Optimized Heart Sampling and Systematic Evaluation of Cardiac Therapies in Mouse Models of Ischemic Injury: Assessment of Cardiac Remodeling and Semi‐Automated Quantification of Myocardial Infarct Size

Mariana Valente1, Ana Araújo2, Tiago Esteves3, Tiago L. Laundos2, Ana G. Freire4, Pedro Quelhas2, Perpétua Pinto‐do‐Ó1, Diana S. Nascimento2

1 Unit for Lymphopoiesis, Immunology Department, INSERM U668, University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Institut Pasteur, Paris, 2 INEB – Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal, 3 FEUP – Faculdade de Engenharia da Universidade do Porto, Universidade do Porto, Porto, Portugal, 4 Department of Developmental and Regenerative Biology and The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140293
Online Posting Date:  December, 2015
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Abstract

Cardiac therapies are commonly tested preclinically in small‐animal models of myocardial infarction. Following functional evaluation, post‐mortem histological analysis is essential to assess morphological and molecular alterations underlying the effectiveness of treatment. However, non‐methodical and inadequate sampling of the left ventricle often leads to misinterpretations and variability, making direct study comparisons unreliable. Protocols are provided for representative sampling of the ischemic mouse heart followed by morphometric analysis of the left ventricle. Extending the use of this sampling to other types of in situ analysis is also illustrated through the assessment of neovascularization and cellular engraftment in a cell‐based therapy setting. This is of interest to the general cardiovascular research community as it details methods for standardization and simplification of histo‐morphometric evaluation of emergent heart therapies. © 2015 by John Wiley & Sons, Inc.

Keywords: mouse‐models of myocardial infarction; systematic evaluation of cardiac therapies; representative heart sampling; quantification of infarct size; software MIQuant

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Representative Sampling of the Murine Heart
  • Basic Protocol 2: Infarct Size Calculation and Morphometric Analysis
  • Support Protocol 1: Assessment of Neovascularization in the Infarcted Myocardium Following Administration of a Therapy
  • Support Protocol 2: Identification of Transplanted Human Cells in the Mouse Heart
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Representative Sampling of the Murine Heart

  Materials
  • Young adult (8 to 12 weeks old) C57BL/6 mice (Mus musculus) sacrificed at 14 days post‐MI (Virag and Murry, )
  • 400 mg/kg pentobarbital (Eutasil, Alliance Healthcare, cat. no. 7412874)
  • 99.8% (v/v) ethanol (see recipe)
  • 4 M potassium chloride (KCl, see recipe)
  • Phosphate‐buffered saline (PBS)
  • 10% (v/v) neutral buffered formalin, pH 7.0 at 25°C (VWR BDH & Prolabo, cat. no. 361387P)
  • Clear‐Rite 3 (Thermo Scientific, cat. no. 6901) or other clearing agent (e.g., xylene)
  • Paraffin wax (56° to 57°C) (Thermo Scientific, cat. no. 6774060)
  • Distilled water (18.2 MΩ.cm dH 2O)
  • 1‐ml syringes with 26‐G, ½‐in., 0.45 × 13–mm needle
  • Dissection tools:
    • Dissection holder (styrofoam plaque, laboratory made)
    • 21‐G, ½‐in., 0.8 × 40–mm needles
    • Fine scissors (fine iris scissors, curved 10.5 cm, Fine Science Tools, cat. no. 14095‐11)
    • Gross forceps (narrow pattern forceps, curved 12 cm, Fine Science Tools, cat. no. 11003‐12)
    • Fine forceps (Dumont no. 7 forceps, Fine Science Tools, cat. no. 11272‐30)
  • Petri dish, 100 × 15–mm, non‐tissue culture
  • 15‐ml conical tubes (Falcon)
  • Histological cassettes (Thermo Scientific, cat. no. 1000957)
  • 500‐ml glass beaker
  • Tissue processor (Thermo Scientific, cat. no. 813150)
  • Paraffin embedding center (Thermo Scientific, cat. no. A81000002)
  • Stainless steel molds, 60 × 45 × 15–mm (Thermo Scientific, cat. no. E10/M8)
  • Cooling plate (part of paraffin embedding center; Thermo Scientific, cat. no. A81000002)
  • 45°C water bath (Medite Medizintechnik, cat. no. TFB 35)
  • Transparent plastic container (dark bottom), ∼200 × 100 mm
  • APES‐coated slides (see recipe) or ready‐to‐use poly‐L‐lysine‐ or silane‐coated slides (e.g., VWR, cat. no. 100490‐312 or cat. no. 100501‐000)
  • Single‐edge razor blade
  • Rotary microtome (Thermo Scientific, cat. no. 902100A)
  • Disposable microtome blade (Thermo Scientific, cat. no. 3051835)
  • Set of brushes for microtome (Bio-Optica, cat. no. 08‐0829)
  • Slide staining holders (Bio‐Optica, cat. no. 10‐42)
  • Slide boxes (Thermo Scientific, cat. no. E39)

