Animal care and drug treatment
A total of 136 adult male CD1 Elite mice (22-28 g, 10-12 weeks; Jining, China) were used in this study. A total of 20 adult male Nrf2 knockout (Nrf2−/−) CD1 male mice were obtained from Dr. Chunyan Li (Second Hospital of Hebei Medical Univercity). The Institutional Animal Care and Use Committee of Jining Medical University approved all the experiments, which were performed according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No.80-23, revised 1978). All efforts were made to reduce animal suffering and minimize the number of animals used for these experiments. The mice were housed in environmentally controlled conditions under a 12:12-h light/dark cycle at 25 °C and were allowed to acclimate to the animal facility for one week before the beginning of the experiment. The mice were then randomly assigned and evenly distributed among one of 5 groups: sham, TBI, TBI + low dose (0.5 µg/kg) of calcitriol, TBI + medium dose (1 µg/kg) of calcitriol, and TBI + high dose (3 µg/kg) of calcitriol. The animals received daily gavage of vehicle or different doses of calcitriol for 14 days. Moreover, to confirm the neuroprotective mechanisms of VitD, CQ (30 mg/kg/day) was co-treated with calcitriol by intraperitoneal injection to block autophagy flux.
Mouse model of TBI
A previously described mouse model of TBI was utilized in this study (10). Briefly, after anesthesia was induced with an intraperitoneal injection of 10% chloral hydrate (3 mL/kg), the head of the mouse was fixed on a stereotactic frame. Aseptic techniques were used throughout the surgery. A 10-mm-long midline scalp incision was made to expose the skull. A 4-mm-diameter bone window was created in the central aspect of the right parietal bone, 1 mm lateral to the sagittal suture. The integrity of the dura was carefully maintained throughout the operation. All the mice (including those in the sham operation and TBI-only groups) underwent anesthesia and the surgical procedure. TBI was induced by hitting the brain surface at the center of the craniotomy with a 2.5-mm-diameter rounded metal tip at a velocity of 4 m/s and a deformation depth of 2 mm using a controlled cortical impact (CCI) device (CCI Model 6.3; Custom Design, USA). The bone flap was immediately replaced and sealed, and the scalp was sutured. The animals were returned to the feeding room after recovering from anesthesia.
Neurological score evaluation
At 1–14 days following TBI, the neurological scores were determined as Neurological Severity Scores, a composite of motor, sensory, reflex, and balance tests (normal score: 2–3; maximal deficit score: 18; Supplementary Table 1).
The Morris water maze (MWM) test
The spatial learning ability of the mice was assessed through the MWM test. The apparatus consisted of a circular black water tank (180 cm in diameter, 50 cm high) filled with water (26 °C) to a depth of 30 cm and divided into four equivalent quadrants: north (N), south (S), east (E), and west (W). An escape platform (diameter 12 cm, height 28 cm, painted opaque) submerged 2 cm below the water surface was placed in the middle of one of the quadrants equidistant from the tank wall and the center of the pool. All the mice were trained to find the platform before the sham operation or the induction of TBI. For each trial, each mouse was randomly placed into a quadrant start point (N, S, E, or W) facing the wall of the pool and was allowed a maximum of 60 s to find the escape platform. The mice that failed to escape within 60 s were placed on the platform for a maximum of 20 s and returned to their cage to await a new trial (intertrial interval, 10 min). Maze performance was recorded using a video camera suspended above the maze and interfaced with a video tracking system (HVS Imaging, Hampton, UK). Probe trials were conducted at 11–14 days following the induction of TBI or the sham operation. The average escape latency of a total of 4 trials was calculated per day for 4 consecutive days. The time spent in the target quadrant and the swim speeds were evaluated on the last day of the test after the platform was removed.
At 14 days after TBI, mice were anesthetized and sacrificed, followed by perfusion with 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The tissues were removed and fixed for 48 h in 4% (w/v) paraformaldehyde. After fixation, the brains were embedded in paraffin and sliced into 4-µm coronal sections at the level of the bregma and stained with hematoxylin and eosin (H&E). Apoptosis was assessed using terminal deoxynucleotidyl transferase-mediated cyanine–dUTP nick-end labeling (TUNEL) following the manufacturer’s protocol. Nuclei were counterstained with DAPI (Beyotime Biotechnology, China). The number of TUNEL–DAPI-positive cells was counted, with the counting area being located in the same position for all the groups. For each group, sections from three different mice were used for quantification.
Transmission electron microscopy (TEM)
Neurons in the cortical region were examined via TEM at 14 days after TBI induction. Following perfusion, the brains were removed and the cortical region was dissected and washed in 0.1 M phosphate buffer. Tissue samples were immersed in 2% glutaraldehyde and 1% osmium tetroxide (Sigma–Aldrich; Merck KGaA) for 2 h at 4 °C, and then dehydrated via a graded ethanol series. Following the displacement of ethanol with propylene oxide, the tissues were embedded in Epon (both from Sigma–Aldrich) and sectioned along the coronal plane with a diamond knife (FernAnclez-hIorln 1953; Ivan Sorvall, Inc., New York, NY, USA) at a thickness of 60 nm. The sections were stained with lead citrate and observed using a CM-120 electron microscope (Philips, Eindhoven, Netherlands). In order to quantify the alteration of the number of the autolysosomes, the area of the cell cytoplasm was measured by using Image-Pro Plus 6.0.
