Mice
All mouse lines were established on a C57Bl/6 background. We used female and male littermate mice that were age-matched between experimental groups. Mice were between 6 and 10 weeks of age. All animal experiments were approved by the local animal welfare committee (Regierungspräsidium Karlsruhe, Germany, permission number: 35–9185.81/G-112/13), conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, and were performed in accordance with the recently published Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines (https://www.nc3rs.org.uk/arrive). All mice were housed at constant room temperature (22 ± 2 °C) and relative humidity (50–55%) on a controlled 12:12 h light-dark cycle, and were provided with standard laboratory chow (LASQCdiet Rod16; LASvendi, Soest, Germany) and water ad libitum. Mice homozygous for a null allele of Ephb2 (Ephb2tm1Paw; Ephb2−/−) [21] and corresponding wild-type (WT) littermates were obtained by mating Ephb2 haploinsufficient (Ephb2+/−) mice. Depletion of EphB2 protein was confirmed by capillary electrophoresis (Additional file 1: Figure S1a). Neural cell-specific ephrin-B2 deficient mice (nEfnb2Δ/Δ) were generated by crossing animals harboring two floxed alleles (exon 2 flanked by loxP sites) of the Efnb2 gene (B6.E14-TgH(efnb2flx/flx)RK; Efnb2fl/fl) [15] with transgenic mice expressing Cre recombinase under control of the promoter and the nervous system-specific enhancer present in the second intron of the rat nestin gene (B6.Cg-Tg(Nes-cre)1Kln) [52]. Cre-mediated excision of floxed exon 2 in the Efnb2 gene was successfully verified on the mRNA level using real-time RT-PCR (Additional file 1: Figure S1b). Mice were genotyped using primers (Eurofins Genomics, Ebersberg, Germany) described in Additional file 2: Table S1. All mice were randomly allocated to experimental groups. Operators and investigators were blinded for mouse genotype in all experiments and analyses. Evaluation of all read-out parameters was done independently and in a blinded fashion.
Experimental stroke model
Mice were used at the age of 7–9 weeks. Female and male mice were anesthetized by a mixture of 2% isoflurane in, 70% N2O and remainder O2, and were maintained by reducing the isoflurane concentration to 1.0–1.5%. To induce focal cerebral ischemia, a 7–0 silicon rubber-coated nylon monofilament (Doccol Corporation, Redlands, USA) was introduced in the left internal carotid artery and pushed toward the left middle cerebral artery (MCA) as previously described [27]. In subgroups of mice laser-Doppler flowmetry (LDF) was used to confirm successful MCA occlusion (MCAO) as reported previously [27]. The intraluminal suture was left for 60 min. Subsequently, animals were re-anesthetized and the occluding monofilament was withdrawn to allow reperfusion for 6–72 h. For sham surgery, the mice underwent the same procedure without vessel occlusion. The animals were maintained at 37 °C during and after surgery until they were fully recovered from anesthesia. Then, mice were returned to their solitary cages in a heated (30 °C) environment with free access to food and water for 12 h. During the remaining time animals were kept under normal conditions as described above. Additional file 2: Table S2 lists the criteria resulting in exclusion from end-point analysis.
Behavioral assessment
Motor coordination and balance were assessed by using the Rotarod performance test. Mice were placed individually on the revolving drum. Once they were balanced, the drum was accelerated from 4 to 40 rpm over the course of 300 s, and the time at which the animal dropped off the drum was determined (maximum testing time 300 s). Mice were trained for three consecutive days (three runs each) and were tested directly before MCAO and 24 h after onset of reperfusion. At indicated time points, neurological function was additionally evaluated using the modified Bederson neurological deficit score, according to the following scoring system: 0, no observable deficit; 1, forelimb flexion; 2, decreased resistance to lateral push; 3, unidirectional circling; 4, no movement [4].
Magnetic resonance imaging (MRI)
MRI was performed on a dedicated small animal scanner with 9.4 Tesla magnetic field strength (BioSpec 94/20 USR, Bruker, Ettlingen, Germany) using a volume coil for RF transmission and a 4-channel phased-array surface receiver coil. An isoflurane evaporator connected to a supply of compressed air was used for anesthesia. Anesthesia was induced at 2% isoflurane and maintained with 1–1.5%. Animals were placed in prone and fixed positions on an animal holder equipped with a headlock and tooth bar to minimize head motion. Body temperature was maintained using a temperature-controlled heating pad. Respiration was monitored externally with an in-house developed program in LabView (National Instruments Corporation, Austin, Texas, USA).
