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 of Materials) and maintain them at 22 &#177; 1 &#176;C, 40-70% relative humidity with a 12:12 h light-dark cycle.</li>
	<li>Place the rats weighing 150-175 g (~6-7 weeks old) on a choline-deficient, high-fat diet with 1% cholesterol (n = 20) or a standard lab rodent chow (n = 20) depending on the study design. 
	&#8203;NOTE: In this study, a total of 40 rats were enrolled with 20 animals per group. At the end of the 6th week, half of the cohort from each group was necropsied for mid-study histological analysis of liver samples. Thus, the sample size was 10 animals per group for 9th and 12th week time points.</li>
</ol>
2. Instrument setup
<ol>
	<li>Set up the imaging area as follows: include a warmed surface to keep the animal warm during imaging (c in Figure 1), and a secured anesthesia nose cone to deliver inhalant anesthesia to maintain a plane of anesthesia throughout the procedure (b in Figure 1).</li>
	<li>Use an ultrasound probe holder to facilitate moving the ultrasound probe to the desired location and to prevent the probe from resting on the animal.
	<ol>
		<li>Use warmed ultrasound gel on the skin where the ultrasound image is acquired.</li>
		<li>Maintain the following settings throughout the procedure, which can be adjusted on the touch screen: Acoustic Power 0.0 dB; Tissue Tuner 1540 m/s; Dynamic Range 60 dB; Elasticity range (for SWE mode) &#60; 30 kPa.</li>
	</ol>
	</li>
	<li>Attach the ultrasound probe to the rail system in the specialized holder (a in Figure 1).</li>
	<li>Switch the instrument on and allow it to boot up. Once the monitor is turned on, note the B Mode image with connected transducer details.</li>
</ol>
3. Subject preparation
<ol>
	<li>Make sure the animals are fasted at least 4 h prior to the imaging procedure to prevent gut content from interfering with image acquisition.
	<ol>
		<li>After at least 4 h of fasting, place a rat in an isoflurane anesthetic induction chamber until a suitable level of anesthesia is reached, confirmed by no response to toe pinch. Expose the animals to 3-5% isoflurane for 3-5 min to induce anesthesia.</li>
		<li>For maintenance anesthesia, keep the animals under 2-3% isoflurane during image acquisition. Apply ophthalmic ointment to protect the eye from drying during anesthesia.</li>
	</ol>
	</li>
	<li>Once anesthesia has been achieved, remove an animal from the induction chamber and place it on a warm hot water circulating blanket. Place an anesthetic nose cone over the snout, and shave the animal on its right side, from the ribcage to pelvis. Use chemical depilation cream to remove all the remaining hair in this area.</li>
	<li>Once hair has been removed, place an animal in left lateral recumbency with upper paws taped above the head on a warm imaging platform (Figure 3A).</li>
	<li>Press the Patient key on the instrument control panel, and identify the subject according to the study design.
	<ol>
		<li>Open the Keyboard function on the instrument by tapping the icon on the touch screen. Type the names as desired.</li>
		<li>Tap Exit to exit the patient name screen. Observe that B-mode reopens on the monitor.</li>
	</ol>
	</li>
</ol>
4. Image acquisition for hepato-renal (HR) index measurement
<ol>
	<li>Apply a small amount of warmed ultrasound gel to the depilated skin region on the animal.</li>
	<li>Move the ultrasound probe to touch the gel-covered area of the subject (Figure 3B). Once a live B-mode image of the subject&#39;s internal organs appears on the monitor, move the ultrasound probe to the area slightly above the hip, just parallel to the lumbar vertebrae (sagittal plane).</li>
	<li>Using the B-mode display on the monitor, locate the right kidney by identifying the large renal artery and cortex/medulla separation (Figure 4A). In addition, observe part of the liver in a single plane of the image.
	<ol>
		<li>Ensure that there are little to no image artifacts such as shadows and air bubbles.</li>
	</ol>
	</li>
	<li>Measure a B Mode ratio to obtain the HR index.
	<ol>
