New approaches to studying morphological details of intramolluscan stages of Angiostrongylus vasorum / Novas abordagens para estudo morfológico dos estágios intramoluscos de Angiostrongylus vasorum

Abstract Angiostrongylus vasorum is a pulmonary artery parasite of domestic and wild canid. On molluscs, intermediate host, first stage larvae (L1) are found after the first day of infection, in the 8th L2 and in the 30 th L3. It was evaluated L1, L2 and L3 recovered by Baermann technique from Achatina fulica infected with 1000 L1. Fifty larvae/stage were incubated with antibodies anti-β-tubulin, anti-α-tubulin, anti- α-actin, anti-β-actin and anti-collagen, and then with Alexa 633. Fifty larvae/stage were observed with picrosirius red and Oil Red O. It was also observed in the anterior region of L1 the beginning of the chitinous stems development, in the initial portion of the intestine and genital primordium. In L2 anterior region, the papillae, chitinous canes juxtaposed to the mouth and intestines bigger than L1. The L3 musculature is well defined, next to the chitinous stems, there are two round distally arranged from each other. It was observed the whole extension of the intestine genital primordium and intense cellularity in the L3 distal portion. With the picrosirius red the L1, L2 and L3 musculature could be observed, as the nerve ganglia on L3. Oil Red O revealed that L1, L2 and L3 store energy on lipid droplets.

Resumo Angiostrongylus vasorum é um parasito de artérias pulmonares dos canídeos domésticos e silvestres. Nos moluscos, hospedeiros intermediários, encontram-se no primeiro dia após a infecção, larvas de primeiro estágio (L1), ao 8° L2 e ao 30° L3. Avaliou-se L1, L2 e L3 recuperadas pela técnica de Baermann de Achatina fulica infectada com 1.000 L1. Incubou-se 50 larvas/estádio com anticorpos anti-β-tubulina, anti-α-tubulina, anti-β-actina e anti-colágeno e, em seguida, com anticorpo Alexa 633. Observaram-se também 50 larvas/estádio com picrosirius red e Oil Red O, na região anterior da L1, o início do desenvolvimento de hastes quitinosas, a porção inicial do intestino e o primórdio genital. Na região anterior de L2, papilas, bastões quitinosos justapostos à boca e ao intestino maior que em L1. A musculatura de L3 é bem desenvolvida, próximo às hastes quitinosas, há duas estruturas redondas dispostas distalmente uma da outra. Observaram-se também toda a extensão do intestino, o primórdio genital e a intensa celularidade na porção distal da L3. Com o picrosirius red observou-se a musculatura de L1, L2 e L3, assim como, gânglios nervosos na L3. Oil Red O revelou que L1, L2 e L3 armazenam energia em gotículas lipídicas.

Collagen fibers in oral submucous fibrosis - A polarizing microscopy study using two special stains

Background: Oral submucous fibrosis (OSMF), a well-recognized oral potentially malignant disorder, results due to increased collagen production and reduced collagen degradation. Aims and Objectives: To qualitatively compare the staining properties of collagen in OSMF using two special stains based on their birefringent property using polarizing microscopy. The study also assessed the distribution and orientation of collagen fibers in different grades of OSMF. Materials and Methods: A total of 73 subjects with different clinical and histopathological staging of OSMF comprised the study population. Histopathological examination was done using hematoxylin and eosin stain, Van Gieson and picrosirius red. Collagen fibers were analyzed for polarization colors, distribution, and orientation. Results: Picrosirius red stained both thick and thin collagen fibers. Irrespective of the histopathological grades reddish orange and yellowish orange were the most predominant colors. Parallel arrangement of fibers was observed when stained with Van Gieson but picrosirius red stained sections showed a majority of parallel type I fibers with perpendicular type III fibers which increased with advancement in the histopathological grade. Yellowish orange and greenish yellow fibers were predominant in the lamina propria, while reddish orange fibers were predominant in the submucosa. Conclusion: Picrosirius red was found to be a better stain. Histopathological grading and polarization colors showed no association with each other. Collagen fibers were more thickly and tightly packed in the submucosa indicating that the process of fibrosis began there. The increase in perpendicular type III fibers with advancing histopathological grades suggested their role in fibrosis.

An Immunohistochemical and Polarizing Microscopic Study of the Tumor Microenvironment in Varying Grades of Oral Squamous Cell Carcinoma

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.

