Animals
In this study, 8-10 weeks old female Sprague-Dawley (SD) rats (200-250g) were selected. The living conditions, surgical methods and postoperative care of all animals were approved by the animal experimental Committee of the First Affiliated Hospital of Harbin Medical University.
Cell isolation, culturing and labeling
Partially purified stem cell–enriched BMSCs were prepared as previously described [28]. Briefly, BMSCs were obtained from the femurs and tibias of 4-week-old female SD rats (Harbin, Heilongjiang, China) and fractionated by density centrifugation using Lymphoprep! density solution (density: 1.077; Nycomed Pharma, Oslo, Norway). After centrifugation, whole mononuclear cells were collected from the interface and extruded with 10mL of DMEM/F12. The remaining cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (Gibco BRL), 3mML-glutamine, 100U=mL penicillin, 100U=mL streptomycin sulfate, and incubated at 37℃ with 95% humidity and 5% CO2. After 48h of incubation, the nonadherent cells were removed by changing the medium. The BMSCs were passed four times before being used in the following experiments.
Before transplanting BMSCs were labeled with the red fluorescent cell tracking dye PKH26 (Sigma-Aldrich) following the manufacturer's instructions. The efficiency of cellular labeling (routinely 100%) was examined with a fluorescence microscope.
Spinal cord injury modle
The rats were anesthetized by an intraperitoneal injection with 5% chloral hydrate (30mg/kg). After adequate amounts of anesthetized, the rats were fixed on the sterile table. Using aseptic techniques, a midline incision was made in the back in the skin and musculature to expose the T9-T11 vertebrae. Then a complete laminectomy was performed on the 10th thoracic spinal cord level exposing the cord using a surgical microscope. After laminectomy, the spinal cord was compressed by placing a 50 g weight on the exposed spinal cord column for 5 min using a rectangular plate which was longitudinally oriented over the spinal cord. The plate had an area of 11.0 mm2 (2.2×5.0 mm) and a concave shape that ensured equal distribution of the pressure on the spinal cord tissue. It was considered that the model was successful to see the spastic tail swing of rats, paralysis of upper and lower extremities after retraction and flapping of both legs and body. The wound site was rinsed with sterile phosphate-buffered saline (PBS) with 0.1% gentamicin, the muscles were sutured in layers, and the skin was closed. During the whole process, the body temperature of the rats were maintained until they woke up. After the operation, the rats were assisted to urinate 3-4 times a day until the function of urination recovered. After the operation, penicillin was injected intraperitoneally for 100 mg/kg/D × 3 D to prevent infection.
Experimental groups and intraperitoneal administration
Eighty rats were divided into four groups: (i) SCI group; (ii) AC-YVAD-CMK treatment group: intraperitoneal injection of AC-YVAD-CMK dissolved by DMSO (10mg/kg) immediately after injury, three times every other day; (iii) BMSCs treatment group: immediately after injury, 1×105 BMSCs were injected into 3ul medium with microinjector to the epicenter of spinal cord contusion, and single dose DMSO with equal volume was injected into abdominal cavity; (iiii) Combined treatment group (AC-YVAD-CMK + BMSCs): immediately after injury, inject AC-YVAD-CMK solution and BMSCs into corresponding parts.
Western blot
Immediately after the rats (n=4 for each group) were deeply anesthetized and transcardially perfused with 0.9% ice-cold saline, the spinal cord tissues around the epicenter of the lesion (10 mm in total length) were carefully isolated at the end of first week. The spinal cord tissues were digested with RIPA lysate containing PMSF and 1% protease inhibitor for 30 min and the protein concentration was determined using the BCA method and stored at −80℃. Different samples with an equal amount of protein were separated using 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocked with 5% skim milk at room temperature for 1h, the members were incubated with primary antibodies against pre-caspase-1 (1:200), active-caspase-1 (1:200), IL-1β (1:100), IL-18 (1:100), and β-actin (1:1000) at 4℃ overnight. After 3 washes with TBST (Tris-buffered saline and Tween 20, PH 7.5), the membranes were appropriately incubated with secondary antibodies (HRP, 1 : 1500 dilutions) at room temperature for 2h. The images were captured by means of the Odyssey Infrared Imaging System (LI-COR, Biosciences) and the band intensities were measured with Odyssey v1.2 software (LI-COR, Biosciences).
