Reagents and Antibodies

Antidepressant drugs: imipramine hydrochloride (Sigma-Aldrich Chemie GmbH, Germany), fluoxetine hydrochloride (Farmacom, Poland), citalopram (Lundbeck, Denmark), reboxetine (Pharmacia, Italy), mirtazapine (Organon, The Netherlands), tianeptine sodium (Servier, France).

Neurobasal A medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), supplement B27 were from Gibco (Invitrogen, Paisley, UK). The Cytotoxicity Detection Kit, BM Chemiluminescence Western Blotting Kit and In Situ Cell Death Detection Kit Fluorescein were from Roche Diagnostic (Mannheim, Germany). Primary antibodies: anti-MAP-2 (sc-51669), antiphospho-Tyr204 ERK 1/2 (pERK, sc-7383), anti-ERK2 (sc-474), anti-spectrin α IIsc-48382), protein markers and appropriate secondary antibodies were from Santa Cruz Biotechnology Inc. (CA, USA). All other reagents were from Sigma (Sigma-Aldrich Chemie GmbH, Germany).

Neuronal Cell Cultures

The experiments were conducted on primary cultures of mouse cortical neurons and on neuronal differentiated human neuroblastoma SH-SY5Y cells as described previously (Jantas et al. 2008; Jantas-Skotniczna et al. 2006).

The protocol for generation of the primary neuronal cultures was concordant with local and international guidelines on the ethical use of animals. Neuronal tissues were taken from Swiss mouse embryos at 15/16th day of gestation and were cultured essentially as described previously (Jantas-Skotniczna et al. 2006). The isolated cortical cells were suspended in Neurobasal medium containing penicillin (0.06 μg/ml) and streptomycin (0.1 μg/ml), supplement B27 without antioxidants and were plated at a density of 1.5 × 105 cells/cm2 onto poly-ornithine (0.05 mg/ml)-coated multi-well plates. This procedure typically yields cultures that contain >90 % neurons and <10 % supporting cells as verified by immuncytochemistry (not shown). The cultures were then maintained at 37 °C in a saturated humidity atmosphere containing 95 % air and 5 % CO 2 for 7 days prior to experimentation and culture medium was exchanged every 2 days.

The human SH-SY5Y neuroblastoma cells (ATCC), passages 5–20 were grown in DMEM supplemented with 10 % heat-inactivated FBS and 100 units/ml of penicillin and 100 μg/ml of streptomycin as described previously (Jantas et al. 2008). Cells were maintained at 37 °C in a saturated humidity atmosphere containing 95 % air and 5 % CO 2 . After reaching an 80 % confluence, cells were seeded onto appropriate multi-well plates at a density of 4 × 104 cells/cm2 and were differentiated to a neuronal phenotype for 7 days by adding retinoic acid (RA) to the culture medium at a final concentration of 10 μM. The culture medium with RA was changed three times during the period of differentiation. One day before the experimental treatment of RA-differentiated SH-SY5Y cells, the culture medium was replaced by DMEM containing antibiotics and 1 % FBS.

Glia Cell Cultures

Cortical glia cells were prepared from 2-day-old Albino Swiss mice according to the protocol described by Johann et al. (2008) with some modifications. Briefly, cerebral cortices were dissected, meninges were removed and tissues were minced separately into small pieces, then digested with trypsin (0.1 % for 30 min at 37 °C), triturated in the presence of 20 % fetal bovine serum and DNAse I (150 Kunitz units/ml), and finally centrifuged for 5 min at 100×g. Cells were resuspended in DMEM containing 20 % FBS and 100 units/ml of penicillin and 100 μg/ml of streptomycin. Finally, cells were seeded into 75-cm2 culture bottles coated with poly-ornithine (0.05 mg/ml) and grown at 37 °C in a humidified incubator with 5 % CO 2 . Culture medium initially contained 20 % of FBS and after 4 days was replaced with medium containing 10 % FBS and was exchanged twice a week. Cells after reaching about 80–90 % confluence were trypsinized and replated at a lower density. This procedure was repeated three times and after the last trypsinization cells were seeded on poly-ornithine-coated 96-well plates. Four- week-old glia cell cultures were used for drug testing. Three days before drug exposure culture medium was replaced with the culture medium used for cortical neurons (Neurobasal medium, antibiotics, and B27 supplement) in order to reduce cell proliferation and to achieve similar experimental conditions for drug treatment as in primary neurons. One day before the cell treatment, Neurobasal culture medium was replaced with fresh one. The obtained glia cultures are characterized by about 90 % homogeneity of glial fibrillary acidic protein (GFAP)-positive cells with no neuronal (MAP-2-positive cells) contamination (not shown).

