It was hypothesized that superparamagnetic nanoparticles encapsulated in a silica shell with a fluorescent dye could be functionalized with axonal tracers and could be used for serial, non-invasive imaging with magnetic resonance imaging (MRI) for axon tract tracing. Nanoparticles functionalized with amine, octadecyl, silica, and biotinylated dextran amine were manufactured and characterized with MRI, scanning electron microscopy, and UV-visible, infrared, and fluorescence spectroscopy. Nanoparticle concentrations of 10 mM were not toxic to adult rat neural progenitor cells (NPCs) and labeled approximately 90% of cells. Nanoparticles were assessed for anterograde and retrograde tract tracing in adult rat models. With MRI and microscopy, the nanoparticles did not appear to trace axons but did provide an MRI signal for up to 3 weeks post implantation. While functionalized nanoparticles did not appear to trace axons, they are not toxic to NPCs and may be used as a MRI contrast agent in the neural axis.

Every year, several thousand individuals suffer spinal cord injury (SCI) in North America, while 1.5 million suffer traumatic brain injury in the U.S.A. alone. Primary mechanical trauma to the CNS is followed by a complex pathology, including vascular dysregulation, ischemia, edema and traumatic hemorrhage. Secondary damage is to a large extent caused by oxidative stress and inflammatory processes, resulting in necrosis and apoptosis of neural cells. If secondary tissue injury could be limited by interference with any of the pathomechanisms involved, preservation of structure and function would increase the potential for functional recovery. Experiments performed in other laboratories have shown that the polyphenolic flavonoid quercetin acts as an anti-oxidant and anti-inflammatory, reduces edema formation and apoptotic cell death. Quercetin is also an excellent iron chelator. This action profile suggested a high therapeutic potential for acute CNS trauma. Therefore, I used models of both spinal cord injury and head trauma in adult male rats to test the hypothesis that administration of quercetin is beneficial for the therapy of acute traumatic CNS injury. While the primary focus of my work was on therapy of acute traumatic spinal cord injury, quercetin was also evaluated in the settings of chronic SCI and acute head trauma. I found that, in a rat model of mid-thoracic spinal cord compression injury, 1) administration of quercetin, starting 1 hr after injury and continued every 12 hr, improved recovery of motor function in the hind limbs in more than half of the injured animals to a degree that allowed previously paraplegic animals to step or walk. The minimum quercetin dose that was efficacious was 5 µmol/kg. The minimum treatment duration for optimal outcome was determined to be 3 days. In control animals, some spontaneous recovery of motor function did occur, but never to an extent that allowed animals to step or walk. Quercetin administration was associated with more efficient iron clearance from the site of injury, decreased inflammatory response as reflected in decrease of myeloperoxidase activity and decreased apoptosis of neural cells at the site of injury. 2) Quercetin administered in the same injury model as late as 2 weeks after injury, given in a higher dose than that used for treatment in the acute phase, still resulted in significant recovery of motor function in 40% of the injured animals, although at a lower level of performance, when compared to early onset of treatment. 3) Quercetin administered after moderate fluid percussion brain injury resulted in decreased oxidative stress, as reflected in higher tissue glutathione levels at the site of injury. In animals receiving quercetin, the amplitude of compound action potentials was significantly better maintained at 24 hr and 72 hr after injury than in saline-treated control animals. My experiments have shown that the flavonoid quercetin is neuroprotective in a rat model of brain trauma and in a rat model of spinal cord injury. My data show that administration of quercetin after CNS trauma promotes iron clearance, decreases oxidative stress and inflammation. Quercetin also decreases apoptotic cell death following neurotrauma. These results suggest that quercetin may be a valuable adjunct in the therapy of acute CNS trauma. There is a possibility that administration of quercetin may be beneficial even in certain settings of chronic CNS trauma. These conclusions are based solely on the results from animal experiments. However, the fact that few adverse reactions have been noted to date in either animal experiments or human trials targeting other diseases is encouraging for the progression to human clinical trials for patients with spinal cord injury.

