Spinal Cord Injury

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. 2019 Mar; 19(3): 1687–1693.
Published online 2019 Jan 4. doi: 10.3892/mmr.2019.9820
PMCID: PMC6390012
PMID: 30628673

Low-frequency pulsed electromagnetic field promotes functional recovery, reduces inflammation and oxidative stress, and enhances HSP70 expression following spinal cord injury

1Department of Rehabilitation Medicine, Tianjin Medical University General Hospital, Tianjin 300052, P.R. China
2Department of Orthopedics, Tianjin Medical University General Hospital, Tianjin 300052, P.R. China
Correspondence to: Professor Shiqing Feng, Department of Orthopedics, Tianjin Medical University General Hospital, 154 Anshan Road, Heping, Tianjin 300052, P.R. China, E-mail: nc.ude.umt@gnefqs
Professor Chunxiao Wan, Department of Rehabilitation Medicine, Tianjin Medical University General Hospital, 154 Anshan Road, Heping, Tianjin 300052, P.R. China, E-mail: moc.361@6222xcw
*Contributed equally
Received 2018 Apr 20; Accepted 2018 Nov 23.
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Associated Data

Data Availability Statement

Abstract

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-?, interleukin-1? and nuclear factor-?B. 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.

Keywords: low-frequency pulsed electromagnetic fields, spinal cord injury, inflammation, oxidative stress, heat shock protein 70

Introduction

Spinal cord injury (SCI) causes severe damage to the central nervous system, resulting in irreversible motor and sensory dysfunction below the injury area (). In modern society, the global incidence of SCI varied from 8 to 246 per million depending on the country or region (), and an increasing number of paralyzed patients survive longer, experiencing a variety of complications (,). Currently there are no effective therapeutic treatments for SCI. Methylprednisolone, which was previously used as first-aid measure for acute SCI, is no longer recommended in the guidelines of AANS/CNS as of 2013 due to an increased occurrence of complications and no strong evidence of clinical efficacy (). Rehabilitation, the only proven effective treatment for SCI, is limited for early application as it is only suitable for less serious injuries and requires active cooperation from patients (). Thus, novel noninvasive treatments are required as early interventions for patients with SCI.

The pathological process of SCI can be divided into two stages; the primary and secondary injury (). The primary injury is the original tissue breakdown caused by contusion or compression, which occurs immediately after SCI. The secondary injury is a series of pathologic changes following the primary injury, including an increase in the permeability of the blood spinal cord barrier, the infiltration of inflammatory cells, excitotoxicity, demyelination and neuronal apoptosis, which lasts for days to months. Two of the most important components of the secondary injury in its early stages are inflammation and oxidative stress. The inflammation includes increased expression of pro-inflammatory factors, including tumor necrosis factor-? (TNF-?) and interleukin-1? (IL-1?), and a reduction in inflammatory mediators (). The oxidative stress involves an increase in the levels of nitric oxide (NO) and reactive oxygen species (ROS) ().

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-1? and TNF-? in cultured nucleus pulposus cells (). Furthermore, LPEMFs can reduce ROS levels and enhance antioxidative stress responses in osteoblasts (). LPEMFs exhibit strong neuroprotective effects in the nervous system. In ischemic stroke, LPEMFs can promote functional recovery by activation of the brain derived neurotrophic factor/tropomyosin receptor kinase B/protein kinase B signaling pathway (). Additionally, LPEMFs can modulate the expression of microRNAs and stimulate tissue regeneration in in vitro models of Alzheimer’s disease (). Recent studies have revealed that extremely low-frequency magnetic fields reduce iron-induced tissue damage following SCI (). However, whether the administration of LPEMFs inhibits the early-stage reaction of SCI secondary injury, inflammation and oxidative stress, requires further exploration. In the present study, neuroprotective effects of LPEMF stimulation on SCI model rats were investigated. The changes in inflammation and oxidative stress molecular markers were then compared, and whether this protective effect was modulated by targeting heat shock protein 70 (HSP70) was examined. The results of the current study may provide a noninvasive alternative therapeutic method for the early treatment of SCI.

Materials and methods

Experimental animals

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 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.

