Animals and rmTBI model
Adult male C57BL/6J mice (age: 10–12 weeks; weight, 20–25 g; Institute of Experimental Animals of the Chinese Academy of Medicine, Beijing, China) were used in this study. Animal care and experimental protocols were reviewed and approved by the Animal Research and Ethics Committee of Tianjin Medical University. The mice were housed at a temperature of 20 °C to 22 °C, humidity of 40% to 70% and a 12-h light/dark cycle. All mice ate and drank ad libitum prior to and following surgery.
A controlled cortical impact (CCI) method was used to establish the rmTBI model in this study . The mice were anaesthetized with 4.6% isoflurane and then positioned in a stereotaxic frame using ear bars. A 3.0 mm diameter craniotomy was performed over the right parietal cortex between the right coronary suture and the herringbone suture and 2 mm lateral to the midline suture. Mild TBI was induced using a CCI device, and injury parameters were set to a controlled velocity of 3 m/s, depth of 1 mm and dwell time of 200 ms. Mice in the sham-operated group were anaesthetized, and a craniotomy was performed without CCI. Mice in the rmTBI group underwent four head impacts with a 24-hour interval between each impact . After trauma or sham surgery, the mice were housed in separate cages until their consciousness was restored.
Experimental groups and treatment
Mice were randomly assigned to three groups (n=48 per group): the sham group,
rmTBI+PBS, and rmTBI+TAK242 groups. TAK242 was dissolved in DMSO and diluted with PBS to a final concentration of 0.4 mg/ml. The rmTBI+TAK242 groups received intraperitoneal injections of TAK242 (3 mg/kg) at 6 h, 1 day, 3 days, 7 days, 14 days, and 21 days post-last impact. The rmTBI+PBS groups received injections with equal volumes of DMSO/PBS. All time points used for detection started from the fourth strike. Mice were euthanized at 3 and 28 days following rmTBI to evaluate the levels of TLR4 and downstream signalling proteins, the polarized phenotype of microglia, inflammatory factors, and pathological proteins. Mice were evaluated for neurological function with the mNSS on days 1, 3, 7, 14, and 21 after rmTBI, and the cognitive function of mice was evaluated using the Morris water maze (MWM) at 28 days after rmTBI (Fig. 1).
Modified neurological severity score (mNSS) test
Each group of mice was evaluated for neurological function using the mNSS, including sensory tests, motor tests, reflexes, and balance tests. Mice were assessed with the mNSS prior to injury and on days 1, 3, 7, 14, and 21 after rmTBI to explore the cumulative effects of repeated injury on neurological function. A higher score indicates a more severe neurological deficit (normal score, 0; maximum score, 18).
Morris water maze (MWM)
The MWM test was conducted in mice to evaluate spatial learning and reference memory, as described previously . The opaque water-filled pool (diameter, 122 cm; height, 55 cm) was divided into four equal quadrants and maintained at a temperature of 22 ± 2 °C. A target platform was placed in the centre of the northeast quadrant, submerged 2 cm below the water surface. The place navigation test started 28 days after rmTBI and was conducted for 5 consecutive days. The mice were placed in the pool at one of four fixed starting points and allowed to swim freely for 90 s or until they reached and stayed on the target platform. Each mouse was trained four times per day at 20-minute intervals. The time to reach the hidden platform (escape latency) was recorded and analysed by a video tracking system, and the values of 4 trials were averaged. A spatial probe test was performed 24 h after the last acquired training session. The platform was removed from the maze, and the mice were allowed to swim freely for 90 s. The swimming path and speed of the mouse, the number of target quadrants crossed, and the percentage of dwell time in the target quadrant were recorded and measured.
For immunofluorescence staining, the mice were sacrificed by transcardial perfusion with cold PBS at 3 and 28 days after rmTBI. The brain tissue was harvested on ice and then postfixed with 4% paraformaldehyde for 24 hours, followed by an incubation with 30% sucrose for 48 hours. After fixation, tissues were embedded in the optimum cutting temperature medium (Sakura, Torrance, CA, USA) on dry ice. Brain tissue at the injury area was sliced into 8 μm coronal sections using a -20℃ frozen microtome.
