Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae
© Malone et al; licensee BioMed Central Ltd. 2007
Received: 20 February 2007
Accepted: 10 July 2007
Published: 10 July 2007
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.
This technique, called laser-scanning velocimetry, was used to quantify the effects of pharmacological, developmental, and genetic modifiers of cardiac function. Laser-scanning velocimetry was applied to analyze the cardiovascular effects of morpholino knockdown of osmosensing scaffold for MEKK3 (OSM), which when mutated causes the human vascular disease cerebral cavernous malformations. OSM-deficient embryos had a constricted aortic arch and markedly increased peak cell velocity, a characteristic indicator of aortic stenosis.
These data validate laser-scanning velocimetry as a quantitative tool to measure cardiovascular performance for pharmacological and genetic analysis in zebrafish, which requires no specialized equipment other than a laser-scanning confocal microscope.
The zebrafish Danio rerio are widely used in genome-wide screens to identify genes that regulate the development and function of the vertebrate cardiovascular system [1–3]. Zebrafish are powerful model organisms to define genes relevant to human cardiovascular disease because of their similarity in genetic complexity to mammals, the ease of selective gene knockdown, and their suitability for large-scale mutagenesis screens [4, 5]. A beating heart and functional circulatory system forms within 26 hours of fertilization, and within 48 hours the heart develops into a two-chambered conformation, complete with functional valves. Because of their transparency, severe morphological abnormalities in cardiac development are easily observed, but the functional consequences of these defects are difficult to quantify. Methods are needed for functional analysis of cardiovascular defects that can be utilized by any investigator studying zebrafish cardiovascular physiology. Using an unmodified, commercially-available laser-scanning confocal microscope, we have developed a novel, non-invasive method of measuring cardiac performance in zebrafish embryos that provides measurements similar to those obtained with Doppler echocardiography. The technique, called laser-scanning velocimetry provides a continuous measurement of blood cell velocity that is necessary for estimating cardiac output (velocity integral) and measuring indices of vascular resistance (peak deceleration) and ventricular contractility (peak acceleration).
We first demonstrate that laser-scanning velocimetry is a sensitive and precise tool for measuring the physiological changes during normal cardiovascular development and drug-induced cardiotoxicity. We then use laser-scanning velocimetry to evaluate the cardiovascular function of zebrafish embryos with reduced expression of osmosensing scaffold for MEKK3 (OSM) – a scaffold protein, whose mutation in humans leads to the development of a vascular disorder called cerebral cavernous malformations . Using microangiography we demonstrate that OSM morphant embryos have a constricted aortic arch. Analysis by laser-scanning velocimetry functionally verified the presence of aortic stenosis through a markedly increased peak cellular velocity. These experiments establish laser-scanning velocimetry as a simple yet powerful technique that allows quantitative physiological analysis of cardiac performance in zebrafish embryos.
Results and discussion
Linescan images depict cell flux and velocity within blood vessels
Measured quantities, their physiological correlates and diagnostic utility
↑ in Aortic Stenosis, ↓ in Heart Failure
Negative with valvular dysfunction
↓ in Heart Failure
Indicative of vascular tone and development
Stroke Volume and Cardiac Output
↓ in Heart Failure
A scan line placed in the middle of the vessel was found to be optimal for velocimetry measurements because the flux of cells crossing the line is much higher than at the extreme periphery (Figure 1c, bottom panel). While blood flow within this long artery should be laminar, significant differences in cellular velocity were not observed as the scan line was moved from the center of the lumen to the periphery. The maximum cellular velocity obtained from a scan line placed in the middle of the lumen of a 48 hpf embryo was 0.2459 ± 0.0102 cm/s, and that obtained from a scan line at the periphery of cell flux was 0.2455 ± 0.0276 cm/s. The apparently constant flow profile may be explained by the requirement of cell motion for laser-scanning velocimetry to measure velocities. While the plasma velocity may be lower very near the vessel wall, velocities in this region cannot be determined by laser-scanning velocimetry because few cells are present (Figure 1c, bottom panel).
