Atherosclerosis is a chronic inflammatory disease of arterial blood vessels, characterized by deposition of lipoproteins in the arterial wall. Atherosclerotic plaques form preferentially in distinct regions of the vasculature such as branch points, curvatures and bifurcations, suggesting that local hemodynamic forces may contribute to disease susceptibility. Shear stress imparted on endothelial cells (ECs) by the flowing blood has been shown to modulate gene expression and remodelling of the artery. In this thesis, an in vitro model was established to recreate the contrasting environments found in atherosclerosis-prone and atherosclerosis-resistant regions of the vasculature to demonstrate a direct causal-relationship between shear stress and expression of endothelial nitric oxide synthase (eNOS) and p65 in ECs. In vitro assessment of cell shape and expression patterns of these anti- and atherogenic genes demonstrated that shear stress can induce cell morphology and gene expression patterns that are similar to ECs in atherosclerosis-prone and atherosclerosis-resistant regions of the mouse vasculature. Regulation of eNOS transcription by shear stress was demonstrated using a transgenic mouse model and in vitro heterogeneous nuclear RNA (hnRNA) quantification. Similar to ECs in atherosclerosis-prone regions, epithelial cells lining the small intestine lumen express high levels of p65. To investigate the effects of p65 expression levels on constitutive and tumour necrosis factor α (TNFα)-induced nuclear factor-κB (NF-κB) signalling, p65 expression was suppressed in HeLa cells by RNA interference. Lower p65 expression resulted in reduced TNFα-induced expression of NF-κB target genes, including many subunits of inhibitor of nuclear factor κB (IκB), demonstrating modulation of NF-κB priming by p65 expression levels. Suppression of p65 also affected constitutive expression levels of IκB, and resulted in re-setting of the NF-κB/IκB equilibrium. Experiments using inhibitors of canonical NF-κB signalling found that basal expression of NF-κB components is independent of nuclear factor κB kinase β (IKKβ) activity and proteasome-mediated degradation of IκBα. Together, these studies elucidate the mechanism of flow-mediated gene regulation and the effect of resulting changes in p65 expression on NF-κB signalling.

In the adult brain, localized increases in neural activity almost always result in increases in local blood flow, a relationship essential for normal brain function. This coupling between neural activity and blood flow provides the basis for many neuroimaging techniques including functional magnetic resonance imaging (fMRI) and near-infrared spectroscopy (NIRS). However, functional brain imaging studies in newborns and children have detected a range of responses, including some entirely inverted with respect to those of the adult. Confusion over the properties of functional hemodynamics in the developing brain has made it challenging to interpret functional imaging data in infants and children. Additionally, developmental differences in functional hemodynamics would suggest postnatal neurovascular maturation and a unique metabolic environment in the developing brain. This thesis begins with a series of studies in which I tracked and characterized postnatal changes in functional hemodynamics in rodent models utilizing high-speed, high-resolution multi-spectral optical intrinsic and fluorescent signal imaging. I demonstrated that in early postnatal development increases in cortical blood flow do not occur in response to somatosensory stimulation. In fact, I observed stimulus-linked global vasoconstrictions in the brain. In slightly older age groups, I observed biphasic hemodynamic responses, with initial local hyperemia followed by global vasoconstriction, eventually progressing with age to recognizable adult-like hemodynamic responses. In these studies, I also found that the postnatal development of autoregulation is a potential confound in the study of early functional activation, and may account for some of the variability seen in prior human studies. Charting this progression led to the hypothesis that anomalous functional responses observed in human subjects are due to the postnatal development of neurovascular coupling itself. To directly assess neurovascular development, I performed a further set of studies in Thy1-GCaMP3 mice, permitting simultaneous observation of the development of neural function and connectivity along with functional hemodynamics. My results demonstrate that the spatiotemporal properties of neural development do not predict observed changes in the hemodynamic response, consistent with the parallel development of neural networks and neurovascular coupling. Confirming the presence of vascularly-uncoupled neural activity in the newborn brain led me to question how the brain supports its energy needs in the absence of evoked hyperemia, prompting the exploration of the potential metabolic bases and consequences of developmental changes in neurovascular coupling. Finally, I explore the cellular and vascular morphological and functional correlates of functional neurovascular development. My results confirm that neurovascular development occurs postnatally, which has critical implications for the interpretation of functional imaging studies in infants and children. My work also provides new insights into postnatal neural, metabolic, and vascular maturation and could have important implications for the care of infants and children, and for understanding the role of neurovascular development in the pathophysiology of developmental disorders.

