Injury to the spinal cord results in the disruption of signal transmission between the brain and distal targets. It often presents with the loss of motor function and sensory perception below the level of injury. There are many obstacles following injury that must be overcome in order to encourage axon regeneration and improve functional recovery. A combinatorial approach is necessary to combat physical and chemical barriers to recovery. The fluid filled cyst that forms in the majority of spinal cord injuries presents a physical barrier that we treat with our electrospun bridges. We implanted our bridges into female Long Evans Hooded rats following a complete transection. Using a molecular fusogen, polyethylene glycol, known to seal damaged membranes in conjunction with our bridges, we were able to increase functional recovery compared to animals treated with a bridge and saline. In Chapter 1, we introduce spinal cord anatomy, the pathological classifications, axon pathology, and our therapeutic strategy. Chapter 2 details the materials and methods. Chapter 3 examines previous uses of polyethylene glycol as a molecular fusogen, previous studies utilizing it in spinal cord injury, and our strategy of fusing damaged axons to improve functional recovery. Finally in Chapter 4, I discuss our behavioral results, compare histology, and detail the future of our research including protocol improvements and future combination therapies that include PEG to improve outcome.

Injury to the spinal cord results in partial or complete loss of sensory perception and motor function. After spinal cord injury (SCI), damaged tissue dies and a cavity will form. This cavity prevents the regeneration of tissue and any functional recovery. One way to address the cavity is the insertion of an electrospun scaffold that our lab has created. This provides a substrate for regenerating tissue to grow on, and it is thought that reestablishing the blood supply within the scaffold will allow cells necessary for regeneration to thrive. This could ultimately lead to meaningful recovery for patients who have suffered SCI. Full spinal cord transections were performed on rats, and the scaffolds were inserted into the lesion site. Two different types of scaffold were tested to see if altering the fiber size in the scaffolds produced more blood vessels, and ultimately better regeneration of tissue.

Master of Science / Department of Anatomy & Physiology / Masaaki Tamura / Rat umbilical cord matrix stem cells (UCMSC) have been shown to exhibit a remarkable ability to control rat mammary adenocarcinoma (Mat B III) cell proliferation both in vivo and in vitro. To study the underlying mechanisms and genes involved in Mat B III growth attenuation, total RNA was extracted from the naïve rat UCMSC alone and those co-cultured with Mat B III in Transwell culture dishes. Gene expression profiles of naive rat UCMSC alone and those cocultured with Mat B III cells were investigated by microarray analysis using an Illumina RatRef- 12 Expression BeadChip. The comparison of gene expression profiles between untreated and cocultured rat UCMSC identified five up-regulated candidate genes (follistatin (FST), sulfatase1 (SULF-1), glucose phosphate isomerase (GPI), HtrA serine peptidase (HTRA1), and adipocyte differentiation-related protein (ADRP)) and two down-regulated candidate genes (transforming growth factor, beta-induced, 68kDa (TGFβI) and podoplanin (PDPN)) based upon the following screening criteria: 1) expression of the candidate genes should show at least a 1.5 fold change in rat UCMSC co-cultured with Mat B III cells; 2) candidate genes encode secretory proteins; and 3) they encode cell growth-related proteins. Following confirmation of gene expression by real time-PCR, ADRP, SULF-1 and GPI were selected for further analysis. Addition of specific neutralizing antibodies against these three gene products individually in co-cultures of 1:20 rat UCMSC:Mat B III cells significantly increased cell proliferation, implying that these gene products are produced under the co-cultured condition and functionally attenuate cell growth. Immunoprecipitation followed by Western blot analysis demonstrated that these proteins are indeed secreted into the culture medium. Individual over-expression of these three genes in rat UCMSC significantly enhanced UCMSC-dependent inhibition of cell proliferation in co-culture. These results suggest that ADRP, SULF-1 and GPI act as tumor suppressor genes, and these genes might be involved in rat UCMSC-dependent growth attenuation of rat mammary tumors.

