We thank Hyesuk Park for the technical help. This research was supported by funding from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH (R01 2DK083283, to NJT), the National Institute on Aging (NIA), NIH (1R01AG060726, to NJT).&#160;We gratefully acknowledge Jon Mulholland and Kitty Lee of the Cell Sciences Imaging Facility in the Beckman Center for technical assistance with the two-photon microscopy imaging.&#160;

Animal experiments are performed according to the experimental procedures approved by the institutional animal care and use committees (IACUCs) of Stanford University and the Veterans Affairs Hospital in Palo Alto. 6-8 week-old male C57BL/6J mice were fed either chow or a fast-food diet supplemented with 4.2% high-fructose corn syrup (see Table of Materials) in drinking water for 14 weeks5. The mice were kept in standard cages at a 12 h dark/light cycle.
...

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	<li>Friedman, S. L., Pinzani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+fibrosis:+2022+unmet+needs+and+a+blueprint+for+the+future.">Hepatic fibrosis: 2022 unmet needs and a blueprint for the future.</a> Hepatology. 75 (2), 473-488 (2021).</li><li>Ye, Q., 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+prevalence,+incidence,+and+outcomes+of+non-obese+or+lean+non-alcoholic+fatty+liver+disease:+A+systematic+review+and+meta-analysis.">Global prevalence, incidence, and outcomes of non-obese or lean non-alcoholic fatty liver disease: A systematic review and meta-analysis.</a> The Lancet Gastroenterology and Hepatology. 5 (8), 739-752 (2020).</li><li>Schwabe, R. F., Tabas, I., Pajvani, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+fibrosis+development+in+non-alcoholic+steatohepatitis.">Mechanisms of fibrosis development in non-alcoholic steatohepatitis.</a> Gastroenterology. 158 (7), 1913-1928 (2020).</li><li>Arteel, G. E., Naba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+matrisome+-+looking+beyond+collagens.">The liver matrisome - looking beyond collagens.</a> JHEP Reports. 2 (4), 100115 (2020).</li><li>Jiang, J. X., 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=Nonphagocytic+activation+of+NOX2+is+implicated+in+progressive+non-alcoholic+steatohepatitis+during+aging.">Nonphagocytic activation of NOX2 is implicated in progressive non-alcoholic steatohepatitis during aging.</a> Hepatology. 72 (4), 1204-1218 (2020).</li><li>Dehnad, A., 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=AGER1+downregulation+associates+with+fibrosis+in+non-alcoholic+steatohepatitis+and+type+2+diabetes.">AGER1 downregulation associates with fibrosis in non-alcoholic steatohepatitis and type 2 diabetes.</a> Journal of Clinical Investigation. 130 (8), 4320-4330 (2020).</li><li>Mayorca-Guiliani, A. 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=Decellularization+and+antibody+staining+of+mouse+tissues+to+map+native+extracellular+matrix+structures+in+3D.">Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.</a> Nature Protocols. 14 (12), 3395-3425 (2019).</li><li>Mazza, 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=Cirrhotic+human+liver+extracellular+matrix+3D+scaffolds+promote+smad-dependent+tgf-beta1+epithelial+mesenchymal+transition.">Cirrhotic human liver extracellular matrix 3D scaffolds promote smad-dependent tgf-beta1 epithelial mesenchymal transition.</a> Cells. 9 (1), 83 (2019).</li><li>Klaas, 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=The+alterations+in+the+extracellular+matrix+composition+guide+the+repair+of+damaged+liver+tissue.">The alterations in the extracellular matrix composition guide the repair of damaged liver tissue.</a> Scientific Reports. 6, 27398 (2016).</li><li>Mattei, 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=Mechanostructure+and+composition+of+highly+reproducible+decellularized+liver+matrices.">Mechanostructure and composition of highly reproducible decellularized liver matrices.</a> Acta Biomaterialia. 10 (2), 875-882 (2014).</li><li>Ren, H., 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=Evaluation+of+two+decellularization+methods+in+the+development+of+a+whole-organ+decellularized+rat+liver+scaffold.">Evaluation of two decellularization methods in the development of a whole-organ decellularized rat liver scaffold.</a> Liver International. 33 (3), 448-458 (2013).</li><li>Piersma, B., Hayward, M. K., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrosis+and+cancer:+A+strained+relationship.">Fibrosis and cancer: A strained relationship.</a> Biochimica et Biophysica Acta - Reviews on Cancer. 1873 (2), 188356 (2020).</li><li>Cox, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+matrix+in+cancer.">The matrix in cancer.</a> Nature Reviews Cancer. 21 (4), 217-238 (2021).</li><li>Mirdamadi, E. S., Kalhori, D., Zakeri, N., Azarpira, N., Solati-Hashjin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+tissue+engineering+as+an+emerging+alternative+for+liver+disease+treatment.">Liver tissue engineering as an emerging alternative for liver disease treatment.</a> Tissue Engineering Part B: Reviews. 26 (2), 145-163 (2020).</li><li>Mazza, 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=Decellularized+human+liver+as+a+natural+3D-scaffold+for+liver+bioengineering+and+transplantation.">Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation.</a> Scientific Reports. 5, 13079 (2015).</li><li>Jia, Z., 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=3D+culture+system+for+liver+tissue+mimicking+hepatic+plates+for+improvement+of+human+hepatocyte+(C3A)+function+and+polarity.">3D culture system for liver tissue mimicking hepatic plates for improvement of human hepatocyte (C3A) function and polarity.</a> BioMed Research International. 2020, 6354183 (2020).</li><li>Shimoda, H., 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=Decellularized+liver+scaffolds+promote+liver+regeneration+after+partial+hepatectomy.">Decellularized liver scaffolds promote liver regeneration after partial hepatectomy.</a> Scientific Reports. 9 (1), 12543 (2019).</li></ol>

