| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Toolbox
1 Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario, Canada, M5G 1X5
2 Sunnybrook and Women’s College Health Sciences Centre, Toronto, Ontario, Canada, M5G 1X5
3 Department of Obstetrics/Gynecology, University of Toronto, Toronto, Ontario, Canada, M5G 1X5
4 Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5G 1X5
5 Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada, M5G 1X5
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONReferences |
|---|
2 days after implantation (7 days gestation) to near term (17.5 days) were obtained using frequencies from 25 to 40 MHz. The ectoplacental cone and early embryonic cavities were visible as were the placenta and embryonic organs throughout development to term. We also evaluated the ability of the biomicroscope to detect important features of heart development by examining embryos from 8.5 to 17.5 day gestation in exteriorized uteri using 55 MHz ultrasound. Cardiac looping, division of the outflow tract, and ventricular septation were visible. In postnatal imaging, we observed the heart and kidney of neonatal mice at 55 MHz, the carotid artery in juveniles (8 g body wt) and adults (25 g body wt) at 40 MHz, and the adult heart, aorta, and kidney at 19 MHz. The coefficient of variation of carotid and aortic diameter measurements was 1–3%. In addition, blisters in GRIP1 -/- embryos and aortic valvular stenosis in two adults were readily visualized. Using image-guided Doppler function, low blood velocities in vessels as small as 100 µm in diameter including the primitive heart tube at day 8.5 were measurable, but high blood velocities (>37.5 cm/s) such as in the heart and large arteries in late gestation and postnatal life were off-scale. Accurate cardiac dimension measurements were impeded by poor temporal resolution (4 frames/s). In summary, the multifrequency ultrasound biomicroscope is a versatile tool well suited to detailed study of the morphology of various organ systems throughout development in mice and for hemodynamic measurements in the low velocity range. Doppler; heart; placenta; embryo; newborn; development; aorta; umbilical cord; kidney; mouse; biomicroscope; carotid artery; juvenile; blood velocity; heart valves
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONReferences |
|---|
30,000 genes in the genome. Methods suitable only for adult mice are insufficient because genetic modifications often cause embryonic or perinatal lethality, thus appropriate technology for rapid and accurate assessment of phenotype in mice at all stages of development is required to accelerate progress in this task.
The current paper evaluates the application of a newly developed multifrequency ultrasound biomicroscope that was designed specifically for imaging and hemodynamic evaluation of mice. Like other ultrasound systems, the biomicroscope provides noninvasive and real-time images, as well as Doppler blood velocity measurements, and is suitable for serial in vivo studies, for instance, to study phenotypic expression during development or following gene induction or ablation. In addition, ultrasound biomicroscopy is the only small-animal imaging method suitable for image-guided injections because the images are available in real time and the imaging apparatus does not interfere with access to the mouse. Although conventional ultrasound systems that use frequencies from 7 to 15 MHz have proven useful for imaging and Doppler studies of cardiac function in adult mice (6) and for Doppler studies in mouse embryonic hearts (11), image resolution and Doppler sample volume size are inadequate for detailed study of morphology and hemodynamics in the developing heart of embryos and juveniles (16). The biomicroscope takes advantage of the limited penetration depths required for mouse imaging (5–15 mm) by exploiting high frequencies so that higher resolution images and smaller Doppler sample volumes can be achieved. Image resolutions are increased 10-fold, which is nearly sufficient to compensate for the 20-fold difference in linear dimensions between adult mice and humans (e.g., aortic diameter 1–1.5 mm in mice, 2–3 cm in humans).
The multifrequency biomicroscope is a new imaging system based on the technology of the 40 MHz prototype instrument used extensively by Turnbull and colleagues for imaging and Doppler studies of mouse embryos between 9.5 and 14.5 days of gestation and neonates between birth and 7 days of age (1, 8, 19, 32, 33), and for image-guided microinjection of mouse embryos (10, 17, 22, 32). Unlike the prototype, the multifrequency biomicroscope operates at a range of frequencies between 19 and 55 MHz and so can be used for imaging mice over a wider range of development. In addition, this system has integrated Doppler capability in the high-frequency range and a relatively small sample volume. This study was conducted to explore the potential applications and evaluate the limitations of the multifrequency ultrasound biomicroscope for examining morphology and hemodynamics during development of mice.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONReferences |
|---|
The biomicroscope.
