Animal stroke model. Male C57BL/6 mice (Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China) weighing 23-26g were used in this study. All protocols for these studies were approved by the Animal Care and Use Committee of the Shanghai Medical College of Fudan University according to National Institutes of Health Guidelines Mice were anesthetized with 1-1.5% isoflurane in 30% oxygen and 70% nitrous oxide. Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery (MCA) for 60 minutes with a siliconized filament38. Cerebral blood flow was monitored by continuous laser doppler flowmetry (Perimed, Stockholm, Sweden) to confirm induction of ischemia and reperfusion. Body temperature was maintained at 37 ± 0.5°C using a temperature control unit (World Precision Instruments, Florida) during surgery. MLN4924 (10mg/ml, 60mg/kg) or vehicle (10% 2-hydroxypropyl-β-cyclodextrin) was injected subcutaneously twice at 1 and 12 hours after MCA occlusion39. Adenoviral vector expressing NF1 shRNA (pDKD-CMV-Puro-U6-(NF1)-shRNA, produced by Obio Technology, Shanghai, China) or control shRNA (pDKD-CMV-Puro-U6-shRNA) (2 µl of 1.34 × 1011 plaque-forming-unit/ml) was injected into three points of cortex (coordinates: 0.3 mm anterior, 0.8 mm and 1.9 mm posterior to bregma, 3.0 mm lateral to midline, and 2.0 mm ventral to skull surface) in the right hemisphere 3 days before stroke40. Blocking antibodies against P-selectin (1.6 mg/kg, 553742, BD Pharmingen, San Jose, CA), anti-intercellular adhesion molecule-1 (ICAM-1) antibodies (200 µg/mouse, YN1/1.7.4, BE0020-1, Bioxcell, NH) or isotype control antibody was administered intravenously immediately before MCAO41, 42. Protein kinase C δ (PKCδ) inhibitor rottlerin (10 mg/kg) or vehicle (2% dimethyl sulfoxide) was injected intraperitoneally 30 minutes before stroke43, 44.
Cranial window surgery and multiphoton microscopy. Cranial windows were prepared as we previously described45, 46. Mice were anaesthetized with 1-1.5% isoflurane in 30% oxygen and 70% nitrous oxide. Body temperature was maintained at 37 ± 0.5°C during surgery. After fixation in a stereotaxic head holder, a craniotomy (5 mm diameter) was created above the right somatosensory cortex (centered 2.5 mm lateral and 2.5 mm posterior to the bregma) using a high-speed micro drill. The window was closed with a sterile cover glass. For multiphoton imaging, Olympus FluoView FVMPE-RS upright multiphoton laser-scanning system with an Olympus XL Plan N 25 × /1.05 WMP ∞/0-0.23/FN/18 dipping objective was used. Multiphoton excitation was performed using MAITAI eHPDS-OL and Spectra Physics InSight DS-OL lasers (Mai Tai, Spectra-Physics, CA). Emitted fluorescence was detected through 495–540 nm and 575-645nm bandpass filters.
For imaging of neutrophils, phycoerythrin (PE)-conjugated monoclonal Ly6G antibody (1A8 clone; 3 µg, 551461, BD Pharmingen) was intravenously injected into mice46. Fluorescein isothiocyanate (FITC)-dextran (0.1 ml of 10 mg/ml, 2000 KDa, Sigma-Aldrich, MO) was used to label the cerebral vasculature. Time-lapse images at 6 µm steps were acquired from 100 to 150 µm below the surface every 6 seconds for 25 minutes. The area scanned was at 900 nm excitation wavelength in a 508 × 508 µm with 512 × 512 pixel resolution. Neutrophil movement was determined by imaging of blood vessels with a diameter between 20 and 40 µm. Images reconstruction was carried out using Olympus FV 10-ASW software. Neutrophil movement analysis was performed using the IMARIS image analysis software (Bitplane AG, Zurich, Switzerland). Forty cells per animal were tracked. Adherent neutrophils were defined as the cells that were remained arrested in the microvessels for at least 30 seconds. The intravascular rolling velocity for neutrophils was calculated by determining the distance neutrophils moved between a certain time. Transmigrated neutrophils were determined in an area reaching out 75 µm to each side of a 100-µm-length vessel (representing 1.5 × 104 µm2 tissue area).
Cerebrovascular permeability based on FITC-dextran (MW = 40 KDa, Sigma-Aldrich) leakage was analyzed as described previously47. In brief, time lapse imaging of FITC-dextran was acquired every 3 minutes for 30 minutes. The fluorescence of randomly chosen 20 × 20 µm2 regions of interest within the vessel and corresponding areas within the perivascular brain parenchyma were recorded.
