In vivo electrophysiological mechanisms underlying cervical epidural stimulation in adult rats.

KEY POINTS: To electrophysiologically determine the predominant neural structures activated with cervical epidural stimulation (ES), well-established electrophysiological protocols (single-pulse, paired-pulse and multiple frequency stimulation) were delivered at rest, during motor activity and under anaesthesia in adult rats. Cervical ES resulted in spinal evoked motor responses with three different waveforms - early response (ER), middle response (MR) and late response (LR). ERs remained unmodulated by repeated stimulation protocols. In contrast, MRs and LRs were modulated by repeated stimulation protocols and volitional motor activity. ERs are consequential to the direct activation of motor efferents; MRs are secondary to type-I sensory afferent activation and LRs result from the engagement of wider spinal interneuronal circuitry with potential influence from supraspinal pathways. Evidence from this work is fundamental in enhancing our understanding of cervical ES, and critical in refining the design of neuromodulation-based rehabilitative strategies and in the construction of neuroprosthetics. ABSTRACT: Epidural stimulation (ES) of the lumbar spinal cord has demonstrated significant improvements in various physiological functions after a traumatic spinal cord injury in humans. Electrophysiological evidence from rodent, human and computational studies collectively suggest that the functional recovery following lumbar ES is mediated via direct activation of sensory afferent fibres. However, the mechanisms underlying cervical ES have not been comprehensively studied, which greatly limits our understanding of its effectiveness in restoring upper limb function. In this work, we determined the predominant neural structures that are activated with cervical ES using in vivo cervical spinal evoked motor responses (SEMRs). Standard electrophysiological protocols (single-pulse, paired-pulse and multiple frequency stimulation) were implemented in 11 awake and anaesthetized rats in four experimental stages. Three distinct types of cervical SEMRs were identified based on latency of their appearance: early response (ER), middle response (MR) and late response (LR). ERs remained unmodulated by repeated stimulation protocols. MRs and LRs were modulated by repeated stimulation protocols and volitional motor activity. Except for LRs being completely abolished under urethane, ketamine or urethane anaesthesia did not affect the appearance of cervical SEMRs. Our data, backed by literature, suggest that ERs are secondary to the direct activation of motor efferents, MRs are elicited by activation of type-I sensory afferents and LRs result from the engagement of interneuronal circuitry with potential influence from supraspinal pathways. The gathered information paves the way to designing motor rehabilitation strategies that can utilize cervical ES to recover upper limb function following neurological deficits.

3D Kinematic Gait Analysis for Preclinical Studies in Rodents.

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

Rostral lumbar segments are the key controllers of hindlimb locomotor rhythmicity in the adult spinal rat.

The precise location and functional organization of the spinal neuronal locomotor-related networks in adult mammals remain unclear. Our recent neurophysiological findings provided empirical evidence that the rostral lumbar spinal cord segments play a critical role in the initiation and generation of the rhythmic activation patterns necessary for hindlimb locomotion in adult spinal rats. Since added epidural stimulation at the S1 segments significantly enhanced the motor output generated by L2 stimulation, these data also suggested that the sacral spinal cord provides a strong facilitory influence in rhythm initiation and generation. However, whether L2 will initiate hindlimb locomotion in the absence of S1 segments, and whether S1 segments can facilitate locomotion in the absence of L2 segments remain unknown. Herein, adult rats received complete spinal cord transections at T8 and then at either L2 or S1. Rats with spinal cord transections at T8 and S1 remained capable of generating coordinated hindlimb locomotion when receiving epidural stimulation at L2 and when ensembles of locomotor related loadbearing input were present. In contrast, minimal locomotion was observed when S1 stimulation was delivered after spinal cord transections at T8 and L2. Results were similar when the nonspecific serotonergic agonists were administered. These results demonstrate in adult rats that rostral lumbar segments are essential for the regulation of hindlimb locomotor rhythmicity. In addition, the more caudal spinal networks alone cannot control locomotion in the absence of the rostral segments around L2 even when loadbearing rhythmic proprioceptive afferent input is imposed.NEW & NOTEWORTHY The exact location of the spinal neuronal locomotor-related networks in adult mammals remains unknown. The present data demonstrate that when the rostral lumbar spinal segments (~L2) are completely eliminated in thoracic spinal adult rats, hindlimb stepping is not possible with neurochemical modulation of the lumbosacral cord. In contrast, eliminating the sacral cord retains stepping ability. These observations highlight the importance of rostral lumbar segments in generating effective mammalian locomotion.

Novel Speed-Controlled Automated Ladder Walking Device Reveals Walking Speed as a Critical Determinant of Skilled Locomotion after a Spinal Cord Injury in Adult Rats.

