© 1996 American Heart Association, Inc.
Articles |
In Vivo Assessment of Embryonic Cardiovascular Dimensions and Function in Day-10.5 to -14.5 Mouse Embryos
NIH SCOR in Pediatric Cardiovascular Diseases, University of Rochester School of Medicine, Strong Children's Research Center, Rochester, NY.
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Abstract |
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Embryonic cardiovascular function has been extensively studied in vivo in the chick embryo. However, the geometry of mammalian and avian hearts differs; the mammalian cardiovascular system is coupled to both yolk sac and placental circulations, and unique murine genetic models associated with structural and functional cardiovascular defects are now available. We therefore adapted techniques validated for the chick embryo to define cardiovascular dimensions and function in the mouse embryo. We bred C3HeB female and C57Bl/J6 male mice and ICR pairs for experiments on embryonic days (EDs) 10.5 to 14.5 (n=130 dams). After maternal anesthesia (pentobarbital, 60 mg/kg IP), laparotomy, and sequential regional hysterotomy, we exposed and then imaged individual embryos at 60 Hz (video) in the ventral and/or left anterior oblique views while maintaining uteroplacental continuity. We measured epicardial chamber dimensions and then calculated right and left ventricular elliptical volumes from areas. In addition, we measured pulsed-Doppler blood velocity across the atrioventricular cushions and ventricular outflow tract. We maintained embryonic temperature with a heated surgical platform, topical oxygenated and warmed buffer, and warming lamps. Embryonic heart rate increased from 124.7±5.2 to 194.3±13.2 bpm from EDs 10.5 to 14.5 (P<.01). Right and left ventricular end-diastolic and end-systolic dimensions increased (P<.05 by ANOVA for each). Maximal ventricular mean inflow and outflow velocities increased from 62.33±4.06 to 106.23±11.59 and from 55.79±6.11 to 91.61±6.93 mm/s, respectively (P<.05 by ANOVA for each). Thus, as has been done for chick and rat embryos, the maturation of murine embryonic cardiovascular function can be quantified in vivo, setting the stage for the investigation of structure-function relations in mouse models of cardiovascular development and disease.
Key Words: mouse embryo • cardiovascular development • ventricular function • video microscopy • pulsed Doppler
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Introduction |
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Numerous investigators interested in the pathogenesis of congenital cardiovascular anomalies have recognized the relationship between cardiac function and form and have focused attention on the period of early cardiac morphogenesis.1 2 3 In the chick embryo, ventricular pressure, ventricular cross-sectional area, and cardiac output can be accurately measured shortly after the onset of cardiac contraction.4 5 In addition, the structurally simple embryonic heart accelerates ventricular growth in response to experimentally increased developed pressure.6 However, the investigation of embryonic cardiovascular function has broadened to include mammalian7 8 9 and other nonmammalian10 species. Because of the morphological and functional differences between the avian and mammalian cardiovascular systems, we have adapted existing technologies for embryonic physiological assessment of the intact murine embryonic circulation.
