Groups of 8–12-week-old C57BL/6 mice were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences of China (Beijing). B6.129S6-Il10tm1Flv/J mice  were from Prof. Jianli Wang at Zhejiang University. In all experiments, age- and sex-matched wild-type (WT) littermates were used for controls. Mice were maintained in a pathogen-free barrier facility. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals and were approved by the Institute of Military Cognition and Brain Sciences. The institutional Ethics Review Committee for Animal Experimentation approved all experimental protocols.
Primary cell culture
Primary astrocytes were prepared from newborn C57BL/6 or B6.129S6-Il10tm1Flv/J pups. After the removal of meninges, the cortical tissues were mechanically minced and dissociated with 0.25% trypsin (Gibco). After trypsin inactivation, the tissue suspension was filtered through a 70-mm nylon cell strainer. Cell pellets were harvested and resuspended in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) (1:1) (Gibco) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin and plated on poly-L-lysine pre-coated culture flasks. Following incubation overnight in a humidified incubator at 37 °C and 5% CO2, the flasks were vigorously shaken to detach neurons and microglia. Culture medium was replaced every 3 days thereafter until the cell monolayer reached confluence. Primary neurons were obtained by microdissection from brains of neonatal C57BL/6 pups using the same protocol but cultured in neurobasal medium supplemented with 1% glutamate and 2% B27 (Gibco). Primary microglial cells were also dissected from neonatal C57BL/6 pups. After removing the meninges, brains were mechanically minced and dissociated with 0.25% trypsin. The tissue suspension was passed through a 70-mm nylon cell strainer. Cell pellets were harvested and resuspended in DMEM supplemented with 10% heat-inactivated FBS and plated on poly-L-lysine precoated culture flasks. Primary microglial cells were harvested by shaking (200 rpm, 20 min) after overnight in culture.
Cells were collected in TRIzol (Sigma-Aldrich), and total RNA were prepared by chloroform extraction and isopropanol precipitation according to the manufacturer’s recommendations (Invitrogen). cDNA was used as a template in quantitative PCR with SYBR Green (Toyobo) to determine specific gene expression. Real-time RT-PCR was performed using a SYBR Green PCR mix and conducted with the FTC-3000. Primer sequences were as follows: Aldh1l1 (forward, AGCAGAGGCCATTCACAACT; reverse, GCCACCAGTCCTGAAGTGTT), Aqp4 (forward, CTCCCTTTGCTTTGGACTCA; reverse, CGATGCTGATCTTTCGTGTG; GFAP: forward, GCCACCAGTAACATGCAAGA; reverse, GGCGATAGTCGTTAGCTTCG), Vim (forward, CTGCACGATGAAGAGATCCA; reverse, AGCCACGCTTTCATACTGCT), Aif1 (forward, CCGAGGAGACGTTCAGCTAC; reverse, GACCAGTTGGCCTCTTGTGT). Cx3cr1 (forward, AGCTCACGACTGCCTTCTTC; reverse, GTCCGGTTGTTCATGGAGTT), Snap25 (forward, GAGCAGGTGAGCGGCATCATC; reverse, CGTTGGTTGGCTTCATCAATTCTGG; Syt1: forward, GACTATGACAAGATTGGCAAGAACGAC; reverse, ATGGCATCAACCTCCTCCTCTACC), H2-T23 (forward, TGATCATCCTTGGAGCTGTG; reverse, TTCTGAGGCCAGTCAGAGGT), Serping1 (forward, GCTCTACCACGCCTTCTCAG; reverse, GGATGCTCTCCAAGTTGCTC). H2-D1 (forward, GTTGCTGTTCTGGGTGTCCT; reverse, CCTGGAGCCAGAGCATAGTC), Gbp2 (forward, GGAGGAGCTGTGTGGTGAAT; reverse, TTAGACGTGGCCCATTGACT; Tgm1: