Design

A randomized, controlled, in vitro cell culture experiment.

Time and setting

Experiments were performed at the Brain Research Center, Ege University and Biochemistry Department, Faculty of Pharmacy, Ege University, Turkey from September 2009 to January 2010.

Materials

Neuronal restricted precursors and glial restricted precursors from embryonic neural stem cells were isolated from embryonic day 13.5 Sprague-Dawley rats (n = 5). The Appropriate Animal Care Committee of Ege University approved the protocol for the experiment. All efforts were made to minimize the number of animals used and their suffering.

Methods

Preparation of embryonic neural stem cells

The preparation of the neuronal restricted precursors and glial restricted precursors has been described previously^[59-60]. Briefly, embryos were isolated in Dulbecco’s Modified Eagle Medium/Nutrient F12 Ham’s (DMEM/F12; Sigma, St. Louis, MO, USA). Trunk segments (approximately 0.96 mm) were incubated in a solution of collagenase type I, dispase II, and Hanks balanced salt for 8 minutes at room temperature to remove the meninges from the brain and to remove all of the spinal cord. Cords were dissociated using a 0.05% trypsin and ethylenediamine tetraacetic acid solution for 20 minutes at 37°C. Cells were plated in the complete medium (DMEM/F12, bovine serum albumin, B27, basic fibroblast growth factor, penicillin-streptomycin, N2, and neurotrophin-3) on poly-L-lysine and laminin-coated dishes. After dissection, neuronal restricted precursors and glial restricted precursors were co-cultured for 5–10 days in the complete medium to generate a mixed population for grafting. Previous studies^[60-61] verified that these cultures contained only the precursors that were devoid of multipotent stem cells and mature cell types. The purity of the culture in lineage-restricted precursors was verified by staining the immature neural marker nestin. The neuronal restricted precursor/glial restricted precursor ratio was determined using embryonic neural cell adhesion molecule and A2B5. Embryonic neural stem cells were cultured at 37°C in 5% CO₂ 95% humidified atmosphere. The cells were cultured in DMEM containing: 25 mM glucose, 1 mM pyruvate,  4.02 mM l-alanyl-glutamine and 10% fetal calf serum (Sigma). The cells were cultured 1 day prior to the treatments. Confluent cells were detached with 0.15% trypsin for 5 minutes. Afterwards, the cells were mixed with 2 mL of complete medium and centrifuged at      1 000 r/min (180 × g) for 5 minutes. Cells were transferred to 96-well microtitre plates (Corning, Acton, MA, USA) with a cell number of 1 × 10⁴ cells per well (at the concentration of 1 × 10⁶/mL).

Cell treatment

Cells were exposed to different concentrations (10–150 µM) of hydrogen peroxide or 100 µM of hydrogen peroxide for 60 minutes to determine the appropriate concentration and exposure time for decreased neuronal survival. Cells were exposed to hydrogen peroxide (100 µM) and resveratrol (5–250 µM) for 30 minutes at 37°C to determine whether resveratrol has cytoprotective effects that attenuate the effects of hydrogen peroxide. After these experiments, the cells were divided into four groups as follows: (1) control group, cells treated with PBS; (2) hydrogen peroxide group, cells treated with  100 µM hydrogen peroxide for 30 minutes only; (3) hydrogen peroxide + resveratrol group, cells treated with hydrogen peroxide (100 µM) and resveratrol (100 µM) for 30 minutes; (4) hydrogen peroxide + trolox group, cells treated with hydrogen peroxide (100 µM) and trolox  (100 µM) for 30 minutes. Resveratrol (5–250 µM) and trolox (100 µM) were dissolved in PBS. Trolox, a well-known antioxidant and oxygen free radical scavenger, was used to calibrate the effects of resveratrol in embryonic neural stem cells. The concentration used for trolox (100 µM) was chosen according to a previous study in which trolox produced significant protection against oxidative stress in cultured hippocampal neurons^[43].