Measurements of Evans blue and BSA vascular leakage. At 23 hours after MCAO, mice were intravenously injected with 4 ml/kg of 4% Evans blue dye (Sigma-Aldrich). After 1 hour, mice were perfused transcardially with phosphate buffer saline (PBS), and ischemic hemispheres were weighted and placed in formamide for 72 hours. After centrifugation, the amount of extravasated Evans blue dye in the supernatants was evaluated by spectrophotometry (Thermo Scientific, MA) at 620 nm48. Alexa fluor 488-conjugated bovine serum albumin (BSA; 66 kDa, 50 µl of 100 mg/ml) was intravenously injected into mice 1 hour before killing49. Brains were collected, fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose in PBS. Coronal brain sections (20 µm thick) were used for fluorescent detection. Images were acquired using an Olympus FV1000 confocal microscope and an Olympus BX 63 microscope, and extravascular BSA fluorescence in tissue sections was quantified using NIH Image J software.
Analysis of extravascular IgG deposition. Coronal brain sections were blocked with 1% BSA in PBS and incubated with goat anti-CD31 antibody (AF3628, R&D Systems, MN) overnight at 4˚C. The sections were washed and incubated with Alexa Fluor 488-conjugated donkey anti-mouse immunoglobulin G (IgG) and Alexa Fluor 594-conjugated donkey anti-goat IgG (Invitrogen, Carlsbad, CA). To quantify extravascular deposits of IgG, the images were contrast enhanced to clearly differentiate positivity from background and quantified using the NIH Image J integrated density analysis tool.
Neurobehavioral test. Forelimb force and rotarod test were carried out by an investigator blinded to the experimental groups as described previously46, 48. In the forelimb force test, a grip strength meter (Bio-Seb, Vitrolles, France) was used to assess the peak force exerted by a mouse when the mouse released the forepaws from a grid. In the rotarod test, mice were placed on an accelerating rotating rotarod cylinder (Ugo Basile, Varese, Italy), and the time the mice remained on the rotarod was recorded. The speed was increased from 5 to 40 rpm within 5 minutes. Before surgery, mice were trained for 3 days.
Measurement of infarct volume and neuronal death. At 24 hours after MCAO, mice were sacrificed. The infarct area was detected by 2% triphenyl-2,3,5-tetrazolium chloride (TTC) staining and measured using the NIH Image J software in a blinded manner. Neuronal cell death in peri-infarct regions was detected using an In Situ Cell Death Detection Kit (11684795910, Roche, Mannheim, Germany) and mouse anti-neuronal nuclei (NeuN, MAB377, Millipore, MA) immunostaining.
Immunoblotting. Brain capillaries and capillary-depleted brain homogenates were prepared as we described previously46, 50. Isolated protein from brain tissues, capillaries, and capillary-depleted brain homogenates was detected by immunoblotting according to standard procedures46, 50. The primary antibodies used were: rabbit anti-NEDD8 (1:1000, 2754), rabbit anti-Bcl-2 (1:1000, 3498), mouse anti-p53 (1:1000, 2524), rabbit anti-Bax (1:1000, 2772), rabbit anti-Cleaved Caspase-3 (caspase-3, 1:1000, 9661), HRP-linked anti-mouse IgG (1:2000, 7076), rabbit anti-phospho-PKCδ (pPKCδ, 1:1000, 9376), rabbit anti-phospho-Myosin Light Chain (pMLC; 1:1000, 3671), rabbit anti-Myosin Light Chain (MLC, 1:1000, 3672), and rabbit anti-β-actin (1:1000, 4970, all from Cell Signaling Technology, Danvers, MA), rabbit anti-APPBP1 (NAE1, 1:1000, SAB1300843, Sigma-Aldrich), rabbit anti-UBA3 (1:1000, ab124728), rabbit anti-UBE2M/UBC12 (1:1000, ab109507), rabbit anti-occludin (1:1000, ab167161), rabbit anti-claudin5 (1:1000, ab15106), rabbit anti-CD144 (vascular endothelial cadherin, VE-cadherin; ab33168), rabbit anti-protein kinase Cδ (PKCδ, 1:1000, ab182126), rabbit anti-phospho-myristoylated alanine-rich C-kinase substrate (pMARCKS, 1:1000, ab81295,all from Abcam, Cambridge, UK), mouse anti-CUL-1 (1:1000, sc-17775), mouse anti-Neurofibromin (NF1, 1:1000, sc-376886), mouse anti-P-Selectin (1:1000, sc-8419), mouse anti-MARCKS (1:1000, sc-100777, Santa Cruz Biotechnology, Dallas, Texas), rat anti-mouse Ly6G (1:1000, 551459, BD Pharmingen), rabbit anti-Zonula occludens-1 (ZO-1; 1:1000, 617300, Invitrogen), goat anti-ICAM-1/CD54 (1:1000, AF796), goat anti-vascular cell adhesion molecule-1 (VCAM-1, 1:1000, AF643, both from R&D Systems, Minneapolis). Blots were visualized by labeling with horseradish peroxidase-conjugated anti-rabbit, anti-goat, anti-mouse, anti-rat secondary antibodies (CST) and incubation with chemiluminescent substrate (Millipore). Blots were processed and analysed with the Image Lab-5.2.1 software (Bio-Rad Laboratories, CA).
