Effect of Hindlimb Unloading on Hamstring Muscle Activity in Rats.

INTRODUCTION: The changes in knee axial rotation play an important role in traumatic and non-traumatic knee disorders. It is known that support afferentation can affect the axial rotator muscles. The condition of innervation of the semitendinosus (ST) and biceps femoris posterior (BFp) has changed in non-terrestrial and terrestrial vertebrates in evolution; thus, we hypothesized this situation might be replayed by hindlimb unloading (HU). METHODS: In the present study, the EMG activity of two hamstring muscles, m. ST and m. BFp, which are antagonists in axial rotation of the tibia, was examined before and after 7 days of HU. RESULTS: During locomotion and swimming, the ST flexor burst activity increased in the stance-to-swing transition and in the retraction-protraction transition, respectively, while that of BFp remained unchanged. Both ST and BFp non-burst extensor activity increased during stepping and decreased during swimming. CONCLUSIONS: Our results show that (1) the flexor burst activity of ST and BFp depends differently on the load-dependent sensory input in the step cycle; (2) shift of the activity gradient towards ST in the stance-to-swing transition could produce excessive internal tibia torque, which can be used as an experimental model of non-traumatic musculoskeletal disorders; and (3) the mechanisms of activity of ST and BFp may be based on reciprocal activity of homologous muscles in primary tetrapodomorph and depend on the increased role of supraspinal control.

Distribution of Spinal Neuronal Networks Controlling Forward and Backward Locomotion.

Higher vertebrates, including humans, are capable not only of forward (FW) locomotion but also of walking in other directions relative to the body axis [backward (BW), sideways, etc.]. Although the neural mechanisms responsible for controlling FW locomotion have been studied in considerable detail, the mechanisms controlling steps in other directions are mostly unknown. The aim of the present study was to investigate the distribution of spinal neuronal networks controlling FW and BW locomotion. First, we applied electrical epidural stimulation (ES) to different segments of the spinal cord from L2 to S2 to reveal zones triggering FW and BW locomotion in decerebrate cats of either sex. Second, to determine the location of spinal neurons activated during FW and BW locomotion, we used c-Fos immunostaining. We found that the neuronal networks responsible for FW locomotion were distributed broadly in the lumbosacral spinal cord and could be activated by ES of any segment from L3 to S2. By contrast, networks generating BW locomotion were activated by ES of a limited zone from the caudal part of L5 to the caudal part of L7. In the intermediate part of the gray matter within this zone, a significantly higher number of c-Fos-positive interneurons was revealed in BW-stepping cats compared with FW-stepping cats. We suggest that this region of the spinal cord contains the network that determines the BW direction of locomotion.SIGNIFICANCE STATEMENT Sequential and single steps in various directions relative to the body axis [forward (FW), backward (BW), sideways, etc.] are used during locomotion and to correct for perturbations, respectively. The mechanisms controlling step direction are unknown. In the present study, for the first time we compared the distributions of spinal neuronal networks controlling FW and BW locomotion. Using a marker to visualize active neurons, we demonstrated that in the intermediate part of the gray matter within L6 and L7 spinal segments, significantly more neurons were activated during BW locomotion than during FW locomotion. We suggest that the network determining the BW direction of stepping is located in this area.

Biomimetic computer-to-brain communication enhancing naturalistic touch sensations via peripheral nerve stimulation.

Artificial communication with the brain through peripheral nerve stimulation shows promising results in individuals with sensorimotor deficits. However, these efforts lack an intuitive and natural sensory experience. In this study, we design and test a biomimetic neurostimulation framework inspired by nature, capable of "writing" physiologically plausible information back into the peripheral nervous system. Starting from an in-silico model of mechanoreceptors, we develop biomimetic stimulation policies. We then experimentally assess them alongside mechanical touch and common linear neuromodulations. Neural responses resulting from biomimetic neuromodulation are consistently transmitted towards dorsal root ganglion and spinal cord of cats, and their spatio-temporal neural dynamics resemble those naturally induced. We implement these paradigms within the bionic device and test it with patients (ClinicalTrials.gov identifier NCT03350061). He we report that biomimetic neurostimulation improves mobility (primary outcome) and reduces mental effort (secondary outcome) compared to traditional approaches. The outcomes of this neuroscience-driven technology, inspired by the human body, may serve as a model for advancing assistive neurotechnologies.

Neural population dynamics reveals disruption of spinal circuits' responses to proprioceptive input during electrical stimulation of sensory afferents.

