Modelo experimental de perfusão pulmonar ex vivo em ratos: avaliação histopatológica e de apoptose celular em pulmões preservados com solução de baixo potássio dextrana vs. solução histidina-triptofano-cetoglutarato

OBJETIVO: Comparar os achados histopatológicos e de apoptose em pulmões de ratos preservados em soluções low-potassium dextran (LPD, baixo potássio dextrana), histidine-tryptophan-ketoglutarate (HTK, histidina-triptofano-cetoglutarato) ou salina normal (SN) em 6 h e 12 h de isquemia pela utilização de um modelo experimental de perfusão pulmonar ex vivo. MÉTODOS: Sessenta ratos Wistar foram anestesiados, randomizados e submetidos à perfusão anterógrada pela artéria pulmonar com uma das soluções preservadoras. Após a extração, os blocos cardiopulmonares foram preservados por 6 ou 12 h a 4ºC, sendo então reperfundidos com sangue homólogo em um sistema de perfusão ex vivo durante 60 min. Ao final da reperfusão, fragmentos do lobo médio foram extraídos e processados para histopatologia, sendo avaliados os seguintes parâmetros: congestão, edema alveolar, hemorragia alveolar, hemorragia, infiltrado inflamatório e infiltrado intersticial. O grau de apoptose foi avaliado pelo método TdT-mediated dUTP nick end labeling. RESULTADOS: A histopatologia demonstrou que todos os pulmões preservados com SN apresentaram edema alveolar após 12 h de isquemia. Não houve diferenças em relação ao grau de apoptose nos grupos estudados. CONCLUSÕES: No presente estudo, os achados histopatológicos e de apoptose foram semelhantes com o uso das soluções LPD e HTK, enquanto a presença de edema foi significativamente maior com o uso de SN.

Porphyromonas gingivalis infection at different gestation periods on fetus development and cytokines profile

Oral Diseases (2012) 18, 648654 Aim: Infective agents may affect pregnancy outcomes by deregulating homeostasis. Objectives: The effects of Porphyromonas gingivalis infection before and at different gestation periods were evaluated. Materials and Methods: Wistar rats infected via subcutaneous with P. gingivalis W83, one week before mating (BM), days 1 (PR1) and 11 of gestation (PR11), and controls were evaluated, and samples were obtained at the end of gestation. P. gingivalis was detected by PCR. Cytokine was determined by ELISA. Results: Infected rats had lower maternal gain of weight. Implantation was not observed in 2/12 BM rats. PR11 presented more fetal-placental resorptions and lower placenta/fetus weight than controls. P. gingivalis was detected in placenta and fetus. IL-6 and TNF-a levels were higher in placenta and serum of infected groups, except for TNF-a in placenta of PR1. IL-1 beta levels were higher in placenta of PR11, but lower in serum and placenta of PR1. There were no differences in IL-10 and PGE2 concentrations among the groups (P < 0.05). Conclusions: The experimental infection by P. gingivalis resulted in alterations in the gestational pattern and in fetal development. The consequences of infection at mid-gestation were more severe than at the beginning, possibly due to the induction of pro-inflammatory cytokines in the fetal compartment.

Metacontingencies, Experimentation and Nonhumans: Searching for Conceptual and Methodological Advances

Metacontingency has been described as the functional relation between interlocking behavioral contingencies, plus their direct and immediate effect, called aggregated product, and a selecting event dependent of such effect, called cultural consequence. The metacontingencies analysis enables the discussion of human behavior complexity in social systems. In the present study, we aimed to review and discuss: (a) the importance of basic behavioral processes analysis for the comprehension of social human phenomena; (b) the necessity of constructing and improving metacontingencies experimental models; (c) the current state of metacontingencies experimental investigations in humans; (d) the use of animal models as a way to control the effects of verbal behavior, among other variables, over cultural selection; (e) a concrete and illustrative proposal of an animal model of metacontingencies.

