Thèse d'exercice : Médecine. Anesthésie-réanimation : Université de Nantes : 2007. / Bibliogr.

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Intrapulmonary arteriovenous anastomoses (IPAVA) have been known to exist as part of the normal pulmonary vasculature for over 50 years but have been underappreciated by physiologists and clinicians. Using a technique called saline contrast echocardiography we and others have demonstrated that during exercise or when breathing low oxygen gas mixtures IPAVA open, but breathing 100% oxygen during exercise prevents them from opening. However, the mechanism(s) for this dynamic regulation and the role IPAVA play in affecting pulmonary gas exchange efficiency remain unknown. In Chapter IV the infusion of epinephrine and dopamine into resting subjects opened IPAVA. While it is possible this opening was due to the direct vasoactive action of these catecholamines, the opening may simply be due to increases in cardiac output and pulmonary artery systolic pressure secondary to the cardiac effects of these drugs. In Chapter V I used Technetium-99m labeled macroaggregated albumin (99mTc-MAA) to quantify blood flow through IPAVA in exercising healthy humans. Initial attempts to correct for attenuation of the emitted signal were unsuccessful due to the time necessary for data acquisition and the resulting accumulation of free-99mTc. However, I used a blood sample to calculate freely circulating 99mTc which could be subtracted from the shunt fraction. Using this procedure I demonstrated for the first time using filtered solid particles that breathing 100% oxygen reduces blood flow through IPAVA during exercise. Finally, in Chapter VI I tackled the elephant in the room surrounding IPAVA in healthy humans: do these vessels play a role in pulmonary gas exchange efficiency? Our data suggest that the efficiency of pulmonary gas exchange is dependent on the driving pressure gradient for oxygen and the distance to blood flowing through the core of IPAVA. As such, with increases in exercise intensity the diffusion distance and transit time of blood at the core of IPAVA prevent complete gas exchange, thus blood flow through IPAVA acts as a shunt. This dissertation includes previously unpublished co-authored material.

Abstract Purpose: This study evaluated the use of hyperspectral imaging for the determination of the resection margin during colorectal resections instead of clinical macroscopic assessment. Methods: The used hyperspectral camera is able to record light spectra from 500 to 1000 nm and provides information about physiologic parameters of the recorded tissue area intraoperatively (e.g., tissue oxygenation and perfusion). We performed an open-label, single-arm, and non-randomized intervention clinical trial to compare clinical assessment and hyperspectral measurement to define the resection margin in 24 patients before and after separation of the marginal artery over 15 min; HSI was performed each minute to assess the parameters mentioned above. Results: The false color images calculated from the hyperspectral data visualized the margin of perfusion in 20 out of 24 patients precisely. In the other four patients, the perfusion difference could be displayed with additional evaluation software. In all cases, there was a deviation between the transection line planed by the surgeon and the border line visualized by HSI (median 1 mm; range - 13 to 13 mm). Tissue perfusion dropped up to 12% within the first 10 mm distal to the border line. Therefore, the resection area was corrected proximally in five cases due to HSI record. The biggest drop in perfusion took place in less than 2 min after devascularization. Conclusion: Determination of the resection margin by HSI provides the surgeon with an objective decision aid for assessment of the best possible perfusion and ideal anastomotic area in colorectal surgery.:Inhaltsverzeichnis Inhaltsverzeichnis................................................................. I 1 Einführung............................................................................. 1 1.1 Anastomoseninsuffizienz...................................................1 1.2 Methodik Hyperspectral Imaging (HSI)............................. 3 1.3 Einsatzbereiche der Hyperspektral-Kamera..................... 5 1.4 Chirurgische Technik........................................................ 6 1.5 Studienplanung................................................................. 7 1.6 Vergleich der HSI-Technik mit weiteren Messmethoden...8 2 Publikation...............................................................................11 3 Zusammenfassung der Arbeit............................................... 21 4 Literaturverzeichnis............................................................... 26 5 Anhang.................................................................................... 30 Darstellung des eigenen Beitrags.........................................34 Eigenständigkeitserklärung...................................................35 Lebenslauf.............................................................................. 36 Danksagung........................................................................... 38

