P6 was synthesized according to Di Nunno et al. (15), whereas mofezolac was synthesized following Micetich’s protocol (16). All the other reagents and solvents were purchased from Sigma-Aldrich (Milan, Italy) and used without any further purification.

Lipopolysaccharide from Escherichia coli serotype 0127:B8 was purchased from Sigma-Aldrich (Milan, Italy). The goat polyclonal a p-IκB (sc-7977) antibody (Ab) was purchased from Santa Cruz Biotechnology (DBA, Milan, Italy); COX-1 (Ab 695) and COX-2 (Ab 15191) Abs were obtained from Abcam (Cambridge, UK). Goat anti-rabbit IgG (sc-2004), goat anti-mouse IgG (sc-2005), and donkey anti-goat IgG (sc-2020) were purchased from Santa Cruz Biotechnology; mouse primary monoclonal antibody (mAb) anti-glial fibrillary acidic protein (GFAP) (Merck Millipore, Milan, Italy), mouse mAb anti-ionized calcium-binding adapter molecule-1 (Iba-1) (Merck Millipore). Elisa kit for PGE₂ evaluation was purchased from Cayman Chemical (Ann Arbor, MI, USA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], 3,3′-diaminobenzidine, and tribromoethanol were obtained from Sigma-Aldrich, Milan, Italy.

BV2 microglia cells (ICLC HTL 03001-Interlab Cell Line Collection) were grown in high glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. They were maintained at 37°C in a humidified 5% CO₂/95% environmental air.

Then, microglial cells were plated, at a density of 25 × 10⁴/well in 6-well plates (Falcon) and treated with the chosen COX inhibitors (P6 and mofezolac) when they reached 80% confluence. Preliminary experiments were conducted to establish the optimal concentration and exposure times necessary for LPS (1 µg/mL) treatment, which were found to be in accordance with other reports (17, 18), as well as to establish the optimal dose of the COX inhibitors and exposure times to detect their effects on LPS-stimulated BV2 microglial cell function. In this regard, a set of experiments was carried out in which microglial cells were 1 h pretreated with the selective COX-1 inhibitors P6 (0.5 and 1.0 µM) or mofezolac (0.1 and 0.5 µM). Cells were then incubated for different times at 37°C with LPS, as pro-inflammatory stimulus. Experiments included cells grown in medium alone (control).

Cell viability of microglial cells was quantified using the MTT reduction assay. The cells (8 × 10³/well) were grown in 96-well plates (Becton Dickinson Labware) in complete medium and treated with different concentration of COX inhibitors, in presence or absence of LPS. Untreated cells were used as a control. A PBS 1× solution of MTT (5 mg/mL) was prepared and added to the cell medium at a final concentration of 0.5 mg/mL. Cells were incubated for 4 h at 37°C and 5% CO₂ to allow the MTT metabolism. The formazan crystals formed (from MTT) into the cells were solubilized with DMSO (Sigma–Aldrich). The levels of MTT formazan were determined measuring the optical density at λ = 560 nm and subtracting the background (λ = 670 nm) with a Victor Multiplate Reader (Wallac). Optical density was directly correlated to cell quantity.

After treatment of cultures as previously described, cells were harvested and lysed by ice-cold lysis buffer [1% Triton X-100, 20 mM Tris–HCl, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin hemisulfate salt, and 0.2 U/mL aprotinin (all from Sigma–Aldrich)] for 30 min on an ice bath.

Substantia nigra pars compacta, hippocampus, frontal lobe, and caudate from mice brains were minced in ice-cold PBS, washed, and then homogenized in a buffer containing lysis buffer (50 mM Tris pH 8, 0.02 g/mL NaCl, 0.2% SDS, 1% Triton-X, 4 U/mL aprotinin, 2 mM leupeptin, and 100 mM phenylmethanesulfonyl fluoride).

