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, Cette partie traite du travail réalisé en collaboration avec le Docteur Carine Karachi de l'équipe du Docteur Brian LAU (Neurochirurgie expérimentale

. Stornetta, Par ailleurs les atlas numériques, basés sur l'acquisition de données IRM ou de tractographie présentent une qualité d'image excellente sans pour autant offrir une délimitation précise des groupes de neurones de la formation réticulée. De façon plus précise, la construction de cet atlas s'inscrit dans ce travail doctoral. La délimitation des frontières anatomiques via l'immunohistochimie des principales structures contrôlant et régulant la CCR devrait permettre de déterminer si l'absence d'amélioration de la ventilation de certaines patientes CCHS sous désogestrel (cf : Introduction, Chapitre 4, III.1.1-Effet des systèmes progeste?onergiques chez l'Homme), L'objectif de cette étude est de développer un atlas numérique, histologique, tridimensionnel et déformable du tronc cérébral humain, visant à cartographier les structures qui contrôlent la respiration, en combinant des techniques d'imagerie et d'histologie. En effet, par analogie avec les données animales qui ont caractérisé les structures respiratoires du tronc cérébral via les neurotransmetteurs qu'elles expriment, 1954.

. Les and . Le-dr-mathieu-santin, Une première acquisition, comparable à celle utilisée chez les patients, a été faite à 3T (Siemens Prisma) pendant 13H30 et a permis d'obtenir la séquence anatomique en T1. Cette étape a été immédiatement suivie par une acquisition IRM à 11,7T (Brucker) pendant 8H, toujours dans du Fluorinert, afin d'effectuer la séquence anatomique T2*

, Ils ont ainsi été transférés de 37°C (température à l'intérieur de la machine IRM) à 4°C, puis -20°C et enfin placés dans la neige carbonique à -78°C pour être placés dans un congélateur à -80°C. La découpe au microtome à congélation (Tetrander, Richert-Jung, Heidelberg) multipuits (Raaco), soit 100 puits en tout, IV-Investigations anatomiques et immunohistochimiques IV.1-Découpe du tissu Après l'acquisition des images IRM, les tissus ont été progressivement refroidis pour être congelés et conservés jusqu'au jour de leur découpe

, Chacune des deux colorations des coupes ont été réalisées à intervalle régulier : toutes les 20 coupes et donc tous les 1000 µM, le long du tronc cérébral, afin d'obtenir pour chaque niveau une représentation de la morphologie et de l'anatomie. Le colorant utilisé dans la coloration de Nissl, le crésyl violet, lie les composés basophiles tels que la molécule d'ADN ou d'ARN permettant ainsi la visualisation au microscope des organites les contenant, en particulier le noyau. La coloration a été effectuée en mettant les coupes en présence d'une solution d'eau déminéralisée contenant du crésyl violet à 1% (C-1893, Coloration Nissl et Luxol En plus des protocoles d'immunomarquage, il a été entrepris de réaliser des colorations histologiques : la coloration Nissl et coloration de la myéline à l'aide du

, Tableau récapitulant les étapes successives d'une coloration Nissl, vol.3

. Guyenet, . Mulkey, and L. Kapur, A noter que les neurones du noyau rétrotrapézoïde ont aussi été décrits par le fait d'être négatifs pour la TH, 2010.

. Kinney, USA) que par un anticorps fourni par le Dr Jean-François Brunet (ENS Biologie, Paris) n'ont pas été assez convaincante pour être utilisées, nous avons donc employé la stratégie décrite ci-dessus. Les groupements sérotoninergiques à savoir, les noyaux du ROb, RPa, RMg ainsi que le noyau arqué bulbaire (qui correspond au groupe parapyramidal chez le rongeur) ont été caractérisés via la détection de la sérotonine, Malgré nos efforts exhaustifs, leur caractérisation aussi bien par à un anticorps anti-PHOX2B commercial (sc-13226, 2006.

, Tableau récapitulant les structures respiratoires et les antigènes ou colorations utilisés pour les caractériser, vol.5

. Smith, Abréviations : NK1R : récepteur neurokinine 1 à la substance P, Chacune des structures listées dans ce tableau est caractérisée par le fait d'être immunoréactive (+) ou non (-) pour chaque marqueur ou coloration donné. Par analogie avec les données animales on attribue aux structures ci-après, décrites chez l'Homme, un rôle dans l'élaboration d'une CCR adaptée : le complexe de pré-Bötzinger, 1971.

, Les marquages ont été effectués sur des séries c'est-à-dire un nombre donné de

. Bryan, La commande ventilatoire du nouveau-né et plus particulièrement du nouveau-né prématuré se caractérise par des apnées ou des bradypnées fréquentes qui engendrent la récurrence d'épisodes d'hypoxiques durant la période postnatale, La problématique de cette étude est en lien avec les caractéristiques de la commande ventilatoire du nouveau-né voire même du prématuré, 1986.

, Les mécanismes à l'origine de la réponse respiratoire à l'hypoxie sont divers

. Vizek, L'hyperventilation transitoire est attribuée à la stimulation des chémorécepteurs périphériques qui engendre une élévation d'activité dans certaines structures du réseau respiratoire bulbo-pontique comme notamment le noyau du tractus solitaire commissural (NTSc), le noyau réticulaire bulbaire ventrolatéral (VLM) mais aussi le noyau rétrotrapézoïde/groupe respiratoire parafacial (RTN/pFRG), 1987.

