A Brain-Machine-Muscle Interface for Restoring Hindlimb Locomotion after Complete Spinal Transection in Rats

A brain-machine interface (BMI) is a neuroprosthetic device that can restore motor function of individuals with paralysis. Although the feasibility of BMI control of upper-limb neuroprostheses has been demonstrated, a BMI for the restoration of lower-limb motor functions has not yet been developed. The objective of this study was to determine if gait-related information can be captured from neural activity recorded from the primary motor cortex of rats, and if this neural information can be used to stimulate paralysed hindlimb muscles after complete spinal cord transection. Neural activity was recorded from the hindlimb area of the primary motor cortex of six female Sprague Dawley rats during treadmill locomotion before and after mid-thoracic transection. Before spinal transection there was a strong association between neural activity and the step cycle. This association decreased after spinal transection. However, the locomotive state (standing vs. walking) could still be successfully decoded from neural recordings made after spinal transection. A novel BMI device was developed that processed this neural information in real-time and used it to control electrical stimulation of paralysed hindlimb muscles. This system was able to elicit hindlimb muscle contractions that mimicked forelimb stepping. We propose this lower-limb BMI as a future neuroprosthesis for human paraplegics.     

Locomotion induced by epidural stimulation in a decerebrated cat after spinal cord injury

The effect of partial and complete spinal cord transection (Th7–Th8) on locomotor activity evoked in decerebrated cats by electrical epidural stimulation (segment L5, 80–100 μA, 0.5 ms at 5 Hz) has been investigated. Transection of dorsal columns did not substantially influence the locomotion. Disruption of the ventral spinal quadrant resulted in deterioration and instability of the locomotor rhythm. Injury to lateral or medial descending motor systems led to redistribution of the tone in antagonist muscles. Locomotion could be evoked by epidural stimulation within 20 h after complete transection of the spinal cord. The restoration of polysynaptic components in EMG responses correlated with recovery of the stepping function. The data obtained confirm that initiation of locomotion under epidural stimulation is caused by direct action on intraspinal systems responsible for locomotor regulation. With intact or partially injured spinal cord, this effect is under the influence of supraspinal motor systems correcting and stabilizing the evoked locomotor pattern.     

Motor mechanisms in the turtle spinal cord

We reveal the intrinsic motor capacity of the spinal cord by examining motor behaviours produced by spinal segments caudal to a complete transection of the spinal cord. The turtle spinal cord generates three forms of the scratch reflex in the absence of neural inputs from supraspinal structures. Each form exhibits a characteristic motor neuron discharge pattern. We test the ability of the spinal cord to generate organized motor patterns in the absence of movement-related sensory feedback by examining motor neuron discharge patterns in spinal preparations that are immobilized with a neuromuscular blocking agent. The motor neuron discharge pattern associate with each form is observed in the spinal immobilized preparation. Each of these motor patterns is therefore generated centrally within the spinal cord.     

Effect of diaphragm small-fiber afferent stimulation on ventilation in dogs

Little is known regarding the role of diaphragm small-fiber afferents (groups III and IV) in the control of breathing. This study was designed to determine whether activation of these afferents with use of capsaicin affects phrenic efferent activity. Capsaicin injections into the phrenic artery were made in 10 alpha-chloralose-anesthetized dogs after each of the following procedures performed in succession: bilateral cervical vagotomy, C7 spinal cord transection, bilateral cervical dorsal rhizotomy. In six of these animals injections were also made after C2 spinal cord transection and removal of the cervical spinal cord. Injections made in the vagotomized animals were associated with apneusis followed by hyperpnea. C7 spinal transection eliminated the hyperpneic response, but the apneusis remained. Cervical dorsal rhizotomy or C2 spinal cord transection failed to abolish the apneusis in response to injection. No diaphragm response was obtained after removal of the cervical spinal cord. Experiments in three additional animals showed that capsaicin does not have a direct excitatory effect on the muscle cells of the crural diaphragm, nor does it potentiate the release of neurotransmitter in the diaphragm. The results of this study indicate that small-fiber afferents in the diaphragm have an excitatory effect on phrenic motoneurons. There is a segmental component to this reflex, since the response is observed after C2 spinal cord transection. The data also suggest that at least some of these afferents enter the spinal cord through the ventral roots.     

