Anti-MMP-9, anti-Tubulin were purchased from Proteintech (Rosemont, IL, USA). Anti-VEGF and β-Actin were purchased from Abcam (330 Cambridge Science Park, Cambridge, UK). Anti-p120-Catenin, anti-β-Catenin, anti-ZO-1, anti-Occludin, and anti-Claudin were purchased from Affinity (OH, USA).
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 (group S); SCI (group M); SCI + TT (group TM).
Rats were anaesthetized with 2% pentobarbital sodium (30 mg/kg) and then were shaved to expose the T10 segment. The exposed site was impinged with a New York University(NYU) Impactor (10 g × 20 cm) in all groups except group S. The lower limb trembling contractions and tail wagging showed that SCI modeling was successful. Finally, the wound was sutured, disinfected with iodine volt, and the bladder was emptied every morning and evening.
Our research group provided the initial designed of an underwater treadmill (Wenzhou Xinglong Stainless Steel Co., LTD, Zhejiang, China) and submitted it for a patent. Rats were given adaptive training for three days before spinal cord injury. One day after SCI, rats in the underwater-treadmill training group began training, which lasted for 7 d or 14 d (10-15 m/min, 5 min/round, 3 rounds in total, 5 min interval between rounds).
Two independent examiners who were blinded to the treatment groups conducted BBB scores on rats in an open field test . To put it simply, the BBB score has a total of 21 points, and the higher the score is, the closer the animal is to normal.
Evaluation of BSCB permeability
At 7 d and 14 d after SCI, 2% sodium pentobarbital was intraperitoneally injected to anaesthetise the animals, and 0.5 cm of the T10 spinal cord was taken after perfusion with 0.9% normal saline. The degree of edema in this segment was measured by the dry and wet weight method.
Evans blue dye assays
According to previously reported methods [4, 8], rats were injected with EB dye (4 ml/kg) by tail vein at 7 d and 14 d after SCI, which was followed by 2% sodium pentobarbital anesthesia 2 hours late and 0.9% saline perfusion. Tissue containing T10 was soaked in N,N’-dimethylformamide at 50°C for 72 hours. The concentration of dyes in the samples were determined based on a standard curve (μg/g). Tissues were cut into 15 μm thick sections with a frozen microtome at 20°C, and then the sections were analysed. Quantitative analysis of data was performed with ImageJ software.
Briefly, tissues were removed from rats at 7 and 14 d after spinal cord injury, and then they were 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 use. HE staining experiments were carried out with the appropriate kits.
Western Blot Analysis
Tissues containing T10 segments were put into a collection tube containing a mixture of PMSF and RIPA (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 kit. The mixed solution was heated to 100°C for 10 min. After electrophoretic transfer to membranes, primary and secondary antibodies were incubated successively. Then, the signal was digitally quantified.
After drying sections, they were washed 3 times for 15 minutes. After being treated with nonimmune goat serum for 1 hour, sections were incubated first with primary antibodies against rabbit anti-occludin antibody (1:100, Affinity, US), and rabbit anti-claudin antibody (1:100, Affinity, US) 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. Cells were incubated with DAPI for 10 minutes, and then washed with PBS. Observation of the fluorescence signal under laser confocal microscopy. Five fields on each of three slides per animal were randomly selected for visualization by light microscopy. Analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Transmission Electron Microscopy
Refer to previous research, fresh spinal cord tissue was removed after administering anaesthesia and performing perfusion. Tissue was quickly 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 the tissue was embedded after dehydration in gradient acetone solution. After semithin sectioning and toluidine blue staining, ultrathin sections were cut and observed by Hitachi TEM.
All experimental data are expressed as the mean ± standard deviation. 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.