Immunohistochemical, tomographic and histological study on onlay bone graft remodeling. Part II: calvarial bone

Objectives Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. Material and methods Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-kappa beta ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. Results The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P < 0.05), and both groups showed high bone density rates at 60 days. This set of evidences is corroborated by the immunohistochemical outcomes indicating that proteins associated with revascularization and osteogenesis (VEGF, OPN, TRAP and ALP) were found in higher levels in the perforated group. Conclusions These findings indicate that the bone volume of calvarial grafts is better maintained when the receptor bed is perforated, probably resulting from more effective graft revascularization and greater bone deposition. The process of bone resorption peaked between 20 and 60 days post-operatively in both groups although significantly less in the perforated group. To cite this article:Pedrosa Jr WF, Okamoto R, Faria PEP, Arnez MFM, Xavier SP, Salata LA. Immunohistochemical, tomographic and histological study on onlay bone grafts remodeling. Part II: calvarial bone.Clin. Oral Impl. Res. 20, 2009; 1254-1264.doi: 10.1111/j.1600-0501.2009.01747.x.

Immunohistochemical, tomographic and histological study on onlay iliac grafts remodeling

The information concerning the molecular events taking place in onlay bone grafts are still incipient. The objective of the present study is to correlate the effects of perforation of resident bone bed on (1) the timing of onlay autogenous graft revascularization; (2) the maintenance of volume/density of the graft (assessed through tomography); and (3) the occurrence of bone remodeling proteins (using immunohistochemistry technique) delivered in the graft. Thirty-six New Zealand White rabbits were subjected to iliac crest onlay bone grafting on both sides of the mandible. The bone bed was drill-perforated on one side aiming at accelerating revascularization, whereas on the other side it was kept intact. After grafts fixation and flaps suture all animals were submitted to tomography on both mandible sites. Six animals were sacrificed, respectively, at 3, 5, 7, 10, 20 and 60 days after surgery. A second tomography was taken just before sacrifice. Histological slides were prepared from each grafted site for both immunohistochemistry analysis [osteopontin, osteocalcin, type I collagen and vascular endothelial growth factor (VEGF) anti-bodies] and histometric analysis. The values on bone volume measured on tomography showed no statistic significance (P >= 0.05) between perforated and intact sites. Grafts placed on perforated beds showed higher bone density values compared with non-perforated ones at 3 days (P <= 0.05). This correlation was inverted at 60 days postoperatively. The findings from VEGF labeling revealed a tendency for earlier revascularization in the perforated group. The early revascularization of bone grafts accelerated the bone remodeling process (osteocalcin, type I collagen and osteopontin) that led to an increased bone deposition at 10 days. The extended osteoblast differentiation process at intermediate stages in the perforated group cooperated for a denser bone at 60 days.

In vitro inhibitory effect of trichostatin A on canine grade 3 mast cell tumor

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect skin and soft tissue in dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. Trichostatin A (TSA), an antifungal antibiotic, has shown inhibitory effects on the proliferation and induction of apoptosis in various types of cancer cells. In order to evaluate the potential of trichostatin A as a therapeutic drug, cells of grade 3 MCT were cultured and treated with concentrations of 1 nM to 400 nM of TSA. MTT assay and trypan blue exclusion assays were performed to estimate cell growth and cell viability, and cell cycle analysis was evaluated. TSA treatment showed a reduction in numbers of viable cells and an increase of cell death by apoptosis. The cell cycle analysis showed an increase of hypodiploid cells and a reduction of G0/G1 and G2/M -phases. According to these results, trichostatin A may be an interesting potential chemotherapeutic agent for the treatment of canine MCT.

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na(+)/K(+)-ATPase

Background: Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na(+)/K(+)-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na(+)/K(+)-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. Methods: Male Wistar rats were injected with venom (0.8 mg/kg, iv.) and renal function was assessed 6.24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na(+)/K(+)-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. Results: Venom caused lobulation of the capillary tufts, dilation of Bowman`s capsular space. F-actin disruption in Bowman`s capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na(+)/K(+)-ATPase alpha(1) subunit were increased 6 h post-venom, whereas Na(+)/K(+)-ATPase activity increased 6 h and 24 h post-venom. Conclusions: Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na(+)/K(+)-ATPase expression and activity in the early phase of renal damage. General significance: Enhanced Na(+)/K(+)-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage. (C) 2011 Elsevier B.V. All rights reserved.

