Animals
GFP transgenic rats (Japan SLC, Inc.; Hamamatsu, Japan) and Sprague-Dawley (SD) wild-type rats, genetically identical to each other except for their transgenes, were used. All experimental procedures employed in this study have been approved by Institutional Animal Care and Use Committees of Shenzhen University.
Culturing of mesenchymal stem cells
Bone marrow-derived MSCs were harvested from GFP transgenic rats according to the procedure described previously [21]. Briefly, after the sacrifice of the animal, the femur and tibia were removed and flushed out with phosphate-buffered saline (PBS). The marrow was collected and spun down, the supernatant discarded, and the resulting cell pellet suspended in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) with 10% fetal bovine serum (Biosera, UK) and antibiotics (100IU/ml penicillin G and 100 μg/ml streptomycin), namely MSC culture medium, and transferred to tissue culture flasks (Costar, USA) at densities ranging from 1× 105/cm2 to 1× 106/cm2. Cultures were incubated at 37oC in 5% CO2. The medium was changed after 48 h and then every 2 to 3 days. Primary cultures were maintained for 7 to 10 days, during which time the non-adherent hematopoietic cell fraction was depleted. After the cells had grown to near confluence, they were passaged 2 to 3 times by digestion with 0.25% trypsin and 0.02% EDTA.
Lentiviral vectors and MSC transfection
The lentiviruses were created using the ViraPower™ Lentiviral Expression System (Invitrogen, Paisley, UK). The coding sequence of luciferase was subcloned into pLenti6/V5-D-TOPO (Invitrogen). pLenti6/V5-DTOPO/luciferase vector and the ViraPower™ Packaging Mix (Invitrogen) were co-transfected using a gene carrier kit (Epoch-Biolabs, Missouri City, TX, USA) into the 293T cell line to produce a lentiviral stock. Forty-eight hours post-transfection, virus-containing supernatant was harvested by collecting the medium. Viral particles were purified by ultracentrifugation through a 20% sucrose cushion. For infecting MSC, cells were cultured in 24-well plates, and when the culture reached 80% confluence, the concentrated lentivirus was added to the culture dishes. After incubation for 48 h, the medium was replaced with selection medium containing 10 μg/ml blasticidin. The selection medium was replaced every 2 days until antibiotic-resistant colonies were identified, and thus a stable cell line of MSC-GFP-Luci was established.
In vitro firefly luciferase assays
Cells were dislodged from culture flasks to be resuspended in PBS. Cell suspensions were divided into a 96-well plate in known concentrations. After administration of D-Luciferin (4.5 μg/ml, Goldbio, USA), peak signal expressed as photons per second per centimeter square per steridian (photons/s/cm2/sr) was measured using a charged coupled device camera (IVIS100, Xenogen, Alameda, CA, USA). All samples were conducted in triplets.
Spinal cord injury (SCI) and postoperative care
Eighty-three male SD rats (240 to 260 g) were used for visualizing the location of transplanted MSCs-GFP-Luci on the whole spinal cord. The lesion induction and cellular transplantation were standardized. Under anesthesia with pentobarbital sodium (50 mg/kg, ip), contusion injury was performed at the mid-thoracic (T8-T9) level of the spinal cord. A standard spinal cord contusion was made using a weight-drop device. A metal rod 8 g in weight and 2.0 mm in diameter was dropped from a height of 10 mm onto the exposed spinal cord for contusion injury. Sixty-nine rats were used for MSCs-GFP-Luci transplantation, and the remaining 14 rats served as controls. Sixty-nine rats for transplantation were divided into three groups: 23 rats were treated with MSCs, 23 rats with the injection of SDF-1 followed by transplantation of MSCs, and the other 23 rats with AMD3100-pretreated MSCs. The control rats were treated with PBS. 3 rats of each experimental group were performed in vivo fluorescence imaging at time points of 1, 3, 5, 7, 11, and 14 days after cell injection, 14 rats were performed RT-PCR and western blotting, while the other 6 rats were respectively undergone transcardiac perfusion with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer.
For the rats pretreated with SDF-1, a Hamilton syringe needle was inserted into the spinal cord to a depth of 2 mm from the surface of the dura mater at a point 3 mm rostral to the injury site, and 5 μl of SDF-1 (500ng/ml) were introduced into the spinal cord over 5 min with an automatic microinjection pump (Muromachi Kikai Co., Tokyo, Japan).
Animals received continuous attention after surgery. Weight control was performed daily. Bladder expression was performed per day until reaching a maximum of 2 ml of urine in the morning expression, or the animal recovered its bladder function. Urine volume, PH, and blood presence were also monitored. Urinary tract infections were treated with the suspension of cefazolin and administration of 12 mg/kg gentamicin for 1 week. Ascorbic acid (60 mg/ml; Merk, Germany) was administered to prevent bacterial growth. Animals were examined daily for signs of autophagia, and treated with acetaminophen 64 mg/kg (baby Tylenol, McNeil, USA) for 1 week to stop self-mutilation.
Cerebrospinal fluid (CSF) collection
For rats, CSF was collected before and 24, 48, 72, 96, and 120 h after SCI. Prior to CSF collection, the fur on the neck region of the rat was removed and anesthetized with 5% halothane. A needle connected to a syringe was inserted horizontally and centrally into the cisterna magna for CSF collection without making any incision at this region. A gentle aspiration will make the CSF flow through the needle. The colorless CSF sample is slowly drawn into the syringe, and the color of the CSF was carefully observed to avoid any possible blood contamination. The non-contaminated sample was drawn into the syringe.