The tissue and cell culture lysates were vortexed for 15–20 s and, then, centrifuged at 12,800 × g for 20 min. The protein concentration in the supernatant was spectrophotometrically determined by Bradford’s protein assay. Protein samples were diluted with sample buffer (0.5 M Tris–HCl pH = 6.8, 10% glycerol, 10% (w/v) SDS, 5% 2-mercaptoethanol, and 0.05% (w/v) bromophenol blue) and then boiled for 3 min. Proteins (25 µg/lane) and prestained standards (BioRad Laboratories, Hercules, CA, USA) were loaded on 7% or 12% SDS precast polyacrylamide gels (BioRad Laboratories).

After electrophoresis, the resolved proteins were transferred from the gel to nitrocellulose membranes. A blotting buffer [20 mM Tris/150 mM glycine, pH = 8.0, and 20% (v/v) methanol] was used for gel and membrane saturation and blotting. Then, membranes were incubated in the dark with the following specific primary Abs reported in reagents section for 60 min at room temperature. The membranes were washed with 0.1% Tween 20-PBS (for 20 min, three times) and then incubated with the secondary Ab diluted 1:2,000 for 60 min. Bands were visualized by chemiluminescence detection (Invitrogen, Milan, Italy). The β-actin level was used as a protein loading control. For tissue analysis, obtained bands were normalized to the level of β-actin performed for each cerebral area tested. For cell cultures, the bands were normalized to the β-actin level of each experimental condition. The bands obtained after immunoblotting were submitted to densitometric analysis using 1D Image Analysis Software (Kodak Digital Science). Results were expressed as arbitrary units.

This study was carried out in strict accordance with the European Council Directive 86/609/EEC and the Italian animal welfare legislation (art. 4 and 5 of D.L. 116/92). Seventy adult male 129S2/Sv mice (22–24 g body mass, 8–10 weeks of age) were purchased from Harlan—Italy and were kept under environmentally controlled conditions (20 ± 2°C, 50–80% humidity, 12 h light/dark cycle, food and water ad libitum) and divided into 7 groups of 10 animals (5 for western blotting and 5 for immunohistochemical procedures).

Mice received either the selective COX-1 inhibitor mofezolac (6 mg/kg, i.p.; indicated as M in all the figures) or vehicle (40% DMSO in 0.1 M phosphate buffer, pH = 7.4; VM in all the figures) once a day for 10 days. Mofezolac amount to be injected was chosen taken into account previous study and its COXs IC₅₀ values. On the seventh day, mice were anesthetized with tribromoethanol (250 mg/kg, i.p.) and positioned in a stereotactic apparatus (Kopf Instruments, Tujunga, CA, USA). Vehicle (sterile saline, 5 µL; V-LPS in all the figures) or 5 µg LPS in 5 µL of sterile saline (LPS in all the figures) was administered into the cerebral lateral ventricle using a fine needle glass syringe (Hamilton, Lyon, France) and a syringe pump (KD Scientific, Holliston, MA, USA) at a rate of 1 µL/min. This LPS dose and time point (72 h) induced the best neuroinflammatory response following a titration (24 up to 60 h) preliminary study (data not shown). Stereotaxic injections coordinates were 2.3 mm dorsal/ventral, 1.0 mm lateral, and 0.5 mm anterior/posterior from the bregma. Mofezolac was given 30 min prior to LPS injection (M + LPS in all the figures).

Mice were transcardially perfused with tris-buffered saline (pH = 7.6) followed by 4% paraformaldehyde in PBS pH = 7.4 at 4°C. Brains were subsequently postfixed in the same fixative, paraffin embedded, and 10 µm slices were obtained with a rotative microtome (Leica, Milan, Italy). Immunohistochemistry was performed following a standard avidin–biotin complex procedure. Briefly, specimens were incubated with mouse primary mAb anti-GFAP at a ratio of 1:1,000 (Merck Millipore, Milan, Italy), or a mouse mAb anti-Iba-1 at a ratio of 1:500 (Merck Millipore) overnight at 4°C and then with an anti-mouse biotinylated secondary Ab (Dako, Milan, Italy), at a 1:1,000 dilution for 1 h at room temperature. The antigen–Ab complexes were visualized by sections incubation for 1 h with extravidin peroxidase (Sigma-Aldrich) diluted 1:1,500 and 3,3′-diaminobenzidine oxidation in the presence of H₂O₂.

