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Purpose Histological analysis of human carotid atherosclerotic plaques is critical in understanding atherosclerosis biology and developing effective plaque prevention and treatment for ischemic stroke. However, the histological staining process is laborious, tedious, variable, and destructive to the highly valuable atheroma tissue obtained from patients. Procedures We proposed a deep learning-based method to simultaneously transfer bright-field microscopic images of unlabeled tissue sections into equivalent multiple sections of the same samples that are virtually stained. Using a pix2pix model, we trained a generative adversarial neural network to achieve image-to-images translation of multiple stains, including hematoxylin and eosin (H&E), picrosirius red (PSR), and Verhoeff van Gieson (EVG) stains. Results The quantification of evaluation metrics indicated that the proposed approach achieved the best performance in comparison with other state-of-the-art methods. Further blind evaluation by board-certified pathologists demonstrated that the multiple virtual stains have high consistency with standard histological stains. The proposed approach also indicated that the generated histopathological features of atherosclerotic plaques, such as the necrotic core, neovascularization, cholesterol crystals, collagen, and elastic fibers, are optimally matched with those of standard histological stains. Conclusions The proposed approach allows for the virtual staining of unlabeled human carotid plaque tissue images with multiple types of stains. In addition, it identifies the histopathological features of atherosclerotic plaques in the same tissue sample, which could facilitate the development of personalized prevention and other interventional treatments for carotid atherosclerosis.

Aim Odontogenic tumors (OTs) and bone lesions of the oral cavity present diverse histological features and varying clinical behavior that makes predicting their biologic behavior difficult. The research undertaken in the current study aims to predict the biological behavior of oral hyalinizing odontogenic and bone lesions (OHO-BL) for the first time by employing four differential stains with clinicopathologic correlation. Materials and methods The study was performed on retrospectively diagnosed formalin-fixed paraffin-embedded cases of OTs (n = 53) and bone lesions (n = 10). The severity of hyalinization (SOH) was assessed from stained tissue sections. Polarizing microscopy was used to analyze hyalinization in tissues stained with differential special stains, namely periodic acid-Schiff (PAS), Safranin-O, Alcian Blue, and Picrosirius red. SOH was also analyzed for possible correlation with recurrence and clinicopathologic correlation in OHO-BL. Results Intense staining was observed with PAS, Alcian Blue, and Safranin-O in OTs with increased SOH with a statistical significance. Polarizing greenish yellow color correlated significantly with the recurrence potential of the OT group. Recurrence in individual lesions of the OT group showed a statistically significant association with SOH. Such individual correlation was not observed in bone diseases. Conclusion PAS, Alcian Blue, Safranin-O, and Picrosirius red are reliable stains to assess hyalinization in OHO-BL. Picrosirius red-polarizing microscopy is a dependable tool for identifying recurrent odontogenic lesions. Clinical significance SOH can be considered a histological predictor of aggressive biologic behavior in oral hyalinizing odontogenic lesions that can enable the surgeon to arrive at an appropriate management protocol.

The excellent adaptability of Callithrix jacchus to life in captivity presents advantages in comparison to other nonhuman primates that are used in experimental models for biomedical research, which explains the increasing scientific interest in investigating the anatomical characteristics of this species. Owing to the relative scarcity of publications on the descriptive morphology of the heart of C. jacchus, the aim of this study was to quantify the presence of collagen in the left and right ventricular myocardium using modified picrosirius red and acid fuchsine colorimetric assays. The myocardium of the right ventricle presented a higher percentage of collagen than that of the left ventricle. No sex-related differences were observed between the groups. Interestingly, the absolute values of collagen were different depending on the method used for quantification (modified picrosirius red vs. acid fuchsine). The level of collagen quantification observed in the ventricular myocardium of C. jacchus was similar to that seen in other nonhuman primates traditionally used in experimental models of cardiac diseases.

Background: Collagen fibers are the main element of gingival connective tissue and contribute a leading role in the preservation of structural integrity and tissue function. Hence, its degradation is regarded as the main marker of periodontal disease progression. Aim: The aim of this study is to analyze and compare collagen fibers, their birefringence pattern in healthy and in diseased gingival tissues stained using picrosirius red stain (PRS) and the polarizing microscope. Materials and Methods: A total of 90 participants screened were divided into the control group (healthy gingiva) and experimental group (moderate periodontitis and severe periodontitis) based on the clinical parameters. Gingival tissue sections were stained with PRS and observed under the polarized microscope to assess the type of collagen fibers in healthy and diseased gingival tissue. Statistical analysis was performed using the one-way ANOVA and Tukey multiple comparison test. Results: The healthy group revealed well-packed collagen in a parallel pattern with a strong birefringence, whereas in severe periodontitis group showed loosely packed collagen fibers in a haphazard pattern suggestive of severe destruction of the extracellular matrix. The moderate periodontitis group had a blended mixture of thick and thin fibers. Conclusions: Collagen fibers showed birefringence property when stained with PRS that helps in a better understanding of normal and pathological conditions.

To examine healing adaptations over 17 weeks post Achilles tendon (AT) rupture in the injured region (IR) compared to an uninjured region (UIR) of the AT. Twenty-four rats were subjected to a complete right-sided AT rupture, while the left side served as a control. ATs were harvested at 1, 2, 8 and 17 weeks post-rupture and stained with antibodies specific to Collagen type I (Col I) and II (Col II) as well as Alcian Blue and Picrosirius Red staining techniques. Histopathological changes, proteoglycan content, collagen alignment and immunoexpression were assessed. Both regions examined, IR and UIR, exhibited over weeks 1–17 similar healing adaptations of increasing collagen alignment, decreasing Col I immunoexpression, as well as increasing proteoglycan content and Col II occurrence. Increased proteoglycan content was found already at week 2 in the UIR, while it first increased at week 8 in the IR. The area positive to Col II was increased compared to controls at week 8 in the UIR, whereas it first raised at week 17 in the IR. Collagen disorganization successively declined to reach control levels at week 17 in the UIR, but was still higher in the IR. This study demonstrated that uninjured areas of the AT remote from the rupture site also undergo pronounced remodeling, although with time-span differences relative to injured AT portions. These changes including the pathologic heterotopic mineralization and chondrogenic differentiation observed in both regions may have implications in the choice of rehabilitation regimes in order to prevent secondary rupture.

Picrosirius red stain has been increasingly used for collagen studies in dental and medical research. Sirius red is an acidic dye which binds specifically to collagen and distinguishes type I and type III collagen fibres. This review depicts earlier uses of picric acid, preparation of the picrosirus red stain, mechanism of action, hazards and safety protocols of handling picric acid in laboratory. Keywords: Birefringence, Collagen fibres, Picrosirius red stain, Polarized microscopy, Picric acid.

Osteons are the structure and the base foundation of the human skeletal system. This cylindrical structure contains blood vessels and nerves that supply the bone matrix in humans. To study bone remodeling and bone diseases, mini pigs and rats have mainly been effective models for humans. This study was conducted to examine mouse bone structures in comparison to human, mini pig, and rat bone structures. This addresses the Three Rs principle of clinical testing on animals and proves that mice should be used as models for humans instead of mini pigs and rats. Many scientists prefer not to use mice as models for studying human bone diseases because it has been suggested that their skeletal systems are morphologically and physiologically different–as seen through aging effects. Because mice are easier to produce and can grow at a faster rate, they are more cost and time efficient to use in labs compared to rats and mini pigs. Aniline blue, Ploton silver, picrosirius red stains and TRAP and ALP cellular assays were conducted to analyze bone structures to compare with humans, mini pigs, and rats. Our data did support the hypothesis as explicit similarities between mice bone and other samples such as rats, mini pig, and human was deduced. This study concluded that there were little limitations present by using murine bone as samples for human when studying bone diseases.

