Primary culture of OPCs
The OPCs primary culture was carried out as our previous study [20]. Briefly, Sprague Dawley (SD) rat (postnatal 1~2 d, provided by Laboratory Animal Center, Xuzhou Medical University) cortical cells were cultured in DMEM/F12 medium (Hyclone, USA) containing 10% fetal bovine serum (FBS) and 0.1% penicillin/streptomycin. Seven days later, the culture flask was shaken on a shaker at 37 ℃ for 1 h and then the supernatant was discarded. Fresh medium was added to the culture flask, OPCs was separated after shaking the flask for 18 h on the shaker. Cells were centrifuged for 5 min at 600 ×g and then resuspended in DMEM/F12 medium (HyClone, USA) supplemented with 10 ng/ml recombinant human basic fibroblast growth factor (bFGF), 10 ng/ml recombinant human platelet-derived growth factor-AA (PDGF-AA), 1% N2 and 2% B27 (all from Gibco, USA). Cells were seeded into a 6-well plate at a density of 1×105 cells/ml, and the medium was changed every 2 d.
Primary culture of human EPCs
Human EPCs were isolated from umbilical cord blood provided by Affiliated Hospital of Xuzhou Medical University. Briefly, the fresh umbilical cord blood was taken and blended with 0.01mol/L phosphate buffer saline (PBS). The mixture was carefully and evenly poured over the Lymphocyte Separation Medium (LSM, Corning, USA). After centrifugation for 30 min at 1800 g, the interphase (white layer) between the plasma and the separation solution was carefully extracted, washed and spin in PBS for 10 min at 600 g twice. The pellet was re-suspended and poured over the LSM and centrifugated at 1200 g for 30 min. The interphase was extracted and washed with PBS. After centrifugation, the pellet was re-suspended with Endothelial Cell Growth Medium-2 (EGM-2MV) BulletKit (LONZA, Switzerland) and the cells were placed into a 25-cm2 culture bottle. The medium was changed every 2~3 d.
HBMECs culture and treatments
Human brain microvascular endothelial cells (HBMECs/ECs) were purchased from ScienCell Research Laboratories, Inc. and cultured in EGM-2MV according to our lab’s protocol [21]. To investigate the influence of OGD on ECs, the cells were divided into control, OGD 3 h, 6 h and 9 h groups. When the ECs are 60~70% confluency in culture dishes, EGM-2MV was replaced by sugar-free DMEM (HyClone, USA) and the cells were subjected to 0, 3, 6 or 9 h of hypoxia in an incubator containing 1% oxygen and 95% nitrogen and then reoxygenated for 24 h.
Fluorescence-activated cell sorting (FACS) was used to detect the apoptosis of ECs. Briefly, the cells were digested and centrifugated at 1000 g for 5 min. After washing and centrifugation twice, the pellets were re-suspended with 300 ml binding buffer, then incubated for 15 min with 5 ml Annexin V-PE and 5 ml 7-AAD (BD-pharmingen, USA). Flow cytometry (FACS Canto II, Becton-Dickinson, USA) and FlowJo software were used for apoptosis analysis and data processing.
Detection of the influence of EPCs on HBMECs
Cell apoptosis test
To observe the effect of EPCs on ECs viability, ECs were divided into three groups: normal control (Ctrl), ECs were exposed to OGD for 6 h, then reoxygenated for 24 h (OGD-EC), and EPCs grown in transwell chamber were co-cultured with OGD-ECs for 24 h (OGD-EC+EPCs). Cell apoptosis was detected by FACS.
Cell migration assay
To investigate the effect of EPCs on the invasion ability of OGD-ECs, ECs were seeded into the upper transwell chamber, which then were inserted into a 24-well plate. EPCs were cultured in another 24-well plate. After OGD for 6 h, the transwell chamber was removed and placed into the 24-well plate grown with EPCs. Meanwhile, the medium derived from OGD-EC was collected and used to incubate the EPCs. ECs and EPCs were co-cultured for 4 h, the invasion ability of ECs was examined by crystal violet (Beyotime, China) staining.
Tube formation assay
EPCs were seeded into the upper transwell chamber, which then were inserted into a 24-well plate. After ECs were exposed to OGD for 6 h, the transwell chamber was moved to the plate grown with OGD-ECs. The OGD-ECs and EPCs were co-cultured for 24 h at 37°C in a humidified atmosphere with 5% CO2, then the ECs were digested and seeded into Matrigel (Corning Inc., USA)-treated 24-well plate. Tube formation was observed with microscopy (Olympus, TKY, Japan) and the images were analyzed by Image J software.
