Microcirculation has become a key factor for the study and assessment of tissue perfusion and oxygenation. Detection and assessment of the microvasculature using videomicroscopy from the oral mucosa provides a metric on the density of blood vessels in each single frame. Information pertaining to the density of these microvessels within a field of view can be used to quantitatively monitor and assess the changes occurring in tissue oxygenation and perfusion over time. Automated analysis of this information can be used for real-time diagnostic and therapeutic planning of a number of clinical applications including resuscitation. The objective of this study is to design an automated image processing system to segment microvessels, estimate the density of blood vessels in video recordings, and identify the distribution of blood flow. The proposed algorithm consists of two main stages: video processing and image segmentation. The first step of video processing is stabilization. In the video stabilization step, block matching is applied to the video frames. Similarity is measured by cross-correlation coefficients. The main technique used in the segmentation step is multi-thresholding and pixel verification based on calculated geometric and contrast parameters. Segmentation results and differences of video frames are then used to identify the capillaries with blood flow. After categorizing blood vessels as active or passive, according to the amount of blood flow, quantitative measures identifying microcirculation are calculated. The algorithm is applied to the videos obtained using Microscan Side-stream Dark Field (SDF) imaging technique captured from healthy and critically ill humans/animals. Segmentation results were compared and validated using a blind detailed inspection by experts who used a commercial semi-automated image analysis software program, AVA (Automated Vascular Analysis). The algorithm was found to extract approximately 97% of functionally active capillaries and blood vessels in every frame. The aim of this study is to eliminate the human interaction, increase accuracy and reduce the computation time. The proposed method is an entirely automated process that can perform stabilization, pre-processing, segmentation, and microvessel identification without human intervention. The method may allow for assessment of microcirculatory abnormalities occurring in critically ill and injured patients including close to real-time determination of the adequacy of resuscitation.

Pela sua alta incidência, morbidade, mortalidade e custos ao sistema de saúde, a sepse se destaca entre as diversas indicações de internação em unidade de terapia intensiva (UTI). A disfunção da microcirculação tem papel central na gênese e manutenção da síndrome séptica, sendo um marco fisiopatológico desta síndrome. Pacientes críticos invariavelmente estão ansiosos, agitados, confusos, desconfortáveis e/ou com dor. Neste contexto, drogas sedativas são amplamente utilizadas na medicina intensiva. A dexmedetomidina, um agonista potente e altamente seletivo dos receptores alfa-2 adrenérgicos, vem conquistando espaço como o sedativo de escolha nas UTIs por seus efeitos de sedação consciente, redução da duração e incidência de delirium e preservação da ventilação espontânea. Apesar destas possíveis vantagens, a indicação de uso da dexmedetomidina na síndrome séptica ainda carece de conhecimentos sobre seus efeitos na microcirculação e perfusão orgânica. Com o intuito de caracterizar os efeitos microcirculatórios da dexmedetomidina em um modelo murino de endotoxemia que permite estudos in vivo da inflamação e disfunção da perfusão microvascular, hamsters Sírios dourados submetidos à endotoxemia induzida por administração intravenosa de lipopolissacarídeo de Escherichia coli (LPS, 1,0 mg.kg-1) foram sedados com dexmedetomidina (5,0 &#956;g.kg.h-1). A microscopia intravital da preparação experimental (câmara dorsal) permitiu a realização de uma análise quantitativa das variáveis microvasculares e do rolamento e adesão de leucócitos à parede venular. Também foram analisados os parâmetros macro-hemodinâmicos e gasométricos (arterial e venoso portal), as concentrações de lactato arterial e venoso portal, a água pulmonar total e a sobrevivência do animal. Animais não-endotoxêmicos e/ou tratados com solução salina a 0,9% serviram como controles neste experimento. O LPS aumentou o rolamento e a adesão de leucócitos à parede venular, diminuiu a densidade capilar funcional e a velocidade das hemácias nos capilares e induziu acidose metabólica. O tratamento com dexmedetomidina atenuou significativamente estas respostas patológicas (p < 0,05). A frequência de pulso dos animais foi significativamente reduzida pela droga (p < 0,05). Outros resultados não foram tão expressivos (estatisticamente ou clinicamente). Estes resultados indicam que a utilização de dexmedetomidina produz um efeito protetor sobre a microcirculação da câmara dorsal de hamsters endotoxêmicos. Efeitos anti-inflamatórios da dexmedetomidina sobre os leucócitos e o endotélio poderiam melhorar a perfusão capilar e representar o mecanismo in vivo de ação da droga na microcirculação. / Due to its high incidence, morbidity, mortality and costs to the healthcare system, sepsis stands out among the many indications for intensive care unit (ICU) admission. The microcirculatory dysfunction plays a central role in the genesis and maintenance of the septic syndrome, being a pathophysiologic milestone in this syndrome. Critically ill patients are invariably anxious, agitated, confused, uncomfortable and/or with pain. In this context, sedative drugs are widely used in intensive care medicine. Dexmedetomidine, a potent and highly selective agonist of alpha-2 adrenergic receptors, is gaining ground as the sedative of choice in ICUs due to its effects of "conscious sedation", reducing the duration and incidence of delirium and preservation of spontaneous ventilation. Despite these potential advantages, the indication of dexmedetomidine in sepsis syndrome still lacks knowledge about its effects on microcirculation and perfusion. To characterize microcirculatory actions of dexmedetomidine in an endotoxemia rodent model that allows in vivo studies of microvascular inflammation and perfusion dysfunction, endotoxemia-submitted Syrian golden hamsters, induced by intravenous Escherichia coli lipopolysaccharide (LPS, 1,0 mg.kg-1) administration, were sedated with dexmedetomidine (5,0 &#956;g.kg.h-1). Intravital microscopy of skinfold chamber preparations allowed quantitative analysis of microvascular variables and venular leukocyte rolling and adhesion. Macro-hemodynamic parameters, arterial and portal venous blood gases and lactate concentrations, pulmonary total water, and animal survival were also analyzed. Non-endotoxemic and/or normal saline treated animals served as controls in this experiment. LPS increased leukocyte rolling and adhesion, decreased functional capillary density and red blood cell velocity, and induced metabolic acidosis. Dexmedetomidine treatment significantly attenuated these pathologic responses (p < 0.05). The pulse rate was significantly reduced by the drug (p < 0.05). Other results were not as expressive (statistically or clinically). These results indicate that the use of dexmedetomidine yields a protective effect on the microcirculation of the dorsal skinfold in endotoxemic hamsters. Anti-inflammatory dexmedetomidine effects on leukocytes and the endothelium, subsequently improving capillary perfusion, could represent the in vivo mechanism of the microcirculatory action of the drug.

O objetivos deste estudo foi avaliar a influência da radiação ionizante na vitalidade do tecido pulpar mensurada através dos níveis de saturação de oxigênio (%SpO2) em pacientes com tumores malígnos intraoral ou de orofaringe, passado de 4-6 anos da radioterapia (RT). Noventa pacientes com tumores malígnos intraoral ou de orofaringe, submetidos de 4-6 anos anteriores a RT foram selecionados para este estudo. Os níveis de oxigenação e sensibilidade pulpar, avaliados através do oxímetro de pulso e do spray refrigerante TFE (tetrafluoretano), foram analisados nos dentes anteriores (superior e inferior) de cada paciente selecionado (n=693),, indiferente do quadrante e da área irradiada. Como grupo controle foram selecionados noventa pacientes saudáveis (nunca submetidos a RT) e os mesmos testes foram empregados (n=693). Todos os dentes foram considerados vitais. 100% mostraram resposta positva ao teste térmico e as médias de %SpO2 foram de 92.7% no grupo dos pacientes irradiados (SD ± 1.83) e 92.6% no grupo dos não-irradiados (SD ± 1.80), sem diferença estatística observada. Houve uma tendência de valores de %SpO2 menores em dentes caninos comparados aos incisivos, entretanto sem diferença estatística significante. Passados de 4-6 anos da RT, as %SpO2 da polpa dental estão dentro dos padrões considerados normais para uma polpa vital e podese assumir que a RT não têm influência danosa a longo prazo sobre a vitalidade do tecido pulpar, assim sugerindo que este tecido pode ser apto a retornar o fluxo sanguíneo normal após a RT. As mudanças observadas na microcirculação pulpar devido a RT parecem ser temporárias, então o tratamento endodôntico preventivo ou a extração dental em pacientes que receberam radiação ionizante são desnecessários. / The aim of this study was to evaluate the influence of radioation on pulp vitality through the measurement of pulpal oxigenation levels (%SpO2) in patients with malignant intraoral and oropharyngeal tumors at 4-6 years after radiotherapy (RT). Ninety patients with malignant tumors in the oral cavity or oropharynx, submitted to RT 4-6 years prior to the study were selected. Pulp oxygenation levels and pulp sensitivity, measured by pulse oximetry and by cold refrigerant spray TFE (tetrafluoroethane), were analyzed in the anterior teeth (upper and lower) of each patient selected (n=693), regardless of the quadrant and the irradiated area. As a control group were selected ninety healthy patients (never submitted to RT) and the same tests were performed (n=693). All teeth were considered vital. 