In order to compare developmental stages in chick,11 12 13 mouse,14 15 16 17 and human18 embryos, rigorous detail must be paid to the timing of gestation and to the identification of external and internal markers of morphogenesis. The external and internal morphological characteristics of the murine embryonic heart have been characterized by scanning electron microscopy starting at embryonic day (ED) 10 and proceeding at 8-hour intervals to ED 17,14 15 which corresponds with days 2 to 8 in the chick embryo.13
Although there are many similarities in the three-dimensional morphogenesis of the embryonic heart across species, there are also important distinctions between species. For example, the embryonic chick heart is larger than the embryonic mouse heart at comparable developmental stages,11 14 19 and there are two pairs of conotruncal ridges forming the outlet septum in the chick versus one pair in the mouse.11 The mouse embryo has a prominent interventricular sulcus present on the exterior surface of the heart that is absent from the chick embryo at comparable developmental stages. In the mouse embryo, the left ventricle is initially prominent; the ventricles appear balanced from ED 11 (16 hours) to ED 13, and then the right ventricle predominates. Overall, the rapid growth of the left ventricle is most prominent in the mouse embryo, whereas the growth of the right ventricle is most prominent in the chick.11
Video microscopy and planimetry of the beating embryonic chick left atrium reveals a smooth-walled endocardium, a spherical shape, and a distinct atrial contraction phase.20 Cinephotography and videography can be used to accurately trace embryonic ventricular epicardial5 21 22 or endocardial23 24 25 borders in order to calculate ventricular volumes. In addition, video images can be combined with measures of ventricular pressure to quantify diastolic and systolic properties in chick and rat embryos.22 26 27 28 Embryonic blood flow has been measured using pulsed-Doppler techniques in a wide range of species. In the chick embryo, ventricular stroke volume measured from the dorsal aorta increases from 0.01 to 0.69 mm3 from stages 12 to 29, and because of the coincident increase in heart rate, cardiac output increases 160-fold.19 Intracardiac blood flow has been characterized by measuring the flow across the atrioventricular (AV) cushions, and ventricular filling has distinct passive and active phases. There is a progressive increase in passive ventricular filling during primary cardiac morphogenesis associated with increasing ventricular compliance.29 In the rat embryo at comparable developmental stages, pulsed-Doppler measures of blood flow reveal similar results.7 22 30 31 Blood flow has been assessed noninvasively in mouse embryos as early as ED 10.5 using maternal transabdominal Doppler.9 The human embryo at comparable stages reveals distinct passive and active ventricular filling phases and a geometric increase in blood flow and contractility with development.8 32
In addition to the investigation of normal embryonic cardiovascular structure and function, cardiac morphogenesis has been experimentally altered and investigated using teratogen, targeted cell, polygenic, and gene-targeting strategies.33 With the advent of the modern molecular era, cardiovascular morphogenesis and function are now under intense investigation using targeted cell and gene techniques.34 Replication-deficient retroviruses have been used to determine myocyte cell fate in the chick embryo.35 36 The iv/iv mouse has been studied extensively in the investigation of asymmetric cardiac looping.37 In the mouse, the identification of chamber-specific promoters has allowed the description of a number of cardiac and skeletal muscle–specific genes and proteins38 39 40 41 42 that are crucial in regulating myocardial growth and function. In addition, a number of targeted reductions of critical genes have resulted in a nonspecific embryo lethal phenotype associated with abnormal cardiac morphogenesis.43 Thus, the quantitative definition of embryonic cardiovascular function and the impact of alterations in basic structure-function relationships are crucial for defining the pathogenesis of altered cardiac phenotypes.
We therefore adapted techniques developed to determine cardiovascular dimensions and function in the chick embryo to the murine embryo. We bred both C3HeB female and C57Bl/J6 male mice and ICR pairs and studied embryos at EDs 10.5 to 14.5. Murine embryonic heart rate increased from 124.7±5.2 to 194.3±13.2 bpm from EDs 10.5 to 14.5 (P<.05). Right ventricular and left ventricular end-diastolic and end-systolic dimensions increased from EDs 10.5 to 14.5 (P<.05 by ANOVA for each). Maximal mean ventricular inflow and outflow velocities increased from 62.33±4.06 to 106.23±11.59 and from 55.79±6.11 to 91.61±6.93 mm/s from EDs 10.5 to 14.5, respectively (P<.05 by ANOVA for each), comparable to increases seen in chick and rat embryos. Thus, the maturation of murine embryonic cardiovascular function can be assessed in vivo, setting the stage for the investigation of structure-function relations in mouse models of cardiovascular development and disease.
