Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

Thesis (Ph.D.) - University of Queensland, 2006. / Includes bibliography.

This study examined the effect of optic tract ablation on retinotectal fiber regeneration in goldfish. Approximately two-thirds of the left optic tract was removed, and, at various times post lesion (10-75 days), the course of regenerating retinotectal fibers was traced using horseradish peroxidase. In all experimental animals, axons were observed regenerating through the visual pathway but at the brachia most of the fibers were channeled through the ventral brachium. We present evidence that fibers in the ventral brachium originated from ganglion cells in all regions of retina and that these fibers grew almost exclusively into ventral half tectum even though some of these fibers would normally synapse in dorsal half tectum. These observations suggest that optic tract ablation does not prevent retinal fiber regeneration but results in aberrant growth through the brachia and significant inhibition of exploratory fiber growth within the tectum.

In the adult brain, the thalamocortical tract conveys sensory information from the external environment to the cortex. The cortex analyzes and integrates this information and sends neural responses back to the thalamus through the corticothalamic tract. To reach their final target both thalamocortical and corticothalamic axons have to cover long distances during embryogenesis, changing direction several times and passing through different brain territories. The ventral telencephalon plays a major role in the early development of these tracts. At least three main axon guidance mechanisms act in the ventral telencephalon. First, two different populations of pioneer neurons in the lateral ganglionic eminence (LGE) (LGE pioneer neurons) and medial ganglionic eminence (MGE) (MGE pioneer neurons) provide scaffolds which allow growing corticothalamic and thalamocortical axons to cross the pallium sub pallium boundary (PSPB) and the diencephalic telencephalic boundary (DTB), respectively. Second, the ventral telencephalon forms a permissive corridor for thalamic axons by tangential migration of Isl1 and Ebf1 expressing cells from the LGE into the MGE. Finally, thalamortical and corticothalamic axons guide each other once they have met in the ventral telencephalon (“handshake hypothesis”). The Gli3 transcription factor has been shown to be essential for normal early embryonic regionalization of the mammalian forebrain, although roles of Gli3 in later aspects of forebrain development, like the formation of axonal connections, have not been investigated previously. Here, I present the analysis of axonal tract development in the forebrain of the Gli3 hypomorphic mutant mouse Polydactyly Nagoja (Pdn). These animals lack the major axonal commissures of the forebrain: the corpus callosum, the hippocampal commissure, the anterior commissure and the fimbria. In addition, DiI injections and neurofilament (NF) staining showed defects in the formation of the corticothalamic and thalamocortical tracts. Although the Pdn/Pdn cortex forms early coticofugal neurons and their axons, these axons do not penetrate the LGE and instead run along the PSPB. Later in development, although a thick bundle of Pdn/Pdn cortical axons is still observed to project along the PSPB, some Pdn/Pdn cortical axons eventually enter the ventral telencephalon navigating along several abnormal routes until they reach thalamic regions. In contrast, Pdn/Pdn thalamic axons penetrate into the ventral telencephalon at early stages of thalamic tract development. However, rostrally they deviate from their normal trajectory, leaving the internal capsule prematurely and only few of them reach the developing cortex. Caudally, an ectopic Pdn/Pdn dorsal thalamic axon tract projects ventrally in the ventral telencephalon not entering the internal capsule at all. These defects are still observed in newborn Pdn/Pdn mutant mice. Next, I investigated the developmental mechanisms causing these pathfindings defects. No obvious defects are present in Pdn/Pdn cortical laminae formation and in the patterning of the Pdn/Pdn dorsal thalamus. In addition, Pdn/Pdn thalamocortical axons are able to respond to ventral telencephalic guidance cues when transplanted into wild type brain sections. However, these axonal pathfinding defects correlate with patterning defects of the Pdn/Pdn LGE. This region is partially ventralized and displays a reduction in the number of postmitotic neurons in the mantle zone due to an elongated cell cycle length of LGE progenitor cells. Finally, Pdn/Pdn mutant display an upregulation of Shh expression and Shh signalling in the ventral telencephalon. Interestingly, these patterning defects lead to the absence of DiI back-labelled LGE pioneer neurons, which correlates with the failure of corticothalamic axons to penetrate the ventral telencephalon. In addition, ventral telencephalic thalamocortical guidance mistakes happen at the same time of abnormal formation of the corridor cells. Taken together these data reveal a novel role for Gli3 in the formation of ventral telencephalic intermediate cues important for the development of the thalamocortical and corticothalamic connections. Indeed, Pdn animals are the first known mutants with defective development of the LGE pioneer neurons, and their study provides a link between early patterning defects and axon pathfinding in the developing telencephalon.

