In the current study, all the mice were randomly assigned to undergo one of the following four experimental procedures in a blinded manner. The number and distribution of animals in each experimental group are shown in Fig. 1.
To determine the effects of CYM-5442 after TBI, mice were assigned to one of three groups: sham, TBI+ vehicle, and TBI+ CYM-5442. CYM-5442 was dissolved in 0.9% saline containing 10% dimethylsulfoxide (DMSO) and 10% Tween-80 and administered daily intraperitoneally. Vehicle animals received the same volume of saline containing 10% DMSO and 10% Tween-80. Neurological outcomes were evaluated at the corresponding time points following TBI before the animals were sacrificed for sample collection. To determine the therapeutic dosage, mice were treated with 0.3, 1 or 3 mg/kg CYM-5442 at 30 min after TBI induction according to their brain water content and neurobehavioural test results. A final CYM-5442 dosage of 3 mg/kg, administered after TBI, was finally adopted, and Western blot (WB), IF, MRI, blood count and other evaluations were performed.
Endogenous expression of Mfsd2a, cav-1, and claudin-5 was evaluated by WB using samples obtained from the pericontusional cortex at the indicated time point after TBI. Brain ultrastructure was detected by transmission electron microscopy (TEM) at 72 h following TBI. The cellular colocalization of Mfsd2a with an EC marker (CD31) in the pericontusional region was evaluated by double-IF staining (IF) at 72 h following TBI.
To enhance or downregulate the protein expression of Mfsd2a, experiment 3 was designed as follows. All mice were randomly divided into 6 groups, the sham, TBI, TBI+Vehicle, TBI+Over-Mfsd2a, TBI+Control-ShRNA and TBI+ShRNA-Mfsd2a groups. All AAVs were administered by intraventricular injection at 4 W before TBI. The mice were euthanized on the third day after TBI, and their brain tissues were harvested for analysis.
The potential mechanism underlying the protective effects of CYM-5442 on BBB integrity through Mfsd2a/CAV-1 was evaluated. The experimental design for the fourth experiment was as follows. All mice were randomly divided into the following six groups: the Vehicle+Sham, Vehicle+TBI+Control-ShRNA, Vehicle+TBI+ShRNA-Mfsd2a, CYM-5442+Sham, CYM-5442+TBI+Control-ShRNA and CYM-5442+TBI+ShRNA-Mfsd2a groups. The mice were euthanized on the third day after TBI, and their brain tissues were harvested for analysis.
All experiments involving animals were approved by the Chongqing Medical University Administrative Panel on Laboratory Animal Care.
Male C57BL/6 mice (8–10 weeks old, 22-26 g) were purchased from the Experimental Animal Center of Chongqing Medical University. The animals were housed on a 12 h light-dark cycle at a controlled room temperature (23 ± 2 °C) and relative humidity (40-60%) and had ad libitum access to food and water. The mice were generally anaesthetized via the intraperitoneal (i.p.) injection of chloral hydrate (3.5%, 350 mg/kg). All surgeries were performed under anaesthesia, and all efforts were made to minimize animal suffering.
A standard protocol and a controlled cortical impact (CCI) device (TBI-0310, Precision Systems and Instrumentation, Fair fax, VA, USA) were utilized to induce brain injuries as described previously. Briefly, after anaesthetization, a circular craniotomy (3 mm in diameter) was performed (1 mm posterior to bregma and 1 mm lateral to the sagittal suture over the right parietal cortex). Following the craniotomy, the CCI model was established with a TBI-0310 TBI model system with the following impact parameters: velocity, 5.0 m/s; depth, 2.0 mm; and dwelling time, 100 ms. As a result, a moderately severe contusion was induced in the right sensorimotor cortex underlying the hippocampus. The mice exhibited pronounced behavioural deficits but none died. Mice in the Sham group underwent identical surgical procedures but without impact. Following the injury, the hole was sealed with bone wax, and the skin incision was sutured. Finally, the mice were kept on an electric blanket to maintain their normal body temperature until the completely awakened.
