Bioactive potential of Bio-C Temp demonstrated by systemic mineralization markers and immunoexpression of bone proteins in the rat connective tissue.

Intracanal medications are used in endodontic treatment due to their antibacterial activity and ability to induce the periapical repair. Among the intracanal medications, the Calen (CAL; SS. White, Brazil) is a calcium hydroxide-based medication that provides an alkaline pH and releases calcium, exerting an antimicrobial activity. Bio-C Temp (BIO; Angelus, Brazil), a ready-to-use bioceramic intracanal medication, was designed to stimulate the mineralized tissues formation. Here, we investigated the bioactive potential of BIO in comparison to the CAL in the rat subcutaneous. Polyethylene tubes filled with medications, and empty tubes (control group, CG) were implanted in the subcutaneous tissue of rats. After 7, 15, 30 and 60 days, the blood was collected for calcium (Ca+2) and alkaline phosphatase (ALP) measurement, and the capsules around the implants were processed for morphological analyses. The data were submitted to two-way ANOVA and Tukey test (p < 0.05). At 7, 15 and 30 days, the ALP level was grater in BIO and CAL than in CG (p < 0.0001). At 7 and 15 days, greater Ca+2 level was seen in the serum of CAL samples. From 7 to 60 days, an increase in the number of fibroblasts, osteocalcin- and osteopontin-immunolabelled cells was observed in BIO and CAL groups (p < 0.0001). In all periods, BIO and CAL specimens showed von Kossa-positive structures. Moreover, ultrastructural analysis revealed globules of mineralization in the capsules around the BIO and CAL specimens. Thus Bio-C Temp caused an increase in the ALP, osteocalcin and osteopontin, which may have allowed the formation of calcite, suggesting bioactive potential.

Role of serotonin, estrogen, and TNF-α in the paroxetine-impaired steroidogenesis and testicular macrophages polarization.

BACKGROUND: Paroxetine, a selective serotonin reuptake inhibitor (SSRI) antidepressant, has caused male sexual dysfunction; however, the paroxetine mechanisms of action in testes are still unclear. OBJECTIVES: Paroxetine serotonergic effects in testes were evaluated, focusing on steroidogenesis and the correlation between macrophages population and possible TNF-α-derived oxidative stress. We also verified whether the changes are reversible following treatment interruption. MATERIALS AND METHODS: Adult rats received paroxetine (PG35 and PG65) or tap water (CG) for 35 days. PG65 was maintained without treatment for 30 more days. Intratesticular testosterone (IT), nitrite, and malondialdehyde concentrations were measured. To confirm serotonergic and estrogenic effects, Htr1b and Esr1 expressions were analyzed. The daily sperm production (DSP), frequency of abnormal seminiferous tubules (ST), SC number, ST area, and Leydig cells nuclear area (LCnu) were evaluated. TUNEL+ germ cells, M1 (CD68+ ), and M2 (Perls+ ) macrophages were quantified. 17ß-HSD7, CYP19A1, NDRG2, oxytocin, TNF-α, and iNOS were evaluated by immunoreactions. Oxytocin and NDRG2 protein levels as well as Tnfa mRNA expression were also analyzed. RESULTS: The Htr1b downregulation in testes confirmed the paroxetine serotonergic effect. The testicular sections showed abnormal ST frequency, ST atrophy and reduction of DSP, LCnu, SC number and Perls+ macrophages. TUNEL+ germ cells and LC were associated with strong NDRG2 immunoexpression. Paroxetine reduced IT levels and 17ß-HSD7 immunoexpression in parallel to increased CYP19A1, oxytocin, TNF-α and iNOS. Esr1 and Tnfa overexpression and increased number of CD68+ macrophages were also observed together with high nitrite and malondialdehyde levels. Most parameters were not recovered in PG65. CONCLUSIONS: Paroxetine serotonergic effect impairs LC steroidogenesis, via aromatization, increasing estrogen/testosterone ratio, which in turn upregulate NDRG2, promoting apoptosis, and impairing sperm production. Serotonin-estrogen pathways may be responsible for M2/M1 polarization, Tnfa upregulation, and induction of oxidative stress. The unrecovered testicular changes after treatment discontinuation are due to persistent paroxetine serotonin/estrogen effects.

Additional Insights Into the Role of Osteocalcin in Osteoblast Differentiation and in the Early Steps of Developing Alveolar Process of Rat Molars.

