The rhythmic activity of locomotion is most frequently modeled as a periodic oscillation coordinated by a spinal Central Pattern Generator (CPG) controlling reciprocal activation of flexor and extensor muscles. Expression of locomotion errors in the form of spontaneous deletions in the motor output has been critical in formulating models of CPG network structure governing locomotion in mammals (Lafreniere-Roula et al 2005, Duysens 2006). Deletions are defined as the disappearance of either antagonist or agonist muscles’ activity along with the simultaneous tonic/rhythmic activity of the corresponding agonist or antagonist muscles. The formulation of a two-layer model of the CPG (Rhythm Generator (RG) layer & Pattern Formation (PF) layer) by Rybak et al (2006) stems from observations of such deletions in the fictive locomotion of the decerebrated cat. The RG functions as a clock controlling the temporal activity of the PF layer which controls the firing pattern of motor neuron pools that activate muscles. The deletion episodes are said to be “resetting” if the EMG activity after the deletion does not return after an integer value of the pre-deletion average period. If the motoneuron activity returns in phase with the pre-deletion “clock”, the deletion period is considered to be “non-resetting”. Multiunit Activity (MUA) recorded from a spinalised air-stepping cat was analyzed against its corresponding EMG activity to investigate the role of MUA in rhythmogenicity, specifically whether or not MUA activity may represent the RG layer of the Central Pattern Generator (CPG) model. This hypothesis would predict that MUA activity should be disrupted in phase or amplitude when and only when deletions episodes are re-setting.. Alternatively, MUA activity may reflect PF layer activity. In this case MUA activity should be disrupted in phase or amplitude during each of the deletions episodes. MUA’s spatio-temporal characteristics were compared to that of the EMG activity during the deletion periods for analysis. From the analysis performed, there was a significant proportion (average more than 25%) of the MUA (collected from the lumbar region of the spinal cord of spinalized cat) that were disrupted in phase or amplitude during non-resetting deletions or undisrupted during resetting episodes, indicating that MUA activity is unlikely to represent the RG layer activity during . In addition, MUA oscillation during the period of deletions was unchanged (amplitude or phase) for more than 25% of the deletion episodes, ruling out the possibility that MUA represents the activity of the PF layer. So although MUA has been found to be highly synchronized throughout the lumbar extent during locomotor activity, it does not appear to act as a “clocking” mechanism for the locomotor rhythm. / Bioengineering

Modulation of the endocannabinoid system by the administration of exogenous agonists and selective antagonists has been shown to have potential to attenuate the contribution of inflammation to secondary injury in the CNS. The two most well-defined receptors are the CB1 and CB2 receptors. CB2, the cannabinoid receptor expressed primarily on hematopoietic cells and activated microglia, mediates the immunoregulatory functions of cannabinoids. The involvement of CB2 in central nervous inflammation has been demonstrated by using both endogenous and exogenous ligands. We showed previously that CB2 selective agonists inhibited leukocyte rolling and adhesion to CNS microvasculature and ameliorate clinical symptom in both chronic and remitting-relapsing EAE models; and our previous studies also demonstrated therapeutic potential of CB2 agonist improving recovery following spinal cord injury in the mouse. The goal of the current investigation was to evaluate the mechanisms through which administration of a selective cannabinoid-2 (CB2) agonist modifies inflammatory responses and helps to improve function following the injury in central nervous system. In the EAE project, we showed that Gp1a, a highly selective CB2 agonist with a four log higher affinity for CB2 than CB1, reduced clinical scores and facilitated recovery in EAE in conjunction with long term reduction in demyelination and axonal loss. We also established that Gp1a affected EAE through at least two different mechanisms, i.e. an early effect on Th1/Th17 differentiation in peripheral immune organs, and a later effect on the accumulation of pathogenic immune cells in the CNS, associated with reductions in the expression of CNS and T cell chemokine receptors, chemokines and adhesion molecules. This is the first report on the in vivo CB2-mediated Gp1a inhibition of Th17/Th1 differentiation. We also confirmed the Gp1a-induced inhibition of Th17/Th1 differentiation in vitro, both in non-polarizing and polarizing conditions. The CB2-induced inhibition of Th17 differentiation is highly relevant in view of recent studies emphasizing the importance of pathogenic self-reactive Th17 cells in EAE/MS. In spinal cord injury project, we showed that spinal cord injury mice CB2 agonist O-1966 (with affinities to the CB1 and CB2 receptors of 5055±984 and 23±2.1 nM, respectively) had improved motor function, autonomic function. They also had significant reductions in CXCL-9, CXCL-11, dramatic reductions in IL-23p19 expression and its receptor IL-23r, and reduction in the number of immunoreactive microglia. The results reported in this thesis, demonstrated that the combined effect on Th17 differentiation and immune cell accumulation into the CNS, may contribute to the usefulness of CB2 selective ligands as potential therapeutic agents in neuroinflammation. / Physiology

