Reagents and experimental drugs
Trimethyltin was purchased from Sigma, USA. Primary antibodies (SYP&Siah-1, β-actin) were respectively purchased from Abcam, England and Sigma, USA. The secondary antibodies were purchased from LI-COR Abcam, England. Materials for Western blot, such as enhanced chemiluminescent (ECL) and Polyvinylidene difluoride (PVDF, 0.2μm) membrane were purchased from Elabscience Biotechnology, Wuhan, China. Zinc (Beyotime Biotechnology, Shanghai, China) reacts with dilute sulfuric acid to form hydrogen, passed the hydrogen into normal saline water placed in ice water for four to six hours to prepare for hydrogen rich water. All other chemical reagents and medicines are purchased through regular channels and are available.
Animals and experimental design
Forty-eight male BALB/c mice weighing 25-35 g were purchased from the animal center of the Chongqing Medical University(license numbers: SCXK(Yu)2012-001), Chongqing, China. Every four mice were kept in one cage, the temperature of the animal room was kept at 21 ± 2°C, and the mice were fed once a day with 12 hours under light (8:00 AM to 8:00 PM). All the animal feeding and raising were carried out according to the Guidelines of the Care and Use of Laboratory Animals of the Chongqing Medical University. All procedures and detection methods adopted in this study were approved and supervised by the Institutional Animal care and use Committee of the Chongqing Medical University.
Animals in control group received normal saline (4 ml/d, i.p.2 ml at 9:00 AM and 5:00 PM respectively) for seven days as vehicle. The HRW group was given hydrogen rich water intraperitoneally (4 ml/d, i.p.2ml at 9:00 AM and 5:00 PM respectively) for seven days which served as HRW control. The TMT group was administrated with TMT single i.p. injection (based as control group, 2.25 mg/kg, i.p. at 12:00 AM) at the seventh day at 12:00 AM. The dose of TMT and used in this study was based on our previous study[13]. In this process, the mice did not show any significant toxicity, including tremor, fighting and apparent damage. The TMT + HRW group animals was injected with TMT on the basis of the pretreatment of HRW (based as HRW group, treated TMT <2.25mg/kg, i.p.> at 12:00 AM at the seventh day), the processing method of this group was similar to the HRW group and TMT group, respectively. All the treatments were performed gently and all efforts were made to minimize animal suffering [27].
Morris water maze task
The Morris water maze task was designed to detect the spatial learning and memory impairment induced by TMT exposure and the protective effect of HRW. The tank (Tai Meng Technology, Chengdu, China) was divided into four equal quadrants, and this tank was 120 cm in diameter, 50 cm in height, the platform was 10 cm in diameter, the temperature of the water was about 24 to 26 ℃.
Before the task, the water was added into some mike powder to make it opaque, and the platform was placed in the center of any quadrant and immersed in water for about 1-2 centimeters. The day before the task, the mice could swim freely in the tank without the platform for one minute, ten times in total, to make them familiar with the Morris water maze. In the second day, every mouse was tested eight times consecutively [28]. The mice had eight chances to search the hidden platform and each chance for 60 seconds, the time of searching would be recorded. If the mice could not find the platform for themselves within 60s, then they were gently guided to the platform and the searching time would be recorded as sixty seconds. After the mice fond the platform, they could stay on the platform for 15 seconds to get familiar with the position of the platform. Completing all the tasks, the mice were dried and put back into the cage.
Immunohistochemical
At the end of the last Morris water maze task, about 0.4ml of 20% Urethane was intraperitoneally injected to anesthetize the mice, and then decapitated, and an appropriate amount of colorless formalin was infused through the cardia. After killing the mice, the brain tissue was quickly removed and fixed in 4% paraformaldehyde for use, or dehydrated by 75%-100% graded ethanol, cleared in xylene and embedded in paraffin. The thickness of the paraffin section was 5μm; HE staining was performed on every five sections.
Before dewaxing, the paraffin sections were placed at room temperature for 60 minutes, then the sections were immersed in xylene for 10 minutes and dehydrated in 100% - 75% graded ethanol. About 40 - 50ul polyclonal antibodies against SYP (1:1000) and Siah-1 (1:1000) were added to the tissue sample and placed them overnight at 4 °C. After placing overnight, rinsed the tissue sample three times with PBS, then added about 40 - 50ul secondary antibody at room temperature for 1 hour. After completing all the processes mentioned above, rinsed with PBS three times again.
SYP and Siah-1 were separately detected by DAB Horseradish Peroxides Color Development Ki. The images of tissue sample were taken by the Leica Microsystems (TYPE DM LB 2, Austria). By photographing the hippocampus and cortex of mice (DG, CA1, CA3), we could obtain the images of positive cells expressing SYP and Siah-1 proteins in the hippocampus and cortex.
Western blot
The cortex and hippocampus of mice were separated and frozen at - 80 ℃ for reserve,
tissue proteins were decomposed by RIPA solution (50 mM TrisHCL(PH7.5),150 mM Nacl, 1% NP-40, 0.5% Triton X-100, 0.1%SDS) and the total protein concentration was measured. The protein was separated by 10% SDS-PAGE and transferred to PVDF/NC membrane, then blocked the membranes with 5% skimmed milk powder. After blocking, added the primary antibodies (mice monoclonal anti-SYP antibody (1:1000), mice monoclonal anti-Siah-1 antibody (1:1000) and mice monoclonal anti-β-actin antibody (1:1000)) to incubate the membranes at 4°C overnight. After that, the membranes were rinsed in TBST (150mM NaCl, 50mM Tris(PH7.0), 0.05% Tween 20) three times. The Donkey anti-Mice IRDye800CW secondary anti-bodies (1:5000) for SYP, Siah-1 and β-actin were added to incubate the membranes at room temperature for one hour, and then rinsed in TBST three times again. Densitometry analysis of target protein expression would be measured by Odyssey Infrared Imaging System (LI-COR).
ICP-MS analysis
The mice were executed and the blood was removed by rinsing the tissues of the mice with ultra-pure water, 0.2mL blood and tissues samples were placed in Teflon digestion tank, 5.0mL digestion solution was added, and the temperature on the heating plate was kept at about 180℃ until the decomposition was colorless and transparent. When the solution is cooled, the nitric acid solution was added to 10mL. NexION 300X (PerkinElmer Company, USA) was used to measure the samples. The sample solution was introduced into the instrument for determination, the standard curve was drawn and the concentration of the target elements in the samples was calculated according to the regression equation. Specific machine parameters refer to other research reports [29].
Statistical analysis
The average escape latency of mice in Morris water maze was compared by repeated measurements of variance analysis. Western Blot results analysis: the gray values of total protein Siah-1 and SYP were compared with the gray values of β-actin, respectively. The rest of the data were analyzed by independent sample T test. All the data were processed by SPSS 22.0 software, expressed as mean±standard deviation, and P<0.05 suggesting that the difference was significant.