Warren J. Alilain

Functional regeneration of respiratory pathways after spinal cord injury

Spinal cord injuries often occur at the cervical level above the phrenic motor pools, which innervate the diaphragm. The effects of impaired breathing are a leading cause of death from spinal cord injuries, underscoring the importance of developing strategies to restore respiratory activity. Here we show that, after cervical spinal cord injury, the expression of chondroitin sulphate proteoglycans (CSPGs) associated with the perineuronal net (PNN) is upregulated around the phrenic motor neurons. Digestion of these potently inhibitory extracellular matrix molecules with chondroitinase ABC (denoted ChABC) could, by itself, promote the plasticity of tracts that were spared and restore limited activity to the paralysed diaphragm. However, when combined with a peripheral nerve autograft, ChABC treatment resulted in lengthy regeneration of serotonin-containing axons and other bulbospinal fibres and remarkable recovery of diaphragmatic function. After recovery and initial transection of the graft bridge, there was an unusual, overall increase in tonic electromyographic activity of the diaphragm, suggesting that considerable remodelling of the spinal cord circuitry occurs after regeneration. This increase was followed by complete elimination of the restored activity, proving that regeneration is crucial for the return of function. Overall, these experiments present a way to markedly restore the function of a single muscle after debilitating trauma to the central nervous system, through both promoting the plasticity of spared tracts and regenerating essential pathways.

Increased chondroitin sulfate proteoglycan expression in denervated brainstem targets following spinal cord injury creates a barrier to axonal regeneration overcome by chondroitinase ABC and neurotrophin-3.

Increased chondroitin sulfate proteoglycan (CSPG) expression in the vicinity of a spinal cord injury (SCI) is a primary participant in axonal regeneration failure. However, the presence of similar increases of CSPG expression in denervated synaptic targets well away from the primary lesion and the subsequent impact on regenerating axons attempting to approach deafferented neurons have not been studied. Constitutively expressed CSPGs within the extracellular matrix and perineuronal nets of the adult rat dorsal column nuclei (DCN) were characterized using real-time PCR, Western blot analysis and immunohistochemistry. We show for the first time that by 2 days and through 3 weeks following SCI, the levels of NG2, neurocan and brevican associated with reactive glia throughout the DCN were dramatically increased throughout the DCN despite being well beyond areas of trauma-induced blood brain barrier breakdown. Importantly, regenerating axons from adult sensory neurons microtransplanted 2 weeks following SCI between the injury site and the DCN were able to regenerate rapidly within white matter (as shown previously by Davies et al. [Davies, S.J., Goucher, D.R., Doller, C., Silver, J., 1999. Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord. J. Neurosci. 19, 5810-5822]) but were unable to enter the denervated DCN. Application of chondroitinase ABC or neurotrophin-3-expressing lentivirus in the DCN partially overcame this inhibition. When the treatments were combined, entrance by regenerating axons into the DCN was significantly augmented. These results demonstrate both an additional challenge and potential treatment strategy for successful functional pathway reconstruction after SCI.

Light-Induced Rescue of Breathing after Spinal Cord Injury

Paralysis is a major consequence of spinal cord injury (SCI). After cervical SCI, respiratory deficits can result through interruption of descending presynaptic inputs to respiratory motor neurons in the spinal cord. Expression of channelrhodopsin-2 (ChR2) and photostimulation in neurons affects neuronal excitability and produces action potentials without any kind of presynaptic inputs. We hypothesized that after transducing spinal neurons in and around the phrenic motor pool to express ChR2, photostimulation would restore respiratory motor function in cervical SCI adult animals. Here we show that light activation of ChR2-expressing animals was sufficient to bring about recovery of respiratory diaphragmatic motor activity. Furthermore, robust rhythmic activity persisted long after photostimulation had ceased. This recovery was accomplished through a form of respiratory plasticity and spinal adaptation which is NMDA receptor dependent. These data suggest a novel, minimally invasive therapeutic avenue to exercise denervated circuitry and/or restore motor function after SCI.

Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

Regeneration of injured adult CNS axons is inhibited by formation of a glial scar. Immature astrocytes are able to support robust neurite outgrowth and reduce scarring, therefore, we tested whether these cells would have this effect if transplanted into brain injuries. Utilizing an in vitro spot gradient model that recreates the strongly inhibitory proteoglycan environment of the glial scar we found that, alone, immature, but not mature, astrocytes had a limited ability to form bridges across the most inhibitory outer rim. In turn, the astrocyte bridges could promote adult sensory axon re‐growth across the gradient. The use of selective enzyme inhibitors revealed that MMP‐2 enables immature astrocytes to cross the proteoglycan rim. The bridge‐building process and axon regeneration across the immature glial bridges were greatly enhanced by chondroitinase ABC pretreatment of the spots. We used microlesions in the cingulum of the adult rat brains to test the ability of matrix modification and immature astrocytes to form a bridge for axon regeneration in vivo. Injured axons were visualized via p75 immunolabeling and the extent to which these axons regenerated was quantified. Immature astrocytes coinjected with chondroitinase ABC‐induced axonal regeneration beyond the distal edge of the lesion. However, when used alone, neither treatment was capable of promoting axonal regeneration. Our findings indicate that when faced with a minimal lesion, neurons of the basal forebrain can regenerate in the presence of a proper bridge across the lesion and when levels of chondroitin sulfate proteoglycans (CSPGs) in the glial scar are reduced.

MK-801 upregulates NR2A protein levels and induces functional recovery of the ipsilateral hemidiaphragm following acute C2 hemisection in adult rats.

Abstract Background: C2 hemisection results in paralysis of the ipsilateral hemidiaphragm. Recent data indicate that an upregulation of the N-methyl-D-aspartate (NMDA) receptor 2A subunit following chronic C2 hemisection is associated with spontaneous hemidiaphragmatic recovery following injury. MK-801, an antagonist of the NMDA receptor, upregulates the NR2A subunit in neonatal rats. Hypothesis: We hypothesized that administration of MK-801 to adult, acute C2-hemisected rats would result in an increase of NR2A in the spinal cord. Furthermore, we hypothesized that upregulation of NR2A would be associated with recovery of the ipsilateral hemidiaphragm as in the chronic studies. Design: To develop a dose-response curve, adult rats were treated with varying doses of MK-801 and their spinal cords harvested and assessed for NR2A as well as AMPA GluRI and GluR2 subunit protein levels. In the second part of this study, C2-hemisected animals received MK-801. Following treatment, the animals were assessed for recovery of the hemidiaphragm through electromyographic recordings and their spinal cords assessed for NR2A, GluRI, and GluR2. Results: Treatment with MK-801 leads to an increase of the NR2A subunit in the spinal cords of adult noninjured rats. There were no changes in the expression of GluRI and GluR2 in these animals. Administration of MK-801 to C2-hemisected rats resulted in recovery of the ipsilateral hemidiaphragm, an increase of NR2A, and a decrease of GluR2. Conclusion: Our findings strengthen the evidence that the NR2A subunit plays a substantial role in mediating recovery of the paralyzed hemidiaphragm following C2 spinal cord hemisection.

Treatments to restore respiratory function after spinal cord injury and their implications for regeneration, plasticity and adaptation

Spinal cord injury (SCI) often leads to impaired breathing. In most cases, such severe respiratory complications lead to morbidity and death. However, in the last few years there has been extensive work examining ways to restore this vital function after experimental spinal cord injury. In addition to finding strategies to rescue breathing activity, many of these experiments have also yielded a great deal of information about the innate plasticity and capacity for adaptation in the respiratory system and its associated circuitry in the spinal cord. This review article will highlight experimental SCI resulting in compromised breathing, the various methods of restoring function after such injury, and some recent findings from our own laboratory. Additionally, it will discuss findings about motor and CNS respiratory plasticity and adaptation with potential clinical and translational implications.

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.

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.

Shedding light on restoring respiratory function after spinal cord injury

Loss of respiratory function is one of the leading causes of death following spinal cord injury. Because of this, much work has been done in studying ways to restore respiratory function following spinal cord injury (SCI) – including pharmacological and regeneration strategies. With the emergence of new and powerful tools from molecular neuroscience, new therapeutically relevant alternatives to these approaches have become available, including expression of light sensitive proteins called channelrhodopsins. In this article we briefly review the history of various attempts to restore breathing after C2 hemisection, and focus on our recent work using the activation of light sensitive channels to restore respiratory function after experimental SCI. We also discuss how such light-induced activity can help shed light on the inner workings of the central nervous system respiratory circuitry that controls diaphragmatic function.

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.