Contributed equally
Low-frequency pulsed electromagnetic fields (LPEMFs) have been reported to be protective for multiple diseases. However, whether the administration of LPEMFs inhibits inflammation and oxidative stress following spinal cord injury requires further investigation. In the current study, a contusion spinal cord injury model was used and LPEMFs administration was applied to investigate the molecular changes, including inflammation, oxidative stress and heat shock protein 70 (HSP70) levels. The results revealed that LPEMFs significantly promoted functional recovery following spinal cord injury, as demonstrated by an increased Basso, Beattie and Bresnahan score. The results demonstrated that LPEMFs decreased the expression of inflammatory factors, including tumor necrosis factor-α, interleukin-1β and nuclear factor-κB. Additionally, LPEMFs exposure reduced the levels of inducible nitric oxide synthase and reactive oxygen species, and upregulated the expression of catalase and superoxide dismutase. Furthermore, treatment with LPEMFs significantly enhanced the expression of HSP70 in spinal cord-injured rats. Overall, the present study revealed that LPEMFs promote functional recovery following spinal cord injury, potentially by modulating inflammation, oxidative stress and HSP70.
Spinal cord injury (SCI) causes severe damage to the central nervous system, resulting in irreversible motor and sensory dysfunction below the injury area (
The pathological process of SCI can be divided into two stages; the primary and secondary injury (
The use of low-frequency pulsed electromagnetic fields (LPEMFs) is a noninvasive therapeutic method for various diseases. Recent evidence has demonstrated that LPEMFs can prevent inflammation and oxidative stress. LPEMF stimulation can suppress the production of IL-1β and TNF-α in cultured nucleus pulposus cells (
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 (
The standard New York University impactor machine was used to induce a spinal cord contusion injury model as described previously (
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 (
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
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 (
NF-κB is an important transcription factor that stimulates inflammation (
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 (
To investigate the protective effect of LPEMFs on ROS production in SCI rats, the ROS levels between groups were measured. As demonstrated
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 (
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 (
Electromagnetic fields (EMFs) have long been deemed relevant for human health (
Inflammatory cascades are activated during secondary injury following SCI. High levels of pro-inflammatory factors are released from spinal cord tissue (astrocytes and microglia) and peripheral cells (neutrophils, monocytes and macrophages) to increase vascular permeability. TNF-α and IL-1β pathophysiological signaling pathways are two of the most important components in SCI inflammatory cascades. The increased expression of TNF-α and IL-1β can suppress cell survival and lead to cell death. The NF-κB signaling pathway has been well established as the center of the pathophysiology of inflammatory reactions induced by SCI (
Following SCI, the production and elimination of oxidative species are imbalanced, which results in tissue oxidative stress (
Heat shock proteins have an important role in transport and folding proteins, which is necessary for numerous biological processes. HSP70 has been considered to protect against cellular stress. HSP70 is also modulates inflammation and oxidative stress (
In conclusion, the findings of the present study revealed that the administration of LPEMFs reduces inflammation and oxidative stress to promote functional recovery following SCI, and the potential mechanism involves the activation of HSP70. The findings provide new perspective for identifying novel noninvasive therapeutic methods for early intervention following SCI.
The authors are grateful for the valuable suggestions of Professor Xiaohong Kong from the 221 Laboratory, School of Medicine, Nankai University (Tianjin, China).
The authors thank the following sources for funding support: the NSFC program (grant nos. 81330042, 81472070, 81772342 and 81620108018), the Ministry of Science and Technology, China (grant no. 2014DFR31210), and the Tianjin Science and Technology Committee, China (grant nos. 13RCGFSY19000 and 14ZCZDSY00044).
The data and materials used or analysed during the current study are available from the corresponding author on reasonable request.
SF, CXW, CYW and YL conceived and designed the experiments. CYW and YL performed the experiments. YW, ZW and DS provided critical reagents and scientific input. GN and QW maintained the animals. CYW, YL, SF and CXW analyzed data and prepared the manuscript.
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.
Not applicable.
The authors declare that they have no competing interests.
BBB scores in Sham group, SCI group and LPEMF group at different time points. *P<0.01 vs. Sham group; #P<0.05 vs. SCI group. BBB, Basso Beattie Bresnahan; SCI, spinal cord injury; LPEMF, low-frequency pulsed electromagnetic fields.
ELISA analysis of expression levels of TNF-α and IL-1β in the spinal cord. The ELISA was conducted at 14 days post-injury. (A) Expression levels of TNF-α. (B) Expression levels of IL-1β. *P<0.01 vs. Sham group; #P<0.05 vs. SCI group. TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; SCI, spinal cord injury; LPEMF, low-frequency pulsed electromagnetic fields.
Expression of NF-κB in the ventral horn of the spinal cord. The NF-κB was detected at 14 days post-injury. (A) Diagram of the spinal cord. Representative image of NF-κB expression in the ventral horn of the spinal cord in the (B) sham group, (C) SCI group and (D) LPEMF group. Scale bar, 50 µm. (E) Statistical comparison among the three groups. *P<0.01 vs. Sham group; #P<0.05 vs. SCI group. NF-κB, nuclear factor-κB; SCI, spinal cord injury; LPEMF, low-frequency pulsed electromagnetic fields.
Effect of LPEMFs on the iNOS expression following SCI. The iNOS was detected at 14 days post-injury. (A) Western blot and (B) quantification was used to detect the expression of iNOS in sham, SCI and LPEMF groups. *P<0.01 vs. Sham group; #P<0.05 vs. SCI group. SCI, spinal cord injury; LPEMF, low-frequency pulsed electromagnetic fields; iNOS, inducible nitric oxide synthase.
Effect of LPEMFs on the ROS level following SCI. ROS were detected at 14 days post-injury. ROS assay kit was used to determine the production of ROS. *P<0.01 vs. Sham group; #P<0.05 vs. SCI group. ROS, reactive oxygen species; SCI, spinal cord injury; LPEMF, low-frequency pulsed electromagnetic fields.
Effect of LPEMFs on the expression of antioxidant enzymes following SCI. SOD and CAT were detected at 14 days post-injury. Expression of (A) SOD and (B) CAT in the spinal cord was measured using ELISA. *P<0.01 vs. Sham group; #P<0.05 vs. SCI group. SOD, superoxide dismutase; CAT, catalase; SCI, spinal cord injury; LPEMF, low-frequency pulsed electromagnetic fields.
Expression of HSP70 in ventral horn of the spinal cord. HSP70 was detected at 14 days post-injury. (A) Diagram of the spinal cord. Representative image of HSP70 expression in the ventral horn of the spinal cord in the (B) sham group, (C) SCI group and (D) LPEMF group. Scale bar, 50 µm. (E) Statistical comparison among the three groups. *P<0.01 vs. Sham group; #P<0.05 vs. SCI group. HSP70, heat shock protein 70; SCI, spinal cord injury; LPEMF, low-frequency pulsed electromagnetic fields.