The horizontal ladder task is an established method to assess skilled locomotor recovery after neurological dysfunction. Walking speed is often used as a standardized measure in locomotor assessment of overground walking in human and pre-clinical studies, but the assessment of walking speed is typically ignored during skilled locomotor tasks. Ample empirical evidence indicates that walking speeds on the horizontal ladder are largely non-uniform after central nervous system trauma, suggesting that it could pose a potential source of variability in assessing motor deficits. Here, we investigate whether walking speed influences the assessment of motor recovery during skilled walking after a spinal cord injury (SCI). We hypothesized that if rats walk at imposed walking speeds, motor deficits and recovery after an SCI will be more reliably assessed than when not controlling walking speeds. To address this, we developed a novel speed-controlled Automated Device for the Assessment and Training of Skilled locomotion (ADATS) as a surrogate device to the horizontal ladder. The ADATS allows testing at user-defined speeds, thereby forcing the rats to step consistently. Our results demonstrate that: 1) the ability to walk (or not) at one or multiple speeds on the ADATS serves as a gross measure of motor dysfunction/recovery after a spinal cord injury and 2) skilled motor deficits are more readily detected at lower than higher walking speeds. We conclude that walking speed is an important factor in the analyses of skilled locomotion and testing at multiple speeds is useful in accurately measuring recovery after neurotrauma in rats.

Development of an Activity-Dependent Epidural Stimulation System in Freely Moving Spinal Cord Injured Rats: A Proof of Concept Study.

Purpose: Extensive pre-clinical and clinical experimentation has yielded data on the robustness and versatility of epidural stimulation (ES) strategies to activate spinal neural circuitry to produce functional benefits. Increasing studies are now reporting that closed-loop electrical stimulation delivery methods significantly enhance the neuromodulation effects of stimulation, to in turn, improve physiological outcomes of the intervention. No studies have yet explored the feasibility and usage of closed-loop systems to neuromodulate the cervical spinal cord using ES. Methods: We developed an activity-dependent system that utilizes electromyography (EMG) activity to trigger epidural stimulation (tES) of the cervical spinal cord in awake, freely moving rats. Experiments were performed on rats that were implanted with chronic forelimb EMG and cervical epidural implants, with (n = 7) and without (n = 2) a complete C4 spinal hemisection. Results: Our results show that the EMG triggered activity-dependent system can be reliably applied and reproduced for: (i) stimulating multiple rats simultaneously throughout the night during free home-cage activity and (ii) use as a mobile system for testing and training during various short-term behavioral testing conditions. The system was able to consistently generate stimulation pulse trains in response to attempted EMG activity that crossed a user-defined threshold in all rats for all experiments, including the overnight experiments that lasts for 7 h/session for 6 days/week through the 3-month period. Conclusion: The developed closed-loop system can be considered to represent a class of bidirectional neural prostheses via a circuit that enables two-way interactions between neural activity (real-time processing of EMG activity) and external devices (such as a stimulator). It can operate autonomously for extended periods of time in unrestrained rats, allowing its use as a long-term therapeutic tool. It can also enable us to study the long-term physiological effects of incorporating electrical stimulation techniques into the nervous system. The system can also be experimented for connecting several neural systems into a Brainet by combining neural signals from multiple rats dynamically and in real-time so as to enhance motor performance. Studies are ongoing in our laboratory to test the usefulness of this system in the recovery of hand function after cervical spinal cord injuries.

Spinal Epidural Stimulation Strategies: Clinical Implications of Locomotor Studies in Spinal Rats.

Significant advancements in spinal epidural stimulation (ES) strategies to enable volitional motor control in persons with a complete spinal cord injury (SCI) have generated much excitement in the field of neurorehabilitation. Still, an obvious gap lies in the ability of ES to effectively generate a robust locomotor stepping response after a complete SCI in rodents, but not in humans. In order to reveal potential discrepancies between rodent and human studies that account for this void, in this review, we summarize the findings of studies that have utilized ES strategies to enable successful hindlimb stepping in spinal rats. Recent clinical and preclinical evidence indicates that motor training with ES plays a crucial role in tuning spinal neural circuitry to generate meaningful motor output. Concurrently administered pharmacology can also facilitate the circuitry to provide near optimal motor performance in SCI rats. However, as of today, the evidence for pharmacological agents to enhance motor function in persons with complete SCI is insignificant. These and other recent findings discussed in this review provide insight into addressing the translational gap, guide the design of relevant preclinical experiments, and facilitate development of new approaches for motor recovery in patients with complete SCIs.

