3.2. Neuroprotection Assessment Assays

The human neuroblastomas cell line SH-SY5Y were cultured in Dulbecco’s: Ham’s F12, 1:1 [vol/vol] containing 3.15 mg/mL glucose, 2.5 mM Glutamax and 0.5 mM sodium pyruvate DMEM/F-12, GlutaMAX™; GIBCO, Life Technologies, Madrid (Spain), 1% Antibiotique-Antimitotic (Gibco; Life Technologies, Madrid, Spain) (containing 100 ui/mL penicillin, 100 mg/mL of streptomycin and 0.25 mg of amphotericine B), 1% gentamicina 15 mg/mL (Sigma-Aldrich, Madrid, España) and 10% Foetal Calf Serum (FCS) (Gibco; Life Technologies, Madrid, Spain) as described [46,49]. Cultures were seeded into flasks containing supplemented medium and maintained at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. Culture media were changed every 2 d. Cells were subcultured after partial digestion with 0.25% trypsin-EDTA. For assays, SHSY5Y cells were subcultured in 96- or 48-well plates at a seeding density of 0.50–1 or 2–2.5 × 10⁵ or cells per well, respectively. When the SHSY5Y cells reached 80% confluence, the medium was replaced with fresh medium containing 0.01–1000 μM compound concentrations or PBS in the controls, as indicated in each assay.

Neuroblastoma cell cultures were exposed to OGD so as to induce cellular damage (experimental ischemia) as described [46]. Cultured cells were washed and placed in glucose-free Dulbecco’s medium (bubbled with 95% N₂/5% CO₂ for 30 min) and maintained in an anaerobic chamber containing a gas mixture of 95% N2/5% CO₂ and humidified at 37 °C at a constant pressure of 0.15 bar. Cells were exposed to OGD for a period of 4 h (OGD 4 h), as indicated. At the end of the OGD period, culture medium was replaced with oxygenated serum-free medium, and cells were placed and maintained in the normoxic incubator for 24 h to recovery (R24h). In the neuroprotection experiments, HTNs 1-3 and PBN (0.01 μM−1 mM) were added at the beginning of recovery period (see below). Control cultures in Dulbecco’s medium containing glucose were kept in the normoxic incubator for the same period of time as the OGD (C4h), and then culture medium was replaced with fresh medium and cells were returned to the normoxic incubator until the end of recovery period (C24h). Control experiments included the same amounts of vehicle (final concentration < 0.01% dimethyl sulfoxide). The experimental procedures were blindly performed, assigning a random order to each assayed nitrone. Nitrones were analyzed independently 3–5 times with different batches of cultures, and each experiment was run in triplicate.

Measurements of cell viability in human SHSY5Y neuroblastoma cells were carried out into 96- well culture plates as described [46,50]. Briefly, control and treated SH-SY5Y neuroblastoma cells (about 0.75–1 × 105 cells/well) were incubated with the XTT solution (Cell Proliferation Kit II (XTT), Sigma, Aldrich, Madrid) at 0.3 mg/mL final concentration for 2 h in a humidified incubator at 37 °C with 5% CO₂ and 95% air (v/v) and the soluble orange formazan dye formed was spectrophotometrically quantified, using a Biotek Power-Wave XS spectrophotometer microplate-reader at 450 nm (reference 650 nm). All XTT assays were performed in triplicate in cells of at least three different cell batches. Control cells treated with DMEM alone were regarded as 100% viability. Controls containing different DMSO concentrations (0.001–1% DMSO) were performed in all assays.

For these assays, cultured neuroblastoma cells grown in 96-well culture dishes at a density of 1.5 × 105 cells/well were used. LDH activity was measured as the rate of decrease of the absorbance at 340 nm, resulting from the oxidation of NADH to NAD+ as described [46,51]. Data are given as the percentage of LDH release with respect to the total LDH content (LDH in the culture medium and LDH inside the cells).

For these assays, cultured neuroblastoma cells grown in 48-well culture dishes, at a density of 2.5 × 10⁵ cells/well, were used. After OGD treatment, cells were treated with different nitrones or indicated positive controls at 1−500 μM concentrations and subjected to 24 h IR. Attached cells were lysed at 4 °C in a lysis medium containing 5 mM Tris/HCl (pH 8.0), 20 mM ethylenediaminetetraacetic acid, and 0.5% Triton X-100 and centrifuged at 13,000g for 10 min. The activity of caspase- 3 was measured using the fluorogenic substrate peptide DEVD-amc (66081; BD Biosciences PharMingen), as described [46,51]. Proteins were measured by the Bradford assay. Results were expressed as arbitrary fluorescence units [(AFU)/μg protein/h].

SHSY5Y human neuroblastoma cells (2 × 10⁵ cells/well) were exposed to OGD for a period of 4 h (OGD4h). At the end of the OGD period, the culture medium was replaced with oxygenated Dulbecco’s modified Eagle’s medium containing glucose and 10% fetal calf serum. Cells were treated in the absence (controls) or presence of indicated concentrations of nitrones or different known neuroprotective agents and maintained at 37 °C in a normoxic incubator for 3 h for recovery. At the end of this period, 20 μM DHE (HEt; Molecular Probes) was added and fluorescence was recorded every 15−30 s during a 15 min period, using an excitation filter of 535 nm and an emission filter of 635 nm in a spectrofluorimeter (Bio-Tek FL 600) as previously described [46,52]. Linear regression of fluorescence data [expressed as arbitrary fluorescence units (AFU)] was calculated for each condition, and the slopes (a) of the best fitting lines (y = ax) were considered as an index of O₂^•− production [52].

Data were expressed as mean ± SEM of results obtained from at least three independent experiments from different cultures, each of which was performed in triplicate. Statistical comparisons between the different experimental conditions were performed using one-way analysis of variance (ANOVA), followed by Holm−Sidak’s post -test when the analysis of variance was significant. A p value < 0.05 was considered statistically significant. Fit curves for EC₅₀ determinations were performed according to the program of SigmaPlot v.11 (Systat Software Inc., Germany), 2012).