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Equine endometrosis is a degenerative and predominantly fibrotic condition resulting from progressive and irreversible multifactorial causes that influence the endometrium of mare. Tissue remodeling in the equine endometrium occurs as part of the pathogenesis of endometrosis, a process characterized by a shift in extracellular matrix (ECM) components. The relationship between matrix metalloproteinases and their specific inhibitors is crucial for the remodeling process. Collagen play a significant role in maintaining a healthy uterus and may promote fibrotic processes. The aim of this study was to quantify endometrial collagen deposition using picrosirius 25 red (PSR) staining, and to evaluate gene expression of collagen type 2 (COL-2) and 3 (COL-3), matrix metalloproteinases 1 (MMP-1) and 2 (MMP-2), their tissue inhibitor (TIMP-2), and tumor necrosis factor (TNF-α) in the endometrium of mares with different grades of fibrosis. The samples (n = 34) were classified into three categories based on the frequency and distribution of fibrosis-related changes in the endometrium: Category I (healthy endometrium, n = 12), Category II (moderate fibrosis, n = 12), and Category III (severe fibrosis, n = 10). Collagen quantification demonstrate a substantial proportional increase (P < 0.0001) in collagen deposition across Category I (11.72 ± 1.39 %), Category II (17.76 ± 1.29 %), and Category III (24.15 ± 1.87 %). In transcript evaluations, higher COL-2 expression was found in Category II than in mares classified as Category I or III. MMP-1 showed increased transcript expression in Category II compared to Category III endometrial samples. Higher expression of MMP-2 was detected in Category III than in Category I and II. TIMP-2 showed lower mRNA expression in Category III vs Category I and II. However, TNF-α gene expression was higher in Category II than in Categories I and III. This study demonstrates that endometrial evaluation using PSR can play an important role in routine analyses for the detection and objective quantification of collagen in endometrial tissues. Additionally, this study demonstrated through gene expression analysis that MMP-1 may be linked to physiological endometrial remodeling. In contrast, MMP-2 could be associated with fibrogenesis in the endometrium, which is regulated by the inhibitor TIMP-2. Furthermore, COL-2 and TNF-α could be considered as biological markers involved in the progression endometrosis in mares. As such, the results of this study may contribute to the development of future antifibrotic therapies that aim to delay or even reverse the pathological remodeling of the extracellular matrix in the uterus, in addition to optimizing the diagnosis and prognosis of endometrial fibrosis in mares. • Increased collagen accumulation in the uterine of mares with endometrosis. • Positive correlation between the animals' age and uterine collagen deposition. • Lower MMP-2 and higher TIMP-2 expression could be key for ECM reorganization. • TNF-α and COL-2 as possible markers of developing endometrosis. [ABSTRACT FROM AUTHOR]

Background: An ambiguity persists regarding variation in clinical aggressiveness of different oral lesions, among which are the giant cell granulomas. Different types of giant cell granulomas can develop in the jaws, including peripheral giant cell granuloma, and central giant cell granuloma, whether aggressive or non-aggressive, each of which show different clinical behavior. Although they show great resemblance histologically on the cellular level, still they may show variation in other histological components as those regarding collagen fibers present. This raises the question about any possible relationship between the clinical behavior and histological components of these lesions. Aim of the Work: This study aimed to investigate and explore the possible correlation between immunohistochemical detection of tumor associated macrophages (TAMs) using CD163 and the density, packing and type of collagen in giant cell granulomas of the jaws and the role of this correlation in the variation of clinical aggressiveness of these lesions. Methods: We measured the immunohistochemical expression of CD163 and quality of collagen fibers, using picrosirius red stain in normal oral mucosa, peripheral giant cell granuloma, non-aggressive and aggressive central giant cell granulomas (10 samples each). Results: Our results indicate that the expression of CD163 showed a statistically significant increase in aggressive central giant granuloma as compared to the non-aggressive samples. Moreover, picrosirius staining revealed that samples from aggressive lesions showed predominant green-yellow birefringence indicating higher expression of collagen type III in these lesions. Conclusion: These findings suggested a possible influencing role of TAMs and increased deposition of collagen type III in the progression and aggressive behavior of giant cell granulomas of the jaws. [ABSTRACT FROM AUTHOR]

The estimation of the post-mortem submersion interval (PMSI) of skeletal remains can provide critical information to forensic investigations, contributing to various aspects such as identification. A fundamental aspect of the PMSI estimation in bone is the degradation of collagen, the most abundant organic component. This degradation can be assessed using various analytical techniques, including the digital analysis of images taken from histological slides. Two open-source software for image digital analysis, ImageJ® and Orbit®, provide novice-friendly and accessible technology to a broad audience of forensic practitioners. However, the absence of established procedures can challenge the consistency of the interpretation of the results, especially between observers. The principal aim of this study was to compare the efficacy of ImageJ® and Orbit® in quantifying the degradation of collagen in images taken from histological slides of mammalian non-human bones submerged for 4 months to 2 years. Despite the inconsistency in the outcomes of intraobserver vs. interobserver testing, this research has demonstrated the promising potential of digital histology. It has also identified the need for further investigation, particularly focusing on the usability of ImageJ® and the refinement of the Orbit® workflow. [ABSTRACT FROM AUTHOR]

The estimation of the post-mortem submersion interval (PMSI) of skeletal remains can provide critical information to forensic investigations, contributing to various aspects such as identification. A fundamental aspect of the PMSI estimation in bone is the degradation of collagen, the most abundant organic component. This degradation can be assessed using various analytical techniques, including the digital analysis of images taken from histological slides. Two open-source software for image digital analysis, ImageJ® and Orbit®, provide novice-friendly and accessible technology to a broad audience of forensic practitioners. However, the absence of established procedures can challenge the consistency of the interpretation of the results, especially between observers. The principal aim of this study was to compare the efficacy of ImageJ® and Orbit® in quantifying the degradation of collagen in images taken from histological slides of mammalian non-human bones submerged for 4 months to 2 years. Despite the inconsistency in the outcomes of intraobserver vs. interobserver testing, this research has demonstrated the promising potential of digital histology. It has also identified the need for further investigation, particularly focusing on the usability of ImageJ® and the refinement of the Orbit® workflow. [ABSTRACT FROM AUTHOR]

Dysregulation of collagen deposition, degradation, and crosslinking in the heart occur in response to increased physiological stress. Collagen content has been associated with ultrasonic backscatter (brightness), and we have shown that the anisotropy of backscatter can be used to measure myofiber alignment, that is, variation in the brightness of a left ventricular short‐axis ultrasound. This study investigated collagen's role in anisotropy of ultrasonic backscatter; female Sprague–Dawley rat hearts were treated with a collagenase‐containing solution, for either 10 or 30 min, or control solution for 30 min. Serial ultrasound images were acquired at 2.5‐min intervals throughout collagenase treatment. Ultrasonic backscatter was assessed from anterior and posterior walls, where collagen fibrils are predominately aligned perpendicular to the angle of insonification, and the lateral and septal walls, where collagen is predominately aligned parallel to the angle of insonification. Collagenase digestion reduced backscatter anisotropy within the myocardium. Collagen remains present in the myocardium throughout collagenase treatment, but crosslinking is altered within 10 min. These data suggest that crosslinking of collagen modulates the anisotropy of ultrasonic backscatter. An Anisotropy Index, derived from differences in backscatter from parallel and perpendicularly aligned fibers, may provide a noninvasive index to monitor the progression and state of myocardial fibrosis. [ABSTRACT FROM AUTHOR]

