Animals and experimental groups
Sprague-Dawley rats (4–6 weeks old, 48 males and 48 females) were obtained from the Experimental Animal Centre of Mudanjiang Medical University of China. The rats were maintained under specific-pathogen-free conditions at 22 ± 2 °C and 45 ± 5% humidity with ad libitum access to food and water. After 7 days acclimation, rats were randomly assigned into 4 groups (24 rats per group): (1) sham group, rats only received a laminectomy; (2) sham + EA group, rats subjected to a laminectomy and treated with EA at GV14 and GV4 acupoints; (3) SCI group, rats underwent spinal cord hemisection injury at the T9 segment; (4) SCI + EA group, rats received EA stimulation at GV14 and GV4 acupoints following spinal cord hemisection injury. All animal experimental procedures were carried out in strict accordance with the recommendation in the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH publications No.8023) revised 1978, and approved by the Animal Care and Use Committee of Mudanjiang Medical University. All efforts were made to minimize the number of animals used and their suffering.
Spinal cord hemisection injury
Spinal cord hemisection injury was produced using the protocol described previously . Briefly, rats were anesthetized using 2% pentobarbital sodium (40 mg kg− 1; Solarbio Science & Technology, Beijing, China) by intraperitoneal injections, and an incision was made at the dorsal midline, followed by cutting the cutaneous tissue and separating the muscle. Then, the laminectomy was performed at the T8 vertebral level. After the dura had been exposed, a hemisection was conducted using an iridectomy scissors at T9 spinal segment and with no tissue removed. The tail was spastically swung and the posterior limb twitching rigidity confirming that the hemisection of the spinal cord was completed. Rats in the sham and sham + EA groups were subjected to a laminectomy without spinal cord hemisection injury. After adequate hemostasis with absorbable gelatin sponge, the musculature, fascia and skin were sutured in layers. During the procedures, rat temperature was recorded and maintained at 37 °C using a heating pad. After surgery, rats received an intramuscular injection of penicillin (100,000 U/d) for 3 days and placed on thick soft bedding in separated cages with sufficient water and food.
EA stimulation was performed once daily at GV14 and GV4 acupoints. According to the Laboratory Animal Acupuncture Atlas developed by the National Acupuncture Society for Experimental Research, GV14 is located on the posterior midline below the spinous process of the seventh cervical vertebra, and GV4 is located on the posterior midline below the spinous process of the second lumbar vertebra [31, 34]. Rats were placed within a specially designed cloth bag without anesthesia during EA stimulation. Sterilized disposable stainless steel needles (0.3 mm in diameter, 25 mm in length, Beijing Zhongyan Taihe Medical Instrument Co., Ltd., Beijing, China) were punctured into GV14 (oblique downwardly) and GV4 (oblique upwardly) as deep as 5 mm for both points. Then, the needles were connected to the output terminals of an EA apparatus (G6805-Ⅱelectric pulse stimulator, Qingdao Xinsheng Co., Ltd., Qingdao, China), and the electric simulation parameters were set at a frequency of 2 Hz and an intensity of 1 mA. EA stimulation started on the second day of surgery and lasted for 56 days. To minimize any discrepancies due to stress, rats in the sham and SCI groups were also immobilized in a manner same as in the EA groups for duration of 30 min.
Motor function assessment
The Basso, Beattie and Bresnahan (BBB) locomotor rating scale  was used to evaluate rat hind limb motor function at 0, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after SCI. Briefly, rats were allowed to walk feely in an open field (80 × 130 × 30 cm) with a pasteboard-covered non-slippery floor. In each testing session, rats were observed individually for 5 min by two trained observers who were blinded to grouping. The BBB rating scale ranges from 0 to 21 points, in which a score of 0 indicates complete paralysis and a score of 21 implies normal motor function. The scores were recorded for analysis. The inclined plane test was performed as previously described . In brief, rats were placed with their body axis perpendicular to an orientation of the plate covered with a rubber mat containing horizontal ridges spaced 3 mm apart, and evaluate the maximum vertical axis of the inclined plate. The maximum angle at which a rat maintained its position for 5 sec without falling was recorded for analysis. Footprint analysis was carried out as previously described . In short, the animal’s forepaws and hindpaws were brushed with red and blue nontoxic dye, respectively. Then, the rats were required to walk across a paper lined runway (3 feet long, 3 inches wide) to obtain an edible treat in a darkened box at the end, and the footprints were scanned for analysis.
