Specification of mouse telencephalic and mid-hindbrain progenitors following heterotopic ultrasound-guided embryonic transplantation.

We have demonstrated the utility of ultrasound backscatter microscopy for targeted intraparenchymal injections into embryonic day (E) 13.5 mouse embryos. This system has been used to test the degree of commitment present in neural progenitors from the embryonic ventral telencephalon and mid-hindbrain region. Many E13.5 ventral telencephalic progenitors were observed to integrate and adopt local phenotypes following heterotopic transplantation into telencephalic or mid-hindbrain targets, whereas mid-hindbrain cells of the same stage were unable to integrate and change fate in the telencephalon. In contrast, many mid-hindbrain cells from an earlier developmental stage (E10.5) were capable of integrating and adopting a forebrain phenotype after grafting into the telencephalon, suggesting that mouse mid-hindbrain progenitors become restricted in their developmental potential between E10.5 and E13.5.

A method for rapid gain-of-function studies in the mouse embryonic nervous system.

We used ultrasound image-guided injections of high-titer retroviral vectors to obtain widespread introduction of genes into the mouse nervous system in utero as early as embryonic day 8.5 (E8.5). The vectors used included internal promoters that substantially improved proviral gene expression in the ventricular zone of the brain. To demonstrate the utility of this system, we extended our previous work in vitro by infecting the telencephalon in vivo as early as E8. 5 with a virus expressing Sonic Hedgehog. Infected embryos showed gross morphological brain defects, as well as ectopic expression of ventral telencephalic markers characteristic of either the medial or lateral ganglionic eminences.

Neonatal cardiomyopathy in mice homozygous for the Arg403Gln mutation in the alpha cardiac myosin heavy chain gene.

Heterozygous mice bearing an Arg403Gln missense mutation in the alpha cardiac myosin heavy chain gene (alpha-MHC403/+) exhibit the histopathologic features of human familial hypertrophic cardiomyopathy. Surprisingly, homozygous alpha-MHC403/403 mice die by postnatal day 8. Here we report that neonatal lethality is caused by a fulminant dilated cardiomyopathy characterized by myocyte dysfunction and loss. Heart tissues from neonatal wild-type and alpha-MHC403/403 mice demonstrate equivalent switching of MHC isoforms; alpha isoforms in each increase from 30% at birth to 70% by day 6. Cardiac dimensions and function, studied for the first time in neonatal mice by high frequency (45 MHz) echocardiography, were normal at birth. Between days 4 and 6, alpha-MHC403/403 mice developed a rapidly progressive cardiomyopathy with left ventricular dilation, wall thinning, and reduced systolic contraction. Histopathology revealed myocardial necrosis with dystrophic calcification. Electron microscopy showed normal architecture intermixed with focal myofibrillar disarray. We conclude that 45-MHz echocardiography is an excellent tool for assessing cardiac physiology in neonatal mice and that the concentration of Gln403 alpha cardiac MHC in myocytes influences both cell function and cell viability. We speculate that variable incorporation of mutant and normal MHC into sarcomeres of heterozygotes may account for focal myocyte death in familial hypertrophic cardiomyopathy.

Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice.

To elucidate the role of cardiac myosin-binding protein-C (MyBP-C) in myocardial structure and function, we have produced mice expressing altered forms of this sarcomere protein. The engineered mutations encode truncated forms of MyBP-C in which the cardiac myosin heavy chain-binding and titin-binding domain has been replaced with novel amino acid residues. Analogous heterozygous defects in humans cause hypertrophic cardiomyopathy. Mice that are homozygous for the mutated MyBP-C alleles express less than 10% of truncated protein in M-bands of otherwise normal sarcomeres. Homozygous mice bearing mutated MyBP-C alleles are viable but exhibit neonatal onset of a progressive dilated cardiomyopathy with prominent histopathology of myocyte hypertrophy, myofibrillar disarray, fibrosis, and dystrophic calcification. Echocardiography of homozygous mutant mice showed left ventricular dilation and reduced contractile function at birth; myocardial hypertrophy increased as the animals matured. Left-ventricular pressure-volume analyses in adult homozygous mutant mice demonstrated depressed systolic contractility with diastolic dysfunction. These data revise our understanding of the role that MyBP-C plays in myofibrillogenesis during cardiac development and indicate the importance of this protein for long-term sarcomere function and normal cardiac morphology. We also propose that mice bearing homozygous familial hypertrophic cardiomyopathy-causing mutations may provide useful tools for predicting the severity of disease that these mutations will cause in humans.

