Reagents and Antibodies
rhFGF21 was supported by the laboratory of Biotechnology Pharmaceutical Engineering at Wenzhou Medical University and synthesized on the basis of the study previously reported [31]. Antibodies in flow cytometry analysis involving CD3-PE (17A2, 100206), CD3-PerCP-Cy5.5 (17A2, 100217), 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-APC (C068C2, 141708), CD206-FITC (C068C2, 141704), CD68-PerCP-Cy5.5 (FA-11, 137014), CD86-PE (GL-1, 105008), CD45-PE/Cy7 (30-F11, 103114), CD11b-PE/Cy7 (M1/70, 101216), CD11b-PerCP-Cy5.5 (M1/70, 101230) 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 (Tokyo, 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)
Animal Groups 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 randomly assigned to the following three groups by a randomized block design: Sham group, MCAO group, MCAO+rhFGF21 group. In the sham group, mice were subjected to the same anesthesia and surgical procedures as the other groups but the filament was not inserted. In the MCAO+rhFGF21 group, the mice were intraperitoneally injected with rhFGF21 once per day at a dose of 1.5 mg/kg for 7 consecutive days beginning at 6 h after reperfusion.
Transient Focal Cerebral Ischemia and Reperfusion Model Preparation
The surgical procedures to establish the MCAO model were based on the intraluminal filament technique [32]. Briefly, the mice were anesthetized by isoflurane and placed on a heating blanket to maintain body temperature at 37±0.5°C. A midline incision was made to expose the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). The CCA was temporarily closed and a monofilament (0.18±0.01 mm, Jialing Biotechnology Company, Guangdong, China) was insert into the ICA through the ECA until it reached the middle cerebral artery, and it was left for 60 min. Laser doppler flowmetry (model P10, Moor Instruments, Wilmington, DE, USA) was used to monitor whether cerebral flow dropped to lower than 20% of the pre-ischemic level. The occluding filament was returned to the ICA to achieve reperfusion after 60 min of occlusion. In the MCAO model, the mortality rate was 9.3% (23 of total 246) and exclusion rate was 10.9% (11 of the total 246 experienced inadequate reperfusion, and 16 of the total 246 reached the criteria limitations set for the modified Neurological Severity Score (mNSS) scoring system, i.e., mNSS scores < 6 or > 13 at 24 h after MCAO were excluded).
Neurological function assessment
The mNSS, rotarod test, corner-turning test and adhesive removal test were performed to assess neurodeficits, motor coordination, sensorimotor asymmetry and feeling functions at 1, 3, 7 and 14 d after surgery. All animals received training for 3 consecutive days before suffering ischemia reperfusion injury. Behavior data were recorded as preoperative data at the second day after training. Subsequently, a transient focal cerebral ischemia and reperfusion model about MCAO was performed on the following day. The assessment procedure was performed by the same investigator who were blinded to the group identity of each mouse.
Quantitative real-time PCR
Total mRNA was isolated from the cortex samples around the infarcted zone using trizol reagent according to the manufacturer’s instructions. The cDNA was synthesized by the PrimeScriptTM RT Reagent Kit (TaKaRa, Shiga, Japan) following the manufacturer’s protocol. PCR assays were performed on a CFX Connect Real-time System (Bio-Rad, Hercules, CA, USA) using SYBR Green. The primers used in this study are shown in Table 1. Additionally, total RNA was extracted from the sorted microglia using the miRNeasy Micro Kit according to the manufacturer’s protocol. cDNA was transcribed with a Reverse Transcription kit and amplified in step one using Gene Expression Assays for TNF-α, IL-6, IL-1β and TGF-β. The reaction volume was set to 20 µl and performed at 50°C for 2 min, 95°C for 20 s, followed by 40 cycles of 1 s at 95°C and 20 s at 60°C. Data were analyzed using the 2-∆∆Ct method , and the expression level of relative mRNA was then reported as the fold difference.
