Animals
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.
Experiment design
Experiment 1
A total seven pregnant mice were used in the in vitro experiment. The primary cortical neurons were randomly assigned into five groups: Sham group, H2O2 group, H2O2 + Au group (50 μg/ml, 100 μg/ml, 200 μg/ml) for protein extraction (n = 3 for each group). In addition, three groups of neurons (Sham group, H2O2 group, H2O2 + Au 200 μg/ml group) were used for immunofluorescence staining and ROS activity analysis (n = 3 for each group).
Experiment 2
We used 120 mice (130 mice underwent the operation, 120 survived). The mice 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 (n = 8 for each group). The other mice were sacrificed on day 3 after trauma to measure brain water content (BWC) (n = 6 for each group).
Experiment 3
A total of 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). Brain tissue 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).
Experiment 4
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 were 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 group, TBI + Au + NC group and TBI + Au + LV group for WB.
Primary cortical neuron culture
Primary cortical neuron culture was performed as previously described[33]. Briefly, the skull, blood, meninges and hippocampus were carefully removed from the fetal mice brain. After cortical tissue digested within 0.25% trypsin for 5 min at 37 °C, the suspensions were passed through filters with 22 μm mesh size and were centrifuged at 1500 rpm for 5 min. The supernatant were discarded and the pellets were resuspended in neurobasal medium supplemented with streptomycin, penicillin, HEPES, glutamate and B27. The cells were distributed in poly-D-lysine-coated plates. We refreshed half of the medium 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[34]. Then, the neurons were collected for WB, IF staining and ROS activity analysis. For in vivo experiments, we used the TBI model described above [11]. In brief, mice were anesthetized 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 the Sham group underwent the same procedures except for the weight drop.
Drug administration
For in vitro studies, Au was dissolved in neuronal culture medium at concentrations of 50 μg/ml, 100 μg/ml, or 200 μg/ml. After stimulating cells with H2O2, the various concentrations of Au were added immediately and given again at intervals of six hours. 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 at 40 mg/kg.
Preparation of paraffin-embedded sections
Anesthetized mice were perfused 0.85% saline solution followed by 4% paraformaldehyde and the brain tissues were removed. After immersion in 4% paraformaldehyde for 24h, the brain tissues were dehydration and vitrification, then made into paraffin blocks, that were cut into 6 μm sections. After dewaxing and antigen repair, sections were used for IF staining, Nissl staining, TUNEL staining and IHC staining.
WB analysis
The neurons and brain tissue were collected for WB analysis. The procedure of nuclear and total protein extraction was performed as previously described[11]. 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), H3 (1:5000), NQO-1 (1:1000), HO-1 (1:1000), B-cell lymphoma-2 (Bcl2, 1:200), Bcl-2 Associated X Protein (Bax, 1:200), cleaved-caspase 3 (CC3, 1:1000), matrix metalloprotein-9 (MMP-9, 1:1000), GPx1 (1:1000), SOD1 (1:200), Iba-1 (1:1000), HMGB1 (1:100), TLR4 (1:200), MyD88 (1:200), NF-κB p65 (1:1000), inducible Nitric Oxide Synthase (iNOS, 1:1000), Cyclooxygenase-2 (COX2, 1:500), Interleukin-1β (IL-1β, 1:500) or β-actin (1:3000) 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) 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.
IF staining
The brain sections with antigen retrieval or cultured cells were treated with blocking buffer for 30 min at room temperature and then incubated with primary antibodies against Nrf2 (1:100), MAP2 (1:200), NeuN (1:200) and Iba-1 (1:100) overnight at 4 °C. The cells or slides were incubated with corresponding secondary antibodies Alexa Fluor 594 and/or Alexa Fluor 488 after washed in phosphate buffered saline-Tween twice for 20 min and counterstained with 4,6-diamidino-2-phenylindole (DAPI) for 5 minutes. Fluorescence was captured on a Zeiss HB050 inverted microscope system.
ROS detection
The procedures of ROS measurement were performed as described previously[35]. The H2O2-induced neurons were incubated with M29,79-dichlorodihydrofluorescein diacetate (DCFH-DA) for 10 min at 37 °C. The pictures were captured using an inverted fluorescence microscope. The mean fluorescence intensity was analyzed using ImageJ software.
IHC staining
The sections were incubated with primary antibodies against HO-1 (1:200), NQO1 (1:200) and 8-hydroxyguanosine (8-OHdG) (1:200) overnight at 4 °C after blocking for 30 min. The slides were washed twice with phosphate-buffered saline (PBS) and incubated with biotinylated secondary antibody and horseradish peroxidase (HRP)-streptavidin reagent. Then, the sections were re-stained with hematoxylin and we measured immunoreactivity using 3,3-diaminobenzidine (DAB). 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 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 our previous studies[33]. The mNSS consists of motor (muscle status and abnormal movement), sensory (visual, tactile and proprioceptive), reflex, and balance tests. The higher score represents the more serious neurological impairment.
MWM test
On the 24th day after the trauma, the mice were administered the MWM test with minor modifications to assess their cognitive functions based on previous experiments[36]. Visual cues of figures were hung on the wall of the tank. During the acquisition phase, the mice were trained to find a submerged platform 1 cm below the surface of the water. The mice subjected to the probe trials on next day after 5 consecutive training days. The platform was removed from the tank, and then mice were placed in the quadrant opposite the platform. The ANY-Maze video tracking system was used to videotape the whole process and data.
Rotarod test
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
TUNEL staining was performed as described previously[33]. In brief, antigen-repaired brain sections were incubated with TUNEL reaction mixture for 1 hours at room temperature. After washed 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. For Nissl staining, the sections were hydrated in 1% toluidine blue, washed with double-water and mounted with permount. The pictures were captured under a light microscope.
Contents of 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 according to the manufacturer’s instructions at 3 d after TBI.
RNA interference
The transfection of LV expressing Nrf2-specific shRNA or negative control shRNA for mice brains in vivo were conducted according to previously described methods[36]. After induction of anesthesia, 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 [37].
Quantitative real-time PCR
Q-PCR was performed as previously described[36]. Total mRNA was isolated from tissues using the Trizol reagent 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.
Statistical analysis
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.