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Imaging Guide: Guide to Small Animal Pancreatic Imaging using the Vevo 2100 Page 1

Guide to Small Animal Pancreatic Imaging using the Vevo® 2100 Imaging System

Course Objectives: This guide is designed to assist the user to:

- recognize pancreatic structures in typical views - position the correct probe to get the appropriate views of the pancreas - performing the measurements of interest to get anatomical and physiological

data Overview of the Research Areas and Anatomy of the Pancreas The pancreas of the mouse is located in the upper left abdominal cavity. It borders the major vessels medially and the spleen laterally. The kidney is inferior to the pancreas while the stomach is superior (Figure 1). In ultrasound, the pancreas has a “brick road” or “cobblestone” appearance.

Figure 1. Schematic showing the abdominal cavity of the mouse with the pancreas exposed and its relative position to surrounding anatomy. http://www.informatics.jax.org/cookbook/figures/figure56.shtml

Imaging Guide: Guide to Small Animal Pancreatic Imaging using the Vevo 2100 Page 2

Overview of the Imaging Modes to be Used in this Guide B-Mode: B-Mode, or brightness mode, imaging is used to acquire two dimensional images of an area of interest. The grayscale image allows for identification of anatomical structures, and the software allows for various measurements in this mode of imaging. A motor can be attached to the ultrasound probe to translate the probe over a defined range with a defined step-size, the software then recompiles the various 2D slices into a 3D volume. The 3D software allows for various measurements and imaging planes in this type of imaging. Power Doppler Mode: Power Doppler Mode is another tool used to analyze blood flow within vessels. Power Doppler Mode color codes flow according to a frequency Doppler shift and signal intensity. This results in less angle dependent color changes and more sensitivity. However, this mode is often used on smaller vessels having slower blood flow where velocity data isn’t important. A Power sample box is placed around the vessels of interest and the system is able to display the Doppler shift in a color (orange to yellow) and is displayed over a B-Mode image. Various Power maps are available to optimize flow data presentation and are user selectable. Please note: Power, Color, and simultaneous B/PW modes display the flow data in the highest resolution/sensitivity as possible in a given physiological state. The B mode resolution will be slightly reduced by the required time sharing found on all ultrasound systems. So high resolution imaging should be done by B-Mode while flow data should be done by PW, Color and Power modes. Color Doppler Mode: Color Doppler Mode is similar to Power Doppler Mode and both utilize many of the same parameters to optimize their sensitivity. Color Doppler Mode displays the mean velocity while power displays a combination of Intensity and Doppler shift. Color mode displays the direction of the Doppler shift so the color is assigned accordingly with blood flowing towards the transducer on a scale from red to white and away from the transducer on a scale from blue to white. BART is an acronym commonly used to remember the colors associated with the direction of flow; Blue Away, Red Towards. Various Power maps are available to optimize flow data presentation and are user selectable. Contrast Mode: Contrast Mode is a mode very similar to B-Mode in that it is used to look at 2D anatomical structures. However, it includes specific analysis tools such as reference subtraction of two images. The result is a copper (or green) contrast overlay used to show the injected contrast agents. Contrast agents are used to enhance the visualization of blood flow in blood vessels including very small vessels such as capillaries. The contrast measurement tools not only produce enhanced flow visualization, but also provides a variety of quantification tools including various hemodynamic parameters.

Imaging Guide: Guide to Small Animal Pancreatic Imaging using the Vevo 2100 Page 3

Overview of the Parameters to be Optimized Presets All modes come with Presets. These are groups of imaging parameters that can be selected by pressing the “Preset” key. This reduces the time required to change imaging parameters individually. Users can define and name their own presets to optimize imaging for their own protocols by pressing the “Save Preset” key. This memorizes the current settings and requires a new name. These presets are probe-specific so each probe must have its own settings. Presets can be imported and exported between users. B-Mode Parameters: There are numerous parameters which can be set to optimize a B-Mode image, outlined below are a few key parameters, for a full list of parameters and how they affect B-Mode imaging please consult our Vevo 2100 Operators Manual.

Display Maps Display maps are used to assign grayscale colors to detected ultrasound signal intensities; this allows optimized grayscale differentiation of specific anatomical targets. The Vevo 2100 system comes with numerous factory settings which can be selected from to provide the best quality images.

Dynamic Range Dynamic range determines the range of grayscale intensities which can be displayed on an image. Together with the display maps, dynamic range can be adjusted to provide the best quality images with less noise.

