Treatment with Pulsed Extremely Low Frequency Electromagnetic Field (PELF-EMF) Exhibit Anti-Inflammatory and Neuroprotective Effect in Compression Spinal Cord Injury Model

Abstract

Background: Spinal cord injury (SCI) pathology includes both primary and secondary events. The primary injury includes the original traumatic event, and the secondary injury, beginning immediately after the initial injury, involves progressive neuroinflammation, neuronal excitotoxicity, gliosis, and degeneration. Currently, there is no effective neuroprotective treatment for SCI. However, an accumulating body of data suggests that PELF-EMF has beneficial therapeutic effects on neurotrauma. The purpose of this study was to test the efficacy of the PELF-EMF SEQEX device using a compression SCI mouse model.

Methods: C57BL/6 mice were exposed to PELF-EMF for 4 h on a daily basis for two months, beginning 2 h after a mild-moderate compression SCI.

Results: The PELF-EMF treatment significantly diminished inflammatory cell infiltration and astrocyte activation by reducing Iba1, F4/80, CD68+ cells, and GAFP at the lesion borders, and increased pro-survival signaling, such as BDNF, on the neuronal cells. Moreover, the treatment exhibited a neuroprotective effect by reducing the demyelination of the axons of the white matter at the lesion's center.

Conclusions: Treatment with SEQEX demonstrated significant anti-inflammatory and neuroprotective effects. Considering our results, this safe and effective rehabilitative device, already available on the market, may provide a major therapeutic asset in the treatment of SCI.

Keywords: electromagnetic field; neuroprotection; rehabilitation; spinal cord injury; treatment.

Figure 1

Decreased microglia activation and astrogliosis in PELF-EMF-treated mice after SCI. Two weeks after SCI (A) Representative images of the lesion site of Iba1 (green) immunostaining; DAPI (blue) demonstrate the lesion site. The scale bar is 200 μm. (B) Quantitation of Iba1 at the lesion site shows a significant decrease in the PELF-EMF-treated compared to the non-treated mice (n = 8 in each group; *** p < 0.001). (C) Representative images of the lesion site of GFAP (green) immunostaining; DAPI (blue) demonstrate the lesion site. The scale bar is 200 μm. (D) Quantitation of GFAP at the lesion site shows a significant decrease in the PELF-EMF-treated compared to the non-treated mice (n = 8 in each group; ** p < 0.01).

Figure 2

Decrease of other microglia activation markers in PELF-EMF-treated mice after SCI. Two weeks after SCI (A) Representative images of the lesion site of CD68 (green) immunostaining; DAPI (blue) demonstrate the lesion site. The scale bar is 100 μm. (B) Quantitation of CD68 at the lesion site shows a significant decrease in the PELF-EMF-treated compared to the non-treated mice. The results are mean ± SD (n = 8/group; *** p < 0.001). (C) Representative images of the lesion site of F4/80 (green) immunostaining; DAPI (blue) demonstrate the lesion site. The scale bar is 100 μm. (D) Quantitation of F4/80 at the lesion site shows a significant decrease in the PELF-EMF-treated compared to the non-treated mice. The results are mean ± SD (n = 8 in each group; *** p < 0.001).

Figure 3

The increased axonal survival and BDNF expression in PELF-EMF-treated mice after SCI. Two months after SCI (A) Representative images of the white matter at the lesion site of the βIII-tubulin immunostaining; the scale bar is 100 μm. (B) Quantitation of the βIII-tubulin density immunostaining at the lesion site shows a significant increase in the PELF-EMF-treated compared to the non-treated mice. The results are mean ± SD (the red box represents the area of analysis; n = 10 in each group; * p < 0.05). (C) Quantitation of the BDNF (green) expression density in neuronal cells (NeuN in red) close to the lesion site shows a significant increase in the PELF-EMF-treated compared to the non-treated mice. The results are mean ± SD (n = 10 in each group; *** p < 0.001). (D) Representative images of lesion site of the BDNF (green) and NeuN (red) immunostaining; DAPI (blue) demonstrate the lesion site. The scale bar is 100 μm.

Figure 4

The reduced axonal demyelination and increased MBP expression in PELF-EMF-treated mice after SCI. Two months after SCI (A) Representative images both sides of the white matter of the spinal cord stained with LFB. The injury epicentre (0) is marked in the red box. The scale bar is 100 μm. (B) Quantitative analysis of residual myelin in different rostral and caudal distances from the epicentre of the white matter in the PELF-EMF and the control treated groups shows a significant decrease in demyelination in the PELF-EMF-treated mice. Data represent the mean ± SD (n = 10/group; * p < 0.05). (C) Representative images of the white matter at the lesion site with MBP immunostaining; the scale bar is 100 μm. (D) Quantitation of the MBP density immunostaining at the lesion site shows a significant increase in the PELF-EMF-treated compared to the non-treated mice. Results are mean ± SD (n = 10/group; *** p < 0.001).

Figure 5

The improved motor function in the PELF-EMF-treated mice after SCI. Two months after SCI, the motor function recovery of the mice was assessed using different behavioral tests. (A) mBBB scores demonstrated a significant improvement in the first week after SCI in the PELF-EMF-treated mice compared to the control; the subsequent weeks showed no difference between the groups (n = 10 animals/group. The results are the mean ± SEM (* p < 0.05; non-parametric Mann–Whitney test, α set to 5%). The CatWalk analysis of swing duration (B), stride length (C), and the base of support (D) (n = 10 animals/group); the data are expressed as the mean ± SEM, one-way ANOVA followed by Bonferroni’s multiple comparison test; * p < 0.05, *** p < 0.001).