Pregnant C57BL/6 mice at 16–18 days' gestation and adult male C57BL/6 mice were purchased from the Animal Center of Drum Tower Hospital, Nanjing, China. All adult male C57BL/6 mice (25-30g) were raised in a 12-h light/dark cycle environment with free access to food and water.
7 pregnant mice and 65 fetuses were used in vitro experiments. The primary cortical neurons were randomly assigned into five groups: Control group, H2O2 group, H2O2 + Au groups (50 μg/ml, 100 μg/ml, 200 μg/ml) for protein extraction (n = 3 for each group). In addition, three groups of neurons: Control group, H2O2 group and H2O2 + group (200 μg/ml) were used for immunofluorescence staining and M29,79-dichlorodihydrofluorescein diacetate (DCFH-DA) staining (n = 3 for each group).
120 mice (130 mice underwent the operation, 120 survived) were used in the experiments. The mice were randomly allocated into four groups: Sham group, TBI group, TBI + Au group (20 mg/kg and 40 mg/kg). We performed modified Neurological Severity Scores (mNSS), rotarod test, and Morris Water Maze (MWM) test, respectively (n = 8 for each group). The other mice were sacrificed 3 d after trauma to measure brain water content (BWC) (n = 6 for each group).
96 mice (107 mice underwent the operation, 96 survived) were randomly assigned to four groups: Sham group, TBI group, TBI + Au group (20 mg/kg and 40 mg/kg). All mice in this experiment were sacrificed at 3 d after trauma. Six mice in each group were used for western blot (WB), quantitative real time polymerase chain reaction (q-PCR) and enzyme linked immunosorbent assay (ELISA), respectively (n = 6 for each group). Brain tissues from the remaining mice were used to make paraffin slices for TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, Nissl staining, immunofluorescence (IF) staining and immunohistochemistry (IHC) staining (n = 6 for each group).
First, 48 mice (56 mice underwent the operation, 48 survived) were divided into four groups (n = 6, each group): Sham group, TBI group, TBI + negative control (NC) group and TBI + lentiviral vectors (LV) group, and sacrificed at 3 d after TBI for WB and q-PCR to verify the effectiveness of the Nrf2-specific shRNA. Then, 30 mice (35 mice underwent the operation, 30 survived) were randomly assigned to five groups (n = 6, each group): Sham group, TBI group, TBI + Au (40 mg/kg) group, TBI + NC+ Au (40 mg/kg) group and TBI + Lv + Au (40 mg/kg) group for WB.
Primary cortical neuron culture
The skull, blood, meninges and were carefully removed from the fetal mice brain. After cortical tissue digested within 0.25% trypsin (Gibco, USA) for 5 min at 37 °C, the suspensions with foetal bovine serum (biological industries, USA) were passed through filters with 22 μm mesh size (Millipore, USA) and then were centrifuged at 1500 rpm for 5 min. The supernatant was discarded and the pellets were resuspended in Dulbecco’s Modified Eagle Medium (Gibco, USA). The cells were distributed in poly-D-lysine-coated plates. Four hours later, the medium was replaced with neurobasal medium supplemented with streptomycin, penicillin, HEPES, glutamate and B27 (Gibco, USA). Half of the neurobasal medium was refreshed every 2 days. After 7- 8 days culture, neurons were used in vitro experiments.
In vitro and in vivo model establishment
For in vitro studies, the primary cortical neurons were incubated with H2O2 dissolved in neuronal culture medium at a final concentration of 100μM for 12 hours according to published research with minor modification. Then, the neurons were collected for WB, IF staining and DCFH-DA staining. For in vivo experiments, we used the TBI model induced by weight-drop. Mice were anesthetized with intraperitoneal sodium pentobarbital (40mg/kg), and then placed onto the platform of the weight-drop apparatus. After disinfection, mice scalps were cut with a longitudinal midline incision to expose the skull. Then, the weight-drop device with a 200 g was released from a height of 2.5 cm, to cause focal trauma on the hemisphere 1.5 mm lateral to the midline on the mid-coronal plane. Mice with scalp incisions were sutured and returned to their cages and awaked from anesthesia. Mice in Sham group underwent the same procedures except for the weight drop.
