JOURNAL OF NEUROTRAUMA 31:1088–1106 (June 15, 2014) ª Mary Ann Liebert, Inc. DOI: 10.1089/neu.2013.3096

Effect of Combined Treadmill Training and Magnetic Stimulation on Spasticity and Gait Impairments after Cervical Spinal Cord Injury Jiamei Hou,1,2 Rachel Nelson,2 Nicole Nissim,2 Ronald Parmer,2 Floyd J. Thompson,1–3 and Prodip Bose1,2,4

Abstract

Spasticity and gait impairments are two common disabilities after cervical spinal cord injury (C-SCI). In this study, we tested the therapeutic effects of early treadmill locomotor training (Tm) initiated at postoperative (PO) day 8 and continued for 6 weeks with injury site transcranial magnetic stimulation (TMSsc) on spasticity and gait impairments after low C6/7 moderate contusion C-SCI in a rat model. The combined treatment group (Tm + TMSsc) showed the most robust decreases in velocity-dependent ankle torques and triceps surae electromyography burst amplitudes that were time locked to the initial phase of lengthening, as well as the most improvement in limb coordination quantitated using threedimensional kinematics and CatWalk gait analyses, compared to the control or single-treatment groups. These significant treatment-associated decreases in measures of spasticity and gait impairment were also accompanied by marked treatmentassociated up-regulation of dopamine beta-hydroxylase, glutamic acid decarboxylase 67, gamma-aminobutyric acid B receptor, and brain-derived neurotrophic factor in the lumbar spinal cord (SC) segments of the treatment groups, compared to tissues from the C-SCI nontreated animals. We propose that the treatment-induced up-regulation of these systems enhanced the adaptive plasticity in the SC, in part through enhanced expression of pre- and postsynaptic reflex regulatory processes. Further, we propose that locomotor exercise in the setting of C-SCI may decrease aspects of the spontaneous maladaptive segmental and descending plasticity. Accordingly, TMSsc treatment is characterized as an adjuvant stimulation that may further enhance this capacity. These data are the first to suggest that a combination of Tm and TMSsc across the injury site can be an effective treatment modality for C-SCI-induced spasticity and gait impairments and provided a pre-clinical demonstration for feasibility and efficacy of early TMSsc intervention after C-SCI. Key words: ankle torque; spasticity; locomotor training; magnetic stimulation, spasticity; spinal cord injury

Introduction

C

ervical spinal cord injury (C-SCI) is the most common level of human SCI and results in a variety of complications, including spasticity and motor impairments. Ninety-three percent of C-SCI graded as American Spinal Injury Association (ASIA) A and 78% graded as ASIA B-D reported symptoms of spasticity.1 Spasticity refers to a significant exaggeration of the velocitydependent lengthening resistance of the affected muscles.2,3 This condition can severely diminish movement, affect posture and balance, disrupt daily activities and sleep patterns, and significantly encumber care giving. Spasticity may be a product of unselective, maladaptive plasticity in the motor and sensory projection systems as part of an unguided attempt for recovery of function after SCI. Although currently available therapies provide some relief, significant enduring disabilities emphasize the need to further develop 1 2

therapy to ameliorate spasticity and improve the motor functions of C-SCI individuals. Research aimed at therapy development has almost exclusively focused on single therapies, all of which were reported to be minimally effective or ineffective in multi-center clinical trials.4 Our recent work, using locomotor exercise therapy, yielded significant, but modest, decreases in measures of spasticity and locomotor disability. These treatment-induced functional changes were accompanied by treatment-induced increased expression of trophic factors, as well as molecules that augment presynaptic inhibition in the spinal cord (SC). Accordingly, these changes were suggested to have contributed to the improvement of spasticity and motor function. Though these data are promising, the modest therapeutic outcomes observed from the single therapeutic approach suggest that the robustness necessary to make significant functional improvements may require amplification of therapeutic effect through the successful combination of complementary

Department of Physiological Sciences, 3Department of Neuroscience, 4Department of Neurology, University of Florida, Gainesville, Florida. BRRC, North Florida/South Georgia Veterans Health System, Gainesville, Florida.

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COMBINED THERAPY IMPROVED SCI DISABILITIES individual therapies.5 Recent observations6–8 have provided exciting ‘‘proofs of principle’’ that epidural stimulation of the SC can improve locomotor performance in an individual with motor complete paralysis. It was proposed that this approach utilized propriospinal input that projected to the lumbosacral SC circuitry to serve as an adjuvant source of neural control.6,7 Accordingly, locomotor activity-based rehabilitation remains a promising therapeutic approach, particularly with stimulation-induced conditioning, to enhance rehabilitation gains. During a recent series of neurophysiological studies in SCI animals, we stimulated the SC with transcranial magnetic stimulation (TMS) at subthreshold, threshold, and suprathreshold intensities to measure conductivity. Although the intent was simply diagnostic evaluation, based upon behavioral observations in these animals, this evaluation procedure appeared to result in a decrease in lowerlimb spasticity that persisted through a testing session the following day. Therefore, we designed the current studies to systematically evaluate the therapeutic potential of this SC TMS (TMSsc) protocol to influence behavioral, electrophysiological, and immunohistochemical (IHC) measures in the setting of C-SCI. Accordingly, these initiating studies were performed using precisely the stimulus parameters that were used for the former diagnostic procedures. These studies were performed in animals with C-SCI; several experimental outcome measures in these animals were compared with those obtained in C-SCI animals receiving no therapy. In addition, we compared these outcomes to those obtained using a therapeutic approach of guided patterned activity induced by walking on a treadmill (Tm). Finally, we tested the potential for TMSsc to serve as an amplifying adjuvant when added to locomotor therapy by assessing the effect of adding TMSsc to a C-SCI/Tm group that had been training for 4 weeks. Accordingly, these studies were designed to test the efficacy of TMSsc as a stand-alone and also as an adjunctive therapy. Although widely used in a broad range of therapeutic applications since its inception in 1985, detailed knowledge of TMS use as a modulator of neural activity has lagged, in part, because of the lack of a broad perspective of risk/benefit data that is typically evaluated in suitable pre-clinical models.9 Currently, it is not known whether TMS of the cervical SC is safe or to what extent this stimulation could enhance the therapeutic effects of locomotor training in the setting of C-SCI. Because the safety of repetitive TMS (rTMS), relative to seizure induction, has been a concern, we chose to use single-pulse TMS, which has been reported to be safer for clinical use.10,11 The aim of this study was to compare the treatment efficacy of TMSsc and Tm locomotor training both as individual as well as combination therapies to reduce the magnitude of SCI-induced lower-limb spasticity. In addition, this study evaluated the potential for these therapies to influence gait parameters obtained during over-ground locomotion on a CatWalk or during Tm locomotion (three-dimensional [3D] kinematics). These studies also tested the potential for these therapies to influence TMS indices of central excitability of the lower-limb motor system. Finally, the potential for these therapies to influence markers of plasticity was tested by evaluating the IHC expression of signal markers for 1) two factors that are well known to be essential for normal patterns of presynaptic and postsynaptic modulation of reflex excitability: gammaaminobutyric acid (GABA)/GABAb receptors and descending noradrenergic (NE) fiber projection using a marker of dopamine beta-hydroxylase (DbH; the intravesicular synthetic enzyme that converts dopamine into NE) and 2) brain-derived neurotrophic factor (BDNF) in the lumbar SC after moderate C-SCI.

1089 Methods Animals A total of 46 female adult Sprague-Dawley rats (251–275 g; Charles River Laboratories, Wilmington, MA) were used in these studies. All procedures were performed in accord with the U.S. Government Principle for the Utilization and Care of Vertebrate Animals and were approved by the institutional animal care and use committees at the North Florida/South Georgia Veterans Health System (Gainesville, FL) and the University of Florida (Gainesville, FL). Spinal cord injury Contusion injuries were produced using the Infinite Horizons impactor device (Precision Scientific Instrumentation, Lexington, KY). The injury was performed under ketamine (100 mg/kg), xylazine (10 mg/kg; 1:3 with normal saline), and glycopyrrolate (200 lL in each animal) anesthesia.5 A laminectomy was performed at the C6/7 segment, exposing the underlying dura. The spinal column was stabilized with serrated Addison forceps. The impactor tip (2.5 mm in diameter) was positioned to 5 mm above the dorsal surface of the SC, and a moderate contusion injury was produced by an impact force of 200 kdynes. The mean impact force (n = 38) and displacement were 205.79 – 1.05 kdynes and 1535.90 – 37.61 lm, respectively. The whole procedure was performed under aseptic conditions. Animals were monitored routinely and postoperative (PO) care was performed as required to address any issue of pain or other discomfort. Two animals died during the SCI surgery (n = 2), and another 2 died as a result of PO complications (n = 2). Four animals were excluded that received injuries made by impact of a force that was 10% less or more than the desired 200 kdynes mean. Therefore, the remaining 38 SCI animals were used for continuing these studies. At PO week (WK) 1, animals were randomly divided into two groups: a Tm training group (Tm; n = 20) and an untrained control group (n = 18). The injured control animals were handled an equal amount of time (i.e., brought to the lab and placed on the Tm) to neutralize any potential unaccounted contribution from animal handling in the treatment groups. After all tests were completed at PO WK 5, half of the animals from each of these two groups received magnetic stimulation across the injury site (C6/7; TMSsc) for another 2 weeks. Thus, a total of four groups were created: a control group (C-SCI); a Tm training-only group (Tm); a magnetic stimulation-only group (TMSsc); and a combined treatment group (Tm + TMSsc). All treatments were completed at PO WK 7. The treatment-based group design and number of animals in each group are shown in Figure 1. Treadmill training A three-runway Tm (Columbus Instruments, Columbus, OH) was used in this study. The training schedule was performed 5 days a week using two sessions per day, 20 minutes each, starting from PO day 8. Details of this protocol were recently reported on.5 Briefly, on the first day of training, rats were given 5 minutes to explore the Tm and then encouraged to walk on the moving belt at a speed of 11 meters per minute (mpm)12 during four 5-minute bouts of walking. Rats were given a minimum of 5 minutes rest between bouts. On the second day of training, rats walked for two bouts of 10 minutes each twice a day. Then, beginning on day 3 through PO WK 7, rats were trained to walk for 20 minutes per session without a rest. The training consisted of two sessions per day with an interval of 2 h between sessions. Partial body-supported manual assistance was provided as needed.5 PO day 8 was chosen as the time to initiate the training based upon our previous studies, which compared locomotor training initiated at PO days 1, 8, and 17 after mid-thoracic SCI. The PO day 8 training group showed the best result in reducing spasticity among these groups.