Laser-scanning velocimetry is a sensitive technique for assessing drug-induced cardiotoxicity
Stroke volume, the volume of blood ejected by the heart in one beat, can be determined by integrating the continuous velocity versus time graphs obtained by laser-scanning velocimetry and multiplying by the cross-sectional area of the aorta. Since the heart is a pump, stroke volume is an excellent functional measure of cardiac performance. Estimates of stroke volume decreased significantly over a concentration range of tricaine that caused little change in heart rate (Figure 2e and 2f). Heart rates were found to remain relatively steady at low to moderate doses of tricaine (Figure 2f). Because heart rates varied little over the dose range tested, cardiac output decreased proportionately with decreases in stroke volume (Figure 2e and 2g). Our assessment of cardiac output by laser-scanning velocimetry (~ 18.6 nL/min at 48 hpf) is in good agreement with volume-approximating methods of estimating cardiac output but our method is based on actual output and does not assume normal heart morphology [11, 12].
Absolute measurements of stroke volume can be obtained when the scan line is placed along the outflow tract prior to any arterial branches. While it is not always possible to image the outflow tract prior to arterial branching, an estimate of stroke volume can be obtained by measuring cellular velocities along the dorsal aorta immediately caudal to the fusion of the lateral dorsal aortas (Figure 1a). Relative comparisons of stroke volume and cardiac output can be made between embryos of the same developmental stage when the scan line is placed in the same position along the artery. To investigate the dependence of stroke volume and cardiac output estimates on scan line position, measurements were acquired along the dorsal aorta in a rostral location (immediately caudal to the fusion of the lateral dorsal aortas) a caudal location (above the cloaca). When the scan line is placed in the caudal position, peak velocity and acceleration are underestimated by 15–20% (see Additional file 1, panels a-c). Interestingly, stroke volume and cardiac output estimates are not significantly changed when measured with either a rostral or a caudal scan line position (see Additional file 1, panels d and e).
Correlating structure and function in the developing heart
At day one, the zebrafish heart exists as an elongated, primitive tube that regularly contracts but has not yet developed into a two-chambered heart . Blood is ejected from the one day heart with gradual but rather sustained changes in velocity (Figure 3a). Both acceleration and deceleration are minimal at this stage (Figure 3d and 3e). By day two the heart has differentiated into a two-chambered heart, has looped into its adult conformation, and the atrio-ventricular valves have formed. Velocity-versus-time peaks for each heart beat are sharper and more frequent than at day one (Figure 3a). Notably, by day two there are significant increases in peak velocity and peak acceleration, as well as in estimates of stroke volume, and cardiac output (Figure 3b, d, g, and 3h). Peak deceleration is an index of vascular resistance and is greatest at this time. This increase in resistance provides enough opposing force to the forward motion of blood that some retrograde motion, indicated by negative minimum velocity values, is observed (Figure 3c and 3e). After day two, vascular resistance decreases and minimum velocities are greater than zero (Figure 3c and 3e). Because there is no reversal of flow direction, peak acceleration actually decreases between days two and three. Thus, it should be noted that, as in ultrasound, peak acceleration is a load-dependent measurement and should be used as an index of contractility only when there are no significant differences in vascular resistance.
Between days 2 and 5 changes in heart chamber orientation and differentiation continue, but no overt structural changes occur . This is reflected by only modest changes in peak velocity, stroke volume, and cardiac output between days two and five (Figure 3b–e, and 3g).
Laser-scanning velocimetry provides functional evidence for the presence of an aortic constriction in OSM-deficient embryos
Additional File 3: restricted circulation movie. Restricted blood flow through constricted aortic arch of control and OSM morpholino-injected embryo (MOV 1 MB)
In human and veterinary cardiology, Doppler echocardiography is used to diagnose aortic stenosis (constrictions). One characteristic change observed in patients with aortic stenosis is an increase in peak velocity because the constricted passage acts as a nozzle to increase the velocity of blood flowing through it . Laser-scanning velocimetry measurements indicated that the peak cellular velocity in OSM morphants was 0.182 ± 0.019 cm/s versus 0.076 ± 0.011 cm/s in control embryos (Figure 4f). These results demonstrate that laser-scanning velocimetry can be used as a diagnostic tool for studying the genetics of cardiac performance as well as identifying genes responsible for congenital heart diseases.