Contusion SCI model

The standard New York University impactor machine was used to induce a spinal cord contusion injury model as described previously (). 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.

LPEMFs treatment

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.

Assessment of locomotor activity

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

Enzyme-linked immunosorbent assay (ELISA)

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-? ELISA kit (cat. no. ab46070; Abcam, Cambridge, UK), IL-1? 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).

Western blot analysis

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-?-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 ?-actin levels.

Immunohistochemistry analysis

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-?B (NF-?B) 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).

ROS assay

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.

Statistical analysis

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

Results

Protective effects of LPEMFs on locomotor recovery following SCI in rats

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).

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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.

Protective effect of LPEMFs on expression of pro-inflammatory cytokines in SCI rats

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-? and IL-1? were assessed. Following SCI, the inflammation markers TNF-? and IL-1? were significantly increased compared with the Sham group (Fig. 2A and B). However, after 2 weeks of LPEMF treatment, the expression of TNF-? and IL-1? 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.

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ELISA analysis of expression levels of TNF-? and IL-1? in the spinal cord. The ELISA was conducted at 14 days post-injury. (A) Expression levels of TNF-?. (B) Expression levels of IL-1?. *P<0.01 vs. Sham group; #P<0.05 vs. SCI group. TNF-?, tumor necrosis factor-?; IL-1?, interleukin-1?; SCI, spinal cord injury; LPEMF, low-frequency pulsed electromagnetic fields.

Suppressive effect of LPEMFs on NF-?B expression in injured spinal cord

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

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Expression of NF-?B in the ventral horn of the spinal cord. The NF-?B was detected at 14 days post-injury. (A) Diagram of the spinal cord. Representative image of NF-?B 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-?B, nuclear factor-?B; SCI, spinal cord injury; LPEMF, low-frequency pulsed electromagnetic fields.

Suppressive effect of LPEMFs on iNOS expression in injured spinal cord

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.

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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.

Protective effect of LPEMFs on ROS production in SCI rats

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.

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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.

Protective effect of LPEMFs on expression of antioxidant enzymes 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.

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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.

Protective effect of LPEMFs on expression of HSP70 SCI rats

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.

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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.

Discussion

Electromagnetic fields (EMFs) have long been deemed relevant for human health (), and recent studies have indicated that LPEMFs exhibit protective effects in multiple pathologies, including Alzheimer’s disease (), stroke (), wound healing () and pain (). The definition of low frequency is <300 Hz and research has demonstrated that pulsed EMFs exhibited greater therapeutic effects when applied with an amplitude <3 mT and frequencies <100 Hz (). In the current study, LPEMFs with a 50 Hz frequency and 2.5 mT amplitude were applied, which are suitable parameters for the exploratory research. However, to the best of our knowledge, the current study was first administration of LPEMFs in a contusion SCI model, which is more clinically relevant than the transection model (,). Unlike other disorders, the pathological changes following SCI are complex due to the microenvironment comprised of neurons, astrocytes, oligodendrocytes, microglial cells and vascular endothelial cells (). It is well established that the secondary injury is far more important than primary injury, due the crucial role of the microenvironment in tissue preservation and regeneration (). In the current study, LPEMF treatment improved the recovery of motor function in the SCI rats and alleviated the inflammation and oxidative stress in the damaged spinal cord, which indicated that LPEMFs promote functional recovery, which may be associated with reduced secondary injury following SCI.

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-? and IL-1? pathophysiological signaling pathways are two of the most important components in SCI inflammatory cascades. The increased expression of TNF-? and IL-1? can suppress cell survival and lead to cell death. The NF-?B signaling pathway has been well established as the center of the pathophysiology of inflammatory reactions induced by SCI (). The NF-?B can be elevated by the pro-inflammatory cytokines and chemokines following injury. The activation of NF-?B, as a transcription factor, is required for the upregulation of TNF-? and IL-1?. Therefore, this positive feedback amplifies the inflammatory reaction and exacerbated the microenvironment following SCI (). Thus, the results of the current study suggest that LPEMFs can reduce the expression of TNF-?, IL-1? and NF-?B to alleviate the inflammatory reaction induced by SCI, and this supports previous reports demonstrating LPEMF exposure exhibits anti-inflammatory effects in synoviocytes, chondrocytes and osteoblasts (). However, in the current study, inflammation was only detected at 14 dpi. It will be necessary to use other time points to validate these results.