Immunocytochemistry

The purity of primary neuronal and glia cultures and the morphological changes in cortical neurons after a particular drug treatment were determined by immunocytochemistry as described by Singh et al. (2010). At particular DIV (7th for neuronal and 4 weeks for glia cells) cells were washed with pre-warmed phosphate-buffered saline (PBS), fixed for 20 min at RT with 4 % paraformaldehyde and washed twice with PBS. Next the cells were permeabilized with PBS-containing 0.25 % Triton X-100 (PBS-TX-100) for 15 min after which the blocking was performed in the presence of 5 % normal goat serum in PBS-TX-100 at room temperature for 60 min. Primary antibodies against neuronal (anti-MAP-2, 1:250) and glia (anti-GFAP, 1:500) markers were added and incubated with cells for the next 60 min at RT. Subsequently, cells were washed three times with PBS and incubated for 60 min at RT in the presence of secondary antibodies: Alexa Fluor®488 labeled goat anti-mouse and Alexa Fluor®568 labeled goat anti-rabbit IgG (Invitrogen, USA) diluted 1:500 in PBS. Cells were washed with PBS three times for 5 min and mounted with ProLong®Gold antifade reagent (Invitrogen, USA). Cells were examined using a fluorescence AxioObserver microscope (Carl Zeiss, Germany) equipped with the software Axiovision 3.1 at excitation wavelengths of 470 nm (Alexa Fluor®488) and 555 (Alexa Fluor®568).

Cell Treatment

Seven days in vitro (DIV) cortical neurons were co-treated with Tian (0.001–10 μM) and St (0.5 μM) or Dox (0.5 μM) for 14–48 h. RA-SH-SY5Y cells were incubated with Tian (0.01–10 μM) and St (0.5 μM) or Dox (1 μM) for 14–48 h. The effective concentrations and time of exposure to St and Dox were chosen on the basis of our previous reports where these pro-apoptotic factors evoked about 50 % reduction in cell viability of cortical neurons and RA-SH-SY5Y cells (Jantas et al. 2008, 2009; Jantas and Lason 2009; Jantas-Skotniczna et al. 2006). For comparative studies between the effects of Tian with other ADs, cortical neurons and RA-SH-SY5Y cells were co-treated with Imi, Flu, Cit, Reb, or Mirt at concentrations from 0.01 to 10 μM and St or Dox. In glia cell cultures, we also tested the protective effect of Tian and other ADs (Imi, Flu, Cit, Reb, Mirt) at concentrations 0.01–10 μM against cell damage induced by Dox (1 μM for 48 h). An involvement of PI-3 K/Akt and MAPK/ERK/1/2 pathways as well as necroptosis in Tian-mediated neuroprotection was tested by using specific inhibitors of these pathways: LY294002 (10 μM), U0126 (10 μM) and necrostatin-1 (10 μM) which were added 30 min before treatment of neuronal cells with Tian and St or Dox. The caspase-3 inhibitor (Ac-DEVD-CHO, 10 μM) was added to cells 20 min before treatment of cells with St or Dox. St (1 mM), Ac-DEVD-CHO (10 mM), LY 294002 (10 mM), necrostatin-1 (100 mM), and U0126 (10 mM) stock solutions were prepared in dimethyl sulfoxide. Mirt stock solution (10 mM) was prepared in distilled water acidified with few drops of 0.1 M HCl followed by pH adjustment to 7.15 with 0.1 M NaOH. Dox, Imi, Flu, Cit, Reb, and Tian were dissolved in distilled water. The chemicals were present in cultures at a final concentration of 0.1 % and vehicle-treated cells were treated with a relevant solvent.

Measurement of Lactate Dehydrogenase (LDH) Release

In order to estimate cell death, the level of lactate dehydrogenase (LDH) released from damaged cells into culture media was measured after 24 and 48 h of treatment of cells with ADs and St or Dox, respectively, as described previously (Jantas-Skotniczna et al. 2006). Cell-free culture supernatants were collected from each well and incubated with the appropriate reagent mixture according to the supplier’s instructions (Cytotoxicity Detection Kit, Roche) at RT for 20 min. The intensity of red color formed in the assay and measured at a wavelength of 490 nm was proportional to LDH activity and to the number of damaged cells. Absorbance of blanks, determined as no-enzyme control, has been subtracted from each value. The data were normalized to the activity of LDH released from vehicle-treated cells (cortical neurons and glia cells) or toxin-treated cells (RA-SH-SY5Y cells) and expressed as a percent of the control ± SEM or a percent of toxin-induced ± SEM established from n = 5 wells per one experiment from three separate experiments.