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Materials and Methods |
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Breeding
We bred both C3HeB female and C57Bl/J6 male mice (The Jackson Laboratory, Bar Harbor, Me, and Charles River Laboratories, Wilmington, Mass) and ICR pairs (Harlan Sprague Dawley, Inc, Indianapolis, Ind) and studied embryos at EDs 10.5 to 14.5 (n=130 dams) under university-approved animal research protocols Nos. 95-3 and 95-100. Each female mouse received an intraperitoneal injection of pregnant mares' serum gonadotropin (5 IU in 0.2 mL of phosphate buffer, catalog No. G-4877, Sigma Chemical Co) on day -2.5 and then an intraperitoneal injection of human chorionic gonadotropin (5 IU in 0.2 mL of phosphate buffer, catalog No. CG-5, Sigma) on day -0.5.44 Female mice were then bred overnight, and the presence of a vaginal plug on the morning after mating signified ED 0.5 of gestation. Breeding success using the hyperovulation protocol varied from 50% to 75% of the dams. Each pregnant mouse had 6 to 12 embryos available for study.
Maternal Preparation
The physiological assessment of embryos from one dam required 1 to 2 hours. We used pentobarbital (60 mg/kg IP) for maternal anesthesia and assessed the plane of anesthesia by the absence of startle and toe pinch reflexes.7 45 Pentobarbital may depress cardiovascular function in the dam or embryos, and future studies will need to address this potential confounder.45 A second dose of 20 mg/kg of pentobarbital was given intraperitoneally, as needed, on the basis of animal activity, although this was rarely required. Once anesthetized, the dam was placed in a supine position on a custom glass maternal/embryo chamber, which included a vertical trough for the pregnant mouse and a lateral, elevated, smaller chamber for dissecting individual embryos (custom design, Radnotti Glass Technologies). Because embryonic heart rate is extremely sensitive to changes in environmental temperature and because embryonic cardiovascular function is profoundly affected by alterations in cycle length,31 46 47 maternal and embryonic temperature were maintained by a 15-L/min flow of 40°C water through the hollow wall of the glass chamber using a circulating water bath (model Ex221, Neslab Instruments, Inc).
Surgical Technique
The lower abdomen was not shaved for these studies. A midline vertical laparotomy was performed from the umbilicus to immediately above the symphysis pubis. The linea alba was then identified and incised, exposing the bicornuate uterus and discrete swellings identifying the developing embryos.7 In order to generate a fluid-filled abdominal well, a 25-mm-diameter plastic dish containing a 10-mm-diameter circular opening was positioned directly over the laparotomy site and sutured to the maternal abdominal wall at 12-, 3-, 6-, and 9-o'clock positions using interrupted 5-0 silk sutures (Express Supply). Each pregnant uterine horn was then sequentially incised 180° from the uterine vascular pedicle in the direction of the exposed myometrium over the distinct swellings associated with developing embryos.7 Upon incision of the uterus, the embryonic yolk sac was gently exposed with the placenta and uterine vessels intact, and the embryo was then released from the yolk sac using microforceps (Roboz Surgical Instruments). Small squares of damp cotton gauze were used to stabilize embryonic position for study. In this fashion, uteroplacental continuity was maintained throughout the investigation of each individual embryo. Embryonic temperature was maintained using ambient heat lamps, the double-walled glass chamber, and topical warmed Krebs-Henseleit buffer (KHB) bubbled with 95% O2/5% CO2 (Air Products). Supplemental topical warmed KHB was used to maintain a fluid surface over the embryo at all times. Stability of an individual mouse embryo varied between 
3 and 10 minutes. Data were recorded in each embryo as soon as the embryonic heart could be fully visualized for video recording or the Doppler crystal could be positioned for blood velocity recording. All measures were not acquired in all embryos. Signs of reduced embryo viability included extreme pallor, irregular heart rate, or evidence of obvious bleeding.
