The effects of conjugated estrogen, raloxifene and soy extract on collagen in rat bones

Objective To evaluate the action of conjugated equine estrogen, raloxifene and isolated or combined genistein-rich soy extracts on collagen fibers in the bones of oophorectomized rats. Materials and methods Seventy female rats received testosterone propionate (0.1 mu g/g) on the 9th day after birth. At 6 months of age, the rats were administered the vehicle (propylene glycol, 0.5 ml/day), and ten of the rats were randomly chosen to comprise the non-oophorectomized control group (GI). The other 60 rats were ovariectomized and randomized into six groups of ten as follows: GII, vehicle; GIII, conjugated equine estrogen (CEE), 50 mu g/kg/day; GIV, raloxifene (RAL), 0.75 mg/kg/day; GV, genistein-rich soy extract (GSE), 300 mg/kg/day; GVI, CEE + GSE, 50 mu g/kg/day + 300 mg/kg/day; and GVII, CEE + RAL, 50 mu g/kg/day + 0.75 mg/kg/day. Three months after surgery, the drugs were administered for 60 consecutive days. All rats were euthanized, and their left tibiae were removed for histological routine. The histological sections were stained with hematoxylin-eosin, and picrosirius for evaluating bone microarchitecture. Types I and II collagen fibers were analyzed by immunofluorescence. Data analysis was carried out with ANOVA and Tukey's test. Results Collagen reduction was significant in the GIII animals when compared to the other groups (p < 0.05). There was no significant difference in the thickness of collagen fibers among the groups. There was a greater quantity of type III collagen in GVI than in the other groups. Conclusion Our data indicate that conjugated equine estrogen improves bone quality because it increases the quantity of type I collagen while reducing the quantity of thin collagen fibers. In addition, the combination of CEE and raloxifene or genistein-rich soy extract is not as efficient as CEE itself to improve bone quality.

Influence of a hypercholesterolemic diet on the collagen composition of the bladder wall extracellular matrix in rats

Purpose: To investigate the effects of hypercholesterolemic diet on the collagen composition of urinary bladder wall. Materials and methods: Forty-five female 4-week-old Wistar rats were divided into three groups: 1) control group fed a normal diet (ND); 2) model of bladder outlet obstruction (BOO) group fed a ND; and 3) group fed a HCD (1.25% cholesterol). Total serum cholesterol, LDL cholesterol and body weight were assessed at baseline. Four weeks later, group 2 underwent a surgical procedure resulting in a partial BOO, while groups 1 and 3 underwent a sham similar surgical procedure. Six weeks later, all animals had their bladders removed; serum cholesterol and LDL cholesterol levels and body weights were measured. Morphological and morphometric analysis was performed by Picrosirius staining and collagen types I and III were identified by immunofluorescence. Statistical analysis was completed and significance was considered when p<0.05. Results: Rats fed an HCD exhibited a significant increase in LDL cholesterol levels (p<0.001) and body weight (p=0.017), when compared to the groups fed a ND during the ten-week study period. Moreover, the HCD induced morphological alterations of the bladder wall collagen, regarding thin collagen fibers and the amounts of type III collagen when compared to the control group (p=0.002 and p=0.016, respectively), resembling the process promoted in the BOO model. Conclusions: A hyper-cholesterolemic diet in Wistar rats promoted morphological changes of the bladder types of collagen, as well as increases in body weight and LDL cholesterol.