The imaging protocol included T2-weighted imaging (TEeff/TR = 66 ms/2650 ms, RARE factor = 8, slice thickness 0.5 mm, 13 slices, matrix 256 × 256, in plane resolution 78 × 78 μm), diffusion-weighted imaging (TEeff/TR = 20 ms/3400 ms, slice thickness 0.7 mm, one measurement with b = 0 and 30 diffusion sensitized directions with a b-value of 1500 s/mm2, field-of-view 12 × 15 mm, matrix 96 × 128, resolution 125 × 117 μm), as well as quantitative T2 measurements (TE increments of 8 ms from 8 ms to 136 ms, TR = 3100 ms, slice thickness 0.5 mm, matrix 172 × 172, resolution 116 × 116 μm). The real signal component from the Multislice Multiple Spin Echo data were fitted after phase correction and SNR optimized multiple coils signal combination on a voxel-by-voxel basis with the monoexponential function S0*e –(TE/T2) using a nonlinear least-squares fit procedure (MATLAB Release 2012b, The MathWorks, Inc., Natick, Massachusetts, United States). S0 is the signal at TE = 0. Apparent diffusion coefficient (ADC) values were obtained from diffusion-weighted images by FSL’s (FMRIB [The Oxford Centre for Functional Magnetic Resonance Imaging of the Brain] Software Library) FDT toolbox (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FDT). Image analysis was performed with the software Amira (Visage Imaging, Inc., San Diego, USA). The ischemic lesion on ADC and T2 maps in basal ganglia and cortex was determined by manual region growing using threshold-based pre-segmentation.
Histopathological analysis
Animals were deeply anesthetized and transcardially perfused with PBS (2 ml/min) for 5 min. Brains were removed and embedded into Tissue-Tek (Sakura Finetek, Staufen, Germany). From each brain, 24 coronal sections (10 μm thickness, 0.4 mm distance) were prepared using a Leica CM1520 cryostat (Leica Biosystems, Wetzlar, Germany) at a constant temperature of − 15 °C, and stained with cresyl violet (Merck Millipore, Darmstadt, Germany, #105235) according to the manufacturer’s instructions. Stained brain slices were digitized, and infarct and edema volume was measured using the image analysis software ImageJ (National Institutes of Health, Bethesda, MD, USA) as described previously [29, 43].
Fluoro-Jade C (FJC) staining of cerebral cryosections (10 μm thickness; + 0.62 to − 0.62 mm relative to Bregma) was used to detect neuronal degeneration following the protocol of manufacturer (Merck Millipore, #AG325). FJC staining was recorded using a Zeiss Axiovert 200 M microscope (Carl Zeiss Microscopy, Göttingen, Germany) with a Hamamatsu ORCA flash 4.0 camera (Hamamatsu Photonics, Herrsching am Ammersee, Germany) by applying TissueFAXS scanning software (TissueGnostics, Vienna, Austria). Nuclei were identified by DAPI staining. Cells showing fluorescent signal for FJC were automatically counted by using TissueQuest 4.0 software (TissueGnostics).
Analysis of cerebrovascular anatomy
Gross anatomical features of the cerebrovascular architecture were determined as described previously [3].
Evaluation of BBB permeability
Mice were anesthetized 2 h prior to the end of the reperfusion time, and 100 μl of a pre-warmed (37 °C) solution containing 2% Evans Blue (Sigma-Aldrich, Steinheim, Germany) in 0.9% NaCl were injected into the tail vein. Mice were transcardially perfused, brains harvested and separated into entire left and right hemispheres. Each hemisphere was suspended in 500 μl ice-cold 50% trichloroacetic acid and homogenized with a grinding ball at 30 Hz for 2 min (Mixer Mill MM301; Retsch, Haan, Germany). Tissue lysates were then incubated for 2 h at 4 °C and centrifuged at 16,000×g for 15 min at 4 °C. Evans Blue in the supernatant was measured using a spectrophotometer (Synergy HT; BioTek, Bad Friedrichshall, Germany) at 610 nm and quantified according to a standard curve.