Purpose: Hodgkin's disease is a common malignant disorder in adolescent patients. Although most patients are cured, approximately 10%-15% of patients experience a relapse or have resistant disease. Furthermore, there are no definitive molecular predictors for early identification of patients at high risk of treatment failure to first line therapy. The aim of this study was to evaluate the deep learning-based classifier model of medical image classification to predict clinical outcome that may help in appropriate therapeutic decisions. Methods: Eighty-three FFPE biopsy specimens from patients with Hodgkin's disease were stratified according to the patient's qPET scores, stained with picrosirius red dye and digitalized by whole slide image scanning. The resulting whole slide images were cut into tiles and annotated by two classes based on the collagen fibers' degree of coloring with picrosirius red. The neural network (YOLOv4) was then trained with the annotated data. Training was performed with 30 cases. Prognostic power of the weakly stained picrosirius red fibers was evaluated with 53 cases. The same neural network was trained with MMP9 stained tissue slides from the same cases and the quantification results were compared with the variant from the picrosirius red cases. Results: There was a weak monotonically increasing relationship by parametric ANOVA between the qPET groups and the percentages of weakly stained fibers (p = .0185). The qPET-positive cases showed an average of 18% of weakly stained fibers, and the qPET-negative cases 10%-14%. Detection performance showed an AUC of 0.79. Conclusions: Picrosirius red shows distinct associations as a prognostic metric candidate of disease progression in Hodgkin's disease cases using whole slide images but not sufficiently as a prognostic device. [ABSTRACT FROM AUTHOR]

Background: Solid multicystic ameloblastoma (SMA) is a locally aggressive, benign odontogenic tumor of odontogenic origin with greater rate of recurrence. Epithelial-mesenchymal interaction plays an important role in tooth morphogenesis that shows complete differentiation of epithelial and ectomesenchymal components to the level of tooth formation. Tumor stroma in ameloblastoma is normal mature collagen that prevents differentiation to the level of tooth formation. Current study evaluates the role of stromal elements in aggressive behavior of SMA using picrosirius red staining with polarizing microscopy and CD44v6 immunohistochemistry (IHC). Objectives: To compare nature of collagen using picrosirius red staining under polarized microscope and IHC expression of CD44v6 marker in SMA and oral squamous cell carcinoma (OSCC). Methods: Thirty blocks were retrieved from departmental archives and subjected to picrosirius red staining and CD44v6 IHC staining. Slides stained with picrosirius red were observed under polarized microscope to report the birefringence pattern. IHC slides were annotated for intensity of staining of tumor cells. Results: In contrast to OSCC's 40% red, 40% yellowish-red, and 20% greenish-yellow birefringence, SMA displayed 87% red, 13% yellowish-red, and 0% greenish-yellow. Compared to OSCC, which had tumor cells stained 9% strongly, 64% moderately, 27% mildly, and 0% negatively, SMA revealed 0% strong, 10% moderate, 60% weak, and 30% negative staining. Conclusion: As opposed to OSCC, which exhibited a greater quantity of greenish-yellow birefringence of immature collagen, SMA showed predominantly red birefringence, which is suggestive of mature collagen with a lack of metastasis. Comparing SMA to OSCC, the lack of significant CD44v6 positivity suggests that there has not been perineural invasion or regional metastases in SMA. [ABSTRACT FROM AUTHOR]

The complexity of the Sunda porcupine skin has become an important topic due to the unique characteristics of its quill follicles. The structure and chemical composition of the skin has affected many physiological and other conditions. Generally, quills are larger, stronger and stiffer than hair; therefore, the skin structure needs to adapt to support their physiology. The strength of the skin is determined by its collagen composition and arrangement; therefore, this study aims to analyse the composition and distribution of thick and thin fibres based on the specific characteristics of Sunda porcupine skin under polarized light using picrosirius red staining. The skin samples used were from the thoracodorsal and lumbosacral regions of eight Sunda porcupine adults. The histological staining was carried out using the picrosirius red method, while the samples were observed under a polarized light microscope and analysed with software. The results showed that the skin is composed of 36%–65% thick fibres, 20%–35% thin fibres and small amounts of other types with the lumbosacral region having higher compositions of thick and thin fibres than those in the thoracodorsal region. Furthermore, the thoracodorsal and lumbosacral regions have the highest composition of thick fibre in the deeper dermis and quill follicle, respectively. These demonstrated that the complexity of the skin structure of Sunda porcupine due to its quill derivates correlated with its collagen composition and distribution. [ABSTRACT FROM AUTHOR]

Mutation of just a single extracellular matrix protein, a receptor or enzyme involved in connective tissue metabolism is sufficient to cause systemic pathologies and failure of tissues that are subjected to strong mechanical stresses. Skin histological and computerized image analyses can provide a good qualitative and quantitative indication of these inherited connective tissue diseases. In this study, skin biopsies from young (10 to 25 years) and middle-aged patients (26 to 50 years) suffering from Ehlers-Danlos syndromes (EDS), Marfan syndrome (MS) or pseudoxanthoma elasticum (PXE) were studied after specific staining of both the collagen and elastic networks. Findings from the histomorphometric analyses conducted on skin sections of the patients with EDS, MS and PXE were compared to skin sections of healthy subjects from the same age groups. Our results show that both the collagen and the elastic networks were affected in all the studied pathological cases, but that the adverse changes to the elastic network in older patients were distinct from the physiological changes observed during aging process for healthy subjects. This degenerative process may be explained by an added phenomenon involving a general connective tissue proteolysis. [ABSTRACT FROM AUTHOR]

Introduction: The morphology and tissue response to macro- and micro-fat grafting have been widely studied in both clinical and experimental settings; the histological effects of the nanofat graft, however, remain largely unexplored. Aims: This study was carried out to evaluate the histological changes leading to scar rejuvenation in a fine scar following nanofat grafting. Materials and Methods: This was an experimental study carried out on guinea-pig fine-line scar models. Nanofat prepared from abdominal fat of the animal was injected into scar on right legs (NFG) at 1 month whereas left acted as controls (CG). Punch biopsies from all scars were analyzed at 2, 4, and 6 months by Hematoxylin&Eosin, Masson's trichrome, and Picrosirius red stains to evaluate dermal/epidermal regeneration, collagen fiber orientation, pattern of distribution, and amount of mature and immature collagen. Results: Nine animals were included in the final analysis of the study. On histological analysis, the amount of inflammatory infiltrate, collagen fiber orientation, pattern and total histological score at 2, 4, and 6 months were similar between the groups. There was an increased trend for earlier appearance of organized and mature forms of collagen in the NFG group. The distribution of collagen was similar at 2 months; however, there was a significant increase in collagen distribution in NFG at 4 months (NFG: 46.11±11.6, CG: 31.16±9.9; P = 0.010) and at 6 months (NFG: 63.48± 6.6, CG: 49.9 ±8.8; P = 0.002). Conclusion: Nanofat grafting is associated with an accelerated and increased production of mature collagen with proper alignment in fine-line scars. [ABSTRACT FROM AUTHOR]