Telencephalic neural progenitors appear to be restricted to regional and glial fates before the onset of neurogenesis.

The contribution of early cell lineage to regional fate in the mammalian forebrain remains poorly understood. Previous lineage-tracing studies using retroviral methods were only begun at mid-neurogenesis and have suffered from region-specific retroviral silencing. We have been able to study cell lineage in the telencephalon from the onset of neurogenesis by using ultrasound backscatter microscopy to label the forebrain neuroepithelium and a modified retroviral lineage library to overcome regional silencing. Our studies suggest that by embryonic day 9.5, forebrain clones are primarily restricted to territories within anatomically demarcated regional boundaries, such as the cortex, striatum and hypothalamus. In addition, we observed a subset of clones that appeared to be composed entirely of glia. These observations suggest that both regional and cell-type restrictions exist within progenitor populations before the first forebrain cells become postmitotic.

Alteration of limb and brain patterning in early mouse embryos by ultrasound-guided injection of Shh-expressing cells.

A basic limitation of the study of development in the mouse is the inaccessibility of the embryos, which are encased in the maternal uterus. We demonstrate the first use of ultrasound backscatter microscopy for guiding injections of cells and other agents into early stage mouse embryos. Cells were injected into the mouse neural tube cavity as early as 9.5 days post coitus (E9.5), and into the developing limb buds as early as E10.5. Furthermore, a cell-line engineered to express the secreted factor Sonic Hedgehog (Shh) was injected into early developing mouse brains or limbs. The Shh-expressing cells were found to induce ectopic expression of the Shh target genes Patched and Hnf3beta in the dorsal brain, and to alter digit patterning in the anterior limb bud. These results show that gene misexpression studies can be performed in mouse embryos using ultrasound-guided injection of transfected cells or retroviruses. In combination with the many available mouse mutants, this method offers a new approach for analyzing genetic interactions through gain-of-function studies performed in mutant mouse backgrounds.

The use of high-frequency ultrasound as a method of assessing the severity of a plaque of psoriasis.

BACKGROUND AND DESIGN: Ultrasound imaging, while initially developed to visualize internal organs, is now being applied to image the skin. In this preliminary study, we used a high-frequency, 40-MHz ultrasound imaging system to provide high-resolution images in psoriasis and examined the relationship between clinical and ultrasound ratings in plaque-type psoriasis. The ultrasound image of a psoriatic plaque demonstrates a superficial echogenic band (band A), followed by a nonchogenic band (band B), and a deeper echogenic band (band C). RESULTS: In psoriatic plaques (N = 145), the severity of the psoriasis as assessed according to the degree of scaling, erythema, and thickness (SET score) correlated best with the width of band B (P < .001, r = 0.86) and less well with the width of bands A (P < .001, r = 0.59) and C (P < .001, r = 0.44). For the treated psoriatic plaques (n = 64), for which paired readings were available before and after therapy, changes in the SET scores correlated best with the change in the width of band B (P < .001, r = 0.96) and less well with the change in the width of bands A (P < .001, r = 0.61) and C (P < .001, r = 0.45). Ultrasound analyses and clinical evaluation were performed by independent raters. CONCLUSIONS: The data suggest that high-frequency ultrasound imaging may prove to be a noninvasive technique that can be used as an adjunct to the clinical evaluation of the lesional severity of psoriatic plaques.

