The Regenerative Effect of Trans-spinal Magnetic Stimulation After Spinal Cord Injury: Mechanisms and Pathways Underlying the Effect

Abstract

Spinal cord injury (SCI) leads to a loss of sensitive and motor functions. Currently, there is no therapeutic intervention offering a complete recovery. Here, we report that repetitive trans-spinal magnetic stimulation (rTSMS) can be a noninvasive SCI treatment that enhances tissue repair and functional recovery. Several techniques including immunohistochemical, behavioral, cells cultures, and proteomics have been performed. Moreover, different lesion paradigms, such as acute and chronic phase following SCI in wild-type and transgenic animals at different ages (juvenile, adult, and aged), have been used. We demonstrate that rTSMS modulates the lesion scar by decreasing fibrosis and inflammation and increases proliferation of spinal cord stem cells. Our results demonstrate also that rTSMS decreases demyelination, which contributes to axonal regrowth, neuronal survival, and locomotor recovery after SCI. This research provides evidence that rTSMS induces therapeutic effects in a preclinical rodent model and suggests possible translation to clinical application in humans.

Keywords: Rehabilitation; glial scar; magnetic stimulation and neuroregeneration; spinal cord injury.

Fig. 1

rTSMS-based treatment modulates spinal scar formation after SCI. (A) Experimental design, SCIs were performed on mice (day 0), then half of the mice has been treated using rTSMS during 2 weeks (from day 1 to day 14). At day 15 and day 90, mice were fixed and immunohistological analyses were performed. At day 15 (B–D, H–J, N–P) and day 90 (E–G, K–M), two groups of mice were fixed and immunohistological analyses were performed. (B, C, E, F, H, I, K, L, N, O) Representative pictures of sagittal spinal cord sections of (B, E, H, K, N) control (SCI) and (C, F, I, L, O) Stm (rTSMS treated) animals 15 and 90 days after SCI. Sections were stained with (B, C, E, F) PDGFRβ, (H, I, K, L) GFAP, (N, O) Iba1, and DAPI. (B–D, E–G) Fibrosis-positive area (PDGFRβ+) and quantification of PDGFRβ+ area 15 and 90 days after SCI. (H–J, K–M) Astrocytic-negative area (GFAP−) and quantification of GFAP− area 15 and 90 days after SCI. (N–P) Iba1-positive area (Iba1+) and quantification of Iba1+ area 15 days after SCI. Scale bars are 200 μm. N = 5 animals per group at 15 days and N = 4 animals per group at 90 days. Quantifications are expressed as average ± SEM. * = p < 0.05; ** = p < 0.01

Fig. 2

rTSMS-based treatment enhances functional recovery. (A) Experimental design, SCIs were performed on mice (day 0), then half of the mice has been treated using rTSMS during 2 weeks (from day 1 to day 14). At day 15 and day 30, functional recovery was analyzed using locotronic test. (B–G) At days 15 and 30, functional recovery was analyzed for the two groups of mice using locotronic test. (B, E) Quantification of the number of back leg errors 15 and 30 days after SCI. (C, F) Quantification of the total back leg error time 15 and 30 days after SCI. (D, G) Quantification of the total crossing time 15 and 30 days after SCI. N = 6–7 animals per group. Dashed lines correspond to the baseline parameters obtained during locotronic habituation (7 days before SCI). Quantifications are expressed as average ± SEM. * = p < 0.05; ** = p < 0.01, *** = p < 0.001

Fig. 3

rTSMS regulates functional protein synthesis and pathways. (A) Workflow for quantitative proteomics and data analyses of spinal cord tissue between stimulated (rTSMS treated) and control (SCI) mice. (B) Modulation of biological functions involved in neural development and cell death from differentially regulated proteins in stimulated mice compared to controls. Analysis was performed using Ingenuity Pathway Analysis (IPA, Qiagen). Statistical significance is proposed through the calculation of p values and z-scores. (C) Functional regulatory effects of upregulated (red) or downregulated (green) proteins belonging to the different pathways associated to neural development and cell death (red arrows: activation; green arrows: inhibition; orange nut: gain of biological function; green nut: decrease of biological function)

Fig. 4

rTSMS inhibits demyelination and enhances neuronal survival and axonal regrowth. (A, B) Representative pictures of sagittal spinal cord sections of (A) control (SCI) and (B) Stm (rTSMS treated) animals 15 days after SCI. Sections were stained with MBP and DAPI. (C) Quantification of MBP-negative area (MBP−) 15 days after SCI. Scale bars are 200 μm. N = 5–6 animals per group. (D, E) Representative pictures of axial spinal cord sections of (D) control and (E) Stm animals 15 days after SCI. Sections were stained with NeuN and DAPI. (F) Quantification of NeuN+ cells 15 days after SCI. Scale bars are 200 μm. Dashed line corresponds to the number of NeuN+ cells from non-injured spinal cord. N = 4 animals per group. (G, H) Design of the BDA-labeled axons experiments. SCIs were performed on mice (day 0), 2 days after mice have been injected with BDA. Then, half of the mice has been treated using rTSMS during 2 weeks (from day 2 to day 15). At day 16, mice were fixed and immunohistological analyses were performed on control and Stm mice. (I, J) Representative pictures of sagittal spinal cord sections of (I) control and (J) Stm mice 16 days after SCI. Sections were stained with BDA and DAPI. Dashed lines demarcate proximal (PB) and distal (DB) borders around lesion core (LC). Images are representative of n = 3 animals per group. Scale bars are 200 μm. Quantifications are expressed as average ± SEM. * = p < 0.05