Background: Odontogenic keratocyst (OKC) is one of the debatable odontogenic lesions. The need for more researches in order to determine the true nature of this lesion was one of the WHO's recommendations in 2016. Stromal reactions to epithelial neoplasms are marked by the appearance of alpha smooth muscle actin (α-SMA) positive myofibroblasts (MFs). Stromal collagen has a vital role in pathogenesis of odontogenic lesions. Objectives: To compare the count of α-SMA positive MFs, collagen quality and packing in OKC, in relation to dentigerous cyst and ameloblastoma. Material and Methods: α-SMA immunohistochemically positive MFs count, as well as the fraction of yellow-green fibers to detect the collagen quality and packing using picrosirius red staining with polarized light microscope, were compared in 11 samples of each of ameloblastoma, OKC and dentigerous cyst. Statistical analysis was performed. Results: The average number of MFs were 53.7, 41.9 and 11.3 in ameloblastoma, OKC and dentigerous cysts respectively with statistically significant differences (P-value <0.001, Effect size = 2.619). The mean area fraction of yellow-green fibers were 65.5%, 47.72% and 6.1% in ameloblastoma, OKC and dentigerous cyst respectively. Differences were statistically significant (P-value <0.001, Effect size = 2.645). Statistically significant direct correlation between MFs cell counts and area fraction of yellow-green fibers was found (ρ = 0.614, P-value <0.001). Conclusions: The immunohistochemical profile of OKC as regard to α-SMA positive MFs as well as histochemical profile as regard to collagen packing and quality were much closer to odontogenic tumors (ameloblastoma) than odontogenic cysts (dentigerous cyst) suggesting the neoplastic nature of this debatable lesion. [ABSTRACT FROM AUTHOR]

Head and neck connective tissue lesions may have diverse calcifications within the fibrous connective tissue stroma. The perplexity involved in the identification and determination of the nature or degree of calcification through routine hematoxylin and eosin (H&E) stains necessitates the usage of a specific, simple, and cost- and time-effective differential staining techniques. The aim of the present study was to develop criteria to distinguish bone formation from bone resorption using methylene blue-acid fuchsin (MB/AF) stain and the role of collagen fibers in the identification of stromal calcifications using polarizing microscopy with picrosirius red stain. Twenty cases with pathological diagnoses for various stromal calcifications in maxillofacial lesions were retrieved from the departmental archives. Decalcified formalin fixed paraffin embedded tissue sections were stained with hematoxylin and eosin, Masson's trichrome (MT), methylene blue-acid fuchsin (MB/AF), and picrosirius red. The stained sections were assessed to identify the calcifications found in the surrounding connective tissue stroma. It was observed that most cases showed maximum staining intensity with MB/AF stain as compared to the other staining methods. Moreover, the results suggested that contrast between calcification and stromal soft tissue was best distinguished with the MB/AF stain except in the case of dystrophic calcifications. Along with this, polarizing microscopy with picrosirius red enables better characterization of stromal components. Although the H&E stain and a connective tissue stain i.e. Masson's trichrome, are employed routinely in histopathology; the use of special stains such as MB-AF and picrosirius red facilitates the identification of calcifications from the stromal tissues. [ABSTRACT FROM AUTHOR]

Cancer is a disease involving interactions between cancer cells and stromal cells. Mast cells and collagen in oral squamous cell carcinoma (OSCC) has been studied individually here. It is not known if mast cells influence collagen in the microenvironment of OSCC, which in turn may influence tumour progression. The data was tabulated and analyzed using Pearson correlation coefficient. It was observed that mast cell count decreased with increasing grade of OSCC. Collagen in Well differentiated OSCC showed a predominant red orange hue, in moderately differentiated OSCC showed a yellow orange hue and in poorly differentiated OSCC showed a greenish yellow hue. The present research concluded that a co-relation exists between the grade of OSCC, mast cells and type of collagen. [ABSTRACT FROM AUTHOR]

In this study, the application of a recently introduced device based on electromagnetic energy transfer by microwaves for fat reduction, permitted to study specifically the modifications of thick fibrous collagen interlobular septa in the subcutaneous adipose tissue, related to the formation of large clusters of adipocytes. The use of Picrosirius red staining associated with circularly polarized microscopy gave evidence of appreciable modifications of the fibrous connective tissue forming septa. Compact fibrotic bundles of collagen I forming interlobular septa appeared reduced or dissolved, in part substituted by the increase of more diffuse and finely reticular collagen III. Remodeling of fibrous collagen, which formed bridles involved in the appearance at the surface of the skin of dimpling/orange peer pattern typical of cellulite, was observed. [ABSTRACT FROM AUTHOR]

The distribution and composition of extracellular matrix (ECM) of the spleen in two species of fruit bats, namely Cynopterus titthaecheilus and Rousettus leschenaultii, were examined by histochemistry and immunohistochemistry. Reticular fibres accompanied by laminin were identified to make up the splenic stromal network. Types I and III collagen were identified in various spleen compartments with varying intensities. Thin and short elastin fibres were scattered in several parts of the spleen. Visualization of the ECM of the spleen can better demonstrate spleen compartmentalization. The alleged vascular space structure in the fruit bats spleen was the sinus structure that was strengthened by the presence of reticular fibres that limit the sinus basement membrane. The present study identified periellipsoidal lymphoid sheath (PELS)‐like structure in fruit bats spleen that had never been identified in mammals before. In addition to describing the structure, this study highlighted the variations in ECM composition of the spleen between species that can provide new insight into the phylogenetic study of spleen morphology. [ABSTRACT FROM AUTHOR]

Microarchitectural features of collagen-rich extracellular matrices provide the mechanical foundation for tissue function and exhibit topographical cues that influence cellular behavior including proliferation, migration and protein expression. Preservation of tissue microarchitecture is required for accurate evaluation of tissue characteristics and pathology. It is unclear whether common tissue preservation methods possess equal ability to preserve microarchitecture. We investigated collagen microarchitecture in samples that had been flash frozen, fixed in formalin or preserved in RNAlater®, and which contained both collagen-rich and collagen-sparse regions. Fibrillar collagen organization was characterized using picrosirius red staining and second harmonic generation (SHG) microscopy. Maintenance of collagen fiber characteristics compared to the gold standard of flash freezing depended on both the method of preservation and the local collagen content of the tissue. Both formalin fixation and RNAlater® preserved collagen fiber characteristics similar to flash freezing in collagen-rich areas of the tissue, but not in collagen-sparse regions. Analysis using picrosirius red staining indicated preservation-dependent changes in overall tissue architecture and suprafibrillar organization. Together with considerations of cost, ease of use, storage conditions and ability to use the preserved tissue for RNA or protein analysis, our quantitative characterization of the effects of preservation method on collagen microarchitecture may help investigators select the most appropriate preservation approach for their needs. [ABSTRACT FROM AUTHOR]

