Facilitation of Stepping with Epidural Stimulation in Spinal Rats: Role of Sensory Input

Surgical procedures.

All surgeries were performed under aseptic conditions. The rats were anesthetized deeply by a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally and were maintained at a surgical level with supplemental doses of ketamine as needed.

Headplug.

A small incision was made at the midline of the skull. The muscles and fascia were retracted laterally, small grooves were made in the skull with a scalpel, and the skull was dried thoroughly. Two amphenol headplugs with Teflon-coated stainless steel wires (AS632; Cooner Wire) were securely attached to the skull with screws and dental cement as described previously (Roy et al., 1991; Ichiyama et al., 2005). A skin incision was made in the mid-dorsal region of the back (see below), and eight wires from the headplug were routed subcutaneously to the most distal end of the opening.

EMG implants.

Skin and fascial incisions were made to expose the bellies of the medial gastrocnemius (MG) and tibialis anterior (TA) muscles bilaterally. Two wires were routed subcutaneously from the back incision to each muscle site. Bipolar intramuscular EMG electrodes were formed and secured into the midbelly of each muscle as described previously (Roy et al., 1991). The EMG wires were coiled near each implant site to provide stress relief. Stimulation through the headplug was used to verify the proper placement of the electrodes in each muscle.

Spinal cord transection, unilateral dorsal rhizotomy, and epidural stimulation electrode implants.

A mid-dorsal skin incision was made between T6 and L4, and the paravertebral muscles were retracted as needed. A partial laminectomy was performed at the T8–T9 level, and the dura was opened longitudinally. Lidocaine (2–3 drops) was applied locally, and the spinal cord was completely transected at a midthoracic (approximately T8) level as described previously (Talmadge et al., 2002). A partial laminectomy was performed from T13 to L2, and the dura was opened from T12 to S1 and from S3 to S5. The dorsal roots on the left side were transected intradurally (as close to their exit from the spinal column as possible) from T12 to S2 (Fig. 1). Two wires from the headplug were passed subcutaneously to the most proximal end of the back incision. One Teflon-coated wire was passed to S1, and a small notch was made in the Teflon coating (∼0.5–1.0 mm) on the surface toward the spinal cord and served as the stimulating electrode. The wire was affixed to the dura (Fig. 1, S1 spinal segment) at the midline using 9.0 sutures as described previously (Ichiyama et al., 2005). The appropriate position of the stimulating electrode was verified postmortem: connective tissue secured the electrode to the dura at the correct location on the spinal cord in all rats and thus ensured a stable position for all stimulation sessions. All wires (for EMG and ES) were coiled in the back region to provide stress relief. The Teflon coating was stripped from the distal centimeter of the second wire, which was then inserted subcutaneously in the back region and served as a common ground.

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Figure 1.

Schematic of the spinal cord transection and unilateral deafferentation surgery. A complete spinal cord transection was performed at approximately the T8 spinal level (between vertebral segments T7 and T8). The dura (gray shading) was opened longitudinally from vertebral level T11 to S3 (leaving a short strip at approximately the L2 level intact), and a unilateral (left side) dorsal rhizotomy (cutting the dorsal root as close to the spinal cord as possible and as close to its exit from the vertebral column as possible) was performed intradurally from the T12 to the S2 spinal segments. A single insulated wire electrode with a small notch (∼0.5–1.0 mm) exposing the wire facing the spinal cord was sutured (asterisks represent the two sutures) on the short strip of intact dura over spinal segment S1 through which electrical stimulation was used to facilitate stepping and induce bilateral reflex responses. The middle arrow points to the transection site, and the bottom arrow, to the epidural electrode implant site. The extent of the unilateral dorsal rhizotomy is identified on the left side with the cut dorsal roots shown as broken lines.

All incision areas were irrigated liberally with warm, sterile saline and closed in layers (i.e., investing fascia and then the skin). All closed incision sites were cleansed thoroughly with saline solution. Analgesia was provided by Buprenex (0.5–1.0 mg/kg, i.m.; three times per day). The analgesics were initiated before completion of the surgery and continued for a minimum of 2 d. The rats were allowed to fully recover from anesthesia in an incubator. The rats were housed individually, and the bladders of the spinal rats were expressed manually three times per day for the first 2 weeks after surgery and twice per day thereafter. The hindlimbs of the spinal rats were moved passively through a full range of motion once per day to maintain joint mobility. The motor responses to pinching of the toes were evaluated daily to test for the presence of afferent input. The procedures for the care of spinal animals have been detailed previously (Roy et al., 1992).

Stimulation and recording procedures.