Delivery of neurotrophins to the injury site via cellular transplants or viral vectors administration has previously been shown to promote recovery of locomotor behavior in the absence of locomotor training in adult spinalized animals. Viral vectors still pose clinical concerns associated to recombinant genetics and the lack of understanding of how they react with the human immune system. Delivery via graft of autologous fibroblast engineered to produce brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3) has been shown as a valuable method; however, the need for multiple invasive surgeries, along with the impossibility of delivering a controlled and constant dosage of protein are serious obstacles to obtaining approval by the FDA. The present study was aimed at evaluating the efficacy of BDNF delivered to the lumbar locomotor centers using a clinically translational delivery method at restoring stepping abilities in a large animal model of spinal cord injury. We wanted to evaluate if intrathecal delivery of BDNF to the lumbar spinal cord would promote a locomotor recovery as effective as delivery to the injury site, even at doses low enough not to trigger the side effects observed at high doses. A programmable and implantable mini-pump was used to intrathecally deliver a 50 ng/day dose of BDNF to the lumbar spinal cord for 35 days after spinal thoracic transection. Kinematic evaluation was conducted before, 3 and 5 weeks after injury/pump implant. Ground reaction forces (GRFs) analysis was performed 5 weeks after injury to evaluate the animals’ ability to weight support during locomotion and standing trials. Results showed that treated cats were capable of executing weight-bearing plantar stepping at all velocities tested (0.3-0.8 m/s). Control cats did not recover stepping ability, especially at higher velocities, and dragged their hind paws on the treadmill. We were also interested in measuring the extent of BDNF diffusion within the lumbar area of the spinal cord and the potential damage to the cord caused by catheter insertion. Immunohistological evaluation showed higher BDNF expression in the dorsal root ganglions, with BDNF Immuno-Histo Chemistry (IHC) extending from L3 to L7 in all treated cats. BDNF was also found within multiple cells of the grey matter, although the levels were not significantly higher than background density. Glial fibrillary acidic protein (GFAP) stain was used to measure the immunohistological reaction of the spinal cord to the implanted catheter, and to establish the safety of the delivery method. Gross examination of the spinal cord post-mortem revealed no damage to the cord or the roots with minimal encapsulation of the catheter/pump. Minimal tissue inflammation was revealed by the GFAP stain, underlying the safety of our method. We also wanted to investigate and characterize changes in the locomotor circuitry induced by BDNF delivery. Comparison of multiunit activity in the lumbar area between BDNF treated and non-treated cats allows a better understanding of the mechanism of action of BDNF on the spinal interneurons. This was accomplished by extracellularly recording lumbar interneuronal firing during air-stepping in a 5 weeks post-injury terminal experiment. The cord was exposed at the lumbar level between the L3 and L7 spinal segments. In-vivo recordings of spinal extracellular signals were conducted using two 64 channels microelectrode arrays inserted at the dorsal root entry zone to depths of ~3000µm and ~1500µm. The ability to record simultaneous activity of multiple single neurons made it possible to study the extent to which spiking activity in a given neuron is related to concurrent ensemble spiking activity. A point process generalized linear model (PP-GLM) approach was used to assess the strength of the connections between spike trains. Interneurons activity was assessed in terms of average firing rate, signal-to-noise ratio (SNR), and number of active units per trial. Although BDNF infusion in the lumbar segments did not show significant effect on strengthening synaptic connections, we did find greater multiunit activity in the treated animals, sign of a potential BDNF-induced increase in interneuronal activation, which could be likely involved in recovery of stepping ability after SCI. Together, findings from these aims demonstrated the therapeutic potential of intrathecal lumbar BDNF delivery in spinalized animals. Constant infusion of BDNF to the locomotor centers promotes locomotor recovery similar to training or delivery to the injury site via cellular transplants after complete SCI. Intrathecal delivery by an implantable/programmable pump is a safe and effective method for delivery of a controlled BDNF dosage; it poses minimal risks to the cord and is clinically usable. Lastly, this study confirmed the major involvement of BDNF in increasing the activity of the interneurons in the locomotor circuitry, opening the door to further investigating the mechanism through which neurotrophins induce recovery of locomotion. / Bioengineering