3 vidéos sont dans des fichiers complémentaires à ce mémoire / Les champignons mycorhiziens à arbuscules (CMA) ont une structure génétique très particulière et certains aspects de leur génétique sont encore incompris et peu documentés. Les CMA se reproduisent par voie asexuée à l’aide de spores multinucléées. Dans cette étude, j’ai cherché à comprendre les mécanismes de l’hérédité génétique des noyaux par la voie de la reproduction asexuée chez les CMA. La première étape était de déterminer le contenu en noyaux des spores matures, ainsi que celui des spores en formation. Des analyses statistiques ont été utilisées pour vérifier le type de relation entre le nombre de noyaux et le diamètre des spores. Quatre espèces du genre Glomus ont été observées au microscope confocal. Les résultats démontrent une hétérogénéité entre les spores dans leur contenu en noyau pour un même diamètre en plus d’une relation positive entre le nombre de noyau et le diamètre de la spore. Afin de vérifier le contenu en noyaux dans les phases extraracinaires, trois différentes structures du mycélium ont été observées au microscope confocal. Aucune structure n’a été retrouvée avec un seul noyau, ce qui permet de conclure que les CMA ne possèdent vraisemblablement pas de stade uninucléé dans leurs phases extraracinaires. Pour étudier l’hérédité des noyaux, deux différentes approches ont été utilisées: (i) Glomus irregulare a été mis sur milieu complémenté avec de l’aphidicoline pour inhiber la mitose. Des observations au microscope confocal ont permis de dénombrer les noyaux qui sont issus des hyphes et non des mitoses. Les résultats indiquent que la population de noyaux présents dans les spores matures provient d’une migration massive de noyaux à l’intérieur des spores en formation suivie d’un nombre faible de mitoses. (ii) La deuxième approche est l’observation microscopique en temps-réel de spores en formation de G. diaphanum qui a permis de confirmer cette affirmation, car il a été possible de voir plusieurs noyaux entrer dans la spore. Dans la dernière partie de cette étude, je me suis intéressée aux échanges génétiques horizontaux chez les CMA qui sont possibles grâce aux anastomoses. Quatre isolats de l’espèce G. irregulare ont été croisés en co-culture par couple de deux isolats (six croisements) pour permettre une proximité propice aux anastomoses et aux échanges génétiques. Ces croisements ont été maintenus pendant deux ans en culture par le repicage des racines colonisées. Des spores des deux différents isolats ont été confrontées sur eau gélifiée, afin d’observer la formation d’anastomose. Un pourcentage de 13% de formation de fusions d’hyphes pour une des confrontations suggère que l’échange des marqueurs parentaux a pu avoir lieu entre les deux isolats grâce aux anastomoses. Un marqueur moléculaire mitochondrial nommé Indel 5 a été développé et utilisé pour l’analyse des spores filles. Ce marqueur possède entre les isolats une délétion de 39 pb et la différence entre les isolats est facilement détectable sur gel d’électrophorèse après amplification PCR. Le génotypage par PCR des spores individuelles a montré que certaines des spores filles issues du croisement possèdent un des deux marqueurs parentaux alors que d’autres spores ont un génotype qui semble posséder les deux marqueurs. Même si la fusion d’hyphes entre spores en germination est possible, d’autres recherches devront être réalisées pour confirmer qu’un échange génétique est possible entre deux isolats très éloignés géographiquement. Le fait qu’il n’existe aucun stade uninucléé au cycle de vie des CMA et qu’il y ait une migration massive de noyaux lors de la formation des spores permet de limiter la dérive génique lors de la reproduction asexuée. Les anastomoses, quant à elles, permettent de rétablir la diversité génétique. Ces deux particularités de la génétique des CMA ont été fort importantes au cours de leur évolution pour permettre de maintenir une variabilité génétique élevée et permettre ainsi une grande adaptation à différents type d’habitats. / Arbuscular Mycorrhizal Fungi (AMF) have a particular and complex genetic structure. Yet, many aspects of their genetics are still misunderstood and poorly documented. These organisms reproduce by asexual multinucleate spores. In this study, I investigated the mechanisms of genetic inheritance of nuclei through asexual reproduction in AMF. First, I determined the number of nuclei in mature and juveniles spores; I used statistical analysis to determine the relationship between the number of nuclei and the spore