V. R. Edgerton

Differential regulation by exercise of BDNF and NT-3 in rat spinal cord and skeletal muscle.

We have investigated the impact of neuromuscular activity on the expression of neurotrophins in the lumbar spinal cord region and innervating skeletal muscle of adult rats. Rats were exercised on a treadmill for 1 day or 5 consecutive days and euthanized at 0, 2 or 6 h after the last bout of exercise. By Day 1, there was no clear evidence of an increase in brain‐derived neurotrophic factor (BDNF) mRNA in the spinal cord or the soleus muscle. By Day 5, there was a significant increase in BDNF mRNA in the spinal cord at 2 h post‐training, and the soleus muscle showed a robust increase between 0 and 6 h post‐training. Immunoassays showed significant increases in BDNF protein in the soleus muscle by training Day 5. Immunohistochemical analyses showed elevated BDNF levels in motoneuron cell bodies and axons in the ventral horn. Neurotrophin‐3 (NT‐3) mRNA was measured to determine whether selected neurotrophins respond with a selective pattern of induction to neuromuscular activity. In the spinal cord, there was a progressive post‐training decrease in NT‐3 mRNA following a single bout of training, while there was a significant increase in NT‐3 mRNA at 2 h post‐training by Day 5. The soleus muscle showed a progressive increase in NT‐3 mRNA by Days 1 and 5 following training. These results show that neuromuscular activity has specific effects on the BDNF and NT‐3 systems, and that repetitive exercise affects the magnitude and stability of these responses.

Endogenous Neurogenesis Replaces Oligodendrocytes and Astrocytes after Primate Spinal Cord Injury

Neurogenesis has been described in various regions of the CNS throughout life. We examined the extent of natural cell division and replacement from 7 weeks to 7 months after cervical spinal cord injury in four adult rhesus monkeys. Bromodeoxyuridine (BrdU) injections revealed an increase of >80-fold in the number of newly divided cells in the primate spinal cord after injury, with an average of 725,000 BrdU-labeled cells identified per monkey in the immediate injury zone. By 7 months after injury, 15% of these new cells expressed mature markers of oligodendrocytes and 12% expressed mature astrocytic markers. Newly born oligodendrocytes were present in zones of injury-induced demyelination and appeared to ensheath or remyelinate host axons. Thus, cell replacement is an extensive natural compensatory response to injury in the primate spinal cord that contributes to neural repair and is a potential target for therapeutic enhancement.

BDNF-exercise interactions in the recovery of symmetrical stepping after a cervical hemisection in rats.

Clinical evidence indicates that motor training facilitates functional recovery after a spinal cord injury (SCI). Brain-derived neurotrophic factor (BDNF) is a powerful synaptic facilitator and likely plays a key role in motor and sensory functions. Spinal cord hemisection decreases the levels of BDNF below the injury site, and exercise can counteract this decrease [Ying Z, Roy RR, Edgerton VR, Gomez-Pinilla F (2005) Exercise restores levels of neurotrophins and synaptic plasticity following spinal cord injury. Exp Neurol 193:411-419]. It is not clear, however, whether the exercise-induced increases in BDNF play a role in mediating the recovery of locomotion after a SCI. We performed a lateral cervical ( approximately C4) hemisection in adult rats. Seven days after hemisection, the BDNF inhibitor trkB IgG was injected into the cervical spinal cord below the lesion ( approximately C5-C6). Half of the rats were exposed to voluntary running wheels for 14 days. Locomotor ability was assessed by determining the symmetry between the contralateral (unaffected) vs. the ipsilateral (affected) forelimb at the most optimum treadmill speed for each rat. Sedentary and exercised rats with BDNF inhibition showed a higher level of asymmetry during the treadmill locomotion test than rats not treated with the BDNF inhibitor. In hemisected rats, exercise normalized the levels of molecules important for synaptic function, such as cyclic AMP response element binding protein (CREB) and synapsin I, in the ipsilateral cervical enlargement, whereas the BDNF blocker lessened these exercise-associated effects. The results indicate that BDNF levels play an important role in shaping the synaptic plasticity and in defining the level of recovery of locomotor performance after a SCI.

BDNF and learning: Evidence that instrumental training promotes learning within the spinal cord by up-regulating BDNF expression.

