Anat Cell Biol.  2015 Jun;48(2):104-113. 10.5115/acb.2015.48.2.104.

The effects of repetitive transcranial magnetic stimulation on proliferation and differentiation of neural stem cells

• Abbasnia K
• Ghanbari A
• Abedian M
• Ghanbari A
• Sharififar S
• Azari H

• KMID: 1845278
• DOI: http://doi.org/10.5115/acb.2015.48.2.104

Abstract

Repetitive transcranial magnetic stimulation (rTMS) is a new method for treating many neurological conditions; however, the exact therapeutic mechanisms behind rTMS-induced plasticity are still unknown. Neural stem and progenitor cells (NS/PCs) are active players in brain regeneration and plasticity but their behavior in the context of rTMS therapy needs further elucidation. We aimed to evaluate the effects of rTMS on proliferation and differentiation of NS/PCs in the subventricular zone (SVZ) of adult mouse brain. Adult male mice (n=30) were divided into rTMS (1-Hz and 30-Hz) and sham groups and treated for 7 or 14 consecutive days. Harvested NS/PCs from the SVZ were cultured in the neurosphere assay for 8 days and the number and size of the resulting neurospheres as well as their in vitro differentiation capacity were evaluated. After one week of rTMS treatment at 1-Hz and 30-Hz compared with sham stimulation, the mean neurosphere forming frequency per brain was not different while this measure significantly increased after two weeks (P<0.05). The mean neurosphere diameter in 1-Hz treatment paradigm was significantly larger compared with sham stimulation at both 1 and 2 weeks. In contrast, 30-Hz treatment paradigm resulted in significantly larger neurospheres only after 2 weeks. Importantly, rTMS treatment at both frequencies increased neuronal differentiation of the harvested NS/PCs. Furthermore, one week in vitro rTMS treatment of NS/PCs with both 1-Hz and 30-Hz increased NS/PCs proliferation and neuronal differentiation. It is concluded that both 1-Hz and 30-Hz rTMS treatment increase NS/PCs proliferation and neuronal differentiation.

Keyword

rTMS; Neural stem and progenitor cells; Subventricular zone; Proliferation; Neurosphere assay

MeSH Terms

Adult
Animals
Brain
Humans
Male
Mice
Neural Stem Cells*
Neurons
Plastics
Regeneration
Stem Cells
Transcranial Magnetic Stimulation*
Plastics

Figure

• 

  Fig. 1 Neurosphere formation and differentiation of the subventricular zone (SVZ) neural stem and progenitor cells (NS/PCs). (A) Isolated neurospheres from the SVZ expressed (upper panel) nestin (green) and glial fibrillary acidic protein (GFAP) as putative neural stem cell markers. Dissociated neurospheres (NS/PCs) were capable of differentiating into astrocytes (lower panels, GFAP positive, green color), neurons (lower left, β-III tubulin positive, red color), and oligodendrocytes (lower right, O4 positive, red color). Cell nuclei were counterstained with DAPI (blue color). (B) Mean neurosphere-forming frequency per brain after 1 week of repetitive transcranial magnetic stimulation (rTMS) treatments. No significant differences were detected between the rTMS and sham treated groups. (C) Mean neurosphere-forming frequency per brain after 2 weeks of rTMS treatment. As evident, neurosphere formation significantly increased in both the 1-Hz and 30-Hz rTMS treated groups (**P<0.01, ***P<0.001). (D) Relative neurosphere numbers in different rTMS groups treated for 1 or 2 weeks. As shown, both treatment paradigms were clearly more effective upon 2 weeks rTMS application (*P<0.05, **P<0.01, ***P<0.001). Data are presented as a percent change relative to matched sham control values. Values are mean±SEM. Scale bar=100 µm (A, upper), 50 µm (A, lower).

• 

  Fig. 2 Mean neurosphere diameter change following in vivo exposure of neural stem and progenitor cells to different repetitive transcranial magnetic stimulation paradigms. (A) Representative neurospheres from the sham, 1-Hz and 30-Hz treated groups from both 1-week and 2-week treatment paradigms after 8 days in neurosphere culture. (B) Percent neurosphere diameter change in 1-week treatment group showed that only 1-Hz group resulted in significantly larger neurospheres (*P<0.05). (C) Percent neurosphere diameter change in 2-week treatment group showed that both 1-Hz and 30-Hz groups resulted in significantly larger neurospheres (*P<0.05, **P<0.01). (D) Comparing percent neurosphere diameter changes in 1-week versus 2-week treatment groups showed that 30-Hz at 2-week treatment group could significantly result in larger neurospheres (*P<0.05).

• 

  Fig. 3 Differentiation of the in vivo repetitive transcranial magnetic stimulation (rTMS) exposed neural stem and progenitor cells. (A) Representative pictures of differentiated neural stem and progenitor cells from the sham, 1-Hz and 30-Hz treated groups from both 1-week and 2-week treatment paradigms. Astrocyte (green) expressed glial fibrillary acidic protein (GFAP) and neuronal cells (red) expressed β-III tubulin. DAPI was used to stain cell nuclei. (B, C) Graphs show the percentage of β-III tubulin immuonreactive (IR) cells relative to sham groups in 1-week (B) and 2-week (C) treatment paradigms. 1-Hz rTMS could only significantly increase neuronal cell differentiation in 2-week paradigm (***P<0.001) but 30-Hz rTMS resulted in statistically significant neuronal differentiation in both 1-week and 2-weeks paradigms (**P<0.01). (D) Comparing percentage of β-III tubulin-IR cells in 1-week versus 2-week treatment paradigms showed that applying 1-Hz rTMS for 2 weeks could significantly result in more neuronal cell differentiation compared to the 1-week treatment paradigm (**P<0.01). Also, applying 30-Hz rTMS for 2 weeks significantly increased neuronal cell differentiation compared to 1-week application of both 1-Hz and 30-Hz rTMS (*P<0.05). (E, F) Graphs show the percentage of GFAP-IR cells in 1-Hz and 30-Hz rTMS treatment paradigms relative to sham groups in 1-week (E) and 2-week (F) treatment paradigms. No significant changes were noticed. (G) Comparing percentage of GFAP-IR cells in 1-week versus 2-week treatment paradigms showed that applying 1-Hz rTMS for 1-week could significantly result in more astrocytic differentiation as compared to the 1-week 30-Hz and 2-week 1-Hz rTMS treatment (*P<0.05).

• 

  Fig. 4 Proliferation and differentiation of the in vitro repetitive transcranial magnetic stimulation (rTMS) exposed neural stem and progenitor cells. (A) Percent change in total number of neural stem and progenitor cells after 7 days of culture in sham, 1-Hz and 30-Hz treated conditions. Both 1-Hz and 30-Hz rTMS treatment significantly increased NS/PCs proliferation as compared to the sham condition (*P<0.05, ***P<0.001). Also, 30-Hz rTMS significantly increased NS/PCs proliferation comparing to 1-Hz rTMS application (*P<0.05). (B) Representative pictures of differentiating NS/PCs cultures that were exposed to the sham, 1-Hz and 30-Hz rTMS for 1 week. Astrocyte (green) expressed glial fibrillary acidic protein (GFAP) and neuronal cells (red) expressed β-III tubulin. DAPI was used to stain cell nuclei. (C) Comparing percentage of β-III tubulin-immunoreactive (IR) cells in sham, 1-Hz and 30-Hz treated conditions showed that both frequencies increased neuronal differentiation of rTMS treated NS/PCs (*P<0.05). (C) Comparing percentage of GFAP-IR cells in sham, 1-Hz and 30-Hz treated conditions showed no significant differences between groups.

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