Despite the widespread use of zebrafish for understanding cardiac development and function, few quantitative tools exist to measure functional cardiovascular phenotypes in embryos. Laser-scanning velocimetry meets this need, using a standard laser-scanning confocal microscope for non-invasive measures of cardiac function. High-resolution ultrasound has been used to measure cardiovascular performance in adult zebrafish , and technically could be applied to the analysis of the zebrafish embryonic cardiovascular system. Unlike confocal microscopes that are available at many research institutions, high-resolution ultrasound requires expensive and highly specialized equipment that are not generally available to many investigators. We have shown that laser-scanning velocimetry provides sensitive and precise measures of cardiac function including contractility, cardiac output, vascular resistance, heart rate, and stroke volume. Our data demonstrate that laser-scanning velocimetry easily measures drug-induced cardiotoxicity, performance changes during cardiovascular development and can diagnose anatomic lesions such as aortic stenosis in genetically-manipulated embryos.
Laser-scanning velocimetry provides quantitative and precise measures of several indices of cardiac performance (summarized in Table 1) and offers significant improvements over existing pre-clinical cardiovascular diagnostic techniques. A recent report by Pan, et. al. combines linescans with correlation spectroscopy to measure the velocity of cells travelling through the dorsal aorta of embryonic zebrafish . While determining velocity vectors by correlation spectroscopy is a significant advance, this technique requires two sequential measurements that severely limit its temporal resolution. Discontinuous measurements of cellular velocities have been made in zebrafish embryos using video microscopy, and in transgenic mouse embryos expressing GFP-labeled erythrocytes using perpendicular linescans [15, 19]. Dual spot cross correlation methods of measuring blood cell velocity have existed since the mid-1970s and should be applicable to measuring blood cell velocity in zebrafish embryos with good temporal resolution [20–23]. These methods use two photodiodes spaced a fixed distance apart along the path of blood flow, and cell velocities are calculated based on the time required for a cell to pass between the two detectors. Because cross-correlation methods only measure the average speed of a cell as it passes between the two detectors, they do not continuously measure velocity. This continuity is required for calculating acceleration and deceleration, which contains important information about contractility and peripheral resistance. The use of a parallel scan line allows us to continuously measure the velocity of the same object so long as it is located within the linear scanning region. This feature is what makes laser-scanning velocimetry unique in its temporal resolution for both velocity and acceleration measurements.
Video microscopy has also been used to measure cardiac output in Xenopus laevis, Gallus gallus, and Danio rerio embryos [11, 12, 24–26]. This technique estimates the volume of blood ejected from the ventricle based on the observed changes in its diameter. Our measurements of cardiac output using laser-scanning velocimetry are in good agreement with these published values . While the cardiac output measurements described here are estimates that assume a circular vessel cross-section, and can be underestimated if linescans are obtained from the aorta after arterial branches, they do not require normal heart morphology and valve function. A more exact determination of cardiac output may be obtained using laser-scanning velocimetry in transgenic zebrafish animals where the vessel dimensions are delineated by an endothelial expressed GFP such as fli1-GFP .
Recently, the genes responsible for two mutant zebrafish embryos that have similar cardiovascular phenotypes, santa and valentine, were mapped to ccm1 and ccm2, respectively . The valentine mutant contains a mutation in OSM and exhibits pericardial edema, a dilated heart, and a complete lack of blood flow . Morpholino knockdown of OSM resulted in significantly reduced circulation relative to control zebrafish. The fact that morpholino knockdown of OSM did not completely inhibit blood flow is probably a result of low level OSM expression in contrast to complete loss of functional OSM protein in the valentine mutant.
