Search

In vivo acquisition of endothelial wall shear stress requires instantaneous depth-resolved whole-field pulsatile flow profile measurements in microcirculation. High-accuracy, quantitative and non-invasive velocimetry techniques are essential for emerging real-time mechano-genomic investigations. To address these research needs, a novel biological flow quantification technique, OCT-μPIV, was developed utilizing high-speed optical coherence tomography (OCT) integrated with microscopic Particle Image Velocimetry (μPIV). This technique offers the unique advantage of simultaneously acquiring blood flow profiles and vessel anatomy along arbitrarily oriented sagittal planes. The process is instantaneous and enables real-time 3D flow reconstruction without the need for computationally intensive image processing compared to state-of-the-art velocimetry techniques. To evaluate the line-scanning direction and speed, four sets of parametric synthetic OCT-μPIV data were generated using an in-house code. Based on this investigation, an in vitro experiment was designed at the fastest scan speed while preserving the region of interest providing the depth-resolved velocity profiles spanning across the width of a micro-fabricated channel. High-agreement with the analytical flow profiles was achieved for different flow rates and seed particle types and sizes. Finally, by employing blood cells as non-invasive seeding particles, in vivo embryonic vascular velocity profiles in multiple vessels were measured in the early chick embryo. The pulsatile flow frequency and peak velocity measurements were also acquired with OCT-μPIV, which agreed well with previous reported values. These results demonstrate the potential utility of this technique to conduct practical microfluidic and non-invasive in vivo studies for embryonic blood flows.

Congenital heart disease (CHD) has the highest incidence and mortality rate of all birth defects in the U.S., occurring in at least 8 of every 1000 live births, and accounting for more than 24% of birth defect related infant deaths. Clinical and experimental data indicate that the hemodynamic environment has a significant role in the etiology of CHD, motivating the development of fetal valvuloplasty to reverse the progression of heart defects in utero. The efficacy of this intervention relies heavily on understanding the biomechanical relationships between blood flow and cardiovascular growth and remodeling. This thesis describes several studies using the chick embryo model designed to quantify vascular morphogenesis in vivo and link the observed trends to hemodynamic forces such as wall shear stress (WSS). Morphogenesis of the embryonic aortic arches, a series of bilaterally paired vessels surrounding the foregut and the precursors to the great vessels, was investigated using fluorescent dye injection and optical coherence tomography imaging. These experimental results were combined with computational fluid dynamics models of flow through the aortic arches, which revealed a correlation between transitioning aortic arch patterns and acute increases in WSS. Additionally, a regression analysis found a strong polynomial relationship between luminal aortic arch growth and deviations in WSS. Transformations of the aortic arches were further investigated using a computational optimization-based growth model. The model demonstrated that selection of the adult single aortic arch was influenced by the rotation of the outflow tract of the heart. The principle of minimum work, combined with this model, accurately predicted the transformation to a single aortic arch configuration. In order to support predictive computational models, quantitative data of vascular growth is required. Morphogenesis of an embryonic vitelline artery was tracked using a time-lapse, long-term optical coherence tomography based imaging system. Global and local growth of the artery was quantitatively analyzed at high spatial and temporal resolution. Finally, a model of hypoplastic left heart syndrome in the chick embryo was used to determine alterations in intracardiac flow patterns that may lead to the progression of this defect. Out of three venous injection sites, two shifted their flow pattern significantly. This change was observed soon after the intervention to generate the defect was performed, suggesting that flow disruption is an early insult leading to hypoplastic left heart syndrome. Together, these studies support the importance of the hemodynamic environment in determining vascular morphogenesis in the embryo. The combined experimental and computational approach provided new quantitative data of embryonic vascular morphogenesis. The results of these studies and the methods established in this thesis lay the foundation for future research on the biomechanics of cardiovascular development.

