Marie-Pascale Côté

Activity-dependent increase in neurotrophic factors is associated with an enhanced modulation of spinal reflexes after spinal cord injury.

Activity-based therapies such as passive bicycling and step-training on a treadmill contribute to motor recovery after spinal cord injury (SCI), leading to a greater number of steps performed, improved gait kinematics, recovery of phase-dependent modulation of spinal reflexes, and prevention of decrease in muscle mass. Both tasks consist of alternating movements that rhythmically stretch and shorten hindlimb muscles. However, the paralyzed hindlimbs are passively moved by a motorized apparatus during bike-training, whereas locomotor movements during step-training are generated by spinal networks triggered by afferent feedback. Our objective was to compare the task-dependent effect of bike- and step-training after SCI on physiological measures of spinal cord plasticity in relation to changes in levels of neurotrophic factors. Thirty adult female Sprague-Dawley rats underwent complete spinal transection at a low thoracic level (T12). The rats were assigned to one of three groups: bike-training, step-training, or no training. The exercise regimen consisted of 15 min/d, 5 days/week, for 4 weeks, beginning 5 days after SCI. During a terminal experiment, H-reflexes were recorded from interosseus foot muscles following stimulation of the tibial nerve at 0.3, 5, or 10 Hz. The animals were sacrificed and the spinal cords were harvested for Western blot analysis of the expression of neurotrophic factors in the lumbar spinal cord. We provide evidence that bike- and step-training significantly increase the levels of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4 in the lumbar enlargement of SCI rats, whereas only step-training increased glial cell-derived neurotrophic factor (GDNF) levels. An increase in neurotrophic factor protein levels that positively correlated with the recovery of H-reflex frequency-dependent depression suggests a role for neurotrophic factors in reflex normalization.

Step training-dependent plasticity in spinal cutaneous pathways.

Plasticity after spinal cord injury can be initiated by specific patterns of sensory feedback, leading to a reorganization of spinal networks. For example, proprioceptive feedback from limb loading during the stance phase is crucial for the recovery of stepping in spinal-injured animals and humans. Our recent results showed that step training modified transmission from group I afferents of extensors in spinal cats. However, cutaneous afferents are also activated during locomotion and are necessary for proper foot placement in spinal cats. We therefore hypothesized that step training would also modify transmission in cutaneous pathways to facilitate recovery of stepping. We tested transmission in cutaneous pathways by comparing intracellular responses in lumbar motoneurons (n = 136) in trained (n = 11) and untrained (n = 7) cats spinalized 3-5 weeks before the acute electrophysiological experiment. Three cutaneous nerves were stimulated, and each evoked up to three motoneuronal responses mediated by at least three different pathways. Overall, of 71 cutaneous pathways tested, 10 were modified by step training: transmission was reduced in 7 and facilitated in 3. Remarkably, 6 of 10 involved the medial plantar nerve innervating the plantar surface of the foot, including two of the facilitated pathways. Because the cutaneous reflexes are exaggerated after spinalization, we interpret the decrease in most pathways as a normalization of cutaneous transmission necessary to recover locomotor movements. Overall, the results showed a high degree of specificity in plasticity among cutaneous pathways and indicate that transmission of skin inputs signaling ground contact, in particular, is modified by step training.

Axon regeneration and exercise-dependent plasticity after spinal cord injury

Current dogma states that meaningful recovery of function after spinal cord injury (SCI) will likely require a combination of therapeutic interventions comprised of regenerative/neuroprotective transplants, addition of neurotrophic factors, elimination of inhibitory molecules, functional sensorimotor training, and/or stimulation of paralyzed muscles or spinal circuits. We routinely use (1) peripheral nerve grafts to support and direct axonal regeneration across an incomplete cervical or complete thoracic transection injury, (2) matrix modulation with chondroitinase (ChABC) to facilitate axonal extension beyond the distal graft–spinal cord interface, and (3) exercise, such as forced wheel walking, bicycling, or step training on a treadmill. We and others have demonstrated an increase in spinal cord levels of endogenous neurotrophic factors with exercise, which may be useful in facilitating elongation and/or synaptic activity of regenerating axons and plasticity of spinal neurons below the level of injury.