diameter. Four species from the genus Glomus were observed with a confocal microscope. The results showed that the number of nuclei has a significant positive relationship with spore diameter and more importantly, surprising heterogeneity in the number of nuclei among sister spores was found. To determine the number of nuclei in extraradical phases, three different structures from the mycelia were carefully examined with a confocal microscope. All the structures possessed more than one nucleus and showed that AMF probably lack a single-nucleus stage during their extraradical phases. To study the nuclei’s heritance, two different approaches were used: (i) Glomus irregulare was grown on medium complemented with aphidicolin to inhibit the mitosis. Observations with a confocal microscope permit to count the nuclei that come from the hyphae and not from the mitosis. The results showed that massive nuclear migration and mitosis are the mechanisms by which AMF spores are formed. (ii) The second approach confirm these results because with time-laps live cellular imaging of young spores of Glomus diaphanum it was possible to see many nuclei to get in the spores. In a second part of this thesis, I studied horizontal gene exchanges among AMF isolates through anastomoses. Thus, four isolates of the species G. irregulare were used in in vitro crossing experiments, in total six combinations using two isolates per crossing experiment. These crossing co-cultures were maintained over two years by subculturing. Spores of two different isolates were confronted in vitro prior to observation of anastomoses. 13% of spores formed anastomoses suggesting the occurrence of genetic exchange between two isolates. A mitochondrial molecular marker referred as Indel 5, was used to genotype individual spores of crossing progenies. A 39 bp deletion occurs in the marker among different isolates and is clearly discriminated by PCR. PCR patterns showed that some spores seem to have both parental markers demonstrating that genetic exchange could occur between the two isolates used in crossing experiment. Even though hyphal fusions occur between germinating spores, subsequent research needs to be done to confirm genetic exchange among different isolates from different geographic areas. The finding that AMF lack a single nuclear stage in their extraradical phases and that mitosis and nuclear migration are the mechanisms by which AMF spores are formed reduce genetic drift that acts on these organisms during asexual reproduction. Anastomoses are likely a mechanism that maintains the genetic diversity in AMF. These genetic characteristics of AMF were very important during their evolution to maintain a high genetic variability that allows their adaptation to many different ecosystems.

Les champignons mycorhiziens à arbuscules (CMA) ont une structure génétique très particulière et certains aspects de leur génétique sont encore incompris et peu documentés. Les CMA se reproduisent par voie asexuée à l’aide de spores multinucléées. Dans cette étude, j’ai cherché à comprendre les mécanismes de l’hérédité génétique des noyaux par la voie de la reproduction asexuée chez les CMA. La première étape était de déterminer le contenu en noyaux des spores matures, ainsi que celui des spores en formation. Des analyses statistiques ont été utilisées pour vérifier le type de relation entre le nombre de noyaux et le diamètre des spores. Quatre espèces du genre Glomus ont été observées au microscope confocal. Les résultats démontrent une hétérogénéité entre les spores dans leur contenu en noyau pour un même diamètre en plus d’une relation positive entre le nombre de noyau et le diamètre de la spore. Afin de vérifier le contenu en noyaux dans les phases extraracinaires, trois différentes structures du mycélium ont été observées au microscope confocal. Aucune structure n’a été retrouvée avec un seul noyau, ce qui permet de conclure que les CMA ne possèdent vraisemblablement pas de stade uninucléé dans leurs phases extraracinaires. Pour étudier l’hérédité des noyaux, deux différentes approches ont été utilisées: (i) Glomus irregulare a été mis sur milieu complémenté avec de l’aphidicoline pour inhiber la mitose. Des observations au microscope confocal ont permis de dénombrer les noyaux qui sont issus des hyphes et non des mitoses. Les résultats indiquent que la population de noyaux présents dans les spores matures provient d’une migration massive de noyaux à l’intérieur des spores en formation suivie d’un nombre faible de mitoses. (ii) La deuxième approche est l’observation microscopique en temps-réel de spores en formation de G. diaphanum qui a permis de confirmer cette affirmation, car il a été possible de voir plusieurs noyaux entrer dans la spore. Dans la dernière partie de cette étude, je me suis intéressée aux échanges génétiques horizontaux chez les CMA qui sont possibles grâce aux