The present protocol shows that decellularization through a low flow rate DOC in situ perfusion preserves the frangible triple-helical and native fibrillar collagen structures, providing a reliable and cost-effective platform to capture dynamic ECM remodeling in NASH liver fibrosis. Although decellularization was performed in normal and fibrotic livers before identifying ECM components or generating biological scaffolds for cell culture, the dynamics of ECM remodeling in liver fibrosis have not been well studied...

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease, affecting 20%-25% of the adult population. 25% of NAFLD patients progress to non-alcoholic steatohepatitis (NASH), where the risk of cirrhosis, liver failure, and hepatocellular carcinoma increases1. In the next 20 years, it is estimated that NASH will account for 2 million liver-related deaths in U.S2. As there are no approved treatments, there is an urgent need to decipher the mechanisms that cause liver fibrosis in NASH patients and develop targeted treatment3.
The extracellular matrix (ECM) is a dynamic, complex microenvironment that exerts bi-directional communication with cells to regulate tissue homeostasis4. The liver ECM is composed of structural elements such as proteoglycans, collagens, fibronectin, elastin, and other non-structural proteins (e.g., olfactomedin and thrombospondin) to provide physical and structural support4.
Liver fibrosis is a chronic wound-healing response to liver damage of various etiologies, including NASH3. It results from an imbalance in the dynamic ECM matrix remodeling process and is characterized by excessive structural proteins in the injured liver4. Fibrogenesis depends on the dynamic cell-cell communication among different hepatic cell types. Hepatic stellate cells (HSCs),&#160;when activated, differentiate into Smooth Muscle Alpha 2 Actin-expressing, migrating, and proliferating myofibroblast-like cells and synthesize ECM proteins as a wound-closing action. Activated HSCs are the central collagen-producing cells in the liver1.
The molecular mechanism of ECM remodeling, patterns of fibrosis, and their relationship with cellular events are not clear. A better understanding of the three-dimensional (3D)&#160;ECM structure is still needed, even though mass spectrometry techniques have helped analyze ECM protein composition4. Traditionally, Masson&#39;s trichrome stain, Picro Sirius Red stains, and second harmonic generation (SHG) imaging have been performed on two-dimensional (2D) thin liver sections. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which extends to zone 3 and is perisinusoidal/pericellular fibrosis5,6. However, there has been a lack of studies focusing on the 3D structure of the native liver, particularly those that do not involve tissue sectioning. Robust imaging approaches to identify patterns and characteristics of fibrosis throughout dynamic ECM remodeling in liver fibrosis would significantly strengthen the understanding of NASH mechanisms and identify new therapeutic targets.
To address these challenges, a fast and efficient protocol was optimized to image the native liver ECM via decellularization7. Whole-liver decellularization is an approach to remove the hepatic cellular content while maintaining the native 3D ECM network through detergent perfusion. Mice were fed either chow or fast-food diet (FFD) for 14 weeks. Decellularization was performed after in situ portal vein perfusion with mild detergent and low flow rates to preserve triple-helical and native fibrillar collagen structures. Two-photon microscopy was applied to analyze changes in collagen structures in ECM. The 3D images of the native ECM structure in normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provides a practical and affordable platform to visualize the dynamic ECM remodeling in the liver.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>4-0 MONOCRYL UNDYED 1 x 18&#34; P-3</td>
