Antibodies
5-bromo-20-deoxyuridine (BrdU), glial fibrillary acidic protein (GFAP), ionized calcium binding adapter molecule 1 (Iba1) and neurofilament-200(NF200) antibodies were purchased from Abcam (MC, UK). Ras homolog gene family, member A (RhoA), anti-C3d, anti-NGR, interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNFα), growth associated protein-43 (GAP43) and anti-GAPDH antibodies were purchased from Affinity (OH, USA). S100 calcium-binding protein A10 (S100A10), anti-ROCK2 were purchased from ABclonal Technology Co., Ltd (Wuhan, China). CSPGs was purchased from Merck Chemicals (Shanghai, China) Co., Ltd. Neuron-specific nuclear protein (NeuN) was purchased from Novus (Co, USA). Anti-Tubulin was purchased from Proteintech (IL, USA).
Animals
A total of 160 adult male SD rats weighing 200–250 g were obtained from Shanghai Laboratory Animal Centre. Rats were randomly divided into five groups: Sham-operated (n=40; group S); 7 d after SCI (n=30; group M7); 14 d after SCI (n=30; group M14); SCI + TT for 7 d (n=30; group TM7); SCI + TT for 14 d (n=30; group TM14). Rats are kept in a controlled environment and fed food and water regularly. All protocols were approved by the Animal Research Committee of Wenzhou Medical University.
SCI Model
Rats were kept anesthetized using 2% pentobarbital sodium (30 mg/kg). Then the T9-T11 area on the back of the rat was quickly scraped off, an incision of about 1 cm was made with a scalpel, and the lamina was peeled off with rongeurs to expose the corresponding spinal cord. A New York University Impactor (10 g × 20 cm) was used to impact the exposed site except for group S. Spinal cord bruising, lower limb trembling and tail wagging indicate the success of the model. Finally, the wound is quickly sutured with absorbable surgical thread. In the following experimental phase, the bladder was emptied and the urethra was cleaned every morning and evening.
Water treadmill training
The rats will have 3 d of adaptive treadmill training before the establishment of the model. The setting of the treadmill (Wenzhou Xinglong Stainless Steel Co, LTD, Zhejiang, China): 10-15 m/min, 5 min/round, 3 rounds in total, 5 min interval between rounds, water temperature is maintained at 30 °C. The following day after SCI, the rats in the TM group were treated with TT for 7 d or 14 d, respectively. Each time before experiment, rats were put into water at 30 °C for defecation. After the experiment, the rats will be cleaned and dried and then put back into the cage.
Behavioural tests
Motor function was conducted by two independent examiners who were blinded to the treatment groups using the Basso-Beattie-Bresnahan (BBB) open-field test, which ranges from 0 (complete paralysis) to 21 (normal locomotion). The animals in each group (n=10) were tested at 1, 3, 7, and 14 d following injury.
Tissue preparation
Rats from each group were sacrificed at 7 and 14 d after SCI. After 2% pentobarbital sodium deep anesthesia, rats were transcardially perfused with 0.9% sodium chloride at 4 °C, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). For the rats to be stained with Brdu, 1% solution (10mg/ml) was injected intraperitoneally 3 times every 4 h before the rats were sacrificed. Then the T9-T11 spinal cord tissue was removed from the rats quickly, stored in the same fixative for 24 hours (4°C), and immersed in 0.1 M phosphate-buffered 20% and 30% sucrose overnight at 4 °C, respectively. Successive sections (15-μm thick) were frozen and stored for subsequent experiments.
Haematoxylin-eosin (HE) and Nissl staining
Prepared sections (n=5 per group) were naturally air-dried for 3 min. Then sections were stained with haematoxylin and eosin for HE staining and cresyl violet for Nissl staining, respectively. Finally, the Olympus BH- 2 microscope (Olympus Optics, London, UK) was used to capture the image and the size of cavity area was quantitatively counted.