Following SCI, the production and elimination of oxidative species are imbalanced, which results in tissue oxidative stress (). ROS and NO are two main end-products of oxidative stress. Superoxides, hydroxylradicals, hydrogen peroxides and peroxynitrites are the principal components of ROS (). NO is synthesized by NOS, and the most abundant isoform is the iNOS, which is located in a variety of cell types in the spinal cord and can represent the production of NO (). Antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), act to reduce oxidative stress. However, damage of the spinal cord impairs their protective ability (). In the current study, the administration of LPEMFs significantly reduced the levels of ROS and iNOS in the injured spinal cord. By contrast, the expression of CAT and SOD were upregulated by LPEMFs, which can protect damaged tissue. A previous study also demonstrated the antioxidant protective effects of LPEMFs in neuronal cell lines by elevating endogenous antioxidant properties (), which was in accordance with the current study.

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 an SCI model, HSP70 was demonstrated to promote the survival of motor neurons (). In the current study, LPEMFs significantly promoted the expression of HSP70 in the ventral horn of the injured spinal cord, where the motor neurons are located. Therefore, the enhanced tolerance to inflammation and oxidative stress may be mediated by upregulation of HSP70 following LPEMF treatment. However, the causal association between HSP70 and inflammation/oxidative stress requires further investigation.

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.

Acknowledgements

The authors are grateful for the valuable suggestions of Professor Xiaohong Kong from the 221 Laboratory, School of Medicine, Nankai University (Tianjin, China).

Funding

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).

Availability of data and materials

The data and materials used or analysed during the current study are available from the corresponding author on reasonable request.

Authors’ contributions

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.