Glial cell cultures
Primary astroglial and microglial cultures were prepared from neonatal WT, Ephb2−/− and nEfnb2Δ/Δ mice (P0-P2). The purity of astrocyte and microglia cultures was about 96% and almost 100%, respectively (Additional file 1: Figure S1c). Details on isolation, culture, treatment, and analysis of microglial phagocytosis are provided in the Supplementary Methods (Additional file 3).
Neuronal cell culture
Primary dissociated cortical cultures were prepared from newborn WT and Ephb2−/− mice (P0). The relative portion of neurons within the mixed cultures was about 83% (Additional file 1: Figure S1c). Detailed experimental procedures for isolation, culture, and treatment of neuronal cultures as well as analysis of mitochondrial/cytoplasmic Ca2+ concentration and mitochondrial membrane potential can be found in the Supplementary Methods (Additional file 3).
Immunofluorescence staining
Immunofluorescence staining techniques were applied to determine abundance and subcellular localization of certain proteins and were used to identify different cell types in brain tissue sections and cellular monolayers by detection of cell-specific marker proteins. A detailed description is given in the Supplementary Methods (Additional file 3; see also Additional file 2: Table S3).
Quantitative real-time RT-PCR analysis
Mice were transcardially perfused with PBS, brains harvested, and a 2-mm-thick tissue slice (− 1.0 to − 3.0 mm relative to bregma) was prepared from each brain and separated into the left and right hemispheres. Total RNA from brain tissue samples or cells was isolated using the TRI reagent (Thermo Fisher Scientific, Dreieich, Germany) according to manufacturer’s instructions. For digestion of residual DNA, 10 μg of total RNA was incubated in a 25 μl reaction mix containing 1x DNase-buffer, 40 U RNasin and 1 U DNase (Promega, Mannheim, Germany) for 30 min at 37 °C. Subsequently, cDNA was synthesized using the Access Reverse Transcription PCR Kit (Promega, #A1260) and quantitative real-time PCR for the target sequences was performed in the Rotor-Gene Q (Qiagen, Hilden, Germany) using the QuantiTect SYBR Green PCR Kit (Qiagen). Fluorescence was monitored (excitation at 470 nm and emission at 530 nm) at the end of the annealing phase. Threshold cycle (Ct) was set within the exponential phase of the PCR. Quantification of the PCR product was done by using the ΔΔCt method. Amplification of the 40S ribosomal protein S12 (Rps12) cDNA served as an internal standard. Primers were purchased from Eurofins Genomics (for primer sequences, see Additional file 2: Table S4).
DNA microarray analysis
Mice were transcardially perfused with PBS, brains extracted, and separated into the left and right hemispheres. Total RNA from brain tissue samples was prepared using the TRI reagent (Thermo Fisher Scientific) according to manufacturer’s instructions followed by additional purification utilizing the RNeasy Mini Kit (Qiagen). RNA was tested by capillary electrophoresis on an Agilent 2100 bioanalyzer (Agilent, Waldbronn, Germany) and high quality was confirmed. Gene expression profiling was performed using arrays of mouse MoGene-2_0-st-type from Affymetrix (Santa Clara, USA). Biotinylated antisense cRNA was then prepared according to the Affymetrix standard labeling protocol with the GeneChip® WT Plus Reagent Kit and the GeneChip® Hybridization, Wash and Stain Kit (both from Affymetrix). Afterwards, hybridization on the chip was performed on a GeneChip Hybridization oven 640, then dyed in the GeneChip Fluidics Station 450 and thereafter scanned with a GeneChip Scanner 3000. All equipment used was from Affymetrix (High Wycombe, UK). A Custom CDF Version 20 with ENTREZ-based gene definitions was used to annotate the arrays [7]. The Raw fluorescence intensity values were normalized by applying quantile normalization and RMA background correction. An ANOVA was performed to identify differentially expressed genes using a commercial software package (SAS JMP10 Genomics, version 7) from SAS (SAS Institute, Cary, NC, USA). A false positive rate of a = 0.05 with FDR correction was taken as the level of significance. Gene Set Enrichment Analysis (GSEA) was used to determine whether defined sets of genes exhibited a statistically significant bias in their distribution within a ranked gene list using the software GSEA [49]. Pathways belonging to various cell functions such as cell cycle or apoptosis were obtained from public external databases (KEGG, http://www.genome.jp/kegg). The raw and normalized data are deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/; accession No. GSE120565).