For in vitro studies, Au (cat# HY-N0664, MedChemExpress, USA) was dissolved in neuronal culture medium at concentrations of 50 μg/ml, 100 μg/ml, or 200 μg/ml. After stimulating cells with 100μM H2O2, the various concentrations of Au were added immediately and given again at 6 h after H2O2 administration. For in vivo studies, Au was dissolved in normal saline to reach final concentrations of 4 mg/ml. The intraperitoneal injection of Au (20 mg/kg or 40 mg/kg) were at 30min, 12h, 24h and 48h after TBI. In the RNA interference experiments, the mice were intraperitoneally injected with Au (40 mg/kg) at 30 min, 12 h, 24 h and 48 h after TBI establishment.
Preparation of paraffin-embedded sections
Anesthetized mice were perfused 0.85% solution followed by 4% paraformaldehyde and the brain tissues were removed. After immersion in 4% paraformaldehyde for 24h, the brain tissues were dehydration in 45%，55%，65%，75%，85%，95% and 100% ethanol for 30 min, respectively. Then, the tissues were vitrification in 50% xylene (diluted with 100% ethanol) for 10 min and 100% xylene twice for 40 min. Then, brain tissues were made into paraffin blocks, that were cut into 6 μm sections. The paraffin sections were dewaxed in 100% xylene twice for 10 min, 100% ethanol twice for 10 min, 95% ethanol for 5 min, 85% ethanol for 5 min and 70% ethanol for 5 min, respectively. After washing two times with distilled water for 10 min, the slices were boiled in a microwave with citrate buffer solution (cat# P0083, Beyotime, China) for 15 min to retrieve antigens, and then were used for IF staining, Nissl staining, TUNEL staining and IHC staining.
The neurons and brain tissue (Additional file 1: Figure S1) were collected for WB analysis. The procedure of nuclear and total protein extraction was according to manufacturer’s instructions. Equal protein amounts were separated using polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes blocked in 5% skim milk for 2 hours at indoor temperature. Then, the membranes were incubated with primary antibodies against Nrf2 (1:1000, cat# ab137550, Abcam), Histone H3 (H3) (1:5000, cat# AF0009, Beyotime, China), NQO-1 (1:1000, cat# ab34173, Abcam), HO-1 (1:1000, cat# ab13248, Abcam), B-cell lymphoma-2 (Bcl2) (1:200, cat# 196495, Abcam), Bcl-2 Associated X Protein (Bax) (1:200, cat# ab32503, Abcam), cleaved-caspase 3 (CC3) (1:1000, cat# 9664, Cell Signaling), matrix metalloprotein-9 (MMP-9) (1:1000, cat# PB10008, Boster), glutathione peroxidase 1 (GPx1) (1:1000, cat# ab22604, Abcam), superoxide dismutase 1 (SOD1) (1:200, cat# sc-101523, Santa Cruz), Iba-1 (1:1000, cat# ab178846, Abcam), HMGB1 (1:100, cat# sc-56698, Santa Cruz), TLR4 (1:200, cat# sc-30002, Santa Cruz), MyD88 (1:200, cat# sc-74532, Santa Cruz), NF-κB p65 (1:1000, cat# 8242, Cell Signaling), inducible Nitric Oxide Synthase (iNOS) (1:1000, cat# 13120, Cell Signaling), Cyclooxygenase-2 (COX2) (1:500, cat# sc-19999, Santa Cruz), Interleukin-1β (IL-1β) (1:500, cat# 12242, Cell Signaling) or β-actin (1:3000, cat# BS6007M, Bioword) overnight at 4 °C. After washing 3 times for 15 min with Tris-buffered saline with Tween 20, the membranes were incubated with corresponding HRP conjugated secondary antibodies (1:5000, Bioword) for 1 h at room temperature. The protein bands were detected using enhanced chemiluminescence (ECL). ImageJ software were used to measure the optical density of protein bands.
