We consider this protocol significant because it demonstrates a non-invasive simple and easy method to assess over time key indicators for the diagnosis of liver disease. This imaging methodology allows for analysis over time of liver statuses, evaluation of the relative blood volume, portal vein diameter, intensity of the vascular network, all important parameters of liver pathology. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with specific markers such as steatosis, inflammation, and fibrosis can help establish new efficient therapy schemes.
This technique aids in the identification of such markers and also upgrades the value of mice in preclinical studies focusing on the development of novel therapies against disease progression. To begin, place the anesthetized animal in the CT scanner cradle. Apply ophthalmic ointment, secure the nose cone, and set the scanning parameters on the CT scanner.
Prepare the tail vein catheter and place the mouse on a bed to warm the tail in warm water. Insert the catheter into the tail vein and administer the first contrast agent via an injection, performed slowly and manually with a duration of one to three minutes. Then, acquire whole body and liver scans at different time points.
Follow the same procedure for the administration of the second contrast agent, 10 days following the final reading with the first contrast agent. After loading the DICOM file of the pre-contrast scan, adjust the contrast to see the liver, spleen, and white adipose tissue clearly. Under the 3D ROI tool, select add ROI to generate multiple ROIs to perform sampling in the areas where the liver, spleen, and adipose tissue appear clear with no apparent blood vessels and fat.
Under paint mode, choose Sphere. Tick the box 2D only and select a diameter of eight pixels from the dropdown menu to paint ROI over data. From the erode dilate feature, choose minus one erode.
Perform sampling by segmenting the 2D ROIs on the areas of interest using the Sphere tool on the transverse plane. Go to navigation and select show table to display the quantification table containing the calculated hounds field unit values for each ROI. Then, plug the values into this equation.
Calculate the percentage of liver fat. Load the eXIA scan DICOM file, and adjust the contrast to see the liver, spleen, and left ventricle clearly. Under the 3D ROI tool, select add ROI to segment multiple ROIS for the liver.
Under paint mode, select 2D only, and choose Sphere add a diameter of eight pixels to paint ROI over data. From the erode dilate feature, choose minus one erode. Specify a name and color for each ROI and select show table to display the quantification table containing the calculated HU values for each ROI.
After repeating these steps to obtain PV organ t0, insert the values into the equation shown on the screen to extract the percentage contrast corresponding to the functional tissue uptake lipid transfer. Load the ExiTron scan DICOM file, and adjust the contrast to see the liver vascular network clearly. Select add ROI to generate a 3D ROI for the liver.
Define the liver ROI by marking the desired area. Then choose the background ROI from the ROI selector. Click on the Perform Cut icon, to remove the background without changing the liver ROI.
Activate the maximum intensity projection viewer. Then, under segmentation algorithms denoted by the magic wand icon, select connected thresholding to resegment the liver ROI. Set the thresholds by entering the minimum and maximum values and obtain the vascular network.
Again, remove the background liver tissue to get a clear representation of the vascular network. After loading the ExiTron scan DICOM file, adjust the contrast to see the liver, spleen, and left ventricle clearly. Select add ROI and segment ROIs for the liver and a large blood vessel.
Define the segmentation layers to generate 2D ROIs for each tissue. Repeat the steps for the pre-contrast DICOM file to obtain the mean brightness of the liver before the contrast agent injection. For the portal vein diameter, locate the transversal planes of three to four slices above the junction of the superior mesenteric and splenic veins, and then go to navigation followed by distance sanitation, select line to measure the exact distance between two points.
The average contrast values are presented by this grouped data. The functional tissue uptake assay indicated a higher accumulation and slower clearance of the eXIA contrast agent in mice with non-alcoholic fatty liver disease compared to healthy controls. These representative results indicate the differences in the percentage of liver fat, hepatic vascular network volume, portal vein diameter, and hepatic relative blood volume between mice with non-alcoholic fatty liver disease and healthy controls.
Micro-CT imaging with without any contrast agent, indicated a higher percentage of liver fat in mice with non-alcoholic fatty liver disease compared to controls. Similarly, a higher hepatic vascular network volume, hepatic relative blood volume, and a larger portal vein diameter were found in mice with non-alcoholic fatty liver disease compared to healthy controls. The most important thing to remember while attempting this technique is to constantly be accurate on the anatomical region selected.
Choosing the wrong anatomical spot can lead in non accurate results. By following this methodology and protocol, an accurate evaluation of liver disease staging can be performed. Thus, it can act as a specified platform for evaluating new drug efficiency.
This technique paves the way for new, easier, and more accurate ways to evaluate liver pathologies and initiate relevant drug development assays.
