The animal protocol used was reviewed beforehand and approved by the INHA University-Institutional Animal Care and Use Committee (INHA-IACUC) with respect to ethicality (Approval Number INHA-141124-337-2). All procedures concerning animals were in accord with the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (2010) and with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guideline . Male Sprague-Dawley (SD) rats (8 weeks) weighing 230–250 g were purchased from Orient Bio Inc. (Gyeonggi, South Korea) and housed under diurnal lighting conditions with ad libitum access to food and tap water for a week before experiments. Male SD rats (9 weeks) were anesthetized with 5% isoflurane in a 30% oxygen/70% nitrous oxide mixture delivered through a close-fitting facemask during surgery. The permanent MCAO-induced focal cerebral ischemia model used was generated as described previously . Briefly, the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were exposed through a midline incision of the neck, and the ECA and CCA were ligated with silk sutures and the ICA was temporarily clipped. A monofilament nylon suture (4–0; AILEE, Busan, Korea) was then inserted into the ICA and slowly advanced from the lumen of the ICA to the MCA; this suture was left in place until sacrifice. All procedures were finished within 15 min. Sham controls underwent CCA, ICA, and ECA exposure only. During the procedure, left femoral arteries were cannulated to obtain blood samples, which were analyzed for pH, PaO2, PaCO2, and blood glucose concentration (I-STAT; Sensor Devices, Waukesha, WI). Laser Doppler flowmeter (Periflux System 5000; Perimed, Jarfalla, Sweden) was used to monitor regional cerebral blood flow (CBF) and relative CBF during experiment, and a thermoregulated heating pad and a heating lamp were used to maintain a rectal temperature of 37.0 ± 0.5 °C during surgeries.
HMGB1 a box or anti-HMGB1 antibody administration
HMGB1 A box (50 μg/kg; HMGbiotech, Milano, Italy; HM-012) was administered intravenously in 0.3 ml PBS after 3 h of pMCAO. Anti-HMGB1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was also administered intravenously at the dose of 200 μg/kg after 3 h of pMCAO. Animals in the sham group were injected with 0.3 ml of PBS intravenously at 3 h after surgery.
Animals were sacrificed at the indicated times after surgery. Brains were isolated and fixed with 4% paraformaldehyde (PFA; Sigma Aldrich, St. Louis, MO) by transcardiac perfusion and then stored in the same solution overnight at 4 °C. Brain sections (40 μm) were prepared using a vibratome and immunological staining was performed using a previously described floating method . The sections were preincubated in blocking solution containing 5% FBS, 5% horse serum, and 2% BSA in PBS. Primary antibodies were diluted 1:500 for anti-ionized calcium binding adaptor molecule-1 (Iba-1) (Wako Pure Chemicals, Osaka, Japan). After washing with PBS containing 0.1% Triton X-100, sections were incubated with anti-rabbit IgG (Vectorlab, Peterborough, UK) in PBS for 1 h at room temperature and visualized using the HRP/3,3-diaminobenzidine (DAB) system and observed under a fluorescence microscope (Axioplan 2; Zeiss, Oberkochen, Germany).
Polymorphonuclear leukocytes (PMNs) were prepared from peripheral blood or CSF and a cytospin was used to immobilize them on slides. After cytospin centrifugation, slides were fixed for 15 min. Polymorphonuclear leukocytes prepared from bone marrow (PMNs-BM) were cultured in 24 well plate and fixed for 15 min. Both PMNs and PMNs-BM were blocked with 1% normal goat serum and incubated overnight with anti-CitH3 antibody (ab18956–100; Abcam, Cambridge, UK) or anti HMGB1 antibody (ab672; Abcam, Cambridge, UK) at 4 °C. Brain sections were prepared as described in immunohistochemistry. The sections were preincubated in blocking solution containing 5% FBS, 5% horse serum, and 2% BSA in PBS. Primary antibodies for anti-CitH3 (ab18956–100; Abcam, Cambridge, UK), anti-Ly6g-FITC (ab2949, Abcam, Cambridge, UK), anti-MPO-FITC (ab18956, Abcam, Cambridge, UK), anti-RECA (MCA970GA, Bio-rad, Kidlington, UK), and anti-lamin (ab78946; Abcam, Cambridge, UK) were diluted 1:200. For double immunostaining, rhodamine anti-rabbit IgG (Jackson ImmunoRes Lab, West Grove, PA) was used as the secondary antibody and incubated for 1 h at room temperature. Brain sections or cytospin slides were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Sigma Aldrich, St. Louis, MO) to visualize nuclei, and observed under a fluorescence microscope (Axioplan 2; Zeiss, Oberkochen, Germany). The numbers of CitH3 positive cells in 0.16 mm2 (0.4 × 0.4 mm) were scored and vessel length and density were analyzed using the AngioTool Software (National Cancer Institute, Gaithersburg, MD, USA).
H & E staining
Brain tissues were fixed overnight in 4% paraformaldehyde (PFA), embedded in paraffin, and cut into 5 μm sections using a microtome. Deparaffinized sections were stained with hematoxylin and eosin (H&E) and observed under a light microscope (Axioplan 2; Zeiss, Oberkochen, Germany).
Rats were sacrificed at 12 h after MCAO and whole brains were dissected coronally into 2-mm slices using a metallic brain matrix (RBM-40000, ASI, Springville, UT). Slices were immediately incubated in saline containing TTC (2, 3, 5-triphenyl tetrazolium chloride, 2%) for 15 min at 37 °C and then stored in 4% PFA.