Embryo Microscopy
Experiments were performed and analyzed at an integrated physiology and morphometry workstation (n=75 dams, n=65 embryos).5 Video images were acquired using a photomacroscope (model M400, Wild Leitz, USA, Inc), a video camera (model 70, Dage-MTI) with a grade 1 Newvicon tube, a fiberoptic light source (Dolan-Jenner Industries), a 60-Hz video recorder (model VR9670, Magnavox), and a time-date generator (model VTG-33, FOR.A). Real time±0.005 seconds was recorded on each field. A 10-µm scale–scribed glass standard was recorded in the plane of each embryo after imaging.5 We studied mouse embryos at 24-hour intervals from ED 10.5 through the completion of cardiac septation at ED 14.5.15 This stage range is similar to stages 14 to 29 in the chick embryo, EDs 12.5 to 15 in the rat embryo, and days 29 to 41 in the human embryo.13 We imaged individual embryos in the ventral position (EDs 11.5 to 14.5) and/or left anterior oblique position (EDs 10.5 to 12.5) while maintaining uteroplacental continuity. Each mouse embryo was scored for developmental stage on the basis of morphological markers, including maturation of the yolk sac circulation, eye and limb bud development, and the external form of the embryonic heart.14 15 16 Embryonic developmental stage varied with embryonic position in the uterine horn, as has been previously noted.14 The heart was imaged through the translucent anterior splanchnopleure for ED-10.5 to -12.5 embryos; however, for ED-13.5 and -14.5 embryos, we incised the anterior chest wall with a U-shaped incision to expose the embryonic heart. In contrast to our previous method in the chick embryo, ventricular tetany was not produced with the topical application of 2 mol/L NaCl because of the concern regarding contamination of the abdominal contents. Therefore, ventricular wall volume could not be calculated or subtracted from total ventricular volume determined by epicardial planimetry.26
Individual video fields were analyzed at workstations that included a minicomputer (Gateway2000 486DX2/50), monitor (Multisync II, NEC Information Systems), frame-grabbing board (Targa model M8, Jandel Scientific), video analysis software (JAVA, Jandel Scientific), and multipurpose video monitor (model PVM1271Q, Sony Corp).5 Intraobserver and interobserver errors of area measurement by planimetry were not significant (P>.29 and P>.96, respectively).5
The video planimetry protocol for each embryo included calibration of measurement software for length (mm) and area (mm2) from the recorded scribed standard image, followed by planimetry of sequential video fields for major and minor cardiac axis diameters and epicardial ventricular cross-sectional area of the right and/or left ventricles for three cardiac cycles (Fig 1
). There is an obvious interventricular sulcus to delineate the primitive murine embryonic right and left ventricles. In the chick embryo, the conotruncal cushions are visible, aiding in the separation of ventricle from outflow chamber. In the mouse, we attempted to transect the right ventricle at a similar point, ie, at the proximal portion of the conotruncal cushions. We used the external feature of the rapid tapering of the right ventricular diameter as the site of proximal conotruncal cushions. This site of division may be somewhat subjective, but we attempted to consistently select the same region between embryos. Right ventricular major- and minor-axis diameters were measured from ventral views, and left ventricular major- and minor-axis diameters were measured from ventral and/or left lateral views (Fig 2). The maximum long axis of the embryonic right ventricle was drawn parallel to ventricular outflow, and the maximal short axis was then drawn perpendicular to the long axis. Because of differences in the shape of the embryonic right and left ventricles in the ventral view, the long axes for the right and left ventricle were not always parallel.
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We calculated heart rate (bpm) from the cycle length of sequential end-diastolic images. We then calculated the maximum and minimum major and minor cardiac diameters and maximum and minimum epicardial cross-sectional areas. Because of the unique and different shapes of the murine embryonic right and left ventricles throughout development14 15 and because of the range of mathematical methods applied to calculating embryonic ventricular volume, we calculated right and left ventricular volumes using two methods. We calculated ventricular volume from measured epicardial cross-sectional area using the model of an ellipsoid of revolution with a fixed aspect ratio. The ellipsoid equation was derived from equations for the cross-sectional area (A) of an ellipsoid, A=(
ab)/4, and the volume (V) of an ellipsoid of revolution, V=(ab2)/6, where a is the semi–major axis diameter, and b is the semi–minor axis diameter. Assuming a fixed aspect ratio of a/b=4/3, then V=0.65A3/2. We also calculated ventricular volume from the measured major-axis (a) and minor-axis (b) diameters using the model of an ellipsoid of revolution. We used each method to calculate maximum and minimum right and left ventricular volumes and right and left ventricular stroke volumes for each day of development.