SDF-1 in CSF was measured by enzyme-linked immunosorbent assay (ELISA) with commercial kits (R&D System, USA) and western blotting. ELISA was carried out according to the manufacturer's recommendations.
For western blotting, the collected CSF or tissue sample was lysed with lysing buffer (Sigma-Aldrich). Protein concentration was measured using the Bradford protein assay. Proteins were denatured by boiling for 3 min in the presence of β-mercaptoethanol. Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, USA). Membranes were blocked in TBST containing 5% nonfat milk at room temperature for 2 h and probed with the rabbit anti-rat polyclonal primary antibody directed against SDF-1 (Santa Cruz, CA) overnight at 4˚C. Then the membranes were blotted with horseradish peroxidase (HRP)-linked mouse anti-rabbit IgG (Bio-Rad, Hercules, CA). β-actin was shown as a control. Proteins were visualized by using Kodak film (Kodak, Rochester, USA)
Cell transplantation and in vivo bioluminescence imaging (BLI)
The experimental rats were randomly divided depending on their treatment after thoracic SCI. For transplantation group, MSCs-GFP-Luci were trypsinized with 0.25 M EDTA and 0.05% trypsin (Invitrogen) on the day of transplantation and resuspended in PBS at 1×10 7 cells/ml; 1×10 6 of cells were injected into the tail vein of SCI-rats. The animals were examined at different time points after the injection using the imaging system (IVIS100, Xenogen). For each time, D-luciferin was dissolved in PBS and given to each rat by intraperitoneal injection at a dose of 150 mg/kg. Rats were imaged 5 min later using a 20-cm field of view and an exposure time of 3 min (3 min exposure; f-stop, 1; binning, 16; the field of view, 15 cm). Bioluminescence values were calculated by measuring photons/s/cm2/sr in the region of interest (ROI). For the control group, the rats received an injection of PBS, the amount of which was similar with that of the transplanted MSC group.
Histological Analysis
Histological evaluation was performed at the time points of the different groups in the experiment. The animals were killed at 1, 3, 7, 14, and 21 days after transplantation surgery. The T8-10 portion of the spinal cord was removed from the vertebral column and was then immersed for 24 h in the fixative containing 4% paraformaldehyde. The tissue was embedded in paraffin and sectioned sagittally (6 μm thickness). All sections were stained with hematoxylin-eosin (HE) for general histology. For assessment of the distribution of transplanted MSCs-GFP-Luci, the fluorescence emitted by GFP was directly observed by fluorescence microscopy (BX51; Olympus Optical, Tokyo, Japan).
Transwell migration assay
The migratory ability of MSCs-GFP-Luci was determined using transwell plates (Corning Costar, Cambridge, MA) that were 6.5mm in diameter with 8 μm pore filters. In brief, cells were suspended in serum-free medium and seeded into the upper well, and different concentrations of SDF-1-containing medium or different concentrations of CSF-containing medium were placed in the lower well of a transwell plate. Following incubation for 4 h at 37oC, cells that had not migrated from the upper side of the filter were scraped off with a cotton swab, and those in the lower surface were fixed with 95% ethanol, stained with hematoxylin. The number of cells that had migrated to the lower side of the filter was counted under a light microscope at 200 magnification in five randomly-selected fields. Each experiment was performed in triplicate. Where indicated, cells were incubated with AMD3100 blocking against CXCR4 or the antibody against SDF-1 (Sigma). Serum-free medium was used as an experimental control.
RT-PCR
Total RNA was extracted with Trizol reagent (Invitrogen). Equal amounts of mRNA were subjected to RT-PCR analysis using a SuperScriptTM One-step RT-PCR kit (Invitrogen) according to the manufacturer's instructions. The endogenous gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also quantified to normalize differences in the added RNA and efficiency of reverse transcription. Following oligonucleotide primers were used: SDF-1 (AF189724), forward 5′-CCCTGCCGATTCTTTGAG-3′, reverse 5′-GTCCTTTGGGCTGTTGTG-3′; GADPH (NM002406), forward 5'-CCA CAGTCCATGCCATCACTG-3', reverse 5'-CGCTGTTGAAGTCAGAGGAGA-3’; CXCR4 (AF452185), forward 5'-GGCAATGGGTTGGTAATC-3', reverse 5'-GACAATGGCAAGGTAGCG-3'. 1 μg of total RNA was reverse transcribed to synthesize cDNA at 50˚C for 30 min, and then the cDNA was subjected to PCR amplification with specific primers in 25μl mixtures. The amplification conditions were 30 cycles at 94˚C for 30 sec, 56˚C for 30 sec, and 72˚C for 45 sec in each cycle using an MJ PCR System. The PCR products were electrophoresed in a 2% agarose gel. All results represented the average density of positive bands obtained from 3 independent experiments.
Behavioral testing
The rats were tested behaviorally to evaluate the effect of MSCs-GFP-Luci on the recovery of motor function of the injured rats. Behavioral testing was performed for each limb using the Basso-Beatie-Bresnahan (BBB) locomotor rating scale at different time points (before the injury and on day 1, 7, 14, 21, 28 post-injury). For BBB assessment, rats were placed in an open field. Hindlimb motor function was scored simultaneously by two independent observers who were blind to the transplantation status of the animals. A 0 score represents no locomotion, and a 21 score represents a normal motor function. The scores for the left and right leg were averaged to generate the actual score for each trial.
Statistical analysis
All experiments were repeated at least three times unless otherwise indicated. Data are presented as mean±SD. Statistical analysis involved the use of the Student t-test. A P<0.05 was considered significant.