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Background: Implementing exercises in the form of video games, otherwise known as exergaming, has gained recent attention as a way to combat health issues resulting from sedentary lifestyles. However, these exergaming apps have not been developed for exercises that can be performed in wheelchairs, and they tend to rely on whole-body movements., Objective: This study aims to develop a mobile phone app that implements electromyography (EMG)-driven exergaming, to test the feasibility of using this app to enable people in wheelchairs to perform exergames independently and flexibly in their own home, and to assess the perceived usefulness and usability of this mobile health system., Methods: We developed an Android mobile phone app (Workout on Wheels, WOW-Mobile) that senses upper limb muscle activity (EMG) from wireless body-worn sensors to drive 3 different video games that implement upper limb exercises designed for people in wheelchairs. Cloud server recordings of EMG enabled long-term monitoring and feedback as well as multiplayer gaming. Bench testing of data transmission and power consumption were tested. Pilot testing was conducted on 4 individuals with spinal cord injury. Each had a WOW-Mobile system at home for 8 weeks. We measured the minutes for which the app was used and the exergames were played, and we integrated EMG as a measure of energy expended. We also conducted a perceived usefulness and usability questionnaire., Results: Bench test results revealed that the app meets performance specifications to enable real-time gaming, cloud storage of data, and live cloud server transmission for multiplayer gaming. The EMG sampling rate of 64 samples per second, in combination with zero-loss data communication with the cloud server within a 10-m range, provided seamless control over the app exergames and allowed for offline data analysis. Each participant successfully used the WOW-Mobile system at home for 8 weeks, using the app for an average of 146 (range 89-267) minutes per week with the system, actively exergaming for an average of 53% of that time (39%-59%). Energy expenditure, as measured by integrated EMG, was found to be directly proportional to the time spent on the app (Pearson correlation coefficient, r=0.57-0.86, depending on the game). Of the 4 participants, 2 did not exercise regularly before the study; these 2 participants increased from reportedly exercising close to 0 minutes per week to exergaming 58 and 158 minutes on average using the WOW-Mobile fitness system. The perceived usefulness of WOW-Mobile in motivating participants to exercise averaged 4.5 on a 5-point Likert scale and averaged 5 for the 3 participants with thoracic level injuries. The mean overall ease of use score was 4.25 out of 5., Conclusions: Mobile app exergames driven by EMG have promising potential for encouraging and facilitating fitness for individuals in wheelchairs who have maintained arm and hand mobility., (©James Enciso, Dhruval Variya, James Sunthonlap, Terrence Sarmiento, Ka Mun Lee, James Velasco, Roxanna N Pebdani, Ray D de Leon, Christine Dy, Stefan Keslacy, Deborah Soonmee Won. Originally published in JMIR Rehabilitation and Assistive Technology (http://rehab.jmir.org), 19.01.2021.)

Previous research by the authors on an animal model showed that bloodstains can contain additional information about their somatic origin in the form of wound cells. Bloodstains produced by a gunshot wound to the head were distinguished from bloodstains produced by a gunshot wound to the chest by testing the stains for a brain microRNA marker. In this study, the effectiveness of the technique was examined on blood drops shed externally from a stab wound to the liver of rat carcasses. Specifically, investigations were conducted on the liver microRNA marker, rno-mir-122-3p, with the QIAGEN miScript System, and PCR analysis. Between the two stabbing methods used, 67% of the scalpel blades and 57% of the blood drops tested positive for rno-mir-122-3p; however, other samples tested negative giving inconclusive results as to the wound-of-origin. The amount of the liver cells in the bloodstains appeared to be related to the extent of trauma., (© 2017 American Academy of Forensic Sciences.)

Background: The application of resistive forces has been used during body weight-supported treadmill training (BWSTT) to improve walking function after spinal cord injury (SCI). Whether this form of training actually augments the effects of BWSTT is not yet known., Objective: To determine if robotic-applied resistance augments the effects of BWSTT using a controlled experimental design in a rodent model of SCI., Methods: Spinally contused rats were treadmill trained using robotic resistance against horizontal (n = 9) or vertical (n = 8) hind limb movements. Hind limb stepping was tested before and after 6 weeks of training. Two control groups, one receiving standard training (ie, without resistance; n = 9) and one untrained (n = 8), were also tested. At the terminal experiment, the spinal cords were prepared for immunohistochemical analysis of synaptophysin., Results: Six weeks of training with horizontal resistance increased step length, whereas training with vertical resistance enhanced step height and movement velocity. None of these changes occurred in the group that received standard (ie, no resistance) training or in the untrained group. Only standard training increased the number of step cycles and shortened cycle period toward normal values. Synaptophysin expression in the ventral horn was highest in rats trained with horizontal resistance and in untrained rats and was positively correlated with step length., Conclusions: Adding robotic-applied resistance to BWSTT produced gains in locomotor function over BWSTT alone. The impact of resistive forces on spinal connections may depend on the nature of the resistive forces and the synaptic milieu that is present after SCI.

