Os músculos extraoculares são responsáveis pela movimentação dos olhos em todos os vertebrados e estão agrupados em quatro músculos retos e dois oblíquos. Porém existem poucas descrições destes músculos para as raias. Neste estudo são descritos e comparados os músculos extraoculares de quatro espécies de raias Mylibatiformes que possuem habitat e hábitos alimentares distintos, sendo elas: Mobula thurstoni (n=10), Pteroplatytrygon violacea (n=10), Daysatis hypostigma (n=10) e Gymnura altavela (n=10). Dasyatis hypostigma, G. altavela e P. violacea possuem o músculo reto dorsal, m. reto ventral, m. reto lateral, m. reto medial, m. obliquo dorsal e o m. obliquo ventral. Em M. thurstoni não foram encontrados dois músculos oblíquos dorsal e ventral e sim apenas um músculo com uma cabeça e duas origens (bíceps). Diferenças significativas como à disposição do olho no condrocrânio, o afunilamento das fibras e local de inserção dos mm. oblíquos próximo ao ponto de inserção; a posição de cruzamento dos músculos reto medial e ventral com o pedículo óptico e a posição da inserção do músculo reto dorsal agruparam as espécies de acordo com seu habitat e modo de vida. Dasyatis hypostigma e G. altavela, raias bentônicas apresentaram o m. oblíquo dorsal mais desenvolvido do que os demais músculos. Em P. violacea, a inserção do músculo oblíquo dorsal ocorre no equador do bulbo e suas fibras não apresentam mudança na direção desde a origem à inserção. Em M. thurstoni, o m. reto lateral está suportado pela ação do músculo reto lateral β. Este músculo pode ser responsável por uma maior ação sinérgica com o músculo oblíquo bíceps. Este estudo mostrou que existem diferenças entre os músculos extraoculares, caindo, portanto a afirmativa de que os músculos extraoculares são \"extraordinariamente constantes\" em todos os vertebrados e abre-se um leque de opções de estudos comparativos para as raias que até então tiveram o estudo dos músculos extraoculares negligenciados. / The extraocular muscles, responsible for the eye movements in all vertebrates, are classically grouped as four rectus muscles: rectus dorsal muscle, rectus ventral muscle, rectus lateral muscle and rectus medial muscle; and two oblique: oblique dorsal muscle and oblique ventral muscle; however, the description of these groups and their possible association with several species habits is very limited. Hence the objective of this study is to demonstrate the differences and singularities of the extraocular muscles in rays of diverse habitats and habits. This study used four species of rays of the Myliobatiformes order: Mobula thurstoni, pelagic stingray and planktofoga. Pteroplatytrygon violacea, pelagic stingray, predator of fish and squid; Dasyatis hypostigma and Gymnura altavela, both benthonic, predators of small fish and invertebrates. Ten heads of each species were decalcified and dissected to characterize and describe the extraocular muscles. The final results followed, qualitatively and quantitatively, the pattern of extraocular muscles found in vertebrate animals, for P. violacea, D. hypostigma e G. altavela species. But this pattern could not be established for M. thurstoni species because of, instead of two oblique muscles, only one muscle with two origins (biceps) was observed. There were also significant differences of the eye disposition in the chondrocranium; fibers narrowing down and on the place of insertion of oblique muscles near to the insertion point; the crossing position of the rectus medial and ventral muscles with the optical pedicle and the insertion position of the rectus dorsal muscle. Furthermore, this study shows that, distinctively from what has been known so far, the extraocular muscles are not the same for all species and present important anatomical differences that allow grouping the studied species according to their feeding behavior. In face of the obtained results, it is safe to conclude that the extraocular muscles are not \"extraordinarily uniform\" in all vertebrates and provide a range of options to comparative studies to various species that, until now, have had their study of extraocular muscles neglected.

Os músculos extraoculares são responsáveis pela movimentação dos olhos em todos os vertebrados e estão agrupados em quatro músculos retos e dois oblíquos. Porém existem poucas descrições destes músculos para as raias. Neste estudo são descritos e comparados os músculos extraoculares de quatro espécies de raias Mylibatiformes que possuem habitat e hábitos alimentares distintos, sendo elas: Mobula thurstoni (n=10), Pteroplatytrygon violacea (n=10), Daysatis hypostigma (n=10) e Gymnura altavela (n=10). Dasyatis hypostigma, G. altavela e P. violacea possuem o músculo reto dorsal, m. reto ventral, m. reto lateral, m. reto medial, m. obliquo dorsal e o m. obliquo ventral. Em M. thurstoni não foram encontrados dois músculos oblíquos dorsal e ventral e sim apenas um músculo com uma cabeça e duas origens (bíceps). Diferenças significativas como à disposição do olho no condrocrânio, o afunilamento das fibras e local de inserção dos mm. oblíquos próximo ao ponto de inserção; a posição de cruzamento dos músculos reto medial e ventral com o pedículo óptico e a posição da inserção do músculo reto dorsal agruparam as espécies de acordo com seu habitat e modo de vida. Dasyatis hypostigma e G. altavela, raias bentônicas apresentaram o m. oblíquo dorsal mais desenvolvido do que os demais músculos. Em P. violacea, a inserção do músculo oblíquo dorsal ocorre no equador do bulbo e suas fibras não apresentam mudança na direção desde a origem à inserção. Em M. thurstoni, o m. reto lateral está suportado pela ação do músculo reto lateral β. Este músculo pode ser responsável por uma maior ação sinérgica com o músculo oblíquo bíceps. Este estudo mostrou que existem diferenças entre os músculos extraoculares, caindo, portanto a afirmativa de que os músculos extraoculares são \"extraordinariamente constantes\" em todos os vertebrados e abre-se um leque de opções de estudos comparativos para as raias que até então tiveram o estudo dos músculos extraoculares negligenciados. / The extraocular muscles, responsible for the eye movements in all vertebrates, are classically grouped as four rectus muscles: rectus dorsal muscle, rectus ventral muscle, rectus lateral muscle and rectus medial muscle; and two oblique: oblique dorsal muscle and oblique ventral muscle; however, the description of these groups and their possible association with several species habits is very limited. Hence the objective of this study is to demonstrate the differences and singularities of the extraocular muscles in rays of diverse habitats and habits. This study used four species of rays of the Myliobatiformes order: Mobula thurstoni, pelagic stingray and planktofoga. Pteroplatytrygon violacea, pelagic stingray, predator of fish and squid; Dasyatis hypostigma and Gymnura altavela, both benthonic, predators of small fish and invertebrates. Ten heads of each species were decalcified and dissected to characterize and describe the extraocular muscles. The final results followed, qualitatively and quantitatively, the pattern of extraocular muscles found in vertebrate animals, for P. violacea, D. hypostigma e G. altavela species. But this pattern could not be established for M. thurstoni species because of, instead of two oblique muscles, only one muscle with two origins (biceps) was observed. There were also significant differences of the eye disposition in the chondrocranium; fibers narrowing down and on the place of insertion of oblique muscles near to the insertion point; the crossing position of the rectus medial and ventral muscles with the optical pedicle and the insertion position of the rectus dorsal muscle. Furthermore, this study shows that, distinctively from what has been known so far, the extraocular muscles are not the same for all species and present important anatomical differences that allow grouping the studied species according to their feeding behavior. In face of the obtained results, it is safe to conclude that the extraocular muscles are not \"extraordinarily uniform\" in all vertebrates and provide a range of options to comparative studies to various species that, until now, have had their study of extraocular muscles neglected.

