Effects of aerobic exercise training on muscle plasticity in a mouse model of cervical spinal cord injury

Abstract

Cervical spinal cord injury (SCI) results in permanent life-altering motor and respiratory deficits. Other than mechanical ventilation for respiratory insufficiency secondary to cervical SCI, effective treatments are lacking and the development of animal models to explore new therapeutic strategies are needed. The aim of this work was to demonstrate the feasibility of using a mouse model of partial cervical spinal hemisection at the second cervical metameric segment (C2) to investigate the impact of 6 weeks training on forced exercise wheel system on locomotor/respiratory plasticity muscles. To measure run capacity locomotor and respiratory functions, incremental exercise tests and diaphragmatic electromyography were done. In addition, muscle fiber type composition and capillary distribution were assessed at 51 days following chronic C2 injury in diaphragm, extensor digitorum communis (EDC), tibialis anterior (TA) and soleus (SOL) muscles. Six-week exercise training increased the running capacity of trained SCI mice. Fiber type composition in EDC, TA and SOL muscles was not modified by our protocol of exercise. The vascularization was increased in all muscle limbs in SCI trained group. No increase in diaphragmatic electromyography amplitude of the diaphragm muscle on the side of SCI was observed, while the contraction duration was significantly decreased in sedentary group compared to trained group. Cross-sectional area of type IIa myofiber in the contralateral diaphragm side of SCI was smaller in trained group. Fiber type distribution between contralateral and ipsilateral diaphragm in SCI sedentary group was affected, while no difference was observed in trained group. In addition, the vascularization of the diaphragm side contralateral to SCI was increased in trained group. All these results suggest an increase in fatigue resistance and a contribution to the running capacity in SCI trained group. Our exercise protocol could be a promising non-invasive strategy to sustain locomotor and respiratory muscle plasticity following SCI.

Figure 1

Extent of injury following a C2 hemisection. (A) Schematic representation of the extent of injury for each animal in SCI SED and SCI TR groups. (B) Graph shows the quantification of the extent of injury in percentage compared to a 100% hemisected spinal cord. The quantification has only been made in the ventral part where the phrenic motoneurons are located. Data are presented as mean ± SD (n = 6). There is no difference between the different groups (t-test, p = 0.4127).

Figure 2

Maximal physical capacity expressed as running time (s). (A) Representative snapshot of a SHAM (upper) and SCI (below) mouse running in forced exercise wheel. (B) Running capacity on forced exercise wheel at different time points (before SCI; 7 days after surgery and 49 days after surgery). Data are presented as mean ± SD (n = 6). Significance: b p < 0.05 compared to sedentary, f p < 0.05 compared to before surgery or 7 days after surgery.

Figure 3

Representative images of fiber type composition in locomotor muscles and analysis of fiber type composition. Extensor digitorum communis (EDC), Tibialis anterior (TA) and Soleus (SOL) cryosections were immunostained to identify Myosin heavy chain (MyHC) isoforms: MyHCSlow (type I in blue), MyHC2A (type IIa in green) co-labeled with an anti-laminin antibody to visualize myofiber boundaries (type IIb/x corresponding to unlabeled fibers). Scale bar = 200 μm.

Figure 4

Representative images of CD31 expression and number of fibers in the skeletal muscles. Cryosections were immunostained to visualize CD31 (green) co-labeled with an anti-Laminin antibody (in red) in Soleus (SOL), while Extensor digitorum communis (EDC), Tibialis (TA) anterior are illustrated in supplemental Figs. 1 and 2, respectively. Scale bar = 200 μm.

Figure 5

Diaphragm activity at 51 days following a C2 hemisection. (A) Representative traces of diaphragm raw electromyography for uninjured animals: NAIVE and SHAM groups (SED and TR) and (B) injured (contralateral diaphragm and ipsilateral diaphragm) animals: SCI groups (SED and TR).

Figure 6

Diaphragm activity analysis at 51 days following a C2 hemisection. Graphs show integrated diaphragm amplitude during (A) spontaneous breathing and (B) mild asphyxia. Graphs represent the width from each EMG bursts of integrated diaphragm signal during (C) spontaneous breathing and (D) mild asphyxia. Data are presented as mean ± SD (n = 6). Significance: f p < 0.05 compared to ipsilateral side.

Figure 7

Representative images of fiber type composition in respiratory muscle. Diaphragmatic muscle cryosections were immunostained to visualize Myosin heavy chain (MyHC) isoforms: MyHCSlow (type I in blue), MyHC2A (type IIa in green) co-labeled with an anti-laminin antibody to visualize myofiber boundaries (type IIb/x corresponding to unlabeled fibers). Scale bar = 200 μm.

Figure 8

Analysis of fiber type composition in the respiratory muscle. (A–C) muscle fiber cross sectional area and (D) percentage of fiber type relative in diaphragmatic muscle. Data are presented as mean ± SD (n = 6). Significance: a p < 0.05 compared to TR group; f p < 0.05.

Figure 9

Analysis of endothelial cells on respiratory muscle. (A). Cryosections were immunostained to visualize CD31 (green) co-labeled with an anti-Laminin antibody (in red) in the diaphragm muscle. Scale bar = 200 μm. Comparison of CD31 expression and number of fibers in the diaphragm muscle represented by SED and TR SCI groups, while NAIVE and SHAM groups (SED and TR) are illustrated in supplemental Figs. 3. Scale bar = 200 μm. (B) Capillary density and (C) Ratio between capillary and fiber. Data are presented as mean ± SD (n = 6). Significance: b p < 0.05 compared to SED group.

Figure 10

Experimental design throughout surgery and low intensity exercise training periods. After 1st incremental test on forced exercise wheel, animals were distributed into six experimental groups (3 sedentary and 3 trained of NAIVE, SHAM and SCI). SHAM and SCI groups underwent cervical laminectomy followed by a 1-week recovery. Next, all groups went through a 6-week aerobic exercise training on forced exercise wheel.