Local neuronal activity in the brain results in increased blood flow and is called neurovascular coupling. Such blood flow changes result in the blood-oxygen level dependent (BOLD) fluctuations detectable by functional magnetic resonance imaging (fMRI). The hemodynamic response is also an essential component of brain health and is impaired in various models of cognitive dysfunction. However, we still do not understand why functional hyperemia in the brain is important. To understand this question, various groups have studied brain metabolic activity as well as the mechanisms underlying neurovascular coupling. Over the years, several cell types have been proposed to contribute to functional hyperemia in the brain, including neurons, astrocytes and pericytes. However, the picture remains incomplete – controversies abound regarding the exact role of astrocytes, and pericytes in neurovascular coupling. Our lab has studies the mechanisms of neurovascular coupling from a mesoscopic perspective, as vasodilation in the rodent cortex involves capillaries and diving arterioles in the brain parenchyma as well as surface vasculature in the brain. We proposed that the vascular endothelium itself might provide a continuous conduit for transmitting vasodilatory signals initiated at the capillary level due to local neuronal activity. Given that systemic endothelial dysfunction could contribute to decreased neurovascular function, this hypothesis raised important concerns regarding endothelial vulnerabilities in common diseases like hypertension and diabetes and its role in diminished cognitive function and neurodegeneration. Based on findings from vascular research in other organ systems, we hypothesized that two distinct mechanisms of endothelium-derived vasodilation significantly contribute to neurovascular coupling the brain. These two mechanisms were expected to consist of fast long-range endothelium-derived hyperpolarization (EDH) dependent vasodilation (conducted vasodilation) and slower, more localized endothelium calcium-wave dependent vasodilation (propagated vasodilation). Together, we expected these mechanisms to shape the spatio-temporal evolution of hemodynamic responses in the brain. This dual mechanism of endothelial control of the hyperemic response in the brain might explain the complex spatiotemporal properties and non-linearities of the fMRI blood oxygen level dependent (BOLD) signal. My initial experiments were conducted in anesthetized rats, where I pharmacologically inhibited endothelial dependent vasodilation during functional hyperemia in the somatosensory cortex under a hind-paw electrical stimulus paradigm. While the results gleaned from these experiments were very revealing, it was important to consider the effect of the pharmacological manipulations on neuronal activity in the brain. In addition, neurovascular coupling and overall brain blood flow in anesthetized animals is dramatically altered when compared to awake animals. In order to accomplish these goals, I built a wide-field optical imaging system that could simultaneously measure fluorescence-based neuronal activity and reflectance-based hemodynamic activity in awake head-restrained mice. I then used non-blood brain barrier permeable pharmacology to study endothelial mechanisms of neurovascular coupling in awake Thy1-GCaMP6f mice, which express the calcium fluorophore in a subset of excitatory neurons in the cortex. I found that using this pharmacology I could dissect out the hypothesized two spatiotemporally distinct components of whisker-stimulus evoked neurovascular coupling in awake mice. With simultaneous recording of the neuronal activity driving this blood flow, I was able to build a mathematical model for neurovascular coupling that accounted for these two mechanisms by allowing for the superposition of a time-invariant, constant hemodynamic response with a hemodynamic response obtained by convolving the underlying neuronal response with a hemodynamic response function (HRF). I was able to linearize these apparent non-linearities in the hemodynamic response by studying the properties of deconvolved HRFs for stimuli of different durations before and after pharmacological manipulation of endothelial activity. Two important considerations remain. Firstly, our wide-field, mesoscopic view of the brain prevents observations of endothelial function (hyperpolarization and calcium activity) and the propagation dynamics of dilation best observed at the microscopic level. To accomplish this task, ongoing experiments currently use our high-speed volumetric imaging technology (SCAPE – Swept Confocally Aligned Planar Excitation microscopy) to study stimulus-evoked vascular dynamics in mouse lines expressing GFP and GCaMP8 in endothelial cells. Secondly, our longitudinal imaging of these animals is ideal for studying the acute and long-term effects of endothelial dysfunction on cognitive function. This requires adequate study of changes in mouse behavior during manipulations of endothelial function longitudinally in awake mice. Future experiments should involve the development and implementation of appropriate task-based behavior experiments, and analysis methods for more carefully exploring changes in neuronal activity in the mouse brain during stimulus and non-stimulus dependent activity.

