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Spinal cord injuries, for which there are limited effective clinical treatments, result in enduring paralysis and hypoesthesia due, in part, to the inhibitory microenvironment that develops and limits regeneration/sprouting, especially during chronic stages. Recently, we discovered that targeted enzymatic modulation of the potently inhibitory chondroitin sulfate proteoglycan (CSPG) component of the extracellular and perineuronal net (PNN) matrix via Chondroitinase ABC (ChABC) can rapidly restore robust respiratory function to the previously paralyzed hemi-diaphragm after remarkably long times post-injury (up to 1.5 years) following a cervical level 2 lateral hemi-transection. Importantly, ChABC treatment at cervical level 4 in this chronic model also elicited rapid, albeit modest, improvements in upper arm function. In the present study, we sought to further optimize and elucidate the capacity for nerve sprouting and/or regeneration to restore gross as well as fine motor control of the forearm and digits at lengthy chronic stages post injury. However, instead of using ChABC, we utilized a novel and more clinically relevant systemic, non-invasive combinatorial treatment strategy designed to both reduce and overcome inhibitory CSPGs simultaneously and spatially extensively. Following a three-month upper cervical spinal hemi-lesion using adult female Sprague Dawley rats, we show that the combined treatment has a profound effect on functional recovery of the chronically paralyzed forelimb and paw, specifically during walking as well as precision movements of the digits. Our exciting pre-clinical findings will begin to enhance our understanding of the basic mechanisms underlying functionally beneficial regenerative events occurring at chronic injury stages for clinically relevant translational benefits.Significance statementOvercoming the persistent axon inhibitory environment following a functionally debilitating incomplete spinal cord lesion has long proven to be an elusive dilemma, especially months to years after the initial spinal injury. Current therapeutic and rehabilitative techniques for patients suffering from chronic cervical spinal insults minimally, if at all, address this structural hindrance and support limited return of crucial behaviors such as voluntary use of the arms and hands. Our investigation into the behavioral and anatomical consequences of systemically perturbing the high-affinity binding interaction between the receptor PTPσ and the extracellular chondroitin sulfate proteoglycans highlight an underlying barrier to the restoration of forelimb/paw walking and eating behavior 12-weeks after a cervical spinal hemi-transection.

It has been reported that intramuscular injection of an Adeno-associated viral vector serotype 1 (AAV1) encoding Neurotrophin-3 (NT3) into hindlimb muscles 24 hours after a severe T9 contusion in rats induced lumbar spinal neuroplasticity, partially restored locomotive function and reduced spasms during swimming. Here we investigated whether a targeted delivery of NT3 to lumbar and thoracic motor neurons 48 hours following a severe contusive injury aids locomotive recovery in rats. AAV1-NT3 was injected into the tibialis anterior, gastrocnemius and rectus abdominus muscles 48-hours following trauma, persistently elevating serum levels of the neurotrophin. NT3 improved trunk stability, accuracy of stepping during skilled locomotive tasks, and alternation of the hindlimbs during swimming, but it had no effect on gross locomotion function in the open field. The number of vGlut1+ (likely proprioceptive afferent) boutons on gastrocnemius α-motor neurons was increased after injury but normalised following NT3 treatment suggestive of a mechanism in which the functional effects may be mediated through proprioceptive feedback. Ex vivo MRI revealed substantial loss of grey and white matter at the lesion epicentre but no effect of delayed NT3 treatment to induce neuroprotection or prevent secondary damage. Spasms and hyperreflexia were not reliably induced in this severe injury model suggesting a more complex anatomical or physiological cause to their induction. We have shown that delayed intramuscular AAV-NT3 treatment can promote recovery in skilled stepping and coordinated swimming supporting a role for NT3 as a therapeutic strategy for spinal injuries potentially through modulation of somatosensory feedback.Key PointsTargeted delivery of NT3 to hindlimb and trunk muscles at a clinically relevant 48h following a severe thoracic contusion aids fine locomotor control and synchronised movement.NT3 mediated improvements in trunk stability, accuracy of stepping during skilled locomotive tasks, and alternation of the hindlimbs during swimming through the normalisation of vGlut1+ boutons on presumptive proprioceptive afferents innervating these muscles.250kDyn thoracic contusion does not reliably result in measurable signs of spasticity.

