Primary microglia were collected from mixed glial cultures that were prepared via homogenization of cortical tissues dissected from rat pups at postnatal day (PD) 1-2 28. The tissues were filtered through a 70-mm nylon filter mesh, and after centrifugation, the cell pellet was re-suspended in DMEM/F-12 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (Gibco). The cells were seeded onto poly-D-lysine (Sigma-Aldrich)-coated T-75 culture flasks and incubated in 5% CO2 at 37°C for 7-8 days. After microglia were collected using the shake-off method, these cells were re-plated onto coverslips in 24-well plates at a density of 5 x 104 cells/well or in 60-mm Petri dishes at a density of 1 x 106 cells/dish in DMEM/F-12 medium containing 10% FBS for 3 h. The cultures were treated with 1 mM TFP (Sigma-Aldrich) in the presence of LPS (10 ng/ml; Sigma-Aldrich) in DMEM containing N1 serum supplement (Thermo Fisher Scientific) for 3 h to examine isolectin B4 (IB4) and immunofluorescence for inducible nitric oxide synthase (iNOS) or for 6 h to investigate TNF-a and IL-1b mRNA expression. Alternatively, mouse mixed glia were prepared from the hypothalamus of pups at the age of PD 1-2 and re-plated onto PDL-coated coverslips in a 24-well plate at a density of 5 x 104 cells per well. After incubation in 5% CO2 at 37°C for 5 days, the cultures were treated in DMEM/F-12 medium without or with 1 mM TFP in the presence of LPS (10 ng/ml) for 3 h. The cultured media were collected for the measurement of TNF-a and IL-1b levels. Animal use for primary microglia preparation was approved by the National Cheng Kung University Institutional Animal Care and Use Committee, Tainan, Taiwan (IACUC approval number: 106060). The methods were performed in accordance with relevant guidelines and regulations.
Eight-week-old male C57BL/6 mice were purchased from National Cheng Kung University Laboratory Animal Center (http://www.ncku.edu.tw/animal/eng/nckulac.html), and randomly paired-housed in cages with free access to food and water ad libitum under standard room conditions (room temperature: 23±2°C; humidity: 58±2%; 12-h light/dark cycle). To induce obesity in mice, the animals were fed a HFD containing 61.6% kcal from fats, 18.1% from proteins and 20.3% from carbohydrates (Rodent Purified Diet #58Y1; TestDiet, St. Louis, MO, USA). The animals receiving a normal diet (Laboratory Rodent Diet #5001; LabDiet, St. Louis, MO, USA) were referred to as the chow group. All the experiments was performed in compliance with ARRIVE (Animal Research: Reporting In Vivo Experiments) Guidelines (https://www.nc3rs.org.uk/arrive-guidelines) and strictly following the 3Rs (Replacement, Reduction and Refinement) to avoid unnecessary sacrifice. Animal experiments through the performance of the following methods were approved by the National Cheng Kung University Institutional Animal Care and Use Committee, Tainan, Taiwan (IACUC approval number: 106060). The animals were anesthetized and sacrificed by i.p. injection with Zoletil 50 (Virbac Taiwan Co., Ltd.; 5X dilution in saline, 0.05-0.06 ml/10 g). The choice of anesthetics was recommended by the veterinary specialist at the university animal center to effectively reduce pain in animals. The experimental protocol is outlined in Fig. S2.
Peripheral injection of LPS and TFP
Injection of LPS (1 mg/kg) by the i.p. route combined with saline or TFP (2 mg/kg) was performed on 8-week-old C57BL/6 mice. Animals that received saline served as the control group (vehicle). Blood samples were collected at 3 h from the retro-orbital sinuses of anesthetized mice using a heparinized capillary tube29. Alternatively, under anesthesia, blood samples were collected at 6 h via cardiac puncture using a 26G needle rinsed with 10 ml of heparin (5000 IU/mL; Leo Pharmaceutical, Ltd., Denmark). Animals were then sacrificed, and hypothalamic tissues were removed for measurement of TNF-a and IL-b mRNA levels.