Evaluation of a nanometer roughness scale resorbable media-processed surface: a study in dogs

Objectives: This study compared the biomechanical fixation and bone-to-implant contact (BIC) of implants with different surfaces treatment (experimental resorbable blasting media-processed nanometer roughness scale surface, and control dual acid-etched) in a dog model. Material and methods: Surface characterization was made in six implants by means of scanning electron microscopic imaging, atomic force microscopy to evaluate roughness parameters, and X-ray photoelectron spectroscopy (XPS) for chemical assessment. The animal model comprised the bilateral placement of control (n = 24) and experimental surface (n = 24) implants along the proximal tibiae of six mongrel dogs, which remained in place for 2 or 4 weeks. Half of the specimens were biomechanically tested (torque), and the other half was subjected to histomorphologic/ morphometric evaluation. BIC and resistance to failure measures were each evaluated as a function of time and surface treatment in a mixed model ANOVA. Results: Surface texturing was significantly higher for the experimental compared with the control surface. The survey XPS spectra detected O, C, Al, and Ti at the control group, and Ca (similar to 0.2-0.9%) and P (similar to 1.7-4.1%) besides O, C, Al, and Ti at experimental surfaces. While no statistical difference in BIC was found between experimental and control surfaces or between 2 and 4 weeks in vivo, both longer time and use of experimental surface significantly increased resistance to failure. Conclusions: The experimental surface resulted in enhanced biomechanical fixation but comparable BIC relative to control, suggesting higher bone mechanical properties around the experimental implants.

Human Adipose-Derived Mesenchymal Stromal Cells Injected Systemically Into GRMD Dogs Without Immunosuppression Are Able to Reach the Host Muscle and Express Human Dystrophin

Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, is the most common and severe form of muscular dystrophies, affecting I in 3,500 male births. Mutations in the DMD gene lead to the absence of muscle dystrophin and a progressive degeneration of skeletal muscle. The possibility to treat DMD through cell therapy has been widely investigated. We have previously shown that human adipose-derived stromal cells (hASCs) injected systemically in SJL mice are able to reach and engraft in the host muscle, express human muscle proteins, and ameliorate the functional performance of injected animals without any immunosuppression. However, before starting clinical trials in humans many questions still need to be addressed in preclinical studies, in particular in larger animal models, when available. The best animal model to address these questions is the golden retriever muscular dystrophy (GRMD) dog that reproduces the full spectrum of human DMD. Affected animals carry a mutation that predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. These dogs present clinical signs within the first weeks and most of them do not survive beyond age two. Here we show the results of local and intravenous injections of hASCs into GRMD dogs, without immunosuppression. We observed that hASCs injected systemically into the dog cephalic vein are able to reach, engraft, and express human dystrophin in the host GRMD dystrophic muscle up to 6 months after transplantation. Most importantly, we demonstrated that injecting a huge quantity of human mesenchymal cells in a large-animal model, without immunosuppression, is a safe procedure, which may have important applications for future therapy in patients with different forms of muscular dystrophies.

Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation

Possa SS, Charafeddine HT, Righetti RF, da Silva PA, Almeida-Reis R, Saraiva-Romanholo BM, Perini A, Prado CM, Leick-Maldonado EA, Martins MA, Tiberio ID. Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. Am J Physiol Lung Cell Mol Physiol 303: L939-L952, 2012. First published September 21, 2012; doi:10.1152/ajplung.00034.2012.-Several studies have demonstrated the importance of Rho-kinase in the modulation of smooth muscle contraction, airway hyperresponsiveness, and inflammation. However, the effects of repeated treatment with a specific inhibitor of this pathway have not been previously investigated. We evaluated the effects of repeated treatment with Y-27632, a highly selective Rho-kinase inhibitor, on airway hyperresponsiveness, oxidative stress activation, extracellular matrix remodeling, eosinophilic inflammation, and cytokine expression in an animal model of chronic airway inflammation. Guinea pigs were subjected to seven ovalbumin or saline exposures. The treatment with Y-27632 (1 mM) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the animals' pulmonary mechanics were evaluated, and exhaled nitric oxide (E-NO) was collected. The lungs were removed, and histological analysis was performed using morphometry. Treatment with Y-27632 in sensitized animals reduced E-NO concentrations, maximal responses of resistance, elastance of the respiratory system, eosinophil counts, collagen and elastic fiber contents, the numbers of cells positive for IL-2, IL-4, IL-5, IL-13, inducible nitric oxide synthase, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta, NF-kappa B, IFN-gamma, and 8-iso-prostaglandin F2 alpha contents compared with the untreated group (P < 0.05). We observed positive correlations among the functional responses and inflammation, remodeling, and oxidative stress pathway activation markers evaluated. In conclusion, Rho-kinase pathway activation contributes to the potentiation of the hyperresponsiveness, inflammation, the extracellular matrix remodeling process, and oxidative stress activation. These results suggest that Rho-kinase inhibitors represent potential pharmacological tools for the control of asthma.