La protéine Tau est une protéine associée aux microtubules, localisée principalement dans les axones. Elle joue un rôle important dans la polymérisation et la stabilisation des microtubules, in vitro. Sa fixation aux microtubules est régulée par de nombreuses kinases et phosphatases. En effet, lorsque Tau est phosphorylée, elle se détache des MTs. Inversement, elle se fixe aux MTs lorsqu’elle est déphosphorylée. Le dysfonctionnement de la protéine Tau est à l’origine de différentes maladies neurodégénératives appelées Tauopathies comme la maladie d’Alzheimer. Dans ce contexte pathologique, Tau est anormalement phosphorylée et s’accumule sous forme de structures neurofibrillaires appelées PFH (paires de filaments hélicoïdaux). Ces structures sont retrouvées dans les neurones en dégénérescence et constituent une des caractéristiques majeures de lésion histopathologique de la MA. Dans le cadre de cette maladie, deux principaux mécanismes de toxicité neuronale induite par la protéine Tau ont été suggérés. La première hypothèse considère que l’hyperphosphorylation de Tau provoque son détachement des microtubules induisant ainsi une déstabilisation du cytosquelette microtubulaire, une altération du transport axonal et une mort neuronale. Selon la seconde hypothèse, la fixation excessive de Tau aux microtubules altèrerait le transport axonal des vésicules et autres organites nécessaires au bon fonctionnement de la synapse. Dans ce cas, l’hyperphosphorylation de Tau et la formation des structures PFH auraient en premier lieu un effet protecteur pour la cellule. Lors de ce travail de thèse, nous avons confronté ces deux théories en utilisant le modèle invertébré : Drosophila melanogaster. Tout d’abord, nous avons étudié l’effet de la perte de fonction de la protéine Tau de drosophile (dTau) sur l’architecture du cytosquelette microtubulaire et sur le transport axonal des neuropeptides. Ce travail nous a permis d’une part, de tester l’hypothèse de l’effet du détachement de la protéine Tau des MTs sur le transport axonal, et d’autre part d’étudier la fonction endogène de la protéine dTau. En effet, le rôle in vivo de la protéine Tau endogène sur la morphologie et la physiologie axonale reste inconnu à ce jour, et ceci probablement dû à une redondance fonctionnelle avec les autres protéines associées aux microtubules (MAPs). Dans cette présente étude nous utilisons le modèle Drosophila melanogaster qui présente l’avantage de n’avoir qu’un seul homologue de la famille Tau/MAP2/MAP4 des mammifères. Nos données montrent pour la première fois, in vivo, que la protéine Tau contrôle la densité des microtubules axonaux, et que la perte de la protéine Tau altère le transport axonal microtubule-dépendant. Cependant, les défauts observés ne semblent pas être suffisant pour induire une neurodégénérescence, mais pourraient néanmoins constituer un défaut apparaissant précocement chez les individus atteints. Dans la seconde partie de cette thèse, nous avons étudié l’hypothèse centrée sur l’effet de la fixation excessive de la protéine Tau humaine aux microtubules. Pour cela, nous avons utilisé des drosophiles transgéniques exprimant différentes isoformes mutées de Tau humain (hTau) mimant différents états de phosphorylation de la protéine Tau et s’attachant différemment aux microtubules. Nos résultats montrent clairement que la fixation de Tau en excès sur les microtubules induit des défauts majeurs du transport axonal et de la libération des neuropeptides. Nous démontrons ainsi que l’un des mécanismes possible de la maladie d’Alzheimer est la fixation précoce excessive de Tau sur les microtubules. Par ailleurs, nos résultats mettent en évidence une limite sérieuse des thérapies visant à inhiber la phophorylation de Tau dans la MA. / Tau is a microtubule associated protein that belongs to the MAP structural family. it polymerizes and stabilizes microtubules, in vitro. Tau is found in high amount in axons. The microtubule binding capacity of Tau is regulated by kinases and phophatases. Indeed, when Tau is phosphorylated it desengages from microtubules and when it is dephosphorylated it binds to microtubules and stabilizes them. Tau is involved in several neurodegenerative disorders called tauopathies like the elderly neuropathy, Alzheimer disease (AD). In this neurodegenerative disorder, Tau is abnormally phosphorylated and aggregates to forme neurofibrillary tangles called