Pulsed-Doppler Velocimetry
We measured the velocity of ventricular filling and ejection in 65 dams and 85 embryos using a 20-MHz directional pulsed-Doppler velocimeter and a 750-µm-diameter piezoelectric crystal positioned to record phasic blood velocity across the AV cushions and/or ventricular outflow tract (model 545, Department of Bioengineering, University of Iowa, Iowa City) as has been described for the rat embryo. To maintain embryonic temperature, the piezoelectric crystal was submerged in warmed KHB buffer when not in use. The piezoelectric crystal was manually positioned over the ventricular apex to measure ventricular filling and ejection velocities with an incident angle of blood flow and an ultrasound beam of <10°.29 The ventricular inflow pattern corresponded visually with atrial contraction, and the ventricular ejection pattern corresponded visually with ventricular ejection, as has been previously described.7 The phasic velocity profile contained ventricular filling and ejection phases (Fig 3). The analog velocity waveform was digitally sampled at 2-ms intervals (model MIO16, National Instruments), viewed with a customized virtual instrument developed using LABVIEW software (National Instruments), and stored on a computer hard disk. This system is linear over a range of 0 to 16 cm/s.4 19 Because of dynamic changes in AV canal cross-sectional area that occur with atrial systole and conotruncal cross-sectional area that occur with ventricular ejection, absolute blood flow could not be accurately calculated from these velocity measurements. We determined peak instantaneous mean velocity for active ventricular filling and ventricular outflow for three to five cardiac cycles for each embryo. We integrated the area under the curve for outflow tract velocity and then divided by the cycle length for three to five cardiac cycles to determine mean outflow tract velocity for each embryo.19
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Statistical Analysis
Embryos were included for analysis if the epicardial area was reproducible for consecutive cardiac cycles and the ventricular velocity waveforms remained stable over the study period. Data were not partitioned by strain before analysis, although strain-associated differences in mature cardiovascular function have been reported.48 Data are reported as mean±SEM. Heart rate, velocity, and dimensional data were analyzed by ANOVA followed by a Bonferroni/Dunn correction test for determining statistical significance at a level of 5% 
error between groups for a single measure, eg, peak outflow tract velocity for ED 10.5 versus ED 14.5. We used unpaired t tests to test the hypothesis that left ventricular dimensions were similar in the ventral and left lateral views. We used paired t tests, ANOVA, and linear regression to determine the difference between ventricular volume calculation methods, eg, ellipsoid left ventricular volume calculated from cross-sectional area with fixed aspect ratio versus ellipsoid left ventricular volume calculated from major- and minor-axis diameters.
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Results |
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As have other investigators, we found the outbred ICR mice to have a higher breeding success. The ICR mice required a shorter period for onset of sedation and less frequent redosing. The short umbilical vascular length and the importance of maintaining uteroplacental continuity required the development of an intra-abdominal investigation of the embryos. Because of the variable position of the exposed embryo and the fragile nature of the embryo, not all video views or velocity patterns were obtained on each embryo. Likewise, the piezoelectric pulsed-Doppler crystal had to be manually manipulated in order to obtain the maximal ventricular inflow and ejection velocities.
Murine embryonic heart rate increased from 124.7±5.2 to 194.3±13.2 bpm from EDs 10.5 to 14.5 for the velocity study (P<.01). We frequently noted a transient decrease in heart rate with the opening of the yolk sac, which reversed with the application of additional warmed KHB buffer. We initially noted a slower heart rate in embryos studied by pulsed-Doppler than by video microscopy; however, after routinely preheating the piezoelectric crystal, heart rates for the embryos studied by pulsed-Doppler were actually faster than for embryos studied by video alone (Tables 1 and 4). In contrast to the embryos studied by pulsed-Doppler, the heart rates of ED-13.5 and -14.5 embryos studied by video microscopy were slower than expected; this was likely due to cooling during dissection and positioning.
