Enhanced effect of combining bone marrow mesenchymal stem cells (BMMSCs) and pulsed electromagnetic fields (PEMF) to promote recovery after spinal cord injury in mice

Abstract

Spinal cord injury (SCI) is a destructive traumatic disease of the central nervous system without satisfying therapy efficiency. Bone marrow mesenchymal stem cells (BMMSCs) therapy promotes the neurotrophic factors' secretion and axonal regeneration, thereby promoting recovery of SCI. Pulsed electromagnetic fields (PEMF) therapy has been proven to promote neural growth and regeneration. Both BMMSCs and PEMF have shown curative effects for SCI; PEMF can further promote stem cell differentiation. Thus, we explored the combined effects of BMMSCs and PEMF and the potential interaction between these two therapies in SCI. Compared with the SCI control, BMMSCs, and PEMF groups, the combinational therapy displayed the best therapeutic effect. Combinational therapy increased the expression levels of nutritional factors including brain-derived neurotrophic factor (BDNF), nerve growth factors (NGF) and vascular endothelial growth factor (VEGF), enhanced neuron preservation (NeuN and NF-200), and increased axonal growth (MBP and myelin sheath). Additionally, PEMF promoted the expression levels of BDNF and VEGF in BMMSCs via Wnt/β-catenin signaling pathway. In summary, the combined therapy of BMMSCs and PEMF displayed a more satisfactory effect than BMMSCs and PEMF therapy alone, indicating a promising application of combined therapy for the therapy of SCI.

Keywords: bone marrow mesenchymal stem cells; motor functional recovery; nutritional factors; pulsed electromagnetic fields; spinal cord injury.

FIGURE 1

BMMSCs and PEMF promote functional recovery and reduce the injury volume in SCI mice. (A) Schematics of PEMF intervention in vivo. (B) Hindlimb locomotion after contusion injury in mice of the sham, SCI control, BMMSCs, PEMF, and BMMSCs+PEMF groups evaluated weekly from 1 to 56 days using the Basso Mouse Score (BMS) (n = 10). (C, D) Photograph of regular horizontal ladder (C) and quantification of error rate of hindlimbs. There was significant difference between SCI control and BMMSCs+PEMF at week 7 and week 8 (n = 5). (E) Representative footprint analysis images of the sham, SCI control, BMMSCs, PEMF, and BMMSCs+PEMF groups at week 8. (F, G) Sagittal images of the T10 injury epicenter were obtained by MRI at week 8. PEMF and BMMSCs+PEMF statistically decreased the injury volume compared with SCI control group (n = 7). (Error bars show mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with SCI control, ^# p < 0.05, compared with BMMSCs group, ^& p < 0.05, compared with PEMF group.)

FIGURE 2

BMMSCs and PEMF reduce the injury volume and increase Nissl bodies in vivo. (A) Representative images of fibrous scar formation in the different groups. Area of fibrous scar was indicated by black dotted line (scale bar 200 μm). (B) Representative images of Nissl bodies in the different groups. Representative Nissl bodies were indicated with arrows (Scale bar 100 μm). (C) Percentage of fibrous scar area in the BMMSCs+PEMF group was significantly decreased compared to SCI control group. (D) Combination of BMMSCs and PEMF statistically increased the number of Nissl bodies compared to SCI control group, PEMF group and BMMSCs group. (Error bars show mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.)

FIGURE 3

BMMSCs and PEMF increase the expression levels of BDNF, NGF and VEGF in vivo. (A) Representative immunofluorescence images of BDNF (red), astroglial maker GFAP (green), and DAPI (blue) in spinal cord lesions at week 8. (B) Representative immunofluorescence images of VEGF (red), astroglial maker GFAP (green) and DAPI (blue) in spinal cord lesions at week 8. (C) Representative immunofluorescence images of NGF (green) and DAPI (blue) in spinal cord lesions at week 8. (D) Quantification of the relative intensity of BDNF showed BMMSCs+PEMF group shows highest expression level of BDNF and had significant difference compared with SCI control group and BMMSCs group. PEMF group also increases BDNF expression compared with SCI control group. (E) Quantification of the relative intensity of VEGF. VEGF expression in the BMMSCs+PEMF group was higher than those in the SCI control, BMMSCs and PEMF group. (F) Quantification of the relative intensity of NGF. There was statistical difference between SCI control and BMMSCs+PEMF group (scale bar 200 μm. Error bars show mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)

