This protocol demonstrates robotic ultrasound (US) as a practical, cost-effective, and quick alternative to traditional non-invasive image modalities.
Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Each method has limitations and advantages, including availability, ease of use, cost, size, and the use of ionizing radiation or magnetic fields. This protocol describes the use of 3D robotic US for in vivo imaging of rodent kidneys and heart, subsequent data analysis, and possible research applications. Practical applications of robotic US are the quantification of total kidney volume (TKV), as well as the measurement of cysts, tumors, and vasculature. Although the resolution is not as high as other modalities, robotic US allows for more practical high throughput data collection. Furthermore, using US M-mode imaging, cardiac function may be quantified. Since the kidneys receive 20%-25% of the cardiac output, assessing cardiac function is critical to the understanding of kidney physiology and pathophysiology.
The most common modalities for in vivo rodent imaging include positron emission tomography (PET), optical imaging (OI), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). These techniques provide high-resolution in vivo images, allowing investigators to quantitatively assess and longitudinally follow disease models non-invasively1. While each imaging modality has limitations, they also provide invaluable tools for preclinical research.
Here, the study details a US system and presents the protocol for robotic and 3D rodent imaging. US waves are produced by a probe called a transducer, which is typically hand-held. Sound waves are reflected back as they interact with tissues, and the echoes are reconstructed into images2. The protocol described here will focus on kidney and cardiac imaging using a robotically controlled transducer and using software that allows rapid 3D reconstruction for quantitative assessment.
Robotic US is a fast, reliable, and non-invasive imaging modality that allows investigators to conduct high-throughput and longitudinal studies. Compared to hand-held US methods, the robotic US method is time-efficient, as up to three animals can be scanned in a matter of minutes. High throughput for kidney measurements suggests that up to 20 mice per hour may be imaged. The robotic transducers are located underneath the acoustic membranes and move independently of the animal with two degrees of freedom (Figure 1A). This allows novice users to obtain high-quality images, whereas hand-held US methods are more susceptible to user error. The coupled software allows efficient, real-time 3D kidney reconstruction. Previously, magnetic resonance imaging (MRI) has been a prevalent method for non-invasive imaging due to the excellent soft-tissue contrast, lack of radioactivity, and penetration depth. However, MRI often requires long acquisition times and is costly to perform. US has been evaluated as a reliable and more rapid alternative to MRI in assessing total kidney volume (TKV)3.
All steps in this protocol comply with the Mayo Clinic (Rochester, MN) animals use guidelines and have been approved by the Mayo Clinic Institutional Animal Care and Use Committee.
1. Animal model
2. Hair removal
3. Animal positioning
4. Ultrasound measurements
5. Kidney analysis (mechanics of analysis)
6. Cardiac analysis
Results of kidney analysis
Surface area and volume data are acquired from the segmentation of the kidneys. This information can be used to compare experimental and control models or track changes over time. The calipers tool is useful for quickly measuring abnormalities (i.e., cysts, tumors) and how they change in length over time. Figure 3 suggests that both the segmentation and caliper methods can be used to measure cyst volumes accurately. Figure 4 demonstrates a clear difference in total kidney volume (TKV) between age-matched control and experimental (Pkd1RC/RC) mice. 3D visualization of these volume renderings may be performed within the system, including rotations within 3D space (Figure 5). These 3D-reconstructions are then used to calculate TKV (mm3; Figure 4) as well as individual large cyst volume.
Results of cardiac analysis
Many useful parameters are acquired from the analysis of M Mode images. These data provide a good snapshot of the left ventricular (LV) cardiac function at that point in time. The data output includes LV internal diameter, LV posterior wall, LV anterior wall diameter, ejection fraction, fractional shortening, stroke volume, heart rate, cardiac output, LV volume, and LV mass. The success of cardiac analysis is dependent on accurate segmentation of the layers on the M Mode image. Most cardiovascular results are calculated by the peak systolic and diastolic phases of the posterior and anterior endocardial layers. The posterior epicardial layer appears bright white and follows a similar pattern to the posterior endocardial layer. The tracing for the posterior endocardial layer should be placed on the lowest contour. The anterior endocardial layer should be traced along the highest contour of that layer. The anterior epicardial layer appears linear at the bottom of the image due to the prone positioning of the animal (Figure 2D). Figure 6 shows an example of one study with no significant difference in cardiac output between experimental and control mice. As with renal imaging, 3D cardiac visualization is possible. Nevertheless, a 4D visualization of the cardiac cycle (Supplemental Figure 1) allows the investigator to visualize and pinpoint both morphologic and cycle-dynamic abnormalities in the assessed animal.