A multifrequency ultrasound biomicroscope (model VS40; VisualSonics, Toronto, Canada; http://www.visualsonics.com) was used in the current study. The biomicroscope has a single transducer with a nominal center frequency of 40 MHz, a diameter of 3 mm, and a focal length of 6 mm. The transducer design has previously been described and characterized (9). One of its features is that it has a wide bandwidth of 120%. This allows the transducer to operate over the frequency range from 16 MHz to 64 MHz. The software of the multifrequency biomicroscope allows the user to select 19, 25, 40, or 55 MHz as the driving frequency for the transducer from a menu on the monitor. Driving frequency can be changed during imaging sessions. In this study, the transducer was mechanically scanned at 4 frames/s to create an 8 x 8 mm two-dimensional (2D) image. The B-scan low-pass filter was set to 80 MHz, and the high-pass filter was "open" (Table 1). The transducer was held stationary to obtain Doppler flow velocity spectra in real time from a sample volume located within the 2D image. The pulsed Doppler default settings [7 cycles per pulse, 40 MHz center frequency, 10 kHz pulse repetition frequency (PRF)] were used. The Doppler sample volume size was dependent on the number of cycles per pulse and the insonation frequency as shown in Table 2. The maximum measurable velocity was 37.5 cm/s, and this would be achieved using a PRF of 20 kHz and an operating frequency of 20 MHz (Table 1). Additional technical specifications and menu options are shown in Table 1.
|
|
|
For transcutaneous embryonic imaging, 14 pregnant mice were studied once between 6.5 and 17.5 days of gestation, and serial observations were conducted daily from 6.5 to 17.5 days of gestation in another 2 pregnant mice. Once anesthetized, the mouse abdomen was shaved and further cleaned with a chemical hair remover to minimize ultrasound attenuation. After prewarming the ultrasound gel, an outer ring of thick gel (Aquasonic 100; Parker Laboratories, Orange, NJ) was filled with a thinner gel (EcoGel 100; Eco-Med Pharmaceutical, Mississauga, Ontario, Canada) over the region of interest, to provide a coupling medium for the transducer. Transcutaneous imaging and Doppler studies of embryos were usually performed at 40 MHz. However, when imaging deep embryos or large embryos in late gestation, a lower frequency (e.g., 25 MHz) was used to obtain greater depth of penetration. We usually imaged two to four embryos in sessions limited to 1-h duration. Sometimes a few embryos were studied thoroughly, or a greater number were examined more selectively. Usually not all embryos in a litter were imaged. Some might be too deep or in an inappropriate orientation to achieve high-quality images in the desired plane of view. We limited time to minimize anesthetic exposure in this serial study, because anesthesia may adversely affect embryonic development (4).
We imaged one pregnant GRIP1 +/- mouse at 12.5 and 13.5 days of gestation to screen embryos for the skin blister phenotype characteristic of GRIP1 -/- mutants (5), to illustrate the use of the transcutaneous approach in phenotype detection. In an additional experiment on one pregnant ICR mouse, an embryo at 10.5 days gestation was imaged transcutaneously using the multifrequency biomicroscope (40 MHz), and the same embryo was then imaged with the conventional ultrasound system (13 MHz) to directly compare the imaging capabilities of the two systems.
Transuterine embryonic imaging was conducted in 10 pregnant mice after surgical exteriorization of the uterus. One mouse was studied on each day from 8.5 to 17.5 days of gestation. In each isoflurane-anesthetized pregnant mouse, one uterine horn (containing 5–7 embryos) was exposed through an elliptical hole cut into the bottom of a petri dish which was filled with warm, circulating phosphate-buffered saline (PBS). To prevent leakage, the petri dish was sealed to the cleanly shaven skin of the maternal abdomen using double-sided tape. Four to six 6-0 silk sutures through the myometrium were tethered to a rubber ring at the edge of the dish to hold the uterus stationary. Care was taken to avoid any tension or pressure on the uterine vessels. Indomethacin (300 µM in PBS) was added to the circulating PBS in the petri dish to inhibit spontaneous uterine contractions (36), which could move embryos through the plane of view. Maternal body temperature was maintained at 36–38°C with the use of a heating pad, a lamp, and a warmer for the circulating PBS. The structure of the embryonic heart was observed using 55 MHz ultrasound, and Doppler flow waveforms were recorded using 40 MHz ultrasound. Several sites of interest within the heart were studied. Two to four embryos were observed in sessions limited to 1.5 h. At the end of the study, mice were killed while still anesthetized.