TFP (2 mg/kg/injection) administration through the i.p. route began at 12 weeks post HFD feeding, and the daily injection continued for 4 weeks. The animal groups includes chow-vehicle, HFD-vehicle, chow-TFP, and HFD-TFP. Blood samples and brains were collected after animals were anesthetized.
Measurement of cytokines
The mixed glial culture media or blood samples from mice were collected at the different experimental time points as described above. After centrifugation, the supernatant was used to measure TNF-a and IL-1b using a murine TNF-a Quantikine ELISA Kit and murine IL-1b/IL-1F2 Quantikine ELISA Kit following the protocol provided by the vendor (R&D system; Table 1).
Assays for metabolic indices
The blood samples used for the measurement of glucose were collected using a heparinized capillary tube from animals after fasting for 15 h. Roche Accu-Chek® blood glucose meters were used to measure the amount of blood glucose in the animal groups. In addition, the blood samples collected from the fasting animals were centrifuged, and the supernatant was used to measure plasma insulin (Table 1) and other metabolic markers, including high-density lipoprotein (HDL), low-density lipoprotein/very-low-density lipoprotein (LDL/VLDL), and triglyceride (TG), using Quantification Colorimetric/Fluorometric Kits (Table 1).
Collection of adipose tissues
After perfusion, white adipose tissue (WAT) was collected from epididymal adipose tissue, and brown adipose tissue (BAT) was collected from interscapular brown adipose tissue for weight measurement on an electronic analytical balance (ATX224; SHIMADZU, Japan).
Quantitative real-time polymerase chain reaction (QPCR)
Animals were anesthetized with Zoletil 50 (Virbac Taiwan Co., Ltd.; 5X dilution in saline, 0.05-0.06 ml/10 gm) by i.p. injection and then perfused with 0.9% saline in diethylpyrocarbonate (DEPC; Sigma)-treated distilled water. Hypothalamic tissues were removed and homogenized in TRIzolTM (Invitrogen) for RNA extraction. cDNA generation using MMLV reverse transcriptase (Invitrogen) and PCR amplification by Fast SYBR® Green Master Mix (Applied Biosystems) were previously described 30. We used Primer-BLAST software provided by the National Center for Biotechnology Information to design primers that were manufactured by Taiwan Genomics. The level of cyclophilin A (CyPA) was used as an internal control. StepOne Software v2.1 (Applied Biosystems) was used to determine the cycle threshold fluorescence values. The expression level of the target genes relative to the internal control is presented as 2−ΔCT, where ΔCT = (Ct target gene - Ct CyPA). The sequences of the specific primers for TNF-a, IL-1b, D2R, and Cyclophilin A (CyPA) are as follows: rat TNF-a (NM_012675): Forward 5’-CATCCGTTCTCTACCCAGCC-3’, Reverse 5’-AATTCTGAGCCCGGAGTTGG-3’; rat IL-1b (NM_031512.2): Forward 5’-CCTATGTCTTGCCCGTGGAG-3’ Reverse 5’-CACACACTAGCAGGTCGTCA-3’ ; rat CyPA (NM_017101.1): Forward 5’-CGTCTGCTTCGAGCTGTTTG-3’, Reverse 5’-GTAAAATGCCCGCAAGTCAA-3’; murine TNF-a (NM_008361.4) Forward 5’-CCGGACTCCGCAAAGTCTAA-3’, Reverse 5’-ACCGTCAGCCGATTTGCTAT-3’; murine IL-1b (NM_008361.4): Forward 5’-TGCCACCTTTTGACAGTGATGA-3’, Reverse 5’-AAGGTCCACGGGAAAGACAC-3’; murine D2R (NM_010077.3): Forward 5’-CCATTGTCTGGGTCCTGTCC-3’, Reverse 5’-CTGCTACGCTTGGTGTTGAC-3’; and murine CyPA (NM_008907.1): Forward 5’-CGTCTGCTTCGAGCTGTTTG-3’, Reverse 5’-GTAAAATGCCCGCAAGTCAA-3’.
