Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

Summary

A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.

Abstract

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

Introduction

Although cardiomyocytes comprise the greatest volume fraction in the heart, cardiac fibroblasts are more plentiful and are critically involved in regulating the baseline structural and reparative features of this organ. Cardiac fibroblasts are highly mobile, mechanically responsive, and phenotypically ranging depending on the extent of their activation. Cardiac fibroblasts are necessary to maintain normal levels of extracellular matrix (ECM), and too little or too much ECM production by these cells can lead to disease^1,2,3. Given their importance in disease, cardiac fibroblas....

Protocol

All experiments involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children’s Hospital Medical Center. The institution is also AAALAC (American Association for Accreditation of Laboratory Animal Care) certified. Mice were euthanized via cervical dislocation, and mice undergoing survival surgical procedures were given pain relief (see below). All methods used for pain management and euthanasia are based on recommendations of the Panel on Euthanasia of the American V.......

Discussion

This article presents a refined method for tissue clearing that allows for visualization of cardiac fibroblasts in vivo, both at baseline and following injury, to characterize and better understand fibroblasts in the mouse heart. This enhanced protocol addresses limitations in existing tissue clearing protocols that have attempted to identify specific cell types in the adult or neonatal heart^12,13,14,

Materials

Name	Company	Catalog Number	Comments
4-0 braided silk	Ethicon	K871H
8-0 prolene	Ethicon	8730H
40% Acrylamide Solution	Bio-Rad	1610140
Angiotensin II	Sigma	A9525-50G
Artificial Tear Ointment	Covetrus	048272
DABCO (1,4-diazabicyclo[2.2. 2]octane)	Millipore Sigma	D27802-25G
GLUture topical tissue adhesive	World Precision Instruments	503763
Heparin	Sigma	H0777
Imaris Start Analysis Software	Oxford Instruments	N/A
Micro-osmotic pumps	Alzet	Model 1002
Nikon Elements Analysis Software	Nikon	N/A
Nikon A1R HD upright microscope	Nikon	N/A
Normal autoclaved chow	Labdiet	5010
Nycodenz, 5- (N-2, 3-dihydroxypropylacetamido)-2, 4, 6-tri-iodo-N, N'-bis (2, 3 dihydroxypropyl) isophthalamide	CosmoBio	AXS-1002424
Paraformaldehyde	Electron Microscopy Sciences	15710
Phenylephrine Hydrochloride	Sigma	P6126-10G
Photoinitiator	Wako Chemicals	VA-044
Rosa26-nLacZ [FVB.Cg-Gt(ROSA)26Sortm1 (CAG-lacZ,-EGFP)Glh/J]	Jackson Laboratories	Jax Stock No:012429
Sodium Azide	Sigma Aldrich	S2002-5G
Sodium Chloride solution	Hospira, Inc.	NDC 0409-4888-10
Tamoxifen	Sigma Aldrich	T5648
Tamoxifen food	Envigo	TD.130860
Tween-20	Thermo Fisher Scientific	BP337-500
Quadrol, N,N,N′,N′-Tetrakis(2-Hydroxypropyl)ethylenediamine, decolorizing agent	Millipore Sigma	122262-1L
X-Clarity electrophoretic clearing chamber	Logos Biosystems	C30001
X-Clarity electrophoretic clearing solution	Logos Biosystems	C13001
X-Clarity electrophoresis tissue basket	Logos Biosystems	C12001
X-Clarity electrophoresis tissue basket holder	Logos Biosystems	C12002