100% showed a positive response to the thermal test and the %SpO2 rates were 92.7% in irradiated group (SD ± 1.83) and 92.6% in non-irradiated group (SD ± 1.80), without statistical difference. There was a trend for lower %SpO2 values in canine teeth compared to incisors, however it was not statistically significant. After 4-6 years of RT the dental pulp %SpO2 are within the normal range considered for a vital pulp and it can be assumed that RT did not have a long term influence on the pulp vitality, therefore suggesting that the pulp tissue may be able to recover normal blood flow after RT. The changes observed in the pulp microcirculation due to RT seems to be temporary, so preventive endodontic treatment or tooth extraction in patients who will receive RT may not be necessary.

La sclérodermie systémique (ScS) est une maladie qui peut engager le pronostic vital des malades de façon très rapide. La pathogénie de la maladie reste encore obscure mais est liée aux atteintes vasculaires probablement en lien avec la fibrose cutanée. La thérapeutique de cette maladie est difficile. Aucun traitement étiologique n'existe et la prise en charge est plutôt axée sur les traitements des différentes atteintes. La iontophorèse cutanée est un dispositif qui permet la diffusion de molécules en solution à travers la peau grâce à une stimulation électrique de manière non invasive. Cette technique a été utilisée initialement comme test physiologique. Notre objectif est d'évaluer et de développer la iontophorèse thérapeutique, notamment avec des molécules vasodilatatrices, pour pouvoir l'utiliser dans des applications thérapeutiques telles que la pris en charge des ulcérations digitales (ou troubles trophiques cutanés) de la ScS. Trois parties ont été développées. La première partie consiste en l'association de deux molécules vasodilatatrices (une per os, le sildenafil, et en iontophorèse, le nitroprussiate de sodium (SNP), en étudiant la tolérance de l'association et son effet sur le flux vasculaire cutané chez le volontaire sain (étude INFLUX-VS). La deuxième partie correspond à un screening de molécules ayant une action vasodilatatrice et étant délivrées en iontophorèse chez le rat : l'étude INFLUX-RAT conclue à l'obtention d'une vasodilatation cutanée chez le rat avec les analogues de la prostacycline. La troisième partie correspond à l'évaluation chez le volontaire sain de la iontophorèse des analogues de la prostacycline : l'étude INFLUX-IT-VS conclue à une dilatation du flux sanguin cutané avec le tréprostinil et avec une bonne tolérance cutanée et systémique. La dernière partie correspond aux avancées parallèles des travaux tant dans le domaine de la reproductibilité des techniques d'acquisition des signaux du flux sanguin cutané que les études réalisées avec les patients sclérodermiques à qui on pourrait espérer un jour voir proposer une iontophorèse thérapeutique. / Systemic scleroderma (ScS) is a rare disease that may be associated with a poor prognosis. The pathogenesis of the disease remains still unclear but comprises vascular abnormalities related to skin fibrosis. ScS disease treatment is difficult. No etiologic therapy is available and patient's management is rather centred on the treatments of the different organs failure symptoms. Skin iontophoresis is a non invasive technique, which allows a transcutaneous diffusion of molecules in solution thanks to an electrical stimulation. This technique was initially used as physiological test. Our objective is to test and to develop therapeutic iontophoresis, in particular with molecules possessing vasodilator properties. The final objective is to use it in therapeutic applications such as the treatment of ScS digital ulcerations. Three parts will be developed in the present work. The first part describes the association of two vasodilatator drugs (sildenafil used per os, associated with sodium nitroprusside used through cathodal iontophoresis. We studied the safety of the association and its effect on the skin vascular flow in healthy volunteers (INFLUX-VS