Adult male Sprague-D
awley rats (weighing 230–260 g) were purchased from the Experimental Animal Center of Fujian Medical University (Fuzhou, Fujian, China) and housed in a clean, temperature-controlled environment (23°C ± 2°C) under a 12-h light/dark cycle with free access to food and water. The experimental protocols of the present study, including all surgical procedures and animal usages, were approved by the Experimental Animal Ethics Committee of Fujian Medical University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Bethesda, MD, USA).
Cell culture and treatment
Murine microglial BV-2 cells were obtained from the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences (Beijing, China) and maintained in Dulbecco's modified Eagle’s medium (Nanjing KeyGen Biotech. Co., Ltd.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin (Nanjing KeyGen Biotech. Co. Ltd.) at 37°C under an atmosphere of 5% CO2/95% air. In vitro, BV2 cells were stimulated with 10 μg/ml of lipopolysaccharide (LPS; Dalian Meilun Biotech Co., Ltd., Dalian, China) for 6 h and 20 ng/ml of exogenous IL-4 (Novoprotein Scientific, Shanghai, China) overnight, respectively, to mimic the M1 and M2 polarization environments of the brain. Then, 50 nM LY2157299 (Selleck Chemicals, Houston, TX,USA), a TGF-βRI kinase inhibitor, was applied to the BV2 cells for further study of the signaling pathways involved in polarization.
To evaluate the effect of ADAM17 on microglia polarization, BV2 cells were transfected with small interfering RNA (siRNA) against ADAM17 and the plasmid pcDNA3.1-Flag-ADAM17, respectively. Mouse ADAM17 siRNA and a negative control were chemically synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Mouse pcDNA3.1-Flag-ADAM17 (open reading frame sequence MR210844) was purchased from PPL (Nanjing KeyGen Biotech. Co. Ltd.). The BV2 cells were transiently transfected with ADAM17 siRNA and the plasmid with a corresponding negative control using Lipofectamine 2000 reagent (Thermo Fisher Scientific Waltham, MA, USA) for 24 h at 37℃ in accordance with the manufacturer’s instructions. Afterward, transfected BV2 cells were treated with 50 nM LY2157299 for 24 h at 37°C for signaling pathway assessment. The target sequences of ADAM17 siRNA and RealTime-PCR primers of ADAM17 are showed in table S1, S2.
Animal model and drug delivery
Rats were randomly assigned to one of four groups: sham, TBI, TBI+Vehicle, or TBI+ TNF-alpha protease inhibitor I (TAPI-1, an ADAM17 inhibitor) (n = 48 each), each of which were further divided into four time-based subgroups (1, 3, 7, and 14 days). The rat TBI model was established as previously described under sodium pentobarbital anesthesia (50 mg/kg by intraperitoneal injection). An incision was made to the scalp and a 5 mm-diameter hole was made to the right side of the coronal suture (anterior-posterior, -2 mm; lateral-anterior, 2 mm). A 40-g hammer was dropped on the brain from a height of 20 cm to simulate TBI. The hole in the posterior bone was then sealed closed with wax. In sham rats, the surgery was performed but the impact was omitted. Rats in the TBI+TAPI-1 group were administered TAPI-1 by intraventricular injection (300 mg/kg/day; Sigma–Aldrich Corporation, St. Louis, MO, USA) at 0.5 h after surgery, while those in the TBI+Vehicle group were administered an equal volume of the vehicle dimethyl sulfoxide as a negative control.
Neurological impairment score
Rats were subjected to exercise (muscular state and abnormal action), sensation (visual, tactile, and balance), and reflex examinations and assigned a modified neurological severity score (mNSS) that was recorded when the rat failed to complete the task or there was no corresponding reflex. The mNSS ranged from 0 to 18 points, where a total score of 18 points indicated severe neurological deficits and a score of 0 indicated normal performance (13–18 points indicated severe injury, 7–12 indicated moderate injury, and 1–6 indicated mild injury). Neurological function was measured at different time points by investigators who were blinded to group information.
Measurement of brain water content and BBB permeability
Brain water content was calculated using the wet weight-dry weight method. Animals were sacrificed after neurological assessment and the brain cortex was removed at the edge of the bone window (200 ± 20 mg). Filter paper was used to remove excess blood and cerebrospinal fluid. The wet weight was measured and the brains were dried in an oven at 100°C for 24 h until a constant weight was achieved, at which point the dry weight was measured. The % brain water content was calculated as: (wet weight − dry weight) / wet weight × 100%.
BBB permeability was investigated by measuring the extravasation of Evans blue dye (2% in saline; 4 mL/kg; Sigma-Aldrich Corporation), which was intravenously injected 2 h prior to sacrifice on post-injury day 3. Following sacrifice, the mice were transcardially perfused with PBS followed by PBS containing 4% paraformaldehyde. Each tissue sample was immediately weighed, homogenized in 1 mL of 50% trichloroacetic acid, and centrifuged. Then, the absorption of the supernatant was measured with a spectrophotometer (UV-1800 ENG 240V; Shimadzu Corporation, Kyoto, Japan) at a wavelength of 620 nm. The quantity of Evans blue dye was calculated using a standard curve and expressed as µg / g of brain tissue.