Phospho-receptor tyrosine kinase array
Mice were transcardially perfused with PBS, brains removed, and a 3-mm-thick tissue slice (+ 2.50 ± 0.5 to 0.00 ± 0.5 mm relative to bregma) was prepared from each brain and separated into the left and right hemispheres. The relative level of tyrosine phosphorylation of 39 different receptor tyrosine kinases (RTK) was determined in brain tissue samples using the Proteome Profiler Mouse Phospho-RTK Array Kit (R&D Systems, Wiesbaden, Germany, #ARY014) according to manufacturer’s instructions. Briefly, 500 μl lysis buffer was added to each brain tissue slice, and tissue samples were homogenized mechanically as described above. Following incubation on ice for 10 min, tissue homogenates were centrifuged for 5 min at 16,100×g at 4 °C. Protein concentration in the supernatant was then quantified by Bradford assay, and 250 μg protein/sample was processed further following the protocol of manufacturer.
Capillary electrophoresis
Mice were transcardially perfused with PBS, brains harvested, and a 2-mm-thick tissue slice (+ 3.0 to + 1.0 mm relative to bregma) was prepared from each brain and separated into the left and right hemispheres. Lysis buffer containing 20 mM Tris (pH 7.6), 250 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM DTT, 1 mM PMSF, and 1% protease inhibitor cocktail (all from Sigma-Aldrich) was added to brain tissue samples or cell monolayer. Tissue samples were homogenized mechanically as reported above. Following incubation on ice for 15 min, tissue and cell homogenates were centrifuged for 15 min at 16,100×g at 4 °C. Protein concentration in the supernatant was then quantified by Bradford assay. Analysis of protein expression was performed according to the Wes User Guide using a Wes instrument from ProteinSimple (San Jose, CA, USA). Briefly, protein samples were diluted with 0.1X sample buffer to a final concentration of 0.5 μg/μl, and were mixed with fluorescent 5x Master Mix and incubated at 95 °C for 5 min. The samples were loaded into the Wes microplate along with a biotinylated protein ladder, blocking reagent, primary antibodies against EphB2 (R&D Systems, #AF467; 1:10) and beta-tubulin (Abcam, Cambridge, UK, #ab6046; 1:500), HRP-conjugated anti-rabbit secondary antibody, luminol peroxide, and washing buffer. The plates and capillary cartridges were loaded into the Wes for electrophoresis and chemiluminescence immunodetection using a CCD camera with default settings: electrophoresis, 375 V, 30 min; blocking, 5 min; primary antibody, 30 min; secondary antibody, 30 min; and camera exposure times, 1 s to 32 s. Compass software (ProteinSimple) was used to acquire and analyze the data and to generate gel images and chemiluminescence signal intensity values. Protein expression is calculated as the chemiluminescence intensity area under the curve.
Enzyme-linked immunosorbent assay (ELISA)
MCP-1 and TNF protein levels in cell supernatants were measured by quantitative ELISA (R&D Systems, Mouse MCP-1 DuoSet ELISA #DY479–05, Mouse TNF-alpha DuoSet ELISA #DY410–05) according to manufacturer’s instructions.
Statistical analysis
If not indicated otherwise, all results are expressed as means and displayed on scattered dot plots ± standard deviation (SD). Differences between 2 independent experimental groups were analyzed by two-tailed Student’s t tests (normally distributed data) or Mann-Whitney U rank-sum tests (ordinal and non-normal data). Differences of one parameter among three or more independent experimental groups were analyzed by either one-way ANOVA followed by a Holm-Sidak’s multiple comparisons test (normally distributed data), or by Kruskal-Wallis H test with Dunn’s post hoc test (ordinal and non-normal data). Differences of two parameters among two or more independent/correlated experimental groups were analyzed by two-way (Repeated Measures) ANOVA followed by a Holm-Sidak’s multiple comparisons test (normally distributed data). A probability value of P < 0.05 was considered statistically significant. Data plotting and statistical analyses were done with Prism 6 (GraphPad Software, La Jolla, CA, USA).