Estudo elaborado para a investigação hemodinâmica das artérias alveolar inferior e mentual, relacionada com pacientes idosos edêntulos (totais e parciais) e dentados (com a presença do primeiro molar inferior bilateral), insento de doença oral e sistêmica, por meio da ultrassonografia modo B e Doppler, como forma de exame complementar. Por tratar-se de um método de diagnóstico preciso e confiável de visualização por meio de imagens, em tempo real, prontamente disponível, não invasivo, relativamente de baixo custo, inócuo, é uma importante ferramenta na avaliação de possíveis alterações da vascularização de diversos segmentos corpóreos, influenciando nas decisões clínicas, aumentando a precisão do diagnóstico complementar. Com o emprego do transdutor linear e endocavitário, de maneira intraoral, em 30 pacientes especificados acima, sendo 15 de cada grupo, foi mensurado as seguintes variáveis: IRV (índice de resistência vascular) o qual não sofreu efeitos de interação com lado (D e E), grupos (edêntulos totais e dentados) e artérias (AAI e AM), porém o ID (índice do diâmetro) e o VPS (velocidade de pico sistólico) apresentaram uma forte relação, isto significou que quando o valor de VPS aumentava no vaso estudado, o ID diminuía, ou seja a VPS e o ID se compensavam para manter a hemodinâmica constante, independentemente do lado (D e E) e do grupo (edêntulos e dentados), variando somente de artéria para artéria (AAI e AM). / Study prepared for the inferior alveolar artery hemodynamic investigation and mentual, related to edentulous and dentate elderly oral pathology free through ultrasonography and Doppler mode B, as a form of additional examination, because it is an accurate and reliable method of real time preview, readily available, non-invasive, relatively low-cost, innocuous, making it an important tool in the evaluation of possible changes in vascularity of various body segments corporals\' influencing clinical decisions by increasing the accuracy of diagnosis. With the use of linear array transducer and endocavity, of 30 patients specified above, being 15 from each group, in a way, intraoral were measured the following variables: VRI (vascular resistance index) which did not suffer the effects of interaction with side (R and L), groups (toothed and edentulous) and arteries (AAI and AM), however the ID (index of the diameter) and PSV (peak systolic velocity) showed a strong relationship, this meant that when the PSV value increased in the studied vessel, the ID decreased, i.e. the PSV and the ID they were compensating for to maintain constant hemodynamic, regardless of the side (R and L) and group (edentulous and serrated), varying only the artery to artery (AAI and AM).

Estudo elaborado para a investigação hemodinâmica das artérias alveolar inferior e mentual, relacionada com pacientes idosos edêntulos (totais e parciais) e dentados (com a presença do primeiro molar inferior bilateral), insento de doença oral e sistêmica, por meio da ultrassonografia modo B e Doppler, como forma de exame complementar. Por tratar-se de um método de diagnóstico preciso e confiável de visualização por meio de imagens, em tempo real, prontamente disponível, não invasivo, relativamente de baixo custo, inócuo, é uma importante ferramenta na avaliação de possíveis alterações da vascularização de diversos segmentos corpóreos, influenciando nas decisões clínicas, aumentando a precisão do diagnóstico complementar. Com o emprego do transdutor linear e endocavitário, de maneira intraoral, em 30 pacientes especificados acima, sendo 15 de cada grupo, foi mensurado as seguintes variáveis: IRV (índice de resistência vascular) o qual não sofreu efeitos de interação com lado (D e E), grupos (edêntulos totais e dentados) e artérias (AAI e AM), porém o ID (índice do diâmetro) e o VPS (velocidade de pico sistólico) apresentaram uma forte relação, isto significou que quando o valor de VPS aumentava no vaso estudado, o ID diminuía, ou seja a VPS e o ID se compensavam para manter a hemodinâmica constante, independentemente do lado (D e E) e do grupo (edêntulos e dentados), variando somente de artéria para artéria (AAI e AM). / Study prepared for the inferior alveolar artery hemodynamic investigation and mentual, related to edentulous and dentate elderly oral pathology free through ultrasonography and Doppler mode B, as a form of additional examination, because it is an accurate and reliable method of real time preview, readily available, non-invasive, relatively low-cost, innocuous, making it an important tool in the evaluation of possible changes in vascularity of various body segments corporals\' influencing clinical decisions by increasing the accuracy of diagnosis. With the use of linear array transducer and endocavity, of 30 patients specified above, being 15 from each group, in a way, intraoral were measured the following variables: VRI (vascular resistance index) which did not suffer the effects of interaction with side (R and L), groups (toothed and edentulous) and arteries (AAI and AM), however the ID (index of the diameter) and PSV (peak systolic velocity) showed a strong relationship, this meant that when the PSV value increased in the studied vessel, the ID decreased, i.e. the PSV and the ID they were compensating for to maintain constant hemodynamic, regardless of the side (R and L) and group (edentulous and serrated), varying only the artery to artery (AAI and AM).

