Repetitive Transcranial Magnetic Stimulation (rTMS) Improves the Gait Disorders of Rats Under Simulated Microgravity Conditions Associated With the Regulation of Motor Cortex

Abstract

In previous studies, it has been proved that repetitive transcranial magnetic stimulation (rTMS) improves dyskinesia induced by conditions such as spinal cord injury, Parkinson diseases and cerebral ischemia. However, it is still unknown whether it can be used as a countermeasure for gait disorders in astronauts during space flight. In this study, we evaluated the effects of rTMS on the rat gait function under simulated microgravity (SM) conditions. The SM procedure continued for consecutive 21 days in male Wistar rats. Meanwhile, the high-frequency rTMS (10 Hz) was applied for 14 days from the eighth day of SM procedure. The behavioral results showed that SM could cause gait disorders such as decreased walking ability and contralateral limb imbalance in rats, which could be reversed by rTMS. Furthermore, rTMS affected the neural oscillations of motor cortex, enhancing in δ (2-4 Hz) band, suppressing in θ (4-7 Hz), and α (7-12 Hz) bands. Additionally, rTMS could activate mTOR in the motor cortex. These data suggests that the improvement effects of rTMS on gait disorders in rats under SM conditions might be associated with its regulation on neural oscillations in the cerebral motor cortex and the expression of some motor-related proteins which may enhance the control of nervous system on muscle function. Based on our results, rTMS can be used as an potential effective supplement in the field of clinical and rehabilitation research to reduce gait disorders caused by the space environment.

Keywords: IGF-1-PI3K-Akt-mTOR signaling pathway; gait disorders; local field potential; repetitive transcranial magnetic stimulation; simulated microgravity.

FIGURE 1

Scheme of the experimental design and effects of rTMS on body weight and muscle weight in SM rats. (A) Scheme of the experimental design. The experimental groups were divided into control (CON, n = 10), simulated microgravity for 3 weeks with sham stimulation for 2 weeks (SM+sham, n = 7) and simulated microgravity for 3 weeks with rTMS for 2 weeks (SM+rTMS, n = 8). Behavioral experiments include open field, swimming, and gait analysis experiments. Biochemical experiments include HE staining and western blot experiment. (B) The broken-line graph shows the weight change rate of rats during the modeling period. (C) Relative mass of Sol muscle. Sol, soleus. (D) Relative mass of MG muscle. MG, musculus gastrocnemius. Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; n.s., no significant, one-way ANOVA.

FIGURE 2

Effects of rTMS on walking and swimming ability of SM rats. (A,G) Schematic diagrams of equipment for open field and swimming experiments. (B) The walking distance of rats in the open field in 5 min. (C) The time of rats rested in the open field within 5 min. (D,J) The trajectories of rats among groups in the open field and swimming process, respectively. (E,F) The average and maximum speed of rats walking in the open field, respectively. (H) Average time for climbing on the platform of three groups, since forelimbs reaching the platform to all the hind limbs on the platform. (I) The distance of rats swimming in the tank in 1 min. (K,L) The average and maximum speed of rats swimming in the tank, respectively. Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., no significant, one-way ANOVA.

FIGURE 3

Effect of rTMS on the gait of SM rats. (A) Diagram of experimental equipment for gait test. (B) The actual trajectory of the rat walking. RF, right front; RH, right hind; LF, left front; LH, left hind. (C) The graph of the time step sequence, which reflects the sequence of different feet walking on the track. Stride length, stand, and swing definitions are shown in the figure. (D) The average speed of the rats walking on the track. (E) The length of rats’ strides during walking. (F) Distance between forefoot and hindfoot of rats. (G,J) The percentage of time spent on contralateral foot or tripod support of rats. (H) The diagram of pressure-time of the each foot of rat, which reflects the pressure of each foot contacting with the ground. The intensity ranges from 0 to 255 MPa. (I) The maximum strength of contact between the contralateral feet and the ground (LF, left front foot; RH, right hind foot). *p < 0.05; **p < 0.01, ***p < 0.001; n.s., no significant, one-way ANOVA. Error bars, SEM. See also Supplementary Figure 1.

FIGURE 4

Brain electrical activity of M1 and M2 regions in SM rats. (A) The M1 and M2 regions of the motor cortex are shown in the brain atlas. (B) Diagram of the position coordinates of craniotomies during surgical procedure. (C,E) The corresponding spectrogram of LFP activity, indicating changes in power at 2–12 Hz in M1 and M2 regions. (D,F) Results of LFP activities in the M1 and M2 regions. Power spectra showed a mean level of LFP activities from all the rats in CON (gray line), SM+sham (red line) and SM+rTMS (blue line). The bar graphs represent the mean level of relative LFP power in the M1 and M2 within a series of frequency ranges: 2–4, 4–7, and 7–12 Hz. *p < 0.05; **p < 0.01; n.s., no significant, one-way ANOVA. Error bars, SEM.

FIGURE 5

Schematic results of HE staining of motor cortex. Morphological changes of nerve cells in motor cortex. The sections of tissue were HE stained. In the schematic diagram, the shape difference and rupture degree of different cells in different groups can be observed. Pictures were all at 20× magnification.

FIGURE 6

Effects of rTMS on the IGF-1-PI3K-Akt-mTOR signaling pathway in the motor cortex. (A) Representative western blot bands using total protein extracts from the motor cortex. (B–F) Quantification of protein expression levels in the motor cortex among the CON, SM+sham, and SM+rTMS group (n > 10, from five rats in each group). *p < 0.05; ***p < 0.001; n.s., no significant, one-way ANOVA. All values are mean ± SEM.