Developmental plasticity in the cardiovascular system of fish,with special reference to the zebrafish

During development the circulatory system of vertebrates typically starts operating earlier than any other organ. In these early stages, however, blood flow is not yet linked to metabolic requirements of tissues, as is well established for adults. While the autonomic nervous system becomes functional only quite late during development, in the early stages control of blood flow appears to be possible by blood-borne and/or local hormones. This study presents methods based on video-imaging techniques and fluorescence microscopy to visualize cardiac activity, as well as the vascular bed of developing lower vertebrates, and tests the idea that environmental factors, such as hypoxia, may modify cardiac activity, or even the early formation of blood vessels in embryos and larvae. In zebrafish larvae, adaptations of cardiovascular activity to chronic hypoxia become visible shortly after hatching, and the formation of some blood vessels is enhanced under chronic hypoxia. Exposure of early larval stages of zebrafish to a constant water current induces physiological adaptations, resulting in enhanced swimming efficiency and increased tolerance towards hypoxia. Furthermore, application of hormones such as NO can modify cardiac activity as well as peripheral resistance, and they can stimulate blood vessel formation. In consequence, even during early development of fish or amphibian larvae, the performance of cardiac muscle and of skeletal muscle can be modified by environmental influences and peripheral resistance can be adjusted. Even blood vessel formation can be stimulated by hypoxia, for example, or by the presence of specific hormones. Thus, at approximately the time of hatching the physiological performance of vertebrate larvae is already determined by the combined action of environmental influences and of genetic information.     

Effect of simulated microgravity induced PI3K-nos2b signalling on zebrafish cardiovascular plexus network formation

Local abnormal angiogenesis and cardiovascular system reorganization have been observed in embryos exposed to a simulated microgravity (SM) environment. In this study, changes in key molecular signals and pathways in cardiovascular development have been investigated under microgravity conditions. In particular, the caudal vein plexus (CVP) network, formed by sprouting angiogenesis has been chosen. Zebrafish embryos were exposed to SM using a ground-based microgravity bioreactor for 24 and 36 h. The SM was observed to have no effect on the zebrafish length, tail width and incubation time whereas it was observed to significantly reduce the heart rate frequency and to promote abnormal development of the CVP network in the embryos. Nitric oxide (NO) content demonstrated that the total proteins in zebrafish embryos were significantly higher in SM than in the control group grown under normal conditions. It was then preliminarily determined how NO signals were involved in SM regulated zebrafish CVP network formation. nos2b MO was injected and CVP network evolution was observed in 36 h post fertilization (hpf) under SM condition. The results showed that the CVP network formation was considerably decreased in the nos2b MO treated group. However, this inhibition of the CVP network development was not observed in control MO group, indicating that nos2b is involved in the SM-regulated vascular development process in zebrafish. Moreover, specific phosphoinositide 3-kinase (PI3K) inhibitors such as LY294002 were also tested on zebrafish embryos under SM condition. This treatment significantly inhibited the formation of zebrafish CVP network. Furthermore, overexpression of nos2b partly rescued the LY294002-caused CVP network failure. Therefore, it can be concluded that SM affects zebrafish CVP network remodeling by enhancing angiogenesis. Additionally, the PI3K-nos2b signaling pathway is involved in this process.     

Zebrafish embryos and larvae: a new generation of disease models and drug screens

Technological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in applied biomedical research, using selected cases. We look at the use of zebrafish embryos as disease models, taking fetal alcohol syndrome and tuberculosis as examples. We discuss advances in imaging, in culture techniques (including microfluidics), and in drug delivery (including new techniques for the robotic injection of compounds into the egg). The use of zebrafish embryos in early stages of drug safety-screening is discussed. So too are the new behavioral assays that are being adapted from rodent research for use in zebrafish embryos, and which may become relevant in validating the effects of neuroactive compounds such as anxiolytics and antidepressants. Readouts, such as morphological screening and cardiac function, are examined. There are several drawbacks in the zebrafish model. One is its very rapid development, which means that screening with zebrafish is analogous to "screening on a run-away train." Therefore, we argue that zebrafish embryos need to be precisely staged when used in acute assays, so as to ensure a consistent window of developmental exposure. We believe that zebrafish embryo screens can be used in the pre-regulatory phases of drug development, although more validation studies are needed to overcome industry scepticism. Finally, the zebrafish poses no challenge to the position of rodent models: it is complementary to them, especially in early stages of drug research.     