Unique Spatiotemporal Neuromodulation of the Lumbosacral Circuitry Shapes Locomotor Success after Spinal Cord Injury.

Spinal cord epidural stimulation has resulted in the initiation of voluntary leg movements and improvement in postural, bladder, and sexual function. However, one of the limitations in reaching the full potential of epidural stimulation for therapeutic purposes in humans has been the identification of optimal stimulation configurations that can neuromodulate the spinal cord for stepping. In the present work, we investigated the mechanisms underlying the specificity of interaction between the rostral and caudal spinal cord circuitries in enabling locomotion in spinal rats (n = 10) by epidural spinal cord stimulation. By using unique spatiotemporal epidural stimulation parameters of the lumbar and sacral spinal cords, a robust stepping pattern in spinal rats was observed with only six training sessions and as early as 3 weeks post-injury. Electrophysiological evidence reveals that in addition to frequency of stimulation pulses at the stimulation sites, the relative timing between stimulation pulses applied at the lumbar (L2) and sacral (S1) segments of the spinal cord heavily impacted stepping performance. Best stepping was established at a higher stimulation frequency (40 Hz vs. 5, 10, 15, and 20Hz) and at specific relative time-intervals between the stimulation pulses (L2 pulse applied at 18-25 msec after the onset of the S1 pulse; S1 pulse applied 0-7 msec after the L2 pulse). Our data suggest that controlling pulse-to-pulse timing at multiple stimulation sources provides a novel strategy to optimize spinal stepping by fine-tuning the physiological state of the locomotor networks. These findings hold direct relevance to the clinician who will incorporate electrical stimulation strategies for optimizing control of locomotion after complete paralysis.

Evaluation of optimal electrode configurations for epidural spinal cord stimulation in cervical spinal cord injured rats.

BACKGROUND: Epidural spinal cord stimulation is a promising technique for modulating the level of excitability and reactivation of dormant spinal neuronal circuits after spinal cord injury (SCI). We examined the ability of chronically implanted epidural stimulation electrodes within the cervical spinal cord to (1) directly elicit spinal motor evoked potentials (sMEPs) in forelimb muscles and (2) determine whether these sMEPs can serve as a biomarker of forelimb motor function after SCI. NEW METHOD: We implanted EMG electrodes in forelimb muscles and epidural stimulation electrodes at C6 and C8 in adult rats. After recovering from a dorsal funiculi crush (C4), rats were tested with different stimulation configurations and current intensities to elicit sMEPs and determined forelimb grip strength. RESULTS: sMEPs were evoked in all muscles tested and their characteristics were dependent on electrode configurations and current intensities. C6(-) stimulation elicited more robust sMEPs than stimulation at C8(-). Stimulating C6 and C8 simultaneously produced better muscle recruitment and higher grip strengths than stimulation at one site. COMPARISON WITH EXISTING METHOD(S): Classical method to select the most optimal stimulation configuration is to empirically test each combination individually for every subject and relate to functional improvements. This approach is impractical, requiring extensively long experimental time to determine the more effective stimulation parameters. Our proposed method is fast and physiologically sound. CONCLUSIONS: Results suggest that sMEPs from forelimb muscles can be useful biomarkers for identifying optimal parameters for epidural stimulation of the cervical spinal cord after SCI.

Plasticity of subcortical pathways promote recovery of skilled hand function in rats after corticospinal and rubrospinal tract injuries.

The corticospinal and rubrospinal tracts are the predominant tracts for controlling skilled hand function. Injuries to these tracts impair grasping but not gross motor functions such as overground locomotion. The aim of the present study was to determine whether or not, after damage to both the corticospinal and rubrospinal tracts, other spared subcortical motor pathway can mediate the recovery of skilled hand function. Adult rats received a bilateral injury to the corticospinal tract at the level of the medullar pyramids and a bilateral ablation of the rubrospinal axons at C4. One group of rats received, acutely after injury, two injections of chondroitinase-ABC at C7, and starting at 7days post-injury were enrolled in daily reaching and grasping rehabilitation (CHASE group, n=5). A second group of rats received analogous injections of ubiquitous penicillinase, and did not undergo rehabilitation (PEN group, n=5). Compared to rats in the PEN group, CHASE rats gradually recovered the ability to reach and grasp over 42days after injury. Overground locomotion was mildly affected after injury and both groups followed similar recovery. Since the reticulospinal tract plays a predominant role in motor control, we further investigated whether or not plasticity of this pathway could contribute to the animal's recovery. Reticulospinal axons were anterogradely traced in both groups of rats. The density of reticulospinal processes in both the normal and ectopic areas of the grey ventral matter of the caudal segments of the cervical spinal cord was greater in the CHASE than PEN group. The results indicate that after damage to spinal tracts that normally mediate the control of reaching and grasping in rats other complementary spinal tracts can acquire the role of those damaged tracts and promote task-specific recovery.

