Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

This protocol allows novice users to obtain high-quality renal, cardiac, and liver data. The integrated robotic ultrasound scanning is also significant, as there is no need to reposition animals or the acoustic probes. The main advantages of this technique are both speed and ease of use of the instrument.

One can easily obtain high-quality images from 20 to 30 mice in an hour. We would expect a novice user to struggle positioning the animal correctly. Our advice would be to use a large ROI box to ensure the entire kidney is imaged.

Begin by use an electric razor to shave the desired skin area for ultrasound, or US, measurements. Apply the depilatory cream to the animal's shaved skin with a cotton-tipped applicator. Wipe the cream off after 30 to 60 seconds using a dry paper towel.

Continue to wipe away the depilatory cream residue using gauze pads moistened with water, ensuring that the hair removal is complete before beginning with the scans. Turn on the US machine and the temperature control above the US platform. Turn on the appropriate anesthesia valves for the corresponding US bays.

For cardiac measurements, place the animal in a prone position and angle it approximately 30 degrees to the right. For kidney measurements, place the animal in a straight and supine position. Gently flatten the animal's body to minimize the air pockets between the animal and the membrane.

In the imaging US software, select US Acquisition and allow the instrument to initialize. To begin with kidney imaging using B Mode, enter a name for the study in the appropriate dialogue box. Then, enter the animal ID in the appropriate dialogue box.

Click on the tab labeled 3D to initialize 3D scanning. Using the live camera feed, move the region of interest, or ROI box, to contain the kidneys. Depending on the type of image desired, select Linear Array or Wobbler to choose the transducer.

Select from Fast Scan or Optimized for either transducer. Click on Live View to see a live image of the desired viewing area. To move the transducer, click on the circular Go To button and then click on the area of the mouse to be imaged.

Use the directional arrows with predetermined step sizes to finely adjust the transducer position. Click on the arrow at the side of the control panel window to open the Settings tray Click on Scan Protocol to open scanning settings. Determine the ideal focal depth and adjust the sliders for the time gain compensation, or TGC, to change the image contrast to best view the kidneys.

After achieving the desired settings, Click on the arrow again to close the Settings tray. Press the Acquire Static button to commence 3D scanning. Once the scanning is complete, check the image quality by clicking on the blue download arrow to open the scan in preview mode.

Move through the 3D frames of the scan by dragging the red arrow at the top of the window. To zoom in or out hold the Control key and use the scroll button on the mouse. Click on the M Mode tab to activate cardiac imaging.

Push the Heart Finder button to locate the heart. Use the heat map to optimize the location of the transducer. Use the circular Go To button to move the transducer to the left ventricle.

Go to the live view and use the arrow buttons to place the transducer between the papillary muscles, and proper placement of the transducer can be verified in the live view. Once the transducer is in the appropriate position, click on Acquire Sequence. When the scans are complete, dry the animals off and return them to their home cages.

Move the colored lines representing various planes to find the kidney in all the planes. Click on Add to add a new segmentation. Provide a markup name for the file.

Use the Surface Cut button to add points around the outer edge of the kidney in multiple planes. Click on Apply to generate segmentation through the entire kidney. Scroll through the different frames in each plane and edit any inaccurate regions by clicking on Edit and dragging the points to the correct location.

Use the Point Placement tool to trace the systole and diastole for each layer of heart tissue. Continue with the other layers of heart tissue. Use the Edit function to modify the traces.

Select Apply to calculate the cardiac parameters. View the Quantify tab and select Export Statistics to export all data as a csv file. It was observed that the surface area and volume data acquired from the segmentation of kidneys, and the caliper tool used to measure abnormalities, could be used to measure cyst volumes accurately.

A clear difference was observed in the total kidney volume between age-matched control and experimental mice. 3D visualization of the volume renderings was performed within the system, including rotations within 3D space. The posterior epicardial layer appeared bright white, and followed a similar pattern to the posterior endocardial layer.

The anterior endocardial layer was traced along the highest contour. The anterior epicardial layer appeared linear at the bottom of the image due to the prone positioning of the animal. A normal mouse kidney was compared to a cystic mouse kidney and a moderately calcified mouse kidney using ultrasound.

The most important step in obtaining high-quality images is ensuring the hair is completely removed. Any remaining hair will cause interference in the images and decrease image quality.