Neuroprotective role of thymoquinone against 1-methyl-4-phenylpyridinium-induced dopaminergic cell death in primary mesencephalic cell culture

Preparation of primary mesencephalic cell culture

This in vitro study was conducted in the Chemistry and Biochemistry Institute, University of Veterinary Medicine, Vienna, Austria between June and August 2013 in accordance with the guidelines of the European Union Council (86/609/EU) for the use of laboratory animals. The work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation. Primary mesencephalic cell cultures were prepared from C57/B16 embryos according to Radad et al.10 To summarize, embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of Dulbecco’s phosphate-buffered saline (DPBS) (Invitrogen, Darmstadt, Germany), 2 ml of 0.2% trypsin solution (Invitrogen, Darmstadt, Germany) and 2 ml of 0.02% DNase I solution (Roche, Berlin, Germany) were added and the tissue was subsequently incubated in a water bath at 37°C for 7 minutes (min). Then, 2 ml of trypsin inhibitor (0.125mg/ml) (Invitrogen, Darmstadt, Germany) were added, the tissue was centrifuged at 100g for 4 min and the supernatant was aspirated. The tissue pellet was triturated 2-3 times with a fire-polished Pasteur pipette, each time 0.02% DNase I (Invitrogen, Darmstadt, Germany) was included in the medium. Dissociated cells were plated at a density of 257,000 cells/cm² in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Aldrich, Hamburg, Germany) supplemented with 4mM glutamine, 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 30 mM glucose, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated fetal calf serum (Sigma Aldrich, Hamburg, Germany). The medium was exchanged on the first day in vitro (DIV) and on the third DIV. On the fifth DIV, half of the medium was replaced by serum-free DMEM containing 0.02 ml B-27/ml (Invitrogen, Darmstadt, Germany) DMEM. Serum-free supplemented DMEM was used for feeding from the sixth DIV, and subsequently replaced every second day.

Study design and treatment of primary mesencephalic cell cultures

A stock solution of TQ (Sigma Aldrich, Hamburg, Germany) (10 mM) was prepared in dimethyl sulfoxide (DMSO). Final concentrations of TQ were prepared in DMEM. The DMSO concentration in the culture medium did not exceed 0.01%. Four sets of cultures were treated as follows: The first set of cultures was treated with DMSO and kept as untreated controls. The second set of cultures was treated with TQ (0.01, 0.1, 1, and 10 µM) on the eighth DIV for 4 consecutive days to investigate the effect of TQ on the survival of dopaminergic neurons. The third set of cultures was treated with 10 µM of MPP⁺ on the tenth DIV for 48 hours (h). The fourth set of cultures was concomitantly treated with TQ (0.01, 0.1, 1, and 10 µ), and 10 µM of MPP⁺ on the tenth DIV for 48 h.

Identification of tyrosine hydroxylase immunoreactive (THir) neurons

Dopaminergic neurons were identified immunocytochemically by staining tyrosine hydroxylase. Cultures were rinsed carefully with phosphate buffered saline (PBS, pH 7.2) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4°C. After washing with PBS, cells were permeabilized with 0.4% Triton X-100 for 30 min at room temperature. Cultures were washed 3 times with PBS and incubated with 5% horse serum (Vectastain ABC Elite kit, Biozol Diagnostica Vertrieb GmbH, Eching, Germany) for 90 min to block nonspecific binding sites. To determine the number of THir in cultures, cells were sequentially incubated with anti-TH primary antibody overnight at 4°C, biotinylated secondary antibody (Vectastain), and avidin-biotin-horseradish peroxidase complex (Vectastain) for 90 min at room temperature and washed with PBS between stages. The reaction product was developed in a solution of diaminobenzidine (1.4 mM) in PBS containing 3.3 mM hydrogen peroxide and stained cells were counted with a Nikon inverted microscope in 10 randomly selected fields per well at 10x magnification.

Measurement of lactate dehydrogenase activity

Cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase (LDH) released from damaged cells into the culture medium. The reaction was initiated by mixing 0.2 ml of cell-free supernatant (diluted 1:1 with aqua dest.) with potassium phosphate buffer containing ß-nicotinamide adenine dinucleotide (NADH) and sodium pyruvate (0.18 and 0.62 mM in potassium phosphate buffer) in a final volume of 0.5 ml in 1 ml cuvettes. The decrease of NADH was spectrophotometrically (NOVASPEC II, GE Healthcare Europe GmbH, Freiburg, Germany) monitored. Reagent blanks were subtracted. The LDH activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min-time period. The LDH release is proportional to the number of damaged or destroyed cells.11,12

Fluorescence staining

LysoTracker^® Deep Red (Life Technologies™, Invitrogen, Grand Island, NY, USA) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes. Cultures were treated with 1 µM of TQ (a concentration that significantly protected dopaminergic neurons in MPP⁺-treated cultures) on the eighth DIV and co-administered with MPP⁺ (10 µM) on the tenth DIV for 2 days. On the twelfth DIV, culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nM LysoTracker^® Deep Red fluorescence dye (Life Technologies™, Invitrogen, Grand Island, NY, USA) for 15-30 min at 37°C. After washing with DPBS, cultured cells were photographed on a Nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set (580/590, G-2A) and a Coolpix 990 digital camera (Nikon, Otawara, Japan). Three photos were taken randomly from each well. All photos were analyzed densitometrically by Adobe Photoshop^® software.

5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine (JC-1) is a lipophilic cationic dye that selectively enters into mitochondria. In healthy cells with high mitochondrial membrane potential (Δψm), JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence. The dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells. The JC-1 red:green ratio is used to estimate changes in Δψm.13 The JC-1 was dissolved in DMSO and further diluted in DMEM (10 µg/ml final concentration). After removal of the culture medium, cells were loaded with JC-1 for 15 min at 37°C, rinsed twice with PBS, and photographed on a Nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set (520 DM/520 BA, B-2A) and a Coolpix 990 digital camera (Nikon, Otawara, Japan). Three photos were taken randomly from each well. Fluorescence intensity of the red:green ratio was determined semi quantitively by using Adobe Photoshop^® software.

4’,6-diamidino-2-phenylindole (DAPI) is a fluorescent stain that binds strongly to DNA. It passes through intact membranes of both live and fixed cells. Cells were fixed with 4% paraformaldehyde for 45 min at 4°C. After washing with PBS (pH 7.2), cells were permeabilized with 0.4% Triton X-100 for 30 min at room temperature. The DAPI solution (2 µM final concentration) was added to the cultures at room temperature for 5 min in the dark. After washing with DPBS, 3 photos were taken randomly from each well with a Coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter (Nikon, Otawara, Japan). Nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with Adobe Photoshop^® software.

Data was obtained from 12 wells (from 2 repeats) for each treatment condition. Data were expressed as mean ± SEM. Comparisons were made using ANOVA and post-hoc Duncan’s test using the Statistical Analysis System program 1998 (SAS Institute Inc., Cary, NC, USA). P<0.05 was considered as statistically significant.