While neurostimulation technologies are rapidly approaching clinical applications for sensorimotor disorders, the impact of electrical stimulation on network dynamics is still unknown. Given the high degree of shared processing in neural structures, it is critical to understand if neurostimulation affects functions that are related to, but not targeted by, the intervention. Here, we approach this question by studying the effects of electrical stimulation of cutaneous afferents on unrelated processing of proprioceptive inputs. We recorded intraspinal neural activity in four monkeys while generating proprioceptive inputs from the radial nerve. We then applied continuous stimulation to the radial nerve cutaneous branch and quantified the impact of the stimulation on spinal processing of proprioceptive inputs via neural population dynamics. Proprioceptive pulses consistently produce neural trajectories that are disrupted by concurrent cutaneous stimulation. This disruption propagates to the somatosensory cortex, suggesting that electrical stimulation can perturb natural information processing across the neural axis.

Postnatal growth of the lumbosacral spinal segments in cat: Their lengths and positions in relation to vertebrae.

Cat is a prominent model for investigating neural networks of the lumbosacral spinal cord that control locomotor and visceral activity. We previously proposed an integral function, establishing the topographical relationship between the spinal cord segments and vertebrae in adult animals. Here, we investigated the dynamic of this topographical relationship through early and middle periods of development in kittens. We calculated the length of each vertebra relative to the total length of the region from 13th thoracic (T) to the 7th lumbar (L) vertebrae (V) as well as the length of each segment relative to the total region from T13 to the three-dimensional sacral (S) segment. As in our previous work, the length and position of VL2 were used to establish relationships between the characteristics of the segments and vertebrae. Cubic regression reliably approximates the lengths of segments relative to VL2 length. As the cat aged, the relative length of VT13 and VL1 decreased while the relative length of VL5 increased. The relative length of the T13 and L3 segments increased while the relative length of the S1-S2 segments decreased. The T13-L2 segments are descended monotonically relative to the VL1-VL2 border. The L3-S1 segments are also descended, though with more complex dynamics. The positions of the S2-S3 segments remained unchanged. To conclude, different spinal segments displayed different developmental dynamics. The revealed relationship between vertebrae and lumbosacral spinal segments may be helpful for clearly defining stimulation regions to invoke particular functions, both in experimental studies on the spinal cord and clinical treatment.

Alteration of Postural Reactions in Rats with Different Levels of Dopamine Depletion.

Dopamine (DA) is the critical neurotransmitter involved in the unconscious control of muscle tone and body posture. We evaluated the general motor capacities and muscle responses to postural disturbance in three conditions: normal DA level (wild-type rats, WT), mild DA deficiency (WT after administration of α-methyl-p-tyrosine-AMPT, that blocks DA synthesis), and severe DA depletion (DAT-KO rats after AMPT). The horizontal displacements in WT rats elicited a multi-component EMG corrective response in the flexor and extensor muscles. Similar to the gradual progression of DA-related diseases, we observed different degrees of bradykinesia, rigidity, and postural instability after AMPT. The mild DA deficiency impaired the initiation pattern of corrective responses, specifically delaying the extensor muscles' activity ipsilaterally to displacement direction and earlier extensor activity from the opposite side. DA depletion in DAT-KO rats after AMPT elicited tremors, general stiffness, and akinesia, and caused earlier response to horizontal displacements in the coactivated flexor and extensor muscles bilaterally. The data obtained show the specific role of DA in postural reactions and suggest that this experimental approach can be used to investigate sensorimotor control in different dopamine-deficient states and to model DA-related diseases.

Radical-regulating and antiviral properties of ascorbic acid and its derivatives.

The ability of ascorbic acid and a number of its derivatives to suppress replication of Herpes simplex virus type I was investigated in human rhabdomyosarcoma cell line. In parallel, interaction of the test compounds with carbon- and oxygen-centered radicals formed on radiolysis of hydroxyl-containing organic compounds was studied using the steady state radiolysis method. It has been shown that 2-O-glycoside of ascorbic acid, displaying marked antiviral properties against Herpes simplex virus type I, is also capable of inhibiting fragmentation and recombination reactions of α-hydroxyl-containing carbon-centered radicals while not affecting processes involving oxygen-centered radicals.

Multi-pronged neuromodulation intervention engages the residual motor circuitry to facilitate walking in a rat model of spinal cord injury.

A spinal cord injury usually spares some components of the locomotor circuitry. Deep brain stimulation (DBS) of the midbrain locomotor region and epidural electrical stimulation of the lumbar spinal cord (EES) are being used to tap into this spared circuitry to enable locomotion in humans with spinal cord injury. While appealing, the potential synergy between DBS and EES remains unknown. Here, we report the synergistic facilitation of locomotion when DBS is combined with EES in a rat model of severe contusion spinal cord injury leading to leg paralysis. However, this synergy requires high amplitudes of DBS, which triggers forced locomotion associated with stress responses. To suppress these undesired responses, we link DBS to the intention to walk, decoded from cortical activity using a robust, rapidly calibrated unsupervised learning algorithm. This contingency amplifies the supraspinal descending command while empowering the rats into volitional walking. However, the resulting improvements may not outweigh the complex technological framework necessary to establish viable therapeutic conditions.

