Regents and Antibodies
rhFGF21 supported by the laboratory of Biotechnology Pharmaceutical Engineering at Wenzhou medical university and synthesized on the basis of the study previously reported [30]. Antibodies in Flow cytometry analysis involving CD3-PE (17A2, 100206), CD8-FITC (53-5.8, 140404), CD4-APC (GK1.5, 100412), F4/80-FITC (BM8, 123108), NK1.1-APC (PK136, 180710), Ly6G-PE (1A8, 127608), Ly6C-APC (HK1.4, 128016), CD45-APC (103112), CD11b-PE (101208), CD206-FITC (C068C2, 141704), CD68-PE (FA-11, 137014), CD86-PE (GL-1, 105008), CD45-PE/Cy7 (30-F11, 103114), CD11b-PE/Cy7 (M1/70, 101216) purchased from BD Biosciences (San Jose, CA, USA) and CD45-APC (OX33, 17046280), CD11b-PE (OX42, 12011080), CD86-FITC (24F, 11086081) purchased from eBioscience (San Diego, CA, USA).
Antibodies applied in Immunofluorescence including CD16/32(AF1460), CD206 (AF2535), purchased from R&D Systems (Minneapolis, MN, USA) and Iba1(019-19741) purchased from wako pure chemical corporation (Japan).
The primary antibodies applied in Western Blot including anti-NF-κB (3033T), anti-FGFR1 (ab824), anti-p-FGFR1 (ab59194), anti-PPAR-γ (ab28364) and anti-β-Actin (ab8227) were purchased from Cell Signaling Technology (Danvers, MA, USA) or Abcam (Cambridge, MA, USA). The secondary antibody used were donkey anti-rabbit IgG H&L (HRP) (ab150075) or goat anti-mouse IgG H&L (HRP) (ab150115), which were commercially purchased from Abcam (Cambridge, MA, USA).
Corresponding reagent or kit applied in this study include Trizol reagent (Qiagen, Duesseldorf, Germany), PrimeScriptTM RT Reagent Kit (TaKaRa, Shiga, Japan), iQTM SYBR Green supermix (Bio-Rad, Hercules, CA, USA), miRNeasy Micro Kit (Qiagen, Duesseldorf, Germany), QuantiTect reverse Transcription kit (Qiagen, Duesseldorf, Germany), TaqMan® Gene Expression Assays (ThermoFisher Scientific, Fremont, CA, USA), Neural Tissue Dissociation Kits (Miltenyi Biotech, Bergisch Gladbach Germany), Fluoroshield mounting medium with DIPI (Abcam, Cambridge, MA, USA)
Animals Group and Drug Administration
C57BL/6 mice (20-25 g) were purchased from the Animal Center of the Chinese Academy of Science (Beijing, China) and all surgical procedures and experimental protocols were approved by the Animal Care and Use Committee of Wenzhou medical university. All animals were blindly and randomly assigned to three groups: Sham, MCAO, MCAO+rhFGF21group. Sham-operated mice under anesthesia and surgical procedure identically but without filament inserted. rhFGF21 (1.5mg/kg) were given to animals intraperitoneally beginning at 6 h after reperfusion for consecutive 7 days.
Transient Focal Cerebral Ischemia and Reperfusion Model Preparation
The surgical procedures to establish MCAO model was based on the method induced by intraluminal filament technique [31]. Briefly, the mice were anesthetized by isoflurane and then placed on a heating blanket to maintain body temperature at 37±0.5℃. A midline incision of mice was made to expose common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). The CCA was temporarily closed and a monofilament was insert into ICA through the ECA until reached the middle cerebral artery and stay for 60 min, laser doppler flowmetry was used to monitor cerebral flow that lower than 20% of pre-ischemic. The occluding filament was returned to ICA to achieve reperfusion after 60 min occlusion.