study). The second part describes the screening of vasodilatator drugs delivered through cutaneous iontophoresis in rats (INFLUX-RAT study). The aim of the study was to select the more potent drugs in term of maximal rat skin vasodilatation. The more potent drugs were prostacyclins analogues. The third part describes the iontophoresis of prostacyclins analogues in healthy volunteers: the INFLUX-IT-VS study. The more potent cutaneous vasodilatation was observed with treprostinil, with a good skin and systemic tolerance. The last part decribes the reproducibility of the techniques used to quantify skin blood flow along with studies using these techniques in scleroderma patients. These are required to enable a reproducible evaluation of the effect of skin iontophoresis in patients with scleroderma

Depuis les observations par Poiseuille au XVIIIe de réseaux microvasculaires de petits vertébrés,la microcirculation sanguine a fait l'objet d'abondantes études. Une spécificité mise en avant par le médecin français est la forte hétérogénéité de la distribution des globules rouges dans ces réseaux. En dépit du lien étroit qui lie la fraction volumique locale des globules rouges(hématocrite) à l'oxygénation des tissus environnants, le couplage entre l'architecture microvasculaire et la micro-hémodynamique est encore mal compris. Le sang est un fluide complexe composé de globules rouges, cellules très déformables, suspendus dans du plasma.Dans les vaisseaux de petits diamètres, i.e. du même ordre de grandeur voire plus petits que la taille caractéristique d'un globule rouge (~10µm), le sang possède des propriétés rhéologiques atypiques induites par la structuration locale de l'écoulement et les hétérogénéités d'hématocrite qui en résultent dans la section droite. Ces hétérogénéités se traduisent, aux bifurcationsdivergentes, par une répartition non proportionnelle des débits de globules rouges et de plasma entre les deux branches filles. L'hématocrite de l'une d'elles est alors plus élevé que celui de labranche d'entrée, alors qu'il est plus faible dans l'autre branche. Cet effet, connu sous le nom d'effet de séparation de phase, induit une très grande hétérogénéité de l'hématocrite à l'échelle du réseau. L'objectif de cette thèse est d'étudier l'apparition de ces hétérogénéités, de l'échelle du vaisseau à l'échelle du réseau, dans des conditions expérimentales contrôlées et pour des régimes de confinement et d'hématocrite représentatifs des écoulement sanguins de la partie terminale du lit microvasculaire (artérioles, capillaires et veinules de diamètres inférieurs à 20 µm).De nombreuses données expérimentales ont été acquises in vivo, mais elles sont sujettes à de fortes incertitudes quant à la forme et aux dimensions de la section droite des vaisseaux, mais aussi soumises aux effets de régulations physiologiques des débits (e.g. par vasoconstriction ou vasodilatation). A notre connaissance, du fait des contraintes expérimentales inhérentes aux régimes de confinement et d'hématocrite des plus petits vaisseaux de la microcirculation, très peu d'études in vitro dans ces conditions ont été menées. Tout d'abord, nous présentons les profils de vitesse des globules rouges et les profils d'hématocrite obtenus grâce à une nouvelle méthode de mesure de concentration développée pendant ce travail, dans des canaux uniques de taille comprise entre 5 et 20µm, et dans une large gamme d'hématocrite. Nous proposons une paramétrisation générale semi-empirique de ces profils, qui prend en compte la présence d'unecouche d'exclusion plasmatique, observée quelle que soit la taille du canal aux faibleshématocrites. Ensuite, nous présentons une étude paramétrique de l’effet de séparation dephase. Nous montrons que les résultats obtenus sont indépendants, dans les régimes étudiés, de l’angle de la bifurcation et du débit de la branche d’entrée. Ces résultats sont en général en bon accord avec un modèle simple qui s’appuie sur la paramétrisation précédente des profils d'hématocrite et de vitesse des globules rouges et suppose l'existence d'une ligne séparatrice de fluides dans la section droite de la branche mère. Ces résultats suggèrent que les globules rouges peuvent être décrits par un fluide équivalent, y compris dans les conditions de très fortconfinement. Enfin, nous reportons pour la première fois des résultats quantitatifs liés à la distribution de l'hématocrite dans des réseaux modèles. Nous montrons notamment quel'asymétrie des profils d'hématocrite dans les branches amonts distribue les globules rouges en enrichissant le cœur du réseau au détriment des bords. Nous comparons nos résultats à ceux d’un modèle non-linéaire de type réseau classique proposant des corrections prenant en compte cette asymétrie.