Body weight-supported treadmill training (BWSTT) developed from animal studies of spinal cord injury (SCI). Evidence that spinal cats (i.e., cats that have a complete surgical transection of the cord) could regain the ability to step on a moving treadmill indicated a vast potential for spinal circuits to generate walking without the brain. BWSTT represented a means to unlock that potential. As the technique was adapted as a rehabilitation intervention for humans with SCI, shortcomings in the translation to walking in the real world were exposed. Evidence that BWSTT has not been as successful for humans with SCI leads us to revisit key animal studies. In this short review, we describe the task-specific nature of BWSTT and discuss how this specificity may pose limits on the recovery of overground walking. Also discussed are more recent studies that have introduced new strategies and tools that adapt BWSTT ideas to more functionally-relevant tasks. We introduce a new device for weight-supported overground walking in rats called Circular BART (Body weight supported Ambulatory Rat Trainer) and demonstrate that it is relatively easy and inexpensive to produce. Future animal studies will benefit from the development of simple tools that facilitate training and testing of overground walking.

Background: Providing weight support facilitates locomotion in spinal cord injured animals. To control weight support, robotic systems have been developed for treadmill stepping and more recently for overground walking., New Method: We developed a novel device, the body weight supported ambulatory rodent trainer (i.e. BART). It has a small pneumatic cylinder that moves along a linear track above the rat. When air is supplied to the cylinder, the rats are lifted as they perform overground walking. We tested the BART device in rats that received a moderate spinal cord contusion injury and in normal rats. Locomotor training with the BART device was not performed., Results: All of the rats learned to walk in the BART device. In the contused rats, significantly greater paw dragging and dorsal stepping occurred in the hindlimbs compared to normal. Providing weight support significantly raised hip position and significantly reduced locomotor deficits. Hindlimb stepping was tightly coupled to forelimb stepping but only when the contused rats stepped without weight support. Three weeks after the contused rats received a complete spinal cord transection, significantly fewer hindlimb steps were performed., Comparison With Existing Methods: Relative to rodent robotic systems, the BART device is a simpler system for studying overground locomotion. The BART device lacks sophisticated control and sensing capability, but it can be assembled relatively easily and cheaply., Conclusions: These findings suggest that the BART device is a useful tool for assessing quadrupedal, overground locomotion which is a more natural form of locomotion relative to treadmill locomotion., (Published by Elsevier B.V.)

Bloodstain pattern analysis to determine the wound-of-origin of bloodstains is problematic with nonspecific patterns. In this proof-of-concept study, the authors examined a molecular approach to correlate bloodstains with injuries using the rat as a model. Specifically, investigations were conducted on the rat brain marker, rno-miR-124-3p, with the QIAGEN miScript System and real-time PCR analysis. Rno-miR-124-3p was detected in brain homogenates diluted 100,000 times; in 3-week-old, room temperature stored, simulated brain-blood stains; and in bloodstains from head gunshot wounds collected with swabs and subsequently frozen for 9-18 months; however, rno-miR-124-3p was not detected in whole blood. Proof-of-principle was demonstrated by the ability to distinguish bloodstains from a gunshot wound to the head versus bloodstains from a gunshot wound to the chest, by the testing of otherwise identical bloodstains from the two patterns for the presence of the marker. The results suggest a viable approach to a longstanding problem in casework., (© 2014 American Academy of Forensic Sciences.)

Loading on the limbs has a powerful influence on locomotion. In the present study, we examined whether robotic-enhanced loading during treadmill training improved locomotor recovery in rats that were spinally transected as neonates. A robotic device applied a force on the ankle of the hindlimb while the rats performed bipedal stepping on a treadmill. The robotic force enhanced loading during the stance phase of the step cycle. One group of spinally transected rats received 4 wk of bipedal treadmill training with robotic loading while another group received 4 wk of bipedal treadmill training but without robotic loading. The two groups exhibited similar stepping performance during baseline tests of bipedal treadmill stepping. However, after 4 wk, the spinally transected rats that received bipedal treadmill training with robotic loading performed significantly more weight-bearing steps than the bipedal treadmill training only group. Bipedal treadmill training with robotic loading enhanced the ankle trajectory and ankle velocity during the step cycle. Based on immunohistochemical analyses, the expression of the presynaptic marker, synaptophysin, was significantly greater in the ventral horn of the lumbar spinal cord of the rats that received bipedal treadmill training with robotic loading. These findings suggested that robotic loading during bipedal treadmill training improved the ability of the lumbar spinal cord to generate stepping. The results have implications for the use of robotic-enhanced gait training therapies that encourage motor learning after spinal cord injury.