Ethics approval and consent to participate

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.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1. Ropper AE, Ropper AH. Acute spinal cord compression. N Eng J Med. 2017;376:1358–1369. doi: 10.1056/NEJMra1516539. [PubMed] [CrossRef[]
2. Furlan JC, Sakakibara BM, Miller WC, Krassioukov AV. Global incidence and prevalence of traumatic spinal cord injury. Can J Neurol Sci. 2013;40:456–464. doi: 10.1017/S0317167100014530. [PubMed] [CrossRef[]
3. Jain NB, Ayers GD, Peterson EN, Harris MB, Morse L, O’Connor KC, Garshick E. Traumatic spinal cord injury in the United States, 1993–2012. JAMA. 2015;313:2236–2243. doi: 10.1001/jama.2015.6250.[PMC free article] [PubMed] [CrossRef[]
4. Miller LE, Herbert WG. Health and economic benefits of physical activity for patients with spinal cord injury. Clinicoecon Outcomes Res. 2016;8:551–558. doi: 10.2147/CEOR.S115103. [PMC free article][PubMed] [CrossRef[]
5. Hurlbert RJ, Hadley MN, Walters BC, Aarabi B, Dhall SS, Gelb DE, Rozzelle CJ, Ryken TC, Theodore N. Pharmacological therapy for acute spinal cord injury. Neurosurgery. 2013;72(Suppl 2):S93–S105. doi: 10.1227/NEU.0b013e31827765c6. [PubMed] [CrossRef[]
6. Herzer KR, Chen Y, Heinemann AW, González-Fernández M. Association between time to rehabilitation and outcomes after traumatic spinal cord injury. Arch Phys Med Rehabil. 2016;97:1620–1627.e4. doi: 10.1016/j.apmr.2016.05.009. [PMC free article] [PubMed] [CrossRef[]
7. Anwar MA, Al Shehabi TS, Eid AH. Inflammogenesis of secondary spinal cord injury. Front Cell Neurosci. 2016;10:98. doi: 10.3389/fncel.2016.00098. [PMC free article] [PubMed] [CrossRef[]
8. Huang JH, Yin XM, Xu Y, Xu CC, Lin X, Ye FB, Cao Y, Lin FY. Systemic administration of exosomes released from mesenchymal stromal cells attenuates apoptosis, inflammation, and promotes angiogenesis after spinal cord injury in rats. J Neurotrauma. 2017;34:3388–3396. doi: 10.1089/neu.2017.5063.[PubMed] [CrossRef[]
9. Xun C, Mamat M, Guo H, Mamati P, Sheng J, Zhang J, Xu T, Liang W, Cao R, Sheng W. Tocotrienol alleviates inflammation and oxidative stress in a rat model of spinal cord injury via suppression of transforming growth factor-? Exp Ther Med. 2017;14:431–438. doi: 10.3892/etm.2017.4505.[PMC free article] [PubMed] [CrossRef[]
10. Zou J, Chen Y, Qian J, Yang H. Effect of a low-frequency pulsed electromagnetic field on expression and secretion of IL-1? and TNF-? in nucleus pulposus cells. J Int Med Res. 2017;45:462–470. doi: 10.1177/0300060516683077. [PMC free article] [PubMed] [CrossRef[]
11. Ehnert S, Fentz AK, Schreiner A, Birk J, Wilbrand B, Ziegler P, Reumann MK, Wang H, Falldorf K, Nussler AK. Extremely low frequency pulsed electromagnetic fields cause antioxidative defense mechanisms in human osteoblasts via induction of •O2 and H2O2Sci Rep. 2017;7:14544. doi: 10.1038/s41598-017-14983-9. [PMC free article] [PubMed] [CrossRef[]
12. Urnukhsaikhan E, Mishig-Ochir T, Kim SC, Park JK, Seo YK. Neuroprotective effect of low frequency-pulsed electromagnetic fields in ischemic stroke. Appl Biochem Biotechnol. 2017;181:1360–1371. doi: 10.1007/s12010-016-2289-z. [PubMed] [CrossRef[]
13. Capelli E, Torrisi F, Venturini L, Granato M, Fassina L, Lupo GF, Ricevuti G. Low-frequency pulsed electromagnetic field is able to modulate miRNAs in an experimental cell model of Alzheimer’s disease. J Healthc Eng 2017. 2017:2530270. [PMC free article] [PubMed[]
14. Dey S, Bose S, Kumar S, Rathore R, Mathur R, Jain S. Extremely low frequency magnetic field protects injured spinal cord from the microglia- and iron-induced tissue damage. Electromagn Biol Med. 2017;36:330–340. doi: 10.1080/15368378.2017.1389750. [PubMed] [CrossRef[]
15. The National Academies Press; Washington, DC: 2011. NIH (National Institutes of Health U.S.A): Guide for the Care and Use of Laboratory Animals; p. 246. []
16. Zhou H, Li X, Wu Q, Li F, Fu Z, Liu C, Liang Z, Chu T, Wang T, Lu L, et al. shRNA against PTEN promotes neurite outgrowth of cortical neurons and functional recovery in spinal cord contusion rats. Regen Med. 2015;10:411–429. doi: 10.2217/rme.14.88. [PubMed] [CrossRef[]
17. Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 1995;12:1–21. doi: 10.1089/neu.1995.12.1. [PubMed] [CrossRef[]
18. Wei ZJ, Zhou XH, Fan BY, Lin W, Ren YM, Feng SQ. Proteomic and bioinformatic analyses of spinal cord injury-induced skeletal muscle atrophy in rats. Mol Med Rep. 2016;14:165–174. doi: 10.3892/mmr.2016.5272. [PMC free article] [PubMed] [CrossRef[]
19. Xu L, Botchway BOA, Zhang S, Zhou J, Liu X. Inhibition of NF-?B signaling pathway by resveratrol improves spinal cord injury. Front Neurosci. 2018;12:690. doi: 10.3389/fnins.2018.00690.[PMC free article] [PubMed] [CrossRef[]
20. Ferroni L, Tocco I, De Pieri A, Menarin M, Fermi E, Piattelli A, Gardin C, Zavan B. Pulsed magnetic therapy increases osteogenic differentiation of mesenchymal stem cells only if they are pre-committed. Life Sci. 2016;152:44–51. doi: 10.1016/j.lfs.2016.03.020. [PubMed] [CrossRef[]
