BMSCs were harvested from the femurs and tibias of three tree shrews as described in previous reports (Hofstetter et al., 2002; Fu et al., 2015). Briefly, the tree shrews were anesthetized intraperitoneally by 2% pentobarbital sodium (30 mg/kg), the femurs and tibias were then dissected in a sterile environment and rinsed with D-Hanks. Epiphyses of the femurs were removed, and the marrow was then extruded using a syringe filled with DMEM/F12 containing 10% fetal bovine serum (FBS, Gibco), and repeatedly beated into a single cell suspension with 5 ml DMEM/F12 containing 10% FBS and penicillin/streptomycin. After centrifuging (1000 r/min, 5 min) and re-suspending, the cells were plated in 75 cm² culture flasks at a density of 1 × 10⁶/ml in an incubator (37°C, 95% humidity, 5% CO₂). Twelve hours after incubation at 37°C and 5% CO₂, supernatant containing nonadherent cells were removed and fresh medium was added. Medium was changed twice a week, when cells were about (4–5) × 10⁶ cells/cm², they were passaged 2–4 times and the suspension cells were discarded. The adherent cells were continually cultivated. Therefore, we got the pure adherent BMSC for further study.

The morphology and proliferation of BMSC (passage four) were observed at day 3, day 5, day 9 and day 12 under an inverted phase contrast microscope (Leica Microsystems Wetzlar GmbH). Cell number (from five fields in each well and five wells of each day) was then calculated for BMSC proliferation evaluation using Image-Pro plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

In order to identify the cultured BMSC and detect the expression of NTFs, corresponding primary and secondary antibodies (Table 2) for immunofluorescent staining were used following a previously reported protocol (Liu et al., 2014). Negative control was incubated with phosphate buffer saline (PBS) to replace the primary antibody. Thereafter, DAPI was used to label the BMSC nucleus; the cells were photographed under the fluorescent microscope (Leica, Germany). Then, the positive rate (mean number of the positive cells/the number of DAPI) was calculated by three investigators blinded to the experimental information using Image-Pro plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

Western blot was used to confirm the true expression of neurotrophins, and detailed protocols were described in a previous report (Liu et al., 2014). Briefly, protein was extracted from BMSC samples, after the protein concentration was certained by BCA protein assay kit (Beyotime Institute), 60 μg samples were separated onto 4%–12% gradient gels and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were then blocked with 5% nonfat milk in TBST for 2 h at room temperature and incubated overnight at 4°C with related primary (Table 3) antibodies, which was followed by incubation with corresponding secondary antibodies (anti-mouse/anti-rabbit, Abcam, 1:5000) for 2 h at room temperature. β-actin was used as a loading control. Afterwards, the immunoblot was revealed with an ECL Western Blot Detection Kit (Amersham Pharmacia Biotech, Buckinghamshire, England). Densitometry analysis was performed using ImageJ software.

All tree shrews were anesthetized intraperitoneally by 2% pentobarbital sodium (30 mg/kg) and placed in the prone position. A midline skin incision was made at the thoracic area (T8–T12), then paravertebral muscles and supraspinal ligaments were separated to expose the T10 spinal segment. Subsequently, the spinal cord at T10 was hemisected by microscissors to establish the tree shrew hSCI. Sham group underwent all the procedures except the T10 hemisection. Then, the surgical wounds were closed with a 3–0 silk suture and the tree shrews were injected intraperitoneally with 5% cefotaxime sodium salt (diluted 10× with normal saline) (0.5 μl per tree shrew) once a day for 7 days. Their bladders were manually massaged three times a day until recovery of micturition reflex.

According to the cell morphology and proliferation, the BMSCs at passage four were collected when they reached 90% of confluence at day 12 in culture and selected for transplantation. In addition, for tracing cells, these BMSCs were stained with Hoechst 33342 (Beyotime Biotechnology, China) for 5 min 2 h before transplantation. Cell morphology was directly observed under a fluorescent microscope (Leica, Germany), then the cells were counted and concentrated with a final concentration of 2.0 × 10⁴/μl.

At 9 days after hSCI (McDonald et al., 1999; Alexanian et al., 2011), each tree shrew was anesthetized as described above and placed in a stereotaxic frame, then the T10 spinal segment was exposed as previously described. Cell suspension was implanted into the spinal cord by the insertion of a pored glass pipette attached to a micro-injector carrying a capillary glass microelectrode (Thermo Scientific, Rockford, IL, USA). Six sites with 2 mm depth were selected at the following coordinates: two sites 5 mm rostral to the injured site, two sites 5 mm caudal to the injured site, one site 0.5 mm left and one site 0.5 mm right to the injured site. Each site was injected with approximately 2 μl cell suspension. Infusion was performed at a rate of 600 nl/min. After injection, the glass pipette was left for an additional 5 min before being slowly retracted. The control group underwent the same procedure using equivalent culture medium. In addition, cyclosporin-A was intraperitoneally (10 mg/kg per day) used from the third day before cell transplantation, and kept till the animals were sacrificed to suppress the immunoreaction. This could provide an environment for BMSCs survival in the host spinal cord. In order to exclude possible effects of immunosuppression on experimental outcome such as signal transducer and activator of transcription 3 (STAT3) mRNA levels, all the tree shrews in the sham, hSCI and hSCI + BMSCs groups received the same cyclosporin-A treatment.

