Embryonic Chick Heart Research Articles

Formation and development of lamellar histoarchitecture of the ventricular myocardium of chick embryos

Background. The existence of several models of the myolamellar structure of the ventricular myocardium, which currently has a number of contradictory provisions, reflects the need for a reasonable integration of the results of different methods. Under these circumstances, the study of those ontogenetic mechanisms that are responsible for the formation and development of the myolamellar architecture of the myocardium is of great interest. The purpose of the study is to determine the ontogenetic transformations of the embryonic chicken heart that ensure the formation and development of the myolamellar structure of the ventricular myocardium. Methods. The work examined the embryos of Cobb500 cross chickens from the beginning of the 10th day to the 21st day of incubation. The lamellar organization of the ventricular myocardium was studied using light and transmission electron microscopy. Results. Starting from the 36th stage according to NN (the beginning of the 10th day of incubation), the active development of the stromal component was observed in the heart of chicken embryos, which led to the division of the tissue of the compact ventricular myocardium into groups of muscle fibers in the form of narrow elongated flat plates containing thicker than 3 to 5 rows of cardiomyocytes. At the 41st and 43rd stages of development, active development of the intercellular matrix and division of the myocardium mass into muscle plates continued as part of the compact ventricular myocardium. The intercellular spaces within the plates narrowed, and between the myolamellae, the perimysium accumulated elements of the microcirculatory bed, functionally active fibroblasts, a large amount of amorphous substance, and bundles of formed collagen fibers. At the final stages of embryogenesis, the muscle plates of the left ventricle acquired a pronounced spiral orientation with a gradual displacement of the long axis of the muscle fibers in the direction from the apex of the ventricle to its base. In the wall of the right ventricle, the location of the myolamella acquired a transverse oblique-circular orientation. Conclusion. A comparison of the structure and geometry of the myolamella made it possible to reveal that starting from the 38th stage of development in the left ventricle, the conditions for the translational-rotational mechanism of chamber contraction were formed and increased, in which the formation of the difference between the systolic and diastolic volumes of the left ventricle is ensured not only by the longitudinal apico-basal vector compression of the cavity, but also by mutual sliding of spirally oriented plates in the ventricular wall. In right ventricle, the contraction mechanism is based on the longitudinal-circular compression of the chamber in accordance with the oblique-circular orientation of the muscle fibers in the composition of the myolamella without displacement in the state of systole.

The Stress Phase Angle Measurement Using Spectral Domain Optical Coherence Tomography.

The stress phase angle (SPA), defined as the temporal phase angle between circumferential stress (CS) in the arterial wall and wall shear stress (WSS), is utilized to investigate the interactions between CS and WSS. SPA serves as an important parameter for the early diagnosis of cardiovascular disease. In this study, we proposed a novel method for measuring SPA using spectral domain optical coherence tomography (SD-OCT). The multi-M-mode scan strategy is adopted for interference spectrum acquisition. The phases of CS and WSS are extracted from the corresponding structural and flow velocity images of SD-OCT. The method is validated by measuring SPA in the outflow tract (OFT) of chick embryonic hearts and the common carotid artery of mice. To the best of our knowledge, this is the first time that OCT has been used for SPA measurement.

Shear and hydrostatic stress regulate fetal heart valve remodeling through YAP-mediated mechanotransduction.

Clinically serious congenital heart valve defects arise from improper growth and remodeling of endocardial cushions into leaflets. Genetic mutations have been extensively studied but explain less than 20% of cases. Mechanical forces generated by beating hearts drive valve development, but how these forces collectively determine valve growth and remodeling remains incompletely understood. Here, we decouple the influence of those forces on valve size and shape, and study the role of YAP pathway in determining the size and shape. The low oscillatory shear stress promotes YAP nuclear translocation in valvular endothelial cells (VEC), while the high unidirectional shear stress restricts YAP in cytoplasm. The hydrostatic compressive stress activated YAP in valvular interstitial cells (VIC), whereas the tensile stress deactivated YAP. YAP activation by small molecules promoted VIC proliferation and increased valve size. Whereas YAP inhibition enhanced the expression of cell-cell adhesions in VEC and affected valve shape. Finally, left atrial ligation was performed in chick embryonic hearts to manipulate the shear and hydrostatic stress in vivo. The restricted flow in the left ventricle induced a globular and hypoplastic left atrioventricular (AV) valves with an inhibited YAP expression. By contrast, the right AV valves with sustained YAP expression grew and elongated normally. This study establishes a simple yet elegant mechanobiological system by which transduction of local stresses regulates valve growth and remodeling. This system guides leaflets to grow into proper sizes and shapes with the ventricular development, without the need of a genetically prescribed timing mechanism.