Results of previous studies raise the question of how timing neuromuscular functional electrical stimulation (FES) to limb movements during stepping might alter neuromuscular control differently than patterned stimulation alone. We have developed a prototype FES system for a rodent model of spinal cord injury (SCI) that times FES to robotic treadmill training (RTT). In this study, one group of rats (n = 6) was trained with our FES+RTT system and received stimulation of the ankle flexor (tibialis anterior [TA]) muscle timed according to robot-controlled hind-limb position (FES+RTT group); a second group (n = 5) received a similarly patterned stimulation, randomly timed with respect to the rats' hind-limb movements, while they were in their cages (randomly timed stimulation [RS] group). After 4 wk of training, we tested treadmill stepping ability and compared kinematic measures of hind-limb movement and electromyography (EMG) activity in the TA. The FES+RTT group stepped faster and exhibited TA EMG profiles that better matched the applied stimulation profile during training than the RS group. The shape of the EMG profile was assessed by "gamma," a measure that quantified the concentration of EMG activity during the early swing phase of the gait cycle. This gamma measure was 112% higher for the FES+RTT group than for the RS group. The FES+RTT group exhibited burst-to-step latencies that were 41% shorter and correspondingly exhibited a greater tendency to perform ankle flexion movements during stepping than the RS group, as measured by the percentage of time the hind limb was either dragging or in withdrawal. The results from this study support the hypothesis that locomotor training consisting of FES timed to hind-limb movement improves the activation of hind-limb muscle more so than RS alone. Our rodent FES+RTT system can serve as a tool to help further develop this combined therapy to target appropriate neurophysiological changes for locomotor control.

Brain-derived neurotrophic factor (BDNF) induces plasticity within the lumbar spinal circuits thereby improving locomotor recovery in spinal cord-injured animals. We examined whether lumbar spinal cord motor neurons and other ventral horn cells of spinally transected (ST) rats were stimulated to produce BDNF mRNA in response to treadmill training. Rats received complete spinal cord transections as neonates (n=20) and one month later, received four weeks of either a low (100 steps/training session; n=10) or high (1000 steps/training session; n=10) amount of robotic-assisted treadmill training. Using combined non-radioactive in situ hybridization and immunohistochemical techniques, we found BDNF mRNA expression in heat shock protein 27-labeled motor neurons and in non-motor neuron cells was greater after 1000 steps/training session compared to the 100 steps/training session and was similar to BDNF mRNA labeling in untrained Intact rats. In addition, there were significantly more motor neurons that contained BDNF mRNA labeling within processes in the ST rats that received the higher amount of treadmill training. These findings suggested that motor neurons and other ventral horn cells in ST rats synthesized BDNF in response to treadmill training. The findings support a mechanism by which postsynaptic release of BDNF from motor neurons contributed to synaptic plasticity., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Adult cats with a complete spinal cord transection at T12-T13 can relearn over a period of days-to-weeks how to generate full weight-bearing stepping on a treadmill or standing ability if trained specifically for that task. In the present study, we assessed short-term (milliseconds to minutes) adaptations by repetitively imposing a mechanical perturbation on the hindlimb of chronic spinal cats by placing a rod in the path of the leg during the swing phase to trigger a tripping response. The kinematics and EMG were recorded during control (10 steps), trip (1-60 steps with various patterns), and then release (without any tripping stimulus, 10-20 steps) sequences. Our data show that the muscle activation patterns and kinematics of the hindlimb in the step cycle immediately following the initial trip (mechanosensory stimulation of the dorsal surface of the paw) was modified in a way that increased the probability of avoiding the obstacle in the subsequent step. This indicates that the spinal sensorimotor circuitry reprogrammed the trajectory of the swing following a perturbation prior to the initiation of the swing phase of the subsequent step, in effect "attempting" to avoid the re-occurrence of the perturbation. The average height of the release steps was elevated compared to control regardless of the pattern and the length of the trip sequences. In addition, the average impact force on the tripping rod tended to be lower with repeated exposure to the tripping stimulus. EMG recordings suggest that the semitendinosus, a primary knee flexor, was a major contributor to the adaptive tripping response. These results demonstrate that the lumbosacral locomotor circuitry can modulate the activation patterns of the hindlimb motor pools within the time frame of single step in a manner that tends to minimize repeated perturbations. Furthermore, these adaptations remained evident for a number of steps after removal of the mechanosensory stimulation.

While neuromuscular electrical stimulation (NMES) has enabled patients of neuromotor dysfunction to effectively regain some functions, analysis of neuromuscular changes underlying these functional improvements is lacking. We have developed an NMES system for a rodent model of SCI with the long term goal of creating a therapy which restores control over stepping back to the spinal circuitry. NMES was applied to the tibialis anterior (TA) and timed to the afferent feedback generated during robotic treadmill training (RTT). The effect of NMES+RTT on modifications in EMG was compared with that of RTT alone. A longitudinal study with a crossover design was conducted in which group 1 (n=7) received 2 weeks of RTT only followed by 2 weeks of NMES+RTT; group 2 (n=7) received 2 weeks of NMES+RTT followed by RTT only. On average, both types of training helped to modulate TA EMG activity over a gait cycle, resulting in EMG profiles across steps with peaks occurring just before or at the beginning of the swing phase, when ankle flexion is most needed. However, NMES+RTT resulted in concentration of EMG activation during the initial swing phase more than RTT only. In conjunction with these improvements in EMG activation presented here, a more complete analyses comparing changes after NMES+RTT vs. RTT is expected to further support the notion that NMES timed appropriately to hindlimb stepping could help to reinforce the motor learning that is induced by afferent activity generated by treadmill training.

