A protocol for in ovo electroporation of chicken and snake embryos to study forebrain development.

In vivo electroporation has become a key technique to study genetic mechanisms of brain development. However, electroporation of the embryonic pallium in oviparous species, interesting for evolutionary studies but distinct from in utero electroporation, is quite infrequent. Here, we detail the in ovo electroporation of the developing pallium in chick and snake embryos. This protocol allows gene manipulation through introducing exogenous DNA into brain progenitor cells and can be adapted to any type of gene manipulation of the embryonic telencephalon. For complete information on the use and execution of this protocol, please refer to Cárdenas et al. (2018).

Multiscale cardiac imaging spanning the whole heart and its internal cellular architecture in a small animal model.

Cardiac pumping depends on the morphological structure of the heart, but also on its subcellular (ultrastructural) architecture, which enables cardiac contraction. In cases of congenital heart defects, localized ultrastructural disruptions that increase the risk of heart failure are only starting to be discovered. This is in part due to a lack of technologies that can image the three-dimensional (3D) heart structure, to assess malformations; and its ultrastructure, to assess organelle disruptions. We present here a multiscale, correlative imaging procedure that achieves high-resolution images of the whole heart, using 3D micro-computed tomography (micro-CT); and its ultrastructure, using 3D scanning electron microscopy (SEM). In a small animal model (chicken embryo), we achieved uniform fixation and staining of the whole heart, without losing ultrastructural preservation on the same sample, enabling correlative multiscale imaging. Our approach enables multiscale studies in models of congenital heart disease and beyond.

The heart is our hardest-working organ and beats around 100,000 times a day, pumping blood through a vast system of vessels to all areas of the body. Specialized heart cells make the heart contract rhythmically, enabling it to work efficiently. Contractile molecules inside these cells, called myofibrils, align within the heart cells, and heart cells align to each other, so that the heart tissue contracts effectively. However, when the heart has defects or is diseased this organization can be lost, and the heart may no longer pump blood efficiently, leading to sometimes life-threatening complications. For example, around one in a hundred newborn babies suffer from congenital heart defects, and despite medical advances, these conditions remain the main cause of non-infectious mortality in children. Many cases of congenital heart disease are diagnosed before a baby is born during an ultrasound scan. However, these scans, as well as subsequent diagnostic tools, lack the precision to detect problems within the heart cells. Now, Rykiel et al. used two complementary imaging techniques known as micro-computed tomography and scanning electron microscopy to acquire pictures of the whole heart as well as of the organization inside the heart cells. This made it possible to capture the structure of the heart tissue at both micrometer (the whole heart) and nanometer resolution (the inside of the cells), and to study what happens within the heart and its cells when the heart has a defect. Rykiel et al. tested the imaging technology on the hearts of chicken embryos, at stages equivalent to a five to six-month-old human fetus, and compared a healthy heart with a heart with a defect called tetralogy of Fallot. They found that the tissues in the heart with a defect had a sponge-like appearance, with increased space in between cells. Moreover, the myofibrils of the heart with a defect were aligned differently compared to those in the normal heart. More research is needed to fully understand what happens when the heart has a defect. However, the imaging technology used in this study offers the possibility of examining the heart at an unprecedented level of detail. This will deepen our understanding of how structural heart defects arise and how they affect the pumping of the heart, and will give us clues to design better treatments for patients with heart defects and other heart anomalies.

Ultrahigh-Field Quantitative MR Microscopy of the Chicken Eye In Vivo Throughout the In Ovo Period.

PURPOSE: Ultrahigh-field MRI (UHF-MRI) with an in-plane spatial resolution of less than 100 µm is known as MR microscopy (MRM). MRM provides highly resolved anatomical images and allows quantitative assessment of different tissue types using diffusion-weighted imaging (DWI). The aim of the present study was to evaluate the feasibility of combined in vivo anatomical and quantitative assessment of the developing chicken eye in ovo. PROCEDURES: Thirty-eight fertilized chicken eggs were examined at 7.1 T (ClinScan, Bruker Biospin, Germany) acquiring a dataset comprising T2-weighted anatomical images, DWI, and diffusion tensor imaging. To reduce motion artifacts, the eggs were moderately cooled before and during MR imaging. Two eggs were imaged daily for the entire developmental period, and 36 eggs were examined pairwise at only one time point of the embryonic period. Development of the eye was anatomically and quantitatively assessed. RESULTS: From the D5 embryonic stage (116-124 h), MRM allowed differentiation between lens and vitreous body. The lens core and periphery were first identified at D9. DWI allowed quantification of lens maturation based on a significant decrease in apparent diffusion coefficient values and course of fractional anisotropy. Repeated moderate cooling had no influence on the development of the chicken embryo. CONCLUSIONS: MRM allows in vivo assessment of embryonic development of the chicken eye in ovo without affecting normal development. The method provides anatomical information supplemented by quantitative evaluation of lens development using DWI. With increasing availability of ultrahigh-field MR systems, this technique may provide a noninvasive complementary tool in the field of experimental ophthalmology.

