Inducing and Characterizing Liver Regeneration in Mice: Reliable Models, Essential “Readouts” and Critical Perspectives

Dimitrios C. Mastellos1, Robert A. DeAngelis1, John D. Lambris2

1 These authors contributed equally to this article., 2 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo130087
Online Posting Date:  October, 2013
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Abstract

Elucidating the molecular circuitry that regulates regenerative responses in mammals has recently attracted considerable attention because of its emerging impact on modern bioengineering, tissue replacement technologies, and organ transplantation. The liver is one of the few organs of the adult body that exhibits a prominent regenerative capacity in response to toxic injury, viral infection, or surgical resection. Over the years, mechanistic insights into the liver's regenerative potential have been provided by rodent models of chemical liver injury or surgical resection that faithfully recapitulate hallmarks of human pathophysiology and trigger robust hepatocyte proliferation leading to organ restoration. The advent of mouse transgenics has undeniably catalyzed the wider application of such models for researching liver pathobiology. This article provides a comprehensive overview of the most reliable and widely applied murine models of liver regeneration and also discusses helpful hints, considerations, and limitations related to the use of these models in liver regeneration studies. Curr. Protoc. Mouse Biol. 3:141‐170 © 2013 by John Wiley & Sons, Inc.

Keywords: liver; partial hepatectomy; hepatotoxicity; carbon tetrachloride; hepatocytes; proliferation; regeneration

     
 
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Table of Contents

  • Liver Regeneration: Overview
  • Basic Protocol 1: Inducing Liver Regeneration in Mice: The 2/3 Partial Hepatectomy Model (Suture Ligation Technique)
  • Basic Protocol 2: The CCl4‐Induced Acute Liver Injury and Regeneration Model
  • Support Protocol 1: Harvesting Mouse Organs and Blood
  • Support Protocol 2: Quantifying Hepatocyte Cell Cycle Re‐Entry by Means of BrdU Immunohistochemistry
  • Support Protocol 3: Preparation of Nuclear Protein Extracts from Mouse Liver
  • Support Protocol 4: Probing the Activation of Transcriptional Networks that Promote Liver Regeneration (Electrophoretic Mobility Shift Assay)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Inducing Liver Regeneration in Mice: The 2/3 Partial Hepatectomy Model (Suture Ligation Technique)

  Materials
  • 8‐ to 14‐week old mice
  • 70% and 95% (v/v) ethanol
  • Isoflurane for anesthesia
  • Iodine/betadine (Ricca Chemical, cat. no. 3955‐16, or equivalent; http://riccachemical.com)
  • 0.9% (w/v) NaCl solution (normal saline) or phosphate‐buffered saline (PBS), sterile
  • Ophthalmic ointment (Valleyvet, cat. no. 37327; http://www.valleyvet.com/)
  • 10% formalin and/or liquid N 2 (if processing resected tissues)
  • Surgical instrument cleaner (Integra Miltex, cat. no. 3‐720; http://www.integralife.com/)
  • 5‐0 silk suture (Roboz, cat. no. SUT‐15‐1) for occluding blood flow to liver lobes (supplied as a spool)
  • 1 Petri dish
  • 1 Autoclavable case or tray for sterilizing surgical instruments (Roboz, cat. no. RS‐9910)
  • 2 Curved microdissecting forceps (Roboz, cat. no. RS‐5135)
  • 1 Operating scissors (Roboz, cat. no. RS‐6702)
  • 3 Hemostatic forceps (Roboz, cat. no. RS‐7291)
  • 1 Microdissecting scissors (preferably with curved blades; Roboz, cat. no. RS‐5913)
  • 1 Small tube or thin gauze roll (∼12 × 75 mm)
  • Gaymar TP500 water pump for heated bed (Molecular Imaging, cat. no. PM‐04‐0009‐NP): the use of a water pump with circulating heated water minimizes the rodents' heat loss during surgery and aids post‐surgical recovery.
  • Germinator dry bead sterilizer (Roboz, cat. no. DS‐401) for instrument sterilization (between surgeries)
  • 6‐in. w. × 8‐in. d. heated hard pad for surgical bed (Molecular Imaging, cat. no. AV‐03‐0053)
  • 2 Paper towels per surgery
  • Transparent tape
  • 1 Plastic or styrofoam pad for prep area bed
  • Isoflurane vaporizer (for mouse anesthesia; SurgiVet)
  • 2 Small beakers (∼50 ml)
  • 1 15‐ or 50‐ml conical tube (e.g., BD Falcon) for normal saline or PBS
  • 1 15‐ or 50‐ml conical tube for 10% formalin (if fixing resected liver tissue)
  • 1 1.5‐ml screw‐cap cryotube (if freezing resected liver tissue)
  • 2‐ to 3‐ml syringes
  • 1 Sub‐Q or 27‐ to 30‐G 1/2‐in. needle (Becton Dickinson)
  • 1 Small chamber (e.g., a desiccator vessel) for initial rodent anesthetization
  • Animal balance
  • Nose cones (Molecular Imaging, cat. no. AS‐01‐0305)
  • 2 Mapelson‐D rebreathers (Molecular Imaging, cat. no. AS‐01‐0500‐07)
  • 2 Mask stabilizers (Molecular Imaging, cat. no. AA‐00‐0322)
  • Electric razor for shaving mouse fur
  • 4 6‐in. cotton swabs per surgery
  • 1 Sterile cotton swab per surgery (Fisher, cat. no. 14‐959‐90)
  • 1 Sterile surgical drape per surgery (8‐in. × 8‐in.; GEPCO, cat. no. 88VCSTF)
  • 2 Pads or rolled‐up aluminum foil (∼2 × 2 × 10 cm) to prop up hemostatic forceps
  • 1 Sterile gauze pad per surgery (Fisher, cat. no. 19808936)
  • 1 Needle holder (Roboz, cat. no. RS‐7832)
  • 1 5‐0 silk suture (Roboz, cat. no. SUT‐1073‐21) with attached 12‐mm cutting needle for muscle and skin closure (one suture can usually be used for two to three surgeries)
  • 2 Trays for washing instruments

