published_or_final_version / Anatomy / Master / Master of Philosophy

Peroxynitrite (PN, ONOO-), formed by nitric oxide radical (•NO) and superoxide radical (O2•-), plays an important role in post-traumatic oxidative damage. In the early work, we determined the temporal characteristics of PN-derived oxidative damage in a rat spinal cord injury (SCI) model. Our results showed 3-nitrotyrosine (3-NT), a specific marker for PN, rapidly accumulated at early time points (1 hr, 3 hrs), after when it plateaued and the high level was sustained to 1 week post injury. The co-localization of 3-NT and lipid peroxidation derived-4-HNE observed in immunohistochemistry indicates PN is involved in lipid peroxidative as well as protein nitrative damage. PN-oxidative damage exacerbates intracellular Ca2+ overload, which activates Ca2+ dependent calpain-mediated cytoskeletal protein (α-spectrin) degradation. The 145 kD fragments of α-spectrin (SBDP 145), which are specifically generated by calpain, increased dramatically as early as 1 hr after injury although the peak increase did not occur until 72 hrs post injury. The high level waned back toward sham level at one week post injury. We then carried out experiments to evaluate the beneficial effects of tempol, a scavenger of PN-derived radicals, following SCI. Three pathological events including PN-induced oxidative damage, mitochondrial dysfunction and cytoskeletal degradation were investigated. Immunoblotting and immunohistochemical studies indicated PN-mediated oxidative damage including protein nitration, protein oxidation and lipid peroxidation, were all reduced by a single dose of tempol (300mg/kg, i.p) after SCI. Spinal cord (SC) mitochondrial dysfunction in terms of the respiratory control ratio (RCR) significantly improved by both 150 mg/kg and 300 mg/kg tempol treatments. Moreover, calpain-mediated proteolysis was significantly decreased by tempol, with greater effects on calpain-specific SBDP 145 observed. Direct PN-scavenging effect of tempol was confirmed in vitro. Exposure of healthy SC mitochondria to SIN-1, a PN donor in vitro, impaired mitochondrial respiration in a dose-dependent manner. Tempol was able to protect mitochondria against SIN-1-induced damage by improving mitochondrial function and decreasing mitochondrial 3-NT formation. These findings strongly support the concept that PN is a crucial player in the secondary damage following SCI. And tempol, by scavenging PN-induced free radicals, provides a promising pharmocotherapeutic strategy for treating acute SCI.

In the hours and days following acute CNS injury, a secondary wave of events is initiated that exacerbate spinal tissue damage and neuronal cell death. A potential mechanism driving these secondary events is opening of the mitochondrial permeability transition pore (mPTP) and subsequent release of several cell death proteins. Previous studies have shown that inhibition of cyclophilin D(CypD), the key regulating component in mPTP opening, was protective against insults that induce necrotic cell death. We therefore hypothesized that CypD-null mice would show improved functional and pathological outcomes following spinal cord injury (SCI) and traumatic brain injury (TBI). Moderate and severe spinal contusion was produced in wild-type (WT) and CypD-null mice at the T-10 level using the Infinite Horizon impactor. Changes in locomotor function were evaluated using the Basso Mouse Scale (BMS) at 3 days post-injury followed by weekly testing for 4 weeks. Histological assessment of tissue sparing and lesion volume was performed 4 weeks post SCI. Calpain activity, measured by calpain-mediated spectrin degradation, was assessed in moderate injury only by western blot 24 hours post SCI. Results showed that following moderate SCI, CypD-null mice had no significant improvement in locomotor recovery or tissue sparing compared to wild-type mice. Following severe SCI, CypD-null mice showed significantly lower locomotor recovery and decreased tissue sparing compared to WT mice. Calpain-mediated spectrin degradation was not significantly reduced in CypD-null mice compared to WT mice 24h post moderate SCI. The lack of protective effects in CypD-null mice suggests that more dominant mechanisms are involved in the pathology of SCI. In addition, CypD may have a pro survival role that is dependent on the severity of the spinal cord injury.