40 MHz Doppler characterization of umbilical and dorsal aortic blood flow in the early mouse embryo.

Physiological study of the developing mouse circulation has lagged behind advances in molecular cardiology. Using an innovative high-frequency Doppler system, we noninvasively characterized circulatory hemodynamics in early mouse embryos. We used image-guided 43 MHz pulsed-wave (PW) Doppler ultrasound to study the umbilical artery and vein, or dorsal aorta in 109 embryos. Studies were conducted on embryonic days (E) 9.5-14.5. Heart rate, peak blood flow velocities, and velocity time integrals in all vessels increased from E9.5-14.5, indicating increasing stroke volume and cardiac output. Heart rate, ranging from 192 bpm (E9.5) to 261 bpm (E14.5), was higher than previously reported. Placental impedance, assessed by the time delay between the peaks of the umbilical arterial and venous waveforms and by venous pulsatility, decreased with gestation. Acceleration time, a load-independent Doppler index of cardiac contractility, remained constant but seemed sensitive to heart rate. High-frequency PW Doppler is a powerful tool for the quantitative, noninvasive investigation of early mouse circulatory development.

40-MHZ echocardiography scanner for cardiovascular assessment of mouse embryos.

Congenital heart disease results from genetic defects that are manifested at early stages of embryogenesis. The mouse is the preferred animal model for studies of mammalian embryonic development and for an increasing number of human disease models. A number of genes identified in the mouse are critical for normal cardiovascular development, but an understanding of the underlying mechanisms regulating heart development is still incomplete, in part because of the lack of methods to measure hemodynamics in live mouse embryos. We describe the development of a 40-MHz ultrasound scanner, which allows image-guided continuous-wave and pulsed Doppler blood flow measurements in mouse embryos, in utero, at the critical early developmental stages. Doppler waveforms acquired from mouse embryonic umbilical vessels, descending aorta, and cardiac ventricles are presented to demonstrate the utility of the method. By combining image-guided ultrasound Doppler with the many available mouse mutants, this approach should lead to new insights into embryonic cardiovascular structure-function relationships.

Ultrasonic characterization of selected renal tissues.

Velocity, attenuation, and backscatter of ultrasound were measured in human renal tissues over a frequency range relevant to clinical imaging (3.5-7 MHz). Normal renal tissues, as well as three types of mass (angiomyolipoma, renal cell carcinoma, and oncocytoma) were studied, and comparisons made of the appearance of the tissues in clinical images to their ultrasonic and pathological properties. The results showed angiomyolipoma had high attenuation and backscatter coefficients due to acoustic impedance differences between fat and smooth muscle components of the tumour. The renal cell carcinomas were indistinguishable from normal kidney tissue, except in one case where infiltration by fatlike macrophages led to high attenuation and backscatter coefficients. This finding also supports the conclusion that fat/nonfat interfaces are a dominant scatter mechanism in renal tissues.

Advances in ultrasound biomicroscopy.

The visualisation of living tissues at microscopic resolution is attracting attention in several fields. In medicine, the goals are to image healthy and diseased tissue with the aim of providing information previously only available from biopsy samples. In basic biology, the goal may be to image biological models of human disease or to conduct longitudinal studies of small-animal development. High-frequency ultrasonic imaging (ultrasound biomicroscopy) offers unique advantages for these applications. In this paper, the development of ultrasound biomicroscopy is reviewed. Aspects of transducer development, systems design and tissue properties are presented to provide a foundation for medical and biological applications. The majority of applications appear to be developing in the 40-60-MHz frequency range, where resolution on the order of 50 microm can be achieved. Doppler processing in this frequency range is beginning to emerge and some examples of current achievements will be highlighted. The current state of the art is reviewed for medical applications in ophthalmology, intravascular ultrasound, dermatology, and cartilage imaging. Ultrasound biomicroscopic studies of mouse embryonic development and tumour biology are presented. Speculation on the continuing evolution of ultrasound biomicroscopy will be discussed.