Imunocompetent Mice Model for Dengue Virus Infection

Dengue fever is a noncontagious infectious disease caused by dengue virus (DENV). DENV belongs to the family Flaviviridae, genus Flavivirus, and is classified into four antigenically distinct serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. The number of nations and people affected has increased steadily and today is considered the most widely spread arbovirus (arthropod-borne viral disease) in the world. The absence of an appropriate animal model for studying the disease has hindered the understanding of dengue pathogenesis. In our study, we have found that immunocompetent C57BL/6 mice infected intraperitoneally with DENV-1 presented some signs of dengue disease such as thrombocytopenia, spleen hemorrhage, liver damage, and increase in production of IFN gamma and TNF alpha cytokines. Moreover, the animals became viremic and the virus was detected in several organs by real-time RT-PCR. Thus, this animal model could be used to study mechanism of dengue virus infection, to test antiviral drugs, as well as to evaluate candidate vaccines.

Effects of lithium on oxidative stress parameters in healthy subjects

Increased neuronal oxidative stress (OxS) induces deleterious effects on signal transduction, structural plasticity and cellular resilience, mainly by inducing lipid peroxidation in membranes, proteins and genes. Major markers of OxS levels include the thiobarbituric acid reactive substances (TBARS) and the enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase. Lithium has been shown to prevent and/or reverse DNA damage, free-radical formation and lipid peroxidation in diverse models. This study evaluates OxS parameters in healthy volunteers prior to and following lithium treatment. Healthy volunteers were treated with lithium in therapeutic doses for 2-4 weeks. Treatment with lithium in healthy volunteers selectively altered SOD levels in all subjects. Furthermore, a significant decrease in the SOD/CAT ratio was observed following lithium treatment, wich was associated with decreased OxS by lowering hydrogen peroxide levels. This reduction in the SOD/CAT ratio may lead to lower OxS, indicated primarily by a decrease in the concentration of cell hydrogen peroxide. Overall, the present findings indicate a potential role for the antioxidant effects of lithium in healthy subjects, supporting its neuroprotective profile in bipolar disorder (BD) and, possibly, in neurodegenerative processes.

Considerations for Assessing Maximum Critical Temperatures in Small Ectothermic Animals: Insights from Leaf-Cutting Ants

The thermal limits of individual animals were originally proposed as a link between animal physiology and thermal ecology. Although this link is valid in theory, the evaluation of physiological tolerances involves some problems that are the focus of this study. One rationale was that heating rates shall influence upper critical limits, so that ecological thermal limits need to consider experimental heating rates. In addition, if thermal limits are not surpassed in experiments, subsequent tests of the same individual should yield similar results or produce evidence of hardening. Finally, several non-controlled variables such as time under experimental conditions and procedures may affect results. To analyze these issues we conducted an integrative study of upper critical temperatures in a single species, the ant Atta sexdens rubropiosa, an animal model providing large numbers of individuals of diverse sizes but similar genetic makeup. Our specific aims were to test the 1) influence of heating rates in the experimental evaluation of upper critical temperature, 2) assumptions of absence of physical damage and reproducibility, and 3) sources of variance often overlooked in the thermal-limits literature; and 4) to introduce some experimental approaches that may help researchers to separate physiological and methodological issues. The upper thermal limits were influenced by both heating rates and body mass. In the latter case, the effect was physiological rather than methodological. The critical temperature decreased during subsequent tests performed on the same individual ants, even one week after the initial test. Accordingly, upper thermal limits may have been overestimated by our (and typical) protocols. Heating rates, body mass, procedures independent of temperature and other variables may affect the estimation of upper critical temperatures. Therefore, based on our data, we offer suggestions to enhance the quality of measurements, and offer recommendations to authors aiming to compile and analyze databases from the literature.