Supraspinal influence on the development of motor behavior in the fetal lamb

To examine the involvement of supraspinal inputs in the maturation of motor activity patterns in the developing fetal lamb, we recorded spontaneous electromyographic activity from spinally innervated muscles at approximately 45, 65, and 95 days gestation (G45, G65, and G95; term = 147 days). At G45, fetal activity occurred in synchronized activity-inactivity cycles of approximately 2 min duration, with the activity phase lasting 22.2 ± 4.8 s and the inactivity phase lasting 95.4 ± 13.3 s (mean ± standard error of the mean, n = 5). At G65 and G95, the organization of activity was clearly different from that at G45 in that it was no longer cyclic, nor was the discharge of different muscles synchronized. By contrast, after spinal cord transection at G62, synchronised cyclic activity occurred in muscles innervated by segmental levels below the transection, both at G65 and G95. At G65 the duration of the activity phase of the cycle was 53.5 ± 6.0 s, while the inactivity phase lasted 171.6 ± 22.1 s; these durations did not alter between G65 and G95. Since spinal cord transection leads to the motor behavior of the G65 fetus reverting to the cyclic pattern characteristic of the G45 fetus, we conclude that supraspinal inputs begin to modulate the output of the spinal pattern generators at some stage between G45 and G65. The observation that spinally transected fetuses generate identical behavior at G65 and G95, both in terms of its cyclic character and the duration of cycles, suggests that spinal circuits undergo little autonomous development overthis period; that is, the altered behavior observed in the developing intact fetus reflects the influence of supra-spinal inputs on the motor circuits of the spinal cord. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 276–288, 1997     

Zebrafish Spinal Cord Repair Is Accompanied by Transient Tissue Stiffening

Severe injury to the mammalian spinal cord results in permanent loss of function due to the formation of a glial-fibrotic scar. Both the chemical composition and the mechanical properties of the scar tissue have been implicated to inhibit neuronal regrowth and functional recovery. By contrast, adult zebrafish are able to repair spinal cord tissue and restore motor function after complete spinal cord transection owing to a complex cellular response that includes axon regrowth and is accompanied by neurogenesis. The mechanical mechanisms contributing to successful spinal cord repair in adult zebrafish are, however, currently unknown. Here, we employ atomic force microscopy-enabled nanoindentation to determine the spatial distributions of apparent elastic moduli of living spinal cord tissue sections obtained from uninjured zebrafish and at distinct time points after complete spinal cord transection. In uninjured specimens, spinal gray matter regions were stiffer than white matter regions. During regeneration after transection, the spinal cord tissues displayed a significant increase of the respective apparent elastic moduli that transiently obliterated the mechanical difference between the two types of matter before returning to baseline values after the completion of repair. Tissue stiffness correlated variably with cell number density, oligodendrocyte interconnectivity, axonal orientation, and vascularization. This work constitutes the first quantitative mapping of the spatiotemporal changes of spinal cord tissue stiffness in regenerating adult zebrafish and provides the tissue mechanical basis for future studies into the role of mechanosensing in spinal cord repair.     

Effect of high level section of the spinal cord on the cyclic AMP system in rat liver

The effect of high level section of the spinal cord upon the hepatic cyclic AMP system was investigated in the rat. We report that transection of the spinal cord dramatically decreases the basal level of cyclic AMP from 0.88 nmol/g liver to 0.36 nmol/g at 1 h and to 0.20 nmol/g at 4 h. This was not due to increased activity of cyclic AMP phosphodiesterase or to decreased activity of basal adenylate cyclase. The sensitivity of adenylate cyclase to its usual effectors in vitro was not impaired. It is proposed that the lowering of liver cyclic AMP below its basal level after high level section of the spinal cord is due to decreased levels of hepatic catecholamines and/or plasma glucagon.     

Effect of high level section of the spinal cord on the cyclic AMP system in rat liver.

The effect of high level section of the spinal cord upon the hepatic cyclic AMP system was investigated in the rat. We report that transection of the spinal cord dramatically decreases the basal level of cyclic AMP from 0.88 nmol/g liver to 0.36 nmol/g at 1 h and to 0.20 nmol/g at 4 h. This was not due to increased activity of cyclic AMP phosphodiesterase or to decreased activity of basal adenylate cyclase. The sensitivity of adenylate cyclase to its usual effectors in vitro was not impaired. It is proposed that the lowering of liver cyclic AMP below its basal level after high level section of the spinal cord is due to decreased levels of hepatic catecholamines and/or plasma glucagon.     

