The experimental procedures used in this study were approved by the Kunming Medical College Institutional Animal Care and Use Committee. In the handling and care of all animals, the International Guiding Principles for Animals Research, as stipulated by the Council for International Organizations of Medical Sciences (1985) and as adopted by the Laboratory Animal Centre, Kunming Medical College, Yunnan, China, were followed. All efforts were made to minimize the number of rats used and their suffering.
P2X4 immunoexpression in amoeboid microglial cells in postnatal rats
Sprague Dawley (SD) rats at postnatal (P) 3 days (d) (P3) (n = 6) were used. The rats were anesthetized with 3% sodium pentobarbital (100 mg/kg) injected intraperitoneally and sacrificed by perfusion transcardially first with Ringer's solution, followed by fixation with 2% paraformaldehyde in 0. 1 M phosphate buffer. The brain was removed, post-fixed for 3 h in the same fixative, and then cryoprotected in 15% sucrose for 24 h. Frozen sections at 30 μm thickness were cut coronally through the forebrain with a cryostat (Leica model CM3050, Germany) and mounted onto gelatin-coated slides and stored at -20°C until use.
For ATP receptor immunofluorescence and double labeling with mouse anti-OX42 (CD11B) monoclonal antibody (1: 200, Milipore, Cat: CBL1512, USA), frozen coronal sections of the brain containing the CC rich in AMC were incubated with 10% horse serum (Vector, Cat: S-2000, USA.) for 40 min and rinsed in phosphate buffered saline (PBS) to block nonspecific protein. The sections were then incubated at 22-24°C with rabbit anti-P2X4 polyclonal primary antibody (1: 200, Alomone, Cat: APR-002, Yiselie), rabbit anti-P2X7 polyclonal primary antibody (1: 200, Alomone, Cat: APR-004, Yiselie), and rabbit anti-P2Y12 polyclonal primary antibody (1: 200, Alomone, Cat: APR-012, Yiselie) overnight. Subsequent antibody detection was carried out with FITC-conjugated donkey anti-rabbit IgG (1: 200, Millipore, Cat: AP182F, USA) for 1 h. After washing with PBS, the sections were incubated with Cy3-conjugated goat anti-mouse IgG (1: 200, Millipore, Cat: AP124C, USA) for OX42, a marker for microglial cells  for 1 h. The sections were then washed in PBS and mounted in a fluorescent mounting medium (DAKO Cytomation, Denmark). Cellular colocolization was then examined in a confocal microscope (Zeiss, LSM510 Axiovert 200 M, Germany).
Hypoxic exposure of postnatal rats
P0 rats (n = 60) were kept in a hypoxia chamber (Chinese Utility Model Patent; patent number: ZL2010 2 01877. 4) filled with a gas mixture of 5% oxygen and 95% nitrogen for 3. 5 h. The rats were allowed to recover under normoxic conditions and killed at 4, 24, 72 h, 7, and 14 d (n = 6 at each time point for immunofluorescence or western blotting analysis) after hypoxia. For normoxic matching control, the rats (n = 6 for each time point for immunofluorescence or western blotting analysis) were kept outside of the chamber. For immunofluorescence study, the rats were sacrificed and the tissues processed as described above. Coronal brain sections were processed for P2X4 immunohistochemistry and immunofluorescence double labeling. For western blotting, the tissue was removed and stored at -80°C until use. The protein was extracted using a protein extraction kit according to the manufacturer's protocol. Western blotting was used to detect the expression levels of P2X4.
Primary microglial cell culture
Highly purified microglial cultures were prepared from a modified method [33, 34] using P2-P3 SD pups. The meninges were carefully removed from the brain and the cortical tissue was minced with a sterile scalpel blade and digested with 0. 25% trypsin/EDTA (HyClone, SH30042. 01, USA) for 15 min at 37°C. Five milliliters of Dulbecco's modified Eagle's medium high glucose (DMEM) (HyClone, Cat: SH30243. 01B, USA) supplemented with 10% charcoal-stripped fetal bovine serum (HyClone, Cat: SV30087. 02, USA) and 1% penicillin/streptomycin (HyClone, Cat: SV30010, USA)(P/S, 100 U/mL penicillin, 100 lg/mL streptomycin, P/S) (DMEM complete) were added to the tissue on ice, and the tissue was triturated with a 5 ml pipette several times, allowing the tissue clumps to settle. The supernatant was removed to a clean 50 ml conical tube on ice between triturations. Triturations were repeated until no tissue clumps were observed. The final volume was diluted to 25 mL medium and centrifuged at 3000 rpm for 5 min. The supernatant was discarded and cells resuspended in medium. A small aliquot of cells was stained for trypan blue exclusion and cells were plated at 10 million cells per 75 cm2 flask. Cultures were maintained at 37°C and 5% CO2. The medium was changed the first time after 24 h, and then twice a week. After 10 days the bottom of flask was confluent with astrocytes and microglia. The flasks containing a mixture of astrocytes and microglia were shaken by hand for 2 min. Microglial cells in the medium were plated into 75 cm2 flask or cover slip for different use. After 15 min, the medium was changed and the cells adherent to the bottom will be kept in the incubator for 24 h until use. About 98% of cells in cultures were identified to be microglia by staining with OX42 antibody, a marker for the microglial CR3/CD11B receptor.