This study investigated whether osteocalcin (OCN) is present in osteoblast precursors and its relationship with initial phases of alveolar process formation. Samples of maxillae of 16-, 18-, and 20-day-old rat embryos (E16, E18, and E20, respectively), and 05-, 10-, and 15-day-old postnatal rats (P05, P10, and P15, respectively) were fixed and embedded in paraffin or araldite. Immunohistochemistry for osterix (Osx), alkaline phosphatase (ALP), and OCN detection was performed and the number of immunolabelled cells was computed. Non-decalcified sections were subjected to the von Kossa method combined with immunohistochemistry for Osx or OCN detection. For OCN immunolocalization, samples were fixed in 0.5% glutaraldehyde/2% formaldehyde and embedded in LR White resin. The highest number of ALP- and OCN-immunolabelled cells was observed in dental follicle of E16 specimens, mainly in basal portions of dental alveolus. In corresponding regions, osteoblasts in differentiation adjacent to von Kossa-positive bone matrix exhibited Osx and OCN immunoreactivity. Ultrastructural analysis revealed OCN immunoreactive particles inside osteoblast in differentiation, and in bone matrix associated with collagen fibrils and within matrix vesicles, at early stages of alveolar process formation. Our results indicate that OCN plays a role in osteoblast differentiation and may regulate calcium/phosphate precipitation during early mineralization of the alveolar process.

Possible role of annexin A1/FPR2 pathway in COX2/NLRP3 inflammasome regulation in alveolar bone cells of estrogen-deficient female rats with diabetes mellitus.

BACKGROUND: Annexin A1 (ANXA1) and the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome play important roles in bone remodeling. However, expression profiles of these factors in bone cells under diabetes mellitus (DM) and estrogen-deficient conditions are poorly understood. This study investigated the immunoexpression of ANXA1 and its formyl peptide receptor 2 (FPR2), as well as NLRP3 inflammasome mediators, during remodeling of the alveolar process in diabetic and estrogen-deficient rats. METHODS: Twenty adult female Wistar rats were divided into four groups (n = 5): Sham-operated (SHAM) and ovariectomized (OVX) rats received a vehicle solution, and SHAM and OVX rats were intraperitoneally administered 60 mg/kg/body weight (BW) of streptozotocin (STZ) to induce DM (SHAM-Di and OVX-Di groups). After 7 weeks, the rats were euthanized and their maxillae were fixed in phosphate-buffered 4% formaldehyde and embedded in paraffin. Sections were stained with hematoxylin/eosin (H&E) and picrosirius red or subjected to immunohistochemical detection of ANXA1, FPR2, NLRP3, interleukin-1ß (IL-1ß), and cyclooxygenase-2 (COX2). RESULTS: Estrogen deficiency and DM were associated with deleterious effects in bone tissue, as evidenced by a lower number of osteocytes and higher number of empty lacunae in the SHAM-Di and OVX-Di groups compared to the nondiabetic groups. Both diabetic groups showed a smaller vascular area and weaker collagen fiber birefringence intensity in alveolar bone tissue. A significantly higher number of ANXA1/FPR2-positive osteoblasts, osteocytes, and osteoclasts was accompanied by a significantly higher number of these cells immunolabeled for COX2, NLRP3, and IL-1ß in the diabetic and OVX groups, especially in both estrogen-deficient and diabetic rats. CONCLUSION: These results indicate a possible role for the ANXA1/FPR2 pathway as a fine-tuning/anti-inflammatory regulator to counterbalance exacerbated COX2/NLRP3/IL-1ß activation in bone cells during bone remodeling under estrogen deficiency and DM.

Venlafaxine increases aromatization, reduces apical V-ATPase in clear cells and induces increased number of mast cells and smooth muscle cells death in rat cauda epididymis.

Depressive disorders (DD) have affected millions of people worldwide. Venlafaxine, antidepressant of the class of serotonin and norepinephrine reuptake inhibitors, has been prescribed for the treatment of DD. In rat testes, venlafaxine induces testosterone (T) aromatization and increases estrogen levels. Aromatase is a key enzyme for the formation of estrogen in the epididymis, an essential organ for male fertility. We investigated the impact of serotonergic/noradrenergic venlafaxine effect on the epididymal cauda region, focusing on aromatase, V-ATPase and EGF epithelial immunoexpression, smooth muscle (SM) integrity and mast cells number (MCN). Male rats were distributed into control (CG; n = 10) and venlafaxine (VFG, n = 10) groups. VFG received 30 mg/kg b.w. of venlafaxine for 35 days. The epididymal cauda was processed for light and transmission electron microscopy (TEM). The expression of connexin 43 (Cx43) and estrogen alpha (Esr1), adrenergic (Adra1a) and serotonergic (Htr1b) receptors were analyzed. Clear cells (CCs) area, SM thickness, viable spermatozoa (VS) and MCN were evaluated. Apoptosis was confirmed by TUNEL and TEM. The following immunoreactions were performed: T, aromatase, T/aromatase co-localization, V-ATPase, EGF, Cx43 and PCNA. The increased Adra1a and reduced Htr1b expressions confirmed the noradrenergic and serotonergic venlafaxine effects, respectively, corroborating the increased MCN, apoptosis and atrophy of SM. In VFG, the epithelial EGF increased, explaining Cx43 overexpression and basal cells mitotic activity. T aromatization and Esr1 downregulation indicate high estrogen levels, explaining CCs hypertrophy and changes in the V-ATPase localization, corroborating VS reduction. Thus, in addition to serotonergic/noradrenergic effects, T/estrogen imbalance, induced by venlafaxine, impairs epididymal structure and function.

