All experimental procedures were approved by the Animal Ethics Committee of Chiba University Hospital (approval No. A28-090, 29 − 10). Ten female Sprague Dawley rats (8–9 weeks old) were used in the present study. A summary of the experimental protocol is shown in Fig. 1.
All rats were anesthetized with 2% sevoflurane (Sevofrane, Maruishi Pharmaceutical Co., Osaka, Japan) delivered via a nose cone and deep level of anesthesia was maintained during surgical procedures. We performed laminectomy at the level of T8-9 and completely transected the spinal cord. To prevent reconnection of the cut ends of the spinal cord, we resected a segment of the spinal cord at the T8-9 level. We used a small cotton pellet and microfibrillar collagen hemostat (Avitene, C. R. Bard, Murray Hill, NJ) for hemostasis. To prevent infection, all rats were treated with cefmetazole (Cefmetazon, Daiichi Sankyo, Tokyo, Japan, 40 mg/kg/day, subcutaneously) for 7 days. We evacuated the bladder of rats twice a day and used hind limb exercises to prevent contracture during the first 7 days after the surgery.
Two weeks after the spinal cord transection, rats were randomly divided into an SCS group and a control group. The rats in the SCS group underwent a laminectomy at the level of L2-3 and an SCS electrode (Pisces-Quad Lead, Medtronic, Minneapolis, MN) was implanted on the dorsal surface of the dural tube at the level of L2. The rats in the control group underwent a laminectomy at the level of L3-4 alone without implanting an electrode.
SCS and treadmill training
Rats underwent SCS and treadmill training with body weight support for 30 min per day, 5 days per week, for four weeks. SCS was delivered at 40 Hz 240 µs pulses following a published protocol . We adjusted intensities between 0.5 and 2.0 V under the threshold for generating hind limb locomotion.
Treadmill training was performed by using a device developed for the present study (Shibata system, Kyoto, Japan). The device had arms to passively move the hind limbs of the rats reciprocally. The device also had a harness for the rats that could be adjusted to support their body weight (Fig. 2). The training was performed with 95–100% of body weight support. Treadmill belt speed was set at 5 cm/s and the arm of the device was adjusted to move the hind limbs of the rats in synchrony with the treadmill.
We evaluated hind limb locomotor function using the Basso–Beattie–Bresnahan scale (BBB) score once a week for 6 weeks . Rats were allowed to move freely in an open field for 5 min and their hind limb locomotion was assessed. We also measured resistance force for full extension of hind limbs to evaluate spasticity once a week for 6 weeks. In brief, we put the rats in a supine position in the harness and measured the maximum force by using a handheld strain gauge (model FGP-0.5; Nidec-Shimpo, Kyoto, Japan). Every measurement was repeated 6 times and the average was considered as the degree of spasticity.
To elucidate the possible mechanism for attenuation of spasticity obtained by SCS and treadmill training, we used immunohistochemistry to determine the expression of glutamic acid decarboxylase-65 (GAD65), a molecular marker for spinal cord inhibitory neurons.
Six weeks after spinal cord transection, the rats were humanely killed with an intraperitoneal overdose of pentobarbital sodium (50 mg/kg of Somnopentyl, Kyoritsuseiyaku, Tokyo, Japan) followed by transcardial perfusion with 4% paraformaldehyde. The lumbar enlargement of the spinal cord was removed and postfixed overnight in 4% paraformaldehyde. Tissues were stored in 20% sucrose overnight for cryoprotection and frozen in TissueTek OCT compound (Sakura Finetek, Tokyo, Japan) at − 70 ℃. Serial sections of the lumbar enlargement at 20 µm thickness were cut on a cryostat and mounted onto MAS coated glass slides (Matsunami Glass Industries, Osaka, Japan).
The sections were permeabilized with 0.3% Triton X-100 (Nacalai Tesque, Kyoto, Japan) in PBS for 1 h, then incubated with primary antibody (mouse anti-GAD65 antibody at 1:200, ab26113, Abcam) at 4 ℃ overnight. After washing with PBS three times, slides were incubated with secondary antibody (goat anti-mouse IgG H and L chain Alexa Fluor 488 at 1:200, ab150113, Abcam). GAD65 staining was visualized using fluorescence microscopy (BX51, Olympus, Tokyo, Japan), and assessed by observers blinded to the treatments who measured the numbers of GAD65-positive cells.
All data are presented as mean ± l SEM. The differences in BBB score and spasticity between SCS and control groups were examined using a repeated measures ANOVA and relative GAD65 expression between SCS and control groups was examined using a t test. All the analyses were conducted using JMP (version 10.0.2, SAS Institute, Cary, NC, USA); p < 0.05 was considered statistically significant.