Body Weight-Supported Treadmill Training Ameliorates Motoneuronal Hyperexcitability by Increasing GAD-65/67 and KCC2 Expression via TrkB Signaling in Rats with Incomplete Spinal Cord Injury

Abstract

Spasticity is a typical consequence after spinal cord injury (SCI). The critical reasons are reducing the synthesis of Gamma-Aminobutyric Acid (GABA), glycine and potassium chloride co-transporter 2 (KCC2) inside the distal spinal cord. The current work aimed to test whether exercise training could increase the expression of glutamic acid decarboxylase 65/67 (GAD-65/67, the key enzymes in GABA synthesis) and KCC2 in the distal spinal cord via tropomyosin-related kinase B (TrkB) signaling. The experimental rats were randomly assigned to the following five groups: Sham, SCI/phosphate-buffered saline (PBS), SCI-treadmill training (TT)/PBS, SCI/TrkB-IgG, and SCI-TT/TrkB-IgG. After that, the model of T10 contusion SCI was used, then TrkB-IgG was used to prevent TrkB activity at 7 days post-SCI. Body weight-supported treadmill training started on the 8^th day post-SCI for four weeks. The Hmax/Mmax ratio and the rate-dependent depression of H-reflex were used to assess the excitability of spinal motoneuronal networks. Western blotting and Immunohistochemistry techniques were utilized for measuring the expression of GAD-65, GAD-67, and KCC2. The findings revealed that exercise training could reduce motoneuronal excitability and boost GAD-65, GAD-67, and KCC2 production in the distal region of the spinal cord after SCI. The effects of exercise training were decreased after the TrkB signaling was inhibited. The present exploration demonstrated that exercise training increases GAD-65, GAD-67, and KCC2 expression in the spinal cord via TrkB signaling and that this method could also improve rats with motoneuronal hyperexcitability and spasticity induced by incomplete SCI.

Keywords: Body-weight supported treadmill training; Glutamic acid decarboxylase 65/67; Potassium chloride co-transporter 2; Spasticity; Spinal cord injury; Tropomyosin-related kinase B.

Fig. 1

Immunohistochemical staining of CREB, p-CREB, GAD-65, GAD-67, and KCC2 in rat lumbar spinal cord horizontal transverse sections. The immunohistochemical staining reveals that A CREB is predominantly expressed in the neuronal cytoplasm. B p-CREB is upregulated in the neuron nucleus. C GAD-65 is widely expressed in gray matter and is associated with immunoreactive punctate structures [29, 30]. D GAD-67 immunopositive cells are primarily rooted in the gray matter, including the neurons in the lamina VII [29, 30], and E KCC2 is predominantly expressed on the cell membrane, dendritic spines and dendritic trunks of neurons in lumbar the spinal cord. F A schematic representation of the spinal cord's gray matter layer at the lumbar spinal cord. Scale bar: 400 μm

Fig. 2

The western blot examination of BDNF and TrkB in the lumbar spinal cord. A Western blot analysis of BDNF and TrkB expression in the lumbar spinal cord. B the relative density of BDNF as a statistical graph. C the relative density of TrkB as a statistical graph. **P < 0.01, ***P < 0.001

Fig. 3

The H-reflex in five groups. A Typical H-reflex waveforms at 0.3, 5, and 10 Hz. B the Hmax/Mmax ratio graph. C the graph of the H-reflex RDD at various stimulus frequencies. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

The immunohistochemical investigation of CREB and p-CREB in the distal spinal cord lamina VII. A Immunohistochemical analysis reveals that CREB is primarily expressed in the cytoplasm of neurons, whereas p-CREB is predominantly expressed in the nucleus of neurons. B and C the statistical graphs of the CREB and p-CREB relative densities. **P < 0.01, **P < 0.01, ***P < 0.001. Scale bar: 50 μm

Fig. 5

The immunohistochemical assessment of CREB and p-CREB in the distal spinal cord lamina IX. A Immunohistochemical analysis reveals that CREB is largely expressed in the cytoplasm of neurons, whereas p-CREB is predominantly expressed in the nucleus of neurons. B and C the statistical graphs of the CREB and p-CREB relative densities. **P < 0.01, **P < 0.01, ***P < 0.001. Scale bar: 50 μm

Fig. 6

The western blot examination of CREB and p-CREB in the lumbar spinal cord. A Western blot picture of the CREB and p-CREB transcription factors. B A graph illustrating the relative density of CREB. C Statistical graph of p-relative CREB's density. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

The immunohistochemical assessment of GAD-65 and GAD-67 in the lumbar spinal cord. A Immunohistochemical staining of GAD-65 in the lumbar spinal cord lamina IX and GAD-67 in the lumbar spinal cord lamina VII. B the statistical graph of the GAD-65's relative density. C a statistical graph of the GAD-67 immunopositive cells. **P < 0.01, ***P < 0.001. Scale bar: 50 μm

Fig. 8

The western blot examination of GAD-65 and GAD-67 in the lumbar spinal cord. A GAD65 and GAD-67 western blot picture. B The relative density of GAD-65 is plotted statistically. C A statistical plot of GAD-67's relative density. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 9

Immunohistochemistry and western blot examination of KCC2 in the lumbar spinal cord. A KCC2 immunohistochemistry staining in lamina IX. B Western blot analysis of the KCC2 protein. C a statistical graph depicts the relative density of KCC2 on the motor neuron membrane. D The relative density of KCC2 in the distal spinal cord as a statistical graph. **P < 0.01, ***P < 0.001. Scale bar: 50 μm