Comparison of the effects of two therapeutic strategies based on olfactory ensheathing cell transplantation and repetitive magnetic stimulation after spinal cord injury in female mice

Abstract

Spinal cord injury (SCI) is a debilitating condition, which leads to a permanent loss of functions below the injury site. The events which take place after SCI are characterized by cellular death, release of inhibitory factors, and inflammation. Many therapies have been studied to cure SCI, among them magnetic stimulation aims to reduce the secondary damages in particular by decreasing apoptosis, while, cellular transplantation promotes neuroregeneration by enhancing axonal regrowth. In the present study, we compared individually primary olfactory ensheathing cell (OEC) transplantation and repetitive trans-spinal magnetic stimulation (rTSMS) and then, we combined these two therapeutic approaches on tissue repair and functional recovery after SCI. To do so, SCIs were performed at Th10 level on female C57BL/6 mice, which were randomized into four groups: SCI, SCI + primary bOECs, SCI + STM, SCI + primary bulbar olfactory ensheathing cells (bOECs) + stimulation (STM). On these animals bioluminescence, immunohistological, and behavioral experiments were performed after SCI. Our results show that rTSMS has beneficial effect on the modulation of spinal scar by reducing fibrosis, demyelination, and microglial cell activation and by increasing the astroglial component of the scar, while, primary bOEC transplantation decreases microglial reactivity. At the opposite, locotronic experiments show that both treatments induce functional recovery. We did not observed any additional effect by combining the two therapeutic approaches. Taken together, the present study indicates that primary bOEC transplantation and rTSMS treatment act through different mechanisms after SCI to induce functional recovery. In our experimental paradigm, the combination of the two therapies does not induce any additional benefit.

Keywords: RRID:AB_10563302: PDGFRβ, Abcam, ab91066; RRID:AB_10643424: PE, poly4064, BioLegend, 406408; RRID:AB_2313568: Jackson ImmunoResearch, 711-166-152; RRID:AB_2340667: Jackson ImmunoResearch, 712-165-153; RRID:AB_2340812: Jackson ImmunoResearch, 715-165-140; RRID:AB_2715913: Alexa 488, MRG2b-85, BioLegend; RRID:AB_306827: p75, Abcam, ab8874; RRID:AB_476889: GFAP Cy3-conjugated Sigma-Aldrich, C9205; RRID:AB_777165:P DGFRβAbcam ab32570; RRID:AB_839504: Iba1, Wako, 019-19741; RRID:AB_94975: MBP, Millipore, MAB386; RRID:IMSR_JAX:008450: L2G85Chco+/+ (FVB-Tg(CAG-luc,-GFP)L2G85Chco/J); glial scar; magnetic stimulation; olfactory ensheathing cells and neuroregeneration; rehabilitation; spinal cord injury.

FIGURE 1

Experimental paradigm illustrating the timeline of the major experimental manipulations

FIGURE 2

Cellular characterization of bOEC primary culture by flow cytometry. Cells are isolated after (a) viability exclusion and (b and c) doublet exclusion. (d) Cells are characterized using anti‐p75 and anti‐PDGFrβ antibodies. Olfactory ensheating cells (OECs) were defined as p75high or p75low positive and PDGFrβ negative cells. Stromal cell was defined as PDGFrβ positive (PDGFrβ+) and p75 negative cells. (e) Cell ratio in the bOEC primary culture. Dot plots represent mean ± standard deviation. N = 4 per group. All experiments were conducted in duplicate

FIGURE 3

The survival of transplanted primary bOECs is not influenced by rTSMS treatment. (a and b) Representative images of luciferase+ (LUX) primary bOECs transplanted in mice stimulated or not at 9d and 16d after SCI. (c–e) Quantification of luciferase signals at (c) 9d, (d) 14d, and (e) 16d after SCI and/or rTSMS treatment. Quantifications are expressed as average ± standard deviation. N = 10 per group. Statistical evaluations were based on Mann–Whitney test (ns = not significant)

FIGURE 4

rTSMS treatment modulates glial and fibrotic scars but not primary bOEC transplantation. Representative pictures of sagittal spinal cord sections 60 days after SCI (a, b, c, d, f, g, h, and I 50× magnification) and border of the spinal cord scar (a', b', c', d', f', g', h', and i 200× magnification). (a and f) SCI control, (b and g) SCI + primary bOECs, (c and h) SCI + STM, (d and i) SCI + primary bOECs + STM. Sections were stained with (a–d) anti‐GFAP antibody, (f–j) anti‐PDGFrβ antibody and DAPI. Dotted lines show (a–d) astrocytic negative area (GFAP‐) and (f–i) PDGFrβ positive area (PDGFrβ+). (e and j) Quantitative analysis of (e) GFAP‐ areas and (j) PDGFrβ + areas. Quantifications are expressed as average ± standard deviation. N = 5 per group. Statistical evaluations were based on Kruskal–Wallis test (*p < 0.05; **p < 0.01)

FIGURE 5

Primary bOEC transplantation and rTSMS treatment decrease microglial cells activation but only rTSMS treatment decreases demyelination. Representative pictures of sagittal spinal cord sections 60 days after SCI (a, b, c, d, f, g, h, and I 50× magnification) and border of the spinal cord scar (a', b', c', d', f', g', h', and i 200× magnification). (a and f) SCI control, (b and g) SCI + primary bOECs, (c and h) SCI + STM, (d and i) SCI + primary bOECs + STM. Sections were stained with (a–d) anti‐MBP antibody, (f–j) anti‐Iba1 antibody and DAPI. Dotted lines show (a–d) demyelinated area (MBP‐). Rectangles show (f–i) Iba1 intensity areas measured. (e and j) Quantitative analysis of (e) MBP‐ area and (j) Iba1 intensity. Quantifications are expressed as average ± standard deviation. N = 5 per group. Statistical evaluations were based on Kruskal–Wallis test (*p < 0.05; **p < 0.01; ***p < 0.001)

FIGURE 6

Primary bOEC transplantation and rTSMS treatment enhance functional recovery after SCI. Quantification of locotronic evaluation at (a–c) 15 days, (d–f) 30 days, and (g–i) 60 days after SCI. Parameters are (a, d, and g) number of back leg errors, (b, e, and h) total back leg error time and (c, f, and i) total crossing time. Quantifications are expressed as average ± standard deviation. N = 5–10 in control group and N = 8–10 in SCI + primary bOECs, SCI + STM and SCI + primary bOECs + STM groups. Statistical evaluations were based on Kruskal–Wallis test (*p < 0.05; **p < 0.01; ***p < 0.001; ^# p < 0.0001)