Y. Q. Zhou, F. S. Foster, D. W. Qu, M. Zhang, K. A. Harasiewicz and S. L. Adamson Physiol Genomics 10:113-126, 2002. First published Jun 18, 2002; doi:10.1152/physiolgenomics.00119.2001 You might find this additional information useful... This article cites 32 articles, 11 of which you can access free at: http://physiolgenomics.physiology.org/cgi/content/full/10/2/113#BIBL This article has been cited by 15 other HighWire hosted articles, the first 5 are: Spiral Arterial Remodeling Is Not Essential for Normal Blood Pressure Regulation in Pregnant Mice S. D. Burke, V. F. Barrette, J. Bianco, J. G. Thorne, A. T. Yamada, S. C. Pang, M. A. Adams and B. A. Croy Hypertension, March 1, 2010; 55 (3): 729-737. [Abstract] [Full Text] [PDF]
Effects of rosuvastatin on cardiovascular morphology and function in an ApoE-knockout mouse model of atherosclerosis J. Gronros, J. Wikstrom, U. Brandt-Eliasson, G. B. Forsberg, M. Behrendt, G. I. Hansson and L. M. Gan Am J Physiol Heart Circ Physiol, November 1, 2008; 295 (5): H2046-H2053. [Abstract] [Full Text] [PDF] Endothelial Nitric Oxide Synthase Gene Expression During Murine Embryogenesis: Commencement of Expression in the Embryo Occurs With the Establishment of a Unidirectional Circulatory System 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, M. I. Cybulsky and P. A. Marsden Circ. Res., July 3, 2008; 103 (1): 24-33. [Abstract] [Full Text] [PDF] Modest maternal caffeine exposure affects developing embryonic cardiovascular function and growth N. Momoi, J. P. Tinney, L. J. Liu, H. Elshershari, P. J. Hoffmann, J. C. Ralphe, B. B. Keller and K. Tobita Am J Physiol Heart Circ Physiol, May 1, 2008; 294 (5): H2248-H2256. [Abstract] [Full Text] [PDF] Updated information and services including high-resolution figures, can be found at: http://physiolgenomics.physiology.org/cgi/content/full/10/2/113 Additional material and information about Physiological Genomics can be found at: http://www.the-aps.org/publications/pg
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Physiological Genomics publishes results of a wide variety of studies from human and from informative model systems with techniques linking genes and pathways to physiology, from prokaryotes to eukaryotes. It is published quarterly in January, April, July, and October by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright Š 2002 by the American Physiological Society. ISSN: 1094-8341, ESSN: 1531-2267. Visit our website at http://www.the-aps.org/.
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Homozygous Missense N629D hERG (KCNH2) Potassium Channel Mutation Causes Developmental Defects in the Right Ventricle and Its Outflow Tract and Embryonic Lethality 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 Circ. Res., December 5, 2008; 103 (12): 1483-1491. [Abstract] [Full Text] [PDF]
Physiol Genomics 10: 113–126, 2002. First published June 18, 2002; 10.1152/physiolgenomics.00119.2001.
toolbox Applications for multifrequency ultrasound biomicroscopy in mice from implantation to adulthood
Received 18 December 2001; accepted in final form 11 June 2002
Zhou, Y. Q., F. S. Foster, D. W. Qu, M. Zhang, K. A. Harasiewicz, and S. L. Adamson. Applications for multifrequency ultrasound biomicroscopy in mice from implantation to adulthood. Physiol Genomics 10: 113–126, 2002. First published June 18, 2002; 10.1152/physiolgenomics.00119. 2001.—A new multifrequency (19–55 MHz) ultrasound biomicroscope with two-dimensional imaging and integrated Doppler ultrasound was evaluated using phantoms and isoflurane-anesthetized mice. Phantoms revealed the biomicroscope’s lateral resolution was between 50 and 100 m, whereas that of a conventional 13 MHz ultrasound system was 200–500 m. This difference was apparent in the markedly higher resolution images achieved using the biomicroscope in vivo. Transcutaneous images of embryos in pregnant mice from ⬃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 m 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 Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org). 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@mshri.on.ca).
