Uncovering and leveraging the return of voluntary motor programs after paralysis using a bi-cortical neuroprosthesis.

Rehabilitative and neuroprosthetic approaches after spinal cord injury (SCI) aim to reestablish voluntary control of movement. Promoting recovery requires a mechanistic understanding of the return of volition over action, but the relationship between re-emerging cortical commands and the return of locomotion is not well established. We introduced a neuroprosthesis delivering targeted bi-cortical stimulation in a clinically relevant contusive SCI model. In healthy and SCI cats, we controlled hindlimb locomotor output by tuning stimulation timing, duration, amplitude, and site. In intact cats, we unveiled a large repertoire of motor programs. After SCI, the evoked hindlimb lifts were highly stereotyped, yet effective in modulating gait and alleviating bilateral foot drag. Results suggest that the neural substrate underpinning motor recovery had traded-off selectivity for efficacy. Longitudinal tests revealed that the return of locomotion after SCI was correlated with recovery of the descending drive, which advocates for rehabilitation interventions directed at the cortical target.

Recovery of locomotion in cats after severe contusion of the low thoracic spinal cord.

Large bilateral contusions of the T10 thoracic spinal cord were performed in 16 adult cats using a calibrated impactor. EMG and video recordings allowed weekly assessments of key locomotor parameters during treadmill training for 5 wk. Thirty-five days postcontusion, several hindlimb locomotor parameters were very similar to the prelesion ones despite some long-term deficits such as paw drag and disrupted fore-hindlimb coupling. Nine out of ten tested cats could step over obstacles placed on the treadmill. Acute electrophysiological experiments showed viable connectivity between segments rostral and caudal to the contusion. At the fifth postcontusion week, a complete spinalization was performed at T13 in 10 cats and all expressed remarkable bilateral hindlimb locomotion within 24-72 h. From our histological evaluation, we concluded that only a small percentage (~10%) of spinal cord pathways was necessary to initiate and maintain a voluntary quadrupedal locomotor pattern on a treadmill and even to negotiate obstacles. Our findings suggest that hindlimb stepping largely resulted from the activity of spinal locomotor circuits, which gradually recovered autonomy week after week. Our histological and electrophysiological evidence indicated that the persistence of specific deficits or else the maintenance of specific functions was related to the integrity of specific supraspinal and propriospinal pathways. The conclusion is that the recovery of locomotion after large spinal contusions depends on a homeostatic recalibration of a tripartite control system involving interactions between spinal circuits (central pattern generator), supraspinal influences, and sensory feedback activated through locomotor training.NEW & NOTEWORTHY The recovery of quadrupedal treadmill locomotion after a large bilateral contusion at the low thoracic T10 spinal level and the ability to negotiate obstacles were studied for 5 wk in 16 cats. Ten cats were further completely spinalized at T13 and were found to walk with the hindlimbs within 24-72 h. We conclude that the extent of locomotor recovery after large spinal contusions hinges both on remnant supraspinal pathways and on a spinal pattern generator.

Ladder Treadmill: A Method to Assess Locomotion in Cats with an Intact or Lesioned Spinal Cord.

