Delayed tooth replantation after root surface treatment with sodium hypochlorite and sodium fluoride: Histomorphometric analysis in rats

In cases of delayed tooth replantation, non-vital periodontal ligament remnants have been removed with sodium hypochlorite in an attempt to control root resorption. Nevertheless, reports of its irritating potential in contact with the alveolar connective tissue have been described. Therefore, this study evaluated the healing process on delayed replantation of rat teeth, after periodontal ligament removal by different treatment modalities. Twenty-four rats, assigned to 3 groups (n=8), had their upper right incisor extracted and left on the workbench for desiccation during 60 min. Afterwards, the teeth in group I were immersed in saline for 2 min. In group II, root surfaces were scrubbed with gauze soaked in saline for 2 min; and in group III, scrubbing was done with gauze soaked in 1% sodium hypochlorite solution. Thereafter, root surfaces were etched with 37% phosphoric acid and immersed in 2% acidulate-phosphate sodium fluoride solution, at pH 5.5. Root canals were filled with a calcium hydroxide-based paste and the teeth were replanted. The animals were sacrificed 60 days postoperatively and the pieces containing the replanted teeth were processed and paraffin- embedded. Semi-serial transversally sections were obtained from the middle third of the root and stained with hematoxylin and eosin for histomorphometric analysis. Data were analyzed statistically using Kruskal-Wallis and Dunn's tests. The results showed that root structure and cementum extension were more affected by resorption in group III (p<0.05). All groups were affected by root resorption but the treatment performed in group III was the least effective for its control. The treatment accomplished in groups I and II yielded similar results to each other.

Avaliação histológica da pele após exposição a gel acrescido de hialuronidase associado ou não a ultra-som

The objective of this work was to verify the effect in the skin of male swines gel (G) containing hyaluronidase (H) associated or not to ultrasound (US). In different areas was applied G; G+US; G+H; G+H+US and mesotherapy (M). Skin fragment was processed in paraffin. To evidence hyaluronic acid (HA) coloration with Alcian Blue (AB) was used and coloration with Hematoxilin/Eosin for morphometry. It was observed that G+H and G+H+US did not reduce coloration for the AB nor presented significant differences for the morphometry. When H was applied mesoteraphycally coloration for the AB diminished. Then, the use of H associated or with US did not seem efficient in the HA reduction.

Effects of fibrin sealer and resorbable gelatin on the repair of osseous defects in rat tibia

Gelfoam® - a biologically resorbable gelatin sponge - has the function of restricting hemorrhage, providing platelet rupture, and supporting fibrin threads. Beriplast® - a fibrinogen-thrombin compound - is used to adhere tissues, to consolidate sutures and in hemostasis. The objective of this study was to perform a histological analysis of the effects of haemostatic agents on osseous repair. These materials were inserted into surgical sites in young rat right and left tibiae. After the observation periods of 7, 14, 30 and 45 days, according to the bioethic protocol, the animals were killed, the tibiae were removed and fixed in 10% formalin and decalcified in equal parts of formic acid and sodium citrate solutions. After routine processing, the specimens were embedded in paraffin for microtomy. Analysis of the results demonstrated that the haemostatic agents are effective in controlling hemorrhage; they stimulate osteogenesis, featuring a pattern of osseous tissue formation similar to the control pattern, although the amount of osseous trabeculae was superior, especially in the Gelfoam group in the periods of 7 and 14 days; 30 days after surgery, the delay in tissue healing in the control group in relation to the experimental groups started to decrease, and the control and experimental groups exhibited similar tissue repair after 45 days, when all the groups exhibited secondary osseous tissue.

Immunohistochemistry assay to detect Turkey coronavirus (TCoV) from experimentally infected poults

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of the biological behavior of wollastonite bioceramics prepared from synthetic and natural precursors

Wollastonite bioceramics prepared from synthetic and natural precursors were implanted in rats in bone and subcutaneous tissues. The implant sites were excised after 7, 30 and 120 days, fixed, dehydrated, embedded in paraffin wax for serial cutting and examined under transmitted light microscope. It was found a very similar behavior for both wollastonite bioceramics. They were biocompatible, bioactive and biodegradable when implanted in rat bone. The synthetic ceramic was more reabsorbable than the one from natural powder. When implanted in subcutaneous rat tissue, both materials elicited a mild initial inflammatory reaction that practically disappeared after 120 days. Both materials were encapsulated with a very thin fibrous capsule and slightly reabsorbed at their surfaces. None of the materials induced ectopic osteogenesis. According to the results, the studied materials seem to be able for manufacturing reabsorbable bone implants.

