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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.
Funding Acknowledgements
Type of funding sources: Public grant(s) – EU funding. Main funding source(s): “Instituto de Salud Carlos III” and “Fondos Europeos de Desarrollo Regional FEDER” and Conselleria de Educación – Generalitat Valenciana.
Introduction
After acute myocardial infarction (AMI), the lack of oxygen and nutrients leads to cardiomyocyte necrosis and eventually to the formation of a collagen-based scar. Infarct scar characteristics, such as collagen bundle orientation, have a relevant influence on scar mechanics, the occurrence of cardiac arrhythmias, left ventricular dilation or aneurysm formation, wall stiffness, and the development of wall rupture or heart failure. However, the most adequate method for collagen bundle orientation (CBO) measurement in myocardial scar is not established.
Purpose
We aim to compare the measurement of collagen bundle orientation in infarct scar by Fourier analysis in three different histopathological techniques.
Methods
Juvenile swine (n=21) were subjected, by means of percutaneous balloon inflation, to a transient 90-min occlusion of mid left anterior descending artery followed by one month of reperfusion (chronic AMI group). Samples were obtained from the infarcted zone and stained with Masson’s trichrome, Picrosirius red and Haematoxylin-Eosin (H-E) standard protocols. Five microphotographs of the myocardial scar were taken at 200x magnification with light, polarised and confocal microscopy, respectively. A single observer measured CBO by means of Fast Fourier Transform analysis using a semi-automated protocol. Comparability between techniques was studied by the Intraclass Correlation Coefficient (ICC), the coefficient of variation (CV) and the Bland-Altman (B&A) plots and limits of agreement.
Results
Measurement of CBO in Masson’s trichrome tended to show higher (more "random-oriented") values than in Picrosirius and H-E+confocal techniques (ICC 0.79 and 0.7, p=0.001 and 0.005; B&A 0.29 to -0.02 and 0.43 to 0.01; CV 6.97% and 12.98%, respectively). However, measurement of CBO in Picrosirius and H-E+confocal techniques showed an "almost perfect" agreement (ICC 0.84, p<0.001; B&A 0.28 to -0.09; CV 17.33%). Selective staining and/or visualization of collagen in these latter techniques may underlie our findings, contrary to non-selective Masson’s trichrome.
Conclusion
Picrosirius red staining (visualized with polarised microscopy) and Haematoxylin-Eosin (visualized with confocal microscopy) are comparable in terms of collagen bundle orientation measurement by Fourier analysis in an animal model of chronic infarct scar. Masson’s trichrome (visualized with light microscopy) tends to show more "random-oriented" values, potentially due to non-specific staining and visualization of non-collagenous structures such as cells, and should not be recommended for this specific purpose.
Introducción: algunas especies animales son utilizadas como modelos experimentales en la práctica clínica y quirúrgica y en los claustros universitarios para la enseñanza de la anatomía animal y comparada. Como un aporte a estos ejercicios, esta investigación enriquece el conocimiento de la morfología exterior del útero de cerda y su irrigación vascular.
Metodología: este estudio descriptivo transversal, se realizó con 60 úteros de porcinos. Las arterias y venas uterinas, previo reparo con seda a nivel del cérvix uterino, fueron perfundidas con resina semisintética (palatal 85 % y estireno 15 %) coloreado con rojo y azul mineral respectivamente. Para el estudio anatómico del órgano se realizó una descripción general de la irrigación uterina. Se tomaron las medidas externas de los ovarios y del útero para su caracterización.
Resultados: la longitud promedio de los ovarios fue de 23,07 +/- 3,93 mm para el derecho, y 23,02 +/- 4,45 mm para el izquierdo. La arteria ovárica derecha presentó un calibre y una longitud de 1,7 +/- 0,31 mm y 36,2 +/- 24,1 mm; mientras que en la izquierda se observó un calibre y una longitud de 1,6 +/- 0,5 mm y 35,6 +/- 16,5 mm. Las tubas uterinas presentaron una longitud de 140,3 +/- 29,3 mm del lado derecho y 135,9 +/- 29,2 mm del lado izquierdo.
Conclusiones: se encontró que los valores morfométricos encontrados en esta investigación resultaron en general menores a lo descrito en estudios previos, posiblemente porque las medidas descritas por otros autores se realizaron en hembras multíparas. Esta consideración invita a realizar otros estudios que incluyan hembras con estas características de paridad, para conocer y precisar si el factor está asociado con las variaciones de las medidas reportadas. Vale la pena resaltar que la morfometría del aparato genital tiene creciente importancia en las investigaciones médicas.
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