archives@tulane.edu / 1 / Genevieve Mattei

Thesis (Ph.D.)--Drexel University, 2007. / Includes abstract and vita. Includes bibliographical references (leaves 70-74).

In an effort to develop an optical-acoustical understanding of ultrasound contrast agent microbubble dynamics in a micro-environment that resembles blood vessels, this thesis presents experimental work on optical trapping and acoustical probing of ultrasound contrast agent microbubbles confined in regenerated cellulose capillaries. First, we showed by acoustical means that the pressure threshold of an individual microbubble shell rupture increases significantly when confined in regenerated cellulose capillaries. We report that the shell rupture threshold in regenerated cellulose capillaries increased by at least 0.3 MPa from 0.8 MPa for unconfined microbubbles. Second, we achieved optical trapping and manipulation of ultrasound contrast agent microbubbles confined in capillaries using Hermite-Gaussian laser beams. / Graduation date: 2012

Purpose: To compare hemodynamic of malignant and benign SRMs on CT and CEUS. Method: Seventy biopsy proven SRM underwent CEUS. Sixty-three had CT. After injection of 0.2 ml of Definity, 3min and after 0.9 ml infusion, 30 sec of data were acquires. Lesion hemodynamics relative to the cortex was evaluated both qualitatively and quantitatively. Results: Considering 15 and 20 HU as enhancement threshold, 10% to 13% of patients did not enhance on CT, while all lesions enhanced on CEUS. Papillary RCCs showed hypovascularity with 100% specificity. In other RCCs, PI, WI slope 5 to45%, 50 to100%, 10 to 90%, WO slope 100 to 50%, 100 to 10%, WO intensity at peak+30 seconds were statistically higher than benign SRMs. Conclusion: All solid SRMs enhance on CEUS, while CT does not show vascularity in 10-13% of solid SRMs. CEUS can differentiate malignant from benign SRMs by evaluating their hemodynamics.

Purpose: To compare hemodynamic of malignant and benign SRMs on CT and CEUS. Method: Seventy biopsy proven SRM underwent CEUS. Sixty-three had CT. After injection of 0.2 ml of Definity, 3min and after 0.9 ml infusion, 30 sec of data were acquires. Lesion hemodynamics relative to the cortex was evaluated both qualitatively and quantitatively. Results: Considering 15 and 20 HU as enhancement threshold, 10% to 13% of patients did not enhance on CT, while all lesions enhanced on CEUS. Papillary RCCs showed hypovascularity with 100% specificity. In other RCCs, PI, WI slope 5 to45%, 50 to100%, 10 to 90%, WO slope 100 to 50%, 100 to 10%, WO intensity at peak+30 seconds were statistically higher than benign SRMs. Conclusion: All solid SRMs enhance on CEUS, while CT does not show vascularity in 10-13% of solid SRMs. CEUS can differentiate malignant from benign SRMs by evaluating their hemodynamics.

The utility of microbubble agents in a variety of diagnostic and therapeutic ultrasound techniques has been widely demonstrated, most notably in Contrast Enhanced Ultrasound (CEUS) imaging. Unfortunately, the underlying mechanisms of their response to ultrasound excitation are poorly understood, restricting the development of promising techniques, such as quantitative perfusion imaging. A significant reason for this is that current microbubble characterisation techniques suffer from one or more of the following limitations: i) large experimental uncertainties, ii) physical restrictions on microbubble response and iii) failure to provide large data sets suitable for statistical analysis. This thesis presents a new technique to overcome these limitations. A co-axial microfluidic device is used to hydrodynamically confine microbubbles through the focal region of a laser and ultrasound field. The magnitude of light scattered by isolated microbubbles during ultrasound excitation is converted to radius using Mie Scattering theory. This technique is capable of obtaining large samples (>10³/min) of microbubbles to be efficiently characterised. The response of a commercial contrast agent, SonoVue®, is first investigated for a range of ultrasound exposure parameters; frequency (2 MHz - 4.5 MHz), peak negative pressure (6 kPa - 400 kPa) and pulse length (3 cycles - 8 cycles). Second the device is used to investigate the effect of composition and fabrication on microbubble response to similar ultrasound conditions. The results demonstrate a very large variability in microbubble response independent of initial size, indicating a significant lack of uniformity of coating properties. This is further supported by quantitative fluorescence imaging and quasi-static pressure chamber measurements. The implications of the findings for CEUS imaging and the development of microbubble contrast agents are discussed, as well as the limitations and suggested improvements of the characterisation technique.