21. Ottani V, De Pasquale V, Govoni P, Franchi M, Zaniol P, Ruggeri A. Effects of pulsed extremely-low-frequency magnetic fields on skin wounds in the rat. Bioelectromagnetics. 1988;9:53–62. doi: 10.1002/bem.2250090105. [PubMed] [CrossRef[]
22. Jorgensen WA, Frome BM, Wallach C. Electrochemical therapy of pelvic pain: Effects of pulsed electromagnetic fields (PEMF) on tissue trauma. Eur J Surg Suppl. 1994:83–86. [PubMed[]
23. Kavand H, Haghighipour N, Zeynali B, Seyedjafari E, Abdemami B. Extremely low frequency electromagnetic field in mesenchymal stem cells gene regulation: Chondrogenic markers evaluation. Artif Organs. 2016;40:929–937. doi: 10.1111/aor.12696. [PubMed] [CrossRef[]
24. Feng SQ, Zhou XF, Rush RA, Ferguson IA. Graft of pre-injured sural nerve promotes regeneration of corticospinal tract and functional recovery in rats with chronic spinal cord injury. Brain Res 1209. 2008:40–48. doi: 10.1016/j.brainres.2008.02.075. [PubMed] [CrossRef[]
25. Feng SQ, Kong XH, Guo SF, Wang P, Li L, Zhong JH, Zhou XF. Treatment of spinal cord injury with co-grafts of genetically modified Schwann cells and fetal spinal cord cell suspension in the rat. Neurotox Res. 2005;7:169–177. doi: 10.1007/BF03033785. [PubMed] [CrossRef[]
26. Kjell J, Olson L. Rat models of spinal cord injury: From pathology to potential therapies. Dis Model Mech. 2016;9:1125–1137. doi: 10.1242/dmm.025833. [PMC free article] [PubMed] [CrossRef[]
27. Cavalli G, Dinarello CA. Suppression of inflammation and acquired immunity by IL-37. Immunol Rev. 2018;281:179–190. doi: 10.1111/imr.12605. [PubMed] [CrossRef[]
28. Ni H, Jin W, Zhu T, Wang J, Yuan B, Jiang J, Liang W, Ma Z. Curcumin modulates TLR4/NF-?B inflammatory signaling pathway following traumatic spinal cord injury in rats. J Spinal Cord Med. 2015;38:199–206. doi: 10.1179/2045772313Y.0000000179. [PMC free article] [PubMed] [CrossRef[]
29. Varani K, De Mattei M, Vincenzi F, Gessi S, Merighi S, Pellati A, Ongaro A, Caruso A, Cadossi R, Borea PA. Characterization of adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes exposed to low frequency low energy pulsed electromagnetic fields. Osteoarthritis Cartilage. 2008;16:292–304. doi: 10.1016/j.joca.2007.07.004. [PubMed] [CrossRef[]
30. Vincenzi F, Targa M, Corciulo C, Gessi S, Merighi S, Setti S, Cadossi R, Goldring MB, Borea PA, Varani K. Pulsed electromagnetic fields increased the anti-inflammatory effect of A2A and A3 adenosine receptors in human T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. PLoS One. 2013;8:e65561. doi: 10.1371/journal.pone.0065561. [PMC free article] [PubMed] [CrossRef[]
31. Ongaro A, Varani K, Masieri FF, Pellati A, Massari L, Cadossi R, Vincenzi F, Borea PA, Fini M, Caruso A, De Mattei M. Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts. J Cell Physiol. 2012;227:2461–2469. doi: 10.1002/jcp.22981. [PubMed] [CrossRef[]
32. Visavadiya NP, Patel SP, VanRooyen JL, Sullivan PG, Rabchevsky AG. Cellular and subcellular oxidative stress parameters following severe spinal cord injury. Redox Biol. 2016;8:59–67. doi: 10.1016/j.redox.2015.12.011. [PMC free article] [PubMed] [CrossRef[]
33. Yang Y, Bazhin AV, Werner J, Karakhanova S. Reactive oxygen species in the immune system. Int Rev Immunol. 2013;32:249–270. doi: 10.3109/08830185.2012.755176. [PubMed] [CrossRef[]
34. Sheng W, Zong Y, Mohammad A, Ajit D, Cui J, Han D, Hamilton JL, Simonyi A, Sun AY, Gu Z, et al. Pro-inflammatory cytokines and lipopolysaccharide induce changes in cell morphology, and upregulation of ERK1/2, iNOS and sPLA2-IIA expression in astrocytes and microglia. J Neuroinflammation. 2011;8:121. doi: 10.1186/1742-2094-8-121. [PMC free article] [PubMed] [CrossRef[]
35. Wang Q, Chen Q, Ding Q, Yang Q, Peng Y, Lu Y, Deng J, Xiong L. Sevoflurane postconditioning attenuates spinal cord reperfusion injury through free radicals-mediated up-regulation of antioxidant enzymes in rabbits. J Surg Res. 2011;169:292–300. doi: 10.1016/j.jss.2009.11.707. [PubMed] [CrossRef[]
36. Vincenzi F, Ravani A, Pasquini S, Merighi S, Gessi S, Setti S, Cadossi R, Borea PA, Varani K. Pulsed electromagnetic field exposure reduces Hypoxia and inflammation damage in neuron-like and microglial cells. J Cell Physiol. 2017;232:1200–1208. doi: 10.1002/jcp.25606. [PubMed] [CrossRef[]
37. Jacquier-Sarlin MR, Fuller K, Dinh-Xuan AT, Richard MJ, Polla BS. Protective effects of hsp70 in inflammation. Experientia. 1994;50:1031–1038. doi: 10.1007/BF01923458. [PubMed] [CrossRef[]
38. Sevin M, Girodon F, Garrido C, de Thonel A. HSP90 and HSP70: Implication in inflammation processes and therapeutic approaches for myeloproliferative neoplasms. Mediators Inflamm 2015. 2015:970242. [PMC free article] [PubMed[]
39. Shabbir A, Bianchetti E, Cargonja R, Petrovic A, Mladinic M, Pilipovi? K, Nistri A. Role of HSP70 in motoneuron survival after excitotoxic stress in a rat spinal cord injury model in vitro. Eur J Neurosci. 2015;42:3054–3065. doi: 10.1111/ejn.13108. [PubMed] [CrossRef[]

