Design
A randomized, controlled experiment.

Time and setting
Experiments were performed at the Research Center of Medicine, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, China from June 2010 to December 2011.
Materials

Adult male BALB/c mice (n = 15, 6–8 weeks of age) weighing 18–20 g were bought from the Laboratory Animal Center of Sun Yat-sen University in China (license No. 0094467). All protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[37].

Methods
Cell stimulation and treatment
BV2 cells, an immortalized murine microglial cell line that exhibits phenotypic and functional properties of reactive microglial cells, were obtained from the Cell Center of Peking Union Medical College in China. The BV2 cell line has been used as a suitable model for the study of resting and activated microglia in vitro ^[38]. BV2 microglial cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium-F12 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Gibco), 100 μg/mL streptomycin and 100 U/mL penicillin at 37°C in a humidified incubator with air/CO₂ (95%/5%, v/v). Cells were passaged every 2–3 days. BV2 cells were pretreated with 50, 100 and 200 ng/mL lipopolysaccharide (Sigma, St. Louis, MO, USA) for 24 hours. Cells pretreated with 100 ng/mL^[39] lipopolysaccharide were used for morphological experiments. Cell culture medium without lipopolysaccharide was used as a control.

Animal preparation and drug intervention
Mice from the lipopolysaccharide plus brilliant blue G group were injected intraperitoneally with brilliant blue G (45 mg/kg per day; Sigma)^[40] dissolved in normal saline 30 minutes prior to lipopolysaccharide injection. A single dose of lipopolysaccharide (5 mg/kg; Sigma)^[41] was intraperitoneally injected in mice from the lipopolysaccharide and lipopolysaccharide plus brilliant blue G groups. Brilliant blue G was administered to mice of the lipopolysaccharide plus brilliant blue G group for 3 consecutive days. Simultaneously, the lipopolysaccharide group received an equal volume of normal saline.

Immunofluorescence staining
Immunohistochemistry: After administration for 3 consecutive days, animals were anesthetized with 10% chloral hydrate (4 mL/kg）and then received intracardiac perfusion with PBS and 4% paraformaldehyde. After perfusion, brains were quickly removed, post-fixed overnight with 4% paraformaldehyde and then dehydrated in gradient sucrose solution (10%–20%–30%) at 4°C. Serial sections of mouse brain (10 μm) were cut using a cryostat microtome and mounted onto microscope slides. These slides were stored temporarily in cryoprotectant solution at –30°C. Frozen sections were blocked in solution containing 5% normal goat serum for 1 hour after rinsing in 0.3% Triton X-100 for 30 minutes at 37°C. After several washes, sections were simultaneously incubated overnight at 4°C with the following primary antibodies: monoclonal rat anti-mouse CD11b (1:100; AbD Serotec, Kidlington, Oxford, UK) and polyclonal rabbit anti-P2X7 receptor (1:200; Alomone Labs, Jerusalem, Israel). Primary antibodies were detected with Alexa-488-conjugated goat anti-rabbit and Alexa-594-conjugated goat anti-rat antibodies (1:500; Jackson ImmunoResearch, Jacksonville, PA, USA) for 1 hour at room temperature. Sections were coverslipped with glycerol and fluorescent signals were then detected using an Olympus BX5 microscope (Tokyo, Japan). P2X7 receptor⁺/CD11b⁺ cells were counted in five random 200 × fields in the cortex for each repeated experiment. Five coronal slices per animal, including the hippocampus (five mice in each group), were counted. The average of all fields for one mouse was included in statistical analysis.
Immunocytochemistry: Prior to staining, BV2 cultures were washed twice with PBS. Cells were fixed with 4% paraformaldehyde for 20 minutes, and then blocked with 5% goat serum albumin for 1 hour. Afterwards, cells were incubated with monoclonal rat anti-mouse CD11b (1:100; AbD Serotec) at 4°C overnight. After repeated washing with PBS three times, cells were incubated with rabbit anti-rat Cy3-conjugated secondary antibodies (1:200; Millipore, Billerica, MA, USA) for 1 hour at 37°C in a thermostat incubator and coverslipped in anti-fluorescence-quenching reagent (Boster, Wuhan, Hubei Province, China). After 4’,6-diamidino-2-phenylindole (Sigma) staining, fluorescent images were detected and analyzed, as mentioned above.