anastomoses. Quatre isolats de l’espèce G. irregulare ont été croisés en co-culture par couple de deux isolats (six croisements) pour permettre une proximité propice aux anastomoses et aux échanges génétiques. Ces croisements ont été maintenus pendant deux ans en culture par le repicage des racines colonisées. Des spores des deux différents isolats ont été confrontées sur eau gélifiée, afin d’observer la formation d’anastomose. Un pourcentage de 13% de formation de fusions d’hyphes pour une des confrontations suggère que l’échange des marqueurs parentaux a pu avoir lieu entre les deux isolats grâce aux anastomoses. Un marqueur moléculaire mitochondrial nommé Indel 5 a été développé et utilisé pour l’analyse des spores filles. Ce marqueur possède entre les isolats une délétion de 39 pb et la différence entre les isolats est facilement détectable sur gel d’électrophorèse après amplification PCR. Le génotypage par PCR des spores individuelles a montré que certaines des spores filles issues du croisement possèdent un des deux marqueurs parentaux alors que d’autres spores ont un génotype qui semble posséder les deux marqueurs. Même si la fusion d’hyphes entre spores en germination est possible, d’autres recherches devront être réalisées pour confirmer qu’un échange génétique est possible entre deux isolats très éloignés géographiquement. Le fait qu’il n’existe aucun stade uninucléé au cycle de vie des CMA et qu’il y ait une migration massive de noyaux lors de la formation des spores permet de limiter la dérive génique lors de la reproduction asexuée. Les anastomoses, quant à elles, permettent de rétablir la diversité génétique. Ces deux particularités de la génétique des CMA ont été fort importantes au cours de leur évolution pour permettre de maintenir une variabilité génétique élevée et permettre ainsi une grande adaptation à différents type d’habitats. / Arbuscular Mycorrhizal Fungi (AMF) have a particular and complex genetic structure. Yet, many aspects of their genetics are still misunderstood and poorly documented. These organisms reproduce by asexual multinucleate spores. In this study, I investigated the mechanisms of genetic inheritance of nuclei through asexual reproduction in AMF. First, I determined the number of nuclei in mature and juveniles spores; I used statistical analysis to determine the relationship between the number of nuclei and the spore diameter. Four species from the genus Glomus were observed with a confocal microscope. The results showed that the number of nuclei has a significant positive relationship with spore diameter and more importantly, surprising heterogeneity in the number of nuclei among sister spores was found. To determine the number of nuclei in extraradical phases, three different structures from the mycelia were carefully examined with a confocal microscope. All the structures possessed more than one nucleus and showed that AMF probably lack a single-nucleus stage during their extraradical phases. To study the nuclei’s heritance, two different approaches were used: (i) Glomus irregulare was grown on medium complemented with aphidicolin to inhibit the mitosis. Observations with a confocal microscope permit to count the nuclei that come from the hyphae and not from the mitosis. The results showed that massive nuclear migration and mitosis are the mechanisms by which AMF spores are formed. (ii) The second approach confirm these results because with time-laps live cellular imaging of young spores of Glomus diaphanum it was possible to see many nuclei to get in the spores. In a second part of this thesis, I studied horizontal gene exchanges among AMF isolates through anastomoses. Thus, four isolates of the species G. irregulare were used in in vitro crossing experiments, in total six combinations using two isolates per crossing experiment. These crossing co-cultures were maintained over two years by subculturing. Spores of two different isolates were confronted in vitro prior to observation of anastomoses. 13% of spores formed anastomoses suggesting the occurrence of genetic exchange between two isolates. A mitochondrial molecular marker referred as Indel 5, was used to genotype individual spores of crossing progenies. A 39 bp deletion occurs in the marker among different isolates and is clearly discriminated by PCR. PCR patterns showed that some spores seem to have both parental markers demonstrating that genetic exchange could occur between the two isolates used in crossing experiment. Even though hyphal fusions occur between germinating spores, subsequent research needs to be done to confirm genetic exchange among different isolates from different geographic areas. The finding that AMF lack a single nuclear stage in their extraradical phases and that mitosis and nuclear migration are the mechanisms by which AMF spores are formed reduce genetic drift that acts on these organisms during asexual reproduction. Anastomoses are likely a mechanism that maintains the genetic diversity in AMF. These genetic characteristics of AMF were very important during their evolution to maintain a high genetic variability that allows their adaptation to many different ecosystems. / 3 vidéos sont dans des fichiers complémentaires à ce mémoire