No description available.

Orientadores: Maria Julia Marques, Humberto Santo Neto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T12:44:34Z (GMT). No. of bitstreams: 1 Pertille_Adriana_D.pdf: 3109380 bytes, checksum: b99b5c31b8c138b7d403dd2de4f6629c (MD5) Previous issue date: 2008 / Resumo: A distrofia muscular de Duchenne (DMD) é caracterizada pela falta de distrofina, proteína estrutural do sarcolema que promove a sua estabilização. Em ausência de distrofina, ocorre aumento da permeabilidade ao cálcio e conseqüente mionecrose. Músculos como tibial anterior, sóleo, diafragma e esternomastóide sofrem ciclos de mionecrose e regeneração muscular. Por outro lado, os músculos extra-oculares (EO) não apresentam degeneração, sendo protegidos da falta da distrofina. A atividade das proteínas ligadas ao Ca++ pode ser um dos mecanismos envolvidos para explicar tal proteção. Nossos resultados revelaram aumento significativo do conteúdo da calmodulina (CaM) e quinase da cadeia leve de miosina (MLCK) no músculos EO mdx quando comparado ao controle. A quantidade da calpaína 1 dos músculos EO distróficos foi igual ao controle, confirmando a ausência do processo de degeneração muscular. Também verificamos se alterações no padrão de distribuição dos receptores de acetilcolina (ACh) e dos terminais nervosos, observadas em junções neuromusculares distróficas, são decorrentes da falta da distrofina ou da regeneração da fibra muscular. O padrão de distribuição dos receptores ACh nos músculos retos e oblíquos distróficos, não mostraram alteração quando comparados ao controle. No músculo retrator do bulbo mdx (parcialmente afetado pela distrofia) 56% dos receptores apresentaram padrão de distribuição alterado. Nossos resultados sugerem que a distrofina ou o complexo distrofina-glicoproteínas (CDG), não estão diretamente envolvidos na organização dos receptores nos músculos EO / Abstract: Duchenne muscular dystrophy (DMD) is characterized by the lack of dystrophin, structural protein that provides stability to the sarcolemma. In the absence of dystrophin, causes increased calcium permeability, leading to myonecrosis. Tibialis anterior, soleus, diaphragm and stermomastoid muscles undergoes myonecrosis and regeneration cycles. However, extraocular muscles (EO) do not show degeneration and are spared of the lack of dystrophin. We investigated whether this protection is related to an activated of calcium-binding proteins. Ours results showed significantly increased of calmodulin (CaM) and of the myosin light chain kinase (MLCK) in the mdx EO compared to control muscles. Calpain quantity in the dystrophic EO was equal of the control, confirmed the lack of the degeneration muscular processed. We also investigated whether changes in acetylcholine (Ach) receptor distribution at the neuromuscular junction and the nerve terminal, showed in the dystrophic neuromuscular junction, which could be correlated to the lack of dystrophy or the muscle fiber regeneration. Distribution ACh receptor in the dystrophic rectus and oblique exhibited no changes compared to control. In mdx retractor bulbi (partial affected by the dystrophy) 56% of the receptor exhibited distribution altered. Taken together, the results suggest the dystrophin or the dystrophin-glycoprotein complex does not influence the distribution of acetylcholine receptors at the neuromuscular junction of spared EO / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

Os músculos extraoculares (EOM), responsáveis pelas rotações oculares, apresentam-se aumentados em suas dimensões na orbitopatia de Graves, o que pode levar o paciente à cegueira (neuropatia óptica). Na prática clínica normalmente mede-se manualmente, em cada imagem coronal de tomografia computadorizada por raios-X (CT), o diâmetro desses músculos para avaliar se estes estão aumentados. A subjetividade e o tempo consumido na aquisição destas medidas são as principais deficiências desses métodos manuais. Dessa forma, apresentamos um método de segmentação dos EOM (MSEG) que supera as falhas, acima citadas. O MSEG proposto é baseado no detector de bordas Laplaciano da Gaussiana (LoG) associado à morfologia matemática. Para determinação do tamanho da máscara LoG levou-se em consideração os efeitos devido ao truncamento e a amostragem. A acurácia das medidas em modelos tridimensionais (3D) é afetada pelo efeito de volume parcial (PVE). Em CT, por exemplo, falsas estruturas de tecidos moles aparecem nas interfaces do osso-para-gordura e do osso-para-ar. Além disso, a pele, que tem número CT (ou escala de Hounsfield) idêntico ao tecido mole, obscurece a renderização deste. A fim de produzir imagens 3D do osso e dos tecidos moles, mais confiáveis para medidas e com melhora de qualidade, foram desenvolvidos dois métodos de classificação dos voxels com PVE (MCLA) baseados num novo modelo de mistura. A remoção da pele é realizada por meio da morfologia matemática. Renderizações volumétricas foram criadas, antes e depois de aplicar os MCLA. Experimentos qualitativo e quantitativo foram conduzidos utilizando fantons matemáticos que simularam diferentes níveis de PVE por adição de ruído e borramento e em dados clínicos de CT. O resultado em 218 pares de medidas de áreas dos EOM realizadas em imagens coronais de CT (3 normais e 2 Graves) revelou uma boa correlação (R=0,92) entre o MSEG e o traçado manual. A medida de taxa de ocupação dos EOM na órbita (TO) feita em 33 pacientes (5 normais e 28 Graves) apresentou o maior valor no grupo Graves com neuropatia óptica, TO=34,3%. Este valor é quase cinco vezes maior que o grupo normal, TO=7,3%. Todos os resultados demonstraram uma melhora de qualidade das imagens 3D depois da aplicação dos MCLA. A análise quantitativa indica que mais de 98% dos voxels com PVE foram removidos por ambos MCLA, e o segundo MCLA têm um desempenho um pouco melhor que o primeiro. Além disso, a remoção da pele torna vívidos os finos detalhes nas estruturas musculares. Medidas em modelos 3D devem ser tomadas com cuidado na radiologia em vista dos artefatos demonstrados neste trabalho, artefatos vindos, principalmente, do PVE. Em nossos experimentos, os erros nas medidas de volume dos EOM foram acima de 25% do valor estimado como "verdadeiro". Imagens volumétricas com PVE resolvidos são apresentadas, e assim medidas mais acuradas são asseguradas. / The extraocular muscles (EOM), which are responsible for the eyes movements, are presented enlarged in their dimensions in Graves’ orbitopathy. These deformations can lead patients to blindness. In clinical routine, physicians normally evaluate, in computer tomography (CT) images, the diameter of the EOM by manual tracing to check if they are enlarged. However, the accuracy of the EOM