Résumé confidentiel / Résumé confidentiel

Ces dernières années, un nouveau composant de l'environnement des tumeurs (ET) a été mis en évidence: les projections des neurones du système nerveux. En effet, les tumeurs sont infiltrées par des axones, ce qui pourrait réguler la progression du cancer.Le cancer du pancréas fait partie des cancers les plus mortels. Les traitements thérapeutiques actuels qui ciblent ce cancer ne sont pas efficaces. Il est donc important de mieux comprendre les différentes composantes de l'ET de ce cancer afin d’identifier de nouvelles cibles thérapeutiques. Nous proposons de décrire l’innervation des tumeurs pancréatiques ce qui est le point de départ pour mieux comprendre l’importance de cette composante de l'ET. Les objectifs ont été d’analyser en 3D les réseaux d'axones qui innervent le pancréas sain et cancéreux, ainsi que leurs relations avec d'autres types cellulaires de l'ET (vaisseaux sanguins (VS)).Pour cela, nous avons utilisé une méthode d'imagerie 3D de pancréas entiers, rendus transparents, qui proviennent de modèles génétiques de souris qui développent des cancers du pancréas similaires à ceux de l'homme. Nous avons observé que les réseaux d'axones sont plus denses et plus complexes dans les régions cancéreuses du pancréas par rapport aux régions saines. Alors que dans les tissus sains les axones sont associés aux VS, ils ne le sont plus dans les régions cancéreuses. Nous avons de plus identifié des groupes morphologiques de réseaux d'axones qui permettent de discriminer une région saine d'une région cancéreuse.L’analyse de la structure en 3D de ces réseaux d'axones pourrait donc représenter une donnée prédictive et pronostique de l'état d'avancé clinique de la maladie. / Cancers are diseases in which cancer cells interact with a complex tumor environment (TE). In recent years, a new component of TE has been highlighted: neuronal projections of the nervous system. Indeed, the axons of neurons innervate the tumors, which could regulate cancer progression.Pancreatic cancer is among the most deadly cancers. Indeed, the current therapeutic treatments that target this cancer are not effective. It is therefore important to better understand the different components of the TE of this cancer in order to identify new potential therapeutic targets.In this thesis, we propose to describe the innervation of pancreatic tumors which is the starting point to better understand the importance of this component of the TE. The objectives were to visualize and analyze in 3D the networks of axons that innervate the healthy and cancerous pancreas, as well as their relations with other cell types of the TE (blood vessels (BV)).For this, we used a method of 3D imaging of whole pancreas, made transparent, which come from genetic models of mice that develop pancreatic cancer similar to that of humans.We observed that axon networks are denser and more complex in cancerous regions of the pancreas compared to healthy regions. Moreover, while in healthy tissue, axons are associated with BV, they are no longer in cancerous areas.We also identified morphological groups of axon networks that discriminate a healthy region from a cancerous region.The analysis of the 3D structure of these axon networks could thus represent a predictive and prognostic value for the progression of the disease.

The nervous system, including the brain, is the most sophisticated organ in the mammalian body. In such a complex network, neuron-glial interaction is essential and controls most developmental processes, such as stem cell fate determination, migration, differentiation, synapse formation, ensheathment and myelination. Many of these events are critical for the developmental process and small errors can lead to growth retardation, malformation or disease. The understanding of the normal progress of nervous system development is fundamental and will help the discovery of new treatments for disease. This thesis discusses three types of neuron-glia interactions at different developmental stages; neural stem/progenitor cell (NSPC) differentiation, building and maintaining the structure of the sciatic nerve, and myelin formation. In Paper I we show that NSPCs, based upon their morphology and expression of specific protein markers, have the capacity to differentiate into cells of either the peripheral nervous system (PNS) or enteric nervous system (ENS) when grown with PNS or ENS primary cell cultures, or fed with conditioned medium from these. This indicates that soluble factors secreted from the PNS or ENS cultures are important for stem cell differentiation and fate determination. The adhesion protein neuronal cadherin (N-cadherin) is implicated in migration, differentiation and nerve outgrowth in the developing PNS. In Paper II N-cadherin was exclusively found in ensheathing glia (nonmyelinating Schwann cells, satellite cells and enteric glia) in contact with each other or with axons. Functional blocking of N-cadherin in dissociated fetal dorsal root ganglia (DRG) cultures led to a decrease in attachment between Schwann cells. N-cadherin-mediated adhesion of nonmyelinating Schwann cells may be important in encapsulating thin calibre axons and provide support to myelinating Schwann cells. In Paper III the inhibitory gamma aminobutyric acid (GABA) and GABAB receptors were studied in the Schwann cell of the adult sciatic nerve and DRG cultures. GABAB receptors were primarily expressed in nonmyelinating Schwann cells and protein levels decreased during development and myelination. Blocking the GABAB receptor in long-term DRG cultures led to decreased levels of mRNA markers for myelin. These results indicate that the GABA and GABAB receptors may be involved in Schwann cell myelination.