MTT Reduction Assay

Cell viability assessment was done after 24–48 h of treatment of cortical neurons, glia cells, and RA-SH-SY5Y cells with particular chemicals. Cell damage was quantified using a tetrazolium salt colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) as described previously (Jantas et al. 2011). The absorbance of each sample was measured at 570 nm in a 96-well plate-reader (Multiscan, Labsystem). The data after subtraction of blanks (absorbance of cells without MTT) were normalized to the absorbance in the vehicle-treated cells (100 %) and expressed as a percent of the control ± SEM established from n = 5 wells per one experiment from three separate experiments.

CalceinAM Staining of Viable Cells

In order to morphologically assess the changes in cell viability in cortical neurons after treatment with Tian and pro-apoptotic agents, we employed a cell-permeable CalceinAM dye according the protocol described previously (Jantas and Lason 2009). The cells were evaluated using an inverted fluorescence microscope (AxioObserver, Carl Zeiss) with excitation wavelength 480 nm (green fluorescence).

Identification of Pyknotic Nuclei by Hoechst 33342 Staining

In order to visually assess the changes in DNA structure after cell treatment with pro-apoptotic factors and Tian in cortical neurons and in RA-SH-SY5Y cells, Hoechst 33342 staining was applied as described previously (Jantas et al. 2011). That dye binds to double-stranded DNA and can be used to visualize highly compacted chromatin of fragmented pyknotic cell nuclei. Images were recorded using an inverted fluorescence microscope (AxioObserver, Carl Zeiss, Germany) with excitation wavelength 350 nm (blue fluorescence). Uniformly stained nuclei were scored as healthy, viable nuclei while those with condensed or fragmented nuclei were identified as damaged one. The number of cells with nuclei having normal and changed (condensed or fragmented) morphology was counted in six randomly chosen fields per a cover slip (150–200 cells); two cover slips per condition from three separate experiments were evaluated. The data were calculated as a percentage of damaged nuclei compared to the total number of cells per one field and presented in histograms as the mean ± SEM.

Identification of Apoptotic Nuclei by TUNEL Method

In order to determine DNA fragmentation in apoptotic cells, the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) technique was applied using the In Situ Cell Death Detection Kit Fluorescein (Roche Diagnostic) as described previously (Jantas et al. 2011). The cortical neurons after Tian and St (24 h) or Dox (36 h) exposure were washed with PBS, fixed with 4 % paraformaldehyde for 20 min and washed two times with PBS. Subsequently, the cells were treated with 0.1 % sodium citrate/0.1 % Triton X-100 for 2 min on ice, and incubated with TUNEL reaction mixture for 60 min at 37 °C. After washing, the TUNEL-labeled nuclei (green points) were examined under excitation 470 nm using inverted fluorescence microscope (AxioObserver, Carl Zeiss). Apoptotic nuclei were counted on recorded images from six randomly chosen fields per a cover slip, two cover slips per condition from three separate experiments and are shown as the mean ± SEM of apoptotic nuclei per one field.

Assessment of Caspase-3 Activity

The caspase-3 protease activity assay in the cortical neuronal samples treated with St (0.5 μM) and Dox (0.5 μM) for 14 and 24 h, respectively, was performed on 96-well plates using colorimetric substrate preferentially cleaved by caspase-3, AcDEVD-pNA (N-acetyl-asp-glu-val-asp p-nitro-anilide) as described previously (Jantas-Skotniczna et al. 2006). As a marker of assay specificity, the cell-permeable caspase-3 inhibitor, AcDEVD-CHO (10 μM) was added to St- or Dox-treated cells during cell treatment. The amounts of p-nitroanilide cleaved by caspase-3 were monitored continuously over 60 min with a plate-reader (Multiscan, Labsystems) at 405 nm. Absorbance of blanks, determined as no-enzyme control, has been subtracted from each value. The data were normalized to the absorbance of vehicle-treated cells (100 %) and expressed as percent of absorbance ± SEM established from n = 5 wells per experiment from three separate experiments.