The brain sections with antigen retrieval or cultured cells were treated with QuickBlock™ Blocking Buffer (cat# P0260, Beyotime, China) for 30 min at room temperature and then incubated with primary antibodies against Nrf2 (1:100, cat# ab137550, Abcam), MAP2 (1:200, cat# sc-32791, Santa Cruz), NeuN (1:200, cat# MAB377, EMD Millipore) and Iba-1 (1:100, cat# ab178846, Abcam) overnight at 4 °C. The cells or slides were incubated with corresponding secondary antibodies Alexa Fluor 594 and/or Alexa Fluor 488 (1:200, Jackson ImmunoResearch Incorporation, West Grove, PA, USA). Then, the cells or slides were washed in phosphate buffered saline-Tween twice for 20 min and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (1:2000, cat# ab104139, Abcam) for 5 minutes. Fluorescence was captured on a Zeiss HB050 inverted microscope system. ImageJ software were used to measure the inflorescence intensity.
The procedures of ROS measurement were according to manufacturer’s instructions. After stimulating with H2O2 for 12 hours, the neurons were incubated with DCFH-DA (cat# S0033, Beyotime, China) for 10 min at 37 °C. Following three washes with serum-free medium (Gibco, USA) for 1 min, the cells were immediately photographed under an inverted fluorescence microscope. The mean fluorescence intensity was analyzed using ImageJ software.
After antigens retrieval, the sections were treated with 3% H2O2 for 15 min and QuickBlock™ Blocking Buffer (cat# P0260, Beyotime, China) for 30 min at room temperature. Then, sections were incubated with primary antibodies against HO-1 (1:200 cat# ab13248, Abcam), NQO1 (1:200, cat# ab34173, Abcam) and 8-hydroxyguanosine (8-OHdG) (1:200, cat# sc-393871, Santa Cruz) overnight at 4 °C. The slides were washed twice with phosphate-buffered saline (PBS) and incubated with biotinylated secondary antibody (1:500, Vector, Burlingame, CA, USA) and horseradish peroxidase (HRP)-streptavidin reagent (Vector, USA). Then, the sections were re-stained with hematoxylin and we measured immunoreactivity using 3,3-diaminobenzidine (DAB, Zsgb-bio, China). Images were pictured by a microscope. ImageJ software was used to analyze the IHC images.
Brain water content
The brain water content was performed at 3 d after TBI. The entire brain was divided into injured hemispheres, cerebellum and brainstem. Each part was weighed immediately to obtain the wet weight. Brain tissue was dried at 80 °C for 72 h and re-weighed again to calculate dry weight. The brain water content was calculated as [(wet weight – dry weight)/wet weight] ×100%.
Neurologic function testing
The mice were assessed using mNSS test on days 1 and 3 after TBI according to previous studies. The mNSS consists of motor (muscle status and abnormal movement), sensory (visual, tactile and proprioceptive), reflex, and balance tests. 1 score is awarded for the inability to perform the test or for the lack of a tested reflex. The higher score represents the more serious neurological impairment (normal score, 0; maximal score, 18). Detailed score criteria was shown in Additional file 1: Figure S2.
On the 24th day after the trauma, the mice were performed the MWM test with minor modifications to assess their cognitive functions. Visual cues of figures were hung on the wall of the tank. During the acquisition phase, the mice were continuously trained for 5 days, with 3 trials per day. In each trial, mice were given 1 min to find a submerged platform 1 cm below the surface of the water. When arrival to the platform, mice were allowed to stay on the platform for 15 s. If failed, mice were guided to the platform and stay for 30 s. The mice were subjected to the probe trials on next day after 5 consecutive training. The platform was removed from the tank, and then mice were placed in the quadrant opposite the platform to seek platform for 1 min. The ANY-Maze video tracking system was used to videotape the whole process and data.
