All animal experiments were approved by the 900th Hospital Ethics Committee (Fuzhou, China) and were performed under strict supervision. Adult male C57BL/6 mice (25 ± 3g) were purchased from the experimental animal facilities of Fujian Medical University. All animals were housed at room temperature (24–26 °C), with a light-dark cycle of 10 h/14 h, and were provided with sufficient water and food.
Experimental model and drug administration
All mice were randomly divided into three groups: a control group, a TBI group, and a TBI+infliximab (IFX) group (n = 18 each). Nine mice in each group were used for neurological assessment and cerebral blood flow monitoring, and the remaining mice were used for histological and molecular studies. The details of the damage caused by controlled cortical impact (CCI) have been described previously. First, anesthesia was induced with 3% isoflurane in a plexiglass container. After the animal was successfully anesthetized, anesthesia was maintained with 1.5% isoflurane delivered using a small-animal anesthetic machine (RWD Life Science Co., Shenzhen, China). Then each mouse was placed in a stereotactic frame. The craniotomy was located approximately midway between bregma and lambda on the right side, with the medial edge of the craniotomy 1 mm lateral to the midline. Mice were subjected to an impact using a 2 mm metal flat-tip impactor (Brain and Spinal Cord Impactor, 68099H, RWD Life Science). The impact central point was drilled 2.5 mm to the right of the sagittal suture and 2.5 mm posterior to the coronal suture. The velocity was 5 m/s, the depth was 3 mm, and the impact duration was 100 ms. Then the scalp was closed with a suture, and the mice were put back into their home cages to recover from the anesthesia. The control group animals received identical surgical procedures without CCI. Pharmacological inhibition of TNF-α was performed using IFX, as previously described. Approximately 30 min after TBI, the TBI+IFX group was intraperitoneally injected with IFX (10 μg/g, cilag Ag) once per day for 3 consecutive days. The remaining groups were injected with the same dose of the vehicle as used in the TBI+IFX group.
The murine microglial cell line BV2 was obtained from the China Infrastructure of Cell Line Resources (Beijing, China) and cultured in a medium comprising 90% Dulbecco’s Modified Eagle’s Medium (Invitrogen, Frederick, MD, USA), 10% fetal bovine serum (Hyclone, Logan, UT, USA), and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 ℃ in a humidified atmosphere of 5% CO2.
Preparation of paraffin-embedded sections
At 72 h after TBI, after deep anesthesia with sodium pentobarbital, the mice were transcardially perfused with 0.01 M phosphate buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde solution. Then, the brains were removed and post-fixed by immersion in the same fixative solution at 4 °C for 24–48 h. After dehydration and vitrification, tissue samples were embedded in paraffin, and 4-μm sections were prepared. The sections were then dewaxed in xylene, rehydrated in graded ethanol and deionized water, and then processed for immunofluorescence, immunohistochemistry, Nissl staining, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining.
Formaldehyde-fixed specimens were embedded in paraffin and cut into 4-μm-thick sections that were deparaffinized with xylene and rehydrated in a graded series of alcohol. Antigen retrieval was carried out by microwaving in citric acid buffer. Sections were incubated with an antibody against ionized calcium-binding adapter molecule (Iba)-1 (1:500; Abcam, Cambridge, UK), ADAM17 (1:200; Boaosen Biotechnology, Beijing, China), NF-κB p65 (1:200; Santa Cruz), or iNOS (1:250; Abcam), washed and then incubated with secondary antibody for 1 h at room temperature. A total of five sections from each animal was used for quantification, and the signal intensity was evaluated as follows: 0, no positive cells; 1, very few positive cells; 2, moderate number of positive cells; 3, large number of positive cells; and 4, the highest number of positive cells.
Enzyme-linked immunosorbent assay (ELISA)
Inflammatory factors and free radicals were measured in brain tissue using ELISA kits (Jingmei Biotechnology, Jiangsu, China) for TNF-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, ROS, RNS, and cyclic guanosine monophosphate (cGMP). According to the manufacturer’s instructions, standards and samples were sequentially incubated with respective monoclonal antibodies, biotinylated anti-rat antibodies, and then horseradish peroxidase. The detected optical density (OD) values were transformed into a concentration.