			<td>MONOCRYL</td>
			<td>Y494G</td>
			<td></td>
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			<td>4-0 suture</td>
			<td>fisher scientific</td>
			<td>10-000-649</td>
			<td>https://www.fishersci.com/shop/products/monomid-nylon-non-absorbable-sutures-7/10000649?keyword=true</td>
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			<td>AnaSed Injection (xylazine)</td>
			<td>AnaSed</td>
			<td>NDC 59399-110-20</td>
			<td>this drug to use by or on the order of a licensed veterinarian.</td>
		</tr>
		<tr>
			<td>BD INSYTE AUTOGUARD I.V. CATHETER WITH BC TECHNOLOGY</td>
			<td>BD</td>
			<td>382612</td>
			<td></td>
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			<td>Chow diet</td>
			<td>Envigo</td>
			<td># 2918</td>
			<td>Control diet. A fixed formula, non-autoclavable diet manufactured with high quality ingredients and designed to support gestation, lactation, and growth of rodents.</td>
		</tr>
		<tr>
			<td>Fast-food diet (AIN76A Western Diet)</td>
			<td>Test Diet</td>
			<td>1810060</td>
			<td>https://www.testdiet.com/cs/groups/lolweb/@testdiet/documents/web_content/mdrf/mdux/~edisp/ducm04_051601.pdf</td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin Stain Kit</td>
			<td>vectorlabs</td>
			<td>H-3502</td>
			<td>https://vectorlabs.com/hematoxylin-and-eosin-stain-kit.html</td>
		</tr>
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			<td>Kent Scientific Rat Surgical Kit</td>
			<td>fisher scientific</td>
			<td>13-005-205</td>
			<td>https://www.fishersci.com/shop/products/rat-surgical-kit/13005205#?keyword=mouse%20surgery%20kit</td>
		</tr>
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			<td>KETAMINE HYDROCHLORIDE INJECTION</td>
			<td>Vedco</td>
			<td>NDC 50989-996-06 - 10 mL - vial.</td>
			<td>KetaVed has been clinically studied in subhuman primates in addition to those species listed under Administration and Dosage.</td>
		</tr>
		<tr>
			<td>Leica SP5 upright Confocal, multi-photon</td>
			<td>Leica</td>
			<td>SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer connector (Three-way stopcock with SPIN-LOCK&#174;)</td>
			<td>bbraun</td>
			<td>D300</td>
			<td>https://www.bbraunusa.com/en/products/b0/three-way-stopcockwithspin-lock.html</td>
		</tr>
		<tr>
			<td>Picrosirius Red Stain Kit</td>
			<td>Polysciences, Inc.</td>
			<td>24901</td>
			<td>https://www.polysciences.com/default/picrosirius-red-stain-kit-40771</td>
		</tr>
		<tr>
			<td>Rayon tipped applicator</td>
			<td>puritan</td>
			<td>25-806 1PR</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>sigmaaldrich</td>
			<td>D6750-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Syrup</td>
			<td>www.target.com</td>
			<td>24 fl oz</td>
			<td>https://www.target.com/p/pancake-syrup-24-fl-oz-market-pantry-8482/-/A-13007801</td>
		</tr>
		<tr>
			<td>Variable Speed Peristaltic Pump</td>
			<td>INTLLAB</td>
			<td>BT100</td>
			<td>https://www.amazon.com/gp/product/B082K97W5W/ref=ox_sc_saved_title_2?smid=A12NUUP87ZRRAR&#38;psc=1</td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium</td>
			<td>vectorlabs</td>
			<td>H-1000-10</td>
			<td>https://vectorlabs.com/vectashield-mounting-medium.html</td>
		</tr>
	</tbody>
</table>

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 collagen fibers were detected with second harmonic generation and two-photon microscopy. The signal is from the frangible triple-helical and native fibrillar collagen structures. Specific antibodies were not used to analyze collagen subtypes; however, this could be added to the imaging&#160;technique.
When the liver tissue is studied without decellularization, it is challenging to get high-resolution images of the collagen network (Figure 5A)....