Cellular immunofluorescence staining
Cells were fixed at room temperature for 15 min with 4% paraformaldehyde (PFA) and rinsed with 0.01mol/L PBS. After blocking non-specific antigens with 5% BSA, OPCs were treated with mouse monoclonal anti-A2B5 antibody (1:200, Sigma, USA), ECs and EPCs were incubated with rabbit anti-CD31 antibody (1:100, Abcam, USA) and rabbit anti-CD133 antibody (1:100, Abcam, USA) overnight at 4℃, respectively. After washing with PBS, Dylight-488 or Dylight-649 conjugated goat anti-mouse IgG (H+L) secondary antibodies (1:500, Abbkine, USA) were added at room temperature for 2 h. Nucleus were stained with 4',6-diamidino-2-phenylindole (DAPI, 1:200, Beyotime, China) for 10 minutes. The images were taken under Olympus Bx60 fluorescence microscope (Olympus, TKY, Japan).
OPCs processing and RNA interference
To investigate the influence of OGD-ECs and EPCs on OPCs, OPCs were divided into normal group (DMEM/F12), CM derived from ECs treated group (EC-CM), CM derived from OGD-EC treated group (OGD-EC-CM) and CM derived from EPCs and OGD-ECs co-culture treated group (OGD-EC+EPCs-CM). OPCs were cultured for 24 h, cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and FACS. The expression of CXCR4 and CXCR7 was analyzed by western blotting.
To examine whether OGD-EC+EPCs-CM alleviates OPCs apoptosis through CXCL12 and its receptors, RNA interference was used to knock down the level of CXCR4 and CXCR7 in OPCs. Briefly, 60~70% confluent OPCs were transfected with CXCR4 or CXCR7 small interfering RNAs (siRNA) (Santa Cruz Biotechnology, USA) duplexes for 6 h using Lipofectamine 3000 (Invitrogen, Carsbad, CA, USA) according to the manufacturer’s instructions. After incubation with the CM for another 24 h, the cells were harvested for FACS analysis.
TUNEL staining
OPCs grown in 48-well plates were fixed at room temperature for 15 min with 4% PFA, then were incubated in 0.3% Triton X-100 and 5% BSA at 37℃ for 30 min. After washing the cells with PBS 2-3 times, TUNEL reagent (KeyGen, China) was added to incubate the cells for 1 h at 37℃. After rinsing the cells with PBS several times, DAPI (Beyotime Biotechnology, China) was added to each well at room temperature for 3-5 min. The photographs were taken under Olympus Bx60 fluorescence microscope.
Enzyme-linked immunosorbent assay (ELISA)
To test the level of CXCL12 derived from ECs, ECs were divided into normal control group (Ctrl-EC), OGD 6 h and then reoxygenation 24 h group (OGD-EC), EPCs co-cultured with OGD-ECs group (OGD-EC+EPCs), and EPCs co-cultured with normal ECs group (Ctrl-EC+EPCs). The medium was collected and centrifuged at 800 g for 5 min to remove debris, the concentration of CXCL12 in the supernatant was measured by ELISA kit (R&D Systems) according to the manufacturer’s instructions. The CXCL12 at the level of ECs was detected by PCR and western blotting.
To further investigate whether OGD-ECs influenced the secretion of CXCL12 from EPCs, EPCs was infected with CXCL12 shRNA or control lentivirus (Lentiviral Particles sc39367-v, Santa Cruz, USA) for 48 h, then were co-cultured with OGD-ECs for another 24 h. The medium was collected and CXCL12 was detect by ELISA kit.
Western blotting
OPCs were dissolved with RIPA splitting buffer (1% deoxycholic acid, 1% Tritonx-100 and 0.1% NaN3) containing 10-micron-benzosulfonyl fluoride and phosphatase inhibitors. The protein concentration was determined by bicondylic acid. The protein lysate was separated by 8% or 10% twelve alkyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose filter membrane. The membrane was incubated with 5% defatted dry milk buffer (dissolved in washing solution) for 1 h and then incubated with mouse anti-CXCR4 (1:200, Santa Cruz, USA) and mouse anti-CXCR7 (1:1000, Abcam, Cambridge, UK) primary antibodies at 4 ℃ overnight. After washing with PBS, the membrane was incubated with goat anti-rabbit or goat anti-mouse (1:5000, LI-COR, USA) in shaking bed for 2 h at room temperature. Finally, Odyssey infrared imager (AGT, SFO, USA) was used to scan the imprinting, and Image J software was used to quantitatively analyze.