At day 1–28 after hSCI, hindlimb motor function of the tree shrews in three groups was assessed with the open-field Basso, Beattie, and Bresnehan (BBB) scoring system, which was graded from 0 point (absence of any hind limb movement) to 21 points (normal mobility). In brief, subjects were acclimated to the open enclosure (99 cm in diameter, 23 cm deep) for 3 days prior to detection, 5 min per day. Then, each subject was observed for 4 min. The final score was the average of these three individual researchers (Basso et al., 1995).

At 28 days after injury, all tree shrews were sacrificed and spinal cords caudal to the injured site (10-mm-long containing injury and graft) were removed for later use. Spinal cords for immunohistochemical and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) observation were dehydrated by 30% sucrose overnight; then, they were sectioned at 10-μm thickness in a freezing microtome (Leica CM1900, Germany). For survival detection, the spinal cord tissue near the injured site was (1 cm below the lesion) harvested, and sectioned into10-μm thickness for later observation under the fluorescent microscope. In addition, the spinal cords caudal to the injured site from the other animals (10-mm-long containing injury and graft) for quantitative real-time PCR (qRT-PCR) were harvested and stored in 1.5 ml EP tube without RNase at −80°C until further required. The sample arrangement was showed in Table 1.

For observing the inflammatory reaction, immunofluorescent staining of interleukin-l β (IL-1β) was performed. In brief, the primary antibody of IL-1β (1:200, Rabbit, Santa), as well as the secondary antibody of Alexa 594 (goat anti-rabbit, 1:200, Zhongshan golden bridge) were applied progressively as previously described (Liu et al., 2014, 2015). Negative control was performed by omitting the primary antibody. Slides were then photographed using a fluorescent microscope (Leica, Germany). The mean density of IL-1β was presented as IOD (integrated optical density) over the total (mm²) area and quantified using Image-Pro plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA; Sun et al., 2013).

TUNEL assay was performed as described in one previous report (He et al., 2016). In brief, a TUNEL reaction mixture of enzyme solution and labeling solution (in situ Cell Death Detection Kit, TMR red; Cat.NO.12156792910) was added at a ratio of 1:9 (v/v). Then the slices were incubated at 4°C overnight in the dark and stained with DAPI for 5 min at room temperature followed by three washes with PBS. Subsequently, pictures were obtained under the fluorescent microscopy (Leica, CM1860, Germany). Finally, cell apoptosis was quantified by calculating the percentage of TUNEL/DAPI using Image-Pro plus 6.0 software.

Enzyme histochemistry was used to observe the glial scar formation (glial fibrillary acidic protein (GFAP) immunohistochemical staining) and motoneurons from lamina IX (Neuronal Nuclei (NeuN) immunohistochemical staining) in the spinal cord tissues. Following three washes in 0.01 mol/L PBS, the slices were incubated with 3% hydrogen peroxide at 37°C for 15 min to block the action of any endogenous peroxidase. Then the sections were incubated with 5% goat serum at 37°C for 30 min. Continually, the tissue sections were incubated overnight at 4°C with GFAP (1:100, rabbit, Bioss) and NeuN (1:100, rabbit, Bioss) primary antibodies respectively. PBS was served as the negative control. Afterwards, sections were separately incubated with PV-9000 reagent 1 (Zhongshan golden bridge) and PV-9000 reagent 2 (Zhongshan golden bridge) for 30 min at 37°C. Subsequently, DAB was incubated as chromogenic agent for 3–7 min in the dark and the sections were counter-stained with hematoxylin. Finally, the positive staining was visualized under an inverted phase contrast microscope imaging system (five sections/animal), the cell area and mean density of GFAP (IOD/area) were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) as above described.

At 28 days after injury, spinal cord caudal to the injured site (10-mm-long containing injury and graft) from BMSC, hSCI and sham groups were collected and homogenized to determine the level of mitogen-activated protein kinase kinase (Merk), phosphatidylinositol 3-kinase (PI3K), STAT3 and CNTF mRNA. Briefly, total RNA was isolated with Trizol reagent (Takara Bio Inc., Otsu, Japan) and was reverse transcribed to cDNA with the Revert Aid™ First Strand cDNA Synthesis kit (Takara Biotechnology, Co., Ltd., Dalian, China). Subsequently, qRT-PCR of cDNA was performed using the specific primers, which were synthesized by TaKaRa Company (Japan), the sequences were described in Table 4. β-actin was used as the internal control. PCR cycling conditions were as follows: initial denaturation at 95°C for 2 min, denaturation at 95°C for 15 s and amplification at 53°C for 20 s, followed by extension 60°C for 30 s for a total of 40 cycles. The threshold cycle (Ct) of each sample was recorded, and data were analyzed by normalization to β-actin values using the 2^−ΔΔCt method (Livak and Schmittgen, 2001; Liu et al., 2014).

Experimental results are expressed as the mean ± standard deviation (SD) and analyzed by one-way ANOVA using SPSS software (version 19.0, SPSS Inc., Chicago, IL, USA). The data had significant differences when p < 0.05.