Effect of axotomy on cholinergic enzyme activities in the spinal cord and sciatic nerve of the dog

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity measured in the ventral and dorsal part of the dog spinal cord (L6-S2) and in the stumps of the sciatic nerve 5, 10, 15 and 21 days after its transection were compared with the corresponding activities in the intact contralateral nerve and in sham-operated animals. AChE was also examined histochemically. Changes in the enzyme activities in the central nerve stump were correlated with activity changes in the spinal cord. In the central nerve stump, a marked (25%) increase in AChE activity was found on the fifth day after transection, but by the 21st day it fell below control value levels; up to the 15th day it showed good correlation with AChE activity in the ventral spinal cord. Histochemically, pronounced reduction of enzymatic activity was found in the ipsilateral part of the spinal cord. On the 15th day, ChAT activity in the ventral spinal cord was also significantly decreased and the accumulation of the enzyme in the central nerve stump was negligible. On the contrary, at the last 21-day interval examined, a significant increase in ChAT activity and a nonsignificant increase in AChE activity was found in the spinal cord, but their activities in the central nerve stump were decreased. In the degenerated peripheral nerve stump ChAT activity dropped by an average of 99% and AChE activity by 48% during the first 15 days after transection but, on the 21st day, AChE activity was 22% higher than at the preceding interval.     

Expression of neural cell adhesion molecule in spinal cords following a complete transection

Neural cell adhesion molecule (NCAM) regulates tissue organization during development and in the adult. NCAM upregulation occurs after an injury to brains and sciatic nerves. However, little is known about NCAM expression after spinal cord injury (SCI). By using a complete spinal cord transection with a 5 mm tissue removal, an increase in the NCAM level is detected in spinal cord stumps proximal and distal to the transection site at 1 d and 3 d post injury, while its expression at 8 d is declined to a lower level than that observed in sham-operated spinal cords. The strong NCAM expression is present in motor neurons at 3 d post transection whereas the intensive NCAM immunostaining is localized in dorsal sensory and corticospinal fiber tracts at 8 d following injury. Collectively, NCAM level is elevated and strongly expressed in dorsal fiber tracts after SCI, implying that the endogenous process for spinal cord regeneration may take place after SCI.     

Neurotrophin expressions in neural stem cells grafted acutely to transected spinal cord of adult rats linked to functional improvement

It is well known that neural stem cells (NSC) could promote the repairment after spinal cord injury, but the underlying mechanism remains to be elucidated. This study showed that the transplantation of NSC significantly improved hindlimb locomotor functions in adult rats subjected to transection of the spinal cord. Biotin dextran amine tracing together with the stimulus experiment in motor sensory area showed that little CST regeneration existed and functional synaptic formation in the injury site. Immunocytochemistry and RT-PCR demonstrated the secretion of NGF, BDNF, and NT-3 by NSC in vitro and in vivo, respectively. However, only mRNA expression of BDNF and NT-3 but not NGF in injury segment following NSC transplantation was upregulated remarkably, while caspase-3, a crucial apoptosis gene, was downregulated simultaneously. These provided us a clue that the functional recovery was correlated with the regulation of BDNF, NT-3, and caspase-3 in spinal cord transected rats following NSC transplantation.     

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.     

Locomotion induced by spinal cord stimulation in the neonate rat in vitro.

The present studies employed the neonate rat brain stem-spinal cord preparation to determine whether electrical stimulation of the lumbosacral enlargement (LE) of the spinal cord itself can be used to elicit locomotion, and whether or not such stimulation persists in inducing locomotion following midthoracic spinal cord transection or hindlimb deafferentation. Results suggest that (1) stimulation of the dorsal columns or ventral funiculus of the LE is effective in inducing airstepping in the neonatal rat brain stem-spinal cord limb-attached preparation; (2) central disconnection by midthoracic spinal cord transection does not alter LE-stimulation-induced airstepping and may lead to an increase in stepping frequency if suprathreshold stimulation is used; and (3) dorsal root section also leads to an increase in the frequency of suprathreshold LE-stimulation-induced locomotion, but there is not further increase in frequency if a spinal cord transection is performed in addition to dorsal rhizotomy.     

