胚胎心脏功能,脊椎动物模型 z-bo 2020-12-11 418

　　Abstract: In recent years, zebrafish has become a powerful vertebrate model for cardiovascular research. Its advantages include easy genetic manipulation, transparency, small size, low cost, and the ability to survive early in development without active cycles. Sequencing the entire genome and using the human genome to identify orthotopic genes makes it possible to induce clinically relevant cardiovascular defects through genetic pathways. For these disease models, it is necessary to assess cardiac function and disturbed hemodynamics in a reliable way to reveal the mechanomechanical biology of the induced defect. This work requires precise determination of blood flow patterns and hemodynamic stress (ie wall shear stress and pressure) levels in the developing heart. The traditional method is to use a time-lapse bright-field microscope to track the movement of cells and tissues, but in recent studies, a fast light-sheet fluorescence microscope was used for this purpose. Combining more complex techniques such as particle image velocimetry and computational fluid dynamics models for hemodynamic analysis will help zebrafish research. We discussed the latest developments in the analysis of zebrafish embryonic heart function and hemodynamics, and summarized our future prospects for dynamic analysis of the zebrafish cardiovascular system.

　　Introduction: The mouse is an established mammalian cardiovascular research model. Knockout mouse models make it possible to study many types of congenital heart defects (CHD). Embryonic chicken is a commonly used vertebrate model. It has the advantages similar to the structure of the human heart. It has a four-chamber/four-petal structure and can perform clinically-related surgical operations. Embryonic zebrafish have recently appeared in cardiovascular research for high-throughput research. Although the zebrafish heart is different from the human heart, it is just a systemic circulation, but the heart structure and physiology of the two are similar. The unique characteristics of zebrafish embryos make them particularly attractive for cardiovascular research. For example, there are some practical methods of genetic interference, such as morphine oligonucleotide injection, to induce heart defects in zebrafish. These embryos are transparent and can be imaged non-invasively during heart development. In the body, the heart cavity and blood vessels and blood flow can be easily observed. In the early stages of development, zebrafish embryos are not dependent on the circulatory system because passive diffusion is sufficient for oxygen. Therefore, embryos with severe heart defects can survive early development. Through this method, we have studied the development of heart valves without blood flow. These studies have proved the important influence of hemodynamics on the development of heart valves. With advances in forward and reverse genetics methods, it is now possible to induce various clinically relevant heart defects in zebrafish embryos. In these animal models, hemodynamic analysis is needed to study the mechanobiological mechanism of inducing defects. For qualitative and quantitative hemodynamic analysis, there are a variety of microscopic imaging and computational modeling methods. These analyses include determining flow patterns, measuring flow rates, calculating cardiac function parameters, and calculating hemodynamic stress levels.

　　Zebrafish heart development: The first organ that forms and starts functioning in a vertebrate embryo is the heart. Unlike the biventricular mammalian heart, the adult zebrafish heart consists of a ventricle, an atrium, an atrioventricular (AV) valve, and an outflow valve. The genetic pathways among vertebrates are conservative, and the heart development process of zebrafish is similar to that of other vertebrates. The precordial cells begin as early as 5 hours after fertilization (hpf) of the anterolateral plate mesoderm, and continue to form the gastrulation, and then form the ectoderm, endoderm and mesoderm. The gastrulation was completed at 10 hpf, followed by fusion of the bilateral hearts at the midline of the embryo at 16 hpf. This forms a heart cone, which then extends forward to become a linear heart tube. The outer layer