After lesions of the CNS, locomotor abilities of animals (mainly cats) are often assessed on a simple flat treadmill (FTM), which imposes little demands on supraspinal structures as is the case when walking on targets. Therefore, the aims of the present work were as follows: (1) to develop a treadmill allowing the assessment of locomotion of intact cats required to place the paws on the rungs of a moving ladder treadmill (LTM); (2) to assess the capability of cats after a unilateral spinal hemisection at T10 to cope with such a demanding locomotor task; and (3) to regularly train cats for 6 weeks on the LTM to determine whether such regular training improves locomotor recovery on the FTM. A significant improvement would indicate that LTM training maximizes the contribution of spinal locomotor circuits as well as remnant supraspinal inputs. Together, we used 9 cats (7 females, 2 males). Six were used to compare the EMG and kinematic locomotor characteristics during walking on the FTM and LTM. We found that the swing phase during LTM walking was slightly enhanced as well as some specific activity of knee flexor muscles. Fore-hindlimb coupling favored a more stable diagonal coupling. These 6 cats were then hemispinalized and trained for 6 weeks on the LTM, whereas the 3 other cats were hemispinalized and trained solely on the FTM to compare the two training regimens. Intensive LTM training after hemisection was found to change features of locomotion, such as the foot trajectory as well as diminished paw drag often observed after hemisection.SIGNIFICANCE STATEMENT This paper introduces a method (ladder treadmill [LTM]) to study the locomotor ability of cats with an intact spinal cord or after a unilateral hemisection to walk with a precise foot placement on the rungs fixed to an ordinary flat treadmill (FTM). Because cats are compared in various conditions (intact or hemisected at different time points) in the same enclosure on the FTM and the LTM, the changes in averaged locomotor characteristics must reflect the specificity of the task and the neurological states. Furthermore, the ladder treadmill permits to train cats repetitively for weeks and observe whether training regimens (FTM or LTM) can induce durable changes in the parameters of locomotion.

Inducing hindlimb locomotor recovery in adult rat after complete thoracic spinal cord section using repeated treadmill training with perineal stimulation only.

Although a complete thoracic spinal cord section in various mammals induces paralysis of voluntary movements, the spinal lumbosacral circuitry below the lesion retains its ability to generate hindlimb locomotion. This important capacity may contribute to the overall locomotor recovery after partial spinal cord injury (SCI). In rats, it is usually triggered by pharmacological and/or electrical stimulation of the cord while a robot sustains the animals in an upright posture. In the present study we daily trained a group of adult spinal (T7) rats to walk with the hindlimbs for 10 wk (10 min/day for 5 days/wk), using only perineal stimulation. Kinematic analysis and terminal electromyographic recordings revealed a strong effect of training on the reexpression of hindlimb locomotion. Indeed, trained animals gradually improved their locomotion while untrained animals worsened throughout the post-SCI period. Kinematic parameters such as averaged and instant swing phase velocity, step cycle variability, foot drag duration, off period duration, and relationship between the swing features returned to normal values only in trained animals. The present results clearly demonstrate that treadmill training alone, in a normal horizontal posture, elicited by noninvasive perineal stimulation is sufficient to induce a persistent hindlimb locomotor recovery without the need for more complex strategies. This provides a baseline level that should be clearly surpassed if additional locomotor-enabling procedures are added. Moreover, it has a clinical value since intrinsic spinal reorganization induced by training should contribute to improve locomotor recovery together with afferent feedback and supraspinal modifications in patients with incomplete SCI.

Plastic Changes in Lumbar Locomotor Networks after a Partial Spinal Cord Injury in Cats.

After an incomplete spinal cord injury (SCI), we know that plastic reorganization occurs in supraspinal structures with residual descending tracts. However, our knowledge about spinal plasticity is rather limited. Our recent studies point to changes within the spinal cord below the lesion. After a lateral left hemisection (T10), cats recovered stepping with both hindlimbs within 3 weeks. After a complete section (T13) in these cats, bilateral stepping was seen on the next day, a skill usually acquired after several weeks of treadmill training. This indicates that durable plastic changes occurred below the lesion. However, because sensory feedback entrains the stepping rhythm, it is difficult to reveal central pattern generator (CPG) adaptation. Here, we investigated whether lumbar segments of cats with a chronic hemisection were able to generate fictive locomotion-that is, without phasic sensory feedback as monitored by five muscle nerves in each hindlimb. With a chronic left hemisection, the number of muscle nerves displaying locomotor bursts was larger on the left than on the right. In addition, transmission of cutaneous reflexes was relatively facilitated on the left. Later during the acute experiment, a complete spinalization (T13) was performed and clonidine was injected to induce rhythmic activities. There were still more muscle nerves displaying locomotor bursts on the left. The results demonstrate that spinal networks were indeed modified after a hemisection with a clear asymmetry between left and right in the capacity to generate locomotion. Plastic changes in CPG and reflex transmission below the lesion are thus involved in the stepping recovery after an incomplete SCI.