Basic Protocol 2: The CCl4‐Induced Acute Liver Injury and Regeneration Model

  Materials
  • Mice, 14‐16 weeks old, (preferable strain: C57BL/6, gender: female)
  • Carbon tetrachloride (CCl 4; Sigma‐Aldrich, cat. no. 289116; avoid breathing vapors)
  • Mineral oil (light oil; Sigma, cat. no. M5904)
  • 70% and 100% ethanol
  • Surgical instrument cleaner (Integra Miltex, cat. no. 3‐720) or laboratory detergent
  • Animal balance
  • 1 15‐ or 50‐ml conical tube for mixing CCl 4 and mineral oil
  • 1 Hamilton microliter syringe with a 0.25 to 0.5 ml calibrated barrel and 22S/2"/2 needle
  • 1 Gauze pad
  • 3 50‐ml conical tubes (e.g., BD Falcon) for holding solutions to wash the syringe

Support Protocol 1: Harvesting Mouse Organs and Blood

  Materials
  • Mouse subjected to partial hepatectomy ( protocol 1) or CCl 4 acute liver injury ( protocol 2)
  • 70% ethanol
  • 10% formalin or 0.9% NaCl (for liver perfusion)
  • 0.5 M EDTA (if plasma is desired and no coated tubes are available)
  • Liquid N 2 (if frozen tissue is desired)
  • 1 1‐ml syringe with 25‐G, 5/8‐in. needle
  • 1 Operating scissors
  • 1 Microdissecting forceps
  • 2 Hemostatic forceps
  • 1 Microdissecting scissors
  • 1 Microtainer tube for blood collection per mouse (with or without a serum separator or EDTA coating, depending on whether plasma or serum is desired; a microcentrifuge tube can be used as an alternative)
  • 1 10‐ml syringe with 25‐G, 5/8 needle
  • 1.5‐ml screw‐cap cryotubes (if frozen tissue is desired)
  • 15‐ or 50‐ml conical tubes (e.g., BD Falcon) for 10% formalin (if tissue fixation is desired)
  • Additional reagents and equipment for isoflurane anesthesia of mice (see relevant steps of protocol 1)

Support Protocol 2: Quantifying Hepatocyte Cell Cycle Re‐Entry by Means of BrdU Immunohistochemistry

  Materials
  • Slides of liver and intestinal tissue previously fixed with 10% formalin ( protocol 3) and paraffin‐embedded
  • BrdU (Sigma)
  • Sterile PBS or 0.9% NaCl (normal saline) for injections
  • PBS, pH 7.4 (500 ml to 1 liter) for immunohistochemistry
  • Horse serum
  • Xylene
  • 95%, 80%, and 70% ethanol
  • Citric acid buffer (see recipe)
  • Methanol
  • 30% Hydrogen peroxide (H 2O 2)
  • Avidin/Biotin Blocking Kit (Vector, cat. no. SP‐2001)
  • Anti‐BrdU antibody (Millipore, clone AH4H7‐1/131‐14871, cat. no. MAB3424)
  • PBT (see recipe)
  • Horse anti‐mouse antibody (Vector, cat. no. BA‐2000, rat‐adsorbed)
  • Vectastain Kit (Vector, cat. no. PK‐4000)
  • DAB Substrate Kit (Vector, cat. No. SK‐4100)
  • Hematoxylin stain, Gill's Formulation #2 (optional for DNA counterstaining)
  • Permount (Fisher, cat. no. SP‐15)
  • Animal balance
  • 1 1‐ml syringe with 25‐ to 27‐G, 3/8‐in. needle
  • Staining jars (50‐ml volume for 1 to 5 slides; 100‐ml volume for 6 to 10 slides)
  • Large tray or Petri dish for humidified chamber (preferably one commercially available; e.g., Scientific Device Laboratory, cat. no. 197‐CR; http://www.scientificdevice.com/)
  • Whatman 3MM paper
  • Serological pipets (if commercially available humidified chamber is not used)
  • 1 Plastic slide rack
  • 1 1‐liter plastic beaker
  • 1 Vacuum trap flask with attached Pasteur pipet
  • 1 Calbiochem Hydrophobic ImmunoPen (EMD Millipore, cat. no. 402176)
  • Parafilm (if blocking slides overnight)
  • 2 15‐ml conical tubes
  • Glass coverslips
  • Additional reagents and equipment for harvesting blood and/or organs ( protocol 3) and intraperitoneal injection (as for CCl 4 injection; see protocol 2, step 6)