Biocompatibility and bioactive potential of an experimental tricalcium silicate-based cement in comparison with Bio-C repair and MTA Repair HP materials.

AIM: To evaluate the tissue reaction of a tricalcium silicate-based repair material associated with 30% calcium tungstate (TCS + CaWO4 ) in comparison to Bio-C Repair (Bio-C; Angelus) and to MTA Repair HP (MTA HP; Angelus). METHODOLOGY: Polyethylene tubes filled with one of the materials or left empty (control group, CG) were implanted into the subcutaneous tissues of rats for 7, 15, 30 and 60 days (n = 32/group). The capsule thickness, number of inflammatory cells, collagen content, interleukin-6 (IL-6), osteocalcin (OCN), von Kossa reaction and analysis under polarized light were evaluated. The data were subjected to generalized linear models for repeated measures, except the OCN. OCN data were submitted to Kruskal-Wallis and Dunn's post hoc test and Friedman followed by Nemenyi's test at significance level of 5%. RESULTS: At all time points, significant differences in the number of inflammatory cells were not observed between TCS + CaWO4 and Bio-C, whereas, at 15, 30 and 60 days, no significant difference was detected between TCS + CaWO4 and MTA HP. At all periods, significant differences were not detected in the number of fibroblasts in TCS + CaWO4 versus MTA HP, and, at 60 days, no significant difference was demonstrated between these groups and CG. Significant differences in the immunoexpression of IL-6 were not detected amongst bioceramic materials at all periods. From 7 to 60 days, significant reduction in the number of inflammatory cells, number of IL-6-immunopositive cells and in the capsule thickness was accompanied by significant increase in the collagen in all groups. OCN-immunolabelled cells, von Kossa-positive structures and amorphous calcite deposits were observed around all materials, whereas, in the CG, these structures were not seen. CONCLUSIONS: These findings indicate that the experimental material (TCS + CaWO4 ) is biocompatible and has a bioactive potential, similar to the MTA HP and Bio-C Repair, and suggest its use as a root repair material.

Participation of fibroblast growth factor-1 and interleukin-10 in connective tissue repair following subcutaneous implantation of bioceramic materials in rats.

AIM: To evaluate whether the bioceramic materials Bio-C Pulpo (Bio-C, Angelus) and mineral trioxide aggregate (MTA) Repair HP (MTA-HP, Angelus) induce fibroblast proliferation and release of interleukin-10 (IL-10), an anti-inflammatory cytokine, stimulating connective tissue remodelling. The tissue response of Bio-C and MTA-HP was compared with the White MTA (WMTA; Angelus) since studies have demonstrated that WMTA induces tissue repair. METHODOLOGY: Bio-C, MTA-HP and WMTA were inserted into polyethylene tubes and implanted in the subcutaneous tissue of Holtzman rats for 7, 15, 30 and 60 days. As a control group (CG), empty tubes were implanted subcutaneously. The number of fibroblasts (FB), Ki-67-, fibroblast growth factor-1- (FGF-1) and IL-10-immunolabelled cells and collagen content in the capsules was obtained. The data were subjected to two-way anova followed by Tukey's test (p ≤ .05). RESULTS: At 7 days, significant differences in the number of FB were not detected amongst Bio-C, MTA-HP and WMTA groups (p Ëƒ .05). The capsules of all groups exhibited a significant increase in the number of FB and content of collagen over time. From 7 to 60 days, a significant reduction in the number of FGF-1- and Ki-67-immunolabelled cells was seen in the capsules of all specimens. In all periods, no significant difference in the number of FGF-1-immunolabelled cells was detected between Bio-C and CG specimens. At 60 days, significant differences in the immunoexpression of FGF-1 were not observed amongst the groups. At 7 and 15 days, the highest immunoexpression for Ki-67 was present in Bio-C specimens whilst, after 30 and 60 days, no significant difference was observed amongst the bioceramic materials. At 7 days, few IL-10 immunolabelled cells were present in the capsules of all specimens whereas, at 60 days, a significant increase in the IL-10-immunostaining was present in all groups. At 60 days, the Bio-C, MTA-HP and WMTA groups showed a greater number of IL-10-immunolabelled cells than in the CG specimens (p < .0001). CONCLUSIONS: Bio-C, MTA-HP and WMTA stimulate fibroblast proliferation, leading to the formation of collagen-rich capsules. FGF-1 and IL-10 may mediate the remodelling of capsules around Bio-C, MTA-HP and WMTA bioceramic materials.