MPO activity assay. Mice were sacrificed, perfused with ice cold PBS and the brains were removed. Ipsilateral brain hemispheres were homogenized in 50 mM potassium phosphate buffer, centrifuged, and resuspended in 0.5% cetyltrimethylammonium bromide (Sigma-Aldrich) in potassium phosphate buffer. The suspensions were sonicated for 30 seconds with 3 cycles of freeze-thaw in liquid nitrogen. After centrifugation, 40 µl of supernatant was mixed with 100 µl tetramethylbenzidine solution (Sigma-Aldrich) in a 96-well plate in duplicates. The reaction was stopped with 100 µl 2N HCl after 15 minutes. The absorbance was measured at 450 nm in a microplate reader (Bio-Tek, Vermont). Myeloperoxidase (MPO) activity was calculated using purified MPO (Sigma-Aldrich) and was expressed as units of MPO per mg protein.
Quantitative real-time polymerase chain reaction. Total RNA was isolated from brain tissue using the TRIzol kits (Invitrogen) according to the manufacturer’s protocol. The polymerase chain reaction (PCR) was performed with equal amounts of cDNA in a Mastercycler ep gradient machine (Eppendorf AG 22331 Hamburg, Germany)45. Relative changes in gene expression levels were calculated as the ratio of target cDNA to GAPDH. The following primers were used: Chemokine (C-X-C motif) ligand 1 (CXCL1) forward 5′-ACCCAAACCGAAGTCATAGCC-3′, reverse 5′-TTGTCAGAAGCCA GCGTTCA-3′, CX3CL1 forward 5′-GCCATTGTCCTGGAGACGAC-3′, reverse 5′-CCAAGGTGATCCCA GGTGTC-3′, chemokine (C-C motif) ligand 2 (CCL2) forward 5′-GCTG-TAGTTTTTGTCA CCAAGC-3′, reverse 5′-AAGGCATCACAGTCCGAGTC-3′, chemokine receptor 1 (CCR1) forward 5′-ACTCTGGAAACACAGACTCACT-3′, reverse 5′-GCCCACCACTCCAATGAT GA-3′, GAPDH forward 5′-AGGTCGGTGTGAACGGATTT G-3′, reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′.
Measurements of interleukin-6, interleukin-1β and TNF-α levels. Protein from brain tissue was extracted in radioimmunoprecipitation (RIPA) lysis buffer (Millipore) containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Protein concentration was determined by bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Quantification of interleukin (IL)-6, IL-1β and tumor necrosis factor-α (TNF-α) proteins were performed using enzyme-linked immunosorbent assay (ELISA) kits (R&D systems) according to the manufacturer’s instructions.
Immunohistochemistry. Mice were deeply anaesthetized with isoflurane and perfused transcardially with PBS followed by 4% paraformaldehyde in PBS. Brains were removed and immersed in 4% paraformaldehyde and cryoprotected in 30% sucrose. Coronal sections of 20 µm thickness were prepared on a cryostat and collected on glass slides. Sections were stained according to standard immunohistochemistry procedures with the following primary antibodies: mouse anti-NEDD8 (1:200, sc-373741), mouse anti-CUL-1 (1:1000, sc-17775), mouse anti-Neurofibromin (NF1, 1:1000, sc-376886, all from Santa Cruz), rat anti-Ly6G (1:200, 551459, BD Pharmingen), goat anti-CD31 (1:200, AF3628, R&D Systems), mouse anti-neuronal nuclei (NeuN, MAB377, Millipore). The secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rat IgG, Alexa Fluor 488-conjugated donkey anti-goat IgG, Alexa Fluor 594-conjugated donkey anti-mouse IgG, Alexa Fluor 488-conjugated donkey anti-mouse IgG, Alexa Fluor 594-conjugated donkey anti-goat IgG, Alexa Fluor 647-conjugated donkey anti-goat IgG and biotin-donkey anti-mouse IgG (all from Invitrogen). DNA was stained with Hoechst 33342 (1:10000, H3570, Invitrogen). For each animal, three fields from the peri-infarct cortex in each section were obtained under × 40 objective. Images were traced (quantitative analyzed) using Image J 1.48v software. The numbers of Ly6G+ neutrophils in the traced area were counted.
Statistics. All values are presented as means ± standard deviation (SD). Statistical analysis for multiple comparisons were performed in Prism 7 software using one-way ANOVA followed by Bonferroni multiple comparison test. Differences between the two groups were assessed by unpaired Student’s t-test. A value of P < 0.05 was considered statistically significant.