Human spinal cord injury (SCI) results in persistent functional deficits as damaged axons in the mature central nervous system (CNS) fail to regenerate after injury. This is due to both growth-inhibiting compounds, e.g., the myelin-associated growth inhibitors and the chondroitin sulfate proteoglycans (CSPGs), in the extracellular environment, and growth-limiting intrinsic factors. Unlike mammals, the primitive sea lamprey robustly recovers swimming and other locomotor behaviors after complete spinal cord transection (TX), despite the presence of homologues of the mammalian growth-inhibiting molecules. This recovery is accompanied by heterogeneous anatomical regeneration of the reticulospinal (RS) system, which, in lampreys, is the dominant descending pathway for motor control. Within the RS system, there are 18 pairs of identifiable neurons that can be classified as “good” or “bad” regenerators based on the likelihood that their axons will regenerate beyond the TX site. Most bad regenerators undergo a delayed form of caspase-mediated cell death. Because both good and bad regenerators project through the same extracellular environment, investigating their divergent responses to axotomy has the potential to reveal the key intrinsic properties that regulate axon regeneration. And, since lampreys share much of the same CNS organization and signaling pathways with higher order mammals, regeneration mechanisms discovered in lampreys may be useful therapeutic targets in humans with SCI. Lampreys do not express myelin, so the CSPGs probably form the principal extracellular inhibitory component of the injured spinal cord. Mammalian in vitro and in vivo studies suggest that CSPGs bind the LAR-family receptor protein tyrosine phosphatases (RPTPs), PTPσ and LAR, leading to growth inhibiting cytoskeletal remodeling and reduced activity of pro-survival pathways via the small GTPAse, RhoA. Intriguingly, preliminary in situ hybridization experiments with antisense riboprobes revealed that PTPσ is preferentially expressed on bad regenerator neurons. Thus, we hypothesized that differential PTPσ expression may be a key signaling determinant of regeneration. Using antisense morpholino oligomers (MOs) applied to the proximal spinal cord stump immediately after TX, we inhibited PTPσ expression among lamprey RS neurons and assessed its effects on regeneration. Contrary to our hypothesis, PTPσ deletion did not promote supraspinal regeneration or enhance behavioral recovery. Most surprisingly, we observed reduced survival of RS neurons at long timepoints post-TX among the PTPσ knockdown cohort. Western blot analysis, using pan-LAR-family receptor antibodies, indicated that the PTPσ knockdown did not affect expression of other LAR-family receptors. Although these results are the opposite of what we expected, there are several potential biological explanations that may explain why the loss of PTPσ antagonizes survival. Notably, these include interactions with the pro-regenerative PTPσ ligands, heparin sulfate proteoglycans (HSPGS), exacerbation of inflammatory processes, reduced synaptogenesis leading to loss of trophic support, and potentially off-target toxicity. These explanations remain under investigation. Notably, pilot studies involving HSPG digestion using bacterial heperainase III did not recapitulate the knockdown phenotype. Following the surprising results of PTPσ knockdown, we stepped back and considered whether simpler factors between good and bad regenerators may contribute to their divergent response to axotomy. We had long noted that bad regenerators tended to be larger than good regenerators, but generally believed this was an epiphenomenon unrelated to axon regeneration. However, a careful reexamination of primary and historic data uncovered an even stronger inverse correlation between soma cross-sectional area and regenerative ability (r = -0.92) than we had suspected. Using a similar approach, we determined that RS neuron soma size is proportional to axon caliber. Because large axons may reseal more slowly following axotomy than smaller axons, we hypothesized that inefficient axolemmal resealing after axotomy may be a key driver of the degenerative processes observed among bad regenerators. Using dye exclusion assays with 10,000 MW fluorescent dextran tracers, we assessed the rate of axolemma resealing for each of the identifiable neurons. Within 2 hours of TX, 75% of axons from small to medium sized neurons (≤ 20 x102 µm2; B5, I3, I5, mth’, M4, B6, I4, I6, M1, B2, I2) were