Myocardial Biomechanics and the Consequent Differentially Expressed Genes of the Left Atrial Ligation Chick Embryonic Model of Hypoplastic Left Heart Syndrome

Left atrial ligation (LAL) of the chick embryonic heart is a model of the hypoplastic left heart syndrome (HLHS) where a purely mechanical intervention without genetic or pharmacological manipulation is employed to initiate cardiac malformation. It is thus a key model for understanding the biomechanical origins of HLHS. However, its myocardial mechanics and subsequent gene expressions are not well-understood. We performed finite element (FE) modeling and single-cell RNA sequencing to address this. 4D high-frequency ultrasound imaging of chick embryonic hearts at HH25 (ED 4.5) were obtained for both LAL and control. Motion tracking was performed to quantify strains. Image-based FE modeling was conducted, using the direction of the smallest strain eigenvector as the orientations of contractions, the Guccione active tension model and a Fung-type transversely isotropic passive stiffness model that was determined via micro-pipette aspiration. Single-cell RNA sequencing of left ventricle (LV) heart tissues was performed for normal and LAL embryos at HH30 (ED 6.5) and differentially expressed genes (DEG) were identified.After LAL, LV thickness increased by 33%, strains in the myofiber direction increased by 42%, while stresses in the myofiber direction decreased by 50%. These were likely related to the reduction in ventricular preload and underloading of the LV due to LAL. RNA-seq data revealed potentially related DEG in myocytes, including mechano-sensing genes (Cadherins, NOTCH1, etc.), myosin contractility genes (MLCK, MLCP, etc.), calcium signaling genes (PI3K, PMCA, etc.), and genes related to fibrosis and fibroelastosis (TGF-β, BMP, etc.). We elucidated the changes to the myocardial biomechanics brought by LAL and the corresponding changes to myocyte gene expressions. These data may be useful in identifying the mechanobiological pathways of HLHS.

Tension-suppressed degradation of collagen controls tissue stiffness scaling with fibrillar collagen.

Extremely soft tissues such as developing hearts or adult brain contain far less collagen than highly stiff adult tissues such as tendons, but cell and molecular mechanisms for such homeostatic differences remain unclear. We hypothesized that cell-generated or exogenous forces combine with tension-suppressed collagen degradation in order to sculpt extracellular matrix (ECM) collagen levels in tissues. For various mature mice tissues and beating embryonic chick hearts, we find collagen-sensitive second harmonic generation (SHG) image intensity scales non-linearly versus tissue stiffness, aligning well with the results from cellularized gels of collagen. Chick hearts beating at ∼5% strain maintain collagen levels until their contractile strain is suppressed by myosin-II inhibition and endogenous matrix metalloproteinases (MMPs) then degrade collagens within ∼30-60 minutes - based on SHG and mass spectrometry proteomics. Although tendons composed of oriented collagen fibrils exhibit heterogeneous strain distributions upon deformation, the addition of exogenous MMP or bacterial collagenase suppresses collagen degradation for strains within physiological limits (i.e., up to ∼5-8%). Sequestration of collagen cleavage sites by tissue strain is a likely mechanism because molecular permeation and mobility prove strain-independent whereas artificial collagen cross-links accelerate strain-dependent collagen degradation via collagen molecular unfolding. Tension-suppressed degradation of collagen thus underlies tissue stiffness scaling.