INTRODUÇÃO: A neuropatia periférica induzida por quimioterapia (NPIQ) está entre os efeitos colaterais mais comuns decorrentes da quimioterapia antineoplásica e uma das principais causas da redução da dose ou interrupção do tratamento. Os sintomas mais prevalentes são a dor e a parestesia, acarretando desconfortos crônicos e perda de habilidades funcionais, interferindo negativamente na qualidade de vida dos pacientes. Estudos recentes têm avaliado o uso da Estimulação Elétrica Nervosa Transcutânea (TENS) nesta patologia, apresentando evidências positivas na redução da dor, porém seu efeito nos sintomas de parestesia e nas atividades de vida diária destes pacientes ainda não foram avaliados. OBJETIVO: Investigar os efeitos da Estimulação Elétrica Nervosa Transcutânea (TENS) nos sintomas de dor, parestesia e nas atividades de vida diária da NPIQ em indivíduos com diagnóstico de câncer de mama e colorretal, submetidos ao tratamento de quimioterapia. MÉTODOS: Trata-se de um ensaio clínico duplo-cego, controlado, randomizado e multicêntrico, com abordagem quantitativa, em pacientes submetidos ao tratamento de quimioterapia, contendo em seu protocolo os seguintes quimioterápicos: paclitaxel e oxaliplatina. Os sujeitos da pesquisa utilizaram o dispositivo terapêutico TENS com modulação de frequência entre 7 e 75 Hz na região distal dos membros, no local de maior desconforto, com intervenções diárias de 60 minutos, durante três ciclos de quimioterapia (45 dias). Os participantes foram divididos em dois grupos: grupo TENS ativa (GTA) e grupo TENS placebo (GTP). A avaliação dos efeitos da TENS foi medida através dos seguintes instrumentos: a Escala Visual Analógica (EVA) para avaliar os sintomas de dor e parestesia e Questionário de Neurotoxicidade Induzida por Anti-neoplásicos (QNIA) para avaliação dos sintomas da NPIQ. RESULTADOS: Finalizaram a pesquisa 24 pacientes. Não se observou uma diferença significativa entre os 2 grupos no que se refere ao desfecho primário de redução dos sintomas de dor (p = 0.666), parestesia (p = 0,673) e impacto da TENS na frequência dos sintomas (p = 0,5906) e atividades de vida diária (p = 0,8565). CONCLUSÃO: Estes resultados sugerem que a TENS aplicada no modo de modulação de frequência não foi eficaz para melhorar os sintomas de neuropatia periférica induzida por quimioterapia, durante os ciclos de quimioterapia. Não houve, porém, agravamento dos sintomas em ciclos subsequentes ao início dos sintomas da doença / BACKGROUND: Peripheral neuropathy induced by chemotherapy (PNIC) is amongst the most common side effects derived from antineoplastic chemotherapy and one of the principal causes of dose reduction or treatment interruption. The most prevalent symptoms are pain and numbness, resulting from chronic discomfort to loss of functional abilities, negatively affecting quality of life and autonomy of patients. Recent studies have evaluated the use of Transcutaneous Electrical Nerve Stimulation (TENS) in this disease, pointing to evidence of pain reduction, but its effect on symptoms of paresthesia and in daily life activities have not yet been evaluated. OBJECTIVE: To investigate the effects of Transcutaneous Electrical Nerve Stimulation (TENS) for reducing the symptoms of pain, paresthesia and the daily activities of PNIC in patients diagnosed with breast cancer and colorectal cancer undergoing chemotherapy treatment. METHODS: It is a double-blind, controlled, randomized, multicenter clinical trial with a quantitative approach in a sample of 24 patients undergoing chemotherapy treatment, containing in its protocol the following chemotherapeutic agents: paclitaxel and oxaliplatin. The research subjects used the TENS therapeutic device with frequency modulation between 7 and 75 Hz in the distal limb, on the location of greatest discomfort with