A 40-100 MHz B-scan ultrasound backscatter microscope for skin imaging.

There is a growing interest in high resolution, subsurface imaging of cutaneous tissues using higher frequency ultrasound, and several commercial systems have been developed recently which operate at 20 MHz. Some of the possible applications of higher frequency skin imaging include tumour staging, boundary definition, and studies of the response of tumours to therapy, investigations of inflammatory skin conditions such as psoriasis and eczema, and basic studies of skin aging, sun damage and the effects of irritants. Investigation of these areas is quite new, and the role of ultrasound skin imaging is continuing to evolve. Lateral resolution in the 20 MHz imaging systems ranges from 200 to 300 microns, which limits imaging applications to cutaneous structures which are relatively large in size. In this paper, a real-time ultrasound backscatter microscope (UBM) for skin imaging is described which operates in the 40-100 MHz range, providing axial resolution between 17 and 30 microns and lateral resolution between 33 and 94 microns. This improvement in resolution over current skin ultrasound systems should prove useful in determining the margins of small skin lesions, and in obtaining more precise, in vivo skin thickness measurements to characterize nonmalignant skin disease. Example images of normal skin, seborrhoeic keratosis and malignant melanoma illustrate the imaging potential of this system.

Ultrasound backscatter microscope analysis of mouse melanoma progression.

The incidence and mortality rate of cutaneous melanoma continue to increase throughout the world, making the study of melanoma biology an important area of current research. While recent breakthroughs in transgenic mouse technology have led to promising mouse skin models of melanoma, there is presently no technique available for quantitatively studying subsurface melanoma progression, in vivo. We demonstrate the first application of an imaging method called ultrasound backscatter microscopy (UBM) for imaging early murine melanomas with spatial resolution of 30 microns axial and 60 microns lateral. Murine B16 F10 melanomas have been imaged from their earliest detection, over several days, until they are 2 to 5 mm in diameter. Melanoma dimensions measured by UBM were found to be in excellent agreement with those determined histopathologically on the excised tumours. The relative rms errors in UBM-determined melanoma height and width were found to be 8.7% and 4.2%, respectively. The mean rate of increase in tumour height of early murine melanoma was found to be 0.37 +/- 0.06 mm/day. Computer-generated volumetric renderings of melanomas have been produced from three-dimensional image data, allowing quantitative comparisons of tumour volumes to be made. Using a priori assumptions of ellipsoid tumour shape, the relative error in UBM-determined volume was shown to be less than 17%. These results should be of considerable interest to investigators studying melanoma biology using mouse skin models, and have implications in the use of high frequency ultrasound imaging for the clinical assessment of cutaneous melanoma.

Fabrication and characterization of transducer elements in two-dimensional arrays for medical ultrasound imaging.

Some of the problems of developing a two-dimensional (2-D) transducer array for medical imaging are examined. The fabrication of a 2-D array material consisting of lead zirconate titanate (PZT) elements separated by epoxy is discussed. Ultrasound pulses and transmitted radiation patterns from individual elements in the arrays are measured. A diffraction theory for the continuous wave pressure field of a 2-D array element is generalized to include both electrical and acoustical cross-coupling between elements. This theory can be fit to model the measured radiation patterns of 2-D array elements, giving an indication of the level of cross-coupling in the array, and the velocity of the acoustic cross-coupling wave. Improvements in bandwidth and cross-coupling resulting from the inclusion of a front acoustic matching layer are demonstrated, and the effects of including a lossy backing material on the array are discussed. A broadband electrical matching network is described, and pulse-echo waveforms and insertion loss from a 2-D array element are measured.

Beam steering with pulsed two-dimensional transducer arrays.