Neurological function assessment
The modified Neurological Severity Score (mNSS), rotarod test, corner-turning test and adhesive move test were performed to assess neurodeficits, motor coordination, sensorimotor asymmetry and feeling functions at multiple time points after surgery. All animals received training before suffering ischemia reperfusion injury. The procedure of assessment was performed by the same investigator who were blinded to surgical groups in order to reduce the variance in experiment.
Quantitative real-time PCR
Total mRNA was isolated from cortex samples around infarcted zone using trizol reagent according to the manufacturer’s instructions. The cDNA was synthesized by PrimeScriptTM RT Reagent Kit. PCR were performed on CFX Connect Real-time System (Bio-Rad, Hercules, CA, USA) using SYBR Green. Primers used in this study were involved in table 1. Additionally, total RNA was extracted from sorted microglia using the miRNeasy Micro Kit according to the manufacturer’s protocol. cDNA was transcribed with Reverse Transcription kit and was amplified in stepone using Gene Expression Assays of TNF-α, IL-6, IL-1β and TGF-β. the volume of reaction was set in 20 µl and performed at 50℃ for 2 min, 95℃ for 20 s, followed by 40 cycles of 1s at 95℃and 20s at 60℃. Data were analyzed using the 2-∆∆Ct method and expression level of relative mRNA were then reported as fold difference.
Flow cytometry
After mice were euthanized, fresh brain, spleen and blood tissues were harvested for single-cell suspension preparation for subsequent single-cell analysis using fluorochrome-conjugated antibodies. Spleen and blood tissues were dissociated into single-cell suspensions as previously described [32, 33]. Splenocytes were dissociated by sieving through a 70-µm filter, then lysing solution (BD Bioscience, CA, USA) was used to deplete red blood cells in the spleen and blood. Brain mononuclear cells were prepared by Neural Tissue Dissociation Kits according to the protocol. Briefly, the ipsilateral hemisphere of the brain was collected and dissected into small pieces. The pieces were pipetted back into an appropriate-sized conical tube, rinsed with cold Hank's balanced salt solution (HBSS), and then centrifuged at room temperature. After the supernatant was carefully aspirated, a preheated enzyme mix was added to digest tissue pieces for cell suspension. Single pellets were isolated by passing through a 30-µm cell strainer. Cell pellets obtained from the spleen, blood and brain were washed and incubated with antibodies targeting the following: CD3, CD8, CD4, F4/80, NK1.1, Ly6G, Ly6C, CD45, CD11b, CD206, CD68 and CD86, and tagged with phycoerythrin (PE), fluorescein isothiocyanate (FTIC), allophycocyanin (APC), PerCP-Cy5.5 or PE-Cy7. Antibody staining was performed following the manufacturer’s protocol. Fluorescence-minus-one (FMO) controls were used to determine the gate of each antibody. Flow cytometry analysis was conducted using a FACS Aria flow cytometer (BD Bioscience, CA, USA), and data were analyzed by FlowJo software (Informer Technologies, USA).
Sorting of microglia and macrophages
Microglia of the mouse brain tissue were sorted by magnetic cell sorting (MACS) in combination with fluorescence-activated cell sorting (FACS). Cell pellets were prepared as described above. Cells were stained with PE-Cy7-conjugated anti-mouse CD45 antibody and PE-conjugated anti-mouse CD11b, followed by incubation with anti-PE microbeads. Cells labeled with primary antibody conjugated to PE were enriched by using MACS columns, and then targeted microglia were gathered using the FACS Aria cell sorting system. Resident microglia were identified as the CD45intCD11b+ population, whereas infiltrated immune cells in the CNS were identified as the CD45highCD11b+population. Isolated cells were collected in Trizol reagent, vortexed and kept at -80°C for further experiments.
