Adult female Wistar rats (230±20 g; n=60) used in the current study were all provided by Tianjin Medical University Animal Research Center (Tianjin, China; permission no. SCXK-2012-0004). All animal experiments were approved by the Animal Welfare Committee of Tianjin Medical University (Tianjin, China), which is based on the NIH Guide for the Care and Use of Laboratory Animals (15). The rats were randomly distributed into three groups: Sham group, SCI group and LPEMF group. In the sham group, rats underwent laminectomy, and the spinal cord was not injured. In the SCI group, the spinal cord was injured using the same procedure used in the LPEMF group on an identical electromagnetic device, but without the application of LPEMFs. In the LPEMF group, Wistar rats received the LPEMFs treatment for 1 h per day from 24 h after SCI. Animals were sacrificed on days 3, 7 and 14 after SCI, and the spinal cords were harvested for further analysis.

The standard New York University impactor machine was used to induce a spinal cord contusion injury model as described previously (16). Rats were anesthetized with chloral hydrate (300 mg/kg), and a laminectomy was performed to expose the T10 spinal cord. A metal rod (10 g, 25 mm) was dropped onto the back side of the spinal cord. A surveillance system was used to control the compression force and velocity to maintain uniformity between animals. Following the operation, the bladders of these rats were manually emptied.

The BG100A-2 pulsed magnet field therapeutic apparatus (Concord Beijing Medical Equipment Co., Ltd., Beijing, China; patent no. ZL00101667.9) was used in the current study. The apparatus contains seven coils arranged end-to-end beneath the treatment table and a magnetic line of force was positioned across the rats longitudinally. The frequency, power and duty cycle are all adjustable and the apparatus can be monitored and controlled by computer system. In the LPEMFs group, rats were exposed to LPEMFs (frequency, 50 Hz; power, 2.5 mT; duty cycle, 40%) at 24 h after SCI. Rats were placed into a transparent plastic chamber with ventilation and were given time to explore for 1 h per day (8:00-9:00 a.m.) for 14 days. In the SCI group, animals were placed in the same chambers on the treatment table for 1 h per day without exposure to LPEMFs.

Functional recovery of the animals was evaluated using the Basso Beattie Bresnahan (BBB) locomotor rating scale (17). The BBB was observed by two groups, and the observers were blinded to the design of current experiment (18). The BBB scores of rats were recorded prior to contusion operation and at 1, 3, 5, 7 and 14 days post-injury (dpi).

The spinal cord tissue (10 mm block of spinal cord surrounding the lesion center) was collected and homogenized at 14 dpi. Then, the tissue homogenate was centrifuged at a speed of 10,000 × g at 4°C for 20 min and the supernatant was collected for determining protein concentration using a Pierce^™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The commercially available ELISA kits were obtained from the following companies: TNF-α ELISA kit (cat. no. ab46070; Abcam, Cambridge, UK), IL-1β ELISA kit (cat. no. RA20422; Bio-Swamp, Wuhan, China), superoxide dismutase (SOD) ELISA kit (cat. no. 706002; Cayman Chemical Company, Ann Arbor, MI, USA), catalase (CAT) ELISA kit (cat. no. 11363727001; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).

After 14 days of treatment or SCI, the spinal cord tissue around the lesion center was collected, harvested and homogenized in radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology, Shanghai, China). The concentration of protein was detected using bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein samples (50 µg) from three individual animals in each group were resolved using SDS-PAGE (12% gel) for separation and then transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk and incubated with anti-inducible nitric oxide synthase (iNOS; 1:250; cat. no. ab15323; Abcam) and anti-β-actin (1:2,000; cat. no. ab8227; Abcam) overnight at 4°C. Then, the membrane was incubated with secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:10,000; cat. no. ab205718; Abcam) at 37°C for 1 h. BeyoECL Star (cat. no. P0018AM; Beyotime Institute of Biotechnology) was used to develop the HRP signal. Signals were captured using a ChemiDoc MP System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and quantified using ImageJ software version 1.32 (National Institutes of Health, Bethesda, MD, USA). The expression levels of iNOS were determined following normalization to β-actin levels.

Spinal cords were collected 14 days after injury and samples were quickly frozen at −40°C immersed in 4% paraformaldehyde. Transverse 10 µm thick sections of spinal cord were used for immunohistochemistry analysis. Following permeabilization in 0.25% Triton X-100/PBS for 10 min at room temperature, sections were blocked with 10% goat serum (cat. no. SL038; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 60 min at room temperature. Sections were stained using anti-nuclear factor-κB (NF-κB) p65 (1:2,000; cat. no. ab16502; Abcam) and anti-HSP70 (1:100; ab79852; Abcam) at 4°C overnight. Samples were incubated with secondary HRP-conjugated goat anti-rabbit antibody (1:10,000; cat. no. ab205718; Abcam) at 37°C for 1 h. The DAB Horseradish Peroxidase Color Development kit (cat. no. P0202; Beyotime Institute of Biotechnology) was used for signal development (sections were incubated at room temperature for 25 min). Then, 3 fields were randomly selected for each sample, and the positive area in each field was collected and quantified using ImageJ software version 1.32 (National Institutes of Health).

