Name: application of ultrasound and shear wave elastography imaging in a rat model of nafld/nash
Uploaded: 2021-04-20T14:00:00
Description: Watch this Scientific Journal Video about Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH at JoVE.com

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&#160;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.

All animal-involved procedures were reviewed and approved by Pfizer&#39;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
<ol>
	<li>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...

<ol>
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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetyl-CoA+carboxylase+inhibition+improves+multiple+dimensions+of+NASH+pathogenesis+in+model+systems.">Acetyl-CoA carboxylase inhibition improves multiple dimensions of NASH pathogenesis in model systems.</a> Cellular and Molecular Gastroenterology and Hepatology. 10 (4), 829-851 (2020).</li><li>Gu, L. H., Gu, G. X., Wan, P., Li, F. H., Xia, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+two-dimensional+shear+wave+elastography+and+texture+analysis+for+monitoring+liver+fibrosis+in+rat+model.">The utility of two-dimensional shear wave elastography and texture analysis for monitoring liver fibrosis in rat model.</a> Hepatobiliary &amp; Pancreatic Diseases International. 20 (1), 46-52 (2020).</li><li>Marshall, R. 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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...

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&#39;s liver. Currently, the gold standard to diagnose NASH in patients is through histological examination of patient-derived liver biopsy samples. Similarly, preclinical researchers who work to understand the pathogenesis of NASH/NAFLD as well as the drug development industry rely on in vivo wedge biopsy of liver samples or terminal euthanasia of satellite cohorts for histology to measure steatosis, inflammation, and fibrosis.
For example, liver wedge biopsy has been a standard technique for assessing steatohepatitis and fibrosis while using the GUBRA NASH model2. The liver wedge biopsy method is invasive and laborious in small animals3. The use of wedge liver biopsy in the middle of a study represents an added experimental variable in a disease model, which often increases the number of animals that are needed. With these factors in mind, non-invasive imaging techniques that can be used to reliably assess steatosis and fibrosis in NASH/NAFLD animal models at early time points prove valuable. Shear wave elastography (SWE) is an ultrasound-based method used to measure the elasticity of soft tissues. The technique measures the propagation of shear waves created by supersonic ultrasound pulses directed at a tissue target, and then calculates a value called E modulus4. The velocity of the shear wave is proportional to the degree of tissue stiffness.
Figure 1 and Figure 2 show the imaging area setup and the SWE instrument. The SWE instrument is a single, wheeled unit with two screens and a control panel shown in Figure 2A. The upper monitor (Figure 2B) acts as the computer monitor and displays images and patient directories. The control panel (Figure 2C) is an array of buttons and dials that control general aspects of image capture: freezing screen, saving images, changing from one mode to another. The lower screen (Figure 2D) is a touch screen with additional controls to change settings and acts as a keyboard to input data as needed. The instrument is equipped with a stylus to use on the touch screen if desired. Ultrasound probes attach to the lower front panel of the device. For B-mode and SWE imaging in rodents, the super-linear 6 to 20 MHz transducer was used. This ability to noninvasively measure tissue stiffness makes SWE a valuable tool for the identification and staging of liver fibrosis5 in NASH patients, decreasing the need for more invasive methods. SWE has, in fact, been used to measure liver fibrosis in patients and is an FDA-approved method to score fibrosis in the clinic6. Using SWE to monitor NASH progression in animal models of the disease would provide a translational tool for the development of treatments and simultaneously improve animal welfare through the reduction of animal subject numbers and refinement of in vivo procedures to minimize pain and distress.