paired helicoidal filament (PHF), witch is one of the hallmark of AD. Hence, two major hypothesis explaining neurodegeneration in this condition have been suggested. The first hypothesis considers that because of Tau hyperphosphorylation, it detaches from microtubules and starts to form aggregates. Tau detachment from microtubules leads to their destabilization and subsequent defects in axonal transport. These defects in axonal transport lead to synaptic dysfonction and neuronal degeneration. The second hypothesis suggests that an excess of Tau binds onto microtubules, induces axonal transport defects and subsequently neuronal loss. The hyperphosphorylation of Tau and PHF formation would represent a protective response of the cell to prevent axonal defects and neurodegeneresence. The aim of our work is to evaluate these two mechanisms using Drosophila melanogaster model. First, we studied the effect of drosophila Tau (dTau) loss of function on microtubule organisation and axonal transport of neuropeptide in vivo. This work allows us to study the first hypothesis of detachment of Tau from microtubules an its consequences, as well as understanding the endogenous function of dTau. Infact, we took the advantage of drosophila lower genetic redundancy in witch dTau is the only homologue of the mamalian Tau/MAP2/MAP family. Our results demontrated that dTau control axonal microtubule number and that the loss of Tau function affects vesicular axonal transport. However, these defects do not seem to be toxic for the neuron but represent an early event that may progressively become toxic. In the second part of this work we evaluated the second hypothesis. It consists of studying the consequences of an excess of hypophosphorylated Tau bound to microtubules on axonal transport. Our results demontrate for the first time a stronger toxicity of hypophosphorylated Tau for neuronal function compared to pseudophosphoryated Tau. These data demonstrate an important mechanism that could probably be implicated in AD. In addition, our work point out a potentiel limit of a current therapeutic strategy aimed at inhibiting Tau phosphorylation.

Os nervos periféricos são extensões do sistema nervoso central e responsáveis pela interação das atividades das extremidades, em suas funções sensitiva e motora. São vulneráveis aos mesmos tipos de traumas que afetam outros tecidos: contusão, compressão, esmagamento, estiramento, avulsão e laceração. Desta forma, a interrupção de continuidade da estrutura do nervo, por algum tipo de trauma, resulta na interrupção de transmissão dos impulsos nervosos e na desorganização de suas atividades funcionais. Para tanto, em vista da evolução tecnológica e o desenvolvimento de equipamentos mais sofisticados, a microcirurgia vem ganhando cada vez mais espaço no campo da investigação experimental referente a recuperação de nervos periféricos lesionados. Em uma secção simples, sem perda tecidual, a neurorrafia denominada término-terminal apresenta bons resultados, contudo, quando ocorre perda de tecido nervoso ou, não se têm mais os cotos distais do nervo, outras técnicas devem ser empregadas, até porque, não se pode de modo algum tencionar o nervo numa tentativa de coaptá-lo novamente. Várias técnicas de tubulização utilizando-se materiais biológicos e não biológicos, para criar um meio por onde ocorrerá a regeneração nervosa já foram ou ainda estão sendo testados com resultados insatisfatórios sob o aspecto funcional.É sabido que em um trauma sem perda tecidual, numa neuropraxia, por exemplo, o nervo recupera espontaneamente de forma satisfatória. É sabido também que em um feixe vásculo-nervoso, o nervo periférico encontra-se em íntimo contato com a adventícia de artérias e veias. A adventícia dos vasos é constituída por tecido conjuntivo frouxo, rico em adipócitos. Assim, em um trauma, os neuritos oriundos do coto proximal do nervo lesado, ficam diretamente em contato com esses adipócitos. Seguindo este raciocínio, e com base em trabalhos anteriores onde foi usada veia preenchida com músculo esquelético a fresco como enxerto, decidimos testar a possibilidade de crescimento axonal por meio de enxerto com tubo de polietileno preenchido por tecido adiposo autólogo. Para tanto será utilizado um tubo com 12 mm de comprimento por 0,25 mm de diâmetro, com poros de 80 µm de diâmetro, preenchido com tecido adiposo in natura retirado das adjacências