Regeneration of monoamine-containing axons in the developing and adult spinal cord of the rat following intraspinal 6-OH-dopamine injections or transections

Summary Growth of descending noradrenaline (NA) and 5-hydroxytryptamine (5-HT) axons in the rat spinal cord during ontogenesis and following mechanical or chemical, 6-hydroxydopamine (6-OH-DA) induced, axotomy, was studied with the Falck-Hillarp histochemical fluorescence method for monoamines.The major NA and 5-HT axon bundles and terminal innervation areas are present already at birth and an essentially mature pattern of innervation is reached after two weeks.Complete degeneration of both 5-HT and NA nerves in the distal segment is obtained by a transection of the spinal cord. Sprouting of the cut monoamine fibers into the necrotic zone and scar tissue is vigorous in both immature and mature animals, but regeneration into the distal segment is very poor.Selective degeneration of the descending NA axons and terminals is obtained by a localized intraspinal 6-OH-DA injection. Thus, the 5-HT fiber systems as well as all other parts of the spinal cord are left intact. The method should therefore prove useful for evaluating the exact functional role of the NA and 5-HT neuron systems in the spinal cord.Reinnervation of the distal part of the spinal cord by new NA fibers following 6-OH-DA induced denervation is described. This process is faster in younger animals but takes place also in adult animals. The present evidence suggests that reinnervation mainly is the result of downgrowth of the axotomized fibers, but growth in the form of collateral sprouting from a few possibly surviving fibers in the distal region may also contribute. Reinnervation lead to a normal innervation pattern within 1–2 months in the various age groups.It is suggested that the poor regeneration of many spinal nerve tracts often reported in the literature following transection of the spinal cord is due to extraneuronal factors such as scar tissue and impaired circulation rather than to the nerves per se since reinnervation by NA nerves was very poor following mechanical transection but good following chemical, 6-OH-DA-induced axotomy.     

Optimal Location and Time for Neural Stem Cell Transplantation into Transected Rat Spinal Cord

Implanted neural stem cells (NSC) could improve neurological functions following spinal cord injury (SCI), but the optimal conditions for NSC transplantation are largely unknown, especially in transected spinal cord. This study investigated the effect and fate of NSC engrafted into spinal cords at different locations and time points following T₉ spinal cord transection. Engrafted NSC could survive and migrate in host spinal cords. Significant improvement in hindlimb locomotor functions associated with NSC survival was found in rats receiving NSC transplantation in the spinal cords rostral to the transection site at the subacute stage (7 days post operation), compared with those caudal to the transection site at the acute stage (at the time of injury). At 4 weeks post operation, CD68 immunohistochemical staining confirmed that macrophages were less in rostrally transplanted sites and in subacute groups than seen in caudal and acute transplanted rats. The present findings indicated that NSC transplantation into spinal cords rostral to transection site at the subacute stage is an optimal strategy for engrafted NSC survival and host behavioral improvement. It therefore would be available to the usage of NSC for the treatment of SCI in the future clinic trial.     

The colour change of the minnow, Phoxinus phoxinus with particular reference to the effect of spinal lesions

An account is given of the anatomy of the spinal cord of the minnow, Phoxinus phoxinus L., and comparisons are made with the spinal cords of other teleosts. The effects on the rapid, neurally controlled colour responses of complete transection of the spinal cord at different levels indicated that, in the particular fish used for this work, the sympathetic pigmento-motor fibres had a localized outflow from the spinal cord around vertebra 13. Partial lesions of the spinal cord at vertebrae 3, 4, 10 and 11 and their effects on the colour responses indicated that, at these spinal levels, the pigmento-motor fibres were dorsomedially located within the dorsal horns. Complete and partial transection of the spinal cord at any single level between vertebrae 12 and 14 and partial transection of the dorsomedial spinal tissues at vertebrae 3, 4, 10 and 11 resulted in differential paling of the entire dorsolateral skin of the fish in response to a change of background from black to white. It would appear that in both cases this differential response of the melanophores was the result of transecting a proportion of the spinal pigmento-motor fibres.     

Regulation of cholinergic expression in cultured spinal cord neurons

Factors regulating development of cholinergic spinal neurons were examined in cultures of dissociated embryonic rat spinal cord. Levels of choline acetyltransferase (CAT) activity in freshly dissociated cells decreased rapidly, remained low for the first week in culture, and then increased. The decrease in enzyme activity was partially prevented by increased cell density or by treatment with spinal cord membranes. CAT activity was also stimulated by treatment with MANS, a molecule solubilized from spinal cord membranes. The effects of MANS were greatest in low-density cultures and in freshly plated cells, suggesting that the molecule may substitute for the effects of elevated density and cell-cell contact. CAT activity in ventral (motor neuron-enriched) spinal cord cultures was similarly regulated by elevated density or treatment with MANS, whereas enzyme activity was largely unchanged in mediodorsal (autonomic neuron-enriched) cultures under these conditions. These observations suggest that development of cholinergic motor neurons and autonomic neurons are not regulated by the same factors. Treatment of ventral spinal cord cultures with MANS did not increase the number of cholinergic neurons detected by immunocytochemistry with a monoclonal CAT antibody, suggesting that MANS did not increase motor neuron survival but rather stimulated levels of CAT activity per neuron. These observations indicate that development of motor neurons can be regulated by cell-cell contact and that the MANS factor may mediate the stimulatory effects of cell-cell contact on cholinergic expression.     