Bioactive potential of Bio-C Pulpo is evidenced by presence of birefringent calcite and osteocalcin immunoexpression in the rat subcutaneous tissue.

As the biocompatibility and bioactive potential of repair materials are desired characteristics in dentistry, the tissue response of Bio-C Pulpo, a bioceramic material launched on the marked by Angelus (Brazil), was compared with Biodentine (Septodont, France) and White MTA (WMTA; Angelus, Brazil). In 32 rats, 148 polyethylene tubes filled with Bio-C Pulpo, Biodentine or WMTA, and empty (CG, control group) were implanted into subcutaneous tissues for 7, 15, 30, and 60 days. The capsule thickness, numerical density of inflammatory cells (IC) and fibroblasts (Fb), amount of collagen, immunohistochemistry detection of interleukin-6 (IL-6) and osteocalcin (OCN), von Kossa and analysis under polarized light were performed. Data were subjected to two-way ANOVA followed by Tukey's test (p ≤ 0.05). At 7 and 15 days, the capsules around Bio-C Pulpo were thicker than in WMTA while, at 30 and 60 days, significant differences were not observed among the groups. Although at 7, 15, and 30 days, a greater number of IL-6-immunostained cells was found in Bio-C Pulpo and Biodentine than in WMTA, no significant difference was detected among the groups at 60 days. In all groups, the number of Fb and collagen content increased significantly over time. The capsules around materials exhibited von Kossa-positive and birefringent structures, and OCN-immunostained cells whereas, in the CG, these structures were not observed. Bio-C Pulpo, similarly to Biodentine and WMTA, is biocompatible, allows the connective tissue repair and presents bioactive potential in connective tissue of rats.

Bilateral asymmetry in bullfrog testes and fat bodies: correlations with steroidogenic activity, mast cells number and structural proteins.

In seasonal breeders, such as amphibians, testicular functions depend on complex processes that change according to seasonality, including Leydig cell (LC) differentiation and lipid-dependent steroidogenesis, extracellular proteins remodeling and actin-dependent cellular dynamics. Speculating that fat bodies (FB) could support some of these processes in L. catesbeianus, we evaluated bilaterally the FB weights, correlating them to testicular parameters such as weight, testosterone (T) immunoexpression, mast cells (MC) number, vascularization and structural proteins. In an attempt to better understand the testicular asymmetry in amphibians, correlations between these different testicular parameters were also established. Right testes (RT), left testes (LT) and associated FB of bullfrogs were weighed, and testes were processed for light and transmission electron microscopy. Collagen content (COL) and MC number were quantified. T and actin immunoexpressions and vascular areas were measured. Statistical analyses and Pearson's correlation were performed. The LT and its associated FB were heavier than the right ones, and showed intense T and actin immunoexpressions, numerous lipid-rich LC, and greater MC number, COL and vascularization than the RT. Positive correlations were detected between: a) FB and testis weights, b) T immunoexpression and testis and FB weights, c) T and actin immunoexpressions and COL. Otherwise, MC number was inversely correlated to T immunoexpression and COL. In right and left sides, the proportional correlation between T immunoexpression and FB weight suggests that FB-stored lipid amount depends on the steroidogenic demand of its associated testis. Thus, the asymmetry in the testes and FB may be associated, at least in part, to the LC steroidogenic activity, which tends to be more intense in LT than in RT. The results also point to a role of COL and mast cells in the LC differentiation and steroidogenesis. Actin was also greater in LT and correlated with T immunoexpression, indicating that the amount of this structural protein depends on androgenic control. Therefore, the testicular asymmetry in bullfrogs seems to be associated to different morphofunctional processes occurring, bilaterally, at different intensities. In this case, there is a tendency of LT, in association with its FB, to be more active than RT. The findings highlight the FB-testis interplay for the comprehension of reproduction in amphibians.

Glioblastoma multiforme targeted delivery of docetaxel using bevacizumab-modified nanostructured lipid carriers impair in vitro cell growth and in vivo tumor progression.