Objectives: The main objective of the current dissertation is to establish a formal shape optimization procedure for a given bypass grafts end-to-side distal anastomosis (ETSDA). The motivation behind this dissertation is that most of the previous ETSDA shape optimization research activities cited in the literature relied on direct optimization approaches that do not guaranty accurate optimization results. Three different ETSDA models are considered herein: The conventional, the Miller cuff, and the hood models. Materials and Methods: The ETSDA shape optimization is driven by three computational objects: a localized collocation meshless method (LCMM) solver, an automated geometry pre-processor, and a genetic-algorithm-based optimizer. The usage of the LCMM solver is very convenient to set an autonomous optimization mechanism for the ETSDA models. The task of the automated pre-processor is to randomly distribute solution points in the ETSDA geometries. The task of the optimized is the adjust the ETSDA geometries based on mitigation of the abnormal hemodynamics parameters. Results: The results reported in this dissertation entail the stabilization and validation of the LCMM solver in addition to the shape optimization of the considered ETSDA models. The LCMM stabilization results consists validating a custom-designed upwinding scheme on different one-dimensional and two-dimensional test cases. The LCMM validation is done for incompressible steady and unsteady flow applications in the ETSDA models. The ETSDA shape optimization include single-objective optimization results in steady flow situations and bi-objective optimization results in pulsatile flow situations. Conclusions: The LCMM solver provides verifiably accurate resolution of hemodynamics and is demonstrated to be third order accurate in a comparison to a benchmark analytical solution of the Navier-Stokes. The genetic-algorithm-based shape optimization approach proved to be very effective for the conventional and Miller cuff ETSDA models. The shape optimization results for those two models definitely suggest that the graft caliber should be maximized whereas the anastomotic angle and the cuff height (in the Miller cuff model) should be chosen following a compromise between the wall shear stress spatial and temporal gradients. The shape optimization of the hood ETSDA model did not prove to be advantageous, however it could be meaningful with the inclusion of the suture line cut length as an optimization parameter.

<p>    Collateral connections in the brain, also known as Leptomeningeal Anastomoses, are connections between blood vessels originating from different arteries. Despite limited knowledge, they are suggested as an important contributor to cerebral stroke recovery that allows additional blood flow through the affected area. However, few databases and algorithms exist for this specific task of locating them. </p> <p>    In this paper, a MATLAB program is developed to find these connections and detect strokes to replace manual labeling by professionals.  The limited data available for this study are 23 2D microscopy images of mice cerebral vascular structures highlighted by dyes. In the images, strokes are shown to diminish the pixel count of vessels below 80\% compared to the healthy brain. Stroke classification error is greatly reduced by narrowing the scope from comparing the entire hemisphere to one smaller region.</p> <p>    A novel way of finding collateral connections is utilizing connected components. Connected components organize all adjacent pixels into a group. All collateral connections can be found on the border of two neighboring arterial flow regions, and belong to the same group of connected components with the arterial source from each side. </p> <p>    Along with finding collateral connections, a newly created coordinate system allows regions to be defined relative to the brain landmarks, based on the brain's center, orientation, and scale.</p> <p>    The method newly proposed in this paper combines stroke detection, brain coordinate system extraction, and collateral connection detection in stroke-affected mouse brains using only image processing techniques.  This allows a simpler, more explainable result on limited data than other techniques such as supervised machine learning.  In addition, the new method does not require ground truth and high image count for training. This automated process was successfully interpreted by medical experts, which allows for further research into automating collateral connection detection in 3D.</p>