Origin of congenital coronary arterio-ventricular fistulae from anomalous epicardial and myocardial development

Coronary Artery Fistulae (CAFs) are cardiac congenital anomalies consisting of an abnormal communication of a coronary artery with either a cardiac chamber or another cardiac vessel. In humans, these congenital anomalies can lead to complications such as myocardial hypertrophy, endocarditis, heart dilatation, and failure. Unfortunately, despite their clinical relevance, the aetiology of CAFs remains unknown. In this work, we have used two different species (mouse and avian embryos) to experimentally model CAFs morphogenesis. Both conditional Itga4 (alpha 4 integrin) epicardial deletion in mice and cryocauterisation of chick embryonic hearts disrupted epicardial development and ventricular wall growth, two essential events in coronary embryogenesis. Our results suggest that myocardial discontinuities in the embryonic ventricular wall promote the early contact of the endocardium with epicardial-derived coronary progenitors at the cardiac surface, leading to ventricular endocardial extrusion, precocious differentiation of coronary smooth muscle cells, and the formation of pouch-like aberrant coronary-like structures in direct connection with the ventricular lumen. The structure of these CAF-like anomalies was compared with histopathological data from a human CAF. Our results provide relevant information for the early diagnosis of these congenital anomalies and the molecular mechanisms that regulate their embryogenesis.

A method for investigating spatiotemporal growth patterns at cell and tissue levels during C-looping in the embryonic chick heart

SummaryWe developed a workflow using multi-scale and multi-disciplinary experimental and computational approaches to analyze C-looping (the first phase of cardiac looping) of the chick across four developing hearts. We provide the first 3D datasets for the C-looping heart with cell to organism level information, including datasets of heart images and segmented myocardial cells within the heart. We used these datasets to investigate, as a proof-of-concept, the differential spatiotemporal patterns of growth at both the cellular and tissue levels, and demonstrate how geometrical changes of C-looping at the tissue level are linked to growth features at the cellular level. Our methodological pipeline provides preliminary results for qualitative and quantitative evidence of various cellular and tissue features as potential candidates regarding the mechanism of C-looping. This pipeline can be used and extended in future studies to include larger specimen samples for detailed analyses of, and potentially new insights into, cardiac C-looping.

Effect of in ovo manganese injection on the embryonic development, antioxidation, hatchability, and performances of offspring broilers under normal and high temperatures

Two experiments were carried out to study the effect of in ovo manganese (Mn) injection on the embryonic development, antioxidation, hatchability, and performances of offspring broilers under normal temperature (NT) and high temperature (HT). Experiment 1 was conducted to investigate the effect of in ovo Mn injection on the embryonic hatchability of Arbor Acres broiler breeders. On D 9 of incubation, a total of 684 fertilized eggs were randomly allocated to 6 treatments: the non-injected positive control (niPC) and treatments injected with 0 (the negative control, iNC), 6.25, 12.5, 25.0, or 50.0 μg Mn/egg as Mn sulfate. Experiment 2 was conducted to investigate the effect of in ovo Mn injection on the embryonic development, antioxidation and performance of offspring broilers under NT and HT. A total of 792 fertilized eggs were randomly allocated to 6 treatments in a 1 (niPC) + 1 (iNC) + 2 (injected Mn sources: Mn sulfate and Mn proteinate) × 2 (injected Mn levels: 12.5 and 25.0 μg/egg) factorial arrangement during the embryonic stage and D1 to 28 at NT. Then, 288 birds were allotted to 12 treatments in a 6 (the above embryonic treatments) × 2 (environmental temperatures: NT-22℃ vs HT-34℃) factorial arrangement from D 29 to 42. The results showed that Mn injection affected (P < 0.03) the hatchability and the maximum level of in ovo injected Mn was 25.0 μg Mn/egg. The Mn injection upregulated (P < 0.05) Mn-containing superoxide dismutase mRNA expression in the embryonic heart compared to the iNC. Hyperthermia decreased (P < 0.05) ADG and ADFI, breast muscle percentage, plsma alkaline phosphatase activity, and red color values of breast and thigh muscles, but increased (P < 0.05) F/G, plasma aspartate aminotransferase and lactic dehydrogenase activities, total cholesterol, uric acid and triiodothyronine contents, abdominal fat, light values of breast and thigh muscles of offspring broilers. The results suggest that in ovo Mn injection can enhance antioxidant ability in the chick embryonic heart.