Articles from Molecular Medicine Reports are provided here courtesy of Spandidos Publications

Spinal Cord. 2009 Jul;47(7):508-18. Epub 2009 Jan 27.

Non-pharmacological treatment and prevention of bone loss after spinal cord injury: a systematic review.

Biering-Sørensen F, Hansen B, Lee BS.

Clinic for Spinal Cord Injuries, Rigshospitalet, Hornbaek, Denmark. finbs@rh.regionh.dk

Abstract

OBJECTIVE: Review the literature on non-pharmacological prevention and treatment of osteoporosis after spinal cord injury (SCI).

METHODS: PubMed, EMBASE and the Cochrane Controlled Trials Register were searched. All identified papers were read by title, abstract and full-length article when relevant. Hand search of the articles’ sources identified additional papers. For included studies, the level of evidence was determined.

RESULTS: No studies conclusively showed an effective intervention. However, there are few randomized controlled trials (RCTs), and those that exist assess interventions and outcome measures that could be improved. Five studies on weight-bearing early post-injury are conflicting, but standing or walking may help retain bone mineral. In the chronic phase, there was no effect of weight bearing (12 studies). One study found that an early commencement of sports after SCI improved bone mineral, and the longer the period of athletic career, the higher the (leg) bone mineral. Early after SCI, there may be some effects of electrical stimulation (ES) (five studies). Chronic-phase ES studies vary (14 studies, including mixed periods after injury), but improvement is seen with longer period of training, or higher frequency or stimulus intensity. Improvements correspond to trabecular bone in the distal femur or proximal tibia. Impact vibration and pulsed electromagnetic fields may have some positive effects, whereas pulsed ultrasound does not. Six studies on the influence of spasticity show inconsistent results.