Mapping of the Spinal Sensorimotor Network by Transvertebral and Transcutaneous Spinal Cord Stimulation.

Transcutaneous stimulation is a neuromodulation method that is efficiently used for recovery after spinal cord injury and other disorders that are accompanied by motor and sensory deficits. Multiple aspects of transcutaneous stimulation optimization still require testing in animal experiments including the use of pharmacological agents, spinal lesions, cell recording, etc. This need initially motivated us to develop a new approach of transvertebral spinal cord stimulation (SCS) and to test its feasibility in acute and chronic experiments on rats. The aims of the current work were to study the selectivity of muscle activation over the lower thoracic and lumbosacral spinal cord when the stimulating electrode was located intravertebrally and to compare its effectiveness to that of the clinically used transcutaneous stimulation. In decerebrated rats, electromyographic activity was recorded in the muscles of the back (m. longissimus dorsi), tail (m. abductor caudae dorsalis), and hindlimb (mm. iliacus, adductor magnus, vastus lateralis, semitendinosus, tibialis anterior, gastrocnemius medialis, soleus, and flexor hallucis longus) during SCS with an electrode placed alternately in one of the spinous processes of the VT12-VS1 vertebrae. The recruitment curves for motor and sensory components of the evoked potentials (separated from each other by means of double-pulse stimulation) were plotted for each muscle; their slopes characterized the effectiveness of the muscle activation. The electrophysiological mapping demonstrated that transvertebral SCS has specific effects to the rostrocaudally distributed sensorimotor network of the lower thoracic and lumbosacral cord, mainly by stimulation of the roots that carry the sensory and motor spinal pathways. These effects were compared in the same animals when mapping was performed by transcutaneous stimulation, and similar distribution of muscle activity and underlying neuroanatomical mechanisms were found. The experiments on chronic rats validated the feasibility of the proposed stimulation approach of transvertebral SCS for further studies.

Rapid prototyping of soft bioelectronic implants for use as neuromuscular interfaces.

Neuromuscular interfaces are required to translate bioelectronic technologies for application in clinical medicine. Here, by leveraging the robotically controlled ink-jet deposition of low-viscosity conductive inks, extrusion of insulating silicone pastes and in situ activation of electrode surfaces via cold-air plasma, we show that soft biocompatible materials can be rapidly printed for the on-demand prototyping of customized electrode arrays well adjusted to specific anatomical environments, functions and experimental models. We also show, with the monitoring and activation of neuronal pathways in the brain, spinal cord and neuromuscular system of cats, rats and zebrafish, that the printed bioelectronic interfaces allow for long-term integration and functional stability. This technology might enable personalized bioelectronics for neuroprosthetic applications.

Site-Specific Neuromodulation of Detrusor and External Urethral Sphincter by Epidural Spinal Cord Stimulation.

Impairments of the lower urinary tract function including urine storage and voiding are widely spread among patients with spinal cord injuries. The management of such patients includes bladder catheterization, surgical and pharmacological approaches, which reduce the morbidity from urinary tract-related complications. However, to date, there is no effective treatment of neurogenic bladder and restoration of urinary function. In the present study, we examined neuromodulation of detrusor (Detr) and external urethral sphincter by epidural electrical stimulation (EES) of lumbar and sacral regions of the spinal cord in chronic rats. To our knowledge, it is the first chronic study where detrusor and external urethral sphincter signals were recorded simultaneously to monitor their neuromodulation by site-specific spinal cord stimulation (SCS). The data obtained demonstrate that activation of detrusor muscle mainly occurs during the stimulation of the upper lumbar (L1) and lower lumbar (L5-L6) spinal segments whereas external urethral sphincter was activated predominantly by sacral stimulation. These findings can be used for the development of neurorehabilitation strategies based on spinal cord epidural stimulation for autonomic function recovery after severe spinal cord injury (SCI).

Prediction Algorithm of the Cat Spinal Segments Lengths and Positions in Relation to the Vertebrae.