Heterogeneous strains in tissue suppress collagen degradation by collagenases

Tissue homeostasis reflects a balance of synthesis and degradation of collagen, and past studies of homogeneously stressed tissues and collagenous materials indicate that collagen degradation is suppressed by strain. We investigated isolated adult mice tendons and beating embryonic chick hearts to study strain dependent of collagen degradation using label-free second harmonic generation (SHG). Tendon fascicles, natively formed by collagen fibrils primarily orient along the tension-axis, were subjected to heterogeneous strains when deformed by either three-point bending plus adhesion and uniaxial tension using novel mechano-bioreactor.

Left-handed cardiac looping by cell chirality is mediated by position-specific convergent extensions

In the embryonic heart development of mammals and birds, a straight initial heart tube undergoes left-handed helical looping, which is a remarkable and puzzling event. We are interested in the mechanism of this chiral helical looping. Recently, observations were reported that myocardial cells in the embryonic chick heart show intrinsic chirality of rotation. The chirality of myocardial cells, via anisotropic polarization of Golgi inside the cells, leads to a left-right (LR) asymmetry of cell shape. On cell boundaries of LR asymmetric cells, phosphorylated myosin and N-cadherin are enriched. Such LR asymmetric cellular circumstances lead to a large-scale three-dimensional chiral structure, the left-handed helical loop. However, the physical mechanism of this looping is unclear. Computer simulations were performed using a cell-based three-dimensional mathematical model assuming an anterior-rightward-biased contractile force of the cell boundaries on the ventral surface of the heart (orientation of a clock hand pointing to 10 to 11 o’clock). An initially straight heart tube was successfully remodeled to the left-handed helical tube via frequent convergent extension (CE) of collective cells, which corresponds to the previously reported observations of chick heart development. Although we assumed that the biased boundary contractile force was uniform all over the ventral side, orientations of the CEs became position specific on the anterior, posterior, right, and left regions on the ventral tube. Such position-specific CEs produced the left-handed helical loop. In addition, our results suggest the loop formation process consists of two distinct phases of preparation and explicit looping. Intrinsic cell properties of chirality in this investigation were discussed relating to extrinsic factors investigated by other researches. Finally, because CE is generally exerted in the axial developmental process across different animal species, we discussed the contribution of CE to the chiral heart structure across species of chick, mouse, Xenopus, and zebrafish.

The early embryonic heart regenerates by compensation of proliferating residual cardiomyocytes after cryoinjury

The adult mammalian heart is non-regenerative because cardiomyocytes withdraw from the cell cycle shortly after birth. Embryonic mammalian hearts, in which cardiomyocytes are genetically ablated in a salt-and-pepper-like pattern, regenerate due to compensation by residual cardiomyocytes. To date, it remains unknown whether or how transmural ventricular defects at the looped heart stage regenerate after cryoinjury. We established a cryoablation model in stage 16 chick embryonic hearts. In hearts at 5h post cryoinjury (hpc), cryoinjury-induced defects were approximately 200µm in width in the primitive ventricle; thereafter, the defect was filled with mesenchymal cells accumulating between the epicardium and endocardium. The defect began to regress at 4days post cryoinjury (dpc) and disappeared around 9 dpc. Immunohistochemistry showed that there were no isl1-positive cells in either the scar tissue or residual cardiomyocytes. BrdU incorporation into residual cardiomyocytes was transiently downregulated in association with upregulation of p27 (Kip1), suggesting that cell cycle arrest occurred at G1-to-S transition immediately after cryoinjury. Estimated cell cycle length was examined, and the results showed that the shortest cell cycle length was 18h at stages 19-23; it increased with development due to elongation of the G2-M-G1 phase and 30h at stages 27-29. The S phase length was constant at 6-8h. The cell cycle length was elongated immediately after cryoinjury, and it reversed at 1-2 dpc. Cryoablated transmural defects in the early embryonic heart were restored by compensation by residual myocytes.

