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Updated: Nov 4, 2025
Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts
Published on: May 16, 2021
Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse
Demetria M Fischesser1, Evan C Meyer2, Michelle Sargent3
1Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine; Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center.
Insights
This study visualizes cardiac fibroblasts in 3-D, revealing their dynamics in heart disease. Fibroblast patterns and death/repopulation were observed in injured mouse hearts, improving our understanding of cardiac fibrosis.
Area of Science:
- Cardiovascular Biology
- Cellular Biology
- Biomedical Imaging
Background:
- Cardiovascular disease is a leading cause of death, often involving cardiac fibrosis and altered heart function.
- Cardiac fibrosis stems from activated resident fibroblasts, but their 3-D behavior in the heart is poorly understood.
- Understanding fibroblast dynamics is crucial for addressing heart disease.
Purpose of the Study:
- To investigate the role and dynamics of cardiac fibroblasts in the 3-D heart.
- To develop and refine a CLARITY-based tissue clearing and imaging method for visualizing fibroblasts.
- To observe fibroblast behavior in healthy and fibrotic mouse heart models.
Main Methods:
- Genetically labeled cardiac fibroblasts using Rosa26-loxP-eGFP reporter and Tcf21-MerCreMer mice.
- Employed a refined CLARITY tissue clearing technique for whole mouse heart imaging.
- Utilized digitized imaging to analyze fibroblast localization and dynamics in 3-D.
Main Results:
- Successfully visualized fluorescently labeled cardiac fibroblasts throughout the entire adult mouse left ventricle.
- Observed unique fibroblast patterns following contractile fiber bands in one injury model.
- Identified fibroblast death and subsequent repopulation from the infarct border zone in ischemic injury models.
Conclusions:
- The refined 3-D cardiac tissue clearing and imaging technique allows unprecedented visualization of cardiac fibroblasts.
- This method overcomes limitations of antibody penetration and fluorescence loss in tissue processing.
- Provides new insights into fibroblast dynamics and their contribution to cardiac fibrosis and heart disease progression.
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.