Spinal Cord Injuries (SCIs) are debilitating injuries where damage to the spinal cord causes a loss of mobility and feeling in muscles innervated below the injury point. Tetraplegia refers to an SCI in the cervical region of the spinal cord that impacts on the functionality of all four limbs. ‘Complete’ tetraplegia results in complete paralysis of the legs, partial or complete paralysis of the arms and trunk, and in the most severe cases, the neck. The independence of people living with tetraplegia is heavily dependent on assistive and mobility devices. Understanding the strength characteristics of people with tetraplegia is crucially important for the suitable and effective design of mobility and rehabilitative devices such as wheelchairs. A study using a stationary dynamometer and video capture measured kinetic and kinematic characteristics of wheelchair propulsion for 15 subjects with C5-C7 tetraplegia. This study differentiated between subjects with different injuries, at two different test resistances, and was more comprehensive than other reported studies on MWC propulsion. Some of the subjects in the study with C5-C6 injuries had no elbow extension capability, while others had undergone a deltoids-to-triceps tendon transfer procedure called TROIDS, which restores some elbow extension capability. No differences were found in any of the push phase metrics between those who had undergone the TROIDs procedure, and those who had not, suggesting that TROIDs provides no significant benefit for mobility. As expected, subjects with C7 tetraplegia recorded velocity and power outputs significantly higher than those for subjects with C5-C6 tetraplegia. To better understand the strength characteristics over the full range of motion in the sagittal plane, and thus potentially modify the design of mobility devices to better suit these characteristics, a novel method for gathering strength data in multiple directions and positions was developed. This method had advantages over other commonly used methods. In particular, it was inclusive of complex muscle and joint interactions that would otherwise be very difficult to build into a model. Sagittal horizontal push strength was measured using this method for 8 able bodied and 4 tetraplegic subjects. There were clear trends in the data from the able-bodied subjects, and a fourth order polynomial (R-squared = 0.8) was fitted to the data for modelling purposes. Data for the tetraplegic subjects varied significantly from the able-bodied data, but inter-individual variation was such that no model would provide a satisfactory fit to the data indicating a very high degree of patient-specific behaviour. One multi-directional data set, consisting 1584 measurements in the sagittal plane, was gathered for an able-bodied subject. The main trends in this measured data were successfully captured by a model consisting of twelve fourth-order polynomials. Building on these measurements, and employing a human model in the constraint modelling environment, SWORDS, this thesis develops a conceptual design tool for comparing the effectiveness of different hand force paths. Initial simulations using hypothetical hand paths indicated that the proposed method for predicting the direction of the applied force needs to be verified, and likely refined, for hand paths that differ significantly from the traditional wheelchair push-rim path. This proposed procedure has the potential to be a powerful tool for optimising and modifying the design of wheelchairs or human powered devices to utilise previously untapped abilities for any given population.