IL-1β and TNF-α expression in primary culture microglial cells after hypoxia
Primary culture microglial cells were seeded in a 75 cm2 TC-treated culture flasks. They were then kept in the hypoxia chamber containing a gas mixture of 3% oxygen, 5%CO2 and 92% nitrogen at 37°C for 4 h. The protein was extracted using a protein extraction kit according to the manufacturer's protocol. Western blotting was used to detect the expression levels of IL-1β and TNF-α.
P2X4 receptor blocking in primary culture microglial cells
Primary culture microglial cells were prepared as described above. Both PAADS (Sigma, Cat: P178, USA) and TNP-ATP (Sigma, Cat: T4193, USA) were used for blockade of P2X4. PAADS block P2X1-3, 5-7 receptors, while TNP-ATP blocks P2X1-7 receptor selectively. By comparing the blockade efficiency of the two blockers, information on the role of P2X4 in regulation of AMC activation is obtained [18, 35].
PPADS at 30 μM and TNP-ATP at 20 μM were added into the primary culture microglial cells, and incubated for 2 h at 37°C in the chamber before hypoxia. After the hypoxic exposure, the protein was collected from the microglial cells for western blot analysis.
Briefly, proteins were extracted from primary cultured microglial cells or postnatal rat brain tissue using a protein extraction kit (Biovision, Cat: K269-500, USA) according to the manufacturer's protocol. Cerebral tissues containing the CC were rapidly removed and immediately placed in dry ice before being stored at -80°C until use. The brain tissue was gently homogenized in ice-cold PBS (pH 7. 4) and centrifuged at 12000 g for 10 min at 4°C. The supernatant was recovered and frozen at -80°C. Total protein was determined using a commercial BCA kit (Pierce, IL, USA). Samples of supernatant containing 25 μg of protein were heated to 95°C for 5 min and were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% gels, in a Mini-Protein 3 apparatus (Bio-Rad Laboratories, Hercules, CA, USA). After separation, proteins were transferred to a polyvinylidene difluoride membrane (Minipore, Cat: IPVH00010, USA) at 0. 8 mA/cm2 of membrane for 2. 5 h in mixture solution of Tris-Glycine buffer (TBS) and 20% (v/v) methanol. After transfer, the membranes were blocked with 5% non-fat dry milk in TBS-Tween 20 0. 1% (TBST) and incubated with rabbit anti-P2X4 (1: 400), rabbit anti-β-actin (1: 5000, Abcam, ab8227, USA), rabbit anti-TNF-α polyclonal IgG (1: 500, Chemicon, Cat: AB1837P, USA) and rabbit anti-IL-1β polyclonal IgG(1: 500, Chemicon, Cat: AB1832P, USA) overnight, at 4°C. The antibodies were detected using horseradish peroxidase conjugated anti-rabbit IgG (1: 2500, KPL, Cat: 14-13-06, USA) secondary antibody and visualized using a chemiluminescence substrate system kit from Super Signal West Pico (KPL, Cat: 54-61-00, USA) on X-ray film (Super RX FujiFilm, Hanimax, QLD, Australia). Precision pre-stained standards (Bio-Rad Laboratories, Cat: 161-0376, USA) were used as molecular weight markers. X-ray films were scanned to quantify the band optical density using an Image J software (National Institutes of Health, USA). Figures with the quantitative western were derived from 6 animals from each group and the hybridizations of each animal were repeated at least 3 times.
A two tails Student's t-test was used to determine the statistical significance of difference in values between the control and experimental preparations. A value of P < 0. 05 or P < 0. 01 was considered significant.