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The external contours of the embryonic right and left ventricles viewed by video microscopy resembled the contours previously described by scanning electron microscopy.14 When viewed ventrally, the embryonic right and left ventricles were exposed, and in contrast to the developing chick heart, a prominent interventricular sulcus was evident (Fig 1
). The ventral view could not be obtained in ED-10.5 embryos because of embryo curvature. When viewed from the left lateral approach, the embryonic left atrium was partially visualized, the left ventricle was fully exposed, and only the anterior portion of the conotruncus was visible (Fig 2). After ED 12.5, the opaque chest wall and left limb bud obscured the left lateral view.
The major ventricular axes for the right and left ventricles were oriented approximately parallel to each other (Fig 1). Data on cardiac dimensions from EDs 10.5 to 14.5 are summarized in Tables 1 through 4. Ventral-view end-diastolic right ventricular major and minor axes increased from 1.18±0.07 to 1.42±0.05 mm and from 0.90±0.03 to 0.97±0.02 mm, respectively, from EDs 11.5 to 14.5 (Fig 4A, Table 1). Ventral-view end-diastolic left ventricular major- and minor-axis diameters increased from 1.05±0.04 to 1.31±0.05 mm and from 0.82±0.05 to 0.91±0.03 mm, respectively, from EDs 11.5 to 14.5 (Table 1). Left ventricular dimensions calculated from ventral and left lateral views were similar for ED-11.5 embryos, but both maximum and minimum minor-axis diameters differed at ED 12.5, resulting in differences between ventral and left lateral calculations of left ventricular volume and stroke volume (Tables 1 and 3).
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Epicardial right and left ventricular end-diastolic and end-systolic cross-sectional areas measured from the ventral and left lateral views increased from EDs 10.5 to 14.5 (Fig 4B, Table 1). The ratio of major-axis–to–minor-axis diameters provided support for the use of an elliptical model for calculating right or left ventricular volume in the mouse embryo (Table 2). However, ellipsoid left ventricular volumes calculated from epicardial cross-sectional area and from measured major and minor axes in the left lateral view resulted in differences in maximum volume, minimum volume, and stroke volume for EDs 10.5 to 12.5. (Fig 5, Table 3).
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Ventricular inflow and outflow velocities were usually measured with the embryo in the right lateral decubitus position. Isovolumic contraction and relaxation time periods were not always evident in the Doppler signal, depending on the location of the Doppler sample volume (Fig 3). Peak instantaneous ventricular mean inflow velocity increased from 62.33±4.06 to 106.23±11.59 mm/s from EDs 10.5 to 14.5 (P<.05, Table 4). Peak instantaneous ventricular mean outflow velocity increased from 55.79±6.11 to 91.61±6.93 mm/s from EDs 10.5 to 14.5 (P<.05, Table 4). The sample volume of the 750-µm-diameter piezoelectric crystal could not selectively measure velocities from the right versus left ventricular inflow or outflow regions in the nearly septated ED-14.5 embryos.
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Discussion |
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Embryonic cardiovascular functional maturation is now under investigation in a broad range of species, including chick,28 rat,7 mouse,9 and numerous lower vertebrate species.49 Although there are structural and functional similarities across species, there are also important differences related to unique metabolic and environmental demands specific to each species. These data are critical in the interpretation of data available at comparable developmental stages in the human embryo.8 Of note, most congenital cardiovascular defects likely occur during the period of primary cardiac morphogenesis.1 Cardiac morphogenesis is completed by stage 36 (day 10 of 21) in the chick embryo, by embryo day 15.5 of 21 in the mouse embryo, and week 8 of 40 in the human embryo.13 Expanding the investigation of embryonic cardiovascular function to the mouse model provides the additional advantage of a wide range of targeted-gene paradigms related to cardiovascular structure and function. Thus, the study of embryonic cardiovascular structural and functional maturation provides a crucial new opportunity for developing information directly relevant to our understanding of normal heart development and to test novel strategies for the early diagnosis and therapy of congenital cardiovascular mal-formations.