Western blot analysis
For in vivo analysis, mice were anesthetized and the cortical region of the brain was homogenized in lysis buffer. Cell lysates were prepared by lysing the rat cortical neuron cultured in 24-well plates with SDS-PAGE buffer. Total protein was collected by centrifugation at 12,000 × g at 4 °C. Cytoplasmic and nucleic components were isolated using the Nuclei Isolation Kit from Sigma (NUC-201, MO, USA), according to the manufacturer's protocol. Proteins (50 µg) were separated by 12% SDS–PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk at room temperature for 2 h and then incubated overnight at 4 °C with primary antibodies against microtubule-associated protein light chain 3 (LC3) (1:500, PM036, MBL, Nagoya, Japan), p62 (1:1,000, ab101266, Abcam, Cambridge, MA, USA), beclin 1 (1:1,000, ab137161, Abcam), Keap1 (1:1,000, ab118285, Abcam), Nrf2 (1:1,000, ab62352, Abcam), beta-actin (1:1,500, A1978, Sigma, USA), and PCNA (1:500, ab92552, Abcam). The next day, the membrane was incubated with secondary antibody at room temperature for 2 h, and an enhanced chemiluminescence reagent kit was used for visualization. The gray values of the protein bands were measured using ImageJ software and normalized to that of β-actin that was used as an internal control.
The animals were anesthetized and perfused. The brains were then removed and postfixed in the same fixative for 1 day at room temperature, and then soaked in 30% sucrose for 2–3 days. The tissues were subsequently embedded in optimal cutting temperature compound. Frozen cross-sections (15 µm) were prepared and examined. Following three 10-min washes with phosphate-buffered saline (PBS), the sections were incubated with 5% donkey serum (Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China) and 0.4% Triton X-100 (Amresco, LLC, Solon, OH, USA) at room temperature for 2 h. The sections were incubated with primary antibodies against Keap1 (1:200, ab139729), p62 (1:200, ab56416), and Nrf2 (1:1,000, ab62352) (all from Abcam) overnight at 4 °C, and then with a mixture of FITC- and TRITC-conjugated secondary antibodies for 2 h at room temperature. After three 10-min PBS washes, the sections were stained with DAPI (Beyotime Biotechnology) to stain the cell nuclei. Images were captured under a ﬂuorescence microscope (Olympus, Japan). All the images were quantified using custom automated macros in Elements: nuclei were identified using a spot detection algorithm, while immunofluorescence-positive cells were identified using a regional maxima detection algorithm followed by global thresholding. The number of positive cells was normalized to the total number of cells imaged. Intracellular puncta were detected using spot detection and normalized to the number of cells imaged. Median cell numbers of 3,000 (cortical sections) and 200 (in vitro) were quantified at 20× magnification; 250 (cortical sections) and 50 (in vitro) at 60× magnification, per group per condition.
Real-time PCR analysis
The animals were anesthetized and the cortical region of the brain was removed. Total RNA was extracted using Trizol reagent (Invitrogen, USA) following the manufacturer's instructions. Quantitative PCR was performed on a Bio-Rad Cx96 Detection System (Bio-Rad, USA) using a SYBR green PCR kit (Applied Biosystems, USA) and gene-specific primers (Supplementary Table 2). Each cDNA was tested in triplicate. The thermocycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min. The relative mRNA levels were normalized to that of β-actin that was used as an internal standard.
Detection of oxidative parameters
The mice were anesthetized and the cortical region of the brain was removed. The generation of reactive oxygen species (ROS) was determined by fluorescence-labeled dihydroethidium (DHE). Frozen cross-sections (15 µm) were incubated in DHE for 30 min at 37 °C in a dark humidified chamber. The sections were rinsed three times in PBS and observed using an inverted fluorescence microscope (Olympus, Japan). The number of DHE–DAPI-positive cells was counted, with the counting area being located in the same position for all the groups. For each group, sections from three different mice were used for quantification. Malondialdehyde (MDA) levels were measured using the thiobarbituric acid reactive substances (TBARS) assay. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) were determined using SOD, CAT, and GSH assay kits, respectively (Nanjing Jiancheng Bioengineering Institute, China).
Primary cortical neuron culture
The skull, blood and meninges were carefully removed from fetal mouse brains. After the cortical tissue was digested in 0.25% trypsin (BI, Israel) for 5 min at 37 °C, the suspensions, containing fetal bovine serum (BI, Israel), were passed through filters with a 0.22-μm pore size (Millipore, USA) and then centrifuged at 1,500 rpm for 5 min. The supernatants were discarded, and the pellets resuspended in Dulbecco’s modified Eagle’s medium (BI, Israel). 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 (BI, Israel). Half of the neurobasal medium was refreshed every 2 days. The cells were exposed to different doses of calcitriol (1nM, 10nM, 100nM, 500nM) for 24 h after the cell density had reached approximately 70–80%. The cells were pre-treated for 6h with 25 μM CQ and then treated with the indicated doses of calcitriol.
Cell viability analysis
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the viability of cells that had been treated with different concentrations of calcitriol for 24 h. Briefly, MTT was added to each well of a 24-well plate followed by incubation at 37 °C for 1 h. The purple formazan crystals formed through the reduction of MTT were then dissolved in 500 µL of dimethyl sulfoxide (DMSO), and the absorbance of the wells was recorded at 590 nm. Cell viability was calculated by the absorbance ratio of the treated group to that of the control.
Autophagic flux analysis
Autophagic flux was detected by using the RFP-GFP-LC3 adenovirus (Hanbio, China). After plating the cells in a 24-well plate at a density of 1 × 104 cells/ dish and incubating with mRFP-GFP-LC3 adenovirus for 24 h. Autophagic flux was observed under an inverted fluorescent microscope (Olympus, Japan). The yellow puncta indicated autophagosomes, and the red puncta indicated autolysosomes.
The results were expressed as means ± SD. All the analyses were performed using SPSS 17.0 software. Statistical significance was determined using one-way analysis of variance (ANOVA), and the Student–Newman–Keuls post hoc test was used to determine differences among different groups. A P-value < 0.05 was considered statistically significant.