The blood and lymphatic vascular systems coordinate to play critical roles in tissue fluid homeostasis and immune function and are fundamentally associated with diseases including inflammation, wound healing, edema, and tumor progression and metastasis. Their coordination during vascular growth and remodeling represents an under-investigated area of research in which a better understanding will provide insights into future therapeutic approaches. Angiogenesis and lymphangiogenesis are the growth of new blood or lymphatic vessels, respectively, from pre-existing vessels. The comprehensive goal of this work was to provide a new perspective on the relationships between angiogenesis and lymphangiogenesis in microvascular networks and develop tools needed to probe the mechanisms involved in lymphatic/blood vessel patterning and identity. The first aim of this study was to characterize the spatiotemporal relationships between lymphatic and blood vessel growth in response to an inflammatory stimulus. We found that lymphangiogenesis temporally lagged angiogenesis during inflammation and that the presence of lymphatic vessels attenuated angiogenesis. We also identified increased lymphatic/blood endothelial cells connections and a novel lymphatic marker. These results motivated the need for a system to probe the multicellular and multisystem interactions suggested by these findings. Our lab recently developed the rat mesentery culture model as an ex vivo model for investigating angiogenesis in the context of intact microvascular networks. The second aim was to determine whether this model can be used to study lymphangiogenesis. We found that vascular endothelial growth factor C stimulated lymphatic sprout formation in the rat mesentery culture model and confirmed the ability to observe angiogenesis and lymphangiogenesis simultaneously in an ex vivo environment. This suggests the rat mesentery culture model can be used to investigate the dynamics of lymphatic/blood vessel patterning and plasticity motivated by our in vivo work. The final aim of this work was to investigate the ability to induce phenotypic plasticity in intact vasculature by using lysophosphatidic acid, an agonist suggested to cause blood-to-lymphatic endothelial cell transition. We demonstrated that while lysophosphatidic acid stimulated angiogenesis, it was not sufficient to reprogram blood vessels to acquire a lymphatic phenotype. These results underscore the necessity of investigating these multisystem relationships in the context of intact microvascular networks. These studies as a whole demonstrate the coordination that exists between blood and lymphatic vessels and reinforce the need for novel models that incorporate the complexities of the entire microvasculature. / acase@tulane.edu

Le choc septique reste grévé d'une mortalité importante malgré les efforts de recherche expérimentale et clinique. Certains protocoles thérapeutiques cependant, ont permis ces dernières années de diminuer cette mortalité. Les mécanismes précis d'amélioration de ce pronostic sont inconnus. Il est possible que ce gain de survie soit du à une atténuation du Syndrome de Défaillance Microcirculatoire et Mitochondriale du Sepsis. Les travaux réunis dans une première partie de ce volume ont permis de mieux caractériser l'effet de différentes thérapeutiques sur la microcirculation, la vasoréactivité. Thérapeutique annexe comme la sédation qui est utilisée en pratique clinique dans nos services de réanimation ou dans nos laboratoires chez nos animaux, qui perturbe la microcirculation. Thérapeutique directe comme la protéine C activée qui est déjà connue pour être bénéfique sur la microcirculation. La protéine C activée dans notre modèle de choc au LPS chez le rat a notamment un effet bénéfique microcirculatoire possiblement par une dégradation moindre du glycocalyx, le manteau endothélial. Ce produit a également un effet bénéfique sur la réponse vasculaire à la noradrénaline, restaurant cette réponse par rapport à des rats septiques. Une amélioration de la fonction vasculaire et cardiaque est constatée avec ce traitement, sans que l'on puisse affirmer que cette amélioration soit le fait d'une action directe bénéfique sur la microcirculation, la cellule musculaire lisse etc ou une amélioration globale de la réponse de l'organisme au choc : baisse concommittante de médiateurs comme le TNF-α, les dérivés nitrés, ou baisse concommitante du stress oxydatif. Dans une deuxième partie du travail, l'implication de la mitochondrie par le biais de la modulation de facteurs proapoptotiques et de la dissipation du potentiel de membrane ouvre des perspectives pour mieux caractériser cette atteinte chez l'homme dans le futur. En effet moduler la dissipation du potentiel membranaire mitochondriale module chez l'animal la mortalité dans notre modèle. Nous proposons pour l'avenir d'essayer de mieux caractériser ces anomalies chez l'homme.

