Recovery of Forearm and Fine Digit Function After Chronic Spinal Cord Injury by Simultaneous Blockade of Inhibitory Matrix Chondroitin Sulfate Proteoglycan Production and the Receptor PTPσ.

Spinal cord injuries (SCI), for which there are limited effective treatments, result in enduring paralysis and hypoesthesia, in part because of the inhibitory microenvironment that develops and limits regeneration/sprouting, especially during chronic stages. Recently, we discovered that targeted enzymatic removal of the inhibitory chondroitin sulfate proteoglycan (CSPG) component of the extracellular and perineuronal net (PNN) matrix via Chondroitinase ABC (ChABC) rapidly restored 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 improvements in gross upper arm function. In the present study, we focused on arm and hand function, seeking to highlight and optimize crude 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 combinatorial treatment strategy designed to simultaneously reduce and overcome inhibitory CSPGs. Following a 3-month upper cervical spinal hemi-lesion using adult female Sprague Dawley rats, we show that the combined treatment had a profound effect on functional recovery of the chronically paralyzed forelimb and paw, as well as on precision movements of the digits. The regenerative and immune system related events that we describe deepen our basic understanding of the crucial role of CSPG-mediated inhibition via the PTPσ receptor in constraining functional synaptic plasticity at lengthy time points following SCI, hopefully leading to clinically relevant translational benefits.

Delayed viral vector mediated delivery of neurotrophin-3 improves skilled hindlimb function and stability after thoracic contusion.

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+ boutons, likely arising from proprioceptive afferents, on gastrocnemius α-motor neurons was increased after injury but normalised following NT3 treatment, suggestive of a mechanism in which functional benefits 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. Lower body spasms and hyperreflexia of an intrinsic paw muscle 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.

New insights into glial scar formation after spinal cord injury.

Severe spinal cord injury causes permanent loss of function and sensation throughout the body. The trauma causes a multifaceted torrent of pathophysiological processes which ultimately act to form a complex structure, permanently remodeling the cellular architecture and extracellular matrix. This structure is traditionally termed the glial/fibrotic scar. Similar cellular formations occur following stroke, infection, and neurodegenerative diseases of the central nervous system (CNS) signifying their fundamental importance to preservation of function. It is increasingly recognized that the scar performs multiple roles affecting recovery following traumatic injury. Innovative research into the properties of this structure is imperative to the development of treatment strategies to recover motor function and sensation following CNS trauma. In this review, we summarize how the regeneration potential of the CNS alters across phyla and age through formation of scar-like structures. We describe how new insights from next-generation sequencing technologies have yielded a more complex portrait of the molecular mechanisms governing the astrocyte, microglial, and neuronal responses to injury and development, especially of the glial component of the scar. Finally, we discuss possible combinatorial therapeutic approaches centering on scar modulation to restore function after severe CNS injury.

Peripherally delivered Adeno-associated viral vectors for spinal cord injury repair.

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.

Substrate Specificity and Biochemical Characteristics of an Engineered Mammalian Chondroitinase ABC.

Chondroitin sulfate proteoglycans inhibit regeneration, neuroprotection, and plasticity following spinal cord injury. The development of a second-generation chondroitinase ABC enzyme, capable of being secreted from mammalian cells (mChABC), has facilitated the functional recovery of animals following severe spinal trauma. The genetically modified enzyme has been shown to efficiently break down the inhibitory extracellular matrix surrounding cells at the site of injury, while facilitating cellular integration and axonal growth. However, the activity profile of the enzyme in relation to the original bacterial chondroitinase (bChABC) has not been determined. Here, we characterize the activity profile of mChABC and compare it to bChABC, both enzymes having been maintained under physiologically relevant conditions for the duration of the experiment. We show that this genetically modified enzyme can be secreted reliably and robustly in high yields from a mammalian cell line. The modifications made to the cDNA of the enzyme have not altered the functional activity of mChABC compared to bChABC, ensuring that it has optimal activity on chondroitin sulfate-A, with an optimal pH at 8.0 and temperature at 37 °C. However, mChABC shows superior thermostability compared to bChABC, ensuring that the recombinant enzyme operates with enhanced activity over a variety of physiologically relevant substrates and temperatures compared to the widely used bacterial alternative without substantially altering its kinetic output. The determination that mChABC can function with greater robustness under physiological conditions than bChABC is an important step in the further development of this auspicious treatment strategy toward a clinical application.

Oxygen transport kinetics underpin rapid and robust diaphragm recovery following chronic spinal cord injury.