Glioblastoma multiforme (GBM) is the most common malignant brain cancer, characterized by high invasiveness and poor prognosis. Docetaxel (DTX) is a chemotherapeutic drug with promising anti-tumor properties. However, conventional intravenous formulations exhibit side effects of systemic biodistribution and low brain bioavailability, limiting their clinical use. The current work aimed to evaluate the effect of DTX-loaded nanostructured lipid carriers (NLC) functionalized with bevacizumab (BVZ-NLC-DTX) against GBM using in vitro and in vivo models. The NLC was obtained by the fusion-emulsification method followed by sonication, with narrow size distribution, negative zeta potential, and low polydispersity index. NLC showed DTX entrapment efficiency above 90%. BVZ coupling efficiency was 62% and BVZ integrity after functionalization was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Calorimetry studies confirmed thermal stability and molecular dispersion of DTX in the lipid matrix. NLC showed a sustained DTX release over 84 h. In vitro anti-tumor assays shown that BVZ-NLC-DTX selectively increased the cytotoxic of DTX in cells overexpressing VEGF (U87MG and A172), but not in peripheral blood mononuclear cells (PMBCs), promoting cell death by apoptosis. BVZ functionalization did not impair cellular uptake. An in vivo orthotopic rat model demonstrated that free-DTX was not capable of reducing tumor growth whereas BVZ-NLC-DTX reduced up to 70% tumor volume after 15-days of treatment. Therefore, this study contributes to understanding new nanotechnology-based vehicles capable of reaching the brain more efficiently and repurposing the use of anti-cancer drugs in GBM treatment.

Diacerein-induced interleukin-1ß deficiency reduces the inflammatory infiltrate and immunoexpression of matrix metalloproteinase-8 in periodontitis in rat molars.

BACKGROUND: Periodontitis is an immunoinflammatory disease that involves the release of cytokines and enzymes, including interleukin-1 (IL-1) and matrix metalloproteinases (MMPs). Diacerein is an anti-IL-1 drug used for the treatment of osteoarthritis. The aim of the study was to evaluate whether diacerein suppresses the inflammatory reaction and reduces the collagen degradation in the gingival connective tissue in periodontitis. METHODS: Fifty-four male rats were distributed into three groups (n = 18 animals/group): 1) periodontitis + diacerein group (PDG), 2) periodontitis + saline group (PSG) and control Group (CG; without treatment). Periodontitis was induced for 7 days in the upper right first molars; after 7 days, the animals of PDG received 100 mg/kg of diacerein while in PSG, the animals received saline solution for 7, 15, and 30 days. The animals were killed and fragments of maxilla containing the right molars were processed for paraffin embedding. In hematoxlin & eosin-stained sections, the volume density of inflammatory cells (VvIC) and fibroblasts (VvFb) in the gingival mucosa, the distances between the junctional epithelium (JE) to the cemento-enamel junction (CEJ) and the CEJ to the alveolar process crest (AP) were obtained. The number of IL-1ß- and MMP-8-immunolabeled cells, and collagen content in the gingival mucosa were computed. Data were subjected to two-way analysis of variance and Tukey post-test (P ≤ 0.05). RESULTS: The PDG and PSG rats showed a significant increase in the distances of JE-CEJ and CEJ-AP. In all periods, the VvIC, the number of IL-1ß- and MMP-8-immunolabeled cells was significantly lower in PDG than in PSG while the collagen content was significantly greater in PDG than PSG. At 30 days, significant differences in the IL-1ß immunoexpression, collagen content, and in the MMP-8 immunostaining were not seen between PDG and CG groups. CONCLUSIONS: Our results show an inhibitory effect of diacerein on IL-1ß in the inflamed gingival mucosa of rat molars, decreasing the inflammatory infiltrate and immunoexpression of MMP-8, and restoring the collagen content.

Hepatic enzymes and immunoinflammatory response to Bio-C Temp bioceramic intracanal medication implanted into the subcutaneous tissue of rats.

Our purpose was to evaluate the biocompatibility and hepatotoxicity of a new bioceramic intracanal medicament, Bio-C Temp (BIO). The biological properties of BIO were compared with calcium hydroxide-based intracanal medicament (Calen; CAL), used as gold pattern. Polyethylene tubes filled with BIO or CAL, and empty tubes (control group, CG) were implanted into subcutaneous tissue of rats. After 7, 15, 30 and 60 days, the samples were embedded in paraffin for morphological, quantitative and immunohistochemistry analyses. At 7 and 60 days, blood samples were collected for analysis of serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels. The data were submitted to two-way ANOVA and Tukey's test (p ≤ 0.05). No significant difference was detected in serum GOT and GPT levels among BIO, CAL and CG specimens. In all periods, BIO specimens exhibited lower number of inflammatory cells and immunoexpression of IL-6, a pro-inflammatory cytokine, than CAL specimens. The reduction of these parameters was accompanied by significant increase in the collagen content and in the immunoexpression of IL-10, a cytokine involved in the tissue repair, over time. Our findings indicate that Bio-C Temp is biocompatible and had no hepatotoxicity effect.

