Construction of mouse model of TBI
Kunming mice (8 weeks old, weight 28 g ~ 32 g) were obtained from Medical Animal Center of Xi’an Jiaotong University and housed in a humidity- and temperature-controlled (40% ~ 60%, 20°C ~ 24°C) environment with a 12 h/12 h light/dark cycle. All animal experimental procedures were conducted in accordance with guidelines established by the Animal Committee of Xi’an Jiaotong University Health Science Center. Three mice were caged together and free access to food and water for the duration of experiments.
Feeney's free-fall epidural impact method was used to construct TBI model. Briefly, mice were anesthetized with pentobarbital sodium (10 mg/kg) via intraperitoneal injection and placed into a stereotaxic frame. Then a 4-mm-diameter craniotomy was performed at a point that was centered 1 mm posterior and 2 mm lateral to the right of bregma. The skull cap was carefully removed without damaging of leptomeninges. The hammer in 80 g dropped from 25 cm high was used to induce craniocerebral injury. After TBI, all mice were allowed to recover on a heat pad to maintain core body temperature at 36°C ~ 37°C. Mice in control group (named as sham) underwent the same surgical procedure but without TBI.
Voluntary exercise strategy
Voluntary wheel running (VWR) was used as the voluntary physical activity. A total number of 32 mice were divided into 4 groups (8 mice for each group) and named as Sham group, TBI group, Sham + VWR group and TBI + VWR group, respectively. Two days before TBI, mice in VWR group were placed in a clean polycarbonate cage with stainless-steel running wheel (12.7 cm in diameter, Lafayette Instrument, USA) individually, to adapt to the environment. Exercise (in Sham + VWR group and TBI + VWR group) was carried out at 2nd day after TBI (Fig. 2A). Running activity was monitored daily and lasted for one week. The running distance and the total number of wheel's rotation were measured by Scurry VitalView Acquisition and Analysis Software (version 1.1). Mice in other two groups were housed in standard cages without running wheel.
Neurological severity score (NSS) test
To assess the motor, sensory, and reflex of mice after TBI and to evaluate the neurofunctional recovery with exercise intervention, NSS test was applied as previously described . The test that was blinded to the two observers was carried out at 2 days after TBI and 7 days after VWR, respectively. The scale ranges from 0 to 10 (normal: score 0; maximal deficit: score 10) and 1 point was awarded for the lack of reflex or for the inability to perform the task. A maximal of 10 points indicated the most severe neurological dysfunction with failure of all tasks.
Motor coordination and balance test
The motor coordination and balance were assessed by the beam walking assay once daily until 9 days after TBI. Blinded evaluation was performed to lessen observer bias during the whole process. Before TBI, all mice were pre-trained until the proficiency to walk across a wooden beam without pausing was achieved. The beam walking test apparatus consists of a flat bench (70 cm x 35 cm) with a wooden beam (100 cm long and 1 cm wide) located 20 cm above the bench and held to it on two posts. The mouse was placed in the same starting position. The experimenter observed the site of falling off and recorded the distance between starting and dropping position.
Behavioral tests for anxiety
Mice were subjected to a series of behavioral tests following VWR. All tests were performed according to the standard procedure and optimized in our lab . All data were analyzed in a double-blind manner by using automated video tracking software (Smart 3.0 video tracking system, USA). Anxiety behavior was evaluated by open-field (OF) test and elevated plus maze (EPM) test.
OF test Each mouse was placed in an OF environment for acclimation and baseline measurements. The mice were individually placed into the center of the apparatus (50 cm × 50 cm) to explore for 5 min. The movements of the animals were recorded by an overhead video camcorder. The traveling time in the software-defined 25 cm × 25 cm center region of the apparatus was recorded for each animal. The time spent in the center region was measured and the total distance traveled were calculated.
EPM test The EPM apparatus consisted of two enclosed arms and two open arms (35 cm in length × 6 cm in width × 15 cm in height; Harvard Apparatus, Holliston, MA) at 90o angle to each other with all arm platforms elevated 50 cm from the floor. At the start of a trial, the mouse was placed in the center with its nose directed toward the same closed arm and allowed to explore the maze freely for 5 min. The total time spent, total distance covered, and distance in and entries into each arm and the center were digitally recorded. The times entered and the duration in the open arms were calculated, showed as a percentage of the total number of entries (sum of open and closed arms entries) and total time (sum of duration in both arms). The anxiolytic effect was represented by an increase in the percentage of the enter times and the time spent in the open arms and the total number of entries and the total time spent remained unchanged .