do referido nervo, na tentativa de se recuperar o nervo isquiático. A certificação do sucesso da recuperação do nervo será feita por meio de técnicas de microcirurgia, microscopia e morfometria. O enxerto de polietileno mostrou ter resistência a pressões, uma vez que não foi observado colapso estrutural. A gordura mostrou ser bom material trófico já que no grupo onde a utilizou apresentou resultados mais próximos do controle final. / Peripheral nerves are extensions of the central nervous system and are responsible for the interaction of the activities of the extremities in their sensory and motor function. They are vulnerable to these types of injuries that affect other tissues: contusion, compression, crush, stretch, tear and avulsion. Thus, the interruption of continuity of the nerve structure, due to some sort of trauma, results in the interruption of transmission of nerve impulses and disruption of their functional activities. Therefore, in view of technological evolution and development of more sophisticated equipment, microsurgery is gaining more space in the field of experimental research concerning the recovery of injured peripheral nerves. In a single section, without loss of tissue, called the end-terminal neurorrhaphy was also good, however, when there is loss of nervous tissue or, if not longer have the distal nerve stumps, other techniques must be employed, because, in any circumstance the nerve should be tensioned as an attempt to coaptation again. Several techniques of tubing, using biological materials and non-biological means to create a place where nerve regeneration have been or are still being tested with unsatisfactory results in the functional aspect. It is known that in a trauma without loss of tissue, a neuropraxia, for example, the nerve recovers spontaneously and satisfactorily. It is also known that in a neurovascular bundle, the peripheral nerve is in close contact with the adventitia of arteries and veins. The adventitia of the vessels is composed of loose connective tissue rich in adipocytes. Thus, in trauma, the neurites from the proximal stump of injured nerve, are directly in contact with these adipocytes. Following this reasoning, and based on previous work where it was used vein filled with fresh skeletal muscle as a graft, decided to test the possibility of axonal growth by grafting polyethylene tube filled with autologous adipose tissue. For that will be used with a tube 12 mm long and 0.25 mm in diameter, with pores of 80 microns in diameter, filled with fresh adipose tissue removed from the vicinity of that nerve, in an attempt to recover the sciatic nerve. Certification of successful nerve recovery will be done through microsurgical techniques, microscopy and morphometry. The graft polyethylene proved to have pressure resistance, since no structural collapse was observed. The fat was found to be good material as trophic group which had used the results closer to the ultimate control.

O traumatismo craniencefálico (TCE) fechado é, classicamente, um modelo de lesão neuronal e axonal monofásica, onde a destruição do parênquima, incluindo neurônios e células gliais, ocorre principalmente no momento do trauma, seguida pela degeneração Walleriana anterógrada e retrógrada nos dias subseqüentes. Há evidências de progressão da perda neuronal e axonal na fase tardia após o trauma, observada principalmente pela evolução da atrofia cerebral, secundária a vários fatores, incluindo a apoptose neuronal. Com o objetivo de testar a hipótese de que as técnicas quantitativas em ressonância magnética (RM) permitem identificar, de modo não invasivo, as variáveis biológicas que estimam a perda neuronal e axonal no cérebro relacionadas ao TCE moderado e grave e à lesão axonal difusa, na fase tardia, foram avaliados 9 pacientes, sendo 5 do sexo masculino e 4 do sexo feminino, com idades variando de 11 a 28 anos (média de 21,1 anos), que foram vítimas de TCE moderado ou grave (Escala de Coma de Glasgow menor que 12 na admissão hospitalar após o TCE) e que tiveram boa recuperação. O tempo médio entre o trauma e o exame de ressonância magnética foi de 3,1 anos (± 0,5 anos). Foram utilizados os índices ventrículo-cerebrais bifrontal (IVCF) e bicaudado (IVCC), a medida do tempo de relaxação T2, o índice de transferência de magnetização (MTR), o coeficiente de difusão aparente (ADC) e a espectroscopia de prótons multi-voxel, com o cálculo