Magister Scientiae (Physiotherapy) - MSc(Physio) / A traumatic spinal cord injury (TSCI) often occurs unexpectedly and causes considerable disability. This condition requires specialized care that is delivered in a time sensitive manner. Data on the incidence, causes, mortality and injury characteristics of TSCI are important for gauging demand for health care and social support services. Unfortunately data on the incidence and causes, as well as functioning such as participation and integration into society, of TSCI are sparse in developing countries such as Tanzania. The overall aim of this study is to determine the incidence, causes, mortality and injury characteristics of TSCI, and to explore the challenges experienced with community reintegration after injury in Tanzania. Both quantitative and qualitative research paradigms were used. The quantitative phase of the study was conducted at Kilimanjaro Christian Medical Centre (KCMC), a referral and teaching hospital in northern Tanzania. The study population was all patients admitted to KCMC with a TSCI from 1 January 2011 to 31 December 2015 (five year period). Quantitative data were collected retrospectively using a data extraction sheet designed by the International Spinal Cord Society which consisted of the following sections: participants’ characteristics (for example age, gender, duration of hospital stay, causes of injury and vertebral injuries) and injury characteristics (location of injury and completeness of injury). Differences between groups (stratified by year) was analysed using both descriptive and inferential statistics. The population for the qualitative phase was based on those included in the quantitative phase. Purposive sampling was used to select adults (over the age of 18 years) with TSCI who were managed at KCMC. Qualitative data were collected by means of individual, semi-structured interviews, and lasted until theoretical saturation was achieved. Thematic analysis was used to derive themes (categories) explaining the latent perceptions of community reintegration and its influences.

Philosophiae Doctor - PhD / Spinal cord injury (SCI) is a devastating condition often affecting young and healthy individuals around the world. This debilitating condition not only creates enormous physical and emotional suffering to individuals but also is a significant financial burden to families and society at large and it affects quality of life. Successful community reintegration following spinal cord injury is considered an important goal of rehabilitation as this has been positively associated with self-esteem, life satisfaction and quality of life. The overall aim of the study was to design a community reintegration programme for individuals who sustained a traumatic spinal cord injury (TSCI) in the Cape Metropolitan Area. The design of this study was a mixed method design including four phases. The first phase of the study was to determine the base line information regarding reintegration into communities after sustaining a TSCI and included 108 participants. Data was collected by use of self-administered/interview administered questionnaire. The results of this phase demonstrated that community reintegration of individuals who sustained a traumatic spinal cord injury was relatively low and employment was found to be a statistically significant variable influencing community reintegration following the injury. The second phase of the study aimed to gain a deeper understanding of how employment and other factors influence reintegration into communities after the injury.

Spinal Cord Stimulation (SCS) is a treatment for lumbar back pain and despitethe proven effcacy of the technology, there is a lack of knowledge in how the treatment outcome varies between different patients groups. Furthermore, since the method is costly, in the sense of material, surgery and follow-up time, a more accurate patient targeting would decrease healthcare costs. Within recent years, Real World Data (RWD) has become a vital source of information to describe the effects of medical treatments. The complexity, however, calls for new, innovative methods using a larger set of useful features to explain the outcome of SCS treatments. This study has employed machine learning algorithms, e.g., Random Forest Classier (RFC) boosting algorithms to finally compare the result with the baseline; Logistic regression (LR). The results retrieved was that RFC tend to classify successful and unsuccessful patients better while logistic regression was unstable regarding unbalanced data. In order to interpret the insights of the models, we also proposed a Soft Accuracy Measurement (SAM) method to explain how RFC and LR differ. Some factors have shown to impact the success of SCS. These factors were age, income, pain experience time and educational level. Many of these variables could also be supported by earlier studies on factors of success from lumbar spine surgery.