Presumptive relationships between ventricular proliferaion and development of the lateral motor columns in the spinal cord of Rana pipiens larvae.

The extent of mitotic activity in the proliferative ventricular zone of the developing frog (Rana pipiens) spinal cord is a function of both the longitudinal cord level and the developmental stage. Counts of mitotic cells in the ventricular zone demonstrated higher levels of proliferation in the dorsal than ventral halves of the spinal cord with decreasing total proliferative activity from the early to late larval (tadpole) stages. Mitoses were virtually absent from the ventricular zone by the conclusion of metamorphosis. Changes in mitotic counts at different levels of the spinal cord can be correlated with the presence or absence of the brachial or lumbosacral pairs of lateral motor columns. A parallel also exists between the caudo-cephalic direction of motor column development and a similar progression of mitotic activity in the ventricular zone, a portion of which gives rise to the spinal motor neurons. It is suggested that proliferation in the ventricular zone during the larval frog stages contributes to the presumptive motor neuron population and migrates into the lateral motor columns during their later maturation.     

Impairment of the mitochondrial respiratory enzyme activity triggers sequential activation of apoptosis-inducing factor-dependent and caspase-dependent signaling pathways to induce apoptosis after spinal cord injury

The mitochondrion participates in caspase-independent or caspase-dependent apoptotic pathways through the release of apoptosis-inducing factor or cytochrome c. Whether both mitochondrial apoptotic cascades are triggered in the injured spinal cord remains unknown. Here, we demonstrated that neurons, astrocytes and microglia in spinal segments proximal to a complete spinal cord transection underwent two phases of apoptotic cell death. The early phase of high-molecular weight (HMW) DNA fragmentation was associated with nuclear translocation of apoptosis-inducing factor, reduction in mitochondrial respiratory chain enzyme activity and decrease in cellular ATP concentration. The delayed phase of low-molecular weight (LMW) DNA fragmentation was accompanied by cytosolic release of cytochrome c , activation of caspases 9 and 3, and resumption of mitochondrial respiratory functions and ATP contents. Microinfusion of coenzyme Q₁₀, an electron carrier in mitochondrial respiratory chain, into the epicenter of the transected spinal cord attenuated both phases of induced apoptosis, and reversed the elicited mitochondrial dysfunction, bioenergetic failure, and activation of apoptosis-inducing factor, cytochrome c , or caspases 9 and 3. We conclude that mitochondrial dysfunction after spinal cord transection represents the initiating cellular events that trigger the sequential activation of apoptosis-inducing factor-dependent and caspase-dependent signaling cascades, leading to apoptotic cell death in the injured spinal cord.     

Experiments on the central pattern generator for swimming in amphibian embryos

The central nervous system of paralysed Xenopus laevis embryos can generate a motor output pattern suitable for swimming locomotion. By recording motor root activity in paralysed embryos with transected nervous systems we have shown that: (a) the spinal cord is capable of swimming pattern generation; (b) swimming pattern generator capability in the hindbrain and spinal cord is distributed; (c) caudal hindbrain is necessary for sustained swimming output after discrete stimulation. By recording similarly from embryos whose central nervous system was divided longitudinally into left and right sides, we have shown that: (a) each side can generate rhythmic motor output with cycle periods like those in swimming; (b) during this activity cycle period increases within an episode, and there is the usual rostrocaudal delay found in swimming; (c) this activity is influenced by sensory stimuli in the same way as swimming activity; (d) normal phase coupling of the left and right sides can be established by the ventral commissure in the spinal cord. We conclude that interactions between the antagonistic (left and right) motor systems are not necessary for swimming rhythm generation and present a model for swimming pattern generation where autonomous rhythm generators on each side of the nervous system drive the motoneurons. Alternation is achieved by reciprocal inhibition, and activity is initiated and maintained by tonic excitation from the hindbrain.