-20 (TBS-T) and 3% BSA. Incubations with primary antibodies (anti-iNOS or anti-GAPDH, 1:500; rabbit anti-phospho-Tyr1173-EGFR, rabbit anti-EGFR, rabbit anti-phospho-ERK1/2 and mouse anti-ERK1/2, 1:1,000) in TBS-T with 1% BSA were performed overnight, at 4°C. Next, the membranes were incubated for 1 h at room temperature with alkaline phosphatase-linked secondary antibodies (anti-rabbit or anti-mouse IgG, 1:20,000) in TBS-T with 1% BSA. After extensive washing in TBS-T with 0.5% BSA, immunoreactive bands were visualized in the VersaDoc 3000 imaging system (BioRad, Hercules, CA, USA), following incubation of the membrane with ECF reagent for 5 min.

Following the various experimental treatments, as detailed in figure legends, the cultures were lysed in 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, 2 mM EGTA, supplemented with 100 μM PMSF, 1 mM dithiothreitol, 1 μg/ml chymostatin, 1 μg/ml leupeptin, 1 μg/ml antiparin, 5 μg/ml pepstatin A, 1 mM sodium orthovanadate, 50 mM NaF, pH 7.0, at 4°C. Protein concentration was determined by the BCA method, and the samples were used for immunoprecipitation of nitrated proteins, using an antibody against 3-NT conjugated with agarose beads. Briefly, equal amounts of sample (250 μg of protein) were incubated with the antibody overnight at 4°C, and then with the beads for 2 h at room temperature. Following rinsing, the supernatant was discarded and the beads were suspended in 2x concentrated sample buffer, boiled for 5 min, and centrifuged using Spin-X centrifuge tube filters (0.45 μm cellulose acetate), to separate the beads from the immunoprecipitates. Equal volumes of immunoprecipitate were loaded onto SDS-PAGE gels, and Western blotted as described above against the EGF receptor.

Data are expressed as mean ± SEM. Statistical significance was determined by using two-tailed t-tests, one-factor or two-factor analysis of variance (ANOVA) as appropriate, followed by post hoc Bonferroni’s or Dunnet’s tests, as indicated in the figure legends and in the text. Differences were considered significant when p < 0.05.

Neural stem cells were isolated from the SVZ and cultured as floating aggregates, also referred as neurospheres. Neurospheres were then dissociated and plated on poly-L-lysine-coated coverslips for 3–5 days, and characterized at this stage. Staining against the transcription factor Sox-2, and against nestin, a neural precursor cell marker, was performed. The percentage of double-labeled cells was approximately 70%, which suggests that the majority of cells remained undifferentiated after plating as shown previously by our group (Carreira et al., 2010).

Microglial cells were seeded on poly-L-lysine-coated coverslips for 3 days and challenged with an inflammatory stimulus, LPS (100 ng/ml) plus IFN-γ (0.5 ng/ml), for 24 h. Cultures were then characterized by evaluating microglial cells morphology, iNOS expression and NO production. In iNOS^+/+ microglial cell cultures, exposure to LPS plus IFN-γ increased iNOS immunoreactivity, but not in iNOS^-/- microglial cell cultures (Figure 1A), as expected. Concomitantly, following treatment with LPS plus IFN-γ, both iNOS^+/+ and iNOS^-/- microglial cells exhibited an activated morphology with ovaloid cytoplasm, marked cellular hypertrophy and retraction of processes. In order to estimate the amount of NO produced by activated microglia in culture, we assessed NO production by measuring nitrite levels in the culture media following challenging with LPS plus IFN-γ. Nitrite levels were higher in treated iNOS^+/+ microglial cell cultures (1.95 ± 0.3 μM, p < 0.001), than in untreated cultures (0.32 ± 0.1 μM), corresponding to a 6-fold increase in NO production above control levels. In iNOS^-/- microglial cell cultures, treatment with LPS plus IFN-γ did not significantly change NO levels, as compared to untreated cultures (Figure 1B).

To investigate how inflammation, particularly NO formed due to microglial activation, could affect the proliferation of SVZ-derived NSCs, mixed cultures were prepared as described in Section “Materials and Methods.” We evaluated the expression of iNOS in iNOS^+/+ mixed cultures, following treatment with LPS plus IFN-γ by immunocytochemistry (Figure 2A) and Western blot analysis (Figure 2B). In iNOS^-/- mixed cultures, iNOS was not detected, as assessed by Western blotting, even after the inflammatory stimulus (Figure 2B). Furthermore, when measuring nitrite levels in the culture media, an increase in NO production was observed in iNOS^+/+ mixed cultures (1.96 ± 0.2 μM, p < 0.001) following treatment with LPS plus IFN-γ, as compared to control cultures (0.39 ± 0.1 μM). On the contrary, treatment with LPS plus IFN-γ for 24 h did not significantly change NO levels in iNOS^-/- mixed cultures, as compared to untreated cultures (Figure 2C).

To determine whether SVZ-derived NSCs contributed to the increase in iNOS levels observed in mixed cell cultures, we also evaluated the presence of iNOS in SVZ-derived NSC alone, and observed a complete absence of immunoreactivity against iNOS following treatment with LPS plus IFN-γ in these cultures (Figure 2D). Moreover, treatment with LPS plus IFN-γ for 24 h did not significantly change NO levels in SVZ-derived NSC cultures, as compared to untreated cultures, as assessed by evaluating nitrite levels in cultures (Figure 2E).

