Controlled cortical impact (CCI) model establishment and grouping
Adult male Sprague-Dawley rats (250–300 g) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). Rats were generally anesthetized via intraperitoneal (i.p.) injection of chloral hydrate (3.5%, 350 mg/kg), and a circular craniotomy (5 mm in diameter) was performed (located 4 mm posterior to bregma and 2.5 mm lateral to the sagittal suture over the right parietal cortex). Following the craniotomy, a CCI model was established with a TBI-0310 TBI model system (Precision Systems and Instrumentation, USA) and the following impact parameters, as previously reported: a velocity of 3.5 m/s, a depth of 2.5 mm, and a dwell time of 200 ms [13]. The sham rats underwent the same procedures as the CCI rats but without impact.
The rats were randomly divided into four groups in this study: the sham group, the TBI + vehicle (vehicle) group, the TBI + JWH133 (JWH) group, and the TBI + SR144528 + JWH133 (SR+JWH) group. The sham group received only a craniotomy, the vehicle group underwent CCI and received an equal volume of vehicle, the JWH group underwent CCI and received a selective CB2 agonist, JWH133 (1.5 mg/kg, MedChemExpress, USA), by i.p. injection at 1 h after operation, and the SR +JWH group underwent CCI and received SR144528 (3 mg/kg, Cayman, USA) by i.p. injection 3 min before receiving JWH133 intraperitoneally. SR144528 is a selective CB2 antagonist. JWH133 and SR144528 were used according to the methods of previous studies[12, 14].
Immunohistochemistry and quantification
Coronal brain sections (20-µm thickness) were subjected to immunofluorescence staining. Briefly, after washing with PBS, the sections were permeabilized with 0.4% Triton X-100 in PBS for 30 min at room temperature, blocked with 10% goat serum for 1 h at 37°C, and subsequently incubated overnight with primary antibodies at 4°C. After three washes in PBS, the sections were incubated with the appropriate secondary antibodies for 1 h at 37°C. The above protocol was repeated once for double staining[14]. The primary antibodies included anti-Iba1 (1:500, Wako, Japan), anti-CD206 (1:100, Santa Cruz Biotechnologies, USA), anti-CD86 (1:100, Santa Cruz Biotechnologies, USA), anti-NG2 (1:100, Biorbyt, UK), anti-APC (1:100, Millipore, USA), anti-myelin basic protein (MBP) (1:100, Millipore, USA), and anti-neurofilament H (NF-H) (1:100, CST, USA) antibodies. Confocal microscopy (Zeiss, LSM780, Germany) was used to capture images. The immunopositive cell numbers were calculated with ImageJ software. All counts were obtained in a blinded fashion[12].
The distribution of microglia around myelin sheaths was assessed by counting the microglial cell bodies in contact with and localized within each myelin sheath in the striatum. The proportion of microglia in contact with myelin relative to the amount of myelin in each image field was calculated. Hence, different amounts of myelin in different fields were considered to represent changes in the redistribution of microglia in relation to myelin[15].
Western blot analysis
Brain tissues were collected, and protein was extracted in RIPA lysis buffer (containing protease and phosphatase inhibitors) prepared as described previously to perform western blotting[11]. Fifty micrograms of prepared protein was loaded into each lane of SDS-PAGE gels. The primary antibodies used in this study included anti-total-PERK (1:1000, CST, USA), anti-phospho-(p-)PERK (1:1000, Invitrogen, USA), anti-total-eIF2α (1:1000, CST, USA), anti-p-eIF2α (1:1000, CST, USA), anti-ATF4 (1:1000, CST, USA), anti-Gadd34 (1:1000, Proteintech, China), anti-total-Akt (1:1000, CST, USA), anti-p-Akt (1:1000, CST, USA), anti-p-inositol-requiring enzyme 1α (p-IRE1α) (1:1000, Proteintech, China), anti-activating transcription factor 6 (ATF6) (1:1000, Proteintech, China), anti-MBP (1:1000, Millipore, USA), anti-NF-H (1:100, Proteintech, China), anti-GAPDH (1:1000, Proteintech, China), and anti-β-tubulin (1:1000, CST, USA) antibodies. Briefly, the polyvinylidene difluoride (PVDF) membranes were blocked in 5% nonfat milk for 1 h at room temperature and then incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. The bands were visualized with a Fusion-FX7 system (Vilber Lourmat, Chongqing, China). ImageJ software was used for densitometric analyses[16].