Fluid mechanics of the left atrial ligation chick embryonic model of hypoplastic left heart syndrome

Left atrial ligation (LAL) of the chick embryonic heart at HH21 is a model of the hypoplastic left heart syndrome (HLHS) disease, demonstrating morphological and hemodynamic features similar to human HLHS cases. Since it relies on mechanical intervention without genetic or pharmacological manipulations, it is a good model for understanding the biomechanics origins of such HLHS malformations. To date, however, the fluid mechanical environment of this model is poorly understood. In the current study, we performed 4D ultrasound imaging of LAL and normal chick embryonic hearts and 4D cardiac flow simulations to help shed light on the mechanical environment that may lead to the HLHS morphology. Results showed that the HH25 LAL atrial function was compromised, and velocities in the ventricle were reduced. The HH25 LAL ventricles developed a more triangular shape with a sharper apex, and in some cases, the atrioventricular junction shifted medially. These changes led to more sluggish flow near the ventricular free wall and apex, where more fluid particles moved in an oscillatory manner with the motion of the ventricular wall, while slowly being washed out, resulting in lower wall shear stresses and higher oscillatory indices. Consequent to these flow conditions, at HH28, even before septation is complete, the left ventricle was found to be hypoplastic while the right ventricle was found to be larger in compensation. Our results suggest that the low and oscillatory flow near the left side of the heart may play a role in causing the HLHS morphology in the LAL model.

Early embryonic exposure to chlorpyrifos-cypermethrin combination induces pattern deficits in the heart of domestic hen.

Exposure to chlorpyrifos-cypermethrin combination during early development resulted in defective looping and ventricular noncompaction of heart in domestic chicken. The study was extended to elucidate the molecular basis of this novel observation. The primary culture of chicken embryonic heart cells showed a concentration-dependent loss of viability when challenged with this combination of technical-grade insecticides. Comet assay, DNA ladder assay, and analyses of appropriate markers at transcript and protein levels, revealed that chlorpyrifos-cypermethrin combination induced cell death by activating apoptosis. Parallelly, the tissues derived from control and experimental group hearts were checked for apoptotic markers, and the result was much similar to that of the in-vitro study. Further analysis showed that chlorpyrifos-cypermethrin combination deranged the expression pattern of the transcriptional regulators of cardiogenesis, namely TBX20, GATA5, HAND2, and MYOCD. This, together with heightened apoptosis, could well be the reason behind the observed structural anomalies in the heart of chlorpyrifos-cypermethrin poisoned embryos.

Msx1 upregulates p27 expression to control cellular proliferation during valvuloseptal endocardial cushion formation in the chick embryonic heart.

Cushion tissues, the primordia of valves and septa of the adult heart, are formed in the atrioventricular (AV) and outflow tract (OFT) regions of the embryonic heart. The cushion tissues are generated by the endothelial-mesenchymal transition (EMT), involving many soluble factors, extracellular matrix, and transcription factors. Moreover, neural crest-derived mesenchymal cells also migrate into the OFT cushion. The transcription factor Msx1 is known to be expressed in the endothelial and mesenchymal cells during cushion tissue formation. However, its exact role in EMT during cushion tissue formation is still unknown. In this study, we investigated the expression patterns of Msx1 mRNA and protein during chick heart development. Msx1 mRNA was localized in endothelial cells of the AV region at Stage 14, and its protein was first detected at Stage 15. Thereafter, Msx1 mRNA and protein were observed in the endothelial and mesenchymal cells of the OFT and AV regions. in vitro assays showed that ectopic Msx1 expression in endothelial cells induced p27, a cell-cycle inhibitor, expression and inhibited fibroblast growth factor 4 (FGF4)-induced cell proliferation. Although the FGF signal reduced the EMT-inducing activities of transforming growth factor β (TGFβ), ectopic Msx1 expression in endothelial cells enhanced TGFβ signaling-induced αSMA, an EMT marker, expression. These results suggest that Msx1 may support the transformation of endothelial cells due to a TGFβ signal in EMT during cushion tissue formation.

Cardiac motion estimation from medical images: a regularisation framework applied on pairwise image registration displacement fields

Accurate cardiac motion estimation from medical images such as ultrasound is important for clinical evaluation. We present a novel regularisation layer for cardiac motion estimation that will be applied after image registration and demonstrate its effectiveness. The regularisation utilises a spatio-temporal model of motion, b-splines of Fourier, to fit to displacement fields from pairwise image registration. In the process, it enforces spatial and temporal smoothness and consistency, cyclic nature of cardiac motion, and better adherence to the stroke volume of the heart. Flexibility is further given for inclusion of any set of registration displacement fields. The approach gave high accuracy. When applied to human adult Ultrasound data from a Cardiac Motion Analysis Challenge (CMAC), the proposed method is found to have 10% lower tracking error over CMAC participants. Satisfactory cardiac motion estimation is also demonstrated on other data sets, including human fetal echocardiography, chick embryonic heart ultrasound images, and zebrafish embryonic microscope images, with the average Dice coefficient between estimation motion and manual segmentation at 0.82–0.87. The approach of performing regularisation as an add-on layer after the completion of image registration is thus a viable option for cardiac motion estimation that can still have good accuracy. Since motion estimation algorithms are complex, dividing up regularisation and registration can simplify the process and provide flexibility. Further, owing to a large variety of existing registration algorithms, such an approach that is usable on any algorithm may be useful.