BDNF and NT-3 assay
In the present study, we also detected the levels of brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) at the end of the first and eighth week after operation. The rats (n=4 for each group per time point) were anesthetized with 1% sodium pentobarbital (40 mg/kg) and transcardially perfused with 200 mL of ice-cold 0.1M PBS, pH 7.2. A 5-mm long segment of spinal cord containing the lesion site and substitutes was excised on dry ice. The segment was weighed and then mechanically homogenized in ice-cold 0.1M PBS. Homogenates were centrifuged for 10 min at 14,000 rpm at 4℃, and the supernatant fluid was detected with a BDNF kit (R&D Systems, Minneapolis, MN) and an NT-3 ELISA kit (Abnova, Taipei, Taiwan), according to the manufacturer’s instructions. The samples were always analyzed in triplicate. Histological analysis The animals were given an overdose of Nembutal (80 mg/kg, i.p.) and were transcardially exsanguinated with 150 mL of physiological saline followed by fixation with ice-cold 4% paraformaldehyde in 0.01M PBS (pH 7.4). Spinal cords were dissected and postfixed and cryoprotected.
Immunohistochemistry
The rats (n=6 for each group) were transcardially perfused with 100ml 0.9% ice-cold saline and 100ml 4% paraformaldehyde under deep anesthesia at the end of the first and eighth week after operation. The spinal cords were harvested and fixed with 4% paraformaldehyde for 24h, then in 10% and 30% sucrose until saturation successively and embedded them in OCT compound and sectioned them frontally at 20 mm on a cryostat. For immunofluorescence staining, the sections were blocked with 10% normal goat serum in PBST (0.1%) for 1h after pushed with PBST for 5 min × 3 times and incubated overnight at 4°C with the primary antibodies: ployclonal anti-mouse antineurofilament protein-200 (NF-200, 1:100) to identify axons. After several pushed with PBST, the sections were incubated with FITC antibody in the darkroom. The images were acquired using inverted fluorescence microscope and confocal microscopy.
Functional assessment and electrophysiological analysis
At 1-day postoperation (1d) and weekly thereafter, the rats were functionally monitored using the well-characterized Basso–Beattie–Bresnahan (BBB) locomotor rating scale, which is graded from 0 (absence of performance) to 21 (completely normal gait performance).24 rats were placed on a 2 × 3 m2 open field and observed for 5 mins by two observers who were blinded to the experimental grouping. They individually scored every rat at the same time, and the ultimate scores of the rats were averaged. The animals survived and exhibited no signs of autophagia throughout the course of the experiment.
Motor-evoked potentials (MEPs, n=6 for each group) were measured at the end of the first day and the eighth week after operation, as described in our previous work.26 Interelectrode impedances were maintained below 3.5–5 kX. The bandpass filter was 10–3000 Hz. Evoked responses were displayed on a Cadwell Excel monitor (S-100, Medtronic, Dantec Company, Denmark).
Quantitative and statistical analysis
For quantification, all photographs were taken using a confocal microscope (LSM 510 Meta, Zeiss, Germany). Quantitative analyses of NF-200-positive fibers, equivalent regions were analyzed for each subject (T10 level). Throughout every eighth section, the length of the excised segment of rat spinal cord was analyzed. Ten areas were chosen randomly from all the sections containing NF-200-labeled axons. Image analysis was performed with Image-Pro Plus software (Version 5.0; Media Cybernetics, The Imaging Expert, Silver Spring, MD). The percentage of each area containing NF-200-immunoreactive axons was measured, and the values in all the sections were averaged. The NF-200 immunoreactivity of each section at T10 level was expressed as a value relative to ones at T9 (taken as 100% of NF-200-labeled axons for each animal) in the experimental groups.
All the data from this study were presented as means with standard deviation (SD). All statistical analyses were performed using SPSS 18.0 (SPSS, Chicago, IL). BBB scores were analyzed using two-way repeated-measures analysis of variance, which showed an overall significant effect of treatment. The other data were compared between groups with one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post hoc q test. A value < 0.05 was considered to be statistically signific.