CCR2-/- mice (B6.129S4-Ccr2tm1Ifc) and wild type C57BL/6 mice were purchased from Jackson Laboratory (Bar harbor, ME) and mice were housed in pathogen-free Animal Experiment Center of Xi’an Jiaotong University’s college of medicine. All experimental procedures were approved by Xi’an Jiaotong University’s Animals Care and Use Committee (ACUC) and performed in accordance with the guide for the care and use of laboratory animals.
Reagents and Antibodies
Dulbecco’s Modified Eagle’s Medium, DMEM/F12 (1:1) and B-27supplement were purchased from Invitrogen (Carlsbad, CA, USA). Basic fibroblast growth factor (bFGF), interferon-γ (IFN-γ), epithelial growth factor (EGF) and anti-TUJ1 antibody were obtained from Millipore (Billerica, MA, USA). Hybridoma cell lines of Mac-2 and F4/80 were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). Anti-MCP-1 antibody, anti-nestin antibody and anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Abcam (Cambridge, MA, USA), and anti-iNOS antibody from BD Biosciences（San Jose, CA, USA）. Antibodies of anti-GAPDH, phosphor-ERK1/2 and ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). All secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA).
Preparation of Mouse Bone Marrow-derived Macrophage
Mouse BMDMs were prepared from C57BL/6 wild type mice as previously described. Briefly, BMDMS were harvested from mice 6-8 weeks of age with 7days culture in DMEM supplemented with 5% newborn calf serum (NCS) (Rocky Mountain Biologicals), 15% conditioned medium from L929 cells (a source of macrophage colony-stimulating factor), and 1% penicillin/streptomyclin-solution (Corning, USA).
Induction of M1 Macrophage and Conditioned medium preparation
To promote polarization into M1 macrophages, BMDMs were treated with 10ng/ml interferon-γ (IFN-γ, Millipore, Billerica, MA, USA) for 24h[19, 20]. M0 macrophages weren’t treated with IFN-γ except for a change in the macrophage medium. After 24h, macrophages were washed three times with phosphate-buffered saline, changed with fresh medium including 2% B-27 supplement (Gibco) and then cultured for 24h. Conditioned medium (CM) from M0 macrophages and M1 macrophages were prepared by centrifuging collected medium at 2500RPM for 19min and filtering through a 0.4-μm filter (Corning). While, culture medium (without BMDMs) was used as control medium in this study.
NSCs Isolation and Culture
Primary NSCs were isolated and cultured from fetal wild type C57BL/6 mice and CCR2-/- mice (B6.129S4-Ccr2tm1Ifc) cortices at embryonic day 14, as previously described. In brief, cerebral cortex was collected and mechanically dissociated into single cell. Then cells were seeded in DMEM/F12 supplemented wiht 20ng/ml EGF, 10ng/ml bFGF, 2% B-27 supplement and 1% penicillin/streptomycin, and incubated at 37°C on a humidified atmosphere containing 5% CO2.
Transwell Migration Assay of NSCs
Migration of NSCs were conducted by using the transwell chamber with a pore size of 8μm (Neuro probe) as described previously. After 8-μm pore membrane coated with 0.01% poly-l-lysine, 20μl of 5×106/ml NSCs were added to upper wells of the transwell chamber. DMEM with 2% B-27 supplement (control medium), conditioned medium from M0 macrophages(M0-CM), conditioned medium from M1 macrophages (M1-CM) and other reagents dissolved in DMEM with 2% B-27 supplement were located at the lower wells of the chamber. After 12h, the number of cells, that migrated through the filters were fixed and subjected to Giemsa staining, were analyzed by counting three independent field of each condition.
MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay was performed to identify whether M1-CM influences the proliferation of NSCs. After treated with M1-CM, M0-CM or control medium for 12h, cells were incubated with MMT solution (5 mg/ml, Sigma-Aldrich, St.Louis, MO, USA) for 5h, and then added 200μl of DMSO to dissolve crystals. The absorbance was measured at 550nm using a microplate reader (Bio-Rad, Hercules, CA, USA) and relative proliferation rates were observed by comparing the optical density value of experimental samples to that of control sample.
