Adult mammalian central nervous system (CNS) neurons are unable to extend axons after injury, partially owing to the inhibitory myelin and chondroitin sulfate proteoglycans (CSPGs) present in the environment. A neuron's intrinsic state is also important for determining its regenerative potential. Peripheral nervous system (PNS) neurons, unlike their CNS counterparts, have increased ability to regrow their axons after injury, even in the presence of inhibitory molecules. With the goal of discovering novel regeneration associated genes, we have isolated the genes differentially expressed by PNS neurons. We then developed a high throughput neuronal transfection method to test whether these genes were sufficient to modify neurite growth in vitro. Using high content screening, we measured the ability of cerebellar neurons to initiate neurite outgrowth on inhibitory and permissive substrates. This combination of technologies (subtractive hybridization, microarray, high throughput electroporation and high content screening) allowed phenotypic examination of neurons after the overexpression of over a thousand genes. Additionally, kinases and phosphatases were assayed for their ability to modify neurite outgrowth in hippocampal neurons. Results from both of these large unbiased screens confirmed many of the existing candidates for neurite growth during development and regeneration. We also discovered many novel genes which promoted neurite outgrowth such as GPX3, EIF2B5, RBMX, CHKA, IRF6, and PKN2. To accurately interpret the large volume of data, new methods of analysis were performed. Finally, we developed novel techniques that took advantage of public databases to cluster genes and determine whether those clusters produced robust changes in neurite growth. In summary, we have provided a vast repository of functional data to study axon development and regeneration after injury as well as developing the tools needed to interpret that data.

Persons with spinal cord injury (SCI) suffer life-long consequences including paralysis, loss of involuntary bodily functions, and chronic pain. A subset of SCI patients develop neuropathic pain (NP), a chronic condition resulting from damage to the spinal cord. Hyperexcitability of spinal cord sensory neurons near damaged tissue is believed to underlie SCI-related NP. Although many therapies have been employed clinically to combat SCI-NP, few give satisfactory long-term relief. Transplantation of cells that release GABA, a molecule that inhibits neuronal activity, is being explored as an alternative to current SCI-NP therapies. My experiments made progress toward preclinical modeling of GABA cell therapy for SCI-NP. First, I sought to determine whether quisqualic acid (QUIS)-induced SCI altered responses to tonic pain stimuli or altered GABAergic neural circuitry in rats. Second, I sought to determine whether a combination of genetic and trophic manipulations could promote a GABAergic phenotype in rat embryonic neural precursor cells (NPCs) in an in vitro culture system. The results revealed that QUIS-SCI rats exhibit unusually prolonged nocifensive responses to hind paw formalin injections. There was no significant difference between QUIS-SCI and sham surgery rats in c-Fos immunolabeling of spinal cord sensory neurons after formalin-induced neuronal activity. However, immunohistochemistry revealed substantial decreases in staining for markers of GABA presynaptic vesicles in injured spinal cord tissue. NPCs were enriched for a neuronal phenotype by combining withdrawal of the growth factor FGF-2 from culture media and overexpression of the transcription factor MASH1 in transfected cells. Although glial marker expression was suppressed in NPCs by these manipulations, expression of neuronal markers none the less declined through time. MASH1-overexpressing NPCs exhibited greater clonal expansion and decreased stress-induced PDI expression after FGF-2 withdrawal as compared to naïve. In light of existing data, these results suggest that the QUIS-SCI model may be useful for testing the efficacy of GABAergic NPC transplantation to reduce neuropathic pain. MASH1 overexpression and FGF-2 withdrawal could serve as a first step toward enriching GABA in NPCs for transplantation. Although the mechanism for MASH1 cytoprotection remains unclear, MASH1 may enhance survival of NPCs grafted into the spinal cord. These experiments contributed to the preclinical basis for application of therapeutic GABAergic stem cell transplantation for NP in human SCI patients.

In individuals with spinal cord injury (SCI) who have disrupted communication between the brain and spinal cord, vibration may mimic functions formerly served by the lost or impaired supraspinal inputs resulting in more normal reflex modulation and improved walking function. Three experiments assessed the effects of vibration on spinal locomotor-generating circuitry, spinal reflex activity, and walking function. In Experiment 1, localized leg vibration was used to elicit air-stepping responses in the lower extremities. We compared responses of individuals with SCI to those of non-disabled (ND) individuals and assessed the influence of severity injury and locomotor training on the air-stepping response in individuals with SCI. Our results indicate that vibration of the tensor fascia latae elicited more consistent and robust responses than vibration of the quadriceps or hamstrings muscles. Individuals with SCI had less consistent and robust responses than ND individuals. In those with SCI, neither severity of injury nor locomotor training influenced the robustness or consistency of the response. In Experiment 2, we investigated the effect of whole-body vibration (WBV) on spasticity, as measured by spinal stretch reflex (SSR) excitability, in individuals with SCI. We also assessed differences in the influence of WBV among individuals who used antispastic medications and those who did not. Subjects were tested before and after participation in a 3 day/week, 12-session WBV intervention. There was a significant reduction in spasticity that persisted for several days following the WBV intervention. The amount by which spasticity was reduced was not different in those who used antispastic agents compared to those who did not use these agents. In Experiment 3, we assessed the effects of the 12-session WBV intervention on walking function. WBV was associated with significant increases in walking speed, cadence, step length of the stronger leg, and consistency of hip-knee intralimb coordination. Increases in cadence and stronger-leg step length correlated with improvements in walking speed. These results suggest that WBV may represent an approach to decreasing spasticity, and may be useful for individuals in whom spasticity interferes with function. Furthermore, vibration appears to have a beneficial effect on walking function, perhaps by influencing spinal locomotor-generating circuitry.

Spinal cord injury (SCI) often results in irreversible paralysis and widespread oligodendrocyte death and white matter damage. While the mechanisms underlying this phenomenon are poorly understood, previous studies from our laboratory indicate that inhibition of astroglial-NF-κB activation reduces white matter damage and improves functional recovery in a mouse model of SCI. Here we provide novel evidence demonstrating that astrocytes directly regulate oligodendrocyte fate after trauma by a glutamate-mediated AMPA receptor dependent mechanism. Following trauma, elevated expression of the SLC39a10 zinc transporter correlated with an increase in zinc uptake by astrocytes, thereby reducing extracellular zinc concentrations required for AMPA receptor inhibition. Stimulation of AMPA receptors on oligodendrocytes by glutamate induced oligodendrocyte toxicity through the activation of the NADPH oxidase enzyme within oligodendrocytes. Genetic and pharmacological inhibition of active NADPH oxidase was sufficient to attenuate oligodendrocyte death in vitro. Following SCI, NADPH oxidase inhibition reduced oligodendrocyte death by ~75%, suggesting that glutamate-mediated oligodendrocyte death is dependent on the activation of the NADPH oxidase enzyme within oligodendrocytes. Combined treatment of the NADPH oxidase inhibitor apocynin and the AMPA receptor inhibitor NBQX significantly improved hind limb locomotor behavior, reduced white matter damage and lesion volume, and significantly spared descending serotonergic fibers. These studies provide a novel mechanism of oligodendrocyte death and may lead to clinically relevant therapeutics after SCI.

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.