Focal cerebral ischemia
Male Sprague–Dawley rats (250–270 g) and C57BL/6 J mice (20–25 g) were reared under a 12-h light/dark cycle at approximately 25 °C and 65% humidity, and were provided with free access to food and water. Transient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) surgery following previous methods [12, 16]. A silicon-coated monofilament (diameter 0.18 mm for mice, 0.36 mm for rats) was used to occlude the right middle cerebral artery for 90 min and subsequently withdrawn for reperfusion. A heating pad was used to maintain the body temperature at 37.0 ± 0.5 °C during surgery. The sham-operated animals underwent the same procedures except that the middle cerebral artery was not occluded. A total of 42 male rats (n = 8 for sham operation; n = 34 for MCAO) and 46 male mice (n = 8 for sham operation; n = 38 for MCAO) were used in this study. All animal experiments were carried out in compliance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) and approved by the ethical standards of Nanjing First Hospital (Permit Number: DWSY-2104285). Maximum efforts have been made to minimize the number of animals used and their suffering.
Primary neuron culture
Primary cortical neurons were isolated from the embryonic C57BL/6 J mice (E16–18) according to previous reports [12]. Briefly, cortical tissues were digested with 0.125% trypsin (Gibco, MD, USA) for 15 min. Then the cortical tissues were pipetted blow and mixed 100 times and filtered with a 100-μm cell strainer (Biologix, Shandong, China) to remove impurities. Subsequently, the cells were centrifuged and suspended in DMEM medium (Hyclone, UT, USA) with 10% FBS (Gibco, USA) and then seeded into flasks. Two hours later, the DMEM medium was replaced with neurobasal medium supplemented with 2% B27 and 1% glutamax (Thermo Fisher Scientific, MA, USA).
BV-2 microglia culture and OGD/R
BV-2 microglial cell line, purchased from China Infrastructure of Cell Line Resources (Beijing, China), was grown in DMEM F12 medium (Gibco, USA) supplemented with 10% FBS (Invitrogen, USA), and 1% penicillin–streptomycin (Invitrogen, USA). oxygen and glucose deprivation/reoxygenation (OGD/R) was conducted according to a previously established protocol [17]. Normal cell culture medium was removed and changed by glucose-free DMEM medium (Gibco, USA). Then, cells were incubated in an oxygen-free chamber equipped with AnaeroPack-Anaero (MGC, Japan) at 37 °C for 2 h. Finally, the cells were returned to normal incubator and incubated with the initial culture medium for reoxygenation.
rTMS
The 10 Hz repetitive transcranial magnetic stimulation for BV2 microglial cells and MCAO rats were conducted according to a previous study [12]. A customized magnetic stimulator (MagPro X 100 with Magoption, Tonica, DK) with a C-100 circular coil (20 mm in inner diameter with 1.9 T peak magnetic stimulator output) was used. For BV2 cells, the magnetic coil was placed and positioned 1 cm away from the cell culture dish to give 10 Hz for two consecutive days. For MCAO rats, rTMS started at 24 h after the reperfusion and lasted for 7 days. The magnetic coil was located above the ipsilateral primary motor cortex (right M1 region). The rTMS stimulation paradigm consisted of stimulation for 3 s followed by 50 s of rest, which was repeated twenty times (600 pulses per day) at a frequency of 10 Hz.
Cerebral infarct volume
The 2, 3, 5-triphenyltetrazoliumchloride (TTC, Sigma, USA) staining was applied to detect infarct volume. After the mice were sacrificed, their brains were cut into 1-mm sections immediately. Then, the brain slices were incubated with 2% TTC solution at 37 °C for 15 min and fixed by 4% paraformaldehyde (PFA) at 4 °C overnight. The relative infarct volume was calculated as previously reported [12].
Neurological deficit evaluation
Neurological deficits of the experimental animals were assessed with the modified neurologic severity score (mNSS) as described [18]. The mNSS scoring system consists of four tests: motor, sensory, balance, reflex tests and abnormal movements, such as seizure. The total scores ranged from 0 to 18, which 0 represents no deficit and 18 represents maximal deficit.
Immunofluorescence
Samples were fixed with 4% paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 10 min, then blocked with 5% BSA in PBS for 60 min at 37 °C and incubated with the indicated primary antibodies at 4 °C overnight. Primary antibodies include: anti-Iba-1 (1:200; # ab178847, Abcam, UK), anti-iNOS (1:100; # ab210823, Abcam, UK), anti-CD206 (1:100; #sc-376232, Santa Cruz, USA). After four washes with PBS, the samples were incubated with corresponding fluorescence-conjugated secondary antibodies (1:500, Jackson Immuno Research, USA) for 2 h, followed by DAPI (Sigma-Aldrich, USA) staining for 10 min. Finally, samples were observed under a FluoView FV10i confocal laser scanning microscope (Olympus, Japan) or an Olympus BX51 microscope. The fluorescently-stained cells were analyzed by Image J software (NIH, USA).