The "beneficial" effects of locomotor training after various types of spinal lesions in cats and rats.

This chapter reviews a number of experiments on the recovery of locomotion after various types of spinal lesions and locomotor training mainly in cats. We first recall the major evidence on the recovery of hindlimb locomotion in completely spinalized cats at the T13 level and the role played by the spinal locomotor network, also known as the central pattern generator, as well as the beneficial effects of locomotor training on this recovery. Having established that hindlimb locomotion can recover, we raise the issue as to whether spinal plastic changes could also contribute to the recovery after partial spinal lesions such as unilateral hemisections. We found that after such hemisection at T10, cats could recover quadrupedal locomotion and that deficits could be improved by training. We further showed that, after a complete spinalization a few segments below the first hemisection (at T13, i.e., the level of previous studies on spinalization), cats could readily walk with the hindlimbs within hours of completely severing the remaining spinal tracts and not days as is usually the case with only a single complete spinalization. This suggests that neuroplastic changes occurred below the first hemisection so that the cat was already primed to walk after the spinalization subsequent to the hemispinalization 3 weeks before. Of interest is the fact that some characteristic kinematic features in trained or untrained hemispinalized cats could remain after complete spinalization, suggesting that spinal changes induced by training could also be durable. Other studies on reflexes and on the pattern of "fictive" locomotion recorded after curarization corroborate this view. More recent work deals with training cats in more demanding situations such as ladder treadmill (vs. flat treadmill) to evaluate how the locomotor training regimen can influence the spinal cord. Finally, we report our recent studies in rats using compressive lesions or surgical complete spinalization and find that some principles of locomotor recovery in cats also apply to rats when adequate locomotor training is provided.

Examination of the combined effects of chondroitinase ABC, growth factors and locomotor training following compressive spinal cord injury on neuroanatomical plasticity and kinematics.

While several cellular and pharmacological treatments have been evaluated following spinal cord injury (SCI) in animal models, it is increasingly recognized that approaches to address the glial scar, including the use of chondroitinase ABC (ChABC), can facilitate neuroanatomical plasticity. Moreover, increasing evidence suggests that combinatorial strategies are key to unlocking the plasticity that is enabled by ChABC. Given this, we evaluated the anatomical and functional consequences of ChABC in a combinatorial approach that also included growth factor (EGF, FGF2 and PDGF-AA) treatments and daily treadmill training on the recovery of hindlimb locomotion in rats with mid thoracic clip compression SCI. Using quantitative neuroanatomical and kinematic assessments, we demonstrate that the combined therapy significantly enhanced the neuroanatomical plasticity of major descending spinal tracts such as corticospinal and serotonergic-spinal pathways. Additionally, the pharmacological treatment attenuated chronic astrogliosis and inflammation at and adjacent to the lesion with the modest synergistic effects of treadmill training. We also observed a trend for earlier recovery of locomotion accompanied by an improvement of the overall angular excursions in rats treated with ChABC and growth factors in the first 4 weeks after SCI. At the end of the 7-week recovery period, rats from all groups exhibited an impressive spontaneous recovery of the kinematic parameters during locomotion on treadmill. However, although the combinatorial treatment led to clear chronic neuroanatomical plasticity, these structural changes did not translate to an additional long-term improvement of locomotor parameters studied including hindlimb-forelimb coupling. These findings demonstrate the beneficial effects of combined ChABC, growth factors and locomotor training on the plasticity of the injured spinal cord and the potential to induce earlier neurobehavioral recovery. However, additional approaches such as stem cell therapies or a more adapted treadmill training protocol may be required to optimize this repair strategy in order to induce sustained functional locomotor improvement.

