No description available.

Le procédé de la métastase cancéreuse et sa compréhension sont devenus un des sujets majeurs de recherche en Biologie. En utilisant des modèles in-vitro en culture statique et dynamiques, nous avons étudié la possibilité de reproduire l’environnement physiologique in-vivo avec ces modèles. Nous avons développé un modèle de coculture hiérarchique dans des plaques à fond en PDMS. Composé d’hépatocytes, de pericytes et de cellules endothéliales. Dans différentes conditions, l’influence de ces cellules sur l’adhésion de cellules cancéreuses ou promyéloblastiques a été analysée ainsi que leur effet sur l’état inflammatoire du système. Afin de reproduire le flux sanguin et les forces de cisaillement présents in-vivo, le modèle a été transféré dans un système microfluidique. Le système se compose de trois canaux séparés par des micro-piliers, pouvant être remplis indépendamment. Les pericytes insérés dans du gel, les hépatocytes, les cellules endothéliales et finalement les cellules cancéreuses ont été injectés de façon successive afin de reproduire l’environnement in-vivo. Les cellules ont été trouvées viables durant toute la culture et des marqueurs relatifs au foie et à l’inflammation exprimés. L’influence des hépatocytes et des pericytes a été analysé. Il a été observé que les cellules cancéreuses adhérées dans le canal du haut étaient attirées par les autres cellules dans les diffèrent canaux. Les modèles établis posent de solides bases pour d’autres systèmes plus complexes et d’intérêt pouvant servir de complément aux modèles in-vivo lors de la recherche de nouvelles substances médicamenteuses. / The cancer metastatic process and its understanding have been a major topic of interest for researchers in the past. Using in-vitro models in both standard culture conditions and in microfluidic devices, we investigated the feasibility of such models in the representation of the physiological in-vivo situation. We developed a hierarchical coculture model in PDMS plates, composed of hepatocytes, pericytes and endothelial cells. In different culture conditions, the influence of the different cells composing the model on the adhesion of cancer cells and promyeloblastic cells was investigated as well as the influence on the inflammatory state of the culture. To reproduce the in-vivo blood flow and shear stress to which the endothelial cells and the adhering cells are subjected, the model was then transferred into a microfluidic biochip. The device was composed of three channels, separated by micropillars and which could be filled independently one from another. Pericytes embedded in a hydrogel, hepatocytes, endothelial cells and finally pancreatic cancer cells could be inserted successively to reproduce the in-vivo hierarchical situation. Cells were found to viable after the culture and markers related to the liver and inflammation to be expressed. The influence of the presence of hepatocytes and pericytes was investigated by varying the culture conditions. It was found that pancreatic cancer cells were attracted by the cells in other channels in coculture. The established models lay the bases for more complex and relevant systems that could complement their in-vivo counterparts in the drug discovery process.

No description available.

Brief periods of skeletal muscle ischemia (ischemic pre-conditioning) alter cellular metabolism in a way that confers protection over subsequent ischemic episodes. The mechanisms behind this effect have been studied indirectly through assays for the byproducts of ATP synthesis and in vitro studies of cellular signaling cascades and ROS generation. There have been no direct, in vivo assessments of the changes in respiration during reperfusion. We employed phosphorescence quenching microscopy in conjunction with a flow-arrest technique to assess the influences of external, pressure-induced 1- to 10-min focal ischemia on interstitial oxygenation (PISFO2) and the consumption of oxygen (VO2) in spinotrapezius muscles of Sprague-Dawley rats. During reperfusion following an ischemic period VO2 was measured by the rate of PISFO2 decline during brief, serial flow-arrest compressions. Our tests of this intermittent compression technique indicate that 5 s of flow-arrest followed by 15 s of flow restoration allow measurement of VO2 without compromising baseline or reperfusion recovery of PISFO2. There was a steady rise in VO2 during early reperfusion which was correlated with increasing ischemic durations. Treatment with cyanide confirmed that at least some of this increase was due to an upregulation of cytochrome c oxidase activity. Nitric oxide (NO) suppressed VO2 during rest and reperfusion, while L-NAME did not influence respiration under normoxic conditions. L-NAME produced a significant rise in VO2 under hypoxic conditions following 10 minutes of ischemia, indicating a greater role of NO in the regulation of respiration during low PISFO2 conditions. We conclude that physiological levels of NO regulate mitochondrial respiration during hypoxia and confirm that pharmacological elevation of [NO] reduces VO2 in a manner consistent with the ischemic pre-conditioning effect.