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
PHENOTYPIC EVALUATION of genetically altered mice presents an important rate-limiting step for physiologists working toward assigning function to each of the ⬃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 imageguided 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
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Y. Q. ZHOU,1,4 F. S. FOSTER,2,5 D. W. QU,1 M. ZHANG,2,5 K. A. HARASIEWICZ,2,5 AND S. L. ADAMSON1,3,4 1 Samuel Lunenfeld Research Institute at Mount Sinai Hospital, and 2Sunnybrook and Women’s College Health Sciences Centre, Departments of 3Obstetrics/Gynecology, 4Physiology, and 5Medical Biophysics, University of Toronto, Toronto, Ontario, Canada, M5G 1X5
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METHODS
Experiments were approved by the animal care committee of Mount Sinai Hospital and were conducted in accord with guidelines established by the Canadian Council on Animal Care. The biomicroscope. A multifrequency ultrasound biomicroscope (model VS40; VisualSonics, Toronto, Canada; http://
Table 1. Instrument specifications for the multifrequency biomicroscope Feature
Specification
B-scan frequency Frame rate Scan format Line spacing B-scan high-pass filtering B-scan low-pass filtering
19, 25, 40, 55 MHz 2, 4, 8 Hz* bidirectional 15.6 m open, 5, 10, 30 MHz 40, 50, 70, 80 MHz
Doppler mode Doppler frequency Pulse repetition frequency Max. unaliased velocity at 0° Minimum velocity Radio frequency cycles per pulse Doppler low-pass filtering Doppler high-pass filtering
pulsed 20–55 MHz 1–20 kHz 37.5 cm / s† ⬍1 mm/s 2–16 100 Hz to 25 kHz (steps of 100 Hz) 6, 60, 100, 320 Hz
Analog-to-digital conversion Micropositioning
125 MHz, 12 bits x, y, z ⫾ 3 m
fo ⫽ 20 MHz, Rlat ⫽ 104 m n
L, m
4 8 12 16
128 257 385 513
DSV,
⫻10⫺3
l
1.09 2.18 3.27 4.36
fo ⫽ 40 MHz, Rlat ⫽ 68 m L, m
DSV, ⫻10⫺3 l
64 128 192 257
0.233 0.466 0.699 0.932
Axial sample volume length (L) and Doppler sample volume (DSV) for different pulse lengths at 20 and 40 MHz. The DSV is modeled as a cylinder with diameter given by the measured lateral resolution (Rlat) and length defined by the number of cycles in the transmitted waveform. The length of the pulse is given by L ⫽ (1⁄2) (cn/1.2 fo) where c is the speed of sound, fo is the operating frequency, and n is the number of cycles in the Doppler radiofrequency (rf) transmit waveform. The Doppler sample volume is then given by DSV ⫽ L (Rlat/2)2 where Rlat is the measured lateral resolution.
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 ⫻ 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. Image resolution and comparative performance. The resolution in the lateral direction of the scanner operating at 19, 25, 40, or 55 MHz was measured in vitro with a calibrated hydrophone. The resolution in the axial direction was calculated using assumed filter settings for a pulse echo bandwidth of 50% (Table 3). In two phantom studies, the image resolution of the biomicroscope was compared with that of a conventional ultrasound system, the Sequoia C256 with a
Table 3. Spatial resolution as a function of frequency for the multifrequency biomicroscope
* The maximum frame rate was 4 Hz at the time of study. † The maximum unaliased velocity measurable at 0° insonation angle is obtained at a pulse repetition frequency of 20 kHz and a Doppler operating frequency of 20 MHz. Physiol Genomics • VOL
Table 2. Doppler sensitivity pattern
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Nominal Frequency, MHz
Lateral Resolution, m
Axial Resolution, m
19 25 40 55
104.2 88.2 68.2 62.5
81.1 61.6 38.5 28
Lateral resolution was measured with a calibrated hydrophone. Axial resolution assumes filter settings for a pulse echo bandwidth of 50%. www.physiolgenomics.org
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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 ⬃20fold 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.
