Establishment of the C5 spinal cord crush injury model.
We first examined whether C5 crush surgery could specifically injure the dorsal corticospinal tract (dCST) in adult mice. As shown (Fig. 1d), compared with the normal mice, only little neurons labeled Mini Ruby was observed in the right motor cortex of the injured mice, suggesting that the dCST could be crushed specifically and completely by the C5 crush surgery, and indicating the successful establishment of the C5 spinal cord crush injury model.
Treadmill exercise promotes skilled motor function recovery and enhances nerve conduction capability after SCI.
We next investigated whether the recovery of skilled motor function was enhanced by treadmill exercise after injury. Date showed all mice performed worse in the behavioral tests at 3 days and 1 week after injury (Fig. 2a-f). After the treadmill exercise for 2 and 4 weeks, we found that the mice in the LEI, MEI and HEI groups showed lower error rate of left limbs in the horizontal ladder test (P < 0.05, Fig. 2b-c) and higher usage rate of left forelimb and both forelimbs in the cylinder rearing test (P < 0.05, Fig. 2e-f) when compared with the control group. More importantly, mice in the MEI and HEI groups performed slightly better than the LEI group, even though, there was no significant difference among three trained groups.
Meanwhile, we asked whether the nerve conduction capability was also enhanced by performing the electrophysiology method to test the motor evoked potential (MEP) in mouse right motor cortex (Fig. 2g-i). Obviously, results showed that the latency of N1 wave (LEI, MEI, HEI vs. control, 6.3 ± 0.2, 6.4 ± 0.2, 6.3 ± 0.2 vs. 6.7 ± 0.4, P < 0.0001) and P1 wave (LEI, MEI, HEI vs. control, 7.2 ± 0.3, 7.1 ± 0.5, 7.2 ± 0.3 vs. 8.0 ± 1.1, P < 0.0001) in the LEI, MEI and HEI groups was significantly shorter than that in the control group, suggesting that the nerve conduction capability was potentiated by the treadmill exercise.
Exercise-induced enhancement of neuroplasticity in motor cortex is necessary for skilled motor function recovery.
To verify effects of the treadmill exercise on neuroplasticity, we then measured the alterations of dendrite complexity, spine density and the morphology in the right motor cortex using Golgi staining. Results showed that the neurite arborization significantly increased after training (Fig. 3a-b). The total length of dendrites was longer (LEI, MEI, HEI vs. control, 2160.45 ± 399.65, 2210.34 ± 388.12, 2109.99 ± 559.94 vs. 690.80 ± 116.92, P < 0.0001; Fig. 3a-c), and the density of dendritic spines was higher (LEI, MEI, HEI vs. control, 0.13 ± 0.02, 0.11 ± 0.01, 0.17 ± 0.03 vs. 0.02 ± 0.01, P < 0.0001; Fig. 3d-e) in the mouse right motor cortex of the LEI, MEI and HEI groups compared with the control group, suggesting that the complexity of neurons was enhanced by the treadmill exercise. Besides, a high correlation was found between the exercise-enhanced cortical neuroplasticity and the mouse performance in the horizontal ladder test (Fig. 3f-i). The longer the total length of cortical dendrites and the higher the density of dendritic spines, the lower the error rate of left limbs in the horizontal ladder test.
Treadmill exercise alone fails to promote axonal regeneration beyond lesion site but enhances axonal sprouting in ipsilateral side after SCI.
We next explored the effects of the treadmill exercise on axonal regeneration and sprouting. As shown (Fig. 4a), the treadmill exercise alone was not enough to promote axonal regeneration beyond the lesion site. But, the enhancement of axonal sprouting in ipsilateral gray matter region 0 ~ 500 µm rostral to the lesion site was observed in the LEI, MEI and HEI groups after 4 weeks treadmill exercise (P < 0.05; Fig. 4b-d). Some of the new axonal sproutings could even elongate to the ipsilateral ventricornu, a dense region of motor neurons, about 1,000 µm far from the spinal dorsal horn region, where dCST converged. It is may be one of the structural basis for the exercise-induced promotion of functional recovery in the cervical SCI mice.
Treadmill exercise facilitates neurotrophic factors expression in mouse motor cortex after SCI.