Mice were received three days of rotarod test training before TBI induction and then placed in a neutral position on an accelerating rotating rod (from 5 to 40 r/min within 5 min). The blinded experimenters recorded the latency to fall of each mouse. An average latency of three trials in one day represented the mouse motor performance. The test was performed before TBI and 0, 3, 7 and 14 days following TBI.
TUNEL staining and Nissl staining
Antigen-repaired brain sections were incubated with TUNEL reaction mixture (cat# C1089, Beyotime, China) for 1 hour at room temperature. After two washes with PBS for 20 min, the slides were incubated with DAPI for 5 min. Then, the brain sections were washed twice again and exposed under an inverted fluorescence microscope. The TUNEL-positive cells were counted by ImageJ software. The number of apoptosis cells to DAPI was regarded as an apoptosis index (apoptosis cells/DAPI). For Nissl staining, the sections were stained in Nissl staining solution (cat# C0117, Beyotime, China) for 5 min, washed with double- distilled water and mounted with permount. The pictures were captured under a light microscope. The neurons with visible nuclear and relatively complete cellular morphology were counted by ImageJ software.
Contents of malondialdehyde (MDA), SOD, ROS, GSH and GSH-Px measurements
Levels of MDA, SOD, ROS, GSH and GSH-Px in serum and brain tissue were measured using ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions at 3 d after TBI.
The transfection of LV expressing Nrf2-specific shRNA or negative control shRNA for mice in vivo were conducted three days prior to TBI modeling. After intraperitoneal anesthesia with sodium pentobarbital (40mg/kg), mice with scalp incisions were placed in a stereotaxic device, then a cranial burr hole (2.5 mm in depth, 1.2 mm lateral from midline, and 0.4 mm posterior from the bregma) was drilled. The mice underwent injection with 4 μL lentiviruses into the lateral ventricles (2 μL/min). The needle remained in place for 30 s after completing the infusion and the scalp incision was sutured. The mice in Sham, TBI and TBI + Au groups received a cranial burr hole, but not intracerebroventricular (ICV) injection. The experimental TBI was established 3 d after ICV injection. The Nrf2 shRNA sequence was 5’-AAGCCTTACTCTCCCAGTGAATCGAAATTCACTGGGAGAGTAAGGCTT-3’ and a non-targeting RNA sequence serving as a negative control.
Quantitative real-time PCR
Q-PCR was performed as previously described. Total mRNA was isolated from tissues (Additional file 1: Figure S1) using the Trizol reagent (TAKARA, Japan) and measured using spectrophotometric analysis (OD260/OD280). After reverse transcription of total mRNA into cDNA, q-PCR analysis was performed with SYBR Green qPCR Master Mix. Nrf2 forward and reverse primers were 5ʹ-CTACTCCCAGGTTGCCCACA -3ʹ and 5ʹ-CGACTCATGGTCATCTACAAATGG-3ʹ, respectively; HO-1 forward and reverse primers were 5′-GCTGGTGATGGCTTCCTTGTA-3′ and 5′-ACCTCGTGGAGACGCTTTACAT-3′, respectively; NQO-1 forward and reverse primers were 5′-ACGACAACGGTCCTTTCCAGA-3′ and 5′-CAGAAACGCAGGATGCCACT-3′, respectively; β-actin forward and reverse primers were 5ʹ-GACAGGATGCAGAAGGAGATTACT-3ʹand 5ʹTGATCCACATCTGCTGGAAGGT-3′, respectively. All samples were analyzed in triplicate with normalization to the β-actin in the Sham group. Relative quantification of mRNA expression was measured using the 2-ΔΔCT method.
The data were expressed as mean ± standard deviation (SD) and analyzed using an analysis of variance (ANOVA) followed by Tukey’s (one-way) or Bonferroni's (two-way) multiple comparisons post-hoc test. Values of P < 0.05 were considered statistically significant. Analyses were performed using SPSS version 20.0 software.