The measurements of caspase-3 activity in RA-SH-SY5Y cells were performed in the same lysis buffer as described for primary neurons (Jantas-Skotniczna et al. 2006) but with some modifications. Cells were cultured in six-well plate and after drug exposure (Tian and St or Dox for 12 and 24 h, respectively), the culture medium was removed, and cells were washed with cold PBS and stored at −20 °C until measurement (no more than 1 week). Next, the cells were thawed on ice and lysed in 150 μl of ice-cold Caspase Assay Buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1 % CHAPS, 1 mM EDTA, 10 % glycerol, and 10 mM dithiothreitol) lysis buffer supplemented with 10 μg/ml of each leupeptin and pepstatin A for 15 min at 4 °C. Next, the lysed cells were scraped and collected in separate tubes and centrifuged at 16,000×g for 20 min at 4 °C. The supernatants were used for determination of caspase-3 activity by the usage of fluorometric substrate Ac-DEVD-AMC (Promega), according to which the amount of fluorochrome 7-amino-4-methyl coumarin (AMC) is released from the substrate (Ac-DEVD-AMC) upon cleavage by caspase-3-like enzymes. A yellow-green fluorescence produced by free AMC is proportional to the caspase-3 activity present in the sample. Cell lysates (50 μl) were incubated with Ac-DEVD-AMC (50 μM) for 60 min at 37 °C in the absence and presence of a specific caspase-3 inhibitor (Ac-DEVD-CHO; 10 μM) and the fluorescence was measured with a plate-reader (Infinite® M1000 PRO, Tecan, Switzerland) at 360 nm excitation and 460 nm emission wavelengths. The measurement was performed in triplicates and mean RFU (relative fluorescence units) were calculated per mg of protein for each experimental sample. The protein concentration in cell lysates was determined with the bicinchoninic acid protein assay kit (BCA1, Sigma). Data were presented as the mean RFU/mg protein ± SEM established from n = 3 wells per one experiment from three independent experiments.

Western Blotting Analysis

For Western blot analysis, cortical neurons were cultured in six-well plates coated with poly-ornithine (0.05 mg/ml) and after 7 DIV were treated for 6 and 18 h with Tian (0.1 and 10 μM) and St or Dox. For preparation of the whole cell lysates, cells were washed with ice-cold PBS and harvested and lysed with ice-cold RIPA buffer (150 mM NaCl, 1.0 % IGEPAL CA-630, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris, pH 8.0) in the presence of a cocktail containing protease inhibitors. For measurement of phosphorylated forms of kinases (pERK), the lysis buffer was enriched with heat-activated sodium orthovanadate and phosphatase inhibitors (cocktail I and II). Cell lysates were centrifuged at 20,000×g for 15 min at 4 °C and the supernatants were stored at −20 °C until further use. Protein amounts were determined with the BCA method and equal amount of proteins was denatured in a modified Laemmli sample buffer (0.25 M Tris–HCl pH 6.8, 10 % SDS, 40 % glycerol, 10 % 2-mercaptoethanol, 0.5 % bromophenol blue) and boiled for 3 min. An equal amount of protein from experimental groups was separated on 10 % SDS–polyacrylamide gel and transferred onto a PVDF membrane. Membranes were blocked for 1 h with 5 % nonfat milk in TBS-T (Tris-buffered solution pH 7.5/0.005 % Tween 20) and incubated overnight with primary antibodies diluted at 1:500 (spectrin α II), 1:1000 (pERK) and 1:2,000 (ERK2) in 1 % nonfat milk in TBS-T. The amount of ERK2 was determined on the same membrane on which the level of pERK and spectrin α II of were measured by stripping and reprobing the membrane as described previously (Jantas et al. 2011). The primary antibody reaction was followed by 1 h incubation with relevant secondary antibodies connected with horseradish peroxidase. Immunocomplexes were detected using an enhanced chemiluminescence detection system (Roche) and band intensities were determined by densitometric analysis of immunoblots (Fuji Film Las 4000). MultiGauge v.3 Software was used for quantification of Western blot signals. Data from duplicate determinations in three independent experiments were normalized to ERK2 level in particular samples and were shown as fold of control (mean ± SEM).

Data Analysis

Data after normalization were analyzed using the Statistica software (StatSoft Inc., Tulsa, OK, USA). The analysis of variance (one-way ANOVA) and post-hoc Tukey’s test for multiple comparisons were used to show statistical significance with assumed p < 0.05.