Antibodies
Anti-MMP-9, anti-MMP-2, and anti-Tubulin antibodies were purchased from Proteintech (Rosemont, IL, USA). Anti-VEGF, anti-BrdU, anti-Laminin and anti-β-Actin antibodies 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 antibodies were purchased from Affinity (OH, USA). An 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 the Shanghai Laboratory Animal Center. The animal-related protocols had been approved by the Animal Research Committee of Wenzhou Medical University. All animals were housed in a controlled environment and regularly fed food and water. Rats were randomly divided into the following three groups: sham-operated (n=41; group S), SCI (n=41; group M), and SCI + TT (n=41; group TM).
SCI modelling
Rats were anaesthetized with 2% pentobarbital sodium (30 mg/kg) and shaved, after which 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 modeling had been successful. Finally, the wound was sutured and disinfected with lodophor, and the rats were allowed to recover from the anaesthetic. Over the following days, the bladder was emptied manually every morning and evening.
Water TT
Our research group provided the initial design for the water treadmill (Wenzhou Xinglong Stainless Steel Co., Ltd, Zhejiang, China) and submitted it for patenting. The rats were given adaptive training for three days before SCI. The surface of the water was adjusted to the xiphoid process of the rat sternum, the water temperature was set at 30 °C, and the speed of the water treadmill was maintained at 10-15 metres per minute. Training started one day after SCI. Rats in the training group(TM) began training that lasted for 7or 14 d (5 min/round, 3 rounds in total, 5-min interval between rounds) (see Additional file 1).
Behavioural tests
Two independent examiners who were blinded to the treatment groups conducted the Basso-Beattie-Bresnahan (BBB) motor functional scale test in an open field. The maximum total BBB score was 21 points, and the higher the score was, the closer the animal’s motor function was to normal [27].
Evaluation of BSCB permeability
Water content
At 7 or 14 d after SCI, 2% sodium pentobarbital was intraperitoneally injected to anaesthetize the animals (n=5), after which the rats underwent cardiac perfusion with 0.9% normal saline and 0.5 cm of the T10 spinal cord segment was removed. The degree of oedema in this segment was assessed by the dry and wet weight method as previously reported [28, 29].
Evans blue (EB) dye assay
According to previously reported methods [4, 8], rats (n=5) were injected with EB dye (4 ml/kg) by the tail vein at 7 or 14 d after SCI to assess BSCB permeability, followed by treatment with 2% sodium pentobarbital anaesthesia 2 hours later and 0.9% saline perfusion. Tissues containing T10 were soaked in N,N’-dimethylformamide at 50°C for 72 hours. The concentration of EB 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 analysed. Quantitative data analysis was performed with ImageJ software.
Haematoxylin-eosin (HE) staining
Briefly, T9-T11 spinal cord tissue was removed from the rats at 7 or 14 d after SCI, and stored in 4% paraformaldehyde for 24 hours (4°C). The spinal cord tissue was immersed in a 0.1 M phosphate buffer solution and 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 microcentrifuged 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, or anti-β-Actin) and secondary antibodies, and the signal was digitally quantified.
Immunofluorescence staining
After the sections had been dried, they were washed 3 times for 15 min. They were treated with nonimmune goat serum for 1 hour and then incubated with the following primary antibodies for 50 min at room temperature: 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. This was followed by incubation 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 as a negative control. We use an in situ cell death detection kit to detect apoptotic cells. The nuclei were coloured by staining with Hoechst or DAPI. The fluorescence signal was observed by laser confocal microscopy. Five fields on each of three slides per animal were randomly selected for visualization and analysis performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Transmission electron microscopy(TEM)
A total of 18 mice were subjected to TEM. Tissue was quickly removed, cut into 1 mm3 pieces on ice and soaked in 2.5% glutaraldehyde. The tissue was then fixed with a 1% oxidizing fixative for 1 hour, stained with 1% uranyl acetate for 2 hours, and embedded after dehydration in a gradient acetone solution. After semi-thin sectioning and toluidine blue staining, ultrathin sections were cut and observed using a Hitachi transmission electron microscope.
Images were taken under the same conditions, including brightness and contrast, to better compare TJs among different groups. According to standard protocols[30, 31], the width and length of the TJs were blindly measured and averaged by two examiners using ImageJ software.
Statistical analyses
All experimental data are expressed as the mean ± standard deviation. The Kolmogorov-Smirnov (K-S) test was used as a normality test, with p > 0.05 indicating a normal distribution, Levene’s test was used as a test of homogeneity of variance, with p > 0.05 used to indicate homogeneous variance, and vice versa. A t-test was used to compare two groups. One-way ANOVA and Dunnett’s test were used to evaluate the data when more than two groups were compared. Statistical analyses were performed with SPSS 16 statistical software, and p < 0.05 was used to indicate statistical significance.