KEY POINTS: Spinal treatment can restore diaphragm function in all animals 1 month following C2 hemisection induced paralysis. Greater recovery occurs the longer after injury the treatment is applied. Through advanced assessment of muscle mechanics, innovative histology and oxygen tension modelling, we have comprehensively characterized in vivo diaphragm function and phenotype. Muscle work loops reveal a significant deficit in diaphragm functional properties following chronic injury and paralysis, which are normalized following restored muscle activity caused by plasticity-induced spinal reconnection. Injury causes global and local alterations in diaphragm muscle vascular supply, limiting oxygen diffusion and disturbing function. Restoration of muscle activity reverses these alterations, restoring oxygen supply to the tissue and enabling recovery of muscle functional properties. There remain metabolic deficits following restoration of diaphragm activity, probably explaining only partial functional recovery. We hypothesize that these deficits need to be resolved to restore complete respiratory motor function. ABSTRACT: Months after spinal cord injury (SCI), respiratory deficits remain the primary cause of morbidity and mortality for patients. It is possible to induce partial respiratory motor functional recovery in chronic SCI following 2 weeks of spinal neuroplasticity. However, the peripheral mechanisms underpinning this recovery are largely unknown, limiting development of new clinical treatments with potential for complete functional restoration. Utilizing a rat hemisection model, diaphragm function and paralysis was assessed and recovered at chronic time points following trauma through chondroitinase ABC induced neuroplasticity. We simulated the diaphragm's in vivo cyclical length change and activity patterns using the work loop technique at the same time as assessing global and local measures of the muscles histology to quantify changes in muscle phenotype, microvascular composition, and oxidative capacity following injury and recovery. These data were fed into a physiologically informed model of tissue oxygen transport. We demonstrate that hemidiaphragm paralysis causes muscle fibre hypertrophy, maintaining global oxygen supply, although it alters isolated muscle kinetics, limiting respiratory function. Treatment induced recovery of respiratory activity normalized these effects, increasing oxygen supply, restoring optimal diaphragm functional properties. However, metabolic demands of the diaphragm were significantly reduced following both injury and recovery, potentially limiting restoration of normal muscle performance. The mechanism of rapid respiratory muscle recovery following spinal trauma occurs through oxygen transport, metabolic demand and functional dynamics of striated muscle. Overall, these data support a systems-wide approach to the treatment of SCI, and identify new targets to mediate complete respiratory recovery.

Secretion of a mammalian chondroitinase ABC aids glial integration at PNS/CNS boundaries.

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.

Regulation of autophagy by inhibitory CSPG interactions with receptor PTPσ and its impact on plasticity and regeneration after spinal cord injury.

Chondroitin sulfate proteoglycans (CSPGs), extracellular matrix molecules that increase dramatically following a variety of CNS injuries or diseases, have long been known for their potent capacity to curtail cell migrations as well as axon regeneration and sprouting. The inhibition can be conferred through binding to their major cognate receptor, Protein Tyrosine Phosphatase Sigma (PTPσ). However, the precise mechanisms downstream of receptor binding that mediate growth inhibition have remained elusive. Recently, CSPGs/PTPσ interactions were found to regulate autophagic flux at the axon growth cone by dampening the autophagosome-lysosomal fusion step. Because of the intense interest in autophagic phenomena in the regulation of a wide variety of critical cellular functions, we summarize here what is currently known about dysregulation of autophagy following spinal cord injury, and highlight this critical new mechanism underlying axon regeneration failure. Furthermore, we review how CSPGs/PTPσ interactions influence plasticity through autophagic regulation and how PTPσ serves as a switch to execute either axon outgrowth or synaptogenesis. This has exciting implications for the role CSPGs play not only in axon regeneration failure after spinal cord injury, but also in neurodegenerative diseases where, again, inhibitory CSPGs are upregulated.

Plasticity Induced Recovery of Breathing Occurs at Chronic Stages after Cervical Contusion.

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.

Rapid and robust restoration of breathing long after spinal cord injury.

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.

Mid-cervical spinal cord contusion causes robust deficits in respiratory parameters and pattern variability.

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.

The Biology of Regeneration Failure and Success After Spinal Cord Injury.

Since no approved therapies to restore mobility and sensation following spinal cord injury (SCI) currently exist, a better understanding of the cellular and molecular mechanisms following SCI that compromise regeneration or neuroplasticity is needed to develop new strategies to promote axonal regrowth and restore function. Physical trauma to the spinal cord results in vascular disruption that, in turn, causes blood-spinal cord barrier rupture leading to hemorrhage and ischemia, followed by rampant local cell death. As subsequent edema and inflammation occur, neuronal and glial necrosis and apoptosis spread well beyond the initial site of impact, ultimately resolving into a cavity surrounded by glial/fibrotic scarring. The glial scar, which stabilizes the spread of secondary injury, also acts as a chronic, physical, and chemo-entrapping barrier that prevents axonal regeneration. Understanding the formative events in glial scarring helps guide strategies towards the development of potential therapies to enhance axon regeneration and functional recovery at both acute and chronic stages following SCI. This review will also discuss the perineuronal net and how chondroitin sulfate proteoglycans (CSPGs) deposited in both the glial scar and net impede axonal outgrowth at the level of the growth cone. We will end the review with a summary of current CSPG-targeting strategies that help to foster axonal regeneration, neuroplasticity/sprouting, and functional recovery following SCI.

Reprint of "Drawing breath without the command of effectors: the control of respiration following 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.

The challenges of respiratory motor system recovery following cervical spinal cord injury.

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.

Drawing breath without the command of effectors: the control of respiration following 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.

The role of the crossed phrenic pathway after cervical contusion injury and a new model to evaluate therapeutic interventions.

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.

Combination treatment with anti-Nogo-A and chondroitinase ABC is more effective than single treatments at enhancing functional recovery after spinal cord injury.

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.

Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells.

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.