		<li>Ensure that both renal cortex and liver parenchyma are in the same plane of focus. If needed, adjust focus and gain control to obtain a clear image.
		<ol>
			<li>Adjust the focus by turning the Focus knob on the control panel. Adjust the gain by pressing the Auto TGC button once.</li>
		</ol>
		</li>
		<li>Press the Freeze key on the control panel. Ensure that the animal is between breaths when freezing the screen to avoid blurry images.</li>
		<li>Once the screen is frozen, tap Measurement Tools on the touch screen. Select B-mode Ratio, a built-in tool that measures relative brightness of a tissue from a selected region of interest. Create a 2 mm circle to select a region of interest (ROI). Adjust the circle size by moving a finger along the outer edge of the trackball on the control panel.</li>
		<li>Place the 2 mm circle on the liver image ROI, which should be located to the right of the kidney. Identify liver tissue based on its homogeneous echogenicity and smooth contour.</li>
		<li>Once the circle is in place, press the Select button on the control panel, and observe the new circle that appears.</li>
		<li>Adjust the size of the new circle to 2 mm, and place it on the image of the kidney cortex. Be sure to keep the depth of the circles on the liver and kidney cortex the same. Once in place, press the Select button on the control panel. Observe the built-in system tool displays the HR index as a B-mode ratio.</li>
		<li>Press Save Image to save the image, and observe the saved images that appear as thumbnails on the right side of the monitor.</li>
		<li>Press the Freeze button on the control panel to unfreeze the image and return to a live B mode image.</li>
	</ol>
	</li>
	<li>Repeat the B Mode ratio measurement 3 times at different depths and planes of tissue. Calculate the average of these three B-mode ratios for each animal and time point.</li>
</ol>
5. Image acquisition for Shear Wave Elastography
<ol>
	<li>Move the probe transversely in the right subcostal area to locate the liver using B Mode. Locate an area of the liver that is mostly parenchyma and free of large blood vessels such as the portal vein and hepatic artery. Once a clear area of the liver has been found, generate a shear elasticity map of the tissue by pressing the SWE button on the control panel.</li>
	<li>Adjust the size and position of the SWE box below the liver capsule in an area that is free of shadows. Identify the capsule as a bright echogenic line near the top of the liver.</li>
	<li>Observe that the SWE box transitions to a color map within 5-10 s. Once the box is full and stable, press the Freeze button on the control panel when the animal is between breaths. 
	NOTE: The minimum amount of box coverage should be 60-80% to accurately assess the elasticity of the liver.</li>
	<li>On the touch screen, tap QBox, a built-in system tool that computes elasticity from an ROI on the shear wave elasticity map. Observe the circle and data box that appear on the monitor. Adjust the position of the QBox by tapping the position icon on the touch screen to the desired setup.</li>
	<li>Adjust the size of the circle to 3 mm by moving a finger along the outer edge of the trackball on the control panel. Using the trackball, position the circle in an area free from shadow with uniform coloring (Figure 5A,B). Take care to avoid known areas of stiffness such as blood vessels or the liver capsule, as well as bleed-down from these structures.</li>
	<li>When an adequate area is found, press Save Image on the control panel to save the image. Repeat this procedure 3 times in different areas of the liver. Move the probe up and down or sideways on the abdomen to gather SWE-mapped images from different areas of the liver.</li>
	<li>Once all images have been collected, press End Exam on the control panel, and make note of the patient information screen that appears on the monitor.</li>
	<li>Remove the tape from the animal&#39;s paws, wipe excess gel away, and remove the animal from the imaging stage. Allow it to recover from anesthesia in a warm, dry cage by itself until fully recovered. Monitor each animal to ensure full recovery from anesthesia, indicated by its ability to maintain sternal recumbency</li>