Fluorescence in situ hybridization (FISH)
FISH was performed in tissue sections using fluorescence in situ hybridization (FISH) kit (Servicebio, Wuhan, China) and the let-7b-5p detection probe (Servicebio, Wuhan, China) by following the manufacturer’s protocol.
Western blot analysis
Western blot analysis was conducted according to previous report [12]. Proteins were extracted from cortical tissues or cultured cells with RIPA lysis buffer (Cell Signaling Technology, MA, USA). The protein concentrations were detected by the BCA protein assay kit (Generay Biotechnology, Shanghai, China). Then, equal amount of protein was applied for SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). After blocking with 10% skim milk for 1 h, the PVDF membranes were cut according to the molecular weight of the proteins as indicated in the instruction manuals, then incubated with primary indicated antibodies: CD206 (1:500; #sc-376232, Santa Cruz, USA), iNOS (1:1000; # ab210823, Abcam, UK), HMGA2(1:1000; ab97276, Abcam, UK), p-IκBα(1:1000, #2859, Cell Signaling Technology, USA), IκBα (1:1000, #9242, Cell Signaling Technology), p-P65(1:1000, #3036, Cell Signaling Technology), P65(1:1000, #4764, Cell Signaling Technology), and β-actin (1:5000, Cell Signaling Technology) overnight at 4 °C. After four washes with PBST, HRP-conjugated secondary antibodies were used for further incubation of the membranes for 120 min at room temperature. Finally, the protein signals were developed by the ECL solution (Millipore, MA, USA). Quantitative analysis of protein blots was analyzed by Image J software. The original unedited blots were presented in Additional file 2.
miRNA sequencing and analysis
Peri-infarct cortex tissue derived from the MCAO and MCAO + rTMS rats (n = 3) were used for miRNA sequencing. The miRNA sequencing was completed by Illumina HiSeqTM 2500 platform and data analysis was performed by Ribo Bio (Co., Ltd., Guangdong, China). Comparisons between groups were performed with two-tailed Student’s t-tests, wherein P < 0.05 and fold changes > 2 were considered significant.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from peri-infarct cortex tissues and BV2 cells using TRIzol regent (Sigma-Aldrich, USA) followed by cDNA synthesized by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). SYBR Green based quantitative PCR was conducted on a Stratagene Mx3000P real-time PCR system (Agilent Technologies). U6 was used as endogenous controls for miRNA, results were calculated using 2−ΔΔCt (Ct, threshold cycle). Primers for qRT-PCR were listed in Additional file 3: Table S1.
miRNA transfection and dual-luciferase reporter assay
Let-7b-5p agomir, antagomir, the entire and mutated 3’ UTR of HMGA2 were generated by Ribo Bio (China). For miRNAs transfection, 293 T or BV2 cells were incubated with 50 nM let-7b-5p agomir, antagomir or non-targeting control sequence using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, USA). Dual-luciferase report assay was conducted according to previous report [17]. Briefly, 293 T cells were plated into 96-well plates and cultured to 90–95% confluence. Cells in each well were then co-transfected with 1 µg of the indicated 3′ UTR luciferase reporter vectors and 100 nM of let-7b-5p agomir or control agomir (Ribo Bio, China). Finally, cells were harvested after 48 h to measure the Renilla and firely luciferase activities by the Dual-Luciferase Reporter Assay System (Promega, MI, USA).
Microglia-conditioned media collection
BV2 microglia-conditioned media (MCM) were obtained as previously reported [12]. After OGD/R and receiving rTMS stimulation, the BV2 culture media were collected at 48 h post-OGD and centrifuged. The MCM were applied to ELISA experiment or used to co-culture neurons.
Concentrated MCM was used for MCM therapy. The concentrated MCM were obtained by centrifugation of MCM in the 10 KDa-membrane centrifuge tubes (Millipore, UFC901024) at 4000g at 21 °C for 30 min.
MCM Therapy
Under anesthesia, each mouse received posterior orbital intravenous injection of 100 μL concentrated MCM at the time of MCAO reperfusion, and at three days post reperfusion.
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of TNF-α and IL-10 in all MCM samples were measured by the specific ELISA kit (Neobioscience, China). These measurements were based on the instructions of manufacturer.
Cell death detection
Propidium iodide (PI)/Hoechst 33342 assay kit (Thermo Fisher Scientific, USA) was used to detect cell death following the manufacturer’s instructions. Firstly, PI/Hoechst 33342 solution was added to the neurons which were cultured with different MCM for 48 h. After incubating for 15 min, the neurons were washed with PBS for five times and observed under an Olympus BX51 microscope. The dead cells were stained with propidium iodide (red), while live cells stained with Hoechst 33342 (blue). The fluorescently-stained cells were calculated by Image J software (NIH, USA).
Statistical analysis
SPSS 22.0 software (IBM, Armonk, NY, USA) was used for data analysis. Differences between groups were compared using two-tailed Student’s t tests and one-way ANOVA followed by Tukey’s post hoc test. All data are expressed as mean ± SD. Statistical significance was determined as P < 0.05.