Watch this Scientific Journal Video about 3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis at JoVE.com

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

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 ...

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1.6K ビュー.  Stanford University. NASHは、世界で最も重要な慢性肝疾患の1つです。それは線維症、肝硬変、そして最終的には肝臓癌に進行する可能性があります。NASHにおけるECMの動的変化を研究することは、線維症をよりよく理解するのを助けることができる。従来のイメージングでは、3D ECM構造の変化を実証できず、肝線維症における動的ECMリモデリングプロセスの理解が制限されています。開?...

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

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...

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

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...

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

investigating the protective effects of platycodin d on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model

Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model

This protocol investigates the protective effects of platycodin D on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro...

Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model

novel in vivo micro-computed tomography imaging techniques for assessing the progression of non-alcoholic fatty liver disease

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...

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

analysis of liver microenvironment during early progression of non-alcoholic fatty liver disease-associated hepatocellular carcinoma in zebrafish

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell...

a human 3d extracellular matrix-adipocyte culture model for studying matrix-cell metabolic crosstalk

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic...

histological analyses of acute alcoholic liver injury in zebrafish

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic...

a 3d quantification technique for liver fat fraction distribution analysis using dixon magnetic resonance imaging

Author Spotlight: A Non-Invasive Tool to Assess and Differentiate Fat Patterns in Liver Using 3D Dixon MRI 

This study introduces a unique 3D quantification method for liver fat fraction (LFF) distribution using Dixon Magnetic Resonance Imaging (Dixon MRI). LFF maps, derived from in-phase and water-phase images, are integrated with 3D liver contours to differentiate LFF patterns between normal and steatotic livers, enabling precise assessment of liver fat...

A Comprehensive 3D-Liver Fat Function Distribution Model Using Dixon MRI

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fabrication of extracellular matrix from mouse model: an effective procedure to prepare decellularized tongue extracellular matrix from murine tongue

Next, in a 3.5-centimeter or 6-centimeter culture dish containing 75% ethanol, use a surgical needle to load each tongue onto a piece of surgical suture and, with a small piece of sterile tin foil, wrap the end of the suture. In a 3.5-centimeter or 6-centimeter culture dish, use 3 milliliters of PBS to rinse each tongue for 30 seconds. Then, in a wide-mouth bottle, prepare 90 micrograms per milliliter of ampicillin in 250 milliliters of sterile ultrapure water and add a stir...

Fabrication of Extracellular Matrix from Mouse Model: An Effective Procedure to Prepare Decellularized Tongue Extracellular Matrix from Murine Tongue

This video demonstrates the preparation of the murine tongue extracellular matrix by using the decellularization method, which involves exposing the harvested tongue to a series of physical, chemical, and enzymatic treatments. The resulting TEM scaffold retains its native extracellular matrix composition, removing the cellular...

longitudinal measurement of extracellular matrix rigidity in 3d tumor models using particle-tracking microrheology

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor...

a medical decision support platform for identifying thrombospondin 1 in non-alcoholic fatty liver disease via immuno-infiltration analysis

This study investigated the relationships between non-alcoholic fatty liver disease (NAFLD) and myocardial infarction (MI) through co-expressed genes, identifying Thrombospondin 1 (THBS1) as a biomarker. Immuno-infiltration analysis revealed CD8+ T cells and neutrophils as key factors, with THBS1 showing potential as a diagnostic tool for NAFLD and...

A Medical Decision Support Platform for Identifying Thrombospondin 1 in Non-alcoholic Fatty Liver Disease via Immuno-Infiltration Analysis

microgel-extracellular matrix composite support for the embedded 3d printing of human neural constructs

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high...

non-invasive pet/mr imaging in an orthotopic mouse model of hepatocellular carcinoma

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose...

non-invasive optical imaging of the lymphatic vasculature of a mouse

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated...

in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye

Author Spotlight: Harnessing Mouse Eye Chambers for Noninvasive Liver Spheroid Studies

Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy.