Intramuscular injection of an Adeno-associated viral vector serotype 1 (AAV1) encoding Neurotrophin-3 (NT3) into hindlimb muscles 24 h after a severe T9 spinal level contusion in rats has been shown to induce lumbar spinal neuroplasticity, partially restore locomotive function and reduce spasms during swimming. Here we investigate whether a targeted delivery of NT3 to lumbar and thoracic motor neurons 48 h following a severe contusive injury aids locomotive recovery in rats. AAV1-NT3 was injected bilaterally into the tibialis anterior, gastrocnemius and rectus abdominus muscles 48-h following trauma, persistently elevating serum levels of the neurotrophin. NT3 modestly improved trunk stability, accuracy of stepping during skilled locomotion, and alternation of the hindlimbs during swimming, but it had no effect on gross locomotor function in the open field. The number of vGlut1

Via the peripheral and autonomic nervous systems, the spinal cord directly or indirectly connects reciprocally with many body systems (muscular, intengumentary, respiratory, immune, digestive, excretory, reproductive, cardiovascular, etc). Accordingly, spinal cord injury (SCI) can result in catastrophe for multiple body systems including muscle paralysis affecting movement and loss of normal sensation, as well as neuropathic pain, spasticity, reduced fertility and autonomic dysreflexia. Treatments and cure for an injured spinal cord will likely require access of therapeutic agents across the blood-CNS (central nervous system) barrier. However, some types of repair within the CNS may be possible by targeting treatment to peripherally located cells or by delivering Adeno-Associated Viral vectors (AAVs) by peripheral routes (e.g., intrathecal, intravenous). This review will consider some future possibilities for SCI repair generated by therapeutic peripheral gene delivery. There are now six gene therapies approved worldwide as safe and effective medicines of which three were created by modification of the apparently nonpathogenic Adeno-Associated Virus. One of these AAVs, Zolgensma, is injected intrathecally for treatment of spinal muscular atrophy in children. One day, delivery of AAVs into peripheral tissues might improve recovery after spinal cord injury in humans; we discuss experiments by us and others delivering transgenes into nerves or muscles for sensorimotor recovery in animal models of SCI or of stroke including human Neurotrophin-3. We also describe ongoing efforts to develop AAVs that are delivered to particular targets within and without the CNS after peripheral administration using capsids with improved tropisms, promoters that are selective for particular cell types, and methods for controlling the dose and duration of expression of a transgene. In conclusion, in the future, minimally invasive administration of AAVs may improve recovery after SCI with minimal side effects.

Schwann cell grafts support axonal growth following spinal cord injury, but a boundary forms between the implanted cells and host astrocytes. Axons are reluctant to exit the graft tissue in large part due to the surrounding inhibitory environment containing chondroitin sulphate proteoglycans (CSPGs). We use a lentiviral chondroitinase ABC, capable of being secreted from mammalian cells (mChABC), to examine the repercussions of CSPG digestion upon Schwann cell behaviour in vitro. We show that mChABC transduced Schwann cells robustly secrete substantial quantities of the enzyme causing large-scale CSPG digestion, facilitating the migration and adhesion of Schwann cells on inhibitory aggrecan and astrocytic substrates. Importantly, we show that secretion of the engineered enzyme can aid the intermingling of cells at the Schwann cell-astrocyte boundary, enabling growth of neurites over the putative graft/host interface. These data were echoed in vivo. This study demonstrates the profound effect of the enzyme on cellular motility, growth and migration. This provides a cellular mechanism for mChABC induced functional and behavioural recovery shown in in vivo studies. Importantly, we provide in vitro evidence that mChABC gene therapy is equally or more effective at producing these effects as a one-time application of commercially available ChABC.

There exists an abundance of barriers that hinder functional recovery following spinal cord injury, especially at chronic stages. Here, we examine the rescue of breathing up to 1.5 years following cervical hemisection in the rat. In spite of complete hemidiaphragm paralysis, a single injection of chondroitinase ABC in the phrenic motor pool restored robust and persistent diaphragm function while improving neuromuscular junction anatomy. This treatment strategy was more effective when applied chronically than when assessed acutely after injury. The addition of intermittent hypoxia conditioning further strengthened the ventilatory response. However, in a sub-population of animals, this combination treatment caused excess serotonergic (5HT) axon sprouting leading to aberrant tonic activity in the diaphragm that could be mitigated via 5HT2 receptor blockade. Through unmasking of the continuing neuroplasticity that develops after injury, our treatment strategy ensured rapid and robust patterned respiratory recovery after a near lifetime of paralysis., Respiratory failure is one of the leading causes of death following spinal cord injury and it is unclear if normal respiratory motor activity can be recovered after chronic injury-induced paralysis. Here, authors show that treatment with chondroitinase ABC induces robust rescue of breathing up to 1.5 years following complete hemidiaphragm paralysis.