CONCLUSIONS: Bone mineral should be measured around the knee; the length and intensity of the treatment should be sufficiently long and high, respectively, and should commence early after SCI. If bone mineral is to remain, the stimulation has to be possibly continued for long term. In addition, RCTs are necessary.

Spine. 2003 Dec 15;28(24):2660-6.

Exposure to pulsed magnetic fields enhances motor recovery in cats after spinal cord injury.

Crowe MJ, Sun ZP, Battocletti JH, Macias MY, Pintar FA, Maiman DJ.

Neuroscience Research Laboratories, The Clement J. Zablocki VA Medical Center, Milwaukee, WI 53295, USA. mcrowe@mcw.edu

STUDY DESIGN: Animal model study of eight healthy commercial cats was conducted.

OBJECTIVE: To determine whether pulsed electromagnetic field (PMF) stimulation results in improvement of function after contusive spinal cord injury in cats. SUMMARY OF

BACKGROUND DATA: PMF stimulation has been shown to enhance nerve growth, regeneration, and functional recovery of peripheral nerves. Little research has been performed examining the effects of PMF stimulation on the central nervous system and no studies of PMF effects on in vivo spinal cord injury (SCI) models have been reported.

MATERIALS AND METHODS: PMF stimulation was noninvasively applied for up to 12 weeks to the midthoracic spine of cats with acute contusive spinal cord injury. The injury was produced using a weight-drop apparatus. Motor functions were evaluated with the modified Tarlov assessment scale. Morphologic analyses of the injury sites and somatosensory-evoked potential measurements were conducted to compare results between PMF-stimulated and control groups.

RESULTS: There was a significant difference in locomotor recovery between the PMF-stimulated and control groups. Although not statistically significant, PMF-stimulated spinal cords demonstrated greater sparing of peripheral white matter and smaller lesion volumes compared to controls. Somatosensory-evoked potential measurements indicated that the PMF-stimulated group had better recovery of preinjury waveforms than the control group; however, this observation also was not statistically significant because of the small sample size.

CONCLUSIONS: This preliminary study indicates that pulsed magnetic fields may have beneficial effects on motor function recovery and lesion volume size after acute spinal cord injury.

J Spinal Cord Med. 1999 Winter;22(4):239-45.

The effect of pulsed electromagnetic fields on osteoporosis at the knee in individuals with spinal cord injury.

Garland DE, Adkins RH, Matsuno NN, Stewart CA.

Rancho Los Amigos Medical Center, Downey, California 90242, USA.

Abstract

The purpose of this study was to determine the effects of pulsed electromagnetic fields on osteoporotic bone at the knee in individuals with chronic spinal injury. The study consisted of 6 males with complete spinal cord injury at a minimum of 2 years duration. Bone mineral density (BMD) was obtained at both knees at initiation, 3 months, 6 months, and 12 months using dual energy X-ray absorptiometry. In each case, 1 knee was stimulated using The Bone Growth Stimulator Model 3005 from American Medical Electronics, Incorporated and the opposite knee served as the control. Stimulation ceased at 6 months. At 3 months BMD increased in the stimulated knees 5.1% and declined in the control knees 6.6% (p < .05 and p < .02, respectively). By 6 months the BMD returned to near baseline values and at 12 months both knees had lost bone at a similar rate to 2.4% below baseline for the stimulated knee and 3.6% below baseline for the control. There were larger effects closer to the site of stimulation. While the stimulation appeared useful in retarding osteoporosis, the unexpected exaggerated decline in the control knees and reversal at 6 months suggests underlying mechanisms are more complex than originally anticipated. The authors believe a local as well as a systemic response was created.

Zh Nevropatol Psikhiatr Im S S Korsakova. 1990;90(7):108-12.

Regional cerebral angiodystonia in the practice of a neurologist and therapist.

[Article in Russian]

Pokalev GM, Raspopina LA.

Altogether 108 patients with regional cerebral angiodystonia were examined using rheoencephalography, measurements of temporal and venous pressure and functional tests (nitroglycerin and bicycle ergometry). Three variants of abnormalities connected with regional cerebral angiodystonia were distinguished: dysfunction of the inflow, derangement of the venous outflow, and initial functional venous hypertonia. The patients were treated with nonmedicamentous therapy (electroanalgesia, magnetotherapy, iontotherapy).