measurements is impaired by the subjectivity of these manual methods. Further, the time consuming is also one of the main drawbacks on these methods. This way we present an EOM segmentation method (MSEG) that overcomes the difficulties pointed above. The MSEG method is based on the Laplacian-of-Gaussian operator (LOG) combined with the mathematical morphology theory. We have taken into account the effect of discretization and numerical truncation during the LOG implementation. In CT, partial volume effects (PVE) cause several artifacts in volume rendering. In order to create 3D rendition more reliable to carry out anatomical measures and also to pursue superior quality of display of both soft-tissue and bone, we introduce two methods for detecting and classifying voxels with PVE (MCLA) based on a new approach. A method is described to automatically peel skin so that PVE-resolved renditions of bone and soft-tissue reveal considerably more details. We have conducted experiments to evaluate quantitatively and qualitatively all methods proposed here. The MSEG method is well correlated with manual tracing in our experiments (R=0,92). Surface renditions are created from EOM CT dataset segmented using the MSEG method. We have also conducted a quantitative evaluation in patients with Graves’ orbitopathy wherein the EOM volume ratio in the orbit (TO) was T=34,3%, which is about five times higher than in normal patient (TO=7,3%). Volume renditions have been created before and after applying the methods for several patient CT datasets. A mathematical phantom experiment involving different levels of PVE has been conducted by adding different degrees of noise and blurring. A quantitative evaluation was performed using the mathematical phantom and clinical CT data wherein an operator carefully masked out voxels with PVE in the segmented images. All results have demonstrated the enhanced quality of display of bone and soft tissue after applying the proposed methods. The quantitative evaluations indicate that more than 98% of the voxels with PVE are removed by the two methods and the second method performs slightly better than the first. Further, skin peeling vividly reveals fine details in the soft tissue structures. 3D renditions should be used with care in radiology in view of artifacts demonstrated in this work coming from PVE. Finally, we have estimated volume errors in the EOM models higher than 25% if PVE is not properly handled.

Os músculos extraoculares (EOM), responsáveis pelas rotações oculares, apresentam-se aumentados em suas dimensões na orbitopatia de Graves, o que pode levar o paciente à cegueira (neuropatia óptica). Na prática clínica normalmente mede-se manualmente, em cada imagem coronal de tomografia computadorizada por raios-X (CT), o diâmetro desses músculos para avaliar se estes estão aumentados. A subjetividade e o tempo consumido na aquisição destas medidas são as principais deficiências desses métodos manuais. Dessa forma, apresentamos um método de segmentação dos EOM (MSEG) que supera as falhas, acima citadas. O MSEG proposto é baseado no detector de bordas Laplaciano da Gaussiana (LoG) associado à morfologia matemática. Para determinação do tamanho da máscara LoG levou-se em consideração os efeitos devido ao truncamento e a amostragem. A acurácia das medidas em modelos tridimensionais (3D) é afetada pelo efeito de volume parcial (PVE). Em CT, por exemplo, falsas estruturas de tecidos moles aparecem nas interfaces do osso-para-gordura e do osso-para-ar. Além disso, a pele, que tem número CT (ou escala de Hounsfield) idêntico ao tecido mole, obscurece a renderização deste. A fim de produzir imagens 3D do osso e dos tecidos moles, mais confiáveis para medidas e com melhora de qualidade, foram desenvolvidos dois métodos de classificação dos voxels com PVE (MCLA) baseados num novo modelo de mistura. A remoção da pele é realizada por meio da morfologia matemática. Renderizações volumétricas foram criadas, antes e depois de aplicar os MCLA. Experimentos qualitativo e quantitativo foram conduzidos utilizando fantons matemáticos que simularam diferentes níveis de PVE por adição de ruído e borramento e em dados clínicos de CT. O resultado em 218 pares de medidas de áreas dos EOM realizadas em imagens coronais de CT (3 normais e 2 Graves) revelou uma boa correlação (R=0,92) entre o MSEG e o traçado manual. A medida de taxa de ocupação dos EOM na órbita (TO) feita em 33 pacientes (5 normais e 28 Graves) apresentou o maior valor no grupo Graves com neuropatia óptica, TO=34,3%. Este valor é quase cinco vezes maior que o grupo normal, TO=7,3%. Todos os resultados demonstraram uma melhora de qualidade das imagens 3D depois da aplicação dos MCLA. A análise quantitativa indica que mais de 98% dos voxels com PVE foram removidos por ambos MCLA, e o segundo MCLA têm um desempenho um pouco melhor que o primeiro. Além disso, a remoção da pele torna vívidos os finos detalhes nas estruturas musculares. Medidas em modelos 3D devem ser tomadas com cuidado na radiologia em vista dos artefatos demonstrados neste trabalho, artefatos vindos, principalmente, do PVE. Em nossos experimentos, os erros nas medidas de volume dos EOM foram acima de 25% do valor estimado como "verdadeiro". Imagens volumétricas com PVE resolvidos são apresentadas, e assim medidas mais acuradas são asseguradas. / The extraocular muscles (EOM), which are responsible for the eyes movements, are presented enlarged in their dimensions in Graves’ orbitopathy. These deformations can lead patients to blindness. In clinical routine, physicians normally evaluate, in computer tomography (CT) images, the diameter of the EOM by manual tracing to check if they are enlarged. However, the accuracy of the EOM measurements is impaired by the subjectivity of these manual methods. Further, the time consuming is also one of the main drawbacks on these methods. This way we present an EOM segmentation method (MSEG) that overcomes the difficulties pointed above. The MSEG method is based on the Laplacian-of-Gaussian operator (LOG) combined with the mathematical morphology theory. We have taken into account the effect of discretization and numerical truncation during the LOG implementation. In CT, partial volume effects (PVE) cause several artifacts in volume rendering. In order to create 3D rendition more reliable to carry out anatomical measures and also to pursue superior quality of display of both soft-tissue and bone, we introduce two methods for detecting and classifying voxels with PVE (MCLA) based on a new approach. A method is described to automatically peel skin so that PVE-resolved renditions of bone and soft-tissue reveal considerably more details. We have conducted experiments to evaluate