SWE imaging in human patients uses a low-frequency ultrasound transducer4, which is not ideal for small animals. Notably, high-frequency SWE techniques have been used to evaluate the efficacy of acetyl-CoA carboxylase inhibition on pathogenesis of NASH in a rat model7, and the utility of this technique has been described in carbon tetrachloride rat models of liver fibrosis with successful results when compared to traditional METAVIR histological scoring methods8. However, existing literature lacks detailed technique and methodology information on the application of SWE imaging in preclinical models of NASH. As described above, liver steatosis is one of the key features of the NAFLD/NASH condition and is an important stage where intervention can be considered. Thus, assessing liver fat accumulation using an imaging modality is as important as assessing liver fibrosis in preclinical models of NASH/NAFLD.
An ultrasound technique known as the HR index, a ratio of tissue brightness of the liver compared to that of the renal cortex, has been used as a surrogate marker of steatosis in the clinic9,10. This approach, however, has not been extensively used in preclinical animal models of NAFLD/NASH. This article describes a method of measuring elasticity as well as the HR index as a surrogate marker of hepatic fibrosis and steatosis, respectively, in a choline-deficient, high-fat diet (CDAHFD) rat model of NAFLD/NASH. This model induces rapid steatosis, liver inflammation, and fibrosis, which is measurable within 6 weeks in mice11. The addition of cholesterol (1%) to this diet has been shown to promote fibrogenesis in rats12, making this model a suitable candidate for validation studies involving shear wave imaging. Overall, this imaging technology can also be applied to a wide range of NASH models/diets where steatosis and/or fibrosis is an endpoint of interest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aixplorer</td>
			<td>Supersonic Imagine</td>
			<td></td>
			<td>Shear Wave Elastography Instrument</td>
		</tr>
		<tr>
			<td>Aixplorer SuperLinear SLH20-6 Transducer</td>
			<td>Supersonic Imagine</td>
			<td></td>
			<td>Transducer for Shear Wave Elastography</td>
		</tr>
		<tr>
			<td>Alpha-dri bedding</td>
			<td></td>
			<td></td>
			<td>rat cages</td>
		</tr>
		<tr>
			<td>Aperio AT2 scanner</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>Digital Pathology Brightfield Scanner</td>
		</tr>
		<tr>
			<td>Compac 6 Anesthesia System</td>
			<td>VetEquip</td>
			<td></td>
			<td>Anesthesia Vaporizer and Delivery System. Any anesthesia delivery system can be used, however.</td>
		</tr>
		<tr>
			<td>Manage Imager Database</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>Digital Pathology</td>
		</tr>
		<tr>
			<td>Mayer&#39;s Hematoxilin</td>
			<td>Dako/Agilent</td>
			<td></td>
			<td>H&#38;E Staining/Histology</td>
		</tr>
		<tr>
			<td>Nair</td>
			<td>Church &#38; Dwight</td>
			<td></td>
			<td>Hair remover</td>
		</tr>
		<tr>
			<td>Oil Red O solution</td>
			<td>Poly Scientific</td>
			<td></td>
			<td>Lipid Staining/Histology</td>
		</tr>
		<tr>
			<td>Picrosirius Red Stain (PSR)</td>
			<td>Rowley Biochemical</td>
			<td>F-357-2</td>
			<td>Collagen Stain/Histology</td>
		</tr>
		<tr>
			<td>Puralube Opthalmic ointment</td>
			<td>Dechra Veterinary Product</td>
			<td></td>
			<td>Lubricatn to prevent eye dryness&#160; during anesthesia</td>
		</tr>
		<tr>
			<td>Tissue-Tek Prisma Plus</td>
			<td>Sakura Finetek USA</td>
			<td></td>
			<td>Automated slide stainer</td>
		</tr>
		<tr>
			<td>VISIOPHARM software</td>
			<td>Visiopharm</td>
			<td></td>
			<td>Digital pathology software</td>
		</tr>
		<tr>
			<td></td>
			<td>Research Diets</td>
			<td>A06071309i</td>
			<td>NASH inducing diet</td>
		</tr>
		<tr>
			<td></td>
			<td>Purina</td>
			<td>5053</td>
			<td>Control animal chow</td>
		</tr>
		<tr>
			<td>Wistar Han rats</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

application of ultrasound and shear wave elastography imaging in a rat model of nafld/nash

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&#160;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.

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...

Watch this Scientific Journal Video about Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH at JoVE.com

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

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 ...

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.

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