Foi realizado estudo experimental com ratos Wistar Albinos, com o objetivo de avaliar diferentes modelos de estudo da lesão da medula espinhal. Os controles foram formados pelos grupos Anestesia, Divulsionamento dos tecidos moles pré-vertebrais e Laminectomia. Os grupos para estudo da lesão da medula espinhal foram formados por animais nos quais o segmento vertebral localizado entre T10 e L1 foi submetido à secção total da medula espinhal, secção parcial (hemissecção), secção total precedida da aplicação local de anestésico e compressão vertebral. Os animais foram avaliados 2 e 24 horas após a realização dos procedimentos, por meio da Escala de Avaliação Locomotora, Teste do Campo Aberto, Teste de Retirada da Cauda, Teste da Placa Quente e Teste do Pinçamento da Pata (reflexos segmentares e supra-segmentares) e também por métodos histológicos, utilizando-se as colorações de Níssl, HematoxHina-Eosina e Glees. Foi observado que as escalas de avaliação locomotora utilizadas foram menos sensíveis à detecção de mudanças funcionais nos animais se comparadas com os testes de comportamento sensório-motores. O teste Exploração no Plano Vertical foi o mais sensível para detectar deficiências sensório-motoras nos diferentes grupos experimentais. Por esse método detectamos uma diminuição da capacidade de exploração do plano vertical nos animais 24 horas após os procedimentos. O teste de função reflexa mais sensível à detecção de deficiências sensório-motoras nos animais após trauma raquimedular leve, moderado e grave, foi o Teste da Placa Quente. Portanto, este estudo demonstrou que, além das Escalas de Avaliação Locomotora, o Teste do Campo Aberto e o Teste da Placa Quente são de utilização fácil, simples, barata, reprodutível e quantitativa, com sensibilidade para detectar deficiências funcionais em animais que tenham sofrido trauma raquimedular leve, moderado ou grave. A análise histológica em segmentos da medula espinhal abrangendo o sítio da lesão permitiu a avaliação da lesão e correlação entre a lesão sensitiva e motora. / An experimental study was carried out using Wistar Albine rats, aiming at an evaluation of different models for the study of spinal injury. Controls included the following groups: Anesthesia, Divulsion of the soft prevertebral tissues and Laminectomy. Groups for the study of spinal injury were composed by animals whose vertebral segment between T10 and L1 was submitted to Total Section of the Spinal cord, Partial Section (hemissection), Total Section Preceded of Application of Local Anesthetic and Vertebral Compression. Animals were evaluated, 2 and 24 hours after the experimental procedures, by means of the Locomotor Evaluation Scale, Open Field Test, Tail Flick Test, Hot Plate Test and Paw Nippering Test (segmentary and suprasegmentary reflex), as well as by means of histological methods using Níssl, Hematoxilin-Eosin and Glees Stainning. lt was observed that the Locomotor Evaluation Scales so far used were less sensitive to detection of functional changes in animals when compared with the sensorio-motor behaviour tests. The vertical Plain Exploration Test was the most sensitive for detection of sensorio-motor deficiences in different experimental groups. By means of this method we could detect a decrease in the ability to explore the Vertical Plain in the animals of 24 hours. The more sensitive reflex function test to detect sensorio-motor deficiences of animals, following light, moderate or severe spinal cord injury, was the Hot Plate Test. Therefore, this study showed that, in addition to the locomotor Evaluation scales, the Open Field Test and Hot Plate Test are of easy, simple, cheap, reproducible and quantitative utilization, showing enough sensitivity to detect functional deficiences in animals previously submitted to light, moderate or severe spinal cord injury. Histological analysis of spinal cord segments which enclosed the injured site allowed the evaluation of the injury and the relationship between sensitive and motor lesions.