There is increasing interest in assessing the role of hemodynamics in aneurysm growth and rupture mechanism. Identification of the indicators of rupture risk can prove very valuable in the clinical management of patients. If rupture risk of aneurysms can be predicted, immediate preemptive treatments can be done for the high risk patients whereas others can avoid the risky intervention. Retrospective studies have been performed in the past to filter out indices that differentiate ruptured aneurysms from unruptured aneurysms. However, these differences may not necessarily translate to differences between aneurysms that present unruptured but fork towards growth/rupture and unruptured aneurysms that are invariably stable. The hypothesis of the present study is that hemodynamic indices of unruptured aneurysms when they first presented can be used to predict their longitudinal outcome. A prospective longitudinal cohort study was designed to test this hypothesis. Four clinical centers participated in this study and a total of 198 aneurysms were recruited. These aneurysms were chosen by the physicians to be kept under watchful waiting. Three-dimensional models of aneurysms and their contiguous vasculature generated using the initial scans of patients were used for computational fluid dynamic (CFD) simulations. Both pulsatile and steady flow analyses were performed for each patient. By collating all the prominent hemodynamic indices available in aneurysm literature and developing a few new indices, 25 hemodynamic indices were estimated for each subject. For statistical analysis, it was hypothesized a priori that low wall shear area is different between stable and unstable aneurysms. All other indices were tested in a post-hoc manner. The longitudinal outcome information of these patients was recorded at the clinical centers and the author was blinded until all analyses were complete. Aneurysms that grew during the follow up period were labeled as "grown" and otherwise they were called "stable" by the radiologists. After the hemodynamic analysis was complete, a non-parametric Mann Whitney U test was performed to determine if any index can statistically differentiate the two groups ("grown" versus "stable"). It was found that none of the indices distinguished the two groups with statistical significance. Comparison of the steady and pulsatile flow analysis suggested that the patient population is stratified in the same order by an index, irrespective of whether the index is computed using a steady or pulsatile flow simulation. Pearson correlation coefficient was obtained between basic geometric indices and hemodynamic indices of this population. No strong correlation was found in between morphology and hemodynamics, suggesting uniqueness of the hemodynamic indices. The hypothesis motivating the present study is that aneurysm blood flow based indices can be used as prognostic indicators of growth and/or rupture risk. This study is the first to analyze intracranial aneurysm hemodynamics of a large cohort in a longitudinal prospective manner. Results of the present study indicate that quantitative hemodynamics cannot be used to predict the longitudinal outcome of an aneurysm. Further studies are needed to gain additional clinical insights.

Cardiac output Increases by 30-50% during mammal Ian pregnancy. This Increase Is reflected by elevation In both heart rate and stroke volume. The primary mechanism of Increased stroke volume appears to be cardiac enlargement, rather than increased preload, afterload, or contractility. Animal studies have shown that enlargement of the heart occurs prior to an Increase In uterine blood flow during pregnancy and this type of enlargement can be mimicked by sex steroid administration. Systemic vascular resistance greatly decreases during pregnancy and with sex steroid administration. It has been postulated that systemic vascular resistance may be a signal for heart size changes. This study attempted to chronically decrease systemic vascular resistance by administration of an arterial vasodllator (hydralazlne) over a three week period to guinea pigs. At the time of study hemodynamlcs were measured which Included, heart rate, arterial pressure, right atrial pressure and cardiac output. In vitro left ventricular pressure volume relationships were also evaluated, as was total plasma volume.