Basic Protocol 2: Infarct Size Calculation and Morphometric Analysis

  Materials
  • Representative sections of LV of infarcted hearts (see protocol 1)
  • Xylene (Merck Millipore, cat. no. 1086611000)
  • 50%, 70%, 96%, and 99.8% ethanol dilutions (see recipe)
  • Distilled water (18.2 MΩ.cm dH 2O)
  • Celestine Blue solution (see recipe, Sigma‐Aldrich, cat. no. 206342‐5G
  • Gill's hematoxylin solution (Sigma‐Aldrich, cat. no. GHS232‐1L)
  • Bouin solution (Sigma‐Aldrich, cat. no. HT10132‐1L)
  • Masson's Trichrome Kit (Sigma, cat. no. HT15; also see recipe for phosphomolybdic/phosphotungstic acid working solution) containing:
    • Biebrich Scarlet acid‐fuchsin solution
    • Phosphotungstic acid solution
    • Phosphomolybdic acid solution
    • Aniline blue solution
  • Mounting medium (Marienfeld Laboratory Glassware, cat. no. 6900002)
  • Slide staining holders (Bio‐Optica, cat. no. 10‐42)
  • Slide staining jars (Bio‐Optica, cat. no. 10‐10)
  • Coverslips (MGF, cat. no. 65.300.14)
  • Stereomicroscope with digital camera
  • Software ImageJ1.42
  • Computer with Windows and Internet access

Support Protocol 1: Assessment of Neovascularization in the Infarcted Myocardium Following Administration of a Therapy

  Materials
  • Slides with representative tissue sections of LV (see protocol 1) from treated and control groups
  • Phosphate‐buffered saline (PBS)
  • 10 mM Tris/1 mM EDTA, pH 9.0 (see recipe)
  • 0.2% (v/v) Triton X‐100 (see recipe)
  • 4% FBS/1% BSA blocking solution (see recipe)
  • Primary antibody: 0.8 μg/ml goat anti‐mouse CD31 IgG (Santa Cruz Biotechnology, cat. no. sc‐1506, see recipe) or 0.8 μg/ml normal goat IgG (Santa Cruz Biotechnology, cat. no. sc‐2028, see recipe)
  • Secondary antibody: 2 μg/ml donkey anti‐goat IgG Alexa Fluor 568 (Life Technologies, cat. no. A11057, see recipe)
  • Vectashield with DAPI (Vector Laboratories, cat. no. H‐1200)
  • 98°C water bath
  • Plastic cuvette (58 × 53.5 × 86 mm, Bio‐Optica, cat. no. 44‐13101)
  • Hydrophobic pen (PAP pen)
  • StainTray slide staining system (humidity chamber, eBioscience, cat. no. 44‐0404‐10)
  • Coverslips
  • Dark slides boxes
  • Inverted fluorescence microscope with MosaiX AxioVision module (Zeiss)
  • Software ImageJ1.42
  • Additional reagents and equipment for dewaxing and hydrating sections (see protocol 2)
NOTE: The microscope must be equipped with emission light sources and filter sets to excite and detect DAPI and Alexa 568, as well as a monochrome, high‐resolution, sensitive camera for image acquisition. If the MosaiX AxioVision module (Zeiss) is not available, the user must acquire a random selection of LV fields, i.e., ten fields of infarcted and ten fields of peri‐infarcted regions per heart section (as described below in step 15).

Support Protocol 2: Identification of Transplanted Human Cells in the Mouse Heart

  Materials
  • Slides with adherent paraffin tissue sections (see protocol 1) from treated and control groups
  • Phosphate‐buffered saline (PBS)
  • 10 mM citrate solution, pH 6.0 (see recipe)
  • 0.4% (v/v) Triton X‐100 solution (see recipe)
  • Vector M.O.M. kit (Vector Laboratories, cat. no. BMK‐2202, see recipe)
  • Primary antibody: 2.5 μg/ml mouse anti‐human nuclei IgG1 antibody (Millipore, cat. no. MAB4383, see recipe) or 2.5 μg/ml mouse IgG1 negative control (Millipore, cat. no. MABC002, see recipe)
  • Secondary antibody: 2 μg/ml donkey anti‐mouse IgG Alexa Fluor 594 (Life Technologies, cat. no. A21203, see recipe)
  • Vectashield with DAPI (Vector Laboratories, cat. no. H‐1200)
  • Plastic cuvette (58 × 53.5 × 86 mm, Bio‐Optica, cat. no. 44‐13101)
  • 98°C water bath
  • Hydrophobic PAP pen
  • StainTray slide staining system (humidity chamber, eBioscience, cat. no. 44‐0404‐10)
  • Coverslips
  • Dark slide boxes
  • Inverted fluorescence microscope
  • Additional reagents and equipment for dewaxing and hydrating sections (see protocol 2)
NOTE: The microscope must be equipped with emission light sources and filter sets to excite and detect DAPI and Alexa 594, as well as a monochrome high‐resolution sensible camera for image acquisition. For further information, see Critical Parameters and Troubleshooting.
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Figures

Videos

Literature Cited

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Internet Resources
  http://paginas.fe.up.pt/∼quelhas/MIQuant/
  This webpage contains instructions and access to MIQuant, including a detailed stepwise manual and associated publications.
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