Prior to the start of the study, approval had been obtained from the Ethics/Review Committee of experimental animal Use and Care in Chengdu General Hospital of the Military Region (2013YG-B005). 80 SD rats (half male and half female; 285 ± 25 g) were purchased from Chengdu Dossy Experimental Animals Co. Ltd. (License number SCXK (Sichuan) 2015-030). The experimental protocol was approved by the Local Ethics Committee for Animal Experiments in General Hospital of Western Theater Command (China) for the care and use of laboratory animals. All animals were maintained in the animal rooms where controlled conventional conditions at temperatures of 24 ± 1℃ and relative humidity of 55 ± 15%, 12 h light-dark cycle, and allowed free water and standard diet. The leader of the project promised that the members of this project had complied with the ethical principles of experimental animals, paid attention to animal welfare, cared for animals, and minimized the damage to animals.
2.2 Establishment of a rat model of acute spinal cord injury
After anesthetized with 3% pentobarbital sodium (30 mg pentobarbital sodium /kg rat body weight) by intraperitoneal injection (14), the rats were fixed on surgery table. The surgical site was shaved and sterilized. Briefly, a longitudinal incision of 3 cm was made to expose the T9, T10 and T11 vertebral plate. Then, the laminectomy was performed at the T10 level to expose the dorsal epidural of spinal cord. The triangular needle was clamped with a needle-holding device so that the blunt end passes through the space between the ventral dura and the vertebral body completely to avoid spinal cord injury and pulling. The aneurysm clip (Yasargil Titan Mini Clips, FT228T, Closing force 70 g) was fixed with the application clip, and after opening the clip, the aneurysm clip was perforated from T10 through the channel to the opposite side to ensure that the aneurysm clip completely crossed the spinal cord. Then the application clip was suddenly released, and the spinal cord was compressed by instant violence, causing spasmodic tremor and spasmodic oscillation in the rat body. After the clip was removed gently for 10 s, subdural congestion or hematoma could be seen, with obvious clip marks outside the dural (15). The wound was then sutured layer by layer. After the surgery, the rats were injected with penicillin for 7 successive days. Auxiliary urination by rubbing the bladder of rats at 4 h after the operation and at 8 am and 8 pm every day until the rats resumed spontaneous urination. For the sham-surgery controls, the rats underwent a T10 laminectomy without transient violent oppression by aneurysm clip.
2.3 Animal grouping and intervention method
80 SD rats were randomly divided into five groups (n = 16 for each), including A group (T6 segment stimulation), B group (T10 segment stimulation), C group (L2 segment stimulation), D group (acute SCI without stimulation), E group (sham surgery). Our magnetic stimulation program follows established safety guidelines for repetitive magnetic stimulation (16, 17). Rats in groups A, B and C were treated with rTSMS corresponding to their different segments from 4 d postoperative using aMagstim Rapid Stimulator (Magstim Co. Ltd, Whitland,Wales, UK), where, group A corresponding to Segment T6, group B corresponding to segment T10, and group C corresponding to segment L2. The center of the "8" shape coil cooled by vacuum was fixed close to the spine and the axis of the coil was perpendicular to the rat spinal segment to be stimulated. During the stimulus in group E (sham surgery), the magnetic coil was placed under other conditions, but the magnetic stimulus was not transmitted. However, the magnetic coil vacuum cooling system still simulated some typical auditory stimuli that occurred during the real stimulus. The rats underwent 20 min of baseline measurement, and 10 min of post-stimulation measurement. During the 20-minute baseline measurement, rats were continuously stimulated by rTSMS or sham stimulation. Treatment in daily at 3 pm, stimulating parameters are as follow: 5 Hz, 75% maximum output intensity (1.5 T), each sequence 5 s, 2 s intermittent time, every time-continuous stimulation 10 series. Duration: 1 time a day, five times a week, continuous treatment for 8 weeks.
