Spinal cord injury (SCI) has presented itself as a multifaceted pathology that is largely inhibitory to regeneration, and therefore to functional recovery, even though spinal cord neurons have been found to be innately regenerative. Thus, having identified the key players in the inhibition of this innate regeneration, SCI researchers have focused on two major types of approaches: (1) blocking inhibitory cues and (2) promoting innate regeneration. Schwann cells (SCs) have long been shown to promote and enhance functional recovery after SCI through providing supplemental myelination and trophic and tropic factors to regenerating axons, though singular approaches rarely address the complex SCI pathology. Guidance channels and scaffolds have been shown to provide physical support and directional cues to regeneration axons. Therefore, a combinatorial approach in which SCs migrate into and throughout a guidance scaffold would be an ideal research focus for treating SCI. However, cell migration into guidance scaffolds has been shown to be problematic. This study attempts to assess and improve two- and three-dimensional SC migration on electrospun scaffolds. Additionally, we evaluate the ability of SCs, seeded on Matrigel-coated electrospun scaffolds, to improve functional recovery in rats with completely transected spinal cords.

This study examines the potential of Schwann cells (SCs) to be manipulated by electric fields (EFs) in order to improve recovery from spinal cord injury (SCI). It had long been believed that the central nervous system (CNS) is incapable of regeneration, but recent studies have proven otherwise. SC transplants are known to be useful in promoting axon regeneration after SCI, but is not sufficient for functional recovery. EFs are known to exist in vivo, and have been known to drastically affect the morphology and behavior of cells in various tissues. It was the hypothesis of this study that the conditioning and observed alignment of SCs was a reproducible phenomenon that could promote the growth of axons. It was found that SCs could be aligned at various field strengths and preliminary data suggest that aligned SCs increased the length and directionality of axons extending from DRG explants.

Introduction - This thesis focuses on the reaction of cardiovascular system to Lokomat therapy in patients with lesion of the spinal cord. It observes reaction of the heart rate and systolic blood pressure and compares with the cardiovascular response to the exercise on an upper body ergometer. Methods - We recruited 25 patients with spinal cord lesion under Th 5 and 4 patients with lesion above Th 5. The blood pressure was measured. The heart rate was measured by by ambulatory blood pressure monitor and an auscultatory method using a mercury manometer and a stethoscope a sporttester. Patients were measured during the Locomat therapy and the exercise on an upper body ergometer during the 6-minute arm test. Results - The parameters of systolic blood pressure and heart rate had a tendency to increase with the peak in 25th minute of the therapy. There was no significant difference between the patients with lesion under or above Th5. The parameters of heart rate were significantly higher on the upper body ergometer than on Lokomat. Conclusion - The Lokomat therapy does have a positive influence to cardiovascular system of patients with complete or incomplete spinal cord injury. When using this approach, the most important benefit is the possibility to activate all parts of body. Keywords: spinal cord injury,...