Postnatal mouse imaging.
Mice were anesthetized with isoflurane as described above. The heart and kidney were imaged at 55 MHz in six mouse neonates (ICR, wild type) at 1–3 days after birth and at 19 MHz in five adult mice (ICR, wild type). During all imaging, the position of the mouse was adjusted to place the structure of interest 6 mm from the transducer, so it would be within the transducer’s focal zone. Each experiment in postnatal mice lasted for about 30–45 min.
In an additional experiment, the hearts of two adult ICR mice (7 wk, 27 and 34 g body wt) were imaged transcutaneously using the multifrequency biomicroscope (19 MHz) and then imaged with the conventional ultrasound system (13 MHz) to directly compare the imaging capabilities of two systems.
Two C57Bl6/C3H hybrid mice were imaged at 19 MHz to test the feasibility of using the system as a secondary screen to investigate the cause of the high peak aortic blood velocities in these mice. Peak blood velocities in the ascending aorta were >200 cm/s, which is twice normal. The abnormal peak velocities were detected in a primary high-throughput screen in our mouse mutagenesis program (http://www.cmhd.ca).
Another five adult mice [ICR, wild type, mean 28 ± 0.8 (SE) g] were imaged at 19 MHz daily over 4 consecutive days to test the reproducibility of aortic inner diameter measurements using the biomicroscope’s on-screen calipers. Aortic diameter during systole (i.e., when the aortic valve was open) was measured at the aortic annulus in the long-axis view and also at the level of the proximal ascending aorta just distal to the aortic sinuses in both long- and short-axis views. On each day, six to eight successive diameter measurements were obtained and were averaged for each site. The coefficients of variation within sessions and in the session means over 4 days were calculated. Inner diameters of the common carotid artery in long-axis views were measured in additional four juvenile (8.1 ± 0.6 g body wt) and six adult (25 ± 2 g body wt) wild-type C57Bl6/J mice. Images were saved when vessels were near their largest diameter (i.e., systole). The mean and coefficient of variation were determined from six to eight successive carotid diameter measurements.
For Doppler recordings, the sample volume was positioned over the structure of interest in the 2D image, and then the biomicroscope was switched to Doppler mode. Doppler velocity spectra appeared in real time on the biomicroscope’s screen but were transferred to a second computer (Doppler Signal Processing Workstation; Indus Instruments, Houston, Texas) for quantitative measurement of velocities and time intervals.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONReferences |
|---|
|
Images of cylindrical cavities cast in agarose showed that the biomicroscope introduced negligible image distortion. The biomicroscope generated images of the cylindrical cavities at all frequencies (19–55 MHz) that, when viewed in cross section (i.e., short axis) were circular and when viewed longitudinally (i.e., long axis) had straight, parallel sides as expected (Fig. 1). Like the biomicroscope, the conventional ultrasound system showed little image distortion of the cylindrical cavities viewed longitudinally. Unlike the biomicroscope, the echoes generated by the lateral walls of the cylindrical cavities were not visible in cross-sectional views so that circular cross sections could not be confirmed (Fig. 1).
Comparison of biomicroscopy and conventional ultrasound imaging in mice.