Trauma-induced spinal cord injury (SCI) is the most prevalent form of spinal cord injury affecting over 80% of the 36,000 Canadians living with this condition. The pathophysiological profile of traumatic SCI consists of an initial stage of direct damage followed by a series of secondary events, including reduced blood flow and increased generation of free radicals that leads to excitotoxicity, oxidative stress, hemorrhagic necrosis, inflammation, and apoptosis. We examined the hypotheses that delayed administration of the flavonoid quercetin inhibits the propagation of secondary events and promotes functional recovery after traumatic SCI by inhibiting inflammatory processes and signaling pathways that promote apoptosis and thereby promoting axon survival. To determine whether delayed quercetin treatment promoted functional recovery following SCI, male Wistar rats were subjected to a spinal cord compression injury by application of a 50 g modified aneurysm clip at the mid thoracic cord level. A treatment regimen of 75 µmol quercetin per kg rat or saline only (controls) was administered for a period of 3 days, 1 week or 2 weeks beginning at 2 weeks post surgery. Delayed quercetin treatment improved locomotion in injured animals although with severe deficit. To determine whether improved functional outcome correlated with improved tissue preservation and reduced scarring, we performed histological examinations at the injury site. In saline treated animals, at 8 weeks post injury we found over 80% of tissue loss with the majority of the remaining cells undergoing apoptosis. However, with 2 weeks delayed quercetin treatment, at least 50% of the tissue was still present at 8 weeks post surgery with a significant reduction of apoptosis. Quercetin treated animals also showed a reduction of reactive gliosis. To determine which intracellular signaling pathways may mediate the protective effects of quercetin, we carried out Western blots and immunocytochemical analyses of a number of potential pro-apoptotic pathways. We found that quercetin reduced the levels of the phosphorylated (activated) forms of the MAPK p38, ERK 1/2 (p42/44) and SAPK/JNK seen after SCI. We conclude that delayed quercetin treatment likely rescues neurons that would otherwise have died between the third and sixth weeks following injury by inhibiting apoptosis of glia cells. Quercetin may be acting via selective inhibition of kinase pathways that have been shown to be involved in apoptosis and cell growth. These findings not only reveal the protective effects of quercetin in reducing secondary damage after chronic SCI but also shed some light on some of the mechanisms underlying its actions.

Objectives: To describe the relationship between sensorimotor function and walking in incomplete SCI. Methods: 25 subjects were assessed using Lower Extremity Motor (LEMS) and Pinprick (LEPS) scores, and 7 walking measures: FIM-Locomotor Score, Assistive Device Score, Walking Index for SCI, 10-metre Walk Test (10mWT), Timed Up and Go (TUG), Six-Minute Walk Test (6MWT) and Walking Mobility Scale. Results: Walking and sensorimotor function varied between subjects. Walking measures significantly correlated with LEMS and individual leg muscles but not LEPS. 21/22 ambulatory subjects had LEMS threshold>20. Non-ambulatory subjects didn’t achieve threshold. Not all subjects completed all walking measures: 10mWT: n=19; TUG: n=14, 6MWT: n=13. Most walking measures were significantly related. 10mWT and 6MWT were highly correlated. Subjects walking0.95 m/s didn’t reach predicted 6MWT. Conclusion: Lower extremity strength is important for walking and should be further examined with other factors in a range of subjects across different measures to fully understand these relationships.

Objective: To describe the walking characteristics of a sample of ambulatory subjects with sub-acute incomplete spinal cord injury (iSCI). Methods: 52 subjects were included in a secondary analysis of clinical and spatiotemporal measures of walking. The study sample was described as a whole and subsequently divided into subgroups on the basis of 3 clinical factors (etiology, severity, and neurological level of injury) and 4 gait factors (gait aid, velocity, symmetry, and variability). Results: Clinical and spatiotemporal parameters were highly variable across the study population. Sub–groups with unique gait features were best identified by velocity and variability. Conclusions: Spatiotemporal measures of walking provide augmented description of walking in the sub-acute iSCI population. Sub-grouping by gait factors warrants further investigation with respect to their ability to act as predictors and modifiers of treatment effect.

Objective: To describe the walking characteristics of a sample of ambulatory subjects with sub-acute incomplete spinal cord injury (iSCI). Methods: 52 subjects were included in a secondary analysis of clinical and spatiotemporal measures of walking. The study sample was described as a whole and subsequently divided into subgroups on the basis of 3 clinical factors (etiology, severity, and neurological level of injury) and 4 gait factors (gait aid, velocity, symmetry, and variability). Results: Clinical and spatiotemporal parameters were highly variable across the study population. Sub–groups with unique gait features were best identified by velocity and variability. Conclusions: Spatiotemporal measures of walking provide augmented description of walking in the sub-acute iSCI population. Sub-grouping by gait factors warrants further investigation with respect to their ability to act as predictors and modifiers of treatment effect.