La locomotion implique l'activité coordonnée de réseaux d'interneurones spinaux inhibiteurs et excitateurs qui génèrent le rythme et le pattern de la décharge des motoneurones et de la contraction musculaire. La maturation de la transmission inhibitrice au niveau des motoneurones chez le rat se produit durant la période périnatale. Dans cette fenêtre temporelle, les projections provenant du tronc cérébral commencent à envahir l'élargissement lombaire de la moelle épinière. Nous avons étudié les effets du blocage de la mise en place des afférences supraspinales dans la moelle lombaire par transection complète de la moelle épinière (SCT) à la naissance (P0), sur l'expression des sous-unités des récepteurs GABAA et glycine (RcGABAA et RcGly) au niveau des motoneurones lombaires. Nous concluons en une plasticité différentielle des récepteurs de l'inhibition en réponse à une section spinale néonatale. La première étape de notre seconde étude était d'évaluer les modifications de la transmission synaptique inhibitrice sur des motoneurones lombaires innervant des muscles fléchisseurs et extenseurs après SCT complète chez des rats adultes. Une étude longitudinale a montré une évolution différentielle de l'expression des RcGly et des RcGABAA au cours des mois qui suivent la lésion mais qui est a peu près identique sur les Mns fléchisseurs et extenseurs. Nos résultats montrent que chez les rats avec SCT, l'entrainement permet d'acquérir une « locomotion spinale » qui résulte d'interactions dynamiques entre un programme moteur dans la moelle sous lésionnelle, et des feedback proprioceptifs. / Maturation of inhibitory postsynaptic transmission onto motoneurons in the rat occurs during the perinatal period, a time window during which pathways arising from the brainstem reach the lumbar enlargement of the spinal cord. There is a developmental switch in miniature IPSCs (mIPSCs) from predominantly long-duration GABAergic to short-duration glycinergic events. We investigated the effects of a complete neonatal [postnatal day 0 (P0)] spinal cord transection (SCT) on the expression of Glycine and GABAA receptor subunits (GlyR and GABAAR subunits) in lumbar motoneurons. In P7 animals with neonatal SCT (SCT-P7), the GlyR densities were unchanged compared with controls of the same age, while the developmental down regulation of GABAAR was prevented. After spinal cord injury, the disruption of flexion/extension and left-right alternations is largely attributed to a deterioration of the inhibitory circuitry below the lesion, but most of the cellular mechanisms are still unknown. Our aim of this was to measure the alteration of the GABA and glycinergic synaptic transmission on lumbar motoneurons (Mns) after spinal cord transection (SCT) in the adult rat, and evaluate the benefit of manual training and stepping recovery on the inhibitory networks. All in all our results show that, the presynaptic and postsynaptic components of the glycinergic synapses are relatively preserved on lumbar Mns. We developed a manual training procedure, based on daily alternate phases of imposed stepping and free walking in enriched environment. Pharmacological treatment with 5-HT2 receptor agonists allowed a standing recovery and alternate stepping.

Thesis title: Pathological consequences of hypokinesis in paraplegic patient and their identification by TVS method The thesis focus: The focus of the thesis was to identify and classify differences of mechanical properties of axial systems between paraplegic patient and people from healthy population. Secondary focus of the thesis was to identify and classify changes of mechanical properties of axial system in paraplegic patient depending on physiotherapeutic intervention. The research methods: Three people, one paraplegic person and two healthy people were measured with TVS (Transfer Vibration Through Spine) method. Paraplegic person was measured twice, before and after one hour long physiotherapeutic intervention (PI). Healthy people were measured once, without PI. TVS method is a diagnostic apparatus that enables qualitative and quantitative classification of properties of axial skeleton and its segments. It is based on fact that velocity of mechanical waves and density of mechanical energy transmitting through axial skeleton is affected by properties of this system. Thus we were able to analyze and identify mechanical properties of measured axial systems according to differences of input and output values of acceleration on particular spinous processes. The results: When we compared frequency...

Philosophiae Doctor - PhD / Given the devastating and debilitating impact of spinal cord injuries (SCI) globally and the effects on any population, its impact extends far beyond just the victim to people and institutions surrounding them and supporting them post-injury. Of growing concern is the increased risk that individuals with SCI have been seen to have a three-fold greater risk of developing cardio-vascular disease (CVD) than their able-bodied counterparts. Prevention strategies to curtail the onset of CVD in the SCI population is limited, and often developed for individuals from developed countries. The overall aim of this study was to assess and explore the need to implement CVD prevention programmes in a regional South African population with individuals after sustaining a traumatic spinal cord injury (TSCI). The study employed a mixed methods approach and was conducted in four (4) phases. Permission and ethics clearance were obtained from the Research Ethics Committee at the University of the Western Cape (UWC) and the Western Cape Department of Health. Phase One of the study utilized a questionnaire to collect TSCI incidence data of which 108 of the eligible 132 cases consented to take part in the study. The demographic findings of this study indicated that a person sustaining a TSCI in the Cape Metropolitan area in South Africa is most likely to be a male, young (20 to 29 years of age), from a Black African or Coloured race group and living in the Cape Flats suburbs. The male to female ratio was 6.2:1. The main cause of TSCI was assault at 58.33% (n=63) with males accounting for the majority of cases (88.89%, n=65). According to the AIS classification, ASIA A and D were the most common classification seen in 38.89% (n=42) and 39.81% (n=43) of the cohort respectively. Phase Two utilized a questionnaire and looked at CVD risk factors of the original cohort. A large portion of the cohort was engaged in high-risk behaviours, i.e. smoking and alcohol consumption. A low number of individuals reported a baseline history of hypertension diagnosis prior to their TSCI (5.56%, n=6). Phase Three of the study emplored semi-structured interviews and a focus group discussion to explore the experiences of persons with a TSCI regarding their ability to be physically active once reintegrated back into the community. Despite understanding the associated benefits of physical activity, several barriers to being physically active were reported; factors within their homes, access within their community, and transportation. The present study’s findings illustrated a growing concern among the SCI population for increased risk for developing CVD due to decreased physical activity. Phase Four of the study utilised a scoping review to identify CVD prevention programmes for individuals with a TSCI. Physical activity has been shown to have numerous health benefits of which reducing the risk of CVD is one. Engaging in physical activity, whether it be structured, unstructred or through a sporting activity can play a major role in combating the onset of CVD. Other tools used in reducing the onset of CVD were seen to be self-management strategies of which contrayer views were seen both for and against their use. Conclusion: Better education during the rehabilitation phase might be a key component to individuals with TSCI injury making more informed decisions about prioritising physical activity as they attempt to reintegrate back into their respective communities. The removal of socio-environmental barriers could allow motivated TSCI individuals better access to choosing how to increase their physical activity levels.