Environmental influences on the development of the cardiac system in fish and amphibians

In poikilothermic animals body temperature varies with environmental temperature, and this results in a change in metabolic activity (Q10 of enzymatic reactions typically is around 2-3). Temperature changes also modify gas transport in body fluids. While the diffusion coefficient increases with increasing temperatures, physical solubility and also hemoglobin oxygen affinity decrease. Therefore, an increase in temperature typically requires adjustments in cardiac activity because ventilatory and convectional transport of respiratory gases usually are tightly coupled in adults in order to meet the oxygen demand of body tissues. Hypoxic conditions also provoke adaptations in the central circulatory system, like the hypoxic bradycardia, which has been described for many adult lower vertebrates, combined with an increase in stroke volume and peripheral resistance. In embryos and larvae the situation is much more complicated, because nervous control of the heart is established only late during development, and because the site of gas exchange changes from mainly cutaneous gas exchange during early development to mainly pulmonary or branchial gas exchange in late stages. In addition, recent studies in amphibian and fish embryos and larvae reveal, that at least in very early stages convectional gas transport of the hemoglobin is not essential, which means that in these early stages ventilatory and convectional gas transport are not yet coupled. Accordingly, in early stages of fish and amphibians the central cardiac system often does not respond to hypoxia, although in some species behavioral adaptations indicate that oxygen sensors are functional. If a depression of cardiac activity is observed, it most likely is a direct effect of oxygen deficiency on the cardiac myocytes. Regulated cardiovascular responses to hypoxia appear only in late stages and are similar to those found in adult species.     

Nitric oxide and vascular reactivity in developing zebrafish, Danio rerio

We used a newly developed digital motion analysis video technique to study the effects of nitric oxide (NO) and epinephrine on the early larval arterial and venous vasculature of zebrafish. Application of the NO donor sodium nitroprusside resulted in a significant increase in both the venous and arterial vessel diameters, whereas N(G)-nitro-L-arginine methyl ester caused a significant decrease in the same diameters. Thus our results show that both the venous and arterial vasculature of the 5- and 6-day-old zebrafish larvae are influenced by endogenously produced NO. By use of immunohistochemistry, NO synthase immunoreactivity was demonstrated in endothelial cells of the dorsal vein. Local application of epinephrine onto the dorsal artery had no effect on vessel diameter. However, if the embryos were preincubated with N(omega)-nitro-L-arginine methyl ester, addition of epinephrine resulted in a significant reduction in both arterial and venous vessel diameters. Thus this study provides increasing evidence that before a functional autonomic innervation of the peripheral vascular system, vascular tone in larval tissue is regulated by a complex interaction of vasoactive substances that are produced locally by vascular endothelial cells.     

Nitrate (NO3) and nitrite (NO2) are endocrine disruptors to downregulate expression of tyrosine hydroxylase and motor behavior through conversion to nitric oxide in early development of zebrafish