The major problem facing the development of 2-D arrays for imaging is the complexity arising from the large number of elements anticipated in such transducers. The authors have undertaken a theoretical investigation of the focusing and steering properties of pulsed 2-D arrays to characterize the parameters required for medical imaging, such as element size, spacing, and number of elements. Details of the computational methods employed are presented, as well as a discussion of the steered beam properties of wideband 2-D arrays. The effects of apodization and element cross-coupling on the beam properties of a 2-D transducer array are examined. The beam properties of various sparse arrays with elements randomly distributed over the aperture of the transducer are discussed.

Characterization of lead zirconate titanate ceramics for use in miniature high-frequency (20-80 MHz) transducers.

The material properties of lead zirconate titanate (PZT) ceramics for operation in the thickness mode at frequencies as high as 80 MHz are reported. Each of the ceramics tested showed a reduction in k (t) with increasing frequency. In a fine-grained PZT, values of k(t) as high as 0.44 were measured at 80 MHz. The effects of grain size were also evident in the measurement of frequency dependent mechanical losses. Experimental and theoretical analysis of a 1 mmx1 mm, 45 MHz PZT transducer verified the validity of the measurements of the properties and demonstrated excellent insertion loss and bandwidth characteristics. The minimum insertion loss of -17.5 dB is in good agreement with theory and is a marked improvement over the performance of polymer devices. Details on the fabrication and testing of high frequency ceramic transducers are described.

In utero ultrasound backscatter microscopy of early stage mouse embryos.

A high resolution ultrasound imaging technique, ultrasound backscatter microscopy (UBM), has previously been shown to be useful for in utero imaging of mouse embryos, and for direct manipulation of mouse embryos through UBM-guided injections. UBM images from mouse embryos staged between 8.5 and 10.5 days of gestation are presented to demonstrate the range of anatomical structures which can be studied with this approach. Ultrasound contrast agents have been injected into the forebrain ventricle of 10.5 day embryos to characterize the resulting three-dimensional distribution of the injected agents. These studies provide important background data relevant to future use of this technique for in utero analysis of early brain and heart development, and for in utero manipulation of mouse embryos through UBM-guided injections.

Simulation of B-scan images from two-dimensional transducer arrays: Part II--Comparisons between linear and two-dimensional phased arrays.

Two-dimensional (2-D) arrays have been proposed as a solution to the degradation in medical ultrasound image quality occurring as a result of asymmetric focusing properties of linear phased array transducers. The 2-D phased transducer array is also capable of electronically steering the symmetrically focused ultrasound beam throughout a three-dimensional volume. In a companion paper the potential of 2-D transducer arrays for medical imaging has been investigated using simulated B-scan images. In this paper, the advantages of 2-D over linear transducer arrays is demonstrated by simulating images of spherical cysts embedded in a large scattering volume. The large elevation beamwidth in the nearfield of a 5 MHz linear phased transducer array results in a severe reduction in the image contrast measured between a 4 mm diameter cyst and the surrounding scattering media. By employing a 2-D array with symmetric focusing, the contrast between the cyst and surrounding scatterers is significantly improved. The use of additional elements in the elevation direction of a linear array is also investigated. In this case the additional elements are included only to focus, but not to steer the ultrasound beam. Using the contrast characteristics of a 4 mm diameter cyst, it is shown that relatively few elevation elements are required to significantly improve the nearfield imaging capability of the linear array.

2D multislice and 3D MRI sequences are often equally sensitive.

A simple theoretical model was developed to compare the sensitivities (i.e., signal-to-noise ratios per unit imaging time) of two-dimensional (2D) multislice and 3D imaging sequences. The model shows that the sensitivities of 3D and 2D multislice MRI sequences are usually similar. Sensitivities are identical in T2-weighted sequences when the T(R)s of the two sequences are the same. In T1-weighted gradient-echo sequences, sensitivities are very similar when Ernst angle excitation is used and the T(R) of the 2D sequence is less than T1. The predictions of the model are confirmed in phantom and animal experiments.