of the linear cardiac catheter is composed of contractile muscle cells, while the inner layer is composed of endocardial cells. The two layers are separated by a cell-free extracellular matrix layer (called heart freeze). After its formation, the linear cardiac catheter began to contract rhythmically at 24 hpf. The first heartbeat of humans starts at 3 weeks, the first heartbeat of mice starts at (e is the day of embryonic development) e8.5, and the first heartbeat of chickens starts at (hh is the developmental period of chickens) hh10. The degree of contraction of the zebrafish embryos in the subsequent stages can be quantified by high-speed time-lapse video microscopy to determine the partial shortening of the ventricles, which is a comparison of the ventricular size during diastole and contraction. Even if there is no valve at this stage, blood is still pumped out at a peak velocity of 250 μm/sec, with no obvious backflow. Soon after, the lumen began to open, and the beat output and heart rate rose rapidly. At 28 hpf, the heartbeat is normal, with a frequency of 2.6 Hz. The heart tube turned left in an S shape at 33 hpf, and the ventricle moved to the right side of the atrium. At 36 hpf, the pumping mechanism changed from slow peristaltic waves to continuous ventricular contractions, suggesting the beginning of the cardiac conduction system. Two days after fertilization, the ventricle swells, and the inside and outside curves are obvious. At this stage, myocardial cells began to stratify from the ventricular wall and began to form trabeculae. At 72 hpf, the ventricle had a clear trabecular ridge. Trabecular myocardium expands to the ventricular cavity with the significant remodeling of the myocardial wall, the proliferation of the dense myocardium and the maturation of the conduction system. Genetic screening studies have revealed the molecular regulation of these early events in zebrafish. GATA factors related to NKx2.5 control the migration of cardiomyocyte progenitor cells and the regulation of the heart by limiting Wnt signaling. Hand2 plays a role in cardiac differentiation and morphogenesis, while Nkx2.5 maintains the characteristics of the cardiac ventricle. Recently, the contraction of the heart and the resulting fluid force have been shown to activate the Notch signal to regulate the zebrafish heart trabeculae. Heart valves composed of leaflets ensure one-way blood flow in zebrafish. There is a valve in the atrioventricular tube between the atrioventricular and the outflow tract between the ventricle and the bulbar artery. Atrioventricular valve development begins with the expression of BMP4, TBX2b and VCANA in the myocardium, and the endocardial expression of Notch1b, Has2 and neuregulin at 37 hpf. Hemodynamics is an important mechanical stimulus for the development of AV valve in zebrafish. For example, the expression of KLF2A depends on the existence of reverse flow, and the shear stress controls the differential expression of MIR-21 in the AV tube. At 40 hpf, the endocardial cushion began to transform into the original valve leaflets. The endocardial cushion was reshaped into the original valve leaflets to prevent complete retrograde at 76 hpf. At this time, the blood flow velocity through the endocardial pad is about 3.0 mm/sec, and the maximum wall shear stress (wss) is 70 dynes/cm². The formation of the endocardial pad occurred at 4.5 weeks in humans, e12 weeks in mice, and hh24 weeks in chickens. In the outflow vessel, the arterial bulb is composed of a thick outer layer of smooth muscle cells and a thin inner layer of endothelial cells. This small cavity is elastic, which can ensure the maintenance of high pressure and prevent aortic reflux. Compared with the three-leafed mammalian aortic valve, the outflow valve has only two leaflets. The flow of zebrafish heart can be measured by simply tracking individual red blood cells. Or, for more detailed flow analysis, Digital Particle Image Velocimetry (DPIV) can generate a spatial flow map based on the particle position correlation between consecutive frames (red blood cell fluorescence in most cases). Both technologies require fast image acquisition of at least 150 frames per second (fps). Red blood cells or plasma should be fluorescently labeled to enhance contrast.