Peripheral Nerve Grafts Support Regeneration after Spinal Cord Injury

SummaryTraumatic insults to the spinal cord induce both immediate mechanical damage and subsequent tissue degeneration leading to a substantial physiological, biochemical, and functional reorganization of the spinal cord. Various spinal cord injury (SCI) models have shown the adaptive potential of the spinal cord and its limitations in the case of total or partial absence of supraspinal influence. Meaningful recovery of function after SCI will most likely result from a combination of therapeutic strategies, including neural tissue transplants, exogenous neurotrophic factors, elimination of inhibitory molecules, functional sensorimotor training, and/or electrical stimulation of paralyzed muscles or spinal circuits. Peripheral nerve grafts provide a growth-permissive substratum and local neurotrophic factors to enhance the regenerative effort of axotomized neurons when grafted into the site of injury. Regenerating axons can be directed via the peripheral nerve graft toward an appropriate target, but they fail to extend beyond the distal graft–host interface because of the deposition of growth inhibitors at the site of SCI. One method to facilitate the emergence of axons from a graft into the spinal cord is to digest the chondroitin sulfate proteoglycans that are associated with a glial scar. Importantly, regenerating axons that do exit the graft are capable of forming functional synaptic contacts. These results have been demonstrated in acute injury models in rats and cats and after a chronic injury in rats and have important implications for our continuing efforts to promote structural and functional repair after SCI.

Exercise Modulates Chloride Homeostasis after Spinal Cord Injury

Activity-based therapies are routinely integrated in spinal cord injury (SCI) rehabilitation programs because they result in a reduction of hyperreflexia and spasticity. However, the mechanisms by which exercise regulates activity in spinal pathways to reduce spasticity and improve functional recovery are poorly understood. Persisting alterations in the action of GABA on postsynaptic targets is a signature of CNS injuries, including SCI. The action of GABA depends on the intracellular chloride concentration, which is determined largely by the expression of two cation-chloride cotransporters (CCCs), KCC2 and NKCC1, which serve as chloride exporters and importers, respectively. We hypothesized that the reduction in hyperreflexia with exercise after SCI relies on a return to chloride homeostasis. Sprague Dawley rats received a spinal cord transection at T12 and were assigned to SCI-7d, SCI-14d, SCI-14d+exercise, SCI-28d, SCI-28d+exercise, or SCI-56d groups. During a terminal experiment, H-reflexes were recorded from interosseus muscles after stimulation of the tibial nerve and the low-frequency-dependent depression (FDD) was assessed. We provide evidence that exercise returns spinal excitability and levels of KCC2 and NKCC1 toward normal levels in the lumbar spinal cord. Acutely altering chloride extrusion using the KCC2 blocker DIOA masked the effect of exercise on FDD, whereas blocking NKCC1 with bumetanide returned FDD toward intact levels after SCI. Our results indicate that exercise contributes to reflex recovery and restoration of endogenous inhibition through a return to chloride homeostasis after SCI. This lends support for CCCs as part of a pathway that could be manipulated to improve functional recovery when combined with rehabilitation programs.

Plasticity in ascending long propriospinal and descending supraspinal pathways in chronic cervical spinal cord injured rats

The high clinical relevance of models of incomplete cervical spinal cord injury (SCI) creates a need to address the spontaneous neuroplasticity that underlies changes in functional activity that occur over time after SCI. There is accumulating evidence supporting long projecting propriospinal neurons as suitable targets for therapeutic intervention after SCI, but focus has remained primarily oriented toward study of descending pathways. Long ascending axons from propriospinal neurons at lower thoracic and lumbar levels that form inter-enlargement pathways are involved in forelimb-hindlimb coordination during locomotion and are capable of modulating cervical motor output. We used non-invasive magnetic stimulation to assess how a unilateral cervical (C5) spinal contusion might affect transmission in intact, long ascending propriospinal pathways, and influence spinal cord plasticity. Our results show that transmission is facilitated in this pathway on the ipsilesional side as early as 1 week post-SCI. We also probed for descending magnetic motor evoked potentials (MMEPs) and found them absent or greatly reduced on the ipsilesional side as expected. The frequency-dependent depression (FDD) of the H-reflex recorded from the forelimb triceps brachii was bilaterally decreased although Hmax/Mmax was increased only on the ipsilesional side. Behaviorally, stepping recovered, but there were deficits in forelimb–hindlimb coordination as detected by BBB and CatWalk measures. Importantly, epicenter sparing correlated to the amplitude of the MMEPs and locomotor recovery but it was not significantly associated with the inter-enlargement or segmental H-reflex. In summary, our results indicate that complex plasticity occurs after a C5 hemicontusion injury, leading to differential changes in ascending vs. descending pathways, ipsi- vs. contralesional sides even though the lesion was unilateral as well as cervical vs. lumbar local spinal networks.