Aim of the study Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation and expansion of cysts within the kidney. Caroli syndrome (CS) is characterized by cystic saccular dilatation of intrahepatic ducts. Kidney and liver images from a model of ADPKD-CS were evaluated to characterize remodeling of the cystically dilated intrahepatic duct wall and the renal cyst wall. Material and methods Archival digitized images from Masson's trichrome-stained renal and Picrosirius red (PSR)-stained renal and hepatic cross-sections were sourced from the PCK rat model of ADPKD-CS, and age-matched Sprague-Dawley rats (wild-type). Cross-sectional areas and wall thicknesses of renal cysts and intrahepatic ducts were measured. Circularly polarized PSR microscopy was utilized to observe accumulation of collagen and identify its subtype. Results In the PCK rat model of ADPKD-CS, renal cysts were relatively thin-walled in comparison to intrahepatic ducts with renal cyst cross-sectional area to wall ratio 47-fold greater than the corresponding ratio in intrahepatic ducts. Increasing intrahepatic duct cross-sectional area was accompanied by a rapid and steep rise in wall thickness. There was a weak but significant direct correlation (r = 0.49, p = 0.037) between renal cyst cross-sectional area and wall thickness. Circularly polarized Picrosirius red microscopy revealed collagen I accumulation within the walls of dilated intrahepatic ducts but not renal cysts. Conclusions These data suggest that unlike renal cysts, cystically dilated intrahepatic ducts undergo collagen-driven wall remodeling in the PCK rat.

Objectives: To explore the polarizing color profile in connective tissue stroma of Picro-Sirius Red (PSR) stained sections of benign and malignant salivary gland tumors (SGT) and to assess the possible utility of this method as a diagnostic tool. Materials and Methods: A total number of 60 Paraffin blocks of benign (30 cases) and malignant (30 cases) salivary gland tumors were selected from the archives of our institute and regional cancer hospital. 30 cases of Mucous Extravasation Phenomenon (MEP) were selected as control. Salivary gland tumors and control were stained with PSR stain and examined under polarizing microscope. We counted 50 thin and 50 thick collagen fibers in each section of benign and malignant SGT and were categorized as Greenish Yellow (GY) or Yellowish Orange (YO). Statistical analysis was performed by using oneway ANOVA and Paired t-test. Results: We found similar presentation of polarizing color arrangement for thin collagen fibers (80-85% GY and 15-20% YO) in both benign and malignant SGT and MEP. In malignant SGT, thick collagen fibers exhibited different polarizing color (80% GY) than benign SGT and MEP (12-14% GY). Polarizing color profile of collagen in malignant SGT displayed a different presentation as compared to benign SGT and control (MEP) and differences were statistically significant(p

Purpose: Dipotassium glycyrrhizinate (DPG) has anti-inflammatory properties, besides promoting the regeneration of skeletal muscle. However, it has not been reported on skin wound healing/regeneration. This research aimed to characterize the effects of DPG in the treatment of excisional wounds by second intention. Methods: Male adults (n=10) and elderly (n=10) Wistar rats were used. Two circular wounds were excised on the dorsal skin. The excised normal skins were considered adult (GAN) and elderly (GIN) naïve. For seven days, 2% DPG was applied on the proximal excision: treated adult (GADPG) and elderly (GIDPG), whereas distal excisions were untreated adult (GANT) and elderly (GINT). Wound healing areas were daily measured and removed for morphological analyses after the 14th and the 21st postoperative day. Slides were stained with hematoxylin-eosin, Masson’s trichrome, and picrosirius red. Results: Histological analysis revealed intact (GAN/GIN) and regenerated(GANT/GINT/GADPG/GIDPG) skins. No differences of wounds’ size were found among treated groups. Epidermis was thicker after 14 days and thinner after 21 days of DPG administration. Higher collagen I density was found in GIDPG (14th day) and GADPG (21st day). Conclusions: DPG induced woundhealing/skin regeneration, with collagen I, being more effective in the first 14 days after injury.

Introduction Intervertebral disc (IVD) degeneration is often associated with low back pain and radiating leg pain. The purpose of this study is to develop a reproducible and standardized preclinical model of painful lumbar IVD degeneration by evaluation of structural and behavioral changes in response to IVD injury with increasing needle sizes. This model can be used to develop new therapies for IVD degeneration. Methods Forty‐five female Sprague Dawley rats underwent anterior lumbar disc needle puncture at levels L4‐5 and L5‐6 under fluoroscopic guidance. Animals were randomly assigned to four different experimental groups: needle sizes of 18 Gauge (G), 21G, 23G, and sham control. To monitor the progression of IVD degeneration and pain, the following methods were employed: μMRI, qRT‐PCR, histology, and biobehavioral analysis. Results T1‐ and T2‐weighted μMRI analysis showed a correlation between the degree of IVD degeneration and needle diameter, with the most severe degeneration in the 18G group. mRNA expression of markers for IVD degeneration markers were dysregulated in the 18G and 21G groups, while pro‐nociceptive markers were increased in the 18G group only. Hematoxylin and Eosin (H&E) and Alcian Blue/Picrosirius Red staining confirmed the most pronounced IVD degeneration in the 18G group. Randall‐Selitto and von Frey tests showed increased hindpaw sensitivity in the 18G group. Conclusion Our findings demonstrate that anterior disc injury with an 18G needle creates severe IVD degeneration and mechanical hypersensitivity, while the 21G needle results in moderate degeneration with no increased pain sensitivity. Therefore, needle sizes should be selected depending on the desired phenotype for the pre‐clinical model., Study Design Overview This study establishes and validates a rat model for lumbar IVD degeneration that is reflected in painful behavior. Based on our findings we conclude that annular disc injury with a 18G needle creates severe, painful IVD degeneration, while 21G needle injury results in moderate degeneration.

Background and objective This study was aimed to evaluate the collagen fibers qualitatively and its correlation with microvascular density in various grades of oral submucous fibrosis (OSMF). Material and methods The present study comprised of total 40 cases of oral submucous fibrosis. Picrosirius red staining was done on all the specimens' sections. They were analyzed for the colour and orientation of collagen fibers. Morphometric measurements were done using image analysis on immunohistochemical stained sections for Factor VIII-related antigen and analyzed for microvascular density. Results Picrosirius red polarizing microscopy results revealed that there was a shift in the colour of collagen fibers from greenish yellow to orange red and red colour as the severity of the oral submucous fibrosis increased. The collagen fibers showed mixed orientation in early oral submucous fibrosis and parallel orientation in advanced oral submucous fibrosis. There was a significant decrease in microvascular density from early to advanced oral submucous fibrosis. Conclusion The change in the colours and orientation of collagen fibers in early and advanced oral submucous fibrosis could be attributed to the fibre thickness, type of collagen, alignment and packing, cross-linking of the fibers and the section thickness. However, in advanced cases the vascularity is reduced which may predispose to epithelial atrophy and subsequent malignant changes.