　　The genetic pathway of zebrafish: Although embryonic chickens are widely used in surgical/mechanical interference research, zebrafish embryos are suitable for genetic interference. The zebrafish genome sequence is known, and the forward/reverse genetic method has been successfully applied to zebrafish embryos to determine the molecular pathways to study the functions of specific genes. Forward genetics is the genetic basis that determines a specific phenotype of an organism. On the other hand, reverse genetics is to selectively manipulate a previously identified gene and analyze the impact of this manipulation on the organism. For zebrafish, radiation or chemical treatment successfully induced random mutations based on forward genetics, and produced stable lines for hybrid offspring. In this way, hundreds of mutants with abnormal cardiovascular development were produced. For example, forward genetic screening identified several mutations that cause valve defects and coarctation of the aorta. Another example of forward genetics is the double mutant line casper. This mutant has no melanocytes and iris cells, which makes it transparent throughout life and capable of non-invasive imaging. Zebrafish embryos can also use several reverse genetic techniques to selectively inhibit gene function. The most commonly used technique is to inject antisense morpholine oligonucleotides (mos) into fertilized eggs. mos can inhibit translation by targeting the transcription start site or targeting splicing connection and inducing abnormal splicing. Effectively prevent protein synthesis of target genes for 3-5 days. Because it is easy to use in zebrafish, mos enables the analysis of gene function to be widely used. It soon became apparent that some mos worked well, and many mos phenotypes effectively reproduced the mutant phenotype without any major side effects. However, mos can induce p53-dependent cell apoptosis and non-targeted cell-specific effects in gene expression, which may affect phenotypic analysis. In a large number of knockout lines, in the corresponding mutants, almost 80% of the Mo variant phenotypes were not observed, which led people to question the wide application of Mo in zebrafish research. It has been suggested that mos should not be overinjected to limit the targeting effect, and stable mutants must be generated and properly characterized to verify the phenotype of mo deformers. Zebrafish recently introduced two alternative reverse genetics technologies: TALENs and CRISPR/CAS9. These technologies affect genomic DNA rather than RNA transcription. Therefore, their molecular effects can be determined at the single embryo level to obtain a clear phenotype/genotype correlation. The non-target effects of these technologies are negligible. These genetic methods were used to produce human cardiovascular diseases in zebrafish embryos to reveal molecular mechanisms. There are two common forms: dilated cardiomyopathy and hypertrophic cardiomyopathy. Forward genetic screening of zebrafish mutants with cardiomyopathy showed that mutations in titin, laminin alpha 4, and integrin-linked kinase can lead to heart failure that mimics human clinical conditions. In a reverse genetics study, after identifying the Eya4 mutation in DCM patients, the zebrafish Eya4 gene was knocked out by morphine oligonucleotide injection. The resulting phenotype has cardiomyopathy, indicating the role of the Eya4 mutation in this situation. Our research team discovered several myosin binding protein C mutations in patients with hypertrophic cardiomyopathy, and summarized these mutations in the zebrafish model by morphine oligonucleotide injection. Cardiac function analysis and hemodynamic evaluation are important in these and similar animal defect models. Below, we will explain the analysis techniques of these zebrafish embryos.

　　Cardiac function analysis of time-lapse image sequence of bright field: In the early stage of zebrafish development (3-4 dpf), the embryo is transparent, and the internal organs including the heart and blood circulation have good visibility. Therefore, at this stage, the video brightfield microscope can be used for quantitative analysis of cardiac function and morphology. The method is based on recording two-dimensional (2D) image sequences for further cardiovascular analysis. The animal is in a lateral position and the ventricles are clearly visible throughout the heart cycle. In this configuration, the atrium is not visible. The use of an automatic video edge detection system can measure the myocardial wall of the ventricular function. The 120-fps video capture speed is sufficient for this application. The contraction wall velocity level is about 200μm/sec at 2 dpf and about 275μm/sec at 6 dpf. Ventricular wall motion can also be actually analyzed by a method similar to M-mode echocardiography. The purpose here is to track the continuous changes in the position of the ventricular wall throughout the heart cycle. This is achieved by first determining the linear region of interest. This area is either the short axis of the ventricle or the long axis of the ventricle. For M-mode images, the intensity value along this line is the Y axis, and each frame of the video is represented on the X axis. In this way, changes in the short and long axis diameters of the ventricles can be measured throughout the heart cycle. M-mode analysis can track myocardial wall thickness and ventricular diameter. Diastolic and systolic minor axis diameters are expressed as dd and ds, and myocardial thickness is expressed as mtd and mts. The image was captured at 250 fps for good time resolution. The blood flow velocity is measured to quantify the cardiovascular function of zebrafish embryos. This can be achieved by simply tracking the movement of red blood cells (RBC) in the embryo, which are easily identified due to the transparent skin. The acceleration, deceleration and peak velocity of red blood cell movement can be calculated for analysis. The movement of red blood cells in the two main blood vessels that pass through the body, in the dorsal aorta and the main vein, can be imaged.