Transcutaneous images of the same mouse embryo, and same adult mouse heart were obtained using the multifrequency biomicroscope and the conventional ultrasound system (Fig. 2). The 8 x 8 mm image generated by the multifrequency biomicroscope was big enough to include the entire conceptus at 10.5 days gestation (Fig. 2). The embryo, amniotic cavity, and placenta were visible, as were the cerebral ventricles, the common atrium, common ventricle, and outflow tract of the embryonic heart. Cardiac movement was detectable despite the low frame rate (4 frames/s) relative to the high embryonic heart rate (3–4 beats/s). The scan distance and the depth of penetration of the conventional ultrasound system was much greater, so that several embryos could be visualized in one image frame (2.5 cm x 2 cm). Embryos were visible within the amniotic cavity, and although details of the embryonic structure were not detectable, the beating of the heart was more readily detected than with the biomicroscope due to the much higher frame rate of the conventional system.
|
Embryonic imaging.
The multifrequency biomicroscope was used at 40 MHz to image early postimplantation development using a noninvasive, transcutaneous approach. At 7 days of gestation, regions where the cross section of the uterus was enlarged were clearly visible within the maternal abdomen (Fig. 3A). The enlarged regions were relatively dark. Near the center of each enlargement was a small, echo-free region that is likely the proamniotic cavity of the developing embryo (20). It was surrounded by a relatively bright region known to be populated by embryonic trophoblast giant cells that invade the maternal decidua during implantation in the mouse (20). By 7.5 days, three dark regions were visible within the conceptus (Fig. 3B). They likely correspond to the ectoplacental, amniotic, and exocoelomic cavities of the developing embryo (20). At 8.5 days, the embryo and amniotic membrane were visible, and the allantois could be seen emerging from the embryo and approaching the ectoplacental cone (where the chorio-allantoic placenta later develops; Ref. 20) (Fig. 3C). The implantation site looked similar whether viewed in vivo using ultrasound (Fig. 3C) or in histological sections (Fig. 3D). Features were visible at even higher resolution when the uterus was exteriorized and 55 MHz ultrasound was used (Fig. 3, E and F). At this stage, a pulsatile blood velocity signal was first detectable in the heart tube, but no signal was detected within the allantois (Fig. 3, E and F).
|
9.5–10.5 days gestation) through to term using the transcutaneous approach. Relatively superficial embryos were imaged at 40 MHz, and deeper embryos (a more common problem near term) were imaged at 19 or 25 MHz. On day 9.5, the amniotic membrane, amniotic and yolk sac cavities, brain, cerebral ventricles, and heart were visible using transcutaneous imaging (Fig. 4A). Later during embryonic development, many other structures could also be visualized using transcutaneous imaging including the developing paw and forelimb, eyes, lung, liver, kidney, vertebrae, and veins (Fig. 4, B–F). The skin blister phenotype observed in GRIP1 -/- embryos (5) was also readily detectable by the transcutaneous approach (Fig. 5). The umbilical cord and placenta were visible and umbilical Doppler blood velocity waveforms were detectable from day 9.5 of gestation. From day 10.5, Doppler blood velocity waveforms could be recorded separately from the umbilical artery and vein.
|
|
14.5 days gestation appeared bright on ultrasound images, whereas this space generally appears dark in postnatal subjects when viewed at conventional frequencies (e.g., 7.5–15 MHz). This was likely due to higher blood echogenicity caused by the short wavelength of high-frequency ultrasound and the relatively large size of embryonic blood cells. Red cell nucleation may also be a factor, because blood appeared less echogenic in embryos near term (e.g., at 16.5 days, Figs. 6H and 4E), when red cells are still large but are no longer nucleated (25). On day 9.5, the U-shaped heart tube was clearly visible and Doppler blood velocity waveforms could be recorded separately from the inflow and outflow regions of the heart tube (Fig. 6, A–D). On day 11.5, it was possible to detect the process of division of the outflow tract into the ascending aorta and main pulmonary artery (not shown). On day 12.5, the separation of the aorta and main pulmonary artery appeared complete (not shown), but the interventricular septum was visibly incomplete, and flow streams from both ventricles could be seen entering the aorta (Fig. 6F). By day 13.5, the embryonic ventricles were fully septated, the atrioventicular valves were visible, and the heart had a mature fetal form (Fig. 6G). After day 15.5, the heart chambers began to darken in the ultrasound image, and the ventricular wall, endocardium, and septum became easier to discern (Fig. 6H). The improved contrast after day 15.5 meant that ventricular chamber dimensions and wall thickness measurements became feasible.