Inhibiting expression of P2X7 receptor by siRNA
BV2 microglia cells were maintained in proliferating medium in 6-well plates for 2 days, at which time they reached 30–50% confluence and were used for transfection of siRNA. The siRNAs targeting mouse P2X7 receptor sequence were chemically synthesized by GenePharma: forward and reverse RNA strands (5’-CCG UAC UCA UCA AGA AUA ATT-3’; 5’-UUA UUC UUG AUG AGU ACG GTT-3’). Control non-specific sequences were as follows: 5’-UUC UCC GAA CGU GUC ACG UTT-3’; 5’-ACG UGA CAC GUU CGG AGA ATT-3’. Briefly, 5 μL of transfectamine (Invitrogen, Carlsbad, CA, USA) was added to Opti-MEM I (Gibco) reduced serum medium to a final volume of 250 μL. A volume of 10 μL of siRNA was added to Opti-MEM I to a final volume of 250 μL and incubated for 5 minutes. The serene and transfectamine solutions were combined and mixed by gentle pipetting and incubated at room temperature for 20 minutes. Cells were washed with Opti-MEM I, and 1 500 μL of fresh Opti-MEM I was added to each well. The transfection agent/siRNA complex was added dropwise into the cells and incubated at 37°C. BV2 cells were treated with P2X7 receptor siRNA or a negative control siRNA for 6 hours, followed by an addition of lipopolysaccharide (100 ng/mL) for 24 hours. Gene and protein expression were detected by real-time PCR and western blot assay, respectively.

Expression of P2X7 receptor, inteleukin-6 and cyclooxygenase-2 mRNA in BV2 cells
P2X7 receptor, interleukin-6 and cyclooxygenase-2 mRNA expression was determined by real-time PCR. BV2 cells were treated and total RNA was extracted using an RNeasy mini kit [TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, Liaoning Province, China]. RNA samples were used for first-strand cDNA synthesis using a SuperScript cDNA kit (TaKaRa). Real-time PCR was performed using a Roche Lightcycle 480 (Roche, Welwyn Garden, Hertfordshire, UK) and a SYBR green real-time kit (TaKaRa). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The ratios of P2X7 receptor/GAPDH, interleukin-6/GAPDH and cyclooxygenase-2/GAPDH for every sample were first calculated, and results were expressed as fold changes compared with control. Primer sequences are shown in Table 1. 

 Western blot analysis for P2X7 receptor and cyclooxygenase-2 expression in BV2 cells
Protein expression of P2X7 receptor and cyclooxygenase-2 was monitored in microglia by western blot assay. Total protein extracts were prepared and quantified from BV2 cells in radioimmunoprecipitation assay lysates. In brief, 60 μg of total proteins for each lane were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane using an electrophoretic transfer apparatus. The membrane was then incubated with primary antibodies, including polyclonal rabbit anti-P2X7 receptor (1:400; Alomone Labs), polyclonal rabbit anti-cyclooxygenase-2 (1:300; Cell Signaling Technology, Beverly, MA, USA), and polyclonal rabbit anti-GAPDH (1:500; Cell Signaling Technology) at 4°C overnight. Subsequently the membrane was incubated with the secondary antibody: goat anti-rabbit IgG-horseradish peroxidase (1:5 000; Cell Signaling Technology) for 1 hour at room temperature. After washing in Tris-buffered saline Tween-20, blots were developed with enhanced chemiluminescence detection reagents (Millipore). The relative absorbance values of bands were analyzed using a gel imaging analysis system (Genetics, Campbell, CA, USA).

Statistical analysis
Statistical analysis was performed using SPSS 16.0 software (SPSS, Chicago, IL, USA). At least three independent experiments were performed for each experimental procedure. Measurement data are expressed as mean ± SD. Differences were assessed using one-way analysis of variance for multiple comparisons followed by the Student-Newman-Keuls test for intergroup comparisons or Student’s t-test for intergroup comparisons. Two-tailed values of P < 0.05 were considered statistically significant.

Funding: This study was supported by the National Natural Science Foundation of China, No. 81072242, and the Excellent Supervisor & Yat-Sen Creative Talent Development Program of Sun Yat-sen University.
Author contributions: Kui Lu, Jue Wang, Junyi Zhou, Ying Peng and Yamei Tang designed the study. Kui Lu, Jue Wang, Bin Hu and Xiaolei Shi conducted the experiments and analyzed the data. Kui Lu wrote the paper. All authors approved the final version of the paper.
Conflicts of interest: None declared.
Ethical approval: All procedures followed the guideline of the Chinese National Institute of Health for Humane Care and protocols were approved by the committee on animal research at Sun Yat-sen University in China.
Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application/funding source disputations.

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