Matrine promotes neural circuit remodeling to regulate motor function in a mouse model of chronic spinal cord injury

Abstract

In chronic phase of spinal cord injury, functional recovery is more untreatable compared with early intervention in acute phase of spinal cord injury. In the last decade, several combination therapies successfully improved motor dysfunction in chronic spinal cord injury. However, their effectiveness is not sufficient. We previously found a new effective compound for spinal cord injury, matrine, which induced axonal growth and functional recovery in acute spinal cord injury mice via direct activation of extracellular heat shock protein 90. Although our previous study clarified that matrine was an activator of extracellular heat shock protein 90, the potential of matrine for spinal cord injury in chronic phase has not been sufficiently evaluated. Thus, this study aimed to investigate whether matrine ameliorates chronic spinal cord injury in mice. Once daily intragastric administration of matrine (100 μmol/kg per day) to spinal cord injury mice were starte at 28 days after injury, and continued for 154 days. Continuous matrine treatment improved hindlimb motor function in chronic spinal cord injury mice. In injured spinal cords of the matrine-treated mice, the density of neurofilament-H-positive axons was increased. Moreover, matrine treatment increased the density of bassoon-positive presynapses in contact with choline acetyltransferase-positive motor neurons in the lumbar spinal cord. These findings suggest that matrine promotes remodeling and reconnection of neural circuits to regulate hindlimb movement. All protocols were approved by the Committee for Animal Care and Use of the Sugitani Campus of the University of Toyama (approval No. A2013INM-1 and A2016INM-3) on May 7, 2013 and May 17, 2016, respectively.

Keywords: Basso Mouse Scale; Body Support Score; Sophora flavescens; axonal growth; chronic spinal cord injury; hindlimb locomotor; immunohistochemistry; matrine; presynapse; synaptogenesis.

Figure 1

Experiment flow chart. Matrine (100 μmol/kg) or vehicle solution (saline) was constitutively administered orally for 154 days from 28 days post-injury.

Figure 2

Chemical structure of matrine.

Figure 3

Matrine improves motor function of hindlimbs in chronic spinal cord injury mice. The Basso Mouse Scale (BMS, A), Body Support Score (BSS, B), and body weight (C) were measured. #P < 0.05, vs. vehicle, drug × day interaction analyzed by two-way repeated measures analysis of variance. *P < 0.05, vs. vehicle, post hoc Bonferroni test. n = 10 and 12 hindlimbs in the vehicle and matrine groups, respectively.

Figure 4

Matrine improves motor function of hindlimbs in chronic spinal cord injury (SCI) mice. The movement of a representative SCI mouse treated with vehicle (A) or matrine (B) was captured at 182 days after injury. The images are sequential for 0.7 seconds. Vehicle-treated mouse hardly touched soles on the floor, whereas matrine-treated mouse walked with touching soles on the floor.

Figure 5

Matrine increases neurofilament-H (NF-H)-positive axons in injured spinal cord. Sagittal sections of the spinal cords of vehicle-treated or matrine-treated were immunostained for NF-H. (A) Representative images at rostral and caudal sits are shown. Scale bar: 25 μm. (B) The diagram of analyzed area is shown. The measured areas in rostral and caudal sites were defined as the area 2.0–3.0 mm apart from the lesion center. (C, D) Ratio of NF-H-positive area to measured area was quantified at the rostral (C) and the caudal site (D). *P < 0.05, unpaired t-test (two-tailed). n = 5 mice (vehicle) or 6 mice (matrine).

Figure 6

Matrine increases presynaptic density on motor neurons in injured spinal cord. Sagittal sections of the spinal cords of vehicle-treated or matrine-treated were immunostained for choline acetyltransferase (ChAT) and bassoon. (A) Representative stains of single motor neuron on lumbar spinal cord are shown. Immunohistological images for ChAT (red) and bassoon (green) are merged. Scale bar: 20 μm. (B) The diagram of analyzed area. Motor neurons in L1–3 segments of the spinal cord were analyzed. (C) Density of bassoon-positive presynapses was quantified on the contour of motor neurons. *P < 0.05, unpaired t-test (two-tailed). n = 5 mice (vehicle) or 5 mice (matrine).