N-acetylcysteine (NAC) Protects Against Acute Kidney Injury (AKI) Following Prolonged Pneumoperitoneum in the Rat

Background. Acute kidney injury (AKI) following prolonged laparoscopy is a documented phenomenon. Carbon dioxide pneumoperitoneum induces oxidative stress. Previous experimental studies have shown that the antioxidant, N-acetylcysteine, protects the rat from AKI following ischemia-reperfusion. The aim of this study was to evaluate the effects of N-acetylcysteine (NAC) on rat renal function after prolonged pneumoperitoneum. Methods. Normal rats treated or not with NAC were submitted to abdominal CO2 insufflation of 10 mmHg, at short and long periods of time of 1 and 3 h, respectively, and evaluated at 24, 72 h, and 1 wk after deinsufflation. Glomerular filtration rate (GFR) was measured by inulin clearance and oxidative stress was evaluated by serum thiobarbituric acid reactive substances (TBARS) Results. No significant alterations in GFR were observed in normal animals submitted to the pneumoperitoneum of 1 h and evaluated after 24 h desufflation. With 3 h of pneumoperitoneum, a significant and progressive decrease in GFR occurred 24 and 72 h after desufflation with an increase in serum TBARS. GFR returned to normal levels a week later. In the NAC-treated rats, a complete protection against GFR drops was observed 24 and 72 h following 3 h of pneumoperitoneum associated with a decrease in TBARS. Conclusion. These results suggest that NAC protects against acute kidney injury following prolonged pneumoperitoneum. These findings have significant clinical implications. (C) 2012 Elsevier Inc. All rights reserved.

Inducible Nitric Oxide Synthase Inhibition Attenuates Physical Stress-Induced Lung Hyper-Responsiveness and Oxidative Stress in Animals with Lung Inflammation

Mechanisms involved in stress-induced asthmatic alterations have been poorly characterised. We assessed whether inducible nitric oxide synthase (iNOS) inhibition modulates the stress-amplified lung parenchyma responsiveness, oxidative stress and extracellular matrix remodelling that was previously increased by chronic lung inflammation. Guinea pigs were subjected to 7 exposures to ovalbumin (1-5 mg/ml) or saline (OVA and SAL groups) over 4 weeks. To induce behavioural stress, animals were subjected to a forced swimming protocol (5 times/week, over 2 weeks; SAL-Stress and OVA-Stress groups) 24 h after the 4th inhalation. 1400W (iNOS-specific inhibitor) was administered intraperitoneally in the last 4 days of the protocol (SAL-1400W, OVA-1400W, SAL-Stress+1400W and OVA-Stress+1400W groups). Seventy-two hours after the last inhalation, animals were anaesthetised and exsanguinated, and adrenal glands were removed. Lung tissue resistance and elastance were evaluated by oscillatory mechanics and submitted for histopathological evaluation. Stressed animals had higher adrenal weights compared to non-stressed groups, which were reduced by 1400W treatment. Behavioural stress in sensitised animals amplified the resistance and elastance responses after antigen challenge, numbers of eosinophils and iNOS+ cells, actin content and 8-iso-PGF2 alpha density in the distal lung compared to the OVA group. 1400W treatment in ovalbumin-exposed and stressed animals reduced lung mechanics, iNOS+ cell numbers and 8-iso-PGF2a density compared to sensitised and stressed animals that received vehicle treatment. We concluded that stress amplifies the distal lung constriction, eosinophilic inflammation, iNOS expression, actin content and oxidative stress previously induced by chronic lung inflammation. iNOS-derived NO contributes to stress-augmented lung tissue functional alterations in this animal model and is at least partially due to activation of the oxidative stress pathway. copyright (C) 2012S. Karger AG, Basel

