Figure 1 was created with BioRender.com. This work was supported by the Hellenic Foundation for Research and Innovation (#3222 to A.C.). Anna Hadjihambi is funded by The Roger Williams Institute of Hepatology, Foundation for Liver Research.

All procedures were carried out by BIOEMTECH&#39;s personnel in accordance with European and national welfare regulations and were approved by national authorities (license number EL 25 BIOexp 45/PN 49553 21/01/20). All experiments were designed and reported with adherence to ARRIVE guidelines26. The mice were purchased from the Hellenic Pasteur Institute, Athens, Greece.
NOTE: Animals were group-housed in individually ventilated cages enriched with rails and cardboard ...

The current recommended method for NAFLD diagnosis and staging in humans is liver biopsy, which harbors the risk of bleeding complexities, as well as sampling inaccuracies40. On the contrary, in animal models, such diagnosis is performed by histology post-mortem, although protocols for survivable liver biopsy are now available and are recommended when the study design allows41. The use of post-mortem histology means that a large number of animals are required to investigate...

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease that affects approximately 25% of the population and &#62;80% of morbidly obese people1. An estimated one-third of these individuals progress to non-alcoholic steatohepatitis (NASH), which is characterized by hepatic steatosis, inflammation, and fibrosis2. NASH is a disease stage with a significantly higher risk for the development of cirrhosis and hepatocellular carcinoma (HCC)3,4. For this reason, NASH is currently the second most common cause of liver transplantation, and it is also expected to soon become the most important predictor of liver transplantation5,6,7. Despite its prevalence and severity, no disease-specific therapy is available for NAFLD, and the existing treatments only aim to tackle disease-associated pathologies such as insulin resistance and hyperlipidemia5,6.
In recent years, the pathophysiological role and adaptations of the endothelium and, in general, of the vascular network of metabolic tissues, such as the adipose tissue and the liver, have been gaining more importance in research, especially during obesity and metabolic dysregulation7,8. The endothelium is a cellular monolayer that lines the vascular network internally, acting as a functional and structural barrier. It also contributes to various physiological and pathological processes, such as thrombosis, metabolite transport, inflammation, and angiogenesis9,10. In the case of the liver, the vascular network is, among other features, characterized by the presence of highly specialized cells, defined as liver sinusoidal endothelial cells (LSECs). These cells lack a basement membrane and have multiple fenestrae, allowing for the easier transfer of substrates between the blood and liver parenchyma. Due to their distinctive anatomical location and characteristics, LSECs likely have a crucial role in the pathophysiological processes of the liver, including the development of liver inflammation and fibrosis during NAFLD/NASH. Indeed, the pathological, molecular, and cellular adaptations that LSECs undergo in the course of NAFLD contribute to the disease progression11. Specifically, the LSEC-dependent hepatic angiogenesis that takes place during NAFLD is significantly associated with the development of inflammation and the progression of the disease to NASH or even HCC12. Besides, obesity-related early NAFLD is characterized by the development of insulin resistance in LSECs, which precedes the development of hepatic inflammation or other advanced NAFLD signs13.
Additionally, LSECs have recently emerged as central regulators of hepatic blood flow and vascular network adaptations during liver disease of several etiologies14,15. Indeed, chronic liver disease is characterized by prominent intra-hepatic vasoconstriction and increased resistance to blood flow, which contribute to the development of portal hypertension16. In the case of NAFLD, several LSEC-related mechanisms contribute to this phenomenon. For instance, LSEC-specific insulin resistance, as mentioned above, is associated with reduced insulin-dependent vasodilation of the hepatic vasculature13. Besides, over the course of the disease, the liver vasculature becomes more sensitive to vasoconstrictors, further contributing to impairment of the hepatic blood flow and leading to the emergence of shear stress, which both result in a disruption of the sinusoidal microcirculation17. These facts suggest that the vasculature is a key target in liver disease. Nevertheless, limiting factors hindering the timely diagnosis and monitoring of NAFLD/NASH, as well as the development of potential therapies, are the inadequacies in the consistent characterization of the hepatic microenvironment and (micro)vascular structure, as well as the scoring of the disease stage in a spatiotemporal and non-invasive manner.
