Published: May 16th, 2021
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.
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.
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 disease1,2,3. Given their importance in disease, cardiac fibroblas....
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.......
Cardiac fibroblasts are essential for baseline function of the heart as well as for response to cardiac injury. Previous attempts to understand the arrangement and morphology of these cells have been conducted largely in 2-D settings. However, a refined cardiac tissue clearing (Figure 2) and 3-D imaging technique has been published, which allows for the advanced, more detailed visualization of cardiac fibroblasts. With this imaging technique, fibroblasts were found to be densely packed and h.......
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 heart12,13,14,
The authors would like to acknowledge the CCHMC Confocal Imaging Core for their assistance and guidance in development of this model, as well as Matt Batie from Clinical Engineering for the design of all 3D printed parts. Demetria Fischesser was supported by a training grant from the National Institutes of Health, (NHLBI, T32 HL125204) and Jeffery D. Molkentin was supported by the Howard Hughes Medical Institute.....
|4-0 braided silk
|40% Acrylamide Solution
|Artificial Tear Ointment
|DABCO (1,4-diazabicyclo[2.2. 2]octane)
|GLUture topical tissue adhesive
|World Precision Instruments
|Imaris Start Analysis Software
|Nikon Elements Analysis Software
|Nikon A1R HD upright microscope
|Normal autoclaved chow
|Nycodenz, 5- (N-2, 3-dihydroxypropylacetamido)-2, 4, 6-tri-iodo-N,
N'-bis (2, 3 dihydroxypropyl) isophthalamide
|Electron Microscopy Sciences
|Rosa26-nLacZ [FVB.Cg-Gt(ROSA)26Sortm1 (CAG-lacZ,-EGFP)Glh/J]
|Jax Stock No:012429
|Sodium Chloride solution
|Thermo Fisher Scientific
|Quadrol, N,N,N′,N′-Tetrakis(2-Hydroxypropyl)ethylenediamine, decolorizing agent
|X-Clarity electrophoretic clearing chamber
|X-Clarity electrophoretic clearing solution
|X-Clarity electrophoresis tissue basket
|X-Clarity electrophoresis tissue basket holder
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