Recovery of locomotion after partial spinal cord lesions in cats: assessment using behavioral, electrophysiological and imaging techniques.

This short review summarizes experimental findings made after spinal cord injury, mainly in cats. After a complete spinal injury, cats re-express hindlimb locomotion after 2-3 weeks because of a spinal locomotor circuitry named the central pattern generator or CPG. To investigate whether such circuits are also implicated in the recovery of locomotion after partial spinal lesions, we have used a dual spinal lesion paradigm. Essentially, after an initial unilateral hemisection, cats spontaneously recover quadrupedal locomotion. When a complete section is then performed 3 weeks after this hemisection, cats can walk with the hindlimbs within 24 hours compared to 2-3 weeks in cats with single complete spinal lesions demonstrating the importance of spinal mechanisms after partial lesions. Using kinematic and electromyographic methods to evaluate the changes throughout the dual lesion paradigm, we could show that the spinal cord reorganizes spontaneously without locomotor training or with training provided between the partial and complete spinal lesion. To assess spinal lesions we have used histology and magnetic resonance imaging (MRI). We will describe some advanced MRI techniques such as diffusion and magnetization transfer, which provide higher specificity to axon degeneration and demyelination. Examples of advanced MRI techniques in cats and humans are described, including the current limitations and perspectives.

Emergence of deletions during treadmill locomotion as a function of supraspinal and sensory inputs.

During locomotion, alternating activity of flexor and extensor muscles is largely regulated by a spinal neuronal network, the central pattern generator, the activity of which is modulated by peripheral and supraspinal inputs. In the absence of these modulatory inputs, for example during fictive locomotion after spinalization and curarization, spontaneous failures of motor activation (deletions) in a muscle can occur without perturbing the rhythmic cycle structure of the antagonists on the same side or the contralateral side. This suggests that the central pattern generator can maintain the locomotor period when motoneuron discharges fail in a given pool of motoneurons. Here we first examined whether such deletions could occur during real locomotion on a treadmill and determined their consequences on the overt locomotor pattern. We also evaluated the role of supraspinal and sensory inputs in modulating the occurrence of failures of rhythmic activity by comparing the same cats in the intact state, then after a partial spinal cord injury (SCI), and finally after a complete SCI at different treadmill speeds. We showed that deletions: (1) are absent in intact animals and occur only after SCI; (2) affect only flexor muscle activity; (3) neither perturb the timing of rhythmic activity of these muscles in subsequent cycles nor interfere with the timing of the ipsilateral and contralateral agonists and antagonists; (4) do not affect significantly the locomotor pattern kinematics; and (5) are sensitive to treadmill speed and lesion severity, suggesting a role for sensory and supraspinal inputs in stabilizing rhythmic output activity.

Treadmill training promotes spinal changes leading to locomotor recovery after partial spinal cord injury in cats.

After a spinal hemisection at thoracic level in cats, the paretic hindlimb progressively recovers locomotion without treadmill training but asymmetries between hindlimbs persist for several weeks and can be seen even after a further complete spinal transection at T13. To promote optimal locomotor recovery after hemisection, such asymmetrical changes need to be corrected. In the present study we determined if the locomotor deficits induced by a spinal hemisection can be corrected by locomotor training and, if so, whether the spinal stepping after the complete spinal cord transection is also more symmetrical. This would indicate that locomotor training in the hemisected period induces efficient changes in the spinal cord itself. Sixteen adult cats were first submitted to a spinal hemisection at T10. One group received 3 wk of treadmill training, whereas the second group did not. Detailed kinematic and electromyographic analyses showed that a 3-wk period of locomotor training was sufficient to improve the quality and symmetry of walking of the hindlimbs. Moreover, after the complete spinal lesion was performed, all the trained cats reexpressed bilateral and symmetrical hindlimb locomotion within 24 h. By contrast, the locomotor pattern of the untrained cats remained asymmetrical, and the hindlimb on the side of the hemisection was still deficient. This study highlights the beneficial role of locomotor training in facilitating bilateral and symmetrical functional plastic changes within the spinal circuitry and in promoting locomotor recovery after an incomplete spinal cord injury.