Hypermethioninemia provokes oxidative damage and histological changes in liver of rats

In the present study we evaluated the effect of chronic methionine administration on oxidative stress and biochemical parameters in liver and serum of rats, respectively. We also performed histological analysis in liver. Results showed that hypermethioninemia increased chemiluminescence, carbonyl content and glutathione peroxidase activity, decreased total antioxidant potential, as well as altered catalase activity. Hypermethioninemia increased synthesis and concentration of glycogen, besides histological studies showed morphological alterations and reduction in the glycogen/glycoprotein content in liver. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose were increased in hypermethioninemic rats. These findings suggest that oxidative damage and histological changes caused by methionine may be related to the hepatic injury observed in hypermethioninemia. (C) 2009 Elsevier Masson SAS. All rights reserved.

Histological analyses of thermal effect caused by 1.2 W diode laser irradiation at rat periodontal pockets

The aim of this in vivo study was to evaluate the thermal effects caused by 810 nm 1.2 W diode laser irradiation of periodontal tissues. Despite all data available concerning the laser application for periodontal treatment, one of the most relevant challenges is to prevent the harmful tissue heating induced by different clinical protocols. Periodontal pockets were induced at molars in 96 rats. Several irradiation powers under CW mode were investigated: 0, 400, 600, 800, 1000, 1200 mW. The pockets were irradiated using a 300 A mu m frontal illumination fiber. The animals were killed at 4 or 10 days after irradiation. The mandible was surgically removed and histologically processed. The histological sections stained with H/E demonstrated that irradiation parameters up to 1000 mW were thermally safe for the periodontal tissues. The sections stained with Brown & Brenn technique evidenced bacteria in the periodontal tissues. Consequently, the diode laser irradiation as a unique treatment was not capable to eliminate bacteria of the biofilm present in the pockets. According to the methodology used here, it was concluded that the thermal variation promoted by a diode laser can cause damage to periodontal tissues depending on the energy density used. The 1.2 W diode laser irradiation itself does not control the bacteria present in the biofilm of the periodontal pockets without mechanical action. The knowledge of proper high intensity laser parameters and methods of irradiation for periodontal protocols may prevent any undesirable thermal damage to the tissues.

Enhanced visualization of histological samples with an adjustable RGB contrast system with application for tissue used in photodynamic therapy

The analysis of histological sections has long been a valuable tool in the pathological studies. The interpretation of tissue conditions, however, relies directly on visual evaluation of tissue slides, which may be difficult to interpret because of poor contrast or poor color differentiation. The Chromatic Contrast Visualization System (CCV) combines an optical microscope with electronically controlled light-emitting diodes (LEDs) in order to generate adjustable intensities of RGB channels for sample illumination. While most image enhancement techniques rely on software post-processing of an image acquired under standard illumination conditions, CCV produces real-time variations in the color composition of the light source itself. The possibility of covering the entire RGB chromatic range, combined with the optical properties of the different tissues, allows for a substantial enhancement in image details. Traditional image acquisition methods do not exploit these visual enhancements which results in poorer visual distinction among tissue structures. Photodynamic therapy (PDT) procedures are of increasing interest in the treatment of several forms of cancer. This study uses histological slides of rat liver samples that were induced to necrosis after being exposed to PDT. Results show that visualization of tissue structures could be improved by changing colors and intensities of the microscope light source. PDT-necrosed tissue samples are better differentiated when illuminated with different color wavelengths, leading to an improved differentiation of cells in the necrosis area. Due to the potential benefits it can bring to interpretation and diagnosis, further research in this field could make CCV an attractive technique for medical applications.

Renal ischemia in rats: Mitochondria function and laser autofluorescence

Ischemia-reperfusion injury is the major cause of organ dysfunction or even nonfunction following transplantation. It can attenuate the long-term survival of transplanted organs. To evaluate the severity of renal ischemia injury determined by histology, we applied laser(442 nm and 532 nm) induced fluorescence (LIF), mitochondria respiration, and membrane swelling to evaluate 28 Wistar rats that underwent left kidney warm ischemia for 20, 40, 60, or 80 minutes. LIF performed before ischemia (control) was repeated at 20, 40, 60, and 80 minutes thereafter. We harvested left kidney tissue samples immediately after LIF determination for histology and mitochondrial analyses: state 3 and 4 respiration, respiration control rate (RCR), and membrane swelling. The association of optic spectroscopy with histological damage showed: LIF, 442 nm (r(2) = 0.39, P < .001) and 532 nm, (r(2) = 0.18, P = .003); reflecting laser/fluorescence-induced, 442 nm (r(2) = 0.20, P = .002) and 532 nm (r(2) = 0.004, P = .67). The associations between mitochondria function and tissue damage were: state 3 respiration (r(2) = 0.43, P = .0004), state 4 respiration (r(2) = 0.03, P = 0.38), RCR (r(2) = 0.28, P = .007), and membrane swelling (r(2) = 0.02, P = .43). The intensity of fluorescence emitted by tissue excited by laser, especially at a wave length of 442 nm, was determined in real time. Mitochondrial state 3 respiration and respiratory control ratio also exhibited good correlations with the grade of ischemic tissue damage.