　　Three-dimensional real-time imaging through a light-sheet fluorescence microscope: The high-resolution imaging of the zebrafish embryonic heart is very demanding, because its heart rate is 2-4 Hz, and its relatively large size is about 250μm. Therefore, imaging the in vivo functions and structures of the zebrafish cardiovascular system requires an advanced microscope, which can record optical sections with high temporal and spatial R values. The traditional confocal laser scanning fluorescence microscope (CLSM) is based on point scanning of the object. The penetration depth is also relatively low; therefore, capturing the beating zebrafish embryonic heart is difficult, especially for older embryos. Light sheet fluorescence microscope (LSFM) was introduced in 2004 to eliminate the limitations of CLSM. This technique is called selective planar illumination microscope (SPIM). Its working principle is to use a thin laser sheet to illuminate the fluorescently labeled sample from the side, only in the focal plane of the detection target to excite fluorescence, while recording the emitted light. The detection system in LSFM includes a CCD/SCMOS camera, instead of scanning as in CLSM Photomultiplier tube detector. Therefore, LSFM provides several important advantages, including improved acquisition speed, high signal-to-noise ratio, low photobleaching and large penetration depth. In addition, in most applications, the sample is placed in a dedicated vertical holder (transparent syringe or capillary) and immersed in an imaging chamber filled with media. Embed the sample in a low-concentration agarose column. Compared with traditional glass slides, the low-concentration agarose column puts less pressure on living biological samples. Vertical installation can also rotate precision samples without deformation, and facilitate 360° imaging. Therefore, LSFM has become the new standard imaging method for zebrafish research because it can achieve fast and high-resolution dynamic imaging in physiological imaging media. LSFM significantly enhanced the heart function analysis of zebrafish embryos. Specific areas within the heart can now be imaged at high resolution in the body. However, in order to perform a complete and accurate functional analysis of the heart, it is necessary to perform a three-dimensional analysis on the three-dimensional reconstructed heart beat image. The continuous rapid movement of the heart in all dimensions makes it very difficult to capture such high-resolution images. One solution is to suppress the heart like a silent heart model. However, this model is not suitable for studying hemodynamics and cardiac contraction during heart development. Another method is to use prospective gating technology to obtain a resting 3D heart. As the heart continues to beat, it will slowly move on the focal plane of the imaging objective. Three-dimensional reconstruction of these images to generate a heart model at this specific stage. The dynamics of the heartbeat cannot be studied in this way. The dynamics of the heart beat can only be fully analyzed by 4D imaging (3d + time), which requires very fast image acquisition and deep penetration. The advancement of post-acquisition synchronization technology makes it possible to capture the dynamics of zebrafish embryo heart beating in 4D. ? In this technique, the dynamic motion of the heart tissue is reconstructed by retrospective time registration of image overlays. A short film of the beating heart is recorded on a continuous optical section across the depth of the heart. Then, retrospectively register the image sequence of the continuous z-plane to make a 4D movie of the heartbeat. Pericardial cells, cardiomyocytes and red blood cells can be fluorescently stained, and 3D movies recorded at 70–85 fps can be used to make 4D movies to achieve detailed and dynamic cardiac function analysis. Recently, the integration of two-photon excitation and LSFM has resulted in enhanced penetration depth and preservation of light plate thickness, thereby achieving high acquisition speed (>70 fps) and low light damage. To further increase the scanning speed, new methods have been developed. One is multi-color LSFM. Realize mixed wavelength excitation and realize fast multi-color two-photon imaging. Through this method, simulated imaging of the pericardium, myocardium and red blood cells labeled with CFP, GFP and DSRED were performed. Fast time series images are collected at 85 frames per second and then used to generate a 4D movie of the heart's periodic motion. Compared with monochromatic LSFM, this technology does not produce additional light damage. Simultaneous imaging of myocardium and red blood cells can improve dynamic analysis. Finally, advances in image capture technology have enabled 4D movies to directly record heart beats. Light sheet microscopes can now record the dynamic movements of the myocardial wall and red blood cells; this advancement has led to the application of advanced technologies, such as particle image velocimetry and computational modeling, and zebrafish cardiovascular hemodynamic analysis.