quantitatively and qualitatively all methods proposed here. The MSEG method is well correlated with manual tracing in our experiments (R=0,92). Surface renditions are created from EOM CT dataset segmented using the MSEG method. We have also conducted a quantitative evaluation in patients with Graves’ orbitopathy wherein the EOM volume ratio in the orbit (TO) was T=34,3%, which is about five times higher than in normal patient (TO=7,3%). Volume renditions have been created before and after applying the methods for several patient CT datasets. A mathematical phantom experiment involving different levels of PVE has been conducted by adding different degrees of noise and blurring. A quantitative evaluation was performed using the mathematical phantom and clinical CT data wherein an operator carefully masked out voxels with PVE in the segmented images. All results have demonstrated the enhanced quality of display of bone and soft tissue after applying the proposed methods. The quantitative evaluations indicate that more than 98% of the voxels with PVE are removed by the two methods and the second method performs slightly better than the first. Further, skin peeling vividly reveals fine details in the soft tissue structures. 3D renditions should be used with care in radiology in view of artifacts demonstrated in this work coming from PVE. Finally, we have estimated volume errors in the EOM models higher than 25% if PVE is not properly handled.

La sclérose latérale amyotrophique (SLA) est une maladie touchant spécifiquement les motoneurones (MN) qui se caractérise par la perte précoce des jonctions neuromusculaires (JNMs) et menant à une paralysie musculaire. La dénervation des JNMs des muscles squelettiques se produit en amont de la mort des MN de la moelle épinière. Des études récentes publiées ont révélé une altération de la transmission synaptique, une instabilité de la morphologie des JNMs ainsi que des mécanismes de réparations de la JNMs inappropriés dans le modèle SOD1, et ce avant l’apparition des symptômes moteurs. De manière intéressante, ces mécanismes sont régulés par les cellules de Schwann périsynaptiques (CSPs), la cellule gliale présente à la JNM suggérant ainsi que l’altération des fonctions des CSPs peut contribuer à la vulnérabilité des JNMs. Tandis que de nombreuses études ont démontré une susceptibilité à la dénervation qui est dépendante du type d’unité motrice (UM), l’innervation des muscles extraoculaires (EOMs) montre une importante résistance à la progression de la maladie. Afin d’investiguer les distinctions dans les JNMs des EOMs menant à cette résistance, nous avons procédé à une analyse de la morphologie des JNMs via microscopie confocale, nous avons étudié les propriétés fonctionnelles des CSPs par imagerie calcique ainsi qu’effectuer une analyse différentielle du protéome entre les JNMs résistantes de l’EOM et les JNMs vulnérables du soleus (SOL) ou de l’extensor digitorum longus (EDL) dans la souris SOD1G37R. Peu de dénervation des JNMs et aucune altération des JNMs sont observées dans l’EOM à un stade tardif de la maladie. Contrairement aux muscles vulnérables, la sensibilité des CSPs suite à l’application locale d’ATP et de muscarine n’est pas altérée dans les EOM. L’analyse du protéome entre l’EDL et l’EOM au stade symptomatique démontre des fonctions cellulaires distinctes. Dans l’EDL, au stade symptomatique, les cascades cellulaires catabolique et reliée au protéosome sont augmentées : reflétant le processus de dénervation en cours dans ce muscle. Dans l’EOM, une diminution de l’expression de SOD1 muté, une augmentation des processus d’oxydoréductions, des protéines importantes pour maintien du repliement des protéines, des neurofilaments ainsi qu’une expression distincte des enzymes régulant les neurotransmetteurs est observée dans les JNMs résistantes. Ainsi, comprendre les fonctions des CSPs ainsi que les profils d’expression protéomique distincte entre les JNMs vulnérables et résistantes durant la progression de la maladie peut nous fournir des informations sur les mécanismes impliqués durant la dénervation et aider à identifier les protéines potentielles qui peut favoriser la réparation et l’intégrité des JNMs. Ainsi, cette étude peut mener à l’identification de biomarqueur musculaire et de cible thérapeutique potentielle pour des perspectives curatives futures. / Amyotrophic lateral sclerosis (ALS) is a motor neuron (MNs) disease characterized by the precocious loss of neuromuscular junctions (NMJs) and muscular paralysis. The denervation of NMJs at striated muscles is an early event that occurs before the loss of spinal cord MNs. Recent data revealed an alteration of synaptic transmission, morphological instability and inappropriate repair in NMJs of SOD1 mice model prior to motor symptoms. Interestingly, these mechanisms are known to be regulated by Perisynaptic Schwann cells (PSCs), glial cells at NMJs, suggesting that the alteration of PSC functions may contribute to NMJ vulnerability. While numerous studies demonstrated a motor unit type-dependent susceptibility to denervation, the extraocular muscles (EOM) innervation shows a prominent resistance to disease progression. We hypothesized that PSCs functions and intrinsic properties at extraocular NMJs contribute to the resistance of the disease progression. NMJ morphological analysis by immunostaining and confocal imaging, functional properties of PSCs by calcium imaging and a differential proteomic analysis using Tandem Mass Tags coupled to quantitative mass spectrometry was performed between the resistant EOM and the vulnerable, soleus (SOL) or Extensor digitorum longus (EDL) muscles in SOD1G37R mice. Fewer denervated NMJs and no alteration of NMJ integrity was observed in the EOM in comparison to the EDL. Sensitivity of EOM PSC to local application of ATP and muscarine are not altered in the EOM SOD1G37R in comparison to WT suggesting an adequate decoding of synaptic activity of PSC. Proteomics analysis between EDL and EOM at symptomatic stage demonstrates distinct cellular pathway. In the EDL, at symptomatic stage, catabolism and proteasome cellular pathways are upregulated reflecting the undergoing denervation processes observed. In the EOM, overall lower expression of SOD1, up-regulation of oxidoreduction process, of mechanism against protein unfolding, of neurofilament and distinct expression of enzymes regulating neurotransmitter homeostasis is observed in the resistant NMJ. Understanding PSC functions and investigating the distinctive protein expression profile between vulnerable and resistant NMJs during disease progression will help provide insights into the denervation mechanisms involved and help identify potential proteins that could favor NMJ repair and integrity. Also, this study may lead to the identification of muscle biomarkers and potential therapeutic targets moving toward curative perspectives.