Objective: The aim of the study was to analyze bone matrix (BMX) organization after bone grafting and repair using a new bioactive glass-ceramic (Biosilicate®) associated or not with particulate autogenous bone graft.Material and methods: Thirty rabbits underwent surgical bilateral parietal defects and divided into groups according to the materials used: (C) control—blood clot, (BG) particulate autogenous bone, (BS) bioactive glass-ceramic, and BG + BS. After 7, 14, and 30 days post-surgery, a fragment of each specimen was fixed in − 80 °C liquid nitrogen for zymographic evaluation, while the remaining was fixed in 10% formalin for histological birefringence analysis.Results: The results of this study demonstrated that matrix organization in experimental groups was significantly improved compared to C considering collagenous organization. Zymographic analysis revealed pro-MMP-2, pro-MMP-9, and active (a)-MMP-2 in all groups, showing gradual decrease of total gelatinolytic activity during the periods. At day 7, BG presented more prominent gelatinolytic activity for pro-MMP-2 and 9 and a-MMP-2, when compared to the other groups. In addition, at day 7, a 53% activation ratio (active form/[active form + latent form]) was evident in C group, 33% in BS group, and 31% in BG group.Conclusion: In general, BS allowed the production of a BMX similar to BG, with organized collagen deposition and MMP-2 and MMP-9 disponibility, permitting satisfactory bone remodeling at the late period.Clinical relevance: The evaluation of new bone substitute, with favorable biological properties, opens the possibility for its use as a viable and efficient alternative to autologous bone graft. [ABSTRACT FROM AUTHOR]

Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin-Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image-Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E-confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland-Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong agreement with H&E-confocal (0.7 < ICC < 0.8). Comparison of measurements between the three techniques for the same observer showed an almost perfect agreement (ICC > 0.8), better with Fourier analysis than with semiquantitative evaluation (single and average). These results in nonpathological skin samples were also confirmed in a preliminary analysis in eight scleroderma skin samples. Our results show that Masson's trichrome and Picrosirius red are consistent with H&E-confocal for measuring collagen bundle orientation in histological samples and could thus be used indistinctly for this purpose. Fourier analysis is superior to average semiquantitative evaluation and should keep being used as the preferred method. [ABSTRACT FROM AUTHOR]

Context:The quantification of total collagen is of major importance in a wide range of research areas, including the study of cutaneous wound healing and new drugs trials. Objective:The total collagen content in skin biopsies was compared by biochemical hydroxyproline assay and by two computer-aided histomorphometric analyses of histological sections. Materials and methods:Two methods were used to evaluate collagen formation: the hydroxyproline assay, as the gold standard and histomorphometric image analysis of the filled areas by corresponding stained collagen fibres, using picrosirius and Gomori’s trichrome staining. The image analyses were determined by digital densitometry recognition using computer-aided ImageJ software. One-way ANOVA, simple linear regression and ANCOVA were applied for the statistical analysis and correlation. Results:In a simple linear regression analysis carried out on the 14th day period after the induction of skin injury, three techniques, picrosirius red (F = 33.57,p = 0.00), Gomori’s trichrome (F = 81.61,p = 0.00) and hydroxyproline content (F = 16.85,p = 0.00) were able to detect collagen production. After scale adjustment, there were no significant differences among either the slopes (F = 1.17,p = 0.32) or the intercepts (F = 0.69,p = 0.51) of the estimated regression lines. It seems that a highly significant correlation exists between the histomorphometrical analysis and hydroxyproline assay. Discussion and conclusion:The morphometric analysis proved to be adequate and can be used as a simple, rapid, low-cost technology for evaluating total collagen in cutaneous wound specimens, compared with the gold standard hydroxyproline assay. [ABSTRACT FROM AUTHOR]

Objective The aim was to investigate collagen fibers in giant cell fibroma, inflammatory fibrous hyperplasia, and oral normal mucosa. Materials and methods Sixty-six cases were stained with picrosirius red. The slides were observed under polarization, followed by the measurement of the area and the percentage of the type I and type III collagens. The age and gender were obtained from the clinical records. Results No differences could be observed in both the area and percentage of the type I and type III collagens within the categories of lesions and normal mucosa. In the giant cells fibroma, a greater area and percentage of type I collagen could be identified in individuals of less than 41.5 years (p < 0.05). Conclusion The distribution of type I and type III collagen fibers in the studied lesions followed a similar pattern to that observed in the normal mucosa, indicating a normal collagen maturation process of type III to I. The study supports that multinucleated and stellate cells of the giant cell fibroma appear to be functional within collagen types III and I turnover. The greater amount of type I collagen identified in giant cell fibroma in individuals of less than 41.5 years reinforce the neoplastic nature of lesion. [ABSTRACT FROM AUTHOR]

Background: Reciprocal interaction between dental epithelium and mesenchyme is thought to be crucial for normal odontogenesis. Thus, the mesenchymal influence of the fibrous capsules may play an important role in the maintenance of epithelial expression. Collagen is the major component of the extracellular matrix and possibly there is an alteration in the nature and structure of collagen in various pathological conditions. Studies by polarizing microscopy have also shown that there is a difference in collagen and probably these differences may play a role in their biologic behaviour. Aim: The purpose of this study was to evaluate the nature of collagen fibers in keratocystic odontogenic tumour (KCOT), dentigerous cyst (DC), unicystic ameloblastoma (UA) and solid/ multicystic ameloblastoma (SMA) and correlating this with their biological behaviour. Materials and Methods: Five diagnosed cases each of UA, SMA, KCOT and DC were taken and stained using Picrosirius red stain kit and evaluated using a polarizing microscope. Statistical Analysis: Chi-square test was used to analyse the results. Results and Conclusion: Collagen fibers in dentigerous cysts showed predominant yellowish-red birefringence and fibers in KCOT and ameloblastomas showed a predominantly greenish-yellow birefringence. Hence, our study suggests that the nature and character of collagen fibers may influence the clinical behaviour of the lesion. Since ours is a pilot study, to corroborate our view, studies with larger sample size are required to substantiate the results. [ABSTRACT FROM AUTHOR]