Histology and immunofluorescence staining
Mouse was perfused transcardially with 0.01 M phosphate buffer saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) after anesthetization at 1.5 h post-TBI or behavioral tests. Whole brain was rapidly dissected and post-fixed in PFA for 24 h at 4°C. After gradient dehydration with shaking overnight, the brain tissues were embedded with OCT and coronal sectioned into slices (10 µm thick) in a cryostat microtome (CM1950; Leica Microsystems, Wetzlar, Germany). Hematoxylin and eosin (H&E) staining was performed, following the standard procedures, to assess the degree of brain tissue damage. For immunofluorescence staining, brain sections were firstly treated with antigen retrieval solution (C1035, Solarbio, China) for 15 min and washed with 0.01 M PBS for 5 min. Then blocked with 5% bovine serum albumin (BSA) in 0.3% Triton X-100 for 2 h at room temperature (RT). The sections were then incubated over night at 4°C with primary antibodies, including rabbit polyclonal anti-Ionized calcium binding adaptor molecule 1 (Iba1, 019-19741, 1:500, Wako, Japan), rat monoclonal anti-CD68 (ab53444, 1:100, abcam, USA), mouse monoclonal anti-NLRP3 (AG-20B-0014, 1:50, Whatman, UK), rabbit polyclonal anti-c-fos (26192-1-AP, 1:50, Proteintech, Wuhan, China). On the following day, sections were incubated at RT in the dark for 2 h with secondary antibodies, including Alexa Fluor 488 and 594 conjugated Donkey anti-rabbit/Donkey anti-mouse (1:600, Invitrogen, USA) and Goat anti-rat (1:300, abcam, USA). After thoroughly wash, the brain sections were then incubated with 4′,6-Diamidino-2-phenylindole (DAPI) for 5 min to label the cell nuclei and mounted with Antifade Mounting Medium (Sigma, USA). All immunofluorescence images were captured under fluorescent microscope (BX57, Olympus Corporation, Japan).
Western blot assay
Molecules related to inflammatory cell signaling pathway were detected by Western blot assay. Protein was extracted from perilesional cortex using the RIPA lysate buffer (PE01, Pioneer, China). The protein was then separated on 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membrane (IPVH00010, Millipore, Billerica, MA, USA). After blocking with 5% (m/v) skim milk (232100, BD Biosciences, USA.) for 3 h at RT, the membrane was incubated with primary antibodies overnight at 4°C. Following antibodies that diluted in skim milk at 1:1000 were used. Rabbit polyclonal anti-caspase-1 (ab179515, 1:1000, abcam, USA), rabbit polyclonal anti-IL-1β (ab283818, 1:1000, abcam, USA), rabbit polyclonal anti-IL-18 (ab207323, 1:1000, abcam, USA) and mouse monoclonal anti-GAPDH (60004-1-Ig, 1:10000, Proteintech, China). On the following day, after washing with Tris-buffered saline-Tween 20 (TBST) buffer for 3 times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse (1:10000, Proteintech, China) secondary antibody for 2 h at RT. The membranes were then washed with TBST and visualized using an enhanced chemiluminescence kit (WBKLS0100, Millipore, USA). Images were digitally scanned and quantified by densitometry using Imaging J software (US NIH).
Quantitative real-time PCR analysis
Quantitative real-time PCR (qRT-PCR) was used to analyze the relative mRNA expression of genes that related to the pro- or anti-inflammatory factors and NLRP3 inflammasome. Total RNA of perilesional cortex was extracted by the Trizol reagent (Takara, Dalian, China) and transcribed into cDNA using reverse transcription kit (K1622, Thermo Scientific, USA). Then qPCR was performed using SYBR green dye with gene specific primer sets and iQ5 PCR thermal cycler (Bio-Rad, Hercules, USA) with following cycle parameters: 95°C for 3 min, 40 cycles at 95°C for 10 s, 60°C for 30 s and 72°C for 30 s. The mRNA expression of target genes were normalized to GAPDH and quantified by the 2−ΔΔCT method. The primer sequences used were as follows: Pro-inflammatory factors: IL-12: sense primer 5’- GTCCTCAGAAGCTAACCATCTCC − 3’, antisense primer 5’- CCAGAGCCTATGACTCCATGTC − 3’; IFN-γ: sense primer 5’- GAACTGGCAAAAGGATGGTGA − 3’, antisense primer 5’- TGTGGGTTGTTGACCTCAAAC-3’; CCL2: sense primer 5’- TTAAAAACCTGGATCGGAACCAA − 3’, antisense primer 5’- GCATTAGCTTCAGATTTACGGGT − 3’. Anti-inflammatory factors: IL-10: sense primer 5’- GGCAGAGAAGCATGGCCCAGAA − 3’, antisense primer 5’- AATCGATGACAGCGCCTCAGCC-3’; TGF-β: sense primer 5’- CTCCCGTGGCTTCTAGTGC − 3’, antisense primer 5’- GCCTTAGTTTGGACAGGATCTG − 3’. Activation of NLRP3 inflammasome: IL-1β: sense primer 5’- TGGGAAACAACAGTGGTCAGG − 3’, antisense primer 5’- CCATCAGAGGCAAGGAGGAA − 3’; NLRP3: sense primer 5’- ATTACCCGCCCGAGAAAGG − 3’, antisense primer 5’- CATGAGTGTGGCTAGATCCAAG − 3’. Housekeeping gene: GAPDH: sense primer 5’- GCCAAGGCTGTGGGCAAGGT − 3’, antisense primer 5’- TCTCCAGGCGGCACGTCAGA − 3’.
Data collection and statistical analysis
Data were expressed as mean ± standard error of the mean (SEM). In each experimental group, at least three samples were selected. Four sections from each brain and three randomly selected fields were imaged and counted. For Western blot assay and qRT-PCR, data from four samples were used. All data were analyzed by using Prism 8 (GraphPad Software, Inc., San Diego, USA) with p ≤ 0.05 considered as statistically significant. Differences between two groups were analyzed by unpaired Student’s t test. Relationship between NLRP3 inflammasome activation and anxiety-like behavior was analyzed by two-tailed Mann-Whitney U-test.