Knowledge mobilization has been described as putting research in the hands of research users. Few studies have examined knowledge mobilization within community-based organizations (CBOs). To address this research gap, this dissertation examines knowledge mobilization within a CBO that supports people with spinal cord injury (SCI). Research suggests that communication channels, or the means by which a message is delivered, may affect knowledge mobilization. This dissertation presents four manuscripts examining how communication channels were used by the CBO to disseminate physical activity guidelines and intervention strategies to people with SCI. Manuscript 1 investigated reach and effectiveness of an event-based knowledge mobilization initiative delivered by the CBO using interpersonal communication channels to disseminate the guidelines to people with SCI. Results indicated that the event-based knowledge mobilization initiative was effective for initial dissemination of the guidelines. However, further long-term efforts are needed. Manuscript 2 describes how the event-based knowledge mobilization initiative was implemented by the CBO. Recognizing that support personnel are important messengers, the reach and effectiveness of the initiative for persuading support personnel to disseminate the guidelines was examined. Results indicated that an event-based knowledge mobilization initiative can be implemented by a CBO and may be an effective strategy for CBOs to disseminate information to support personnel. Manuscript 3 further examined the role of communication channels in the process of knowledge mobilization within the CBO using network analysis. Results indicated that CBO staff and volunteers’ integration within interpersonal communication channels was associated with greater knowledge of evidence-based physical activity resources and engagement in physical activity promotion behaviours. Manuscript 4 examined the feasibility of training peers with SCI to learn an evidence-based approach to physical activity promotion called Brief Action Planning. Findings indicated that Brief Action Planning is a tool that can be feasibly learned and potentially used by peers to promote physical activity to their mentees through interpersonal communication channels. Overall this dissertation contributes to a small but emerging body of literature examining knowledge mobilization in CBOs. Findings indicate that through a community-university multidisciplinary partnership and appropriate communication channels, a CBO can effectively and feasibly disseminate evidence-based physical activity information. / Thesis (Ph.D, Kinesiology & Health Studies) -- Queen's University, 2013-08-28 17:27:32.841

Parkinson’s disease, the second most common neurodegenerative disorder,is characterized by loss of nigrostriatal dopaminergic neurons. To date,there is no defined cause and cure for the disease. An ideal treatmentstrategy is to replace the lost neurons by transplanting fetal dopaminergicneurons to the brain of parkinsonian patients. Clinical trials have beenperformed and the outcome was variable where one significant obstaclewas the limited graft reinnervation of the host brain. To study this issue,organotypic tissue culture can be utilized to monitor dopaminergic nervefiber outgrowth in vitro and their association with astrocytes. Using thisculture technique, dopaminergic nerve fibers appear in twomorphologically and temporally different types. The early appearing nervefibers are formed in the absence of astrocytes, reach long distances, andare called non-glial-associated tyrosine hydroxylase (TH) -positive nervefibers. After a few days, the second sequence of nerve fibers, the glialassociatedTH-positive nerve fibers, are formed, and their growth arelimited to the presence of astrocytes, that migrate and form a monolayersurrounding the plated tissue. The aim of this thesis was to study theinteraction between nerve fiber formation and astrocytes with a specialfocus on the long-distance growing nerve fibers. Ventral mesencephalic(VM) organotypic slice cultures from embryonic day (E) 12, E14, and E18were incubated for 14, 21, 28, and 35 days in vitro (DIV). The resultsrevealed that the two morphologically different processes were found incultures from the younger stages, while no non-glial-associated growthwas found in cultures of tissue from E18. Instead neurons had migratedonto the migrating astrocytes. Astrocytes migrated longer distances intissue from older stages, and the migration reached a plateau at 21 DIV.Co-cultures of E14 VM tissue pieces and cell suspension of matureastrocytes promoted migration of neurons, as seen in E18 cultures. Thus,9the maturity of the astrocytes was an important factor for nerve fiberoutgrowth. Hence, targeting molecules secreted by astrocytes might bebeneficial for regeneration. Chondroitin sulfate proteoglycan (CSPG), amember of proteoglycan family, is produced by the astrocytes and has adual role of being permissive during development and inhibitory afterbrain injury in adult brain. Cultures were treated with chondroitinase ABC(ChABC) or methyl-umbelliferyl-β-D-xyloside (β-xyloside) in twodifferent protocols, early and late treatments. The results from the earlytreated cultures showed that both compounds inhibited the outgrowth ofnerve fibers and astrocytic migration in cultures from E14 tissue, while β-xyloside but not ChABC promoted the non-glial-associated growth incultures derived from E18 fetuses. In addition, β-xyloside but not ChABCinhibited neuronal migration in E18 cultures. Taken together, β-xylosideappeared more effective than ChABC in promoting nerve fiber growth.Another potential candidate, integrin-associated protein CD47, was studiedbecause of its role in synaptogenesis, which is important for nerve fibergrowth. Cultures from E14 CD47 knockout (CD47-/-) mice were plated andcompared to their wildtypes. CD47-/- cultures displayed a massive and longnon-glial-associated TH-positive nerve fiber outgrowth despite theirnormal astrocytic migration. Blocking either signal regulatory protein-α(SIRPα) or thrombospondin-1 (TSP-1), which bind to CD47, had nogrowth promoting effect. In conclusion, to promote nerve growth, youngertissue can grow for longer distances than older tissue, and inhibiting CSPGproduction promotes nerve growth in older tissue, while gene deletion ofCD47 makes the astrocytes permissive for a robust nerve fiber growth.