Experimental diabetes modulates collagen remodelling of joints in rats

The aim of this study was to evaluate extracellular matrix components in articular cartilage, ligaments and synovia in an experimental model of diabetes. Young Wistar rats were divided into a streptozotocin-induced (STZ; 35 mg/kg) diabetic group (DG; n=15) and a control group (CG; n=15). Weight, blood glucose and plasma anti-carboxymethyllysine were measured 70 days after STZ infusions. Knee joints, patellar ligaments, and lateral and medial collateral ligaments were isolated and stained with hematoxylineosin and Picrosirius. The total collagen content was determined by morphometry. Immunofluorescence was employed to evaluate types I, III, and V collagen in ligaments and synovial tissues and types II and XI collagen in cartilage. Results: Higher blood glucose levels and plasma anti-carboxymethyllysine were observed in DG rats when compared to those in CG rats. The final weight was significantly lower in the DG rats than in the CG rats. Histomorphometric evaluation depicted a small quantity of collagen fibers in ligaments and articular cartilage in DG rats, as well as increased collagen in synovial tissue. There was a decrease in cartilage proteoglycans in DG rats when compared with CG rats. Immunofluorescence staining revealed an increase of collagen III and V in ligaments, collagen XI in cartilage, and collagen I in synovial tissue of DG rats compared with CG rats. Conclusion: The ligaments, cartilage and synovia are highly affected following STZ-induced diabetes in rats, due the remodeling of collagen types in these tissues. This process may promote the degradation of the extracellular matrix, thus compromising joint function. Our data may help to better understand the pathogenesis of joint involvement related to diabetes.

Anti-inflammatory Effects of Aerobic Exercise in Mice Exposed to Air Pollution

VIEIRA, R. D. P., A. C. TOLEDO, L. B. SILVA, F. M. ALMEIDA, N. R. DAMACENO-RODRIGUES, E. G. CALDINI, A. B. G. SANTOS, D. H. RIVERO, D. C. HIZUME, F. D. T. Q. S. LOPES, C. R. OLIVO, H. C. CASTRO-FARIA-NETO, M. A. MARTINS, P. H. N. SALDIVA, and M. DOLHNIKOFF. Anti-inflammatory Effects of Aerobic Exercise in Mice Exposed to Air Pollution. Med. Sci. Sports Exerc., Vol. 44, No. 7, pp. 1227-1234, 2012. Purpose: Exposure to diesel exhaust particles (DEP) results in lung inflammation. Regular aerobic exercise improves the inflammatory status in different pulmonary diseases. However, the effects of long-term aerobic exercise on the pulmonary response to DEP have not been investigated. The present study evaluated the effect of aerobic conditioning on the pulmonary inflammatory and oxidative responses of mice exposed to DEP. Methods: BALB/c mice were subjected to aerobic exercise five times per week for 5 wk, concomitantly with exposure to DEP (3 mg.mL (1); 10 mu L per mouse). The levels of exhaled nitric oxide, reactive oxygen species, cellularity, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) were analyzed in bronchoalveolar lavage fluid, and the density of neutrophils and the volume proportion of collagen fibers were measured in the lung parenchyma. The cellular density of leukocytes expressing IL-1 beta, keratinocyte chemoattractant (KC), and TNF-alpha in lung parenchyma was evaluated with immunohistochemistry. The levels of IL-1 beta, KC, and TNF-alpha were also evaluated in the serum. Results: Aerobic exercise inhibited the DEP-induced increase in the levels of reactive oxygen species (P < 0.05); exhaled nitric oxide (P < 0.01); total (P < 0.01) and differential cells (P < 0.01); IL-6 and TNF-alpha levels in bronchoalveolar lavage fluid (P < 0.05); the level of neutrophils (P < 0.001); collagen density in the lung parenchyma (P < 0.05); the levels of IL-6, KC, and TNF-alpha in plasma (P < 0.05); and the expression of IL-1 beta, KC, and TNF-alpha by leukocytes in the lung parenchyma (P < 0.01). Conclusions: We conclude that long-term aerobic exercise presents protective effects in a mouse model of DEP-induced lung inflammation. Our results indicate a need for human studies that evaluate the pulmonary responses to aerobic exercise chronically performed in polluted areas.