TUNEL staining
Prepared sections (n=5 per group) were collected for TUNEL staining. Permeabilized the slides for 10 min at room temperature and washed the slides three times for 15 min. Then an In Situ Cell Death Detection Kit (Roche Molecular Biochemicals) was used to detect the apoptotic cells. Using a microscope (BX51, Olympus) to observe the images. The apoptosis positive cells were counted quantitatively using Image-Pro Plus 6.0 analysis software.
RT-PCR
The primer sequences were 5′TGAGGAGAAGAAGGGCGAAGGG 3′ (Forward), 5′AGGACGGCGAGTTATCAGTGGTAG3′ (Reverse) for GAP43, 5′ACGCTGCAGTCAGAGGAGTG3′ (Forward), 5′CTGCCGCCGGTACTCAGTTA 3′ (Reverse) for NF200. High Capacity RNA-to-cDNA Master Mix (A3500, Promega) was applied to reverse transcribed the one microgram of total RNA. Real-time amplification was performed using SYBR Green (QPK-212, Tokyo, Japan) and a Light Cycler480 (Roche, USA). To amplify genomic DNA, PCR was performed under the following conditions: reverse transcription at 50 °C for 3 min, DNA polymerase activation and RT enzyme inactivation at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 10 s, primer annealing at 60 °C for 10 s, and elongation at 72 °C for 10 s. Comparative mRNA expression levels were expressed as 2-ΔΔCt.
Western blot Analysis
At 7 and 14 d after injury, rats (n=5 each group) were sacrificed and a 5-mm length of T10 spinal cord was obtained. After the tissue is fully homogenized, the supernatant is taken. Then the extracted protein concentration was quantified by the bicinchoninic acid (BCA) assay kit. A separation gel suitable for the molecular weight of the protein was prepared for electrophoresis, and separated proteins were transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). After a 90-minute 5% milk closure, the membrane was washed three times with Tris-buffered saline solution with Tween (TBST)and incubated with the corresponding primary antibodies at 4 °C for 16-24 h: GAP43 (1:1000), NF200 (1:1000), IL-1β (1:500), IL-6 (1:1500), TNFα (1:500), GFAP (1:1000), Iba1 (1:1000), C3d (1:500), S100A10 (1:500), CSPGs (1:500), NGR (1:500), RhoA (1:1000), ROCK2 (1:500), Tubulin (1:1000), GAPDH (1:1000). The secondary antibodies were incubated for 2 h and washed with TBST 3 times. All experiments were repeated three times. Images were visualized by the ChemiDicTM XRS + Imaging System (Bio-Rad).
Immunofluorescence Staining
Prepared sections were naturally air-dried and washed three times in 0.01 M PBS, blocked with 10% normal goat serum at room temperature for 1 h, and incubated with rabbit anti-GAP43 (1:200), mouse anti-NF200 (1:200), mouse anti-Brdu (1:200), goat (rabbit) anti-NeuN (1:100), mouse (rabbit) anti-GFAP (1:500), rabbit anti-Iba1(1:300), rabbit anti-C3d (1:200), rabbit anti-S100A10 (1:20), mouse anti-CSPGs (1:200), rabbit anti-ROCK2(1:100), rabbit anti-RhoA (1:200) and rabbit anti-NGR (1:200) overnight at 4 °C. The second day, the sections were incubated with secondary antibodies: cy3-conjugated goat anti-rabbit (1:300), Alexa Flour 488-conjugated goat anti-mouse (1:400), cy3-conjugated goat anti-mouse (1:300), Alexa Flour 488-conjugated goat anti-rabbit (1:400) at room temperature for 50 min. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). The fluorescence signal was observed under 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).
Statistical Analysis
All experimental values are presented as the mean ± SD. The Kolmogorov-Smirnov
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. When there were two experimental groups, Student’s t-test was used. Statistical differences among groups were analyzed using two-way ANOVA followed by Tukey’s test. BBB scores were detected using repeated measurement two-way ANOVA with group and time as factors, followed by Tukey’s test to detect differences between groups. SPSS 16 statistical software was used for statistical analysis, and p < 0.05 was considered statistically significant.