acase@tulane.edu

Modeling interstitial fluid flow involves processes such as fluid diffusion, convective transport in the extracellular matrix, and extravasation from blood vessels. In all of these processes, computational fluid dynamics can play a crucial role in elucidating the mechanisms of fluid flow in solid tumors and surrounding tissues. To date, microvasculature flow modeling has been most extensively studied with simple tumor shapes and their capillaries at different levels and scales. With our proposed numerical model, however, more complex and realistic tumor shapes and capillary networks can be studied. First, a mathematical model of interstitial fluid flow is developed, based on the application of the governing equations for fluid flow, i.e., the conservation laws for mass and momentum, to physiological systems containing solid tumors. Simulations of interstitial fluid transport in a homogeneous solid tumor demonstrate that, in a uniformly perfused tumor, i.e., one with no necrotic region, the interstitial pressure distribution results in a non-uniform distribution of drug particles. Pressure distribution for different values of necrotic radii is examined, and two new parameters, the critical tumor radius and critical necrotic radius, are defined. In specific ranges of these critical dimensions the interstitial fluid pressure is relatively lower, which in turn leads to a diminished opposing force against drug movement and a subsequently higher drug concentration and potentially enhanced therapeutic effects. In this work, the numerical model of fluid flow in solid tumors is further developed to incorporate and investigate non-spherical tumor shapes such as prolate and oblate ones. Using this enhanced model, tumor shape and size effects on drug delivery to solid tumors are then studied. Based on the assumption that drug particles flow with the interstitial fluid, the pressure and velocity maps of the latter are used to illustrate the drug delivery pattern in a solid tumor. Additionally, the effects of the surface area per unit volume of the tissue, as well as vascular and interstitial hydraulic conductivity on drug delivery efficiency, are investigated. Using a tumor-induced microvasculature architecture instead of a uniform distribution of vessels provides a more realistic model of solid tumors. To this end, continuous and discrete mathematical models of angiogenesis were utilized to observe the effect of matrix density and matrix degrading enzymes on capillary network formation in solid tumors. Additionally, the interactions between matrix-degrading enzymes, the extracellular matrix and endothelial cells are mathematically modeled. Existing continuous and discrete models of angiogenesis were modified to impose the effect of matrix density on the solution. The imposition has been performed by a specific function in movement potential. Implementing realistic boundary and initial conditions showed that, unlike in previous models, the endothelial cells accelerate as they migrate toward the tumor. Now, the tumor-induced microvasculature network can be applied to the model developed in Chapters 2 and 3. Once the capillary network was set up, fluid flow in normal and cancerous tissues was numerically simulated under three conditions: constant and uniform distribution of intravascular pressure in the whole domain, a rigid vascular network, and an adaptable vascular network. First, governing equations of sprouting angiogenesis were implemented to specify the different domains for the network and interstitium. Governing equations for flow modeling were introduced for different domains. The conservation laws for mass and momentum, Darcy’s equation for tissue, and a simplified Navier Stokes equation for blood flow through capillaries were then used for simulating interstitial and intravascular flows. Finally, Starling’s law was used to close this system of equations and to couple the intravascular and extravascular flows. The non-continuous behavior of blood and the adaptability of capillary diameter to hemodynamics and metabolic stimuli were considered in blood flow simulations through a capillary network. This approach provided a more realistic capillary distribution network, very similar to that of the human body. This work describes the first study of flow modeling in solid tumors to realistically couple intravascular and extravascular flow through a network generated by sprouting angiogenesis, consisting of one parent vessel connected to the network. Other key factors incorporated in the model for the first time include capillary adaptation, non-continuous viscosity blood, and phase separation of blood flow in capillary bifurcation. Contrary to earlier studies which arbitrarily assumed veins and arteries to operate on opposite sides of a tumor network, the present approach requires the same vessel to run and from the network. Expanding the earlier models by introducing the outlined components was performed in order to achieve a more-realistic picture of blood flow through solid tumors. Results predict an almost doubled interstitial pressure and are in better agreement with human biology compared to the more simplified models generally in use today.

Modeling interstitial fluid flow involves processes such as fluid diffusion, convective transport in the extracellular matrix, and extravasation from blood vessels. In all of these processes, computational fluid dynamics can play a crucial role in elucidating the mechanisms of fluid flow in solid tumors and surrounding tissues. To date, microvasculature flow modeling has been most extensively studied with simple tumor shapes and their capillaries at different levels and scales. With our proposed numerical model, however, more complex and realistic tumor shapes and capillary networks can be studied. First, a mathematical model of interstitial fluid flow is developed, based on the application of the governing equations for fluid flow, i.e., the conservation laws for mass and momentum, to physiological systems containing solid tumors. Simulations of interstitial fluid transport in a homogeneous solid tumor demonstrate that, in a uniformly perfused tumor, i.e., one with no necrotic region, the interstitial pressure distribution results in a non-uniform distribution of drug particles. Pressure distribution for different values of necrotic radii is examined, and two new parameters, the critical tumor radius and critical necrotic radius, are defined. In specific ranges of these critical dimensions the interstitial fluid pressure is relatively lower, which in turn leads to a diminished opposing force against drug movement and a subsequently higher drug concentration and potentially enhanced therapeutic effects. In this work, the numerical model of fluid flow in solid tumors is further developed to incorporate and investigate non-spherical tumor shapes such as prolate and oblate ones. Using this enhanced model, tumor shape and size effects on drug delivery to solid tumors are then studied. Based on the assumption that drug particles flow with the interstitial fluid, the pressure and velocity maps of the latter are used to illustrate the drug delivery pattern in a solid tumor. Additionally, the effects of the surface area per unit volume of the tissue, as well as vascular and interstitial hydraulic conductivity on drug delivery efficiency, are investigated. Using a tumor-induced microvasculature architecture instead of a uniform distribution of vessels provides a more realistic model of solid tumors. To this end, continuous and discrete mathematical models of angiogenesis were utilized to observe the effect of matrix density and matrix degrading enzymes on capillary network formation in solid tumors. Additionally, the interactions between matrix-degrading enzymes, the extracellular matrix and endothelial cells are mathematically modeled. Existing continuous and discrete models of angiogenesis were modified to impose the effect of matrix density on the solution. The imposition has been performed by a specific function in movement potential. Implementing realistic boundary and initial conditions showed that, unlike in previous models, the endothelial cells accelerate as they migrate toward the tumor. Now, the tumor-induced microvasculature network can be applied to the model developed in Chapters 2 and 3. Once the capillary network was set up, fluid flow in normal and cancerous tissues was numerically simulated under three conditions: constant and uniform distribution of intravascular pressure in the whole domain, a rigid vascular network, and an adaptable vascular network. First, governing equations of sprouting angiogenesis were implemented to specify the different domains for the network and interstitium. Governing equations for flow modeling were introduced for different domains. The conservation laws for mass and momentum, Darcy’s equation for tissue, and a simplified Navier Stokes equation for blood flow through capillaries were then used for simulating interstitial and intravascular flows. Finally, Starling’s law was used to close this system of equations and to couple the intravascular and extravascular flows. The non-continuous behavior of blood and the adaptability of capillary diameter to hemodynamics and metabolic stimuli were considered in blood flow simulations through a capillary network. This approach provided a more realistic capillary distribution network, very similar to that of the human body. This work describes the first study of flow modeling in solid tumors to realistically couple intravascular and extravascular flow through a network generated by sprouting angiogenesis, consisting of one parent vessel connected to the network. Other key factors incorporated in the model for the first time include capillary adaptation, non-continuous viscosity blood, and phase separation of blood flow in capillary bifurcation. Contrary to earlier studies which arbitrarily assumed veins and arteries to operate on opposite sides of a tumor network, the present approach requires the same vessel to run and from the network. Expanding the earlier models by introducing the outlined components was performed in order to achieve a more-realistic picture of blood flow through solid tumors. Results predict an almost doubled interstitial pressure and are in better agreement with human biology compared to the more simplified models generally in use today.

