Experimental design, mice anesthesia, BrdU treatment and iron therapy
All animal experiments were performed in accordance with ethical standards and the procedures were approved by the Hebei normal university ethics committee. Postnatal 14 days C57BL/6 male mice were fed in stainless steel rust-free cages at 22–24° C and a 12-h light/dark cycle and were provided free access to the food and distilled water. The mice were assigned into four groups: control group, Sev treatment group, iron treatment group, Sev+iron treatment group. Anesthesia treatment was according to our previous report. Briefly, the mice in the anesthesia groups were placed in an anesthetic induction chamber filled with Sev (2%) for approximately 5 min until they became unconscious. The mice were then removed and attached to one of the nose cones of the anesthetizing apparatus in order to be exposed to the same amount of anesthetic. The mice were treated with gases of 2% Sev and 100% oxygen within the anesthetic chamber for 6 hours and were continuously monitored (Ohmeda Excel 210 SE anesthetic machine, Datex Instrumentarium Corp., Helsinki, Finland). BrdU labeling, behavioral test, and iron therapy of mice are shown in Figure 1.
Cell culture and anesthesia
NE4C cells, which were neuron stem cells from mouse, were grown in MEM supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. Cells were maintained at 37 °C in a humidified 5% CO 2 /95% air incubator. To investigate the effect of Sev on the NE4C cells, when the cells were in logarithmic phase, we treated the NE4C cells with 2% Sev for 6 h in an anesthetic chamber in a carbon dioxide incubator with 5% CO 2 at 37 °C. Then the cells were harvested to be assayed.
Morris water maze (MWM) test
MWM test was done according to our previous report . Briefly, for mice in control group, each mouse was given swimming training one time a day for 6 days. For Sev group mice, the mice were first anesthetized and then they were given the MWM test. Then, these mice were given swimming training one time a day for 6 days. On the sixth day, the platform was removed to test the times of crossing platform. For each mouse, the escape latency was recorded as a maximum of 120 s.
Immunohistochemistry studies and immunofluorescence assay
For immunohistochemistry assay, the brains were carefully dissected, removed, post-fixed, and then transferred to 30% sucrose for 2 days. The coronal brain sections were cut and frozen with a thickness of 15μm, and then sections were washed with 0.01 M PBS three times for 5 min each time. The sections were incubated with 3%
H2O2 for 20 min and then were washed with 0.01 M PBS three times for 5 min each time. The sections were then incubated with goat serum at 37 °C for 60 min. The samples were incubated overnight at 4 °C with the rabbit polyclonal anti-MBP antibody (Cat. No.10458-1-AP, Proteintech, Wuhan, China) and anti–CC1 antibody (1:250; Cat. No. ab16794, Abcam, Massachusetts, USA). Next, they were incubated with biotinylated secondary goat anti-rabbit IgG at 37 °C for 1 h. After being washed with PBS three times for 5 min each time, the sections were incubated in horseradish peroxidase (HRP)-labeled streptavidin reagent for 1 h at room temperature and then were stained using a DAB kit (SK-4100; VECTOR, Kronshagen, Germany) for 40 s. Finally, the sections were dehydrated and mounted. Images were captured using a ZEISS LSM710 (LSM710; ZEISS, Germany).
For the immunofluorescence assay, mouse monoclonal anti-NeuN antibody (1:200; Cat. No. ab104224, Abcam, Massachusetts, USA), rabbit monoclonal anti-FtL (1:400; Cat. No. ab109373, Abcam, Massachusetts, USA), and rabbit monoclonal anti-FtH (1:400; Cat. No. ab183781, Abcam, Massachusetts, USA) were used as primary antibodies. FITC-conjugated and rhodamine-conjugated secondary antibodies were used. Images were captured using a ZEISS LSM710 (LSM710; ZEISS, Germany).
Perl's iron staining
The brain slices were washed with 0.01 mol/L PBS (pH 7.4) for three times, for 5 min each, and then the slices were incubated with 3% H2O2 for 20 min. The slices were washed with 0.01 mol/L PBS (pH7.4) for three times, 5 min each again. The slices were stained using Perl's dye liquor for 8 h and then were washed with 0.01 mol/L PBS (pH7.4) for three times, 5 min each. Finally, the slices were strengthened using dyeing with DAB-H2O2 solution and then were washed by double-distilled water for 30 min. After being washed with gradient of ethanol and xylene, the slices were packaged by neutral gum, and the images were taken with the microscope (ZEISS Axio imager).
Western blot analysis
The hippocampal and cortex tissues were homogenized in RIPA buffer followed by centrifugation at 12,000 g for 20 min at 4 °C. The supernatant containing proteins was collected and its was measured content using protein quantification kit (KangWei, Beijing, China). The samples were resolved by 8–12% of SDS–PAGE, respectively, and then transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The target proteins FtH, FtL, NeuN, Ki67, and CC1 (Abcam, USA), MBP (Proteintech, Wuhan, China), TfR1 (ThermoFisher Scientific, USA)，Pax6 (Biolegend, USA), IBA1, and GFAP (Millipore, USA) were detected by their primary antibodies. The relative expression quantity of proteins was normalized to that of β-actin (KangWei, Beijing, China).
All experiments were performed at least in triplicate. One-way ANOVA or t-test was used to estimate the overall significance, followed by the post hoc Tukey's test corrected for multiple comparisons. The data are presented as means ± standard error of mean (SEM). A probability level of 95% (P < 0.05) was considered significant.