Real-time polymerase chain reaction
Total RNA was extracted from each group of brains using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. One microgram of RNA was reverse-transcribed into cDNA using a PrimeScriptTM RT kit (Vazyme, Nanjing, China). RT-PCR systems were set up in 10 µl volumes using a SYBR Premix Ex Taq II kit (Vazyme, Nanjing, China), and RT-PCR was performed on a CFX-96 Real-Time PCR Detection System (Bio-Rad, USA). The program included 30 s at 95°C, 40 cycles of 5 s at 95°C and 30 s at 60°C, and melt curve analysis. Gene expression was quantified and normalized to that of standard samples (GAPDH)[12]. The sequences of the primers for M1-phenotype genes were as follows: CD16 forward sequence, ATCCTAGACCCTGAATGGG; CD16 reverse sequence, GCGAGATGAGGCTTTTGT; CD32 forward sequence, CAAAGCCAACCACAGTCA; CD32 reverse sequence, CAATACCGGCAACGAACT; CD86 forward sequence, TGTTGGCCTATCTGCTCTC; and CD86 reverse sequence, AGCTCACTCGGGCTTATG. The sequences of the primers for M2-phenotype genes were as follows: CD163 forward sequence, GGGGTTCCGTCTGTGAT; CD163 reverse sequence, TGTCCGCTTCCTTCTCC; CD206 forward sequence, CAACCAAAGCTGACCAAAG; CD206 reverse sequence, AAGAAATAGGCCGCAACA; Arg1 forward sequence, CAGTATTCACCCCGGCTA; and Arg1 reverse sequence, CCTCTGGTGTCTTCCCAA.
Laser speckle contrast imaging (LSCI)
LSCI was conducted to image cerebral perfusion and record cerebral blood flow (CBF) 3 d after TBI. The rat body temperature was maintained at 37 ± 0.5°C during the procedure. First, a midline incision was made to expose the skull. Tampons were used to keep the exposure area clean and dry. The head of a PeriCam PSI System was adjusted to ensure that the red cross (indicator laser, 660 nm) was located at the center of the brain, and the measurement distance was kept at 10 cm. The test area was adjusted with PIMSoft. Cerebral blood signals were collected at 785 nm and transferred into blood perfusion images via PIMSoft. Perfusion images were collected with a PeriCam high-resolution LSCI instrument (PSI System, Perimed) with a 70 mW built-in laser diode for illumination and a CCD camera installed 10 cm above the skull. Cerebral perfusion was calculated in the region of interest (ROI) located between the bregma and lambda in the hemisphere containing the contusional and pericontusional cortex using PIMSoft software. The CBF change is presented as the mean perfusion value [17, 18].
Electrophysiologic recording
To record the motor evoked potentials (MEPs) elicited by transcranial electrical stimulation, rats were anesthetized with 1% pentobarbital sodium (25 mg/kg i.p.)[19]. A stimulation needle electrode (DSN1620, Medtronic, USA) was inserted subcutaneously at the base of the nose with the tip touching the scalp, acting as the anode. The cathode was placed at the midpoint of an imaginary line between the two ears. The ground electrode was placed subcutaneously at the base of the tail. The recording electrode was inserted into the contralateral gastrocnemius muscle to record MEPs. Electrical stimulation was performed with a stimulator to excite the brain (Keypoint, Medtronic, USA). A single pulse of stimulation (10 mA, 0.1 ms, 1 Hz) was delivered by a single electrode. The electrical stimulation was repeated at least five times in each rat with an interval of 15 s. The base-to-peak amplitude of a single stimulation was recorded as the MEP. The MEP and the latency were recorded for analysis[20].