abstract: There is a robust association between psychosis and cannabis use, but the mechanisms underlying this relation are poorly understood. Because both psychosis and cannabis use have been linked to cardiovascular problems, it is possible that cannabis use exacerbates an underlying vascular vulnerability in individuals prone to psychosis. To investigate microvascular differences in individuals with psychotic symptoms and cannabis use, the current study tested associations between psychotic-like experiences, cannabis use, and retinal vessel diameter in 101 young adults (mean age=19.37 years [SD=1.93]). Retinal venular diameter did not differ between participants with (M=218.08, SD=15.09) and without psychotic-like experiences (M=216.61, SD=16.18) (F(1, 97)=0.01, p=.93) or between cannabis users (M=218.41, SD=14.31) and non-users (M=216.95, SD=16.26) (F(1, 97)=0.37, p=.54). Likewise, mean retinal arteriolar diameter did not differ between participants with (M=157.07, SD=10.96) and without psychotic-like experiences (M=154.88, SD=9.03) (F(1, 97)=0.00, p=.97). However, cannabis users had statistically significantly wider retinal arterioles (M=159.10, SD=9.94) than did non-users (M=154.29, SD=10.20) (F(1, 97)=5.99, p=.016), and this effect was robust to control for covariates. There was no evidence of an interaction between psychotic-like experiences and cannabis use in predicting retinal vessel diameter. These results indicate that cannabis use is associated with microvascular differences in young adulthood. Given current trends toward legalization of recreational cannabis use, future research should explore these differences and their potential consequences for cardiovascular health. / Dissertation/Thesis / Masters Thesis Psychology 2016

The structure and ultrastructure of the microvascular bed of fatty bone marrow and the relationships of this vascular bed to endosteal bone surfaces was studied in adult beagles. The vascular volume of fatty bone marrow, as demonstrated by India ink-gelatin perfusion, appears less than the vascular volume of red bone marrow. The capillaries in fatty bone marrow are found between the large fat cells in a loose reticular connective tissue and have a continuous endothelial lining and basal lamina. Capillaries are frequently found adjacent to bone surfaces in these fatty marrow sites, yet are separated from these surfaces by very thin bone-lining cells. The nuclei of bone-lining cells are often found near these capillaries. The association of bone-lining cells to the microvasculature suggests that these cells may play a role in partitioning the extracellular fluids from the bone.

L'accident vasculaire cérébral (AVC) représente la première cause de handicap acquis de l'adulte. A l'heure actuelle, moins de 10% des patients peuvent bénéficier de la thrombolyse, et aucun traitement, en dehors de la rééducation, ne permet de réduire efficacement le handicap. Il existe donc un réel besoin de disposer de nouvelles thérapeutiques permettant d'améliorer la récupération et pouvant être administrées dans un délai élargi par rapport à celui de la thrombolyse. Nos travaux expérimentaux chez le rat, associant imagerie IRM de la microvascularisation, analyse de l'expression des gènes de l'angiogenèse et étude comportementale, ont permis de définir une phase de transition (J3-J7) suivie d'une phase subaigüe (J7-J25) post-AVC. Ces deux phases sont apparues comme des fenêtres thérapeutiques potentielles pour l'administration de traitement pro-angiogéniques. Depuis près de 20 ans, de nombreuses équipes se sont tournées vers la thérapie cellulaire, notamment par cellules souches/stromales mésenchymateuses humaines (CSMh), comme thérapie réparatrice dans les AVC avec un triplement du nombre d'essais cliniques au cours des 10 dernières années. Cependant, les données de la littérature ne permettent pas de bien comprendre le mécanisme d'action des CSMh, particulièrement après une administration à la phase subaigüe. Nos travaux ont permis de progresser dans la compréhension de l'effet microvasculaire des CSMh, administrées dans les conditions d'un essai clinique de phase II qui se déroule actuellement à Grenoble (ISIS : Intravenous Stem Cells After Ischemic Stroke). Nous avons montré que la récupération sensori-motrice et cognitive post-ischémique observée après administration intraveineuse de CSMh était liée à une augmentation de l'angiogenèse. Les facteurs angiogéniques Ang2, Ang1, SDF-1 et TGFβ1, dont la sécrétion endogène est augmentée par les CSMh, semblent participer à une meilleure stabilisation vasculaire et pourrait expliquer l'effet bénéfique de ces cellules. Dans le cadre du développement des CSMh en tant que médicament de thérapie innovante, nous avons montré l'absence de potentiel tumorigène des CSMh par une étude toxicologique de tumorigénicité in vivo. Par analyse rétrospective des CSMh produites dans le cadre de l'essai clinique de phase II, nous avons montré la faisabilité de la production de CSMh conformes aux spécifications et en quantité suffisante par l'Unité de Thérapie Cellulaire. Par ailleurs, ces CSMh cultivées ex vivo peuvent présenter des anomalies caryotypiques erratiques, non clônales. Ces anomalies semblent être liées au maintien en culture, plus qu'au procédé lui-même. Une composante "donneur" semble également contribuer à l'apparition de ces anomalies. / Stroke is the leading cause of disability in adult. Less than 10% of patients can be treated with thrombolysis. Except rehabilitation, no effective treatment exists to improve functional recovery after the acute phase. Therefore, there is a wide need to develop an effective therapy applicable after several days or weeks following stroke. Using a multiparametric approach (microvascular MRI, analysis of angiogenic genes expression and behavioral study) in rat ischemic stroke model, we defined a transition stage (D3-D7) followed by a subacute phase (D7-D25) during post-stroke remodeling. These two phases represent an interesting target time-window for administration of pro-angiogenic therapies. Since 20 years, cell therapy, notably by human mesenchymal stem/stromal cells (hMSC), emerged as a “regenerative treatment” with threefold increase in clinical trial during the last 10 years. However, still limited data are available regarding the mechanisms by which hMSC benefit, especially at the subacute phase. We progressed in understanding the microvascular plasticity that occurs after an intravenous injection of hMSC in a rat model of transient focal cerebral ischemia. Our preclinical studies were carried out simultaneously with a phase II clinical trial that currently goes on in Grenoble (ISIS: Intravenous Stem Cells After Ischemic Stroke). We reported a sustained functional and cognitive long-term benefit of hMSC IV injected at the subacute stage correlated to an increase of angiogenesis. Ang2, Ang1, SDF-1 and TGFβ1, whose endogenous level tends to be overexpressed by hMSC, would enhance stabilization and survival of newborn vessels, accounting for benefit of these cells. As part of the hMSC development as an advanced therapy medicinal product, we realized an in vivo tumorigenicity assay and showed the absence of tumor development after hMSC injection. We also retrospectively analyzed hMSC produced for the phase II clinical trial. We confirmed the feasibility to produce hMSC, conformed to specifications and in adequate quantity, in the Cell Therapy Unit. In addition, we showed that ex vivo expanded hMSC can present, non clonal, erratic chromosomal abnormalities. Such chromosomal abnormalities appeared to be more related to the maintenance in culture than to the manufacturing process. A “donor” component may also contribute to emergence of such abnormalities.