With a view to consider the increasing concern over nitrogen pollution in the aquatic environment, we investigated effects of nitrate (NO₃⁻) and nitrite (NO₂⁻) on the activity of dopaminergic neuron in zebrafish embryos and larvae. Both nitrate and nitrite exposure decreased the expression of tyrosine hydroxylase (TH) in dopaminergic neurons at 48 hpf. Only nitrite decreased the response to tactile stimulation at 72 hpf, whereas both nitrate and nitrite decreased the swimming activity at 6 dpf. When the embryos were exposed to nitrate or nitrite together with an estrogen receptor blocker (ICI 182,780), the decreases in TH expression and motor behavior caused by nitrate or nitrite alone were reversed suggesting the effects of nitrate and nitrite were mediated through estrogen receptor (ER). The result of co-incubation with an oxidoreductase inhibitor, diphenyleneiodonium, indicated the conversion to nitric oxide (NO) is likely to be responsible for the effects of nitrate and nitrite, which was further supported by the increased staining for NO after exposure. The present study demonstrates that nitrate and nitrite are neurotoxicants acting as an endocrine disruptor possibly through conversion to NO to downregulate the activity of dopaminergic neuron in early development of zebrafish.     

Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation: An Alternative Model System to Study FASD

Background

The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines.

Methodology/Principal Findings

In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification.

Conclusion

Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development.     

Cyclosporin A Disrupts Notch Signaling and Vascular Lumen Maintenance

Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos.     

How does blood cell concentration modulate cardiovascular parameters in developing zebrafish (Danio rerio)?

Blood flow and shear forces are considered to be important parameters possibly stimulating angiogenesis or cardiovascular remodeling. The main objective of this study was to test the hypothesis that a significant reduction in shear forces as a consequence of a significant isovolemic anemia induced by microsurgical techniques during early larval development of the zebrafish might induce a compensatory stimulation of erythropoiesis and/or induce a modification of cardiac activity or even the formation of the heart and may influence the shaping of the vascular bed. Blood from 2 day old zebrafish larvae was withdrawn and replaced by zebrafish Ringer's solution, so that the blood cell concentration was reduced by at least 75%. At 5 days post fertilization (dpf) a partial recovery in blood cell concentration was observed and reached a value of 814.55+/-85.42 cells/nL, while in control animals blood cell concentration amounted to 1856.00+/-131.59 cells/nL. At 7 dpf the value of blood cell concentration was 1023.89+/-95.75 cells/nL versus 1701.54+/-146.03 cells/nL in control animals. Compared to control animals, heart rate and cardiac output were significantly reduced in anemic animals and alterations in the formation of the vascular bed were also observed. A significant decrease in the end-diastolic volume suggested that ventricular volume was reduced. Thus, within a few days zebrafish larvae were nearly able to compensate for an isovolemic anemia by an enhanced erythropoiesis. However, several changes in cardiovascular system indicated that phenotypic plasticity is established even at an early developmental stage.     

Zebrafish on a Chip: A Novel Platform for Real-Time Monitoring of Drug-Induced Developmental Toxicity

Pharmaceutical safety testing requires a cheap, fast and highly efficient platform for real-time evaluation of drug toxicity and secondary effects. In this study, we have developed a microfluidic system for phenotype-based evaluation of toxic and teratogenic effects of drugs using zebrafish (Danio rerio) embryos and larvae as the model organism. The microfluidic chip is composed of two independent functional units, enabling the assessment of zebrafish embryos and larvae. Each unit consists of a fluidic concentration gradient generator and a row of seven culture chambers to accommodate zebrafish. To test the accuracy of this new chip platform, we examined the toxicity and teratogenicity of an anti-asthmatic agent-aminophylline (Apl) on 210 embryos and 210 larvae (10 individuals per chamber). The effect of Apl on zebrafish embryonic development was quantitatively assessed by recording a series of physiological indicators such as heart rate, survival rate, body length and hatch rate. Most importantly, a new index called clonic convulsion rate, combined with mortality was used to evaluate the toxicities of Apl on zebrafish larvae. We found that Apl can induce deformity and cardiovascular toxicity in both zebrafish embryos and larvae. This microdevice is a multiplexed testing apparatus that allows for the examination of indexes beyond toxicity and teratogenicity at the sub-organ and cellular levels and provides a potentially cost-effective and rapid pharmaceutical safety assessment tool.     

Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae

Background  

The zebrafish Danio rerio is an important model system for drug discovery and to study cardiovascular development. Using a laser-scanning confocal microscope, we have developed a non-invasive method of measuring cardiac performance in zebrafish embryos and larvae that obtains cardiovascular parameters similar to those obtained using Doppler echocardiography in mammals. A laser scan line placed parallel to the path of blood in the dorsal aorta measures blood cell velocity, from which cardiac output and indices of vascular resistance and contractility are calculated.     

Malachite green induces cardiovascular defects in developing zebrafish (Danio rerio) embryos by blocking VEGFR-2 signaling

Malachite green (MG) is a triphenyl methane dye used in various fields that demonstrates high toxicity to bacteria and mammalian cells. When bud stage zebrafish embryos were treated with MG at 125, 150, and 175 ppb for 14 h, the development of trunk including intersomitic vessels was inhibited in MG-treated flk-1-GFP transgenic embyos. MG clearly induced whole growth retardation. MG induced severe cell death in trunk intersomite region of zebrafish embryos and in human vascular endothelial cells in a dose-dependent manner. MG inhibited heart rates and cardiac looping. MG attenuated whole blood formation and inhibited vascular endothelial growth factor (VEGF)-induced receptor (R)-2 phosphorylation in vascular endothelial cells. In conclusion, MG significantly alters the cardiovascular development causing growth retardation in zebrafish through the blocking VEGFR-2 activation in early cardiovascular development. It suggests that MG may be an environmental toxic agent with the potential to induce embryonic cardiovascular defects in vertebrates.     

PFOS Induces Behavioral Alterations,Including Spontaneous Hyperactivity That Is Corrected by Dexamfetamine in Zebrafish Larvae

Perfluorooctane sulfonate (PFOS) is a widely spread environmental contaminant. It accumulates in the brain and has potential neurotoxic effects. The exposure to PFOS has been associated with higher impulsivity and increased ADHD prevalence. We investigated the effects of developmental exposure to PFOS in zebrafish larvae, focusing on the modulation of activity by the dopaminergic system. We exposed zebrafish embryos to 0.1 or 1 mg/L PFOS (0.186 or 1.858 µM, respectively) and assessed swimming activity at 6 dpf. We analyzed the structure of spontaneous activity, the hyperactivity and the habituation during a brief dark period (visual motor response), and the vibrational startle response. The findings in zebrafish larvae were compared with historical data from 3 months old male mice exposed to 0.3 or 3 mg/kg/day PFOS throughout gestation. Finally, we investigated the effects of dexamfetamine on the alterations in spontaneous activity and startle response in zebrafish larvae. We found that zebrafish larvae exposed to 0.1 mg/L PFOS habituate faster than controls during a dark pulse, while the larvae exposed to 1 mg/L PFOS display a disorganized pattern of spontaneous activity and persistent hyperactivity. Similarly, mice exposed to 0.3 mg/kg/day PFOS habituated faster than controls to a new environment, while mice exposed to 3 mg/kg/day PFOS displayed more intense and disorganized spontaneous activity. Dexamfetamine partly corrected the hyperactive phenotype in zebrafish larvae. In conclusion, developmental exposure to PFOS in zebrafish induces spontaneous hyperactivity mediated by a dopaminergic deficit, which can be partially reversed by dexamfetamine in zebrafish larvae.     

Effect of Copper Ions on Early Developmental Stages of the Mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> (Bivalvia)

The effect of exposure to copper in seawater (0.005, 0.01, and 0.02 mg/liter) on the development of Mytilus trossulus was examined in the stages of fertilization, blastula, early veliger, veliger, and veliconch. Copper in a concentration of 0.01 and 0.02 mg/liter inhibited the development and growth of the embryos and larvae. At 0.005 mg Cu/liter, the embryos and larvae were capable of adaptation. If M. trossulus embryos and larvae were maintained for 1–2 days in seawater containing 0.01 mg Cu/liter and then transferred to clean water, the consequences were largely dependent on the developmental stage at which the exposure to copper took place. The early developmental stages were more sensitive to the effect of copper than veliger larvae.Original Russian Text Copyright ¢ 2005 by Biologiya Morya, Yaroslavtseva, Sergeeva.     

