Animals and experimental groups
Adult Sprague-Dawley (SD) rats (4–6 weeks old, 24 males and 24 females) were used for model of SCI. Ten 1-week-old SD rats were used for BMSCs preparation. All rats were obtained from Mudanjiang Medical University Experimental Animal Center, and maintained under specific-pathogen-free conditions at 22 ± 1 °C and 45 ± 5% humidity with ad libitum access to food and water. Animal care and all experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 85 − 23, revised 1996), and approved by the Animal Care and Use Committee of Mudanjiang Medical University. All efforts were made to minimize the number of animals used and their suffering.
Isolation, culture, and identification of BMSCs
Isolation and culture of BMSCs were performed as previously described . Briefly, ten 1-week-old SD rats were sacrificed by cervical dislocation after ether inhalation. Under sterile conditions, tibias and femurs were collected, and the ends of bones were excised after the adherent soft tissue was removed. Then, the bone marrow was flushed into a culture dish using Dulbecco’s modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA). The fluid was disaggregated by gentle pipetting several times, and filtered through a 200 mesh screen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). After centrifuging, the supernatant was discarded, and cell pellets were resuspended in DMEM supplemented with 10% fetal bovine serum, 1% glutamine and 1% penicillin/streptomycin at a density of 1 × 106 cells/mL. Cells were cultured in plastic dishes at 37 °C in humidified atmosphere under 5% CO2, and the culture medium was renewed every 3 days. Cell morphology was observed using inverted light microscope (DM2000, Leica Microsystems, Wetzlar, Germany). To identify the cell phenotype, the molecular markers of BMSCs including CD90, CD29 and CD34 (Proteintech, Chicago, IL, USA) were detected by immunofluorescence staining. Cells used in the experiments were after three passages.
NRG1-overexpressing BMSCs generation
The plasmid pcDNA3.1(+)-NRG1-IRES/eGFP expressing the GFP-tagged NRG1 gene was synthesized by Cyagen Biosciences Inc. (Guangzhou, China). The BMSCs were seeded into six-well plates and divided into 3 groups: BMSCs, empty-vector BMSCs and NRG1-BMSCs. In the empty-vector BMSCs and NRG1-BMSCs groups, a mixture consisting of 94µL DMEM, 6µL FuGen6 reagent (Roche, Inc. USA) and 3µL empty pcDNA3.1(+) plasmid or pcDNA3.1(+)-NRG1 (1 µg/µL) were added respectively. In the BMSCs group, 94µL DMEM and 6µL FuGen6 reagent were added. After 6 h of culture, the medium was replaced with a complete culture medium. To obtain stable transfectants, BMSCs were cultured in selection medium containing G418 (200 µg/mL; Sigma-Aldrich, St. Louis, MO), and the levels of NRG1 were examined by ELISA as described below.
NRG1 levels in culture medium and cell lysates were measured by using ELISA Kit (USCNLIFE, Wuhan, China) according to the manufacturer’s instructions. Briefly, the culture medium supernatant and cell lysates of BMSCs, empty-vector BMSCs and NRG1-BMSCs were collected at 1, 2, 3, 4, 7, 14, 21 and 28 day, and transferred to 96-well plates (Corning, New York, USA). After incubation for 2 h at 37 °C, the plates were then incubated with the biotin-conjugated primary detection antibody followed by the horseradish peroxidase-conjugated secondary antibody for 1 h at 37 °C. After addition of the chromogenic substrate, the plates were incubated for 30 min at 37 °C. The optical density (OD) was detected using an iMark microplate reader (Bio-Rad, Hercules, CA, USA).
Co-culture of BMSCs with oxygen-glucose deprivation PC12 cells
To mimic injured conditions of SCI, PC12 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) subjected to oxygen-glucose deprivation (OGD) were used as an in vitro model of SCI. Briefly, PC12 cells were seeded onto the bottom wells of transwell chambers (BD Biosciences, Mississauga, Ontario, Canada) at a density of 5 × 104 cells/well. Cultures were maintained in glucose-free DMEM (Invitrogen, Carlsbad, CA, USA), which were bubbled with N2 for 5 min prior to use. Then, the transwell chambers were placed into an incubator filled with anaerobic gas mixture (1% O2, 94% N2 and 5% CO2) at 37 °C for 2 h. After OGD treatment, the empty-vector BMSCs and NRG1-BMSCs were seeded respectively onto the upper inserts of transwell chambers (5 × 105 cells/well), and co-cultured with OGD PC12 cells in DMEM supplemented with 10% FBS for 72 h. The PC12 cells maintained in a normoxic condition (5% CO2 and 95% air) with regular DMEM were as the control cultures.