dos índices metabólicos N-acetilaspartato/creatina (NAA/Cre) e colina/creatina (Cho/Cre). Foram estudados a substância branca (SB) frontal e parietal bilateralmente, o joelho e o esplênio do corpo caloso (CC) e a substância cinzenta (SC). As médias dos valores medidos foram comparadas às de um grupo controle formado por 9 pessoas sadias, pareadas pela idade e sexo. Observou-se aumento estatisticamente significativo (p ≤ 0,05) do IVCF e do IVCC nos pacientes, devido ao aumento ventricular secundário à atrofia subcortical; aumento no tempo de relaxação T2 na SB e no CC, que reflete o aumento da concentração de água por provável perda axonal e gliose; aumento do ADC e redução do MTR na SB e no CC, que demonstram lesão das fibras axonais mielinizadas, e redução do índice NAA/Cre no CC, indicando perda axonal. Não houve diferença estatisticamente significativa nas medidas realizadas na SC e nem no índice Cho/Cre (p › 0,05). Os resultados encontrados mostram que as técnicas quantitativas em RM foram capazes de detectar, de modo não invasivo, o dano neuronal e axonal na substância branca e no corpo caloso de cérebros humanos, secundário ao TCE moderado e grave. / Closed traumatic brain injury (TBI) is a classic model of monophasic neuronal and axonal injury, where tissue damage mainly occurs at the moment of trauma, followed by anterograde and retrograde Wallerian degeneration in the subsequent days. There are some evidences of delayed progression of the neuronal and axonal loss after TBI, mainly shown by gradual development of cerebral atrophy, due to many factors, including neuronal apoptosis. For the purpose of testing the hypothesis that quantitative magnetic resonance techniques are able to assess the biological variables which estimate neuronal and axonal loss in brain, related to moderate or severe TBI and diffuse axonal injury, nine patients (age range 11 – 28 years; mean age 21,1 years; 5 male and 4 female), who sustained a moderate or severe TBI (initial Glasgow Coma Scale less than 12), with good recovery, were evaluated in a mean of 3,1 years after trauma (± 0,5 year). The following techniques were applied: bicaudate (CVIC) and bifrontal (CVIF) cerebroventricular indexes; T2 relaxation time measurement (T2 relaxometry); magnetization transfer ratio (MTR); apparent diffusion coefficient (ADC); multivoxel proton magnetic resonance spectroscopy, using N-acetylaspartate/creatine (NAA/Cre) and choline/creatine (Cho/Cre) ratios; measured in the frontal and parietal white matter (WM) of both cerebral hemispheres, in the genu and splenium of the corpus callosum (CC) and in the gray matter (GM). The results were compared with those of a control group constituted by 9 healthy volunteers with a matched age and sex distribution. The CVIC and CVIF mean values were significantly increased (p ≤ 0,05) in patients due to ventricular enlargement secondary to subcortical atrophy; an increase in T2 relaxation time was observed in the WM and CC, reflecting an enhancement in water concentration, probably secondary to axonal loss and gliosis; increased ADC mean values and reduced MTR mean values were found in the WM and CC, showing damage in the myelinated axonal fibers; and decreased NAA/Cre ratio mean values in the CC, indicating axonal loss. No significant differences were observed in the mean values measured at the GM or in the Cho/Cre ratio mean values (p › 0,05). These quantitative magnetic resonance techniques were able to non-invasively demonstrate the neuronal and axonal damage in the WM and CC of human brains, secondary to moderate or severe TBI.

Os nervos periféricos são extensões do sistema nervoso central e responsáveis pela interação das atividades das extremidades, em suas funções sensitiva e motora. São vulneráveis aos mesmos tipos de traumas que afetam outros tecidos: contusão, compressão, esmagamento, estiramento, avulsão e laceração. Desta forma, a interrupção de continuidade da estrutura do nervo, por algum tipo de trauma, resulta na interrupção de transmissão dos impulsos nervosos e na desorganização de suas atividades funcionais. Para tanto, em vista da evolução tecnológica e o desenvolvimento de equipamentos mais sofisticados, a microcirurgia vem ganhando cada vez mais espaço no campo da investigação experimental referente a recuperação de nervos periféricos lesionados. Em uma secção simples, sem perda tecidual, a neurorrafia denominada término-terminal apresenta bons resultados, contudo, quando ocorre perda de tecido nervoso ou, não se têm mais os cotos distais do nervo, outras técnicas devem ser empregadas, até porque, não se pode de modo algum tencionar o nervo numa tentativa de coaptá-lo novamente. Várias técnicas de tubulização utilizando-se materiais biológicos e não biológicos, para criar um meio por onde ocorrerá a regeneração nervosa já foram ou ainda estão sendo testados com resultados insatisfatórios sob o aspecto funcional.