After spinal cord transection, the generation of stepping depends on neurotransmitter systems entirely contained within the local lumbar spinal cord. Glutamate and glycine likely play important roles, but surprisingly little is known about how the content of these two key neurotransmitters changes to achieve weight-bearing stepping after spinal cord injury. We studied the levels of glutamate and glycine in the lumbar spinal cord of spinally transected rats. Rats (n = 48) received spinal cord transection at 5 days of age, and 4 weeks later half were trained to step using a robotic treadmill system and the remaining half were untrained controls. Analyses of glutamate and glycine content via high-performance liquid chromatography showed training significantly raised the levels of both neurotransmitters in the lumbar spinal cord beyond normal. The levels of both neurotransmitters were significantly correlated with the ability to perform independent stepping during training. Glutamate and glycine levels were not significantly different between Untrained and Normal rats or between Trained and Untrained rats. There was a trend for higher expression of VGLUT1 and GLYT2 around motor neurons in Trained versus Untrained rats based on immunohistochemical analyses. Training improved the ability to generate stepping at a range of weight support levels, but normal stepping characteristics were not restored. These findings suggested that the remodeling of the lumbar spinal circuitry in Trained spinally transected rats involved adaptations in the glutamatergic and glycinergic neurotransmitter systems. These adaptations may contribute to the generation of novel gait patterns following complete spinal cord transection.

Intensive weight-supported treadmill training (WSTT) improves locomotor function following spinal cord injury. Because of a number of factors, undergoing intensive sessions of training may not be feasible. Whether reduced amounts of training are sufficient to enhance spinal plasticity to a level that is necessary for improving function is not known. The focus of the present study was to assess differences in recovery of locomotor function and spinal plasticity as a function of the amount of steps taken during WSTT in a rodent model of spinal cord injury. Rats were spinally transected at 5 days of age. When they reached 28 days of age, a robotic system was used to implement a weight-supported treadmill training program of either 100 or 1000 steps/training session daily for 4 weeks. Antibodies for brain-derived neurotrophic factor (BDNF), TrkB, and the pre-synaptic marker, synaptophysin, were used to examine the expression of these proteins in the ventral horn of the lumbar spinal cord. Rats that received weight-supported treadmill training performed better stepping relative to untrained rats, but only the rats that received 1000 steps/training session recovered locomotor function that resembled normal patterns. Only the rats that received 1000 steps/training session recovered normal levels of synaptophysin immunoreactivity around motor neurons. Weight-supported treadmill training consisting of either 100 or 1000 steps/training session increased BDNF immunoreactivity in the ventral horn of the lumbar spinal cord. TrkB expression in the ventral horn was not affected by spinal cord transection or weight-supported treadmill training. Synaptophysin expression, but not BDNF or TrkB expression was correlated with the recovery of stepping function. These findings suggested that a large amount of weight-supported treadmill training was necessary for restoring synaptic connections to motor neurons within the locomotor generating circuitry. Although a large amount of training was best for recovery, small amounts of training were associated with incremental gains in function and increased BDNF levels.

Robotic devices have been developed to assist body weight-supported treadmill training (BWSTT) in individuals with spinal cord injuries (SCIs) and stroke. Recent findings have raised questions about the effectiveness of robotic training that fully assisted (FA) stepping movements. The purpose of this study was to examine whether assist-as-needed robotic (AAN) training was better than FA movements in rats with incomplete SCI. Electromyography (EMG) electrodes were implanted in the tibialis anterior and medial gastrocnemius hindlimb muscles of 14 adult rats. Afterward, the rats received a severe midthoracic spinal cord contusion and began daily weight-supported treadmill training 1 wk later using a rodent robotic system. During training, assistive forces were applied to the ankle when it strayed from a desired stepping trajectory. The amount of force was proportional to the magnitude of the movement error, and this was multiplied by either a high or low scale factor to implement the FA (n = 7) or AAN algorithms (n = 7), respectively. Thus FA training drove the ankle along the desired trajectory, whereas greater variety in ankle movements occurred during AAN training. After 4 wk of training, locomotor recovery was greater in the AAN group, as demonstrated by the ability to generate steps without assistance, more normal-like kinematic characteristics, and greater EMG activity. The findings suggested that flexible robotic assistance facilitated learning to step after a SCI. These findings support the rationale for the use of AAN robotic training algorithms in human robotic-assisted BWSTT.