. Simakajornboon, Les changements de la ventilation induits par l'hypoxie ont été appréciés par la technique de pléthysmographie (Bartlett and Tenney, 1970) sur une population de 12 souris nouveau-nées. Cette série expérimentale avait vocation à vérifier les caractéristiques de la réponse ventilatoire des souris nouveau-nées au regard des données de la littérature, 2004.

, II.2-Induction de c-FOS et procédures d'immunohistochimiques et d'immunohistofluorescence Deux groupes d'animaux ont été utilisés : des souris placées dans une enceinte et soumises à un challenge hypoxique, pp.11-13

. Bodineau, Danneman and Mandrell, 1997) et les troncs cérébraux prélevés. Après post-fixation et cryoprotection, les blocs ont été découpés et les coupes obtenues engagées dans les protocoles d'immunodétection : c-FOS seul, c-FOS/TH, c-FOS/5-HT et c-FOS/PHOX2B en suivant les techniques couramment utilisées et maitrisées au laboratoire, A l'issue des 90 minutes soit d'hypoxie, soit de normoxie, les souris nouveau-nées ont été profondément anesthésiées au froid, 2011.

. Berquin, Le nombre de neurones c-FOS-positifs a été quantifié dans diverses structures à l'aide d'atlas (Paxinos and Franklin, 2001) et de repères anatomiques précis afin de standardiser les procédures, 2000.

V. Nts, . Rtn/pfrg, and . Nunez-abades, noyau parabrachial médian (mPB) et lPB) et dans des structures ayant été impliquées dans la régulation de la commande respiratoire ((groupe parapyramidal (PP), raphé magnus (RMg), noyau subcoeruleus (dSubC)). Les co-détections nous ont permis de déterminer que : 1/ virtuellement aucun ou quasiment des neurones c-FOS-positifs en hypoxie au niveau du NTS et de la VLM n'était catécholaminergique ; 2/ qu'aucun des neurones c-FOS-positifs en hypoxie du RTN/pFRG n'était PHOX2B-positif ; 3/ qu'au niveau du PP et du RMg, respectivement 30% et 15% des neurones c-FOS-positifs en hypoxie étaient sérotoninergiques 4/ que 45% des neurones c-FOS-positifs du dSubC étaient catécholaminergiques. précédemment été décrit, Les données obtenues ont mis en évidence une élévation du nombre de neurones c-FOS-positifs dans diverses structures du réseau respiratoire bulbo-pontiques, 1992.

. Teppema, chez la souris nouveau-née ne concerne pas les neurones PHOX2B-positifs du RTN/pFRG mais des neurones 5-HT positifs au sein du PP. L'absence d'élévation de l'expression de c-fos au sein des neurones PHOX2B-positifs du RTN/pFRG suggère que malgré l'hypoventilation observée chez la souris nouveau-née, celle-ci ne soit pas suffisante pour induire une élévation du CO2 qui stimulerait les neurones CO2/H + -sensibles, III.3-La surface ventrale du bulbe rachidien présente une augmentation du nombre de neurones c-FOS-positifs au sein de neurones sérotoninergiques mais pas PHOX2B-positifs L'augmentation du nombre de neurones au niveau de la surface ventrale du bulbe rachidien, 1997.

. Paterson, il nous apparait pertinent de la mettre en relation avec le fait que cette structure, le PP, est considérée comme l'homologue de position du noyau raqué bulbaire chez l'Homme qui a été décrit comme l'objet d'hypoplasie chez des enfants décédés de mort subite du nourrisson (Filiano and Kinney, 1992.

, IV-Conclusion Pour conclure, notre étude a permis de cartographier avec précision les modifications d'activité suggérées par l'expression de c-fos au sein du tronc cérébral d'un organisme très immature, la souris nouveau-née de 1 jour

, FOS/5-HT c-FOS was detected using a rabbit polyclonal antibody against c-FOS (sc-253 Santa Cruz Biotechnology Inc, vol.1, p.500

, Sections were mounted in sequential caudo-rostral order on silane-treated slides as for the single immunohistochemical detection of c-FOS. They were air-dried, dehydrated with absolute alcohol, :500) or goat anti-rabbit (Vector Laboratories, vol.1

, Dual Immunohistofluorescence for c-FOS/PHOX2B

, 1:500; 48 h, 4 ? C), then with an Alexa 488-labeled donkey anti-goat antibody (Molecular Probes, Eugene, OR, 2 h at room temperature), Sections were first incubated with a primary antibody against PHOX2B (sc-13226, Santa Cruz biotechnology INC, p.253

, Santa Cruz Biotechnology Inc, vol.1, p.4000

, and then with an Alexa 555-labeled goat anti-rabbit antibody concomitantly with DAPI at 1:4000 (Molecular Probes, Eugene, OR, 2 h at room temperature). Sections were then washed, mounted in sequential caudo-rostral order on silanetreated slides, air-dried, and coverslipped using AquaPolyMount

, Serotoninergic character of hypoxic c-FOS-positive cells of one-day-old mice. Drawings illustrating the distribution of cells immunoreactive for c-FOS (white points) or both c-FOS and 5-HT (black points) in the PP (A) and RMg (B) under hypoxic conditions. Scale bar = 100 µm. Photomicrographs of sections double-immunolabeled for c-FOS (gray) and 5-HT (brown) in the PP (C) and RMg (D) corresponding to the regions outlined by the black rectangles of (A,B), respectively. Scale bar = 100 µm. (E,F) photomicrographs representing an enlargement of the black rectangles in (C,D), respectively. Scale bar = 50 µm. Black arrows indicate c-FOS, Quantitative Analysis of the Effect of Hypoxia on the Number of c-FOS-Positive Cells and Their Characterization FIGURE 5 |

. Rmg,

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