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

Summary

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.

Abstract

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 the development of novel therapies against disease progression.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease that affects approximately 25% of the population and >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 s....

Protocol

All procedures were carried out by BIOEMTECH'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 tubes in a room at 20-22 °C, with a relative humidity of 50%-60% and a 12 h light/dark cycle (light 07:00 am-07:00 pm). A combination of a ....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
eXIA160	Binitio Biomedical, Inc.		https://www.binitio.com/?Page=Products
High fat diet with 60% of kilocalories from fat	Research Diets, New Brunswick, NJ, USA	D12492
High-fructose corn syrup	Best flavors, CA	hfcs-1gallon
Lacrinorm ophthalmic ointment	Bausch & Lomb
Normal diet with 10% of kilocalories from fat	Research Diets, New Brunswick, NJ, USA	D12450
Viscover ExiTron nano 12000	Milteny Biotec, Bergisch Gladbach, Germany	130-095-698
VivoQuant	Invicro
X-CUBE	Molecubes, Belgium		https://www.molecubes.com/systems/