Significance of peripheral feedback in stepping movement generation under epideral spinal cord stimulation

In acute experiments on decerebrated and spinalized cats, the role of peripheral afferent input from hindlimbs in stepping patterns formation under epidural spinal cord stimulation (ESCS), was investigated. The hindlimb muscles' electromyographic activity and kinematic parameters of evoked stepping were analyzed. It has been shown that epidural stimulation (20-100 microA, 5 Hz) of L4-L5 spine segments induced coordinated stepping on the treadmill belt. In conditions of weight-bearing support (stopped treadmill, hindlimbs lifted above the treadmill), the stepping rhythmic was unstable, stepping cycle period and its internal structure having changed as well. With increased speed of locomotion the stepping frequency increased due to the duration of the support phase decreasing. Forward stepping could be reversed to backward stepping by changing the direction of the treadmill belt movement. In 2-4 hours after complete spinal transection (T8-T9), the epidural stimulation elicited stepping movements on a moving treadmill only. It was found that the influence of peripheral feedback on initiation of the stepping after spinalization increased. Peripheral feedback seems to play a major role in determining the fundamental features of motor output during the ESCS.     

大鼠脊髓诱发电位与脊髓损伤的关系——Ⅲ.脊髓局部损毁后电位的变化及电位来源分析

本文描述了大鼠脊髓L_1节段后柱、后索、侧索和前角的诱发电位及其损伤后的变化,并观察了切断L_4、L_5脊神经背、腹根与横断高位颈髓对电位的影响,以进行行电位来源分析。结果可见,上述四个区域的诱发电位基本由早反应三相波和晚反应组成。分别电解损毁这些部位后,电位波幅均普遍降低,晚期反应较早反应降低明显。后柱或后索受损对电位影响最大。局部损毁后可见L_1及T_(13)水平的硬膜上电位改变明显,尤其晚反应减弱、波峰平坦。反应时值与潜伏时未见明显改变。切断L_4脊神经背、腹根后、电位基本消失。去大脑对电位未见明显影响。结果表明,刺激坐骨神经诱发的脊髓电位起源于低位腰段传入神经和脊髓内多通路的兴奋传导,在一定程度上受腹根逆行活动的影响,与大脑及脊髓下行传导束活动无直接联系。脊髓诱发电位的幅度与波形改变可作为脊髓损伤的判断指标之一。     

大鼠脊髓损伤后感觉、运动及电生理变化

在应用磁控机械夹断法复制的大鼠脊髓损伤模型上,动态地观察了脊髓损伤后的感觉及运动机能变化,并进行了电生理学研究。结果表明,0.3A电流未能导致永久性瘫痪。术后2周,后肢的感觉及运动功能逐渐恢复;可记录到体感诱发电位(SEP)。0.4,0.5和0.8A电流均能导致大鼠永久性瘫痪;倾斜板及开阔场地行走分数均显著低于0.3A组;术后4周这些大鼠可产生行走样动作,于损伤部位再次切断脊髓后仍能出现这些动作;0.4A组可记录到早期SEP,再次切断脊髓后SEP消失。结果提示:(1)脊髓不全横断后,由于残留纤维活动,可在相当程度上导致大鼠感觉和运动机能的恢复;(2)脊髓完全横断后,后肢的上行冲动可能经再生的神经纤维向中枢端传导至脑;(3)大鼠脊髓内可能存在行走中枢模式发生器(CPG),适当刺激可激发其活动,并产生行走样运动。     

大鼠脊髓诱发电位与脊髓损伤的关系——Ⅱ、皮肤、椎板和硬膜上诱发电位的比较

从大鼠的背侧皮肤表面和椎板分别记录刺激坐骨神经诱发的脊髓电位,并与硬膜上电位进行了比较。结果表明:皮肤表面电位与硬膜上直接记录具有相同的节段性特征。从硬膜上经椎板至皮肤表面、反应潜伏时延长、电位幅度递减。各波峰潜伏时也相应增加。电位的波形、幅度与记录方式有关,但反应潜伏时不受影响。     

The role of skin afferent in regulation of locomotion evoked by electrical epidural stimulation of spinal cord in decerebrated cats