In vivo (31)P NMR spectroscopy assessment of skeletal muscle bioenergetics after spinal cord contusion in rats.

PURPOSE: Muscle paralysis after spinal cord injury leads to muscle atrophy, enhanced muscle fatigue, and increased energy demands for functional activities. Phosphorus magnetic resonance spectroscopy ((31)P-MRS) offers a unique non-invasive alternative of measuring energy metabolism in skeletal muscle and is especially suitable for longitudinal investigations. We determined the impact of spinal cord contusion on in vivo muscle bioenergetics of the rat hind limb muscle using (31)P-MRS. METHODS: A moderate spinal cord contusion injury (cSCI) was induced at the T8-T10 thoracic spinal segments. (31)P-MRS measurements were performed weekly in the rat hind limb muscles for 3 weeks. Spectra were acquired in a Bruker 11 T/470 MHz spectrometer using a 31P surface coil. The sciatic nerve was electrically stimulated by subcutaneous needle electrodes. Spectra were acquired at rest (5 min), during stimulation (6 min), and recovery (20 min). Phosphocreatine (PCr) depletion rates and the pseudo first-order rate constant for PCr recovery (k PCr) were determined. The maximal rate of PCr resynthesis, the in vivo maximum oxidative capacity (V max) and oxidative adenosine triphosphate (ATP) synthesis rate (Q max) were subsequently calculated. RESULTS: One week after cSCI, there was a decline in the resting total creatine of the paralyzed muscle. There was a significant reduction (~24 %) in k PCr measures of the paralyzed muscle, maximum in vivo mitochondrial capacity (V max) and the maximum oxidative ATP synthesis rate (Q max) at 1 week post-cSCI. During exercise, the PCr depletion rates in the paralyzed muscle one week after injury were rapid and to a greater extent than in a healthy muscle. CONCLUSIONS: Using in vivo MRS assessments, we reveal an acute oxidative metabolic defect in the paralyzed hind limb muscle. These altered muscle bioenergetics might contribute to the host of motor dysfunctions seen after cSCI.

Use of quadrupedal step training to re-engage spinal interneuronal networks and improve locomotor function after spinal cord injury.

Can lower limb motor function be improved after a spinal cord lesion by re-engaging functional activity of the upper limbs? We addressed this issue by training the forelimbs in conjunction with the hindlimbs after a thoracic spinal cord hemisection in adult rats. The spinal circuitries were more excitable, and behavioural and electrophysiological analyses showed improved hindlimb function when the forelimbs were engaged simultaneously with the hindlimbs during treadmill step-training as opposed to training only the hindlimbs. Neuronal retrograde labelling demonstrated a greater number of propriospinal labelled neurons above and below the thoracic lesion site in quadrupedally versus bipedally trained rats. The results provide strong evidence that actively engaging the forelimbs improves hindlimb function and that one likely mechanism underlying these effects is the reorganization and re-engagement of rostrocaudal spinal interneuronal networks. For the first time, we provide evidence that the spinal interneuronal networks linking the forelimbs and hindlimbs are amenable to a rehabilitation training paradigm. Identification of this phenomenon provides a strong rationale for proceeding toward preclinical studies for determining whether training paradigms involving upper arm training in concert with lower extremity training can enhance locomotor recovery after neurological damage.

Variability in step training enhances locomotor recovery after a spinal cord injury.

Performance of a motor task is improved by practicing a specific task with added 'challenges' to a training regimen. We tested the hypothesis that, in the absence of brain control, the performance of a motor task is enhanced by training using specific variations of that task. We utilized modifications of step performance training to improve the ability of spinal rats to forward step. After a complete thoracic spinal cord transection, 20 adult rats were divided randomly to bipedally step on a treadmill in the forward, sideward, or backward direction for 28 sessions (20 min, 5 days/week) and subsequently tested for their ability to step in the forward direction. Although the animals from all trained groups showed improvement, the rats in the sideward-trained and backward-trained groups had greater step consistency and coordination along with higher peak amplitudes and total integrated activity of the rectified electromyographic signals from selected hindlimb muscles per step during forward stepping than the rats in the forward-trained group. Our results demonstrate that, by retaining the fundamental features of a motor task (bipedal stepping), the ability to perform that motor task can be enhanced by the addition of specific contextual variations to the task (direction of stepping). Our data suggest that the forward stepping neuronal locomotor networks are partially complemented by synchronous activation of interneuronal/motoneuronal populations that are also a part of the sideward or backward stepping locomotor networks. Accordingly, the overlap and interaction of neuronal elements may play a critical role in positive task transference.

