Fourier analysis of collagen bundle orientation in myocardial infarction scars

Abstract

Collagen bundle orientation (CBO) in myocardial infarct scars plays a major role in scar mechanics and complications after infarction. We aim to compare four histopathological methods for CBO measurement in myocardial scarring. Myocardial infarction was induced in 21 pigs by balloon coronary occlusion. Scar samples were obtained at 4 weeks, stained with Masson’s trichrome, Picrosirius red, and Hematoxylin–Eosin (H&E), and photographed using light, polarized light microscopy, and confocal microscopy, respectively. Masson’s trichrome images were also optimized to remove non-collagenous structures. Two observers measured CBO by means of a semi-automated, Fourier analysis protocol. Interrater reliability and comparability between techniques were studied by the intraclass correlation coefficient (ICC) and Bland–Altman (B&A) plots and limits of agreement. Fourier analysis showed an almost perfect interrater reliability for each technique (ICC ≥ 0.95, p < 0.001 in all cases). CBO showed more randomly oriented values in Masson’s trichrome and worse comparability with other techniques (ICC vs. Picrosirius red: 0.79 [0.47–0.91], p = 0.001; vs. H&E-confocal: 0.70 [0.26–0.88], p = 0.005). However, optimized Masson’s trichrome showed almost perfect agreement with Picrosirius red (ICC 0.84 [0.6–0.94], p < 0.001) and H&E-confocal (ICC 0.81 [0.54–0.92], p < 0.001), as well as these latter techniques between each other (ICC 0.84 [0.60–0.93], p < 0.001). In summary, a semi-automated, Fourier-based method can provide highly reproducible CBO measurements in four different histopathological techniques. Masson’s trichrome tends to provide more randomly oriented CBO index values, probably due to non-specific visualization of non-collagenous structures. However, optimization of Masson’s trichrome microphotographs to remove non-collagenous components provides an almost perfect comparability between this technique, Picrosirius red and H&E-confocal.

Meta-Analysis of Extracellular Matrix Dynamics after Myocardial Infarction Using RNA-Sequencing Transcriptomic Database

“…Traditionally, research has focused on evaluating type I and type III collagen, considered the main components of the infarcted heart at the fibrotic phase [ 1 , 3 , 6 ]. Indeed, their amount and spatial orientation is considered crucial to ensure proper mechanical and electrical functions in the repaired myocardium [ 28 , 29 ]. However, little is known about the pathophysiological implications of other subunits of this molecule.…”

Novel Fibrillar and Non-Fibrillar Collagens Involved in Fibrotic Scar Formation after Myocardial Infarction

“…Human and murine myocardium tissue was fixed in 4% paraformaldehyde acid, embedded in paraffin, sectioned, and mounted on double gelatin-coated glass slides. Hematoxylin–eosin and Masson’s Trichrome staining was performed to corroborate the presence of infarcted areas [ 35 , 36 ].…”

“…Images were morphometrically analyzed using Image ProPlus 7.0 software (Media Cybernetics Inc., Rockville, MD, USA) by a technician who was unaware of the study groups. Collagen presence was quantified as the percentage of area occupied by the selected object and the number of positive fibers [ 36 , 37 ]. The range used for segmentation is indicated in Table 8 .…”

“…Regarding proteolytic-induced epitope retrieval, samples were incubated with proteinase K (1 µg/mL, Sigma Aldrich, St. Louis, MO, USA) for 10 min at 37 • C. Subsequently, after unmasking and peroxidase blocking (0.3% H 2 O 2 ), sections were incubated overnight (4 • C) with the specific primary antibody diluted in PBS/0.1% BSA. For immunohistochemistry, specific labeling was detected with a biotin-conjugated goat anti-mouse or goat anti-rabbit secondary antibody (1:500 dilution, Dako, Glostrup, Denmark) [35,36]. Further information about the primary antibodies, antigen retrieval method, and antibody concentration is detailed in Table 7.…”

Fabrication of a tailor-made conductive polyaniline/ascorbic acid-coated nanofibrous mat as a conductive and antioxidant cell-free cardiac patch

“…A large number of cardiomyocytes are lost during MI due to the lack of oxygen and nutrients [3]. As a result, the inflammatory responses and recruitment of local fibroblasts happen which leads to the formation of collagen-rich scar tissue [4]. The introduction of injectable hydrogels, cardiac patches, and cardiac tissue engineering scaffolds may be a powerful tool to address this issue.…”