Effects of the essential oil from Citrus aurantium L. in experimental anxiety models in mice

Citrus aurantium L. is popularly used to treat anxiety, among other indications suggesting central nervous system action. Previous studies showed anxiolytic effect in the essential oil from peel in mice evaluated on the elevated plus maze [Carvalho-Freitas, M.I.R., Costa, M., 2002. Anxiolytic and sedative effects of extracts and essential oil from Citrus aurantium L. Biological and Pharmaceutical Bulletin 25, 1629-1633.]. In order to better characterize the activity of the essential oil, it was evaluated in two other experimental models: the light-dark box and the marble-burying test, respectively related to generalized anxiety disorder and to obsessive compulsive disorder. Mice were treated acutely by oral route 30 min (single dose) or once a day for 15 days (repeated doses) before experimental procedures. In light-dark box test, single treatment with essential oil augmented the time spent by mice in the light chamber and the number of transitions between the two compartments. There were no observed alterations in the parameters evaluated in light-dark box after repeated treatment. Otherwise, single and repeated treatments with essential oil were able to suppress marble-burying behavior. At effective doses in the behavioral tests, mice showed no impairment on rotarod procedure after both single and repeated treatments with essential oil, denoting absence of motor deficit. Results observed in marble-burying test, related to obsessive compulsive disorder, appear more consistent than those observed in light-dark box. (c) 2005 Elsevier B.V. All rights reserved.

Theoretical and experimental characterisation of energy in an electrostatic discharge

Electrostatic discharges have been identified as the most likely cause in a number of incidents of fire and explosion with unexplained ignitions. The lack of data and suitable models for this ignition mechanism creates a void in the analysis to quantify the importance of static electricity as a credible ignition mechanism. Quantifiable hazard analysis of the risk of ignition by static discharge cannot, therefore, be entirely carried out with our current understanding of this phenomenon. The study of electrostatics has been ongoing for a long time. However, it was not until the wide spread use of electronics that research was developed for the protection of electronics from electrostatic discharges. Current experimental models for electrostatic discharge developed for intrinsic safety with electronics are inadequate for ignition analysis and typically are not supported by theoretical analysis. A preliminary simulation and experiment with low voltage was designed to investigate the characteristics of energy dissipation and provided a basis for a high voltage investigation. It was seen that for a low voltage the discharge energy represents about 10% of the initial capacitive energy available and that the energy dissipation was within 10 ns of the initial discharge. The potential difference is greatest at the initial break down when the largest amount of the energy is dissipated. The discharge pathway is then established and minimal energy is dissipated as energy dissipation becomes greatly influenced by other components and stray resistance in the discharge circuit. From the initial low voltage simulation work, the importance of the energy dissipation and the characteristic of the discharge were determined. After the preliminary low voltage work was completed, a high voltage discharge experiment was designed and fabricated. Voltage and current measurement were recorded on the discharge circuit allowing the discharge characteristic to be recorded and energy dissipation in the discharge circuit calculated. Discharge energy calculations show consistency with the low voltage work relating to discharge energy with about 30-40% of the total initial capacitive energy being discharged in the resulting high voltage arc. After the system was characterised and operation validated, high voltage ignition energy measurements were conducted on a solution of n-Pentane evaporating in a 250 cm3 chamber. A series of ignition experiments were conducted to determine the minimum ignition energy of n-Pentane. The data from the ignition work was analysed with standard statistical regression methods for tests that return binary (yes/no) data and found to be in agreement with recent publications. The research demonstrates that energy dissipation is heavily dependent on the circuit configuration and most especially by the discharge circuit's capacitance and resistance. The analysis established a discharge profile for the discharges studied and validates the application of this methodology for further research into different materials and atmospheres; by systematically looking at discharge profiles of test materials with various parameters (e.g., capacitance, inductance, and resistance). Systematic experiments looking at the discharge characteristics of the spark will also help understand the way energy is dissipated in an electrostatic discharge enabling a better understanding of the ignition characteristics of materials in terms of energy and the dissipation of that energy in an electrostatic discharge.

