Animals
All animal experiments were approved by the 900th Hospital Ethics Committee (Fuzhou, China) and conducted under its strict supervision. Adult male C57BL/6 mice weighing 22–28g were purchased from the experimental animal facility of Fujian Medical University. During the experimental period, all animals were housed at room temperature (24–26°C) under a 10h/14h light/dark cycle with free access to standard rodent chow and water.
Experimental Animal Grouping
Mice were randomly divided into five groups (n = 18 mice per group): control, TBI, TBI + ω-3 PUFA, TBI + ω-3 PUFA + NGF inhibitor, and TBI + ω-3 PUFA + PMA (Adam17 activator). Nine mice per group received neurological assessment and the remaining mice were used for histological and molecular studies. A TBI mice model was established using a controlled cortical impact (CCI) device [23]. Briefly, mice were deeply anesthetized with 3% isoflurane in a Plexiglas container and maintained under anesthesia with 1.5% isoflurane using a small animal anesthesia machine (RWD Life Science Co., Shenzhen, China). The anesthetized mice were placed in a stereotaxic frame and the skull was exposed by cutting the scalp along the midline. A hole of approximately 3.0 mm in diameter was drilled midway between the right fontanelle and the lambda and 2.0 mm lateral to the midline. The exposed cortical surface was struck with a 2 mm metal flat-head impactor (Brain and Spinal Cord Impactor, 68099H, RWD Life Science) at 5 m/s to a depth of 3 mm and with a strike dwell time of 100 ms. After the strike, the scalp was sutured and the mouse placed on a heating pad until recovery from anesthesia. Control animals underwent the same surgical procedure but without cortical impact. Thirty minutes after TBI, the TBI + Omega-3 PUFA, TBI + Omega-3 PUFA + PMA, and TBI + Omega-3 PUFA + NGF inhibitor groups were given intraperitoneal injections of Omega-3 PUFA (3 mg/kg docosahexaenoic acid [DHA]), NGF inhibitor (Ro 08-2750, 13.75 mg/kg), and (or) ADAM17 activator (PMA, 100 µg/kg) as indicated, while the remaining groups were injected with the same volumes of 0.9% NaCl as a control.
Bv2 Cell Culture And Lps Stimulation
The mouse microglia cell line BV2 was obtained from the Chinese Cell Line Resource Infrastructure (Beijing, China) and cultured at 37°C in humidified air containing 5% CO2. The medium was composed of 90% Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, Frederick, MD, USA), 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), and 1% antibiotics (100 U/mL penicillin and 100 g/mL streptomycin). Cells were activated by treatment with LPS (100 ng/mL)
Culture Of Primary Neurons
Primary neurons were isolated from the cortex of sham, TBI, and TBI + Omega-3 PUFA group mice, seeded on 6-well plates coated with poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) at approximately 7 × 105 cells/well, and cultured in DMEM. Four hours after seeding, the medium was changed to Neurobasal medium (Gibco, Carlsbad, NM) supplemented with B-27 (Gibco). Cells were incubated in a humidified incubator at 37°C under a 5% CO2 atmosphere. Cultures were used for experiments 7–10 days after seeding.
Isolation, Identification, And Labeling Of Microglia-derived Exosomes
BV2 cells were washed twice with PBS and grown in medium which removed any spontaneously released exosomes. The vehicle group was treated with PBS, the LPS group with LPS (200 ng/mL), and the LPS + ω-3PUFA group with LPS (200 ng/mL) and DHA (200 ng/mL) for 5 h. After 48 h of cell culture, the supernatant was collected and serially ultracentrifuged at 4°C. Exosomes were isolated from supernatant using a whole exosome isolation kit (4478359, Invitrogen, USA)
Exosome concentration and size distribution were measured by Brownian motion using a NanoSight LM10 nanoparticle analyzer (Malvern) equipped with a Blue488 nm laser and high-sensitivity sCMOS camera at a constant temperature of 25°C. The syringe pump speed was set to 20 Units. The exosome suspension was allowed to settle for 90s before acquisition and then three measures were acquired for 30s. The three repeated measures were averaged to determine the particle size distribution and nanoparticle concentration. Exosomes were also labelled using a PKH26 kit, washed and diluted with PBS, and then examined by transmission electron microscopy (JEM-2100F, Japan) for exosome uptake experiments.
