1. Animal Preparation
All procedures relate to animals were approved by the Animal Care and Use Committee of Jinling Hospital. Adult male Institute of Cancer Research (ICR) mice (28–32 g) were purchased from the Animal Center of Qinglongshan (Nanjing, China). The mice were kept in cages with constant room temperature (25 ± 2ºC) and air humidity (50 ± 10%). They could have free access for food and water and the light/dark cycle was 12 h.
2. Experimental designs
This study was divided into two parts: in vivo experiments and in vitro experiments.
2.1 In vivo experiments
First, the cortical expression of S100A8 after TBI was assessed. Twenty-one adult male ICR mice weighing 28 to 32 g were randomly divided into 7 groups: the Sham (n = 3/group) and TBI [3, 6, 9, 12, 24, 72 hours (h)] (n = 3/group) groups. The sham mice were euthanized at 24 h after surgery and the TBI mice were euthanized at the indicated time-point after TBI. Brain samples were collected for Western Blot analysis. Six other mice were added into the Sham (n = 3) and TBI (n = 3) groups, which were euthanized at 24 h after TBI to collect the whole brains for immunofluorescence staining.
Then, one time point(24 h) was selected for further experiments according to the time-course of S100A8 expression. We randomly divided 105 male mice into 5 groups: (1) Sham group, (2) TBI group, (3) TBI + TAK-242 group, (4) S100A8 group, (5) S100A8 + TAK-242 group (n = 21 for each group). The sham mice were euthanized at 24 h after surgery. The mice in the TBI group were euthanized as mentioned later. TAK-242(MCE; Monmouth Junction, NJ, USA) was administered intraperitoneally to the mice in TBI + TAK-242 and S100A8 + TAK-242 groups at 0.5 h before TBI. In the S100A8 and S100A8 + TAK-242 groups, S100A8 recombinant protein was injected into the lateral ventricle of the brain according to the previous report [25]. Brain samples were collected for Western Blot (n = 3) and enzyme-linked immunosorbent assay (ELISA) (n = 3). Nissl staining (n = 3 for each group) was also performed. Besides, brain water content was measured using the wet/dry method (n = 6) as previously described[26] and neurological deficits (n = 6) were evaluated before they were euthanized.
2.2 In vitro experiments
BV-2 cells cultured in the six well plates were randomly divided into 5 groups: (1) Sham group, (2) S100A8 group, (3) S100A8 + TAK-242 group, (4) LPS group, (5) LPS + TAK-242 group. Cells were collected for Western Blot analysis (n = 3), and the cell-free supernatants were collected for ELISA (n = 3).
3. TBI model in mice
The weight-drop model of TBI in this study was followed Flierl et al. [27]. In brief, after the mouse was anesthetized, it was placed onto the platform under the weight-drop device. To expose the skull, a 1.5 cm midline longitudinal scalp incision in the mouse’s head was made. The target area, 1.5 mm lateral to the midline on the mid-coronal plane, was impact by an object which is 200 g weight. We used 0.9% normal saline solution to perfuse the mouse transcardially after it was deeply anesthetized via inhalation of isoflurane. At the end, the brain was collected for subsequent analyses.
4. Neurological scoring
The modified Garcia’s method [28] was used to calculate neurological score at 24 h after TBI by two investigators blinded to the grouping. The modified Garcia method consists of 6 tests, which were Spontaneous activity (in cage for 5 min), symmetry in the movement of four limbs, Symmetry of forelimbs (outstretching while held by tail), Climbing wall of wire cage, Reaction to touch on either side of trunk, and Response to vibrissae stimulation and its scores range from 3 to 18 (Table 1).
Table 1
Neurological Evaluation in Sham, S100A8, S100A8 + TAK-242, TBI and TBI + TAK-242 groups
Test | Score |
0 | 1 | 2 | 3 |
Spontaneous activity (in cage for 5 min) | No movement | Barely moves | Moves but does not approach at least three sides of cage | Moves and approaches at least three sides of cage |
Symmetry in the movement of four limbs | Left side: no movement | Left side: slight movement | Left side: moves slowly | Both sides: move symmetrically |
Symmetry of forelimbs (outstretching while held by tail) | Left side: no movement, no outreaching | Left side: slight movement to outreach | Left side: moves and outreaches less than right side | Symmetrical outreach |
Climbing wall of wire cage | … | Fails to climb | Left side is weak | Normal climbing |
Reaction to touch on either side of trunk | … | No response on left side c | Weak response on left side | Symmetrical response |
Response to vibrissae touch | … | No response on left side | Weak response on left side | Symmetrical response |
5. Brain water content
The brain water content was measured by the dry-wet weight method [26]. The anesthetized mice’s brain was removed. After removed the right cerebral hemispheres, brain stem and cerebellum, the remaining left cerebral hemispheres were collected and obtained the wet weight (WW). After that, we obtained the dry weight (DW) after the left hemispheres were dried at 72 ºC for 72 h. The results of brain water content were calculated as: (WW - DW)/WW × 100%.