impermeable to dye compared to only 5% of axons from the larger bad regenerator RS neurons (B1, M3, M2, B4, Mth, B3, I1). Indeed, many of these large bad regenerators remained permeable to dextran dye for more than 24 hours after injury. Importantly, approximately 65% of neurons with axons that remained dye permeable at 24 hours post-TX were positive for active caspases at +2 weeks, compared to only 10% of neurons with sealed axons (p<0.0001***). When axon resealing was artificially induced with the fusogen, polyethylene glycol (PEG), caspase activation was inhibited, suggesting that slow axolemma causatively promotes degeneration among lamprey RS neurons. Although this study did not investigate the underlying mechanisms, we suspect that prolonged influx of toxic mediators in the extracellular environment, particularly calcium, may drive the degenerative response. Together, these results demonstrate that axon regeneration and cell survival after spinal cord TX is a complex process strongly shaped by the intrinsic characteristics of the neurons themselves. Selective expression of putative inhibitory or pro-growth molecules may regulate the regeneration process in ways that can be difficult to predict a priori and with effects that vary among taxa. Because lampreys are one of the few vertebrates to recover after complete SCI, they remain an essential model organism to study true axon regeneration in the CNS. / Neuroscience

<p>Body image is an important component of physical and psychological health. Cash's (2002) cognitive-behavioural model of body image is useful for understanding factors related to body image, such as body composition and leisure time physical activity (LTPA). Changes in body composition and LTPA following spinal cord injury (SCI) may increase the risk of developing an unhealthy body image and may jeopardize the overall health and well-being of people with SCI. Unfortunately, little research had been conducted to explore factors related to body image in the SCI population.</p><p>The object of the current thesis was to examine body image in people with SCI, within the framework of Cash's (2002) model. Specifically, we examined the relationships between appearance and functional satisfaction, LTPA, and body composition in 61 men and women with SCI. Pearson's correlations were calculated to examine the relationship between body composition and body satisfaction. In order to compare body satisfaction between men and women who engaged in some LTPA versus no LTPA, separate 2 (sex) x 2 (LTPA) ANOVAs were calculated for appearance satisfaction and functional satisfaction.</p><p>We hypothesized that body composition would be negatively related to appearance satisfaction and functional satisfaction such that higher body weight, percentage body fat and waist circumference would be associated with lower levels of satisfaction. Further, we hypothesized that after controlling for body composition, participants who engaged in LTPA would have more appearance and functional satisfaction than those who did not.Finally, we hypothesized that body satisfaction would be more strongly related to moderate-intensity LTPA versus mild-intensity LTPA, and resistance-LTPA versus aerobic-LTP A.</p><p>In partial support of our hypothesis, body composition was significantly related to appearance satisfaction for women only. Body composition was not significantly related to functional satisfaction for men or women. The results of the ANOVAs indicate that women were more dissatisfied with their appearance than men. However, contrary to hypothesis, appearance satisfaction did not differ between men or women who engaged in . LTPA versus those who did not. For men and women, those who engaged in mild or moderate intensity LTPA were less satisfied with their physical function than those who did not. Further, participants who engaged in resistance-LTPA were less satisfied with their physical function that those who did not.</p><p>Results are discussed within the framework of Cash's model. Findings suggest that people who engage in LTPA are less satisfied with their bodies than people who do not. This finding was surprising because LTPA has been shown to improve body image in the SCI population (Hicks et al., 2003). However, dissatisfaction with physical appearance or physical function may motivate individuals to engage in LTPA. The theoretical and practical implications of the current thesis are discussed. Prospective research is necessary to further understand the directionality of the relationship between the constructs of Cash's (2002) model, and to test SCI-specific variables as potential mediators and moderators of the relationship between LTPA and body image.</p> / Thesis / Master of Science (MSc)