Ectopic release of nitric oxide modulates the onset of cardiac development in avian model.

Heart development is one of the earliest developmental events, and its pumping action is directly linked to the intensity of development of other organs. Heart contractions mediate the circulation of the nutrients and signalling molecules to the focal points of developing embryos. In the present study, we used in vivo, ex vivo, in vitro, and in silico methods for chick embryo model to characterize and identify molecular targets under the influence of ectopic nitric oxide in reference to cardiogenesis. Spermine NONOate (SpNO) treatment of 10 μM increased the percentage of chick embryos having beating heart at 40th h of incubation by 2.2-fold (p < 0.001). In an ex vivo chick embryo culture, SpNO increased the percentage of embryos having beats by 1.56-fold (p < 0.05) compared with control after 2 h of treatment. Total body weight of SpNO-treated chick embryos at the Hamburger and Hamilton (HH) stage 29 was increased by 1.22-fold (p < 0.005). Cardiac field potential (FP) recordings of chick embryo at HH29 showed 2.5-fold (p < 0.001) increased in the amplitude, 3.2-fold (p < 0.001) increased in frequency of SpNO-treated embryos over that of the control group, whereas FP duration was unaffected. In cultured cardiac progenitors cells (CPCs), SpNO treatment decreased apoptosis and cell death by twofold (p < 0.001) and 1.7-fold (p < 0.001), respectively. Transcriptome analysis of chick embryonic heart isolated from HH15 stage pre-treated with SpNO at HH8 stage showed upregulation of genes involved in heart morphogenesis, heart contraction, cardiac cell development, calcium signalling, structure, and development whereas downregulated genes were enriched under the terms extracellular matrix, wnt pathway, and BMP pathway. The key upstream molecules predicted to be activated were p38 MAPK, MEF2C, TBX5, and GATA4 while KDM5α, DNMT3A, and HNF1α were predicted to be inhibited. This study suggests that the ectopic nitric oxide modulates the onset of cardiac development.

Small RNA sequencing reveals miRNAs important for hypoxic adaptation in the Tibetan chicken

ABSTRACT 1. The Tibetan chicken, which is an indigenous breed living on the Tibetan Plateau, exhibits hypoxic adaptations to its high-altitude environment. However, the molecular mechanism behind this hypoxic adaptation is still unclear. This study aimed to investigate differentially expressed miRNAs involved in hypoxic adaptation through high-throughput RNA sequencing. 2. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the differentially expressed miRNAs and their target genes in chicken embryonic heart tissues and fibroblasts. Luciferase reporter assays were performed to confirm the relationship between miRNAs and target genes. 3. The study identified 37 differentially expressed miRNAs in Tibetan chicken embryonic heart tissues, including 20 up- and 17 down-regulated miRNAs, compared to those found in lowland chickens. Differentially expressed miRNAs were mainly involved in biological processes, such as cell cycle arrest, toll-like receptor signalling pathways, and I-kappa B kinase/NF-kappa B signalling. The data showed that gga-miR-34 c-5p was significantly upregulated in Tibetan chicken tissues and hypoxic fibroblasts, while EHHADH, a target gene of gga-miR-34 c-5p, was downregulated. Moreover, gga-miR-34 c-5p dramatically decreased the luciferase activity of the wild EHHADH, whereas no effect on the mutational EHHADH was found. 4. This study identified miRNA expression profiles in the Tibetan chicken and suggested that miR-34 c-5p acts as a novel miRNA associated with hypoxic adaptation. This facilitates the understanding of molecular mechanisms that underlie long-term exposure to hypoxia.

What Is the Optimal Light Source for Optical Mapping Using Voltage- and Calcium-Sensitive Dyes?