Support Protocol 3: Preparation of Nuclear Protein Extracts from Mouse Liver

  Materials
  • Homogenization buffer (see recipe)
  • Buffer C (see recipe)
  • Buffer D (see recipe)
  • Mouse liver tissue portion (best if fresh, but snap‐frozen tissue can be used)
  • PBS, ice cold
  • Laboratory detergent
  • 95% ethanol
  • 1 Large glass Dounce homogenizer tube and pestle per liver (or reuse after cleaning)
  • 1 Glass petri dish per liver
  • 1 Razor blade per liver
  • 1 50‐ml conical tube (e.g., BD Falcon)
  • Beckman ultracentrifuge with SW41 rotor
  • Swinging buckets for SW41 rotor
  • Beckman TL‐100 tabletop ultracentrifuge with TLA‐45 rotor and tubes (if this is not available, a Beckman J2‐21 centrifuge with JA‐20 rotor or similar, with 15‐ml Corex tubes and screw‐top microcentrifuge tubes can be used)
  • 1 Plastic SW41 ultracentrifuge tube (14 × 89 Ultra Clear) per liver (if an odd number of livers are processed, one extra tube will be required as a balance)
  • Balance (optional; Mettler)
  • 1 Glass petri dish per liver
  • 1 Razor blade per liver
  • 1 Large glass Dounce homogenizer tube and pestle per liver (or reuse after cleaning)
  • Overhead stirrer
  • 1 Spatula per liver
  • 1 Small plastic homogenizer tube and pestle (Kontes) per liver
  • Dialysis tubing membrane (MWCO 20,000 or smaller)
  • Microdialyzer (Spectrum)
  • Magnetic stirrer and stir bar
  • Additional reagents and equipment for protein assay (Simonian and Smith, )

Support Protocol 4: Probing the Activation of Transcriptional Networks that Promote Liver Regeneration (Electrophoretic Mobility Shift Assay)

  Materials
  • Double‐stranded, desalted, HPLC‐purified oligonucleotide probe (175 pmol to 50 nmol, purchased commercially or synthesized at a core facility)—the following sequences can be used:
  • NF‐κB = 5′‐AGTTGAGGGGACTTTCCCAGGC‐3′; consensus sequence (Promega Corp., cat. no. E3291)
  • STAT3 = 5′‐GATCCTCCAGCATTTCCCGTAAATCCTCCAG‐3′; cis‐inducible factor (SIF) binding element in the c‐fos promoter
  • E2 = 5′‐GGTTCCAGACCGCGATTGGTGGCTGGA‐3′; used as a loading control
  • DNA 5′‐end labeling kit (Promega)
  • 10,000 µCi/ml [γ ‐32P]ATP (sp. act., 3000 Ci/mmol; GE Life Sciences, cat. no. AA0068)
  • Appropriate 2× gel shift reaction buffer (see recipe)
  • 30% (w/v) acrylamide
  • 0.5× and 10× TBE buffer (see recipe)
  • 80% (v/v) glycerol
  • 10% (w/v) ammonium persulfate (APS)
  • TEMED
  • Liver nuclear extract ( protocol 5)
  • Poly(deoxyinosinic‐deoxycytidylic) acid sodium salt (Poly dI:dC; Sigma, cat. no. P4929, or equivalent)
  • Appropriate antibody for “super‐shift”
  • G‐25 Sephadex spin column (GE Life Sciences, cat. no. 27‐5325‐01)
  • Scintillation counter
  • Electrophoresis equipment (gel box and power supply)
  • Gel dryer
  • Whatman 3MM filter paper
  • Autoradiography film
  • Automatic X‐ray film developer (for autoradiography) or developer/fixer solutions for manual processing of the film in a darkroom
  • Additional reagents and equipment for gel electrophoresis (Gallagher, )
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Figures

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Literature Cited

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Key Reference
  Mitchell and Willenbring, 2008. See above.
  This article provides an authoritative overview of the 2/3 partial hepatectomy technique and a comprehensive outline of the surgical procedure for inducing liver regeneration in mice. It also discusses critical parameters affecting experimental design, including limitations and potential caveats that should be taken into consideration when designing similar studies.
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