For western blotting and ELISA, the mice were sacrificed using the same method described above. The injured cerebral cortex with a 5-mm diameter and injured hippocampus were isolated immediately and combined. The separated brain tissue was stored in liquid nitrogen after removing microvessels for protein extraction.
Immunofluorescence staining was performed to observe the expression of TLR4 and the polarization of microglia. After air drying, the sections were incubated with PBS containing 3% BSA for 30 min at 37 °C to block nonspecific staining. Then, sections were incubated with the primary antibody (anti-TLR4 antibody, 1:500, GeneTex, USA) overnight at 4 °C, followed by an incubation for 1 h at 37 °C with an appropriate secondary antibody. The nuclei were counterstained with DAPI. For the quantitative analysis, digital images of sections were captured under an immunofluorescence microscope (ZEISS, Germany) and quantified by a researcher who was blinded to the experiment. Five separate slides were selected from each brain sample. Digital images of positively labelled cells in three randomly selected 400× fields in each slide from the lesion boundary were captured. The lesion boundary was defined as the region surrounding the centre of the injury (0.5–1.5 mm to injured core), as previously reported. The mean number of positively labelled cells per visual field of each slide was counted for statistical analyses.
Double-label immunofluorescence staining for TLR4-Iba-1 and CD16/32-CD206 was performed to further observe the expression of TLR4 on microglia and the M1/M2 polarization of microglia. After blocking with BSA, the sections were incubated overnight at 4 °C with anti-TLR4 antibody (1:500, GeneTex, USA) and anti-Iba-1 antibody (1:500, Abcam, MA, USA) or anti-CD16/32 antibody (1:100, Abcam, MA, USA) and anti-CD206 antibody (1:100, Abcam, MA, USA). The remaining steps were the same as described above.
Western blots were performed using a previously reported method . The cerebral cortex and hippocampus on the injured side were separated and stored in liquid nitrogen until use. Total proteins were extracted, and then the protein concentration was measured using a BCA protein assay kit (Solarbio Science, Beijing, China). Protein samples were separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, Bedford, MA). The blots were blocked with 5% non-fat milk for 2 h at room temperature, then immersed overnight at 4 ℃ in a solution containing the primary antibodies: anti-TLR4 (1:500, GeneTex, USA), anti-APP (1:500, CST, USA), anti-p-Tau(1:500, CST, USA), anti-MyD88 (1:1000, CST, USA), anti-NF-κB p65 (1:1000, CST, USA), anti-CD16/32 (1:1000, Abcam, MA, USA), and anti-CD206(1:1000, Abcam, MA, USA). Afterwards, membranes were washed with TBST and incubated for 1 h at room temperature with HRP goat anti-mouse and HRP goat anti-rabbit secondary antibodies (all diluted 1:5000, ZhongShan, Beijing, China). The blots were treated with enhanced chemiluminescence (ECL) reagents and visualized with a ChemiDocTM XRS + Image System (Bio-Rad, CA, USA). The intensity of each band was analysed using ImageJ software.
The frozen brain tissue was weighed, homogenized in RIPA buffer containing protease inhibitors, and subsequently centrifuged for 20 minutes at 13,000 rpm at 4 ℃. The protein content of the supernatant was detected using the BCA Protein Assay Kit (Solarbio Science, Beijing, China). The protein levels of inflammatory mediators in all samples, including TNF-α, IL-10, IL-4 and IL-1β, were measured using ELISA kits (SBJ, Nanjing, China) according to the manufacturer’s protocols. The optical density (OD) of each well was measured at 450 nm with a microplate reader (Thermo Forma, USA). After generating a standard curve, the OD value of the protein sample was substituted into the standard curve, and the actual concentration of the protein was quantified.
All data were obtained from at least three independent experiments. The data are presented as the means ± SD and were analysed using SPSS 17.0 software. For comparisons among multiple groups, one-way ANOVAs followed by Fisher's LSD post hoc test, were used to analyse the data. The results from the place navigation test in the MWM were compared using repeated measures ANOVAs. Differences between the two groups were analysed using unpaired t-tests. Statistical significance was set to p< 0.05.