The role of hindpaw skin afferent input in the locomotor pattern formation induced by epidural spinal cord stimulation was investigated in decerebrated cats. Locomotor activity was evoked by continuous 3-5Hz stimulation of dorsal surface of L4-L5 spinal segments. Kinematic and electromyographic activity (EMG) of m. Quadriceps, m. Semitendinosus, m. Tibialis anterior an m. Gastrocnemius lateralis before and after blocking of skin receptors in one hind limb were recorded. In addition, reflex responses in the hind limb muscles to epidural stimulation with frequency 0.5 Hz were analysed. Blocking of skin receptors of the foot with chlorothane paw irrigation or 2 % lidocaine administrated into the hind paw was performed. After blocking of skin receptors of the foot the stepping pattern changed. Stepping with dorsal foot placement and dragging during swing phase was observed. Duration of stance phase significantly decreased. Inhibition of polysynaptic activity of proximal and distal extensor muscles and distal flexor muscles of hind paw during locomotion was found. Monosynaptic responses after blocking of skin receptors of the foot changed insignificantly.     

Extrapyramidal descending influences on neurons of the spinal cord in a superreflexia state

The spinal superreflexia state was modeled in experiments on rats using preliminary transection of the spinal cord and injection (in the course of the acute experiment) of 4-aminopyridine. An extremely high (reaching 15–20 mV) amplitude of monosynaptic reflex discharges (MRs) evoked by stimulation of the dorsal root and recorded from the ventral root (VR) L ₄ and the presence of an additional component in the above discharges were phenomena indicative of the development of the above state. Under such conditions, the amplitudes of the discharges evoked in the VR by electrical stimulation of the round window of the labyrinth (vestibular stimulation) and of the discharges elicited by stimulation of the motor cortex under conditions of bilateral transection of the pyramids increased several times. Thresholds of the VR responses to vestibular and cortical stimulations demonstrated an about threefold drop; latencies of the mass responses and responses of single spinal moto-and interneurons decreased about twofold, on average. The pattern of vestibular conditioning effects on the VR MRs changed: in intact animals vestibular stimulation induced inhibition of the VR MRs, while in animals with superreflexia such stimulation led to facilitation of the MRs. Cortical stimulation under conditions of pyramidotomy in both intact animals and animals with superreflexia resulted in facilitation of the VR MRs of a nearly the same intensity. The levels of convergence of the segmental and supraspinal effects on interneurons and motoneurons of the rat spinal cord dramatically increased under superreflexia conditions. The possible mechanisms of augmentation of the descending influences on spinal neuronal systems under the above conditions are discussed. Neirofiziologiya/Neurophysiology, Vol. 38, No. 2, pp. 140–149, March–April, 2006.     

Spinal and sensory neuromodulation of spinal neuronal networks in humans

The present experiments were designed to gain additionally insight into how the spinal networks process direct spinal stimulation and peripheral sensory inputs to control posture and locomotor movements. We have developed a plantar pressure stimulation system that can deliver naturalistic postural and gait-related patterns of pressure to the soles of the feet to simulate standing and walking, thereby activating and/or modulating the automated spinal circuitry responsible for standing and locomotion. In the present study we compare the patterns of activation among selected motor pools and the kinematic consequences of these activation patterns in response to patterned heel-to-toe mechanical stimulation of the soles of the feet, and/or transcutaneous electrical spinal stimulation, for postural and locomotion regulation. The studies were performed in healthy individuals (n = 12) as well as in subjects (n = 2) with motor complete spinal cord injury. We found that plantar pressure stimulation and/or spinal stimulation can effectively facilitate locomotor output in the subjects placed with their legs in gravity neutral position. We have shown synergistic effects of combining sensory and spinal cord stimulation, suggesting that the two networks are different, but complementary. Also we provide evidence that plantar stimulation could serve as a novel neuro-rehabilitation tool alone or as part of a multi-modal approach to restoring motor function after complete paralysis due to SCI.     