Improving in vivo models of fracture fixation associated osteomyelitis

This project’s aim was to create new experimental models in small animals for the investigation of infections related to bone fracture fixation implants. Animal models are essential in orthopaedic trauma research and this study evaluated new implants and surgical techniques designed to improve standardisation in these experiments, and ultimately to minimise the number of animals needed in future work. This study developed and assessed procedures using plates and inter-locked nails to stabilise fractures in rabbit thigh bones. Fracture healing was examined with mechanical testing and histology. The results of this work contribute to improvements in future small animal infection experiments.

Development of a novel experimental model to investigate the influence of mechanics on bone healing

This dissertation proposed a novel experimental model combining a defect configuration with an active instrumented fixation device to investigate the influence of mechanics on bone healing. The proposed defect configuration aimed to minimise physiological loading within an experimental fracture gap and the instrumented fixator was used for the application of controlled displacements and in vivo stiffness monitoring of the healing process. This thesis has provided a novel approach to advance current knowledge and understanding of mechanobiology, which has been limited in previous experimental models.

The Pathobiology of the Saccular Cerebral Artery Aneurysm Rupture and Repair : A Clinicopathological and Experimental Approach

Backround and Purpose The often fatal (in 50-35%) subarachnoid hemorrhage (SAH) caused by saccular cerebral artery aneurysm (SCAA) rupture affects mainly the working aged population. The incidence of SAH is 10-11 / 100 000 in Western countries and twice as high in Finland and Japan. The estimated prevalence of SCAAs is around 2%. Many of those never rupture. Currently there are, however, no diagnostic methods to identify rupture-prone SCAAs from quiescent, (dormant) ones. Finding diagnostic markers for rupture-prone SCAAs is of primary importance since a SCAA rupture has such a sinister outcome, and all current treatment modalities are associated with morbidity and mortality. Also the therapies that prevent SCAA rupture need to be developed to as minimally invasive as possible. Although the clinical risk factors for SCAA rupture have been extensively studied and documented in large patient series, the cellular and molecular mechanisms how these risk factors lead to SCAA wall rupture remain incompletely known. Elucidation of the molecular and cellular pathobiology of the SCAA wall is needed in order to develop i) novel diagnostic tools that could identify rupture-prone SCAAs or patients at risk of SAH, and to ii) develop novel biological therapies that prevent SCAA wall rupture. Materials and Methods In this study, histological samples from unruptured and ruptured SCAAs and plasma samples from SCAA carriers were compared in order to identify structural changes, cell populations, growth factor receptors, or other molecular markers that would associate with SCAA wall rupture. In addition, experimental saccular aneurysm models and experimental models of mechanical vascular injury were used to study the cellular mechanisms of scar formation in the arterial wall, and the adaptation of the arterial wall to increased mechanical stress. Results and Interpretation Inflammation and degeneration of the SCAA wall, namely loss of mural cells and degradation of the wall matrix, were found to associate with rupture. Unruptured SCAA walls had structural resemblance with pads of myointimal hyperplasia or so called neointima that characterizes early atherosclerotic lesions, and is the repair and adaptation mechanism of the arterial wall after injury or increased mechanical stress. As in pads of myointimal hyperplasia elsewhere in the vasculature, oxidated LDL was found in the SCAA walls. Immunity against OxLDL was demonstrated in SAH patients with detection of circulating anti-oxidized LDL antibodies, which were significantly associated with the risk of rupture in patients with solitary SCAAs. Growth factor receptors associated with arterial wall remodeling and angiogenesis were more expressed in ruptured SCAA walls. In experimental saccular aneurysm models, capillary growth, arterial wall remodeling and neointima formation were found. The neointimal cells were shown to originate from the experimental aneurysm wall with minor contribution from the adjacent artery, and a negligible contribution of bone marrow-derived neointimal cells. Since loss of mural cells characterizes ruptured human SCAAs and likely impairs the adaptation and repair mechanism of ruptured or rupture-prone SCAAs, we investigated also the hypothesis that bone marrow-derived or circulating neointimal precursor cells could be used to enhance neointima formation and compensate the impaired repair capacity in ruptured SCAA walls. However, significant contribution of bone marrow cells or circulating mononuclear cells to neointima formation was not found.

