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Collagen and Picrosirius red staining: A polarized light assessment of fibrillar hue and spatial distribution

Department of Anesthesiology, University of Massachusetts Medical School, Worcester, MA, USA
J. morphol. Sci 01/2005; 22:97-104.

ABSTRACT Collagen plays a vital role in maintaining structural integrity and in determining tissue function. Therefore, methods to detect, quantify, and analyze collagen are valuable. Nevertheless, stains historically employed to detect collagen have disadvantages, principally a poor specificity for thin fibers. Conversely, picrosirius red, which has the capability to detect thin fibers, although frequently used, is seldom exploited to the fullest extent. Our goal was, using picrosirius red staining, circularly polarized light, and image-analysis software, not only to identify fibers and quantify collagen content, but also to assess fiber hue and the spatial distribution of the different colors. To assess collagen content, we used a subtraction technique to remove interstitial space and non-collagen elements from images of skin wounds, myocardial scars, and arterial tissue. The hue component of the resulting image was obtained, and the number of red, orange, yellow, and green (the colors of collagen fibers in order of decreasing thickness) pixels calculated. Finally, we assessed the spatial distribution of individual colors by the application of color threshold filters. Skin wound analysis demonstrated good inter-observer agreement for collagen content and fiber color. In myocardial scars, collagen content increased from 1 (61%) to 5 weeks (95%) after injury. The proportion of green (thin) fibers decreased (43 to 4%), while the proportion of orange (thick) fibers increased (13 to 65%). Color threshold application revealed regional variation in fiber color within subintimal arterial lesions. These methods increase the amount of structural information obtained from picrosirius red-stained sections. INTRODUCTION Collagen fibers play a vital role not only in maintaining structural integrity, but also in determining tissue function. For example, collagen degradation and loss after myocardial infarction is associated with infarct expansion and subsequent functional decline [33]. On the other hand, although collagen confers tensile strength, excess accumulation is often detrimental. For instance, increased fibrosis after kidney transplant leads to a decrease in renal function and eventual graft failure [6,9], and hence quantification of fibrosis has been suggested as a means to predict graft survival. In such examples, insight into pathological structure-function relationships depends upon accurate identification of collagen fibers.

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    • "Only the hue component was retained and a histogram of hue frequency was obtained from the resolved 8-bit hue images, which contain 256 colors. We used the following hue definitions; red 0e51, green 52e120 [21] [22]. The number of pixels within each hue range was determined and expressed as a percentage of the total number of collagen pixels, which in turn was expressed as a percentage of the total number of pixels in the image. "
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    • "Quantified collagen content in the scar area was computed using ImageJ and plotted in a graph shown on the right panel of Fig. 3. To characterize the collagen structure further, we viewed the picrosirius red stained sections under polarized light (see the Material and methods section). This reveals the thickness of the collagen fibrils, with the thinnest ones appearing green, intermediates yellow to orange, and the thickest ones red [34]. The images show not only the increased collagen content in the scar (Figs. "
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