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative qui affecte spécifiquement les motoneurones (MNs) supérieurs et inférieurs conduisant à une paralysie musculaire. La dénervation des jonction neuromusculaires (JNMs) se produit en amont de la mort des MNs de la moelle épinière chez les patients atteint de la SLA et dans de nombreux modèles murins de la maladie. Récemment, des chercheurs ont révélé une altération de la transmission synaptique, une instabilité morphologique, et une réparation inappropriée des JNMs dans le modèle de souris SOD1 en amont de l’apparition des désordres moteurs. Tandis que notre laboratoire a étudié les trois éléments synaptiques, ce mémoire porte une attention particulière aux cellules de Schwann périsynaptiques (CSPs), les cellules gliales à la JNM, considérant leurs rôles fondamentaux dans la régulation de la structure et la fonction de la JNM. Alors que de nombreuses études ont démontré une susceptibilité à la dénervation dépendante du type d’unité motrice, où certaines serait plus vulnérables au processus de dénervation que d’autres, les propriétés altérées des CSPs ont été généralisé à tous les types de JNMs étudiés. Notamment, des études réalisées dans le laboratoire ont rapporté une capacité inappropriée des CSPs à décoder l’information basée sur une augmentation de l’activation des récepteurs muscariniques (mAChRs). Les fonctions des mAChRs des CSPs sont d’une importance particulière puisque leur activité est essentielle à la stabilité des JNMs et à leur réparation et est régulé par l’activité synaptique. De manière importante, nous avons observé que la diminution chronique in vivo de l’activation des mAChRs des CSPs chez les souris SOD1G37R favorise la réparation de la JNM et améliore les fonctions motrices chez l’animal. Ainsi, la moindre altération dans les propriétés des CSPs pourrait contribuer directement à la vulnérabilité des NMJs dans la SLA. Considérant le rôle crucial des cellules gliales dans la maintenance et la réparation des JNMs, nous avons émis l’hypothèse que les CSPs contribuent à la différence de vulnérabilité observée dans la SLA. Nous avons postulé que l’hyperactivité muscarinique des CSPs contribue à l’instabilité des JNMs vulnérables, alors qu’une activité muscarinique normale contribue à la stabilité des JNMs résistantes. Pour mieux comprendre les différences dans les propriétés des CSPs contribuant à cette différence de vulnérabilité, nous avons étudié les propriétés fonctionnelles des CSPs par imagerie calcique afin de caractériser la signature muscarinique des CSPs aux JNMs d’un muscle vulnerable, l’extensor digitorum longus (EDL). Nous avons évalué l’intégrité des JNMs par un triple marquage immunohistochimique. De manière intéressante, nos résultats ont montré que L’utilisation d’un outil chémogénétique nous a permis d’augmenter l’excitabilité des AChRs des CSPs aux JNMs résistantes des MEOs. L’évaluation de l’intégrité des JNMs par un triple marquage immunohistochimique a montré que le traitement au CNO induit de l’instabilité au niveau des JNMs et nous avons observé des signes de dénervation. Établir un potentiel rôle des CSPs dans la résistance des JNMs a permis de souligner un nouveau facteur important dans la pathophysiologie de la SLA et a fourni des connaissances dans les mécanismes de résistance sélective/vulnérabilité à la dénervation. Cela permet d’ouvrir le champ à de nouvelles cibles thérapeutiques ciblant les cellules gliales à la JNM. De plus, ce nouveau contexte conceptuel de susceptibilité des JNMs peut être transposé à d’autres maladies neuromusculaires. / Amyotrophic lateral sclerosis (ALS) is a fatal late-onset neurodegenerative disease characterized by progressive loss of upper and lower motor neurons (MNs) leading to muscular paralysis. Denervation of the neuromuscular junction (NMJ) is an early pathological event that occurs before the loss of spinal cord MNs in ALS patients and various murine models of the disease. Recently, authors revealed an alteration of synaptic transmission, morphological instability, and inappropriate repair in NMJs of SOD1 mice model prior to motor impairments. While our laboratory studied all three synaptic elements, we put a particular attention to Perisynaptic Schwann cells (PSC), glial cells at the NMJ, owing to their fundamental roles in regulating NMJ structure and function. While numerous studies demonstrated a motor-unit type dependent susceptibility to denervation where some motor units (MUs) would be more vulnerable than others, altered PSC properties were generalized among all types of NMJ studied. Notably, studies performed in the laboratory reported an inappropriate PSC decoding capability based on an enhanced activation of mAChRs. PSC mAChR functions is of particular importance since it is essential for the management of NMJ stability and repair and is regulated by synaptic activity. Importantly, we observed that chronic in vivo dampening of PSC muscarinic activation in SOD1G37R fostered NMJ repair and improved motor function in the ALS mouse model. Hence, any alteration of PSC properties may directly contribute to NMJ vulnerability in ALS. Owing to the critical roles of glial cells for the maintenance and repair of NMJs, we hypothesized that PSC contribute to the differential vulnerability observed in ALS. We proposed that the hyperactive muscarinic excitation of PSCs contributes to NMJ instability at vulnerable NMJs while the normal muscarinic activity contributes to their stability in resistant ones. To better understand the distinctions in PSCs properties contributing to a difference in NMJ vulnerability, we studied the PSC functional properties by calcium imaging to characterize the muscarinic signature of PSCs at the NMJ of a vulnerable muscle, the extensor digitorum longus (EDL). We assessed the integrity of the NMJ by a triple immunostaining. Interestingly, our data revealed that altering PSC properties at resistant NMJs by enhancing the muscarinic excitation of PSCs using a viral strategy created NMJ instability with signs of denervation. Determining the potential role of PSC in the resistance of NMJs highlighted a novel important factor underlying the pathophysiology of ALS and provided significant insights into the mechanisms of selective resistance/vulnerability to denervation. This could pave the way to novel therapeutic targets and strategies targeting glial cells at the NMJ. Furthermore, this novel conceptual context may be carried over to NMJ susceptibility for other neuromuscular diseases.