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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 M 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 highthroughput 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, www.physiolgenomics.org
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model 15L8 transducer operating at 13 MHz (Acuson, Mountainview, CA; http://www.acuson.com), that is currently used for echocardiography in mice at the University of Toronto and elsewhere (see Ref. 6). A phantom with parallel glass filaments separated by 1,000, 500, 200, 100, and 50 m was imaged in the short-axis view within the focal zone of the transducer. The array of filaments was either perpendicular or parallel to the ultrasound beam to determine lateral or axial resolution, respectively. The mean percent discrepancy [(兩xm ⫺ xa兩)/xa ⫻ 100, where xm is the measurement and xa is the actual dimension] was determined from five caliper measurements per separation distance per frequency for both ultrasound systems. Image distortion was tested by imaging another phantom with two parallel, fluid-filled cylindrical cavities 1,000 m and 460 m in diameter in an agarose block. Both short- and long-axis views were obtained. Mouse embryonic imaging. Timed-pregnant ICR (CD-1) mice (Harlan Sprague Dawley, Indianapolis, IN) were studied. Day 0.5 of pregnancy was defined as 12 noon on the day a vaginal plug was found after overnight mating. Implantation in this strain occurs approximately day 5.0, and day 18.5 is full term. During studies, all mice were lightly anesthetized with isoflurane in oxygen by face mask and were warmed using a heated pad and heat lamp. Heart rate and rectal temperature of adult mice were monitored (model THM100; Indus Instruments, Houston, TX), and heating was adjusted to maintain rectal temperature between 36 and 38°C. 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
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Texas) for quantitative measurement of velocities and time intervals. RESULTS
In vitro validation studies. The lateral resolution of the multifrequency biomicroscope measured in vitro using a hydrophone is shown in Table 3. Lateral resolution ranged from 62 m at 55 MHz to 104 m at 19 MHz. These results are in good agreement with the image resolution of the biomicroscope determined using a glass filament phantom, which showed the lateral resolution of the biomicroscope was between 50 and 100 m at all frequencies (Fig. 1). The percent discrepancies in caliper measurements of filament separation distances were 0% at 200-, 500-, and 1,000-m separa-
tions and 10% at 100 m for all frequencies. The lateral resolution of the conventional ultrasound system was between 200 and 500 m. The mean percent discrepancy in caliper measurements was 3% at 1,000 m and 4% at 500 m. The 200-m separation was not detectable (Fig. 1). The calculated axial resolution of the multifrequency transducer is shown in Table 3. The axial resolution ranged from 28 m at 55 MHz to 81 m at 19 MHz. At 40 and 55 MHz, the axial resolution of the biomicroscope determined using a glass wire phantom in vitro (Fig. 1) was somewhat less than calculated (Table 3) in that the 50-m separation was not detectable at these frequencies. However, the 100-m separation distance was detectable at all frequencies as predicted. The
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Fig. 1. Images of phantoms obtained using the conventional ultrasound system (left column; scale bars are 1 mm) and the multifrequency biomicroscope operating at 40 MHz (right column; smallest divisions on scale bars are 100 m) are shown at approximately same scale. In all cases, the area of interest was placed in the focal zone of the transducer. The same glass filament phantom was imaged in the lateral direction in A and B and the axial direction in C and D. Crosshairs show the calipers of the conventional ultrasound system (in A and C), and plus signs show the calipers as aligned by the operator on the monitor of the multifrequency biomicroscope (B, D, F, and H). As shown in B, filament separation distances were 1,000 m (caliper markers 1 and 2), 500 m (caliper markers 2 and 3), 200 m (caliper markers 3 and 4), and 100 m (caliper marker 4 and the fifth unlabeled marker). The final filament is 50 m further to the right, but this separation interval cannot be resolved by the multifrequency biomicroscope in either the lateral (B) or axial (D) direction. In the axial direction, the most distant marker is not detected by the multifrequency biomicroscope at the low gain used for this image (D), whereas it is detectable using the conventional ultrasound system (C). With the conventional ultrasound system, only the first 2 intervals can be resolved (1,000 m and 500 m) in the lateral direction (A), whereas intervals ⱖ100 m can be resolved in the axial direction (C). In F, the circular cross sections of the cylindrical cavities (1,000 m and 460 m diameter) are easily detected and measured in the lateral and axial directions using the multifrequency biomicroscope, whereas lateral echoes are difficult to detect with the conventional ultrasound system (E). In longitudinal section, the phantom appears as two straight, parallel echoes when viewed by both ultrasound systems (G and H).