Spinal cord injury (SCI) pathophysiology occurs as a primary traumatic event followed by secondary injury, resulting in the loss of neurons, oligodendrocytes and demyelination of residual axons. Unfortunately, endogenous spontaneous regeneration of oligodendrocytes is minimal. Previously, a method to generate multi-potential stem cells (MPSC) from umbilical cord blood (UCB) has been reported using lineage negative cells (Linneg) grown in fibroblast growth factor 4 (FGF4), stem cell factor (SCF) and fms-like tyrosine kinase receptor-3 ligand (Flt-3l) supplemented serum free medium. These MPSC have the ability to differentiate into bone, muscle and endothelial cells. In this thesis, the ability of MPSC to differentiate into oligodendrocytes was investigated as a potential treatment for SCI. Culturing MPSC under conditions that mimic normal timing of oligodendrocyte differentiation resulted in cells that expressed oligodendrocyte markers in vitro and were morphologically similar to them. I next investigated the ability of MPSC to improve functional recovery in a SCI compression injury model. Although the cells did not differentiate into oligodendrocytes in vivo as we initially hypothesised, a modest but significant improvement in hindlimb function was observed. A cytokine assay revealed that MPSC secrete elevated levels of anti-inflammatory, angiogenic and neurotrophic factors, possibly contributing to indirect mechanisms of repair by reducing secondary injury. Shiverer mouse neonates were next used as an alternative non-injury model to investigate the differentiation potential of MPSC. We hypothesised that transplanting MPSC into a host with an immature immune system and an actively myelinating environment would lead to engraftment and differentiation into oligodendrocytes. However no MPSC that differentiated into oligodendrocytes could be detected. Altogether, our in vitro data adds support for the reprogramming of cells, with further studies needed to test the functionality of resulting oligodendrocyte-like cells. Although MPSC failed to differentiate in both in vivo models, several potential therapeutic targets to treat SCI were found.

Objectives: To describe the relationship between sensorimotor function and walking in incomplete SCI. Methods: 25 subjects were assessed using Lower Extremity Motor (LEMS) and Pinprick (LEPS) scores, and 7 walking measures: FIM-Locomotor Score, Assistive Device Score, Walking Index for SCI, 10-metre Walk Test (10mWT), Timed Up and Go (TUG), Six-Minute Walk Test (6MWT) and Walking Mobility Scale. Results: Walking and sensorimotor function varied between subjects. Walking measures significantly correlated with LEMS and individual leg muscles but not LEPS. 21/22 ambulatory subjects had LEMS threshold>20. Non-ambulatory subjects didn’t achieve threshold. Not all subjects completed all walking measures: 10mWT: n=19; TUG: n=14, 6MWT: n=13. Most walking measures were significantly related. 10mWT and 6MWT were highly correlated. Subjects walking0.95 m/s didn’t reach predicted 6MWT. Conclusion: Lower extremity strength is important for walking and should be further examined with other factors in a range of subjects across different measures to fully understand these relationships.

Spinal cord injury (SCI) pathophysiology occurs as a primary traumatic event followed by secondary injury, resulting in the loss of neurons, oligodendrocytes and demyelination of residual axons. Unfortunately, endogenous spontaneous regeneration of oligodendrocytes is minimal. Previously, a method to generate multi-potential stem cells (MPSC) from umbilical cord blood (UCB) has been reported using lineage negative cells (Linneg) grown in fibroblast growth factor 4 (FGF4), stem cell factor (SCF) and fms-like tyrosine kinase receptor-3 ligand (Flt-3l) supplemented serum free medium. These MPSC have the ability to differentiate into bone, muscle and endothelial cells. In this thesis, the ability of MPSC to differentiate into oligodendrocytes was investigated as a potential treatment for SCI. Culturing MPSC under conditions that mimic normal timing of oligodendrocyte differentiation resulted in cells that expressed oligodendrocyte markers in vitro and were morphologically similar to them. I next investigated the ability of MPSC to improve functional recovery in a SCI compression injury model. Although the cells did not differentiate into oligodendrocytes in vivo as we initially hypothesised, a modest but significant improvement in hindlimb function was observed. A cytokine assay revealed that MPSC secrete elevated levels of anti-inflammatory, angiogenic and neurotrophic factors, possibly contributing to indirect mechanisms of repair by reducing secondary injury. Shiverer mouse neonates were next used as an alternative non-injury model to investigate the differentiation potential of MPSC. We hypothesised that transplanting MPSC into a host with an immature immune system and an actively myelinating environment would lead to engraftment and differentiation into oligodendrocytes. However no MPSC that differentiated into oligodendrocytes could be detected. Altogether, our in vitro data adds support for the reprogramming of cells, with further studies needed to test the functionality of resulting oligodendrocyte-like cells. Although MPSC failed to differentiate in both in vivo models, several potential therapeutic targets to treat SCI were found.

It was hypothesized that superparamagnetic nanoparticles encapsulated in a silica shell with a fluorescent dye could be functionalized with axonal tracers and could be used for serial, non-invasive imaging with magnetic resonance imaging (MRI) for axon tract tracing. Nanoparticles functionalized with amine, octadecyl, silica, and biotinylated dextran amine were manufactured and characterized with MRI, scanning electron microscopy, and UV-visible, infrared, and fluorescence spectroscopy. Nanoparticle concentrations of 10 mM were not toxic to adult rat neural progenitor cells (NPCs) and labeled approximately 90% of cells. Nanoparticles were assessed for anterograde and retrograde tract tracing in adult rat models. With MRI and microscopy, the nanoparticles did not appear to trace axons but did provide an MRI signal for up to 3 weeks post implantation. While functionalized nanoparticles did not appear to trace axons, they are not toxic to NPCs and may be used as a MRI contrast agent in the neural axis.