Venlafaxine-induced adrenergic signaling stimulates Leydig cells steroidogenesis via Nur77 overexpression: A possible role of EGF.

Venlafaxine, a norepinephrine and serotonin reuptake inhibitor, impairs rat sperm parameters, spermatogenesis and causes high intratesticular estrogen and testosterone levels, indicating that Leydig cells (LCs) may be a venlafaxine target. We evaluated the effect of venlafaxine treatment on rat LCs, focusing on adrenergic signaling, EGF immunoexpression and steroidogenesis. Germ cells mitotic/meiotic activity and UCHL1 levels were also evaluated in the seminiferous epithelium. Eighteen adult male rats received 30 mg/kg of venlafaxine (n = 9) or distilled water (n = 9). The seminiferous tubules, epithelium and LCs nuclear areas were measured, and the immunoexpression of Ki-67, UCHL1, StAR, EGF, c-Kit and 17ß-HSD was evaluated. UCHL1, StAR and EGF protein levels and Adra1a, Nur77 and Ndrg2 expression were analyzed. Malondialdehyde (MDA) and nitrite testicular levels, and serum estrogen and testosterone levels were measured. Venlafaxine induced LCs hypertrophy and Ndrg2 upregulation in parallel to increased number of Ki-67, c-Kit- and 17ß-HSD-positive interstitial cells, indicating that this antidepressant stimulates LCs lineage proliferation and differentiation. Upregulation of Adra1a and Nur77 could explain the high levels of StAR and testosterone levels, as well as aromatization. Enhanced EGF immunoexpression in LCs suggests that this growth fact is involved in adrenergically-induced steroidogenesis, likely via upregulation of Nur77. Slight tubular atrophy and weak Ki-67 immunoexpression in germ cells, in association with high UCHL1 levels, indicate that spermatogenesis is likely impaired by this enzyme under supraphysiological estrogen levels. These data corroborate the unchanged MDA and nitrite levels. Therefore, venlafaxine stimulates LCs steroidogenesis via adrenergic signaling, and EGF may be involved in this process.

Impaired macrophages and failure of steroidogenesis and spermatogenesis in rat testes with cytokines deficiency induced by diacerein.

The role of cytokines in testicular function under normal conditions has not been completely understood. Here, we evaluated testicular macrophages (TM), steroidogenesis by Leydig cells (LC) and seminiferous tubules integrity in cytokines-deficient rat testes induced by diacerein, an anti-inflammatory drug that inhibits interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α). Male rats received daily 100 mg/kg of diacerein (DIAG; n = 8) or saline (CG; n = 8) for 30 days. Serum testosterone (T) levels were measured and the seminiferous tubule (ST) area, epithelial area (EA), frequency of damaged ST and number of Sertoli cells (SC) were evaluated. TUNEL method and immunoreactions for detection of pro-IL-1ß, TNF-α, steroidogenic acute regulatory protein (StAR), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), androgen receptor (AR) and scavenger receptor for hemoglobin-haptoglobin complexes (CD163), a TM marker, were performed. Testicular AR, 17ß-HSD and IL-1ß levels were detected by Western blot. Data were submitted to Student t test (p ≤ 0.05). In DIAG, T and testicular AR, 17ß-HSD and IL-1ß levels decreased significantly (p < 0.05). The number of TUNEL-positive interstitial cells increased and LC showed weak StAR, 17ß-HSD and AR immunoexpression in association with reduced IL-1ß immunoexpression and number of CD163-positive TM in the interstitial tissue from diacerein-treated rats. Numerous damaged ST were found in DIAG, and reduction in the EA were associated with germ cells death. Moreover, the number of SC reduced and weak AR and TNF-α immunoexpression was observed in SC and germ cells, respectively. The cytokines deficiency induced by diacerein impairs TM, LC and spermatogenesis, and points to a role of IL-1ß in steroidogenesis under normal conditions. In the ST, the weak AR and TNF-α immunoexpression in SC and germ cells, respectively, reinforces the idea that TNF-α plays a role in the SC androgenic control.

Spatio-temporal immunolocalization of VEGF-A, Runx2, and osterix during the early steps of intramembranous ossification of the alveolar process in rat embryos.