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percent discrepancy in caliper measurement in the axial direction was 10% at 100 m at all frequencies and 4% or less for greater separation distances at all frequencies, confirming the accuracy of caliper measurements. The axial resolution of the conventional ultrasound system, like that of the multifrequency biomicroscope, was between 50 and 100 m. The percent discrepancy in caliper measurement in the axial direction on the Sequoia apparatus was 24% at 100 m and 3% or less for greater separations. 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
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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 ⫻ 8 mm image generated by the multifrequency biomicroscope was big enough to include the entire
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Fig. 2. The same pregnant mouse at day 10.5 of gestation (A–C) and the same adult mouse heart (D–I) were viewed transcutaneously using the conventional ultrasound system (13 MHz; 1st and 2nd columns) and using the multifrequency biomicroscope (3rd column). The 1st column (A, D, G) shows the image as viewed on the monitor of the conventional ultrasound system; the 2nd column (B, E, H) shows an enlargement of the region of interest so that the scale is the same as that for images obtained using the multifrequency biomicroscope in the 3rd column (C, F, I). Shown are long-axis sections through embryos in the 1st row, through the adult left ventricle in the 2nd row, and through the adult aortic root in the 3rd row. The multifrequency biomicroscope was operated at 40 MHz for embryo imaging and 19 MHz for adult imaging. Scale bars in 2nd column ⫽ 1 mm; smallest divisions on scale bars in 3rd column ⫽ 100 m. Am, amniotic cavity; Ao, aorta; AV, aortic valve; B, brain; CA, common atrium, CV, common ventricle; Em, embryo; En, endocardium; LV, left ventricle; OFT, outflow tract; Pl, placenta; S, skin. Physiol Genomics • VOL
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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). The multifrequency biomicroscope proved useful for imaging many embryonic structures including the heart from the stage at which the embryo first depends on cardiovascular function for survival (⬃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. Higher resolution images of the developing heart were obtained by exteriorizing the uterus and imaging at 55 MHz (Fig. 6). As observed previously (28), the space within the embryonic heart and blood vessels viewed at ⱕ14.5 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 www.physiolgenomics.org
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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 ⫻ 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. The heart of the adult mouse completely filled the image frame of the biomicroscope in long-axis view (Fig. 2). Penetration depth was just sufficient to visualize the posterior wall of the left ventricle of this normal mouse. The mitral, aortic, and pulmonary valves were visible, and the diameters of the pulmonary artery and aorta were readily measurable. However, despite the high spatial resolution of the images, the relatively low frame rate of the biomicroscope lead to distortion artifacts, especially in phases of the cardiac cycle when the ventricular wall was moving rapidly, and it was also not possible to capture images for caliper measurements at precise time points during the cardiac cycle. The conventional ultrasound system had much better temporal resolution, so cardiac movements did not introduce distortion artifacts, but it was not possible to see the mitral or aortic valves, so these structures could not be used to detect phases of the cardiac cycle. However, spatial resolution was sufficient to visualize the left ventricular myocardium and endocardium and the ascending aorta, so ventricular wall thickness and aortic diameters were measurable as shown previously (6, 35). The pulmonary artery was difficult to visualize even in short-axis view in contrast to the biomicroscope, where it was readily imaged. The scan length and depth of field of the conventional system were more than sufficient to visualize the entire heart of the adult mouse (Fig. 2). 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