Molecular mechanisms of hypertension-induced heart failure : Experimental studies with special emphasis on local renin-angiotensin system, cardiac metabolism and levosimendan

Hypertension is a major risk factor for stroke, ischaemic heart disease, and the development of heart failure. Hypertension-induced heart failure is usually preceded by the development of left ventricular hypertrophy (LVH), which represents an adaptive and compensatory response to the increased cardiac workload. Biomechanical stress and neurohumoral activation are the most important triggers of pathologic hypertrophy and the transition of cardiac hypertrophy to heart failure. Non-clinical and clinical studies have also revealed derangements of energy metabolism in hypertensive heart failure. The goal of this study was to investigate in experimental models the molecular mechanisms and signalling pathways involved in hypertension-induced heart failure with special emphasis on local renin-angiotensin-aldosterone system (RAAS), cardiac metabolism, and calcium sensitizers, a novel class of inotropic agents used currently in the treatment of acute decompensated heart failure. Two different animal models of hypertensive heart failure were used in the present study, i.e. hypertensive and salt-sensitive Dahl/Rapp rats on a high salt diet (a salt-sensitive model of hypertensive heart failure) and double transgenic rats (dTGR) harboring human renin and human angiotensinogen genes (a transgenic model of hypertensive heart failure with increased local RAAS activity). The influence of angiotensin II (Ang II) on cardiac substrate utilization and cardiac metabolomic profile was investigated by using gas chromatography coupled to time-of-flight mass spectrometry to detect 247 intermediary metabolites. It was found that Ang II could alter cardiac metabolomics both in normotensive and hypertensive rats in an Ang II receptor type 1 (AT1)-dependent manner. A distinct substrate use from fatty acid oxidation towards glycolysis was found in dTGR. Altered cardiac substrate utilization in dTGR was associated with mitochondrial dysfunction. Cardiac expression of the redox-sensitive metabolic sensor sirtuin1 (SIRT1) was increased in dTGR. Resveratrol supplementation prevented cardiovascular mortality and ameliorated Ang II-induced cardiac remodeling in dTGR via blood pressure-dependent pathways and mechanisms linked to increased mitochondrial biogenesis. Resveratrol dose-dependently increased SIRT1 activity in vitro. Oral levosimendan treatment was also found to improve survival and systolic function in dTGR via blood pressure-independent mechanisms, and ameliorate Ang II-induced coronary and cardiomyocyte damage. Finally, using Dahl/Rapp rats it was demonstrated that oral levosimendan as well as the AT1 receptor antagonist valsartan improved survival and prevented cardiac remodeling. The beneficial effects of levosimendan were associated with improved diastolic function without significantly improved systolic changes. These positive effects were potentiated when the drug combination was administered. In conclusion, the present study points to an important role for local RAAS in the pathophysiology of hypertension-induced heart failure as well as its involvement as a regulator of cardiac substrate utilization and mitochondrial function. Our findings suggest a therapeutic role for natural polyphenol resveratrol and calcium sensitizer, levosimendan, and the novel drug combination of valsartan and levosimendan, in prevention of hypertension-induced heart failure. The present study also provides a better understanding of the pathophysiology of hypertension-induced heart failure, and may help identify potential targets for novel therapeutic interventions.