Fig. 5

rTSMS increases proliferation and modulates differentiation of spinal cord stem cells. (A) Experimental design, SCIs were performed on mice (day 0), then half of the mice has been treated using rTSMS during 2 weeks (from day 1 to day 14). At day 15, mice were fixed and neurosphere assays (B) or immunohistological analyses (C–K) were performed. (B) Quantification of the total number of neurospheres derived from 100,000 cells from control (SCI) and Stm (rTSMS treated) mice 15 days after injury. N = 4 animals per group. (C) Quantification of mean fluorescence intensity of tomato+ cells 15 days after SCI. (D, E, F, G, I, J) Representative pictures of axial spinal cord sections of (D, F, I) control and (E, G, J) Stm animals 15 days after SCI. Sections were stained with tomato and DAPI (D, E). Scale bars are 250 μm. (F–K) Identification of cells derived from Tomato+ recombined ependymal cells were identified by GFAP (F, G) and Sox10 (I, J). (H, K) Quantification of double positive recombined cells in the spinal cord 15 days after SCI. Scale bars are 100 μm. N = 5 animals per group. Quantifications are expressed as average ± SEM. * = p < 0.05

Fig. 6

rTSMS-based treatment enhances functional recovery in a chronic condition. (A) Experimental design, SCIs were performed on mice (day 0), then 11 days after the same mice have been treated using rTSMS during 2 weeks (from day 11 to day 24). At days 25 and 40, functional recovery was analyzed using locotronic test. At day 90, mice were fixed and immunohistological analyses were performed. (B) Quantification of the number of back leg errors 25 days and 40 days after SCI. (C) Quantification of the total back leg error time 25 days and 40 days after SCI. (D) Quantification of the total crossing time 25 and 40 after SCI. N = 9 animals at 25 days and N = 6 animals at 40 days. Dashed lines correspond to the baseline parameters obtained during locotronic habituation (7 days before SCI). (E–J) At day 90, mice were fixed and immunohistological analyses were performed and Stm (rTSMS treated) animals were compared to control (SCI) mice. (E, F, H, I) Representative pictures of sagittal spinal cord sections of (E, H) control and (F and I) Stm mice 90 days after SCI. Sections were stained with (E, F) PDGFRβ and (H, I) GFAP and DAPI. (E–G) Fibrosis-positive area (PDGFRβ+) and quantification of PDGFRβ+ area 90 days after SCI. (H–J) Astrocytic-negative area (GFAP−) and quantification of GFAP− area 90 days after SCI. Scale bars are 200 μm. N = 4 animals per group. Quantifications are expressed as average ± SEM. * = p < 0.05; ** = p < 0.01

Fig. 7

rTSMS-based treatment enhances functional recovery and modulates scar formation in a chronic condition in juvenile mice. (A) Experimental design, SCIs were performed on mice (day 0), then 11 days after the same mice have been treated using rTSMS during 2 weeks (from day 11 to day 24). At day 25 and 40, functional recovery was analyzed using locotronic test. At day 90, mice were fixed and immunohistological analyses were performed. (B) Quantification of the number of back leg errors 25 days and 40 days after SCI. (C) Quantification of the total back leg error time 25 days and 40 days after SCI. (D) Quantification of the total crossing time 25 days and 40 days after SCI. N = 7 animals. Dashed lines correspond to the baseline parameters obtained during locotronic habituation (7 days before SCI). (E–G) At day 90, juvenile mice were fixed and immunohistologic analyses were performed and Stm (rTSMS treated) animals were compared to control (SCI) mice. (E, F) Representative pictures of sagittal spinal cord sections of (E) control and (F) Stm juvenile mice 90 days after SCI. Sections were stained with PDGFRβ, GFAP, and DAPI. (E–G) Fibrosis-positive area (PDGFRβ+) and quantification of PDGFRβ+ area 90 days after SCI. Scale bars are 200 μm. N = 5 animals per group. Quantifications are expressed as average ± SEM. * = p < 0.05; ** = p < 0.01, *** = p < 0.001

Fig. 8

rTSMS-based treatment enhances functional recovery and modulates scar formation in a chronic condition in aged mice. (A) Experimental design, SCIs were performed on mice (day 0), then 11 days after the same mice have been treated using rTSMS during 2 weeks (from day 11 to day 24). At days 25 and 40, functional recovery was analyzed using locotronic test. At day 90, mice were fixed and immunohistological analyses were performed. (B) Quantification of the number of back leg errors 25 days and 40 days after SCI. (C) Quantification of the total back leg error time 25 days and 40 days after SCI. (D) Quantification of the total crossing time 25 and 40 after SCI. N = 7 animals at 25 days and N = 5 animals at 40 days. Dashed lines correspond to the baseline parameters obtained during locotronic habituation (7 days before SCI). (E–G) At day 90, aged mice were fixed and immunohistological analyses were performed and Stm (rTSMS treated) animals were compared to control (SCI) mice. (E, F) Representative pictures of sagittal spinal cord sections of (E) control and (F) Stm aged mice 90 days after SCI. Sections were stained with PDGFRβ, GFAP, and DAPI. (E–G) Fibrosis-positive area (PDGFRβ+) and quantification of PDGFRβ+ area 90 days after SCI. Scale bars are 200 μm. N = 4 animals per group. Quantifications are expressed as average ± SEM. ns = not significant * = p < 0.05; ** = p < 0.01