Made available in DSpace on 2018-12-11T16:55:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-10-01 Objectives: To evaluate alterations from different therapies in muscular injury using the Fractal Dimension (FD) method. Methods: 35 animals were allocated in Control Group (C), Injury Control Group (IC), Injury Low Level Laser Therapy Group (ILT), Injury Platelet Rich Plasma Group (IP), and Injury LLLT and PRP Group (ILP). The animals suffered a stretch injury in gastrocnemius muscle and after that IP and ILP groups received PRP application. The ILT and ILP groups received daily LLLT applications for seven days. After seven days the animals were euthanized and the gastrocnemius muscle removed and frozen. The muscles were stained with Hematoxylin and Eosin (HE) and Picrosirius Red, for observation of the morphology of the injury and semi-quantitative and quantitative analysis through the Fractal Dimension (FD) method. Results: In the qualitative and semi-quantitative analysis, in relation to IC group, the ILT presented a reduction in rounded fibers and the IP in angular fibers. The ILP group demonstrated a reduction in both polymorphic fibers and inflammatory infiltrate. The FD of the muscles stained with HE was higher in the groups that suffered the injury when compared to the C group (p < 0.05); the FD of the collagen demonstrated no statistical difference between the groups. Conclusion: Both treatments were able to accelerate injury repair, and the association of both presented better results than the isolated applications. However, the FD method showed no sensitivity to differentiate the treatments, either in the histological aspects or the injury in collagen. Universidade Estadual de Campinas (UNICAMP) Programa de Pós-graduação em Ciências da Cirurgia Universidade de São Paulo (USP) Programa de Pós-graduação em Ciências Morfofuncionais Universidade Estadual Paulista (UNESP) Departamento de Fisioterapia Universidade Estadual Paulista (UNESP) Departamento de Fisioterapia

Introduction The analysis of frozen muscle biopsies has become a routine method in the evaluation of muscle structure in health and disease. However, the technique for frozen muscle specimens is not widely available in countries with limited medical facilities. The present study aimed to elucidate a reproducible formalin-fixed and paraffin-embedded (FFPE) method for this type of analysis in postmortem muscles. Methods Diaphragm muscle was obtained within 1 hour of sudden death. Diaphragm strips were washed in saline solution, fixed in 10% formalin, frozen at 4°C in a refrigerator, and stored for 24 hours. Then, the tissue samples were processed into paraffin-embedded blocks. Transversal sections were cut from each paraffin block and stained with hematoxylin and eosin, Picrosirius red, Verhoeff-Van Gieson, and Congo red for the qualitative analysis. Results Our analysis indicated a well-preserved muscle. Conclusion In summary, we demonstrate a simple technique for a reproducible FFPE method in postmortem muscle tissues.

The present study was aimed to evaluate and analyze orientation and birefringence pattern of collagen fibers histopathologically in different grades of OSMF using the special stain (Picrosirius red) under polarizing microscope and relate these factors with the severity of OSMF.45 confirmed cases of OSMF were taken from the archives. The selection of clinical cases was done according to classification by J.N Khanna and Andrade (1999). These cases were correlated histopathologically as per the criteria given by Pindborg J.J and Sirsat S.M (1966). The sections were stained with HE and Picrosirius red stains and assessment was done accordingly. 10 cases of normal mucosa were taken as control.In clinical grade I, 60% of the cases showed histopathological grade II, while 53.4% cases in clinical grade II correlated with histopathological grade III and 33.4% of clinical grade III cases showed histopathological grade IV. Upon application of Fisher's exact test a significant P value.01 was obtained suggesting no statistical significant correlation between these two parameters. However, the orientation pattern revealed a parallel orientation as OSMF advanced. Also as the severity of the disease increased, there was a gradual shift from green- greenish yellow colour of the fibres and a shift to orange red-red colour.Upon correlation of clinical grades with histopathological grades, no statistically significant difference was found. Birefringence pattern of collagen revealed similar findings as other studies in case of clinical grades but histopathological grades showed a contrast finding.

BACKGROUND Invasion of epithelial cells into the connective tissue brings about massive morphological and architectural changes in the underlying stroma. Myofibroblasts reorganize the stroma to facilitate the movement of tumor cells leading to metastasis. The aim of this study was to determine the number and pattern of distribution of myofibroblasts and the qualitative and quantitative change that they cause in the collagen present in the stroma in various grades of oral squamous cell carcinoma (OSCC). METHODS The study was divided into two groups with group I (test group, 65 cases) consisting of 29 cases of well-differentiated squamous cell carcinoma, 25 moderately differentiated SCC, and 11 poorly differentiated SCC, and group II (control group) consisting of 11 cases of normal mucosa. Sections from each sample were stained with anti-α-smooth muscle actin (α-SMA) antibodies, hematoxylin and eosin, and Picrosirius red. Several additional sections from each grade of OSCC were stained with Masson's trichrome to observe the changes in collagen. For the statistical analysis, Fisher's exact test, Tukey's post hoc honest significant difference test, ANOVA, and the chi-square test were used, and p < .05 was considered statistically significant. RESULTS As the tumor stage progressed, an increase in the intensity α-SMA expression was seen, and the network pattern dominated in more dedifferentiated carcinomas. The collagen fibers became thin, loosely packed, and haphazardly aligned with progressing cancer. Additionally, the mean area fraction decreased, and the fibers attained a greenish yellow hue and a weak birefringence when observed using polarizing light microscopy. CONCLUSIONS Myofibroblasts bring about numerous changes in collagen. As cancer progresses, there isincrease in pathological collagen,which enhances the movement of cells within the stroma.

BACKGROUND AND OBJECTIVE The aging of human skin includes intrinsic aging and photo-aging, which are characterized by a decrease in collagen and the deposition of abnormal elastic fibers. Intense pulsed light (IPL) sources are widely used in medicine to treat various cosmetic problems, including photo-damaged skin. Few studies have examined the microscopic changes produced by IPL. The objective of this study was to quantitatively evaluate the effects of IPL on collagen and elastic fibers in mice. MATERIALS AND METHODS Forty female BALB/c mice were divided into four subgroups. Group 1 was the control group (n = 10), and groups 2, 3, and 4 were treatment groups (n = 10 in each group). Group 2 received one treatment, group 3 received two treatments, and group 4 received three treatments every 2 weeks. Skin tissue was obtained from irradiated areas 24 hours after the last treatment in each mouse. Collagen fibers were identified using the picrosirius red method. Elastic fibers were marked by Weigert-oxone stain. All samples were analyzed and quantified by a light microscope using analyzer system images. RESULTS Group 4, which received three IPL treatments, showed significant quantitative increases in both collagen fibers (P

An increased understanding of the reparative process in fetal membrane following surgical techniques may be helpful to decrease the risks to mother and fetus and avoid adverse pregnancy outcomes. The present study discusses histological evaluation of the fetal membrane following open fetal surgery. Chorioamniotic membranes (n=10) were obtained following birth from pregnancies that underwent open fetal surgery for myelomeningocele. The collagen distribution was quantified using picrosirius-polarization method comparing the suture site with non-suture site. The differences between the collagen fiber percentages at the two sites was evaluated by the paired t-test with P

Collagen is a part of the extracellular matrix proteins that form an integral part of the connective tissue stroma. Collagen plays an important role in maintaining the structural and functional int...

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.

