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, 59 (8), 769-79

Quantification of Ceroid and Lipofuscin in Skeletal Muscle

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Quantification of Ceroid and Lipofuscin in Skeletal Muscle

Hatice Tohma et al. J Histochem Cytochem.

Abstract

Ceroid and lipofuscin are autofluorescent granules thought to be generated as a consequence of chronic oxidative stress. Because ceroid and lipofuscin are persistent in tissue, their measurement can provide a lifetime history of exposure to chronic oxidative stress. Although ceroid and lipofuscin can be measured by quantification of autofluorescent granules, current methods rely on subjective assessment. Furthermore, there has not been any evaluation of variables affecting quantitative measurements. The article describes a simple statistical approach that can be readily applied to quantitate ceroid and lipofuscin. Furthermore, it is shown that several factors, including magnification tissue thickness and tissue level, can affect precision and sensitivity. After optimizing for these factors, the authors show that ceroid and lipofuscin can be measured reproducibly in the skeletal muscle of dystrophic mice (ceroid) and aged mice (lipofuscin).

Conflict of interest statement

The author(s) declared no potential conflicts of interest with respect to the authorship and publication of this article.

Figures

Figure 1.
Figure 1.
Fluorescence images of transverse frozen sections of quadriceps muscle. Example images for ceroid analysis are for 3-month-old (A) mdx and (B) C57 mice. Example images for lipofuscin analysis are for (C) 27-month-old and (D) 3-month-old C57Bl/6J mice. Fluorescent images were collected using a 450- to 490-nm excitation and 505- to 520-nm emission filter with a 3-sec exposure. Scale bar is 25 µm.
Figure 2.
Figure 2.
Representative intensity plots of images. (A) Expanded pixel intensity plots from one image collected from mdx (solid line) and one image from C57 (dashed line) muscle sections. (B) Expanded pixel intensity plots of five randomly selected images from C57 muscle sections. Inserts show the complete pixel intensity plots. Data are from images collected at ×20 magnification.
Figure 3.
Figure 3.
Representative quantile–quantile plots. The plots contrast data from pixel intensity plots for (A) C57 and (B) mdx images with normally distributed theoretical data. The diagonal line indicates where normally distributed experimental data would fall. Data are from images collected at ×10 magnification.
Figure 4.
Figure 4.
Use of standard deviation multipliers to estimate percentage area of ceroid. (A) Percentage area of signals from C57 and mdx images (left axis) and the difference between mdx and C57 percentage area (right axis) according to threshold standard deviation multipliers of 5 to 10 (n = 5). (B) Percentage area of signals from young and aged images (left axis) and the difference between aged and young percentage area (right axis) according to threshold standard deviation multipliers of 3 to 10 (n = 5). For A and B, a solid line shows the measured percentage area and a dashed line shows the difference of the area. (C) Quantile–quantile plots for 3 mdx images (1, 2, 3) with the pixel intensity threshold shown as a horizontal line (threshold multiplier = 6). (D) Quantile–quantile plots for three images from aged mouse tissue (1, 2, 3) with the pixel intensity threshold shown as a horizontal line (threshold multiplier = 4). (E) Quantile–quantile plots for three C57 images (1, 2, 3) with the pixel intensity threshold shown as a horizontal line (threshold multiplier = 6). (F) Quantile–quantile plots for three images from young mouse tissue (1, 2, 3) with the pixel intensity threshold shown as a horizontal line (threshold multiplier = 4).
Figure 5.
Figure 5.
Comparison of methods to measure autofluorescent granule content. (A) Ceroid content was estimated in five mdx images using a threshold multiplier of 6 and by manually tracing all visible granules. (B) Ceroid content was estimated in 5 aged images using a threshold multiplier of 4 and by manually tracing all visible granules.
Figure 6.
Figure 6.
Precision of the estimated ceroid value in mdx tissue sections. (A) Variability in estimated ceroid content from 30 images captured from a single mdx quadriceps section using a threshold standard deviation multiplier of 6. (B) Variation in quantifying ceroid when using different numbers of images. Boxes show first and third quantiles. Vertical lines show minimum and maximum values. Horizontal bars in each box are median values. Dashed line represents the value of ceroid content estimated by the analysis of all usable images from three sections. (C) Reproducibility of ceroid measurement performed on three different days (10-µm thickness, n = 24). Data are from images collected at ×10 magnification. Bar indicates p < 0.05 relative to Day 21.
Figure 7.
Figure 7.
Ceroid signal at different magnifications. Pixel intensity plots of C57 (n = 3) and mdx (n = 3) images at (A) ×10, (B) ×20, and (C) ×40 magnification. Heat maps of ceroid spots in images at (D) ×10, (E) ×20, and (F) ×40 magnification. Images collected for tissue of 10-µm thickness, with a 3-sec exposure. Scale bar 25 µm.
Figure 8.
Figure 8.
Variability in estimated granule content. (A) Percentage area of ceroid in mdx tissue sections at three magnifications (n = 30). (B) Ceroid content in sections at different thickness taken from the same tissue level (×10 magnification, n = 24). (C) Calculated lipofuscin content at different tissue thickness (n = 24). Bar indicates p < 0.05.
Figure 9.
Figure 9.
The effect of photobleaching on quantification of ceroid and lipofuscin. An image taken from mdx mouse tissue (A) before and (B) after exposure to blue light. Arrows indicate the same patch of ceroid. (C) Reduction in the integrated optical density of the images taken from mdx and aged tissue following continuing exposure to blue light. Calculated percentage area of (D) ceroid and (E) lipofuscin in images taken from mdx and aged tissue sections, respectively, with a standard deviation multiplier of 6. Scale bar is 25 µm.
Figure 10.
Figure 10.
Quantification of ceroid and lipofuscin in dystrophic and aged muscles. Measurement of ceroid and lipofuscin content throughout quadriceps muscle from (A) mdx and (B) aged mouse (n = 24 images for each level). Ceroid content (C) was estimated from images taken at ×10 magnification for 3-month-old mdx mice (n = 6 mice). To minimize measurement variability, image collection for each mdx mouse was matched with image collection from a 3-month-old normal (C57) mouse to estimate background signal. Lipofuscin content (D) was estimated from images taken at ×40 magnification in 27-month-old C57Bl/6J mice (n = 4 mice). Image collection for each aged mouse was matched with image collection from a young (3 month old) mouse to estimate background signal. Bar indicates p< 0.05.

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