Metastatic epidural spinal cord compression (MESCC) occurs when tumour invades the epidural space and compresses the spinal cord. Despite Level 1 evidence that surgery is the most effective treatment for MESCC, there is controversy regarding the role of surgery because of fear that patients who have a short survival will spend a large fraction of their remaining life recovering from surgery and potential complications. This controversy could be resolved by decision-analysis of MESCC treatments using quality-adjusted-life-years (QALYs). There have been two barriers to conducting decision-analysis of QALYs for MESCC: (a) lack of utility data, and (b) skepticism regarding decision-analysis. The first four research chapters in this thesis address these barriers. The final research chapter reports a decision-analysis of QALYs on the role of surgery in MESCC. Chapter 1 provides background information on the controversy regarding surgical treatment for MESCC and the rationale for each of the subsequent chapters. Chapter 2 reports a psychometric validation study of a web-based utility valuation module for MESCC. In Chapter 3, application of this module to a general population utility valuation study with a market research panel is described. In Chapter 4, the beneficial properties of Bayesian statistical analysis to minimizing “arbitrariness” in probabilistic sensitivity analysis are described in relation to prognostication for MESCC. Chapter 5 presents a strategy for simplifying and enhancing the transparency of Markov cohort simulation. Finally, the work presented in the research chapters is applied in Chapter 6 to conduct Markov cohort simulation to determine if patients with short survival derive net health-related quality-of-life benefit from surgery. Pragmatic research around barriers to decision-analysis of QALYs for MESCC was conducted to resolve the controversy regarding the role of surgery in the treatment of MESCC. Under most circumstances, MESCC patients who can ambulate prior to treatment derive net HRQoL benefit from surgery, even if prognosis is poor. Non-ambulatory patients can derive net HRQoL benefit but only if the morbidity of surgery is relatively low. It is my hope that the work used to address barriers to decision-analysis of QALYs will be disseminated and applied in other clinical problems.