Oral squamous cell carcinoma (OSSC) is the 3rd most common form of malignancy in developed countries[1]. The tumour is composed of two basic components i e, the neoplastic cells and the reactive stroma. The stromal changes which indicate the propensity of tumour cells to infiltrate and metastasise can also be used as one of the prognostic indicator[4,5]. Use of Picrosirius red (PSR) stain along with circular polarising microscope is helpful to detect spread of malignancy and guides the treatment plan and assess prognosis[11]. The present study is aimed to analyse the various aspects of collagen fibres in relation with the different grades OSSC. 30 patients of OSCC of various grades and 10 cases of normal mucosa were evaluated and data analysed statistically using sigma scan pro image analysis software(SPSS). Observation: Thirty patients (male-19 and female -11) of OSCC of age group 47-60 years were analysed. There were 14 well differentiated (WDSCC), 12 moderately differentiated (MDSCC) and 4 poorly differentiated (PDSCC) cases. When examined under polarising microscope majority (85.7%) of WDSCC and 41.67% of MDSCC showed reddish orange (RO) collagen fibres whereas in PDSCC (50%) showed green yellow (GY) collagen fibres which is statistically significant (P=0.02).In WDSCC 57.14% and in MDSCC 50% had swirl(S) arrangement whereas in PDSCC 50% had crosss hatchet (CH) and bundle arrangement. This is again statistically significant (P=0.031).While examining the orientation of collagen fibres in WDSCC 71.43% and in MDSCC, 83.33% had parallel orientation whereas in PDSCC 75% showed non parallel orientation. This orientation is statistically significant (p.008). On comparing the nature of birefringence maximum cases of WDSCC (85.71%) and MDSCC (83.33%) revealed strong birefringence whereas in PDSCC 75% exhibited weak. (statistically significant p.001). Conclusion: PSR stain under polarising microscope is an adjunct to the routine staining for studying stromal changes. The colour, arrangement, orientation & birefringence changes reflect its gradual shift from WDSCC to PDSCC and may help as a diagnostic and prognostic tool in OSCC.

Background Actinic cheilitis is induced by chronic exposure to ultraviolet radiation and shows solar elastosis, a feature that has been associated with mast cell infiltrates. This study aimed to investigate the area of solar elastosis, collagen loss, and mast cell infiltrates in a series of actinic cheilitis. Methods We evaluated the epithelial dysplasia in 52 cases of actinic cheilitis and the solar elastosis with Weigert's resorcin-fuchsin. Collagen loss was evaluated with Picrosirius red, analyzed under polarized microscopy, and scored from 1 to 3. Elastosis proportionate area (EPA) was calculated with image software. Mast cells were highlighted with toluidine blue stain. Results EPA varied from 2% to 45%, with a mean of 17.1% in the cases, with no differences among epithelial dysplasia degrees. Most cases presented collagen loss scores of 2 or 3, and higher loss of type I collagen was associated with older age. Mast cell density was higher in severe epithelial dysplasia (P = 0.002) and in high-risk cases (P = 0.01). Conclusion Actinic cheilitis presented variable EPA and marked loss of type I collagen; however, these features were not associated with the degrees of epithelial dysplasia. Besides, mast cell density increased with epithelial dysplasia worsening and this was not associated with elastosis area or collagen loss.

Mineral smectite granules have traditionally been used for diarrhea and gastrointestinal bleeding; however, the wound healing ability of mineral smectite granules has not been investigated in detail. Therefore, the present study explored the efficacy and safety of mineral smectite granules on wound healing. An excision wound rat model was established. The experimental group was treated with smectite granules, the positive control group was treated with starch and the blank control group was left untreated. Wound closure rates, wound healing times and histopathological analysis were compared among the three groups. The mechanism underlying the effects of mineral smectite granules on wound healing was assessed by performing picrosirius red staining and immunohistochemical staining and measuring hydroxyproline content. Based on wound closure rates, wound healing times and histopathological analysis, the results indicated that rats treated with smectite granules displayed increased wound healing efficacy compared with the other two groups. Investigation of the underlying mechanism suggested that mineral smectite granules could promote wound healing by inducing collagen fiber synthesis and increasing the number of neovessels. The present study indicated the efficacy and safety of mineral smectite granules on wound healing and provided a theoretical basis for its application in a clinical setting.

Made available in DSpace on 2019-10-06T16:41:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) This study aimed to investigate collagen (Coll-I, III, IV) and elastin in canine normal prostate and prostate cancer (PC) using Picrosirius red (PSR) and Immunohistochemical (IHC) analysis. Eight normal prostates and 10 PC from formalin-fixed, paraffin-embedded samples were used. Collagen fibers area was analyzed with ImageJ software. The distribution of Coll-I and Coll-III was approximately 80% around prostatic ducts and acini, 15% among smooth muscle, and 5% surrounding blood vessels, in both normal prostate and PC. There was a higher median area of Coll-III in PC when compared to normal prostatic tissue (p = 0.001 for PSR and p = 0.05 for IHC). Immunostaining for Coll-IV was observed in the basal membrane of prostate acini, smooth muscle, blood vessels, and nerve fibers of normal and PC samples. Although there was no difference in Coll-IV area between normal tissue and PC, tumors with Gleason score 10 showed absence of Coll-IV, when compared to scores 6 and 8 (p = 0.0095). Elastic fibers were found in the septa dividing the lobules and around the prostatic acini of normal samples and were statistically higher in PC compared to normal tissue (p = 0.00229). Investigation of ECM components brings new information and should be correlated with prognosis in future studies. Department of Veterinary Pathology School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) Department of Veterinary Surgery and Anesthesiology School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) Department of Veterinary Pathology School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) Department of Veterinary Surgery and Anesthesiology School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) CNPq: 422139/2018-1 CNPq: 443884/2014-5

Chronic kidney disease (CKD) is a relevant disease in feline clinic. The tubulointerstitial damage, with collagen deposition and fibrosis, is an important result of this process. The aim of this study was to quantify and correlate the deposition of collagen and severity of interstitial fibrosis (IF) in the kidney from cats in different stages of CKD. Kidney fragments from 10 adult cats with CKD were analyzed and stained by Masson's trichrome (MT) and Picrosirius red (PSR) for circular polarized microscopy. Random quantitative analysis was performed on MT sections to classify the degree of IF, per field area, with and without circular polarization. Statistics correlations were performed by Spearman's (ρ; p

A predictable rehabilitation of severely atrophied alveolar ridge defects still remains a matter of concern in implant dentistry. Objective: The aim of this study was to compare histologically biomaterials of bovine (Genox®) and equine (Bio-Gen®) origin associated or not to platelet-rich plasma (PRP), analyzing bone maturity, amount of mature (type I) and immature (type III) collagen present at sixty and ninety days in post-extraction alveolus. Materials and Methods: Four Beagle dogs were used from which the six mandibular premolars were extracted. The alveolus contained respectively: right side, Bio-Gen ® (group B), preserved the clot (group C), Genox ® (group G); left side, Bio-Gen ® + PRP group (BP), Clot + PRP (group CP) and Genox ® + PRP (group GP). After sixty and ninety days, two dogs were sacrificed at each date and the pieces were histologically processed and stained with picrosirius red, a specific stain for analysis of different types of collagen. Results: Comparisons of the averages of mature collagen at 60 days indicated significant differences between Group G when compared to Groups C and B. The biomaterial of bovine origin demonstrated higher amounts of mature collagen in 90 days while the biomaterial of equine origin had a higher average of immature collagen in 90 days. Conclusion: Groups associated to PRP were effective in the production of mature and immature collagen. Genox® associated with PRP demonstrated a more advanced stage of bone regeneration presenting as an alternative to fill post-extraction alveolus.

Dimethyl sulfoxide (DMSO) is one of the commonly used vehicles to dissolve water insoluble drugs, including used for wound healing purposes, for different experiments. The present study was aimed to investigate the gross effects and histopathological changes produced by topical DMSO on granulation tissue at wound site. Open excision cutaneous wounds were created on the back of ten male rats and equally divided into two groups. Distilled water and DMSO (10 %) were topically applied once daily for 21 days on wounds of Group I (control group) and Group II, respectively. Grossly, wounds revealed that DMSO facilitated wound closure on day 7 and 12. On day 21, the wound was still not closed in DMSO treated group in comparison to control group. The H & E stained sections of control group showed formation of complete, immature and thin epithelial layer, collagen deposition and infiltration of abundant number of mononuclear cells. Sections of DMSO treated group showed blood vessels, scattered infiltration of inflammatory cells, collagen fibers and partially formed epithelial layer. Semi-quantitative score for inflammatory cells and epithelialization was significantly lower in DMSO treated group. Picrosirius red stained sections showed dominance of orange-red (thicker) collagen fibers in control group and yellow-green (thinner) collagen fibers in DMSO treated group. DMSO treated group showed higher and lower thin collagen and thick collagen fiber fraction, respectively in comparison to control group. Thus, it might be concluded that DMSO facilitates wound closure during the early stages and delays the wound closure during at later stages of wound healing.