Monoclonal antibodies anti-TGF beta 1 and anti-VEGF inhibit the experimental pleurodesis induced by silver nitrate

Background. The mechanisms underlying pleural inflammation and pleurodesis are poorly understood. We hypothesized that the cytokines transforming growth factor beta (TGF beta 1) and vascular endothelial growth factor (VEGF) play a major role in pleurodesis after intrapleural silver nitrate (SN) injection. Method. Forty rabbits received intrapleurally 0.5% SN alone or 0.5% SN + anti-TGF beta 1, anti-IL-8, or anti-VEGF. After 28 days, the animals were euthanized and macroscopic pleural adhesions, microscopic pleural fibrosis, and collagen deposition were analyzed for characterization of the degree of pleurodesis (scores 0-4). Results. Scores of pleural adhesions, pleural fibrosis, total collagen, and thin collagen fibers deposition after 28 days were significantly lower for 0.5% SN + anti-TGF beta 1 and 0.5% SN + anti-VEGF. Significant correlations were found between macroscopic adhesion and microscopic pleural fibrosis with total collagen and thin collagen fibers. Conclusions. We conclude that both TGF beta 1 and VEGF, but not IL-8, mediate the pleural inflammatory response and pleurodesis induced by SN.

Extracellular Matrix Composition of the Cricopharyngeus Muscle

The aim of this study was to analyze the presence and distribution of total collagen, type I and type III collagen, elastic fibers, fibronectin, and versican in the endomysium of cricopharyngeus muscles from adults of various ages. The study was a cross-sectional analysis of human cricopharyngeus muscles. Twenty-seven muscles obtained from autopsies of men and women ranging in age from 28 to 92 years were analyzed with the Picrosirius method, oxidized Weigert resorcin-fuchsin, immunohistochemistry, and image analysis. Collagen had the highest density among the analyzed components. Elastic fibers surrounded each muscle cell; they were aligned longitudinally by their long axis and associated with traversing fibers, thereby forming a fiber network with embedded muscle cells. The fibronectin and versican contents varied widely among the specimens. We found no statistically significant differences between the proportion of extracellular matrix (ECM) components and factors such as gender and race. We conclude that the higher proportion of type I and type III collagen is compatible with the cricopharyngeus muscle's sphincteric behavior, and the arrangement of the elastic fibers may also contribute to the muscle's elasticity. We found no statistically significant correlation between the ECM components and age.

Experimental Adhesives with Different Hydrophilicity: Microshear Test in after 1, 7, and 90 Days' Storage

Purpose: To assess the microshear bond strength of 3 experimental adhesives with different degrees of hydrophilicity after 1, 7 and 90 days of storage. Materials and Methods: The bonding effectiveness of three experimental two-step etch-and-rinse adhesives (bis-GMA, bis-EMA/bis-GMA, polybutadiene [C6H12]) and one commercial adhesive (Single Bond) to sound hydrated dentin was determined using the nnicroshear test with delimitation of the adhesive area after 1, 7, and 90 days of storage in water at 37 degrees C. Two-way ANOVA was performed at the 0.05 probability level. The fractures were classified as adhesive, cohesive in dentin, cohesive in resin, and mixed. Results: The experimental adhesives showed values in the range of 11.31 to 12.96 MPa, with polybutadiene (PBH) showing the lowest bond strengths, bis-GMA the highest, and bis-EMA/bis-GMA intermediary values. Single Bond yielded bond strengths of approximately 24 MPa. Water storage decreased the bond strength in all adhesives. Adhesive fractures were predominant in experimental adhesives, while mixed fractures were the most frequent type in the Single Bond group. Conclusion: The experimental dentin adhesives of this study were able to form resin tags, but they could not penetrate into the collagen fibers and form hybrid layers. The resulting low bond strength decreased with increasing length of storage.