6. BV-2 microglial cell culture
BV-2 microglial cells were purchased from Cobioer BioScience Co., Ltd (Nanjing, China) and were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) which contains 5% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (pen/strep; Gibco, Grand Island, NY, USA); TAK-242 was added to the cells at the concentration of 10 nM just before the stimulation of S100A8 protein or LPS. Cells were treated with the S100A8 recombinant protein at final concentrations of 0.5µ l/ml; LPS (Lipopolysaccharide, Sigma-Aldrich, St. Louis, MO, USA) was added to stimulate the BV-2 cell line at the final concentration of 0.1 µg/mL according to the previous study [30].
7. Western Blot analysis
In vivo study, the brain tissue located over the injure site was collected. The Total Protein Extraction Kit (Beyotime, Nantong, China) was used to extract protein according to the manufacture’s instruction. The BCA Protein Assay Kit (Beyotime, Nantong, China) was adopted to determine the protein concentrations. Equal amounts of protein were subjected to 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by being transferred to polyvinylidene–difluoride (PVDF) membranes. The membranes were blocked with 5% freshly prepared skim milk-1 for 2 h and then incubated overnight at 4 °C with following primary antibodies: S100A8 (1:1000; Abcam, Cambridge, MA, USA), TLR4 (1:200; Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA), MyD88 (1:500; Abcam, Cambridge, MA USA), p-p65 (1:200; Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA), β-actin (1:5000, Bioworld Technology, Bloomington, MN, USA). At the end, we use the horseradish peroxidase (HRP)-linked secondary antibodies to incubate the membranes for 1 h. The blotted protein bands were visualized by enhanced chemiluminutesescence (ECL) kit (EMD Millipore, Billerica, MA, USA) and were recorded by Tanon 5500 Chemiluminescence Imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China). Band density was quantified using Image J Software (NIH, Bethesda, MA, USA), with normalization to β-actin.
Similarly, Western Blot analysis was done in the cultured BV2 cells. Cells were lysed in lysis buffer containing Phosphatase Inhibitor Cocktail 2(Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were quantified by a BCA protein kit. Samples (50 µg protein per lane) were loaded on 12% SDS-PAGE gels. The following steps were consistent with the experiments in vivo.
8. ELISA
For in vivo study, to quantify the release of cytokines from the brain after TBI (n = 3/group), mice were sacrificed at 24 h after TBI, brain homogenates were centrifuged at 5000 g for 5 min at 4ºC. The cytokines interleukin (IL)-1β and tumor necrosis factor(TNF)- α were quantified in the supernatant used commercial ELISA kits (both purchased from Multisciences, Hangzhou, China). All procedures are performed according to manufacturers’ protocols.
For in vitro study, cell-free supernatants were collected by centrifugation at 1000 g for 20 min and assayed by IL- 1β and TNF-α ELISA kits.
9. Immunofluorescence staining
Immunofluorescence staining was performed as previously described[31]. Briefly, the brain sections were incubated overnight at 4ºC with the antibody against S100A8 (1:200; Abcam, Cambridge, MA, USA). After wished with phosphate buffered saline (PBS), Iba-1 (1:200; EMD Millipore, Billerica, MA, USA) was added and incubated overnight. The goat anti-rabbit IgG (diluted 1:200) was used to incubate the brain sections at room temperature for 10 min after washed with PBS. Then, we incubated the sections at room temperature for 5 min with the DAPI dye solution (Kaiji Biological, Nanjing, China). At the end, anti-fluorescence quenching was used to slightly dry and seal the sections. A fluorescence microscope was used to observe, then the images were collected.
10. Nissl Staining and Cell Counting
To assess neuronal cell death, Nissl staining was performed as previously described [32]. The fixed brain samples were dehydrated, paraffin-embedded, and sliced into 10-mm thick, followed by dewaxing 3 times in xylene for 5 min each time and then placing them in anhydrous ethanol for 5 min, 90% ethanol for 2 min, 70% ethanol for 2 min, and distilled water for 2 min. Specimens underwent Nissl staining for 10 min and then were rinsed twice with distilled water for a few seconds each time. They were then dehydrated twice in 95% ethanol for 2 min each time and made transparent by treatment with xylene twice for 5 min each time, followed by sealing with neutral gum. Specimens were viewed under a light microscope. Neurons have big cell body and rich cytoplasm under normal conditions. In contrast, shrunken cell body and many empty vesicles could be detected in damaged neurons. Cell counting was calculated within the brain cortex. Six random high power fields (x400) were randomly chosen to calculate the mean number of surviving neurons in each coronary section. Every third coronary section, starting from 3.0 mm posterior to the optic chiasma, was selected and we collected 4 sections in each animal for quantification. At the end, the average number of the 12 sections from three individual mice’s brain was considered as the data for each group. We presented the data as the number of surviving neurons per high-power field.
10. Statistical analysis
All data in this study were expressed as mean ± SD. Statistical analysis was performed by the one-way ANOVA according to Tukey’s post hoc tests. P < 0.05 was considered statistically significant.