Mast cell tumours (MCTs) constitute almost 25% of cutaneous neoplasms in dogs. Their biological behaviour is predicted using histopathological grading which is based on several subjective criteria that are vulnerable to intra- and interobserver variability. To improve the prediction of the biological behaviour, several complementary markers have been studied. The integrity of the extracellular matrix (ECM) may play a protective role against tumoral progression, and favour cellular proliferation, angiogenesis, invasion and metastases when altered.To evaluate the quantification of collagen and elastic fibres as prognostic markers for MCTs.Thirty-eight random cases of canine cutaneous MCT surgically treated with wide margins were included.Intratumoral collagen and elastic fibres were identified and quantified on histological sections stained with Masson's trichrome, Picrosirius red and Verhoeff; the results were compared with histopathological grades, mortality due to the disease and postsurgical survival.Morphometric analysis revealed a significant relationship between histopathological grade and intratumoral collagen index (CoI). In addition, the CoI was considered an independent indicator for mortality and postsurgical survival.These results support the importance of the CoI in the grading and prognosis of MCTs, suggesting that preservation and/or synthesis of collagen have the potential to become targets for MCT therapeutics.Les mastocytomes (MCTs) représentent presque 25% des tumeurs cutanées du chien. Leur évolution biologique est prédictible par gévaluation histopathologique basée sur plusieurs critères subjectifs qui sont vulnérables d'un point de vue variabilité intra et inter-observateur. Pour améliorer la prédiction de l’évolution biologique, plusieurs marqueurs complémentaires ont été étudiés. L'intégrité de la matrice extracellulaire (ECM) pourrait jouer un rôle protecteur contre la progression tumorale et son altération pourrait favoriser la prolifération cellulaire, l'angiogenèse, l'invasion et les métastases. HYPOTHÈSES/OBJECTIFS: Evaluer la quantification des fibres de collagène et des fibres élastiques comme facteurs pronostics des MCTs.Trente huit cas de MCT canins traités par chirurgie avec de larges marges ont été inclus. MATÉRIEL ET MÉTHODE: Le fibres élastiques et de collagène intra-tumorales ont été identifiées et quantifiées sur des sections histopathologiques colorées au trichome de Masson, au rouge Picrosirius et Verhoeff; les résultats étaient comparés avec les grades histopathologiques, la mortalité due à la maladie et au temps de survie post-chirurgical. RÉSULTATS: Une analyse morphométrique a révélé un lien significatif entre le grade histopathologique et l'index de collagène inntratumoral (CoI). En outre, le CoI a été considéré comme un indicateur indépendant pour la mortalité et la survie post-chirurgicale.Ces résultats soutiennent l'importance du CoI dans le grading et le pronostic des MCTs suggérant que la préservation et/ou la synthèse du collagène a le potentiel de devenir une cible pour le traitement des MCTs.INTRODUCCIÓN: los mastocitomas (MCTs) constituyen casi el 25% de las neoplasias cutáneas en perros. Su comportamiento biológico se predice utilizando una clasificación histopatológica que se basa en varios criterios subjetivos que son susceptibles de variabilidad intra e interobservador. Para mejorar la predicción del comportamiento biológico, se han estudiado varios marcadores complementarios. La integridad de la matriz extracelular (ECM) puede jugar un papel protector contra la progresión tumoral y favorecer la proliferación celular, la angiogénesis, la invasión y las metástasis cuando se alteran. HIPÓTESIS/OBJETIVOS: Evaluar la cuantificación del colágeno y las fibras elásticas como marcadores de pronóstico para los MCTs. ANIMALES: se incluyeron 38 casos al azar de MCT cutáneo canino tratados quirúrgicamente con amplios márgenes. MÉTODOS Y MATERIALES: el colágeno intratumoral y las fibras elásticas se identificaron y cuantificaron en secciones histológicas teñidas con tricrómico de Masson, rojo de Picrosirius y Verhoeff; Los resultados se compararon con los grados histopatológicos, la mortalidad debida a la enfermedad y la supervivencia postquirúrgica. RESULTADOS: el análisis morfométrico reveló una relación significativa entre el grado histopatológico y el índice de colágeno intratumoral (CoI). Además, el CoI se consideró un indicador independiente de mortalidad y supervivencia postquirúrgica. CONCLUSIONES E IMPORTANCIA CLÍNICA: estos resultados apoyan la importancia del CoI en la clasificación y el pronóstico de los MCTs, lo que sugiere que la preservación y/o la síntesis de colágeno tienen el potencial de convertirse en objetivos para el tratamiento de MCTs.Mastzelltumore (MCTs) stellen fast 25% der Hautneoplasien bei Hunden dar. Ihr biologisches Verhalten wird mittels histopathologischem „Grading“ vorhergesagt, welches auf verschiedenen subjektiven Kriterien beruht, die empfindlich sind in Bezug auf Intra- und Interobserver Variabilität. Um die Vorhersage des biologischen Verhaltens zu verbessern wurden mehrere komplementäre Marker untersucht. Die Integrität der extrazellulären Matrix (ECM) könnte eine schützende Rolle bei der Tumorprogression spielen und die zelluläre Proliferation, die Angiogenese, Invasion und Metastasen favorisieren, wenn sie geändert wird.Eine Evaluierung der Quantifizierung von Kollagen und elastischen Fasern als prognostische Marker für MCTs.Achtunddreißig zufällige Fälle von kutanem MCT bei Hunden, die chirurgisch mit großen Rändern behandelt worden waren, wurden in die Studie aufgenommen.Es wurden intratumorales Kollagen und elastische Fasern identifiziert und auf histologischen Schnitten, die mit Masson´s Trichrom, Picrosirius und Verhoeff gefärbt worden waren, quantifiziert; die Ergebnisse wurden mit den histopathologischen Graden, der Sterberate aufgrund der Erkrankung und die Überlebensdauer nach der Operation verglichen.Die morphometrische Analyse zeigte eine signifikante Beziehung zwischen den histopathologischen Graden und dem intratumoralen Kollagenindex (CoI). Zusätzlich wurde der CoI als unabhängiger Indikator für die Sterblichkeit und die Überlebensdauer nach der Operation betrachtet.Diese Ergebnisse unterstützen die Wichtigkeit von CoI beim „Grading“ und bei der Prognose von MCTs, was darauf hinweist, dass die Erhaltung und/oder die Synthese von Kollagen ein Potential darstellen, Ziele für MCT Therapeutika zu werden.背景: 肥満細胞腫(MCT)は、犬における皮膚腫瘍の約25%を占める。MCTの生物学的挙動は、観察者内および観察者間に対するいくつかの不安定な主観的基準に基づく病理組織学的グレーディングによって予測される。生物学的挙動の予測を改善するため、いくつかの相補的マーカーが研究されてきた。細胞外マトリックス(ECM)の健常性は、腫瘍の進行に対して保護的な役割を果たし、そして改変した場合、細胞増殖、血管形成、浸潤および転移に有利に働き得る。 仮説/目的: 本研究の目的は、MCTの予後マーカーとしてコラーゲンおよび弾性繊維の定量化を評価することである。 被験動物: 広範なマージンで外科的に治療された犬の皮膚MCTの無作為症例38例を供した。 材料と方法: 腫瘍内コラーゲンと弾性繊維を、マッソントリクローム染色、ピクロシリウスレッド染色、ヴァーホフ染色で染色した組織切片上で同定し、定量した。そして、その結果を病理組織学的グレード、疾患による死亡率および術後生存率と比較した。 結果: 形態計測解析は、病理組織学的グレードと腫瘍内コラーゲン指数(CoI)との間の有意な関係を明らかにした。さらに、CoIは死亡率および術後生存率に対し独立した指標であると考えられた。 結論および臨床的重要性: 本研究結果は、MCTグレーディングおよび予後におけるCoIの重要性を支持しており、コラーゲンの保持および/または合成がMCT治療の標的になる可能性を示唆している。.背景: 肥大细胞瘤(MCTs)占犬皮肤肿瘤的近25%。通过组织病理学分级预测它们的生物学行为,该分类基于几个主观标准,这些标准易受观察者自身和观察者间变化影响。为了提高对生物学行为的预测,研究了几种互补标记。细胞外基质(ECM)的完整性可以起到对肿瘤发展的保护作用,并在改变时有利于细胞增殖、血管生成、侵袭和转移。 假设/目标: 评估胶原和弹性纤维作为MCT预后标志物的量。 动物: 包括38例随机手术治疗的多处犬皮肤MCT的随机病例。 方法和材料: 将组织切片进行Masson三色、Picrosirius红和Verhoeff染色,随后鉴定和定量肿瘤内胶原和弹性纤维; 将结果与组织病理学分级、疾病引起的死亡率和术后生存率进行比较。 结果: 形态学分析显示组织病理学分级与肿瘤内胶原指数(CoI)之间有显著关系。此外,CoI被认为是死亡率和术后生存的独立指标。 结论和临床价值: 这些结果支持CoI在MCT分级和预后中的重要性,提示胶原蛋白的保存和/或合成有可能成为MCT治疗的靶点。.Os mastocitomas (MCTs) constituem quase 25% das neoplasias cutâneas em cães. O seu comportamento biológico pode ser predito utilizando uma graduação histopatológica baseada em diversos critérios subjetivos que são vulneráveis a variação intra e interobservador. Para melhorar a previsão do comportamento biológico, diversos marcadores complementares têm sido estudados. É possível que a integridade da matriz extracelular (ECM) possua uma função protetora contra a progressão tumoral, e favoreça a proliferação celular, angiogênese, invasão e metástase quando alterada. HIPÓTESE/OBJETIVOS: Avaliar a quantificação de colágeno e fibras elásticas como marcadores de prognóstico para MCTs.Trinta e oito casos aleatórios de MCTs cutâneos em cães tratados cirurgicamente com margens amplas inclusas. MÉTODOS E MATERIAIS: Colágeno intratumoral e fibras elásticas foram identificadas e quantificadas nos cortes histopatológicos corados com corante tricromo de Masson, Picrosirius vermelho e Verhoeff; os resultados foram comparados com as graduações histopatológicas, mortalidade devido à doença e sobrevida pós-cirúrgica.A análise morfométrica revelou uma relação significativa entre a graduação histopatológica e o índice de colágeno tumoral (CoI). Além disso, o CoI foi considerado um indicador independente de mortalidade e sobrevida pós-cirúrgica. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: Estes resultados corroboram com a importância do CoI na classificação e no prognóstico de MCTs, sugerindo que a preservação e a síntese de colágeno têm o potencial de se transformarem em alvos para o tratamento de MCTs.

