CORM-2 (288144) and N-acetyl-cysteine (NAC) (A7250-10G) were purchased from Sigma-Aldrich (St. Louis, MO, USA).CORM-2 was dissolved in dimethyl sulfoxide, and then diluted in culture media to achieve the required concentrations. Inactivated CORM-2 (iCORM-2) was prepared by dissolving CORM-2 under the same conditions for 3 days at room temperature to liberate all CO from the molecule . NSC C17.2, a stable, fully characterized, mouse stem cell line , was kindly provided by Prof. Jin WL (Institute of Nano Biomedicine and Engineering, Shanghai Jiao Tong University, Shanghai, China). Ferrous chloride (FeCl2) was purchased from Sinopharm Chemical Reagent (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit (C1062), Diamidino-2-phenylindole dihydrochloride (DAPI) (C10005) and Reactive Oxygen Species Assay Kit (S0033) were from Beyotime Biotechnology (Shanghai, China). The primary antibodies Nrf2 (C-20) (SC-722) and NF-κB P65 (SC-372) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-NQO1 antibody (ab2346) was from Abcam (Cambridge, UK). ß-Actin (4970),Tublin(2148) and H3 (12167), were from Cell Signaling Technology (USA).
In vitro experiment
Cell Culture And Treatment
C17.2 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, USA) containing10 % (v/v) fetal bovine serum (Gibco, Carlsbad, CA, USA), 5 % (v/v) horse serum (Gibco), and 2 mM L-glutamine (Gibco) at 37°C in a humidified incubator supplemented with 5 % (v/v) CO2. C17.2 cells were pretreated with or without 50μM CORM-2 /iCORM-2 for 6 h prior to stimulation with 500μM FeCl2 for 24 h.
Western blot analysis
Western blotting was performed using standard techniques. Briefly, C17.2 cells were grown in 6-well cell culture plates (2×105 cells/mL). After treatment, Protein was loaded on 8 % (w/v) or 10 % (w/v) SDS-polyacrylamide gel and protein levels determined by Western blotting using Nrf2 (1:1000),NF-κB p65(1:1000),NQO-1(1:1000) and HRP-conjugated secondary antibody (1:5000).H3(1:5000) antibodies, and Tublin(1:5000) antibodies were used as marker proteins for nuclear and cytosolic extracts respectively.
RNA interference by small interfering RNA (siRNA) of Nrf2
Pre-designed siRNAs against mouse Nrf2 and nontargeting control-pool siRNA were purchased from GenePharma (Shanghai, China). For Nrf2-siRNA transfection, C17.2 cells were grown in 6-well plates (2 × 105 cells per well) until the confluence of cells reached approximately 50%. The cells were then subjected to transient transfection with Nrf2-negative control siRNA, Nrf2-siRNA using the siRNA transfection reagent Lipofectamine™ 2000, following the manufacturer’s protocol. After 48 h, the transfected cells were exposed to CORM-2/ iCORM-2 for 6 h, followed by lysis buffer for western blot analysis. Nontargeting siRNA construct (NC) was used as negative control.
Intracellular reactive oxygen species (ROS) measurement
Detection of intracellular oxidative states was performed by using the fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCFH-DA)  Briefly, C17.2 cells were grown in 6-well cell culture plates (2×105 cells/mL) and pretreated with CORM-2 (50μM) or iCORM-2 for 6 h prior to stimulation with 500μM FeCl2 for 24 h. the DCFH-DA solution (10μM) was added to the suspension of the cells. After incubation with DCFH-DA for for 20 min at 37°C, the fluorescence of DCF was quantified using fluorescence microscopy at excitation and emission wavelengths of 488 and 525 nm, respectively. ROS production was expressed as a percentage of the control. The fold-increase of ROS generation was compared with the control cells, which were arbitrarily considered as 1-fold.
Apoptosis was determined by using Annexin V-FITC apoptosis kits (Beyotime, China). The assays were performed according to the manufacturer’s instructions. Briefly, the cells were pretreated with CORM-2 (50μM) or iCORM-2 for 6 h prior to stimulation with 500μM FeCl2 for 24 h. After treatment, single cell suspension was incubated with 5 μL of Annexin V-FITC and 10 μL of PI in 195 μL of binding buffer for 15 min at room temperature in the dark. Then, the rates of apoptosis were analyzed by a flow cytometry (BD Biosciences, San Jose, CA). A minimum of 10,000 events were acquired for each sample.