Transmission electron microscopy (TEM)
TEM was used to measure the thickness of myelin in the corpus callosum (CC)/external capsule (EC) area. Rats were initially perfused with ice-cold saline and subsequently perfused with 4% PFA and 2.5% glutaraldehyde in 0.1 mol/L PBS buffer. The CC/EC tissue near the CCI site was microdissected into 1-mm3 specimens. Then, these samples were immersion-fixed for 24 h in 2% glutaraldehyde, washed in PBS and fixed in 1% osmium tetroxide in 0.1 M PBS for 45 min. The specimens were dehydrated in increasing concentrations of acetone and embedded in Araldite resin. Ultrathin 60-nm sections were cut on a Leica UCT ultramicrotome with a diamond knife (Diatome, Wetzlar, Germany). The prepared ultrathin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and viewed with a JEM-1400Plus TEM (H-7500, Hitachi Ltd., Japan). Images were acquired in randomly selected CC/EC areas from each section at a magnification of 20,000×. The g-ratios of the myelinated fibers were calculated using ImageJ software by an investigator blinded to the experimental groups, and at least 60 myelinated fibers from each rat were analyzed. The g-ratio was calculated as the ratio of the inner diameter (axon) to the outer diameter (axon and myelin sheath)[21].
DTI scanning and analyses
DTI was used to evaluate white matter integrity after TBI (7, 8). Rats were initially anesthetized with 3% isoflurane and then positioned on an animal cradle with a stereotaxic head holder. Anesthesia was maintained with between 1% and 1.5% isoflurane. Respiration and temperature were monitored continuously during the scanning process. A DTI dataset covering the entire brain was collected with the following parameters: TE=0.85 ms, TR=5000 ms, 256 × 128 matrix, a total of 32 diffusion sampling directions, b= 826.59 s/mm2, in-plane resolution = 0.12 mm, and slice thickness =0.8 mm. These parameters have been established for acquisition of high-resolution DTI scans in small animals[21].
ParaVision version 5.0 (Bruker BioSpin) was used for analysis of the DTI data. The ROI was drawn manually in a blinded manner to encompass the CC/EC in the ipsilesional hemisphere in order to determine the fractional anisotropy (FA) and radial diffusivity (RD) values. The RD value was calculated using the equation and the FA value was calculated using the equation Directionally encoded color (DEC), FA, and RD maps were generated by ParaVision version 5.0 (Bruker BioSpin) software[22].
Behavioral tests
Behavioral tests were conducted by individuals blinded to the experimental groups.
Morris water maze test. Cognitive function was analyzed with the Morris water maze as described previously[21]. Briefly, the rats were trained to search for a hidden platform by using a series of prominent extramaze cues. Nontoxic ink was used to make the water opaque, and the water temperature was maintained at 22–26°C. In the learning test, the rats were tested for 5 consecutive days, and the circular platform (diameter =12 cm, height=20–35 cm) was submerged 2 cm beneath the water surface in a circular pool (diameter = 150 cm, height=60 cm). Each rat was placed into the pool at one of three locations and allowed to locate the hidden platform for 60 s. Each animal was trained in 3 trials (with randomly assigned starting positions) per day to locate the platform. At the end of each trial, the rat was placed on the platform or allowed to stay on the platform for 15 s with prominent spatial cues displayed around the room. The time spent reaching the platform (learning phase) was recorded. A memory test was performed on day 14. The platform was removed, and a single 60 s probe trial was conducted. The time spent in the target quadrant where the platform was previously located was recorded. The swim speed of each rat was also recorded to assess gross motor skills during the experiments. The motion trial and behavioral data from the spatial learning test and memory test were recorded and analyzed using a video-based tracking system (ANY-maze, Stoelting, USA).
Open field test. Anxiety-like behavior was evaluated two weeks after TBI in an open field apparatus (100 cm ×100 cm ×40 cm box). Briefly, a rat was placed in the center of the open field box, and activity was measured and recorded for 5 consecutive minutes using a video-based tracking system (ANY-maze, Stoelting, USA). The total movement time and the time spent in the center of the open field box were analyzed by an investigator who was blinded to the experimental groups[23].
Cell culture
Primary microglial culture. Primary microglial cultures were prepared as previously described with slight modifications[24]. Briefly, pups were sacrificed, and their brains were isolated. Then, the cerebellum was dissected away from each brain, and the meninges and choroid plexus tissue were separated from the cortical and ventricular surfaces. The cortices were dissected away from the diencephalon, cut into 2–3-mm2 pieces and digested with papain and DNase (Sigma) in a 37°C water bath for 10 min. After centrifugation (1000 rpm for 5 min) and resuspension, the cells were plated in poly-L-lysine (10%, Solarbio, China)-precoated cell culture flasks and cultured in high-glucose DMEM (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Solarbio). After culturing for 10 d in a humidified atmosphere at 37°C containing 5% CO2 and 95% air, the cells formed a confluent monolayer (medium changes were performed every 3 d). The microglia were separated from the astrocytes by shaking the flask for 4 h at 220 rpm to detach the layer of loosely adherent astrocytes. The purity of the microglia was > 98%, as verified using anti-Iba-1 immunofluorescence staining. Subsequently, the microglia were seeded on poly-L-lysine-precoated coverslips for immunofluorescence analysis.