Functional electrical stimulation (FES) is used to assist spinal cord injury patients during walking. However, FES has yet to be shown to have lasting effects on the underlying neurophysiology which lead to long-term rehabilitation. A new approach to FES has been developed by which stimulation is timed to robotically controlled movements in an attempt to promote long-term rehabilitation of walking. This approach was tested in a rodent model of spinal cord injury. Rats who received this FES therapy during a 2-week training period exhibited peak EMG activity during the appropriate phase of the gait cycle; whereas, rats who received stimulation which was randomly timed with respect to their motor activity exhibited no clear pattern in their EMG profile. These results from our newly developed FES system serve as a launching point for many future studies to test and understand the long-term effect of FES on spinal cord rehabilitation.

Rats receiving a complete spinal cord transection (ST) at a neonatal stage spontaneously can recover significant stepping ability, whereas minimal recovery is attained in rats transected as adults. In addition, neonatally spinal cord transected rats trained to step more readily improve their locomotor ability. We hypothesized that recovery of stepping in rats receiving a complete spinal cord transection at postnatal day 5 (P5) is attributable to changes in the lumbosacral neural circuitry and not to regeneration of axons across the lesion. As expected, stepping performance measured by several kinematics parameters was significantly better in ST (at P5) trained (treadmill stepping for 8 weeks) than age-matched non-trained spinal rats. Anterograde tracing with biotinylated dextran amine showed an absence of labeling of corticospinal or rubrospinal tract axons below the transection. Retrograde tracing with Fast Blue from the spinal cord below the transection showed no labeled neurons in the somatosensory motor cortex of the hindlimb area, red nucleus, spinal vestibular nucleus, and medullary reticular nucleus. Retrograde labeling transsynaptically via injection of pseudorabies virus (Bartha) into the soleus and tibialis anterior muscles showed no labeling in the same brain nuclei. Furthermore, re-transection of the spinal cord at or rostral to the original transection did not affect stepping ability. Combined, these results clearly indicate that there was no regeneration across the lesion after a complete spinal cord transection in neonatal rats and suggest that this is an important model to understand the higher level of locomotor recovery in rats attributable to lumbosacral mechanisms after receiving a complete ST at a neonatal compared to an adult stage., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Treadmill training is known to improve stepping in complete spinal cord injured animals. Few studies have examined whether treadmill training also enhances locomotor recovery in animals following incomplete spinal cord injuries. In the present study, we compared locomotor recovery in trained and untrained rats that received a severe mid-thoracic contusion of the spinal cord. A robotic device was used to train and to test bipedal hindlimb stepping on a treadmill. Training was imposed for 8 weeks. The robotic device supported the weight of the rats and recorded ankle movements in the hindlimbs for movement analyses. Both the trained and untrained rats generated partial weight bearing hindlimb steps after the spinal cord contusion. Dragging during swing was more prevalent in the untrained rats than the trained rats. In addition, only the trained rats performed step cycle trajectories that were similar to normal step cycle trajectories in terms of the trajectory shape and movement velocity characteristics. In contrast, untrained rats executed step cycles that consisted of fast, kick-like movements during forward swing. These findings indicate that spinal cord contused rats can generate partial weight bearing stepping in the absence of treadmill training. The findings also suggest that the effect of treadmill training is to restore normal patterns of hindlimb movements following severe incomplete spinal cord injury in rats.

The nervous system can adapt to external forces that perturb locomotion by correcting errors in limb movements. It is believed that supraspinal structures mediate these adaptations, whereas the spinal cord contributes only reflexive responses to perturbations. We examined whether the lumbar spinal cord in postnatal day 5 neonatal spinally transected (ST) rats corrected errors in hindlimb coordination through repetitive exposure to an external perturbation. A robotic device was used to deliver a viscous (velocity-dependent) force that opposed only the forward movement of the ankle in one hindlimb while the ST rats performed hindlimb stepping on a treadmill. We measured the interval between paw contact in the perturbed hindlimb and toe off in the unperturbed hindlimb. Before the force was activated, a normal pattern of coordination occurred: paw contact in the perturbed hindlimb occurred before toe off in the unperturbed hindlimb. This sequence was initially disrupted when the force was activated and the unperturbed hindlimb initiated swing during the swing phase of the perturbed hindlimb. Within five step cycles of exposure to the unilateral viscous force, however, the ST rats regained the preforce pattern of hindlimb coordination. These findings suggest that in the absence of supraspinal input, the lumbar spinal circuitry is capable of processing a complex ensemble of sensory information to maintain locomotor stability. Thus, the lumbar spinal circuitry may play a greater role in generating locomotor adaptations than previously thought.