Severe midcervical contusion injury causes profound deficits throughout the respiratory motor system that last from acute to chronic time points post-injury. We use chondroitinase ABC (ChABC) to digest chondroitin sulphate proteoglycans within the extracellular matrix (ECM) surrounding the respiratory system at both acute and chronic time points post-injury to explore whether augmentation of plasticity can recover normal motor function. We demonstrate that, regardless of time post-injury or treatment application, the lesion cavity remains consistent, showing little regeneration or neuroprotection within our model. Through electromyography (EMG) recordings of multiple inspiratory muscles, however, we show that application of the enzyme at chronic time points post-injury initiates the recovery of normal breathing in previously paralyzed respiratory muscles. This reduced the need for compensatory activity throughout the motor system. Application of ChABC at acute time points recovered only modest amounts of respiratory function. To further understand this effect, we assessed the anatomical mechanism of this recovery. Increased EMG activity in previously paralyzed muscles was brought about by activation of spared bulbospinal pathways through the site of injury and/or sprouting of spared serotonergic fibers from the contralateral side of the cord. Accordingly, we demonstrate that alterations to the ECM and augmentation of plasticity at chronic time points post-cervical contusion can cause functional recovery of the respiratory motor system and reveal mechanistic evidence of the pathways that govern this effect.

This chapter describes a unilateral cervical spinal cord contusion model that causes ipsilateral respiratory and/or forelimb motor deficits. Additional techniques are presented to assess forelimb function via grooming and paw placement tasks, as well as respiratory activity using additional lesion techniques that remove descending compensatory respiratory motor control. Cervical injury is the most common type of human spinal cord injury. Modeling functions of highest priority for this spinal cord injured population (i.e. respiratory and arm/hand control) provides a translational approach for the evaluation of potentially therapeutic interventions.

Mid-cervical spinal cord contusion disrupts both the pathways and motoneurons vital to the activity of inspiratory muscles. The present study was designed to determine if a rat contusion model could result in a measurable deficit to both ventilatory and respiratory motor function under “normal” breathing conditions at acute to chronic stages post trauma. Through whole body plethysmography and electromyography we assessed respiratory output from three days to twelve weeks after a cervical level 3 (C3) contusion. Contused animals showed significant deficits in both tidal and minute volumes which were sustained from acute to chronic time points. We also examined the degree to which the contusion injury impacted ventilatory pattern variability through assessment of Mutual Information and Sample Entropy. Mid-cervical contusion significantly and robustly decreased the variability of ventilatory patterns. The enduring deficit to the respiratory motor system caused by contusion was further confirmed through electromyography recordings in multiple respiratory muscles. When isolated via a lesion, these contused pathways were insufficient to maintain respiratory activity at all time points post injury. Collectively these data illustrate that, counter to the prevailing literature, a profound and lasting ventilatory and respiratory motor deficit may be modelled and measured through multiple physiological assessments at all time points after cervical contusion injury.

Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury.

The maintenance of blood gas and pH homeostasis is essential to life. As such breathing, and the mechanisms which control ventilation, must be tightly regulated yet highly plastic and dynamic. However, injury to the spinal cord prevents the medullary areas which control respiration from connecting to respiratory effectors and feedback mechanisms below the level of the lesion. This trauma typically leads to severe and permanent functional deficits in the respiratory motor system. However, endogenous mechanisms of plasticity occur following spinal cord injury to facilitate respiration and help recover pulmonary ventilation. These mechanisms include the activation of spared or latent pathways, endogenous sprouting or synaptogenesis, and the possible formation of new respiratory control centres. Acting in combination, these processes provide a means to facilitate respiratory support following spinal cord trauma. However, they are by no means sufficient to return pulmonary function to pre-injury levels. A major challenge in the study of spinal cord injury is to understand and enhance the systems of endogenous plasticity which arise to facilitate respiration to mediate effective treatments for pulmonary dysfunction.

High cervical spinal cord injury (SCI) typically results in partial paralysis of the diaphragm due to intrusion of descending inspiratory drive at the level of the phrenic nucleus. The degree to which such paralysis occurs depends on the type, force, level, and extent of trauma produced. While endogenous recovery and plasticity may occur, the resulting respiratory complications can lead to morbidity and death. However, it has been shown that through modification of intrinsic motor neuron properties, or altering the environment localized at the site of SCI, functional recovery and plasticity of the respiratory motor system can be facilitated. The present review emphasizes these factors and correlates it to the treatment of SCI at the level of the somatic nervous system. Despite these promising therapies, functional respiratory motor system recovery following cervical SCI is often minimal. This review thus focuses on possible directions for the field, with emphasis on combinatorial treatment.

The maintenance of blood gas and pH homeostasis is essential to life. As such breathing, and the mechanisms which control ventilation, must be tightly regulated yet highly plastic and dynamic. However, injury to the spinal cord prevents the medullary areas which control respiration from connecting to respiratory effectors and feedback mechanisms below the level of the lesion. This trauma typically leads to severe and permanent functional deficits in the respiratory motor system. However, endogenous mechanisms of plasticity occur following spinal cord injury to facilitate respiration and help recover pulmonary ventilation. These mechanisms include the activation of spared or latent pathways, endogenous sprouting or synaptogenesis, and the possible formation of new respiratory control centres. Acting in combination, these processes provide a means to facilitate respiratory support following spinal cord trauma. However, they are by no means sufficient to return pulmonary function to pre-injury levels. A major challenge in the study of spinal cord injury is to understand and enhance the systems of endogenous plasticity which arise to facilitate respiration to mediate effective treatments for pulmonary dysfunction.