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FIG. 1.

Experimental design. PO D, postoperative day; PO WK, postoperative week.

Transcranial magnetic stimulation treatment Animals were immobilized in a custom-designed trunk restraint device that was developed and used regularly for spasticity measures.5,13,14 Because computational models for the neurobiological application of magnetic stimulation are not currently available, a brief description of the stimulus procedure is provided. A 25-mm figure-of-eight Magstim Coil (internal diameter, 25 mm; average inductance, 15.25 lH; maximum magnetic field strength, 4.0T; maximum number of stimulation at 50% power, 212; The Magstim Company Limited, Whitland, UK) was used for delivering singlepulse TMS. This coil was polyurethane coated to reduce enclosure thickness (compared to the standard use of plastic), allowing the stimulating coils to be placed in close proximity to the targeted site of stimulation. Traditionally these 25-mm figure-of-eight coils have been used clinically to enhance positioning accuracy for stimulation of small targets (e.g., facial nerve, median nerve, ulnar nerve, F-wave and reflexes, and so on). To confirm the specifications supplied by the manufacturer, we calibrated and mapped the magnetic field produced by our coil with assistance from University of Florida McKnight Brain Institute Advanced Magnetic Resonance Imaging and Spectroscopy Facility engineers (see details about this facility at: http://amris.mbi.ufl.edu/). The central point of the figure-of-eight coil (where the magnetic field strength was determined to be the greatest) was placed at the C6/C7 vertebral level, slightly touching the skin. For consistent and systematic targeting of the stimulus site, the skin was marked with a tattoo referenced to vertebral spinus process landmarks. Placement of the coil was stabilized by hand and repositioned as needed to maintain accurate placement. Five single, biphasic magnetic pulses (pulse duration, 400 lsec with 3-sec interpulse interval) were delivered using an intensity ladder (recruitment paradigm) of 30% (five pulses), 40% (five pulses), 50% (five pulses), 60% (five pulses), and 70% (five pulses) of maximum output (five-step intensity ladder). One minute separated each step change in intensity. These percentages (30%, 40%, 50%, 60%, and 70%) correspond to field intensities of 1, 1.4, 1.8, 2.2, and 2.4T, respectively. This recruitment sequence was repeated three times (total, 75 pulses). Based on our preliminary studies of motor-evoked potentials (MEPs) of triceps muscles, these TMS intensities represent prethreshold (30%), threshold (40%), and suprathreshold (50%, 60%, and 70%) intensities. Previously, these specific doses were applied for an MEP study in SCI animals; subsequently, we observed significant improvements in

gait and significant reductions in spasticity in these animals. Therefore, these dosage intensities were chosen to systematically study and extend these unanticipated findings. Because the magnetic field generated by the coil had an amplitude of 12.0 mm and a half width of 10.0 mm, ascending and descending spinal tracts above, through, and below the lesion site were well within the magnetic field of the applied TMS. To assess the relative effectiveness of our TMSsc methodology, we conducted MEPs evoked by the coil positioned on the cervical SC over the lesion (C6/C7; Fig. 2C), the cranium of a normal and a C-SCI (Fig. 2A and B respectively), and caudal to the lesion on the lower thoracic/upper lumbar T11-L1 (Fig. 2D). Single magnetic pulses of 60% of maximum (suprathreshold intensity) was applied to test the site specificity of the magnetic coil to activate MEPs from stimulus sites above (Fig. 2A,B), through (Fig. 2C), and below (Fig. 2D) the lesion site. The magnetic pulse shock artifact is the first vertical spike that appeared at 80 msec from left to right (Fig. 2). The latency of soleus MEP in a normal animal was *6 msec and appeared as a well-synchronized burst (Fig. 2A). Stimulation at the cranium in this particular C-SCI animal did not evoke an MEP of the soleus muscle (Fig. 2B). It is presumed that SCI damaged a portion of the descending pathways that were critical for the activation of MEP in this animal. TMS across the injury site (C6/7) in this animal evoked a compound MEP (Fig. 2C). Note, there are multiple peaks in this MEP (Fig. 2C). This complex waveform (compared to that evoked in Fig. 2A) might represent activation of descending pathways with different latencies, such as antidromic activation of primary afferent collaterals coursing in the dorsal columns and orthodromic activation of ventral funicular fibers. Moreover, stimulation of the T11-L1 area (in this C-SCI animal) produced a robust synchronized MEP of soleus muscle (Fig. 2D) with a shorter latency (*2.5 msec) and a complex second peak with a much longer latency (*17 msec). This second peak may be mediated by repetitive/bursting discharges of the soleus motoneurons or may be a result of inputs from multiple pathways to the soleus motor neuron pool. The therapeutic stimulation was applied on every other day from PO WK 5 to PO WK 7 (total, seven sessions). The TMSsc was initiated at PO WK 5 based upon two considerations: 1) safety and 2) unequivocal demonstration of efficacy when initiated at this time point in a setting of spasticity. In regard to safety, the acute period after C-SCI is known to include challenges to homeostatic stability resulting from injury-related changes in descending control of

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FIG. 2. Soleus motor-evoked potentials at single magnetic pulses of 60% of maximum input: (A) normal, cranial stimulation; (B) CSCI, cranial stimulation; (C) C-SCI, stimulation across the SCI injury site; and (D) C-SCI, stimulation caudal to the lesion on the lower thoracic/upper lumbar T11-L1 segments.

the thoracolumbar components of the autonomic nervous system (ANS). Accordingly, we chose to wait a conservative 5 weeks before initiating TMSsc that could potentially interact with these anatomically contiguous components of the ANS. In regard to initiating TMSsc in a setting of spasticity, our previous studies have shown that, by PO WK 5, a fixed pattern of spasticity developed after SCI.13,15,16 Thus, to test the effectiveness of TMSsc as an individual treatment on a fixed pattern of spasticity, the PO WK 5 interval was chosen to initiate this treatment. In addition, to test the potential for TMSsc to provide an adjuvant effect, TMSsc was applied in a combination with Tm training. The timing of Tm and TMSsc therapies was controlled and systematic. TMSsc was applied as the first procedure of the morning (9:30–10:00 am). Then, immediately after completion of the TMSsc session (within 15 min), the animal was provided Tm training. Velocity-dependent ankle torques and electromyography recording The lengthening resistance of the triceps surae muscles was measured indirectly by quantifying velocity-dependent ankle torques (VDAT) and electromyograms (EMGs) during 12-degree dorsiflexion ankle rotation.13,14,17 Briefly, rats were immobilized in a custom-designed trunk restraint device. The hindlimbs were secured above the ankles to permit a normal range of ankle rotation. A transcutaneous EMG electrode was inserted in a skin fold over the distal convergence of the medial gastrocnemius, lateral gastrocnemius, and soleus muscles at the origin of the tendonocalcaneous, whereas a reference electrode was placed in a skin fold over the greater trochanter. The foot was secured within a form-fitted cradle that was aligned with the dorsal edge of the central footpad 2.6 cm distal from the ankle joint. A controlled 12-degree ankle rotation was produced using an electromechanical shaker (model 405; Ling Dynamic Systems, Royston Herts, UK). A length displacement transducer (Shaevitz) was placed around the output shaft of the electromechanical shaker to record displacement. A force transducer (model FT-03; Grass Instruments, Quincy, MA) was placed in series between the output shaft of the ling shaker and the central footpad of the foot. Raw EMG and root mean square (RMS) of EMG bursts were recorded simultaneously with ankle torque and ankle rotation. A series of 12-degree ankle rotations were produced at 3-sec intervals at eight different velocities (49, 136, 204, 272, 350, 408, 490, and 612 degrees/sec). Animals typically adjusted to the restraint device without detectable discomfort. The data recording session began when the animal was relaxed and the protocol required approximately 45 min. At each test velocity, five consecutive sets of waveforms, five waveforms per set (a total of 25 waveforms per velocity) were recorded and saved for subsequent analysis. The data were acquired, digitized, and signal averaged using a digital acquisition system with Lab-