Die moderne Gefäßchirurgie bedient sich bei hohen Stadien der pAVK, spezieller Gefäßrekonstruktionen in Form von distalen End-zu-Seit-Gefäßanastomosen. Das langfristige Versagen der Gefäßanastomose hängt primär von der Entstehung einer subendothelialen Intimahyperplasie (IH) ab. Diese IH-Gebiete befinden sich je nach Anastomosengeometrie im Gebiet der Hauben- und Fersenzone sowie am Boden der Anastomose. // Mit Hilfe der Particle Image Velocimetry-Technik wird eine Taylor-Patch-, eine Miller-Cuff-Anastomose und eine femoro-crurale Patch-Prothese bezüglich ihrer Flussmuster sowie ihrer hämodynamischen Eigenschaften wie Geschwindigkeit, Scherstress und Rotation in z-Richtung (Vorticity) untersucht. // In einem hydrodynamischen Kreislaufmodell werden elastische, transparente Silikonmodelle der Anastomosen hergestellt und mit einem blutanalogen Newtonschen Fluid (Glycerol-Wasser-Gemisch) unter Simulation der femorocruralen Druckkurve, pulsatil bei Variation der Strömungsbedingung perfundiert. Der periphere Widerstand beträgt 0,5 mmHg/ml/min (PRU) und die Phasenverschiebung -12 Grad (zwischen Druck- und Flusskurve). // Die Flussmuster variieren zwischen den unterschiedlichen Ausstromverhältnissen erheblich. Bei den unterschiedlichen Flussstärken hingegen ähneln sich die Flussmuster. Alle drei Modelle zeigen ausgeprägte Flussseparationszonen im Hauben- und Fersengebiet sowie geometrieabhängig auch eine Stagnationszone am Boden. Diese Bereiche wiesen die geringsten Fluidgeschwindigkeiten, deutlich unter normalem Wandscherstressniveau liegende Scherstressverhältnisse sowie geringe Vorticitywerte auf. Im Bereich der Übergangszonen finden sich hohe Scherstress- sowie Vorticitywerte. Geschwindigkeitsunterschiede des Fluids zeigten sich im Bereich der Ausstromsegmente. Variable Stressverteilungen zeigen sich auch innerhalb der Separationszonen. Eine Erklärung für die unterschiedlich beschriebenen Offenheitsraten der drei Anastomosenformen wird durch diese Arbeit nicht gefunden. / Modern vascular surgery uses special termino-lateral anastomoses for treating high levels of peripheral arterial disease (PAD). Long term stenoses and occlusions of vascular anastomoses mostly depend on the development of subendothelial myointimal hyperplasia (MIH). There are characteristic areas within the anastomoses, where this process can be examined: The heel, the tow and the floor zone. // This examination observes local hemodynamics like velocity, shear stress and vorticity (rotation in z-direction) and flow patterns of a Taylor-Patch-, a Miller-Cuff-Anastomosis and a feroro-crural patch prothesis (FCPP) with the usage of a Particle Image Velocimetry. In a hydrodynamic circulation model various elastic, transparent silicon phantoms of termino-lateral anastomoses are perfused with a Newton fluid blood analogon (glycerol-water mixture) while simulating the femorocrural pressure curve in a pulsatile manner under variation of the flow conditions. The outflow resistance is 0.5 mmHg/ml/min (PRU, peripheral resistance units) and a phase shift of -12° between flow and pressure curve is simulated. // The flow patterns differed extremely in accordance of the various outflow ratios. Using different flow intensity, the flow patterns are very similar. // All three anastomoses show characteristic heel and toe separation zones. In the FCPP centre a stagnation zone on the floor can not be examined. Shear stress inside the flow separations was significantly lower than normal wall shear stress. High shear stress levels were found inside the transition zones between flow separation and high velocity mainstream. An explanation for the different stenoses and occlusions time of the three different anastomoses can not be found.

<p>In birds like the Zebra finch (Taeniopygia guttata) the female, but not the male develop a brood patch upon incubation of eggs. The brood patch functions to increase heat exchange between the bird and the eggs. Development of the brood patch includes de-feathering, increased vascularization and edema formation. The increased vascularization is due to the development of arteriovenous anastomoses, AVA. The AVA are thermoregulatory vessels involved in cold induced vasodilation, CIVD, demonstrated to occur in the brood patch. Nitric oxide, NO, which is a well known vasodilator is a candidate substance for involvement in CIVD. In this study a NO-generating gel was applied to the brood patch of male and female zebra finches. Vasodilation was found to be markedly larger in females than in males. The larger vasodilation in the female brood patch is probably because NO vasodilate AVA selectively more than any other vessels. The study also investigated whether vasodilation would cause an increase in brood patch temperature. No definite changes in brood patch temperature could be observed and no conclusions could be drawn in the matter.</p>