Canine endocrinopathies, such as hypothyroidism and hyperadrenocortism,induce typical dermatological alterations. Collagen fibers are significant for the maintenance of structural integrity,as well as in the determination of tissue function. This study aimed at assessing the coloration caused by Picrosirius Red staining under circular polarization and Masson Trichrome staining, as tools to quantify the total collagen in the skin of dogs exhibiting endocrine dermatopathies. Skin samples taken from dogs with hypothyroidism and hyperadrenocorticism were stained using Hematoxylin and Eosin (HE), Masson’s Trichrome (MT) and Picrosirius Red (PSR). The animals with hyperadrenocorticism revealed a higher percentage of collagen area than did the animals with hypothyroidism PSRp (hypothyroidism = 43.76 ± 0.8255 and hyperadrenocorticism = 47.08 ± 0.8584). The percentage of the collagen area using MT between the groups is given in parenthesis (hypothyroidism = 52.06 ± 0.9371, hyperadrenocorticism = 61 ± 0.7529 and control = 56.88 ± 0.64) (p

Introduction Collagen, the most abundant protein in the body, is not only responsible for maintaining the functional integrity of tissues but is also thought to play an important role in pathogenesis and invasion in oral squamous cell carcinoma (OSCC). Aim The study aimed to assess the thickness as well as maturation of collagen fibres in erosive and infiltrative patterns of OSCC in order to determine the possible role of stromal components (collagen) in bone invasion. Materials and methods Histometric analysis was performed on 1350 collagen fibres in 14 cases of OSCC exhibiting either an erosive or an infiltrative pattern of mandibular invasion using polarizing microscope under oil immersion. Two consecutive sections of 5 μm thickness from each sample were taken and stained with haematoxylin and eosin (H&E) and picrosirius red stain, respectively, to enable visualization and counting of thickness of fibres. Results A significantly higher percentage of thick fibres and a greater amount of green/no colour birefringence were observed in cases of OSCC that exhibited an infiltrative pattern of bone invasion. Conclusion The stromal component plays an important role in the biological behaviour of a tumour. It is probable that in OSCC the infiltrative pattern of bone invasion induces increased production of loosely packed procollagens, intermediates, or pathological collagen which further aids in the invasion of the tumour into the surrounding bone, worsening the prognosis.

Background and Aim Traditional pathological scoring systems for liver fibrosis progression are predominantly based on the description of architectural changes with no consideration of the amount of collagen fiber deposition. Our purpose was to explore a true histological standard in accordance with the liver stiffness measured by point shear wave elastography (PSWE) in patients with chronic hepatitis B. Methods A total of 78 patients with liver neoplasms underwent liver stiffness measurements with PSWE as well as biochemical investigations within 3 days before partial hepatectomy. One tissue section of the liver specimens was stained with HE trichrome and evaluated traditionally with the Scheuer scoring system. The other tissue section was stained with picroSirius red and was evaluated according to the semiquantitative Chevallier et al. scoring system. In addition, this second tissue section was evaluated for the collagen proportionate area (CPA) with computer-assisted digital image analysis. The reproducibility of PSWE technology was explored through the intra-class correlation coefficient of a reliability analysis. Results The PSWE technology revealed good reproducibility in liver stiffness measurements, and the PSWE values increased with the pathological severity of liver fibrosis on both the Scheuer scoring system and the semiquantitative Chevallier et al. scoring system. PSWE values exhibited more reasonable relationships with CPA (r = 0.628, P = 0.00