É sabido que em um trauma sem perda tecidual, numa neuropraxia, por exemplo, o nervo recupera espontaneamente de forma satisfatória. É sabido também que em um feixe vásculo-nervoso, o nervo periférico encontra-se em íntimo contato com a adventícia de artérias e veias. A adventícia dos vasos é constituída por tecido conjuntivo frouxo, rico em adipócitos. Assim, em um trauma, os neuritos oriundos do coto proximal do nervo lesado, ficam diretamente em contato com esses adipócitos. Seguindo este raciocínio, e com base em trabalhos anteriores onde foi usada veia preenchida com músculo esquelético a fresco como enxerto, decidimos testar a possibilidade de crescimento axonal por meio de enxerto com tubo de polietileno preenchido por tecido adiposo autólogo. Para tanto será utilizado um tubo com 12 mm de comprimento por 0,25 mm de diâmetro, com poros de 80 µm de diâmetro, preenchido com tecido adiposo in natura retirado das adjacências do referido nervo, na tentativa de se recuperar o nervo isquiático. A certificação do sucesso da recuperação do nervo será feita por meio de técnicas de microcirurgia, microscopia e morfometria. O enxerto de polietileno mostrou ter resistência a pressões, uma vez que não foi observado colapso estrutural. A gordura mostrou ser bom material trófico já que no grupo onde a utilizou apresentou resultados mais próximos do controle final. / Peripheral nerves are extensions of the central nervous system and are responsible for the interaction of the activities of the extremities in their sensory and motor function. They are vulnerable to these types of injuries that affect other tissues: contusion, compression, crush, stretch, tear and avulsion. Thus, the interruption of continuity of the nerve structure, due to some sort of trauma, results in the interruption of transmission of nerve impulses and disruption of their functional activities. Therefore, in view of technological evolution and development of more sophisticated equipment, microsurgery is gaining more space in the field of experimental research concerning the recovery of injured peripheral nerves. In a single section, without loss of tissue, called the end-terminal neurorrhaphy was also good, however, when there is loss of nervous tissue or, if not longer have the distal nerve stumps, other techniques must be employed, because, in any circumstance the nerve should be tensioned as an attempt to coaptation again. Several techniques of tubing, using biological materials and non-biological means to create a place where nerve regeneration have been or are still being tested with unsatisfactory results in the functional aspect. It is known that in a trauma without loss of tissue, a neuropraxia, for example, the nerve recovers spontaneously and satisfactorily. It is also known that in a neurovascular bundle, the peripheral nerve is in close contact with the adventitia of arteries and veins. The adventitia of the vessels is composed of loose connective tissue rich in adipocytes. Thus, in trauma, the neurites from the proximal stump of injured nerve, are directly in contact with these adipocytes. Following this reasoning, and based on previous work where it was used vein filled with fresh skeletal muscle as a graft, decided to test the possibility of axonal growth by grafting polyethylene tube filled with autologous adipose tissue. For that will be used with a tube 12 mm long and 0.25 mm in diameter, with pores of 80 microns in diameter, filled with fresh adipose tissue removed from the vicinity of that nerve, in an attempt to recover the sciatic nerve. Certification of successful nerve recovery will be done through microsurgical techniques, microscopy and morphometry. The graft polyethylene proved to have pressure resistance, since no structural collapse was observed. The fat was found to be good material as trophic group which had used the results closer to the ultimate control.