daily interventions lasting 60 minutes for three chemotherapy cycles (45 days). Participants were divided into two groups: active TENS group (ATG) and placebo TENS group (PTG). The assessment of the effects of TENS was measured by the following instruments: The Visual Analogue Scale (VAS) to assess the symptoms of pain and numbness and Questionnaire for Neurotoxicity Induced by Anti-neoplastic (QNIA) to assess the symptoms of PNIC. RESULTS: A 24-patient study was completed. There was no significant difference between the two groups regarding the primary endpoint of reduced pain symptoms (p = 0.666) and paresthesia (p = 0.673), neither any measurable impact of TENS in the frequency of symptoms (p = 0.5906) or activities of daily living (p = 0.8565). CONCLUSION: These results suggest that TENS applied in frequency modulation mode is not effective for ameliorating the symptoms of peripheral neuropathy induced by chemotherapy during chemotherapy cycles. There was, however, no worsening of symptoms in subsequent cycles after the onset of symptoms

Foi caracterizada uma amostra de 81 doentes com dor relevante crônica pélvica de origem indefinida e na ausência de afecções viscerais, segundo a expressão sintomática, aspectos clínicos e síndromes álgicas. A média das idades foi de 48,6 anos. Foram identificadas anormalidades miofasciais pélvica em 55 doentes (67,9%), neuropáticas em 10 (12,3%) e miofasciais e neuropáticas em 16 (19,8%). A descrição verbal da dor e as regiões acometidas pela dor, impactos funcionais, a evidenciação de pontos dolorosos e em gatilhos e os aspectos psicossociais dos doentes foram estabelecidos para melhorar a compreensão da condição clínica dos doentes. Traumatismos decorrentes de traumatismos cirúrgicos estavam relacionados à ocorrência da dor em 36 (44,4%) doentes, sendo dor miofascial em 15 doentes (18,5%), 6 (7,4%) como neuropática e 15 (18,5%) como dor miofascial e neuropática / Eighty-one chronic pelvic pain patients (mean of ages 48.6 years old) without visceral abnormalities and uncertain etiology were characterized according to the symptomatic expression, clinical findings and pain syndromes. It was observed that in 55 patients there was pelvic miofascial pain (67.9%), in 10 patients neuropathic (12.3%) conditions and 16 (19.8%) had miofascial and neuropathic abnormalities. The verbal description of pain, pain aspects as localization, aggravating and improvement factors, functional impact, the occurrence of trigger or tender points and the psychosocial aspects were determined to improve the comprehension of the clinical presentation of the patients. Surgical traumas were related to pain in 26 (32.0%) patients, miofascial pain in 11 (13.6%), neuropathic pain in 5 (6.2%) and miofascial and neuropathic pain in 10 (12.3%)

Introdução: A síndrome Spoan é uma forma de paraplegia espástica complicada de herança recessiva recentemente identificada em indivíduos originários do sudoeste do estado do Rio Grande do Norte. O quadro clínico é caracterizado por atrofia óptica congênita, paraplegia crural espástica de caráter progressivo e neuropatia axonal levando a perda da função motora em membros superiores. A caracterização fenotípica dessa doença não está completa, e não foram realizados estudos quantitativos e funcionais, que poderiam mensurar a intensidade e contribuir para a definição de uma estratégia de reabilitação. Objetivos: caracterizar o desempenho motor e as habilidades funcionais de indivíduos acometidos pela Spoan. Casuística e metodologia: participaram do estudo 61 indivíduos com diagnóstico clínico de Spoan com idade entre 5 e 72 anos. Avaliou-se a força de preensão palmar por meio do dinamômetro hidráulico de Jamar e a sensibilidade a pressão profunda e protetora dos pés e mãos por meio dos monofilamentos de náilon de Semmes-Weinstein. Definiu-se