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

All animal experiments were performed under European legislation (Directive 2010/63/EU) and Dutch government guidelines and approved by the Animal Ethical Committee of Leiden University Medical Center.

1. Mouse preparation for UFUS imaging (Timing ~ 10 min)

1. Preparation of imaging setup
   1. Centrifuge ultrasound transmission gel in 50 mL tubes at 100 x g for 3-5 min to prevent air bubble formation.
   2. Turn on the heating pad and warm up to 35 °C. Fill the 3D-printed water tank with deionized water.
      NOTE: Water bubbles or small particles present in the water may disturb the Doppler measurement.
2. Anesthetization of the animal
   1. Weigh the animal. Male and female litter mates that are 8-16 weeks old with a bodyweight of 19-32 g were used. Prepare an anesthesia mix of Ketamine with an administration dose of 100 mg/kg and Xylazine 10 mg/kg in 0.9% NaCl. Prepare the mixture sterile and fresh every day because the mixture is not stable and may lose potency.
      NOTE: Preferred animal body weight is between 23-30 g.
   2. Disinfect the injection site, i.e., lower right or left quadrant of the abdomen, using gauze and 70% ethanol. Inject 10 µL/g body weight intraperitoneal (ip) with a 27G needle and a 1 mL disposable syringe or a 30G insulin syringe.
   3. Place the animal back in a cage on a heating pad without closing the cage lid. Start timer.
      NOTE: Deep anesthesia should last for approximately 40 min. If a longer imaging session is desired, consider using reversible anesthesia, such as mixtures of Ketamine 60 mg/kg and Medetomidine 0.4 mg/kg in 0.9% NaCl followed by reversal with subcutaneous injection of Atipamezole 1 mg/kg. Injection anesthesia is preferred over inhalation anesthesia because of the imaging setup.
   4. Check the anesthesia's effectiveness. Confirm whether the anesthesia is working by gently pinching the hind paws after approximately 5 min. If the animal still reacts, wait another 2-3 min to achieve full anesthesia.
   5. Apply ophthalmic ointment to prevent corneal drying and eye trauma when the animal is deep asleep. Check the reflexes again and begin the procedure only when the animal shows no reaction to pinching.
3. Preparation of the imaging area (Figure 1)
   1. Trim the belly of the animal gently with a veterinarian trimmer while it is placed on a heating pad. Perform the trimming very gently without applying compression to the chest or abdomen.
   2. Apply depilatory cream for no more than 2 min.
      CAUTION: Do not extend the incubation time of the depilatory cream, as it may cause skin inflammation. This not only affects imaging quality but also may cause discomfort for the animal.
   3. Remove depilatory cream with some paper tissues and clean gently with warm water.
4. Position the animal and imaging setup for Doppler measurements (Figure 2A).
   1. 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.
   2. Apply the centrifuged ultrasound transmission gel directly on the shaved skin using a 20 mL syringe.
      NOTE: Air bubbles may affect Doppler imaging and cause artifacts.
   3. Place the 3D-printed water tank filled with water above the animal. Ensure the height of the water tank is suitable for the animal, approximately 1 mm above it, and does not squeeze the animal.
   4. Check for any air between the ultrasound transmission gel and the Polymethylpentene thermoplastic (PMP) of the water tank.