Use of animal models for the imaging and quantification of angiogenesis.

Angiogenesis is the process of developing new blood vessels from the original vascular network; it is necessary for normal physiological processes, such as embryonic development and wound healing. Angiogenesis is also involved in pathological events, including myocardial ischemia and tumor growth. To investigate the molecular mechanisms of this important process, a variety of methods and models are employed. These strategies can also be used to provide insight into the etiology of angiogenesis-related diseases, thereby contributing to the development of new diagnostics and treatments. Commonly used animal models include the chorioallantoic membrane and yolk sac membrane of chick embryos, the mouse retina and aortic ring, and angiogenesis reactors implanted into mice. These animal models have been instrumental in the study of the angiogenic process. For example, the chorioallantoic membrane undergoes robust angiogenesis during the development of chick embryos, and, because its surface is easily accessible, this membrane provides a convenient model for experimentation. Here, we discuss the methods that employ animal models for the imaging and quantification of angiogenesis. In addition, we propose potential novel directions for future investigations in this area.

3D Reconstruction of Chick Embryo Vascular Geometries Using Non-invasive High-Frequency Ultrasound for Computational Fluid Dynamics Studies.

Recent animal studies have provided evidence that prenatal blood flow fluid mechanics may play a role in the pathogenesis of congenital cardiovascular malformations. To further these researches, it is important to have an imaging technique for small animal embryos with sufficient resolution to support computational fluid dynamics studies, and that is also non-invasive and non-destructive to allow for subject-specific, longitudinal studies. In the current study, we developed such a technique, based on ultrasound biomicroscopy scans on chick embryos. Our technique included a motion cancelation algorithm to negate embryonic body motion, a temporal averaging algorithm to differentiate blood spaces from tissue spaces, and 3D reconstruction of blood volumes in the embryo. The accuracy of the reconstructed models was validated with direct stereoscopic measurements. A computational fluid dynamics simulation was performed to model fluid flow in the generated construct of a Hamburger-Hamilton (HH) stage 27 embryo. Simulation results showed that there were divergent streamlines and a low shear region at the carotid duct, which may be linked to the carotid duct's eventual regression and disappearance by HH stage 34. We show that our technique has sufficient resolution to produce accurate geometries for computational fluid dynamics simulations to quantify embryonic cardiovascular fluid mechanics.

Subharmonic, non-linear fundamental and ultraharmonic imaging of microbubble contrast at high frequencies.

There is increasing use of ultrasound contrast agent in high-frequency ultrasound imaging. However, conventional contrast detection methods perform poorly at high frequencies. We performed systematic in vitro comparisons of subharmonic, non-linear fundamental and ultraharmonic imaging for different depths and ultrasound contrast agent concentrations (Vevo 2100 system with MS250 probe and MicroMarker ultrasound contrast agent, VisualSonics, Toronto, ON, Canada). We investigated 4-, 6- and 10-cycle bursts at three power levels with the following pulse sequences: B-mode, amplitude modulation, pulse inversion and combined pulse inversion/amplitude modulation. The contrast-to-tissue (CTR) and contrast-to-artifact (CAR) ratios were calculated. At a depth of 8 mm, subharmonic pulse-inversion imaging performed the best (CTR = 26 dB, CAR = 18 dB) and at 16 mm, non-linear amplitude modulation imaging was the best contrast imaging method (CTR = 10 dB). Ultraharmonic imaging did not result in acceptable CTRs and CARs. The best candidates from the in vitro study were tested in vivo in chicken embryo and mouse models, and the results were in a good agreement with the in vitro findings.

Dynamic behaviour of selected PET tracers in embryonated chicken eggs.