Morphofunctional characteristics of the rat distal spinal cord after its complete experimental transection with subsequent animal treadmill training

Motor activity of rats was studied after experimental complete transection of the spinal cord at lower thoracic level. Treadmill training 1 day after the surgery was shown to lead to the appearance of movements in hindlimbs and restoration of the body weight support function. According to our data, the key moment in initiation of locomotor movements is stimulation of foot. Morphoimmunohistochemical investigation of the lumbar enlargement (study of proliferating cell nuclear protein, synaptophysin, and glial fibrillary acidic protein immunohistochemistry) revealed a rearrangement of motoneurons, interneurons, and the afferent chain in the distal part of the transected spinal cord. In the trained animals, there was observed the normal structure of motoneurons and the appearance of aggregates of the synaptophysin-immunoreactive structures lost after the surgery.     

Fictive locomotion and scratching inhibit dorsal horn neurons receiving thin fiber afferent input

In decerebrate paralyzed cats, we examined the effects of two central motor commands (fictive locomotion and scratching) on the discharge of dorsal horn neurons receiving input from group III and IV tibial nerve afferents. We recorded the impulse activity of 74 dorsal horn neurons, each of which received group III input from the tibial nerve. Electrical stimulation of the mesencephalic locomotor region (MLR), which evoked fictive static contraction or fictive locomotion, inhibited the discharge of 44 of the 64 dorsal horn neurons tested. The mean depth from the dorsal surface of the spinal cord of the 44 neurons whose discharge was inhibited by MLR stimulation was 1.77 +/- 0.04 mm. Fictive scratching, evoked by topical application of bicuculline to the cervical spinal cord and irritation of the ear, inhibited the discharge of 22 of the 29 dorsal horn neurons tested. Fourteen of the twenty-two neurons whose discharge was inhibited by fictive scratching were found to be inhibited by MLR stimulation as well. The mean depth from the dorsal surface of the cord of the 22 neurons whose discharge was inhibited by fictive scratching was 1.77 +/- 0.06 mm. Stimulation of the MLR or the elicitation of fictive scratching had no effect on the activity of 22 dorsal horn neurons receiving input from group III and IV tibial nerve afferents. The mean depth from the dorsal surface of the cord was 1.17 +/- 0.07 mm, a value that was significantly (P < 0.05) less than that for the neurons whose discharge was inhibited by either MLR stimulation or fictive scratching. We conclude that centrally evoked motor commands can inhibit the discharge of dorsal horn neurons receiving thin fiber input from the periphery.     

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16 °C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 °C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.     

Initiation of Bladder Voiding with Epidural Stimulation in Paralyzed,Step Trained Rats

The inability to control timely bladder emptying is one of the most serious challenges among the several functional deficits that occur after a complete spinal cord injury. Having demonstrated that electrodes placed epidurally on the dorsum of the spinal cord can be used in animals and humans to recover postural and locomotor function after complete paralysis, we hypothesized that a similar approach could be used to recover bladder function after paralysis. Also knowing that posture and locomotion can be initiated immediately with a specific frequency-dependent stimulation pattern and that with repeated stimulation-training sessions these functions can improve even further, we reasoned that the same two strategies could be used to regain bladder function. Recent evidence suggests that rats with severe paralysis can be rehabilitated with a multisystem neuroprosthetic training regime that counteracts the development of neurogenic bladder dysfunction. No data regarding the acute effects of locomotion on bladder function, however, were reported. In this study we show that enabling of locomotor-related spinal neuronal circuits by epidural stimulation also influences neural networks controlling bladder function and can play a vital role in recovering bladder function after complete paralysis. We have identified specific spinal cord stimulation parameters that initiate bladder emptying within seconds of the initiation of epidural stimulation. The clinical implications of these results are substantial in that this strategy could have a major impact in improving the quality of life and longevity of patients while simultaneously dramatically reducing ongoing health maintenance after a spinal cord injury.     