Effect of locomotor training in completely spinalized cats previously submitted to a spinal hemisection.

After a spinal hemisection in cats, locomotor plasticity occurring at the spinal level can be revealed by performing, several weeks later, a complete spinalization below the first hemisection. Using this paradigm, we recently demonstrated that the hemisection induces durable changes in the symmetry of locomotor kinematics that persist after spinalization. Can this asymmetry be changed again in the spinal state by interventions such as treadmill locomotor training started within a few days after the spinalization? We performed, in 9 adult cats, a spinal hemisection at thoracic level 10 and then a complete spinalization at T13, 3 weeks later. Cats were not treadmill trained during the hemispinal period. After spinalization, 5 of 9 cats were not trained and served as control while 4 of 9 cats were trained on the treadmill for 20 min, 5 d a week for 3 weeks. Using detailed kinematic analyses, we showed that, without training, the asymmetrical state of locomotion induced by the hemisection was retained durably after the subsequent spinalization. By contrast, training cats after spinalization induced a reversal of the left/right asymmetries, suggesting that new plastic changes occurred within the spinal cord through locomotor training. Moreover, training was shown to improve the kinematic parameters and the performance of the hindlimb on the previously hemisected side. These results indicate that spinal locomotor circuits, previously modified by past experience such as required for adaptation to the hemisection, can remarkably respond to subsequent locomotor training and improve bilateral locomotor kinematics, clearly showing the benefits of locomotor training in the spinal state.

Incomplete spinal cord injury promotes durable functional changes within the spinal locomotor circuitry.

While walking in a straight path, changes in speed result mainly from adjustments in the duration of the stance phase while the swing phase remains relatively invariant, a basic feature of the spinal central pattern generator (CPG). To produce a broad range of locomotor behaviors, the CPG has to integrate modulatory inputs from the brain and the periphery and alter these swing/stance characteristics. In the present work we raise the issue as to whether the CPG can adapt or reorganize in response to a chronic change of supraspinal inputs, as is the case after spinal cord injury (SCI). Kinematic data obtained from six adult cats walking at different treadmill speeds were collected to calculate the cycle and subphase duration at different stages after a first spinal hemisection at T(10) and after a subsequent complete SCI at T(13) respectively aimed at disconnecting unilaterally and then totally the spinal cord from its supraspinal inputs. The results show, first, that the neural control of locomotion is flexible and responsive to a partial or total loss of supraspinal inputs. Second, we demonstrate that a hemisection induces durable plastic changes within the spinal locomotor circuitry below the lesion. In addition, this study gives new insights into the organization of the spinal CPG for locomotion such that phases of the step cycle (swing, stance) can be independently regulated for adapting to speed and also that the CPGs controlling the left and right hindlimbs can, up to a point, be regulated independently.

Kinematic study of locomotor recovery after spinal cord clip compression injury in rats.

After spinal cord injury (SCI), precise assessment of motor recovery is essential to evaluate the outcome of new therapeutic approaches. Very little is known on the recovery of kinematic parameters after clinically-relevant severe compressive/contusive incomplete spinal cord lesions in experimental animal models. In the present study we evaluated the time-course of kinematic parameters during a 6-week period in rats walking on a treadmill after a severe thoracic clip compression SCI. The effect of daily treadmill training was also assessed. During the recovery period, a significant amount of spontaneous locomotor recovery occurred in 80% of the rats with a return of well-defined locomotor hindlimb pattern, regular plantar stepping, toe clearance and homologous hindlimb coupling. However, substantial residual abnormalities persisted up to 6 weeks after SCI including postural deficits, a bias of the hindlimb locomotor cycle toward the back of the animals with overextension at the swing/stance transition, loss of lateral balance and impairment of weight bearing. Although rats never recovered the antero-posterior (i.e. homolateral) coupling, different levels of decoupling between the fore and hindlimbs were measured. We also showed that treadmill training increased the swing duration variability during locomotion suggesting an activity-dependent compensatory mechanism of the motor control system. However, no effect of training was observed on the main locomotor parameters probably due to a ceiling effect of self-training in the cage. These findings constitute a kinematic baseline of locomotor recovery after clinically relevant SCI in rats and should be taken into account when evaluating various therapeutic strategies aimed at improving locomotor function.