Neurite outgrowth assay
After co-culture for 72 h, PC12 cells were observed with an inverted phase contrast microscope (Olympus, Tokyo, Japan). Images of three fields per well were taken. Neurite length was measured manually tracing neurite by using Image ProPlus 6.0 imaging software (Media Cybernetics UK, Marlow, UK) and referenced to a known length. Cell processes were defined as neurites when they were longer than the diameter of cell body. A researcher blinded to the experimental protocol counted the number of neuritis for 10 independent cells per field with Image ProPlus 6.0 imaging software as previously studies described .
Cell apoptosis analysis
Cell apoptosis was detected with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL; Roche Boehringer-Mannheim, IN, USA) and Caspase-3 Activity Assay Kit (Beyotime Institute of Biotechnology Haimen, China), respectively. Briefly, PC12 cells were fixed in 4% paraformaldehyde, rinsed in PBS and then incubated for 60 min at 37 °C with TUNEL reaction mixture. The nuclei were counterstained with DAPI (Sigma, Saint Louis, MO, USA). The numbers of total PC12 cells and TUNEL positive cells was counted by an operator blind to the experimental protocol using Image ProPlus 6.0 imaging software. The apoptosis rate was defined as ratio of apoptotic cells to total cells. For assay of Caspase-3 activity, PC12 cells were harvested and rinsed with PBS, and the cells were lysed with lysis buffer. The lysate was collected, centrifuged, and incubated with the mixture composed of 50 µL of cell lysate, 40 µL reaction buffer and 10 µL of 2 mM Caspase-3 substrate (Ac-DEVD-pNA) in 96-well plates at 37 °C for 4 h, the absorbance of p-nitroanilide was measured at 405 nm by using a microplate reader. Caspase-3 activity was defined as a ratio of caspase-3 activation level to control level.
Cell viability assessment
Cell viability was assessed by using Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology Haimen, China) according to the manufacturer’s instructions. Briefly, PC12 cells from each group were seeded in 96-well plates (4 × 104 cells/well) with three replicates, and cultured for 12 h under humid conditions in 5% CO2 and 95% atmospheric air at 37 °C. After that, 10 µL of CCK-8 solution was added to each culture well and incubated at 37 °C for 4 h. The OD values were measured at 450 nm with a microplate reader. Cell viability was calculated for the OD value of the ODG, empty-vector BMSCs and NRG1-BMSCs groups as a percent of the OD value of the control group.
Spinal cord hemi-section injury and cell transplantation
Animals were subjected to spinal cord hemi-section injury as described previously . Briefly, rats were anesthetized using 2% pentobarbital (40 mg/kg), and an incision was made at the dorsal midline, followed by cutting the cutaneous tissue and separating the muscle. The laminectomy was performed at the T9 segment. After spinal cord had been exposed, a dorsal hemi-section was conducted using an iridectomy scissors, and removed any residual fibers at the lesion site. The tail was spastically swung and the posterior limb twitching rigidity, confirming that the hemi-section of the spinal cord was completed. Gelatin foam was used to stop bleeding and the muscular and skin was sutured in layers. Rats were divided into: SCI, empty-vector BMSCs, NRG1-BMSCs, and the control (laminectomy only) groups.
On the day of transplantation, empty-vector BMSCs and NRG1-BMSCs were digested with 0.25% trypsin containing 0.02% EDTA to generate cell suspension. At 7 days post-injury, rats in the SCI, empty-vector BMSCs and NRG1-BMSCs groups were re-anesthetized and the injury site was surgically re-exposed. Then, a total of three sites were injected and each site was injected with 3µL DMEM, empty-vector BMSCs or NRG1-BMSCs suspension (1 × 107 cells/mL), respectively. The detailed procedure for the injection was described below, Hamilton syringe was vertically penetrated into the lesion center and 3µL of cell suspension was injected. In addition, 3µL of cell suspension was injected at 1 mm cranial to and caudal to the lesion center and 0.6 mm laterally from midline at a depth of 1.3 mm. Cells were delivered under the control of a microsyringe pump controller (World Precision Instruments, Sarasota, FL, US) at a rate of 0.5 mL/min. To minimize cellular reflux, the needle was left in place for 3 min following the completion of injections.