During inpatient rehabilitation, arm-ergometry training is utilized to improve the physical capacity of patients with a sub-acute spinal cord injury (SCI) to a level that is desirable for performing activities of daily living (ADLs). Previous work has demonstrated that ≥ 20 minutes of moderate-intensity continuous training (MICT) during inpatient rehabilitation, at a frequency of ≥ 3 times per week, is useful for increasing the physical capacity of these patients. However, considering that inpatient rehabilitation is an intensive program, and given the trend towards a shortened length of stay during inpatient rehabilitation, performing MICT on the arm-ergometer can consume a valuable amount of therapy time. Low-volume sprint interval training (SIT) is a time-efficient alternative to MICT for improving indices of physical fitness in healthy and diseased populations. To date, however, there are no published studies comparing SIT to MICT in persons with sub-acute SCI undergoing inpatient rehabilitation.The purpose of this thesis was to evaluate the efficacy of a five-week, thrice weekly low-volume SIT protocol on the arm-ergometer and compare fitness outcomes to traditional MICT in patients with sub-acute SCI undergoing inpatient rehabilitation. Participants with sub-acute SCI undergoing inpatient rehabilitation were recruited and randomly allocated to the SIT or MICT training group. Both types of training utilized the same 2 min. warm-up and 3 min. cool-down. SIT consisted of 3 x 20 sec. “all-out” cycle sprints (≥ 100% of peak power output [POpeak]), interspersed with 2 min. of low activerecovery (≈ 10% of POpeak; total time commitment, 10 mins). MICT involved 20 min. of arm cycling (45 – 60% of POpeak; total time commitment, 25 mins). SIT elicited a higher relativheart rate response, and ratings of perceived exertion than MICT. Following training, we found similar improvements in maximal and sub-maximal physical capacity across groups. Both exercise modes were equally well tolerated, and enjoyable, and there were no differences in self-efficacy across groups. The significance of this work is that it is the first randomized-controlled trial comparing SIT to MICT on the arm-ergometer in individuals with sub-acute SCI undergoing inpatient rehabilitation. The fact that SIT is palatable and can promote similar increases in physical capacity as MICT, despite less than half the time commitment and training volume, means that clinical rehabilitation specialists can now offer a new, more time-efficient, exercise training strategy to elicit improvements in their patients. / Thesis / Master of Science (MSc)

Chronic pain is a frequent and debilitating comorbidity of SCI (Ravenscroft et al., 2000). Although exercise is an effective strategy for managing pain in other chronic pain populations (e.g., Ettinger et al., 1997), exercise training has not been previously examined in the SCI population. In a RCT of 34 sedentary men and women with traumatic SCI, the effects of exercise on perceived pain and physical and psychological well-being were examined. Additionally, the efficacy of exercise as a pain management strategy was assessed. Exercisers performed aerobic and resistance training twice weekly over 9-months. Controls maintained their usual level of activity. Measures of pain (Ware & Sherbourne, 1992), physical well-being (Reboussin et al., 2000), stress (Cohen et al., 1992), depression (Radlof, 1977) and subjective well-being (Cantril 1965; Patrick et al., 1988) were administered at baseline and at the 3, 6 and 9 months points of the intervention. A series of ANCOV As adjusted for baseline scores revealed a significant group main effect for the measures of pain, stress, depression and subjective well-being which reflected improvement in all of these domains for the exercisers (i.e., decreased pain, stress and depression and increased subjective well-being) and decrement in all of these domains for the controls (ps<.05). Hierarchical linear regression analyses (cf. Baron & Kenny, 1986) revealed that change in physical well-being partially mediated change in pain, change in pain mediated change in stress and subjective well-being and change in stress mediated change in depression. These findings suggest that variables mediating exercise-induced change should be targeted to maximize the effectiveness of exercise as a pain management strategy for individuals with SCI. The therapeutic and theoretical implications of these findings are discussed. / Thesis / Master of Science (MS)

This study investigated potential alterations in brain white matter after spinal cord injury (SCI). Individuals with SCI underwent magnetic resonance diffusion tensor imaging (DTI) and alterations were assessed based on dynamic changes in fractional anisotropy (FA) and apparent diffusion coefficient (ADC) parameters. Three contrast analyses compared FA and ADC values between person with SCI and healthy controls for the Boston cohort, the Denver cohort, and the grouped cohort. Persons with SCI were found to have lower FA and higher ADC values than health controls across all cohorts. These results are consistent with established correlations between DTI values and alteration of brain white matter in other chronic neurodegenerative conditions. This suggests that DTI is a useful tool for measuring white matter changes in the brain following SCI.