Positron emission tomography/computer tomography (PET/CT) is an established method in preclinical research in small animal disease models and the clinical diagnosis of cancer. It combines functional information of the positron-emitting biomarker with the anatomical data obtained from the CT image. Thus, it allows for 4D in vivo investigation of biological processes. Recently, PET/CT was used to monitor bone growth of chicken embryos using (18)F-fluoride as a bone-seeking tracer. We are interested in investigating the adequacy of additional PET/CT tracers in chicken embryos as an in vivo model system. For this reason, we evaluated several positron emitting compounds typically used in clinical tests or if these were not commercially available, we synthesised them. We studied the properties of these (18)F- and (68)Ga-labelled tracers and of (64)Cu-chloride in catheterised eggs via small animal microPET/CT. 2-Deoxy-2-[(18)F]fluoroglucose ([(18)F]FDG) was primarily absorbed at the sites of bone growth. (64)Cu chloride and a (68)Ga-labelled amyloid-fibril-binding antibody accumulated in the liver, while the (68)Ga-albumin desferrioxamine conjugate signal in liver decreased over time. These results indicate that these biomarkers can potentially be used for the monitoring of biological processes in chicken eggs as an animal model.

In vivo characterization of ultrasound contrast agents: microbubble spectroscopy in a chicken embryo.

The dynamics of coated microbubbles was studied in an in vivo model. Biotinylated lipid-coated microbubbles were prepared in-house and were injected into a chick embryo chorioallantoic membrane (CAM) model on the fifth day of incubation. The microbubbles, ranging between 1.0 and 3.5 µm in diameter, were insonified in the frequency range of 4-7 MHz. Two amplitudes of acoustic pressure were applied: 300 kPa and 400 kPa. The fundamental and subharmonic responses were recorded optically with an ultra-fast camera (Brandaris 128) at 20 million frames per second. A subharmonic response was observed for 44% of the studied bubbles. From the data the frequency of the maximum fundamental and subharmonic response was derived for each individual bubble and resulted in the resonance curves of the microbubbles. All the bubbles showed shell (strain) hardening behavior for a higher acoustic pressure. We conclude that the subharmonic oscillations observed in this study belonged to the transmit at resonance (TR) regime.

Avian models in teratology and developmental toxicology.

The avian embryo is a long-standing model for developmental biology research. It also has proven utility for toxicology research both in ovo and in explant culture. Like mammals, avian embryos have an allantois and their developmental pathways are highly conserved with those of mammals, thus avian models have biomedical relevance. Fertile eggs are inexpensive and the embryo develops rapidly, allowing for high-throughput. The chick genome is sequenced and significant molecular resources are available for study, including the ability for genetic manipulation. The absence of a placenta permits the direct study of an agent's embryotoxic effects. Here, we present protocols for using avian embryos in toxicology research, including egg husbandry and hatch, toxicant delivery, and assessment of proliferation, apoptosis, and cardiac structure and function.

Microbubble contrast imaging of the cardiovascular system of the chick embyro.

Ultrasound imaging of the chick embryo cardiovascular system is limited to B-scan and Doppler technologies. This study demonstrates microbubble contrast imaging of the embryonic cardiovascular anatomy and physiology. Day 8-19 (Hamburger & Hamilton Stage 34-43) chick embryos are examined in ovo using high-frequency ultrasound imaging through an opening in the blunt end (air cell) of the egg. A chorioallantoic vein is cannulated, and small boluses of octofluoropropane lipid microspheres (Definity®) are injected to visualize the chick embryo cardiovascular system. The entire chick embryo cardiovascular system including the two embryologic arteriovenous (AV) shunts can be visualized. More accurate physiologic measurements of ejection fractions and cardiac output measurements can be obtained using this technology. Microbubble contrast ultrasound imaging in the chick embryo greatly expands the ability to study cardiovascular development. Also, the two natural embryonic A-V shunts provide an excellent model to study the bioeffects of microbubbles in the arterial system.

Quantitative three-dimensional analysis of embryonic chick morphogenesis via microcomputed tomography.

Embryonic development is a remarkably complex and rapidly evolving morphogenetic process. Although many of the early patterning events have been well described, understanding the anatomical changes at later stages where clinically relevant malformations are more likely to be survivable has been limited by the lack of quantitative 3D imaging tools. Microcomputed tomography (Micro-CT) has emerged as a powerful tool for embryonic imaging, but a quantitative analysis of organ and tissue growth has not been conducted. In this study, we present a simple method for acquiring highly detailed, quantitative 3D datasets of embryonic chicks with Micro-CT. Embryos between 4 and 12 days (HH23 and HH40) were labeled with osmium tetroxide (OT), which revealed highly detailed soft tissue anatomy when scanned at 25 µm resolution. We demonstrate tissue boundary and inter-tissue contrast fidelity in virtual 2D sections are quantitatively and qualitatively similar to those of histological sections. We then establish mathematical relationships for the volumetric growth of heart, limb, eye, and brain during this period of development. We show that some organs exhibit constant exponential growth (eye and heart), whereas others contained multiple phases of growth (forebrain and limb). Furthermore, we show that cardiac myocardial volumetric growth differs in a time and chamber specific manner. These results demonstrate Micro-CT is a powerful technique for quantitative imaging of embryonic growth. The data presented here establish baselines from which to compare the effects of genetic or experimental perturbations. Quantifying subtle differences in morphogenesis is increasingly important as research focuses on localized and conditional effects.