We have previously shown that the spinal cord is capable of learning a sensorimotor task in the absence of supraspinal input. Given the action of brain-derived neurotrophic factor (BDNF) on hippocampal learning, the current studies examined the role of BDNF in spinal learning. BDNF is a strong synaptic facilitator and, in association with other molecular signals (e.g. cAMP-response element binding protein (CREB), calcium/calmodulin activated protein kinase II (CaMKII) and synapsin I), important for learning. Spinally transected rats given shock to one hind leg when the leg extended beyond a selected threshold exhibited a progressive increase in flexion duration that minimized shock exposure, a simple form of instrumental learning. Instrumental learning resulted in elevated mRNA levels of BDNF, CaMKII, CREB, and synapsin I in the lumbar spinal cord region. The increases in BDNF, CREB, and CaMKII were proportional to the learning performance. Prior work has shown that instrumental training facilitates learning when subjects are tested on the contralateral leg with a higher response criterion. Pretreatment with the BDNF inhibitor TrkB-IgG blocked this facilitatory effect, as did the CaMKII inhibitor AIP. Intrathecal administration of BDNF facilitated learning when subjects were tested with a high response criterion. The findings indicate that instrumental training enables learning and elevates BDNF mRNA levels within the lumbar spinal cord. BDNF is both necessary, and sufficient, to produce the enabling effect.

Local and Remote Growth Factor Effects after Primate Spinal Cord Injury

Primate models of spinal cord injury differ from rodent models in several respects, including the relative size and functional neuroanatomy of spinal projections. Fundamental differences in scale raise the possibility that retrograde injury signals, and treatments applied at the level of the spinal cord that exhibit efficacy in rodents, may fail to influence neurons at the far greater distances of primate systems. Thus, we examined both local and remote neuronal responses to neurotrophic factor-secreting cell grafts placed within sites of right C7 hemisection lesions in the rhesus macaque. Six months after gene delivery of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) into C7 lesion sites, we found both local effects of growth factors on axonal growth, and remote effects of growth factors reflected in significant reductions in axotomy-induced atrophy of large pyramidal neurons within the primary motor cortex. Additional examination in a rodent model suggested that BDNF, rather than NT-3, mediated remote protection of corticospinal neurons in the brain. Thus, injured neural systems retain the ability to respond to growth signals over the extended distances of the primate CNS, promoting local axonal growth and preventing lesion-induced neuronal degeneration at a distance. Remote cortical effects of spinally administered growth factors could “prime” the neuron to respond to experimental therapies that promote axonal plasticity or regeneration.

Gravitational unloading effects on muscle fiber size, phenotype and myonuclear number

The effects of gravitational unloading with or without intact neural activity and/or tension development on myosin heavy chain (MHC) composition, cross-sectional area (CSA), number of myonuclei, and myonuclear domain (cytoplasmic volume per myonucleus ratio) in single fibers of both slow and fast muscles of rat hindlimbs are reviewed briefly. The atrophic response to unloading is generally graded as follows: slow extensors > fast extensors > fast flexors. Reduction of CSA is usually greater in the most predominant fiber type of that muscle. The percentage of fibers expressing fast MHC isoforms increases in unloaded slow but not fast muscles. Myonuclear number per mm of fiber length and myonuclear domain is decreased in the fibers of the unloaded predominantly slow soleus muscle, but not in the predominantly fast plantaris. Decreases in myonuclear number and domain, however, are observed in plantaris fibers when tenotomy, denervation, or both are combined with hindlimb unloading. All of these results are consistent with the view that a major factor for fiber atrophy is an inhibition or reduction of loading of the hindlimbs. These data also indicate that predominantly slow muscles are more responsive to unloading than predominantly fast muscles.

Plasticity of functional connectivity in the adult spinal cord

This paper emphasizes several characteristics of the neural control of locomotion that provide opportunities for developing strategies to maximize the recovery of postural and locomotor functions after a spinal cord injury (SCI). The major points of this paper are: (i) the circuitry that controls standing and stepping is extremely malleable and reflects a continuously varying combination of neurons that are activated when executing stereotypical movements; (ii) the connectivity between neurons is more accurately perceived as a functional rather than as an anatomical phenomenon; (iii) the functional connectivity that controls standing and stepping reflects the physiological state of a given assembly of synapses, where the probability of these synaptic events is not deterministic; (iv) rather, this probability can be modulated by other factors such as pharmacological agents, epidural stimulation and/or motor training; (v) the variability observed in the kinematics of consecutive steps reflects a fundamental feature of the neural control system and (vi) machine-learning theories elucidate the need to accommodate variability in developing strategies designed to enhance motor performance by motor training using robotic devices after an SCI.