Evaluation of Fibrotic Changes in OSMF:A Retrospective Study Using Special Stains and Polarizing Microscopy

Background: Oral submucous fibrosis (OSMF), a potentially malignant oral disorder has the highest rate of malignant transformation of about 7-13%. The connective tissue changes that occur in this disease are characteristic and are stained with special stains. Objective: The study was done to compare common and special stains under light microscopy and polarizing microscopy to evaluate the levels of fibrosis in oral submucous fibrosis and assess the type of collagen present in the stromal area. Materials and Methods: Fifty tissue blocks were selected from the archives and were prepared and stained with H&E, Masson's trichrome, Van Gieson and Picrosirius red and studied under light microscope and polarizing microscope respectively. Results: H and E stained slides were useful in diagnosing the lesion but was not able to highlight the level of fibrosis. Masson's trichrome and Van Gieson stained slides showed the depth of the lesion which extended even to the deeper muscle layer. The type of collagen present was definitively seen by the birefringence in polarizing microscopic study. Interobserver variation was less and all the values regarding the effectiveness of the special stains in detecting the level of fibrosis were statistically significant. Conclusion: Special stains can be used routinely in laboratories to demonstrate connective tissue lesions especially in cases of OSMF. Depth of the lesion and the area of involvement help in treatment planning to be delivered. Large scale studies with more categories and inclusion criteria are required along with the special stains to assess the other alterations in OSMF

Quantification of collagen fibers in canine uteri treated with medroxyprogesterone acetate

Collagen plays essential roles in remodeling uterine tissue during decidualization, implantation, pregnancy and involution. To understand whether the progestational agent medroxyprogesterone acetate (MPA) can modify the organization and deposit of collagen in the uteri of normal bitches (Canis Tlupus familiaris), we assessed uterine tissues by histochemistry. Uteri were grouped as: nulliparous (n=11), multiparous (n=11) and treated with MPA (n=11; nulliparous with two treatments; 5mg/kg; i.m.). The amount, location and birefringence of interstitial collagen types I and III in the fold and base of the endometrial stroma and the myometrial muscular layers were studied on sections stained with Picrosirius Red by polarized light microscopy and evaluated by ANOVA. No differences were observed in the endometrium. In the myometrium, differences were observed in collagen type I between MPA-treated and nulliparous uteri vs. multiparous (p<0.05), and differences in collagen type III between nulliparous and multiparous uteri vs. MPA-treated (p=0.0001). In conclusion, two doses of MPA had no significant effect on the investigated collagens in the extracellular matrix.(AU)

Morphological analysis of collagen and elastic fibers in oral squamous cell carcinoma using special stains and comparison with Broder’s and Bryne’s grading systems.

Objectives: Morphologic detection of connective tissue fiber changes in oral squamous cell carcinoma (OSCC) using special stains remains less documented. The aims of the present study were to study the collagen and elastic fibers in different stages of OSCC and to correlate these changes with two grading systems ‑ Broder’s and Bryne’s. Study Design: Forty‑eight cases of OSCC were studied using hematoxylin and eosin, Verhoeff’s ‑ Van Gieson stain for elastic fibers and picrosirius red stain for collagen fibers. The changes were compared with all the grades of carcinoma. Normal mucosa was taken as control. Results: Statistical analysis using Chi‑square and ANOVA, showed significant association between the grades of carcinoma and extracellular matrix changes. Greenish‑yellow collagen fibers were found to be significantly increased in the poorly differentiated/Grade 3 cases (P < 0.0001) where as well‑differentiated/Grade 1 cases showed predominantly reddish‑orange and yellowish‑orange birefringence of collagen fibers. Chi‑square analysis showed a significant amount of fragmented pattern of elastic fibers in poorly differentiated OSCC (χ2 = 104.45, P = 0.009)/Grade 3 OSCC (χ2 = 94.81, P = 0.016). Conclusion: The study of the connective tissue stromal changes can be used as an adjunct to histological grading and thereby helping the surgeon to determine the amount of marginal clearance.

Stromal characterization and comparison of odontogenic cysts and odontogenic tumors using picrosirius red stain and polarizing microscopy: A retrospective and histochemical study.

INTRODUCTION: Odontogenic lesions represent a range of conditions, the features of which probably depend on the stage of induction towards tooth formation reached prior to neoplastic or hamartomatous proliferation. It has been also suggested that inductive changes may allow progression from one type of odontogenic tumor to another. The epithelium also plays an important role in the pathogenesis of these lesions; even stroma is likely to play an equally important role in the pathogenesis and biological behavior. So, this study was performed to investigate, compare, and correlate different types of collagen fibers in odontogenic cysts and odontogenic tumors. MATERIALS AND METHODS: Thirty each pre‑diagnosed odontogenic cysts and tumors were histochemically analyzed using a special stain (Picrosirius red stain) and polarizing microscopy. RESULTS: Seven cases (99%) of inflammatory cysts exhibited predominantly greenish‑yellow birefringence indicating procollagen, intermediate, or pathologic collagen fibers suggestive of loosely packed collagen fibers. Predominant yellowish‑orange birefringence exhibited by 21 cases (99%) of developmental cysts was comparable to the yellowish‑orange and orangish‑red to red birefringence exhibited by odontogenic tumors suggesting tightly packed fibers. CONCLUSIONS: The Picrosirius red stain in conjunction with polarizing microscopy serves as a specific and sensitive tool in characterizing collagen fibers in odontogenic cysts and odontogenic tumor.