Formaldehyde-fixed specimens were embedded in paraffin and cut into 4-μm-thick sections that were deparaffinized with xylene and rehydrated in a graded series of alcohol, followed by antigen retrieval. Sections were incubated overnight at 4 °C with antibodies against Iba-1 (1:500; Abcam), TNF-α (1:100; Abcam), CD31 (1:200, Abcam), NeuN (1:500; Abcam), NF-κB p65 (1:200; Santa Cruz), iNOS (1:250; Abcam), α-smooth muscle actin (α-SMA; 1:500; Cell Signaling Technology, Danvers, MA, USA), ZO-1 (1:200; Abcam), or occludin (1:100; Abcam). After washing, the sections were incubated with secondary antibodies for 1 h at room temperature. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured with a fluorescence microscope (Leica, Wetzlar, Germany).
For the lipopolysaccharide (LPS) group, BV2 cells were stimulated with LPS (0.5 μg/mL) for 24 h, while the control group was not treated with LPS. Then the coverslips were washed with PBS three times and fixed with 4% paraformaldehyde for 10 min at room temperature. Then BV2 cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 10 min, blocked with 5% bovine serum albumin (BSA) for 60 min, and incubated overnight with primary antibodies against Iba-1 (1:500; Abcam) and TNF-α (1:100; Abcam). After incubation with the secondary antibody and DAPI, images were captured with a fluorescence microscope (Leica, Wetzlar, Germany).
The supernatants were collected, and the concentrations of the cytokine TNF-α were measured using an ELISA kit (Jingmei Biotechnology) according to the manufacturer’s instructions.
Western blot analysis
Cultured cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Santa Cruz Biotechnology), then supplemented with protease and phosphatase inhibitors, scraped off the flasks, and collected for protein extraction. Tissue samples were collected around the injury from the cortex and were extracted with RIPA lysis buffer. Lysates were incubated on ice and supernatants were collected after centrifugation. The protein concentration was determined using a BCA protein assay kit (Abcam). Then 30 μg of total protein was loaded on a gel and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently proteins were transferred to polyvinylidene difluoride membranes and probed with primary antibodies against NFκB p65 (1:500; Santa Cruz), p-NFκB p65 (1:500; Santa Cruz), iNOS (1:500; Abcam), 3-nitrotyrosine (3-NT; 1:3,000; Abcam), α-SMA (1:1,000; Cell Signaling Technology), cleaved caspase3 (1:3,000; Abcam), Bax (1:1,000; Abcam), Bcl2 (1:2,000; Abcam), platelet derived growth factor receptor (PDGFR) β (1:1,000; Abcam), neuron-glial (NG) 2 (1:1,000; Abcam), ZO-1 (1:500; Abcam), occludin (1:1,000; Abcam), claudin-5 (1:500; Thermo Fisher Scientific, Waltham, MA, USA), or aquaporin (AQP) 4 (1:1,000; Abcam) followed by incubation with appropriate horseradish peroxidase-conjugated IgG (1:5,000, Boster Biotech) secondary antibodies. Immunoblots were visualized using the Millipore ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or Lamin B1 (1:3,000, Cell Signaling Technology).
Cortical tissue from lesioned areas was fixed in formaldehyde, embedded in paraffin, and cut into 4-μm sections. Slices went through xylene dewaxing and an alcohol gradient rehydration as above and were stained with Nissl solution (Boster Biotech, Wuhan, China) for 5 min. Compared to normal neurons, the cell bodies of injured neurons were shrunken and/or contained vacuoles and the nuclei stained darker. A pathologist who was blinded to the experiments randomly selected five random regions of interest (ROIs) under a high magnification optical microscope (×400; Leica, Wetzlar, Germany) to observe positively stained cells surrounding injured areas. Five random ROIs were selected for quantification, and the mean (%) was used for the statistical analysis.