Micro-computed tomography (CT) imaging is currently the gold-standard non-invasive imaging method for accurately depicting anatomical information within a living organism. Micro-CT and MRI represent two complementary imaging methods that can cover a vast range of pathologies and provide exceptional resolution and detail in the imaged structures and tissues. Micro-CT, in particular, is a very fast and accurate tool that is often used for studying pathologies such as bone diseases and associated bone surface changes18, assessing the progression of pulmonary fibrosis over time19, diagnosing lung cancer and its staging20, or even examining dental pathologies21, without any special preparation (or destruction) of the samples being imaged.
The imaging technology of micro-CT is based on the different attenuation properties of various organs in terms of the interaction of X-rays with matter. Organs presenting high X-ray attenuation differences are depicted with high contrast in CT images (i.e., the lungs appear dark and the bones light). Organs presenting very similar attenuation properties (different soft tissues), are challenging to distinguish on CT images22. To address this limitation, specialized contrast agents based on iodine, gold, and bismuth have been extensively investigated for in vivo use. These agents alter the attenuation properties of the tissues in which they accumulate, are cleared slowly from the circulation, and enable the uniform and stable opacification of the entire vascular system or chosen tissues23.
In human diagnostics, CT imaging and comparable techniques, such as MRI-derived proton density fat fraction, are already in use for the determination of hepatic fat content24,25. In the context of NAFLD, high soft tissue contrast is essential to accurately distinguish pathological lesions or small vessels. For this purpose, contrast agents providing enhanced contrast of the liver tissue characteristics are utilized. Such tools and materials allow for the study of multiple liver characteristics and possible pathology expressions, such as the architecture and density of the vascular network, lipid deposition/steatosis, and functional tissue uptake/lipid (chylomicron) transfer in the liver. Additionally, hepatic relative blood volume and portal vein diameter can also be evaluated. In a very short scan time, all these parameters provide different and complementary information on the evaluation and progression of NAFLD, which can be used to develop a non-invasive and detailed diagnosis.
In this article, we provide a step-by-step protocol for the use of novel in vivo micro-CT imaging techniques as a non-invasive method to assess the progression stages of NAFLD. Using this protocol, the longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network, can be performed and applied in mouse models of liver disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>eXIA160</td>
			<td>Binitio Biomedical, Inc.</td>
			<td></td>
			<td>https://www.binitio.com/?Page=Products</td>
		</tr>
		<tr>
			<td>High fat diet with 60% of kilocalories from fat</td>
			<td>Research Diets, New Brunswick, NJ, USA</td>
			<td>D12492</td>
			<td></td>
		</tr>
		<tr>
			<td>High-fructose corn syrup&#160;</td>
			<td>Best flavors, CA</td>
			<td>hfcs-1gallon</td>
			<td></td>
		</tr>
		<tr>
			<td>Lacrinorm ophthalmic ointment&#160;</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal diet with 10% of kilocalories from fat&#160;</td>
			<td>Research Diets, New Brunswick, NJ, USA</td>
			<td>D12450</td>
			<td></td>
		</tr>
		<tr>
			<td>Viscover ExiTron nano 12000&#160;</td>
			<td>Milteny Biotec, Bergisch Gladbach, Germany</td>
			<td>130-095-698</td>
			<td></td>
		</tr>
		<tr>
			<td>VivoQuant</td>
			<td>Invicro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-CUBE&#160;</td>
			<td>Molecubes, Belgium</td>
			<td></td>
			<td>https://www.molecubes.com/systems/</td>
		</tr>
	</tbody>
</table>

novel in vivo micro-computed tomography imaging techniques for assessing the progression of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

In this representative study, micro-CT imaging without any contrast agent indicated a higher percentage of liver fat in mice with NAFLD compared to controls (Table 2), confirming the pathology. Using the ExiTron contrast agent and the hepatic vascular network architecture and density analysis described above, the total volume density of the hepatic vascular network was found to be higher in mice with NAFLD compared to healthy controls (Figure 6, Table 2). Mi...

Watch this Scientific Journal Video about Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease at JoVE.com

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective ...

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.

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901 Views.  Foundation for Liver Research. 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 w...