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

a mouse model of chronic liver fibrosis for the study of biliary atresia

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA...

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in...

non-contact, label-free monitoring of cells and extracellular matrix using raman spectroscopy

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate...

isolation of tissue extracellular vesicles from the liver

Isolation of Tissue Extracellular Vesicles from the Liver

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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.

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 ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse models, are applied to advance the understanding of the disease pathogenesis and to identify potential therapeutic targets. Testing novel drug candidates to block AAA growth in these models generally requires repeated drug administration during the time course of the experiment. Here, we describe a compiled protocol for AAA induction, insertion of an intravenous catheter to facilitate prolonged therapy, and serial AAA monitoring by 3D ultrasound. Aneurysms are induced in apolipoprotein E (ApoE) deficient mice by angiotensin II release over 28 days from osmotic mini-pumps implanted subcutaneously into the mouse back. Subsequently, the surgical procedure for external jugular vein catheterization is conducted to allow for daily intravenous drug treatment or repeated blood sampling via a subcutaneous vascular access button. Despite the two dorsal implants, the monitoring of AAA development is readily facilitated by sequential semi-automated 3D ultrasound analysis, which yields comprehensive information on the expansion of aortic diameter and volume and on aneurysm morphology, as illustrated by experimental examples.

This protocol describes the consecutive implantation of an osmotic pump to induce abdominal aortic aneurysm by angiotensin II release in apolipoprotein E (ApoE) deficient mice and of a vascular access port with a jugular vein catheter for repeated drug treatment. Monitoring of aneurysm development by 3D ultrasound is effectively conducted despite dorsal implants.

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse ...

The occurrence of non-alcoholic fatty liver disease (NAFLD) has been increasing at an alarming rate worldwide. Platycodon grandiflorum is widely used as a traditional ethnomedicine for the treatment of various diseases and is a typical functional food that can be incorporated into the everyday diet. Studies have suggested that platycodin D (PD), one of the main active ingredients in Platycodon grandiflorum, has high bioavailability and significantly mitigates the progress of NAFLD, but the underlying mechanism of this is still unclear. This study aims to investigate the therapeutic effect of PD against NAFLD in vitro. AML-12 cells were pretreated with 300 &#181;M palmitic acid (PA) for 24 h to model NAFLD in vitro. Then, the cells were either treated with PD or received no PD treatment for 24 h. The levels of reactive oxygen species (ROS) were analyzed using 2&#8242;,7&#8242;-dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining, and the mitochondrial membrane potential was determined by the JC-1 staining method. Moreover, the protein expression levels of LC3-II/LC3-I and p62/SQSTM1 in the cell lysates were analyzed by western blotting. PD was found to significantly decrease the ROS and mitochondrial membrane potential levels in the PA-treated group compared to the control group. Meanwhile, PD increased the LC3-II/LC3-I levels and decreased the p62/SQSTM1 levels in the PA-treated group compared to the control group. The results indicated that PD ameliorated NAFLD in vitro by reducing oxidative stress and stimulating autophagy. This in vitro model is a useful tool for studying the role of PD in NAFLD.

This protocol investigates the protective effects of platycodin D on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model.

The occurrence of non-alcoholic fatty liver disease (NAFLD) has been increasing at an alarming rate worldwide. Platycodon grandiflorum is widely used as a ...

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or regenerative strategies. An essential component of drug development is determining how a novel therapeutic is to be administered. Physiologically relevant large animal models are of critical importance in assessing the feasibility and efficacy of various therapeutic delivery strategies. Due to their similarities to humans in cardiovascular physiology, coronary vascular anatomy, and heart weight to body weight ratio, swine is one of the preferred species in the preclinical evaluation of new therapies for myocardial infarction. The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. After percutaneously induced myocardial infarction, female landrace swine received treatment with novel agents through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. The procedures employed for each technique are reproducible, resulting in reliable cardioactive drug delivery. These models can be easily adapted to suit individual study designs, and each of these delivery techniques can be used to investigate a variety of possible interventions. Therefore, these methods are a useful tool for translational scientists pursuing novel biological approaches in cardiac repair following myocardial infarction.

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or ...

Liver Transplantation in Mouse and Analyzing Allograft Patency

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

Author Spotlight: Enhancing Transplantation Research Through MicroCT Angiography in Murine Models 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...