Focal Zones The Vevo 2100 system can have one, two, or three focal zones, with the spacing of the two and three focal zones being narrow or wide. Using additional focal zones provides an image which is focused over a broader depth, however increasing the focal zones will decrease the maximum frame rate. The number of focal zones and their placement should be selected based on the anatomical structures to be imaged and the amount of image motion.

Line Density Line density allows the user to change the space between lines on the ultrasound image. Increasing the line density will increase the resolution, texture, and overall will increase the quality of the image; however it will decrease the maximum frame rate at which the system can image. Pulsed-Wave, Color and Power Doppler Mode Parameters: There are various parameters which can be set to optimize Doppler imaging in PW and Color Doppler imaging modes. Please see our Quick-Start Guide: Imaging in Color Doppler, Power Doppler and Power Doppler 3D Modes for an explanation of these parameters and how they will affect the image quality.

Imaging Guide: Guide to Small Animal Pancreatic Imaging using the Vevo 2100 Page 4

Selecting the Appropriate Probe for Imaging Transducer (probe) selection involves many factors. Higher frequencies provide the highest resolution but limit both penetration, imaging depth, and image width. Lower frequency probes have the best Doppler sensitivity with the least aliasing. So, using one probe for the best imaging while using another probe for the best Doppler isn’t uncommon and highly recommended. To image the pancreas of a mouse the MS550D is the ideal probe to use, but the MS550S and MS400 may also be used. When imaging rat pancreas, B-Mode images should be acquired with the MS400 probe, while the Doppler images are best acquired with the MS250 or MS200 probes. Software Measurements When imaging the pancreas, some of the measurements that can be made are: Overall Pancreatic Blood Flow (Color Doppler, Power Doppler and Contrast Modes) – To obtain flow within the pancreas using Color Doppler Mode and/or Power Doppler Mode, consult the Quick-Start Guide: Imaging in Color Doppler, Power Doppler and Power Doppler 3D Modes To obtain micro-vasculature flow within the pancreas, please see the VisualSonics Application Protocol – Image Enhancement by Bolus Injection using untargeted MicroMarker™ contrast agents. Animal Preparation The animal should be anaesthetized to a level appropriate to a surgical procedure. Typically isofluorane is used at a concentration of 1.5-2%. After the animal is anaesthetized in the knock down box, it should be positioned on the platform in an optimal position to inject the target structure. Eye lubricant should be placed on each eye to prevent drying of the area. A small amount of ECG gel should be placed on the copper leads on the platforms and the paws taped to them, this will provide the ECG and respiratory physiology. A rectal probe should be inserted to monitor the temperature of the animal during the imaging session. The hair on the abdomen should be removed using a depilatory cream, such as Nair, and rinsed with water.

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B-Mode Imaging To image the pancreas, the animal platform should be level and the probe should be placed transverse – that is, the probe lies across the stomach from left to right as opposed to head-to-toe. Place the probe to the animal below the ribs and slightly to the left side. Using the Y-axis manipulator, move the animal to bring the kidney and spleen into view. When both are visible, the tissue in between the two is the pancreas (Figure 2). It has a very typical “brick road” or “cobblestone” appearance in ultrasound, but no defining wall; it is more of a diffuse structure. To ensure the liver is not in view, scan towards the head and the liver will appear – it is darker in appearance in ultrasound and has a capsule enclosing it.

Figure 2. Mouse pancreas in the transverse view showing relative position to kidney and spleen.

Imaging Guide: Guide to Small Animal Pancreatic Imaging using the Vevo 2100 Page 6

Color and Power Doppler Mode Imaging Turn on the Color (Figure 3) or Power Doppler (Figure 4) Mode and optimize the setting by the “Preset” key. Typical presets for pancreas might be: Very Slow, Slow Flow. Find the highest velocity flow in the Color regional area of interest.

Figure 3. Color Doppler Mode image of the mouse pancreas in transverse view showing relative position to kidney and spleen. After the vessel is located in Color Doppler Mode, PW Doppler Mode can be used to quantify blood flood in this vessel. Press “PW” from the mode buttons and this will bring up the sample volume for PW Doppler Mode. Moving the cursor will move the sample volume. Place the sample volume in the vessel of interest. Adjust the Doppler Angle to align with blood flow. Press “PW” one more time to bring up the PW Doppler window. If the waveform is too big or too small, adjust the display by turning the “Velocity” dial. If the waveform is positioned too high or too low for the display window, it can be moved up or down with the “Baseline” button.

Imaging Guide: Guide to Small Animal Pancreatic Imaging using the Vevo 2100 Page 7

Figure 4. Power Doppler Mode image of the mouse pancreas in transverse view showing relative position to kidney and spleen.

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