Microparticle image velocimetry (μPIV) is an evolving quantitative methodology to closely and accurately monitor the cardiac flow dynamics and mechanotransduction during vascular morphogenesis. While PIV technique has a long history, contemporary developments in advanced microscopy have significantly expanded its power. This chapter includes three new methods for μPIV acquisition in selected embryonic structures achieved through advanced optical imaging: (1) high-speed confocal scanning of transgenic zebrafish embryos, where the transgenic erythrocytes act as the tracing particles; (2) microinjection of artificial seeding particles in chick embryos visualized with stereomicroscopy; and (3) real-time, time-resolved optical coherence tomography acquisition of vitelline vessel flow profiles in chick embryos, tracking the erythrocytes.

BACKGROUND: Hypoplastic left heart syndrome (HLHS) is a major human congenital heart defect that results in single ventricle physiology and high mortality. Clinical data indicate that intracardiac blood flow patterns during cardiac morphogenesis are a significant etiology. We used the left atrial ligation (LAL) model in the chick embryo to test the hypothesis that LAL immediately alters intracardiac flow streams and the biomechanical environment, preceding morphologic and structural defects observed in HLHS. RESULTS: Using fluorescent dye injections, we found that intracardiac flow patterns from the right common cardinal vein, right vitelline vein, and left vitelline vein were altered immediately following LAL. Furthermore, we quantified a significant ventral shift of the right common cardinal and right vitelline vein flow streams. We developed an in silico model of LAL, which revealed that wall shear stress was reduced at the left atrioventricular canal and left side of the common ventricle. CONCLUSIONS: Our results demonstrate that intracardiac flow patterns change immediately following LAL, supporting the role of hemodynamics in the progression of HLHS. Sites of reduced WSS revealed by computational modeling are commonly affected in HLHS, suggesting that changes in the biomechanical environment may lead to abnormal growth and remodeling of left heart structures.

The Fontan procedure is employed as the final-stage palliation in single-ventricle congenital heart patients and results in diversion of venous blood directly to the pulmonary arteries. Fontan patients have been known to suffer from postoperative systemic venous hypertension, which in turn is associated with pleural effusions and protein losing enteropathy, leading to a decreased duration and quality of life. Despite the ongoing debate on its benefits, a circular fenestration hole (typically 4 mm) establishing a venous shunt to the common atrium is traditionally employed to relieve venous pressure in the Fontan conduit and improve early postoperative Fontan hemodynamics. However, these improvements come at the cost of reduced oxygen saturation due to excessive right-to-left shunting if the fenestration is permanent. The ideal "selective" fenestration would therefore limit or eliminate shunt flow at tolerable systemic venous pressures and allow increased flow at high pressures. The objective of this study is to introduce new fenestration designs that exhibit these desirable pressure-flow characteristics. Novel plus-shaped and S-shaped fenestration designs with leaflets are introduced as alternatives to the traditional circular fenestration, each having identical effective orifice areas at the fully open states. In vitro steady leakage flow tests were performed for physiological flow-driving pressures in order to obtain pressure-drop versus flow-rate characteristics. In addition, the leaflet opening kinematics of the plus-shaped fenestration was investigated computationally using finite element simulation. Fluid-structure interaction analysis was performed to determine leaflet displacements and pressure-flow characteristics at low pressures. Further, a lumped parameter model of the single-ventricle circuit was created to simulate pulsatile flow conditions For the plus-shaped fenestration, the flow rate was found to increase nonlinearly with increased driving systemic venous pressures at high physiological-pressure drops which did not cause the leaflets to fully open, and linearly for low driving pressures. These results indicate that leaflets of the plus-shaped fenestration design activated passively after a critical systemic venous pressure threshold. This feature is ideal for minimizing undesirable excessive venous shunting. A large variability in shunting flow rate may be obtained by changing the shape, thickness, size, and material of the fenestration to suit requirements of the patient, which can further limit shunt flow in a controlled manner.