forward, CCCTGGATGACAATGGAGTT; reverse, GAATAGCCGGTGCGTAGGTA), Ptx3 (forward, TTCCTGAGGGTGGACTCCTA; reverse, CCGATCCCAGATATTGAAGC), S100a10 (forward, GTGCTCATGGAACGGGAGT; reverse, AAAGCTCTGGAAGCCCACTT). Gpc4 (forward, GGTGACTGTGAAGCCCTGTT; reverse, TCTCTGCCACCATCAGCATA), Gpc6 (forward, AGCATTGCCCTACACCATCT; reverse, AGCCCATCGTTCATGATCTC; Sparcl1: forward, AAACCATCCCAGTGACAAGG; reverse, TCCTCATCCTTCAGGTCCAC), Thbs1 (forward, TGATGACTACGCTGGCTTTG; reverse, TGAGTATCCCTGAGCCCTTG), Thbs2 (forward, CTGGTGCAGACAGCAAACTC; reverse, CAAAGCCAGCGTAGTCATCA). Megf10 (forward, CTGGCGCTGTTCATCATCTA; reverse, CAGGGTTTCTGCGATGGTAT), Mertk (forward, CACGAGAACAACCTTCGTGA; reverse, TCCTCCGGTAAGACACCATC; Gas6: forward, AGGCCACCCTAGAAGTGGAT; reverse, CAGGCCTCCAACATAGGTGT), Axl (forward, GGAAGGTCAGCTCAATCAGG; reverse, CCTTCATGCAGACAGCTTCA), IL-1β (forward, GTGGCTGTGGAGAAGCTGTGG; reverse, CGGAGCCTGTAGTGCAGTTGTC), IFNγ (forward, CAGGCCATCAGCAACAACATAAGC; reverse, AGCTGGTGGACCACTCGGATG; IL-6: forward, ACTTCCATCCAGTTGCCTTCTTGG; reverse, TTAAGCCTCCGACTTGTGAAGTGG), TNFα (forward, GATGGGTTGTACCTTGTCTACT; reverse, CTTTCTCCTGGTATGAGATAGC), iNOS (forward, CAGCTGGGCTGTACAAACCTT; reverse, CATTGGAAGTGAAGCGTTTCG), GAPDH (forward, TCACCATCTTCCAGGAGCGAGAC; reverse, AGACACCAGTAGACTCCACGACATAC).
Brain processing and immunostaining were performed on free-floating sections. Mice were anesthetized with 2,2,2-tribromoethanol (350 mg/kg, Sigma-Aldrich, T48402) and transcardially perfused with 0.9% saline, and brains were removed and fixed in phosphate-buffered 4% paraformaldehyde for 48 h before cryoprotection with 30% sucrose. Mouse brain sections (30 μm) or fixed cultured cells were washed three times with PBS, and antigen retrieval was performed using citrate buffer (pH 7.0); samples were then permeabilized and blocked in PBS containing 0.5% Triton X-100 and 10% normal goat serum at room temperature for 1 h. Sections were incubated with primary antibodies in blocking buffer overnight at 4 °C. After washing, secondary antibodies were added to the blocking buffer and incubated for 1 h. Samples were then washed and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired under a fluorescence microscope (Nikon AZ-100 multipurpose microscope). Primary antibodies used for immunostaining include GFAP (mouse, 1:500; Abcam, ab10062), IBA1 (Rabbit, 1:300; Abcam, ab178847), and MAP2 (goat, 1:500; Abcam, ab32454). Donkey anti-mouse/rabbit 488/594 secondary antibodies (1:1000) and mounting medium with DAPI were purchased from Invitrogen.
Cells were lysed with lysis buffer, and protein samples were separated on 12% SDS-polyacrylamide gel, and then the proteins were transferred to 0.45 μm polyvinylidene fluoride blotting membranes. The membrane was blocked in 5% non-fat dry milk for 1 h and was then probed overnight at 4 °C with mouse anti-GFAP (1:1000, Abcam, ab10062), rabbit anti-p-STAT3 (1:1000, Cell Signaling Technology, #9167), mouse anti-STAT3 (1:1000, Cell Signaling Technology, #9139), or rabbit anti-GAPDH (1:1000, Cell Signaling Technology, #1228). Blots were incubated with HRP-conjugated secondary antibodies at 1:5000 for 2 h at room temperature and developed using enhanced chemiluminescence substrate (Thermo Fisher, Waltham, MA, USA). Visualization and imaging of blots was performed with a FluorochemQ System (ProteinSimple).