Peripheral Nerve Grafts after Cervical Spinal Cord Injury in Adult Cats

Peripheral nerve grafts (PNG) into the rat spinal cord support axon regeneration after acute or chronic injury, with synaptic reconnection across the lesion site and some level of behavioral recovery. Here, we grafted a peripheral nerve into the injured spinal cord of cats as a preclinical treatment approach to promote regeneration for eventual translational use. Adult female cats received a partial hemisection lesion at the cervical level (C7) and immediate apposition of an autologous tibial nerve segment to the lesion site. Five weeks later, a dorsal quadrant lesion was performed caudally (T1), the lesion site treated with chondroitinase ABC 2 days later to digest growth inhibiting extracellular matrix molecules, and the distal end of the PNG apposed to the injury site. After 4-20 weeks, the grafts survived in 10/12 animals with several thousand myelinated axons present in each graft. The distal end of 9/10 grafts was well apposed to the spinal cord and numerous axons extended beyond the lesion site. Intraspinal stimulation evoked compound action potentials in the graft with an appropriate latency illustrating normal axonal conduction of the regenerated axons. Although stimulation of the PNG failed to elicit responses in the spinal cord distal to the lesion site, the presence of c-Fos immunoreactive neurons close to the distal apposition site indicates that regenerated axons formed functional synapses with host neurons. This study demonstrates the successful application of a nerve grafting approach to promote regeneration after spinal cord injury in a non-rodent, large animal model.

Chapter 2 – The spinal generation of phases and cycle duration

During walking, an increase in speed is accompanied by a decrease in the stance phase duration while the swing phase remains relatively invariant. By definition, the rhythm generator in the lumbar spinal cord controls cycle period, phase durations, and phase transitions. Our first aim was to determine if this asymmetry in the control of locomotor cycles is an inherent property of the central pattern generator (CPG). We recorded episodes of fictive locomotion, that is, locomotor patterns in absence of reafference, in decerebrate cats with or without a complete spinal transection (acute or chronic). In fictive locomotion, stance and swing phases typically correspond to extension and flexion, respectively. In the vast majority of locomotor episodes, cycle period varied more with extensor phase duration. This could be observed without phasic sensory feedback or supraspinal structures or pharmacology. In a few experiments, we stimulated the mesencephalic locomotor region or selected peripheral nerves during fictive locomotion and both could alter the phase/cycle period relationship. We conclude that there is a built-in asymmetry within the spinal rhythm generator for locomotion, which can be modified by extraneous factors. Locomotor and scratching rhythms are characterized by alternation of flexion and extension phases within one hindlimb, which are mediated by rhythm-generating circuitry within the spinal cord. Our second aim was to determine if rhythm generators for locomotion and scratch have similar control mechanisms in adult decerebrate cats. The regulation of cycle period during fictive scratching was evaluated, as were the effects of specific sensory inputs on phase durations and transitions during pinna-evoked fictive scratching. Results show that cycle period during fictive scratching varied predominantly with flexion phase duration, contrary to spontaneous fictive locomotion. Ankle dorsiflexion greatly increased extension phase duration and cycle period during fictive locomotion but did not alter cycle period during scratching. These data indicate that cycle period, phase durations, and phase transitions are not regulated similarly during fictive locomotion and scratching, with or without sensory inputs, providing evidence for the existence of distinct interneuronal components of rhythm generation within the mammalian spinal cord.

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.

Enhancing neural activity to drive respiratory plasticity following cervical spinal cord injury

Cervical spinal cord injury (SCI) results in permanent life-altering sensorimotor deficits, among which impaired breathing is one of the most devastating and life-threatening. While clinical and experimental research has revealed that some spontaneous respiratory improvement (functional plasticity) can occur post-SCI, the extent of the recovery is limited and significant deficits persist. Thus, increasing effort is being made to develop therapies that harness and enhance this neuroplastic potential to optimize long-term recovery of breathing in injured individuals. One strategy with demonstrated therapeutic potential is the use of treatments that increase neural and muscular activity (e.g. locomotor training, neural and muscular stimulation) and promote plasticity. With a focus on respiratory function post-SCI, this review will discuss advances in the use of neural interfacing strategies and activity-based treatments, and highlights some recent results from our own research.