|
|
|
|
2.5 and 2.0 mm). Histological examination revealed marked aortic valve leaflet dysmorphology (Fig. 9C) characterized by markedly increased amounts of myxomatous connective tissue in the stroma of the leaflets. The valve dysmorphology likely resulted in aortic valvular stenosis, leading to high ascending aortic blood velocities, and a poststenotic aortic dilatation.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONReferences |
|---|
5 to 15 mm). The system would also be suitable for other applications with similar penetration depth and resolution requirements (e.g., other small animal species, superficial structures in larger animals). The other main advance of the multifrequency biomicroscope is the integration of pulsed Doppler capability at these frequencies. Implementation of high-frequency Doppler permits low blood velocities such as in the early embryonic cardiovascular system and in the microcirculation of mice at all ages to be studied. The same transducer is used for 2D imaging and pulsed Doppler, and the Doppler sample volume is small (e.g., 70 µm lateral x 250 µm axial) relative to conventional ultrasound systems (e.g., 350 µm lateral x 1,000 µm axial). These capabilities allow the sample volume to be located within small structures in 2D images. Using phantoms, we showed that the lateral resolution of a conventional ultrasound system operating at 13 MHz was between 200 and 500 µm, and the axial resolution was between 50 and 100 µm. Given the small size of cardiac structures in adult mice and the even smaller size of structures in mouse embryos, this resolution is not ideal. Specialized clinical instruments are available that use higher frequencies for imaging the human eye (10 or 50 MHz; model P45; Paradigm Medical Industries, San Diego, CA; http://www.paradigm-medical.com), the human peripheral vasculature (20 MHz, model AU5; Biosound Esaote, Indianapolis, IN; http://www.advanced-ultrasound.com), or the human skin (20 MHz; EPISCAN, Longport International, Silchester, UK; http://www.longport-intl.com). However, these instruments do not support Doppler ultrasound recordings, nor do they allow frequencies to be selected throughout the range best suited for mouse imaging (i.e., 19–55 MHz), so these instruments are less versatile than the multifrequency biomicroscope.
It is not uncommon for mutant mice to die in the early postnatal period or to first express cardiac phenotypes as juveniles, but in vivo study in this age group was limited due to the dearth of technology. The multifrequency biomicroscope enables the researchers to image the mutant mice in vivo and assess functional defects in this age group.
The multifrequency biomicroscope can be used to visualize intrauterine early postimplantation placental and embryonic development (e.g., day 6.5 to 8.5). There was a close correspondence between histology and ultrasound images at this stage, and histological artifacts (e.g., shrinkage during fixation and dehydration), avoided by in vivo imaging approach, may account for some of the differences. Very bright echoes were located near the implantation site where large, polyploid trophoblast giant cells are known to be localized (24). These bright echoes strongly delineated the placental margin. Fluid-filled cavities within the developing embryo were also visible. Thus the multifrequency biomicroscope may be useful for studying morphological development of the embryo and placenta in normal pregnancies and in mutants that die in the early postimplantation period.
The multifrequency biomicroscope was also able to record morphology and hemodynamics in detail within embryos between day 8.5 and 10.5 (3–4 wk in the human; Ref. 27). At 40 MHz, images were similar to those previously published using the 40 MHz prototype biomicroscope (28). This period is important because it includes critical developmental events. The embryo everts so that the inner concave surface of the U-shaped embryo becomes the outer convex, dorsal surface, the open cephalic neural folds close to form the fluid-filled neural tube, and the heart is transformed from a straight tube to a looped heart with atrial and ventricular septation underway. Furthermore, the embryo first becomes dependent on a functional placenta and cardiovascular system during this interval, and so it is an important stage to examine when searching for causes of intrauterine lethality in mutant mice (7). In this regard, the larger image size (more embryos per view) and better time resolution (heart motion easier to detect) of the conventional ultrasound system makes it better than the biomicroscope for quickly evaluating embryo number and viability and hence for determining the gestational age of intrauterine lethality in mutant mice. The higher spatial resolution and smaller image size of the multifrequency biomicroscope suggests that it may be better suited to more detailed studies such as investigating the cause of such deaths.