Protein Mass Spectrometry
The isolated and purified exosome samples were diluted 8-fold in 50 mM ammonium bicarbonate (Sigma) and digested overnight using mass spectrometry grade Trypsin/LysC (Promega) at a 1:10 (v/v) enzyme concentration. The next day, samples were acidified to pH < 4.0 using 1% TFA (Sigma) and the release peptide contents identified by either a Q-accurate HF (Thermo Scientific) or an orbital Lapp Lumos hybrid mass spectrometer, both coupled on-line to an EASY-nLC II 1200 chromatography system. Briefly, samples were loaded onto a 50 cm fused silica emitter with ReproSIL-Pur C18-oven. The peptides were eluted in Q-Exactive HF using a two-step gradient of 80% acetonitrile (solvent B) at a flow rate of 300 nL/min for 125 min, 2–20% 0.1% formic acid over 73 min, and 41% formic acid for 93 min. The peptides were injected into the mass spectrometer by electrospray using a nanoelectrospray ion source (Thermo Scientific), and an active background ion reduction device (ABIRD, ESI source solution) was used to reduce potential airborne contaminants. Data were acquired in positive ion mode using Data Dependent Acquisition (DDA) and Xcalibur software (Thermo Scientific). The mass range for the full scan was set to 375–1500 m/z with a resolution of 60,000 Units. The injection time was set to 20 ms and the target value was 3E6 ions. The MS files were processed by MaxQuant software version 1.6.1.0 for all experiments.
Cell Survival Assay
Cell viability was determined using Cell Counting Kit-8 solution (CCK-8, Dojindo, Kumamoto, Japan). The CCK-8 solution was added to each well for 2 hours and absorbance measured at 450 nm using a microplate reader as an estimate of viable cell number.
Western Blotting
Western Blotting
Proteins were extracted from tissue samples surrounding the cortical injury site using Radioimmunoprecipitation Assay (RIPA) Lysis Buffer (Santa Cruz Biotechnology, Dallas TX, USA). Briefly, lysates were incubating on ice and centrifuged to isolate soluble proteins in the supernatant. Protein concentrations in supernatant were determined using a BCA protein assay kit (Abcam, Cambridge, UK), then equal amounts per gel lane (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for 2 h at room temperature and then incubated with primary antibodies targeting CD16+ (1:500; Santa Cruz), ADAM17 (1:500; Abcam), and TNF-α (1:500; Santa Cruz), TNFR1 (1:500; Santa Cruz), p-IκB (1:500; Santa Cruz), NF-κB p65 (1:500; Santa Cruz), p-NFκB p65(1:500; Santa Cruz), caspase-3 (1:3,000; Abcam), Bax (1:1,000; Abcam), Bcl2 (1:2,000; Abcam), NGF (1:1000, Abcam), TrkA (1:1,000; Abcam), and p-TrkA (1:1,000; Abcam). After incubation with an appropriate secondary antibody, immunolabeling was visualized using the Millipore ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Target band density (gray level) was measured using UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA) and normalized to β-actin (1:2000, Abcam) or GAPDH (1:2000, Abcam) density as the gel loading control.
Immunofluorescence
Cultured cells treated as indicated were fixed in formaldehyde, embedded in paraffin, cut into 4-µm thick sections, dewaxed in xylene, rehydrated in gradient alcohol, then subjected to antigen repair, and incubated at 4°C with antibodies against CD16 (1:500, Abcam), NeuN (1:500; Abcam), and NF-κB p65 (1:500; Santa Cruz). After washing, sections were incubated with secondary antibodies for 1 h at room temperature. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunopositive cells in 5 selected areas were counted under a fluorescence microscope by an observer blinded to treatment history.