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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. Postnatal imaging. After birth, the neonatal heart was still sufficiently superficial to image at 55 MHz, whereas 19 MHz was required to get sufficient penetration depth to image the adult mouse heart (Fig. 7). In both cases, resolution was adequate for measuring dimensions of the ventricular wall and chambers, and the mitral, aortic, and pulmonary orifices, and the ascending aorta and main pulmonary artery and their main branches (Fig. 7). In adults, the diameter of the left and right coronary arteries were measurable (Fig. 7F), but coronary blood velocities were not, because vessel movement during cardiac contractions was excessive. The low scan rate of the transducer (4 frames/s) relative to the high heart rate of mouse neonates (e.g., 240 min⫺1, which is 1 beat/frame) and adults (e.g., 480 min⫺1, which is 2 beats/frame) meant Physiol Genomics • VOL
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that cardiac movement during a single scan often distorted the images. In addition, end-diastolic and endsystolic images were difficult to pinpoint. In postnatal mice, blood velocities in the aorta, pulmonary artery, and heart were too high to be recorded using the multifrequency biomicroscope. On the other hand, mice have a large thymus that provides a good acoustic window for clearly imaging the ascending aorta, aortic arch, and its branches. Moreover, with the exception of a shadowed region near the thoracic inlet, the entire course of left and right carotid arteries could be readily followed from their origin past their bifurcation into the internal and external carotid arteries (Fig. 7, C and D). Inner diameters of the aorta and common carotid arteries were measured with the multifrequency biomicroscope. Aortic diameter was measured during systole (i.e., when aortic valves were open) using 19 MHz ultrasound in adult mice (25–30 g body wt). Systolic diameter is the most relevant when calculating volume flow rate at this site, because it is during systole that cardiac ejection (i.e., flow) occurs. Bright echoes corresponding to the near and far walls appeared only to be observed when the aorta was viewed with the image www.physiolgenomics.org
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Fig. 3. Embryonic development from postimplantation to the beating heart. Transcutaneous cross-sectional images of the pregnant uterus at 40 MHz are shown at day 7.0 (A), day 7.5 (B), and day 8.0 (C) of gestation. Inside the uterus, the decidua is relatively dark, and the eccentrically located conceptus is surrounded by a region of brighter echoes. The fluid-filled cavities of the conceptus appear black. The implantation site at day 8 looks remarkably similar in C using ultrasound and D using histology. In E and F, transuterine crosssectional images of the exteriorized uterus at 55 MHz are shown at day 8.5 of gestation. In E, the embryo is viewed in cross section at the level of the heart. Open embryonic head folds are visible to the right of the heart, as is the amniotic membrane that surrounds the embryo. The Doppler sample volume (indicated by the rectangle on the vertical line) has been placed over the heart and the recorded Doppler signal is shown below. The signal is negative indicating that flow is away from the transducer. Blood velocity is ⬃5 cm/s assuming flow is parallel to the Doppler beam (indicated by the vertical line). In F, the Doppler sample volume has been placed over the allantois, but no blood velocity signal was detectable (as shown in the Doppler recording below). The smallest division on the scale bar to the right of all ultrasound images is 100 m, and on the time scale beneath Doppler recordings is 0.1 s. In E and F, the y-axis is the Doppler shift frequency in Hz (0 to ⫺2,500 Hz is indicated by the vertical white bar to the left). Al, allantois; Am, amniotic cavity; AM, amniotic membrane; D, decidua; EC, ectoplacental cone; Em, embryo; EPC, ectoplacental cavity; Ex, exocoelomic cavity; H, heart; PA, proamniotic cavity; S, maternal skin; Ut, uterus.
