Neuroscience 2012 Spinal Cord Injury Therapeutic Strategies and Recovery Abstracts

Chondroitinase ABC treatment promotes respiratory motor recovery following cervical contusion injury

Authors: *B. I. AWAD1, M. DEPAUL2, M. CLARK1, M. P. STEINMETZ1, J. SILVER2, W. J. ALILAIN1;
1Neurosci. Dept., Metro-Health Med. Ctr., Cleveland, OH; 2Neurosciences, Case Western Reserve Univ. Sch. of Med., Cleveland, OH

Abstract:

Respiratory motor dysfunction is the leading cause of morbidity and mortality following traumatic cervical spinal cord injury (SCI). Prior studies have utilized the C2 hemisection model to induce paralysis of the ipsilateral hemidiaphragm by interrupting the bulbospinal inputs to the phrenic nucleus (PN). We have previously shown that following this injury there is a distal upregulation of the perineuronal net (PNN) and chondroitin sulfate proteoglycans (CSPGs) around phrenic motor neurons. This is important because the PNN and CSPGs are potently inhibitory to regeneration and plasticity. However, we further demonstrated that the digestion of these inhibitory extracellular matrix molecules with Chondroitinase ABC (ChABC) can promote sprouting, plasticity, and respiratory motor recovery. While the contusion-type injury in the spinal cord is one of the most common forms of human SCI, few studies have evaluated or characterized the histopathological effects of such an injury on cervical spinal respiratory circuitry and its impact on breathing. We previously hypothesized that the immediate injection of ChABC after a C3 unilateral contusion injury will promote the respiratory plasticity of spared tracts and improve diaphragmatic activity (diaEMG). Additionally, we proposed that application of the respiratory stimulant, theophylline, will further augment activity. In this study, we extended these initial studies and performed immunohistochemical analysis one week after C3 contusion. We revealed reduced expression of CS-56 and WFA expression after treatment. These results indicate that acute ChABC administration is effective at diminishing the inhibitory influence mediated by the PNN and CSPGs at the cellular level and may provide an additional neuroprotective therapeutic strategy to improve diaEMG and promote respiratory plasticity.
Chondroitinase ABC treatment promotes respiratory motor recovery following cervical contusion injury
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Induced regeneration via peripheral nerve transplantation in combination with aFGF, and chondroitinase improves urinary function in complete spinal cord transected adult mice

Authors: M. DEPAUL1, H.-H. JIANG2, C.-Y. LIN3, J. SILVER1, *Y.-S. LEE3;
1Neurosciences, Case Western Reserve Univ., Cleveland, OH; 2Biomed. Engin., 3Neurosci, Cleveland Clin., CLEVELAND, OH

Abstract:

The loss of lower urinary tract (LUT) control is a ubiquitous consequence of complete spinal cord injuries (SCI). Previous work in our lab has utilized a complete T8 spinal cord transection (TX) model in adult rats resulting in a loss of LUT function. When treated with peripheral nerve grafts (PNG), acidic fibroblast growth factor (aFGF) and chondroitinase ABC (ChABC), LUT control can be improved as measured by urodynamic and metabolic cage activity.
The purpose of this study was to determine whether PNG+aFGF+ChABC can improve bladder control after complete T8 spinal cord transection in adult mice. Mice, unlike rats, never develop a micturition reflex after injury and may be better suited as a human model to study the functional recovery of the LUT after SCI. Adult mice were divided into 3 groups: (1) Sham control (laminectomy only), (2) TX only, and (3) TX+PNG+aFGF+ChABC. After T8 transection, ChABC was injected into both cord stumps followed by auto-transplantation of PNG from intercostal nerves. Fibrin glue with aFGF was applied on top of the PNG prior to wound closure. Urodynamic and external urethral sphincter (EUS) electromyogram (EMG) activity were recorded on restrained, awake animals 18 weeks post spinal cord surgery and then spinal cords and bladders were collected to conduct immunocytochemistry. Sham animals displayed long inter-contraction intervals, with each contraction leading to large changes in bladder pressure and a micturition event. TX only animals displayed hyperactive bladders with short ICIs (define ICI, small changes in bladder pressure during contraction, and many non-voiding contractions. EUS-EMG also revealed a tonic activity during the entire recording period. The TX+PNG+aFGF+ChABC treated group showed (1) longer ICIs with fewer non-voiding contractions, (2) a greater change in pressure during bladder contractions, and (3) a better coordination between bladder contraction and EUS-EMG when compared to TX only. Additionally, TX+PNG+aFGF+ChABC animals showed significant reduction of bladder weight and improvements in bladder and EUS histology compared to Tx only animals. These results suggest our mouse model of LUT dysfunction and recovery recapitulates the results found in the rat model and now allows us to utilize transgenic strategies to further improve regeneration.
Induced regeneration via peripheral nerve transplantation in combination with aFGF, and chondroitinase improves urinary function in complete spinal cord transected adult mice