In this study the role of passive mechanism for deformation of neutrophils, namely the effect of mechanical properties, was studied using microgels as model system. Both alginate-poly(N-isopropylacrylamide) interpenetrating polymer network (IPN) microgels and agarose microgels were synthesized in microfluidic device. The Young’s modulus and relaxation time of the IPN microgels were studied using atomic force microscopy equipped with a tipless cantilever. The lower limits of the elasticity found in this study were within the range of the elasticity reported for neutrophils. Agarose microgels were also prepared with a range of elastic shear modulus similar to neutrophils, and their flow under constrained geometries was studied. The flow profiles of four agarose microgel samples in a microchannel containing a constriction were analyzed. It was found that the stiffness of the microgels affected their velocity before, in and after the constriction.

In this study the role of passive mechanism for deformation of neutrophils, namely the effect of mechanical properties, was studied using microgels as model system. Both alginate-poly(N-isopropylacrylamide) interpenetrating polymer network (IPN) microgels and agarose microgels were synthesized in microfluidic device. The Young’s modulus and relaxation time of the IPN microgels were studied using atomic force microscopy equipped with a tipless cantilever. The lower limits of the elasticity found in this study were within the range of the elasticity reported for neutrophils. Agarose microgels were also prepared with a range of elastic shear modulus similar to neutrophils, and their flow under constrained geometries was studied. The flow profiles of four agarose microgel samples in a microchannel containing a constriction were analyzed. It was found that the stiffness of the microgels affected their velocity before, in and after the constriction.

Heart transplantation is, for certain types of cardiovascular disease, the only form of treatment resulting in patient survival. Its clinical application is, however, limited by the shortage of donor organs. This shortage is largely due to the inability to consistently preserve adequate myocardial function over prolonged ischemic periods. It is the goal of this research to provide information which may contribute to techniques for heart preservation, thus improving graft survival following preservation and transplantation. Current methods for myocardial preservation generally involve the arrest and immersion of the heart in cold cardioplegic solution, the composition of which is designed to provide for the reduced metabolic demands of the cold, arrested muscle. These methods have extended the preservation period to approximately 6 hours; however, hearts cannot be held longer than this period because, although metabolism has been slowed by hypothermia, alterations take place which compromise functional recovery upon reperfusion. A variety of perfusates and perfusion techniques have been developed to protect the myocardium from the damage thought to occur as a consequence of ischemic storage of the isolated heart. However, a consistently successful technique for long-term preservation of the heart remains undefined. A growing body of knowledge has led to the hypothesis that injury to the microcirculation may result in myocardial ischemia during preservation and decreased contractile function following preservation. To test this hypothesis, standard Langendorff techniques for the measurement of left ventricular function were combined with biochemical, histological and morphological techniques to determine: (1) whether loss of microvascular function occurs in isolated hearts hypothermically perfused with an oxygenated solution; (2) the impact of microvascular dysfunction during the preservation period on the functional recovery of hearts; and (3) which mechanisms contribute to decreased microvascular function during preservation. This experimental approach will allow for characterization of the role of the microvasculature in decreased contractility of preserved hearts and will provide information regarding the contribution of specific mechanisms to the compromised contractility of preserved hearts. Systematic evaluation of mechanisms thought to be responsible for decreased contractility of isolated hearts could contribute to improved myocardial preservation techniques that can be applied to clinical transplantation.