Animals and tumor cell lines
Male Wistar rats (8 weeks old, 170–180 g) and female BALB/c nude mice (8 weeks old, 20–24 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animals were maintained under standard conditions at 22 °C to 25 °C with a 12-h light-dark cycle and were fed a normal diet. All procedures were conducted in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (China).
Rat C6 glioma cells and human U87 cells were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA). Cells were routinely cultured at 5% CO2 and 37 °C in DMEM/F12 medium (Gibco, NY, USA) supplemented with 10% fetal calf serum (Gibco, NY, USA) and 1% penicillin and streptomycin (Gibco, NY, USA).
Rat C6 glioma model
Rats were fasted for 1 day before the experiments. They were then anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL/kg; Sigma, USA) and fixed in a stereotactic apparatus. The skin of the scalp was then incised in the midline of the skull with surgical scissors. Then, a hole was made in the cranial bone 1 mm posterior to the bregma and 3 mm lateral to the sagittal suture. A 30-gauge needle with a microsyringe was inserted to a depth of 7 mm from the skull surface; C6 glioma cells (1 × 106) in 10 μL phosphate-buffered saline (PBS) were injected stereotactically. The injection was conducted over 10 min, after which the needle was held in position for 5 min and then gradually withdrawn over 3 min to prevent the backward flow of the solutions. After implantation of glioma cells, the rats were randomly divided into two groups including the hydrogen inhalation group (HI) and control group (CTRL).
Mouse U87 subcutaneous model
BALB/c nude mice were injected subcutaneously with 1 × 106 viable U87 cells. After injection, the mice were randomly assigned to two groups including the hydrogen inhalation group (HI) and control group (CTRL). Tumor volumes were measured on a weekly basis using the following formula: volume = width2 × length × 0.4.
Inhalation of hydrogen gas
A transparent closed box (20 cm × 18 cm × 15 cm) was connected to an AMS-H-3 hydrogen-oxygen nebulizer machine (Asclepius Meditec Inc., Shanghai, China), which produces 67% H2 and 33% O2 (V/V). Hydrogen treatment was given on the second day of the rat C6 glioma or mouse U87 subcutaneous model establishment until the end of the experiment. Animals were placed in this box and inhaled the mixed air for 1 h two times per day. During this inhalation, mice were awake and freely moving. Thermal trace GC ultra-gas chromatography (Thermo Fisher, MA, USA) was used to monitor the concentration of hydrogen gas in the closed box.
Hydrogen-rich medium treatment
A hydrogen-rich medium was produced by placing a metallic magnesium stick (Doctor SUISOSUI®; Friendear Inc., Tokyo, Japan) into DMEM/F12 medium (final hydrogen concentration 0.55–0.65 Mm). The hydrogen concentration was monitored by using a needle-type Hydrogen Sensor (Unisense A/S, Aarhus, Denmark).
MR imaging studies were performed with the Bruker 7.0 T ClinScan high-field small-animal MRI system (Bruker BioSpin, Ettlingen, Germany). The tumor-bearing rats were anesthetized with 2% isoflurane in 2 L min−1 of oxygen and maintained at a normal body temperature. The T2-weighted MR images were acquired for each rat on days 17 and 26 after tumor transplantation using a conventional spin-echo sequence with the following parameters: TR = 3140 ms, TE = 37 ms, bandwidth = 130 Hz, flip angle = 180°, FOV = 4 cm × 4 cm, and slice thickness = 1 mm.
Histology and immunohistochemistry
Glioma tissues were embedded in paraffin and then cut into 8-μm-thick sections. Sections were treated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidases, followed by incubation with 10% normal goat serum. After the blocking serum was removed, sections were incubated overnight at 4 °C with primary antibodies including anti-CD34 (1:200; Abcam), anti-Ki67 (1:200; Abcam), anti-CD133 (1:50; Biobyt), and anti-Nestin (1:100; Millipore). Detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. All data were evaluated by blinded investigators.
Cells were seeded on coverslips, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 1% BSA for 1 h. Subsequently, cells were incubated overnight at 4 °C with primary antibodies including anti-GFAP (1:200; Abcam) and anti-CD133 (1:50; Biobyt). Cells were then incubated with FITC-conjugated secondary antibody (Abcam) for 1 h. The nuclei were stained with Hoechst, and the fluorescence images obtained with an Olympus IX71 inverted microscope.
The expression of CD133 in cultured cells was analyzed by flow cytometry. In brief, cells were incubated for 10 min with 10% horse serum in PBS at 4 °C, followed by incubation with primary anti-CD133 (1:200; Biobyt) at 4 °C for 30 min. After centrifugation, the collected cells were incubated with FITC-conjugated secondary antibody (Abcam) for 20 min. After staining, cells were subjected to flow cytometry for analysis using a flow cytometer (Guava easyCyte 8HT; Millipore).
Sphere formation assay
Cells were collected and washed to remove serum, then suspended in serum-free DMEM or MEM/EBSS medium at a density of 2.0 × 103/mL. Five hundred cells per well were counted and seeded onto ultra-low attachment six-well plates (Corning, Corning, NY) for the sphere formation assay. Cells were cultured in normal serum-free medium or hydrogen-rich serum-free medium with B27 supplement (GIBCO, Grand Island, NY), 20 ng/mL of epidermal growth factor (EGF) (Pepro Tech Inc., Rocky Hill, NJ), and 20 ng/mL of basic fibroblast growth factor (bFGF) (Pepro Tech). The medium was changed every 3 days. Cells were incubated for 10 days, and spheres with a diameter > 50 μm were counted.
Cell migration assay
The cell migration ability was examined using a wound-healing assay. Briefly, cells were seeded in six-well plates at a concentration of 1.0 × 106/well and cultured for 24 h. A plastic pipette tip was used to scratch a line across the cell surface in each plate. The remaining cells were washed three times with PBS to remove the floating cells and debris, followed by incubation for 48 h in normal complete medium or hydrogen-rich medium. The images of the healing process were photographed digitally at the time point of immediately following and 24 h after wounding. The wound-healing assay was performed in three independent experiments.
Cell invasion assay
The cell invasion ability was determined using BD matrigel invasion chambers according to the manufacturer’s protocol. Briefly, the top chambers with polycarbonate filters (8-μm pore size; Costar, Acton, MA) were coated with 50 μL of Matrigel (0.8 μg/μL, 37 °C, 2 h; BD Biosciences, San Diego, CA). 1 × 105 cells in a 100-μL serum-free medium were seeded to the top chamber, and a 650-μL normal complete medium with or without hydrogen was added to the bottom. The cells were allowed to migrate through the porous membrane at 37 °C for 48 h. Then, cells in the upper surface of the chamber were completely removed by cotton swabs. The cells on the lower surface were stained with 0.1% (w/v) crystal violet after fixation, and five random fields from each insert were counted at × 100 magnifications. The invasion assay was conducted in triplicate-independent experiments.
Colony formation assay
One thousand cells per well were counted and seeded in six-well plates. The plates were incubated for 14 days in a normal complete medium either with or without hydrogen, and then the cells were fixed by 4% paraformaldehyde and stained using 0.1% crystal violet. Colonies were counted only if they included at least 15 cells. Triplicate-independent experiments were performed, and all the visible colonies were calculated manually.
Groups from cell culture and in vivo experiments were compared using two-tailed Student’s t tests, and results are presented as means ± SEM. All statistical analyses were performed using GraphPad Prism 6.01. A value of p < 0.05 was considered significant.