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Fig. 4. Embryonic structures visualized by transcutaneous imaging at 40 MHz from day 9.5 to 16.5 of gestation. A: a longitudinal cross-sectional view of an embryo at day 9.5 showing the cephalic vesicle within the brain on the left, the bright heart region near the center, and the lower body to the right. B: the forelimb and paw, and placenta of a mouse embryo at day 11.5. C: embryonic eyes and brain at day 13.5. D: cross section of the embryonic head at day 13.5 showing cerebral ventricles in the brain. E: blood in the hepatic segment of inferior vena cava of the embryo at day 16.5 of gestation appears dark, whereas blood-filled spaces earlier in gestation appear bright as shown in A above. The liver and lung are also visible in E. F: the lung, liver, kidney, and vertebrae of an embryo at day 16.5 viewed in longitudinal section. The smallest division on the scale bar to the right of all ultrasound images is 100 m. ASLV, anterior portion of superior horn of lateral cerebral ventricle; B, brain; CV, cephalic vesicle; E, eye; Em, embryo; FV, fourth cerebral ventricle; H, heart; IVC, inferior vena cava; Li, liver; Lu, lung; P, paw; Pl, placenta; RK, right kidney; S, maternal skin; TV, third cerebral ventricle; Ut, uterus; V, vertebrae.
plane exactly though the midline of the aortic lumen (Fig. 7G). The easiest and most reproducible measurements were obtained from longitudinal views of the proximal ascending aorta just distal to the aortic sinuses (Table 4, Fig. 7G). Measurements were always possible at this site, but were sometimes difficult in the aortic annulus, probably due to higher attenuation and less optimal angulation. The coefficient of variation for aortic diameter measurement was between 1 and 3% (Table 4). The coefficient of variation in inner diameter measurements of the common carotid artery was 3–3.5% in juvenile and adult mice (Table 4). In neonates, the whole kidney was visible within the dimensions of one image, and blood flow velocity was Physiol Genomics • VOL
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measurable from the renal hilus region where the renal artery enters the kidney (Fig. 8, A and B). In adults, only the cortex of the kidney was sufficiently superficial to be imaged in detail. However, flow velocity waveforms could be obtained from the small terminal vessels in the cortical region (Fig. 8, C and D). The multifrequency biomicroscope was used to image the ascending aorta and heart of two adult mice that had abnormally high aortic flow velocities. Both mice had aortic valves that were irregular in shape and hyperechoic and which did not open normally during systole (Fig. 9, A and B). In addition, the ascending aorta appeared to be abnormally dilated in both mice (diameters ⬃2.5 and ⬃2.0 mm). Histological examinawww.physiolgenomics.org
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Fig. 5. Two-dimensional images of GRIP1 ⫺/⫺ mouse embryos. A: a skin blister (arrow) on the embryonic head at 12.5 days gestation is shown (imaged at 40 MHz). B: skin blisters (arrows) on the surface of the lower body and tip of the limb bud are shown at 13.5 days gestation (imaged at 55 MHz). Smallest division on scale bar ⫽ 100 m. E, eye; B, brain; L, limb; S, maternal skin.