o grau de dependência dos indivíduos afetados por meio do Índice de Barthel modificado. Considerou-se para a descrição do desempenho motor: 1. quantificação da espasticidade, por meio da escala modificada de Ashworth; 2. grau de disfunção, de acordo com a escala ponderada de paraplegia espástica descrita por Schule e a escala funcional de paraplegia espástica hereditária descrita por Fink; 3. grau da capacidade de deambulação, por meio do índice de deambulação 4. grau da capacidade de sentar, por meio da escala de avaliação motora. Resultados: constatou-se fraqueza de preensão manual em todos os indivíduos e os valores obtidos indicam correlação inversa moderada entre a idade e a força manual. A sensibilidade mostrava-se anormal em 100% dos indivíduos avaliados em pelo menos seis pontos dos pés e mãos. O grau de dependência foi mínimo em 3,3%, médio em 23,3%, grave em 46,6% e total em 26,6% dos pacientes. Na escala de Schule, 60% dos indivíduos obtiveram entre 40/52 e 52/52 pontos e na escala de Fink detectou-se grau 5 (máximo) de disfunção em 71% dos pacientes. O grau de espasticidade teve uma distribuição bimodal, em média, de 30,5% com grau 1 e 37,7% grau 4. A capacidade de deambulação mostrou-se reduzida, com 83% dos indivíduos restritos a cadeira de rodas e 11% acamados. A habilidade de sentar-se estava preservada em todos os pacientes, sendo que 53% o faziam apenas com apoio. Conclusão: A síndrome Spoan é uma forma grave de paraplegia espástica hereditária, responsável por incapacidade progressiva e duradoura. / INTRODUCTION: Spoan syndrome is a complex form of spastic paraplegia of recessive inheritance recently identified in individuals from Southwest of Rio Grande do Norte state. Clinical features are characterized by congenital optic atrophy, progressive spastic paraplegia, and axonal neuropathy, resulting in severe handicap. Phenotypic description of this disease is nevertheless not complete; functional and quantitative studies, that would help planning a rehabilitation strategy, have not been undertaken. OBJECTIVES: To evaluate the motor performance and functional abilities of individual with Spoan syndrome. CASUISTIC AND METHODS: 61 individuals with confirmed diagnosis of Spoan, with ages ranging from 5 and 72 years were evaluated. Hand grip strength was measured with a Jamar hydraulic dynamometer and the sensitivity to deep pressure and protective hands and feet with Semmes-Weinstein nylon monofilaments. Functional abilities were verified by the Modified Barthel Index. For motor performance, the following procedures were performed: 1. Spasticity quantification, according to modified Ashworth scale; 2. Dysfunction level, according to the spastic paraplegia rating scale described by Schule and functional scale of hereditary spastic paraplegia described by Fink; 3. Gait ability, verified with deambulation index; 4.Sitting ability, using motor assessment scale. RESULTS: grip hand weakness was reduced in all patients, with a moderate inverse correlation between age and hand strength. Sensibility was abnormal in 100% of evaluated individuals in at least six points of hands and feet. Dependency level was minimum in 3.3%, moderate in 23.3%, severe in 46.6%, and total in 26.6% of individuals. According to Schule s scale , 60% of individuals scored between 40/52 and 52/52 points; in Fink s scale,71% achieved level 5 (maximum) of dysfunction. Spasticity level had a bimodal distribution, with 30,5% achieving level 1 and 37,7% level 4. Gait ability was reduced, with 83% of individuals being wheelchair bound and 11% bedridden. Sitting ability was preserved in all patients, but 53% were able to sit only with support. CONCLUSION: Spoan syndrome is a severe form of hereditary spastic paraplegia that is responsible for progressive and long lasting handicap.