Embryonic aortic arches (AA) are initially bilaterally paired, transitional vessels and failures in remodeling based on hemodynamic and growth-related adaptations cause a spectrum of congenital heart disease (CHD) anatomies. Identifying regulatory mechanisms and cross-talk between the genetic elements of these vessels are critical to understand the ethiology of CHD and refine predictive computational models. This study aims to screen expression profiles of fundamental biological pathways in AA at early stages of chick embryo morphogenesis and correlate them with our current understanding of growth and mechanical loading. Reverse transcription-quantitative PCR (RT-qPCR) was followed by correlation and novel peak expression analyses to compare the behaviour and activation period of the genes. Available protein networks were also integrated to investigate the interactions between molecules and highlight major hierarchies. Only wall shear stress (WSS) and growth-correlated expression patterns were investigated. Effect of WSS was seen directly on angiogenesis as well on structural and apoptosis-related genes. Our time-resolved network suggested that WSS-correlated genes coordinate the activity of critical growth factors. Moreover, differential gene expression of left and right AA might be an indicator of subsequent asymmetric morphogenesis. These findings may further our understanding of the complex processes of cardiac morphogenesis and errors resulting in CHD., European Research Council (ERC) Starting Grant - Vascular Growth project; European Molecular Biology Organization (EMBO); European Union (European Union); Horizon 2020; European Research Council (ERC)

The role of hemodynamic forces within the embryo as biomechanical regulators for cardiovascular morphogenesis, growth, and remodeling is well supported through the experimental studies. Furthermore, clinical experience suggests that perturbed flow disrupts the normal vascular growth process as one etiology for congenital heart diseases (CHD) and for fetal adaptation to CHD. However, the relationships between hemodynamics, gene expression and embryonic vascular growth are poorly defined due to the lack of concurrent, sequential in vivo data. In this study, a long-term, time-lapse optical coherence tomography (OCT) imaging campaign was conducted to acquire simultaneous blood velocity, pulsatile micro-pressure and morphometric data for 3 consecutive early embryonic stages in the chick embryo. In conjunction with the in vivo growth and hemodynamics data, in vitro reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to track changes in transcript expression relevant to histogenesis and remodeling of the embryonic arterial wall. Our non-invasive extended OCT imaging technique for the microstructural data showed continuous vessel growth. OCT data coupled with the PIV technique revealed significant but intermitted increases in wall shear stress (WSS) between first and second assigned stages and a noticeable decrease afterwards. Growth rate, however, did not vary significantly throughout the embryonic period. Among all the genes studied, only the MMP-2 and CASP-3 expression levels remained unchanged during the time course. Concurrent relationships were obtained among the transcriptional modulation of the genes, vascular growth and hemodynamics-related changes. Further studies are indicated to determine cause and effect relationships and reversibility between mechanical and molecular regulation of vasculogenesis., European Research Council (ERC); Starting Grant; Vascular Growth project; European Molecular Biology Organization YIP; EMBO; European Union

Hemodynamics of the fetal to neonatal transition are orchestrated through complex physiological changes and results in cardiovascular adaptation to the adult biventricular circulation. Clinical practice during this critical period can influence vital organ physiology for normal newborns, premature babies and congenital heart defect patients. Particularly, the timing of the cord clamping procedure, immediate (ICC) vs. delayed cord clamping (DCC), is hypothesized to be an important factor for the transitory fetal hemodynamics. The clinical need for a quantitative understanding of this physiology motivated the development of a lumped parameter model (LPM) of the fetal cardio-respiratory system covering the late-gestation to neonatal period. The LPM was validated with in vivo clinical data and then used to predict the effects of cord clamping procedures on hemodynamics and vital gases. Clinical time-dependent resistance functions to simulate the vascular changes were introduced. For DCC, placental transfusion (31.3ml) increased neonatal blood volume by 11.7%. This increased blood volume is reflected in an increase in preload pressures by ~20% compared to ICC, which in turn increased the cardiac output (CO) by 20% (CO.sub.ICC =993ml/min; CO.sub.DCC =1197ml/min). Our model accurately predicted dynamic flow patterns in vivo. DCC was shown to maintain oxygenation if the onset of pulmonary respiration was delayed or impaired. On the other hand, a significant 25% decrease in oxygen saturations was observed when applying ICC under the same physiological conditions. We conclude that DCC has a significant impact on newborn hemodynamics, mainly because of the improved blood volume and the sustained placental respiration., National Science Foundation; European Research Council (ERC); European Union; VASCULARGROWTH; ERC-2012-StG_20111012; FP7; Scientific and Technological Research Council of Turkey (TÜBİTAK)