Cardiovascular malformations induced by bromodeoxyuridine in the chick embryo

For the study of morphogenesis and early embryonic development, 5-bromodeoxyuridine (BUdR), a halogenated analogue of thymidine, is incorporated into replicating DNA and serves as a valuable tool. To study the teratogenicity of BUdR on the developing chick cardiovascular system, we topically administered graded doses of BUdR (32.6-325.6 nmol) in ovo during Hamburger-Hamilton stages 15 to 16. We also administered to a parallel group of embryos corresponding nanomole doses of thymidine during identical stages of development. In the thymidine-treated group, survival rates and cardiovascular anomaly rates did not differ statistically from those in the chick Ringer's control group. Both survival rates and cardiovascular anomaly rates in the BudR-treated group were dose-responsive. Among 78 embryos with cardiovascular anomalies induced by BUdR, vascular malformations were found in 96%. These anomalies included interruption of the right fourth aortic arch, absence or hypoplasia of the right and/or left sixth aortic arch, and persistence of the left fourth aortic arch. Interruption of the right fourth aortic arch was always associated with intracardiac anomalies. Intracardiac anomalies were found in 54% of the embryos; these included ventricular septal defect, double outlet right ventricle, and persistent truncus arteriosus. Subclavian artery malformations were noted in 95% of the embryos. Possible mechanisms for BUdR-induced malformations in the cardiovascular system of the chick are discussed.     

Changes in the antioxidant metabolism in the embryonic development of the common South American toad Bufo arenarum: differential responses to pesticide in early embryos and autonomous-feeding larvae

Amphibians may be critically challenged by aquatic contaminants during their embryonic development. Many classes of compounds, including organophosphorus pesticides, are able to cause oxidative stress that affects the delicate cellular redox balance regulating tissue modeling. We determined the progression of antioxidant defenses during the embryonic development of the South American common toad, Bufo arenarum. Superoxide dismutase (SOD) and catalase (CAT) activities were high in the unfertilized eggs, and remained constant during the first stages of development. SOD showed a significant increase when the gills were completely active and opercular folds began to form. Reductase (GR) activity was low in the oocytes and increased significantly when gills and mouth were entirely developed and the embryos presented a higher exposure to pro-oxidant conditions suggesting an environmental control. Reduced glutathione (GSH) content was also initially low, and rose continuously pointing out an increasing participation of GSH-related enzymes in the control of oxidative stress. GSH peroxidases and GSH-S-transferases showed relatively high and constant activities, probably related to lipid peroxide control. B. arenarum embryos have plenty of yolk platelets containing lipids, which provide the energy and are actively transferred to the newly synthesized membranes during the early embryonic development. Exposure to the pro-oxidant pesticide malathion during 48 h did not significantly affect the activity of antioxidant enzymes in early embryos, but decreased the activities of CAT, GR, and the pool of GSH in larvae. Previous work indicated that lipid peroxide levels were kept low in malathion-exposed larvae, thus we conclude that oxidative stress is overcome by the antioxidant defenses. The increase in the antioxidant metabolism observed in the posthatching phase of development of B. arenarum embryo, thus constitutes a defense against natural and human-generated pro-oxidants present in the aquatic environment.     

Phenylthiourea disrupts thyroid function in developing zebrafish

Thyroid hormone (T4) can be detected in thyroid follicles in wild-type zebrafish larvae from 3 days of development, when the thyroid has differentiated. In contrast, embryos or larvae treated with goitrogens (substances such as methimazole, potassium percholorate, and 6-n-propyl-2-thiouracil) are devoid of thyroid hormone immunoreactivity.Phenythiourea (PTurea; also commonly known as PTU) is widely used in zebrafish research to suppress pigmentation in developing embryos/fry. PTurea contains a thiocarbamide group that is responsible for goitrogenic activity in methimazole and 6-n-propyl-2-thiouracil. In the present study, we show that commonly used doses of 0.003% PTurea abolish T4 immunoreactivity of the thyroid follicles of zebrafish larvae. As development of the thyroid gland is not affected, these data suggest that PTurea blocks thyroid hormone production. Like other goitrogens, PTurea causes delayed hatching, retardation and malformation of embryos or larvae with increasing doses. At doses of 0.003% PTurea, however, toxic side effects seem to be at a minimum, and the maternal contribution of the hormone might compensate for compromised thyroid function during the first days of development.     