	<li>Repeat steps in sections 4-5 for each animal in the cohort to be imaged.</li>
</ol>
6. Image data retrieval and analysis
<ol>
	<li>When images for all animals have been collected, turn off the anesthesia.</li>
	<li>To pull image data from the machine, press the Review button on the control panel, and observe all the scans performed on that instrument that appear on the monitor. Search for the desired scans using the search window in the upper corner of the screen.</li>
	<li>Select all scans needed for data analysis by checking the box next to the name of the patient via the trackball and Select button. Once all needed scans are highlighted, select Export JPEGs on the touch screen. Export data to a network drive or a portable Universal Serial Bus (USB) drive. Locate the USB ports on the back of the instrument.</li>
	<li>Once the files have been exported, open individual jpg files of each scan on a workstation computer. Observe all the data on the right side of the image: B Mode Ratio-collect the B Ratio number; Q Box-collect the mean elasticity (kPa) value.</li>
	<li>Enter all data into a spreadsheet or other database management software, and perform the desired statistical analyses.</li>
</ol>
7. Histological analysis of liver samples
<ol>
	<li>At the end of the 6th week, perform necropsy on half of the cohort from each group for mid-study histological analysis of the liver samples. Similarly, euthanize the rest of the cohort of animals, and collect liver samples for histological analysis at the 12th week time point.</li>
	<li>For ORO staining, fix liver sections in 10% neutral-buffered formalin, and cryopreserve them with sucrose using a refrigerated 30% sucrose solution overnight at a minimum. Cryo-embed the sections in optimal cutting temperature compound, and cryo-section them onto charged slides to prepare for ORO staining.</li>
	<li>Place the cryo-sections in 100% propylene glycol for 2 min followed by an overnight incubation in 0.5% ORO solution. After removal from the ORO solution, differentiate the sections in 85% propylene glycol for 1 min, rinse in deionized water, and counterstain with Mayer&#39;s Hematoxylin-Lillie&#39;s modification for 1 min.
	<ol>
		<li>Place coverslips on the slides using an aqueous mounting medium and dry them at room temperature.</li>
	</ol>
	</li>
	<li>For PSR, deparaffinize formalin-fixed, paraffin-embedded liver section slides, place them overnight in Bouin Fluid, and then stain them using an automated slide stainer as per the manufacturer&#39;s protocol with some optimized steps (1% phosphomolybdic acid for 5 min; 0.1% Sirius Red in saturated picric acid for 90 min; 2 x 30 s wash in 0.5% acetic acid). Automatically dehydrate the slides and then mount them with a permanent mounting medium.</li>
	<li>Capture images of the ORO- and PSR-stained slides using the digital microscopy scanner at 20x magnification, save them in .svs format, and store in the slide manager image database.</li>
	<li>Analyze the images using custom algorithms created in digital pathology software. Uniformly apply digital pathology software applications with threshold parameters to identify and quantify the liver sections area as well as the ORO- and PSR-stained areas. Export the measurements to a spreadsheet for area percentage calculations.</li>
</ol>
8. Statistical analysis
<ol>
	<li>Perform statistical analysis of the imaging data with two-way ANOVA using Sidak&#39;s multiple comparisons test to assess the difference between groups at different time points. Assume significant differences between groups for probability values p&#160;&#8804; 0.001. In addition, perform correlation of imaging readouts with histological analyses.</li>
	<li>Use non-parametric statistics to analyze the histological analysis results from this study. Report the group values as median &#177; semi-interquartile range (sIQR). Assume significant differences between groups for probability values p &#8804; 0.001. Use a Mann-Whitney test to compare the quantity of PSR and ORO histochemical stain between different groups.</li>
</ol>

