Fibrotic remodeling of the heart is a frequent condition linked to various diseases and cardiac dysfunction. Collagen quantification is an important objective in cardiac fibrosis research; however, a variety of different histological methods are currently used that may differ in accuracy. Here, frequently applied collagen quantification techniques were compared. A porcine model of early-stage heart failure with preserved ejection fraction was used as example. Semi-automated threshold analyses were imprecise, mainly due to inclusion of non-collagen structures or failure to detect certain collagen deposits. In contrast, collagen assessment by automated image analysis and light microscopy (LM)-stereology was more sensitive. Depending on the quantification method, the amount of estimated collagen varied and influenced the inter-group comparisons. Picrosirius red, Masson's Trichrom and Azan staining protocols yielded similar results, whereas the measured collagen area increased with increasing section thickness. While none of the LM-based methods showed significant differences between the groups, electron microscopy (EM)-stereology revealed a significant collagen increase between cardiomyocytes in the experimental group, but not at other localizations. In conclusion, in contrast to the staining protocol, the section thickness and the quantification method directly influence the estimated collagen content and thus possibly inter-group comparisons. Electron microscopy in combination with stereology is a precise and sensitive method for collagen quantification if certain prerequisites are considered. For subtle fibrotic alterations, consideration of collagen localization may be necessary. Among LM methods, LM-stereology and automated image analysis are appropriate to quantify fibrotic changes, the latter depending on careful control of algorithm and comparable section staining.
- cardiac fibrosis
- collagen quantification
- automated image analysis
- Copyright © 2016, Journal of Applied Physiology