The organization of the respiratory network has traditionally been described through descending medullary tracks. We have determined central and peripheral mechanisms auxiliary to the descending su...

High cervical spinal cord injury (SCI) typically results in partial paralysis of the diaphragm due to intrusion of descending inspiratory drive at the level of the phrenic nucleus. The degree to which such paralysis occurs depends on the type, force, level, and extent of trauma produced. While endogenous recovery and plasticity may occur, the resulting respiratory complications can lead to morbidity and death. However, it has been shown that through modification of intrinsic motor neuron properties, or altering the environment localized at the site of SCI, functional recovery and plasticity of the respiratory motor system can be facilitated. The present review emphasizes these factors and correlates it to the treatment of SCI at the level of the somatic nervous system. Despite these promising therapies, functional respiratory motor system recovery following cervical SCI is often minimal. This review thus focuses on possible directions for the field, with emphasis on combinatorial treatment.

Anti-Nogo-A antibody and chondroitinase ABC (ChABC) enzyme are two promising treatments that promote functional recovery after spinal cord injury (SCI). Treatment with them has encouraged axon regeneration, sprouting and functional recovery in a variety of spinal cord and central nervous system injury models. The two compounds work, in part, through different mechanisms, so it is possible that their effects will be additive. In this study, we used a rat cervical partial SCI model to explore the effectiveness of a combination of anti-Nogo-A, ChABC, and rehabilitation. We found that spontaneous recovery of forelimb functions reflects the extent of the lesion on the ipsilateral side. We applied a combination treatment with acutely applied anti-Nogo-A antibody followed by delayed ChABC treatment starting at 3 weeks after injury, and rehabilitation starting at 4 weeks, to accommodate the requirement that anti-Nogo-A be applied acutely, and that rehabilitation be given after the cessation of anti-Nogo-A treatment. We found that single treatment with either anti-Nogo-A or ChABC, combined with rehabilitation, produced functional recovery of similar magnitude. The combination treatment, however, was more effective. Both single treatments produced increases in sprouting and axon regeneration, but the combination treatment produced greater increases. Anti-Nogo-A stimulated growth of a greater number of axons with a diameter of > 3 μm, whereas ChABC treatment stimulated increased growth of finer axons with varicosities. These results point to different functions of Nogo-A and chondroitin sulfate proteoglycans in axonal regeneration. The combination of anti-Nogo-A, ChABC and rehabilitation shows promise for enhancing functional recovery after SCI.

More than 50% of all spinal cord injury (SCI) cases are at the cervical level and usually result in the impaired ability to breathe. This is caused by damage to descending bulbospinal inspiratory tracts and the phrenic motor neurons which innervate the diaphragm. Most investigations have utilized a lateral C2 hemisection model of cervical SCI to study the resulting respiratory motor deficits and potential therapies. However, recent studies have emerged which incorporate experimental contusion injuries at the cervical level of the spinal cord to more closely reflect the type of trauma encountered in humans. Nonetheless, a common deficit observed in these contused animals is the inability to increase diaphragm motor activity in the face of respiratory challenge. In this report we tested the hypothesis that, following cervical contusion, all remaining tracts to the phrenic nucleus are active, including the crossed phrenic pathway (CPP). Additionally, we investigated the potential function these spared tracts might possess after injury. We find that, following a lateral C3/4 contusion injury, not all remaining pathways are actively exciting downstream phrenic motor neurons. However, removing some of these pathways through contralateral hemisection results in a cessation of all activity ipsilateral to the contusion. This suggests an important modulatory role for these pathways. Additionally, we conclude that this dual injury, hemi-contusion and post contra-hemisection, is a more effective and relevant model of cervical SCI as it results in a more direct compromise of diaphragmatic motor activity. This model can thus be used to test potential therapies with greater accuracy and clinical relevance than cervical contusion models currently allow.

Chondroitin sulfate is a glycosaminoglycan composed of N-acetylgalactosamine and glucuronic acid. It attaches to a core protein to form chondroitin sulfate proteoglycan (CSPG). Being a major component of the brain extracellular matrix, CSPGs are involved in neural development, axon pathfinding and guidance, plasticity and also regeneration after injury in the nervous system. In this review, we shall discuss the structure, the biosynthetic pathway, its functions in the nervous system and how we can improve regeneration in the nervous system by modulating its structure and binding properties.