VIEW graphic programming (version 8.2; National Instruments, Austin, TX). Animals were tested at preinjury, PO WK 1, PO WK 5, and PO WK 7. Transcranial magnetic motor-evoked potentials Magnetic pulse stimulation was applied to the cranium to elicit transcranial magnetic motor-evoked potentials (tcMMEPs) in triceps surae muscles. The center of the 25-mm figure-of-eight coil was positioned on the scalp over the cranial region bregma to apply the magnetic field to a region that systematically included the underlying motor-sensory cortex. It has been reported that tcMMEPs utilize fibers coursing in the ventral lateral SC,18 and it is presumed that this pathway is, in part, comprised of corticoreticular, reticulospinal fibers. This magnetic field stimulation was generated by a Magstim Rapid TMS model (Magstim). A recruitment curve of tcMMEPs response amplitudes was produced using single biphasic magnetic pulses with 400-lsec pulse duration, beginning at 40% and increasing in 10% intervals through 70% of maximum. The threshold and amplitude of these tcMMEPs were compared in each of the experimental conditions. The onset latency time and amplitude of the tcMMEPs were measured in potentials recorded from triceps surae muscles using the same intracutanious electrodes placed during the recording of ankle torque and EMGs.13 These experiments were performed immediately after completion of the VDAT and EMGs in the same setting. Data were recorded using National Instruments signal acquisition hardware and software (version 8.2; National Instruments; PCI-6251 DAQmx card and LabVIEW software modified locally; the card and software operate in a standard PC) with the analog to digital sampling rate set to 5 kHz. CatWalk gait analysis Preinjured animals were trained for 1 day to become familiar with the walk path of a 10-cm-wide by 120-cm-long CatWalk apparatus (Noldus Information Technology, Leesburg, VA). Three nonstop runs were acquired for each animal and the distance traveled before stopping was kept the same for each animal in preand postinjury conditions. The enclosed walkway was formed by a glass plate, which was internally reflected by a green laser that entered at the long edge of the plate. Light was able to escape only at the areas where the rat’s paws made contact with the glass plate. The paw prints were captured by a high-speed video camera that was positioned beneath the walkway. The digital images were then transferred to the computer for data analysis. Three continuous steps for each animal were used for analyses using the gait analyses software (CatWalk XT version 8.1; Noldus Information Technology). Animals were tested at preinjury and PO WK 7, when all treatments were ended.

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Three-dimensional kinematics gait analysis

Statistical analysis

3D kinematic gait analysis (Vicon, Denver, CO) were performed at preinjury and PO WK 7. At least 1 week before recording, animals were anesthetized with ketamine and a permanent marker was tattooed on the shaved skin at the points of shoulder, elbow, wrist, and the fourth metacarpal joints of the forelimb. The iliac crest, hip, knee, ankle, and the fourth metatarsal joint positions were also tattooed to maintain permanent landmarks for these joint positions. Immediately before a recording session, miniature video-sensitive reflectors were placed on to these marked points. Animals then walked on the Tm at a speed of 11 mpm, and a 10second digital video (100 frames per second) was obtained during the walking session. The video frames were digitized and analyzed using Vicon Motus software (Vicon). Three consecutive step cycles of fore- and hindlimbs were averaged and normalized for analysis of the joint angles of shoulder, elbow, wrist, hip, knee, and ankle at specific phases of the step cycle. As a potential marker for representative injury-induced change in kinematic form, the distal joint angles at the conspicuous phase transition, paw liftoff, were compared in all groups.

Two-sample t-tests were used to compare differences between preinjury and each time point after injury for different groups; a single-factor analysis of variance was also used to analyze the different groups at the same time point (e.g., PO WK 7). Wilcoxon’s rank-sum test was used to analyze data that did not pass the normality test. The paired t-test was used to compare the percentage change of ankle torque and EMG-RMS at each time point with preinjury level for individual groups. The p values less than 0.05 were considered significant. In addition, pair-wise correlations between spasticity and kinematic assessments, spasticity, and IHC expression density of specific markers were tested by plotting and comparing treatment and control groups’ spasticity data (ankle torque data at 612 degrees/sec), kinematic data (lift-off data from ankle angles), and densitometry data from IHC.

Immunohistochemistry and histology After all the tests were completed, animals were deeply anesthetized with ketamine and sacrificed by transcardiac perfusion with phosphate-buffered saline (PBS; 0.01 M, pH 7.4) followed by 4% paraformaldehyde (PFA) in phosphate buffer (0.1 M, pH 7.4, 4C). SCs were dissected and postfixed with 4% PFA for 24–48 h. The lumbar SC segments L3–4 (n = 6 for C-SCI and TMSsc groups; n = 5 for Tm and Tm + TMSsc groups, randomly selected) were then further processed and placed into 30% sucrose for 3 days and then cut serially into 40-lm sections rostrocaudally by cryostat, as described in our previous reports.5,19 A total of four sets of sections were made for four antigens (the 67-kDa isoform of glutamate decarboxylase [GAD67], GABAb, DbH, and BDNF). Each set contained equally spaced sections. Floating sections were washed three times with PBS and blocked in 5% goat serum in PBS for 1 h at room temperature. Sections were then incubated with primary antibodies [Abs; rabbit anti-BDNF, 1:2000 [Millipore, Billerica, MA]; guinea pig anti-GABAb, 1:4000 [Millipore]; mouse anti-GAD67, 1:4000 [Millipore]; mouse anti-DbH, 1:2000 [Millipore]) overnight at 4C in 1% goat serum in PBS. Sections were then washed three times in PBS to remove unbound primary Ab. Sections were incubated with secondary Abs (Alexa Fluor 488 goat anti-rabbit immunoglobulin G [IgG], 1:1000 for BDNF; Alexa Fluor 594 goat anti-guinea pig IgG, 1:1000 for GABAb; Alexa Fluor 488 goat anti-mouse IgG, 1:1000 for GAD67 and DbH) for 2 h at room temperature. After washing three times in PBS, sections were mounted on the slides and cover-slipped with Vectashield (Vector Laboratories, Burlingame, CA). Slides were then viewed using a Zeiss Imager Z2 microscope equipped with Axiovision software (version 4.8.2; Carl Zeiss, Jena, Germany). The number of immunopositive cells (for GABAb and BDNF) and fibers (for GAD67 and DbH) were examined qualitatively in all sections throughout the gray matter. However, for quantitative analyses, stained sections were used to quantify the number of immunopositive cells or fibers in the ventral horn corresponding to Rexed laminae VII, VIII, and IX using a customized densitometry program (Densitometry Wizard, Axiovision software, version 4.8.2; Carl Zeiss), following the cell counting procedure we have reported previously.20 For qualitative histological study, the cervical spinal segments, which included the injury epicenter, were embedded with paraffin, then cut into 10-lm sections by microtome, and mounted on gelatin subbed glass slides. Sections were then processed with hematoxylin and eosin (H&E) staining. Slides were then viewed using a Zeiss Imager Z2 microscope equipped with Axiovision software (Carl Zeiss).

Results VDAT and time-locked EMGs were tested across a wide range of velocities (612–49 degrees/sec) to permit quantitation of tonic (low-velocity) and dynamic (high-velocity) contributions to lowerlimb spasticity at PO WK 1, WK 5, and WK 7, compared with values recorded at preinjury. Cervical spinal cord injury and treadmill postoperative week 5 At PO WK 5, all injured control animals revealed significantly increased amplitudes of VDAT during rotation at each of the eight ankle rotation velocities, compared with preinjury levels (Fig. 3A). In addition, EMG-RMS amplitudes, which were time locked to these angular rotations of the ankle, were significantly increased in the C-SCI animals, compared with the recordings in preinjury animals (Fig. 3B). In contrast, after 4 weeks of Tm training, the Tm group showed significantly decreased VDAT amplitudes at all velocities (Fig. 3A) and significantly decreased EMG-RMS amplitudes (Fig. 3B) at 612, 136, and 49 degrees/sec, compared to mean values recorded in the untrained C-SCI group. These data suggested that Tm locomotor training initiated at PO WK 1 significantly reduced the development of spasticity in these animals after C-SCI. Postoperative week 7 Cervical spinal cord injury and individual treatment groups. At PO WK 7, all injured control animals revealed significantly increased amplitudes of VDAT and EMG-RMS during rotation at each of the eight ankle rotation velocities, compared with preinjury levels (Fig. 3C). At PO WK 7, the Tm group and TMSsc groups also showed significant increases in VDAT and EMG-RMS, compared with the preinjury level, at all test velocities. However, the Tm group and the TMSsc treatment groups showed significant decreases in VDAT and EMG-RMS, compared to the C-SCI group, at all eight velocities (Fig. 3C). Moreover, in both treatment groups, the EMGRMS magnitudes were observed to be significantly decreased, compared to those observed in the C-SCI group, at all velocities, except at 490 degrees/sec in the Tm group (Fig. 3D). There were no significant differences in VDAT and EMG-RMS between the Tm and TMSsc groups at any of the tested velocities (Fig 3C). The combined treatment group (Tm + TMSsc) showed the greatest reduction in velocity-dependent ankle torques and time-locked EMGs, compared to the nontreatment group or individual treatment groups. The combined treatment group revealed significantly decreased VDAT, compared to either single-treatment group (Fig. 3C).