In vivo biological behavior of calcium phosphate cements with different Ca/P ratio

Bioceramics with different Ca/P ratio were prepared from a mechanical mixture of NaPO3, CaCO3, Ca(OH)2 and phosphate buffer solution and implanted in rats subcutaneous tissues. The cements were characterized by Thermo gravimetric analysis (TG-TDA), X-ray diffraction and 31P-NMR. The implant sites were excised after 1, 4 and 16 weeks, fixed, dehydrated, included in paraffin wax for serial cutting and examined under the light transmitted microscope. They were biocompatible and biodegradable when implanted in rat subcutaneous. None of the materials induced ectopic osteogenesis. According to the results, the studied materials seem to be able for manufacturing reabsorbable bone implants.

Observation of tags and hybrid layer of a single bottle conventional adhesive system and a self-etching adhesive system, on sound dentin.

This experimental light microscopy study investigated the formation of a hybrid layer and resin tags on sound dentin, after utilization of conventional and self-etching adhesive systems. After restorative procedures, the specimens were decalcified in a formic acid and sodium citrate solution, embedded in paraffin, sectioned at 6-microm thickness and stained by the Brown & Brenn method for analysis and measurement by light microscopy (AXIOPHOT) (400x). The results were statistically analyzed by analysis of variance, at a significance level of 5%. Based on the results, it could be concluded that the conventional adhesive allowed the formation of a thicker hybrid layer than the self-etching adhesive, with similar penetration into the dentinal tubules (resin tags).

Cimetidine-induced vascular cell apoptosis impairs testicular microvasculature in adult rats

Cimetidine, an H2 receptor antagonist used for treatment of gastric ulcers, exerts antiandrogenic and antiangiogenic effects. In the testes cimetidine impairs spermatogenesis, Sertoli cells and peritubular tissue, inducing apoptosis in the myoid cells. Regarding the importance of histamine and androgens for vascular maintenance, the effect of cimetidine on the structural integrity of the testicular vasculature was evaluated. Adult male rats received cimetidine (CMTG) and saline (CG) for 50 days. The testes were fixed in buffered 4% formaldehyde and embedded in historesin and paraffin. In the PAS-stained sections, the microvascular density (MVD) and the vascular luminal area (VLA) were obtained. TUNEL method was performed for detection of cell death. Testicular fragments embedded in Araldite were analyzed under transmission electron microscopy. A significant decrease in the MVD and VLA and a high number of collapsed blood vessel profiles were observed in CMTG. Endothelial cells and vascular muscle cells were TUNEL-positive and showed ultrastructural features of apoptosis. These results indicate that cimetidine induces apoptosis in vascular cells, leading to testicular vascular atrophy. A possible antagonist effect of cimetidine on the H2 receptors and/or androgen receptors in the vascular cells may be responsible for the impairment of the testicular microvasculature.

Primordial germ cells (spermatogonial stem cells) of bullfrogs express sex hormone-binding globulin and steroid receptors during seasonal spermatogenesis

In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers-alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor)-and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)β was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERβ expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERβ, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus. © 2012 S. Karger AG, Basel.

Comparative study of the behavior of p53 immunoexpression in smoking associated lesions: Reinke's edema and laryngeal carcinoma

Objective: To assess the behavior of the immunoexpression of protein p53 in Reinke's edema and laryngeal squamous cell carcinoma. Study design: retrospective. Methods: we recovered the histological paraffin blocks of patients who were subjected to Reinke's edema and laryngeal squamous cell carcinoma surgery in 2000-2011. The paraffin blocks were cut into 3-μm sections; the specimens were prepared in silanized slides (one slide for each paraffin block) and subjected to immunohistochemical reaction according to the Avidin Biotin Peroxidase method. Monoclonal primary anti-p53 antibodies were used at 1:50 dilution. Slides were examined under a light microscope at different magnitudes and results were interpreted based on the degree of brown staining in the nuclei of epithelial cells and in the extent of the fragment by using a semi-quantitative score from 0 to 3. Results: 67 slides of Reinke's edema and 60 slides of laryngeal squamous cell carcinoma were included. Scores 2 and 3 for staining of the nuclei of epithelial cells were recorded for 46 slides of Reinke's edema (68.65%) and for 57 slides of laryngeal squamous cell carcinoma (95%). As to the extent of the fragment, scores 2 and 3 were recorded for 74% slides of Reinke's edema and for 95% slides of carcinomas. Conclusion: the positive immunoexpression for protein p53, positive in 95% carcinomas and 74% Reinke's edemas, makes us aware of the possible preneoplastic condition of the latter lesion. Further studies are needed to identify and reveal the genetic changes that lead to these results. © 2013 Informa Healthcare USA, Inc.