Bakgrund: Ryggmärgens huvudsakliga funktion är att skicka och ta emot nervimpulser för rörelse och känsel. När en ryggmärg skadas innebär det förluster eller nedsättningar av kroppsliga funktioner och förmågor. Omfattningen av funktionsnedsättningen är beroende på vilken nivå i ryggmärgen som skadan sker. Vid en traumatisk ryggmärgsskada har omvårdnaden en stor betydelse för den fortsatta hälsan och sjuksköterskor har beskrivit att de har en mångsidig roll i arbetet. Att ha en funktionsnedsättning kan innebära ett behov av hjälp från andra personer, samt upplevelser av utanförskap och en exkludering från samhälleliga arenor. Syfte: Att belysa personers upplevelser av att leva med en traumatisk ryggmärgsskada. Metod: En litteraturöversikt baserad på tolv vetenskapliga artiklar; elva kvalitativa och en med mixad metod från databaserna CINAHL complete och Psycinfo. Vid sökning i databaserna användes ämnesord som Spinal Cord Injuries, Patients attitudes. Artiklarna har analyserats i enlighet med Fribergs metod. Resultat: Två huvudteman identifierades utifrån Roys adaptionsmodell, Adaptiv respons och ineffektiv respons. Varje huvudtema har tillhörande underteman. Under adaptiv respons återfanns, anhörigas roll i vardagslivet, hoppets betydelse, nya insikter och förändrade perspektiv och betydelsen av meningsfulla aktiviteter. Under ineffektiv respons återfanns upplevelser av att vara beroende, upplevelser av en förändrad identitet, viljan att avsluta sitt liv, upplevelser av ett förlorat hopp och livets ständiga kamp. Diskussion: I metoddiskussionen diskuteras metodens styrkor respektive svagheter. Under rubriken resultatdiskussion diskuteras litteraturöversiktens viktigaste resultat som var anhöriga, hopp, psykisk ohälsa och aktiviteter mot litteraturöversiktens bakgrund, ny litteratur samt mot Roys adaptionsmodell. / Background: The Spinal cord's main function is to send and receive nerve impulses for movement and sensation. Damage of the spinal cord means loss or impairment of bodily functions and abilities. The extent of the disability depends on the level of the spinal cord injury. At a traumatic spinal cord injury, care has a great importance for the continued health. Nurses have described that they have a versatile role in that work. Having a disability can mean a need for help from other people and the experiences of alienation and exclusion from social arenas. Aim: To illustrate people's experiences of living with a traumatic spinal cord injury. Method: A literature review based on twelve scientific articles; eleven qualitative and one with mixed method from the databases CINAHL Complete and PsycINFO. When searching in the databases, some of the terms that was used were Spinal Cord Injuries, Patients, Attitudes. The articles were analyzed according to Fribergs method. Results: Two main themes were identified from the Roy Adaptation Model, adaptive response and ineffective response. Each main theme has related sub-themes. Sub themes in adaptive response are, relatives role in everyday life, importance of hope, new insights and altered perspectives and the importance of meaningful activities. In ineffective response there are experiences of being dependent, experiences of an altered identity, the desire to end one’s life, experiences of a lost hope and life's constant struggle. Discussion: In the method discussion, strengths and weaknesses of the method are discussed. Under the heading discussion of results, the most important results of the literature overview that were relatives, hope, mental health and activities are discussed in comparison with the results overview background, new literature and Roy's adaptation model.