Conventional ultrasound systems are inadequate for imaging the internal anatomy of the embryonic mouse heart and the Doppler sample volume is too large to separately record inflow and outflow waveforms in embryos throughout gestation (11, 16). In contrast, especially at 55 MHz, the resolution of the multifrequency biomicroscope was sufficient to detect division of the common outflow tract into the aorta and pulmonary artery, septation of the ventricle, as well as the change from a common atrioventricular canal to two separate ventricular inflow tracts. The multifrequency biomicroscope can also be used to assess cardiovascular function using Doppler velocity waveforms throughout this period of development. It is noteworthy that at gestational ages <15.5 days, the internal anatomy of the embryonic heart is still somewhat difficult to discern because embryonic blood earlier than this stage is echogenic, so there is poor contrast between the myocardium and the cardiac chambers. On the positive side, the echogenic blood enabled visualization of intracardiac flow streams, which was helpful in identifying flow channels and for choosing appropriate locations for monitoring flow velocity waveforms.
Although embryonic development can be studied using serial observations in intact mice using the transcutaneous approach, this approach does have important limitations. With the exception of the first one or two embryos near the cervix, it is difficult to positively identify embryos in subsequent exams or for later tissue collection, and some embryos may be too deep or in an inappropriate orientation for detailed examination. This may be especially problematic when embryos within a litter differ in phenotype and/or genotype. Exteriorization of the uterus and the use of either a two time-point longitudinal design [as in survival microinjection studies (17, 22)] or a cross-sectional study design avoids these problems. Exteriorization also allows some adjustment of embryo orientation to optimize the imaging view and improves image resolution by eliminating ultrasound attenuation caused by intervening maternal skin, muscle, and viscera as shown previously (32). Furthermore, the reduction in required penetration allowed the multifrequency biomicroscope to be used at a higher insonation frequency, thereby further enhancing image resolution. Exteriorization necessitates careful temperature maintenance for stable embryonic cardiovascular function (21). It also tended to exacerbate embryonic movement caused by uterine contractile activity; however, indomethacin added to the bath appeared to minimize this effect. Movements due to maternal breathing were usually very small whether using transcutaneous or transuterine imaging. Even in the presence of some maternal movements, images with adequate quality could be found in the cineloop of eight images that are automatically stored with each "save" command. In future work, the higher image resolution and improved embryo stability possible with this method should enhance the accuracy of image-guided embryonic injections (10, 17, 22, 32) and the alignment of sequential ultrasound images used for three-dimensional reconstructions (32, 34). Further studies are required to determine the embryonic effects of uterine exteriorization and indomethacin exposure; however, this approach is much less invasive than studying mouse embryos directly using intravital microscopy and pulsed Doppler probes (14, 18, 30).
The multifrequency biomicroscope has lower temporal resolution and a smaller image size than that of conventional ultrasound systems. The frame rate was 4 frames/s during this study, although 8 frames/s is now supported. Frame rates are relatively slow because the image is created using a single, mechanically driven ultrasound transducer. By comparison, conventional ultrasound instruments use multiple transducer elements arranged in an array to achieve frame rates 
120 frames/s (6). Low temporal resolution is a limitation when imaging the adult mouse heart, which typically beats at 500 min-1, as well as in embryos where, at a typical heart rate of 250 min-1, there is only 1 image per cardiac cycle at 4 frames/s (23). The image size of the multifrequency biomicroscope (8 x 8 mm) was adequate for the normal adult mouse heart but may be inadequate when imaging very large mice or mice with cardiac hypertrophy. Thus, for applications requiring a high frame rate, and/or large image size, conventional ultrasound systems may be preferred despite the decrement in image resolution.
The biomicroscope’s Doppler system is ideal for measuring low blood velocities in veins, small arteries, or arterioles or in the embryonic vasculature. For instance, blood flow velocities <1 cm/s were detectable in the umbilical vein within 1 day of umbilical cord formation, as well as within the early primitive heart tube. The ability to detect low blood velocities in small vessels is one strength of the system, but the fact that high blood velocities are off-scale is a limitation. The maximum measurable velocity was calculated to be 37.5 cm/s, assuming 0° between flow and beam directions, 20 kHz PRF, and a Doppler operating frequency of 20 MHz. However, blood flow velocities in the heart and major arteries often exceed this value even in neonatal mice.