Drug-targeting in combined cancer chemotherapy: tumor growth inhibition in mice by association of paclitaxel and etoposide with a cholesterol-rich nanoemulsion

Lipid nanoemulsions (LDE) may be used as carriers of paclitaxel (PTX) and etoposide (ETP) to decrease toxicity and increase the therapeutic action of those drugs. The current study investigates the combined chemotherapy with PTX and ETP associated with LDE. Four groups of 10-20 B16F10 melanoma-bearing mice were treated with LDE-PTX and LDE-ETP in combination (LDE-PTX + ETP), commercial PTX and ETP in combination (PTX + ETP), single LDE-PTX, and single LDE-ETP. PTX and ETX doses were 9 mu mol/kg administered in three intraperitoneal injections on three alternate days. In two control groups mice were treated with saline solution or LDE alone. Tumor growth, metastasis presence, cell-cycle distribution, blood cell counts and histological data were analyzed. Toxicity of all treatments was evaluated in mice without tumors. Tumor growth inhibition was similarly strong in all treatment groups. However, there was a greater reduction in the number of animals bearing metastases in the LDE-PTX + ETP group (30 %) in comparison to the PTX + ETP group (82 %, p < 0.05). Reduction of cellular density, blood vessels and increase of collagen fibers in tumor tissues were observed in the LDE-PTX + ETP group but not in the PTX + ETP group, and in both groups reduced melanoma-related anemia and thrombocytosis were observed. Flow cytometric analysis suggested that LDE-PTX + ETP exhibited greater selectivity to neoplastic cells than PTX-ETP, showing arrest (65 %) in the G(2)/M phase of the cell cycle (p < 0.001). Toxicity manifested by weight loss and myelosuppression was markedly milder in the LDE-PTX + ETP than in the PTX + ETP group. LDE-PTX + ETP combined drug-targeting therapy showed markedly superior anti-cancer properties and reduced toxicity compared to PTX + ETP.

Histology, histochemistry and stereology of the adipose fin of Prochilodus lineatus

The adipose fin is small, nonpared, and usually located medially between the dorsal and caudal fin. Its taxonomic occurrence is very restrict; thus, it represents an important trace for taxon distinction. As it does not play a known vital physiological roll and it is easily removed, it is commonly used in marking and recapture studies. The present study characterizes the adipose fin of Prochilodus lineatus, as it is poorly explored by the literature. The adipose fin consists basically of a loose connective core, covered by a stratified epithelium supported by collagen fibers. At the epithelium, pigmented cells and alarm substance cells are found. Despite the name, adipocytes or lipid droplets are not observed on the structure of the fin. Microsc. Res. Tech., 2012. (c) 2011 Wiley Periodicals, Inc.

Immunophenotyping and extracellular matrix remodeling in pulmonary and extrapulmonary sarcoidosis

Objective: To investigate the significance of cellular immune markers, as well as that of collagen and elastic components of the extracellular matrix, within granulomatous structures in biopsies of patients with pulmonary or extrapulmonary sarcoidosis. Methods: We carried out qualitative and quantitative evaluations of inflammatory cells, collagen fibers, and elastic fibers in granulomatous structures in surgical biopsies of 40 patients with pulmonary and extrapulmonary sarcoidosis using histomorphometry, immunohistochemistry, picrosirius red staining, and Weigert's resorcin-fuchsin staining. Results: The extrapulmonary tissue biopsies presented significantly higher densities of lymphocytes, macrophages, and neutrophils than did the lung tissue biopsies. Pulmonary granulomas showed a significantly higher number of collagen fibers and a lower density of elastic fibers than did extrapulmonary granulomas. The amount of macrophages in the lung samples correlated with FVC (p < 0.05), whereas the amount of CD3+, CD4+, and CD8+ lymphocytes correlated with the FEV1/FVC ratio and VC. There were inverse correlations between TLC and the CD1a+ cell count (p < 0.05), as well as between DLCO and collagen/elastic fiber density (r = -0.90; p = 0.04). Conclusions: Immunophenotyping and remodeling both showed differences between pulmonary and extrapulmonary sarcoidosis in terms of the characteristics of the biopsy samples. These differences correlated with the clinical and spirometric data obtained for the patients, suggesting that two different pathways are involved in the mechanism of antigen clearance, which was more effective in the lungs and lymph nodes.