Detailed knowledge of the topographic organization and precise access to the spinal cord segments is crucial for the neurosurgical manipulations as well as in vivo neurophysiological investigations of the spinal networks involved in sensorimotor and visceral functions. Because of high individual variability, accurate identification of particular portion of the lumbosacral enlargement is normally possible only during postmortem dissection. Yet, it is often necessary to determine the precise location of spinal segments prior to in vivo investigation, targeting spinal cord manipulations, neurointerface implantations, and neuronal activity recordings. To solve this problem, we have developed an algorithm to predict spinal segments locations based on their relation to vertebral reference points. The lengths and relative positions of the spinal cord segments (T13-S3) and the vertebrae (VT13-VL7) were measured in 17 adult cats. On the basis of these measurements, we elaborated the estimation procedure: the cubic regression of the ratio of the segment's length to the lengths of the VL2 vertebra was used for the determination of segment's length; and the quadratic regression of the ratio of their positions in relation to the VL2 rostral part was used to determine the position of the segments. The coefficients of these regressions were calculated at the training sample (nine cats) and were then confirmed at the testing sample (eight cats). Although the quality of the prediction is decreased in the caudal direction, we found high correlations between the regressions and real data. The proposed algorithm can be further translated to other species including human. Anat Rec, 302:1628-1637, 2019. © 2018 American Association for Anatomy.

Brain-controlled modulation of spinal circuits improves recovery from spinal cord injury.

The delivery of brain-controlled neuromodulation therapies during motor rehabilitation may augment recovery from neurological disorders. To test this hypothesis, we conceived a brain-controlled neuromodulation therapy that combines the technical and practical features necessary to be deployed daily during gait rehabilitation. Rats received a severe spinal cord contusion that led to leg paralysis. We engineered a proportional brain-spine interface whereby cortical ensemble activity constantly determines the amplitude of spinal cord stimulation protocols promoting leg flexion during swing. After minimal calibration time and without prior training, this neural bypass enables paralyzed rats to walk overground and adjust foot clearance in order to climb a staircase. Compared to continuous spinal cord stimulation, brain-controlled stimulation accelerates and enhances the long-term recovery of locomotion. These results demonstrate the relevance of brain-controlled neuromodulation therapies to augment recovery from motor disorders, establishing important proofs-of-concept that warrant clinical studies.

Configuration of electrical spinal cord stimulation through real-time processing of gait kinematics.

Epidural electrical stimulation (EES) of the spinal cord and real-time processing of gait kinematics are powerful methods for the study of locomotion and the improvement of motor control after injury or in neurological disorders. Here, we describe equipment and surgical procedures that can be used to acquire chronic electromyographic (EMG) recordings from leg muscles and to implant targeted spinal cord stimulation systems that remain stable up to several months after implantation in rats and nonhuman primates. We also detail how to exploit these implants to configure electrical spinal cord stimulation policies that allow control over the degree of extension and flexion of each leg during locomotion. This protocol uses real-time processing of gait kinematics and locomotor performance, and can be configured within a few days. Once configured, stimulation bursts are delivered over specific spinal cord locations with precise timing that reproduces the natural spatiotemporal activation of motoneurons during locomotion. These protocols can also be easily adapted for the safe implantation of systems in the vicinity of the spinal cord and to conduct experiments involving real-time movement feedback and closed-loop controllers.

Spatiotemporal neuromodulation therapies engaging muscle synergies improve motor control after spinal cord injury.

Electrical neuromodulation of lumbar segments improves motor control after spinal cord injury in animal models and humans. However, the physiological principles underlying the effect of this intervention remain poorly understood, which has limited the therapeutic approach to continuous stimulation applied to restricted spinal cord locations. Here we developed stimulation protocols that reproduce the natural dynamics of motoneuron activation during locomotion. For this, we computed the spatiotemporal activation pattern of muscle synergies during locomotion in healthy rats. Computer simulations identified optimal electrode locations to target each synergy through the recruitment of proprioceptive feedback circuits. This framework steered the design of spatially selective spinal implants and real-time control software that modulate extensor and flexor synergies with precise temporal resolution. Spatiotemporal neuromodulation therapies improved gait quality, weight-bearing capacity, endurance and skilled locomotion in several rodent models of spinal cord injury. These new concepts are directly translatable to strategies to improve motor control in humans.

Biomaterials. Electronic dura mater for long-term multimodal neural interfaces.

The mechanical mismatch between soft neural tissues and stiff neural implants hinders the long-term performance of implantable neuroprostheses. Here, we designed and fabricated soft neural implants with the shape and elasticity of dura mater, the protective membrane of the brain and spinal cord. The electronic dura mater, which we call e-dura, embeds interconnects, electrodes, and chemotrodes that sustain millions of mechanical stretch cycles, electrical stimulation pulses, and chemical injections. These integrated modalities enable multiple neuroprosthetic applications. The soft implants extracted cortical states in freely behaving animals for brain-machine interface and delivered electrochemical spinal neuromodulation that restored locomotion after paralyzing spinal cord injury.