Computer-supported angiogenesis quantification using image analysis and statistical averaging.

Angiogenesis is a complex process, involving multiple crosstalks among tumor, endothelial, and stromal cells in order to establish a biochemical network for oxygen and nutrients supply, necessary for the promotion of tumor growth. In this sense, measuring angiogenic activity is considered an informative marker of tumor growth or its inhibition. One of the most popular testbeds for the study of angiogenesis is developing chick embryo and its chorioallantoic membrane (CAM). In this paper, an automated image analysis and statistical processing method for the extraction of features informative for the angiogenic process is proposed and a Web-based tool that provides an unbiased quantification of the microvessel density and growth in angiogenic CAM images is described. The applicability of the tool is tested in two datasets, concerning: 1) the quantification and subsequent detection of tumor growth at different stages of embryonic development and 2) the inhibitory effect of dexamethasone (i.e., an inhibitor of the angiogenesis phenomenon) over a series of CAM samples. Experimental results presented in this paper indicate the efficiency of the automated angiogenesis quantification method regarding both tumor growth and inhibition detection.

High frequency ultrasound imaging of the growth and development of the normal chick embryo.

The purpose of this study is to delineate with high frequency ultrasound imaging the normal growth and development of the chick embryo throughout its incubation period. White Leghorn chick embryos were imaged through an opening in the egg air cell from incubation day 0-19 (Hamburger & Hamilton stage 1-45) using a 13 MHz clinical high frequency linear small parts transducer. Multiple anatomic growth parameters were measured. Normal growth was confirmed with Hamburger and Hamilton staging. A timeline was constructed showing when each anatomic growth parameter could be visualized. Means and standard deviations of each parameter were plotted against incubation days studied to create nomograms and numerical tables of normal growth and development of the chick embryo. With this set of data, abnormal growth and development of the chick embryo can now be assessed.

Preliminary investigation of the use of high frequency ultrasound imaging in the chick embryo.

BACKGROUND: The purpose of this study was to investigate the feasibility of using high frequency ultrasound to study the chick embryo in a noninvasive and longitudinal fashion. METHODS: A total of 10 SPF White Leghorn chick embryos (GDs 11-17; Hamburger and Hamilton stage 37-43) were consecutively examined with a GE Logiq 400 ProSeries ultrasound unit using an 11-MHz small parts ultrasound probe. Access for ultrasound visualization of the embryos was accomplished by opening a 2-3-cm window either in the air cell over the blunt end of the egg or laterally over the embryo-dependent side of the egg. Warmed ultrasound coupling gel was used for imaging, and thermal regulation was maintained with infant heel warmers. The ultrasound images were recorded directly on digital video using a Sony TRV 900 DV camcorder. The images were directly converted to jpeg and mjeg2 files for further analysis. RESULTS: Effective visualization of each embryo was possible on each day of the study period. The embryos were best visualized through the opening made in the air cell at the blunt end of the egg. The extent of the anatomic survey of the chick embryo was dependent upon the position of the embryo in the egg relative to the opening in the air cell. Doppler color flow mapping studies were obtained of the embryonic and extraembryonic circulation. CONCLUSIONS: This preliminary investigation clearly shows the feasibility of high frequency ultrasound imaging to study chick embryo development in a longitudinal and noninvasive fashion. Further studies are presently ongoing regarding earlier embryo development, as well as to determine the stability and dynamics of the methodology.

Lumped parameter estimation for the embryonic chick vascular system: a time-domain approach using MLAB.

We have evaluated several lumped parameter analog models for the early chick embryonic vascular system that may be used to infer loading characteristics of the developing heart. We measured dorsal aortic pressure and flow simultaneously with a servo-null pressure system and a pulsed Doppler velocimeter. Four different analog circuit models were chosen for comparisons. We formulated the time-domain differential equations specifying the relations between pressure and flow in the models, and then estimated the lumped parameters that produced the best fit. The MLAB mathematical modeling software was used for solving differential equations, and for minimizing the difference between model-predicted values and experimental data. The traditional three-element Windkessel model with an added inductance term was most often the best-fitting model. This is compatible with the previous study using a frequency-domain approach. The procedures developed for the current study are adaptable for the study of a variety of nonlinear models, and distributed parameter models for mammalian cardiovascular development with mechanically, pharmacologically, or genetically altered conditions.