伸长细胞移植对大鼠脊髓损伤后运动功能及运动诱发电位的影响

目的:研究伸长细胞是否可以促进成年大鼠脊髓损伤后传导束再生。方法:采用Wistar大鼠脊髓T8全横断模型,移植传代培养的伸长细胞,以未移植脊髓损伤组为对照,观察两组损伤后第12周末BBB评分,损伤平面以下红核-脊髓运动诱发电位,和横断部位组织学染色结果。结果:第12周末伸长细胞移植组红核脊髓运动诱发电位总峰值显著高于对照组(MD=133.2μV,P0.01),峰潜伏期较对照组缩短(MD=0.061ms,P=0.040);第12周末伸长细胞移植组BBB评分显著高于对照组(MD=5.0000,P0.01);第12周末脊髓横断部位HE染色显示伸长细胞移植组脊髓损伤处结构较完整。结论:伸长细胞移植可以促进大鼠脊髓损伤后神经传导的恢复。     

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.     

Intraspinal serotonergic signaling suppresses locomotor activity in larval zebrafish

Serotonin (5HT) is a modulator of many vital processes in the spinal cord (SC), such as production of locomotion. In the larval zebrafish, intraspinal serotonergic neurons (ISNs) are a source of spinal 5HT that, despite the availability of numerous genetic and optical tools, has not yet been directly shown to affect the spinal locomotor network. In order to better understand the functions of ISNs, we used a combination of strategies to investigate ISN development, morphology, and function. ISNs were optically isolated from one another by photoconverting Kaede fluorescent protein in individual cells, permitting morphometric analysis as they developed in vivo. ISN neurite lengths and projection distances exhibited the greatest amount of change between 3 and 4 days post‐fertilization (dpf) and appeared to stabilize by 5 dpf. Overall ISN innervation patterns were similar between cells and between SC regions. ISNs possessed rostrally‐extending neurites resembling dendrites and a caudally‐extending neurite resembling an axon, which terminated with an enlarged growth cone‐like structure. Interestingly, these enlargements remained even after neurite extension had ceased. Functionally, application of exogenous 5HT reduced spinally‐produced motor nerve bursting. A selective 5HT reuptake inhibitor and ISN activation with channelrhodopsin‐2 each produced similar effects to 5HT, indicating that spinally‐intrinsic 5HT originating from the ISNs has an inhibitory effect on the spinal locomotor network. Taken together this suggests that the ISNs are morphologically mature by 5 dpf and supports their involvement in modulating the activity of the spinal locomotor network. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018     

超早期高压氧对脊髓完全横断损伤大鼠血液生化及后肢运动功能的影响

目的探讨超早期高压氧（HBO）治疗对脊髓完全横断损伤模型血液生化及后肢运动功能的影响。方法 55只SD大鼠随机分为A组（假手术组,15只）、B组（模型组,20只）及C组（高压氧组,20只）,A组仅行椎板切除术,其余2组均行T10椎板水平脊髓完全横向切断术。B、C组均予常规护理,C组于术后3 h置于动物舱内开始高压氧治疗,10 d一疗程,共3疗程。分别于建立模型后第1～6周末,用BBB运动功能评分法评价并比较两组大鼠后肢运动功能恢复程度,术后第6周过量麻醉处死大鼠,以40 g/L多聚甲醛行心室-主动脉灌注,取脊髓损伤区标本,光镜观察损伤脊髓的组织病理学改变。检测血钙（Ca）、血磷（P）、血清碱性磷酸酶（ALP）改变情况。结果 B、C两组大鼠术后第1～6周BBB运动功能评分逐渐增高,C组在3～6周末的BBB运动功能评分均明显高于B组。B组、C组血钙、血磷在术后1、3周高于A组,血清碱性磷酸酶（ALP）术后1、3周低于A组;C组血钙、血磷在术后5、6周低于B组。病理组织切片观察C组较B组组织水肿减轻,炎性细胞浸润减轻。结论超早期高压氧治疗能促进脊髓完全横断损伤大鼠后肢运动功能的部分恢复,降低血钙、血磷含量,对脊髓完全横断损伤大鼠具有保护和治疗作用。     

Spinal cord stimulation as a tool for physiological research

The use of spinal cord stimulation for alleviation of disabilities due to motor neuron lesions has provided the opportunity to explore a new approach to measurement of spinal cord physiology. Externalized leads of epidural electrodes provide the possibility of recording evoked spinal cord activity, while both externalized or implanted leads can be used to study cortical evoked responses and twitches induced by spinal cord stimulation. The use of such electrophysiological techniques can be expected to expand greatly the applicability of the technique for alleviating motor disabilities, through a better definition of the degree, nature and extent of the lesion.     