We further investigated the changes of molecular pathway in the cervical SCI mice after the treadmill exercise. As shown (Fig. 5), BDNF, IGF-1, phosphorylated tyrosine kinase receptor B (p-TrkB) and insulin-like growth factor 1 receptor (p-IGF1-R) were up-regulated in the mouse right motor cortex after chronic treadmill exercise in the MEI and HEI groups compared with the control and LEI groups (P < 0.05). And there was no significant difference between the control group and the LEI group. These results suggested that the upregulated neurotrophic factors are intensity-dependent, and are related to the exercise-enhanced neuroprotective and nerve repair effect.
Treadmill exercise activates cortical mTOR pathway and this exercise-induced activation can be inhibited by using rapamycin.
As the co-downstream of TrkB and IGF1-R, mTOR pathway plays an important role in modulating axonal regeneration and sprouting, plasticity and so on. Therefore, we further explored the effects of treadmill exercise on the activation of cortical mTOR pathway. Consistent with the changes of neurotrophic factors (Fig. 5), elevated phosphorylated levels of ribosomal protein S6 (p-S6) and protein kinase B (p-AKT) were only observed in the MEI and HEI groups (P < 0.05; Fig. 6a-d), indicating the activation of cortical mTOR pathway by the moderate and high intensity treadmill exercise. And as further evidence, elevated neuronal p-S6 in the mouse right motor cortex (Fig. 6f) was also only found in the MEI and HEI groups by immunofluorescence staining (P < 0.05; Fig. 6e-g).
According to the facts that the activation of cortical mTOR pathway was not observed in the LEI group (Fig. 6) and the HEI group showed decreased exercise tolerance and underwent the most total electric shocks during the treadmill exercise (P < 0.05; Fig. 7b-c), all of which leads to vulnerability. We therefore adopted the moderate intensity treadmill exercise in following experiment. To investigate the role of mTOR activation, a mTOR pathway inhibitor, rapamycin, was used. As expected, compared with the SCI + E group, the levels of p-S6 and p-AKT decreased after intraperitoneally injecting rapamycin in mice (P < 0.05; Fig. 7d-g). And a similar result was also validated by the immunofluorescence staining (P < 0.05; Fig. 7h-i).
Exercise-induced promotion of skilled motor function recovery and enhancement of nerve conduction capability after SCI are associated with the activation of mTOR pathway.
More importantly, rapamycin assay further demonstrated that the exercise-induced activation of cortical mTOR pathway is necessary for the exercise-enhanced functional recovery (Fig. 8). Compared with the SCI + E group, the exercise-induced skilled motor function recovery was attenuated in the SCI + E + R group as indicated by lower percentage of performance improvements in the horizontal ladder test (P < 0.05; Fig. 8a-b) and the cylinder rearing test (P < 0.05; Fig. 8c-d). And the exercise-enhanced nerve conduction capability was also eliminated by rapamycin, as proved by the results that the latency of N1 (SCI + E + R vs. SCI + E, 7.2 ± 0.2 vs. 6.5 ± 0.4, P < 0.0001; Fig. 8f) and P1 (SCI + E + R vs. SCI + E, 8.8 ± 0.4 vs. 7.8 ± 0.2, P < 0.0001; Fig. 8g) waves became longer in the SCI + E + R group compared with the SCI + E group.
Enhanced neuroplasticity in motor cortex and potentiated axonal sprouting are associated with exercise-induced activation of mTOR pathway.
We also found that the changes of cortical neuroplasticity are associated with the exercise-induced activation of cortical mTOR pathway (Fig. 9). Compared with the SCI + E group, the exercised-enhanced formation of dendrites (SCI + E + R vs. SCI + E, 735.88 ± 24.43 vs. 1827.29 ± 457.98, P < 0.0001; Fig. 9a-c) and dendritic spines (SCI + E + R vs. SCI + E, 0.03 ± 0.01 vs. 0.12 ± 0.02, P < 0.0001; Fig. 9d-e) were also attenuated after injecting rapamycin in the SCI + E + R group. Consistent with those results, the exercise-induced recovery of skilled motor function was removed as well (Fig. 8). What’s more, compared with the SCI + E group, the exercise-induced enhancement of axonal sprouting was attenuated by the injection of rapamycin in the SCI + E + R group as well (P < 0.05; Fig. 10).
Together, the expression of neurotrophic factors and the activation of cortical mTOR pathway in an intensity dependent manner. Activation of the cortical mTOR pathway induced by the treadmill exercise plays an important role in motor cortex and spine remodeling, including the enhanced complexity of neurons, potentiated nerve conduction capability, and increased axonal sprouting in the cervical SCI mice, all of which further contribute to better motor function recovery.