The present study describes the surgical technique and equipment necessary to measure murine embryonic cardiovascular function in vivo. Of note, our surgical technique differs from previous methods described for the rat embryo22 in that we attempted to preserve uterine blood flow and uteroplacental continuity rather than to remove the uterine segment from the abdomen as a tissue block before visualization and instrumentation of the embryo. For the present study, we did not assist maternal ventilation, in contrast to the approach described to assess embryonic cardiac function in retinoid receptor–deficient mouse embryos.43 After pentobarbital sedation, dams breathed regularly with a respiratory effort qualitatively unchanged from the unrestrained state. Clearly, additional research is needed to define the ventilatory drive, requirements, and reserve of the pregnant mouse and the impact on anesthetic agents on maternal and embryonic cardiovascular function.
The present study also provides new data on heart rate, ventricular chamber dimensions, and ventricular inflow and ejection velocities in the mouse embryo during the period of rapid cardiac morphogenesis. The range of mouse embryos studied, EDs 10.5 to 14.5, is similar to the periods of primary cardiac morphogenesis in chick, rat, and human embryos.13 The measured murine embryonic heart rates were similar to the rates of 114±4 to 208±5 bpm observed in stage-14 to -29 chick embryos19 and 122±3 to 198±5 bpm observed in day-12 to -15 rat embryos.7 Obviously, maximal physiological heart rate occurs at the intrinsic temperature and in the absence of cardiovascular stress. Because the ED-10.5 to -11.5 embryos are very fragile, video imaging of cardiac dimensions was performed almost immediately after embryonic visualization, resulting in higher heart rates for embryos when more time and manipulation were required for Doppler assessment. In contrast, ED-13.5 and -14.5 embryos were assessed by Doppler rather rapidly, resulting in higher heart rates, whereas video microscopy required more time and more surgical manipulation to completely expose the ventricular borders, resulting in lower heart rates. However, we included the full heart rate ranges for each set of embryos. In general, the preinnervated embryonic heart does not alter heart rate to compensate for dramatic changes in ventricular preload and afterload28 ; thus, a "normal" heart rate is insufficient to prove that our experimental technique for the mouse embryo did not significantly disturb the embryo's hemodynamic state.
We describe a progressive increase in right and left ventricular dimensions during primary cardiac morphogenesis in the mouse embryo, as has been reported previously for numerous species.5 14 15 48 In contrast to the lack of external markers for the developing interventricular septum in the early chick embryo, the mammalian heart has a distinct interventricular sulcus that aids in the measurement of right and left ventricular volumes. Our measures of minimum and maximum left ventricular cross-sectional area for the ED-11.5 to -14.5 mouse embryo were in the same range as values published for the stage-14 to -24 chick embryo (0.21±0.01 to 0.94±0.03 mm2 and 0.34±0.03 to 1.26±0.07 mm2, respectively) and day-12 rat embryo (0.5 to 0.7 mm2).22 These data on epicardial measures are not directly comparable to data generated from planimetry of the endocardial border viewed by light24 25 or fluorescence microscopy.43 Although the major-axis and minor-axis aspect ratios were similar for the embryonic right and left ventricles in the ventral views, the aspect ratios of the left ventricle differed in the ventral and left lateral views, with the left ventricle appearing more spherical in the lateral view.