Les modifications induites par les cellules tumorales sur leur environnement ont pour but de permettre leur développement en remodelant le tissu la soutenant et en créant un nouveau réseau vasculaire (angiogenèse). Plusieurs thérapies anti-angiogéniques inhibant le développement du réseau vasculaire tumoral ont obtenu l’autorisation de mise sur le marché et sont actuellement utilisées en clinique. Ces thérapies induisent de fortes modifications fonctionnelles au sein de la tumeur mais le simple suivi de l’évolution du volume tumoral n’est pas suffisant pour rendre compte de ces modifications. L’objectif principal de la thèse a consisté à utiliser différentes modalités d’imagerie ultrasonore afin d’évaluer leur sensibilité aux modifications générées dans des tumeurs murine (carcinome colorectal et pulmonaire) au cours de plusieurs types de thérapie (chimique : cytotoxique, anti-angiogénique / physique : plasma froid, sono-sensibilisation). Les modifications de la distribution spatiale des micro-vaisseaux et leur fonctionnalité ont été caractérisées à l’aide de l’imagerie de contraste ultrasonore (CEUS), l’altération de la microstructure de la tumeur a été évaluée grâce à l’analyse spectrale des signaux radiofréquences, connu comme « quantitative ultrasound » (QUS) et enfin les variations des propriétés mécaniques des tissus tumoraux ont été mesurées en élastographie à l’aide de la technique « Shear Wave Elastography » (SWE). Afin de comprendre l’origine des modifications observées in vivo, des paramètres standard comme les niveaux de fibrose ou de nécrose ont été caractérisés ex vivo dans le tissu tumoral, grâce à l’immunohistochimie, une technique de référence. / Tumor development is complex process made possible thanks to the microenvironment surrounding tumor cell. Modifications induced by tumor cells on their environment enable their own development by remodeling tissues sustaining them and by creating a new vascular network (angiogenesis). The use of several antiangiogenic therapies, inhibiting the sprout of a new vascular network, has been authorized in clinic. These therapies induce strong modifications in tumors at the functional level and following tumor size changes are is not sufficient to fully characterize tumor modifications. The main goal of this thesis was to use different ultrasound-based imaging modalities in order to assess their sensitivity to modifications induced in murine tumor model (colorectal and lung carcinomas) during different type of therapy (chemical: cytotoxic, antiangiogenic / physical: cold plasma, sonosensitization). Modifications of the spatial distribution of microvessels and their functionality were characterized using contrast-enhanced ultrasound (CEUS), alteration of tumor microstructure was assessed using spectral analysis of radiofrequency signal, known as quantitative ultrasound (QUS) and finally variations of mechanical properties in tumor tissues were measured in shear wave elastography (SWE). In order to better understand the origin of the modifications observed in vivo, standard parameters such as level of fibrosis and necrosis were characterize ex vivo in tumor tissue using immunochemistry as gold standard.