Vascular endothelial growth factor A (VEGF-A) is expressed by several cell types and is a crucial factor for angiogenic-osteogenic coupling. However, the immunolocalization of VEGF-A during the early stages of the alveolar process formation remains underexplored. Thus, we analyzed the spatio-temporal immunolocalization of VEGF-A and its relationship with Runt-related transcription factor 2 (Runx2) and osterix (Osx) during the early steps of intramembranous ossification of the alveolar process in rat embryos. Embryo heads (E) of 16, 18 and 20-day-old rats were processed for paraffin embedding. Histomorphometry and immunohistochemistry to detect VEGF-A, Runx2, and Osx (osteoblast differentiation markers) were performed. The volume density of bone tissue including bone cells and blood vessels increased significantly in E18 and E20. Cells showing high VEGF-A immunoreactivity were initially observed within a perivascular niche in the ectomesenchyme; afterwards, these cells were diffusely located near bone formation sites. Runx2-and Osx-immunopositive cells were observed in corresponded regions of cells showing strong VEGF-A immunoreactivity. Although these immunostained cells were observed in all specimens, this immunolocalization pattern was more evident in E16 specimens and gradually decreased in E18 and E20 specimens. Double immunofluorescence labelling showed intracellular co-localization of Osx and VEGF-A in cells surrounding the developing alveolar process, indicating a crucial role of VEGF-A in osteoblast differentiation. Our results showed VEGF-A immunoexpression in osteoblasts and its precursors during the maxillary alveolar process formation of rat embryos. Moreover, the VEGF-A-positive cells located within a perivascular niche at the early stages of the alveolar process development suggest a crosstalk between endothelium and ectomesenchymal cells, reinforcing the angiogenic-osteogenic coupling in this process.

Characterization of Female External Genitalia and Eggs of Four South American Species of the Triatoma Laporte, 1832 Genus (Hemiptera: Reduviidae: Triatominae).

Triatoma is the most diversified and one of the most important genera from an epidemiological perspective. Given the difficulty in identifying some species of the Triatoma genus, morphological, histological, and morphometric studies were performed to provide new characters that make it possible to differentiate T. garciabesi, T. guasayana, T. patagonica, and T. sordida sensu stricto, triatomines that overlap geographically and have vector potential. Through the external female genitalia, as well as morphology, morphometry, and histology of eggshells, it was possible to discriminate the four species. In addition, this study reinforces the taxonomic validity of T. garciabesi and provides new data for discussion on systematic issues of T. guasayana and T. patagonica.

Comparison of Bio-C Pulpo and MTA Repair HP with White MTA: effect on liver parameters and evaluation of biocompatibility and bioactivity in rats.

AIM: To evaluate the tissue response promoted by Bio-C Pulpo (Bio), MTA Repair HP (MTA-HP) and White MTA (WMTA) and whether these materials cause liver changes in a rat experimental model. METHODOLOGY: Polyethylene tubes filled with Bio, MTA-HP and WMTA, and empty tubes (control group, CG) were implanted into the subcutaneous tissues of rats for 7, 15, 30 and 60 days. Inflammatory reaction score (IRS), capsule thickness, number of inflammatory cells (IC), von Kossa reaction, interleukin-6 (IL-6) and alkaline phosphatase (ALP) immunohistochemistry reactions were performed. Combined methods, von Kossa followed by immunohistochemistry for detection of ALP, were performed. At 60 days, the serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels were measured and liver fragments were collected for histological analysis; the data were assessed by one-way ANOVA analysis followed by Sidak's post-test. The biocompatibility and bioactivity data were subjected to the two-way ANOVA analysis followed by Tukey post hoc test, except the IRS. The IRS data were subjected to the Kruskal-Wallis ANOVA non-parametric test followed by Dunn's test (p ≤ .05). RESULTS: No significant difference was detected in serum GOT and GPT concentrations and in the number of hepatocytes among the experimental and CG samples. Although Bio-C Pulpo had the highest IC and IL-6-immunolabelled cells (p < 0.0001) at all periods, no significant difference was observed in the IRS among the materials, except at 60 days. In this period, the WMTA had lower IRS. All groups had a significant reduction in the capsule thickness and in the number of IC and IL-6-immunolabelled cells over time. Bio-C Pulpo, MTA-HP and WMTA specimens had greater immunoexpression of ALP than CG (p < .0001). At all periods, von Kossa-positive and birefringent structures were observed in the capsules around the materials. ALP-immunolabelled cells were also seen near von Kossa-positive structures. CONCLUSIONS: Bio-C Pulpo, MTA-HP and WMTA materials did not cause morphological changes in the liver and no significant alteration in the serum GOT and GPT levels. Moreover, these bioceramic materials were biocompatible and exhibited bioactive potential. However, Bio-C Pulpo induced greater inflammatory infiltrate than MTA-HP and WMTA at all periods.

Cimetidine-induced androgenic failure causes cell death and changes in actin, EGF and V-ATPase immunoexpression in rat submandibular glands.