Neural mechanisms generating locomotion studied in mammalian brain stem-spinal cord in vitro

The neural control system for generation of locomotion is an important system for analysis of neural mechanisms underlying complex motor acts. In these studies, a novel experimental model using neonatal rat brain stem and spinal cord in vitro was developed for investigation of the locomotor system in mammals. The in vitro brain stem and spinal cord system was shown to retain functional circuitry for locomotor command generation, motor pattern generation, and sensorimotor integration. This system was exploited to investigate neurochemical mechanisms involved in neurogenesis of locomotion. Evidence was obtained for peptidergic and gamma-amino-butyric acid-mediated mechanisms in brain-stem circuits generating locomotor commands. Cholinergic, dopaminergic, and excitatory amino acid-mediated mechanisms were shown to activate spinal cord circuits for locomotor pattern generation. Endogenous N-methyl-D-aspartic acid receptors in spinal networks were found to play a central role in the generation of locomotion. The chemically induced patterns of motor activity and rhythmic membrane potential oscillations of spinal motoneurons were characteristic of those during locomotion in other mammals in vivo. The in vitro brain stem and spinal cord model provides a versatile and powerful experimental system with potentially broad application for investigation of diverse aspects of the neurobiology of mammalian motor control systems.     

Segmental release of amino acid neurotransmitters from transcranial stimulation

The present study used microdialysis techniques in an intact rabbit model to measure the release of amino acids within the lumbar spinal cord in response to transcranial electrical stimulation. Dialysis samples from the extracellular space were obtained over a stimulation period of 90 minutes and were examined using high pressure liquid chromatography. Neuronal excitation was verified by recerding corticomotor evoked potentials (CMEPs) from the spinal cord. A significant increase in the release of glycine and taurine compared to sham animals was measured after 90 minutes of transcranial stimulation. Glutamate and aspartate release was not significantly elevated. GABA concentrations were consistently low. CMEP components repeatedly showed adequate activation of descending fiber pathways and segmental interneuron pools during dialysis sampling. Since glycine, and to a lesser extent taurine, have been shown to inhibit motor neuron activity and are closely associated with segmental interneuron pools, suprasegmental modulation of motor activity may be, in part, through these inhibitory amino acid neurotransmitters in the rabbit lumbar spinal cord.     

Long-Term Changes in Spinal Cord Evoked Potentials After Compression Spinal Cord Injury in the Rat

SUMMARY 1. After traumatic spinal cord injury (SCI), histological and neurological consequences are developing for several days and even weeks. However, little is known about the dynamics of changes in spinal axonal conductivity. The aim of this study was to record and compare repeated spinal cord evoked potentials (SCEP) after SCI in the rat during a 4 weeks’ interval. These recordings were used: (i) for studying the dynamics of functional changes in spinal axons after SCI, and (ii) to define the value of SCEP as an independent outcome parameter in SCI studies.2. We have used two pairs of chronically implanted epidural electrodes for stimulation/recording. The electrodes were placed below and above the site of injury, respectively. Animals with implanted electrodes underwent spinal cord compression injury induced by epidural balloon inflation at Th8–Th9 level. There were five experimental groups of animals, including one control group (sham-operated, no injury), and four injury groups (different degrees of SCI).3. After SCI, SCEP waveform was either significantly reduced or completely lost. Partial recovery of SCEPs was observed in all groups. The onset and extent of recovery clearly correlated with the severity of injury.There was good correlation between quantitated SCEP variables and the volumes of the compressing balloon. However, sensitivity of electropohysiological parameters was inferior compared to neurological and morphometric outcomes.4. Our study shows for the first time, that the dynamics of axonal recovery depends on the degree of injury. After mild injury, recovery of signal is rapid. However, after severe injury, axonal conductivity can re-appear after as long as 2 weeks postinjury.In conclusion, SCEPs can be used as an independent parameter of outcome after SCI, but in general, the sensitivity of electrophysiological data were worse than standard morphological and neurological evaluations.