Traumatic injuries to the spinal cord and brachial plexus induce a significant inflammatory response in the nervous tissue with progressive degeneration of neurons and glial cells, and cause considerable physical and mental suffering in affected patients. This thesis investigates the effects of the antioxidants N-acetyl-cysteine (NAC) and acetyl-L- carnitine (ALC) on the survival of motoneurons in the brainstem and spinal cord, the expression of pro-apoptotic and pro-inflammatory cell markers, axonal sprouting and glial cell reactions after spinal hemisection in adult rats. In addition, a novel MRI protocol has been developed to analyse the extent of neuronal degeneration in the spinal cord. Rubrospinal neurons and tibial motoneurons were pre-labelled with the fluorescent tracer Fast Blue one week before cervical C3 or lumbar L5 spinal cord hemisection. The intrathecal treatment with the antioxidants NAC (2.4mg/day) or ALC (0.9 mg/day) was initiated immediately after injury using Alzet2002 osmotic mini pumps. Spinal cord injury increased the expression of apoptotic cell markers BAX and caspase 3, induced significant degeneration of rubrospinal neurons and spinal motoneurons with associated decrease in immunoreactivity for microtubule-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker glial fibrillary acidic protein and microglial markers OX42 and ED1 was markedly increased. Treatment with NAC and ALC attenuated levels of BAX, caspase 3, OX42 and ED1 expression after 2 weeks postoperatively. After 4-8 weeks of continuous intratheca ltreatment, NAC and ALC rescued approximately half of the rubrospinal neurons and spinal motoneurons destined to die, promoted axonal sprouting, restored the density of MAP2 and synaptophysin immunoreactivity and reduced the microglial reaction. However, antioxidant therapy did not affect the reactive astrocytes in the trauma zone. The inflammation modulating properties of ALC were also studied using cultures of human microglial cells. ALC increased the microglial production of interleukin IL-6 and BDNF, thereby possibly mediating the anti-inflammatory and pro-regenerative effects shown in vivo. To study degeneration in the spinal cord following pre-ganglionic and post-ganglionic brachial plexus injuries, adult rat models of ventral root avulsion and peripheral nerve injury were used. A novel MRI protocol was employed and the images were compared to morphological changes found in histological preparations. Ventral root avulsion caused degeneration of dendritic branches and axonal terminals in the spinal cord, followed by significant shrinkage of the ventral horn. Extensive astroglial and microglial reactions were detected in the histological preparations. Peripheral nerve injury reduced the density of dendritic branches but did not cause shrinkage of the ventral horn. Quantitative analysis of MRI images demonstrated changes in the ventral horn following ventral root avulsion only, thus validating the developed MRI technique as a possible tool for the differentiation of pre-ganglionic and post-ganglionic nerve injuries.

Peripheral nerve stimulation (PNS) is commonly used to promote use-dependent cortical plasticity for rehabilitation of motor function in spinal cord injury. Pairing transcranial magnetic stimulation (TMS) with PNS has been shown to increase motor evoked potentials most when the two stimuli are timed to arrive in the cortex simultaneously. This suggests that a mechanism of timing-dependent plasticity (TDP) may be a more effective method of promoting motor rehabilitation. The following thesis is the result of applying a brain-computer interface to apply PNS in closed-loop simultaneously to movement intention onset as measured by EEG of the sensorimotor cortex to test whether TDP can be induced in incomplete spinal cord injured individuals with upper limb motor impairment. 4 motor incomplete SCI subjects have completed 12 sessions of closed-loop PNS delivered over 4-6 weeks. Benefit was observed for every subject although not consistently across metrics. 3 out of 4 subjects exhibited increased maximum voluntary contraction force (MVCF) between first and last interventions for one or both hands. TMS-measured motor map volume increased for both hemispheres in one subject, and TMS center of gravity shifted in 3 subjects consistent with studies in which motor function improved or was restored. These observations suggest that rehabilitation using similar designs for responsive stimulation could improve motor impairment in SCI.

