Static Magnetic Field Stimulation Enhances Oligodendrocyte Differentiation and Secretion of Neurotrophic Factors

Abstract

The cellular-level effects of low/high frequency oscillating magnetic field on excitable cells such as neurons are well established. In contrast, the effects of a homogeneous, static magnetic field (SMF) on Central Nervous System (CNS) glial cells are less investigated. Here, we have developed an in vitro SMF stimulation set-up to investigate the genomic effects of SMF exposure on oligodendrocyte differentiation and neurotrophic factors secretion. Human oligodendrocytes precursor cells (OPCs) were stimulated with moderate intensity SMF (0.3 T) for a period of two weeks (two hours/day). The differential gene expression of cell activity marker (c-fos), early OPC (Olig1, Olig2. Sox10), and mature oligodendrocyte markers (CNP, MBP) were quantified. The enhanced myelination capacity of the SMF stimulated oligodendrocytes was validated in a dorsal root ganglion microfluidics chamber platform. Additionally, the effects of SMF on the gene expression and secretion of neurotrophic factors- BDNF and NT3 was quantified. We also report that SMF stimulation increases the intracellular calcium influx in OPCs as well as the gene expression of L-type channel subunits-CaV1.2 and CaV1.3. Our findings emphasize the ability of glial cells such as OPCs to positively respond to moderate intensity SMF stimulation by exhibiting enhanced differentiation, functionality as well as neurotrophic factor release.

Figure 1

Schematic Illustration of the SMF stimulation set-up. (a) Two parallel magnets with opposite polarity produce a uniform static magnetic field in the central core. The magnetic induction range represented by the shaded blue columns. The field intensity varies from 0.2 T at the center line (white dashed) to 0.4 T at the base of the two magnets. The OPCs are placed at a distance of 1 cm from the base to expose them to 0.3 T magnetic field. (b) The characterisation of the magnetic field strength with respect to the vertical distance from the center line towards the base of the two magnets.

Figure 2

Characterisation of the oligodendrocyte precursor cells. (a) Representative immunofluorescence images of OPCs stained for O4 and Olig 1, (b) Estimate of the percentage of cells staining positive for OPC markers. (c) Representative immunofluorescence images of mature OLs stained for CNP and MBP, (d) Estimate of the percentage of cells staining positive for OL markers. Data represented as mean ± SEM. Scale bar = 2 µm.

Figure 3

Effects of SMF on oligodendrocyte differentiation and neurotrophic factor release. (a) Quantification of gene fold changes in SMF stimulated oligodendrocytes as compared to control (non-stimulated), (b) mRNA expression levels of neurotrophins BDNF and NT3 in SMF stimulated oligodendrocytes as compared to control, (c,d) Cumulative release profile of BDNF and NT3 (*p < 0.05, **p < 0.01) in SMF stimulated and non-stimulated cells.

Figure 4

Effects of SMF on OPC proliferation. (a) Representative images of OPC stained with O4 and Ki67 markers, (b) Quantification of Ki67 positive cells in SMF stimulated OPCs and control, (c) mRNA expression level of proliferative marker Ki67 in SMF stimulated OPCs as compared to control OPCs. Data represented as mean ± SEM. Scale bar = 20 µm.

Figure 5

SMF effects on intracellular calcium levels and L-type channel subunits. (a) Representative scans from the time-lapse calcium imaging experiments showing some responding cells (white arrows) post KCL stimulation at 30 seconds, (b) Representative calcium response traces, grey arrow represents the KCL stimulation at 30 sec (c) Summary data showing significantly higher calcium transient amplitudes in SMF stimulated OPCs (n = 12 cells), (d) Quantification of mRNA expression levels of CaV1.2 and CaV1.3 in SMF stimulated OPCs as compared to control OPCs. Scale bar = 20 µm. *p < 0.05, **p < 0.01. Error bars indicate SEM.

Figure 6

Investigation of myelination potential of SMF stimulated OPCs in DRG microfluidic chamber. (a) Illustration of microfluidic chamber with axonal and soma compartments connected with 10 μm wide and 500 μm long microchannels. DRG neurons (stained with Calcein) were plated in the somatic chamber and only axons (no cell body) extends to the axonal chamber. (b) Representative image demonstrating the wrapping of axons stained with Neurofilament-200 by MOBP+ oligodendrocytes. 3-D analysis of the z-stack images indicate co-localisation of myelinating cells (green) with axons (red) in the z-domain, (c) Representative image demonstrating an increase in myelinating cells (grey arrow) and decrease in nude axons (yellow arrow) in chambers containing SMF stimulated oligodendrocytes as compared to non-stimulated oligodendrocytes. The control OL chamber has fewer cells making contact with the axons. (d) Quantification of number of MOBP+ oligodendrocytes and nude axons. Scale bar = 20 µm, (**p < 0.01).