Laser speckle contrast imaging (LSCI)
Cerebral cortical blood flow was monitored at baseline and at 3, 24, and 72 h post-TBI using laser speckle techniques as described previously. Mice were anaesthetized by isoflurane, and an incision was made along the midline to separate the skin from the skull. The mouse body temperature was maintained at 37° ± 0.5 °C throughout the experiment. The exposure area was kept clean and dry using a tampon during image collection. The PeriCam PSI head was adjusted to ensure that the red cross (indicator laser, 660 nm) was located at the centre of the brain, and the measurement distance was kept at 10 cm. The test area was adjusted with PIM Software version 1.5. Cerebral blood signals were collected at 785 nm and translated 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. Laser speckle blood flow images were recorded and used to identify the regions of interest (ROIs). The cerebral blood flow (CBF) changes are presented as the mean perfusion values.
Magnetic resonance imaging
Serial MRI scanning was used to assess lesion volumes and brain oedema on a 7.0T animal scanner (Bruker Biospin, Germany) at 1 and 3 days after the induction of brain injury. The mice were anaesthetized with isoflurane (3% for induction and 1-1.5% for maintenance) and positioned on an animal cradle with a stereotaxic head holder. The respiration and temperatures of the mice were monitored continuously during the scanning process. To monitor brain oedema evolution, T2-weighted images were acquired at each imaging time point. Brain oedema changes were quantified by measuring the T2-hyperintense area using Weasis software. Each whole-brain scan was composed of 28 slices.
Brain water content
After anaesthetization and euthanasia, the mice were decapitated at 24 and 72 h after TBI, and their brains were immediately removed and divided into three parts: the right hemisphere, left hemisphere, and cerebellum for experiment 1 and the whole cerebrum for experiments 3 and 4. Each brain region was weighed immediately to determine the wet weight and then dried for 24 h at 100 °C to obtain the dry weight. The percentage of brain water content was calculated as follows: (wet weight - dry weight)/wet weight × 100%. The percentage of water content was calculated by 2 trained investigators who were blinded to the animal grouping.
Behavioural tests and neurological scoring
Neurological deficit score
The acute neurologic deficits score was determined at 1, 3, 5, and 7 days (n=10) after the administration of CYM-5442 using a 28-point scoring system as described previously[13, 27]. Body symmetry, gait, climbing, circling behaviour, front limb symmetry, compulsory circling, and whisker responses were assessed. All data were recorded by 2 observers blinded to the mouse grouping, and the average score of the subscales was used as the final score of each mouse.
Hanging wire test
In this study, grip strength was assessed by placing mice on an apparatus consisting of a steel wire (50 cm long; 2 mm in diameter) pulled between two vertical supports and suspended 40 cm over a flat surface; a soft surface was placed below the wire to prevent physical trauma to the mice. The mice were placed on the wire midway with two forepaws and were observed for 60 s in 3 trials. The mice were evaluated as follows: 0, fell off; 1, hung onto the wire with 2 forepaws; 2, same as for 1 with an added attempt to climb onto the wire; 3, hung onto the wire with two forepaws and 1 or both hind paws; 4, hung onto the wire with forepaws and with tail wrapped around the wire; and 5, escaped to one of the platforms. The average score of three successive trials was taken for each animal. All tests were performed by two investigators blinded to the experimental groups.
In the beam-walking test, the mice were placed at one end of a wooden beam (12 mm in diameter, 1 m long, and 50 cm high) and allowed to traverse the beam into a black box located at the end of the beam. The number of foot faults and the time to complete the task were documented. A foot fault was defined as any paw slipping off the top surface of the beam. Data were analysed by the primary experimenter and confirmed by a second experimenter via video recording. Before surgery, the mice were trained for 3 days, and the performance was measured on days 1, 3, 5, and 7 post-CCI. All experimenters and animal handlers were blinded to the treatment groups.