Mineralogy and trace-element geochemistry of the high-grade iron ores of the Aguas Claras Mine and comparison with the Capao Xavier and Tamandua iron ore deposits, Quadrilahero Ferrifero, Brazil

Several major iron deposits occur in the Quadrilatero Ferrifero (QF), southeastern region of Brazil, where metamorphosed and heterogeneously deformed banded iron formation (BIF) of the Caue Formation, regionally called itabirite, was transformed into high- (Fe >64%) and lowgrade (30%grade ores hosted within dolomitic itabirite. Two distinct types of high-grade ore occurred at the mine: soft and hard. The soft ore was the most abundant and represented more than 85% of the total ore mined until it was mined out in 2002. Soft and hard ores consist essentially of hematite, occurring as martite, anhedral to granular/tabular hematite and, locally, specularite. Gangue minerals are rare, consisting of dolomite, sericite, chlorite, and apatite in the hard and soft ores, and Mn-oxides and ferrihydrite in the soft ore where they are concentrated within porous bands. Chemical analyses show that hard and soft ores consist almost entirely of Fe(2)O(3), with a higher amount of detrimental impurities, especially MnO, in the soft ore. Both hard and soft ores are depleted in trace elements. The high-grade ores at the Aguas Claras Mine have at least a dual origin, involving hypogene and supergene processes. The occurrence of the hard, massive high-grade ore within ""fresh"" dolomitic itabirite is evidence of its hypogene origin. Despite the contention about the origin of the dolomitic itabirite (if this rock is a carbonate-rich facies of the Caue Formation or a hematite-carbonate precursor of the soft high-grade ore), mineralogical and geochemical features of the soft high-grade ore indicate that it was formed by leaching of dolomite from the dolomitic itabirite by meteoric water. The comparison of the Aguas Claras, Capao Xavier and Tamandua orebodies shows that the original composition of the itabiritic protore plays a major role in the genesis of high- and low-grade soft ores in the QF. Under the same weathering and structural conditions, the dolomitic itabirite is the more favorable to form high-grade deposits than siliceous itabirite. Field relations at the Aguas Claras and Capao Xavier deposits suggest that it is not possible to form huge soft high-grade supergene deposits from siliceous itabirite, unless another control, such as impermeable barriers, had played an important role. The occurrence in the Tamandua Mine of a large, soft, high-grade orebody formed from siliceous itabirite and closely associated with hypogene hard ore suggests that large, soft, high-grade orebodies of the Quadrilatero Ferrifero, which occur within siliceous itabirite, have a hypogene contribution in their formation.

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model

Background: The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. Objectives: We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. Methods and results: TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. Conclusion: Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Histological evaluation of chitosan-based biomaterials used for the correction of critical size defects in rat`s calvaria

Chitosan, a biopolymer obtained from chitin, and its derivates, such as chitosan hydrochloride, has been reported as wound healing accelerators and as possible bone substitutes for tissue engineering, and therefore these Substances could be relevant in dentistry and periodontology. The purpose of this investigation was to make a histological evaluation of chitosan and chitosan hydrochloride biomaterials (gels) used in the correction of critical size bone defects made in rat`s calvaria. Bone defects of 8 mm in diameter were surgically created in the calviria of 50 Holtzman (Rattus norvegicus) rats and filled with blood clot (control), low molecular weight chitosan, high molecular weight chitosan, low molecular weight chitosan hydrochloride, and high molecular weight chitosan hydrochloride, numbering 10 animals, divided into two experimental periods (15 and 60 days), for each biomaterial. The histological evaluation was made based on the morphology of the new-formed tissues in defect`s region, and the results indicated that there was no statistical difference between the groups when the new bone formation in the entire defect`s area were compared (p > 0.05) and, except in the control groups, assorted degrees of inflammation Could be Seen. In Conclusion, chitosan and chitosan hydrochloride biomaterials used in this study were not able to promote new bone formation in critical size defects made in rat`s calvaria. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 93A: 107-114, 2016

Cathepsin S as a biomarker of low-grade inflammation, insulin resistance, and cardiometabolic disease risk

