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Etude de la structure nanométrique et de la viscosité locale de l’espace extracellulaire du cerveau par microscopie de fluorescence de nanotubes de carbone uniques / A study of the nanoscale structure and local viscosity of the brain extracellular space by single carbon nanotubes fluorescence microscopyDanné, Noémie 30 October 2018 (has links)
Le cerveau est composé de neurones et de cellules gliales qui jouent un rôle de soutien et de protection du réseau cellulaire. L’espace extra-cellulaire (ECS) correspond à l’espace qui existe entre ces cellules. Les modifications de sa structure peuvent dépendre de plusieurs paramètres comme l’âge, l’apprentissage ou les maladies neuro-dégénératives. Le volume de l’ECS correspond à environ 20$%$ du volume total du cerveau et les neurotransmetteurs et autres molécules circulent dans cet espace pour assurer une communication neuronale optimale. Cependant, les dimensions et la viscosité locale de cet espace restent encore mal-connues. L’ECS est composé entre autres de protéoglycans, de glycoaminoglycans (acide hyaluronique…) et de fluide cérébrospinal. Nous avons proposé dans cette thèse une stratégie pour mesurer les dimensions et les propriétés rhéologiques de l’espace extra-cellulaire de tranches de cerveaux de rats maintenue en vie à l’aide du suivi de nanotubes de carbone individuels luminescents. Pour ces applications, nous avons étudier la biocompatibilité et le rapport signal sur bruit de nos échantillons de nanotubes afin de les détecter en profondeur dans les tranches de cerveaux et de pouvoir mesurer leurs propriétés de diffusion. / The brain is mainly composed of neurons which ensure neuronal communication and glialcells which play a role in supporting and protecting the neural network. The extracellular space corresponds to the space that exists between all these cells and represents around 20 %of the whole brain volume. In this space, neurotransmitters and other molecules circulate into ensure optimal neuronal functioning and communication. Its complex organization whichis important to ensure proper functioning of the brain changes during aging, learning or neurodegenerative diseases. However, its local dimensions and viscosity are still poorly known.To understand these key parameters, in this thesis, we developed a strategy based on the tracking of single luminescent carbon nanotubes. We applied this strategy to measure the structural and viscous properties of the extracellular space of living rodent brains slices at the nanoscale. The organization of the manuscript is as follows. After an introduction of the photoluminescence properties of carbon nanotubes, we present the study that allowed us to select the optimal nanotube encapsulation protocol to achieve our biological applications. We also present a quantitative study describing the temperature increase of the sample when laser irradiations at different wavelengths are used to detect single nanotubes in a brain slice.Thanks to a fine analysis of the singular diffusion properties of carbon nanotubes in complex environments, we then present the strategy set up to reconstruct super-resolved maps (i.e. with resolution below the diffraction limit) of the brain extracellular space morphology.We also show that two local properties of this space can be extracted : a structural complexity parameter (tortuosity) and the fluid’s in situ viscosity seen by the nanotubes. This led us to propose a methodology allowing to model the viscosity in situ that would be seen, not by the nanotubes,but by any molecule of arbitrary sizes to simulate those intrinsically present or administered in the brain for pharmacological treatments. Finally, we present a strategy to make luminescent ultra-short carbon nanotubes that are not intrinsically luminescent and whose use could be a complementary approach to measure the local viscosity of the extracellular space of the brain.
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