Neuromuscular interaction is required for neurotrophins-mediated locomotor recovery following treadmill training in rat spinal cord injury

Abstract

Recent results have shown that exercise training promotes the recovery of injured rat distal spinal cords, but are still unclear about the function of skeletal muscle in this process. Herein, rats with incomplete thoracic (T10) spinal cord injuries (SCI) with a dual spinal lesion model were subjected to four weeks of treadmill training and then were treated with complete spinal transection at T8. We found that treadmill training allowed the retention of hind limb motor function after incomplete SCI, even with a heavy load after complete spinal transection. Moreover, treadmill training alleviated the secondary injury in distal lumbar spinal motor neurons, and enhanced BDNF/TrkB expression in the lumbar spinal cord. To discover the influence of skeletal muscle contractile activity on motor function and gene expression, we adopted botulinum toxin A (BTX-A) to block the neuromuscular activity of the rat gastrocnemius muscle. BTX-A treatment inhibited the effects of treadmill training on motor function and BDNF/TrKB expression. These results indicated that treadmill training through the skeletal muscle-motor nerve-spinal cord retrograde pathway regulated neuralplasticity in the mammalian central nervous system, which induced the expression of related neurotrophins and promoted motor function recovery.

Keywords: BDNF; Motor neurons; Neuromuscular activity; Neurotrophins; Spinal cord injury; Treadmill training.

Figure 1. Experimental design and functional assessments.

(A) The experimental procedures of the dual rat spinal lesion model and the time course in all rats are shown. T10: the 10th thoracic spinal cord, T8: the 8th thoracic spinal cord, TT: treadmill training. (B) The experimental procedures of the BTX-A injection and the time course in all rats are shown. FG: Fluoro Gold, TT: treadmill training, Sal: saline. (C) Treadmill-training significantly improved Basso, Beattie, and Bresnahan (BBB) scores starting from the second week up to the seventh week after SCI as compared to non-trained rats. Statistically significant differences were found among the three groups two days after spinal cord transection (SCT). (D) Treadmill-training also significantly increased the angle of the inclinedplane from the third week up to the seventh week after SCI as compared to non-trained rats. No statistically significant difference was found among the three groups two days after SCT. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 2. Treadmill training alleviates secondary injury of the lumbar spinal motor neurons after SCI.

(A) Nissl staining of the lumbar spinal cord transverse section. The blue arrow indicates the normal motor neurons, and the green arrow indicates the abnormal motor neurons. Scale bars, 50 µm. (B) Shows the statistical graph of the mean number of ventral horn motor neurons among the three groups. (C) Shows the statistical graph of the relative area of motor neurons (% of sham group) among the three groups. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 3. Treadmill training enhances the expression of both BDNF/TrkB in the lumbar spinal cord after SCI.

(A) The expression of BDNF and TrkBin the lumbar spinal cord was determined by immunofluorescence staining among the three groups. Scale bars, 100 µm. (B) Shows the statistical graph of the relative density of BDNF and TrkB (% of sham group) among the three groups. ^∗P < 0.05. (C) Western blotting analysis of BDNF and TrkB expression in the lumbar spinal cord among the three groups.

Figure 4. The improvement in motor function after treadmill training was inhibited with BTX-A injection.

(A) The first row of the three images shown are of low magnification immunofluorescence staining; FG⁺ motor neurons (white), NeuN⁺ motor neurons (green), FG⁺/NeuN⁺ motor neurons (lightgreen). The second and the third row show high magnification immunofluorescence staining images of the four groups. The yellow arrow shows FG^-/NeuN⁺ motor neurons, and the white arrow shows FG⁺/NeuN⁺ motor neurons. Scale bars, 500 µm. (B) Shows the statistical graph of the number of NeuN⁺ cells in the lumbar spinal cord of FG⁺ and FG^- motor neurons. (C) The expression of c-fos in the lumbar spinal cord FG⁺ and FG^- motor neurons was determined by immunofluorescence staining among the four groups. In addition, c-fos labeling (red) in FG-positive motor neurons (white) is shown. Overlap of c-fos and FG labeling (pink) indicates FG positive motor neurons that expressed c-fos. Scale bars, 50 µm. (D) Shows the statistical graph of the relative mean density of c-fos (% of SCI/Sal FG^-). (E) Shows Western blot analysis of NeuN and c-fos expression in the lumbar spinal cord among the four groups. ^∗P < 0.05.

Figure 5. The expression of BDNF and TrkB after treadmill training was inhibited after BTX-A injection.

(A) The expression of BDNF in the lumbar spinal cord FG⁺ and FG^- motor neurons was determined by immunofluorescence staining among the four groups. BDNF labeling (red) in FG-positive motor neurons (white) is also shown. The overlap of BDNF and FG labeling (pink) indicates FG positive motor neurons expressing BDNF. Scale bars, 50 µm. (B) Shows the statistical graph of the relative mean density of BDNF in the motor neurons (% of SCI/Sal FG^-). (C) The expressionof TrkBin the lumbar spinal cord FG⁺ and FG^- motor neurons was determined by immunofluorescence staining among the four groups. TrkB labeling (red) in FG-positive motor neurons (white) is also shown. Overlap of TrkB and FG labeling (pink) indicates FG positive motor neurons expressing TrkB. Scale bars, 50 µm. (D) Shows the statistical graph of the relative mean density of TrkB in the motor neurons (% of SCI/Sal FG^-). (E) Western immuno-blotting analysis of BDNF and TrkB expression in lumbar spinal cord among the four groups. ^∗P < 0.05.