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Gene delivery of chondroitinase ABC promotes functional repair following spinal cord injury

Authors: *N. D. JAMES1, K. BARTUS1, J. SHEA1, K. BOSCH1, J. H. ROGERS2, S. B. MCMAHON1, B. L. SCHEIDER3, E. M. MUIR2, E. J. BRADBURY1;
1King’s Col. London, London, United Kingdom; 2Univ. of Cambridge, Cambridge, United Kingdom; 3Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

Abstract:

Spinal cord extracellular matrix is densely packed with growth inhibitory chondroitin sulphate proteoglycans (CSPGs), which become more abundant after injury. Thus, matrix modification has become a leading experimental strategy for promoting repair following spinal cord injury. Despite the beneficial effects that have been achieved by digesting CSPGs with the bacterial enzyme chondroitinase ABC (ChABC), the potential for achieving long term efficacy in traumatic injuries that mimic a human spinal cord injury has not yet been realised. Gene therapy offers a route to achieving stable continuous delivery of ChABC and therefore, here we deliver genetically modified ChABC via a lentiviral vector (LV-ChABC) to the adult rat spinal cord and assess the efficacy of chronic gene delivery using a spinal contusion injury model. Contusion injury represents the most common form of spinal cord injury in humans and, therefore, provides a clinically relevant tool for assessing the efficacy of potential therapeutic interventions. Adult rats received a moderate severity thoracic (T10) contusion injury and LV-ChABC or a control LV-GFP was immediately injected rostral and caudal to the injury site. We demonstrate prolonged and widespread CSPG degradation with LV-ChABC and, using both behavioural and electrophysiological outcome measures, we show improved function in animals treated with LV-ChABC. We saw a dramatic increase in spinal conduction through the injury site as well as a significant improvement in performance on the horizontal ladder test. In addition we will determine if the early functional improvements observed using the horizontal ladder test correlate with changes in spinal conduction at early time points. In order to enhance the potential clinical applications of this study we are now assessing the effects of LV-ChABC in a moderate severity cervical (C5) contusion injury. Approximately 50% of all human spinal cord injuries occur at the cervical level making this injury model of particular clinical relevance as well as allowing us to assess a number of additional functional outcomes such as forelimb grip-strength, sensory and motor function during sticky-tape removal, and proprioception using the inclined plane. Thus, we demonstrate the potential advantages of gene delivery of ChABC for achieving sustained and widespread CSPG degradation and that this is associated with functional improvements following contusion injury.
Gene delivery of chondroitinase ABC promotes functional repair following spinal cord injury

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Activation of the spinal locomotor network using exteroceptive stimulation allows the re-expression of locomotor pattern in chronic spinalized adult rats

Authors: *O. ALLUIN, H. DELIVET-MONGRAIN, S. ROSSIGNOL; Dept. of Physiol., Univ. of Montreal, Montreal, QC, Canada

Abstract:

Recent studies in adult rats with complete thoracic spinal cord injury (SCI) have shown that a re-expression of locomotor pattern is possible using systemic administration of serotoninergic drugs associated with epidural electrostimulation, robotic assistance and treadmill training. Unlike the cats, which are capable to spontaneously execute spinal locomotion on a treadmill 2-3 weeks after a complete SCI, it is commonly accepted that strong invasive stimulations are needed to obtain similar results in rats. This consensus suggests that the basic compensatory mechanisms occurring within the lumbosacral spinal cord after SCI could be different between mammals. In the work reported here, we proposed to study the capability of adult spinalized rats to re-express hindlimb locomotion using only mechanical stimulation of the perineum.
A complete SCI was made at T9 in 6 adult female Wistar rats (300-325 g). Rats were then trained to walk in a natural posture (i.e. horizontal) on a treadmill 10 minutes daily, 5 days a week at different velocities (from 14 to 26 m.min-1) for 6 weeks using only manual perineal stimulation while the forepaws were supported on a fixed platform. Throughout this period, movements were recorded using conventional video or fluoroscopy (X-ray) for kinematic analyses. Electromyography of selected hindlimb muscles was also acquired at the end of experiments.
Our results show the re-expression of intrinsic locomotor capability in all rats of the study. Two days after SCI, strong perineal stimulation was necessary to elicit some erratic flexion/extension alternations of the hindlimbs. Then, in the course of the next 3 weeks the locomotor pattern gradually improved while the perineal stimulation intensity needed to produce locomotion decreased. Three to 6 weeks after SCI, all rats were able to perform more than 10 consecutive, well-defined, alternating and normally coordinated step cycles with plantar placements. Six weeks after SCI, no significant difference with normal state was found during swing phase in the hindlimb flexion capability, phase duration and the velocity of the paw. However, at the end of experiments, the amplitude of knee and ankle joints remained significantly decreased and increased respectively.
Thus, as in other vertebrates, the locomotor network within the lumbosacral cord of adult paraplegic rats can re-express hindlimb locomotion on a treadmill without requiring pharmacological or other types of invasive stimulation. However, exteroceptive stimulation is still required. Therefore, this intrinsic locomotor capability should be taken into account when assessing various forms of extrinsic treatments after SCI.
Activation of the spinal locomotor network using exteroceptive stimulation allows the re-expression of locomotor pattern in chronic spinalized adult rats