Neuron survival assays
Astrocytes were treated with or without LPS (100 ng/mL, L-4391, Sigma-Aldrich) for 24 h, and then, supernatants were collected. Neurons were plated at 150,000 cells per well on PDL-coated plastic coverslips in 24-well cell culture plates. Supernatants were added to neurons and viability assessed at indicated time point.
For phagocytosis assays, astrocytes pretreated with or without LPS (100 ng/mL for 24 h) were incubated with aggregated 400 nM Hilyte555-Aβ42 (Anaspec, Fermont, USA). Cells were analyzed at indicated time points following addition of Aβ to the culture.
Lipopolysaccharide (LPS, L-4391, Sigma-Aldrich) was dissolved in physiological saline. The dose of LPS (0.83 mg/kg) and the time point (24 h) for behavioral tests after LPS intraperitoneal injection (i.p.) were selected based on previous investigations .
Hippocampus and cortex protein extraction
Snap-frozen hippocampus and cortex were homogenized in tissue protein extraction reagent (T-PER, Thermo Fisher Scientific) containing a mixture of protease and phosphatase inhibitors (Thermo Fisher Scientific). Homogenates were centrifuged, and the supernatants were evaluated for the quantification of cytokines.
ELISA quantification of cytokines
Cytokines were quantified with custom Mouse ProcartaPlex™ Panel according to the manufacturer’s instructions (eBioscience). Data acquisition was performed with a MAGPIX (Luminex). Each sample was measured in duplicate.
IL-10 and fluorocitrate injection
Mice were anesthetized with 2,2,2-Tribromoethanol (350 mg/kg, Sigma-Aldrich, T48402) intraperitoneal injection, then placed into a stereotaxic frame. A small Hamilton syringe (33-gauge) was used to slowly inject 1 μl of saline or IL-10 (20 ng, Peprotech) or fluorocitrate (FC, 1 nmol, Sigma-Aldrich) per mouse into intracerebroventricular (i.c.v). After injection, the syringe was held in place for 5 min to avoid back-flow of cerebrospinal fluid (CSF). The stereotaxic coordinates were 1.34 mm posterior, 0 mm lateral to the bregma, and 2.3 mm ventral to the bregma.
Forced swim test (FST)
Mice were individually placed in a glass cylinder (25 cm height, 15 cm diameter) filled with room temperature water (23 °C–25 °C). Mice were allowed to swim inside the cylinder and videotaped for 6 min. The duration of immobility in the last 4 min was counted by an observer blinded to the animal treatments. Immobility was defined as time when animals remained floating or motionless with only movements necessary for keeping balance in the water.
Morris water maze test (MWM)
The water maze used in this study comprised a circular tank 120 cm in diameter with a platform filled with tap water at a temperature of 22 ± 2 °C. Reference cues with different colors and shapes were posted along the walls surrounding the tank. Within the tank was a fixed platform (diameter, 10 cm) located in a target quadrant. A camera was mounted above the maze to record the swimming traces in the water maze. During testing, the platform was submerged, and 1–2 cm below the water surface, mice were placed into the maze at one of four points (N, S, E, W) facing the wall of the tank. Mice were provided 60 s to search for the platform. If a mouse failed to find the platform, it was guided to the platform and maintained on the platform for 10 s. Four trials per day were conducted with an intermission of 1 h minimum between trials. Between the trails, mice were gently padded dry and warmed on a heating pad. The escape latency, or the time it took for the mouse to reach the platform, was scored for each trial and averaged per testing day. On day 6, the platform was removed and a probe test was performed. The percentage of time spent in each of the four quadrants and the number of platform area crossings, mean speed, and total distance were recorded.
All data were analyzed with Prism 5.0 software (GraphPad Software, San Diego, CA, USA) and are presented as means ± standard deviations (SDs). Statistical significance was assessed by unpaired two-tailed Student’s t tests. For multiple comparisons, two-way ANOVA was performed, followed by Bonferroni multiple comparison. (*p < 0.05; **p < 0.01).