Rodent estrous cycle response to incomplete spinal cord injury, surgical interventions, and locomotor training.

Estrous cycle disruption after spinal cord injury (SCI) in female rats is a common phenomenon. It remains unknown, however, if the aberrant estrous cycle is a result of an injury to the spinal cord itself or due to the general stress associated with surgical interventions. We addressed this issue by determining estrous cyclicality in female rats after a spinal cord hemisection (HX), implantation of EMG wires into selected hind limb muscles, and/or injections of tracer dyes into the spinal cord. Because it is known that aerobic exercise can enhance the recovery of locomotor function in rodents with an incomplete SCI, we also determined if locomotor training positively impacts the disrupted estrous cycle after an HX. Estrous cycle assessments were made during a 5-8 week period in 27 female rats before and after HX, EMG, and/or dye injection surgeries and in HX rats that recovered spontaneously or underwent locomotor training. Our results show that estrous cyclicality was disrupted (cycle length >5 days) in approximately 76%, 46%, and 50% of the rats after HX, EMG, and dye injection surgeries, respectively. The cyclicality, however, was disrupted for a longer period after HX than after EMG or dye injection surgeries. Furthermore, estrous cycle mean length was shorter in the trained than nontrained HX group. These results suggest that estrous cycle disruption after a major SCI is a consequence of both the direct injury to the spinal cord and to the associated stress. Moreover, moderate aerobic exercise initiated early after a spinal cord HX returns the duration of the estrous cycle toward normal.

Lower-extremity muscle cross-sectional area after incomplete spinal cord injury.

OBJECTIVES: (1) To quantify skeletal muscle size in lower-extremity muscles of people after incomplete spinal cord injury (SCI), (2) to assess differences in muscle size between involved lower limbs, (3) to determine the impact of ambulatory status (using wheelchair for community mobility vs not using a wheelchair for community mobility) on muscle size after incomplete SCI, and (4) to determine if differential atrophy occurs among individual muscles after incomplete SCI. DESIGN: Case-control study. SETTING: University research setting. PARTICIPANTS: Seventeen people with incomplete SCI and 17 age-, sex-, weight-, and height-matched noninjured controls. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Maximum cross-sectional area (CSA) of individual lower-extremity muscles (soleus, medial gastrocnemius, lateral gastrocnemius, tibialis anterior, quadriceps femoris, hamstrings) as assessed by magnetic resonance imaging. RESULTS: Overall, subjects with incomplete SCI had significantly smaller (24%-31%) average muscle CSA in affected lower-extremity muscles as compared with control subjects (P<.05). Mean differences were highest in the thigh muscles ( approximately 31%) compared with the lower-leg muscles ( approximately 25%). No differences were noted between the self-reported more- and less-involved limbs within the incomplete SCI group. Dichotomizing the incomplete SCI group showed significantly lower muscle CSA values in both the wheelchair (range, 21%-39%) and nonwheelchair groups (range, 24%-38%). In addition, the wheelchair group exhibited significantly greater plantarflexor muscle atrophy compared with the dorsiflexors, with maximum atrophy in the medial gastrocnemius muscle (39%). CONCLUSIONS: Our results suggest marked and differential atrophic response of the affected lower-extremity muscles that is seemingly affected by ambulatory status in people with incomplete SCI.

Eshkol-Wachman movement notation in diagnosis: the early detection of Asperger's syndrome.

The diagnostic criteria of Asperger's syndrome (AS), considered a part of the autistic spectrum disorder, are still unclear. A critical marker, which distinguishes AS from autism, is the presence of language. The ability of a child with AS to acquire and use language early results in the fact that AS usually is diagnosed much later than autism. Autism is not usually diagnosed until around the age of 3, whereas AS usually is not diagnosed until the child is 6 or 7 years of age. In the present article, using Eshkol-Wachman movement notation, we present evidence that abnormal movement patterns can be detected in AS in infancy. This finding suggests that AS can be diagnosed very early, independent of the presence of language. As shown earlier by us, almost all of the movement disturbances in autism can be interpreted as infantile reflexes "gone astray"; i.e., some reflexes are not inhibited at the appropriate age in development, whereas others fail to appear when they should. This phenomenon appears to apply to AS as well. Based on preliminary results, a simple test using one such reflex is proposed for the early detection of a subgroup of children with AS or autism.