INTRODUÇÃO: Após uma lesão de um nervo periférico, o seu reparo é realizado com suturas epineurais dos cotos utilizando-se fio de náilon. O processo inflamatório provocado pela passagem da agulha e pela presença do material de sutura com consequente formação de tecido cicatricial na linha de sutura torna-se um obstáculo à regeneração axonal após o reparo do nervo. Existe na literatura a hipótese que a utilização de um menor número de pontos necessários para assegurar a união dos dois cotos correlaciona-se com uma melhor regeneração axonal. Foi avaliada a influência do diferente número de suturas epineurais terminoterminais no nervo ciático do rato na regeneração axonal, além de avaliar o remodelamento da matriz extracelular através da caracterização dos tipos de colágenos tipo I e III presentes no local de sutura de acordo com o número de pontos adotados. MÉTODOS: Neste estudo experimental foram utilizados trinta ratos machos Wistar submetidos à exposição do nervo ciático direito e divididos em três grupos. Em dois grupos, o nervo foi seccionado e imediatamente reparado com três (Grupo 3P, n=10), ou seis (Grupo 6P, n=10) suturas epineurais usando fio de náilon 10.0. Um grupo (Grupo C, n=10) foi utilizado como controle para determinar os valores normais de todos os parâmetros avaliados. Cada animal pertencente aos grupos de reparo foram submetidos à avaliação eletrofisiológica previamente a secção do nervo e após um intervalo de oito semanas após a neurorrafia, consistindo de registro do potencial de ação motor do músculo gastrocnêmio. Segmentos do nervo foram utilizados para avaliação histomorfométrica da regeneração axonal, avaliada pela contagem de axônios e medida do diâmetro das fibras, e para caracterização e quantificação do colágeno no local de reparo. Imunofluorescência foi utilizada para caracterização dos colágenos tipo I e II no epineuro e endoneuro. RESULTADOS: O índice de regeneração axonal (IR) foi menor no grupo submetido à sutura epineural com seis pontos (IR=1,18±0,26) que no grupo de três pontos (IR=1,32±0,25) e, naquele mesmo grupo, houve uma diminuição mais acentuada da velocidade de condução do potencial de ação do nervo. Os animais submetidos à sutura epineural com seis pontos apresentaram uma diferença significativa na produção do colágeno epineural tipo I (p=0,014) e de colágeno epineural tipo III (p=0,002) quando comparados com os animais submetidos à sutura com três pontos. Não houve diferença significativa na produção de colágeno tipo I ou tipo III endoneural. CONCLUSÕES: O grupo submetido a um maior número de suturas epineurais apresentou maior quantidade de colágenos tipo I e III no epineuro, mas não no endoneuro, que se correlacionou com um menor índice de regeneração axonal e uma diminuição mais acentuada da velocidade de condução do potencial de ação do nervo. / PURPOSE: After an injury to a peripheral nerve, its repair is performed with epineural sutures of the stumps using nylon. The inflammatory process caused by the passage of the needle and the presence of suture material with the consequent formation of scar tissue at the suture line becomes an obstacle to axonal regeneration after nerve repair. In the literature there is a hypothesis that the use of a smaller number of points needed to ensure the union of the two stumps is correlated with better axonal regeneration. It was evaluated the influence of different number of end-to-end epineural sutures in rat sciatic nerve on axonal regeneration and the remodeling of the extracellular matrix through the characterization of collagen type I and III present at the suture according to the number of stitches. METHODS: Thirty male Wistar rats were operated on to exposure the right sciatic nerve and were divided in three groups. In two groups the nerve was sectioned and immediately repaired by means of three (Group 3P, n=10) or six (Group 6P, n=10) epineurium sutures using 10-0 monofilament nylon. One group (Group C, n=10) was used as a control to assess normal values of all evaluated parameters. Each animal from repaired groups underwent electrophysiologic evaluation previously to nerve section and at 8-week interval after neurorrhaphy, consisting of motor action potential recording from the gastrocnemius muscle. Nerve biopsy specimens were used for histomorphometric assessment of axonal regeneration, evaluated by axonal counting and measurement of fiber diameter, and for collagen characterization and quantification at repair site. Immunofluorescence was used for characterization of types I and III collagen at epineurium and endoneurium. RESULTS: The axonal regeneration index (RI) was lower in the group submitted to suture with six stitches (RI = 1.18 ± 0.26) than in the group of three stitches (RI = 1.32 ± 0.25) and the group submitted to six stitches showed a greater reduction in conduction velocity of nerve action potential. Animals submitted to suture with six stitches showed a significant difference in the production of epineural collagen type I (p = 0.014) and type III (p = 0.002) compared to animals submitted to suture with three stitches. There was no significant difference in production of endoneural collagen type I or type III. CONCLUSIONS: The group submitted a greater number of epineural sutures had a higher amount of collagen type I and III in the epineurium but not in the endoneurium, which correlated with a lower rate of axonal regeneration and a greater reduction in conduction velocity of action potential nerve.