Volume estimates from video microscopy need to be compared with volume calculations from three-dimensional data sets in order to determine the appropriate method for estimating ventricular volume.28 Regarding the validation of video-determined cardiac dimensions, the most valuable reference dimensions are the resting dimensions of each individual embryo. Individual tracings are reproducible on the basis of interobserver and intraobserver error measurements.5 In addition, it is extremely important to note that each component of the video imaging system must be optimized in order to visualize a crisp epicardial border of the nearly translucent embryonic heart. Many recordings that appear during the acute experiment to be satisfactory for analysis are later found to have inadequate gray-scale contrast for edge detection. However, with the proper equipment and methods, the ventricular borders are distinct and readily determined across a broad range of heart rates47 and filling volumes.27 Recognizing the limitations of our calculation of embryonic ventricular volume, we noted that the right ventricle was larger than the left ventricle from EDs 10.5 to 14.5 in the mouse. The ability to determine unique right and left ventricular volumes is of particular value, because the phenotype of numerous congenital cardiovascular malformations includes hypoplasia of the right or left ventricle. In addition, the murine model of retinoic acid–induced transposed great vessels displays marked right ventricular hypoplasia.50
Doppler velocimetry can be applied in both invasive and noninvasive methods for serial examinations of numerous embryo species. Pulsed-Doppler velocimetry methods include the use of a single crystal 20-MHz system developed at the University of Iowa that provides a phasic signal of instantaneous mean blood velocity (for review, see Keller28 ) and the use of pediatric cardiology clinical ultrasound equipment that provides a phasic signal containing the full instantaneous velocity spectrum. The combination of integrated mean velocity during a single cardiac cycle and the orifice cross-sectional area allows the calculation of blood flow.4 In contrast, the clinical peak instantaneous velocity measure is related to the instantaneous pressure difference across an orifice. Thus, a critical validation and comparison of Doppler velocimetry techniques is a prerequisite for the interpretation of data on embryonic cardiovascular functional maturation.
Our measured values of maximal instantaneous mean filling velocities in the mouse embryo during this developmental period, 62.33±4.06 to 106.23±11.59 mm/s, were similar to the instantaneous mean active filling velocities of 11.21±1.62 to 82.46±16.78 mm/s in stage-14 to -27 chick embryos (N. Hu, unpublished data, 1996). Distinct passive and active ventricular filling phases have been noted in the chick embryo by stage 14,29 in the rat embryo at day 12,22 and in the mouse embryo by ED 10.9 Based on passive/active filling ratios and passive ventricular filling volumes, the avian embryonic heart is more compliant than mammalian embryos at comparable stages.22 Because of the dynamic cross-sectional area of the AV canal region, blood velocity data from this region cannot be directly converted into blood flow data; instead, the area under the curve of passive and active filling velocities has been used to estimate ventricular filling volumes.19 Thus, the simultaneous measurement of dorsal aortic blood flow and AV blood velocity will be necessary to determine ventricular filling volumes in the mouse embryo.
Our measured values of maximal instantaneous mean outflow tract velocities in the mouse embryo were similar to the instantaneous mean outflow tract velocity of 45 to 50 mm/s in the day-12 rat embryo30 and values of 18.29±2.66 to 41.55±4.17 mm/s for instantaneous mean dorsal aortic velocity from stage-14 to -27 chick embryos (N. Hu, unpublished data, 1996). In contrast to instantaneous mean values, our mean outflow velocity over one cardiac cycle was 10.21±0.97 to 23.40±3.05 mm/s from EDs 10.5 to 14.5 in the mouse embryo versus 7.4±0.6 mm/s in the day-12 rat embryo7 and versus dorsal aortic mean velocity ranges from 1.89±0.12 to 15.33±0.62 mm/s in stage-14 to -29 chick embryos, respectively.19
Thus, as has been done for several experimental species, the maturation of murine embryonic cardiovascular function can be assessed in vivo, setting the stage for the investigation of structure-function relations in mouse models of cardiovascular development and disease. A comprehensive understanding of the mechanisms that regulate embryonic function, growth, and morphogenesis will require the quantitative comparison of normal and experimentally altered heart development that is only available in animal models. These experimental paradigms will be crucial to improve the sensitivity and specificity of in utero Doppler assessments of cardiovascular structure and function in human embryos.
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Acknowledgments |
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This study was supported by National Institutes of Health Specialized Center of Research Award P50-HL-51498, by National Institutes of Health Physician Scientist Training Award K11-HL-02498 (Dr Keller), and by a pilot grant from the Strong Children's Research Center.
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Footnotes |
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Reprint requests to Bradley B. Keller, MD, NIH SCOR in Pediatric Cardiovascular Diseases, University of Rochester School of Medicine, 601 Elmwood Ave, Box 631, Rochester, NY 14642. E-mail bkeller@cvdev.rochester.edu.
Received January 26, 1996; accepted May 13, 1996.
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References |
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