Locomotion recovery assay
The Basso, Beattie and Bresnahan (BBB) locomotor rating scale  was used to evaluate rat motor functions at 0, 7, 14, 21, 28, 35, 42, 49 and 56 days after injury. Briefly, the rats were allowed to walk feely in an open field (80 × 130 × 30 cm) with a pasteboard-covered nonslippery floor. In each testing session, the rats were observed individually for 4 min by two trained observers. The hindlimb locomotion was scored from 0 (complete paralysis) to 21 points (normal locomotion). The means of scores were used for analysis. The inclined plane test was performed at 0, 7, 14, 21, 28, 35, 42, 49 and 56 days as the method described previously. In brief, rats were placed with their body axis perpendicular to an orientation of the plate covered with a rubber mat containing horizontal ridges spaced 3 mm apart, and evaluate the maximum vertical axis of the inclined plate. The maximum angle at which a rat maintained its position for 5 sec without falling was recorded for analysis. Footprint analysis was carried out as previously described . In short, the animal’s forepaws and hindpaws were brushed with red and blue nontoxic dye, respectively, and the rats were required to walk across a paper lined runway (3 feet long, 3 inches wide) to obtain an edible treat in a darkened box at the end, and the footprints were scanned for analysis.
At 56 days after SCI, rats from each group were anesthetized with 10% chloral hydrate (3.0 mL/kg, i.p.), transcardially perfused with heparinized saline, and followed with 4% paraformaldehyde. The spinal cord segments around the injured site (2.0 cm) were collected, fixed in 4% paraformaldehyde overnight, and then embedded in paraffin, followed by preparation of transverse sections (5 µm). The sections were incubated in 0.1% Cresyl violet Nissl staining solution (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Neuronal chromatolysis (defined as dispersion or loss of Nissl bodies) was detected, and the chromatolytic proportions (neurons versus total neurons) were calculated. The integrated optical density (OD) of Nissl bodies were measured using Image Pro Plus 6.0 imaging software by an observer blind to the groups, as previous study described .
The immunofluorescence staining was performed as previously described . Briefly, the spinal cord segments were collected, fixed in 4% paraformaldehyde, and then serial 10 µm transverse sections were cut using a cryostat microtome (Leica, Nussloch, Germany). The sections of the injury epicenter were selected and incubated with a blocking buffer (1% bovine serum albumin diluted in 0.1% PBS, with Triton X-100) for 2 h at 4 °C. The sections were incubated with growth-associated protein 43 (GAP43; 1:400, Cell signaling technology, Danvers, MA, USA) at 4 °C overnight. On the next day, the sections were incubated with the secondary antibody for 2 h at 37 °C. Cell nuclei were counterstained with DAPI solution. The sections were photographed using a fluorescence microscope (Olympus BX51TR, Olympus Corp., Tokyo, Japan), and the camera parameters were kept constant during imaging. For quantitative analysis of the GAP43 expression, integral fluorescence density was measured with Image-Pro Plus 6.0 software by an operator blind to the groups, as previous study described .
Western blot analysis
At 56 days after SCI, the spinal cord segments around the injured site were collected, and homogenized in RIPA buffer (Beyotime Institute of Biotechnology Haimen, China) with 1% Phenylmethylsulfonyl fluoride and protease inhibitor at 4 °C. After centrifugation at 12,000 rpm for 10 min, the supernatant was collected and quantified by BCA Protein Assay Kit. An equal amount of total protein (30 mg) was loaded and separated by sodium dodecyl sulfate polyacrylamide gel and transferred onto polyvinylidenefluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in TBS-T (10 mmol/L Tris, 150 mmol/L NaCl and 0.1% Tween 20, pH 7.5) for 2 h at 37 °C, and then the membranes were incubated at overnight with GAP43 (1:1000), Cleaved-caspase-3, Bcl2, Bax (1:1000; proteintech, Chicago, IL, USA) and β-actin (1:2000; Cell Signaling Technology). On the following day, the membranes were washed in TBS-T and incubated with the appropriate secondary antibodies for 2 h at 37 °C. The target protein bands were visualized using chemiluminescence luminol reagents (Millipore, Billerica, MA, USA). Blots were imaged by Molecular Image®ChemiDocTM XRS+ with Image LabTM Software (Bio-Rad, Hercules, CA, USA). Quantitative data were obtained by Image J 1.50b Gel Analyzer (National Institutes of Health, Washington, DC, USA). Western blot data were normalized relative to the density of the β-actin bands.
All data were presented as mean ± S.E.M. A multifactorial analysis of variance (ANOVA) for repeated measurement was employed for analyzing BBB scores and maximum angles of the inclined plane test. One-way ANOVA was employed for analyzing other data followed by LSD (equal variances assumed) or Dunnett's T3 (equal variances not assumed) post hoc test with SPSS 17.0 software package. Statistical significance was set at P < 0.05.