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TMSsc versus Tm + TMSsc. Compared to the TMSsc group, the combined therapy group revealed significantly decreased VADT at 350, 272, 136, and 49 degrees/sec. Also, compared to TMSsc, the EMG amplitudes recorded in the Tm + TMSsc group were significantly less at 612, 204, and 136 degrees/sec, respectively. Preinjury versus Tm + TMSsc. Moreover, compared with preinjury, the combined treatment group (Tm + TMSsc) showed significant increases only at the upper two velocities. At 350 and 272 degrees/sec, the Tm + TMSsc group showed significant decreases in EMG-RMS amplitudes, compared with preinjury level. Collectively, These data suggested that either Tm or TMSsc applied as individual therapy reduced the SCI-induced spasticity, but that the combination of Tm and TMSsc therapy produced a reduction of VDAT to preinjury level at the lower six test velocities. To further contrast the influence of injury and treatment on the measures of spasticity, the percentage changes of VDAT (Supplementary Table 1) (see online supplementary material at http:// www.liebertpub.com) and EMG-RMS (Supplementary Table 2) (see online supplementary material at http://www.liebertpub.com), relative to preinjury levels, were calculated for each group at PO WK 1, PO WK 5, and PO WK 7. For example, notice the contrast in percentages at PO WK 7 for C-SCI, Tm, TMSsc, and Tm + TMSsc. Effects of locomotor training and/or magnetic stimulation on gait impairments CatWalk gait analysis. Gait analysis was performed using CatWalk (Noldus Information Technology) to evaluate the influence of injury and treatment on interlimb movement sequences (coordination and timing of fore- to hindlimb) during over-ground walking. This analysis consisted of averaging three nonstop walking sequences acquired preinjury and at PO WK 7 for each animal. Phase dispersion. Compared with mean preinjury values (15.96 – 0.89), at PO WK 7, the C-SCI, TMSsc, and Tm groups showed significant increases in the phase dispersions for right frontleft hindlimb (RF-> LH; 42.69 – 4.32, 33.26 – 4.98, and 39.26 – 7.14, respectively). However, the combined treatment group (Tm + TMSsc) did not show significant changes in the fore-/hindlimb phase dispersions 26.97 – 5.17 (Fig. 4A). Moreover, C-SCI and TMSsc groups showed significant changes in right front-right hindlimb (RF- >

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Tm versus Tm + TMSsc. Compared to the Tm group, VDATs in the Tm + TMSsc group were significantly decreased at all except 490 degrees/sec. In addition, compared to Tm, the EMG amplitudes recorded in the Tm + TMSsc group were significantly less at 612, 272, 204, and 136 degrees/sec.

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FIG. 3. Velocity-dependent ankle torque (VDAT) (A and C) and time-locked EMG-RMS amplitudes (B and D) of C-SCI-untreated and -treated animals at PO WK 5 and PO WK 7. All treatment groups showed significant improvement (decreases) in VDAT and corresponding EMGs, compared to values recorded in the C-SCI group. However, the combined treatment group, Tm + TMSsc, exhibited VDAT and EMG-RMS amplitudes levels similar to those that were recorded at the preinjury condition, except the torque values at the upper two velocities (C) and EMG-RMS values at the 350- and 272-degree/sec velocities (D) at PO WK 7. *p < 0.05; **p < 0.01; ***p < 0.001, compared with preinjury; ! p < 0.05; !!p < 0.01; !!!p < 0.001, compared to C-SCI. ^p < 0.05; ^^ p < 0.01; ^^^p < 0.001, compared to the Tm + TMSsc group. CSCI, cervical spinal cord injury; PO WK, postoperative week; TMSsc, transcranial magnetic stimulation on spinal cord; Tm, treadmill training; EMG-RMS, electromyography root mean square.

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RH) phase dispersions, compared with preinjury, and the Tm + TMSsc group showed a significant difference, compared to the C-SCI group (Fig. 4B). Average speed. The average speed of 30.26 – 1.07 cm/sec in the preinjured animals was observed to be significantly decreased to 23.49 – 1.55 cm/sec in C-SCI animals and 24.07 – 2.22 in TMSsc-treated animals. However, the Tm and the Tm + TMSsc groups revealed walking speeds (27.78 – 1.88 and 27.59 – 1.95 cm/ sec, respectively) that were not significantly different from values observed in preinjury (Fig. 4C). Stand index. The mean absolute value for hindpaw (HP) stand index of 4.6 in preinjured animals was observed to be significantly decreased to 3.76 in C-SCI animals and 3.56 in TMSsc-treated animals. However, the Tm and the Tm + TMSsc groups revealed stand indices (4.24 and 4.09, respectively) that were not significantly different from the values obtained preinjury. The stand index values (the speed at which the paw loses contact with the plate) reflected the pattern observed for average walking speed (Fig. 4D). Percent index of dispersion. A measure of the consistency of locomotor pattern was revealed by calculating the percentage index of dispersion (r2/l * 100) of the mean velocity for each group. This measure of pattern consistency observed in the preinjury group (37.76% – 3.17%) was observed to nearly double (67.30% – 12.65%) when calculated for control C-SCI animals and was significantly increased (53.41% – 5.19%) in TMSsc-treated animals. However, the percentage index of dispersion calculated for the Tm (41.50% – 6.93%) and Tm + TMSsc (39.53% – 6.69%) groups were not significantly different from those observed preinjury (Fig. 4E). Foot-print area. The C-SCI group showed significantly decreased HP print area, compared with preinjury animals, whereas the treatment groups did not show significant differences (Fig. 4F). Collectively, the combined treatment group (Tm + TMSsc) gait parameters showed significant normalization of gait patterns in the fundamental index of interlimb coordination (Fig. 4A) and locomotor velocity (Fig. 4C). Although the combined treatment showed improvements in base of support and stride length (data not shown here), those values did not reach significant differences, when compared to values obtained from C-SCI controls or Tm or TMSsctreated groups. Three-dimensional angular kinematics. Collectively, the combined treatments group (Tm + TMSsc) showed the least deviation of fore- and hindlimb joint angles and coordinated sequences of movement. 3D angular kinematic analyses were performed while animals walked at 11 mpm on the treadmill. PO WK 7 plots of joint angles in C-SCI and C-SCI-treated animals were compared with preinjury recordings as reference frames (Supplementary Figs. 1 and 2) (see online supplementary material at http://www.liebertpub.com). The wrist joint angles of C-SCI animals that were analyzed during the course of averaged three consecutive step cycles revealed marked open joint angles during the latter portion of the step cycle. Similarly, joint angle plots of ankle angle data acquired in C-SCI animals demonstrated marked deviations from the movement patterns observed before injury. These patterns revealed marked open angles near the end of the step cycle related to the inverted foot position used during stance and the delayed transition from stance to swing phases of stepping. However, the wrist and ankle angles

FIG. 4. Several gait parameters were analyzed using CatWalk footprints obtained from animals at preinjury and PO WK 7. At PO WK 7, in general, the combined treatment and Tm groups showed better improvement in these gait parameters, except for the right front-left hindlimb phase dispersions, where only the Tm + TMSsc group showed normalized result (A). The Tm and Tm + TMSsc groups showed normalized right front-right hindlimb phase dispersions (B), average speed (C), absolute value of hindpaw stand index (D), and index of dispersion (E), when compared with corresponding values that were obtained from the preinjury condition. All treatment groups resulted in normalized hindpaw print area (F), when compared with the values obtained at preinjury. It is important to note here that base of support and stride length were not significant among groups and thus not included in this figure. *p < 0.05; **p < 0.01; ***p < 0.001, compared with preinjury; !p < 0.05, compared to C-SCI. n = 9 for C-SCI and TMSsc groups; n = 10 for Tm and Tm + TMSsc groups; n = 38 for preinjury. C-SCI, cervical spinal cord injury; PO WK, postoperative week; TMSsc, transcranial magnetic stimulation on spinal cord; Tm, treadmill training.

revealed pronounced normalization in the treated animals particularly, as a function of Tm + TMSsc training. Note that the congruency of the ankle angle plots for the Tm + TMSsc group are reflective of the pronounced normalization of the stepping pattern. It is important to note here that preinjury reference frames are

