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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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Comparing results of quantitative Cx43 distribution and abundance using layer-by-layer and maximum projection imaging quantification.A. Representative sequential confocal images of Cx43 (green) and N-Cad (red) double immuno-stained young rabbit LA from focal layer 1 to layer 6. B. Histogram of quantified Cx43 immuno-stained signals from the maximum projection image. C-D. Summarized data of quantified Cx43E-E and Cx43S-S on young rabbit LA (n = 3; 53 images of each rabbit LA section) using layer-by-layer (B) and maximum projection (C) imaging quantification approaches. D. Pooled data of quantified Cx43E-E and Cx43S-S in young and aged rabbit LA using Layer-by-Layer imaging quantification from Z-stack confocal images (n = 4, 5; *p<0.001).
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pone-0104357-g006: Comparing results of quantitative Cx43 distribution and abundance using layer-by-layer and maximum projection imaging quantification.A. Representative sequential confocal images of Cx43 (green) and N-Cad (red) double immuno-stained young rabbit LA from focal layer 1 to layer 6. B. Histogram of quantified Cx43 immuno-stained signals from the maximum projection image. C-D. Summarized data of quantified Cx43E-E and Cx43S-S on young rabbit LA (n = 3; 53 images of each rabbit LA section) using layer-by-layer (B) and maximum projection (C) imaging quantification approaches. D. Pooled data of quantified Cx43E-E and Cx43S-S in young and aged rabbit LA using Layer-by-Layer imaging quantification from Z-stack confocal images (n = 4, 5; *p<0.001).

Mentions: A processed maximum projection confocal image from sequential Z-stack confocal images is commonly used to quantify Cx43 abundance in IHC stained sections. However, the amount of ID associated Cx43 pixels can vary in distribution and abundance at different confocal scanning layers due to changing fiber orientation in different focal planes. Fig. 6A shows apparently varied abundance of double immuno-stained Cx43 and N-Cad signals at different confocal scanning layers of Z-stack sequential confocal images obtained from a young rabbit atrial tissue section. To determine quantitative differences between using maximum projection image and layer-by-layer image processing, we processed 53 sets of Z-stack confocal images obtained from three young rabbit LA tissue sections. We first quantified Cx43E-E and Cx43S-S layer-by-layer on each image of a total of 6–10 sequential Z-stack confocal images. A maximum projection image processed from the same Z-stack sequential images was also quantified. Before comparing the quantitative results from using the two approaches, all the quantified Cx43 data were normalized with N-Cad as an internal normalization procedure. Fig. 6A shows the representative sequential confocal images of Cx43 (green) and N-Cad (red) double immuno-stained young rabbit LA. Fig. 6B shows that the quantitative amount of Cx43E-E from the maximum projection images was higher than the sum result of using layer-by-layer quantification approach, while Cx43S-S was lower than that of layer-by-layer quantification approach (Fig. 6C). These results suggest that a layer-by-layer quantification approach could minimize overestimation or underestimation of Cx43E-E and Cx43S-S compared to the maximum projection image quantification method.


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)

Comparing results of quantitative Cx43 distribution and abundance using layer-by-layer and maximum projection imaging quantification.A. Representative sequential confocal images of Cx43 (green) and N-Cad (red) double immuno-stained young rabbit LA from focal layer 1 to layer 6. B. Histogram of quantified Cx43 immuno-stained signals from the maximum projection image. C-D. Summarized data of quantified Cx43E-E and Cx43S-S on young rabbit LA (n = 3; 53 images of each rabbit LA section) using layer-by-layer (B) and maximum projection (C) imaging quantification approaches. D. Pooled data of quantified Cx43E-E and Cx43S-S in young and aged rabbit LA using Layer-by-Layer imaging quantification from Z-stack confocal images (n = 4, 5; *p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104357-g006: Comparing results of quantitative Cx43 distribution and abundance using layer-by-layer and maximum projection imaging quantification.A. Representative sequential confocal images of Cx43 (green) and N-Cad (red) double immuno-stained young rabbit LA from focal layer 1 to layer 6. B. Histogram of quantified Cx43 immuno-stained signals from the maximum projection image. C-D. Summarized data of quantified Cx43E-E and Cx43S-S on young rabbit LA (n = 3; 53 images of each rabbit LA section) using layer-by-layer (B) and maximum projection (C) imaging quantification approaches. D. Pooled data of quantified Cx43E-E and Cx43S-S in young and aged rabbit LA using Layer-by-Layer imaging quantification from Z-stack confocal images (n = 4, 5; *p<0.001).
Mentions: A processed maximum projection confocal image from sequential Z-stack confocal images is commonly used to quantify Cx43 abundance in IHC stained sections. However, the amount of ID associated Cx43 pixels can vary in distribution and abundance at different confocal scanning layers due to changing fiber orientation in different focal planes. Fig. 6A shows apparently varied abundance of double immuno-stained Cx43 and N-Cad signals at different confocal scanning layers of Z-stack sequential confocal images obtained from a young rabbit atrial tissue section. To determine quantitative differences between using maximum projection image and layer-by-layer image processing, we processed 53 sets of Z-stack confocal images obtained from three young rabbit LA tissue sections. We first quantified Cx43E-E and Cx43S-S layer-by-layer on each image of a total of 6–10 sequential Z-stack confocal images. A maximum projection image processed from the same Z-stack sequential images was also quantified. Before comparing the quantitative results from using the two approaches, all the quantified Cx43 data were normalized with N-Cad as an internal normalization procedure. Fig. 6A shows the representative sequential confocal images of Cx43 (green) and N-Cad (red) double immuno-stained young rabbit LA. Fig. 6B shows that the quantitative amount of Cx43E-E from the maximum projection images was higher than the sum result of using layer-by-layer quantification approach, while Cx43S-S was lower than that of layer-by-layer quantification approach (Fig. 6C). These results suggest that a layer-by-layer quantification approach could minimize overestimation or underestimation of Cx43E-E and Cx43S-S compared to the maximum projection image quantification method.

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