As lesões medulares acometem anualmente milhares de pessoas e animais em todo o mundo, causando diversos prejuízos econômicos e psicológicos. As principais causas das lesões medulares são traumas automobilísticos e doenças do disco intervertebral. Muito embora a medicina esteja bastante avançada no campo da neurocirurgia, a cura para esse tipo de lesão ainda esta longe de ser obtida. O avanço das pesquisas no campo da terapia celular em lesões medulares surge como uma esperança para os pacientes crônicos. Neste trabalho buscamos avaliar a reposta do tratamento com células-tronco de medula óssea fetal canina em cães com lesão medular crônica toracolombar. Nosso trabalho se baseia em parâmetros clínicos, comportamentais, de imagem e fisioterápicos. Antes de adentrar no experimento, todos os cães foram submetidos a vários exames pré-operatórios (hemograma, exames bioquímicos, eletrocardiograma) para então serem encaminhados para o exame de ressonância nuclear magnética visando um diagnostico mais preciso da lesão. Após esse exame, os cães foram avaliados por fisioterapeutas veterinários que não pertenciam ao nosso grupo de pesquisa para se estabelecer uma pontuação no teste comportamental de Olby. Os animais também tiveram alguns outros reflexos testados (dor profunda, reflexo de panículo). Após a primeira avaliação, os animais foram submetidos à procedimento cirúrgico de descompressão da medula espinhal e aplicação de células-tronco da medula óssea fetal canina. Durante o procedimento foram injetados 1x106 células diretamente em 3 pontos distintos da medula espinhal. Após o procedimento os cães foram encaminhados para a fisioterapia, e por 3 meses, foram submetidos a diversos exercícios de reabilitação com o intuito de potencializar um possível efeito benéfico da terapia celular. Durante a fisioterapia, os animais foram filmados com o intuito de acompanhar a sua evolução, e após o termino da fisioterapia foram novamente avaliados pelos fisioterapeutas. Ao final do experimento 7 animais foram operados e os resultados obtidos demonstraram um aumento do reflexo de marcha em 6 deles. O único animal que não apresentou essa melhora da marcha foi aquele acometido por outra patologia associada à compressão medular. Esses resultados nos levam a sugerir uma ação benéfica da terapia celular em cães portadores de lesão medular crônica. Por outro lado sugere continuar recrutando animais com o objetivo de aprimorar as técnicas utilizadas, para conseguir resultados cada vez melhores. / Spinal cord injuries annually involve thousands of people and animals worldwide, causing economic and psychological damages. The main causes of spinal cord injuries are vehicular traumas and intervertebral disc diseases. Although medicine is very advanced in neurosurgery field, the cure for this type of injury is still far from being achieved. Research progresses in cell therapy for spinal cord injuries appear to be a hope for chronic patients. In this article we aimed to evaluate clinical responses to the treatment using canine fetal bone marrow stem cells in dogs with thoracolumbar chronic spinal cord injury. Our study was based on the evaluation of clinical signs, animal behavior, imaging and physiotherapy aspects. Before clinical-surgery trial, all dogs underwent to preoperative tests as hemogram, blood chemistry, electrocardiogram and to nuclear magnetic resonance exam, in order to stablish more accurate diagnosis of the injury. Following clinical exams, Olby score was determined by evaluating animal behavior. Olby tests were performed by external scientific research group, composed by veterinary phisiotherapists, in order to guarantee blind evaluation. Animals were also tested to deep pain and panniculus reflexes. s, After being evaluated, animals underwent surgical spinal cord decompression and to a 1x106 stem cells injection in three different sites of the spinal cord. After the procedure, dogs were referred to physiotherapy for three months, undergoing to a variety of rehabilitation exercises in order to improve cell therapy hypothetical benefic effect. During therapy, animals were filmed in order to monitor their evolution, and after the end of physiotherapy they were re-evaluated by physiotherapists. At the end of the experiment, 7 animals were operated and had resulted in an increase of reflex motion in 6 of them. The only animal that showed no such improvement was the one whos had other pathology associated with the spinal cord compression, these results lead us to believe in a beneficial action of cell therapy in dogs with chronic spinal cord injury and suggests continue recruiting animas with the aim of improving the techniques used to achieve better results.

A estimulação química das fibras aferentes nociceptivas através da injeção de diferentes substâncias álgicas vem sendo estudada em modelos de dor muscular em animais. A carragenina é um dos agentes que produz inflamação aguda e é freqüentemente utilizada em modelos de dor muscular. O nosso objetivo é aprofundar nossa compreensão e explorar em maior detalhe a participação das células gliais (astrócitos e microglia), de citocinas pró-inflamatórias, prostanóides e do óxido nítrico, após a indução de miosite aguda, correlacionando com modelos comportamentais nociceptivos. Para tanto, serão avaliadas as atividades dos mediadores, previamente descritos, na medula espinal dos ratos com e/ ou sem indução de miosite, através do método de Western blotting. Tais investigações poderão vir a ser uma abordagem totalmente inovadora para tentar entender quais são alguns dos mediadores envolvidos neste modelo de dor. Assim, os dados obtidos neste projeto poderão elucidar os mecanismos envolvidos na dor músculo-esquelética, a qual se enquadra nas patologias de difíceis tratamentos e de grande relevância clínica. / Chemical stimulation of nociceptive afferent fibers through the injection of different substances has been studied in different models of muscle pain in animals. Carrageenan is one of the agents that produce inflammation and is often used in models of muscle pain. Our goal is to explore in greater detail the involvement of glial cells (astrocytes and microglia), pro-inflammatory cytokines, prostanoids and nitric oxide after induction of acute myositis and correlate it with nociceptive behavioral models. It will be assessed the activity of these mediators in the spinal cord after induction of myositis in rats, using Western blotting assay. Such investigations may be a totally new approach to try to understand how some mediators are involved in this kind of pain. Thus, the data obtained in this project will elucidate the mechanisms involved in musculoskeletal pain, which are of difficult treatment and of great clinical relevance.