O traumatismo craniencefálico (TCE) fechado é, classicamente, um modelo de lesão neuronal e axonal monofásica, onde a destruição do parênquima, incluindo neurônios e células gliais, ocorre principalmente no momento do trauma, seguida pela degeneração Walleriana anterógrada e retrógrada nos dias subseqüentes. Há evidências de progressão da perda neuronal e axonal na fase tardia após o trauma, observada principalmente pela evolução da atrofia cerebral, secundária a vários fatores, incluindo a apoptose neuronal. Com o objetivo de testar a hipótese de que as técnicas quantitativas em ressonância magnética (RM) permitem identificar, de modo não invasivo, as variáveis biológicas que estimam a perda neuronal e axonal no cérebro relacionadas ao TCE moderado e grave e à lesão axonal difusa, na fase tardia, foram avaliados 9 pacientes, sendo 5 do sexo masculino e 4 do sexo feminino, com idades variando de 11 a 28 anos (média de 21,1 anos), que foram vítimas de TCE moderado ou grave (Escala de Coma de Glasgow menor que 12 na admissão hospitalar após o TCE) e que tiveram boa recuperação. O tempo médio entre o trauma e o exame de ressonância magnética foi de 3,1 anos (± 0,5 anos). Foram utilizados os índices ventrículo-cerebrais bifrontal (IVCF) e bicaudado (IVCC), a medida do tempo de relaxação T2, o índice de transferência de magnetização (MTR), o coeficiente de difusão aparente (ADC) e a espectroscopia de prótons multi-voxel, com o cálculo dos índices metabólicos N-acetilaspartato/creatina (NAA/Cre) e colina/creatina (Cho/Cre). Foram estudados a substância branca (SB) frontal e parietal bilateralmente, o joelho e o esplênio do corpo caloso (CC) e a substância cinzenta (SC). As médias dos valores medidos foram comparadas às de um grupo controle formado por 9 pessoas sadias, pareadas pela idade e sexo. Observou-se aumento estatisticamente significativo (p ≤ 0,05) do IVCF e do IVCC nos pacientes, devido ao aumento ventricular secundário à atrofia subcortical; aumento no tempo de relaxação T2 na SB e no CC, que reflete o aumento da concentração de água por provável perda axonal e gliose; aumento do ADC e redução do MTR na SB e no CC, que demonstram lesão das fibras axonais mielinizadas, e redução do índice NAA/Cre no CC, indicando perda axonal. Não houve diferença estatisticamente significativa nas medidas realizadas na SC e nem no índice Cho/Cre (p › 0,05). Os resultados encontrados mostram que as técnicas quantitativas em RM foram capazes de detectar, de modo não invasivo, o dano neuronal e axonal na substância branca e no corpo caloso de cérebros humanos, secundário ao TCE moderado e grave. / Closed traumatic brain injury (TBI) is a classic model of monophasic neuronal and axonal injury, where tissue damage mainly occurs at the moment of trauma, followed by anterograde and retrograde Wallerian degeneration in the subsequent days. There are some evidences of delayed progression of the neuronal and axonal loss after TBI, mainly shown by gradual development of cerebral atrophy, due to many factors, including neuronal apoptosis. For the purpose of testing the hypothesis that quantitative magnetic resonance techniques are able to assess the biological variables which estimate neuronal and axonal loss in brain, related to moderate or severe TBI and diffuse axonal injury, nine patients (age range 11 – 28 years; mean age 21,1 years; 5 male and 4 female), who sustained a moderate or severe TBI (initial Glasgow Coma Scale less than 12), with good recovery, were evaluated in a mean of 3,1 years after trauma (± 0,5 year). The following techniques were applied: bicaudate (CVIC) and bifrontal (CVIF) cerebroventricular indexes; T2 relaxation time measurement (T2 relaxometry); magnetization transfer ratio (MTR); apparent diffusion coefficient (ADC); multivoxel proton magnetic resonance spectroscopy, using N-acetylaspartate/creatine (NAA/Cre) and choline/creatine (Cho/Cre) ratios; measured in the frontal and parietal white matter (WM) of both cerebral hemispheres, in the genu and splenium of the corpus callosum (CC) and in the gray matter (GM). The results were compared with those of a control group constituted by 9 healthy volunteers with a matched age and sex distribution. The CVIC and CVIF mean values were significantly increased (p ≤ 0,05) in patients due to ventricular enlargement secondary to subcortical atrophy; an increase in T2 relaxation time was observed in the WM and CC, reflecting an enhancement in water concentration, probably secondary to axonal loss and gliosis; increased ADC mean values and reduced MTR mean values were found in the WM and CC, showing damage in the myelinated axonal fibers; and decreased NAA/Cre ratio mean values in the CC, indicating axonal loss. No significant differences were observed in the mean values measured at the GM or in the Cho/Cre ratio mean values (p › 0,05). These quantitative magnetic resonance techniques were able to non-invasively demonstrate the neuronal and axonal damage in the WM and CC of human brains, secondary to moderate or severe TBI.