<ol>
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All authors are employees of Pfizer, Inc.

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><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&#039;s Hematoxilin</td><td>Dako/Agilent</td><td></td><td>H&amp;amp;E Staining/Histology</td></tr><tr><td>Nair</td><td>Church &amp;amp; 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>Lubrication to prevent eye dryness 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>Vevo imaging station</td><td>Fujifilm VisualSonics</td><td></td><td>The Vevo imaging station is used for holding the ultrasound transducer during imaging.</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...

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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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

3.9K Views.  Pfizer Inc.. 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. 

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

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Medicine

Assessing Hepatic Steatosis with 3D Fluorescence Lipid Droplet Reconstruction

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Author Spotlight: Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

Biochemistry

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Immunology and Infection

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

Quantitative Analysis of Liver Stiffness Distribution

A Three-Dimensional Digital Model for Early Diagnosis of Hepatic Fibrosis Based on Magnetic Resonance Elastography

Hepatic fibrosis is an early stage of liver cirrhosis, and there are no better non-invasive and convenient methods for the detection and evaluation of the disease. Despite the good progress made with the liver stiffness map (LSM) based on magnetic resonance elastography (MRE), there are still some limitations that need to be overcome, including manual focus determination, manual selection of regions of interest (ROIs), and discontinuous LSM data without structural information, which makes it impossible to evaluate the liver as a whole. In this study, we propose a novel three-dimensional (3D) digital model for the early diagnosis of hepatic fibrosis based on MRE.
MRE is a non-invasive imaging technique that employs magnetic resonance imaging (MRI) to measure the liver stiffness at the scanning site through human-computer interaction. Studies have indicated a significant positive correlation between the LSM obtained through MRE and the degree of hepatic fibrosis. However, for clinical purposes, a comprehensive and precise quantification of the degree of hepatic fibrosis is necessary. To address this, the concept of Liver Stiffness Distribution (LSD) was proposed in this study, which refers to the 3D stiffness volume of each liver voxel obtained by the alignment of 3D liver tissue images and MRE indicators. This provides a more effective clinical tool for the diagnosis and treatment of hepatic fibrosis.

Author Spotlight: Advancing Hepatic Fibrosis Diagnosis Using Magnetic Resonance Elastography and AI 

The objective of this study was to develop a novel three-dimensional digital model for the early diagnosis of hepatic fibrosis, which includes the stiffness of each voxel in the patient's liver and can thus, be used to calculate the distribution ratio of the patient's liver at different fibrosis stages.

Hepatic fibrosis is an early stage of liver cirrhosis, and there are no better non-invasive and convenient methods for the detection and evaluation of the ...

Ultrafast Doppler Ultrasound for Hepatic Microvasculature Analysis in Mice

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the microvasculature using Doppler ultrasound methods, imaging modalities employing the concept of ultrafast imaging are preferred. By increasing the frame rate of the ultrasound scanner to thousands of frames per second, it becomes possible to improve the sensitivity of the blood flow down to 2 mm/s and to obtain functional information about the microcirculation in comparison to a sensitivity of around 1 cm/s in conventional Doppler modes. While Ultrafast Doppler ultrasound (UFUS) imaging has become adopted in neuroscience, where it can capture brain activity through neurovascular coupling, it presents greater challenges when imaging the vasculature of abdominal organs due to larger motions linked to breathing. The liver, positioned anatomically under the diaphragm, is particularly susceptible to out-of-plane movement and oscillating respiratory motion. These artifacts not only adversely affect Doppler imaging but also complicate the anatomical analysis of vascular structures and the computation of vascular parameters. Here, we present a qualitative and quantitative imaging analysis of the hepatic vasculature in mice by UFUS. We identify major anatomical vascular structures and provide graphical illustrations of the hepatic macroscopical anatomy, comparing it to an in-depth anatomical assessment of the hepatic vasculature based on Doppler readouts. Additionally, we have developed a quantification protocol for robust measurements of hepatic blood volume of the microvasculature over time. To contemplate further research, qualitative vascular analysis provides a comprehensive overview and suggests a standardized terminology for researchers working with mouse models of liver disease. Furthermore, it offers the opportunity to apply ultrasound as a non-invasive complementary method to inspect hepatic vascular defects in vivo and measure functional microvascular alterations deep within the organ before unraveling blood vessel anomalies at the micron scale levels using ex vivo staining on tissue sections.

