Abstract
Microstructural characterization of cardiac tissue and its remodeling in disease is a crucial step in many basic research projects. We present a comprehensive approach for three-dimensional characterization of cardiac tissue at the submicrometer scale. We developed a compression-free mounting method as well as labeling and imaging protocols that facilitate acquisition of three-dimensional image stacks with scanning confocal microscopy. We evaluated the approach with normal and infarcted ventricular tissue. We used the acquired image stacks for segmentation, quantitative analysis and visualization of important tissue components. In contrast to conventional mounting, compression-free mounting preserved cell shapes, capillary lumens and extracellular laminas. Furthermore, the new approach and imaging protocols resulted in high signal-to-noise ratios at depths up to 60 µm. This allowed extensive analyzes revealing major differences in volume fractions and distribution of cardiomyocytes, blood vessels, fibroblasts, myofibroblasts and extracellular space in control vs. infarct border zone. Our results show that the developed approach yields comprehensive data on microstructure of cardiac tissue and its remodeling in disease. In contrast to other approaches, it allows quantitative assessment of all major tissue components. Furthermore, we suggest that the approach will provide important data for physiological models of cardiac tissue at the submicrometer scale.
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Acknowledgements
This study was supported by the Nora Eccles Harrison Treadwell Foundation (FBS, TS), AHA grant 14POST19820010 (TS), NIH grant R01 HL094464 (FBS), the Studienstiftung des deutschen Volkes (JCE) and Stiftung Familie Klee (JCE). The authors thank Mrs. Jayne Davis and Mrs. Nancy Allen for technical support.
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Associate Editor Jane Grande-Allen oversaw the review of this article.
Thomas Seidel and J.-C. Edelmann have contributed equally to this work.
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Seidel, T., Edelmann, JC. & Sachse, F.B. Analyzing Remodeling of Cardiac Tissue: A Comprehensive Approach Based on Confocal Microscopy and 3D Reconstructions. Ann Biomed Eng 44, 1436–1448 (2016). https://doi.org/10.1007/s10439-015-1465-6
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DOI: https://doi.org/10.1007/s10439-015-1465-6