Vascular effects of endothelin-1 in stage 21 chick embryos.

Endothelin-1 is a very potent vasoconstrictor, but its function has not yet been investigated in the early stage of cardiovascular development. The purpose of the present study was to clarify whether endothelin-1 exerts a hemodynamic effect in stage 21 chick embryos. We measured vitelline artery blood pressure with a servo-null micropressure system and blood flow velocity at the dorsal aorta with a 20 MHz pulsed Doppler velocity meter. The vitelline vessels were directly measured with a microscope video system. While monitoring these parameters, endothelin-1 was infused into a vein by a microinjector and data were collected. Endothelin-1 increased the blood pressure and heart rate, but decreased the dorsal aortic blood flow. Only the vitelline veins with a diameter of between 100 and 200 microm constricted after endothelin infusion, but smaller or larger veins and the arteries did not show any significant change in size, although the resistant arteries could not be measured by this method. In conclusion, endothelin-1 has apparent constrictive effects in the selected vessel in the early stages of cardiovascular development when the endocrine and autonomic nervous systems have not yet developed.

Ultrasonography as a tool for monitoring in ovo chicken development. 1. Technique and morphological findings.

Preliminary studies were performed to develop a method for using real-time, B-mode ultrasonography (US) to directly image the internal morphology of the chicken egg and developing embryo. Different soft tissue interfaces will reflect US waves differentially. These reflected waves, or echoes are then converted into a two-dimensional image of internal morphology. A major limitation of diagnostic US is its inability to penetrate through gas or hard tissue (bone, shell) interfaces. Methodology development to overcome the acoustic obstacle presented by the eggshell and air cell constituted the initial part of the preliminary study. An acoustical window was achieved by creating a 2-cm fenestration through the large end of the eggshell, then filling the air cell with sterile saline. Morphological features of the yolk and embryo were recorded at 0, 2, 6, 9, 14, and 17 days of incubation. The second part of the preliminary study explores whether the acoustic window, once created, could then be closed, and if closed, whether egg viability could be maintained. A second concurrent trial was conducted with 32 eggs that were fenestrated, imaged, recorded, reclosed, and incubated. Two methods of closure were attempted: one using dialysis membrane and tape; the other using an eggshell allograft. Hatchability was partially retained with both window closure methods.

Ultrasonography as a tool for monitoring in ovo chicken development. 2. Effects of eggshell alteration and ultrasonography on embryonic and posthatch development.

The influence of creating and closing acoustic windows on embryonic and posthatch development for the purpose of chicken embryogenesis monitoring by real-time ultrasonography (US) was evaluated at 2, 6, 9, 14, and 17 days of incubation. Acoustic windows were closed using either a porous dialysis film and tape (FM) or an eggshell allograft attached with collodion (CP). Results from eggs closed in each manner with and without concurrent nonsterile US examination were compared with two control groups. Window creation reduced hatchability. The hatchability reduction was caused primarily by bacterial contamination. Contamination was more common in the FM eggs than in the CP eggs and was greater in eggs that also underwent US. Hatchability increased and contamination decreased when US was performed closer to hatch. Egg weight loss was increased after Day 6 of incubation in FM + US eggs treated on Day 2 and after Day 9 in all eggs with windows except CP eggs treated on Day 9 and CP + US eggs treated on Days 6 and 9. Hatch weight decreased in chicks from eggs that had windows, particularly in FM eggs.

Effect of ultrasound on axonal outgrowth in the sympathetic nervous system of the chick.

The effects of ultrasound exposure on the rate and specificity of sympathetic preganglionic axonal outgrowth were examined in the chick embryo. Using a technique which allows for exact quantitation of exposure for exact quantiation of exposure conditions, embryos were irradiated in ovo for 5 min daily on 3 consecutive days at an intensity of 1 W/cm2 Spatial Average, Temporal Average (SATA), with a frequency of 1.1 MHz pulsed at 1 kHz and a pulse width of 75 microsecond(s). Our results show no significant difference between irradiated and sham-irradiated embryos. In addition, we have examined the distributions of several major extracellular matrix molecules (fibronectin, laminin and collagen IV) in irradiated and sham-irradiated embryos using immunofluorescent staining. No difference in the staining pattern was found. Finally, we found no increase in the incidence of gross abnormalities and no evidence of lesions and malformations in irradiated embryos.