Antibodies
Anti-MMP-9, anti-MMP-2, anti-Tubulin were purchased from Proteintech (Rosemont, IL, USA). Anti-VEGF, anti-Brdu, anti-Laminin and anti-β-Actin were purchased from Abcam (330 Cambridge Science Park, Cambridge, UK). Anti-p120-Catenin, anti-β-Catenin, anti-ZO-1, anti-Occludin, anti-Claudin-5 and anti-CD31 were purchased from Affinity (OH, USA). In situ cell death detection kit was purchased from Roche Molecular Biochemicals.
Animals
A total of 123 adult male Sprague-Dawley rats (200–250g) were purchased from Shanghai Laboratory Animal Center. The protocols were approved by the Animal Research Committee of Wenzhou Medical University. All animals were housed in a controlled environment and regularly fed with food and water. Rats were randomly divided into the following three groups: Sham (n=41; group S); SCI (n=41; group M); SCI + TT (n=41; group TM).
SCI Model
Rats were anaesthetized with 2% pentobarbital sodium (30 mg/kg), then were shaved and a 2 cm incision was made to expose the T10 segment of the spinal cord [26]. The exposed site was impacted with a New York University (NYU) Impactor (10 g × 20 cm) in all groups except group S. Lower limb trembling contractions and tail wagging showed that SCI was successful. Finally, the wound was sutured, disinfected with lodophor, and the rats allowed to recover from the anesthetic. During the following days, the bladder was emptied manually every morning and evening.
Water treadmill training
Our research group provided the initial design for the water treadmill (Wenzhou Xinglong Stainless Steel Co., LTD, Zhejiang, China) and submitted it for a patent. Rats were given adaptive training for three days before SCI. The surface of the water was adjusted to the xiphoid process of the sternum of the rat, the water temperature was set at 30 °C, and the speed of the water treadmill was maintained at 10-15 meters per minute. Training started one day after SCI, for rats in the training group(TM) began and lasted for 7 d or 14 d (5 min/round, 3 rounds in total, 5 min interval between rounds) (see Additional file 1).
Behavioral tests
Two independent examiners who were blinded to the treatment groups conducted the Basso-Beattie-Bresnahan (BBB) motor rating scale in an open field test. The BBB score has a total of 21 points, and the higher the score, the closer the animal is to normal[27].
Evaluation of BSCB permeability
Water content
At 7 d or 14 d after SCI, 2% sodium pentobarbital was intraperitoneally injected to anesthetize the animals (n=5), they were perfused cardiac perfusion with 0.9% normal saline and 0.5 cm of the T10 spinal cord segment was removed. The degree of edema in this segment was assessed by the dry and wet weight method as previously reported [28, 29].
Evans blue dye assays
According to previously reported methods [4, 8], rats (n=5) were injected with Evans blue (EB)dye to assess BSCB permeability (4 ml/kg) by tail vein at 7 d or 14 d after SCI, followed by 2% sodium pentobarbital anesthesia 2 hours later and 0.9% saline perfusion. Tissue containing T10 was soaked in N, N’-dimethylformamide at 50°C for 72 hours. The concentration of dye in the samples was determined based on a standard curve (μg/g). Tissues were cut into 15 μm thick sections with a freezing microtome at -20°C, and then the sections analysed. Quantitative analysis of data was performed with ImageJ software.
Haematoxylin-eosin (HE) Staining
Briefly, T9-T11 spinal cord tissue was removed from rats at 7 or 14 d after SCI, and stored in 4% paraformaldehyde for 24 hours (4°C). The spinal cord was immersed in a 0.1 m phosphate buffer solution and a 30% sucrose solution overnight (4°C). Successive sections (15 μm thick) were frozen and stored for subsequent HE staining.
Western Blot Analysis
Tissues containing T10 segments were put into a collection tube containing a mixture of phenylmethanesulfonyl fluoride (PMSF) and RIPA lysis buffer (100:1) and then were microfuged at 12,000 rpm for 5 min at 4°C. We extracted the supernatant and calculated the protein concentration with a BCA protein assay kit. The mixed solution was heated to 100°C for 10 min. After electrophoretic transfer to membranes, they were incubated with the appropriate primary (Anti-p120-Catenin, anti-β-Catenin, anti-ZO-1, anti-Occludin, anti-Claudin-5, anti-MMP-9, anti-MMP-2, anti-VEGF, anti-Tubulin, β-Actin) and secondary antibodies and the signal was digitally quantified.
Immunofluorescence Staining
After drying sections, they were washed 3 times for 15 minutes. They were treated with nonimmune goat serum for 1 hour and incubated first with primary antibodies against rabbit anti-occludin antibody (1:100), rabbit anti-claudin-5 antibody (1:100), rabbit anti-p120-Catenin (1:200), rabbit anti-β-Catenin (1:100), anti-CD31(1:100), anti-Brdu (1:100) and anti-Laminin (1:100) at 4°C, and then with Alexa Fluor 488 Affinipure goat anti-rabbit IgG (H+L) (1:200, Yeasen, China) for 50 min at room temperature. Phosphate-buffered saline (PBS) was used in place of the primary antibody in the negative control. We use an in situ Cell Death Detection Kit to detect apoptotic cells. The nuclei were colored by Hoechst or DAPI. The fluorescence signal was observed with laser confocal microscopy, five fields on each of three slides per animal were randomly selected for visualization and analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Transmission Electron Microscopy(TEM)
Tissue was quickly removed and cut into 1 mm3 pieces on ice and soaked in 2.5% glutaraldehyde. The tissue was fixed with a 1% oxidizing fixative for 1 h and stained with 1% uranyl acetate for 2 h, and then embedded after dehydration in gradient acetone solution. After semi-thin sectioning and toluidine blue staining, ultrathin sections were cut and observed using Hitachi TEM.
Statistical Analysis
All experimental data are expressed as the mean ± standard deviation. Kolmogorov-Smirnov (K-S) test was used for a normality test, p > 0.05 indicated a normal distribution; Levene’s test was used for a test of homogeneity of variance, p > 0.05 was considered homogeneity of variance, and vice versa. When comparing the two groups, a t-test was used. One-way ANOVA and Dunnett’s test were used to evaluate the data when comparing more than two groups of components. SPSS 16 statistical software for statistical analysis, and p < 0.05 was considered statistically significant.