Neuroprotective and immunomodulatory properties of the new PPARγ agonist MDG548: an in vitro and in vivo study in PD models

Neuroinflammation is a key component of Parkinson’s disease (PD) neuropathology. Skewed microglia activation with pro-inflammatory prevailing over anti-inflammatory phenotypes may contribute to neurotoxicity via the production of cytokines and neurotoxic species. Therefore, microglia polarization has been proposed as a target for neuroprotection. The peroxisome proliferator-activated receptor gamma (PPARγ) is expressed in microglia and peripheral immune cells, where it is involved in macrophages polarization and in the control of inflammatory responses, by modulating gene transcription. Several studies have shown that PPARγ agonists are neuroprotective in experimental PD models in rodents and primates. however safety concerns have been raised about PPARγ agonists thiazolidinediones (TZD) currently available, prompting for the development of non-TZD compounds. Aim of this study was to characterize a novel PPARγ agonist non TZD, MDG548, for its potential neuroprotective effect in PD models and its immunomodulatory activity as the underlying mechanism of neuroprotection. The neuroprotective activity of MDG548 was assessed in vivo in the subacute MPTP model and in the chronic MPTP/probenecid (MPTPp) model of PD. MDG548 activity on microglia activation and phenotype was investigated in the substantia nigra pars compacta (SNc) via the evaluation of pro- (TNF-α and iNOS) and anti-inflammatory (CD206) molecules, with fluorescent immunohistochemistry. Moreover, cultured murine microglia MMGT12 were treated with MDG548 in association with the inflammagen LPS, pro- and anti-inflammatory molecules were measured in the medium by ELISA assay and phagocytosis was evaluated by fluorescent immunohistochemistry for CD68. MDG548 arrested dopaminergic cells degeneration in the SNc in both the subacute MPTP and the chronic MPTPp models of PD, and reverted MPTPp-induced motor impairment. Moreover, MDG548 reduced microglia activation, iNOS and TNF-α production, while induced CD206 in microglia. In cultured unstimulated microglia, LPS increased TNF-α production and CD68 expression, while decreased CD206 expression. MDG548 reverted LPS effect on TNF-α and CD206 restoring physiological levels, while strongly increased CD68 expression. Results suggest that the PPARγ agonist MDG548 is neuroprotective in experimental models of PD. MDG548 targets microglia polarization by correcting the imbalance between pro- over antiinflammatory molecules, offering a novel immunomodulatory approach to neuroprotection.

Cement mantle fatigue failure in total hip replacement: Experimental and computational testing

One possible loosening mechanism of the femoral component in total hip replacement is fatigue cracking of the cement mantle. A computational method capable of simulating this process may therefore be a useful tool in the preclinical evaluation of prospective implants. In this study, we investigated the ability of a computational method to predict fatigue cracking in experimental models of the implanted femur construct. Experimental specimens were fabricated such that cement mantle visualisation was possible throughout the test. Two different implant surface finishes were considered: grit blasted and polished. Loading was applied to represent level gait for two million cycles. Computational (finite element) models were generated to the same geometry as the experimental specimens, with residual stress and porosity simulated in the cement mantle. Cement fatigue and creep were modelled over a simulated two million cycles. For the polished stem surface finish, the predicted fracture locations in the finite element models closely matched those on the experimental specimens, and the recorded stem displacements were also comparable. For the grit blasted stem surface finish, no cement mantle fractures were predicted by the computational method, which was again in agreement with the experimental results. It was concluded that the computational method was capable of predicting cement mantle fracture and subsequent stem displacement for the structure considered. (C) 2006 Elsevier Ltd. All rights reserved.

Independent and Interdependent Immunoregulatory Effects of IL-27, IFN-β, and IL-10 in the Suppression of Human Th17 Cells and Murine Experimental Autoimmune Encephalomyelitis