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. Samples were treated with Nissl staining solution for 5 min. Damaged neurons were shrunken or contained vacuoles, whereas normal neurons had a relatively large and full soma and round, large nuclei. Five randomly selected areas were examined by microscopy by investigators who were blinded to the experimental group.
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 (Ab) against ADAM17 at a dilution of 1:200 (Abcam plc, Cambridge, UK), washed, and then incubated with a secondary Ab for 1 h at room temperature. The negative control was prepared without adding the anti-ADAM17 Ab. Five randomly selected visual fields were analyzed. Signal intensity was evaluated as follows: 0, no positive cells; 1, very few positive cells; 2, moderate number of positive cells; 3, many positive cells; and 4, the highest number of positive cells.
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 Abs against neuronal nuclei (1:100; Wuhan Boster Biological Technology, Ltd., Wuhan, China), ionized calcium-binding adapter molecule (Iba)-1 (1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), glial fibrillary acidic protein (GFAP) (1:200; Santa Cruz Biotechnology, Inc.), cluster of differentiation (CD)16, CD206, TGF-βRII, and p-Smads (1:100; Wuhan Boster Biological Technology, Ltd.). After washing, the sections were incubated with secondary Abs for 1 h at room temperature. The cell nuclei were stained with 4',6-diamidino-2-phenylindole. Immunopositive cells in five randomly selected fields were counted under a microscope (Leica, Wetzlar, Germany) at 400× magnification by investigators who were blinded to the experimental group.
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay
Apoptotic cells were detected using a TUNEL kit (Roche Diagnostics, Indianapolis, IN, USA) in accordance with the manufacturer’s instructions. Indicators of apoptosis included a shrunken cell body, irregular shape, nuclear condensation, and brown diaminobenzidine staining, as observed by microscopy at 400× magnification. Positive cells in five random fields per section were counted.
Enzyme-linked immunosorbent assay (ELISA)
Inflammatory factors (i.e., TNF-α, IL-1 β, IL-6, and IFN-γ) of brain tissues and BV2 cell culture supernatants were detected using mouse ELISA kits (Nanjing KeyGen Biotech. Co., Ltd.) at an optical density of 450 nm using a microplate reader (SpectraMax M3; Molecular Devices, Inc., San Jose, CA, USA).
Western blot analysis
The samples, including brain tissues and BV2 cells, were prepared using nuclear and cytoplasmic protein purification assays (Nanjing KeyGen Biotech. Co., Ltd.) with modified radioimmunoprecipitation lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate). The protein concentrations of the cell nuclear and cytosolic lysates were determined separately with a bicinchoninic acid assay (Beyotime Institute of Biotechnology, Shanghai, China). Approximately 25 µg of protein were loaded to each well of a polyacrylamide gel, separated by electrophoresis, and then transferred to polyvinylidene difluoride (PVDF) membranes, which were incubated with primary Abs against B cell lymphoma (Bcl)-2 (1:2000; Abcam plc), Bcl-2-associated X factor (Bax) (1:5000; Abcam plc), cleaved caspase-3 (1:3000), Iba-1 (1:1000), CD16, CD206, IL-1β, Arg-1 (1:2000), TGF-β1, TGF-βRI, TGF-βRII, TGF-βRIII, p-Smads, and Smads (1:1000) (all purchased from Abcam, Shanghai, China), followed by incubation with appropriate secondary Abs. Immunoreactivity was visualized with the ECL Western Blotting Detection System (EMD Millipore Corporation, Billerica, MA, USA). Grey value analysis was conducted with UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA). Expression levels were normalized against β-actin (1:5000; Wuhan Boster Biological Technology, Ltd.)
Co-immunoprecipitation (Co-IP) analysis
BV2 cells were homogenized in IP lysis Buffer (Nanjing KeyGen Biotech. Co., Ltd.) and then incubated with 1 μg of TGF-β1 (Santa Cruz Biotechnology, Inc.) or TGF-βR II (Abcam plc) Abs or immunoglobulin G (Abcam plc) for 1 h at 4℃. A 10-μl volume of protein A agarose beads (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) was added to the sample lysate mixture and incubated overnight with primary Abs at 4℃. After immunoprecipitation and centrifugation, the agarose beads were washed three times with lysis buffer and used for immunoblotting to detect expression of the TGF-βRII and TGF-β1 proteins in order to estimate the expression level of the TGF-β1/TGF-βRII complex.
Data were analyzed using PASW Statistics for Windows, version 18.0. (SPSS Inc., Chicago, IL, USA). All experiments were performed in triplicate unless otherwise noted and the results are expressed as the mean ± standard deviation. Comparisons between groups were made with the unpaired Student’s t-test. Multiple-group comparisons were assessed by one-way analysis of variance and post hoc multiple comparisons were performed using the Student–Newman–Keuls test. A probability (p) value of < 0.05 was considered statistically significant.