Cathepsin S is a protease important in major histocompatibility complex (MHC) class II antigen presentation and also in degrading the extracellular matrix. Studies, most of them experimental, have shown that cathepsin S is involved in different pathological conditions such as obesity, inflammation, atherosclerosis, diabetes, and cancer.    The overall hypothesis of this report is that high levels of circulating cathepsin S, is a biomarker that reflects pathology induced by inflammation and obesity. The overall aim of this report was to investigate possible associations between circulating cathepsin S, inflammation, glucometabolic disturbance, and its associated diseases in the community. As cathepsin S appears to be a novel risk marker for several pathological conditions, we also wanted to examine the effect of dietary intervention on circulating cathepsin S concentrations.    This thesis is based on data from three community-based cohorts, the Uppsala longitudinal study of adult men (ULSAM), the prospective investigation of the vasculature in Uppsala seniors (PIVUS), and a post-hoc study from the randomized controlled NORDIET trial.    In the first study, we identified a cross-sectional positive association between serum cathepsin S and two markers of cytokine-mediated inflammation, CRP and IL-6. These associations were similar in non-obese individuals. In longitudinal analyses, higher cathepsin S at baseline was associated with higher CRP and IL-6 levels after six years of follow-up. In the second study, we identified a cross-sectional association between increased serum levels of cathepsin S and reduced insulin sensitivity. These associations were similar in non-obese individuals. No significant association was observed between cathepsin S and insulin secretion. In longitudinal analysis, higher cathepsin S levels were associated with an increased risk of developing diabetes during the six-year follow-up. In the third study, we found that higher serum levels of cathepsin S were associated with increased mortality risk. Moreover, in the ULSAM cohort, serum cathepsin S was independently associated with cause-specific mortality from cardiovascular disease and cancer. In the fourth study, we identified that adherence to an ad libitum healthy Nordic diet for 6 weeks slightly decreased the levels of plasma cathepsin S in normal or marginally overweight individuals, relative to the control group. Changes in circulating cathepsin S concentrations were correlated with changes in body weight, LDL-C, and total cholesterol.    Conclusion: This thesis shows that circulating cathepsin S is a biomarker that independently reflects inflammation, insulin resistance, the risk of developing diabetes, and mortality risk. Furthermore, a Nordic diet moderately reduced cathepsin S levels in normal-weight and overweight men and women. This effect may be partially mediated by diet-induced weight loss and possibly by reduced LDL-C concentrations. 

Meeting the Standards: An Analysis of Eight Grade Educational Assesment Test Scores in Maine

This paper examines the impact of socioeconomic factors on eighth grade achievement test scores in the face of federal and state initiatives for educational reform in Maine. We use student-level data over a five year period to provide a framework for understanding the policy implications of these initiatives. We model performance on standardized tests using a seemingly unrelated regressions approach and then determine the likelihood of meeting the standards defined by the adequate yearly progress requirements of the No Child Left Behind Act and Maine Learning Results initiatives. Our results indicate that the key factors influencing a student’s test scores include the education of a student’s parents, special services received for learning disabilities, and alternative measures of academic achievement.

Utilização da frutose-1, 6-Bisfosfato como um protetor celular na preservação de fígados para transplante

A solução de preservação da Universidade de Wisconsin (UW) é considerada a solução padrão para preservação de fígados, rins e pâncreas. A frutose-1,6-bisfosfato (FBP) é uma substância que apresenta efeito protetor do fígado contra injúrias provocadas por agentes químicos e ocorridas durante o período de isquemia-reperfusão. O objetivo deste estudo foi avaliar os efeitos da FBP na composição de soluções para preservação de fígados para transplante. Neste estudo experimental, a perfusão e a preservação dos fígados foram realizadas em cada grupo com as soluções de UW, UW contendo 10 mmol/L de FBP (UWM) e FBP 10 mmol/L (FBPS), respectivamente. Os fígados foram armazenados em um recipiente plástico contendo solução de preservação a 4oC por 24 horas. As mensurações bioquímicas de AST, ALT, LDH e TBARS foram realizadas em amostras das soluções de preservação nos tempos de 0, 12, 18 e 24 horas. A análise histológica foi realizada em fígados preservados por 24 horas. O grau de preservação observado com as soluções de UW e FBPS foi similar até 18 horas de armazenamento. A adição de 10 mmol/L de FBP à solução de UW provocou um aumento das injúrias ocorridas e uma pior preservação quando comparado ao grupo armazenado em UW. A FBP protegeu os fígados contra danos causados pelos radicais livres por tempos de preservação inferiores a 18 horas. Não houve diferença significativa entre os grupos na análise histológica dos fígados preservados no tempo de 24 horas. A FBP utilizada em solução foi eficaz na preservação de fígados, podendo ser um importante constituinte para outras formulações.