The cell cultures were fixed in PBS containing 4% paraformaldehyde for 10 min and then treated with 0.1% Triton-X-100 in PBS at room temperature for 30 min. Rat microglia were incubated with rabbit anti-iNOS antibody (1:400) and biotin-conjugated IB4 (1:200) in PBS containing 5% horse serum overnight at 4°C, followed by biotinylated anti-rabbit IgG (1:200) for anti-iNOS antibody and Alexa Fluor 488 (1:200) for IB4 for 1 h at room temperature. The cultures were incubated with PBS containing Cy3–avidin (1:200) for another 45 min. Alternatively, mouse mixed glial cells were incubated with anti-GFAP antibody (1:400) and biotin-conjugated IB4 (1:200) overnight at 4°C. The cells were reacted with biotinylated anti-rabbit IgG (1:200) and Alexa Fluor 488 (1:200) at room temperature for 1 h and then Cy3-avidin (1:200) for another 45 min. The immunostained cells were visualized and photographed under a Nikon E800 fluorescence microscope equipped with a CCD camera. The antibodies are listed in Table 1.
Brain tissue immunostaining
The brain samples removed from animals were fixed overnight in 4% paraformaldehyde and then transferred to tubes containing 30% (w/v) sucrose in PBS until the tissues sank to the bottom of the tube. The tissues were mounted in Tissue Tek optimal cutting temperature compound (Electron Microscopy Sciences, Torrance, CA, USA) and then sectioned at 20-mm thickness. The floating brain sections were treated with 1% Triton-X-100 in PBS at 4°C overnight, followed by incubation with primary antibodies in PBS containing 0.1% Triton X-100 and 1% horse serum at 4°C overnight. The tissue sections were then incubated with biotinylated secondary antibodies for 1 h and with Alexa Fluor 488 or Cy3–avidin (1:200) for another 45 min. The immunostained tissues were observed under an Olympus FLUOVIEW FV1000 confocal laser scanning microscope (FV1000, Japan) at wavelengths of 405, 488 or 594 nm. The primary antibodies used in the study are listed in Table 1. Alternatively, brain sections were permeabilized in 0.3% Triton-X-100 in PBS for 30 min, incubated with rabbit anti-D2R antibody (Table 1) in PBS at 4°C overnight, and treated with biotinylated anti-rabbit IgG (1:200) in PBS for 1 h. The brain sections were then reacted with Vectastain ABC reagent (Vector Labs; Cat# PK-6100) containing 3,3'-diaminobenzidine and nuclear counterstaining by hematoxylin. The immunostained sections were visualized and photographed under a Nikon E800 microscope equipped with a CCD camera.
Determination of microglial activation and astrocyte hypertrophy
The measurement of microglia and astrocyte activation was performed as previously described12,30. The cell number and cell body size of Iba1+ microglia and the intensity of GFAP immunoreactivity in the ARC were analyzed using National Institutes of Health (NIH) ImageJ analysis software31. In general, randomly selected images per brain section were merged and captured in multiple 1-mm-thick steps using an Olympus FLUOVIEW FV1000 confocal laser scanning microscope. Images that were used for quantification were captured from 7-9 brain sections for Iba1 immunostaining and 6 brain sections for GFAP immunostaining from three animals per group. The microglial cell numbers are shown as the number of cells in an image with a size of 0.1 mm2. The GFAP intensity data were normalized to those of the chow group (100%).
The presence of significant differences between each group (chow-vehicle, chow-TFP, HFD-vehicle, HFD-TFP) at different time points observed in this study was determined using one-way ANOVA with Sidak’s multiple comparisons for the in vivo study or two-tailed Student’s t test for in vitro experiments. Data are presented as the mean ± SEM. The statistical significance was set as P < 0.05.