Bioelectromagnetics. 1987;8(2):159-64.

Pulsed subcutaneous electrical stimulation in spinal cord injury: preliminary results.

Ellis W.

The treatment of long-term, stable para- and quadriplegics with pulsed electrical stimulation for pain control resulted in, anecdotally, a significant number of these individuals showing increased motor function as well as sensory awareness. This small pilot study was conducted in order to assess the hypothesis that pulsed electrical fields can effect diseased neurological function. Thirteen para- and quadriplegic subjects with 18 months of stable neurological signs and symptoms were exposed daily to pulsed electrical stimulation for a 6-month period and assessed for any improvement in motor function or sensory perception. The hypothesis is that pulsed electromagnetic fields can normalize viable but dysfunctional neuronal structures. Results were encouraging.

Surg Neurol. 1987 Oct;28(4):269-76.

Effect of alternating current stimulation of the spinal cord on recovery from acute spinal cord injury in rats.

Wallace MC, Tator CH, Gentles WM.

The therapeutic value of electrical stimulation of the spinal cord was studied in rats injured by acute compression of the spinal cord. Twenty adult Wistar rats underwent cord compression at T6-7 by the extradural clip compression technique at a force of 125 g for 1 minute. After injury and group randomization, stimulating electrodes were placed extradurally, proximal and distal to the injury site, and attached to a small, implantable receiver-stimulator. The receiver was secured to the paraspinal muscles and implanted subcutaneously, overlying the thoracic spine. The animals were maintained in specially designed cages with encircling antennae attached to radio frequency transmitters. The 10 treatment animals were subjected to a 460-kHz electromagnetic field, pulsed at a frequency of 10 Hz. The receivers converted the pulsed radio frequency into square-wave pulses at the cord electrodes (width 1 ms, frequency 10 Hz). The 10 control animals were exposed to a similar field but with a frequency below the range of the tuned receiver, and therefore they did not receive the square-wave pulse. Clinical recovery was assessed by the inclined plane technique which measures the maximum angle of inclination attained without falling. After 15 weeks of continuous spinal cord stimulation, the inclined plane performance was not significantly different between the two groups (treatment group mean, 44.4 +/- 5.4; control group mean, 41.7 +/- 7.9). This is the first experimental study of the effect of long-term continuous electrical stimulation on spinal cord recovery in mammals. The methods required and the technical aspects involved in achieving continuous stimulation, and the guidelines for future study of this modality are discussed.

Paraplegia. 1976 May;14(1):12-20.

Experimental regeneration in peripheral nerves and the spinal cord in laboratory animals exposed to a pulsed electromagnetic field.

Wilson DH, Jagadeesh P.

Peripheral nerve section and suture was performed in 132 rats. Postoperatively half the animals were exposed to a pulsed electromagnetic field each day and half were kept as controls. Nerve conduction studies, histology and nerve fibre counts all indicated an increased rate of regeneration in the treated animals. A similar controlled study of spinal cord regeneration following hemicordotomy in cats has been started, and preliminary results indicate that when the animals are sacrificed three months after the hemicordotomy, the pulsed electromagnetic therapy has induced nerve fibre regeneration across the region of the scar.

Neurosci Behav Physiol. 1998 Sep-Oct;28(5):594-7.

Magnetic and electrical stimulation in the rehabilitative treatment of patients with organic lesions of the nervous system.

Tyshkevich TG, Nikitina VV.

A. L. Polenov Russian Science Research Neurosurgical Institute, St. Petersburg.

Studies were performed on 89 patients with organic lesions of the nervous system in which the leading clinical symptoms consisted of paralysis and pareses. Patients received complex treatment, including pulsed magnetic fields and an electrical stimulation regime producing multilevel stimulation. A control group of 49 patients with similar conditions was included, and these patients received only sinusoidal currents. Combined treatment with magnetic and electrical stimulation was more effective, as indicated by radiographic and electromyographic investigations.