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FIG. 5. Knee-ankle angle-angle graphs are shown here by using 3D angular kinematic analyses. At PO WK 7, C-SCI animals showed the greatest loss of coordination (A). The plots of data acquired in the C-SCI animals were translated on the ankle (x) axis, revealing the greatest loss of coordination in the distal joints. The plots of data acquired in the Tm and Tm + TMSsc animals showed less plot translation at the distal joint and a more similar pattern to preinjury than the plots in C-SCI or TMSsc alone (B, TMSsc; C, Tm; D, Tm + TMSsc). ‘‘X’’ represents the transition from stance to swing and ‘‘O’’ denotes the transition from swing to stance; ‘‘+’’ represents centroid of each step cycle standardized as 100 units and averaging over multiple steps within preinjury and within PO WK 7. PO WK, postoperative week. necessary for a meaningful comparison between pre- and postinjury performance in each rat because each animal has unique movement patterns. Plotting knee against ankle provided a cycloplot perspective of changes in the coordinated sequence of movement at one joint as a function of movement at the other (Fig. 5). In C-SCI animals, the plots were translated on the ankle (x) axis, revealing that the greatest loss of coordination was evident in the distal joints (Fig. 5A). The plots of data acquired in Tm and Tm + TMSsc animals showed less plot translation at the distal joint and a more similar pattern to preinjury than the plots in C-SCI or TMSsc alone (TMSsc, Fig. 5B; Tm, Fig. 5C, and Tm + TMSsc, Fig. 5D). The centroids for the plots shown in Figure 5 were consistent with the injury-associated translation and noncongruity, except for the plots in Figure 5A. To further distinguish injury- or treatment-related

changes in the locomotor patterns, the distal limb joint angles at liftoff were measured at PO WK 7 and percentage changes, relative to preinjury, were compared (Table 1). All injured animals showed significant increases in the wrist and ankle lift-off angles at PO WK 7, compared to those measured in preinjured animals. The changes in the ankle joint were significantly increased in each group, compared to corresponding changes in the wrist joint. However, at PO WK 7, the Tm + TMSsc group showed significant decreases in wrist angles, compared to C-SCI and TMSsc groups, and significant decreases in ankle angles, compared to the C-SCI group. The percentage change in wrist and ankle angles at PO WK 7 were compared to those measured in preinjured animals. The Tm group and, especially, the Tm + TMSsc group, showed significantly smaller changes in wrist and ankle angles, when compared to the C-

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Table 1. Wrist and Ankle Angles at Paw/Toe Liftoff Wrist and ankle angle at lift-off Group

Time

Wrist

Ankle

C-SCI

Preinjury PO WK 7 % change Preinjury PO WK 7 % change Preinjury PO WK 7 % change Preinjury PO WK 7 % change

143.26 – 5.45 167.92 – 4.25{ 18.21 – 4.42 153.21 – 3.27 167.55 – 1.87{ 9.68 – 2.17 153.73 – 3.01 161.72 – 2.38* 5.40 – 1.77{ 149.84 – 3.20 155.59 – 3.49{,x,II 3.84 – 0.65{,**

81.92 – 3.15 128.43 – 6.16{ 56.84 – 4.97 83.65 – 3.58 119.13 – 2.92{ 45.12 – 8.51 82.66 – 2.56 114.04 – 2.88{ 39.12 – 5.38{ 84.39 – 1.94 107.72 – 4.96{,x 27.68 – 5.27#

TMSsc

Tm

Tm + TMSsc

Transcranial magnetic motor-evoked potentials

Wrist and ankle angles at paw/toe lift-off moment at preinjury and PO WK 7 for each group are shown here. Percentage changes of preinjury at PO WK 7 are also shown. *p < 0.05, {p < 0.01, and {p < 0.001, compared with preinjury; xp < 0.05, compared to C-SCI at PO WK 7; kp < 0.01, compared to TMSsc at PO WK 7; {p < 0.05 and #p < 0.001, compared to percentage changes of the C-SCI group; **p < 0.05, compared to percentage changes of the TMSsc group. C-SCI, cervical spinal cord injury; TMSsc, transcranial magnetic stimulation on spinal cord; Tm, treadmill training; PO WK, postoperative week.

SCI group. Moreover, the Tm + TMSsc group also showed significantly smaller changes in wrist angle, compared to the TMSsc group (Table 1). Collectively, on PO WK 7, Tm and Tm + TMSsc animals showed a similar flexion-extension cycling pattern as preinjury animals, except for the overall enlarged knee angles resulting from increased knee extension. By comparison, the TMSsc-only–treated animal also showed the increase of knee angle during the step cycle, and the pattern of intralimb movement was more irregular and showed an increase in ankle motion range as a result of an increased period of foot dragging. However, the most irregular ankle-knee movement pattern and the most obvious increased ankle motion range were observed in the plots of data acquired from the untreated C-SCI animals. Further, instead of the enlarged knee angle observed in the treatment animals, C-SCI animals revealed decreased ankle angles resulting from increased ankle flexion (Fig. 5A). These data suggested that the Tm training played an important role in recovery and/or compensation of gait impairments. As an ad-

Transcranial magnetic motor-evoked potential excitability (probability for appearance). In intact animals, the tcMMEP appeared as a biphasic compound EMG waveform time locked to the presentation of the TMS and appeared in parallel with the visual inspection of a twitch contraction of the triceps surae muscles. The probability for appearance of the tcMMEP was 52.63% at 40% maximum TMS intensity and appeared with a probability of 100% at higher intensities (e.g., 50%, 60%, and 70%; Table 2). By comparison, the probability for evoking tcMMEPs in C-SCI animals at PO WK 5 was 0%, 33.3%, 66.7%, and 66.7% using TMS intensities of 40%, 50%, 60%, and 70% maximum, respectively. The probability for evoking tcMMEP was observed to be increased after application of either Tm or TMSsc treatment and, particularly, after the combination of Tm and TMSsc. For example, at PO WK 7, the probability for evoking tcMMEP using 40% TMS was 11.1% in SCI animals, but was observed to be 6 times greater (e.g., 66.7%) in the TMSsc and Tm + TMSsc treatment groups. These responses, and the response probabilities for each of the groups, is summarized in Table 2. Amplitude. In normal animals, the mean amplitude of the tcMMEP was 5.95 mV, but was observed to be decreased by 80% after C-SCI when tested at 5 and 7 weeks postinjury. The tcMMEP amplitude values were similar in the C-SCI and the C-SCI/Tm groups when tested at 5 and 7 weeks postinjury, respectively. The tcMMEP amplitudes measured in the TMSsc group were significantly larger than recorded in this group at PO WK5 (prestimulus) and was also observed to be significantly larger than recorded in the Tm group at PO WK 7 (Supplementary Fig. 3A) (see online supplementary material at http://www.liebertpub.com). Latency. In the intact animals, the latency for the tcMMEP was 5.32 msec. The latencies were observed to be increased in all of the animals after SCI (e.g., to a mean of 9.32 for the C-SCI group), and there was not any significant difference in the latencies between the injured and treatment groups. The latencies were observed to be decreased by approximately 20% across the groups when recorded at PO WK 7, for example, to a mean of 7.38 in the

Table 2. Probability of Motor-Evoked Potential Induced by Different TMS Intensity Probability of motor evoked potential induced by different TMS Intensity

Normal C-SCI TMSsc Tm Tm + TMSsc

40%

50%

60%

70%

20/38 (52.63%)

38/38 (100%)

38/38 (100%)

38/38 (100%)

PO WK 5 0/9 2/9 2/9 2/9

(0%) (22.2%) (22.2%) (22.2%)

PO WK 7 1/9 6/9 5/9 6/9

(11.1%) (66.7%) (55.6%) (66.7%)

PO WK 5 3/9 3/9 4/9 5/9

(33.3%) (33.3%) (44.4%) (55.6%)

PO WK 7 6/9 9/9 9/9 9/9

(66.7%) (100%) (100%) (100%)

PO WK 5 6/9 5/9 5/9 8/9

(66.7%) (55.6%) (55.6%) (88.9%)

PO WK 7 6/9 9/9 9/9 9/9

(66.7%) (100%) (100%) (100%)

PO WK 5 6/9 5/9 5/9 8/9

(66.7%) (55.6%) (55.6%) (88.9%)

PO WK 7 8/9 9/9 9/9 9/9

(88.9%) (100%) (100%) (100%)

The probability of motor-evoked potential induced at 40%, 50%, 60%, and 70% of maximum TMS intensity of normal animals and C-SCI, Tm, TMSsc, and Tm + TMSsc groups at PO WK 5 and PO WK 7 are shown. TMS, transcranial magnetic stimulation; C-SCI, cervical spinal cord injury; TMSsc, transcranial magnetic stimulation on spinal cord; Tm, treadmill training; PO WK, postoperative week.

COMBINED THERAPY IMPROVED SCI DISABILITIES C-SCI group. There were no significant differences between groups in the percent change in latency (details of these data are shown in Supplemental Fig. 3B) (see online supplementary material at http://www.liebertpub.com). Immunohistochemistry All treatment groups showed up-regulation of GABAb receptors, GAD67, BDNF, and DbH immunoreactivity (IR) in the lumbar ventral horn. The combined treatment group showed the most upregulation of GAD67, BDNF, and DbH in the lumbar ventral horn. IHC was performed on tissues from the C-SCI (untreated) and treated C-SCI animals to assess the possibility that treatment could alter the expression of molecules known to be involved in 1) processes that regulate excitability (e.g., GABAergic presynaptic inhibition [markers: GABAb and GAD67]), 2) neuroplasticity (marker: neurotrophic molecule [BDNF], and 3) the noradrenergic descending system (marker: DbH) in lumbar (SC) (Figs. 6–9). Tissues from animals that received treatments applied individually or in combination revealed marked increases in expression of each of the IHC markers, compared to the intensities observed in tissues from C-SCI untreated animals (Figs. 6–9). Quantitative analyses revealed that all three treatment groups showed significant up-regulations of GABAb, compared to C-SCI (Fig. 6), and there were no significant differences for GABAb expression among these three treatment groups. Further, the combination treatment (Tm + TMSsc) group showed the most robust and significant up-regulation of GAD67 (Fig. 7) and BDNF (Fig. 8), compared to the other two treatment groups or injured control group. Although all treatment groups showed significant up-regulation of DbH (Fig. 9) IR fiber density, compared to the SCI control group, there was, however, a clear trend of greater IR values of DbH fibers in the combined treatment group (Fig. 9).