Morphological observations of ampullae of lorenzini in Squatina guggenheim and S. occulta (chondrichthyes, elasmobranchii, squatinidae)

We have conducted a morphological study of the ampullae of Lorenzini on two shark species from Squatina Genus. In both species, S. guggenheim and S. occulta, the ampullae were observed like small pores scattered in the head region similar to other species of the Chondrichthyes Class. However, differently of the other species a greatest density of ampullae of Lorenzini was observed along of the body surface. After fixation using 10% formaldehyde, the ampullae were removed and processed for light and scanning electron microscopy. Macroscopically, the two shark species differed by the presence of dorsal spines that appeared from the head to the first dorsal fin in S. guggenheim and were absent in S. occulta. Microscopically, there were no differences between the ampullae of Lorenzini channels in these two species. The wall of the ampulla was formed by a simple squamous epithelium. Bands of connective tissue, hyaline cartilage and collagen fibers were found between the ampulla and the skeletal striated muscle layer. Nerve branches responsible for conducting signal pulses to the central nervous system were visible between the muscle and connective tissue layers. Using scanning electron microscopy and histological analysis, we found that the channels were twisted and positioned parallel to the skin. The inside of the channels contained a large amount of a gelatinous secretion composed by polysaccharides. Therefore, we conclude that the morphological combination of extended distribution of the ampullae of Lorenzini and the body shape may represent an adaptation of these species to their way of life. Microsc. Res. Tech. 75:12131217, 2012. (C) 2012 Wiley Periodicals, Inc.

Qualitative study of young, adult, and aged wistar rats temporomandibular synovial membrane employing light, scanning, and transmission electron microscopy

The aim of this study was to analyze the rat temporomandibular joint (TMJ) synovial membrane at different ages using light, scanning, and transmission electron microscopy. Under light microscopic analysis, the TMJ structures were observed such as condyle, capsule, disk, the synovial membrane collagen type, and cells distribution. In the scanning electron microscopy, the synovial membrane surface exhibited a smooth aspect in young animals and there was an increase with ageing in the number of folds. The transmission electron microscopic analysis showed more synoviocytes in the synovial layer in the young group and still a great number of vesicles and cisterns dilation of rough endoplasmic reticulum in the aged group. In the three groups, a dense layer of collagen fibers in the synovial layer and cytoplasmic extensions were clearly seen. It was possible to conclude that synovial membrane structures in aged group showed alterations contributing to the decrease in joint lubrication and in the sliding between disk and joint surfaces. These characteristic will reflect in biomechanics of chewing, and may cause the TMJ disorders, currently observed in clinical processes. Microsc. Res. Tech. (c) 2012 Wiley Periodicals, Inc.

Rat subcutaneous tissue response to MTA Fillapex® and Portland cement

The aim of this study was to evaluate the response of rat subcutaneous tissue to MTA Fillapex® (Angelus), an experimental root canal filling material based on Portland cement and propylene glycol (PCPG), and a zinc oxide, eugenol and iodoform (ZOEI) paste. These materials were placed in polyethylene tubes and implanted into the dorsal connective tissue of Wistar rats for 7 and 15 days. The specimens were stained with hematoxylin and eosin, and evaluated regarding inflammatory reaction parameters by optical microscopy. The intensity of inflammatory response against the sealers was analyzed by two blinded and previously calibrated examiners for all experimental periods (kappa=0.96). The histological evaluation showed that all materials caused a moderate inflammatory reaction at 7 days, which subsided with time. A greater inflammatory reaction was observed at 7 days in the tubes filled with ZOEI paste. Tubes filled with MTA Fillapex presented some giant cells, macrophages and lymphocytes after 7 days. At 15 days, the presence of fibroblasts and collagen fibers was observed indicating normal tissue healing. The tubes filled with PCPG showed similar results to those observed in MTA Fillapex. At 15 days, the inflammatory reaction was almost absent at the tissue, with several collagen fibers indicating normal tissue healing. Data were analyzed by the nonparametric Kruskal-Wallis test (?=0.05). Statistically significant difference (p<0.05) was found only between PCPG at 15 days and ZOEI at 7 days groups. No significant differences were observed among the other groups/periods (p>0.05). MTA Fillapex and Portland cement added with propylene glycol had greater tissue compatibility than the PCPG paste.

