Développement de microtechnologies pour l'étude du guidage axonal

Abstract : Axonal guidance is a very important process during brain development, allowing to give it its structure and organization. The neuroscience scientific community has a growing interest in it during the last years. Several tools belonging to the field of microtechnologies, microfluidics and micropatterning are of important help to study axonal guidance in vitro. They allow to confine neurons and their axons and to apply gradients of guidance molecules. During this thesis, my goal was to develop a system to study the effect of guidance molecules gradients on axonal guidance. For that, I tested several configurations of microfluidic devices, micropatterns and combinations of both.First, we used two approaches to isolate dissociated neurons axons from their somas. Our goal was to study the effect of the molecular environment on neurons growth cones, with a high throughput. The first approach consisted in growing neurons on different proteins patterns. It also allowed to show their capacity to adhere on these patterns. The second one consisted in seeding neurons in a microfluidic device in which, during their growth, axons are separated from somas by microchannels. Then we studied the effect, on the axons, of guidance molecules gradients. To begin, we measured the effect of two guidance molecules: ephrin and semaphorin, by culturing neurons in the presence of patterned gradients of these two molecules. After that, we studied another model where neurons are closer from their environment in vivo, explants growing on laminin patterns containing a gradient. To help the explant positioning, we polymerized hydrogels. Then, we put explants next to patterned gradients of ephrin. Finally, we tried to obtain a soluble gradient of guidance molecules, over a long period of time (days), closer to existing gradients in vivo. In that goal, we wanted to build a microfluidic device enabling the application of a soluble gradient of guidance molecules on neurons. To obtain a constant gradient, we also cultured neurons next to cells expressing netrin, another guidance molecule. Finally, we cultured dissociated neurons and glial cells to study their interactions.All these experiments did not allow to obtain a reliable device to study the effect of molecules on axons growth and guidance. Nevertheless, the configuration consisting in a coculture of neurons next to cells releasing netrin allows us to obtain promising preliminary results. We thus drew up a group of methods that will enable us to finalize the development of a system to study axonal guidance, functional and efficient.
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Yohan Lecomte. Développement de microtechnologies pour l'étude du guidage axonal. Neurosciences [q-bio.NC]. Université de Bordeaux, 2019. Français. ⟨NNT : 2019BORD0093⟩. ⟨tel-02278820⟩

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