Cerca de 0,005% da população mundial sofre de lesão medular. O processo regenerativo do tecido nervoso apresenta limitada capacidade para repor as células danificadas (JOHANSSON et al., 1999) e produzir inibidores de crescimento dos axônios associados com a mielina para formação de cicatriz glial (OLSON, 2002). Apesar de resultados promissores, ainda existem controversas quanto ao uso de células-tronco. A eliminação da cicatriz glial, os benefícios de sua formação em diferentes fases e a avaliação da liberação de inibidores de crescimento axonal podem ser parâmetros de análise para o tratamento medular. A enzima condroitinase ABC atua nessa lesão. Neste trabalho avaliamos a interrupção do processo de liberação de inibidores axonais da cicatriz da glia em um tempo não agudo de 7 dias da lesão, sem descartar seus benefícios na fase de formação. Nosso objetivo foi utilizar terapia celular e estabelecer um protocolo de tratamento eficaz, criando uma linha de pesquisa nos estudos da lesão medular. Foi utilizado um grupo de coelhos experimental com realização de hemissecção dorsal e instituído o uso da condroitinase ABC, por aplicação, com micro injeção a curto prazo da lesão. Foi aplicada célula-tronco mesenquimal no foco da lesão após o tratamento da cicatriz da glia com a enzima. Avaliamos por imunohistoquimica a liberação de glial fibrillary acidic protein (GFAP) e sulfato de condroitina proteoglicano (SCPg) nos tecidos após o tratamento no qual foi pretendido fechar algumas lacunas e evitar falhas já descritas, e abrir uma nova esperança no tratamento de pacientes com lesão medular. Nossos resultados ainda mostraram um entendimento superficial sobre a enzima e sua ação sobre cicatrização da glia em associação com o implante celular. Foi aberta uma nova linha de questionamento sobre os benefícios causados à regeneração medular previamente a aplicação de células-tronco. / Around 0,005% of global human population is affected by Spinal Cord Injury (SCI). The regenerative process of neural tissue shows a limited capacity to replace damaged cells (JOHANSSON et al., 1999) and to produce growth inhibitors of associated axons with myelin to create glial scar (OLSON, 2002). Plenty of studies are being developed with stem cell and, despite successful results, there still are controversial opinions. The elimination of the glial scar, the benefits of its growth at different stages and the assessment of axonal growth inhibitors\' release can be parameters of analysis for treating spinal cord. The enzyme chondroitinase ABC acts in this lesion. In this paper we evaluated the release interruption of axonal inhibitors of glial scar in a non-acute 7 days term from injury, not disregarding its benefits during growth. Our goal was to use cell therapy and establish an effective treatment protocol, creating a research line for studies of spinal cord injury and its treatment. A group of rabbits was used under experimental model, conducting dorsal hemisection and application of chondroitinase ABC with micro injection in short-term injury. Mesenchymal stem cells were applied in the lesion focus after the glial scar treatment with the enzyme. Immunohistochemically, we evaluated the release of glial fibrillary acidic protein (GFAP) and sulfate chondroitin proteoglycan (SCPg) in tissues after treatment which was intended to close some gaps and avoid failures described above, and open a new hope in the treatment of patients with spinal cord injury. Our results also showed superficial understanding of the enzyme and its action on glial scarring in association with cell implant. It has opened a new line of questioning about the benefits due to spinal cord regeneration prior to application of stem cells.

A lesão medular é uma injúria do sistema nervoso central que resulta em grande déficit funcional. É uma condição relacionada ao desenvolvimento industrial e tecnológico e acomete principalmente adultos jovens em idade produtiva. Apesar de algumas tentativas terapêuticas estarem sob investigação, ainda não há tratamento eficaz que garanta recuperação funcional. A maioria das estratégias utilizadas visa estimular eventos de neuroplasticidade. A cúrcuma é um composto dietético que tem potencial terapêutico por interferir em processos inflamatórios, oxidativos e imunológicos. O presente estudo investigou possíveis efeitos terapêuticos da cúrcuma após hemissecção medular em ratos. O protocolo experimental foi aprovado pelo Comitê de Ética em Uso de Animais da Escola de Artes, Ciências e Humanidades. Os animais foram submetidos à hemissecção medular após a medula espinhal ser exposta por laminectomia. Os animais foram divididos em 2 grupos experimentais: i) lesionado com cúrcuma (LC); ii) lesionado sem tratamento (LS). Os testes comportamentais BBB Score, Combined Behavior Score, plano inclinado e Beam Walking foram realizados 24h, 72h e 7 dias depois da lesão para avaliar o reaprendizado medular e a recuperação funcional. Os resultados demonstraram melhora do comportamento sensitivo-motor dos animais, provavelmente envolvendo neuroproteção das vias de tato, dor e pressão. As propriedades anti-inflamatórias e antioxidantes da cúrcuma podem ter levado à melhora funcional. Análises celulares e moleculares são necessárias para entendermos melhor os efeitos no epicentro da lesão depois do tratamento com a cúrcuma. Um dos possíveis efeitos prováveis é sobre a conversão de alguns ácidos graxos envolvidos na inflamação, como o ácido araquidônico, diminuindo sua ação e gerando ganhos funcionais. O uso da cúrcuma na fase aguda da hemissecção medular interferiu positivamente na recuperação sensitivo-motora deflagrando, portanto, eventos de neuroproteção e neuroplasticidade / The spinal cord injury is a condition that results in severe functional deficit. It is related to the industrial and technological development and affects mainly young adults. Despite some therapeutic trials are under investigation, there is no treatment that guarantees functional recovery. Most of those strategies for recovery involve neuroplasticity. Curcumin is a dietetic compound with known therapeutic potential, interfering on inflammatory, oxidative and immunological processes. The present study investigated the effects of the treatment with curcumin on acute phase of spinal cord hemi-section in rats. The protocol has been approved by Ethics Committee of School of Arts, Sciences and Humanities. The animals were submitted to hemi-section after laminectomy and were divided in two groups: i) injured with curcumin (LC); ii) injured without treatment (LS). The following behavioral tests were performed 24h, 72h and 7 days after lesion: BBB Score, Combined Behavior Score, inclined plane and Beam Walking test aiming to evaluate medular relearning and functional recovery. The tests indicated an improvement of the sensitive-motor behavior probably by neuroprotection of tactile, pain and pressure pathways. The anti antiinflammatory and antioxidant properties of curcumin could result in functional improvement. Cellular and molecular analysis are necessary to better understand the effects could be over the conversion of some fatty acids involved on inflammation, such as arachidonic acid, reducing its action and promoting functional gain. The use of curcumin in the acute phase of spinal cord hemi-section seems to interfere positively in sensorial-motor recovery triggering neuroprotection and plasticity