The present study aims to elucidate the mechanisms underlying locomotor recovery following spinal cord injury (SCI) through the investigation of Brain-Derived Neurotrophic Factor (BDNF) delivery, and inflammatory responses associated with different spinal transection methods.Chapter 2 focuses on characterizing lumbar interneurons' activity during air- stepping following chronic intrathecal BDNF delivery to the lumbar spinal cord. BDNF has demonstrated the potential to elicit full locomotor recovery in untrained spinal animals, suggesting therapeutic benefits for SCI patients. However, the effects of BDNF on large populations of neurons responsible for this recovery are not well understood. The hypothesis is that intrathecal BDNF delivery will result in significantly increased neuronal activity in the L3-L4 segments during air-stepping. A programmable, implantable mini- pump was used to deliver BDNF at 50 ng/day for 35 days post-transection. Kinematic data was collected before and after BDNF delivery, and multiunit extracellular recordings were be obtained using 64-channel microelectrode arrays. Results from analysis suggest that while BDNF evidently increases neuronal excitability in treated cats, development of locomotor recovery seems to be achieved through subtle changes in neuronal activity. Chapter 3 investigates the mechanisms behind instances of spontaneous locomotor recovery observed in the literature, which could involve endogenous BDNF or other beneficial mechanisms. It compares locomotor recovery between open-dura and closed- dura spinal transection methods in cats. Previous studies have reported inconsistent outcomes regarding spontaneous recovery and the need for treadmill training. The ii hypothesis is that an open-dura transection will lead to better recovery during treadmill locomotion in untrained spinal cats. Kinematic data and ground reaction forces were measured to assess locomotor parameters and weight-bearing abilities, providing a quantitative analysis of recovery. The results show that an open-dura transection is associated with the development of spontaneous locomotor recovery in untrained spinal cats. Chapter 3 also examines differences in the inflammatory response at the lower thoracic cord between the two spinal transection methods, given the significant role of inflammation in CNS repair and recovery. The hypothesis is that the open-dura method will result in a higher inflammatory response, characterized by increased macrophages, microglia, and BDNF levels caudal to the transection site. Immunohistochemistry (IHC) and RNA in-situ hybridization assays were used to analyze the cellular and molecular environment near the injury site. Open dura animals show a decrease inflammatory response to injury and show no evidence of endogenous BDNF caudal to the injury. These results suggest the development of spontaneous locomotor recovery associated with a transected dura can be elicited through inflammatory mechanisms alone without the need for neurotrophic intervention. / Bioengineering

Body weight supported treadmill training (BWSTT) is being promoted as an effective means of restoring ambulatory abilities among individuals with incomplete spinal cord injuries. The emphasis of this thesis is on the description of the metabolic demands and the EMG profiles of able-bodied persons and individuals with a spinal cord injury (SCI) while walking under the identical conditions on a body weight support (BWS) treadmill. The secondary purpose was to contrast the metabolic and muscular responses between the two groups. Two separate chapters describing the metabolic demands and EMG profiles respectively follow the review of the literature. The metabolic results indicate that raising the speed and/or decreasing the amount of BWS increase the intensity of BWS treadmill walking, with speed having a more profound effect. The SCI group was less efficient and they had greater metabolic rates of oxygen consumption than the controls for all conditions examined. This led to the conclusion that walking on the treadmill, for the SCI group can provide an effective aerobic exercise stimulus. The EMG profiles suggest that speed and BWS affect the phasic characteristics of the muscular activity while walking for both groups. Furthermore, abnormalities, omissions and inappropriate levels of activity were observed in the SCI group when compared to the controls. These irregularities suggest that the SCI participants have adopted altered motor strategies while walking, relative to the control group. Nonetheless, the SCI participants showed evidence of appropriate modulations in their EMG activity to meet the demands of the task as they changed from one condition to the next. / Thesis / Master of Science (MSc)