Nicola Zerbinati,1 Alberto Calligaro2 1Department of Surgical and Morphological Sciences, University of Insubria (Varese) and Polyspecialist Medical Center, Pavia, 2Department of Public Health, Experimental and Forensic Medicine, Unit of Histology and Embryology, University of Pavia, Pavia, Italy Background: Calcium hydroxylapatite (CaHA, Radiesse®) is a biocompatible, injectable filler for facial soft-tissue augmentation that provides volume to tissues, followed by a process of neocollagenesis for improved skin quality. Objective: To examine the effects of CaHA treatment on the molecular organization of collagen using a combination of picrosirius red staining and circularly polarized light microscopy.Methods: Five subjects received subdermal injection of 0.3 mL of CaHA in tissues scheduled for removal during abdominoplasty 2 months later. Tissue specimens from the CaHA injection site and a control untreated area were obtained from excised skin at the time of surgery. Processed tissue sections were stained with picrosirius red solution 0.1% and visualized under circularly polarized light microscopy for identification of thick mature (type I) and thin newly formed (type III) collagen fibers. Pixel signals from both the control and CaHA-treated areas were extracted from the images, and morphometric computerized hue analysis was performed to provide a quantitative evaluation of mature and newly formed collagen fibers.Results: Under picrosirius red staining and circularly polarized light microscopy, green/yellow areas (thin newly formed collagen type III) were visible among the collagen fibers in tissue sections from the area of CaHA injection. In contrast, the majority of the collagen fibers appeared red (thick mature collagen type I) in control tissues. Morphometric analysis confirmed that, following CaHA treatment, the proportion of fibers represented by thin newly formed collagen type III increased significantly (p

Graphical abstract, Picrosirius red (PSR) staining is a commonly used histological technique to visualize collagen in paraffin-embedded tissue sections. PSR stained collagen appears red in light microscopy. However it is largely unknown that PSR stained collagen also shows a red fluorescence, whereas live cells have a distinct green autofluorescence. Both emission patterns can be detected using standard filter sets as found in conventional fluorescence microscopes. Here we used digital image addition and subtraction to determine the relative area of the pure collagen and live cell content in heart tissue in a semi-automated process using standard software. This procedure, which considers empty spaces (holes) within the section, can be easily adapted to quantify the collagen and live cell areas in healthy or fibrotic tissues as aorta, lung, kidney or liver by semi-automated planimetry exemplified herein for infarcted heart tissue obtained from the mouse myocardial infarction model. • Use of conventional PSR stained paraffin-embedded tissue sections for fluorescence analysis. • PSR and autofluorescence images are used to calculate area of collagen and area of live cells in the tissue; empty spaces (holes) in tissue are considered. • High throughput analysis of collagen and live cell content in tissue for statistical purposes.

The purpose of this study was to evaluate the effects of low-level laser therapy (LLLT) on morphological aspects, IL-6 and IL-1β expressions, as well as the distribution and organization of collagen in the tibialis anterior (TA) muscle of elderly rats submitted to cryoinjury. Histological photomicrographs were taken of TA muscles stained with HE and picrosirius red. Immunohistochemistry was used for the evaluation of IL-6 and IL-1β. Male Wistar rats, aged 20 months, were distributed into three groups: (1) control animals not injured or treated with LLLT (n = 5), (2) cryoinjury without LLLT treatment (n = 15), and (3) cryoinjury treated with infrared LLLT (n = 15). LLLT was applied to the TA 2 h after of the injury induction and consisted of daily applications until the sacrifice (1, 3, and 7 days). The following parameters were used: λ = 780 nm, power density 1 W/cm2, output power 40 mW, 10 s per point, 8 points, and 3.2 J of total energy. In the histomorphological analysis, the treated group exhibited a significant decrease in inflammatory infiltrate (p

AIM The aim of the present study was to: (a) analyze the nature of collagen with respect to cohesive and discohesive invasive tumor front (ITF) of oral squamous cell carcinoma (OSCC); (b) analyze the clinicopathological correlation with the nature of collagen at the ITF of OSCC; and (c) correlate the nature of collagen with Broder's and Bryne's histological grading system of OSCC. METHODS Tissue sections of 29 OSCC with ITF were stained with hematoxylin-eosin and picrosirius red staining for evaluation under a polarized microscope. RESULTS Tumors with a cohesive front had a thick collagen fiber, are predominantly organized, red-yellow in color, well packed, and show strong birefringence (P

Collagen is one of the foremost components of tissue extracellular matrix (ECM). It provides strength, elasticity and architecture to the tissue enabling it to bear the wear and tear from external factors like physical stress as well as internal stress factors like inflammation or other pathological conditions. During normal pregnancy or pregnancy related pathological conditions like preterm premature rupture of membranes (PPROM), collagen of the fetal membrane undergoes dynamic remodeling defining biochemical properties of the fetal membrane. The protocol in this article describes the histochemical method to stain total collagen by Picrosirius red stain which is a simple, quick and reliable method. This protocol can be used on paraformaldehyde (PFA) and formaldehyde fixed paraffin embedded tissue sections. We further describe the staining and distribution of collagen in different mouse reproductive tissues and also demonstrate how this technique in combination with polarization microscopy is useful to detect the distribution of different subtypes of collagen.

In their article entitled “Sex-Related Discordance Between Aortic Valve Calcification and Hemodynamic Severity of Aortic Stenosis: Is Valvular Fibrosis the Explanation?,” Simard et al1 report that in severe aortic stenosis, women (compared with men) have less valvular calcification (by computed tomography), but more valve fibrosis (by histology: Picrosirius red and Masson’s trichrome) than men. Aortic stenosis is common and therapies are required that alter the trajectory of valve narrowing. Our understanding of the pathophysiology is of mechanical valve leaflet stress triggering endothelial damage, and cascade of inflammation, fibrosis and calcification—but this explanation seems incomplete, and it does not explain the sex dimorphism and discordance of valve calcification and fibrosis. We wondered whether valve amyloid …

Background: Solid tumors such as oral squamous cell carcinoma (OSCC) are composed of malignant epithelial cells and the stroma in which these cells are dispersed. As the tumor progresses, the extracellular matrix undergoes dramatic morphological and architectural changes. Special stains make analysis easy and less erroneous by highlighting the area of interest and can be used to study these changes. Aim: The aim of the study was to analyze morphological changes in collagen fibers in various histological grades of OSCC using Masson's trichrome (MT) and Picrosirius red (PSR). Study Design: The study comprised 74 tissue samples, divided into two groups: Group I consisted of 63 cases of histologically proven OSCC (39 cases of well-differentiated squamous cell carcinoma [WDSCC], 17 moderately differentiated squamous cell carcinoma [MDSCC] and 7 poorly differentiated squamous cell carcinoma [PDSCC]) and Group II consisted of 11 cases of normal mucosa as controls. Materials and Methods: Sections were stained with hematoxylin and eosin, MT and PSR and observed under light and polarizing microscope, respectively. Statistical Analysis: ANOVA, Tukey's honestly significant difference post hoc multiple comparison test, Chi-square test and paired t-test were used for the statistical analysis. Results: As the grade of OSCC progressed, collagen fibers became thin, loosely packed and haphazard. The mean area fraction also decreased. They exhibited orange–red hue and strong birefringence in WDSCC, yellowish-orange hue and strong birefringence in MDSCC and greenish-yellow hue and weak birefringence in PDSCC. Conclusion: Initially, there is a reorganization of the collagen fibers in an attempt to prevent the invasion of tumor cells, but as cancer progresses, the stromal change enhances movement of the tumor cells within it, leading to metastasis.

Interstitial fibrosis is a hallmark structural correlate of progressive and chronic kidney disease. There remain many uncertainties about how to best measure interstitial fibrosis both in research settings and in evaluations of renal biopsies performed for management of individual patients. Areas of uncertainty include determination of the composition of the matrix in a fibrotic parenchyma, the definition of how the interstitium is involved by fibrosing injuries, the choice of histologic stains for evaluation of renal fibrosis, and the reproducibility and robustness of measures currently employed by pathologists, both with and without the assistance of computerized imaging and assessments. In this review, we address some of these issues while citing the key studies that illustrate these difficulties. We point to future approaches that may allow a more accurate and meaningful assessment of renal interstitial fibrosis.