Lactate dehydrogenase assay

Viability of the cultures following hydrogen peroxide alone or hydrogen peroxide and resveratrol treatment was assessed using a lactate dehydrogenase efflux assay^[62-63]. Culture medium (90 µL) was assayed by adding 100 µL PBS containing pyruvate (1.54 mM) and nicotinamide adenine dinucleotid (0.5 mM). The conversion of nicotinamide adenine dinucleotid to oxidized nicotinamide adenine dinucleotide (NAD⁺) was monitored at 340 nm on a microplate reader (Molecular Devices Versamax BN 03252, Sunnyvale, CA, USA). Absorbance was recorded every minute for 5 minutes and the change in absorbance (ΔA_340nm) was determined. In the absence of media or lysate samples, ΔA_340nm/min was negligible over 5 minutes. Total lactate dehydrogenase activity was determined by adding ΔA_340nm/min in lysate and media samples. Lactate dehydrogenase release was determined by dividing ΔA_340nm/min in the media sample by total ΔA_340nm/min. Viability was determined by dividing total ΔA_340nm/min in treated samples by total ΔA_340nm/min for control samples, and expressed as a percentage. The neuronal survival following hydrogen peroxide alone or hydrogen peroxide and resveratrol treatment is represented as a percentage of control.

Catalase activity in embryonic neural stem cell cultures

The activity of catalase was determined spectrophotometrically^[64]. The reaction mixture consisted of 1 mL of PBS (50 mM, pH 7.0) and 2 mL of diluted cell homogenate. The mixture was incubated at 25°C for    3 minutes. The reaction was started by the addition of   1 mL of 30 mM hydrogen peroxide. The decomposition of hydrogen peroxide was followed directly by a decrease in absorbance at 240 nm at 25°C, as measured using a temperature-controlled Shimadzu UV-1601 spectrophotometer (Kyoto, Japan). The results are expressed as U/mg protein. One unit of activity is equal to 1 mM of hydrogen peroxide degraded per minute and is expressed as units per mg of protein.

Superoxide dismutase activity in embryonic neural stem cell cultures

We used xanthine and xanthine oxidase to generate superoxide radicals, which react with 2-(4-iodophenyl)-3- (4-nitropheno)-5-phenyltetrazolium-chloride (INT) to form red formazan dye^[65]. Superoxide dismutase activity was measured based on inhibition of this reaction and expressed as U/mg of protein. One unit of superoxide dismutase was defined by a 50% inhibition of the rate of reduction of INT under the conditions of the assay.

Glutathione peroxidase activity in embryonic neural stem cell cultures

Glutathione reductase and nicotinamide-adenine dinucleotide phosphate convert oxidized glutathione to its reduced form through oxidation of nicotinamide- adenine dinucleotide phosphate to NADP^+[66]. The decrease in absorbance at 340 nm was measured spectrophotometrically and expressed as U/mg of protein. One unit of glutathione peroxidase induces formation of  1 μmol NADP⁺ from nicotinamide-adenine dinucleotide phosphate per minute at pH 8.0 and 25°C.

Nitric oxide synthase activity in embryonic neural stem cell cultures

Nitric oxide synthase activity was measured using the nitric oxide synthase activity kit (Bioxytech, Oxis Inc., Dublin, USA), which determines total nitrite as an indicator of nitric oxide synthase activity. Nitrite production was measured during the timed reaction (60 minutes) and compared with heat-inactivated control samples. Nitrate reductase was used for the enzymatic reduction of nitrate to nitrite. Spectrophotometric quantitation of nitrite was performed using Griess reagents^[57]. In acidic solution, nitrite converts into HNO₂ (diazotied sulfanilamide). Sulfanilamide-diazonium salt reaction with N-(1-naphthyl)-ethylenediamine leads to chromophore production which was measured at 540 nm and expressed as nmol/mg protein/min^[57].

Measurement of nitric oxide levels in embryonic neural stem cells cultures

Chemical reduction of nitrate to nitrite was performed with the kit (Assay Designs, New York, NY, USA) following the manufacturer’s protocol. Afterwards, nitrite was measured with Griess reagent. In acidic solutions, nitrite converts to HNO₂. We used a sulfanilamide- diazonium salt reaction with N-(1-naphthyl)- ethylenediamine to produce the chromophore, which was measured at 540 nm and expressed as nmol/mg of protein^[57].