Submandibular gland (SMG) is responsive to androgens via androgen receptor (AR). We verified whether cimetidine induces androgenic dysfunction in SMG, and evaluated the structural integrity, cell death and immunoexpression of actin, EGF and V-ATPase in androgen-deficient SMG. Male rats received cimetidine (CMTG) and control animals (CG) received saline. Granular convoluted tubules (GCTs) diameter and number of acinar cell nuclei were evaluated. TUNEL and immunofluorescence reactions for detection of AR, testosterone, actin, EGF and V-ATPase were quantitatively analysed. In CG, testosterone immunolabelling was detected in acinar and ductal cells cytoplasm. AR-immunolabelled nuclei were observed in acinar cells whereas ductal cells showed AR-immunostained cytoplasm, indicating a non-genomic AR action. In CMTG, the weak testosterone and AR immunoexpression confirmed cimetidine-induced androgenic failure. A high cell death index was correlated with decreased number of acinar cells, GCTs diameter and EGF immunoexpression under androgenic dysfunction. Actin immunofluorescence decreased in the SMG cells, but an increased and diffuse cytoplasmic V-ATPase immunolabelling was observed in striated ducts, suggesting a disruption in the actin-dependent V-ATPase recycling due to androgenic failure. Our findings reinforce the androgenic role in the maintenance of SMG histophysiology, and point to a potential clinical use of cimetidine against androgen-dependent glandular tumour cells.

Biocompatibility and bioactive potential of the NeoMTA Plus endodontic bioceramic-based sealer.

OBJECTIVES: This study evaluated the biocompatibility and bioactive potential of NeoMTA Plus mixed as a root canal sealer in comparison with MTA Fillapex. MATERIALS AND METHODS: Polyethylene tubes filled with NeoMTA Plus (n = 20), MTA Fillapex (n = 20), or nothing (control group, CG; n = 20) were inserted into the connective tissue in the dorsal subcutaneous layer of rats. After 7, 15, 30 and 60 days, the specimens were processed for paraffin embedding. The capsule thickness, collagen content, and number of inflammatory cells (ICs) and interleukin-6 (IL-6) immunolabeled cells were measured. von Kossa-positive structures were evaluated and unstained sections were analyzed under polarized light. Two-way analysis of variance was performed, followed by the post hoc Tukey test (p ≤ 0.05). RESULTS: At 7 days, the capsules around NeoMTA Plus and MTA Fillapex had more ICs and IL-6-immunostained cells than the CG. However, at 60 days, there was no significant difference in the IC number between NeoMTA Plus and the CG (p = 0.1137) or the MTA Fillapex group (p = 0.4062), although a greater number of IL-6-immunostained cells was observed in the MTA Fillapex group (p = 0.0353). From 7 to 60 days, the capsule thickness of the NeoMTA Plus and MTA Fillapex specimens significantly decreased, concomitantly with an increase in the collagen content. The capsules around root canal sealers showed positivity to the von Kossa stain and birefringent structures. CONCLUSIONS: The NeoMTA Plus root canal sealer is biocompatible and exhibits bioactive potential.

Immunoexpression pattern of autophagy mediators in alveolar bone osteoclasts following estrogen withdrawal in female rats.

It is known that estrogen deficiency increases osteoclast formation and activity. Autophagy, a cell survival pathway, has been shown to be crucial for osteoclast function. However, little is known about the effects of estrogen depletion on osteoclast autophagy. Here, we evaluated the effects of estrogen deficiency in the immunoexpression of autophagy mediators in alveolar bone osteoclasts of ovariectomized rats. Twelve adult female rats were ovariectomized (OVX-group) or SHAM-operated (SHAM-group). After three weeks, the rats were euthanized and maxillary fragments containing alveolar bone of the first molars were processed for light microscopy or transmission electron microscopy (TEM). Paraffin-sections were subjected to the TRAP method (osteoclast marker) or to the immunohistochemical detections of beclin-1, LC3α, and p62 (autophagy mediators); araldite-sections were processed for TEM. The number of TRAP-positive osteoclasts and the number of immunolabeled-multinucleated cells (MNCs) along the alveolar bone surface of the first molar were computed. The number of TRAP-positive osteoclasts and the number of beclin-1-, LC3α- and p62-immunolabelled osteoclasts were significantly higher in OVX-group than the SHAM-group. MNCs were frequently located juxtaposed to Howship lacunae along the alveolar bone surface, indicating that these cells are osteoclasts. TEM revealed osteoclasts exhibiting autophagosomes. Our data indicate that autophagy plays an important role during estrogen deficiency-induced osteoclastogenesis. Thus, our results contribute to a better understanding on the role of autophagy on osteoclasts under estrogenic deficiency, and reinforce the idea that modulation of autophagy may be a useful tool to inhibit excessive oral bone resorption in post-menopausal women.