High-resolution digitalizing of histology slides facilitates the development of computational alternatives to manual quantitation of features of interest. We developed a MATLAB-based quantitative histological analysis tool (QuHAnT) for the high-throughput assessment of distinguishable histological features. QuHAnT validation was demonstrated by comparison with manual quantitation using liver sections from mice orally gavaged with sesame oil vehicle or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD; 0.001–30 μg/kg) every 4 days for 28 days, which elicits hepatic steatosis with mild fibrosis. A quality control module of QuHAnT reduced the number of quantifiable Oil Red O (ORO)-stained images from 3,123 to 2,756. Increased ORO staining was measured at 10 and 30 μg/kg TCDD with a high correlation between manual and computational volume densities ( Vv), although the dynamic range of QuHAnT was 10-fold greater. Additionally, QuHAnT determined the size of each ORO vacuole, which could not be accurately quantitated by visual examination or manual point counting. PicroSirius Red quantitation demonstrated superior collagen deposition detection due to the ability to consider all images within each section. QuHAnT dramatically reduced analysis time and facilitated the comprehensive assessment of features improving accuracy and sensitivity and represents a complementary tool for tissue/cellular features that are difficult and tedious to assess via subjective or semiquantitative methods.

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.

Background: Percutaneous collagen induction (PCI) through dermal roller breaks old collagen strands, promotes removal of damaged collagen and induces more collagen formation. Collagen fibers can be assessed by traditional stains or by polarized light assessment of Picrosirius red stain. Objective: The purpose of the current study is the clinical and histopathological evaluation of percutaneous collagen formation in atrophic acne scars after dermal roller application. Patients and Methods: Total study duration was 26 weeks in which 12 patients received seven sessions of PCI at 3-weeks interval, 3 mm punch biopsy specimens of scars were obtained before and after treatment (at 18 and 26 weeks). Microscopic examination of pre and post operative biopsies were done, using routine stains and Picrosirius red stain. Results: PCI induced notable improvement in the appearance of acne scars with significant reduction in the score from 123.3 ± 24.5 to 74.16 ± 16.49 (P = 0.00) after 26 weeks. Polarized light assessment of Picrosirius red stain clarified the gradual replacement of old thick orange-red birefringent collagen fibers by newly synthesized thin green-yellow birefringent ones postoperatively. Conclusion: Skin needling is a simple and minimally invasive procedure. The polarized light assessment of Picrosirius red stain clarified the change of the optical properties of collagen fibers according to the maturation process.

Adam P Strange,1 Sebastian Aguayo,1 Tarek Ahmed,1 Nicola Mordan,1 Richard Stratton,2 Stephen R Porter,3 Susan Parekh,4 Laurent Bozec1 1Department of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 2Centre for Rheumatology and Connective Tissue Diseases, Royal Free Hospital, UCL Medical School, 3UCL Eastman Dental Institute, 4Department of Pediatrics, UCL Eastman Dental Institute, London, UK Abstract: Scleroderma (or systemic sclerosis, SSc) is a disease caused by excess crosslinking of collagen. The skin stiffens and becomes painful, while internally, organ function can be compromised by the less elastic collagen. Diagnosis of SSc is often only possible in advanced cases by which treatment time is limited. A more detailed analysis of SSc may provide better future treatment options and information of disease progression. Recently, the histological stain picrosirius red showing collagen register has been combined with atomic force microscopy (AFM) to study SSc. Skin from healthy individuals and SSc patients was biopsied, stained and studied using AFM. By investigating the crosslinking of collagen at a smaller hierarchical stage, the effects of SSc were more pronounced. Changes in morphology and Young’s elastic modulus were observed and quantified; giving rise to a novel technique, we have termed “quantitative nanohistology”. An increase in nanoscale stiffness in the collagen for SSc compared with healthy individuals was seen by a significant increase in the Young’s modulus profile for the collagen. These markers of stiffer collagen in SSc are similar to the symptoms experienced by patients, giving additional hope that in the future, nanohistology using AFM can be readily applied as a clinical tool, providing detailed information of the state of collagen. Keywords: rheumatology, adjunct diagnosis, picrosirius red, collagen, nanohistology

Introduction Forensic dentistry is a branch of dentistry which in collaboration with legal profession serves an important role to maintain justice system of a country. Forensic dentists play a major role in identification of an individual. Within the literature various methods have been found to be useful in gender differentiation. An attempt was made for differentiation of gender using picrosirius red and polarization microscopy. Aim To evaluate picrosirius red and polarization microscopy as a tool for gender differentiation by observing birefringence pattern and distribution of thick and thin collagen fibers in males and females. Materials and methods Labial mucosal tissue obtained from 30 deceased individuals (18 male and 12 female) during autopsy was fixed in 10% formalin at 12th hour. Tissue was processed, sectioned and stained using picrosirius red stain and the birefringence pattern of collagen fibers were studied with polarization microscope. The results were statistically analyzed using Z-test and one-way ANOVA to draw the significance. Results The proportion of thick and thin fibres among males and females were compared. It was found that there was statistically significant difference in proportion of thick and thin fibers between male and female. Thick fibres in males were (78.13%) more than females (65.74%) and thin fibres were more in females (34.24%) than males (21.32%). Conclusion Picrosirius red and polarization microscopy may be used as a tool for gender differentiation. Yet the manner of death has to be considered during gender differentiation using this method, as in the present study out of 30 cases studied three cases of death due to debilitating diseases and poison consumption showed altered collagen distribution.

This study aimed to evaluate the inflammatory response of the subcutaneous tissue of twenty rats when the use of iodoform with different drugs or not associated with laser photobiomodulation. In the dorsum of rats were introduced into tubes containing: Iodoform and calcium hydroxide (Ca (OH)2); Iodoform and Ca(OH)2, associated with Laser; Iodoform and Otosporin; Iodoform and Otosporin, associated with Laser. The animals were euthanized eight and fifteen days after surgery, the parts were removed and processed in the laboratory with staining with Hematoxylin-Eosin (HE) and Picrosirius Red. The statistical significances of the inflammatory infiltrate and quantification of collagen fibers were measured by ANOVA-Tukey test. The association between Ca (OH)2 and iodoform with determined the best response in the host tissues. The low level laser therapy was effective in modulating the inflammatory response and deposit collagen fibers.

Objectives: The Brown Norway rat is highly susceptible to cerebral haemorrhage when hypertension is induced experimentally, compared with the Long Evans. The aims of the study were to compare the myogenic properties and also the collagen-staining profile of the middle cerebral artery (MCA) and a small systemic artery (cremaster) from Brown Norway and Long Evans rats. Methods: In-vitro pressure myography was used to compare the myogenic properties and the distensibility of MCA and cremaster arteries from Brown Norway rat, with those of the Long Evans rat. Histologically prepared arterial sections were stained with picrosirius red to compare the collagen-staining profile of MCA and cremaster from these strains of rat. Results: In the presence of myogenic tone, the active pressure–diameter relationship (20–200 mmHg) was significantly different in MCA from the Brown Norway, but not cremaster arteries, compared with the Long Evans, characterized by in the lack of a myogenic range in the Brown Norway. Midwall collagen staining was significantly increased in MCA from the Brown Norway rat, compared with the Long Evans rat; this difference between rat strains was not observed in the cremaster arteries. However, the stress–strain relationship of MCA and cremaster arteries from the Brown Norway rat was shifted to the right, indicating an increased distensibilty of arteries from both vascular beds, compared with the Long Evans. Conclusion: These data demonstrate impaired myogenic properties and differences in the collagen-staining profile of MCA but not cremaster arteries from the Brown Norway rat, compared with the Long Evans. The impaired myogenic properties of MCA from the Brown Norway rat compared with the Long Evans may explain their increased susceptibility to cerebral haemorrhage when hypertension is induced experimentally.