Immunohistochemical features of multifocal melanoacanthoma in the hard palate: A case report

Background: Melanoacanthoma (MA) has been described in the oral mucosa as a solitary lesion or, occasionally, as multiple lesions. MA mainly affects dark skinned patients and grows rapidly, showing a plane or slightly raised appearance and a brown to black color. The differential diagnosis includes oral nevi, amalgam tattoos, and melanomas. We report here the case of a 58-year-old black woman who presented multiple pigmented lesions on the hard palate. Case presentation. Based on the differential diagnosis of melanoma, a punch biopsy (4 mm in diameter) was performed. The material was fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin-eosin or submitted to immunohistochemical analysis. Immunohistochemistry using antibodies against protein S-100, melan-A, HMB-45, MCM-2, MCM-5, Ki-67 and geminin was performed. Immunohistochemical analysis revealed strong cytoplasmic immunoreactivity of dendritic melanocytes for proteinS-100, HMB-45 and melan-A.Positive staining for proliferative markers (MCM-2, MCM-5, Ki-67) was only observed in basal and suprabasal epithelial cells, confirming the reactive etiology of the lesion. The diagnosis was oral Melanoacanthoma (MA). Conclusion: The patient has been followed up for 30 months and shows no clinical alterations. MA should be included in the differential diagnosis of pigmented lesions of the oral cavity. © 2013 das Chagas e Silva de Carvalho et al.; licensee BioMed Central Ltd.

Immunoexpression of aromatase and estrogen receptors β in stem spermatogonia of bullfrogs indicates a role of estrogen in the seasonal spermatogonial mitotic activity

Bullfrog stem spermatogonia, also named primordial germ cells (PGCs), show strong testosterone immunolabeling in winter, but no or weak testosterone immunoexpression in summer. Thus, the role of testosterone in these cells needs to be clarified. In this study, we proposed to evaluate whether PGCs express aromatase and estrogen receptors, and verify a possible role of estrogen in PGCs seasonal proliferation. Testes of male adult bullfrogs, collected in winter (WG) and summer (SG), were fixed and embedded in historesin, for quantitative analysis, or paraffin for immunohistochemistry (IHC). The number of haematoxylin/eosin stained PGCs/lobular area was obtained. Proliferating cell nuclear antigen (PCNA), aromatase, estrogen receptor β (ERβ) and PCNA/ERβ double immunolabeling were detected by IHC. The number of PCNA-positive PGCs and the histological score (HSCORE) of aromatase and ERβ immunolabeled PGCs were obtained. Although the number of PGCs increased significantly in WG, a high number of PCNA-positive PGCs was observed in summer. Moreover, aromatase and ERβ HSCORE was higher in SG than WG. The results indicate that PGCs express a seasonal proliferative activity; the low mitotic activity in winter is related to the maximal limit of germ cells which can be supported in the large lobules. In SG, the increased ERβ and aromatase HSCORE suggests that testosterone is converted into estrogen from winter to summer. Moreover, the parallelism between the high PGCs mitotic activity and ERβ immunoexpression suggest a participation of estrogen in the control of the PGCs seasonal proliferative activity which guarantee the formation of new germ cysts from summer to next autumn. © 2012 Elsevier Inc.

Increased apoptosis in osteoclasts and decreased RANKL immunoexpression in periodontium of cimetidine-treated rats

It has been demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H1- and H2-receptors. Cimetidine is a H2-receptor antagonist used for treatment of gastric ulcers that seems to prevent bone resorption. In this study, a possible cimetidine interference was investigated in the number of alveolar bone osteoclasts. The incidence of osteoclast apoptosis and immunoexpression of RANKL (receptor activator of nuclear factor κB ligand) was also evaluated. Adult male rats were treated with 100mg kg-1 of cimetidine for 50days (CimG); the sham group (SG) received saline. Maxillary fragments containing the first molars and alveolar bone were fixed, decalcified and embedded in paraffin. The sections were stained by H&E or submitted to tartrate-resistant acid phosphatase (TRAP) method. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) method and immunohistochemical reactions for detecting caspase-3 and RANKL were performed. The number of TRAP-positive osteoclasts, the frequency of apoptotic osteoclasts and the numerical density of RANKL-positive cells were obtained. Osteoclast death by apoptosis was confirmed by transmission electron microscopy (TEM). In CimG, TRAP-positive osteoclasts with TUNEL-positive nuclei and caspase-3-immunolabeled osteoclasts were found. A significant reduction in the number of TRAP-positive osteoclasts and a high frequency of apoptotic osteoclasts were observed in CimG. Under TEM, detached osteoclasts from the bone surface showed typical features of apoptosis. Moreover, a significant reduction in the numerical density of RANKL-positive cells was observed in CimG. The significant reduction in the number of osteoclasts may be due to cimetidine-induced osteoclast apoptosis. However, RANKL immunoexpression reduction also suggests a possible interference of cimetidine treatment in the osteoclastogenesis. © 2012 The Authors Journal of Anatomy © 2012 Anatomical Society.

