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(2):97-104.


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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    • "Picrosirius red staining of the femur was used to assess collagen content (Fig. 8A) as described previously [39]. Mecp2 stop/y mice showed a significant decrease (−24%) in collagen content compared to Wt mice (Fig. 8B; Wt = 65.1 ± 8.6%; Mecp2 stop/y = 48.8 "
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    ABSTRACT: Rett syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene are the primary cause of the disorder. Despite the dominant neurological phenotypes, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as scoliosis, reduced bone mineral density and skeletal fractures are also common and important clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any defects, we have conducted a series of histological, imaging and biomechanical tests of bone in a functional knockout mouse model of RTT. Both hemizygous Mecp2(stop/y) male mice in which Mecp2 is silenced in all cells and female Mecp2(stop/+) mice in which Mecp2 is silenced in similar to 50% of cells as a consequence of random X-chromosome inactivation, revealed significant reductions in cortical bone stiffness, microhardness and tensile modulus. Microstructural analysis also revealed alterations in both cortical and cancellous femoral bone between wild-type and MeCP2-deficient mice. Furthermore, unsilencing of Mecp2 in adult mice cre-mediated stop cassette deletion resulted in a restoration of biomechanical properties (stiffness, microhardness) towards wild-type levels. These results show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies. (C) 2014 The Authors. Published by Elsevier Inc.
    Bone 10/2014; 71. DOI:10.1016/j.bone.2014.10.008 · 3.97 Impact Factor
    • "For example, yellow-red strong birefringence would be assigned to collagen type I, whereas collagen type III would display a weak birefringence associated with a greenish color (Junqueira et al. 1979; Montes and Junqueira 1991). Controversially, other authors reported that the polarized colors of Picrosirius red staining depend only on fiber thickness and packing, not on the composition of the specific collagen type within collagen bundles (Pierard 1989; Dayan et al. 1989; Rich and Whittaker 2005; Coleman 2011). But, associated with morphometric image analysis, this dyeing procedure remains the most powerful method to study and quantify collagen network remodeling (Malkusch et al. 1995; Berton et al. 2000; Séguier et al. 2000) As establishing collagen type by picrosirius red staining is still largely reported, we wanted to illustrate through simple and clear examples that phenotypic collagen determination by this method is unsuitable. "
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    ABSTRACT: Specific staining of the extracellular matrix components is especially helpful in studying tissue remodeling, particularly in the case of connective tissue pathologies. As developed by Junqueira and colleagues in 1979, specific staining by Picrosirius red is one of the most important stains to study collagen networks in different tissues. Under polarized light, collagen bundles appear green, red or yellow, and are easily differentiated from the black background, thus allowing for quantitative morphometric analysis. As Junqueira and colleagues point out, many studies use color staining to differentiate collagen bundles and to specify collagen types, yet other studies report that polarized colors only reflect fiber thickness and packing. Using a simple histological example, our study illustrates the inability of Picrosirius red staining to differentiate collagen types, since the absorbed amount of polarized light by this dye strictly depends on the orientation of the collagen bundles.
    Acta histochemica et cytochemica official journal of the Japan Society of Histochemistry and Cytochemistry 07/2014; 62(10). DOI:10.1369/0022155414545787 · 1.39 Impact Factor
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    • "Although type III collagen fibers are usually thinner than type I fibers, it would be presumptuous to state that green (thin) fibers and orange-red (thick) fibers are indicative of type III and type I collagen, respectively. The same authors also speculated the possibility of a green fiber being an immature, thin type I fiber or a thick type I fiber being “smeared” by a sectioning artifact, thereby decreasing its thickness.[9] "
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    ABSTRACT: Introduction:Radiation, commonly employed as neoadjuvant, primary, and adjuvant therapy for head and neck cancer causes numerous epithelial and stromal changes, prominent among which is fibrosis with its early and late consequences. Very little is known about the true nature of the fibrosed tissue and the type of fibers accumulated. Radiotherapy affects the supporting tumor stroma often resulting in a worsening grade of tumor post-radiation.Aim:To study epithelial, neoplastic, stromal, and glandular changes in oral cavity induced by radiation therapy for oral squamous cell carcinoma (OSCC) using special stains.Materials and Methods:The study included 27 samples of recurrent OSCC following completion of radiotherapy (recurrence within an average span of 11 months), and 26 non-irradiated cases of OSCC. Patients with a history of combined radiotherapy and chemotherapy were not included in the study. The epithelial changes assessed included epithelial atrophy, apoptosis, necrosis, dysplasia, and neoplasia. The connective tissue was evaluated for amount of fibrosis, quality of fibers (using picrosirius red staining), fibrinous exudate, necrosis, pattern of invasion, vessel wall thickening, and salivary gland changes. The aforementioned changes were assessed using light and polarizing microscopy and tabulated.Statistical Analysis:Epithelial and connective tissue parameters were compared between the irradiated and non-irradiated cases using chi square and t-tests.Results:Epithelial and connective tissue parameters were found to be increased in irradiated patients. Pattern of invasion by tumor cells varied from strands and cords between the two groups studied. The effect of radiation was seen to reflect on the maturity of fibers and the regularity of their distribution.
    South Asian Journal of Cancer 07/2014; 3(3):159-62. DOI:10.4103/2278-330X.136785
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