Objetivou-se com esta pesquisa descrever a topografia vértebro-medular do jabuti de patas vermelhas, com o intuito de assentar bases morfológicas que possam ser utilizadas em pesquisas aplicadas como a anestesiologia, assim como, desenvolver técnica de anestesia espinhal que possa ser utilizada na rotina clínico-cirúrgica veterinária. Para tanto, este trabalho foi dividido em duas etapas. A primeira etapa, correspondeu aos estudos morfológicos (anatomia), e a segunda, ao desenvolvimento/avaliação da técnica de anestesia espinhal. Para os estudos morfológicos utilizaram-se seis animais adultos provenientes do estado do Maranhão, os quais vieram a óbito por causas naturais. Canulou-se a artéria carótida comum, fazendo-se a perfusão do sistema arterial com solução salina aquecida a 37ºC, procedendo-se então com a fixação do material com solução de formaldeído a 20%. As peças então foram submetidas à ação de solução descalcificadora modificada, por um período de 72 horas, e em seguida, iniciou-se a dissecção com a abertura dorsal da carapaça mediante auxílio de cizel e martelo ortopédico, e posteriormente a musculatura dorsal era dissecada e os arcos vertebrais seccionados, para acesso à medula espinhal. A segunda etapa que consistiu no desenvolvimento/avaliação da técnica de anestesia espinhal, foi realizada no Orquidário Municipal de Santos - São Paulo, onde foram utilizados oito animais, os quais foram submetidos ao emprego da técnica de anestesia espinhal, que consistiu na administração de lidocaína (2%) no volume de 0,2mL/10cm de carapaça, em um dos espaços articulares compreendidos entre a 15ª a 22ª vértebras coccígeas. Os parâmetros avaliados foram o período de latência, o relaxamento muscular e analgesia das regiões de membro pélvico, cauda e cloaca. Os resultados evidenciam que a medula espinhal do G.carbonaria, apresentou-se como uma massa alongada, de coloração esbranquiçada, situada dentro do canal vertebral, preenchedo-o até a articulação entre a penúltima e a última vértebra caudal. A intumescência cérvico-torácica apresentou-se localizada entre os segmentos medulares de C5-T1, enquanto a intumescência tóraco-sacral pelos segmentos medulares de T6-Ca1. A técnica de anestesia espinhal, apresentou-se como técnica segura e eficaz, promovendo relaxamento muscular e analgesia satisfatória para as regiões de membro pélvico e cauda/cloaca, podendo ser utilizada como alternativa anestésica bastante eficiente para procedimentos cirúrgicos nestas regiões. / The objective of this study was to describe the topographic anatomy of spinal cord of the Red Footed Tortoise in order to obtain morphologic bases that can be used in an applied researches as anesthesiology as well as to develop spinal anesthesia techniques that can be used in a clinic and surgery veterinary routine. The first part was the anatomy morphologic study and the second was the evaluation development of the technique spinal anesthesia. It was used six adult animals that died for natural causes, for the morphologic studies from Maranhão State. It was dissected the common carotid artery and the arterial system was perfusion with warm saline solution at 37ºC. The material was fixed with formaldenyde solution at 20%. Then the pieces were submitted by modify decalcified solution action, for 72 hours. The dissection started by the dorsal shell opening with help of a chizel and an orthopedic hammer. The dorsal muscle was dissected and the vertebral arch was sectioned for the spinal cord access. The second part, consisting in a development / valuation of the spinal anesthetic technique, was realized at Orquidário Municipal in Santos - SP where it was used eight animals submitted at this technique. The evaluation parameters were the latent period, the muscle relaxation and the analgesia of the pelvic member region, tail and cloaca. The results proved that the G. carbonaria spinal cord showed as an elongated mass with whitish color, situated in the vertebral duct, between the penultimate and the last caudal vertebrae. The cervical swelling was localizated between the medular segment (C5 to T1) while the sacral swelling was placed between the T6 to Ca1 medular segments. The spinal anesthetic technique showed to be a safe and effective technique, promoting muscular relaxation and satisfactory analgesia for the pelvic member, tail and cloaca regions, being suitable to use as an anesthetic alternative quite efficient for the surgical procedures in this regions.