2. Probe operation and plane selection (Timing ~ 10 min)

1. Start the software and create an experimental session on the system. Start the scanning software.
   NOTE: Scanning software is integrated into the system.
2. Center the motors by clicking on Yes in the pop-up window. A motorized system is used to remotely control the probe by scanning software. By centering the motors, the probe will automatically move in three directions, that is, vertically, laterally, and rotated. Make sure that no objects hinder the trajectory of the probe to avoid damaging the equipment.
   NOTE: It is crucial to center the motors; failure to do so can lead to software dysfunction.
3. Create a new experimental session by selecting Session from the menu. Fill in the required information in the session panel, including Project, Subject, Session, Username, Comment, and Tag. Click Validate.
   NOTE: The session needs to be created before acquiring or saving imaging data. Session information can be changed during the experiment, but keep in mind it will also alter the destination for saving data.
4. Position the probe. The linear 15 MHz ultrasonic probe with 128 elements is connected to the system through a motorized system composed of a four-axis motor module, that is, three translational and one rotational movement direction.
   1. Go to the Move probe menu and use the computer-controlled positioning system to move the probe into the water in the 3D-printed water tank. Utilize the control buttons indicated in the Probe position to move the motors, defining the steps in mm for translational movement (X, Y, and Z) or in ° for rotational movement (R). To prevent accidents in the Z direction, define the Z limit in Safety.
      NOTE: It is crucial that the probe's elements are in contact with the water. However, avoid moving the probe completely into the water, as this may harm the equipment. If the motors cannot reach the position, a warning Out of workspace is displayed.
   2. Save the coordinates as a reference position by clicking on Set Home. Do not modify translations and rotations concurrently because R is relative to X, Y, and Z. Always click on Set Home prior to modifying R.
   3. Define imaging and visualization parameters. Go to the Live view menu and click on Play to select the plane of interest.
      CAUTION: Launch only a Live view or an acquisition when the elements of the ultrasound probe are surrounded by a medium matching the acoustic impedance to ensure the lifespan of the equipment and to prevent overheating.
5. Chose imaging parameters in Preset.
   1. Load a 10 ms Doppler ultrasensitive ultrasound sequence with the Load Sequence button, for example, 11 compounded plane waves (angles from -10° to 10°) with an imaging voltage of 25 V and a Doppler framerate of 10 ms.
      NOTE: There is no need to use a fast Doppler sequence (e.g., 10 ms) in order to image and filter the respiratory movements of the organ during the analysis.
   2. Define imaging depth between 1 mm and 50 mm, with a maximum imageable depth of 50 mm. The default setting is a depth of 9 mm from 1 mm to 10 mm, with the recommended depth between 2 mm and 12 mm.
      NOTE: Depending on the size of the animal and the water tank, imaging depth can be tailored to specific needs.
6. Select visualization parameters.
   1. Choose Doppler to image the vasculature.
      NOTE: Pixel intensity in A.U. represents blood volume and should be compared only when using the same probe and sequence.
   2. Choose Averaging for applying temporal averaging to optimize signal-to-noise ratio.
      NOTE: Temporally averaging the images increases image quality but reduces the frame rate. Depending on the objective, a suitable trade-off should be made.
   3. Choose Image compression: Logarithmic (high), Square root (intermediate), or Linear (low). Alternatively, adapt the contrast range automatically or manually using sliders for high and low contrast options.
      NOTE: Data is not affected by the choice of image compression; it only affects the display of the real-time imaging.
   4. Select the Color Map scale from black to white by checking the Grey option or from reddish-black to yellow by checking Hot.
7. Plane selection (Figure 2B)
   1. Select the imaging plane on anatomical landmarks. Switch between the visualization of Doppler and B-mode to familiarize with the anatomy of the liver lobes and vasculature.
   2. Stop the Live view by clicking on the Stop icon.
   3. Go to the Move probe menu to navigate the probe and select a plane by clicking on Set Home to save the coordinates as a reference position. Ensure that the vascular tree is well-defined and visible with as little out-of-plane signal as possible.
   4. Go back to the menu Live view and click on the Stop icon.