The spinal cord tissue (5 mm block of spinal cord surrounding the lesion center) was collected and homogenized at 14 dpi. Subsequently, the tissue homogenate was centrifuged (1,500 × g at 4°C for 20 min), and the supernatant was collected. A ROS assay kit (cat. no. D6883; Sigma-Aldrich; Merck KGaA) was used to determine the production of ROS. The supernatant was incubated with 2,7-dichlorodihydrofluorescein diacetate for 1 h and then washed twice with PBS in ice. Fluorescence levels were detected at 480/530 nm.

The experimental data are expressed as the mean ± standard error. A one-way analysis of variance followed by Tukey's post hoc tests for multiple comparisons were used to analyze data (SPSS 19.0 software; IBM Corp., Armonk, NY, USA). P<0.05 were considered to indicate a statistically significant difference.

To evaluate whether the LPEMF treatment has protective effects on motor function in SCI rats, the BBB locomotor rating scale was used to measure behavior for 2 weeks. As presented in Fig. 1, all the animals exhibited full marks (21 points) in BBB scoring prior to the injury. At dpi 1, the BBB scores dropped to 0. Following SCI, rats exhibited spontaneous functional recovery over time and there were no significant differences between the SCI group and the LPEMF group before dpi 7. However, the rats exhibited better motor function recovery after 7 dpi under LPEMF treatment compared with SCI rats, and the BBB scores were significantly different ay dpi 7 and 14 (P<0.05). These results suggested that LPEMF exposure can promote locomotor recovery in SCI rats (Fig. 1).

To determine whether LPEMFs suppressed inflammatory reaction by decreasing the secretion of pro-inflammatory cytokines in the injured spinal cord, the expression levels of TNF-α and IL-1β were assessed. Following SCI, the inflammation markers TNF-α and IL-1β were significantly increased compared with the Sham group (Fig. 2A and B). However, after 2 weeks of LPEMF treatment, the expression of TNF-α and IL-1β were decreased in comparison with the SCI group (Fig. 2A and B). These results indicated that LPEMF treatment, to a certain extent, can alleviate the inflammatory reaction following SCI.

NF-κB is an important transcription factor that stimulates inflammation (19). To determine whether LPEMF treatment could suppress the expression of NF-κB in the injured spinal cord, particularly in the ventral horn, NF-κB protein levels were evaluated using immunohistochemistry (Fig. 3A). In the sham group, the NF-κB was difficult to detect (Fig. 3B). However, after 2 weeks of injury, there was a strong, positive NF-κB signal in the ventral horn of the spinal cord, which contains motor neurons (Fig. 3C). By contrast, the administration of LPEMFs significantly reduced the immunoreactivity of NF-κB in SCI rats (Fig. 3D). The quantified result was consistent with observation of sections (Fig. 3E).

To determine the iNOS expression following SCI, the iNOS was detected using western blot analysis. Significantly higher levels of the iNOS protein were detected following SCI compared with the Sham group. By contrast, the LPEMF treatment suppressed the iNOS protein expression in the injured spinal cord (Fig. 4A and B). The decreased iNOS expression indicated that the LPEMFs can alleviate oxidative stress following SCI.

To investigate the protective effect of LPEMFs on ROS production in SCI rats, the ROS levels between groups were measured. As demonstrated Fig. 5, the injury induced strong ROS production in spinal cord tissue compared with those in the uninjured Sham group. However, the LPEMF administration reduced the ROS level significantly compared with SCI rats. This result indicated that LPEMFs can alleviate the oxidative stress by reducing ROS production following SCI.

To explore whether LPEMFs can alleviate oxidative stress through upregulation of antioxidant enzymes, the expression of SOD and CAT in spinal cord was measured using ELISA. Compared with the intact spinal cord, the injured tissue exhibited decreased expression of SOD and CAT. By contrast, the treatment of LPEMFs can reversed this reduction to a certain extent (Fig. 6A and B). These results provided evidence that the protective effect of LPEMFs on oxidative stress may be attributed to the upregulation of antioxidant enzymes.

HSP70 is deemed as the protective agent for inflammation and oxidative stress during tissue damage. In the current study, the expression of HSP70 in the spinal cord following injury with and without the administration of LPEMFs was examined. Following SCI, the expression of HSP70 became scattered in the ventral horn compared with the spinal cord in the Sham group. However, the LPEMF treatment significantly increased the expression of HSP70 in motor neurons (Fig. 7A-D). The quantified result was consistent with observations (Fig. 7E). These results indicated that the anti-inflammatory and antioxidative stress effects of LPEMFs may associated with the high expression of HSP70.