Sodium benzoate exposure downregulates the expression of tyrosine hydroxylase and dopamine transporter in dopaminergic neuronsin developing zebrafish

BACKGROUND: Recent data have demonstrated that treatment with sodium benzoate (SB) leads to significant developmental defects in motor neuron axons and neuromuscular junctions in zebrafish larvae, thereby implying that SB can be neurotoxic. This study examined whether SB affects the development of dopaminergic neurons in the zebrafish brain. METHODS: Zebrafish embryos were exposed to different concentrations of SB for various durations, during which the survival rates were recorded, the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the neurons in the ventral diencephalon were detected by in situ hybridization and immunofluorescence, and the locomotor activity of larval zebrafish was measured. RESULTS: The survival rates were significantly decreased with the increase of duration and dose of SB-treatment. Compared to untreated clutch mates (untreated controls), treatment with SB significantly downregulated expression of TH and DAT in neurons in the ventral diencephalon of 3-day post-fertilization (dpf) zebrafish embryos in a dose-dependent manner. Furthermore, there was a marked decrease in locomotor activity in zebrafish larvae at 6dpf in response to SB treatment. CONCLUSIONS: The results suggest that SB exposure can cause significantly decreased survival rates of zebrafish embryos in a time- and dose-dependent manner and downregulated expression of TH and DAT in dopaminergic neurons in the zebrafish ventral diencephalon, which results in decreased locomotor activity of zebrafish larvae. This study may provide some important information for further elucidating the mechanism underlying SB-induced developmental neurotoxicity. Birth Defects Res (Part B)86: 85-91, 2009. © 2009 Wiley-Liss, Inc.     

Analysis of early embryonic great-vessel microcirculation in zebrafish using high-speed confocal μPIV

In the developing cardiovascular system, hemodynamic vascular loading is critical for angiogenesis and cardiovascular adaptation. Normal zebrafish embryos with transgenically-labeled endothelial and red blood cells provide an excellent in vivo model for studying the fluid-flow induced vascular loading. To characterize the developmental hemodynamics of early embryonic great-vessel microcirculation in the zebrafish embryo, two complementary studies (experimental and numerical) are presented. Quantitative comparison of the wall shear stress (WSS) at the first aortic arch (AA1) of wild-type zebrafish embryos during two consecutive developmental stages is presented, using time-resolved confocal micro-particle image velocimetry (μPIV). Analysis showed that there was significant WSS difference between 32 and 48 h post-fertilization (hpf) wild-type embryos, which correlates with normal arch morphogenesis. The vascular distensibility of the arch wall at systole and the acceleration/deceleration rates of time-lapse phase-averaged streamwise blood flow curves were also analyzed. To estimate the influence of a novel intermittent red-blood cell (RBC) loading on the endothelium, a numerical two-phase, volume of fluid (VOF) flow model was further developed with realistic in vivo conditions. These studies showed that near-wall effects and cell clustering increased WSS augmentation at a minimum of 15% when the distance of RBC from arch vessel wall was less than 3 μm or when RBC cell-to-cell distance was less than 3 μm. When compared to a smooth wall, the WSS augmentation increased by a factor of ~1.4 due to the roughness of the wall created by the endothelial cell profile. These results quantitatively highlight the contribution of individual RBC flow patterns on endothelial WSS in great-vessel microcirculation and will benefit the quantitative understanding of mechanotransduction in embryonic great vessel biology, including arteriovenous malformations (AVM).