Use of ultrasound and shear wave elastography enables researchers to measure the changes in liver fat and fibrosis implicit in models of nonalcoholic steatohepatitis. This imaging modality is a non-invasive, high-throughput, and clinically translatable technique, and can be used to assess the disease stage and efficacy in non-alcoholic steatohepatitis models during drug discovery. To begin, place six-to seven-week-old male Wistar Han rats weighing 150 to 175 grams in pairs in individually-ventilated cages with proper bedding at approximately 22 degrees Celsius and 40 to 70%relative humidity with a 12-to-12 hour light/dark cycle.
Provide 20 rats with a choline deficient, high-fat diet with 1%cholesterol, and another 20 rats with the standard lab rodent chow. To set up the imaging instrument, place a warmed surface in the imaging area to keep the animal warm during imaging, and fix the anesthesia nose cone for delivering the inhalant anesthesia. Use the ultrasound probe holder to move the ultrasound probe to the desired location and to prevent the probe from resting on the animal.
Once the rat is completely anesthetized, transfer it from the induction chamber to a warm hot water circulating blanket. Use chemical depilation cream to remove the hair from the rib cage to the pelvis on the right side of the rat. Place the rat in the left lateral recumbent position and tape the upper paws above the head on a warm imaging platform.
Press the patient key on the instrument control panel and identify the subject according to the study design. Apply a small amount of warmed ultrasound gel on the depilated skin region of the rat. Move the ultrasound probe to touch the gel-covered area.
Once the live B mode image of the internal organs appears on the screen, move the ultrasound probe to the area slightly above the hip, just parallel to the lumbar vertebrae. Use B mode display to locate the right kidney by identifying the large renal artery and cortex medulla separation. Observe a part of the liver in a single plane of the image and ensure that air bubbles or shadows are not present in the image.
Adjust the focus and make sure that the renal cortex and liver parenchyma are in the same plane to obtain a clear image. Ensure that the animal is between breaths when freezing the screen to avoid capturing blurry images. Select the B mode ratio to measure the relative brightness of tissue from the selected region of interest.
Create a two-millimeter circle and place it on the region of interest in the image of the liver, which is located to the right of the kidney. Place a new circle on the image of the kidney cortex, keeping the depths of the circles on the liver and kidney cortex the same. Press the select button on the control panel to display the hepato-renal index as a B mode ratio and repeat the measurement three times at different depths and planes of the tissue, then calculate the average.
Move the probe transversely in the right subcostal area to locate the liver using B mode. Locate a clear area of the liver that is mostly parenchyma and free of large blood vessels, such as the portal vein and hepatic artery, then press the SWE button on the control panel to generate a shear elasticity map of the tissue. Adjust the size and position of the SWE box below the liver capsule in an area that is free of shadows.
When the box is full and stable, press the freeze button on the control panel while the rat is in-between breaths. Tap the Q-Box on the touch display of the instrument to compute the elasticity from the region of interest on the shear wave elasticity map. A circle and data box will appear.
Position the circle in the shadow-free area with uniform coloring, avoiding areas of stiffness. Repeat the procedure three times by moving the probe up and down or sideways on the abdomen to gather SWE-mapped images from different areas of the liver. When finished, remove the tape from the paws and wipe the excess gel.
Place the rat back in a warm and dry cage and allow it to recover fully. Select all scans required for data analysis by checking the box next to the patient's name using the track ball and Select button. After exporting the files, open an individual JPG file of each scan and observe the data on the right side of the image.
Copy all data into a spreadsheet or other database management software, and perform the desired statistical analyses. The representative images of hepato-renal indices from the control and choline-deficient, high-fat diet groups show that the brightness of the liver in the renal cortex was almost the same, and the hepato-renal index was less than one in the control group. In the high-fat diet group, the brightness of the liver increased, and the hepato-renal index was elevated up to 1.91 at six weeks and 1.79 at 12-week time points.
The hepato-renal index values of the high-fat diet group rose quickly throughout the first three to six weeks, before reaching a plateau. Liver sections stained with oil red O dye showed a significantly increased stained area in the high-fat diet group compared to the control group. A correlation was also found between the percent stained area and the hepato-renal index.
Tissue stiffness increased gradually in the high-fat diet group over 12 weeks, and reached 23.1 kilopascals due to fibrosis. The liver samples were stained with picrosirius red to localize collagen as an indicator of fibrosis. A significant increase of the percent stained area was observed in the high-fat diet group compared to the control group.
And a strong correlation was also found between the percent-stained area and the shear wave E modulus numbers. It is important to place the transducer and measurement circles or boxes properly. Avoiding artifacts in the images will allow for repeatable and consistent measurements.
After the procedure, ex vivo measurements of liver triglycerides and collagen gene expression can also be performed on necropsy tissue to confirm the results seen in the imaging.