Microglial cells were cultured in 6-well plates at a density of 3 × 10⁶ cells/well. Then, the cells were pretreated with selective COX-1 inhibitors P6 or mofezolac for 1 h and, subsequently stimulated with LPS (1 µg/mL). The cultures were maintained at 37°C for 24 and 48 h in a humidified air containing a 5% CO₂. PGE₂ levels were determined in the supernatant using a competitive binding immunoassay (Cayman Chemical, Ann Arbor, MI, USA) following the manufacturer’s instructions. Unstimulated cells were included as a control. PGE₂ amount determination in the brain was performed in the tissue extracts, according to the manufacturer’s instructions. The optical density was measured at λ = 405–420 nm with precision microplate reader and the amount of PGE₂ (ng/mL) was calculated using a PGE₂ standard curve.

Student’s t-test and analysis of variance (one-way ANOVA) on the results of at least five independent biological replicates were performed. Values of p < 0.05 were considered statistically significant.

MTT assay was used to quantitatively evaluate cell viability. This was performed to verify whether the tested selective COX-1 inhibitors (P6 and mofezolac) caused toxicity in LPS-treated BV2 cell line. Preliminarily, the effect of two different concentrations of P6 (0.5 and 1 µM) and mofezolac (0.1 and 0.5 µM) on BV2 microglial cell viability was evaluated. No cell toxicity was exerted by either P6, mofezolac, and LPS alone or a combination of LPS and each of the two inhibitors at 24 h. The two concentrations of P6 and mofezolac were chosen based on the basis of previous studies and their COXs IC₅₀ values (14).

Cell viability was found to be significantly (p ≤ 0.001) lower, after 48 h of LPS treatment (percentage cell viability equal to 78.4 ± 0.15 vs 99.8 ± 0.07 of the control), than untreated cells (control). None of the inhibitors used at the above indicated doses were toxic to BV2 microglial cells, when used in the absence of LPS (percentage cell viability ranging between 95.5 ± 0.22 and 98.2 ± 0.16 vs control). In addition, inhibitors used at the same doses, resulted protective toward BV2 microglial cells after 48 h LPS treatment (1 µg/mL), being the percentage of cell viability comparable to that observed in control.

Astroglial activation was characterized by immunoreactivity and immunoblotting analysis of the GFAP expression, a marker used to distinguish astrocytes from other glial cells of the CNS. LPS treatment determined an increase of immunoreactive cell bodies in comparison to untreated mice suggesting astrocyte activation in different brain regions. In particular, the caudate, frontal lobe, hippocampus, and substantia nigra were selected to evaluate the astrocyte activation after different animal treatment (Figures 2A–D). Images report GFAP reactivity in samples of mice control group (CTR), mice treated with LPS vehicle (V-LPS), LPS alone (LPS) or in combination with mofezolac (M + LPS). In CTR sections, there is a physiological astroglial distribution with few immunoreactive elements surrounding the blood vessels as part of the brain–blood barrier, which means that there are no sign of reactive astrogliosis. In V-LPS-treated mice, the expression is similar to CTR, with perivascular immunoreactivity and the presence of a few astrocytes in the tissue, due to inflammation probably caused from the injection. In LPS-treated mice, images show a marked increase of the immunoreactive elements, whereas in vivo administration of mofezolac reduced the presence of GFAP immunoreactive cells in all the tested brain areas (Figures 2A–D).

Ionized calcium-binding adapter molecule-1 immunoreactivity, a marker of activated microglia, was also evaluated (Figure 3). LPS-treated mice Iba-1 positive cells were more numerous, showing a more intense immunoreactivity, as well as a ramified phenotype, in comparison to untreated mice (Figures 3A–D). Mofezolac administration determined the reduction of Iba-1 immunoreactivity in LPS-injected mice in all the tested brain areas (Figures 3A–D), suggesting that mofezolac reduces, at least in part, the microglial activation induced by the neurotoxic insult.

Immunoblotting analysis was also performed to semiquantitatively evaluate both astrocyte and microglia activation in samples derived from mice groups previously described. In LPS-treated mice, a significant increase of GFAP expression was detected in all the brain regions tested when compared to controls or vehicle-LPS (Figure 4). Similar results were obtained for Iba-1 expression analysis (Figure 4).