NF-kB overexpression and decreased immunoexpression of AR in the muscular layer is related to structural damages and apoptosis in cimetidine-treated rat vas deferens

Background: Cimetidine, histamine H2 receptors antagonist, has caused adverse effects on the male hormones and reproductive tract due to its antiandrogenic effect. In the testes, peritubular myoid cells and muscle vascular cells death has been associated to seminiferous tubules and testicular microvascularization damages, respectively. Either androgen or histamine H2 receptors have been detected in the mucosa and smooth muscular layer of vas deferens. Thus, the effect of cimetidine on this androgen and histamine-dependent muscular duct was morphologically evaluated.Methods: The animals from cimetidine group (CMTG; n=5) received intraperitoneal injections of 100 mg/kg b.w. of cimetidine for 50 days; the control group (CG) received saline solution. The distal portions of vas deferens were fixed in formaldehyde and embedded in paraffin. Massońs trichrome-stained sections were subjected to morphological and the following morphometrical analyzes: epithelial perimeter and area of the smooth muscular layer. TUNEL (Terminal deoxynucleotidyl-transferase mediated dUTP Nick End Labeling) method, NF-kB (nuclear factor kappa B) and AR (androgen receptors) immunohistochemical detection were also carried out. The birefringent collagen of the muscular layer was quantified in picrosirius red-stained sections under polarized light. The muscular layer was also evaluated under Transmission Electron Microscopy (TEM).Results: In CMTG, the mucosa of vas deferens was intensely folded; the epithelial cells showed numerous pyknotic nuclei and the epithelial perimeter and the area of the muscular layer decreased significantly. Numerous TUNEL-labeled nuclei were found either in the epithelial cells, mainly basal cells, or in the smooth muscle cells which also showed typical features of apoptosis under TEM. While an enhanced NF-kB immunoexpression was found in the cytoplasm of muscle cells, a weak AR immunolabeling was detected in these cells. In CMTG, no significant difference was observed in the birefringent collagen content of the muscular layer in comparison to CG.Conclusions: Cimetidine induces significant damages in the epithelium; a possible antiandrogenic effect on the basal cells turnover should be considered. The cimetidine-induced muscle cells apoptosis confirms the susceptibility of these cells to this drug. The parallelism between enhanced cytoplasmic NF-kB immunolabeling in the damaged muscular tissue and muscle cell apoptosis suggests that this drug may avoid the translocation of NF-kB to the nucleus and interfere in the control of NF-kB-mediated smooth muscle cell apoptosis. The decreased immunoexpression of ARs verified in the damaged muscular tissue reinforces this possibility. © 2013 Koshimizu et al.; licensee BioMed Central Ltd.

A useful panel for the diagnosis of Hirschsprung disease in rectal biopsies: calretinin immunostaining and acetylcholinesterase histochesmistry

The pathological evaluation of rectal biopsies for the diagnosis of Hirschsprung disease has been a challenging issue. We analyzed prospectively the usefulness of calretinin immunostaining and acetylcholinesterase (AChE) histochesmistry in rectal biopsies for the diagnosis of Hirschsprung disease. Frozen tissue samples from 43 patients were used for AChE histochemistry and paraffin-embedded sections for calretinin immunohistochemistry and conventional histology (hematoxylin and eosin [H&E]). Activity for AChE, was demonstrated in 13 of 43 cases, and the absence of immunoreactivity for calretinin was observed in 14 of 43 cases. Conventional histology (H&E) did not reveal ganglion cells in 24 of 43 cases. The results on calretinin were in good agreement with AChE according to the κ index (0.946; P < .001) and presented significantly higher specificity (96.7 × 63.3; P < .002) and accuracy (97.6 × 74.4; P < .003) when compared with conventional histology (H&E). The final diagnosis of Hirschsprung disease was confirmed in 13 of 43 patients who were submitted to surgical treatment. The results of the present study indicate that calretinin can be a good tool in ruling out the diagnosis of Hirschsprung disease, by showing positive staining in ganglion cells and intrinsic nerve fibers, whereas AChE is useful in confirming the diagnosis of Hirschsprung disease, by revealing activity of this enzyme in hypertrophied nerve fibers. The association between calretinin and AChE can be a useful panel for the histopathologic evaluation of rectal biopsies for the diagnosis of Hirschsprung disease. © 2013 Elsevier Inc. All rights reserved.