Orientador: Alexandre Leite Rodrigues de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T17:19:57Z (GMT). No. of bitstreams: 1 Simoes_GustavoFerreira_D.pdf: 65115196 bytes, checksum: 18f51cababf3e248cceb806b8faef631 (MD5) Previous issue date: 2012 / Resumo: Atualmente, muito se sabe sobre o acometimento muscular na DMD, mas poucos estudos estão voltados para os efeitos no Sistema Nervoso Central (SNC), mais especificamente no microambiente do motoneurônio medular. Sabe-se que durante a evolução da doença, o terminal axonal, na junção neuromuscular, entra em um ciclo de denervação (retração) e reinervação (brotamento). A possibilidade de modulação do MHC I se apresenta como uma nova estratégia de influenciar positivamente o processo de plasticidade sináptica após lesões do Sistema Nervoso Periférico (SNP) e SNC. Tal modulação pode ser realizada através da utilização ou desenvolvimento de drogas específicas. O fator estimulador de colônias glanulocitárias (G-CSF) é uma glicoproteína que foi descrita há mais de vinte anos, possui aprovação do ANVISA (Agência Nacional de Vigilância Sanitária) e é comumente utilizada para tratar neutropenia, ou para transplantes de medula óssea. O GCSF possui um efeito neuroprotetor aparentemente multimodal, incluindo-se a atividade anti-apoptóptica em neurônios, regeneração da vascularização, efeito antiinflamatório e estimulação de neurogênese endógena, sendo capaz de atuar efetivamente no processo de regeneração do sistema nervoso. No presente trabalho, foram utilizados camundongos MDX. Os camundongos foram distribuídos em 4 grupos (axotomia + G-CSF; Axotomia; Controle + G-CSF e Controle), com n=10. Incluiu-se para imunoistoquímica o grupo placebo, onde os animais receberam uma dose diária de 200?m, via subcutânea, de glicose a 25%. Nossos resultados indicam que redução de sinapses nos motoneurônios alfamedulares e aumento da astrogliose circunjacente aos neurônios alfa-medulares, seja decorrente da desconexão parcial entre o orgão alvo e o corpo neuronal durante o período de ciclos de degeneração/regeneração muscular que ocorrem a partir das primeiras semanas de vida nos camundongos MDX. Estes ciclos podem repercutir retrogradamente nos corpos celulares dos motoneurônios alfa-medulares, provocando uma série de alterações denominadas cromatólise. A axotomia do nervo isquiático resulta num aumento significativo da expressão de MHC I nas duas linhagens estudadas. Contudo, nos animais MDX, este aumento é menor, comparativamente à linhagem C57BL/10. Quando tratados com G-CSF a expressão de MCH I ficou maior em relação aos grupos não tratados e, isso pode indicar um papel ativo da droga no potencial regenerativo após a lesão. Também podemos sugerir que, apesar dos animais MDX apresentarem uma menor função motora em relação aos animais controle, os resultados indicam que o tratamento com G-CSF é capaz de reduzir os efeitos inflamatórios e atuar positivamente no processo de regeneração nervosa periférica após esmagamento do nervo isquiático / Abstract: Currently, much is known about the muscular involvement in DMD, but few studies have focused on the effects on the central nervous system (CNS), specifically in the microenvironment of spinal motor neurons. It is known that during the course of the disease, the axon terminal at the neuromuscular junction, enters a cycle of denervation (retraction) and reinnervation (sprouting). The possibility of modulation of MHC I presents itself as a new strategy to positively influence the process of synaptic plasticity after injury Peripheral Nervous System (PNS) and CNS. Such modulation may be accomplished through the use or development of special drugs. The granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which was first described more than twenty years, has approval from ANVISA (Agência Nacional de Vigilância Sanitária) and is commonly used to treat neutropenia, or bone marrow transplants. The G-CSF has a multimodal neuroprotective effect l, including the anti-apoptotic activity in neurons, regeneration of vascularization, anti-inflammatory effect and stimulation of endogenous neurogenesis, being able to act effectively in the process of regeneration of the nervous system. In this study, we used MDX mice. The mice were divided into 4 groups (axotomy + G-CSF; axotomy, Control + G-CSF and Control), with n = 10. Included immunohistochemistry to the placebo group, where the animals received a daily dose of 