Recovery of hindlimb locomotion after incomplete spinal cord injury in the cat involves spontaneous compensatory changes within the spinal locomotor circuitry.

After incomplete spinal cord injury (SCI), compensatory changes occur throughout the whole neuraxis, including the spinal cord below the lesion, as suggested by previous experiments using a dual SCI paradigm. Indeed, cats submitted to a lateral spinal hemisection at T10-T11 and trained on a treadmill for 3-14 wk re-expressed bilateral hindlimb locomotion as soon as 24 h after spinalization, a process that normally takes 2-3 wk when a complete spinalization is performed without a prior hemisection. In this study, we wanted to ascertain whether similar effects could occur spontaneously without training between the two SCIs and within a short period of 3 wk in 11 cats. One day after the complete spinalization, 9 of the 11 cats were able to re-express hindlimb locomotion either bilaterally (n = 6) or unilaterally on the side of the previous hemisection (n = 3). In these 9 cats, the hindlimb on the side of the previous hemisection (left hindlimb) performed better than the right side in contrast to that observed during the hemispinal period itself. Cats re-expressing the best bilateral hindlimb locomotion after spinalization had the largest initial hemilesion and the most prominent locomotor deficits after this first SCI. These results provide evidence that 1) marked reorganization of the spinal locomotor circuitry can occur without specific locomotor training and within a short period of 3 wk; 2) the spinal cord can reorganize in a more or less symmetrical way; and 3) the ability to walk after spinalization depends on the degree of deficits and adaptation observed in the hemispinal period.

Effects of localized intraspinal injections of a noradrenergic blocker on locomotion of high decerebrate cats.

Previous studies demonstrated that neuronal networks located in midlumbar segments (L3-L4) are critical for the expression of locomotion in cats following complete spinalization. In the present study the importance of several thoracolumbar segments (T8-L7) for the generation of spontaneous hindlimb locomotion in decerebrate cats was evaluated. Experiments were performed in high decerebrate cats (n = 18) walking spontaneously. Yohimbine, an alpha2-noradrenergic antagonist, was microinjected intraspinally in various thoracolumbar segments. Locomotor performance was evaluated with kinematics and electromyographic (EMG) recordings before and after each injection. When and if spontaneous locomotion (SL) was abolished, skin or perineal stimuli (exteroceptive stimuli) were used to trigger locomotion (exteroceptive-induced locomotion [EL]). Yohimbine injections at L3 or L4 completely inhibited SL and EL. In contrast, injections at T8 did not interfere with SL or EL. Injections at T10, T11, T12, L5, L6, and L7 inhibited SL but EL could still be evoked. Injections at T13, L1, and L2 had similar effects except that the quality of locomotion evoked by exteroceptive stimulation declined. Combined injections at T13, L1, and L2 abolished SL and EL, in contrast to injections restricted to the same individual segments. Simultaneous injections at L5, L6, and L7 also abolished SL but EL could still be induced. These results suggest that noradrenergic mechanisms in L3-L4 segments are involved in the expression of locomotion in decerebrate cats, whereas antagonizing noradrenergic inputs in individual rostral or caudal segments may alter the expression and overall quality of the locomotor pattern without abolishing locomotion.