Blood flow calculated from noninvasive Doppler blood velocity and vessel diameter measurements is an especially attractive method in mice, where their small size, small blood volume, and large surface area mean that standard invasive methods for blood flow determination are much more difficult and error-prone than in larger species [e.g., flow probes (13), labeled microspheres (26), thermodilution (15)]. Conventional ultrasound systems have been used to calculate cardiac output in adult mice from ascending aortic blood velocity and diameter measurements (35). However, accuracy was limited by the relatively large Doppler sample volume and low spatial resolution of conventional ultrasound. This limitation is even more critical in juveniles and neonates. In our experience, when the multifrequency biomicroscope is used for diameter measurements in conjunction with a separate high-frequency pulsed Doppler system, blood flow can be measured in vessels such as the carotid artery and aorta even in mouse neonates and juveniles. We use a separate 20 MHz transcutaneous Doppler system (Indus Instruments, Houston, TX) to record blood velocities up to 200 cm/s in mice from birth to adulthood from various sites (e.g., ascending aorta, main pulmonary artery, mitral and tricuspid orifices) as described previously for adult mice (12, 29). The high spatial resolution of the multifrequency biomicroscope enables accurate, noninvasive vessel diameter measurements. Accuracy of diameter measurement is extremely important when calculating volume blood flow, because diameter is squared when blood flow is calculated. The multifrequency biomicroscope can also be used to image the region of interest to establish vessel angle and depth (e.g., useful for angle correction and setting pulsed Doppler range).
There is limited information on bioeffects of high-frequency ultrasound. Diagnostic ultrasound at conventional frequencies during pregnancy is regarded as safe, although, under certain conditions, some effects on fetal development have been observed in animal models (e.g., on growth and hematopoiesis) (31). The multifrequency biomicroscope’s mechanical index is 1 (peak pressure is 6 MPa at 40 MHz) and thus would generally be considered safe and unlikely to elicit mechanical tissue damage (3). There are also two studies using the prototype ultrasound biomicroscope that report that transcutaneous scanning of anesthetized pregnant mice at 40 MHz does not adversely affect embryonic development (28, 34). In the current study, we scanned two pregnant mice using 40 MHz ultrasound daily from 6.5 to 17.5 days of gestation with no apparent embryonic loss and no obvious abnormality in the neonates. Nevertheless, the major risk of ultrasound exposure, heating (2), needs to be directly assessed over the relevant frequency range (19–55 MHz). In addition, stress due to handling and/or anesthetic exposure during pregnancy may also affect embryonic development (4) and therefore also needs to be further explored.
In summary, the multifrequency biomicroscope operates over a frequency range appropriate for imaging mice with high spatial resolution throughout development from implantation to adulthood and for recording low blood flow velocities in the order of a few millimeters per second in vessels as small as 100 µm. Conventional ultrasound systems may nevertheless be preferred in applications requiring a larger image size, greater depth of penetration, and/or greater temporal resolution, and for applications where blood velocity exceeds 37.5 cm/s.
|
ACKNOWLEDGMENTS |
|---|
We thank the Richard Ivey Foundation for funding the purchase of the multifrequency biomicroscope, the Canadian Institutes of Health Research for operating grant support, and the Ontario Research and Development Challenge Fund for Fellowship support for Y. Q. Zhou.
F. S. Foster acknowledges a financial interest in the VisualSonics company.
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: S. L. Adamson, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Rm. 138P, 600 Univ. Ave., Toronto, Ontario, Canada, M5G 1X5 (E-mail: adamson{at}mshri.on.ca).