Sample collection and processing
At 56 days after behavioral tests, rats were sacrificed with 2% pentobarbital sodium (40 mg kg− 1; i.p.), and approximately a 0.5 cm segment of spinal cord centered at the injury cite was harvested. Then, the spinal cord segments were immediately preserved in liquid nitrogen for fluorescent staining, western blot and biochemical analysis, or placed in RNA Later for quantitative real-time polymerase chain reaction (qRT-PCR). For paraffin sectioning, rats were anaesthetized and transcardially perfused with 37 °C heparinized saline, followed by 4% paraformaldehyde, and then the spinal cord tissues around the injured site were removed for hematoxylin-eosin or Nissl staining.
After fixed in 4% paraformaldehyde overnight, the spinal cord segments were processed and embedded in paraffin, followed by preparation of transverse sections with 5 µm thick at the injury site. The spinal cord segments were cut at intervals of 50 µm. For each level, two sections were obtained. One section was stained with hematoxylin-eosin according to standard techniques, and observed under a light microscope (DM2000, Leica Microsystems, Wetzlar, Germany). The cavity sizes of each section were measured manually with Image Pro Plus 6.0 imaging software (Media Cybernetics UK, Marlow, UK) by an observer blind to the groups, as previously described .
The other section was incubated in 0.1% Cresyl violet Nissl staining solution (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The sections were examined with a light microscope, and images were captured with an Mshot color video camera (MD50, Beijing, China). Neuronal chromatolysis, defined as dissolution or loss of Nissl bodies, was evaluated. The chromatolytic and total neurons were counted, and the proportions of chromatolytic neurons versus total neurons were calculated in each group. Integrated optical density (OD) of Nissl bodies in each section was analyzed as previous study described .
Intracellular ROS staining
Intracellular ROS was measured by using a fluorescent dye dihydroethidium (DHE) commercial kit (keyGEN BioTECH, Nanjing, China) in accordance with the manufacturer’s instruction . Briefly, the DHE (5 mg) were dissolved in 3.17 mL dimethylsulfoxide to make a 5 mM DHE reagent stock solution. Then the DHE reagent stock solution was diluted in PBS to make a 50 µM DHE reagent working solution. The frozen spinal cord segments were transversely sectioned at 14 µm, and incubated with DHE reagent working solution for 30 min at 37 °C in a humidified chamber protected from light, and then fluorescent images were captured using a fluorescence microscope (Olympus BX51TR, Olympus Corp., Tokyo, Japan). All imaging parameters were kept constant during imaging. Fluorescent intensity was quantified with Image Pro-Plus 6.0 software as previous study described .
Glutathione and total antioxidant capacity assay
The contents of reduced glutathione (GSH) in spinal cord homogenates were measured using a GSH assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions. Briefly, spinal cord segment was homogenized independently in ice cold phosphate buffer and centrifuged at 3500 rpm for 10 min at 4 °C, the supernatants were collected and incubated with a reaction mixture for 10 min at 37 °C, and then the GSH contents were assayed by colorimetric analysis.
Total antioxidant capacity (T-AOC) was measured with a T-AOC assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. In brief, the spinal cord homogenates were fully blend and kept still for 10 min, and then the supernatants were collected and measured spectrophotometrically at 520 nm. The protein concentration for each sample was determined by a BCA Protein Assay Kit (Beyotime, Nanjing, China).
Enzyme-linked immunosorbent assay
The levels of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in spinal cord tissues were assessed using a commercial ELISA kit (Wuhan USCN Business Co., Ltd, Wuhan, China) according to the manufacturer’s instructions. Briefly, the spinal cord segment was homogenized in RIPA buffer (Beyotime, Nanjing, China), centrifuged at 12,000 rpm for 10 min, and the protein concentrations were measured by BCA Protein Assay Kit. Subsequently, the supernatants were added to 96-well ELISA plates and incubated at 37 °C for 30 min. The conjugate reagent was added to the plates. After rinsing, the plates were incubated with color agent at 37 °C for 30 min, and then the reaction was terminated by stop buffer. The OD was measured with a microplate reader at 450 nm. The levels of 8-OHdG in the samples were calculated based on a standard curve, and the results were reported as pg/mg protein.