IFN-ß, IL-27, and IL-10 have been shown to exert a range of similar immunoregulatory effects in murine and human experimental systems, particularly in Th1- and Th17-mediated models of autoimmune inflammatory disease. In this study we sought to translate some of our previous findings in murine systems to human in vitro models and delineate the interdependence of these different cytokines in their immunoregulatory effects. We demonstrate that human IL-27 upregulates IL-10 in T cell-activated PBMC cultures and that IFN-ß drives IL-27 production in activated monocytes. IFN-ß-driven IL-27 is responsible for the upregulation of IL-10, but not IL-17 suppression, by IFN-ß in human PBMCs. Surprisingly, IL-10 is not required for the suppression of IL-17 by either IL-27 or IFN-ß in this model or in de novo differentiating Th17 cells, nor is IL-27 signaling required for the suppression of experimental autoimmune encephalomyelitis (EAE) by IFN-ß in vivo. Furthermore, and even more surprisingly, IL-10 is not required for the suppression of Th17-biased EAE by IL-27, in sharp contrast to Th1-biased EAE. In conclusion, IFN-ß and IL-27 both induce human IL-10, both suppress human Th17 responses, and both suppress murine EAE. However, IL-27 signaling is not required for the therapeutic effect of IFN-ß in EAE. Suppression of Th17-biased EAE by IL-27 is IL-10-independent, in contrast to its mechanism of action in Th1-biased EAE. Taken together, these findings delineate a complex set of interdependent and independent immunoregulatory mechanisms of IFN-ß, IL-27, and IL-10 in human experimental models and in murine Th1- and Th17-driven autoimmunity.

Modelo experimental de lesões intra-epiteliais e de adenocarcinoma ductal pancreático induzidos por 7,12-dimetilbenzantraceno em camundongos

INTRODUÇÃO: O adenocarcinoma de pâncreas apresenta um mau prognóstico. A utilização de modelos experimentais é necessária para a compreensão do comportamento biológico tumoral, principalmente das lesões precoces (neoplasias intra-epiteliais pancreáticas - NIPan) e para o desenvolvimento de opções terapêuticas. OBJETIVO: Avaliar a carcinogênese pancreática induzida por 7,12-dimetilbenzantraceno (DMBA), em camundongos, aplicando a classificação das neoplasias intra-epiteliais pancreáticas. MÉTODOS: 90 camundongos machos, mus musculus, da cepa CF1, foram submetidos à laparotomia mediana e 1 mg de DMBA foi implantado na porção cefálica do pâncreas. Os animais foram divididos em dois grupos, com eutanásia em 30 e 60 dias. Em seguida, o pâncreas foi retirado, fixado em formalina e foram confeccionadas lâminas coradas com hematoxilina eosina. Os cortes histológicos foram avaliados por dois patologistas de acordo com os seguintes critérios: pâncreas normal, hiperplasia reacional, NIPan 1A, NIPan 1B, NIPan 2, NIPan 3 e carcinoma. As alterações inflamatórias também foram analisadas. RESULTADOS: A avaliação patológica evidenciou, no grupo de 30 dias: 4 (16,7%) animais com hiperplasia reativa, 16 (66,6%) com NIPan e 4 (16,7%) com adenocarcinoma. No grupo de 60 dias: 10 (27,1%) animais com hiperplasia reativa, 13 (35,1%) com NIPan e 14 (37,8%) com adenocarcinoma. A diferença entre os grupos apresentou significância estatistística (P < 0,05 – teste exato de Fisher). A prevalência de alterações inflamatórias em 30 dias foi: pancreatite aguda (n=11), pancreatite crônica (n=5) e inflamação dependente da bolsa (n=8). No grupo de 60 dias 11 espécimes apresentavam pancreatite aguda e 26 pancreatite crônica. CONCLUSÕES: O modelo experimental com DMBA em camundongos, induz neoplasia intra-epitelial pancreática e adenocarcinoma ductal histologicamente semelhantes ao carcinoma pancreático em humanos. Este modelo pode ser utilizado na investigação da carcinogênese com enfoque na progressão molecular das lesões precursoras até o adenocarcinoma.