Arterial pulse pressure is critically important to a number of diseases such as isolated systolic hypertension, coronary artery disease and heart failure. Determining the cause of increased pulse pressure has been hampered for two reasons. First, pulse pressure results from contraction of the heart and the load formed by the complex arterial tree. Pressure pulses travel from the heart to the peripheral arteries. As they reach a bifurcation or change in arterial wall properties, some of the pulses get reflected and propagate retrograde towards the heart. Second, two different modeling approaches (0-D and 1-D) describe the arterial system. The Windkessel model ascribed changes in pulse pressure to changes in total arterial compliance (Ctot) and total arterial resistance, whereas the transmission model ascribed them to changes in the magnitude, timing and sites of reflection. Our investigation has addressed both these limitations by finding that a complex arterial system degenerates into a simple 2-element Windkessel model when wavelength of the propagated pulse increases. This theoretical development has yielded three practical results. First, isolated systolic hypertension can be viewed as a manifestation of a system that has degenerated into a Windkessel, and thus increased pulse pressure is due to decreased Ctot. Second, the well-discussed Augmentation Index does not truly describe augmentation of pulse pressure by pulse reflection. Third, the simple 2-element Windkessel can be used to characterize the interaction among heart, arterial system and axial-flow left ventricular assist device analytically. The fact that arterial systems degenerate into Windkessels explains why it becomes much easier to estimate total arterial compliance in hypertension?total arterial compliance is the dominant determinant of pulsatile pressure.

Each year, more than one million prosthetic vascular grafts are implanted. Well-over 50 % of these artificial vessels are of the small caliber variety with an inner diameter less than or equal to 10 mm. The challenge rests in implanting these synthetic substitutes into a hemodynamic environment with a high downstream resistance and low rates of flow. Over the course of four interrelated studies, we investigated the healing properties of small caliber prosthetic vascular grafts. All of these studies were conducted using baboons. First, we documented the difference in healing response between three different types of vascular grafts: (1) autologous artery (2) allogeneic vessel (3) prosthetic ePTFE. This comparison furnished an important model of graft healing. Proliferating endothelial cells were localized to the top 10 % of the neointima, while the proliferating smooth muscle cells were identified within the lower 10 % of the neointima. Secondly, we examined the effects of changing a prosthetic grafts material properties and how that change impacts healing of the grafts surface. These ultrastructural changes were introduced by radially stretching a porous 60 mm ePTFE vascular graft. Radially stretching the graft material decreased the void fraction, reduced the potential for transmural ingrowth, and changed the healing characteristics of the implanted vessels. Thirdly, we investigated the effect of a changing hemodynamic environment upon the healing of a vascular graft with uniform material properties. The changing hemodynamics were generated with a stenotic model. Under sub-acute conditions, an inverse relationship failed to exist between intimal thickening and wall shear stress. Lastly, the details of this hemodynamic environment were documented with computational fluid dynamics (CFD). The computational grids were constructed using three sets of geometric information: (1) incorporating the ideal material dimensions of the implanted vessel (2) utilizing contour information from pressure-perfused histologic cross-sections (3) applying geometric information form detailed MRI imaging. MRI imaging information provided the best description of the vessels hemodynamic environment. With this computational information, correlations were made between the intimal thickening and hemodynamic parameters.

Current designs of bileaflet mechanical heart valves (BMHVs) and trileaflet polymeric heart valves(TPHVs) are plagued by unacceptable levels of hemolysis and thrombus formation in critical areas thereby producing mediocre clinical performance. The objective of this study is: (1) to investigate the influence of BMHV designs on hinge flow characteristics, (2) to quantify the influence of hinge gap width tolerance in a BMHV design, and (3) to investigate the influence of TPHV design on flow characteristics. St. Jude Medical (SJM) provided four transparent mitral BMHVs: one 23 mm CarboMedics (CM), one 27 mm SJM Standard and two 27 mm prototype BHMVs with altered hinge gap widths. Aortech Inc. provided three 23 mm aortic prototype TPHVs. Laser Doppler velocimetry and Particle Image velocimetry were used to measure flow velocity inside these valve prostheses. The flows through the valves were maintained within physiological limits. All valves revealed Reynolds shear stress (RSS) levels greater than 200 Pa far exceeding the threshold for platelet activation and hemolysis. MHV hinge flows in the mitral position were characterized by a strong recirculation during ventricular diastole while leakage jets over and adjacent to leaflets were prominent during ventricular systole. CM hinge flow had higher RSS than in the SJM hinge. The large gap width hinge had the largest leakage jet size and highest RSS (>400 Pa) during ventricular diastole. The Standard gap width hinge showed better washout during systole and provided optimum hemodynamic performance than the prototype designs. In aortic prototype PHVs, elevated RSS conducive to hemolysis was observed along the central jet during systole and the leakage jet at the high central region inside the valve during diastole. This study showed that hinge geometry designs and hinge gap width tolerance governed the success of the bileaflet MHV design. Also the performance of the three aortic PHVs is dependent on commissural designs and leaflet thicknesses. Owing to the critical nature flow fields on clinical outcomes studies such as the current study should be conducted in the pre-clinical evaluation phase for all new MHV or PHVs.