Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

Our research focuses on non-invasive ultrafast ultrasound imaging of the liver using a commercially available ultrasound platform. It aims to streamline imaging and analysis processes, provide an anatomical references, and enhance quantitative and qualitative assessment of the liver vasculature. Recent developments in our field include ultrafast ultrasound, which detects small blood flow in small vessels, enhance sensitivity with the contrast agent, and improve spatial resolution.

This is achieved through plane wave transmissions, increasing frame rate, and providing detailed blood volume estimates in each pixel. Current technologies found in research in our field are ultrafast ultrasound imaging and 3D micro-CT. These tools, combined with the advanced understanding of the liver anatomy, improved accuracy, and reproducibility of ultrasound imaging in murine models.

We have established a non-invasive, ultrafast ultrasound imaging protocol for mouse liver vasculature, enabling precise blood volume measurements without contrast agents. Our findings include a standardized procedure for data analysis, robust vascular parameterification, and clear visualization of anatomical structures, ensuring accurate and reproducible microcirculation assessments. Our protocol offers non-invasive, high-sensitivity liver imaging without the need for contrast agents, capturing slow blood flow and small blood vessels.

It improves accuracy by removing motion artifacts, standardized quantification, and a clear anatomical overview and show precise, reproducible measurements. To begin, position the prepared anesthetized mouse on the heating pad. Gently trim the belly of the animal with a veterinarian trimmer without applying compression to the chest or abdomen.Then.

apply depilatory cream for no more than two minutes. Remove the depilatory cream and clean gently with warm water. Next, position the animal dorsally on a heating pad and gently secure the paws using tape.

Moisturize the skin with a small amount of warm water. Using a 20 milliliter syringe, apply the centrifuged ultrasound transmission gel directly on the shaved skin. Then, place the 3D printed water tank above the animal.

Check for any air between the ultrasound transmission gel and the polymethylpentene thermoplastic of the water tank, then add water to the tank. After preparing the mouse for ultrafast Doppler ultrasound, start the software, create an experimental session on the system, and launch the scanning software. Click yes in the popup window to center the motors.

To create a new experimental session, select session from the menu. In the session panel, fill in the necessary information, including project, subject, session, username, comment and tag. Click validate.

Next, load a 10 millisecond Doppler ultrasensitive ultrasound sequence with the load sequence button. Define the imaging depth between one and 50 millimeters, with a maximum imageable depth of 50 millimeters. To select visualization parameters, choose Doppler to image the vasculature.

Choose the image compression method, logarithmic, square root, or linear. Alternatively, adjust the contrast range automatically or manually using sliders for high and low contrast options. To select the color map scale from black to white, check the gray option, or from reddish black to yellow, check hot.

Select the imaging plane based on anatomical landmarks. Switch between the visualization of Doppler and B mode to get familiar with the anatomy of the liver lobes and vasculature. Click on the stop icon to stop the live view.

Now, navigate to the move probe menu to adjust the probe and select a plane. To begin, prepare the animal for imaging and position the probe in the water tank. Perform probe operation and plane selection, then, launch a Doppler acquisition with a 10 millisecond frame rate.

Go to the fUS 2D menu to image hemodynamic variations over time at a specific position. In the preset tab, set the time between images to zero seconds to choose a continuous mode with no pause between images, then define the total recording duration in seconds. Then, press the record button to start the 2D acquisition.

Once the recording is over, press the stop button. Provide a name for the acquired recording to save the file in a scan format in the session folder. A popup window will appear at the end of each recording to save the data automatically.

Open the analysis script gui_analysis.m. Select the scan and set 10 regions of interest with a voxel size of two by two. Choose 40 milliseconds for each frame to perform the Doppler integration.

Then, draw the vascular periphery of the organ in a way that the 10 ROIs are automatically prepositioned between clearly visible arteries and can be adjusted manually if needed. To quantify mean hepatic blood volume in arbitrary units of the total 10 ROIs, automatically estimate the mean and standard deviation of the quiet frame values within the 30 second recording.