　　 Shear stress analysis based on Digital Particle Image Velocimetry (DPIV): WSS is the friction force acting on the vessel wall and valve leaflet surface in the cardiovascular system. It is a product of the shear rate (the velocity derivative related to the radius of the blood vessel) and the dynamic viscosity of the blood (the fluid viscosity is a measure of its ability to resist gradual stress deformation). Quantitative and qualitative analysis of WSS in the cardiovascular system is important because its size and orientation are thought to contribute to cardiac development. There is important evidence that WSS on cardiovascular endothelial cells can significantly affect the development of blood vessels and valves, as well as the pathogenesis of adult organism vascular and valve diseases. Flows with different hydrodynamic properties (ie, stable and unstable, laminar and turbulent, and anterograde and oscillating) show different regulation of specific gene expression pathways in endothelial cells. Therefore, accurate determination of shear stress mode and shear stress level is of great significance for clinical and in vivo studies of cardiovascular diseases. Since WSS is proportional to the axial velocity gradient of the container wall, the velocity gradient at this position should be determined to calculate WSS. However, this is not an easy task for several reasons: complex geometry, unstable pulse behavior, moving boundaries (ie, vessel walls and valve leaflets) and non-Newtonian behavior of blood (blood viscosity changes with shear Changes in shear rate, shear thinning characteristics). Therefore, for WSS calculations, simplified assumptions are usually used, such as ideal geometry, steady flow, simple parabolic velocity profile, Newtonian behavior, etc., but these will lead to unreliable results. DPIV has become a powerful tool for in vivo studies to determine the velocity vector and WSS level in the cardiovascular system. For small geometries like zebrafish cardiovascular flow, DPIV requires microscopic analysis of the flow field. Here, the diameter of the tracer particles is very important, so these particles should be large enough to be individually identified, and small enough to follow the local flow. For DPIV of zebrafish blood flow, red blood cells can be tracked or beads can be injected. In the groundbreaking study of Hove et al., red blood cells were tracked to determine speed and WSS levels.