Studies have shown that treadmill training with body weight support is effective for enhancing locomotor recovery following a complete spinal cord transection (ST) in animals. However, there have been no studies that have investigated the extent that functional recovery in ST animals is dependent on the amount of activity imposed on the hindlimbs during training. In rats transected as neonates (P5), we used a robotic device to impose either a high or a low amount of hindlimb activity during treadmill training starting 23 days after transection. The rats were trained 5 days per week for 4 weeks. One group (n = 13) received 1000 steps/training session and a second group (n = 13) received 100 steps/training session. During training, the robotic device imposed the maximum amount of weight that each rat could bear on the hindlimbs, and counted the number of stepping movements during each session. After 4 weeks of training, the number of steps performed during treadmill testing was not significantly different between the two groups. However, the quality of stepping in the group that received 1000 steps/training session improved over a range of levels of weight bearing on the hindlimbs and at different treadmill speeds. In contrast, little improvement in the quality of stepping was observed in the group that received only 100 steps/training session. These findings indicate that the ability of the lumbar spinal cord to adjust to load- and speed-related sensory stimuli associated with stepping is dependent on the number of repetitions of the same activity that is imposed on the spinal circuits during treadmill training.

The purpose of this study was to determine if robotic-assisted treadmill training improved hindlimb stepping in complete spinal cord transected (ST) rats. In addition, we examined whether chronic quipazine treatment would enhance the effectiveness of robotic-assisted training. Hindlimb stepping was examined in four groups of ST rats: trained + quipazine; trained + vehicle; untrained + quipazine; and untrained + vehicle. To train the rats to step, a robotic device was used that moved the hindlimbs in a semi-fixed trajectory during treadmill stepping. The robotic device was also used to assess treadmill stepping. Quipazine or vehicle was administered to the lumbar spinal cord using an intrathecal cannula. The groups that received robotic-assisted training performed more stepping movements on the treadmill than the untrained groups 10 weeks after ST. However, no differences were found between the robotic-assisted and untrained groups 16 weeks after ST. Kinematic analyses revealed that abnormally small step cycles were performed by all of the groups of ST rats. There was no significant effect of combining robotic-assisted training and quipazine treatment on stepping recovery. These data suggest that robotic-assisted training may generate hindlimb sensory stimuli that are effective in enhancing the ability of the lumbar spinal cord to generate hindlimb stepping. However, the effectiveness of robotic-assisted training may be limited to the early stages of recovery following spinal cord transection.

There is a critical need to develop objective, quantitative techniques to assess motor function after spinal cord injury. Here, we assess the ability of a recently developed robotic device (the "rat stepper") to characterize locomotor impairment following contusion injury in rats. In particular, we analyzed how the kinematic features of hindlimb movement during bipedal, weight-supported treadmill stepping change following contusion, and whether these changes correlate with the recovery of open field locomotion. Female, Sprague-Dawley rats (n=29, 8 weeks of age) received mid thoracic contusion injuries of differing severities (11 mild, nine moderate, nine severe, and four sham). In a first experiment, 16 of the animals were evaluated weekly for 12 weeks using the robotic stepping device. In a second experiment, 17 of the animals were evaluated every other day for 4 weeks. The contused animals recovered open field locomotion based on the Basso, Beattie, and Bresnahan Scale (BBB) analysis, with most of the recovery occurring by 4 weeks post-injury. Analysis of 14 robotic measures of stepping revealed that several measures improved significantly during the same 4 weeks: swing velocity, step height, step length, hindlimb coordination, and the ability to support body weight. These measures were also significantly correlated with the BBB score. The number of steps taken during testing was not directly related to intrinsic recovery or correlated to the BBB score. These results suggest that it is the quality of weight-supported steps, rather than the quantity, that best reflects locomotor recovery after contusion injury, and that the quality of these steps is determined by the integrity of extensor, flexor, and bilateral coordination pathways. Thus, by measuring only a few weight-supported steps with motion capture, a sensitive, valid measure of locomotor recovery following contusion injury can be obtained across a broad range of impairment levels.

We have developed a robotic device (the "rat stepper") for evaluating and training locomotor function of spinal cord injured rodents. This paper provides a detailed description of the device design and a characterization of its robotic performance capabilities.

Motor function is severely disrupted following spinal cord injury (SCI). The spinal circuitry, however, exhibits a great degree of automaticity and plasticity after an injury. Automaticity implies that the spinal circuits have some capacity to perform complex motor tasks following the disruption of supraspinal input, and evidence for plasticity suggests that biochemical changes at the cellular level in the spinal cord can be induced in an activity-dependent manner that correlates with sensorimotor recovery. These characteristics should be strongly considered as advantageous in developing therapeutic strategies to assist in the recovery of locomotor function following SCI. Rehabilitative efforts combining locomotor training pharmacological means and/or spinal cord electrical stimulation paradigms will most likely result in more effective methods of recovery than using only one intervention.