200?m, subcutaneously, glucose 25%. Our results indicate that reduction of synapses in the alpha motoneurosn and increased astrogliosis , either due to partial disconnection between the target organ and the neuronal body during the cycles of degeneration /regeneration muscle that occur from first weeks of life in MDX mice. These cycles can pass retrogradely in alpha motoneurons cell bodies, causing a series of changes called chromatolysis. The sciatic nerve axotomy results in a significant increase of MHC I expression in both strains studied. However, in MDX strain, this increase is smaller, compared to C57BL/10. After treatment with G-CSF the expression of MCH I got bigger compared to untreated groups, and this may indicate an active role in the regenerative potential of the drug after injury. Also we suggest that while the animals present MDX a smaller motor function compared to control animals, the results indicate that treatment with G-CSF is capable of reducing the inflammatory effects and act positively on peripheral nerve regeneration process after nerve crush sciatic. Also our results indicate that treatment with G-CSF is able to reduce the inflammatory effects and act positively on peripheral nerve regeneration process after nerve crush sciatic / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.

<div>The present thesis was completed to satisfy two functions in our laboratory: (1) explore carbon-black (CB) as an additive for electrodeposited intrinsically conductive polymers (ICPs) to improve electrical properties across the electrode-electrolyte interface for use in neuromodulation; and (2) design a histology protocol that will analyze peripheral nerve system (PNS) tissue following implantation of conventional metal and modified conventional metal electrodes with the ICP poly(3,4-ethylenedioxythiophere):poly(styrenesulfonate)/carbon-black (PEDOT:PSS/CB). It would appear that the functions explored may seem unrelated, however, these two topics play a crucial role in designing a viable electrode for use in acute and chronic neuromodulation and the subsequent analysis required to determine the mechanical properties and overall biocompatibility of design.</div><div><br></div><div><div>A series of experiments with different PEDOT:PSS solutions containing varying amounts of suspended CB (n=19; 0 mg/mL to 2 mg/mL) were explored. Solutions were characterized using cyclic voltammetry (CV) using the intended electrode for deposition, composed of stainless steel (SS), as the working electrode (WE) to determine respective redox potentials. SS was chosen because of its inherently bad electrochemical properties, meaning that improved functionality post electrodeposition would be easy to identify. Immediately following CV, stainless-steel electrodes were electrodeposited using one of two techniques: (1) potentiostat, allowing the cell to rest at the redox potential required for bipolaron formation (0.9 V); or (2) galvanostat, where the electrode was submitted to a constant current of 200 mA and allowed to coat. Rapid electrochemical impedance spectroscopy was performed prior to and immediately following coating to determine the pre-electrochemical and post-electrochemical impedance characteristics. Results indicate that there was a positive relationship between the amount of CB additive and the relative impedance drop between the uncoated and coated counterparts. Furthermore, the modified electrochemical interfaces are substantially improved for use in frequency ranges of 10 Hz to 50 kHz, which encompass the ranges of our labs recently discovered low frequency alternating current (LFAC) for use in neuromodulation; thus indicating that PEDOT:PSS/CB modification may be used to improve impedance characteristics during our future LFAC experiments. This protocol, the one that contains the ideal concentration of carbon-black, was then recorded and will be used in our lab.</div></div><div><br></div><div><div>Histology protocols were developed to improve our labs capabilities of post-mortem analysis of PNS tissue. Processing and embedding preparations that explored included paraffin, acrylic, and frozen. Subsequently, staining protocols were developed; however, they varied as a function of the embedding media used; staining protocols developed incorporated progressive and regressive hematoxylin and eosin (H&E) staining as well as toluidine blue (TB). Tissue was sectioned and observed using light microscopy.</div></div>