10.1152/physiolgenomics.00119.2001.
|
References |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONReferences |
|---|
cardiac myosin heavy chain gene. J Clin Invest 103: 147–153, 1999.[Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  G. Q. Teng, X. Zhao, J. P. Lees-Miller, F. R. Quinn, P. Li, D. E. Rancourt, B. London, J. C. Cross, and H. J. Duff Homozygous Missense N629D hERG (KCNH2) Potassium Channel Mutation Causes Developmental Defects in the Right Ventricle and Its Outflow Tract and Embryonic Lethality Circ. Res., December 5, 2008; 103(12): 1483 - 1491. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gronros, J. Wikstrom, U. Brandt-Eliasson, G. B. Forsberg, M. Behrendt, G. I. Hansson, and L. M. Gan Effects of rosuvastatin on cardiovascular morphology and function in an ApoE-knockout mouse model of atherosclerosis Am J Physiol Heart Circ Physiol, November 1, 2008; 295(5): H2046 - H2053. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.-M. Teichert, J. A. Scott, G. B. Robb, Y.-Q. Zhou, S.-N. Zhu, M. Lem, A. Keightley, B. M. Steer, A. C. Schuh, S. L. Adamson, et al. Endothelial Nitric Oxide Synthase Gene Expression During Murine Embryogenesis: Commencement of Expression in the Embryo Occurs With the Establishment of a Unidirectional Circulatory System Circ. Res., July 3, 2008; 103(1): 24 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Momoi, J. P. Tinney, L. J. Liu, H. Elshershari, P. J. Hoffmann, J. C. Ralphe, B. B. Keller, and K. Tobita Modest maternal caffeine exposure affects developing embryonic cardiovascular function and growth Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2248 - H2256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Mu, D. Qu, A. Bartczak, M. J. Phillips, J. Manuel, W. He, C. Koscik, M. Mendicino, L. Zhang, D. A. Clark, et al. Fgl2 deficiency causes neonatal death and cardiac dysfunction during embryonic and postnatal development in mice Physiol Genomics, September 11, 2007; 31(1): 53 - 62. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gronros, J. Wikstrom, U. Hagg, B. Wandt, and L.-m. Gan Proximal to Middle Left Coronary Artery Flow Velocity Ratio, As Assessed Using Color Doppler Echocardiography, Predicts Coronary Artery Atherosclerosis in Mice Arterioscler Thromb Vasc Biol, May 1, 2006; 26(5): 1126 - 1131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shen, L. Leatherbury, J. Rosenthal, Q. Yu, M. A. Pappas, A. Wessels, J. Lucas, B. Siegfried, B. Chatterjee, K. Svenson, et al. Cardiovascular phenotyping of fetal mice by noninvasive high-frequency ultrasound facilitates recovery of ENU-induced mutations causing congenital cardiac and extracardiac defects Physiol Genomics, December 14, 2005; 24(1): 23 - 36. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. Stennard and R. P. Harvey T-box transcription factors and their roles in regulatory hierarchies in the developing heart Development, November 15, 2005; 132(22): 4897 - 4910. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. B. Keller Developmental structure-function insights from Tbx5del/+ mouse model of Holt-Oram syndrome Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H975 - H976. [Full Text] [PDF]
|
|
|
|
|
|
Y.-Q. Zhou, Y. Zhu, J. Bishop, L. Davidson, R. M. Henkelman, B. G. Bruneau, and F. S. Foster Abnormal cardiac inflow patterns during postnatal development in a mouse model of Holt-Oram syndrome Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H992 - H1001. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-Q. Zhou, F. S. Foster, B. J. Nieman, L. Davidson, X. J. Chen, and R. M. Henkelman Comprehensive transthoracic cardiac imaging in mice using ultrasound biomicroscopy with anatomical confirmation by magnetic resonance imaging Physiol Genomics, July 8, 2004; 18(2): 232 - 244. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H-D Liang and M J K Blomley The role of ultrasound in molecular imaging Br. J. Radiol., December 1, 2003; 76(suppl_2): S140 - S150. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-Q. Zhou, F. S. Foster, R. Parkes, and S. L. Adamson Developmental changes in left and right ventricular diastolic filling patterns in mice Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1563 - H1575. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. K. L. Phoon and D. H. Turnbull Ultrasound biomicroscopy-Doppler in mouse cardiovascular development Physiol Genomics, June 24, 2003; 14(1): 3 - 15. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