Isolation of primary microglia
Primary rat microglia culture was isolated as previously reported[34]. In brief, the cerebral cortices separated from neonatally 1-day-old rats and meninges were removed. Trypsinization was used to digest the striped cortical tissues for 30 min and 70 µm nylon mesh cell strainer was used to obtain the mixed cortical cells. Cells were maintained in DMEM/F12 with fetal bovine serum (FBS), penicillin and streptomycin (Gibco, Grand Island, NY). Culture media were changed every three days until achieving a confluent monolayer at about 15 days. For the isolation of primary microglia, mild trypsinization was added to isolate microglia from the mixed glial cells. Purified microglia were cultured at 37℃ in atmosphere condition for future experiment.
Oxygen-glucose deprivation (OGD)
To establish ischemic-like condition in vitro, primary microglia subjected to OGD as previously reported [35]. Briefly, microglia cultured with serum-glucose-deprived cultures and placed in hypoxic chamber with 95% nitrogen and 5%CO2 for 5min and sealed tightly. Subsequently, the chamber moved to an incubator under 5% CO2/37℃for 3 h. After OGD treatment, serum and glucose-free medium were exchanged by glucose-containing medium with or without rhFGF21, followed by incubating with 95% air and 5%CO2 for 5 hours and then analyzed by qRT-PCR.
Cell culture and treatment
Primary cultured microglia and BV2 cell line were used to characterize the effect of rhFGF21 on microglial polarization, inflammation cytokine release and activation of NF-κB and PPARγ signaling. Cells exposure to lipopolysaccharide (LPS) to induced polarised microglia and inflammatory secretion[36]. Corresponding gene assays (IL-1β, iNOS, TNF-α, IL-6, CD86, CD206, Arg-1, IGF-1 and IL-10) were detected in LPS-stimulated or OGD-treated primary microglia by qRT-PCR and the effects of rhFGF21 on transcriptional activity of NF-κB in primary microglia were detected using Immunofluorescence. Additionally, the polarization of microglia was analyzed by assessing the expression of the M1 maker CD86, identified by FACS staining.
Western blot
Total proteins of LPS-treated BV2 cells were purified by RIPA lysis supplemented with a protease and phosphatase inhibitor mixture. Protein concentrations were measured with a Bradford Protein Detection Kit. 60 µg protein of samples and positive controls were separated on a Sodium Dodecyl Sulfate (SDS) polyacrylamide by electrophoresis. Subsequently, proteins were transferred onto PVDF membranes followed by blocking with primary antibodies FGFR1 (1:1000), p-FGFR1 (1:1000), NF-κB (1:1000), PPAR-γ (1:400), and β-Actin (1:500) overnight at 4℃. Then the membranes were incubated with second antibody donkey anti-rabbit IgG or goat anti-mouse IgG with a 1:10000 dilution for 1 hour at room temperature. Finally, the protein bands were detected with Image Lab software using Gel Doc Imager (Bio-Rad, Hercules, CA, USA) and the expression of target proteins were normalized against β-Actin.
Immunofluorescence analysis
Immunofluorescence staining was performed on paraffin brain sections as previously described [37]. Briefly, non-specific binding of antibodies were blocked with 5% BAS for 1 h at 37℃ and sections were then incubated with one or more primary antibodies against CD16/32, CD206, Iba1 or NF-κB with a dilution following their instruction at 4℃overnight. After washing, secondary antibody conjugated with adequate fluorochrome were added to visualise the expressions of corresponding protein and DAPI were used to stain nuclei. Images were captured using confocal laser sacnning microscope (Laika, Japan) located in peri-infarct cortex of ipsilateral hemisphere. Date were analyzed with Image J for calculating fluorescence intensity or counting numbers of recognized cells per field.
Statistical analysis
All statistical analyses of date were processed with prism 7.0 software (GraphPad, San Diego, CA, USA) in a blinded manner. Data from individual groups are expressed as mean±SEM and characterized by ordinary one-way ANOVE for multiple comparisons or student’s test (and nonparametric tests) with exception of behavior assessment. Statistical analyses based on behavioral data were determined by two-way ANOVE for multiple comparisons. Statistical significance is considered at P<0.05 level.