At 3 h, 24 h, 72 h and 7 days after TBI, blood was collected from the retro-orbital venous plexus of anaesthetized mice into EDTA-coated capillary tubes from, and no drugs were administered on the day of blood collection except for from mice in the group at 3 h after TBI. Blood samples were analysed using an autoanalyzer (XT-2000i, Sysmex Corporation) to detect lymphocytes and leukocytes.
BBB permeability assays
To measure BBB permeability, 2% Evans Blue (EB, 4 mL/kg) in sterile saline was injected through the tail vein 1 h before the animals were sacrificed. The mice were transcardially perfused with saline, and their brains were dissected and weighed. The samples were then homogenized in PBS (1 ml/300 g), sonicated for 2 min, and centrifuged at 15,000 rpm for 5 min at 4 °C, and the supernatant was then collected in aliquots. Next, 500 μL of 50% trichloroacetic acid was added to each 500 μL of supernatant and incubated overnight at 4 °C. Finally, these samples were centrifuged at 15,000 rpm for 30 min at 4 °C. The samples were detected with a spectrometer at 610 nm and quantified using a standard curve that was normalized to tissue weight (μg/g). Then, to assess the fluorescence intensity, the brains were removed in preparation for coronal brain sectioning. Red autofluorescence of EB was observed on the slides as previously described. The mean red autofluorescence of EB was evaluated by 2 observers blinded to the mouse treatment groups.
After deep anaesthetization, the mice were perfused with PBS and 4% PFA. Their brains were removed and immersed in 4% paraformaldehyde, 20% sucrose, and 30% sucrose in sequence for fixation and dehydration, and the tissues were embedded in optimal cutting temperature (OCT; Sakura) compound. Coronal brain sections (20-µm thickness) were subjected to immunofluorescence (IF) staining. After washing with PBS and PBS+0.4% Triton X-100, the brain sections were blocked with 10% goat serum for 1 h at 37 °C, incubated with primary antibodies overnight at 4 °C and washed three times with PBST. Then, the cryosections were incubated with secondary antibodies conjugated to Alexa Fluor 488/594/647 for 1 h at room temperature. The above protocol was repeated once for double staining. The primary antibodies included anti-Mfsd2a (1:50, Invitrogen, USA), anti-caveolin-1 (1:200, CST, USA), anti-claudin-5 (1:100, Invitrogen, USA), anti-CD31 (1:50, Genetex, UK), and anti-albumin (1:200, Abcam, USA). Images were captured by confocal microscopy (Zeiss, LSM780, Germany) and processed using ImageJ and Adobe Photoshop.
Brain tissues were collected, and protein samples from pericontusional tissues were extracted in RIPA lysis buffer (containing protease and phosphatase inhibitors) as described previously to perform Western blotting. Equal amounts of protein samples (50 μg) were separated by SDS–PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked in 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: anti-Mfsd2a (1:50, Invitrogen, USA), anti-Cosolin-1 (1:200, CST, USA), anti-claudin-5 (1:100, Invitrogen, USA), anti-albumin (1:200, Abcam, USA), and anti-GAPDH (1:1000, Proteintech, China). The following day, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. Immunoblots were visualized with a Fusion-FX7 system (Vilber Lourmat, Chongqing, China) and quantified with optical methods using ImageJ software. All WB experiments were repeated 3-5 times.