Brain tissues or PMNs on cytospin slides were washed twice with cold PBS and lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 0.5% Triton X-100, 0.5% NP-40, 0.25% sodium-deoxycholate, 150 mM NaCl, and complete Mini protease inhibitor cocktail tablets (1 tablet in 10 ml) (Roche, Basel, Switzerland). Lysates were centrifuged for 15 min at 14000 rpm at 4 °C and supernatants were loaded into 10–12% SDS PAGE gels. Primary antibodies for anti-CitH3 (ab18956–100; Abcam, Cambridge, UK), anti-HMGB1 (ab67282; Abcam, Cambridge, UK), and anti-GAPDH (2118; Cell Signaling Technology, Danvers, MA) were diluted 1:2000–10,000. The signals were detected using a chemiluminescence kit (Merck Millipore, Darmstadt, Germany).
Preparation of blood and serum
Blood samples were collected via cardiac puncture procedure using 23G syringe without thoracotomy. For serum samples, blood samples (1 ml) were left for 30 min at room temperature. To remove clot, the blood samples were centrifuged for 15 min at 2000 g at 4 °C. and supernatant was transferred immediately to ice-cold new tube. The sample was divided into 100 ul aliquots and stored in EDTA-coated vacutainer (BD Bioscience, Franklin Lakes, NJ).
Isolation of circulating neutrophils
Neutrophil was isolated from rat blood using Histopaque (Sigma Aldrich, St. Louis, MO) gradients as previously described . Briefly, Histopaque 1077 (3 ml) was layered on Histopaque 1119 (3 ml) in 15 ml tube and rat blood (4 ml) was carefully placed on the top of the Histopaque mixture, which formed a three-step gradient (Histopaque 1119/Histopaque1077/blood). The tube was then centrifuged at 400 g for 30 min using a swinging rotor. The first ring, which contained mononuclear cells was slowly aspirated, and the second ring was transferred to another 15 ml tube containing PBS-BG (phosphate buffered solution, 0.1% bovine serum albumin, and 10% glucose) and centrifuged at 1500 g for 10 min. The pellet so obtained was suspended in 3 ml of PBS-BG, placed on Histopaque-1119 (3 ml), and centrifuged at 1500 g for 10 min at 4 °C. The ring that included neutrophils was then suspended in RPMI (Gibco BRL, Gaithersburg, MD) containing 1% FBS.
Primary neuron culture
Mixed cortical cells were obtained from embryonic day 15.5 (E15.5) mouse cortices and cultured as described in a previous report . Dissociated cortical cells were plated at a density of five hemispheres per 24-well poly-d-lysine (100 μg/ml) and laminin (100 μg/ml)-coated plate (~ 4 × 105 cells per well). Cultures were maintained without antibiotics in MEM containing 5% FBS and 5% horse serum. At day 7 in vitro (DIV 7), when astrocytes had reached confluence underneath neurons, cytosine arabinofuranoside (ara-C, Sigma Aldrich, St. Louis, MO) was added to a final concentration of 10 μM, and culture was maintained for 2 d to halt microglial growth. Fetal bovine serum (FBS) and glutamine were not supplemented from DIV 7, and the medium was changed twice weekly after DIV 7. Cultures were used at DIV 12–14.
Treatment of neutrophils with NMDA conditioned media (NCM) or of neurons with NCM-treated neutrophil
Primary cortical neurons (4 × 105/well) were treated with MEM (21 mM glucose) containing 300 μM NMDA (Sigma, St. Louis, MO) for 30 min, washed twice with PBS, cultured in fresh MEM for 4 h. Neutrophils were then treated with this NMDA-conditioned medium (NCM, 1.6 × 106 /4 wells) for the indicated times. NCM-treated neutrophils (5 × 105/well) were co-cultured with primary cortical neurons using a transwell co-culture device (pore size 3 μm) (SPL Lifescience, Gyeonggi, South Korea). Neutrophils were pretreated with AMD3100 (Sigma, St. Louis, MO), TLR-IN-C34 (Sigma, St. Louis, MO), or Cl-amidine (PAD inhibitor; Merk Millipore, Burlington, MA) for 30 min before NCM treatment. NCM was preincubated with anti-HMGB1 antibody or HMGB1 A box (HMGbiotech, Milano, Italy; HM-012) for 30 min and then treated to neutrophils. For propidium iodide (PI) staining, PI (1 μg/ ml) was added to co-culture and incubation continued for 30 min. Primary cortical neurons were then fixed in 4% PFA for 20 min and fluorescence was visualized under a fluorescence microscope (Axioplan 2; Carl Zeiss, Oberkochen, Germany). The numbers of PI positive cells in 250 μm × 250 μm were counted and.
Sample sizes for animal experiments were determined by using power calculation software (http://www.gpower.hhu.de/) and the levels of significance are 5% with 80% power as a minimum. Differences in the parameters was performed by using analysis of variance (ANOVA) followed by the Newman-Keuls test. For a non-parametric statistics test, Kruskal-Wallis H non parametric test was performed, followed by Tukey’s test on SPSS package 18. Simple comparisons for histological data were carried out using Student’s t-tests. All results are presented as means±SEMs, and statistical difference was accepted at P- value < 0.05.