Optical mapping is a fluorescence-based physiological method to image spreading of action potential in excitable tissues, such as the heart and central nervous system. Because of the requirements for high speed imaging in low light conditions, highly sensitive high-speed cameras together with an optical system with maximum photon efficiency are required. While the optimization of these two components is relatively straightforward, the choice of the perfect light source is less simple; depending on the other (usually fixed) components, various parameters may acquire different weight in decision-making process. Here we describe the rationale for building an optical mapping setup and consider the relative advantages and disadvantages of three different commonly available light sources: mercury vapor lamp (HBO), xenon lamp (XBO), and light emitting diode (LED). Using the same optical system (fluorescence macroscope) and high-speed camera (Ultima L), we have tested each of the sources for its ability to provide bright and even illumination of the field of view and measured its temporal fluctuations in intensity. Then we used each in the actual optical mapping experiment using isolated, perfused adult mouse heart or chick embryonic heart to determine the actual signal to noise ratio at various acquisition rates. While the LED sources have undergone significant improvements in the recent past, the other alternatives may still surpass them in some parameters, so they may not be the automatic number one choice for every application.

Organ Dynamics and Hemodynamic of the Whole HH25 Avian Embryonic Heart, Revealed by Ultrasound Biomicroscopy, Boundary Tracking, and Flow Simulations

Congenital heart malformations occur to substantial number of pregnancies. Studies showed that abnormal flow biomechanical environments could lead to malformations, making it important to understand the biomechanical environment of the developing heart. We performed 4D high-frequency ultrasound scans of chick embryonic hearts at HH25 to study the biomechanics of the whole heart (atria and ventricle). A novel and high-fidelity motion estimation technique, based on temporal motion model and non-rigid image registration algorithm, allowed automatic tracking of fluid-structure boundaries from scan images, and supported flow simulations. Results demonstrated that atrial appendages were the most contractile portion of the atria, having disproportionately high contribution to atrial blood pumping for its volume in the atria. However, the atria played a small role in blood pumping compared to the ventricle, as it had much lower ejection energy expenditure, and as the ventricle appeared to be able to draw inflow from the veins directly during late diastole. Spatially and temporally averaged wall shear stresses (WSS) for various cardiac structures were 0.062–0.068 Pa, but spatial-averaged WSS could be as high as 0.54 Pa in the RV. WSS was especially elevated at the atrial inlet, atrioventricular junction, regions near to the outflow tract, and at dividing lines between the left and right atrium and left and right side of the ventricle, where septation had begun and the lumen had narrowed. Elevated WSS could serve as biomechanics stimulation for proper growth and development.

Extracting physiological information in experimental biology via Eulerian video magnification

BackgroundVideographic material of animals can contain inapparent signals, such as color changes or motion that hold information about physiological functions, such as heart and respiration rate, pulse wave velocity, and vocalization. Eulerian video magnification allows the enhancement of such signals to enable their detection. The purpose of this study is to demonstrate how signals relevant to experimental physiology can be extracted from non-contact videographic material of animals.ResultsWe applied Eulerian video magnification to detect physiological signals in a range of experimental models and in captive and free ranging wildlife. Neotenic Mexican axolotls were studied to demonstrate the extraction of heart rate signal of non-embryonic animals from dedicated videographic material. Heart rate could be acquired both in single and multiple animal setups of leucistic and normally colored animals under different physiological conditions (resting, exercised, or anesthetized) using a wide range of video qualities. Pulse wave velocity could also be measured in the low blood pressure system of the axolotl as well as in the high-pressure system of the human being. Heart rate extraction was also possible from videos of conscious, unconstrained zebrafish and from non-dedicated videographic material of sand lizard and giraffe. This technique also allowed for heart rate detection in embryonic chickens in ovo through the eggshell and in embryonic mice in utero and could be used as a gating signal to acquire two-phase volumetric micro-CT data of the beating embryonic chicken heart. Additionally, Eulerian video magnification was used to demonstrate how vocalization-induced vibrations can be detected in infrasound-producing Asian elephants.ConclusionsEulerian video magnification provides a technique to extract inapparent temporal signals from videographic material of animals. This can be applied in experimental and comparative physiology where contact-based recordings (e.g., heart rate) cannot be acquired.