Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Summary

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45⁺ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Abstract

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45⁺ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Introduction

During the evolution of light microscopy, many specialized forms appeared, all of them developed to minimize limitations in the visualization process for particular specimens. One such method is light-sheet fluorescence microscopy (LSFM). First developed in 1903 by Siedentopf and Zsigmondy¹ and finding its first fundamental biological applications in the early 1990s², LSFM has become the most powerful microscopic tool to date for the visualization of large specimens, such as intact mouse organs, with a fluorescence signal resolution down to the cellular level. Because of these benefits, in combination with its potential ....

Protocol

All animal experiments were conducted in accordance with EU guidelines and were approved by the relevant local authorities in Essen (AZ 84-02.04.2014.A036 - Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Essen, Germany). The animals were used and housed under specific pathogen-free (SPF) conditions.

1. Induction of Myocarditis by Diphtheria Toxin (DTX)

1. Prepare the DTX solution for the induction of myocarditis by diluting the DTX stock solution with phosphate-buffered saline (PBS) to a 1 µg/mL working solution.
2. Apply 100 µL of this solution intratracheally (i.t.) into 8 to ....

Results

The presented LSFM approach analyzes the leukocyte distribution and amount in murine hearts upon induction of severe myocarditis. Figure 1A explains the pre-treatment protocol for the transgenic CD11c.DTR mice for myocarditis induction. This step represents the necessary trigger for the recruitment of leukocytes to the myocardium. After successful DTX application, the animals develop severe disease symptoms, such as general weakness, anorexia, and weight loss within the r.......

Discussion

In modern life science, the microscopic visualization of biological processes plays an increasingly important role. In this context, many developments have been achieved during the last two centuries that help to answer questions not addressable up to this point, . First, there has been a clear tendency to fundamentally increase the resolution ability of light microscopes. With structured illumination microscopy (SIM)^21,22, stimulated emission-depletion (STED) mi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
diphtheria toxin	Sigma Aldrich	D0564
phosphate buffered saline	Biochrom	L182-10
ketamine [50 mg/mL]	Inresa	PZN 4089014
xylazine [2 %]	Ceva
syringe	Braun	REF 9161502	Braun Omincan F
indwelt venous catheter	Becton Dickinson	REF 381923	BD Insyte Autoguard
small animal respirator	Harvard Apparatus	73-0044	MiniVent
anit-CD45 antibody (AF647)	BioLegend	103124
ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate	Carl Roth	8043.2
catheter (21 G)	BD Biosciences	REF 387455	BD valu-set
paraformaldehyde	Sigma Aldrich	P6148
PE tube (5 mL)	Carl Roth	EKY9.1	Rotilabo
ethanol	Carl Roth	9065.4
dibenzyl ether	Sigma-Aldrich	108014
tube rotator	Miltenyi Biotec	130-090-753	MACSmix
agarose (low gelling)	Sigma Aldrich	A4018
mold (15 x 15 x 5 mm)	Tissue Tek	4566	Cryomold
light sheet microscope system	LaVision Biotec		Ultramicroscope
microscope body	Olympus		MVX10
objective	Olympus		0.5 NA MVPLAPO 2XC, WD 5.5 mm
sCMOS camera 5.5MP	Andor Technology
488 nm OPSL (50mW) laser	Coherent
647 nm  diode laser (50mW)	Coherent
3D image processing & analysis software	Bitplane		IMARIS Ver. 8.3
transgenic mouse strain	Steffen Jung et al.		CD11c.DTR
wt mouse strain	Envigo		Balb/c Ola Hsd J
Laser Module	LaVision Biotec
MVPLAPO 2XC Objective Lens