Epidural stimulation of the spinal cord in spinal cord injury: current status and future challenges

A spinal cord injury can occur at any point along its length and can have a wide range of severities and varying effects on sensory and motor function, depending largely on these two factors. While considerable function can be recovered if the injury is ‘incomplete’ – that is, there remain some functional connections between the brain and the spinal cord segments below the lesion – there has been little success in improving function after a motor and/or sensory complete lesion. Recently reported functional gains in an individual with complete motor paralysis was possible because most of the cellular structure below the lesion remains intact [1]. It is this network of neurons that can actually control very complicated movements when this spinal network (not the brain itself) receives the appropriate sensory input, and when the excitability of this network is enhanced with a very specific pattern of modest levels of stimulation with epidurally placed electrodes. We refer to this intervention as electro-enabling motor control. Based on extensive animal experimentation of this spinal network, the possibility of regaining locomotor function after complete paralysis in human subjects becomes an important issue. To begin to examine this issue, we performed similar experiments in a single subject with complete motor paralysis. The issues are: is the human spinal cord circuitry without any input from the brain as smart as that of the rat or cat, can it be neuromodulated to stand and to step using epidural stimulation as demonstrated successfully in the rat and cat, and can proprioception serve as a source of control of the spinal circuitry? We made a series of observations demonstrating the feasibility of using epidural stimulation to facilitate the recovery of a series of consciously controlled motor functions, as well as other functions, some of which are largely considered to be autonomically controlled. After implanting an electrode array, recovery of several functions occurred following repeated sessions of epidural stimulation and training to stand and to step, and to exert voluntary control of the lower limbs. The subject is a 23-year-old man with paraplegia from a C7–T1 subluxation as a result of a motor vehicle accident who has had a complete loss of clinically detectable voluntary motor function and partial preservation of sensation below the T1 cord segment. After 170 locomotor training sessions over 26 months, a 16-electrode array was surgically placed on the dura (L1–S1 cord segments). The results from this single subject provide a wakeup call for a change in the perception of the potential for recovery of function using activity-dependent interventions [2]. They also highlight the potential advantage of quantitative assessment of multiple parameters in fewer individuals in lieu of the commonly accepted ‘primary outcome’ measure, often with limited objectivity, in many subjects.

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.

Locomotor training maintains normal inhibitory influence on both alpha- and gamma-motoneurons after neonatal spinal cord transection.

Spinal cord injuries lead to impairments, which are accompanied by extensive reorganization of neuronal circuits caudal to the injury. Locomotor training can aid in the functional recovery after injury, but the neuronal mechanisms associated with such plasticity are only sparsely known. We investigated ultrastructurally the synaptic inputs to tibialis anterior motoneurons (MNs) retrogradely labeled in adult rats that had received a complete midthoracic spinal cord transection at postnatal day 5. A subset of the injured rats received locomotor training. Both γ- and α-MNs were studied. The total number of boutons apposing γ-MNs, but not α-MNs, was reduced after neonatal spinal cord transection. The proportion of inhibitory to excitatory boutons, however, was increased significantly in both α-MNs and γ-MNs in spinally transected rats, but with locomotor training returned to levels observed in intact rats. The specific densities and compositions of synaptic boutons were, however, different between all three groups. Surprisingly, we observed the atypical presence of both C- and M-type boutons apposing the somata of γ-MNs in the spinal rats, regardless of training status. We conclude that a neonatal spinal cord transection induces significant reorganization of synaptic inputs to spinal motoneurons caudal to the site of injury with a net increase in inhibitory influence, which is associated with poor stepping. Spinal cord injury followed by successful locomotor training, however, results in improved bipedal stepping and further synaptic changes with the proportion of inhibitory and excitatory inputs to the motoneurons being similar to that observed in intact rats.