1097 ventrolateral funiculus). Accordingly, these mild-to-moderate injuries, with preservation of intact ventromedial white matter, enabled Tm locomotion, although the patterns were altered, compared to those observed preinjury. The images of 1 representative animal from each of the four groups are shown in Figure 10. Discussion This study evaluated treatment induced changes in C-SCI associated lower limb spasticity, gait impairment, and alterations in the IHC of certain receptors, transmitters, and neurotrophic substances. Findings in animals with C-SCI were compared with findings in animals that received two post-injury rehabilitation therapies, Tm training and magnetic stimulation, both as individual and combined treatments. The underlying premise of the experimental rehabilitation asserts that recovery is based upon adaptive neuroplasticity and that the potential for recovery can be amplified by activity-induced plasticity. Our data suggested that Tm training and TMSsc across the injury site can be an effective and feasible treatment modality for C-SCI induced spasticity and gait impairments and that the combination of these two therapies was significantly more effective than either treatment tested as individual therapy. Ankle extensor stretch reflex after cervical spinal cord injury: (unique pattern of cervical spinal cord injury spasticity)

Polynomial regression analysis revealed predictable direct relationships in the amplitude of ankle torque and alterations in the kinematic pattern of ankle angle at liftoff (see Supplementary Fig. 4A) (see online supplementary material at http://www.liebertpub .com) as well as predictable inverse relationships in the amplitude of ankle torque and intensity of GAD67 IR fiber density (Supplementary Fig. 4B (see online supplementary material at http:// www.liebertpub.com). The polynomial regression analysis also showed a predictable relationship between spasticity and kinematic gait data (R2 = 0.51) and spasticity and GAD67 IR fiber density (Supplementary Fig. 4B; R2 = 0.55) (see online supplementary material at http://www.liebertpub.com). Similar analyses yielded a predictive relationship between spasticity and IR GABAb (R2 = 0.68), spasticity and BDNF IR-positive cells (R2 = 0.50), and spasticity and DbH IR fiber density (R2 = 0.56; graphs not shown). These regressions also clearly revealed that the data clusters of the treatment groups were separated from the data cluster of the injured untreated controls (e.g., see Supplementary Fig. 4 (see online supplementary material at http://www.liebertpub.com).

We previously reported that subsequent to mid-thoracic contusion SCI (T-SCI), significant increases in VDAT and EMGs appeared during rotation at the upper range of ankle rotation velocities that were consistent with expression of a spastic hyperreflexia. This high-velocity spasticity was progressive in onset and, once developed, was permanent.13,21 However, compared with TSCI spasticity, there were some significant differences in the pattern and magnitude of C-SCI spasticity. After C-SCI, untreated control animals showed significantly increased VDAT (and timelocked EMGs) at all tested velocities (not just the upper range of velocities observed after T-SCI), compared with preinjury levels, at all tested time points. In addition, C-SCI animals showed a greater magnitude of VDAT than observed after T-SCI. These two observations led us to conclude that C-SCI produced a greater range and magnitude of spastic interference than produced by T-SCI. It is not clear what accounts for these significantly different spasticity patterns, although, in moderate contusion injures, both gray and white matter of the SC in either cervical or mid-thoracic sites remained. Among the several possibilities, it is proposed that these differences in C-SCI versus T-SCI spasticity are, in part, the result of the prominent role of cervical gray matter in interlimb coordination.22–24 Selective lesion experiments in T-SCI have emphasized the importance of the lateral and ventrolateral white matter, as well as a less-prominent role of thoracic gray matter in locomotor disability after T-SCI.25–28 Accordingly, injury of the cervical neural network may result in additional neurobiological sources of lower-limb spasticity.

Hematoxylin and eosin staining of cervical spinal cord epicenter

Spasticity improvement after cervical spinal cord injury by treadmill and spinal cord transmagnetic stimulation

H&E staining of the cervical SC epicenters revealed that the injuries in all groups were characterized by loss of gray matter, but majority sparing of the surrounding white matter (e.g., the

At PO WK 5, animals completing 4 weeks of Tm training showed a significant decrease in spasticity, evidenced by a 43% reduction in VDAT. An additional 2 weeks of Tm training did not

Correlative analyses between spasticity, gait kinematic, and immunohistochemistry

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FIG. 6. Immunohistochemical staining of GABAb in lumbar enlargement (L3–4) segments of C-SCI and C-SCI-treated animals. The gray matter of one side of the spinal cord is shown here. The GABAb-positive (immunoreactive) cells in lamina VII, VIII, and IX of the spinal cord were counted (see text for details). All treatment groups showed significant up-regulations of GABAb, compared to C-SCI. Scale bar, 200 lm. ***p < 0.001, compared to C-SCI. C-SCI, cervical spinal cord injury; TMSsc, transcranial magnetic stimulation on spinal cord; Tm, treadmill training; GABAb, gamma-aminobutyric acid B. significantly change the VDAT. However, 2 weeks of TMSsc initiated at PO WK 5 in untrained animals produced a significant (59%) reduction in VDAT, compared with the pretreatment values recorded at PO WK 5. More interesting, the addition of TMSsc to Tm training to form a combined treatment group (C-SCI Tm +

TMSsc) showed the most effective results in reducing spasticity at PO WK 7. The VDAT level was similar to preinjury magnitudes at all ankle rotation velocities, except the two upper velocities. Bodyweight–supported Tm training was reported to improve spasticity in patients after chronic SCI.29 Moreover, rTMS, applied at the

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FIG. 7. Immunohistochemical staining of GAD67 in lumbar enlargement segments (L3–4) of C-SCI and C-SCI-treated animals. The gray matter of one side of the spinal cord is shown here. The total immunoreactive GAD67 fiber density in lamina VII, VIII, and IX of the spinal cord were measured (see text for details). The average total GAD67 fiber density of each group is shown in the graph below. Although all treatment types resulted in significant up-regulation of GAD67, compared to C-SCI, the Tm + TMSsc group showed the robust upregulations, which was significantly greater than any other treatment groups. Scale bar, 200 lm. **p < 0.01 and ***p < 0.001, compared to C-SCI; ^^^p < 0.001, compared to the TMSsc group, !!p < 0.01, compared to the Tm group. C-SCI, cervical spinal cord injury; TMSsc, transcranial magnetic stimulation on spinal cord; Tm, treadmill training; GAD67, the 67-kDa isoform of glutamate decarboxylase. spinal level30 or on the motor cortex,31 has been reported to improve management of spasticity in multiple sclerosis (MS) patients. A combination of exercise with intermittent TMS applied to the leg region of the motor cortex was recently reported to achieve greater reduction in spasticity than produced by exercise alone in 30 MS patients.32

Gait improvement after cervical spinal cord injury by treadmill and spinal cord transmagnetic stimulation After C-SCI, animals exhibited significantly decreased walking speed and significantly increased variability in quantitated gait parameters in inter- and intralimb coordination. But, interestingly,

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FIG. 8. Immunohistochemical staining of BDNF in lumbar enlargement segments (L3–4) of C-SCI and C-SCI-treated animals. The gray matter of one side of the spinal cord is shown here. The BDNF immunopositive cells in lamina VII, VIII, and IX of the spinal cord were counted (see details in the text). The average total number of each group is shown in the graph below. Although all treatment types resulted in significant up-regulation of BDNF, compared to C-SCI, the Tm + TMSsc group showed the robust up-regulations, which was significantly greater than any other treatment groups. Scale bar, 200 lm. ***p < 0.001, compared to C-SCI; ^^p < 0.01, compared to the TMSsc group; !!p < 0.01, compared to the Tm group. C-SCI, cervical spinal cord injury; TMSsc, transcranial magnetic stimulation on spinal cord; Tm, treadmill training; BDNF, brain-derived neurotrophic factor. at PO WK 7, the Tm and, especially, the Tm + TMSsc group showed significant recovery of inter- and intralimb coordination, as well as overall gait function, compared to injured untreated controls. Kinematic measurements revealed that the Tm + TMSsc group showed improved step cycles (ankle-knee flexion-extension cycling pattern and ankle-wrist angular rotation pattern), which

revealed cycloplots that were similar to preinjury step cycles recorded before SCI. Moreover, improvements in gait cycle, especially lift-off data, were correlated with improvement in spasticity level. Interestingly, although the TMSsc treatment group showed recovery in certain parameters in CatWalk assessment (Fig. 4), this group did not show improvement in interlimb coordination. The

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FIG. 9. Immunohistochemical staining of DbH in lumbar enlargement segments (L3–4) of C-SCI and C-SCI-treated animals. The gray matter of one side of the spinal cord is shown here. The total immunoreactive DbH fiber density in lamina VII, VIII, and IX of the spinal cord was measured (see details in the text). The average total DbH fiber density of each group is shown in the graph below. All treatment types resulted in significant up-regulation of DbH, compared to C-SCI, although the Tm + TMSsc group showed the robust up-regulations; however, the value did not reach significant difference, compared to values in the other two treatment groups. Scale bar, 200 lm. *p < 0.05 and **p < 0.01, compared to C-SCI. C-SCI, cervical spinal cord injury; TMSsc, transcranial magnetic stimulation on spinal cord; Tm, treadmill training; DbH, dopamine beta-hydroxylase.