Flow cytometry
After the 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 [33, 34]. Splenocytes were dissociated by sieving through a 70-µm filter, and 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 (Miltenyi Biotech, Bergisch Gladbach Germany) according to its protocol. Briefly, the ischemic 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 (300 g, 2 min) at room temperature. After the supernatant was carefully aspirated, preheated enzyme mix1 (37°C, 10 min) in a Neural Tissue Dissociation Kit was added to digest tissue pieces for 15 min, and then preheated enzyme mix 2 (37°C, 10 min) was added to the tissue sample for 10 min. Subsequently, HBSS was used and 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: 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). Single-cell suspensions of the brain tissue were prepared as described above (“Flow cytometry” section). Cells were stained with APC-conjugated anti-mouse CD45 antibody and PE-conjugated anti-mouse CD11b for 30 min at 4°C. Unstained antibody was washed in PBS and cells were incubated with anti-PE microbeads at 4°C for 15 min. HBSS was used to wash off unlabeled microbeads. Cells labeled with primary antibody conjugated to PE were enriched by using MACS columns (Miltenyi Biotech, Bergisch Gladbach Germany) according to the explanatory memorandum, and then targeted microglia were gathered using the FACS Aria cell sorting system. Resident microglia were identified as the CD45intCD11b+ population, whereas infiltrated macrophages in the CNS were identified as the CD11b+CD45highF4/80+ population. In addition, cell sorting of macrophages from the spleen was performed following the method in the “Flow cytometry” section to obtained the cell suspensions of spleen. Then, cells were stained with APC-conjugated anti-mouse CD45 antibody, PE-conjugated anti-mouse CD11b and FITC-conjugated anti-mouse F4/80 30 min at 4°C. Unstained antibody was washed in PBS, and then targeted macrophages defined as the CD45highCD11b+ F4/80+ population were gathered using the FACS Aria cell sorting system. 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 [35]. 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, USA). Culture media were changed every three days until achieving a confluent monolayer at approximately 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°C under atmosphere condition for further experiments.
Oxygen-glucose deprivation (OGD)
To establish an ischemic-like condition in vitro, primary microglia were subjected to OGD as previously reported [36]. Briefly, microglia were 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°C for 3 h. After the OGD treatment, serum and glucose-free medium were exchanged by glucose-containing medium with or without rhFGF21, which was followed by incubating with 95% air and 5% CO2 for 5 hours and then analysis by qRT-PCR.
Cell culture and treatment
Primary cultured microglia and the BV2 cell line were used to characterize the effect of rhFGF21 on microglial polarization, inflammation cytokine release and NF-κB and PPAR-γ signaling activation. Cells were exposed to lipopolysaccharide (LPS) to induce polarized microglia and inflammatory secretion [37]. Briefly, microglia were treated with LPS (250 ng/mL) in the presence and absence of rhFGF21 (100 nM) or PD173074 (10 μM) for 4 h. Gene assays (involving IL-1β, iNOS, TNF-α, IL-6, CD86, CD206, Arg-1, IGF-1 and IL-10) were then 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 marker CD86, which was 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. Then, 60 µg of proteins from the samples and positive controls were separated on sodium dodecyl sulfate (SDS) polyacrylamide gels 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 4°C. Then, the membranes were incubated with secondary antibody donkey anti-rabbit IgG or goat anti-mouse IgG at 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 was normalized against β-Actin.
Immunofluorescence analysis
Immunofluorescence staining was performed on paraffin brain sections as previously described [38]. Briefly, non-specific binding of antibodies were blocked with 5% BAS for 1 h at 37°C and the sections were then incubated with one or more primary antibodies against CD16/32, CD206, Iba1 or NF-κB in a dilution following the manufacturer’s instruction at 4°C overnight. After washing, secondary antibodies conjugated with adequate fluorochrome were added to visualize the expression of corresponding proteins and DAPI was used to stain the nuclei. Images of the penumbra of the infarct cortex were captured using confocal laser scanning microscope (Laika, Japan). Data were analyzed with ImageJ (NIH Image, Bethesda, MD, USA) to calculate the fluorescence intensity or counting number of recognized cells per field.
Statistical analysis
All statistical analyses of the data were processed with Prism 7.0 software (GraphPad, San Diego, CA, USA) in a blinded manner. Data from individual groups were expressed as mean ± SEM and characterized by a one-way ANOVA for multiple comparisons or Student’s t-test (and nonparametric tests). Behavioral data were statistically analyzed by a two-way ANOVA for multiple comparisons. Statistical significance was considered at P<0.05 level.