The observation that OSM plays a role in aortic arch lumen formation has important implications for the pathobiology of cerebral cavernous malformations. Mice homozygous for a gene-trap insertion of β-galactosidase into the ccm1/Krit1 locus also have a constricted aortic arch , indicating that a loss of Krit1 in mouse phenocopies a loss of OSM in zebrafish. When viewed in light of evidence that OSM and Krit1 physically associate with each other at sites of actin reorganization , these data suggest that Krit1 and OSM may act as a complex to organize developmental signals required for proper aortic arch lumen formation.
Zebrafish are a popular model for drug screens because of their genetic similarity to humans, the speed of screening in a physiological context and cost-effectiveness . In addition to providing a diagnostic tool for molecular geneticists, laser-scanning velocimetry has obvious applications in pre-clinical drug development. For example, laser-scanning velocimetry could easily be used to identify anti-arhythmic compounds capable of reversing the fibrillating phenotype of the tremblor or reggae mutants . Similarly, the potency and efficacy of potential inotropic drugs, which increase the force of ventricular contractions in heart failure, could easily be screened in wild-type zebrafish embryos to find those compounds that best increase the peak acceleration of blood cells. As demonstrated in Figure 2, in addition to supporting pre-clinical efficacy studies, laser-scanning velocimetry can also be used to readily identify molecules that possess cardiotoxicity.
Laser-scanning velocimetry quantifies the effects of genetic and pharmacological modifiers of cardiovascular function. With the increasing popularity of zebrafish in basic science and the pharmaceutical industry, the development of additional techniques to correlate physiological function with genes and their chemical modifiers promises to accelerate drug discovery and development in the post-genomic area.
Zebrafish (Danio rerio) embryos used for velocimetry measurements and morpholino injections were obtained by natural spawning of a wild-type AB* line. Embryos were raised at 28.5°C in 30% Danieau solution(17 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO4, 0.18 mM Ca(NO3)2, 1.5 mM HEPES pH 7.6). Zebrafish were handled in accordance with IACUC approved protocols.
Embryos for imaging were placed in a 4-well coverglass-bottom chamber slide (Lab-Tek) in 30% Danieau solution containing the anaesthetic tricaine at a final concentration of 100 μg/ml or as indicated. After a three-minute delay to ensure a steady-state level of anaesthesia, images were captured using an Olympus Fluoview FV1000 laser-scanning microscope with an Olympus PlanApo N (60 ×/1.42 oil) lens using DIC optics. Scan lines were oriented parallel to the flow of blood within the dorsal aorta, just above the yolk ball. Approximately 15,000 lines were acquired per sample at a rate of 0.488 ms/line allowing a maximum velocity of 4.10 cm/s to be measured. Using a 20 μm line and the optical system described above, a minimum scan rate of 8 ms/line is required to record the maximum cellular velocity of 0.25 cm/s observed in these experiments. Cell paths within parallel linescans were traced using the NeuronJ plugin v.1.1.0 for ImageJ v.1.37 . High-quality DIC images and proper adjustment of NeuronJ parameters were critical to obtaining quality tracings. Good results were obtained with the following NeuronJ settings: Hessian smoothing scale = 1.0, cost weight factor = 0.7, snap window size = 1 × 1, path-search window size = 800 × 800, tracing smoothing range = 10, tracing subsampling factor = 1, and line width = 1. NeuronJ trace coordinates were exported as text files then processed to calculate average velocity and acceleration versus time data with Laser-Scanning Velocimetry Data Analyzer software we developed using Visual Basic v6.0 and Access 2003 (Microsoft). Briefly, the position versus time data exported from NeuronJ was smoothed using a moving average function. The instantaneous slope of this smoothed trace was determined for each time point. Where multiple traces were measured for a single point in time, the calculated velocities were averaged. Acceleration data were obtained from the instantaneous slope of velocity-versus-time profiles. A moving average function was used throughout the calculation workflow to minimize noise. The interval of the moving average can be specified as smoothing factors in the data analyzer software. Higher smoothing factors will underestimate measured values but will increase precision. Data are only comparable when calculated using the same smoothing factors and have been specified in the figure legends.