Orbitale Erkrankungen stellen für mehrere klinische Fachdisziplinen in Bezug auf Diagnostik und Therapie ein großes Problem dar. Die hochauflösende MRT (HR-MRT) ist in der Lage, eine detailierte Übersicht für die Orbitaanatomie und deren Abnormalitäten zu liefern. Aus diesem Grunde ist diese Studie durchgeführt worden, in der hochauflösenden MRT-Charakteristika von 224 Patienten mit orbitalen Erkrankungen beurteilt worden ist. Die Kriterien der Beurteilung sind: Lokalisation, Größe, Form, Rand, Ausdehnung und Veränderung der Nachbarstrukturen sowie die Signalintensitäten der Erkrankungen. Die Lokalisationskriterien sind sehr nützlich für die Differentialdiagnose orbitaler Erkrankungen der Augenmuskeln, dem extrakonalen und subperiostalen Kompartiment mittels der HR-MRT mit Oberflächenspule. Durch die Zuordnung zu einem Kompartiment kann die Differentialdiagnose grob eingeengt werden. Das subperiostale Kompartiment kann in Sinus, Meningen, orbitalen Knochen und subperiostalen Spaltraum weiter unterteilt werden. Mukozelen und Nasennebenhöhlentumoren sind nur in den Sinus zu finden, sämtliche Keilbeinmeningeome weisen einen Befall der Meningen auf. 83,3% Epidermoide und Dermoide liegen im orbitalen subperiostalen Raum mit einer engen Nachbarschaft zu einer Sutur. Die meisten Muskelbefälle der endokrinen Orbitopathie betreffen den inferioren und medialen geraden Muskel, die Verdickung des Muskelbefalls ist typischerweise nur im Muskelbauch ohne Muskelsehnebefall. Myositiden haben häufiger einen Muskelsehnebefall. Wenn eine Verdickung eines einzelnen Muskels den M. rectus lateralis betrifft, ist die Diagnose einer endokrinen Orbitopathie nicht wahrscheinlich. Rhabdomyosarkome liegen häufig im oberen inneren Quadranten der Orbita. Lymphome liegen meistens im frontalen Anteil des extrakonalen Kompartiment in Nachbarschaft zum Septum orbitale. Für eine weitere Differentialdiagnose sollen die morphologischen Kriterien verwendet werden. Auch die Große, Form und der Rand der orbitalen Erkrankungen können den wichtigen Hinweis zur Differentialdiagnose liefern. Ein reduziertes Muskelvolumen ist bei der Muskelatrophie und dem Trauma mit Muskel Einklemmung gesehen worden. Bei 11 von 128 endokrinen Orbitopathien ist das Volumen normal. Die meiste Erkrankungen gehen mit einer Volumenzunahme einher. Keilbeinmeningeome, Karziome der Nasennebenhöhlen, Rhabdomyosarkome und Lymphome haben große Volumina. Myositis und endokrine Orbitopathie mit Muskelbefall haben meist geringe oder mittlere Volumenzunahmen. Mukozelen, Epidermoid, Dermoid, Metastasen und Hämangiom können in allen Größenstufen gefunden werden. Epidermoide, Dermoide, Mukozelen, Hämangioperizytome, Hämangiome, Rhabdomyosarkome und Metastasen haben meist runde oder elliptische Formen, eine längliche Form wird bei orbitaler Myositis und endokriner Orbitopathie mit Muskelbefall beobachtet. Karzinome der Nasennebenhöhlen, Keilbeinmeningeome, Lymphome, endokrine Orbitopathie mit Fettbefall, Pseudotumoren und Phlegmone zeigen sich unregelmäßige Formen. Epidermoide, Dermoide, Mukozelen, Myositis, endokrine Orbitopathie, Hämangiome und Hämangioperizytome haben meistens einen scharfen Rand. Dagegen weisen Karziome der Nasennebenhöhlen, Lymphome, Keilbeinmeningeome, Rhabdomyosarkome, Metastasen, Pseudotumoren und Phlegmonen undeutliche Ränder auf.Die Verwendung der MRT-Signalintensit?ten zur Diffenrenzierung ist eingeschränkt, weil die meisten Läsionen sehr ähnliche Signalintensitäten zeigen. Es gibt aber einige Läsionen, die aufgrund ihrer Zusammensetzung besondere Signalintensitäten aufweisen, z.B. Blutungen, Melanin (paramagnetisch), Fett, Proteine, Wasser und Nekrosen. Mit einer Kontrastmittelgabe und dem Verhalten des Enhancements kann eine weitere Charakterisierung erfolgen. Bei den Veränderungen der Nachbarstrukturen sind vor allem die Knochenveränderungen am aussagekräftigsten. Epidermoide, Dermoide und Mukozelen sind meist mit Knochendefekten oder Kompressionsveränderungen verbunden, Keilbeinmeningeome stehen eng in Zusammenhang mit Knochenhyperplasien, Nasennebenhöhlenkarzinome sind immer mit Knochendestruktionen verbunden und Frakturen werden nur bei Traumata gesehen. Die raumfordernde Wirkung von Lymphomen ist nicht sichtbar, denn hier zeigt sich eine Diskrepanz zwischen einer oft charakteristischen, ausgedehnten Infiltration und nur einer geringen Verlagerung anderer Orbitastrukturen. Die klinische Krankengeschichte ist auch wesentlich zur Findung der Differentialdiagnose. Alle hier untersuchten Patienten mit Phlegmone und Trauma haben besondere Krankengeschichten. 70% Patienten mit Metastasen haben einen gesicherten Primärtumor in der Krankengeschichte. Alle Patienten mit endokriner Orbitopathie haben in der Anamnese eine Schilddrüsenerkrankung. Von diesen Patienten sind etwa 80% weiblich und 73% zwischen 30 und 60 Jahre alt. Rhabdomyosarkome treten hauptsächlich in der Kindheit auf. Pseudotumoren sprechen schnell und außerordentlich gut auf Steroidtherapien an. Somit kann eine Verbindung von Anamnese und MRT-Bildgebung wertvolle Informationen zur Differentialdiagnose liefern.Zur Differenzierung endokriner Orbitopathie und Myositis ist es nützlich die Lokation des Muskelbefalls (unilateral oder bilateral, Muskelsehne- oder Muskelbauchbefall, Einzelbefall oder Befall mehrerer Muskeln und Bevorzugung bestimmter Muskeln), die Ausdehnung (mit oder ohne Fettbefall), die Verlagerung des Augapfels, MRT-Signalintensitäten und die zeitliche Anamnese zu berücksichtigen. Zur Differenzierung Rhabdomyosarkom und Lymphome sollen das Alter, die Form der Läsionen und der Muskelbefall berücksichtigt werden. Zur Differenzierung zwischen