Primary OL culture. Primary OLs were collected from 1-d-old postnatal rats as described previously with slight modifications[25]. Briefly, pups were sacrificed, and their brains were isolated. Then, the cerebellum was dissected away from each brain, and the meninges and choroid plexus tissue were separated from the cortical and ventricular surfaces. The cortices were dissected away from the diencephalon and cut into 2–3-mm2 pieces. Papain and DNase (Sigma) were used for tissue digestion in a 37°C water bath for 10 min with occasional inversion. More DNase was added if the tissue became gel-like. The cells were centrifuged at 1000 rpm for 5 min and resuspended in growth medium (90% DMEM/F12 (Gibco), 10% FBS (Gibco), and 1% penicillin-streptomycin (Solarbio)). Dissociated cells were plated in T-75 culture flasks precoated with poly-L-lysine (10%, Solarbio, China). Medium changes were performed every 3 d. On day 6, the medium was replaced with OL differentiation medium (described below).
To prepare OL differentiation medium, B104 cells were first cultured in medium containing 90% DMEM/F12 (Gibco), 10% FBS (Gibco), and 1% penicillin-streptomycin (Solarbio) until they covered 70–80% of the flask. The medium was then replaced with N2 medium (98% DMEM/F12 (Gibco), 1% penicillin-streptomycin (Solarbio), 1% N2 (Gibco)). Over 3–4 d, the N2 medium slowly became yellowish, indicating that the supernatant contained cytokines. After centrifugation (1000 rpm for 30 min) and filtering (0.22 µm, Millipore, USA), differentiation medium was prepared with the supernatant (90% DMEM/F12 (Gibco), 10% B104 supernatant, and 1% penicillin-streptomycin (Solarbio)). Differentiation medium changes were performed every 3 d. On day 9, OLs were obtained for purification.
A transwell system was used to allow communication via diffusible factors and simple conditioned medium (CM) transfer from microglial cultures to OL cultures (Fig 7I). OLs were subjected to oxygen-glucose deprivation (OGD) for 2 h and subsequently returned to normal medium for 24 h (some OLs were treated with JWH133 (OGD+JWH group) or with vehicle (OGD+ vehicle group) directly). Primary microglia were incubated with vehicle (sham group), tunicamycin (3 µg/ml)+vehicle (TM+ vehicle group), tunicamycin (3 µg/ml) + JWH133 (4 µM) (TM+JWH group) or tunicamycin (3 µg/ml) + SR144528 (1 µM) +JWH133 (4 µM) (TM+SR+JWH group) for 6 h and then cocultured with OLs in a transwell system for 24 h[14, 26, 27]. Subsequent experiments (flow cytometry for apoptosis and immunocytochemical staining for MBP, Iba1 and p-PERK) were performed 24 h later.
Measurement of volumetric tissue loss and MBP loss
Rats were euthanized and perfused with PBS followed by 4% PFA. Subsequently, samples were removed and postfixed in 4% PFA overnight, transferred to a 25% sucrose solution until the tissue was submerged at 4°C, and transferred to a 30% sucrose solution until the tissue was submerged under the same conditions. Once gradient dehydration was completed, the samples were cut into 25-µm-thick sections with a cryostat and stored at -20°C. Six equally spaced coronal brain sections encompassing the injury site were selected and stained for MBP (1:100, Millipore, USA) with the same method described previously. Loss of tissue and MBP was analyzed by a blinded observer using NIH ImageJ software, and the % of volumetric tissue and MBP loss was calculated as the ratio of the cortical lesion volume to the entire contralateral cortex volume [21, 23].
Statistical analysis
GraphPad Prism software (version 8.1.0, CA, USA) was used for statistical analyses. The data are presented as the mean ± SEM. Differences between two groups were analyzed by Student’s t test (two-tailed), and differences among multiple groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. In all analyses, P ≤ 0.05 was considered to indicate statistical significance.