La maladie d’Alzheimer est la maladie neurodégénérative la plus fréquente dans le monde industrialisé. Sur le plan neuropathologique, cette maladie est caractérisée par deux types de lésions :les plaques séniles et les dégénérescences neurofibrillaires. <p>Des observations morphologiques précédentes ont permis de mettre en évidence des anomalies du transport axoplasmique dans les neurones chez les patients atteints de la maladie d’Alzheimer. Les mécanismes menant à cette perturbation du transport axoplasmique ne sont pas encore bien établis. La glycogen synthase kinase-3β (GSK-3β) et la cyclin-dependent kinase 5 (Cdk5) associée à son activateur pathologique p25, sont deux kinases clés dont la dérégulation intervient dans la pathogenèse de la maladie d’Alzheimer (MA). Nous avons émis l'hypothèse que ces kinases pourraient jouer un rôle dans la perturbation du transport axoplasmique dans cette maladie.<p><p>Dans la première partie de notre travail, nous nous sommes intéressés à l’effet de la GSK-3β et de Cdk5/p25 sur la croissance des neurites (un processus dépendant du transport axoplasmique) dans un modèle cellulaire, les PC12 différenciées prétraitées au NGF. <p>La surexpression de GSK-3β et de p25 provoque une importante réduction de la croissance neuritique dans ces cellules. Par western blot, nous avons montré que cette réduction est associée à des modifications post-traductionnelles des protéines impliquées dans la régulation du cytosquelette. Ces modifications sont la phosphorylation de la protéine tau et des neurofilaments et l’acétylation de la tubuline α.<p>Cette étude indique donc que la GSK-3β et la protéine p25 contrôlent négativement la croissance neuritique.<p><p>Dans la deuxième partie de notre travail, afin d’étudier la relation entre ces kinases et le transport axoplasmique, nous avons analysé dans des neurones en culture l’effet d’une augmentation d’activité de la GSK-3β et de Cdk5/p25 sur le transport des mitochondries.<p>Pour étudier le déplacement des mitochondries, les neurones en cultures ont été doublement transfectées avec deux plasmides :un marqueur mitochondrial combiné avec la GSK-3β ou p25. Après transfection, le mouvement des mitochondries a été enregistré grâce à la technique du time-lapse.<p>L’étude de la fréquence de trois comportements (mouvement antérograde, mouvement rétrograde et état stationnaire) nous a indiqué que les mitochondries sont normalement en position immobile pendant 70 % de leur temps. La surexpression de GSK-3β ou de p25 augmente la fréquence de cet état stationnaire et diminue de manière plus importante les mouvements antérogrades que rétrogrades sans affecter la vitesse des mitochondries. L’observation au microscope électronique a permis de démontrer la persistance du réseau de microtubules dans les cellules surexprimant GSK-3β ou p25.<p>Le transport des mitochondries est un processus actif faisant intervenir les moteurs moléculaires (kinésine et dynéine) dont le rôle est le transport d’organelles qui repose sur un réseau intact de microtubules.<p>Cette étude suggère donc que la GSK-3β et p25 contrôlent négativement le transport des mitochondries en agissant au niveau des moteurs moléculaires (kinésine et dynéine) plutôt qu’en détruisant le réseau de microtubules.<p><p>Dans la troisième partie de notre travail, nous nous sommes intéressés à l’expression et à la localisation dans le cortex frontal humain et dans le cortex cérébelleux de deux protéines appartenant aux moteurs moléculaires responsables des transports axoplasmiques antérograde et rétrograde :la chaîne légère de la kinésine (KLC1) et la chaîne intermédiaire de la dynéine (DIC). <p>Nous avons observé une diminution du niveau d’expression de la KLC1 et de la DIC dans le cortex frontal (une zone atteinte dans la MA) mais pas dans le cortex cérébelleux chez les patients atteints de la maladie d’Alzheimer par rapport à des sujets contrôles. Une diminution du niveau d’expression de la tubuline-β3 et de la synaptophysine -deux marqueurs neuronaux- a aussi été observée dans le cortex frontal mais pas dans le cortex cérébelleux. Nous avons aussi démontré une hausse de l’état de phosphorylation de la KLC1 dans un modèle cellulaire surexprimant la GSK-3β. Dans le cortex frontal dans la MA, nous avons observé une augmentation de la forme active de la GSK-3β, et une hausse de la phosphorylation de la KLC1. Cette phosphorylation accrue de la KLC1 diminue son activité de transport des organelles.<p>Ces anomalies de l’expression et de la phosphorylation des moteurs moléculaires pourraient jouer un rôle dans les perturbations des transports axoplasmiques dans la MA.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished