Blood Vessel Diameter Estimation System Using Active Contours

The study and analysis of blood vessel geometry has become the basis of medical applications related to early diagnosis and effective monitoring of therapies in vascular diseases. This paper presents a new method to trace the outline of blood vessels from imperfect images and extract useful information about their dimensions in an automated manner. The system consists of a segmentation procedure that uses two Active Contours to detect blood vessel boundaries and a novel approach to measure blood vessel diameters directly as the distance between two points. We have succeeded in designing and implementing an automated, robust, measurement method that is not only accurate (it takes away human error) but also user-friendly and requires very little image pre-processing. The system is tested with a set of grey scale images of blood vessels. Results of all the aspects of the design and implementation are presented along with graphs and images

Novel Techniques For Investigating The Regulation Of Skeletal Muscle Hemodynamics

The effect of the sympathetic nervous system (SNS) on blood flow distribution within skeletal muscle microvasculature is conditional upon regional activation of SNS receptors. Due to a lack of appropriate experimental models and techniques, no study has systematically evaluated the effect of SNS receptor activation in continuously branching skeletal muscle arteriolar trees. In line with previous work, we hypothesize that there will be a spatially-dependent distribution of sympathetic receptor activation along the arteriolar tree. Specifically, we anticipate a progressive decrease of adrenergic activation and a progressive increase of peptidergic and purinergic activation with increasing arteriolar order. We developed a novel rat gluteus maximus (GM) muscle preparation which provided access to a large vascular network, from which we developed an experimental method for collecting cell velocity profiles in fast-flowing arterioles. Using these data, we derived an empirical relationship between velocity ratio (VMax/VMean) and arteriolar diameter, collected novel data on cell free layer width and estimated wall shear rates, and derived a wall shear rate equation from experimental data that can be used for calculating wall shear rates in skeletal muscle microvasculature. We evaluated SNS receptor activation (α1R, α2R, NPY1R, and P2X1R) in continuously branching arteriolar trees in the rat GM, as a function of network topology. A computational flow model estimated the total flow, resistance, and red blood cell flow heterogeneity. For the first time, we highlight effects of SNS receptor activation on network hemodynamics, where proximal arterioles responded most to adrenergic activation, while distal arterioles responded most to Y1R and P2X1R activation. Our data highlight the functional consequences of topologically-dependent SNS receptor activation. The tools developed in this thesis are beneficial for computing hemodynamic parameters from in vivo data, as well as providing input variables to and validation of computational flow models

Behavioral immune landscapes of inflammation.

Transcriptional or proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues. These approaches, however, fail to describe dynamic scenarios in which cells can change their biochemical properties and downstream “behavioral” outputs every few seconds or minutes. Here, we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamism of individual leukocytes at sites of active inflammation. By analyzing over 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioral descriptors of individual cells and used these high-dimensional datasets to build behavioral landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and inside blood vessels uncovered a continuum of neutrophil states, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioral in vivo screening of thousands of cells from 24 different mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and genetic or pharmacological interference of Fgr protected from inflammatory injury. Thus, behavioral landscapes report unique biological properties of dynamic environments at high cellular, spatial and temporal resolution.pre-print4302 K

Modeling Oxygen Transport in Three-Dimensional Capillary Networks

The purpose of this thesis was to examine how the use of real 3-dimensional (3D) capillary network geometries affect models of oxygen transport to tissue. Software was developed to reconstruct microvascular geometry in 3D from intravital video. Characterization of 3D reconstructions demonstrated that capillary density, length and capillary diameter were consistent with previous findings. Using reconstructed capillary networks a strategy was devised that utilized red blood cell (RBC) supply rate (SR) as a metric for flow modeling. Applying the RBC SR based flow model on baseline and perturbed flow conditions demonstrated that RBC SR is a major determinant of oxygen delivery that is insensitive to changes in flow distribution. The resulting flow solutions were used for comparing oxygen transport in 3D networks and synthetic parallel arrays. A variety of physiological conditions were simulated and it was determined that parallel arrays resulted in oxygen transport solutions with higher mean PO2 due to homogeneous distribution of vessels in the volume. Lastly, to investigate oxygen transport in a complex pathology a model of sepsis was used to investigate how incremental perfusion loss, consumption increase and change in RBC SR affect oxygen delivery. It was shown that perfusion loss did not markedly impair oxygen delivery provided that RBC SR was maintained. These results have improved our understanding of oxygen transport to tissue in normal and diseased conditions; the use of reconstructed networks and measurements of blood flow & oxygen saturation in computer models provides different solutions than those using statistical averages and synthetic networks

Ultrasound-driven microbubble dynamics in microvessels

Ultrasound and microbubble induced blood-brain barrier opening has shown success in clinical trials as a promising method to deliver drugs to the brain. Shelled gas bubbles, a few micrometres in diameter, are administered intravenously, and distribute throughout the cardiovascular system. When ultrasound is applied to the brain, the microbubbles expand and contract within the vasculature, temporarily disrupting the blood-brain barrier, and allowing drugs to pass through. While this technique has been shown to be effective at delivering drugs, its mechanisms remain relatively poorly understood. Better understanding how microbubbles interact with tissues could enable refinement of therapies. This thesis investigates the fundamental physical interactions between microbubbles and soft tissues using two distinct but related experimental platforms that utilise high-speed microscopy. Firstly, microbubbles within soft tissue-mimicking hydrogel channels are observed during exposure to typical therapeutic ultrasound pulses. The primary radiation force is shown to be significant, and can cause bubbles to deform the soft gels by several micrometres. Microbubbles are also investigated in brain tissue, using acute cortical slices from the brains of juvenile rats, transcardially perfused post-mortem with a concentrated solution of SonoVue®. This technique is shown to be an effective method of observing microbubbles using optical microscopy within the microvasculature of live brain tissue. Radial oscillations of bubbles within brain microvessels can deform surrounding tissue at both microsecond and millisecond time scales. Extravasation of microbubbles due to the primary radiation force can occur during typical ultrasound pulses, and is common at higher ultrasound pressures (mechanical index of 0.6 and above). These results demonstrate the significance of both radial oscillations and the primary radiation force as ways in which microbubbles can physically impact their surroundings. Additionally, acute brain slices are shown to be a valuable tool to investigate microbubble behaviours and mechanisms of drug delivery in a physiologically relevant environment.Open Acces

Tumor vasculature and microenvironment during progression and treatment : insights from optical microscopy

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, February 2010.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.In addition to cancer cells, solid tumors consist of a variety of cell types and tissues defining a complex microenvironment that influences disease progression and response to therapy. To fully characterize and probe the tumor microenvironment, new tools are needed to quantitatively assess microanatomical and physiological changes during tumor growth and treatment. Particularly important, is the metabolic microenvironment defined in tumors by hypoxia (low p02) and acidity (low pH). These parameters have been shown to influence response to radiation therapy and chemotherapy. However, very little is known about spatio-temporal changes in p02 and pH during tumor progression and therapy. By modifying the technique of intravital multiphoton microscopy (MPM) to perform phosphorescence quenching microscopy, I developed a non-invasive method to quantify oxygen tension (p02) in living tissue at high three-dimensional resolution. To probe functional changes in the metabolic microenvironment, I measured in vivo P02 during tumor growth and antiangiogenic (vascular targeted) treatment in preclinical tumor models. Nanotechnology is rapidly emerging as an important source of biocompatible tools that may shape the future of medical practice. Fluorescent semiconductor nanocrystals (NCs), also known as quantum dots, are a powerful tool for biological imaging, cellular targeting and molecular sensing.(cont.) I adapted novel fluorescence resonance energy transfer (FRET) -based nanocrystal (NC) biosensors for use with MPM to qualitatively measure in vivo extracellular pH in tumors at high-resolution. While intravital multiphoton microscopy demonstrates utility and adaptability in the study of cancer and response to therapy, the requisite high numerical aperture and exogenous contrast agents result in a limited capacity to investigate substantial tissue volumes or probe dynamic changes repeatedly over prolonged periods. By applying optical frequency domain imaging (OFDI) as an intravital microscopic tool, the technical limitations of multiphoton microscopy can be circumvented providing unprecedented access to previously unexplored, critically important aspects of tumor biology. Using entirely intrinsic mechanisms of contrast within murine tumor models, OFDI is able to simultaneously, rapidly, and repeatedly probe the microvasculature, lymphatic vessels, and tissue microstructure and composition over large volumes. Using OFDI-based techniques, measurements of tumor angiogenesis, lymphangiogenesis, tissue viability and both vascular and cellular responses to therapy were demonstrated, thereby highlighting the potential of OFDI to facilitate the exploration of pathophysiological processes and the evaluation of treatment strategies.by Ryan M. Lanning.Ph.D

Deformability-induced effects of red blood cells in flow

To ensure a proper health state in the human body, a steady transport of blood is necessary. As the main cellular constituent in the blood suspension, red blood cells (RBCs) are governing the physical properties of the entire blood flow. Remarkably, these RBCs can adapt their shape to the prevailing surrounding flow conditions, ultimately allowing them to pass through narrow capillaries smaller than their equilibrium diameter. However, several diseases such as diabetes mellitus or malaria are linked to an alteration of the deformability. In this work, we investigate the shapes of RBCs in microcapillary flow in vitro, culminating in a shape phase diagram of two distinct, hydrodynamically induced shapes, the croissant and the slipper. Due to the simplicity of the RBC structure, the obtained phase diagram leads to further insights into the complex interaction between deformable objects in general, such as vesicles, and the surrounding fluid. Furthermore, the phase diagram is highly correlated to the deformability of the RBCs and represents thus a cornerstone of a potential diagnostic tool to detect pathological blood parameters. To further promote this idea, we train a convolutional neural network (CNN) to classify the distinct RBC shapes. The benchmark of the CNN is validated by manual classification of the cellular shapes and yields very good performance. In the second part, we investigate an effect that is associated with the deformability of RBCs, the lingering phenomenon. Lingering events may occur at bifurcation apices and are characterized by a straddling of RBCs at an apex, which have been shown in silico to cause a piling up of subsequent RBCs. Here, we provide insight into the dynamics of such lingering events in vivo, which we consequently relate to the partitioning of RBCs at bifurcating vessels in the microvasculature. Specifically, the lingering of RBCs causes an increased intercellular distance to RBCs further downstream, and thus, a reduced hematocrit.Um die biologischen Funktionen im menschlichen Körper aufrechtzuerhalten ist eine stetige Versorgung mit Blut notwendig. Rote Blutzellen bilden den Hauptanteil aller zellulären Komponenten im Blut und beeinflussen somit maßgeblich dessen Fließeigenschaften. Eine bemerkenswerte Eigenschaft dieser roten Blutzellen ist ihre Deformierbarkeit, die es ihnen ermöglicht, ihre Form den vorherrschenden Strömungsbedingungen anzupassen und sogar durch Kapillaren zu strömen, deren Durchmesser kleiner ist als der Gleichgewichtsdurchmesser einer roten Blutzelle. Zahlreiche Erkrankungen wie beispielsweise Diabetes mellitus oder Malaria sind jedoch mit einer Veränderung dieser Deformierbarkeit verbunden. In der vorliegenden Arbeit untersuchen wir die hydrodynamisch induzierten Formen der roten Blutzellen in mikrokapillarer Strömung in vitro systematisch für verschiedene Fließgeschwindigkeiten. Aus diesen Daten erzeugen wir ein Phasendiagramm zweier charakteristischer auftretender Formen: dem Croissant und dem Slipper. Aufgrund der Einfachheit der Struktur der roten Blutzellen führt das erhaltene Phasendiagramm zu weiteren Erkenntnissen über die komplexe Interaktion zwischen deformierbaren Objekten im Allgemeinen, wie z.B. Vesikeln, und des sie umgebenden Fluids. Darüber hinaus ist das Phasendiagramm korreliert mit der Deformierbarkeit der Erythrozyten und stellt somit einen Eckpfeiler eines potentiellen Diagnosewerkzeugs zur Erkennung pathologischer Blutparameter dar. Um diese Idee weiter voranzutreiben, trainieren wir ein künstliches neuronales Netz, um die auftretenden Formen der Erythrozyten zu klassifizieren. Die Ausgabe dieses künstlichen neuronalen Netzes wird durch manuelle Klassifizierung der Zellformen validiert und weist eine sehr hohe Übereinstimmung mit dieser manuellen Klassifikation auf. Im zweiten Teil der Arbeit untersuchen wir einen Effekt, der sich direkt aus der Deformierbarkeit der roten Blutzellen ergibt, das Lingering-Phänomen. Diese Lingering-Ereignisse können an Bifurkationsscheiteln zweier benachbarter Kapillaren auftreten und sind durch ein längeres Verweilen von Erythrozyten an einem Scheitelpunkt gekennzeichnet. In Simulationen hat sich gezeigt, dass diese Dynamik eine Anhäufung von nachfolgenden roten Blutzellen verursacht. Wir analysieren die Dynamik solcher Verweilereignisse in vivo, die wir folglich mit der Aufteilung von Erythrozyten an sich gabelnden Gefäßen in der Mikrovaskulatur in Verbindung bringen. Insbesondere verursacht das Verweilen von Erythrozyten einen erhöhten interzellulären Abstand zu weiter stromabwärts liegenden Erythrozyten und damit einen reduzierten Hämatokrit