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Fig. 6. The embryonic heart from day 9.5 to 16.5 of gestation viewed at 55 MHz through the exteriorized uterus. A: the U-shape of the tubular embryonic heart at day 9.5 is visible. The Doppler sample volume (rectangle on vertical line) has been placed over the atrioventricular canal in A, and the corresponding Doppler blood velocity waveform is shown in B. B: ventricular inflow velocity in the atrioventricular canal is unidirectional and negative, indicating all flow is away from the transducer. Peak velocity is ⬃3 cm/s, assuming flow is parallel to the Doppler beam. The waveform has a double peak and therefore is similar in shape to that of postnatal animals. In C, the Doppler sample volume has been placed over the outflow tract, and the corresponding Doppler waveform is shown in D. D: blood velocity in the ventricular outflow tract is primarily unidirectional (toward the transducer) with a peak velocity of ⬃5 cm/s, assuming flow is parallel to the beam. E: transverse view of the day 10.5 embryonic heart. The common atrium, common ventricle, and common atrioventricular canal are visible. F: a transverse view of the heart at day 12.5. The ventricular septum is forming but incomplete (the septum is the dark region visible between the left and right ventricles). Flow streamlines from the left and right ventricles (i.e., echoes streaks) can be seen leading into the aorta especially in enlarged inset (arrows highlight streamlines). G: four-chamber view of the heart at day 13.5 showing complete ventricular septation and visible mitral valve leaflets. H: long-axis view of the ventricles on day 16.5. Blood in ventricular chambers appears dark relative to earlier in gestation, and internal cardiac morphology is now visible. The smallest division on the scale bar ultrasound images is 100 m, and on the time scale beneath Doppler recordings is 0.1 s. In B and D, the y-axis is the Doppler shift frequency in Hz (0 to ⫺2,500 Hz is indicated by the vertical white bar to the left). Ao, aorta; AVC, atrioventricular canal; CA, common atrium; CV, common ventricle; LA, left atrium; Lu, lung; LV, left ventricle; MV, mitral valve; OFT, outflow tract; RA, right atrium; RV, right ventricle; Ut, uterus.
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Fig. 7. Transcutaneous images of the heart and nearby vessels in postnatal mice. A: long-axis view of heart at 3 days postnatal age imaged at 55 MHz. The left ventricular chamber and walls, mitral valve leaflets, aortic annulus, and left atrium are visible. B: short-axis view of heart at 2 days postnatal age insonated at 55 MHz, showing left and right ventricular chambers, ventricular walls, and interventricular septum. C: long-axis view of left common carotid artery from aortic origin to beyond the bifurcation into internal and external carotid arteries; imaged with a transducer frequency of 40 MHz in a 3-wk-old mouse. D: long-axis view of the left ventricular outflow tract, ascending aorta, and the innominate artery (first branch from the aortic arch); imaged at 19 MHz in an adult mouse. Length of ascending aorta from aortic annulus to the origin of the innominate is 3.72 mm. E: transverse view showing the aorta in cross section and the pulmonary trunk and pulmonary arteries in longitudinal section in an adult mouse insonated at 19 MHz. Arrow points to a pulmonary valve leaflet. F: view of the left coronary artery branching off at the aortic root of an adult mouse viewed at 19 MHz. Coronary artery diameter is 0.32 mm at the site where the measurement calipers are shown. G: long-axis view of the left ventricular outflow tract of an adult mouse insonated at 19 MHz showing the ascending aorta with caliper markers in place. The left ventricle, left atrium, and an aortic valve (arrow) leaflet are also visible. H: short-axis view of an adult mouse heart insonated at 19 MHz showing the right ventricle, the left ventricular chamber and wall, the interventricular septum, and papillary muscles (arrow). The smallest division on the scale bars is 100 m. AA, ascending aorta; Ao, aorta; IA, innominate artery; LA, left atrium; LCA, left coronary artery; LCCA, left common carotid artery; LV, left ventricle; LVOT, left ventricular outflow tract; MPA, main pulmonary artery; RV, right ventricle; S, skin; Th, thymus.