Single-cell gel electrophoresis (the comet assay)

Single-cell gel (comet) assay was carried out following the protocol described by Tice et al ^[41]. Slides were prepared in duplicate for each treatment. Thus, a volume of 10 µL of treated or control cells (~1 × 10⁴ cells) were added to 120 µL of 0.5% low melting point agarose at 37°C, layered onto a precoated slide with 1.5% regular agarose, and covered with a coverslip. After brief agarose solidification in a refrigerator, the coverslip was removed and slides immersed in lysis solution (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid; 10 mM Tris-HCl buffer pH 10; 1% sodium sarcosinate; with 1% Triton X-100 and 10% dimethyl sulfoxide) for approximately 1 hour. Prior to the electrophoresis, the slides were left in alkaline buffer (0.3 mM NaOH and    1 mM ethylenediaminetetraacetic acid, pH > 13) for    20 minutes and electrophoresed for another 20 minutes at 25 V (0.86 V/cm) and 300 mA. After electrophoresis, the slides were neutralized with 0.4 M Tris-HCl buffer  (pH 7.4) and stained using a silver-based method^[38]. The dried microscope slides were covered with a cover-glass prior to analysis with a light microscope (Olympus, Japan). The microscope was connected to a charge-coupled device camera and a computer with the Comet Assay Software Program version 1.2.2 installed to determine the extent of DNA damage after electrophoretic migration of DNA fragments in the agarose gel.

The three standard parameters of the assay, percentage of tail DNA, tail length (in μm) and the tail moment, were scored. Tail length is the measurement from the center of the head area (nucleus) towards the end of the tail, percentage of tail DNA is the percentage of DNA in the tail, and tail moment is a parameter calculated using the formula: Tail moment = tail length × percentage of tail DNA^[67]. The results are expressed as a percentage of tail DNA, tail moment, and tail length. All of the steps described above were conducted in the dark to prevent additional DNA damage. Throughout this study, several diluted and treated aliquots were tested for viability by trypan blue exclusion^[68-69].

Mitochondrial DNA isolation and agarose gel electrophoresis

Mitochondrial DNA was isolated from control and treated cells with a BioVision mitochondrial DNA isolation kit (BioVision, Mountain View, CA, USA) following the manufacturer’s protocol^[70]. The concentration of mitochondrial DNA was determined by measuring absorbance at A_260nm. The purity of the mitochondrial DNA was determined by the ratio of absorbance at A_260nm/280nm. The mitochondrial DNA was separated by agarose gel electrophoresis as described previously^[70], with some modifications. Briefly, mitochondrial DNA    (2 µg/µL) was dissolved in 5 µL of 6xgel loading buffer and then loaded into a well of a 0.5% agarose gel in a tris, boric acid, and ethylenediaminetetraacetic acid buffer (89 mM Tris boric acid, 2 mM ethylenediaminetetraacetic acid, pH 8.0). Electrophoresis was performed for 50 minutes at 80 V. After electrophoresis, the gel was stained with ethidium bromide (0.5 µg/mL in a tris, boric acid, and ethylenediaminetetraacetic acid buffer) and photographed. Densitometric analysis was performed using Biocapt software (Vilber-Lourmat, France).

Statistical analysis

All results are expressed as mean ± SD from five independent experiments for all indices. Comparisons of means between groups were performed by one-way analysis of variance followed by Tukey’s post hoc test. A P value less than 0.05 was considered statistically significant. The statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS) Version 13.0 for Windows (SPSS Inc, Chicago, IL, USA).

1 Resveratrol treatment can prevent hydrogen peroxide-induced oxidative stress due to the altered activities of catalase, glutathione peroxidase, nitric oxide synthase and nitric oxide levels.
2 Resveratrol treatment can also provide a protection against hydrogen peroxide-induced nuclear DNA or mitochondrial DNA damage in embryonic neural stem cells.
3 Resveratrol treatment has a neuroprotective potential against oxidative stress-induced damages in embryonic neural stem cells.
1 白藜芦醇可拮抗过氧化氢诱导的过氧化氢酶和谷胱甘肽过氧化物酶活性上升的作用。
2 白藜芦醇可拮抗过氧化氢诱导的一氧化氮合酶活性和一氧化氮水平上调的作用。
3 白藜芦醇可拮抗过氧化氢诱导的胚胎干细胞核DNA和线粒体DNA损害作用。

实验流程图：