Estudio histoquímico de la distribución de las fibras de colágeno en hueso sesamoídeo distal de equinos con y sin síndrome navicular / Histochemical study of the distribution of collagen fibers on distal sesamoid bone in equine with and without navicular syndrome

La significancia funcional de cualquier relación que exista entre la orientación de las fibras de colágeno y la tensión ejercida sobre una estructura ósea dependerá de la influencia de estos parámetros microestructurales sobre las propiedades biomecánicas del hueso y su capacidad de adaptarse. Se estudió la localización del colágeno en el hueso sesamoídeo distal (HSD) de la mano en equinos con Síndrome Navicular para observar cómo este refleja las demandas biomecánicas ejercidas por la tensión que ejerce el tendón del músculo flexor digital profundo sobre la articulación interfalángica distal. Se utilizaron las tinciones hematoxilina-eosina-azul alcián, y Rojo Picrosirius de Junqueira. La birrefringencia de colágeno óseo fue determinada utilizando microscopía de luz polarizada. La remodelación del HSD resultó en la formación de osteonas secundarias transversales orientados en una dirección lateral a medial y el colágeno óseo se orientó de manera similar. Estos resultados proporcionan evidencia de la existencia de una relación entre la función mecánica de un hueso con su arquitectura, incluso demuestra que esta se extiende hasta el nivel molecular.

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. Hematoxylin-eosin-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.

Stromal differences in odontogenic cysts of a common histopathogenesis but with different biological behavior: A study with picrosirius red and polarizing microscopy.

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.

Effects of different skin expansion ways on collagen and actin / 实用口腔医学杂志

Objective: To understand the effects of different expansion ways on collagen and actin of skin and to provide an experiment foundation for the suitable expansion method clinically. Methods: The rabbits were divided into five groups. The expanders were embedded in experimental group. The sirius-red specific staining method was applied to observe the changes of type I collagen and type III collagen and their proportion. Immunochemistry method was used to detect the expression of actin. Results: 1: In group A, B and C, type I collagen arranged turbulently and was thicker and type III collagen decreased relatively at earlier maintaining period. After 4 week, type I collagen arranged regularly and type III collagen was relatively increased. 2: Comparisons of the area about type I, III collagen: At the same maintaining period, there were no significant difference among the group A,B and C. With the extension of maintaining period, the area of type I, III collagen increased gradually, but there was inclined to stabilize at the fourth week in group A, B and at the third week in group C. Comparison of the proportion of collagen I and collagen III: There were significant difference between group A, B and group D, but no significant difference was observed after the fourth week. There were significant difference between group C and control group, but no significant difference was observed after three week. 3. With the extension of maintaining period, the positive expression rates of actin decreased gradually in group A, B, C. At the same maintaining period, significant difference were observed among group A, B, C. The positive expression rates of actin in group C were lower than that of group A at the same maintaining period. Conclusion: The repeated rapid expansion can not only shorten the clinical period but also promote the quality of skin flap. It is a safe and feasible method in clinic.

Comparative study of clinicofunctional staging of oral submucous fibrosis with qualitative analysis of collagen fibers under polarizing microscopy.

Objective : The aim of the study was to assess the severity of the disease in oral submucous fibrosis (OSF), correlate the clinical, functional staging with histopathological staging, and analyze collagen distribution in different stages of OSF using the picrosirius red stain under polarizing microscopy. Materials and Methods : The study included randomly incorporated 50 subjects, of whom 40 were patients with OSF, and 10 were in the control group. Clinical, functional staging in OSF cases was done depending upon definite criteria. A histopathological study was conducted using the hematoxylin and eosin stain and picrosirius red stain. Collagen fibers were analyzed for thickness and polarizing colors. Furthermore, clinical, functional, and histopathological stages were compared. Statistical Analysis : Descriptive data which included mean, SD, and percentages were calculated for each group. Categorical data were analyzed by the chi-square test. Multiple group comparisons were made by one-way ANOVA followed by Student's t-test for pairwise comparisons. For all tests, a P-value of 0.05 or less was considered for statistical significance. Results : As the severity of the disease increased, clinically, there was definite progression in subjective and objective symptoms. Polarized microscopic, examination revealed, there was a gradual decrease in the green-greenish yellow color of the fibers and a shift to orange red-red color with increase in severity of the disease. Thereby, it appeared that the tight packing of collagen fibers in OSF progressively increased as the disease progressed from early to advanced stages. We observed that the comparison of functional staging with histopathological staging was a more reliable indicator of the severity of the disease. Conclusion : In the present study, we observed that mouth opening was restricted with advancing stages of OSF. The investigation also points to the importance of assessing the cases of OSF, especially with regard to functional and histological staging in planning the treatment.