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FIG. 10. Hematoxylin and eosin staining of cervical spinal cord injury epicenter of 1 representative animal from each group. The higher magnification images of the marked ventrolateral funiculus are shown next to the corresponding lower magnification images. The severity of injury was qualitatively assessed. Injury severity was graded as mild to moderate. Most gray matter disappeared after injury, but the majority of white matter remained in all of the animals. Scale bar of the lower magnification images is 200 lm, and the scale bar of the higher magnification images is 50 lm. C-SCI, cervical spinal cord injury; TMSsc, transcranial magnetic stimulation on spinal cord; Tm, treadmill training. injured untreated control animals showed the least recovery on gait parameters based on CatWalk and kinematics measurement. Our data are consistent with the growing evidence that shows that the adult mammalian SC is plastic and its neural circuitry can be modified by repetitive and specific activation.6,7,33–39 New approaches to facilitate locomotor recovery in individuals after SCI have been explored that utilize the sensory information related to locomotion to improve stepping on a Tm and/or over-ground walking.6,7,35 Progressively, studies have reported that Tm training can promote the recovery of walking by optimizing the activitydependent neuroplasticity of the nervous system.5,40,41 Neuronal circuits, stimulated by task-appropriate activation of peripheral and central afferents through locomotor training or stimulation, may also reorganize by strengthening existing and previously inactive descending connections and local neural circuits.40–44 In this study, the TMSsc group did not show measurable improvement in gait. However, TMSsc, applied as an adjuvant after 4 weeks of Tm training, and continued as a combination therapy group, produced the greatest improvement in gait parameters. Though the mechanism is unclear, we hypothesize that Tm training can both induce synaptic plasticity and guide plasticity, resulting in more-appropriate gait patterns. Further, these data suggest that application of magnetic stimulation across the injury site can amplify up-regulation of neuroplasticity factors (e.g., BDNF, GABA, and amines) that act as adjuvants to enhance the rehabilitation benefits induced by locomotor therapy. Although there are several pathways through which the TMSsc-induced effects could have influenced the lumbar spinal cord, it has been reported that nonspecific activation of descending propriospinal neurons (PSNs) may partly activate the locomotor central pattern generator, possibly by a nonspecific increase of overall motoneuron excitability.45 In addition, it is possible that magnetic stimulation could enhance the activity of descending monoamine projection fibers as well as PSNs, in our case, at C6-T2, which

could further potentiate the therapeutic efficacy of companion recovery strategies, such as locomotor training. Transcranial magnetic motor-evoked potentials Although TMSsc was used as therapy, tcMMEPs were recorded to test the functional integrity of neural pathways that transmitted TMS signals to the triceps surae muscles after injury and treatments. The probability for evoking tcMMEPs was 100% in the normal (preinjury) animals when tested using 50% maximal TMS intensity, but dropped to 67% after C-SCI at PO WK 7. However, the probability for evoking tcMMEPs at 50% intensity was observed to be 100% in all treatment groups at this PO WK. These data suggest that the treatment groups showed a greater probability of evoking tcMMEPs than observed in untreated animals. The possibilities that account for the treatment-induced increased tcMMEP appearance range from increased conduction integrity of activated descending tissue to increased probability for discharge of the triceps motoneurons. Though the specific mechanisms are not revealed by these data, the treatment increased the production of trophic factors (NE, BDNF, and GAD67; Figs. 7–9) known to induce neuroprotective, repair, and activity induced remodeling of neurons and glia, to enhance functional recovery after SCI.46,47 Although statistically significant, it is unclear whether the differences in the treatment versus nontreatment tcMMEP amplitudes have functional relevance. On the other hand, the treatment-related changes in the probability for appearance of the tcMMEPs does appear to be relevant. Therapeutic targets for spasticity therapy SCI interrupts extensive connectivity that contributes to the direct and indirect regulation of motoneuron excitability utilizing an array of pre- and postsynaptic processes.

COMBINED THERAPY IMPROVED SCI DISABILITIES Presynaptic regulatory processes. Presynaptic processes were among the first to provide effective targets for the management of spasticity. Presynaptic processes include the participation of GABAb receptor modulation of primary afferent transmitter release generated by activation of axo-axonic synapses of GABAergic interneurons on the terminals of afferent fibers.48–51 Altered patterns of motoneuron excitability induced by SCI were uncovered, in part, by neurophysiological studies of reflex activation patterns. A unique feature of GABAb-mediated inhibition is the prolonged time course of action, lasting thousands of milliseconds. Even GABAa-mediated inhibition, which lasts 300 msec, is short by comparison. Therefore, the status of GABAb modulation of sensory input to motoneurons could be neurophysiologically assessed by measuring low-frequency rate depression of monosynaptic reflexes.15,16,52,53 Studies in animals15,53 and humans54 revealed that significant loss of lowfrequency depression of monosynaptic reflexes (H-reflexes) after SCI coincided with the appearance of hyper-reflexia. SCI-associated loss of descending modulation of GABAergic/GABAb regulation has been augmented by antispastic medications, such as l-baclofen, to enhance GABAb regulatory processes.55 Because of widespread central nervous system (CNS) effects induced by orally administered baclofen, focal treatment of the SC using intrathecal administration provides a solution to treat spasticity and avoid the generalized CNS side effects. Accordingly, it is of significant therapeutic interest that the exercise and TMS treatments, individually and, particularly, in combination, enhanced the expression of markers for GABAb and GABA synthesis. It remains to be tested whether adjuvant pharmacologic treatment could further amplify these therapeutic outcomes. Postsynaptic regulatory processes. Progressively, details regarding postsynaptic mechanisms that regulate the input/output gain of motoneuron discharge have been reported on.56–64 These investigators found that the gain of synaptic inputs can be amplified by brainstem monoaminergic inputs that regulate dendritic persistent inward currents (PICs) utilizing sodium and calcium channels. The higher the PICs, the higher the synaptic gain and consequent burst rate of the motoneurons. Therefore, in the intact SC, monoamine regulation of PICs produces a significant influence on motoneuron excitability. In this regard, Bennett and colleagues reported on the important experimental finding that during the acute to subchronic period after SCI, the PICs normally elicited by selective afferent inputs were substantially reduced.65 This corresponded to a period of hypotonia that lasted for 3 or more weeks. However, after a few weeks of postinjury hypotonia, recorded motoneurons were observed to reacquire the PICs elicited by afferent inputs, which had disappeared immediately after SCI.65 However, these recovered PICs were easily initiated by a much wider array of segmental inputs. These unregulated PICs amplified the bistable properties of motoneurons.61 This emphasized that SCI-diminished monoamine regulation of PICs led to a maladaptive plasticity in which PICs, inappropriately triggered, could produce spasms and spastic interference contractions in lower-limb muscles. The decrease in monoamine levels resulting from injury of monoamine projection fibers is known to be an important source of motor/sensory disability after SCI. Within a few weeks after SCI, most measured neurotransmitter levels have been reported to be significantly decreased,66 leading to direct and indirect alterations in SC neuronal excitability.67 Descending fibers containing NE course in the ventrolateral white matter of the SC and are widely distributed in the spinal gray matter to influence pre- and postsynaptic processes that regulate the excitability of spinal reflexes.67–69 In addition to the important contribution to motoneuron

1103 excitability by dendritic PICs described above, NE neurons have inhibitory physiological actions on SC components throughout the stretch reflex circuitry.70–72 Collectively, these studies revealed the importance of both preand postsynaptic alterations that contributed to post-SCI spasticity. In this context, it is important to point out that these pre- (e.g., GABA/GABAb) and postsynaptic processes (e.g., noradrenergic) provide important targets for development and testing of experimental treatments to manage SCI-induced spasticity. Thus, activity and TMSsc-induced increases in expression of GABA/GABAb and NE may significantly contribute to normalization of regulatory processes that relate directly to decreasing spasticity and improving locomotion. Accordingly, we interpret that locomotor training in combination with TMSsc might increase the action of NE neurons that exert important modulations of preand postsynaptic regulatory processes that produced a significant reduction of spasticity in the SCI locomotor-trained, TMSsc, or combined treated animals. Locomotor training is viewed as a strategy that provided patterned activity that guided the nervous system plasticity toward correct task performance and decreased the influence of maladaptive spontaneous recovery. The enhanced expression of IHC markers for BDNF, GABA/GABAb receptors, and NE fiber sprouting supports the concept of activity-induced plasticity that has been ascribed to enhanced expression in other related models. Immunohistochemistry In this regard, these immunohistological studies of the lumbar enlargement showed an up-regulation of GABAergic molecules (GABAb and GAD67) or DbH, which catalyzed the conversion of dopamine to NE in all treatment animals, compared to control animals. Thus, the reduction in spasticity development that accompanied locomotor training could, in part, be a result of improved inhibitory processes that regulate afferent inputs, as well as prevention of maladaptive changes in motoneuron PICs.61 Activity-related reorganization of segmental circuitry, including descending inputs, segmental synaptic inputs, and local interneurons, might contribute in this improvement as well. At PO WK 5, the time of the onset of PIC recovery, which has been proposed to contribute to the development of late onset of spasticity,61 applying at-injury–level magnetic stimulation may further induce the release of monoamines, such as NE, and contribute to the preservation of normal PICs. Therefore, magnetic stimulation could act in synchrony to enhance the scope of adaptive plasticity to include both improved inhibitory regulation of afferent input and decreased maladaptive postsynaptic (PICs) changes. It is known that neurotrophic molecules (such as BDNF) play an important role in the regulation of SC function and in modulation of neuroplasticity. Although several molecular systems could participate in the benefits of locomotor exercise on the CNS, it is noteworthy that NE- and GABA-mediated signaling might be important in the modulation of BDNF gene expression by locomotor exercise.73,74 Recent studies further illustrate that physical activity increases expression of BDNF and neurotrophin-3 (NT-3) in the intact SC.5,75–77 Other related evidence shows that BDNF also stimulates hindlimb stepping.78 Our IHC study of the lumbar enlargement sections also showed the up-regulation of BDNF in the treatment groups, compared to the untreated C-SCI. Collectively, these data suggested that the beneficial effects of locomotor training and TMSsc observed on decreased spasticity and improved gait could be associated with up-regulation of the GABAergic and