Load-related afferent information modifies the magnitude and timing of hindlimb muscle activity during stepping in decerebrate animals and spinal cord-injured humans and animals, suggesting that the spinal cord mediates load-related locomotor responses. In this study, we found that stepping on a treadmill by adult rats that received complete, midthoracic spinal cord transections as neonates could be altered by loading the hindlimbs using a pair of small robotic arms. The robotic arms applied a downward force to the lower shanks of the hindlimbs during the stance phase and measured the position of the lower shank during stepping. No external force was applied during the swing phase of the step. When applied bilaterally, this stance force field perturbed the hindlimb trajectories so that the ankle position was shifted downward during stance. In response to this perturbation, both the stance and step cycle durations decreased. During swing, the hindlimb initially accelerated toward the normal, unperturbed swing trajectory and then tracked the normal trajectory. Bilateral loading increased the magnitude of the medial gastrocnemius electromyographic (EMG) burst during stance and increased the amplitude of the semitendinosus and rectus femoris EMG bursts. When the force field was applied unilaterally, stance duration decreased in the loaded hindlimb, while swing duration was decreased in the contralateral hindlimb, thereby preserving interlimb coordination. These results demonstrate the feasibility of using robotic devices to mechanically modulate afferent input to the injured spinal cord during weight-supported locomotion. In addition, these results indicate that the lumbosacral spinal cord responds to load-related input applied to the lower shank during stance by modifying step timing and muscle activation patterns, while preserving normal swing kinematics and interlimb coordination.

We have developed a robotic device (e.g. the rat stepper) that can be used to impose programmed forces on the hindlimbs of rats during stepping. In the present paper we describe initial experiments using this robotic device to determine the feasibility of robotically assisted locomotor training in complete spinally transected adult rats. The present results show that using the robots to increase the amount of load during stance by applying a downward force on the ankle improved lift during swing. The trajectory pattern during swing was also improved when the robot arms were programmed to move the ankle in a path that approximated the normal swing trajectory. These results suggest that critical elements for successful training of hindlimb stepping in spinal cord injured rats can be implemented rigorously and evaluated using the rat stepper.

The ability to perform stepping and standing can be reacquired after complete thoracic spinal cord transection in adult cats with appropriate, repetitive training. We now compare GAD(67)A levels in the spinal cord of cats that were trained to step or stand. We confirmed that a complete spinal cord transection at approximately T12 increases glutamic acid decarboxylase (GAD)(67) in both the dorsal and ventral horns of L5-L7. We now show that step training decreases these levels toward control. Kinematic analyses show that this downward modulation is correlated inversely with stepping ability. Compared with intact cats, spinal cord-transected cats had increased punctate GAD(67) immunoreactivity around neurons in lamina IX at cord segments L5-L7. Compared with spinal nontrained cats, those trained to stand on both hindlimbs had more GAD(67) puncta bilaterally in a subset of lamina IX neurons. In cats trained to stand unilaterally, this elevated staining pattern was limited to the trained side and extended for at least 4 mm in the L6 and L7 segments. The location of this asymmetric GAD(67) staining corresponded to the motor columns of primary knee flexors, which are minimally active during standing, perhaps because of extensor-activated inhibitory interneuron projections. The responsiveness to only a few days of motor training, as well as the GABA-synthesizing potential in the spinal cord, persists for at least 25 months after the spinal cord injury. This modulation is specific to the motor task that is performed repetitively and is closely linked to the ability of the animal to perform a specific motor task.

We have developed a robotic device that can record the trajectory of the hindlimb movements in rats. The robotic device can also impose programmed forces on the limbs during stepping. In the present paper we describe experiments using this robotic device, i.e. the rat stepper, to determine whether step training improves the locomotor capacity of adult rats that received complete spinal cord transections as neonates. We also determined to what extent the locomotor patterns can be maintained when the step cycle is physically perturbed by the robotic device. The results of the present study demonstrate that a robotic device can be used effectively to quantify the improvements in the locomotor capacity of spinal transected rats that occurs over a period of step training. The present results also demonstrate that when an external force is imposed to disrupt the step cycle, the spinal cord has the neural control elements necessary to normalize the kinematics over a number of steps, in the face of the disrupted forces.

Adult spinal cats were trained initially to perform either bipedal hindlimb locomotion on a treadmill or full-weight-bearing hindlimb standing. After 12 wk of training, stepping ability was tested before and after the administration (intraperitoneal) of the glycinergic receptor antagonist, strychnine. Spinal cats that were trained to stand after spinalization had poor locomotor ability as reported previously, but strychnine administration induced full-weight-bearing stepping in their hindlimbs within 30-45 min. In the cats that were trained to step after spinalization, full-weight-bearing stepping occurred and was unaffected by strychnine. Each cat then was retrained to perform the other task for 12 wk and locomotor ability was retested. The spinal cats that were trained initially to stand recovered the ability to step after they received 12 wk of treadmill training and strychnine was no longer effective in facilitating their locomotion. Locomotor ability declined in the spinal cats that were retrained to stand and strychnine restored the ability to step to the levels that were acquired after the step-training period. Based on analyses of hindlimb muscle electromyographic activity patterns and kinematic characteristics, strychnine improved the consistency of the stepping and enhanced the execution of hindlimb flexion during full-weight-bearing step cycles in the spinal cats when they were trained to stand but not when they were trained to step. The present findings provide evidence that 1) the neural circuits that generate full-weight-bearing hindlimb stepping are present in the spinal cord of chronic spinal cats that can and cannot step; however, the ability of these circuits to interpret sensory input to drive stepping is mediated at least in part by glycinergic inhibition; and 2) these spinal circuits adapt to the specific motor task imposed, and that these adaptations may include modifications in the glycinergic pathways that provide inhibition.