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) analysis
The ribonucleic acid (RNA) of ECs was extracted using the cold Trizol according to the manufacturer’s instructions. The primer sequences of human CXCL12 (F 5′-TGCCAGAGCCAACGTCAAG, R 5′-CAGCCGGGCTACAATCTGAA) and human GAPDH (F 5'- GCAAATTCCATGGCACCGT, R 5' -TCGCCCCACTTGATTTTGG) were used. Total RNA was then reverse transcribed into cDNA using the RevertAid first strand cDNA reverse synthesis kit (Takara). The qRT-PCR reaction was performed using a Light Cycler 480 SYBR green I Master Mix, and the thermocycling conditions were as follows: 95°C for 10 min warm boot, 40 cycles 95°C for 15 sec and 60°C for 45 sec and then 72°C for 30 sec. Relative gene expression was calculated using the 2-△△ct method.
Neonatal rat ischemia-hypoxia model and treatment
SD rat pups (P3) obtained from the Center of Experimental Animals of Xuzhou Medical University were randomly divided into the normal control group, the sham group, the ischemia-hypoxia (HI) with vehicle-treated group, HI with EPCs graft group, HI with EC-pEPCs graft group, and EC-pEPCs graft with AMD3100 treated group. HI was produced as our previously described [9,22]. Briefly, the P3 rat pups were anesthetized by gaseous anesthesia with isoflurane and O2. The right common carotid artery was carefully isolated from the surrounding tissue and ligated. Then, the wound was sutured, and the pups were allowed to recover for 2 h before exposing to 8% O2 (92% N2 saturation) at 37 ℃ for 90 min in a humidified chamber. The sham group underwent the same procedures without ligation of the carotid artery. The pups were exposed to room air after hypoxia.
For transplantation, the HI pups (P3+4) were anesthetized with 2.5% isoflurane (Shandong Keyuan Pharmaceutical Co., Shandong Province, China) in a mixture of air and 100% oxygen (1:1) and positioned in a stereotaxic apparatus. According to the coordinate (bregma: AP -1.0mm, ML 0.5mm, and DV 2.0 mm), a 26-gauge needle attached to a 5-µl Hamilton syringe was lowered into the right corpus callosum and 2µl EPCs or EC-pEPCs (1×105 cells/ml Hank’s Balanced Salt Solution, HBSS) or vehicle was released at a rate of 0.2 µl/min, after which the needle was left in place for 10 min to ensure complete diffusion. After operations were finished, the pups were placed on a warm pad carefully for 30min and then returned to their mother rats.
For the immunosuppression, transplanted pups received tacrolimus (TAC, 4 mg/kg/d) and sirolimus (SIR, 3 mg/kg/d; Sigma-Aldrich) once daily by intraperitoneal injection for 3 weeks, then by drinking water (both 100µg/ml) till sacrifice. For observing the influence of CXCR4 antagonist on transplanted EC-pEPCs, AMD3100 (5 µg/kg body weight, 10ug/ml, Selleck, USA) was intraperitoneally injected once per day for 2 weeks. All procedures in the experiment were consistent with Chinese legislation on the use and care of laboratory animals and were approved by Xuzhou Medical University Committee for animal experiments.
Morris water maze (MWM) test.
MWM test was performed during the sixth week after transplantation as previously described [9,22]. The experimental apparatus consisted of a circular water tank (100 cm in diameter, 35 cm in height), containing water (23 ± 1 °C) to a depth of 15.5 cm, which was rendered opaque by adding black nontoxic carbon ink. A platform (4.5 cm in diameter, 14.5 cm in height) was submerged 1 cm below the water surface and placed at the midpoint of one quadrant. The pool was located in a test room that contained various prominent visual cues. Each rat received four training periods per day for 4 consecutive days. The latency to escape from the water maze (by finding the submerged escape platform) was calculated for each trial. On day 5, the probe test was performed by removing the platform and allowing each rat to swim freely for 60 s. The time that rats spent swimming in the target quadrant (where the platform was located during hidden platform training) and in the three nontarget quadrants (right, left and opposite quadrants) was measured, respectively. For the probe trials, the number of times each rat crossed over the platform site was also measured and calculated. All data were recorded with a computerized video system.
