A feasibility study for noninvasive measurement of shear wave speed in live zebrafish
Introduction
Zebrafish are currently developing animal models for human diseases such as cardiomyopathy or neuroblastoma. Owing to a nearly fully sequenced genome and genetic similarity with respect to human genomes [1], zebrafish are an ideal vertebrate model for human diseases research [2]. For example, zebrafish models have been developed for cardiomyopathies such as doxorubicin-induced cardiomyopathy or diabetes-induced cardiomyopathy [3], [4], and neuroblastoma including a recently developed MYCN model [5], [6]. Current methods of phenotyping these zebrafish models include echocardiography for cardiomyopathy and microscope imaging for neuroblastoma. There are no noninvasive techniques to measure elastic properties of zebrafish.
Vibro-acoustography is a technique based on the acoustic radiation force of ultrasound [7]. In vibro-acoustography, the acoustic radiation force of two focused ultrasound beams with slightly different frequencies generates a vibration at an object at a lower different frequency; the acoustic response of the object to this force is detected by a hydrophone. Vibro-acoustography has been tested on various tissues including breast, prostate, arteries, liver, and thyroid [8]. We also developed an ultrasound stimulated optical vibrometry technique to analyze arterial tube vibration [9] and elastic modulus [10]. This technique uses the acoustic radiation force of ultrasound to locally generate an arterial tube and uses a scanning laser system to detect the resulting vibration or wave propagation in the arterial tube. Sonoelastography was developed in 1990 by applying low frequency vibration to induce oscillations within soft tissues and detecting the tissue motion using a Doppler ultrasound based technique [11]. This technique is technically complex.
We developed ultrasound vibro-elastography (UVE) techniques to study various tissue diseases. UVE is a safe and noninvasive technique for generating and measuring surface wave propagation on the skin and shear wave propagation in the subcutaneous tissue. In UVE, a local 0.1-second harmonic vibration at a frequency is generated on the skin using a handheld shaker. A small tissue motion in tens of µm is enough for sensitive ultrasound detection of the generated tissue motion. The excitation of the shaker on the skin is significantly less than 1 N. Patients feel very gentle motion on their skin and experience no discomfort. An ultrasound probe is aligned about 5 mm away with the shaker indenter to measure the generated tissue motion at each pixel of the ultrasound imaging plane. Particle velocity in the axial direction of the ultrasound beam is analyzed using an autocorrelation method [12]. The resulting surface wave on the skin surface is used to study the skin, and the shear wave in the subcutaneous tissue is used to study internal tissues. Both the surface wave and shear wave measurements are obtained in a single test. The wave speed measurement depends on the local elastic property of tissue and is independent of the location of wave excitation. UVE has been used for assessing interstitial lung disease [13], [14], [15], [16], [17], ocular disorders such as glaucoma [18], [19], and erectile dysfunction [20]. However, the zebrafish size is much smaller than human organs. The purpose of this research was to study the feasibility of zebrafish ultrasound vibro-elastography (ZUVE) for measuring shear wave speed in this vertebrate model.
Section snippets
Materials and methods
Adult female WIK zebrafish were obtained under approved Institutional Animal Care and Use Committee (IACUC) protocol A0000351100 and maintained at 28.5 °C. The zebrafish were anesthetized using 0.016% Tricaine-S (MS-222, Syndel, Ferndale, Washington 98248, USA) in fish water. A zebrafish was considered anesthetized when its respiration rate decreased as visually noticead by its gill movement. Three minutes of anesthesia was designed for the zebrafish study, because the zebrafish experimental
Results
Fig. 2 (b), (c), and (d) show, respectively, for zebrafish 1the shear wave speed of 3.13 ± 1.20 (m/s) (mean ± standard devivation) in the ROI for 300 Hz, 4.28 ± 1.36 (m/s) in the ROI for 400 Hz, and 5.07 ± 1.45 (m/s) in the ROI for 500 Hz.
Fig. 3(a) show wave speed dispersion from 300 Hz to 500 Hz for zebrafish 1. Three measurements were performed at each frequency. Fig. 3(b) show wave speed dispersion from 300 to 500 Hz for zebrafish 2. Fig. 3(c) shows wave speed dispersion from 300 to 500 Hz
Discussion
The aim of this study was to prove the feasibility of using the ultrasound vibro-elastography technique for noninvasively and safely measuring the shear wave speed of live zebrafish. Because a zebrafish is very small, a high frequency 18 MHz ultrasound probe was used. To reduce the wavelength of generated wave propagations, wave propagation in zebrafish was studied at 300, 400, and 500 Hz. In human studies, we typically measured wave speeds at 100, 150, and 200 Hz.1
Conclusions
The feasibility of zebrafish ultrasound vibro-elastography (ZUVE) was demonstrated for measuring shear wave speed. An adult female zebrafish was anesthetized for three minutes for the ZUVE testing. A 0.1 s gentle harmonic vibration was generated on the tail of the zebrafish using a sphere tip indenter with 3 mm diameter. Shear wave propagation in the zebrafish was measured using a high frequency 18 MHz ultrasound probe. Shear wave speeds were measured at 300, 400, and 500 Hz. Shear wave speeds
Acknowledgement
We thank Mrs. Jennifer Poston for editing this manuscript.
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