Adult spinal cats were trained to perform bipedal hindlimb locomotion on a treadmill for 6-12 wk. After each animal acquired the ability to step, locomotor training was withheld, and stepping was reexamined 6 and 12 wk after training ended. The performance characteristics, hindlimb muscle electromyographic activity patterns, and kinematic characteristics of the step cycle that were acquired with training were largely maintained when training was withheld for 6 wk. However, after 12 wk without training, locomotor performance declined, i.e., stumbling was more frequent, and the ability to consistently execute full weight-bearing steps at any treadmill speed decreased. In addition, the height that the paw was lifted during the swing phase decreased, and a smaller range of extension in the hindlimbs occurred during the E3 phase of stance. When three of the spinal cats underwent 1 wk of retraining, stepping ability was regained more rapidly than when trained initially. The finding that stepping ability in trained adult spinal cats can persist for 6 wk without training provides further evidence that training-induced enhancement of stepping is learned in the spinal cats and that a memory of the enhanced stepping is stored in the spinal networks. However, it appears that the spinal cord can forget how to consistently execute stepping if that task is not practiced for 12 wk. The more rapid learning that occurred with retraining is also consistent with a learning phenomenon. These results in conjunction with our earlier findings suggest that the efficacy of the neural pathways that execute a motor task is highly dependent on the periodic activation of those pathways in a sequence compatible with that motor task.

Behavioral and physiological characteristics of standing were studied in nontrained spinal cats and in spinal cats that received daily stand training of the hindlimbs for 12 wk. Training consisted of assisting the cats to stand with full weight support either on both hindlimbs or on one hindlimb (30 min/day, 5 days/wk). Extensor muscle electromyographic (EMG) amplitude and extension at the knee and ankle joints during full weight bearing recovered to prespinal levels in both stand-trained and nontrained spinal cats. However, full weight bearing of the hindquarters was sustained for up to approximately 20 min in the spinal cats that received bilateral stand training compared with approximately 4 min in cats that were not trained to stand. Unilateral stand training selectively improved weight bearing on the trained limb based on ground reaction forces and extensor muscle EMG activity levels measured during bilateral standing. These results suggest that the capacity of the adult lumbar spinal cord to generate full weight-bearing standing can be improved by as much as fivefold by the repetitive activation of selected neural pathways in the spinal cord after supraspinal connectivity has been eliminated. Given that stepping is improved in response to step training, it appears that the recovery of standing provides another example of training-specific motor learning in the spinal cord, i.e., the spinal cord learns to perform hindlimb standing by practicing that specific task.

Locomotor performance, hindlimb muscle activity and gait patterns during stepping were studied in step-trained and non-trained female, adult spinal cats. Changes in locomotor characteristics relative to prespinalization bipedal and quadrupedal stepping patterns were used to evaluate the effects of step training on the capacity to execute full weight-bearing stepping after spinalization. Step training consisted of full weight-bearing stepping of the hindlimbs at the greatest range of treadmill speeds possible at any given stage of locomotor recovery. In the initial stages of training the limbs were assisted as needed to execute successful steps. On the basis of two behavioral criteria, the maximum speed of treadmill stepping and the number of successful steps per unit time, the ability to step was at least 3 times greater in animals trained to step versus those allowed to recover spontaneously, i.e., the non-trained. The greater success in stepping was reflected in several physiological and kinematic properties. For example, the amplitude of electromyograph (EMG) bursts in the tibialis anterior (an ankle dorsiflexor), the amount of extension at the end of both the stance (E3) and swing (E1) phases of the step cycle, and the amount of lift of the hindlimb during swing were greater in step-trained than in non-trained spinal cats. The changes that occurred in response to training reflected functional adaptations at specific phases of the step cycle, e.g., enhanced flexor and extensor function. The improved stepping capacity attributable to step training is interpreted as a change in the probability of the appropriate neurons being activated in a temporally appropriate manner. This interpretation, in turn, suggests that step training facilitated or reinforced the function of extant sensorimotor pathways rather than promoting the generation of additional pathways. These results show that the capacity of the adult lumbar spinal cord to generate full weight-bearing stepping over a range of speeds is defined, in large part, by the functional experience of the spinal cord after supraspinal connectivity has been eliminated. These results have obvious implications with regards to 1) the possibility of motor learning occurring in the spinal cord; 2) the importance of considering "motor experience" in assessing the effect of any postspinalization intervention; and 3) the utilization of use-dependent interventions in facilitating and enhancing motor recovery.