The goal of this study was to create pre-formed neural spheroid networks (NSN) on a polydimethyl siloxane (PDMS) concave microwell array for eventual implantation into the rat brain. Recent studies have shown that stem cells have great potential in treating various neurological insults of the central nervous system, ranging from traumatic brain and spinal cord injury, to neurodegenerative disorders. However, the use of stem cell lines in research are controversial due to the method of obtaining cells, in their formation of teratomas and degeneration into cancer cells, their non-specific differentiation, and lastly in their inability to control the location of neural connections. A novel approach to address this issue utilizes pre-formed neural networks consisting of neural spheroids on polymer scaffolds for the implantation into the rat brain. Yet, it was observed that the cylindrical shape of the wells hindered the transfer process. This study aimed to overcome the lack of neural spheroid network detachment by utilizing concave well structures, using a simple method developed in this laboratory. Primary neurons were isolated from pregnant Sprague Dawley rats at 16 ~ 17 days of gestation. Isolated neurons were cultured in PDMS wells with a concave structure and interconnected by rounded micro channels. It was reported previously that a concave structure enabled an easier and more efficient formation of spheroids, not to mention the ease in extraction of spheroid cells. Various studies have demonstrated the effectiveness of guidance channels in promoting neurite growth. Therefore, micro channels were integrated in the micro array design, and served as a guidance conduit to enhance neurite growth, and by association, spheroid interconnection. The primary neurons formed a spheroid structure after 3 days, upon which they began to sprout new neurites. By day 8, neurite connections peaked. Spheroid diameter underwent an initial decrease then stabilized on day 2. Various well diameters (300~700 um) and channel lengths (1.5 x diameter ~ 3 x diameter) were evaluated, with a 300 um well diameter and 450 um center-to-center channel length found to be optimal. The completed network was assessed for interconnection using calcium imaging and showed coordinated calcium signals between the neural spheroids. The network was then successfully transferred to a collagen matrigel and cultured for a week. The methodology showed an improvement in the transfer of networks, with about a 90% extraction rate. The viability of the NSN on the matrigel was assessed using a Live/Dead assay, and cells were found to have greater than 95% viability. The optimal hydrophilicity was determined for neurite extension and transfer of NSNs onto the matrigel. It was found that an incubation time between 4~6 hours was optimal. Future studies will involve the implantation of the NSN into the rat brain. Additionally, the use of neural progenitor and stem cell lines may provide an autologous source of cells which are immunocompatible with the host. In particular, marrow stromal cells are interesting in that they may also address the ethical concerns. A long term goal is to refine the methodology and apply this research to enable studies in the treatment of patients suffering from spinal cord injury and other neurodegenerative disorders.