2.4 Collection of spinal cord tissue specimens
After 8 weeks, 8 rats were randomly removed from each group and anesthetized by intraperitoneal injection with an excess of 10% chloral hydrate (6 ml/1 kg). The dorsal skin of the rats was then snipped off. With the spinal cord at T10 as the center, the surrounding vertebral plates and spinous processes were cut off, the spinal cord was completely exposed, and the spinal nerve roots on both sides were removed and cut off. With the damaged area as the center, spinal cord tissue about 1 cm length was taken, and 4 samples were randomly selected from each group and then placed in the cryopreservation tube. After being labeled, the samples were put into liquid nitrogen. The remaining 4 samples in each group were prefixed in 3% glutaraldehyde solution and observed by TEM. After the samples were stored, 4 rats were randomly selected from each group and spinal tissue was extracted by cardiac perfusion. The spinal tissue about 1 cm length was removed from the injured area. 10% chloral hydrate solution (3 ml/1 kg) was injected intraperitoneally. After successful anesthesia, the abdomens of rats were cut along the costal margin. The pericardium was removed to fully expose the heart and the ascending aorta. The left ventricle (apex) was rapidly perfused with 150 ml of normal saline. Subsequently, the ascending aorta was perfused with a universal tissue fixator (neutral, 4% paraformaldehyde + PBS). Perfusion was stopped when the liver turned white and the limbs twitched and writhed. According to the modeling method, the skin, fascia, muscle and other tissues on the back of rats were cut along the spinal cord direction to expose the damaged spinal cord. The spinal cord was fully exposed. The spinal nerve roots on both sides of the spinal cord were carefully dissected with the glass minute needle, and the spinal nerve roots about 1 cm length were cut off. Finally, the tissues were fixed in the prepared 4% paraformaldehyde fixative for 24 h, then dehydrated, paraffin-embedded and sliced.
2.5 Quantification of Basso, Beattie, and Bresnahan (BBB) Scores
The BBB 22-point of open-field locomotor rating scale was used for evaluating hind limb motor function. 0 point indicates the total paralysis of the hind limbs, and 21 points are fully functioning. Behavioral analyses were conducted by two investigators who were blind as to the treatment at the indicated time points. Briefly, the BBB scores range from 0 points (complete paralysis) to 21 points (normal locomotion). 0 point indicates the complete paralysis of the hind limbs, and 21 points indicate normal locomotion. The rats were evaluated at 1d before surgery and 1 w, 2 w, 4 w, 6 w and 8 w after surgery.
2.6 Material and specimen handling
At 8 weeks post-operation, 8 rats were randomly selected from each group, the rats were anesthetized with 3% pentobarbital sodium (30 mg pentobarbital sodium /kg rat body weight) by intraperitoneal injection, and sections of spinal cord (1 cm long) were harvested from the epicenter of the injury. Four specimens preserved at -80℃ for molecular experiment, the other four specimens were fixed in 3% glutaraldehyde for transmission electron microscopy assay. Then, 4 rats were randomly selected from each group, the rats were anesthetized with 3% pentobarbital sodium (30 mg pentobarbital sodium /kg rat body weight) by intraperitoneal injection, and the chest was opened and aortic cannulation was carried out using normal saline via the left ventricle of the rat until liquid became clear. Following this, 4% paraformaldehyde in PBS (300 ml) was used for continuous perfusion, and the perfusion was stopped when the liver was white and the limbs and trunk were stiff. Then, the spinal cord (1 cm long) was harvested from the epicenter of the injury and fixed with 4% paraformaldehyde for 24 h for immunohistochemistry (IHC) staining.
2.7 Transmission electron microscopy (TEM)
The spinal cord tissues (1 cm long) originally stored in 3% glutaraldehyde solution were removed and trimmed according to the specification of 0.5 ~ 1.0 mm2. Then, the spinal cord (0.5 ~ 1.0 mm2) was fixed with 3% glutaraldehyde for 2 h, followed by dehydration in a series of acetone solutions. The dehydrated tissues were successively treated with penetrant dehydrating agent: epoxy resin at the ratios of 3:1, 1:1 and 1:3, respectively, for 30 to 60 minutes at each step. Next, tissue was embedded for ultrathin sectioning by electron microscopy. The sections were stained with uranyl acetate and lead citrate for 15 min at room temperature respectively, and then observed under Hitachi H-600IV transmission electron microscope (Hitachi, Japan) and photographed.