The structure of the colonic wall relies on collagen, distributed within the submucosa and the muscularis externa. A recent analysis of total collagen in human ascending colon (AC) suggests that the muscularis externa is more susceptible to age-related increases in collagen among the elderly. However, it is not clear if this change also occurs in the descending colon (DC) or if the circular and longitudinal muscle layers are similarly affected in either region of colon. The aim of this study is to determine the total collagen content in the DC and its distribution between the circular muscle (CM) and taenia coli (TC) of the AC and DC of adults and compare the same with tissue from the elderly. Masson's trichrome and Picrosirius red were used to assess total collagen content in the AC and DC; aged 22 – 91 years. Macroscopically normal AC from 22 patients (adults: 22–60 years; 6 male, 6 female; elderly: 70 – 91 years; 6 male, 4 female) and DC from 23 patients (adults: 23–63 years; 6 male, 7 female; elderly: 66 – 88 years; 6 male, 4 female) were obtained following surgery for non-obstructed bowel cancer. The total hydroxyproline content in DC samples was also evaluated. In the DC, tinctorial staining demonstrated an increased occurrence of total collagen fibres in the submucosa of the elderly (159.8 ± 9.6 in elderly vs. 126.9 ± 6.1 in the adults; p 0.05) and in the muscularis externa (respectively 37.4 ± 4.1 vs. 18.8 ± 2.4; p 0.01). In the adult AC and DC, there were no statistically significant differences in the amount of collagen within the CM and TC. In the elderly, the total collagen fibres within the TC was greater in the AC (mean grey intensity: 63.4 ± 3.9% in the elderly vs. 36.6 ± 1.6% in adults; p 0.05) and DC (mean grey intensity: respectively, 59.82 ± 2.4 vs. 40.2 ± 0.9%; p 0.05). In both AC and DC of the elderly samples, several thickened collagen fibrils were microscopically identified within the TC infiltrating to the myenteric plexus. In the TC of the elderly AC, the total collagen fibres were increased by approximately 4% compared to that of the DC. The total collagen concentration in the elderly DC assessed by hydroxyproline assay was increased by approximately 15% compared to the adult. Sex related differences were not found when data combined. We concluded that the total collagen content in the muscularis externa particularly of the TC of human colon increases with age. The subtle change in collagen distribution with age between AC and DC may differentially affect the tensile strength of the colon. [ABSTRACT FROM AUTHOR]

[Display omitted] • Young dentin collagen fibrils are more coherently aligned. • Aged collagen is more bundled to suggest increased cross-linking. • Collagen distribution remains consistent with age and site within a tooth. • Type III collagen is detectable in peritubular dentin. • Dentin tubules contain large collagen fibers enveloped by mineral-like clusters. To investigate age and site-related changes to human dentin collagen, sound human teeth collected from donors aged 13–29 (young) and 50–74 (aged) years (n = 9/group) were cut to shallow and deep sites. Dentin collagen orientation and fibril bundling was investigated using the Picrosirius Red (PSR) stain observed under cross-polarized light microscopy (Pol), and collagen distribution was investigated using Confocal Laser Scanning Microscopy (CLSM). Collagen types III to I distribution in peritubular dentin (PTD) was revealed using Herovici stain and brightfield microscopy. Image analysis software and linear mixed modelling quantified outcomes. In situ dentin collagen was observed using Xenon Plasma Focussed Ion Beam Scanning Electron Microscopy (Xe PFIB-SEM). The PSR-Pol analysis revealed less coherently aligned and more bundled collagen fibrils in aged dentin (P = 0.005). Deep inner dentin collagen in both groups were less coherently aligned with reduced bundling. Regardless of age, CLSM showed collagen distribution remained stable; and more collagen type III was detectable in PTD located in inner dentin (Young: P = 0.006; Aged: P = 0.008). Observations following Xe PFIB-SEM cross-sectioning showed apatite-like deposits surrounding large intratubular collagen fibers, and evidence of smaller intertubular dentin collagen fibrils in situ. In conclusion, aging changes collagen network architecture, but not distribution or content. [ABSTRACT FROM AUTHOR]

The functional significance of any relationship exists between the orientation of the collagen fibers and the strain on a bone structure depend on the influence of these microstructural parameters on the biomechanical properties of bone and its ability to adapt. Localization of collagen was studied in the distal sesamoid bone (DSB) in in equine foot with Navicular Syndrome to see how this reflects the biomechanical demands by the tension exerted by the tendon of the deep digital flexor muscle on the joint distal interphalangeal. Hematoxylineosin-alcian blue staining, and Red Picrosirius of Junqueira were used. The birefringence of bone collagen was determined using polarized light microscopy. The remodeling of DSB resulted in the formation of transverse secondary osteons oriented lateral to medial and bone collagen was oriented in similar direction. These results provide evidence for the existence of a relationship between the mechanical function of a bone with the architecture, and shows further that this extends up to the molecular level. [ABSTRACT FROM AUTHOR]

Introduction. Morphological studies with standard and special histological techniques provide basic information to the dentist and physician clinical about the nature of the disease process, which allows to raise the quality in histopathological diagnosis. Furthermore, picrosirius red staining collagen types possible to differentiate if the microscope using polarized light. Objective. The aim of this paper is to show our experience in the use of polarization microscopy and staining Picrosirius in human salivary gland tumors. Methods. We used tissue sections of benign salivary tumors (pleomorphic adenoma) and malignant (adenoid cystic carcinoma) stained with Picrosirius colored red. Were observed with microscope NIKON ECLIPSE LV 100POL to differentiate type I collagen (birefringence yellowish orange to orange and red) of type III collagen (green to yellow-green birefringence) in neoplastic stroma. Results. The results show a different profile of birefringence of collagen fibers of the stroma of benign and malignant tumors analyzed. In the stroma of malignant tumors predominance of collagen I was observed, compared with the benign tumor predominantly investigated collagen III. Conclusions. The staining method Picrosirius and observation network with polarized light microscopy is a simple, safe, specific, sensitive and inexpensive technique that allows us to analyze the collagens of salivary gland tumors. Collagen is one of the components of the tumor stroma; latter plays a critical role during carcinogenesis and its quantity and quality differs from one tumor to another in relation to prognosis and tumor progression. [ABSTRACT FROM AUTHOR]

BACKGROUND: Giant cell fibroma (GCF) is a distinct type of fibroma with characteristic large, stellate mononuclear or multinucleated giant fibroblasts; the stroma of GCF is relatively unexplored. The Picrosirus red polarizing microscopy technique is used to characterize the collagen fibers. The aim of this study was to evaluate the staining properties of collagen fibers in GCF and to correlate it with fibroma using Picrosirius red under the polarizing microscope and van Gieson under bright field microscope. METHODS: In the present study van Gieson and picrosirius red stained slides of 7 cases each of GCF and fibroma were compared for the staining properties of collagen. Using picrosirus red polarizing microscopy; colors noted in fibroma included yellow, yellowish-orange and green, whereas the GCF showed predominantly yellow and orange colors. In Van Gieson stained sections it was observed that the collagen in GCF was densely packed and arranged perpendicular to the epithelium while the collagen in fibroma was loosely packed and arranged parallel to the epithelium. CONCLUSION: Observable differences in the stroma of the collagen of GCF and fibroma were noted. Collagen in GCF was more mature and dense. The Picrosirius red polarizing technique can be used to assess the collagen in GCF. [ABSTRACT FROM AUTHOR]