3. Doppler scan acquisition (Timing ~ 20 min)

1. Launch a Doppler acquisition with a 10 ms framerate.
   1. Go to fUS 2D menu to image hemodynamic variations over time in a specific position (Figure 3). To increase the contrast, administer a bolus of 50 µL intravenously (iv) injected microbubbles with a concentration of 2 x 10⁸ microbubbles per mL to the tail vein.
   2. In the Preset tap, define the total recording duration in seconds (30 s). Choose a continuous mode with no pause between the images by setting Time between images to 0 s.
   3. Press the Record button to start the 2D acquisition. The recording can be canceled by pressing the Stop button. Data may be lost when acquisition is interrupted before completion.
2. Visualize and adapt the acquired recording.
   1. Modify the contrast and colormap if desired in tap Visualization. Replay recording if needed by scrolling through the frames with the slider, which is included in the function Time.
3. Save the data.
   1. Provide a name for the acquired recording to save the file in a .scan format in the session folder. A pop-up window to save the data is automatically proposed at the end of every recording. Data is saved automatically.
4. Launch another acquisition by clicking on the Preset tab and repeat from step 3.1.1 onwards.
5. Remove the imaging setup and take care of the animal.
   1. Go to the Move probe menu to vertically move the probe up outside the 3D-printed water tank. Remove the setup and ultrasonic gel and unfix the animal.
   2. Euthanize the animal when the experimental design reaches the endpoint; otherwise, continue with the following steps.
   3. Apply some moisturizing cream on the shaved skin area of the animal if longitudinal measurements are intended.
   4. Place the animal back in the cage and let it wake up from anesthesia under a heat lamp to prevent hypothermia. Monitor the animal while recovering.
   5. Place the animal back in the individually ventilated cage rack when it walks normally again. Finish the laboratory animal experiments before analyzing the data set.

4. Quantitative analysis (Timing ~ 1 h)

1. Open the analysis script gui_analysis.m available in Supplementary File 1.
2. Select the scan and set the quantitative parameters in settings: 10 regions of interest (ROIs) with a voxel size of 2 x 2. Choose 40 ms for each frame to perform the Doppler integration.
3. Draw the vascular periphery of the organ; the 10 ROIs are automatically prepositioned between clearly visible arteries and can be adjusted manually if needed (Figure 4A). Close the floating dialog box when satisfied with the positioning. Ensure a stable acquisition (Figure 4B) for robust blood volume computation, avoiding acquisitions affected by strong motion artifacts (Figure 4C).
4. Quantify mean hepatic blood volume in A.U. of the total 10 ROIs by automatically estimating the mean and standard deviation of the quiet frame values within the 30 s recording (Figure 4D).
   NOTE: The data table of intensities of ROIs can be copied/pasted directly to a spreadsheet file.
5. Repeat analysis for at least two additional planes, -0.3 mm, and -0.6 mm, in a caudal direction. Quantify all the acquisition data before performing the statistical analysis.
6. Check the standard deviation of intensities on the entire image and determine a cutoff as the overall mean (µ) with 2 standard deviations (2σ) to objectively exclude measurements susceptible to motion artifacts (Figure 4E).
   NOTE: The mean and standard deviation of the quiet frame values estimated over the entire image are provided in the first row of the data table.
   CAUTION: Motion artifacts prevent robust quantification and should be objectively identified based on analyzing the entire temporal image.
7. Take the average of all mean hepatic blood volume quantified over 3 different planes in 30 ROIs per animal in step 4.3 (Figure 4F). Apply the ROUT test 1% in a statistical analysis software to identify outlier ROIs before averaging 30 ROIs per animal. To improve the estimation of the blood volume, record additional planes per animal.
8. To administrate vehicle treatment, disinfect the injection site, i.e., lower right or left quadrant of the abdomen, using gauze and 70% ethanol. Inject 20 µL of saline or DMSO ip with a 27G needle and a 1 mL disposable syringe or a 30G insulin syringe or administer 100 µL of (Hydroxypropyl) methylcellulose by gavage using a reusable feeding needle 30G with a round tip. Plot data of 3 cohorts receiving saline or DMSO by ip or oral administration (po) of cellulose (Figure 4G). Perform analysis in a blind way without revealing the condition and/or received treatment.
   NOTE: Vehicle administration has no impact on blood volume measurements.