Tricaine dose-response treatment
A single zebrafish embryo at 30 hpf was placed in a coverglass-bottom chamber slide containing 30% Danieau solution without tricaine. Parallel linescans were acquired from the free-floating, non-anesthetized embryo to obtain baseline values of an untreated embryo. After a baseline image was collected, the 30% Danieau was sequentially replaced with 30% Danieau solutions containing increasing concentrations of tricaine from 300 μM to 1 mM. After each solution change, the embryo was allowed to equilibrate with the drug for three minutes prior to imaging.
Stroke volume, heart rate, and cardiac output calculations
Borrowing principles from human ultrasonic Doppler Imaging, stroke volume and cardiac output were extracted from linescans of the dorsal aorta close to the heart, where there is little influence from peripheral vessels. Stroke volume (SV), the volume of blood ejected by the ventricle in one heartbeat, was determined by multiplying the cross sectional area of the aorta (Aaorta.) by the distance blood is pumped along the aorta in one beat (dx) (Equation 1).
SV = Aaorta × dx
The integral of v(t)dt was approximated by calculating the area under the curve of a plot of velocity as a function of time for each heart beat using Origin v.6.0 (Microcal). Cardiac output, the volume of blood ejected by the heart in one minute, was calculated by multiplying the stroke volume by the heart rate (Equation 5).
CO = SV × HR
Heart rates were determined by dividing 60 seconds by the average duration of a heart beat, which was measured by the average peak-to-peak distance of the velocity-versus-time profile.
Morpholino knockdown of OSM
Morpholino oligonucleotides targeting the translation initiation site of the Danio rerio ccm2/osm mRNA were purchased from Gene Tools, Inc. Two OSM-specific morpholinos were used (MO#1: ATCTAATACAGCGAAAATGAAGAGC; MO#2: ATTTGTACGTAGAGATGGAGGAGGA). Morpholino sequences were designed from the +75 to -25 bp region surrounding the initiating ATG of Danio rerio ccm2/osm cDNA (IMAGE Clone 7396516) by Gene Tools, Inc. A non-targeting morpholino or 1× Danieau solution (57 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES pH 7.6) were used as controls. Unless otherwise specified, morpholinos were dissolved to 2 ng/nl in 1× Danieau solution. Approximately 5 nl of morpholino solution were injected into embryos between the 1- and 4-cell stage. Repression of OSM protein was confirmed by immunoblotting using a rabbit polyclonal anti-OSM antibody produced against recombinant full-length mouse OSM .
Capped OSM mRNA synthesis and rescue
Capped, in vitro transcribed ccm2/osm mRNA was synthesized using Mmessage Mmachine T7 kit (Ambion) from the coding region of a Danio rerio OSM cDNA (IMAGE Clone 7396516). Embryos between the 1- and 4-cell stages were injected with 5 nl ccm2/osm mRNA (20–100 pg/nl) together with an OSM morpholino that targets the 5' UTR (MO#1) at 2–4 ng/nl).
In silico microangiography is an image processing approach to representing motion in a series of two-dimensional images. A DIC movie of cells flowing through the vasculature of anesthetized embryos at 28 or 48 hpf was collected as a set of 100 12-bit tiff images recorded at 15 ms intervals (X, Y).t. Using Metamorph IP software (Molecular Devices) a difference image stack was created by dividing adjacent time points; that is, for time t = n, Difference.n = (X, Y).n+1. *1000/(X, Y).n.. The resultant difference stack has intensity values only where changes occurred from one time point to the next. Maximum intensity projections through the differences stack produce an image correlating to blood cell movement over the duration of the original movie.
The authors are grateful to Sue Donerly for technical assistance with zebrafish husbandry and embryo manipulation. Funding was generously provided by the National Institutes of Health to MHM (HL084971) and GLJ (GM30324 and GM68820).
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