Introduction: Fibro-osseous lesions, along with few reactive lesions of the jaws exhibit an overlapping histo-morphologic spectrum with respect to the nature of calcifications and stromal components. This causes difficulty in assessing the origin, pathogenesis and diagnosis of these lesions. Aim: The present study analyses the mineralized components, cellularity, stromal density and stromal composition (nature of collagen, presence of elastic and oxytalan fibres) in cases of ossifying fibroma (OF), fibrous dysplasia (FD) and peripheral ossifying fibroma (POF). Materials and Methods: The study included a histochemical evaluation of six cases each of FD, OF and POF. Five consecutive sections of each case were stained with hematoxylin and eosin, picrosirius red (to assess maturation of fibres in polarizing light), van Gieson (for area fraction and collagen density) and aldehyde fuchsin (for elastic and oxytalan fibres) respectively. Results: Significantly higher amounts of mature bone were seen in FD while cementicles having microlamellar pattern were predominant in OF and POF (p < 0.001). Area fraction, collagen density and immature stromal fibre content was higher in POF followed by FD and OF (p= 0.039). Oxytalan and elastic fibres were absent in FD. Conclusion: Higher cellularity of the stroma in OF was indicative of its neoplastic behaviour. Higher composition of oxytalan and elastic fibres in OF and POF supports their periodontal ligament origin. FD was distinct with more mature fibres in a lamellated bone and absence of oxytalan fibres. [ABSTRACT FROM AUTHOR]

Aging is accompanied by morphological and mechanical changes to the intramuscular connective tissue (IMCT) of skeletal muscles, but whether physical exercise can influence these changes is debated. We investigated the effects of aging and exercise with high or low resistance on composition and mechanical properties of the IMCT, including direct measurements on isolated IMCT which has rarely been reported. Middle-aged (11 months, n = 24) and old (22 months, n = 18) C57BL/6 mice completed either high (HR) or low (LR) resistance voluntary wheel running or were sedentary (SED) for 10 weeks. Passive mechanical properties of the intact soleus and plantaris muscles and the isolated IMCT of the plantaris muscle were measured in vitro. IMCT thickness was measured on picrosirius red stained cross sections of the gastrocnemius and soleus muscle and for the gastrocnemius hydroxyproline content was quantified biochemically and advanced glycation end-products (AGEs) estimated by fluorometry. Mechanical stiffness, IMCT content and total AGEs were all elevated with aging in agreement with previous findings but were largely unaffected by training. Conclusion: IMCT accumulated with aging with a proportional increase in mechanical stiffness, but even the relatively high exercise volume achieved with voluntary wheel-running with or without resistance did not significantly influence these changes. • Isolated intramuscular connective tissue is stiffer in old plantaris muscle. • Voluntary wheel-running does not affect intramuscular connective tissue. • Endomysium thickness does not differ between fast and slow muscle fibers. [ABSTRACT FROM AUTHOR]

Abstract: We conducted a comprehensive study of all lipomatous tumors of the oral mucosa (1996–2008) accessioned at the Department of Oral Pathology, Tel Aviv University, collected demographic data and analyzed multiple histomorphological features. Furthermore, we examined the immunostaining of aP2 (adipocyte lipid binding protein) and the polarization colors of picrosirius red (PSR)-stained collagen fibers in order to test their potential in differentiating between benign and malignant tumors. All cases were immunohistochemically stained with aP2 antibody; only tumors with considerable collagenous stroma were selected for the PSR staining. A total of 77 tumors were included in the study, 91% benign and 9% malignant. Fibrolipoma (37.7%) and lipoma (36.4%) were the most frequent tumors. Atypical lipomatous tumor (ALT) was the only type of malignancy. The most common location for the benign tumors was the buccal mucosa and for ALT, the tongue. Histomorphological features characteristic of malignant tumors were occasionally present also in the benign entities. Expression of aP2 was similar in all tumors, while the polarization colors of the PSR-stained collagen fibers differed significantly between ALT and benign tumors (p <0.05). Benign and malignant tumors occasionally show overlapping histomorphological features that require a meticulous examination. PSR staining with polarization microscopy could aid in differentiating malignant from benign tumors in equivocal cases. [Copyright &y& Elsevier]

Background: Fibrosis is associated with various cardiac pathologies and dysfunction. Current quantification methods are time-consuming and laborious. We describe a semi-automated quantification technique for myocardial fibrosis and validated this using traditional methods. Methods: Pulmonary Hypertension (PH) was induced in adult Wistar rats by subcutaneous monocrotaline (MCT) injection(40 mg/kg). Cryosections of myocardial tissue (5 μm) of PH rats ( n = 9) and controls ( n = 9) were stained using Picrosirius red and scanned with a digital microscopic MIRAX slide scanner. From these sections 21 images were taken randomly of each heart. Using ImageJ software a macro for automated image analysis of the amount of fibrosis was developed. For comparison, fibrosis was quantified using traditional polarisation microscopy. Both methods were correlated and validated against stereology as the gold standard. Furthermore, the method was tested in paraffin-embedded human tissues. Results: Automated analysis showed a significant increase of fibrosis in PH hearts vs. control. Automated analysis correlated with traditional polarisation and stereology analysis ( r = 0.92 and r = 0.95 respectively). In human heart, lungs, kidney, and liver, a similar correlation with stereology ( r = 0.91) was observed. Time required for automated analysis was 22% and 33% of the time needed for stereology and polarisation analysis respectively. Conclusion: Automated quantification of fibrosis is feasible, objective, and time-efficient. [ABSTRACT FROM AUTHOR]

OBJECTIVES: The present study was undertaken to detect and compare the pattern of collagen fibers in odontogenic cysts and also to find out if this methodology could be used to predict the aggressive nature of odontogenic cysts by comparing with the odontogenic tumors. MATERIALS AND METHODS: The collagen in the wall of 11 odontogenic keratocysts, 14 dentigerous cysts and 14 radicular cysts was studied histochemically by staining sections with picrosirius red and examining under polarizing microscope. This was compared to 10 cases of odontogenic tumors using Z test of proportion at 1% and 5%. RESULTS: In dentigerous cysts, odontogenic keratocysts and odontogenic tumors, the predominant color of collagen fibers birefringence was found to be orangish red, whereas in radicular cysts the collagen fiber was of green color. CONCLUSIONS: Similar birefringence pattern of collagen fibers between dentigerous cysts, odontogenic keratocysts and odontogenic tumors may indicate that these lesions have a common histogenesis with a broad spectrum of biological behavior and belong to the same group, i.e., are developmental in origin. Different patterns of radicular cysts suggest different biological behavior and a positive role of inflammation on polarization color of collagen fibers. [ABSTRACT FROM AUTHOR]

J Oral Pathol Med (2011) : 257-262 The purpose of this study was to evaluate the nature of collagen in the connective tissue walls of odontogenic cysts, like the odontogenic keratocyst (OKC), dentigerous cyst and radicular cyst using picrosirius red stained sections. Furthermore, it was intended to assess if the capsular connective tissue can affect the nature of overlying epithelium, thus emphasizing the role of epithelial-mesenchymal interactions in biological behaviour of the cysts. The material for the study included 51 formalin-fixed paraffin-embedded tissue blocks (15 odontogenic keratocyst, 15 dentigerous cysts, 15 radicular cysts and four normal mucosa and two dental follicular tissue as controls), retrieved from the Department of Oral Pathology and Microbiology, MCODS, Manipal. Tissue blocks were sectioned at 5-μm thickness, stained with picrosirius red stain and observed with polarization and light microscopy. Few sections of OKC and dentigerous cyst exhibited greenish-yellow birefringence in sub-epithelial region, whereas others showed a yellowish-orange birefringence under polarization microscopy. Most radicular cysts had yellowish-orange to orange birefringence. Shift in colour in case OKC and dentigerous cyst was attributed to the presence of inflammation in those sections. These regions also exhibited either a change in phenotype or thickness of overlying epithelium. This technique can be used to study the nature of collagen fibres in odontogenic cyst walls. Further studies with an increased sample size and using various epithelial and mesenchymal markers and ssDNA antibodies should be carried out to confirm the effect of epithelial-mesenchymal interactions on the nature of epithelium of odontogenic cysts. [ABSTRACT FROM AUTHOR]