Testing procedures were started 1 week after surgery and were performed at 1, 3, and 7 weeks after surgery. All testing was performed weekly during ES with and without quipazine when the rats were fully awake. EMG activity was collected during stepping with a frequency of 2 kHz using a custom-made Labview program, and the duration and mean amplitude of identified EMG bursts were determined (Roy et al., 1991; Courtine et al., 2008). The EMG signals were led from the animal through long shielded wires to amplifiers (A-M Systems) with a frequency response between 10 Hz and 1 kHz.

Stimulation was performed using a Grass S88 Stimulator and a stimulus isolation unit (Grass SIU5; both from Grass Instruments). Epidural stimulation at S1 was performed via single stimuli (duration, 0.5 ms). Spinal cord reflexes were tested initially by stimulation at 0.2 Hz to determine the stimulus intensity–response amplitude curve. Averages of 10 responses were collected at stimulation intensities ranging from 0.5 to 10 V at 0.5 V intervals (Lavrov et al., 2006a).

To test for locomotor ability, the spinal rats were secured in an upper body harness support system, and the hindlimbs were placed on a moving treadmill belt as described previously (Ichiyama et al., 2005). Spinal cord stimulation at S1 was induced by continuous ES at 40 Hz (25 ms interpulse interval) and a pulse duration of 0.2 ms. At 7 weeks after surgery, to improve the stepping performance, the rats were injected with the 5-HT agonist quipazine (0.3 mg/kg, i.p.) as described previously (Ichiyama et al., 2005). An automated body weight support system was used to provide the optimum amount of body weight support necessary to enable walking (Ichiyama et al., 2005).

Analysis of reflex data.

All recordings were performed and analyzed as described previously (Lavrov et al., 2006a). To identify the latencies and amplitudes of EMG responses to epidural stimulation, stimulus intensities between 0.5 and 10 V were used. During each test session, it was determined that three responses were modulated differentially and were clearly voltage dependent. We also used rate-dependent inhibition to separate the different spinal cord motor-evoked responses as described previously (Lavrov et al., 2006a). Short-latency (between 1.5 and 6.5 ms) early responses remained unchanged under all frequencies of stimulation and correspond to direct activation of motoneurons. Middle (monosynaptic) responses with latencies between 6.5 and 9.5 ms were partially inhibited with 5 and 10 Hz stimulation. Late responses with latencies between 9.5 and 13.5 ms that may reflect the activation of polysynaptic circuits were completely inhibited with 5 and 10 Hz stimulation (Gerasimenko et al., 2006; Lavrov et al., 2006a).

Kinematics recordings.

Three-dimensional (3D) video recordings (100 Hz) were made using four cameras (two cameras on each side at 100 fps; Basler Vision Technologies) oriented at 45 and 135° with respect to the direction of the locomotion (i.e., the animal's sagittal plane on both sides). Reflective markers were attached bilaterally to the shaved skin overlying the following bony landmarks: the anterior superior iliac spine (Il), the greater trochanter (GT), the knee joint (K), the malleolus (M), the fifth metatarsal (MT), and the tip of toe (T). The SIMI motion capture software (SIMI Reality Motion Systems) was used to obtain 3D coordinates of the markers.

Stepping data analysis.

The body was modeled as an interconnected chain of rigid segments (i.e., Il-GT for the pelvis, GT-K for the thigh, K-M for the shank, M-MT for the foot, and MT-T for the fifth digit), and the joint angles were generated as described previously (Courtine et al., 2005). A cycle was defined as the time interval between two successive paw contacts. Successive paw contacts were identified with an accuracy of ±1 video frame. Data for a duration to acquire ∼10 step cycles on the non-deafferented side were extracted from a continuous sequence for each animal. Limb kinematics were determined as described by Courtine et al. (2005).

Muscle weights.

At the end of the experimental period, the rats were deeply anesthetized with sodium pentobarbital (125 mg · kg⁻¹; Sigma) and transcardially perfused with 1 ml · g⁻¹ body weight of PBS, pH 7.4, followed by 2 ml · g⁻¹ body weight of 4% paraformaldehyde in PBS (Sigma). The following muscles were removed bilaterally, cleaned of excess fat and connective tissue, and weighed: the soleus, MG, and plantaris as representative plantarflexors, and the TA and extensor digitorum longus as representative dorsiflexors.

Statistical analyses.

All data are reported as mean ± SEM. Statistically significant differences were determined using a one-way repeated-measures ANOVA. Values that were not normally distributed were analyzed using the nonparametric Kruskal–Wallis rank test. Paired Student's t tests and nonparametric Wilcoxon tests were used to assess statistical differences between the left and right sides. The criterion level for statistical significance was set at p < 0.05 for all analyses.