<p>Chronic pain is a distressing, prevalent, and expensive condition that is not well understood and difficult to treat. Spinal cord stimulation (SCS) has emerged as a viable means of managing chronic pain when conventional therapies are ineffective, but the efficacy of SCS has improved little since its inception. The mechanisms underlying SCS, in particular the neuronal responses to SCS, are not well understood, and prior efforts to optimize SCS have focused on electrode design and spatial selectivity without considering how the temporal aspects of SCS (stimulation frequency, pattern) may affect neuronal responses to stimulation. The lack of a biophysical basis in prior attempts to optimize therapy may have contributed to the plateau in the clinical efficacy of SCS over time. This dissertation combines computational modeling and in vivo electrophysiological approaches to investigate the effects of SCS on sensory neuron activity in the dorsal horn and uses the insights gained from these experiments to design novel temporal patterns for SCS that may be more effective than conventional therapy.</p><p>To study the mechanisms underlying SCS, we constructed a biophysically-based network model of the dorsal horn circuit consisting of interconnected dorsal horn interneurons and a wide dynamic range (WDR) projection neuron and representations of both local and surround receptive field inhibition. We validated the network model by reproducing cellular and network responses relevant to pain processing including wind-up, A-fiber mediated inhibition, and surround receptive field inhibition. To quantify experimentally the responses of spinal sensory projection neurons to SCS, we recorded the responses of antidromically identified sensory neurons in the lumbar spinal cord during 1-150 Hz SCS in both healthy rats and neuropathic rats following chronic constriction injury (CCI). In a subset of rats, we additionally assessed the impact of GABAergic inhibition on spinal neuron responses to SCS by conducting SCS experiments following the intrathecal administration of bicuculline, a GABAA receptor antagonist, and CGP 35348, a GABAB receptor antagonist. Finally, we used the computational model to design non-regular temporal patterns capable of inhibiting sensory neuron activity more effectively than conventional SCS and at lower equivalent stimulation frequencies than clinical standard 50 Hz SCS, and we experimentally validated model predictions of the improved efficacy of select patterns against conventional SCS.</p><p>Computational modeling revealed that the response of spinal sensory neurons to SCS depends on the SCS frequency; SCS frequencies of 30-100 Hz maximally inhibited the model WDR neuron consistent with clinical reports, while frequencies under 30 Hz and over 100 Hz excited the model WDR neuron. SCS-mediated inhibition was also dependent on GABAergic inhibition in the spinal cord: reducing the influence GABAergic interneurons by weakening their inputs or their connections to the model WDR neuron reduced the range of optimal SCS frequencies and changed the frequency at which SCS had a maximal effect. Experimentally, we observed that the relationship between SCS frequency and projection neuron activity predicted by the Gate Control circuit described a subset of observed SCS-frequency dependent responses but was insufficient to account for the heterogeneous responses measured experimentally. In addition, intrathecal administration of bicuculline, a GABAA receptor antagonist, increased spontaneous and evoked activity in projection neurons, enhanced excitatory responses to SCS, and reduced inhibitory responses to SCS, consistent with model predictions. Finally, computational modeling of dual frequency SCS, implemented by delivering two distinct frequencies simultaneously to distinct fiber populations, revealed frequency pairs that were more effective at inhibiting sensory neuron activity than equivalent conventional SCS and at lower average frequencies than clinically employed 50 Hz SCS. Experimental assessments of the effect of dual frequency SCS on spinal sensory neurons confirmed model predictions of greater efficacy at lower equivalent stimulation frequencies and suggest the use of non-regular temporal patterns as a novel approach to optimizing SCS. The outcomes of this dissertation are an improved understanding of the mechanisms underlying SCS, computational and experimental tools with which to continue the development and improvement of SCS. The insights and knowledge gained from the work described in this dissertation may result in translational applications that significantly improve the therapeutic outcomes of SCS and the quality of life of individuals affected by chronic pain.</p> / Dissertation