1104 descending NE systems. The neurotrophic molecules (e.g., BDNF) may also have contributed an important role in the modulation of spinal reflexes. We recently reported that GABA (especially GAD67) and BDNF colocalized in the SC, including the ventral horn, were up-regulated after locomotor exercise.5 Therefore, therapy-enhanced BDNF production could have contributed to the adaptive plasticity in the SC underlying the reduction in spasticity and improved gait performance. In our present experiments, decreases in spasticity were correlated with GABA (GAD67), GABAb, BDNF, and NE (DbH) expression in the ventral horn of the SC. This study revealed that when applied as individual therapies, exercise or TMSsc provided sufficient therapeutic effect to reduce spasticity by approximately 50%, compared to no treatment. Whereas it is envisioned that locomotor exercise enhanced and guided the patterns of remodeling to enhance adaptive plasticity, the adjuvant therapy, TMSsc, may have further augmented the exercise-related benefits by up-regulating neuroprotective molecules. In conclusion, our study showed that locomotor training in combination with TMSsc produced a decrease in spasticity that led to an overall improvement in gait. Summary and Conclusions Collectively, the present work represents data that support the conclusion that a combination of Tm locomotor exercise with TMSsc therapy yielded significant locomotor improvement and spasticity reduction that was greater than either treatment tested alone. The combination therapy revealed a profound therapeutic reduction of spasticity toward preinjury levels. The therapeutic improvements in gait and spasticity with each treatment modality were correlated with significantly amplified expression of the IHC markers for GABAb receptors, GAD67, DbH, and BDNF. However, the combination therapy produced a greater expression of these markers. Accordingly, it is natural to conclude that although TMSsc may enhance the trophic environment, without behaviorally relevant guided neural signaling, little normalization of gait confirmation was produced by TMSsc. On the other hand, TMSsc perhaps provided an increased unguided ascending and descending activity related to pre- and postsynaptic inhibition, which resulted in significant improvement of spasticity. In contrast, Tm training, as stand-alone therapy, revealed significant improvement in spasticity and normalization of all of the gait parameters. The Tm locomotor therapy perhaps increased guided segmental, ascending, and descending plasticity. These findings suggest that taskappropriate therapy was essential for recovery of gait confirmation. It is natural therefore to propose that the robustness necessary to make significant functional improvements in spasticity and gait may require amplification of therapeutic effect through the successful combination of complementary individual therapies. Although TMS has been used in a broad range of therapeutic applications since its inception in 1985, many questions remain regarding the mechanisms of its beneficial outcome. In this regard, these studies indicate that TMSsc may enhance the trophic environment through BDNF and GAD67 and the upregulation of markers for pre- and postsynaptic inhibition (GABAb receptors, GAD67, and NE). Acknowledgments This work was supported by Merit Review #B5037R, Department of Veteran Affairs, Rehabilitation R&D. The authors thank Dr. Samuel Wu and Yunfeng Dai for their kind assistance in sta-

HOU ET AL. tistical analysis of the data. The authors also thank Susie Sennhauser, Jonathan Keener, Amy Nguyen, and Katrina Obleada for their assistance in treadmill training, kinemetics video digitizing, and histological processing of some tissue. Author Disclosure Statement No competing financial interests exist. References 1. Adams, M.M., and Hicks, A.L. (2005). Spasticity after spinal cord injury. Spinal Cord 43, 577–586. 2. Lance, J.W. (1980). The control of muscle tone, reflexes, and movement: Robert Wartenberg Lecture. Neurology 30, 1303–1313. 3. Young, R.R. (1989). Treatment of spastic paresis. N. Engl. J. Med. 320, 1553–1555. 4. Dobkin, B., Barbeau, H., Deforge, D., Ditunno, J., Elashoff, R., Apple, D., Basso, M., Behrman, A., Harkema, S., Saulino, M., and Scott, M. (2007). The evolution of walking-related outcomes over the first 12 weeks of rehabilitation for incomplete traumatic spinal cord injury: the multicenter randomized Spinal Cord Injury Locomotor Trial. Neurorehabil. Neural Repair 21, 25–35. 5. Bose, P.K., Hou, J., Parmer, R., Reier, P.J., and Thompson, F.J. (2012). Altered patterns of reflex excitability, balance, and locomotion following spinal cord injury and locomotor training. Front. Physiol. 3, 258. 6. Edgerton, V.R., and Harkema, S. (2011). Epidural stimulation of the spinal cord in spinal cord injury: current status and future challenges. Expert Rev. Neurother. 11, 1351–1353. 7. Harkema, S., Gerasimenko, Y., Hodes, J., Burdick, J., Angeli, C., Chen, Y., Ferreira, C., Willhite, A., Rejc, E., Grossman, R.G., and Edgerton, V.R. (2011). Effect of epidural stimulation of the lumbosacral spinal cord on voluntary movement, standing, and assisted stepping after motor complete paraplegia: a case study. Lancet 377, 1938–1947. 8. Edgerton, V.R., Kim, S.J., Ichiyama, R.M., Gerasimenko, Y.P., and Roy, R.R. (2006). Rehabilitative therapies after spinal cord injury. J. Neurotrauma 23, 560–570. 9. Wassermann, E.M., and Zimmermann, T. (2012). Transcranial magnetic brain stimulation: therapeutic promises and scientific gaps. Pharmacol. Ther. 133, 98–107. 10. Rossi, S., Hallett, M., Rossini, P.M., and Pascual-Leone, A. (2009). Safety, ethical considerations, and application guidelines for the use of transcranial magnetic stimulation in clinical practice and research. Clin. Neurophysiol. 120, 2008–2039. 11. Groppa, S., Oliviero, A., Eisen, A., Quartarone, A., Cohen, L.G., Mall, V., Kaelin-Lang, A., Mima, T., Rossi, S., Thickbroom, G.W., Rossini, P.M., Ziemann, U., Valls-Sole, J., and Siebner, H.R. (2012). A practical guide to diagnostic transcranial magnetic stimulation: Report of an IFCN committee. Clin. Neurophysiol. 123, 858–882. 12. Kunkel-Bagden, E., Dai, H.N., and Bregman, B.S. (1993). Methods to assess the development and recovery of locomotor function after spinal cord injury in rats. Exp. Neurol. 119, 153–164. 13. Bose, P., Parmer, R., and Thompson, F.J. (2002). Velocity-dependent ankle torque in rats after contusion injury of the midthoracic spinal cord: time course. J. Neurotrauma 19, 1231–1249. 14. Wang, D.C., Bose, P., Parmer, R., and Thompson, F.J. (2002). Chronic intrathecal baclofen treatment and withdrawal: I. Changes in ankle torque and hind limb posture in normal rats. J. Neurotrauma 19, 875– 886. 15. Thompson, F.J., Reier, P.J., Lucas, C.C., and Parmer, R. (1992). Altered patterns of reflex excitability subsequent to contusion injury of the rat spinal cord. J. Neurophysiol. 68, 1473–1486. 16. Thompson, F.J., Reier, P.J., Parmer, R., and Lucas, C.C. (1993). Inhibitory control of reflex excitability following contusion injury and neural tissue transplantation. Adv. Neurol. 59, 175–184. 17. Thompson, F.J., Browd, C.R., Carvalho, P.M., and Hsiao, J. (1996). Velocity-dependent ankle torque in the normal rat. Neuroreport 7, 2273–2276. 18. Loy, D.N., Talbott, J.F., Onifer, S.M., Mills, M.D., Burke, D.A., Dennison, J.B., Fajardo, L.C., Magnuson, D.S., and Whittemore, S.R.

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Address correspondence to: Prodip Bose, MD, PhD North Florida/South Georgia Veterans Health System Malcolm Randall VA Medical Center Brain Rehabilitation Research Center (151) 1601 SW Archer Road Gainesville, FL 32608-1135 E-mail: [email protected]