Abstract: Cardiac ventricular trabeculae are widely used in the study of cardiac muscle function, primarily because their myocytes are axially-aligned. However, their collagen content has not been rigorously determined. In particular, it is unknown whether the content of collagen differs between specimens originating from the left (LV) and right (RV) ventricles and whether, indeed, either corresponds to the collagen content of the ventricular walls themselves. In order to redress this deficit of knowledge, we have used the techniques of fluorescence confocal microscopy and environmental scanning electron microscopy to quantify the proportion of perimysial collagen comprising the cross-sectional area of trabeculae carneae. In trabeculae from both the RV and LV of adult rat hearts, collagen may occupy as little as 1% or as much as 100% of the cross-section. For specimens of dimensions typically used experimentally, there was no difference in average collagen content (6.03±5.14%, n =33) of preparations from the two ventricles. [ABSTRACT FROM AUTHOR]

Background: Fibrosis is associated with various cardiac pathologies and dysfunction. Current quantification methods are time-consuming and laborious. We describe a semi-automated quantification technique for myocardial fibrosis and validated this using traditional methods. Methods: Pulmonary Hypertension (PH) was induced in adult Wistar rats by subcutaneous monocrotaline (MCT) injection (40 mg/kg). Cryosections of myocardial tissue (5 μm) of PH rats (n=9) and controls (n=9) were stained using Picrosirius red and scanned with a digital microscopic Mirax slide scanner. From these sections 21 images were taken randomly of each heart. Using ImageJ software a macro for automated image analysis of the amount of fibrosis was developed. For comparison, fibrosis was quantified using traditional polarisation microscopy. Both methods were correlated and validated against stereology as the gold standard. Furthermore, the method was tested in paraffin-embedded human tissues. Results: Automated analysis showed a significant increase of fibrosis in PH hearts vs. control. Automated analysis correlated with traditional polarisation and stereology analysis (r2=0.92 and r2=0.95, respectively). In human heart, lungs, kidney and liver, a similar correlation with stereology (r2=0.91) was observed. Time required for automated analysis was 22 and 33% of the time needed for stereology and polarisation analysis, respectively. Conclusion: Automated quantification of fibrosis is feasible, objective and time-efficient. [ABSTRACT FROM AUTHOR]

Abstract: Collagen-based three-dimensional (3D) in vitro models that recapitulate the structural and functional context of normal and malignant tissues provide a relevant surrogate to animal models in the study of developmental and carcinogenic processes. Human breast epithelial MCF10A cells embedded in a collagen gel formed both acinar and tubular structures only when the gel was detached (floating) from the cell culture plate''s well, and allowed to be contracted by the cells. Epithelial phenotype depended upon the time and the location within the gel, as ducts formed exclusively on the upper layer of the gel while ductal branching occurred earlier in the central area of the gel, and gradually progressed toward the periphery. The addition of fibroblasts accelerated tubulogenesis. MCF10A cells facilitated the organization of thick collagen fibers packed into large bundles at the tip of the ducts and parallel to the direction of ductal elongation. In gels that were not detached from the well''s wall, MCF10A cells organized in monolayer and collagen fibers were aligned along the axis of outstretched sprouts stemming from those cellular aggregates. Partial gel release induced uniaxial tubulogenesis associated with orderly aligned collagen fibers. These results suggest that proper collagen organization is necessary for epithelial morphogenesis to occur, and that biomechanical interactions between fibers and cells mediated duct formation, elongation and branching. [Copyright &y& Elsevier]

Aims: To determine the effect of bilateral pelvic neurectomy (BPN) on cervical ripening in pregnant rats by measuring cervical extensibility and changes in collagen cross-linkages. Methods: Timed-pregnant rats were randomly laparotomized on days 9 or 10 of gestation and the pelvic nerves were exposed and either bilaterally transected, or left intact in sham control animals. The rats were sacrificed on day 18 and the uterine cervices obtained. Cervical ripening was assessed by cervical resistance-to-stretch, light-induced autofluorescence (LIF) of cross-linked collagen, and collagen changes analyzed by picrosirius polarization microscopy. Results: Measurements of extensibility and collagen cross-linkages indicated that after BPN the cervix was significantly more ripened than the cervix from sham control animals. Conclusion: BPN stimulates cervical ripening instead of inhibiting this event as previously proposed. Further studies in this area could be critical for developing treatments for dystocia, preterm labor, and cervical insufficiency. [ABSTRACT FROM AUTHOR]

Histochoice™® is a proprietary nontoxic, non-cross-linking fixative designed by the manufacturer to replace formaldehyde based fixation protocols. We compared Histochoice and formalin fixation for several cartilaginous tissues including, articular and growth plate cartilage, meniscus and intervertebral disc. The tissues were stained with general histology stains including toluidine blue for tissue proteoglycans, picrosirius red to evaluate collagenous organization, and hematoxylin and eosin to assess cell morphology. The chondroitin sulfate and heparin sulfate substituted proteoglycans aggrecan and perlecan were also immunolocalized in some of the tissues to provide a comparison. Histochoice did not fix deep into the tissue blocks resulting in focal loss of aggrecan and other matrix components from the more central regions of the blocks. This was evident in toluidine blue stained sections of immature tibial articular cartilage where loss of glycosaminoglycan was significant in Histochoice™® fixed tissues. Histochoice fixation worked well, however, in the aggrecan and perlecan immunohistology applications where its non-cross-linking traits were conducive to epitope retrieval and identification by primary antibodies to extracellular matrix components. [ABSTRACT FROM AUTHOR]

