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>
	<li>Younossi, Z. M., 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=Global+epidemiology+of+nonalcoholic+fatty+liver+disease-Meta-analytic+assessment+of+prevalence,+incidence,+and+outcomes.">Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes.</a> Hepatology. 64 (1), 73-84 (2016).</li><li>Boland, M. L., 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=Towards+a+standard+diet-induced+and+biopsy-confirmed+mouse+model+of+non-alcoholic+steatohepatitis:+Impact+of+dietary+fat+source.">Towards a standard diet-induced and biopsy-confirmed mouse model of non-alcoholic steatohepatitis: Impact of dietary fat source.</a> World Journal of Gastroenterology. 25 (33), 4904-4920 (2019).</li><li>Oldham, S., Rivera, C., Boland, M. L., Trevaskis, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+a+survivable+liver+biopsy+procedure+in+mice+to+assess+non-alcoholic+steatohepatitis+(NASH)+resolution.">Incorporation of a survivable liver biopsy procedure in mice to assess non-alcoholic steatohepatitis (NASH) resolution.</a> Journal of Visualized Experiments: JoVE. (146), e59130 (2019).</li><li>Bercoff, J., Tanter, M., Fink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supersonic+shear+imaging:+a+new+technique+for+soft+tissue+elasticity+mapping.">Supersonic shear imaging: a new technique for soft tissue elasticity mapping.</a> IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control. 51 (4), 396-409 (2004).</li><li>Bavu, E., 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=Noninvasive+in+vivo+liver+fibrosis+evaluation+using+supersonic+shear+imaging:+a+clinical+study+on+113+hepatitis+C+virus+patients.">Noninvasive in vivo liver fibrosis evaluation using supersonic shear imaging: a clinical study on 113 hepatitis C virus patients.</a> Ultrasound in Medicine &amp; Biology. 37 (9), 1361-1373 (2011).</li><li>Ferraioli, G., 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=Accuracy+of+real-time+shear+wave+elastography+for+assessing+liver+fibrosis+in+chronic+hepatitis+C:+a+pilot+study.">Accuracy of real-time shear wave elastography for assessing liver fibrosis in chronic hepatitis C: a pilot study.</a> Hepatology. 56 (6), 2125-2133 (2012).</li><li>Ross, T. 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. H., Eissa, M., Bluth, E. I., Gulotta, P. M., Davis, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatorenal+index+as+an+accurate,+simple,+and+effective+tool+in+screening+for+steatosis.">Hepatorenal index as an accurate, simple, and effective tool in screening for steatosis.</a> American Journal of Roentgenology. 199 (5), 997-1002 (2012).</li><li>Webb, M., 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=Diagnostic+value+of+a+computerized+hepatorenal+index+for+sonographic+quantification+of+liver+steatosis.">Diagnostic value of a computerized hepatorenal index for sonographic quantification of liver steatosis.</a> American Journal of Roentgenology. 192 (4), 909-914 (2009).</li><li>Tous, M., Ferre, N., Camps, J., Riu, F., Joven, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+apolipoprotein+E-knockout+mice+with+cholesterol+and+fat+enriched+diets+may+be+a+model+of+non-alcoholic+steatohepatitis.">Feeding apolipoprotein E-knockout mice with cholesterol and fat enriched diets may be a model of non-alcoholic steatohepatitis.</a> Molecular and Cellular Biochemistry. 268 (1-2), 53-58 (2005).</li><li>Kirsch, R., 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=Rodent+nutritional+model+of+non-alcoholic+steatohepatitis:+species,+strain+and+sex+difference+studies.">Rodent nutritional model of non-alcoholic steatohepatitis: species, strain and sex difference studies.</a> Journal of Gastroenterology and Hepatology. 18 (11), 1272-1282 (2003).</li><li>Journal of Ultrasound in Medicine. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2018+Scientific+Program.">2018 Scientific Program.</a> Journal of Ultrasound in Medicine. 37 (1), 1 (2018).</li><li>Engelmann, G., Quader, J., Teufel, U., Schenk, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+and+opportunities+of+non-invasive+liver+stiffness+measurement+in+children.">Limitations and opportunities of non-invasive liver stiffness measurement in children.</a> World Journal of Hepatology. 9 (8), 409-417 (2017).</li><li>Piscaglia, F., Salvatore, V., Mulazzani, L., Cantisani, V., Schiavone, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+shear+wave+elastography+for+liver+disease.+a+critical+appraisal+of+the+many+actors+on+the+stage.">Ultrasound shear wave elastography for liver disease. a critical appraisal of the many actors on the stage.</a> Ultraschall in der Medizin. 37 (1), 1-5 (2016).</li><li>Singh, S., Loomba, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+two-dimensional+shear+wave+elastography+in+the+assessment+of+chronic+liver+diseases.">Role of two-dimensional shear wave elastography in the assessment of chronic liver diseases.</a> Hepatology. 67 (1), 13-15 (2018).</li></ol>

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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Biology

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.