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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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).

Abstract

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 "chicken wire," which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson'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.

Introduction

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) ....

Protocol

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.

.......

Representative Results

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 technique.

When the liver tissue is studied without decellularization, it is challenging to get high-resolution images of the collagen network (Figure 5A)........

Discussion

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.......

Acknowledgements

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). 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. 

....

Materials

NameCompanyCatalog NumberComments
4-0 MONOCRYL UNDYED 1 x 18" P-3MONOCRYLY494G
4-0 suturefisher scientific10-000-649https://www.fishersci.com/shop/products/monomid-nylon-non-absorbable-sutures-7/10000649?keyword=true
AnaSed Injection (xylazine)AnaSedNDC 59399-110-20this drug to use by or on the order of a licensed veterinarian.
BD INSYTE AUTOGUARD I.V. CATHETER WITH BC TECHNOLOGYBD382612
Chow dietEnvigo# 2918Control diet. A fixed formula, non-autoclavable diet manufactured with high quality ingredients and designed to support gestation, lactation, and growth of rodents.
Fast-food diet (AIN76A Western Diet)Test Diet1810060https://www.testdiet.com/cs/groups/lolweb/@testdiet/documents/web_content/mdrf/mdux/~edisp/ducm04_051601.pdf
Hematoxylin and Eosin Stain KitvectorlabsH-3502https://vectorlabs.com/hematoxylin-and-eosin-stain-kit.html
Kent Scientific Rat Surgical Kitfisher scientific13-005-205https://www.fishersci.com/shop/products/rat-surgical-kit/13005205#?keyword=mouse%20surgery%20kit
KETAMINE HYDROCHLORIDE INJECTIONVedcoNDC 50989-996-06 - 10 mL - vial.KetaVed has been clinically studied in subhuman primates in addition to those species listed under Administration and Dosage.
Leica SP5 upright Confocal, multi-photonLeicaSP5
Luer connector (Three-way stopcock with SPIN-LOCK®)bbraunD300https://www.bbraunusa.com/en/products/b0/three-way-stopcockwithspin-lock.html
Picrosirius Red Stain KitPolysciences, Inc.24901https://www.polysciences.com/default/picrosirius-red-stain-kit-40771
Rayon tipped applicatorpuritan25-806 1PR
Sodium deoxycholatesigmaaldrichD6750-100G
Syrupwww.target.com24 fl ozhttps://www.target.com/p/pancake-syrup-24-fl-oz-market-pantry-8482/-/A-13007801
Variable Speed Peristaltic PumpINTLLABBT100https://www.amazon.com/gp/product/B082K97W5W/ref=ox_sc_saved_title_2?smid=A12NUUP87ZRRAR&psc=1
VECTASHIELD Antifade Mounting MediumvectorlabsH-1000-10https://vectorlabs.com/vectashield-mounting-medium.html

References

  1. Friedman, S. L., Pinzani, M. Hepatic fibrosis: 2022 unmet needs and a blueprint for the future. Hepatology. 75 (2), 473-488 (2021).
  2. Ye, Q., et al. Glo....

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Keywords 3D ImagingLiver Extracellular MatrixMouse ModelNon alcoholic SteatohepatitisNASHFibrosisCirrhosisLiver CancerECM RemodelingPortal Vein CatheterizationPerfusionSodium Deoxycholate

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