Metastasen und Hämangiome können ihre klinische Krankengeschichte (mit oder ohne frühere Tumoren), der Rand der Läsionen, Muskelbefall und ihr MRT-Signal und das Ausmaß des Enhancements wertvolle Informationen liefern. Die einzige Differenzierungsmöglichkeit zwischen Epidermoid und Dermoid ist die MRT-Signalintensität (fettiger oder wässriger Inhalt der Läsion). Die Lokation ist am wichtigsten für die Differenzierung zwischen Epidermiod und Mukozelen. Für eine Differenzierung zwischen Keilbeinmeningeomen und Masennebenhöhlenkarzinome können ihre Lokationen, ihre Signalintensität, das Kontrasmittelverhalten und Veränderungen der Nachbarstrukturen den wichtigen Hinweis liefern. / The clinical diagnosis of orbital diseases is especially difficult because of the variety of tissues that built up the orbit and present with similar clinical presentation. High-resolution MRI (HR-MRI) has become an important modality for evaluation of the orbital diseases, due to its superb soft tissue resolution, direct multiplanar capability and lack of ionizing radiation. In this study, 224 patients with pathologically identified orbital diseases were evaluated retrospectively. The imaging characteristics of the orbital diseases on HR-MRI with surface coil were assessed. The analyzed criteria were: location, size, shape, margins, extension, adjacent structure, and signal intensity. Locazilation criteria in muscles, extraconal and subperiosteal compartments are very useful in the differential diagnosis of orbital tumors and other disease. If their locations are subdivided into greater detail, the differential diagnosis will be narrowed considerably. For example, the subperiosteal compartment can be subdivided into sinus, meninges, bone and subperiosteal space. Mucocele and carcinoma of nasal sinuses always occur in the nasal sinuses. Sphenoidal meningiomas occur in the meninges, and epidermoid and dermoid are found to 83.3% in the orbital subperiosteal space near the bone sutures and show bony defects or thinning and sclerosis. The most frequently involved muscles in thyroid orbitopathy are the inferior rectus and the medial rectus muscles. Typically, the muscle enlargement involves the muscle belly and spares its tendinous portion. While orbital myositis most frequently involves the lateral rectus muscles in particular the muscles' tendon. When isolated lateral rectus muscle enlargement is present, another etiology rather than thyroid orbitopathy should be considered. Within the extraconal compartment the upper inner quadrant is the most common site of rhabdomyosarcoma. While lymphomas mostly occur in the anterior orbit, posterior to the orbital septum. For further differentiation, morphological criteria can be employed, such as the size, shape, and margin help to make the differential diagnosis. Reduced muscle volume is characteristic in the atrophic muscle. The shortened muscle length occurs in traumas connected with muscle incarcerations. Disease without volume changes may happen in thyroid orbitopathy with the involvement of the muscle or the fat tissue. All the other diseases have a enlarged volume of lesion. The size of Sphenoidal meningioma, carcinoma of nasal sinuses, lymphoma and rhabdomyosarcoma is greatly enlarged. While the size-distribution of mucoceles, epidermoids and dermoids show no difference. But orbital myositis and thyroid orbitopathy have slight to moderate enlarged volume of involved muscles. Concerning the pathology shape mucoceles, dermoids, epidermoids, haemangiomas, and metastases are round or elliptical. The orbital myositis and thyroid orbitopathy with muscle involvement show long shapes. Lymphomas and the thyroid orbitopathy with fat tissue involvement have mostly irregular shapes. The poor-defined margin is often seen in malignant lesions, infections or inflammations, such as a paranasal carcinomas, lymphomas, rhabdomyosarcomas, pseudotumors and phlegmon. While the sharp delineation suggests a benign process, such as a mucocele, dermoid, epidermoid, haemangioma, thyroid orbitopathy and myositis. The use of MRI signal intensity for a differential criterion may be limited, because most diseases have the same signal intensity on MR imaging. Thus it seems to be that signal intensity patterns are usually not specific for the differentiating of diagnose. However, MRI may have some specificity for certain lesions based on signal intensity patterns, especially in those containing hemorrhage, paramagnetic melamin, fat, protein, water, enlarged vessels, and necrosis. With contrast enhancement MRI further contributes to the characterization of various types of lesions within the orbit. It can be made a definitive diagnosis for the dermoid (contains fat), cholesterolcyste, meningioma, lymphangioma (contain hemorrhage), haemangioma, metastasis of melanoma, thyroid orbitopathy with active edema and chronic fibrosis, hematoma and emphysema due to a trauma. Another criterium is the adjacent structure changes. Out of them, the bone changes are very important. Epidermoid, dermoid, and mucocele are often accompanied by bone defects or pressure changes. The sphenoidal meningioma is frequently accompanied by bone hyperplasia. Bone fractures are seen in traumas. Malignant tumors are almost associated with bone destruction when they involve the orbit, e.g., the carcinoma of the nasal sinuses. The effect of occupying space in lymphomas causes mismatch with the size, which often has greater volume. It mostly infiltrates tissue, but it seldom produces mechanical shift to adjacent structures. Clinical history is essential and occasionally it provides a clue that alters the boundary