Immunohistochemistry
Brain tissue samples were fixed in formaldehyde, embedded in paraffin, cut into 4-µm thick sections, dewaxed in xylene, rehydrated in gradient alcohol, and incubated with an antibody against ionized calcium-binding adapter molecule (Iba)-1 (1:500; Abcam, Cambridge, UK). Stained sections were then washed, incubated with secondary antibody for 1 h at room temperature, and examined under fluorescence microscopy. Five sections from each animal were graded for fluorescent signal intensity as follows [28]: 0, no positive signal; 1, very few positive cells; 2, moderate positive cells; 3, large number of positive cells; 4, maximum number of positive cells.
Nissl Staining
Tissue specimens encompassing the TBI area were fixed in formaldehyde, embedded in paraffin, cut into approximately 4-µm thick sections, dewaxed in xylene, rehydrated in gradient alcohol, and treated with Nissl staining solution for five minutes. The number of surviving neurons was counted in 5 randomly selected regions of interest (ROIs) by a researcher who was unaware of the experimental group.
Tunel Staining
Apoptotic cells were counted by TUNEL assay using the Apoptosis Cell Kit (Roche Inc., Indianapolis, IN, USA). Sections were washed in PBS and incubated with NeuN (1:500; Abcam) overnight at 4°C and then with the TUNEL reaction mixture for 1 h at 37°C. The number of TUNEL-positive neurons in the area surrounding the TBI site was counted under high-powered microscopy (×400) in five randomly selected ROIs.
1.1 Enzyme-linked immunosorbent assays (ELISAs)
Concentrations of TNF-α, IL-1β, IL-6, and IFN-γ were measured in brain tissue using ELISA kits (Jingmei Biotechnology, Jiangsu, China). Standards and samples were incubated with horseradish peroxidase (HRP) after binding to the respective antibodies according to the manufacturer's instructions. Optical density (OD) values were converted to concentrations according to standard curves.
Blood–brain Barrier Permeability Measures By Evans Blue Staining
Blood–brain barrier permeability was estimated by the extravasation of Evans Blue (EB) following cerebrovascular injection. Briefly, EB solution (Sigma-Aldrich; 2% in saline; 5mL/kg) was injected through the common carotid artery 3 days after TBI and 2 h prior to sacrifice [29]. After sacrifice, mice were perfused with refrigerated PBS (4 ± 1°C) via the heart until no blood flowed out, and then with PBS containing 4% paraformaldehyde. Brains were removed, weighed, and homogenized in 600 µL of 7.5% (w/v) trichloroacetic acid solution. Homogenates were centrifuged and absorption of the supernatant was measured by spectrophotometry at 620 nm. The amount of Evans blue was calculated from a standard curve and expressed as micrograms of per gram of brain tissue (wet weight).
Analysis Of Cerebral Edema
At 72 hours after TBI, brains were quickly removed and weighed on pre-weighed aluminum foil. The samples were then baked in an oven at 90°C for a further 72 h and re-weighed. Brain water content was calculated as (total wet brain weight - dry brain weight) / total wet brain weight × 100%.
Assessment Of Motor And Neurobehavioral Function
Mice were examined for motor (muscle status and abnormal movements), sensory (visual, tactile and balance sensations), and reflex responses using a modified neurological severity score (mNSS) [28]. Each test was scored 0 (normal) or 1 (impaired) for a maximum total score of 18. Performance was then gaged as normal (total score of 0), mild impairment (1–6), moderate impairment (7–12) or severe impairment (13–18).
The Garcia test of neurological function was also used to evaluate spontaneous movement, axial sensation, touch proprioception, limb symmetry, lateral transfer, forelimb extension, and climbing ability. Each test was scored from 0 (poor) to 3 (normal) for a maximum total score of 21, with higher scores indicating better overall performance. Both tests were conducted at different times by experimenters unaware of the grouping information.
Statistical Analysis
All statistical analyses were performed using SPSS 23.0 (SPSS Inc., Chicago, IL, USA). Results are expressed as mean ± standard deviation. Two groups were compared by independent-samples t-test and more than two groups by one-way analysis of variance (ANOVA) with post hoc Student-Newman-Keuls tests for pair-wise comparisons. A p < 0.05 (two-tailed) was considered statistically significant for all tests.