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Novel methods of automated quantification of gap junction distribution and interstitial collagen quantity from animal and human atrial tissue sections.

Yan J, Thomson JK, Wu X, Zhao W, Pollard AE, Ai X - PLoS ONE (2014)

Bottom Line: This approach allowed segmentation between ID-associated and non-ID-associated Cx43.Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts.The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.

ABSTRACT

Background: Gap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies.

Methods and results: Separate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.

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Matlab-based algorithm designed to detect interstitial collagen deposition.A. Representative image of Masson's Trichrome (MT) stained rabbit LA tissue (collagen  =  blue, myocardium  =  red). B-D. Representative grayscale images of myocardium (B), unstained white space (C), and blue stained collagen (D). E. Summarized data of quantified interstitial collagen (blue area) in healthy human LA with increasing age (n = 18).
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pone-0104357-g007: Matlab-based algorithm designed to detect interstitial collagen deposition.A. Representative image of Masson's Trichrome (MT) stained rabbit LA tissue (collagen  =  blue, myocardium  =  red). B-D. Representative grayscale images of myocardium (B), unstained white space (C), and blue stained collagen (D). E. Summarized data of quantified interstitial collagen (blue area) in healthy human LA with increasing age (n = 18).

Mentions: MT stains collagen blue, myoplasm red and nuclei brown. When MT stained RGB images were converted into grayscale images, each pixel was differentiated according to the total amount of emitted light, since less light produces dark pixels and more light generates brighter pixels. Each image of a single color channel consisted of discrete pixels with various brightness intensities from 0 to 255. We therefore thresholded individual channels in each RGB image to initially segment tissue pixels from those occupying open spaces that resulted from sectioning of heterogeneous atrial muscle and to further segment tissue pixels into collagen and non-collagen groups. The intensity values of each pixel (I) in the R (red), G (green) and B (blue) channel were denoted as IR, IG and IB, respectively. Thresholds were based on mean (μ) and standard deviation (σ) of pixel intensity within each image. Because unstained empty white space of the RGB images was composed of the pixels with higher intensity values on all three channels, unstained white pixels were identified as those with IG>μG+0.9σG and IR>μR+0.9σR. Muscle/nuclei pixels had relatively high IR with low IB and IG (IB<μB+0.3σB and IG<μG-0.1σG). Collagen pixels had relatively high IB (IB>μB+0.9σB) and low IR (IR<μR+0.25σR) as well as medium IG (μG+0.75σG<IG <μG+2σG). Any pixels identified as being both collagen and muscle/nuclei were removed from the collagen group to avoid overestimation of IC. IC was then taken as the fraction of collagen pixels relative to total tissue pixels. Fig. 7 shows an example of the procedure with one image from an MT-stained section (A) segmented for assembly of grayscale images including tissue/nuclei (B), unstained white space (C) and collagen (D) from the original image.


Novel methods of automated quantification of gap junction distribution and interstitial collagen quantity from animal and human atrial tissue sections.

Yan J, Thomson JK, Wu X, Zhao W, Pollard AE, Ai X - PLoS ONE (2014)

Matlab-based algorithm designed to detect interstitial collagen deposition.A. Representative image of Masson's Trichrome (MT) stained rabbit LA tissue (collagen  =  blue, myocardium  =  red). B-D. Representative grayscale images of myocardium (B), unstained white space (C), and blue stained collagen (D). E. Summarized data of quantified interstitial collagen (blue area) in healthy human LA with increasing age (n = 18).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126721&req=5

pone-0104357-g007: Matlab-based algorithm designed to detect interstitial collagen deposition.A. Representative image of Masson's Trichrome (MT) stained rabbit LA tissue (collagen  =  blue, myocardium  =  red). B-D. Representative grayscale images of myocardium (B), unstained white space (C), and blue stained collagen (D). E. Summarized data of quantified interstitial collagen (blue area) in healthy human LA with increasing age (n = 18).
Mentions: MT stains collagen blue, myoplasm red and nuclei brown. When MT stained RGB images were converted into grayscale images, each pixel was differentiated according to the total amount of emitted light, since less light produces dark pixels and more light generates brighter pixels. Each image of a single color channel consisted of discrete pixels with various brightness intensities from 0 to 255. We therefore thresholded individual channels in each RGB image to initially segment tissue pixels from those occupying open spaces that resulted from sectioning of heterogeneous atrial muscle and to further segment tissue pixels into collagen and non-collagen groups. The intensity values of each pixel (I) in the R (red), G (green) and B (blue) channel were denoted as IR, IG and IB, respectively. Thresholds were based on mean (μ) and standard deviation (σ) of pixel intensity within each image. Because unstained empty white space of the RGB images was composed of the pixels with higher intensity values on all three channels, unstained white pixels were identified as those with IG>μG+0.9σG and IR>μR+0.9σR. Muscle/nuclei pixels had relatively high IR with low IB and IG (IB<μB+0.3σB and IG<μG-0.1σG). Collagen pixels had relatively high IB (IB>μB+0.9σB) and low IR (IR<μR+0.25σR) as well as medium IG (μG+0.75σG<IG <μG+2σG). Any pixels identified as being both collagen and muscle/nuclei were removed from the collagen group to avoid overestimation of IC. IC was then taken as the fraction of collagen pixels relative to total tissue pixels. Fig. 7 shows an example of the procedure with one image from an MT-stained section (A) segmented for assembly of grayscale images including tissue/nuclei (B), unstained white space (C) and collagen (D) from the original image.

Bottom Line: This approach allowed segmentation between ID-associated and non-ID-associated Cx43.Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts.The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.

ABSTRACT

Background: Gap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies.

Methods and results: Separate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.

Show MeSH
Related in: MedlinePlus