Morphology assessment
For quick and inexpensive assessment, US can effectively monitor physiological parameters longitudinally. However, many studies wish to additionally determine finer morphologic characteristics, e.g., number and sizes of cysts, calcifications (kidney stones), vascularization, or degree of fibrosis. Figure 7 compares a normal mouse kidney to a cystic mouse kidney to a moderately calcified mouse kidney. By increasing the US center frequency (10 MHz with the linear array) to 35 MHz (wobbler amplifier), pictures of increasing detail may be obtained.
Figure 1: Ultrasound system and mouse placement. (A) Diagram of ultrasound system and location of transducers. (B) View of mice in supine position on ultrasound platform. (C) Example of region of interest (ROI)s in place for area of interest (kidneys) with animal IDs. Please click here to view a larger version of this figure.
Figure 2: Cardiac ultrasound imaging to obtain physiological parameters. (A) Use of the Heart Finder heatmap image to position the transducer in the left ventricle for M-Mode imaging. The transducer location in the left ventricle is indicated by the large green dot. (B) View of the transducer when placed correctly over papillary muscles (dotted box). (C) Example view of layers needed to measure cardiac parameters. (D) View of live M-Mode image with layers designated as in panel C. (Layers from top to bottom: posterior epicardial, posterior endocardial, anterior endocardial, and anterior epicardial.) (E) Example output of statistics generated from cardiac measurements. Please click here to view a larger version of this figure.
Figure 3: Using segmentation and calipers tools to measure kidneys and cyst. (A) Example segmentations (axial view) of both kidneys (blue and orange shading) and a large cyst (yellow) with volumes listed below. Non-segmented views are shown underneath so that the unobscured US may be viewed. (B) Example use of calipers to measure the same cyst (sagittal view) from Figure 3A with measurements below. The volume was calculated using the formula for an ellipse (volume = (4/3)π x a x b x c, where a, b, c are relative x, y, z, respectively). Please click here to view a larger version of this figure.
Figure 4: TKV distributions of WT and cystic mouse kidneys. Representation of TKVs for wild-type (WT) (C57BL/6J) and diseased (Pkd1RC/RC) mice. n = 22 (WT) n = 9 (Pkd1RC/RC); Results of two-tailed t-test: p < 0.0001. Box shows 25-75th percentile values and whiskers show 1.5 times interquartile range. Please click here to view a larger version of this figure.
Figure 5: Animated 3D reconstruction of segmented kidneys and cyst. Using the software, the 3D projections of the kidneys and cyst may be rotated or rocked in the 3D space (blue = left kidney; yellow = large cyst; orange = right kidney). Please click here to download this figure.
Figure 6: Cardiac physiological parameters from US measurements. Representation of cardiac output (mL/min) for WT and diseased (Pkd1RC/RC) mice. n = 22 (WT) n = 9 (Pkd1RC/RC). The lower tabulated data show that there is no significant difference for these two groups in ejection fraction, stroke volume, heart rate (HR), or cardiac output (CO). Results of two-tailed t-test: p > 0.05. Box shows 25-75th percentile values. Please click here to view a larger version of this figure.
Figure 7: Comparison of US sagittal sections of normal and two pathologies. (A) Wild-type (C57BL/6J strain) kidney (TKV = 143.202 mm3). (B) Cystic kidney with increased TKV (Pkd1RC/RC mouse) (TKV = 333.158 mm3). Cysts are indicated by yellow arrows. (C) Kidney with vascular calcifications (Model = Low-Density Lipoprotein Receptor Deficient, Apolipoprotein B100-only mouse fed a Western diet for 12 months5) (TKV = 127.376 mm3). Renal stones are indicated by green arrows. Please click here to view a larger version of this figure.
Supplemental Figure 1: 4D cardiac cycle movie from US measurements. Using the software, a representation of the beating heart is captured in 3D US and projected through the cardiac cycle. The green arrow indicates the aortic valve. (Model = Low-Density Lipoprotein Receptor Deficient, Apolipoprotein B100-only mouse, fed a Western diet for 12 months5). This model generates vascular calcifications enabling easier visualization of the heart and valves due to the greater acoustic reflectivity of the calcifications in US. Similar 4D reconstructions are possible with WT mice; however, the captured acoustic contrast will not be as high. Please click here to download this File.
Ultrasound utilizes sound waves, and any barriers to sound wave propagation will interfere with image quality. Thus, complete hair removal of the area to be imaged is critical. It is also important to ensure complete removal of depilatory cream as it can cause burns/irritation of the animal's skin and discolor the transparent membrane of the scanner. Adequate water levels in the bays are necessary for optimal sound wave propagation, thus required for obtaining the highest image resolution. However, when the animal is in a prone position, ensure the animal's snout is above the water level or the animal is at risk of water inhalation. Optimization of imaging parameters, especially focal depth, is critical for obtaining high-quality images. Modifications to parameters may be necessary for individual animals.