of differential diagnoses and redirects the investigation toward possibilities that had not been previously entertained. Patients with metastases have a definite primary tumor history to 70%. The knowledge of previous malignancy is important for the diagnoses of orbital metastasis. All the patients with thyroid orbitopathy, phlegmon, pseudotumor and trauma have also special clinical histories or symptoms. The rhabdomyosarcoma and capillary hemangioma are the most common orbital tumors in children, while the cavernous hemangioma is the most common orbital vascular tumor in adults. Thyroid orbitopathy occurs with highest prevalence in females with middle age. Orbital pseudotumors usually show a dramatic resolution under steroid therapy. In fact, its final diagnosis is often based on response to steroids. When combined with clinical history and examination, radiologic imaging can provide valuable information regarding the diagnosis and differential diagnoses. For the differentiation of the thyroid orbitopathy and myositis it is useful to take their locations (unilateral or bilateral, tendon involvement or not, single or multiple muscles involvement, and which muscles involvement), sizes, extensions (with or without fat-infiltration), and MRI signal intensities into account; For the differentiation of rhabdomyosarcomas and lymphomas clinical history (ages), the shape of the lesions, and the adjacent structure changes (muscles infiltration) are of diagnostic interest. Clinical history (with or without previous malignancy), the margin of the lesion, their adjacent structure changes (muscles infiltration), and MRI signal intensities help to discriminate between metastases and hemangiomas. The only difference between epidermoid and dermoid is their MRI signal intensities (with or without fat). Location is important to differentiate the epidermiod and mucocele. Locations, adjacent structure changes and MRI signal intensities can provide valuable information to distinguish between sphenoidal meningioma and carcinoma of nasal sinuses. The different shape, margin, adjacent structure change, and MRI signal intensity between the carcinoma of nasal sinuses and mucocele are of diagnostic help.

Background: Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder. ALS has been traditionally believed to be primarily a motor neuron disease. However, accumulating data indicate that loss of contact between the axons and the muscle fibres occurs early; long before the death of motor neurons and that muscle fibres may initiate motor neuron degeneration. Thus, the view of ALS is changing focus from motor neurons alone to also include the muscle fibres and the neuromuscular junctions (NMJs). While skeletal muscles are affected in ALS, oculomotor disturbances are not dominant features of this disease and extraocular muscles (EOMs) are far less affected than limb muscles. Why oculomotor neurons and EOMs are capable to be more resistant in the pathogenetic process of ALS is still unknown. The overall goal of this thesis is to explore the pathophysiology of ALS from a muscle perspective and in particular study the expression and distribution of key neurotrophic factors (NTFs) and Wnt proteins in EOMs and limb muscles from ALS donors and from SOD1G93A transgenic mice. Comparisons were made with age-matched controls to distinguish between changes related to ALS and to ageing. Results: Brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4) were present in EOMs and limb muscles at both mRNA and protein levels in control mice. The mRNA levels of BDNF, NT-3 and NT-4 were significantly lower in EOMs than in limb muscles of early and/or late control mice, indicating an intrinsic difference in NTFs expression between EOMs and limb muscles. qRT-PCR analysis showed significantly upregulated mRNA levels of NT-3 and GDNF in EOMs but significantly downregulated mRNA levels of NT-4 in limb muscles from SOD1G93A transgenic mice at early stage. The NTFs were detected immunohistochemically in NMJs, nerve axons and muscle fibres. The expression of BDNF, GDNF and NT-4 on NMJs of limb muscles, but not of EOMs, was significantly decreased in terminal stage ALS animals as compared to the limb muscles of the age-matched controls. In contrast, NTFs expression in intramuscular nerve axons did not present significant changes in either muscle group of early or late ALS mice. NTFs, especially BDNF and NT-4 were upregulated in some small-sized muscle fibres in limb muscles of late stage ALS mice. All the four Wnt isoforms, Wnt1, Wnt3a, Wnt5a and Wnt7a were detected in most axon profiles in all human EOMs with ALS, whereas significantly fewer axon profiles were positive in the human limb muscles except for Wnt5a. Similar differential patterns were found in myofibres, except for Wnt7a, where its expression was elevated within sarcolemma of limb muscle fibres. β-catenin, a marker of the canonical Wnt pathway was activated in a subset of myofibres in the EOMs and limb muscle in all ALS patients. In the SOD1G93A mouse, all four Wnt isoforms were significantly decreased in the NMJs at the terminal stage compared to age matched controls. Conclusions: There were clear differences in NTF and Wnt expression patterns between EOM and limb muscle, suggesting that they may play a role in the distinct susceptibility of these two muscle groups to ALS. In particular, the early upregulation of GDNF and NT-3 in the EOMs might play a role in the preservation of the EOMs in ALS. Further studies are needed to determine whether these proteins and the pathways they control may be have a future potential as protecting agents for other muscles.