tion 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
Relative to existing ultrasound technology used for mouse imaging, the main advance of the current biomicroscope is that insonation frequency is adjustable over Physiol Genomics • VOL
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a range of frequencies from 19 to 55 MHz. This frequency range enables the highest possible resolution given the penetration depths required for imaging mice throughout development from implantation to adulthood (⬃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 highfrequency Doppler permits low blood velocities such as www.physiolgenomics.org
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Table 4. Intraluminal vessel diameter measurement % Coefficient of Variation Vessel
View
Aorta Aorta Annulus
long axis short axis long axis
Diameter, mm (mean ⫾ SD)
Within Session*
Between Sessions†
Adult ICR mice, 25–30 g body wt, N ⫽ 5 1.411 ⫾ 0.029 1.428 ⫾ 0.043 1.485 ⫾ 0.042
1.3 2.6 3.0
2.1 3.0 2.9
Juvenile C57B16/J mice, 3 wk, 7–10 g body wt, N ⫽ 4 Carotid
long axis
0.421 ⫾ 0.016
3.1
not done
Adult C57B16/J mice, 8 wk, 19–30 g body wt, N ⫽ 6 Carotid
long axis
0.500 ⫾ 0.049
3.5
not done
* Variation for 6–8 measurements per session. † Variation in session means, one session per day over 4 days
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
Fig. 8. Kidney and renal blood velocity waveforms in postnatal mice. A: long-axis view of the right kidney of a 2-day-old neonate imaged at 55 MHz. Arrows show the border between the kidney and the liver. B: Doppler blood velocity waveform recorded from the renal hilus region of the kidney shown in A. Peak velocity is ⬃4 cm/s, assuming flow is parallel with the Doppler beam. C: short-axis view of the right kidney of an adult mouse imaged at 19 MHz. Arrows show the outer surface of the kidney. The hilus region is too deep to visualize. The Doppler sample volume (rectangle on vertical line) has been placed over a renal cortical artery to obtain the Doppler blood velocity waveform shown in D. Peak velocity is ⬃6 cm/s, assuming flow is parallel with the Doppler beam. The smallest division on the scale bar in ultrasound images is 100 m, and on the time scale beneath Doppler recordings is 0.1 s. In B and D, the y-axis is the Doppler shift frequency in Hz (0 to ⫺2,500 Hz is indicated by the vertical white bar to the left). Li, liver; RK, right kidney, S, skin.
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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 m lateral ⫻ 250 m axial) relative to conventional ultrasound systems (e.g., ⬃350 m lateral ⫻ 1,000 m 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 m, and the axial resolution was between 50 and 100 m. 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
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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 Ushaped 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 Physiol Genomics • VOL
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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 www.physiolgenomics.org
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Fig. 9. The aortic annulus region of an adult mouse with a peak ascending aortic blood velocity ⬎200 cm/s (more than twice normal) imaged at 19 MHz. A: longitudinal view of the left ventricle and aorta showing bright, coarse echoes (between arrows) in the aortic orifice (contrast with normal mouse shown in Fig. 7G). Diameter at the aortic annulus was 1.3 mm but more distally the ascending aorta was dilated (diameter 2.0 mm). B: short-axis view of the aortic annulus showing bright echoes in the valve region (contrast with normal mouse shown in Fig. 7E). C: histological section through the aortic valve of the same mouse showing abnormally thickened aortic valve leaflets. The smallest division on the scale bar in A and B is 100 m. AA, aortic annulus; Ao, ascending aorta; LA, left atrium; LV, left ventricle; LCA, left coronary artery; MPA, main pulmonary artery; AV, aortic valve.
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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 highfrequency 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 highfrequency 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 m. Conventional ultrasound systems may nevertheless be preferred in applications requiring a larger image size, www.physiolgenomics.org
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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 ⱖ120 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 ⫻ 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
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greater depth of penetration, and/or greater temporal resolution, and for applications where blood velocity exceeds 37.5 cm/s.
18. 19.
20.
21. 22.
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We thank Shathiyah Kulandavelu, Carol Kolb, Donna Johnston, and Kathie Whiteley for assistance during experiments, Yong Lu for sharing his anatomic expertise and archive of histological sections of mouse embryos with us during this work, and Friedholm Bladt for the opportunity to image GRIP1 ⫺/⫺ embryos. We also thank Dr. Colin McKerlie and the Pathology Core of the Centre for Modeling Human Disease (http://www.cmhd.ca) for pathology and histology services for the adult mouse with abnormal aortic valves. We thank VisualSonics for technical support and the Heart and Stroke/Richard Lewar Centre of Excellence for lending us their Acuson Sequoia for this work. 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.
17.
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