To determine how NO released from microglial cells affected the proliferation of SVZ-derived NSCs, we evaluated the incorporation of EdU in iNOS^+/+ or iNOS^-/- mixed cultures, following treatment with LPS plus IFN-γ. In iNOS^+/+ mixed cultures, we observed that EdU incorporation significantly decreased to 7.0 ± 1.09% (p < 0.001), 24 h following exposure to LPS plus IFN-γ, as compared to control cultures (17.3 ± 0.88%), but the same stimulus had no effect in EdU incorporation either in iNOS^-/- mixed cultures or in SVZ-derived NSC cultures alone (Figure 3A).

Moreover, we observed that SVZ-derived NSCs, but not microglia, incorporated EdU, as illustrated by the presence of immunoreactivity against EdU in the GFP-positive SVZ cells, but not in microglial cells, strongly suggesting that these are the dividing cells in mixed cultures (Figure 3B). We also confirmed that LPS plus IFN-γ for 24 h did not affect cell viability in iNOS^+/+ and in iNOS^-/- mixed cultures, or in SVZ-derived NSC cultures (Tables 1 and 2, respectively).

We next investigated the possible mechanism underlying the observed antiproliferative effect of inflammation in mixed cultures. The main regulatory pathway for NSC proliferation is the signaling cascade of MAPK, particularly the ERK1/2 pathway. Within this scenario, we evaluated whether inflammation, particularly NO from inflammatory origin, could affect the activation and signaling through the ERK/MAPK pathway. We found that LPS plus IFN-γ decreased the phosphorylation of ERK1/2 in iNOS^+/+ mixed cultures (Figure 4, p < 0.001), whereas in iNOS^-/- mixed cultures no changes were observed in the levels of phospho-ERK1/2 following exposure to LPS plus IFN-γ (Figure 4).

To better understand whether the antiproliferative effect of inflammation in mixed cultures could be due to the production of highly reactive species, such as ONOO^-, we used MnTBAP, a superoxide scavenger, thus preventing the production of ONOO^-, and FeTMPyP, which catalyzes the degradation of ONOO^-. We observed that the decreased phosphorylation of ERK1/2 in iNOS^+/+ mixed cultures following treatment with LPS plus IFN-γ was prevented either by treatment with MnTBAP or FeTMPyP (Figure 5A, p < 0.001 or p < 0.05, respectively). Moreover, when MnTBAP or FeTMPyP were present during the inflammatory stimulus, cell proliferation was rescued to 15.8 ± 0.4% or 14.7 ± 0.8%, respectively, in iNOS^+/+ mixed cultures (Figures 5B,C).

We also investigated the effect of high levels of NO, an event occurring during inflammation, in the proliferation of SVZ-derived NSC cultures. For that, SVZ-derived NSCs were exposed to a NO donor, NOC-18 (100 μM), for 48 h. We observed a decreased phosphorylation of ERK1/2, following exposure to 100 μM NOC-18 for 48 h, which was prevented by MnTBAP (Figure 6A, p < 0.05). When assessing NSC proliferation by evaluating BrdU incorporation following treatment with NOC-18 (100 μM) for 48 h, we observed that NOC-18 significantly decreased BrdU incorporation to 4.9 ± 0.2% (p < 0.001), as compared to BrdU incorporation in control cultures (7.7 ± 0.2%). Interestingly, MnTBAP rescued cell proliferation (7.9 ± 0.66%), when present during the treatment with NOC-18 for 48 h (Figure 6B). Furthermore, we also confirmed that NOC-18 for 48 h did not affect cell viability in SVZ-derived NSC cultures (Table 2).

We next evaluated whether the inflammatory stimulus induced the nitration of the EGF receptor in mixed cultures, an event that could be linked to the inhibition of the downstream signaling through the ERK/MAPK pathway in mixed cultures treated with LPS plus IFN-γ. Nitration of tyrosine residues of the EGFR was evaluated in iNOS^+/+ and iNOS^-/- mixed cultures. In iNOS^+/+ mixed cultures, treatment with LPS plus IFN-γ for 24 h increased the nitration of the EGFR in immunoprecipitates of nitrated proteins (127.4 ± 5.4%, p < 0.01), as compared to untreated cultures. Moreover, MnTBAP and FeTMPyP were able to prevent the nitration of the EGFR in these cultures, after incubation with LPS plus IFN-γ, reducing EGFR nitration to 68.2 ± 7.2% (p < 0.001) or 84.9 ± 2.4% (p < 0.001) of the control, respectively (Figure 7A, left pannel). On other hand, this effect was not observed in iNOS^-/- mixed cultures, where treatment with LPS plus IFN-γ did not cause increased nitration of the EGFR (Figure 7A, right panel).

Next, we analyzed the phosphorylation of the tyrosine residue 1173, which is involved in the activation of the EGFR. LPS plus IFN-γ caused a decrease in the phosphorylation status of the EGFR in iNOS^+/+ mixed cultures, an event that was prevented either by MnTBAP or FeTMPyP (Figure 7B, left panel). As expected, in iNOS^-/- mixed cultures no changes were observed in the phosphorylation status of the EGFR following exposure to LPS plus IFN-γ for 24 h (Figure 7B, right panel).