Quantitative real-time polymerase chain reaction analysis
The qRT-PCR analysis was conducted following the method described in previous work . Briefly, total RNA was isolated from one half of the spinal cord segment (the spinal cord segment was bisected longitudinally along the middle line) by using TRIzol® reagent (Invitrogen, Thermo Fisher Scientific, Inc., CA, USA), and the RNA concentration and purity were quantified by a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, USA). The RNA was reverse transcribed into cDNA using Thermo Scientific Revert Aid First Strand cDNA kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The qRT-PCR was performed using SsoFast EvaGreen® Supermix (Bio-Rad Laboratories, Hercules, CA, USA) with a PCR Detection System (Applied Biosystems, ABI Prism 7300, USA). The final volume of the PCR reaction mixture was 20 µL, which consisted of 10 µL 2 × SYBR supermix, 0.5 µL 10 µM forward primer and reverse primer, 1 µL cDNA and 8 µL Rnase/DNase-free water. The qRT-PCR was conducted as follows: 95 °C for 30 sec, followed by 40 cycles of 95 °C for 5 sec and 60 °C for 5 sec. The quantitation values of target genes were analyzed by using the relative quantification method. Briefly, a comparative quantification cycle (Cq) was used to determine gene expression level relative to the endogenous β-actin control gene. Amplification specificity was checked using melting curves. The relative change in gene expression level was calculated using the 2−ΔΔCq values of the different groups. The primer sequences as follows: p66Shc, 5′-GCCGAGTATGTCGCCTATGT-3′ (forward), 5′-GGGTGGGTTCCTGAGGTAT T-3′ (reverse); β-actin, 5′-GGTGAAGGTCGGAGTCAACG-3′ (forward), 5′-CCAGTAGGTACTGTTGAAAC-3′ (reverse).
Western blot analysis
The western blot analysis was performed as previous study described . Briefly, the remaining half of spinal cord segment was homogenized in RIPA buffer with 1% phenylmethylsulfonyl fluoride (Beyotime, Nanjing, China) and protease inhibitor (Sigma Aldrich, St Louis, MO, USA) according to the manufacturer’s instructions. Samples were centrifuged at 12,000 rpm for 10 min, and then the protein concentrations were determined by BCA Protein Assay Kit. For electrophoresis, protein samples (30 µg) were dissolved in the loading buffer and boiled for 5 min. Samples were then resolved on 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in TBST (10 mmol/L Tris, 150 mmol/L NaCl and 0.1% Tween 20, pH 7.5) for 2 h at 37 °C. Subsequently, the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies were as follows: 3-nitrotyrosine (3-NT, 1:1000), 4-hydroxynonenal (4-HNE, 1:1000) and ATF6 (1:1000) (Abcam, Cambridge, MA, USA); p66Shc (1:1500), GRP78 (1:3000), CHOP (1:1000), X-box binding protein 1 (XBP1, 1:800), ATF4 (1:1000) and β-actin (1:5000) (Proteintech, Chicago, IL, USA). After three washes in TBST, the membranes were then incubated for 2 h with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature. Following rinsing with TBST, the target proteins were visualized with chemiluminescence luminol reagents (Millipore, Billerica, MA, USA). Blots were imaged by Molecular Image® ChemiDocTM XRS+ with Image Lab™ Software (Bio-Rad, Hercules, CA, USA). The β-actin served as an internal control. The gray values of bands were analyzed by Image J 1.50 b Gel Analyzer (National Institutes of Health, Washington, DC, USA). The gray values of target proteins were divided by that of internal control to correct the error, which resulted due to the relative content of the target protein in the sample.
All data were presented as mean ± SEM. A multifactorial analysis of variance (ANOVA) for repeated measurement was employed for analyzing BBB scores and maximum angles of the inclined plane test. One-way ANOVA was employed for analyzing other data followed by LSD (equal variances assumed) or Dunnett's T3 (equal variances not assumed) post hoc test with SPSS 17.0 software package. Statistical significance was set at P < 0.05.