Enzyme-linked immunosorbent assay (ELISA)
Quantitative measurement of MCP-1 was analyzed and determined using MCP1 ELISA kit (Abcam, Cambridge, MA, USA) following the manufacturer’s instruction. The absorbance was measured at 450nm and MCP-1 concentration was observed by comparing absorbance to standard curves.
After treated with M1-CM, NSCs were washed with ice-cold PBS and lysed in RIPA buffer containing a phosphatase inhibitor and proteinase inhibitor cocktail (Amresco, Solon, OH, USA). Western blot assay was carried out following standard procedure. Briefly, total NSCs proteins were adjusted to equal protein concentrations, loaded onto SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes. After blocking in 5% milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at RT, membranes were incubated with primary antibodies solution overnight at 4°C. Membranes were then placed in appropriate secondary antibody for 1 h at room temperature after rinsing in TBST. Blots were visualized with the ECL plus western blot detection system (GE Healthcare, UK).
The distribution and expression of F-actin was performed using CF488-conjugated phalloidin (89427-132, Biotium, Fremont, CA, USA) according to the manufacturer’s protocol. Briefly, NSCs were treated with control medium and M1-CM, then washed three times with PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature. Following by permeabilization with 0.5% Triton X-100 in PBS for 10 min at room temperature, cells was washed and incubated with phalloidin and DAPI at room temperature for 20 min. Finally, the coverslips were washed, mounted and viewed under EVOS fluorescent microscope (Life technologies, Carisbad, CA USA).
SCI Model and NSCs Transplantation
Contusion SCI was induced using a NYU impactor. Adult female C57BL/6 mice (20±2.5g in body weight) were anesthetized with 100mg/kg ketamine and 10mg/kg xylazine through intraperitoneal injection. The tenth thoracic (T10) spinal cord segment was exposed and a contusion injury of the T10 vertebral cord was induced by dropping a 5-g rod 6.25mm onto the spinal cord. Postoperative care such as manual bladder emptying was performed as previously described.
Then, NSCs were transplanted into mice with SCI to examine their migration in vivo. In brief, CCR2-/- NSCs and WT NSCs were pre-labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE, Sigma-Aldrich, St.Louis, MO, USA) according to manufacture’s protocol before cells transplantation. 7days after SCI, a total volume of 0.5μl of cell suspension containing 5×104 NSCs was microinjected at either side of the spinal cord contusion 2mm away using a Hamilton syringe.
Real Time PCR
Mice were sacrificed and spinal cords were immediately extracted 7days after SCI (n=5 each group). Total RNA was isolated from the indicated tissues via TRI reagent (Sigma-Aldrich, St.Louis, MO, USA) and then reverse-transcribed into cDNA using Fist strand cDNA Synthesis kits (Roche, Basel, Switzerland). The following primer sets were as following: MCP-1 (5'-ATGCAGGTCCCTGTCATGCTT-3' and 5'-CATTGGGATCATCTTGCTGGT-3') and GAPDH (5'- ATCAACGACCCCTTCATTGACC-3' and 5'- CCAGTAGACTCCACGACATACTCAGC-3'). We assayed the expression of target gene mRNA by using the ABI 7900HT Fast real-time PCR detection system (Applied Biosystems, UK) and expression was normalized to GAPDH for each sample.
The spinal cord was fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4°C, incubated with 30% sucrose solution at 4°C until tissue sunk, and then frozen in OCT compound. 8um-thick parasagittal sections were prepared by using a cryostat microtome and attached to poly-l-lysine-coated glass slides. For immunofluorescence staining, samples were blocked with 1% BSA in PBS containing 0.3% Triton X-100 at room temperature for 1h, and incubated with primary antibodies at 4°C for 12 h. After washed with PBS, the sections were incubated with secondary antibodies at room temperature for 1h. All the procedures for negative controls were performed in the same manner, just except incubation of primary antibodies. EVOS fluorescent microscope (Life technologies, Carisbad, CA USA) was used to observe and capture the images.
The results were reported as mean± standard deviation (SD). All statistical data was analyzed using SPSS 22.0 software program (StatSoft, Tulsa, OK). The statistical significance was evaluated using Student’s unpaired t-test and Statistical significance was assumed if P<0.05.