Infection with recombinant adeno-associated virus
The recombinant HBAAV2/9-CMV-m-Mfsd2a-3xflag-Null (Mfsd2a overexpression) and HBAAV2/9-ZsGreen control viruses were generated by Hanbio Technologies (Shanghai, China). For in vivo infection, an AAV overexpressing Mfsd2a (5 μl, 1.2*1012 viral genomes/mL) or a control virus (5 μl, 1.3*1012 viral genomes/mL) was administered intracerebroventricularly using a syringe with a 5-gauge needle. Similarly, the recombinant HBAAV2/9-m-Mfsd2a shRNA2-Null (downregulated Mfsd2a) and HBAAV2/9-mCherry NC control viruses were generated by Hanbio Technologies (Shanghai, China). For in vivo infection, an AAV with Mfsd2a knockdown (5 μl, 1.7*1012 viral genomes/mL) or a control virus (5 μl, 1.3*1012 viral genomes/mL) was administered intracerebroventricularly using a syringe with a 5-gauge needle. TBI was induced 4 weeks after recovery from the intraventricular virus injection. The infectivity of the purified viruses was evaluated by fluorescence microscopy and WB.
S1P measurement by enzyme-linked immunosorbent assay (ELISA)
The S1P concentration was measured according to previous experiments . Five milligrams of tissue was transferred into a silicified glass tube, cut into 1 mm3 pieces and incubated in 0.4 ml of PBS at 4 °C. After 20 min, the supernatant was separated from the tissue fragments, which were again homogenized in 0.4 ml (1:1) of methanol/water. The amount of S1P in the supernatant was measured by a mouse S1P ELISA kit (MEIKE Jiangsu Sumeike Biological Technology Co., Ltd) according to the manufacturer’s instructions. The amount of S1P was normalized to the protein content.
Transmission electron microscopy
After anaesthetization, mice were transcardially perfused with ice-cold saline followed by 4% PFA and 2.5% glutaraldehyde with 0.1 mol/L PBS buffer. The pericontusional cortex tissues were microdissected into 1 mm3 specimens. Then, these samples were immersion-fixed in 2% glutaraldehyde for 24 h. After washing with PBS, they were 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-thick sections were cut using a diamond knife on a Leica UCT ultramicrotome (Diatome, Wetzlar, Germany). The prepared ultrathin sections were mounted on a copper grid, stained with uranium acetate and lead citrate, and observed with a JEM-1400Plus TEM (H-7500, Hitachi LTD., Japan). TEM imaging of horseradish peroxidase (HRP) injection was performed as previously described. Mice were anaesthetized, and HRP type II (Sigma Aldrich, P8250-50KU, 0.5 mg/g body weight dissolved in 400 μL of PBS) was injected through the tail vein. The brain was collected after 30 min of circulation, and the brain tissue was soaked in a 0.1 m cacodylate-buffer mixture (5% glutaraldehyde and 4% PFA) for 1 h at room temperature, rinsed overnight with 4% PFA/0.1 m sodium cacodylate at 4 °C and cut into 50-µm-thick free-floating sections using a vibrotome. Sections were incubated in 3-3' diaminobenzidine (DAB, ZSGB-BIO, ZLI-9018, 5 mg/10 mL in 0.05 M Tris-HCl pH 7.6 buffer) with 0.01% hydrogen peroxide for 45 min at room temperature. After DAB staining, the sections were postfixed in 1% osmium tetroxide and 1.5% potassium ferrocyanide, dehydrated and embedded in epoxy resin. Ultrathin sections (80 nm) were then cut from the block surface, collected on copper grids, stained with Reynold’s lead citrate and examined under a Hitachi-7500 electron microscope. Twenty cortical vessels from each mouse that were comparable in size were analysed for vesicle quantification.
All statistical analyses were performed with Prism 9 software (Prism, GraphPad, San Diego, USA). Data are expressed as the mean ± standard error (SEM). The Pearson test and Shapiro-Wilk test were used to analyze the normality and homogeneity of the data. Differences between two groups were analysed by Student’s t test (two-tailed), and multiple group comparisons were made using a one-way ANOVA followed by a post hoc Tukey’s test. Differences in means across groups with repeated measurements over time were analyzed using the repeated-measures ANOVA. When the ANOVA showed significant differences, pairwise comparisons between means were tested by post hoc Bonferroni tests. P<0.05 indicates statistical significance, and ns indicates not significant.