NSCs cultured on eight-well chamber slides poly-D-lysine-coated glass slips in 24-well dishes were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. Then, cells were incubated for 1 h in blocking solution (PBS containing 3% bovine serum albumin and 0.3% Triton X-100). Cells were then incubated with antibodies against Nrf2 (1:200), NF-κB p65 (1:200) at 4 ℃ overnight. After three washes, samples were incubated with donkey anti-rabbit IgG (A21207; Alexa Fluor 594, 1:500; Invitrogen) or goat anti-rabbit IgG (A11008; Alexa Fluor 488, 1:500; Invitrogen) at 37 ℃ for 2 h. Samples were then washed with PBS and cover slipped with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlington, ON, Canada) nuclear counterstain. Samples were mounted onto slides with anti-fade solution and examined under a confocal laser-scanning microscope.
In vivo experiments
All animal procedures were carried out in accordance with the guideline of the institutional animal care and use committee of The Affiliated Wuxi No.2 Peoples' Hospital of Nanjing Medical University, Wuxi, China. Donate NSCs were harvested from homozygous red fluorescent protein transgenic (RFP Tg) mice (C57BL/6- RFP Tg; Model Animal Research Center of NanJing University, Nanjing, China). Wild-type C57BL/6 mice (Slac laboratory animal CO.LTD, Shanghai, China) were for HS models. All animals were housed on a 12: 12-h light/dark cycle with environmental temperatures at 18-22°C. Food and water were freely available.
Isolation and culture of fetal neural stem cells
NSCs were isolated from the subventricular zones of RFP Tg fetal mice at 14 days of gestation, as described previously . In brief, bilateral subventricular zones were dissected and mechanically dissociated. After collection, the cells were resuspended in Neuro-basal-A medium (Invitrogen, Carlsbad, CA, USA) containing B-27 supplement (Invitrogen), L-glutamine (Invitrogen), 20ng/ml mouse fibroblast growth factor-basic (PeproTech, Rocky Hill, NJ, USA), and 10 ng/ml mouse epidermal growth factor (PeproTech, Rocky Hill, NJ, USA). Cells were grown as suspending neurosphere. The medium was half-changed every 2 days and cells were passaged weekly. Cells that had been passaged 5 to 10 times were used for the experiments.
HS model and experiment group
We used an experimental HS procedure, described previously . Briefly, male C57BL/6 mice (8 weeks old, 20to25g) were anesthetized with ketamine/xylazine (100 mg/10 mg/kg, Sigma, St. Louis, MO), and positioned in the stereotactic frame.After a midline scalp incision, a hole was drilled in the right side of the skull (0.0mm anterior and 2.5mm lateral to the bregma). Blood (20 μl) without any anticoagulant was collected from the tail tip for injection. A 30-gauge needle attached to a 50-ml Hamilton micro-syringe was inserted 3.5mm ventral from the surface of the skull. Ten microliters of blood was injected over 10 minutes. The remaining 10 ml of blood was injected using the same procedure. After infusion, the needle was left in place for 25 minutes and then slowly removed.
Experimental groups were non-precondition group, transplantation of RFP-NSCs (3*105/2μl; n=15); iCORM-2-precondition group, transplantation of iCORM-2 preconditioned RFP-NSCs (3*105/2μl; n=15) and CORM-2-precondition group, transplantation of CORM-2 preconditioned RFP-NSCs (3*105/2μl; n =15). All methods and assessments described below were carried out by individuals blinded to the groups.
CO preconditioning and Intracerebral transplantation
CORM-2/iCORM-2 dissolved in DMSO was added to the cell culture medium (final concentration: 50 μM) for 6 h prior to transplantation, followed by drug washout before transplanting. NSCs were transplanted on the 3rd day after HS as described previously .
Assessment of survival of donate NSCs
HS animals were sacrificed on the thirtieth days after transplantation to detect the implanted NSCs. The survival of donate RFP Tg NSCs were assessed on 12 serial coronal sections per brain (0.25mm apart) using unbiased computational stereology.
Data were analyzed by using SPSS version 18 software. The significance of the difference among mean values was determined by the Student–Newman–Keuls test or two-way analysis of variance followed by the Bonferroni post hoc test. P values<0.05 were accepted to be statistically significant.