Tissue preparation
Briefly, rats were perfused intracardially with PBS followed by 4% cold PFA at the seventh week after transplant. The brains were post-fixed in 4% PFA for 6 h and incubated in 30% sucrose buffer for 48 h at 4 °C, and then were embedded in Optimal Cutting Temperature medium (Leica Microsystems, Nussloch, Germany) for cryosection. Serial coronal sections (20 μm) were cut from the bregma anterior-posterior coordinates +1.0 to −0.2 for immunofluorescence and TUNEL staining.
TUNEL and immunofluorescence staining
For immunofluorescence, the brain sections were blocked with 10% goat serum and/or 0.3% Triton X-100 in 0.01 mol/L PBS for 40 min at 37°C, followed by incubation with primary antibodies for MBP (1:1000, rabbit IgG, Abcam, Cambridge, UK) and platelet-derived growth factor receptor α (PDGFR-α, 1:1000, rabbit IgG, Abcam, Cambridge, UK). Claudin-5 (1:1000, rabbit IgG, Sigma), CD133 (rabbit IgG, 1:100, Abcam, USA), human CD31 (1:100, rabbit IgG, Sino Biology) and vWF (1:200, mouse IgG, Abcam, USA). After washing with PBS, the sections were incubated with the goat anti-mouse IgG (H + L) Alexa Fluor ®488 or 555-conjugated or goat anti-rabbit IgG (H + L) Alexa Fluor ®488 or 555 (Invitrogen, Eugene, OR, USA) secondary antibodies for 1 h. The specificity of the staining was assessed by omitting the primary antibody. After washing with PBS, TUNEL reaction fluid was added to the sections incubated with anti-PDGFRa antibody for 1 h at 37°C. Finally, the sections were incubated with DAPI for 10 min and mounted.
The number of CD133-positive cells was counted in the corpus callosum in three sections per rat from the same levels, at every 9–12th section between bregma levels +1.0 and -0.2 mm. For quantitative analysis of the Claudin-5 or MBP immunofluorescence staining, integral optical density (IOD) were measured by Image-Pro Plus 6.0 software. Values (three slides for each brain) of optical density in individual cells represented the quantity of objective protein and were calculated using the following equation: Σ IOD/Σ DAPI. The apoptotic rate was calculated using the following formula: apoptosis rate = TUNEL/ PDGFRa double positive cells/total PDGFRa positive cells per field ×100%.
Electron microscopy.
For electron microscopic examination, Epon embedding was performed as previously described [23]. In brief, the rats were transcardially perfused with 2% glutaraldehyde (Gla) and 2.5% PFA in 0.1 mol/L PBS. Brains were quickly removed and placed on ice. The corpus callosum was dissected and placed in 3% Gla in 0.1 M cacodylate buffer (pH 7.4) at 4 °C overnight and transferred to 1% osmium tetroxide in the same buffer for 1 h at room temperature. Tissue was transversely cut into 1 mm blocks that were fixed in osmium tetroxide at 4 °C overnight, dehydrated through ascending ethanol washes, and embedded in epoxy resin. To study the remyelinated axons of the corpus callosum, serial 1 μm-thick semi-thin sections were cut with a diamond knife, stained with 1% toluidine blue, and examined by light microscopy. To analyze myelin sheaths in the corpus callosum, 60–70 nm-thick ultra-thin sections were stained with uranyl acetate and lead citrate prior to examination by tEM (FEI Tecnai G2 T12, USA), and the image was analyzed by TEM Imaging and Analysis, TIA. For morphometric analysis, the axonal diameter (d) as the shortest distance across the center of the axon was measured. The axonal diameter plus the total myelin sheath thickness on both sides was defined as the fiber diameter (D). The g-ratio was calculated using the d/D ratio. The mean of the average g-ratios from the three brain samples was determined for each group.
Statistical analysis
All statistical analyses were conducted by observers blinded to the treatment. All data shown represent means ± SEM. from triplicate experiments performed in a parallel manner. Statistical analysis was performed with GraphPad Prism® software. Group differences in the MWM test were analyzed using two-way analysis of variance (ANOVA). The other data were evaluated by one-way ANOVA and Student- Newman-Keuls (SNK) tests with homogeneity of variance or by Dunnett’s post hoc test with square differences. P < 0.05 was considered to be statistically significant.