The group from Montreal, Canada took completely spinalised rats (complete cord transection at T8) and trained them on a treadmill for 11 weeks for 10 mins per day whilst mechanically stimulating the perineal area with a pinch. The mechanical and sensory information flows to the cord and stimulating the lumbar region containing the central pattern generators for locomotion. It was interesting to see the data and video evidence that this simple technique brought about robust recovery of alternating, coordinated hind limb motion. Alluin explained the intensity of the pinch needs to be quite strong at the beginning of training but that this could be reduced significantly as time went on. One obvious extension of this will be to test whether external electrical stimulation in the perineal region also elicits a response.
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Calcium dynamics influence axonal degeneration and recovery following contusion spinal cord injury in vivo

Authors: *P. R. WILLIAMS1, S. E. BEYER1, O. GRIESBECK2, M. KERSCHENSTEINER3, T. MISGELD1;
1Inst. for Neurosci., Tech. Univ. of Munich, Munich, Germany; 2Inst. für Neurobiologie, Max-Planck, Martinsried, Germany; 3Inst. of Clin. Neuroimmunology, Ludwig-Maximilians Univ., Munich, Germany

Abstract:

Following contusion injury to the spinal cord, axons of passage degenerate over a delayed time course. Even axons within very close proximity to one another can demonstrate differential outcomes despite experiencing a very similar physical injury and microenvironment. It is thus difficult to ascertain what determines the fate of individual axons following contusion injury due to the dynamic nature of the lesion. Using in vivo microscopy approaches in transgenic mice, we have directly observed the morphological and ionic dynamics of spinal axons in response to contusion injury during the acute phase (up to 4 hr post injury). We have focused primarily on the role of calcium homeostasis, since rises in intra-axonal calcium have been related to axon degeneration in traumatic brain injury and ex vivo spinal pathology models.
Although a number of axons appear to be directly damaged by the spinal contusion, a considerable fraction undergo swelling and subsequent delayed fragmentation on the order of hours. Interestingly, not all swollen axons degenerate during our observation window, and many axons can be seen to recover normal morphology. We have found considerable variability and dynamics of calcium levels in individual axons. Axons with raised intracellular calcium are more likely to degenerate than those without; however, axonal swellings can develop in the absence of elevated intraaxonal calcium. More surprisingly, axonal calcium rises can spontaneously return to normal levels, which might protect axons from subsequent fragmentation. Furthermore, morphological recovery appears more frequently in axons that maintain normal intracellular calcium levels than in axons with calcium dysregulation. Taken together, our experiments demonstrate that damaged axons can spontaneously recover from both morphological and calcium disturbances. A better understanding of the mechanisms underlying spontaneous axonal recovery relative to axonal breakdown (e.g. differential calcium sources and downstream activation) may allow for interventions that tip the balance from axonal demise to rescue.
Calcium dynamics influence axonal degeneration and recovery following contusion spinal cord injury in vivo

When the cord is injured by an impact some axons that cross the injury site are seen to degenerate over time. Williams wanted to understand why one axon might befall this fate whilst a near neighbour doesn’t even after apparently experiencing a similar trauma – what causes this?

Using an elegant 2-photon microscopy technique he was able to look at what happens early during the first few hours after injury at the resolution of individual axons. He saw spontaneous and previously unappreciated dynamics of damage. Some axons could be seen to swell or “bleb” up before degenerating entirely. Others would bleb but return to normal. When he used a dye to measure calcium concentration he saw that blebbing was most often accompanied by high calcium concentrations in the axon; he was able to demonstrate the cause of this high calcium concentration was most likely due to the axon having holes in the membrane as a result of the trauma allowing calcium to flood in. This was not always the case. Sometimes axons would bleb and return to normal but this was most frequently found in those axons that had maintained their calcium levels near normal.

Overall it was a stunning piece of fundamental research and gave some insight as to importance of maintaining tight control over calcium concentrations in cells (calcium homeostasis) and perhaps even some potential for this technique to screening compounds that shift the balance in these dynamics in the favour of axonal survival after injury.

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