2.8 Western Blot assay
The total protein was extracted by using radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd.). The protein was quantified with a BCA Protein Assay kit (NanJing KeyGen Biotech Co., Ltd.). The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a poly-vinylidene fluoride (PVDF; Millipore, USA) membrane. Then, the PVDF membrane was sealed with 5% BSA at room temperature for 2 h and incubated with rabbit anti-Nogo-A (ab180847; 1:1,000; abcam, USA), rabbit anti-Nogo (ab184556; 1:10,000; abcam, USA), mouse anti-GAP43 (ab129990; 1:1,000; abcam, USA), rabbit anti-Caspase-3 (ab13847; 1:500; abcam, USA), rabbit anti-Caspase-7 (ab255818; 1:1,000; abcam, USA), rabbit anti-Caspase-9 (ab2013; 1:1,000; abcam, USA), rabbit anti-Cleaved Caspase-3 (ab214430; 1:5000; abcam, USA), rabbit anti-Cleaved Caspase-7 (ab256469; 1:1,000; abcam, USA), rabbit anti-Cleaved Caspase-9 (ab2324; 1:1,000; abcam, USA), rabbit anti-Bax (ab263897; 1:1,000; abcam, USA), mouse anti-β-actin (ab8226; 1:5,000; abcam, USA), goat anti-mouse IgG (ab205719; 1:10,000; abcam, USA) and goat anti-rabbit IgG (ab205718; 1:5,000; abcam, USA). β-actin was used as inner loading control. Protein bands were visualized using an ECL chemiluminescence kit (EMD Millipore) and analyzed by Image-Pro Plus software.
2.9 Immunohistochemical assay
Immunohistochemical staining was performed according to the manufacturer’s instructions. Briefly, paraffin-embedded spinal cord tissues were cut into 4 µm and deparaffinized with xylene. Then, antigen retrieval by 3% methanol hydrogen peroxide at room temperature for 10 min, and sealed with goat serum at room temperature for 20 min. The sections were incubated with mouse anti-GAP43 (ab129990; 1:500; abcam, USA) at 4℃ overnight and incubated with goat anti-mouse IgG (ab205719; 1:5,000; abcam, USA) for 30 min at 37℃. Then, the sections were incubated with DAB (Boster, Wuhan, China) for 2 min, and counterstained with hematoxylin. Positive immune staining was presented as brown or yellow granules in cytoplasm or nucleus. PBS was adopted to substitute for primary antibody as negative control group. Five visual fields were randomly selected and assessed for immunoreactive areas at x400 magnification using BA200Digital image system. The image was analyzed by Image-Pro Plus software.
2.10 Immunofluorescence staining of 5-HT
The spinal cord tissues were dissected from rats, rapidly frozen and used for the preparation of longitudinal section (20 µm) by using cryostat (Leica, Wetzlar, Germany). Pretreatment, blocking, and primary and secondary antibody reactions were performed as described previously (18). Briefly, samples were incubated with rabbit anti-5-HT (ab221181; 1:400; abcam, USA) primary antibodies. Fluorochrome-conjugated anti-rabbit (ab221181; 1:1000; abcam, USA) secondary antibodies were added to the primary antibody-probed sections and incubated. After successive washes, the images were analyzed under a fluorescence microscope ((Nikon, Kona, Japan).
2.11 TUNEL staining
Apoptosis of spinal cord tissues from rats was measured by TUNEL staining using a TUNEL Apoptosis Assay kit (Beyotime Institute of Biotechnology, Inc). TUNEL stained apoptotic nuclei; DAPI and fluorescein-dUTP stained all nuclei. The apoptotic index (AI) = the number of TUNEL-positive cells/ the total number of cells. AI was evaluated in 15 randomly selected fields.
2.12 Statistical analysis
Statistical analysis was performed using SPSS19.0 software (IBM Corp., Armonk, NY, USA). Kolmogorov–Smirnov test was used to test the normal distribution of measurement variables, and the normal distribution data were described as mean ± standard error. Behavioral data were compared by repeated-measures ANOVA. The other data among multiple groups were compared by one-way analysis of variance (ANOVA) with Dunnett’s post-tests or two-way ANOVA with Bonferroni’s post-tests. The differences were considered statistically significant at p < 0.05 and p < 0.01.