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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH

Published: April 20th, 2021

DOI:

10.3791/62403

1Comparative Medicine, Pfizer Inc., 2Digital Medicine & Translational Imaging, Pfizer Inc., 3Internal Medicine Research Unit, Pfizer Inc., 4Drug Safety Research & Development, Pfizer Inc.
* These authors contributed equally

This protocol describes the use of an enhanced ultrasound technique to non-invasively observe and quantify liver tissue changes in rodent models of nonalcoholic fatty liver disease.

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat accumulation (steatosis) and inflammation leading to fibrosis. Preclinical models closely recapitulating human NASH/NAFLD are essential in drug development. While liver biopsy is currently the gold standard for measuring NAFLD/NASH progression and diagnosis in the clinic, in the preclinical space, either collection of whole liver samples at multiple timepoints during a study or biopsy of liver is needed for histological analysis to assess the disease stage.

Conducting a liver biopsy mid-study is an invasive and labor-intensive procedure, and collecting liver samples to assess disease level increases the number of research animals needed for a study. Thus, there is a need for a reliable, translatable, non-invasive imaging biomarker to detect NASH/NAFLD in these preclinical models. Non-invasive ultrasound-based B-mode images and Shear Wave Elastography (SWE) can be used to measure steatosis as well as liver fibrosis. To assess the utility of SWE in preclinical rodent models of NASH, animals were placed on a pro-NASH diet and underwent non-invasive ultrasound B-mode and shear wave elastography imaging to measure hepatorenal (HR) index and liver elasticity, measuring progression of both liver fat accumulation and tissue stiffness, respectively, at multiple time points over the course of a given NAFLD/NASH study.

The HR index and elasticity numbers were compared to histological markers of steatosis and fibrosis. The results showed strong correlation between the HR index and percentage of Oil Red O (ORO) staining, as well as between elasticity and Picro-Sirius Red (PSR) staining of livers. The strong correlation between classic ex vivo methods and in vivo imaging results provides evidence that shear wave elastography/ultrasound-based imaging can be used to assess disease phenotype and progression in a preclinical model of NAFLD/NASH.

Non-alcoholic fatty liver disease (NAFLD) is a metabolic condition characterized by an excessive buildup of fat in the liver and is quickly becoming a leading liver ailment worldwide with a recently reported global prevalence of 25%1. Non-alcoholic steatohepatitis (NASH) is a more progressed stage of the spectrum of NAFLD, characterized by excess liver fat with progressive cellular damage, inflammation, and fibrosis. These ailments are often silent, undetected via blood tests or routine examinations, until considerable damage has already occurred to a patient's liver. Currently, the gold standard to diagnose NASH in patients is through hist....

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All animal-involved procedures were reviewed and approved by Pfizer's Institutional Animal Care and Use Committee (IACUC) and conducted in an AAALAC (Assessment and Accreditation of Laboratory Animal Care) International accredited facility.

1. Disease induction

  1. Use male Wister Han rats (150-175 g; ~ 6-7 weeks old; total 40 rats) that are free of known rat adventitial pathogens. House the rats in pairs in individually ventilated caging with paper bedding (see the Table o.......

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One hallmark of animals fed CDAHFD is steatosis. Accumulation of fat in the liver changes the echogenic properties of the tissue, which can be quantified by measuring the brightness of the liver and normalizing it to the brightness of the renal cortex from a B-mode image taken in the same plane. The quantified value is expressed as an HR index, which is an indirect measure of steatosis. In Figure 4A, a representative liver image from a control animal shows approximately equal or less brightn.......

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Ultrasound-based imaging, including SWE, can be an invaluable tool for the longitudinal assessment of liver steatosis and stiffness in preclinical models of NAFLD/NASH. This paper describes detailed methodologies on how to acquire high-quality B-mode as well as SWE images of livers for the measurement of the HR index and elasticity using a CDAHFD diet-induced rat model of NASH. Further, the results show excellent correlation of the HR index and elasticity with the gold standard of evaluation-histological assessment of li.......

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The authors would like to thank the Pfizer Comparative Medicine Operations Team for their hard work caring for and ensuring the health of the study animals as well as assisting with some of the techniques. Also, thanks are owed to Danielle Crowell, Gary Seitis, and Jennifer Ashley Olson for their help with tissue processing for histological analyses. In addition, authors would like to thank Julita Ramirez for reviewing and providing valuable feedback during preparation of this manuscript.

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Name Company Catalog Number Comments
Aixplorer Supersonic Imagine Shear Wave Elastography Instrument
Aixplorer SuperLinear SLH20-6 Transducer Supersonic Imagine Transducer for Shear Wave Elastography
Alpha-dri bedding rat cages
Aperio AT2 scanner Leica Biosystems Digital Pathology Brightfield Scanner
Compac 6 Anesthesia System VetEquip Anesthesia Vaporizer and Delivery System. Any anesthesia delivery system can be used, however.
Manage Imager Database Leica Biosystems Digital Pathology
Mayer's Hematoxilin Dako/Agilent H&E Staining/Histology
Nair Church & Dwight Hair remover
Oil Red O solution Poly Scientific Lipid Staining/Histology
Picrosirius Red Stain (PSR) Rowley Biochemical F-357-2 Collagen Stain/Histology
Puralube Opthalmic ointment Dechra Veterinary Product Lubricatn to prevent eye dryness  during anesthesia
Tissue-Tek Prisma Plus Sakura Finetek USA Automated slide stainer
VISIOPHARM software Visiopharm Digital pathology software
Research Diets A06071309i NASH inducing diet
Purina 5053 Control animal chow
Wistar Han rats Charles River Laboratories

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