In vivo evaluation of tissue response to new endodontic sealers

Introduction: The sealers can be in direct contact with the periapical tissues. Thus, these materials must have appropriate physical and biological properties, providing conditions for repair to occur. Objective: The aim of this study was to evaluate the response of rat subcutaneous tissue to endodontics sealers. Material and methods: Three materials comprised the groups: group I – Zinc Oxide, Eugenol and Iodoform paste, group II – Portland cement with propylene glycol, and group III – MTA Fillapex® (Angelus). These materials were placed in polyethylene tubes and implanted into dorsal connective tissue of Wistar rats for seven and 15 days. The specimens were stained with hematoxylin and eosin and evaluated regarding to inflammatory reaction parameters through a light microscope. The data were compared using Kruskal-Wallis test with significance level of 5%. The intensity of inflammatory response against the sealers was analyzed by two blinded and previously calibrated observers for all experimental periods. Results: The histological evaluation showed that all the materials caused a moderated inflammatory reaction at seven days which decreased with time. A greater inflammatory reaction was observed at seven days in group I. The other specimens had significantly less inflammatory cells when compared to this group. Tubes with MTA Fillapex® presented some giant cells, macrophages and lymphocytes after seven days. At 15 days, the presence of fibroblasts and collagen fibers was observed indicating normal tissue healing. The group II showed similar results to those observed in MTA Fillapex® already at seven days. At 15 days the inflammatory reaction presented was almost absent at the tissue, with many collagen fibers indicating normal tissue healing. Statistical analysis showed a significant statistical difference amongst the group I (seven days) and II (15 days) (p < 0.05). In the other groups no (Continue)

In vivo evaluation of tissue response to new endodontic sealers

Introduction: The sealers can be in direct contact with the periapical tissues. Thus, these materials must have appropriate physical and biological properties, providing conditions for repair to occur. Objective: The aim of this study was to evaluate the response of rat subcutaneous tissue to endodontics sealers. Material and methods: Three materials comprised the groups: group I – Zinc Oxide, Eugenol and Iodoform paste, group II – Portland cement with propylene glycol, and group III – MTA Fillapex® (Angelus). These materials were placed in polyethylene tubes and implanted into dorsal connective tissue of Wistar rats for seven and 15 days. The specimens were stained with hematoxylin and eosin and evaluated regarding to inflammatory reaction parameters through a light microscope. The data were compared using Kruskal-Wallis test with significance level of 5%. The intensity of inflammatory response against the sealers was analyzed by two blinded and previously calibrated observers for all experimental periods. Results: The histological evaluation showed that all the materials caused a moderated inflammatory reaction at seven days which decreased with time. A greater inflammatory reaction was observed at seven days in group I. The other specimens had significantly less inflammatory cells when compared to this group. Tubes with MTA Fillapex® presented some giant cells, macrophages and lymphocytes after seven days. At 15 days, the presence of fibroblasts and collagen fibers was observed indicating normal tissue healing. The group II showed similar results to those observed in MTA Fillapex® already at seven days. At 15 days the inflammatory reaction presented was almost absent at the tissue, with many collagen fibers indicating normal tissue healing. Statistical analysis showed a significant statistical difference amongst the group I (seven days) and II (15 days) (p < 0.05). In the other groups no significant statistical differences were observed. Conclusion: MTA Fillapex® and Portland cement with propylene glycol were more biocompatible than the other tested cements.