FIGURE 4

BMMSCs and PEMF promote neuron preservation in vivo. (A) Immunofluorescence staining of astroglial maker GFAP (green), NeuN (red), and DAPI (blue) in spinal cord lesions at week 8 (transverse section). High magnifications of boxed area are displayed below (A–J) (scale bar 100 μm). (B) Immunofluorescence staining of astroglial maker GFAP (green), NeuN (red), and DAPI (blue) in spinal cord lesions at week 8 (longitudinal section). High magnifications of boxed area are displayed below (K–O) (scale bar 200 μm). (C) Quantification of the relative expression levels of NeuN (transverse section). BMMSCs+PEMF significantly enhanced the expression of NeuN after SCI. (D) Quantification of the relative expression levels of NeuN (longitudinal section). BMMSCs+PEMF significantly enhanced the expression of NeuN after SCI. (Error bars show mean ± SD; *p < 0.05.)

FIGURE 5

BMMSCs and PEMF alleviate axon destruction and promote neurofilament preservation in vivo. (A) Representative immunofluorescence images of MBP (green) and DAPI (blue) in the sham, SCI control, BMMSCs, PEMF, and BMMSCs+PEMF groups (scale bar 200 μm, left; Scale bar 200 μm, right). (B) Representative immunofluorescence images of NF‐200 (red), astroglial maker GFAP (green) and DAPI (blue) in spinal cord lesions at week 8 (scale bar 200 μm). (C) Representative TEM images of the Sham, SCI control, BMMSCs, PEMF and BMMSCs+PEMF groups (scale bar = 2 μm). (D) Quantification of the relative expression levels of MBP. BMMSCs+PEMF and PEMF increased the expression of MBP. (E) The expression level of NF‐200 in the BMMSCs+PEMF group was significantly higher than those in the SCI control, BMMSCs, and PEMF groups. PEMF also increased NF‐200 expression compared with SCI control and BMMSCs group. (F) Quantitative analysis of G‐ratio values. There was statistical difference between SCI control and BMMSCs+PEMF group. (Error bars show mean ± SD; *p < 0.05, **p < 0.01, ****p < 0.0001.)

FIGURE 6

BMMSCs and PEMF increase the expression levels of nutritional cytokines and neuronal markers. (A) The protein levels of BDNF, NGF, and VEGF were investigated by western blotting. GAPDH or β‐actin was used as the loading control. (B) Quantification of the relative expression levels of BDNF, NGF, and VEGF. (C) The protein levels of NF‐200, NeuN, and MBP were analyzed by western blotting. (D) Quantification of the relative expression levels of NF‐200, NeuN, MBP. (E) The protein levels of β‐catenin, integrin β1, and integrin α5 were evaluated by western blotting. (F) Quantification of the relative expression levels of Wnt3a, β‐catenin, integrin β1, and integrin α5. (Error bars show mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)

FIGURE 7

PEMF promotes the expression levels of BDNF and VEGF in BMMSCs in vitro through Wnt/β‐catenin signaling pathway. (A) Representative immunofluorescence images of BMMSCs that were stained for BDNF, NGF, VEGF, and Ki67 (scale bar = 20 μm). (B) The protein levels of BDNF, NGF, and VEGF were analyzed by western blotting. (C) Quantification of the relative expression levels of BDNF, NGF, and VEGF. PEMF 3d group displayed highest expression levels of BDNF among all groups. VEGF was significantly increased in the PEMF 3d group compared with other groups. (D) The protein levels of β‐catenin were analyzed by western blotting. (E) Quantification of the relative expression levels of β‐catenin showed PEMF 3d significantly increased the expression of β‐catenin compared with control group. (F) Co‐IP analysis showed the enhanced interaction between LRP6 and Frizzled5 after PEMF intervention in 293T cells. (G) Representative immunofluorescence images of β‐catenin (red), phalloidine (green), and DAPI (blue) in BMMSCs for all groups. Wnt signaling pathway inhibitor (IWR‐1) significantly inhibited the expression of β‐catenin (scale bar = 20 μm). (H) Wnt signaling pathway inhibitor (IWR‐1) successfully inhibited the expression of β‐catenin. (I) Quantification of the expression levels of β‐catenin. (J) The protein levels of BDNF and VEGF were analyzed by western blotting. (K) The effect of PEMF on the expression of BDNF and VEGF in BMMSCs was partially abolished by a Wnt signaling pathway inhibitor (IWR‐1). (Error bars show mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)