Study of Liver Surface Imaging Marker to Monitor Chronic Liver Disease Progression

Ph.DDOCTOR OF PHILOSOPH

Magnetic resonance imaging and the development of vascular targeted treatments for cancer.

The main subject of the work presented in this thesis is the further development of magnetic resonance imaging (MRI) as a non-invasive method of investigating tumour microcirculation. Two different MR techniques were used: dynamic contrast enhanced (DCE)-MRI and Blood Oxygen Level Dependent (BOLD)-MRI. Intravital microscopy was used to help interpret BOLD-MRI results. The ultimate aims were to determine whether MRI methods could be relied upon to define a drug as having vascular disrupting activity and to develop techniques to predict the effectiveness of vascular disruptive agents (VDA). In DCE-MRI, tissue enhancement is continuously monitored over several minutes after intravenous injection of contrast medium. Modelling of contrast agent kinetics generates quantitative parameters related to tissue blood flow rate and permeability, e.g. Ktrans (transfer constant). In a clinical study, patients had DCE-MRI examinations before and 24 hours after cytotoxic chemotherapy to establish whether any acute ami-vascular effects could be detected. No acute reductions in Ktrans were seen. In this project, the acute effects of the VDA, combretastatin A-4-phosphate, were investigated using DCE-MRI in SW1222 tumours in mice. Responses were seen both at a clinically relevant dose and at higher doses, and a dose-response relationship established. BOLD-MRI can detect changes in oxygenation and blood flow within tumours using deoxygenated haemoglobin as an intrinsic contrast agent. Tumours contain a variable proportion of immature vessels, which may explain differential sensitivity to VDAs. In this project, BOLD-MRI was used to assess tumour vessel maturity using consequent vasoreactivity to angiotensin II and carbon dioxide (as air-5%C02 or as carbogen) in an animal model. Intravital microscopy was used to directly observe response to these agents in mouse window chambers. Results suggest that response to vasoactive agents is useful for assessing vascular maturity in tumours but that more sensitive non-invasive imaging methods than BOLD-MRI are required for clinical use

Tumour-associated macrophages act as a slow-release reservoir of nano-therapeutic Pt(IV) pro-drug

Therapeutic nanoparticles (TNPs) aim to deliver drugs more safely and effectively to cancers, yet clinical results have been unpredictable owing to limited in vivo understanding. Here we use single-cell imaging of intratumoral TNP pharmacokinetics and pharmacodynamics to better comprehend their heterogeneous behaviour. Model TNPs comprising a fluorescent platinum(IV) pro-drug and a clinically tested polymer platform (PLGA-b-PEG) promote long drug circulation and alter accumulation by directing cellular uptake toward tumour-associated macrophages (TAMs). Simultaneous imaging of TNP vehicle, its drug payload and single-cell DNA damage response reveals that TAMs serve as a local drug depot that accumulates significant vehicle from which DNA-damaging Pt payload gradually releases to neighbouring tumour cells. Correspondingly, TAM depletion reduces intratumoral TNP accumulation and efficacy. Thus, nanotherapeutics co-opt TAMs for drug delivery, which has implications for TNP design and for selecting patients into trials.National Cancer Institute (U.S.) (Grant RO1-CA034992