Robotic US provides many advantages over traditional hand-held US modalities. First, the system uses a simple point-and-click camera-based interface. This feature addresses the complexity of conventional US and produces consistent data even when operated by a novice user6. Second, the system allows the use of water rather than traditional US gel as an acoustic medium. Previously, the use of US gel allowed the formation of bubbles that interfered with quality image acquisition. Also, the US gel is messy and provides challenges for clean-up. Further, the water is warmed by the heat lamp and helps maintain the animal's body temperature. Third, the robotic US is faster, so artifacts from respiratory motion are not problematic. The increased imaging speed allows for the practical completion of high throughput data collection. Fourth, the robotic US obtains 3D images, and therefore makes 3D reconstruction of objects simple (Figure 4). MRI and other modalities are expensive, time-consuming, and not always available. Importantly, the robotic US system fits on a table or bench and is more cost-efficient. Finally, prior work demonstrated that robotic US could provide comparable measurement data to more expensive modalities, such as MRI3.
While the image quality and resolution of the robotic US system described in this work were adequate for the proposed application (Figure 7), there are several ways that image quality can be improved in the future. For instance, utilizing higher frequency transducers (e.g., 50-70 MHz) would result in higher resolution images with better feature definition. While utilizing higher frequencies would result in a poorer depth of penetration, the images should be sufficient for in vivo imaging of superficial organs, such as the mouse kidney. As with other imaging modalities, contrast agents may be used to enhance specific features. For US, this typically means using something highly reflective of sound waves. Intravascular microbubbles in which lipids surround very small gas bubbles are one such agent. The micron-sized gas bubbles are highly reflective and thus provide a second distinct signal that translates into a high-resolution of vasculature7. While this acoustic contrast technique may be quite useful, it can have several downsides. First, the microbubbles must be made fresh and only persist in vivo for 5-10 min. Second, in vivo intravascular injection typically requires tail veil catheterization for injection, and this can be technically challenging. Under certain circumstances and pulsing regimes, microbubble imaging can itself lead to renal vasculature damage8.
There are also some more general limitations of the particular US system used. First, only one linear array (centered at 18 MHz) is included in the robotic chassis, so switching to higher or lower frequency probes is not currently possible. This may impact the breadth of models (either larger or smaller) that can be evaluated with the system. Future iterations of the instrument should include multiple linear arrays to cover the full range of preclinical animal models. Second, the transducer angle relative to the animal subject cannot be controlled. Therefore, performing angle-dependent imaging techniques, such as Doppler, or achieving alternative in-plane views of certain organs (e.g., long axis view of kidney) requires repositioning of the animal and can be difficult to achieve. Additional degrees-of-freedom could be added to the robotic movement to ameliorate this challenge. Third, on occasion, we have observed reverberation artifacts arising from the acoustic membrane that separates the animal from the transducer that can obscure visualization of superficial features and boundaries. In these cases, using a gel standoff to elevate the animal away from the membrane can remedy the situation. Finally, temperature control via heat lamp is imprecise, and therefore close attention must be paid to the animal's core body temperature while imaging. More controlled heating mechanisms, such as an integrated heating pad, will likely improve homeostasis management and imaging throughput.
The use of robotic US may be applicable to various fields of research. This technology enables visualization of gross tissue structures, thus may be used to track tumor progression and potential therapies6,9 as well as renal morphology as presented here. The ability to segment the specific features of the images makes it an attractive tool for studying models of polycystic kidney disease (PKD)3. M-mode images allow for simple quantification of many important cardiac parameters enabling in vivo assessment of cardiac physiology. As the kidneys receive 20%-25% of the cardiac output10, understanding cardiac function during the longitudinal assessment of renal pathology is important. Through these US protocols, we have tried to illustrate that US imaging is not only practical for in vivo and longitudinal kidney studies but also that increasingly US tools enable both morphologic as well as physiologic assessment of mice in preclinical studies.
The authors have nothing to disclose.
This work was supported by the NIH (R43-DK126607, TJC, TLK, MFR) and the Mayo Foundation.
Electric Razor | Braintree Scientific, Inc | CLP-9868 14 | |
C57bk6j | The Jackson laboratory | https://www.jax.org/ | |
Cotton gauze pads | Fisher Scientific | ||
Cotton tipped applicators | Fisher Scientific | ||
Depilatory cream | N/a | N/a | This study used Nair |
Heat lamp | Included with SonoVol Vega system | ||
Robotic Ultrasound System | SonoVol Inc | SonoVol Vega system includes anesthesia system | |
SonoEQ Software | SonoVol | Included with SonoVol Vega system | |
TERRELL Isoflurane | Piramal Critical Care, Inc | NDC 66794-019-10 |