Conversely, in mofezolac-treated animals previously injected with LPS, the GFAP as well as Iba-1 levels resulted significantly reduced in comparison to the animals that received LPS alone (Figure 4).

The effect of mofezolac and P6 on COX-1 expression in LPS-treated microglial cells was evaluated by western blotting analysis. After 24 h, no significant difference between LPS-stimulated cells in the presence or absence of both COX-1 inhibitors was observed (data not shown). Interestingly, LPS-stimulated BV2 cells exhibited, at 48 h, increased levels of COX-1 expression in comparison to untreated cells (Figure 5). The COX-1 expression levels evaluated at 48 h of incubation with LPS was significantly reduced in 1 h pretreated cells with either selective COX-1 inhibitor P6 or mofezolac (Figure 5). Therefore, COX-1 expression was found to be time dependently reduced by both selective inhibitors tested.

The expression of COX-2 in the same cell lysates was also evaluated. Interestingly, COX-2 protein levels were unaffected by the presence of P6 or mofezolac. In these conditions, COX-2 expression resulted comparable to the level observed in cells stimulated with LPS alone (Figure 5).

Therefore, in LPS-treated BV2 microglial cell line, P6 and mofezolac were able to reduce COX-1 expression without affecting COX-2.

Cyclooxygenases expression was also in vivo evaluated, assaying different brain regions (Figure 4). LPS-challenged mice exhibited increased levels of COX-1 both in comparison to controls and vehicle of LPS (vLPS) administered animals in all the different areas tested (Figure 4). Interestingly, LPS-challenged mice that received mofezolac treatment exhibited a significantly reduction of COX-1 expression in all brain areas tested. Protein levels comparable to controls were detected in mice treated with vLPS + mofezolac, whereas in mice treated with the vehicle of mofezolac + LPS, COX-1 levels resulted comparable to those detected in LPS-treated mice (Figure 4).

Immunoblotting assay on tissues of the same brain areas was performed to test the in vivo effects of mofezolac administration on COX-2 expression extent. COX-2 resulted overexpressed in all tested brain regions in LPS-administrated mice. Interestingly, mofezolac exhibited no effect on COX-2 modulation in LPS-treated mice, being the protein levels almost comparable to LPS-treated mice (Figure 4).

The PGE₂ biosynthesis extent was evaluated in supernatants of cell cultures at 48 h incubation time (Table 1A). PGE₂ production in LPS-stimulated cells was significantly higher than its basal level present in the untreated cells (control). Interestingly, both COX-1 inhibitors were able to reduce in a dose-dependent manner PGE₂ release in LPS-treated cells (Table 1A).

In the tested brain regions of LPS-treated mice, PGE₂ levels were significantly increased with respect to control animals or mice receiving the vLPS (Table 1B). Interestingly, a significant reduction of PGE₂ levels was detected in mofezolac-treated mice before LPS challenging (Table 1B).

Since the phosphorylation and degradation of IkBα, the inhibitory complex of NF-kB, is an essential step for NF-kB activation, the expression levels of the phosphorylated form of IkBα (p-IkBα) were evaluated. In this context, BV2 microglial cells exposed to LPS exhibited a significant increase of p-IkBα in comparison with no stimulated cells (control) (Figure 5). Densitometric analysis revealed little p-IkBα in the untreated cells, whereas pretreatment with all tested COX-1 inhibitors, significantly reduced IkBα phosphorylation in LPS-stimulated cells (Figure 5).

Similar results were obtained in in vivo model. In fact, in all tested brain areas of LPS-treated mice, an increase of p-IkBα, in comparison to control animals as well as to vLPS administered mice, was detected. Conversely, in mice receiving mofezolac (M), a significant reduction of p-IkBα in all tested areas was detected (Figure 4).

p-IkBα levels comparable to controls were detected in mice treated with vLPS + mofezolac, whereas in mice treated with vehicle of mofezolac + LPS, p-IkBα levels were comparable to those detected in LPS-treated mice (Figure 4).