Comportamento hemodinâmico e metabólico do choque hemorrágico: estudo experimental no cão

JUSTIFICATIVA E OBJETIVOS: Diversos modelos experimentais têm sido utilizados para ilustrar as alterações hemodinâmicas e metabólicas que ocorrem durante o choque hemorrágico. O objetivo da pesquisa é o de observar os comportamentos hemodinâmicos e metabólicos que acontecem em um modelo seqüencial e progressivo de choque hemorrágico no cão, verificando quais índices alteram-se mais precocemente. MÉTODO: O estudo foi realizado em 13 cães sob anestesia venosa total com pentobarbital sódico, em normoventilação e previamente esplenectomizados. Os animais não foram hidratados e a velocidade do sangramento foi ditada pela pressão arterial em que o animal se encontrava. Os atributos estudados foram divididos em hemodinâmicos (freqüência cardíaca - FC, pressão arterial média - PAM, índice de resistência vascular sistêmica - IRVS, índice sistólico - IS, índice cardíaco - IC, índice de choque - I.choque, índice de trabalho sistólico do ventrículo esquerdo - ITSVE, pressão capilar pulmonar - PCP, pressão venosa central - PVC) e metabólicos (saturação venosa mista - SvO2, pressão venosa de oxigênio - PvO2, transporte de oxigênio - DO2, consumo de oxigênio - VO2, extração de oxigênio - TEO2, lactato sérico). A coleta de dados e os atributos foram estudados em 6 momentos distintos, sendo M1, o momento controle e os outros momentos correspondentes a decréscimos gradativos de 10% da volemia calculada para cada animal. RESULTADOS: A hemorragia determinou diminuição significativa da FC somente em M6; queda da PAM, IC, IS e ITSVE a cada momento estudado; discreta alteração da PVC e PCP em cada momento; diminuição da PvO2 e da SvO2 nos momentos estudados; redução do DO2, estabilização do VO2 e elevação da TEO2 nos momentos; o índice de choque apresentou elevação até M3, diminuição em M4 e nova elevação até M6; o IRVS elevou-se até M6, ficou inalterado em M5 e apresentou diminuição significativa em M6; o lactato apresentou elevações a partir de M5 e M6. CONCLUSÕES: Considerou-se que a pressão arterial média, freqüência cardíaca, pressão venosa central e pressão capilar pulmonar não refletem o real estado volêmico dos cães no nosso modelo experimental e que o transporte, consumo e a taxa de extração de oxigênio são parâmetros úteis na determinação da reversibilidade e prognóstico do choque hemorrágico.

Animal models for clinical and gestational diabetes: maternal and fetal outcomes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Hipertensão arterial experimental e prenhez em ratas: uso do modelo Goldblatt I (1 rim - 1 clipe)

Objetivo: desenvolver o modelo experimental de hipertensão tipo Goldblatt I (1 rim - 1 clipe), em ratas, para estudar a interação entre hipertensão e prenhez. Métodos: o experimento foi dividido em 5 períodos: adaptação (2 semanas), cirúrgico (1 semana), desenvolvimento da hipertensão (6 semanas), acasalamento e estabilização da pressão arterial (6 semanas) e prenhez (3 semanas). Foram utilizadas 82 ratas virgens da raça Wistar, pesando entre 180-240 gramas e com idade entre 3 e 4 meses. As ratas foram sorteadas para compor os 4 grupos experimentais (controle, manipulação, nefrectomia e hipertensão) e estudadas em 15 momentos distintos (M1 a M15). A hipertensão foi induzida experimentalmente pela técnica de Goldblatt I (1 rim, 1 clipe), que consiste na constrição da artéria renal esquerda e nefrectomia contralateral. Posteriormente, foram realizadas medidas periódicas da pressão arterial pelo método da pletismografia de cauda (PAC). Resultados: os animais sem tratamento cirúrgico (controle) e com manipulação não apresentaram alterações na PAC durante o experimento. A nefrectomia determinou discreta elevação da PAC. Nos grupos de ratas prenhes, observou-se tendência a discreta diminuição da PAC, que se acentuou no final da prenhez. Conclusões: o modelo experimental foi adequado para o objetivo de nosso estudo, pois permitiu a obtenção de animais hipertensos.

Differential regulation of MMP-13 expression in two models of experimentally induced periodontal disease in rats

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

Máxima fase estável de lactato é ergômetro-dependente em modelo experimental utilizando ratos

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