　　 Hemodynamic analysis through computational fluid dynamics (CFD): CFD models are very useful for studying complex fluid behavior in cardiovascular research, and experimental measurements can only provide limited information. The CFD model has also been used in in vivo studies to study the hemodynamics of the developing heart. For this reason, embryonic chick models have been widely used. Zebrafish is another animal model commonly used in cardiovascular research. Previous studies have shown that, similar to other animal systems, mechanical signals contribute to the development of the zebrafish heart. More specifically, fluid shear stress and transmural pressure affect the morphology of blood vessels, cavities and valves by triggering mechanobiological mechanisms in endothelial cells. The blood flow through the developing heart is disturbed, resulting in changes in heart development, similar to human coronary heart disease. However, it is interesting that there is still a lack of CFD studies for detailed hemodynamic studies of zebrafish models. In a study, a two-dimensional CFD model was developed for a simplified heart geometry (approximately 4.5 dpf zebrafish heart). The model is a linear channel with two staggered ventricles protruding from the opposite side of the channel, representing the atria and ventricles. Endocardial pads are present on the dorsal and ventral sides of the inflow tract, AV tube, and outflow tract. ? Two different methods are used for blood flow simulation: The first method is to define the velocity inlet condition at the inlet boundary through rigid geometry. In the second method, the atria and ventricles are defined as elastic materials. The contracted atria force blood flow, and the contracted atria stretch the ventricles elastically. By solving the control fluid flow equation, the velocity streamline diagram and WSS value acting on the wall and endocardial cushion are obtained. The analysis of streamlines shows that moving boundaries should be considered to accurately capture streamlines. In the rigid model, the maximum normal stress of the wall is 0.02 dynes/cm2, and the maximum WSS of the endocardial cushion is 2 dynes/cm2. Simulations of different endocardial pad heights show that the development of these pads significantly affects the distribution and size of WSS in the heart and the pressure level of the ventricular wall. The highest WSS is located on the cushion, and as the height increases, the WSS will increase. Because the narrow atrioventricular channel has higher flow resistance, as the height of the cushion increases, the normal pressure on the chamber wall also increases significantly. In the subsequent research, the zebrafish hemodynamic boundary modeling method was applied to the CFD model based on real geometry. Transgenic zebrafish embryos are used, which contain green fluorescent protein-labeled endothelial/endocardial cells and GATA1-labeled red blood cells. The GFP marker can track the boundary of the lumen wall, and the GATA1 marker can track blood flow (via DPIV), so blood flow velocity can be measured. Zebrafish embryos were included in the study from 20-30 hpf to 110-120 hpf. A fluorescence microscope was used to collect a two-dimensional image of the embryonic heart at 20 fps. In each image, the luminal boundary is determined by tracking fluorescent endothelial cells. A practical method was established to estimate the WSS level in the zebrafish heart. This method combines in-vivo confocal imaging with CFD and is based on estimating WSS levels from heart wall dynamics rather than directly from red blood cell movement. This study used 48-hpf zebrafish embryos. Real-time imaging of GATA1-DSRED labeled RBCs at 120 fps to generate 4D heart views. On this two-dimensional geometry, the hydrodynamic centerline is defined, which provides one-dimensional parameterization for cardiac anatomy. These works show the importance of computational fluid dynamics in the analysis of zebrafish hemodynamics, but in order to better understand the mechanism of heart development, more work needs to be done. The computer model needs to be developed in 3D and must be accurately analyzed by combining wall dynamics and fluid dynamics (fluid-structure interaction method).

　　 Conclusion: In the past few decades, the zebrafish model has developed into a very powerful model to study heart development. Advances in forward and reverse genetic interference technologies have made it possible to induce human cardiovascular defects in these animals. Cardiovascular dynamics has been studied in these disease models, most commonly through basic 2D bright-field microscopy analysis, which involves errors in the calculation of cardiac function. The light sheet fluorescence microscope was specially developed for zebrafish embryos. These microscopes can track the movement of the myocardial wall and red blood cells in real time. Therefore, these microscopes can now be used to make 4D beating zebrafish heart movies. The rapid acquisition of images through LSFM makes it possible to apply advanced technologies such as DPIV and computational modeling to zebrafish research. However, due to the difficulties in imaging and modeling of the zebrafish heart's small size and fast-moving boundaries, most current studies involve hemodynamic analysis of the two-dimensional heart plane. The development of a three-dimensional micro DPIV system with the working principle of synchronous image acquisition of different planes will help direct and fast visualization of the zebrafish heart in three dimensions. Ultrasound biomicroscopy and optical coherence tomography systems have been successfully applied to other embryonic animal systems, as well as adult zebrafish. These are potentially very useful ways of imaging embryonic zebrafish. Due to its very small size, current commercial systems are not suitable for zebrafish embryos. We hope to develop new probes to image embryonic zebrafish in the near future. According to CFD modeling, future models should be based on the fluid-structure interaction method, combining wall dynamics and hemodynamics. Integrating technology to produce zebrafish defect models will open new horizons for researchers.