A TUNEL assay was performed using an apoptosis kit according to the manufacturer’s instructions (Roche Inc., Indianapolis, IN, USA). Slices were incubated with NeuN (1:500; Abcam) overnight at 4 °C, and after washing in PBS, the samples were incubated with TUNEL reaction mixture for 1 h at 37 °C. TUNEL-positive neurons around the injured area were observed and counted with a microscope at high magnification (×400). Five ROIs were selected for quantification and averaged for statistical analysis.
Assessment of neurological injury
Nerve injury was assessed by modified neurological severity score (mNSS) and Garcia test[22, 42]. The mNSS included motor, sensory, and reflex tests in mice. The neurological injury was recorded when a task was not completed successfully or when the corresponding reflex was lost. The mNSS test was graded on a scale of 0–18, in which a score of 0 indicated normal performance and a total score of 18 points indicated severe neurological deficits, 1–6 indicated mild injury, 7–12 indicated mean-moderate injury, and 13–18 indicated severe injury. The Garcia test consisted of seven evaluations: spontaneous activity, axial sensation, vibrissae proprioception, and limb symmetry, as well as the ability to perform lateral turning, forelimb outstretching, and climbing. Each test received a score between 0 (worst performance) and 3 (best performance), and a total Garcia score was calculated as the sum of all subtests (maximum = 21 points). The evaluation was performed pre-injury and post-injury (24, 48, and 72 h) by investigators who were blinded to the experiments.
Evans blue (EB) extravasation assay
BBB permeability was investigated by measuring the extravasation of EB. EB (Sigma-Aldrich; 2% in saline; 5 mL/kg) was injected via the common carotid artery 2 h prior to sacrifice 72 h after TBI. Mice were transcardially perfused with ice-cold PBS (pH 7.2–7.4) immediately after sacrifice, followed by 4% paraformaldehyde in PBS. The brains were then removed, dissected, weighed, and homogenized in 600 μl 7.5% (w/v) trichloroacetic acid. The samples were then centrifuged. The absorption of the supernatant was measured using a spectrophotometer at a wavelength of 620 nm. The quantity of Evans blue was calculated according to a standard curve and expressed as micrograms of Evans blue/g of brain tissue.
Analysis of cerebral edema
The wet/dry weight method is used to evaluate brain water content, which is a reliable method for brain edema. Briefly, 72 h post-TBI, brains were rapidly removed from the skull, the brain tissue was removed from the injured side with a fixed weight and put in the pre-weighed aluminum foil, and samples were then placed in an oven for 72 h at 90 ℃ and reweighed for dry weight content. Brain water content was calculated according to the following formula: brain water content (%) = (total wet weight of brain - dry weight of brain)/total wet weight of brain × 100%.
Laser speckle contrast imaging (LSCI)
Cortical blood flow was monitored using the laser speckle technique as described previously. The laser speckle imaging system (Wuhan SIM Opto-technology Co., Wuhan, China) consisted of a continuous wavelength (λ = 785 nm) laser source, an Olympus ZS61 microscope, a charge-coupled-device camera, and a computer. The selection of the ROIs in the LSCI was performed by tools provided by the software, and the values obtained were the average blood-flow values in the region. Before making the model, the regional CBF was recorded as baseline. LSCI was used to observe the relative blood flow values in the capillary areas surrounding the impinging lesion in mice before and after craniotomy, and post-injury (5 min, 24 h, 72 h). Before inducing the model, the regional CBF was recorded as the baseline.
All statistical analyses were performed using SPSS 23.0 statistical software (SPSS Inc., Chicago, IL, USA). The results are expressed as mean ± standard deviation. The comparison between two groups was performed using an independent samples t-test, while the statistical difference between each group was evaluated by one-way analysis of variance (ANOVA) with Bonferroni correction for post hoc multiple comparisons. Differences with p < 0.05 were considered statistically significant.