La quantification de la perfusion tissulaire à partir de données dynamiques d’échographie de contraste repose sur une modélisation appropriée de la cinétique de la concentration en agent de contraste dans le tissu étudié. De nombreux facteurs, expérimentaux ou physiologiques, rendent la comparaison inter ou intra-individu de ces paramètres de perfusion difficile. Dans cette thèse, la reproductibilité et la comparaison de différentes méthodes de quantification ont été étudiées dans le cadre d’une étude préclinique de test-retest et sur des simulations numériques. Les méthodes étudiées ont été : le modèle log-normal, le modèle compartimental avec fonction d’entrée et le modèle compartimental avec tissu de référence. Les études précliniques ont montré la difficulté d’estimation d’une fonction d’entrée artérielle et la nécessité de corriger localement le temps d’arrivée de l’agent de contraste dans le tissu pour que l’approximation des cinétiques expérimentales par le modèle soit de qualité suffisante. Une estimation linéaire sous contrainte des paramètres du modèle compartimental avec tissu de référence tirant profit de différentes zones d’intérêt dans l’image a été ensuite proposée pour obtenir à l’échelle régionale et/ou locale des valeurs relatives cohérentes de débit sanguin tissulaire et de volume sanguin tissulaire, exprimées par rapport aux valeurs dans le tissu de référence. Il a été montré que cette approche est la plus robuste et la plus reproductible. L’influence des facteurs tels que la durée d’acquisition, la fréquence d’échantillonnage, le nombre de régions utilisées et l’amplitude du bruit a été étudiée sur des simulations et a permis de formuler des recommandations pour l’acquisition et le traitement des études en échographie de contraste. / Quantification of tissue perfusion from dynamic contrast-enhanced ultrasound data relies on appropriate modeling of the curve representing the evolution of the contrast-agent concentration inside the studied tissue. Many factors, experimental or physiological, make inter-subject or intra-subject comparison of these perfusion parameters difficult. In this thesis, the reproducibility and the comparison of various quantification methods was investigated through preclinical test-retest experiments and through simulations. The investigated methods were: the log-normal model, the one-compartment model using an arterial input function, and the one-compartment model using a reference tissue. The preclinical experiments revealed the difficulty to estimate an arterial input function directly from the image, as well as the necessity to locally correct for the time of arrival of the contrast agent in the tissue in order to ensure the model accurately fits the experimental enhancement curves. A regularized linear estimation of the parameters of the one-compartment model using a reference tissue taking advantage of multiple tissue regions was then proposed to obtain homogeneous relative values of the tissue blood flow and tissue blood volume, expressed relatively to the parameter value inside the reference tissue. The improved robustness and reproducibility of the method was demonstrated. The influence of factors such as the exam duration, the sampling frequency, the number of tissue regions in the analysis, and the noise amplitude were investigated through simulations, and allowed us to formulate recommendations regarding the acquisition and the analysis of contrast-enhanced ultrasound studies.

The harmonic generation due to the nonlinear behavior of Ultrasound Contrast Agents (UCAs) must be exploited for improved efficiency when imaging vascular targets in the neighborhood of highly echogenic tissue. One may even further improve the efficiency by focusing on the subharmonic generation of the UCAs, which is an even more exclusive property than the generation of higher harmonics, for improved Contrast-to-Tissue ratio (CTR). The aim of this work was first, the design of a set-up for nonlinear imaging of Poly-Vinyl Alcohol (PVA) based UCAs on The Verasonics Research System with special focus on nondestructive Subharmonic Imaging. The second part of the work addressed the evaluation of the subharmonic response provided by the agents in the developed setup. Six different imaging techniques were developed. These were Fundamental B-mode imaging (FB), Pulse Inversion imaging (PI), and a Contrast Pulse Sequence based on three pulses (CPS3), with and without a focus on the subharmonic component by the implementation of a Linear Bandpass Filter (LBF). Experiments were performed on a tissue mimicking flow phantom and the performance of the agents for each technique was determined in terms of CTR and CNR. The PVA agents provided a backscattering enhancement of the order of 23 dB through FB imaging. However, the performance of the FB technique was unsatisfactory in terms of CTR. The CPS3 sequence performed best of the six techniques with an improvement of 14 dB and 13 dB in CTR and CNR, respectively, compared with the FB technique. Combining the LBF around the subharmonic component with the multi-pulse techniques of PI and CPS3 resulted in a degraded CTR performance due to significant amount of signals from tissue around the subharmonic component and insufficient subharmonic detection from the PVA agents.

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