Background/Aims: The urinary concentration of the monocyte chemoattractant protein-1 (uMCP-1) chemokine is increased in several proteinuric and/or inflammatory renal diseases. In the present study, we evaluated the association between uMCP-1 and renal function, proteinuria, glomerular and interstitial macrophage infiltration, and renal fibrosis in patients with primary and secondary glomerulopathies diagnosed by renal biopsy. Methods: Thirty-seven patients aged 32.6 ± 7.7 years were studied. uMCP-1 was determined by ELISA. Renal macrophage expression (CD68 positive cells) is reported as number of macrophages/104 μm2 of the cortical tubulointerstitial (TI) area or of glomerular capillary tuft area. Cortical interstitial fibrosis was quantitated by PicroSirius red staining under polarized light by a computerized manner. Results: The uMCP-1 ratio (pg/ml/urinary creatinine mg/ml) was positively correlated (Spearman coefficient) with proteinuria (r = 0.4629; p < 0.005) and number of macrophages in the cortical TI area (r = 0.64; p = 0.0005), and negatively correlated with creatinine clearance (r = –0.4877; p < 0.001). The uMCP-1 ratio was not significantly correlated with number of macrophages/glomerular capillary tuft area (r = 0.27; p = 0.19) or with percent cortical interstitial fibrosis (r = 0.08; p = 0.62). Conclusions: The uMCP-1 excretion is a biomarker of the inflammatory activity of the TI area, and does not reflect chronic interstitial damage. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Denervation or inactivity is known to decrease the mass and alter the phenotype of muscle and the mechanics of tendon. It has been proposed that a shift in the collagen of the extracellular matrix (ECM) of the muscle, increasing type Ill and decreasing type I collagen, may be partially responsible for the observed changes. We directly investigated this hypothesis using quantitative real-time PCR on muscles and tendons that had been denervated for 5 wk. Five weeks of denervation resulted in a 2.91-fold increase in collagen concentration but no change in the content of collagen in the muscle, whereas in the tendon there was no change in either the concentration or content of collagen. The expression of collagen I, collagen III, and lysyl oxidase mRNA in the ECM of muscle decreased (76 ± 1.6%, 73 ± 2.3%, and 83 ± 3.2%, respectively) after 5 wk of denervation. Staining with picrosirius red con- firmed the earlier observation of a change in staining color from red to green. Taken with the observed equivalent decreases in collagen I and III mRNA, this suggests that there was a change in orientation of the ECM of muscle becoming more aligned with the axis of the muscle fibers and no change in collagen type. The change in collagen orientation may serve to protect the smaller muscle fibers from damage by increasing the stiffness of the ECM and may partly explain why the region of the tendon closest to the muscle becomes stiffer after inactivity. [ABSTRACT FROM AUTHOR]

Summary: Salivary gland neoplasms – pleomorphic adenoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma – share a common histogenetic trait, but differ markedly in their biological properties. The objective of the study was to assess the polarization colors of picrosirius red-stained stromal collagen fibers in these salivary gland neoplasms to evaluate their possible role in the histopathogenesis of the tumors and to evaluate the potential usefulness of this approach as a diagnostic tool. Ten cases of each tumor type and 10 cases of mucous extravasation phenomenon (control) were examined using picrosirius red staining and polarizing microscopy. In each case, at least 50 thin (∼0.8μm) and 50 thick (1.6–2.4μm) collagen fibers were counted and classified as green-yellow or yellow-orange, the mean percentage was calculated and statistical differences analyzed by one-way ANOVA. Results showed a similar thin fiber distribution in all tumor types and controls (82–88% green-yellow, 12–18% yellow-orange, p>0.05). Thick fibers showed a different distribution in polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma (approximately 50% green-yellow) compared to pleomorphic adenoma and mucous extravasation phenomenon (approximately 13% green-yellow) (p=0.001). Thick fiber distribution was similar in polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma (p>0.05). We conclude that with picrosirius red staining and polarizing microscopy, stromal collagen fibers differ significantly in pleomorphic adenoma from those in polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma, but not from mucous extravasation phenomenon. Similarity between polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma may indicate that these tumor types represent a single entity with a broad spectrum of biological behavior. [Copyright &y& Elsevier]

Ventricular myocardium has a complex three-dimensional structure which has previously been inferred from two-dimensional images. We describe a technique for imaging the 3D organization of myocytes in conjunction with the collagen network in extended blocks of myocardium. Rat hearts were fixed with Bouin's solution and perfusion-stained with picrosirius red. Transmural blocks from the left ventricular free wall were embedded in Agar 100 resin and mounted securely in an ultramicrotome chuck. Confocal fluorescence laser scanning microscopy was used to obtain 3D images to a depth of 60 μm in a contiguous mosaic across the surface. Approximately 50 μm was then cut off the surface of the block with an ultramicrotome. This sequence was repeated 20 times. Images were assembled and registered in 3D to form an extended volume 3800 × 800 × 800 μm3 spanning the heart wall from epicardium to endocardium. Examples are given of how digital reslicing and volume rendering methods can be applied to the resulting dataset to provide quantitative structural information about the 3D organization of myocytes, extracellular collagen matrix and blood vessel network of the heart. [ABSTRACT FROM AUTHOR]

Current understanding of fibrosis remains incomplete despite the increasing burden of related diseases. Preclinical models are used to dissect the pathogenesis and dynamics of fibrosis, and to evaluate anti-fibrotic therapies. These studies require objective and accurate measurements of fibrosis. Existing histological quantification methods are operator-dependent, organ-specific, and/or need advanced equipment. Therefore, we developed a robust, minimally operator-dependent, and tissue-transposable digital method for fibrosis quantification. The proposed method involves a novel algorithm for more specific and more sensitive detection of collagen fibers stained by picrosirius red (PSR), a computer-assisted segmentation of histological structures, and a new automated morphological classification of fibers according to their compactness. The new algorithm proved more accurate than classical filtering using principal color component (red-green-blue; RGB) for PSR detection. We applied this new method on established mouse models of liver, lung, and kidney fibrosis and demonstrated its validity by evidencing topological collagen accumulation in relevant histological compartments. Our data also showed an overall accumulation of compact fibers concomitant with worsening fibrosis and evidenced topological changes in fiber compactness proper to each model. In conclusion, we describe here a robust digital method for fibrosis analysis allowing accurate quantification, pattern recognition, and multi-organ comparisons useful to understand fibrosis dynamics. [ABSTRACT FROM AUTHOR]

The collagen in the walls of 15 keratocysts was studied histochemically by staining sections with picrosirius red and examining them with polarizing microscopy. This was compared to 15 cases of dentigerous cyst and 15 cases of radicular cyst. Polarization colours of the collagen fibres were recorded according to their width. No differences were found between the polarization colours of thin fibres (< 0.8 μm) in all three lesions; the polarization colours of thick fibres (1.6-2.4 μm) in keratocysts were significantly more greenish-yellow when compared with those of dentigerous cysts and radicular cysts. The staining of the collagen fibres in the keratocysts in similar to that reported in odontogenic neoplasms, which suggests that the stroma of keratocysts could be regarded not just as a structural support of the cyst wall, but as playing a part in the neoplastic behavior of the cyst. [ABSTRACT FROM AUTHOR]

The central odontogenic fibroma (COF) is an uncommon odontogenic tumor in which the microscopic appearance is similar to that of the hyperplastic dental follicle (HDF). Distinction between these lesions is based mainly on their clinical and radiologic features. The collagen of 5 cases of COF and 13 cases of HDF was studied histochemically by staining sections with Picrosirius red and examining them by polarizing microscopy. Polarization colors of the collagen fibers of the lesions were recorded according to their width. While no differences were seen between the polarization colors of thin fibers (< 0.8 mm) in both lesions, the polarization colors of thick fibers (1.6 2 mm) of COF were significantly more green and greenish-yellow as compared with those of HDF. These findings suggest that the Picrosirius red polarization method can be used as a diagnostic tool to differentiate between COF and HDF. [ABSTRACT FROM AUTHOR]