Ultrafast Doppler ultrasound (UFUS) with high spatial resolution (100 &#956;m) and sensitivity represents a suitable noninvasive imaging modality for obtaining a comprehensive qualitative overview of the hepatic vasculature. UFUS also facilitates quantitative measurements of the microvasculature, aiming to enhance our understanding of vascular disease mechanisms.

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the ...

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.

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.

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 ...

Ultrasound II: Endoscopic Ultrasound and FibroScan

Endoscopic Ultrasound (EUS) and FibroScan are valuable diagnostic tools in gastroenterology and hepatology, each with specific applications and techniques.
Endoscopic Ultrasound (EUS):
<ol style="list-style-type: circle;">
	<li>Technological Integration: EUS is a sophisticated integration of optical endoscopy and ultrasonography. The endoscope visualizes the mucosal surface directly, while the ultrasound component penetrates deeper to visualize organs and structures beyond the gastrointestinal wall.</li>
	<li>Image Formation: The ultrasound component emits high-frequency sound waves ranging from 5 to 20 MHz. These waves reflect differently from various types of tissues, creating detailed cross-sectional images of the internal structures.</li>
	<li>Layered Visualization: EUS is particularly good at visualizing the five distinct layers of the gastrointestinal wall, which is crucial for staging gastrointestinal cancers, especially esophageal, gastric, and rectal cancers.</li>
	<li>Fine Needle Aspiration (FNA): EUS can be combined with FNA, in which a thin needle is passed via the endoscope to collect tissue samples from lymph nodes or masses. This technique is invaluable for staging cancer, diagnosing unexplained masses, guiding treatment decisions, and tailoring therapeutic strategies.</li>
	<li>Therapeutic Applications: Beyond diagnostics, EUS can be used for therapeutic procedures like draining cysts or pseudocysts, performing nerve blocks for chronic pancreatitis pain management, and delivering medication directly into tumors.</li>
</ol>
FibroScan (Transient Elastography):
<ol style="list-style-type: circle;">
	<li>Principle of Operation: FibroScan measures liver stiffness by emitting mechanical vibrations of mild amplitude and low frequency (approximately 50 Hz). It measures the velocity of the shear wave (elastic wave) generated by these vibrations.</li>
	<li>Elasticity Measurement: The speed of the shear wave correlates with liver tissue stiffness. The stiffer the tissue (as in fibrosis), the faster the shear wave travels. This speed is converted into a liver stiffness measurement (LSM), usually expressed in kilopascals (kPa).</li>
	<li>CAP (Controlled Attenuation Parameter): Some FibroScan models also feature CAP, which estimates the liver&#39;s degree of steatosis (fat content). It provides a quantitative assessment that is particularly useful for diagnosing and monitoring non-alcoholic fatty liver disease (NAFLD).</li>
	<li>Diagnostic Utility: FibroScan provides quantitative metrics critical for evaluating the severity of liver fibrosis, especially in chronic liver diseases like hepatitis B and C. It helps monitor disease progression and assess the effectiveness of antifibrotic therapies.</li>
	<li>Advantages Over Liver Biopsy: FibroScan is noninvasive, has a minimal risk of complications compared to liver biopsy, and is more patient-friendly. It can also sample a larger volume of liver tissue, potentially providing a more representative assessment of liver stiffness than a biopsy. However, it may be less accurate in cases of severe obesity or ascites.</li>
</ol>
Both EUS and FibroScan represent significant advancements in gastroenterology and hepatology. EUS offers a unique combination of endoscopic visualization and deep tissue ultrasound imaging, making it an essential tool for diagnosing and staging gastrointestinal diseases. Meanwhile, FibroScan provides a quick, non-invasive, and reliable method for assessing liver health, particularly in fibrosis and steatosis.

Endoscopic ultrasound is a minimally invasive diagnostic procedure that combines endoscopy and ultrasound to visualize the digestive tract and nearby ...

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

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Chapters in this video

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

A Three-Dimensional Digital Model for Early Diagnosis of Hepatic Fibrosis Based on Magnetic Resonance Elastography

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

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

Ultrasound II: Endoscopic Ultrasound and FibroScan