Effects of Hepatocyte Growth Factor Injection and Reinjection on Healing in the Rabbit Vocal Fold

Objectives/Hypothesis. Hepatocyte growth factor (HGF) is a multifunctional polypeptide that plays various roles in embryogenesis and tissue regeneration and exhibits marked antifibrotic activity. The present study sought to assess the effects of HGF injection and reinjection coinciding with its peak of activity on collagen density, vessel density, inflammatory reaction in the lamina propria, and mean epithelial thickness in the injured rabbit vocal fold. Study Design. Prospective, controlled, experimental animal study. Methods. Fourteen rabbits were subdivided into two groups and underwent injury of the vocal folds. Immediately after injury, animals in group 1 received HGF injections into the right vocal fold (RVF), whereas those in group 2 received bilateral HGF injections and a single reinjection into the RVF 10 days after the first, to coincide with the peak of HGF activity. The left vocal folds (LVFs) served as controls in both groups. Histological assessment of laryngeal specimens was performed at 30 and 40 days, respectively. Results. In both groups, collagen density was lower in the right (treated) vocal folds than in the left (control) folds (P = 0.018). Vessel density was higher in the RVFs in group 2 (P = 0.018). Differences were found in mean epithelial thickness and inflammatory reaction in the lamina propria but did not reach statistical significance. Conclusions. In the scarred rabbit vocal fold, HGF injection is associated with decreased collagen density in the lamina propria, whereas reinjection after 10 days produces decreased collagen density and higher vessel density.

Early and late neurodegeneration and memory disruption after intracerebroventricular streptozotocin

Glucose metabolism and insulin signaling disruptions in the brain have been proposed as a likely etiology of Alzheimer's disease. The aim of the present study was to investigate the time course of cognitive impairments induced by intracerebroventricular injection of streptozotocin (STZ) in rats and correlate them with the ensuing neurodegenerative process. Early and late effects of STZ were evaluated by using the reference and working memory versions of the Morris' water maze task and the evaluation of neurodegenerative markers by immunoblotting and the Fluoro-jade C histochemistry. The results revealed different types of behavioral and neurodegenerative responses, with distinct time courses. We observed an early disruption on the working memory as early as 3 h after STZ injections, which was followed by degenerative processes in the hippocampus at 1 and 15 days after STZ injections. Memory disruption increases over time and culminates with significant changes in amyloid-beta peptide and hyperphosphorylated Tau protein levels in distinct brain structures. These findings add information on the Alzheimer's disease-like STZ animal model and on the mechanisms underlying neurodegenerative processes. (C) 2012 Elsevier Inc. All rights reserved.

Evidence supporting a protective role for Th9 and Th22 cytokines in human and experimental periapical lesions

Introduction: The development of periapical granulomas is dependent on the host response and involves Th1, Th2, Th17, and Treg-related cytokines. The discovery of new Th9 and Th22 subsets, with important immunomodulatory roles mediated by interleukin (IL)-9 and IL-22, respectively, emphasizes the need for reevaluation of current cytokine paradigms in context of periapical lesions. We investigated the expression of IL-9 and IL-22 in active and stable human granulomas and throughout experimental lesion development in mice. Methods: Periapical granulomas (N = 83) and control specimens (N = 24) were evaluated regarding the expression of IL-9 and IL-22 via realtime polymerase chain reaction. Experimental periapical lesions were induced in mice (pulp exposure and bacterial inoculation) and the lesions evolution correlation with IL-9 and IL-22 expression kinetics was evaluated. Results: IL-9 and IL-22 mRNA expression was higher in periapical lesions than in control samples; higher levels of IL-9 and IL-22 were observed in inactive than in active lesions. In the experimental lesions model, increasing levels of IL-9 and IL-22 mRNA were detected in the lesions, and inverse correlations were found between IL-9 and IL-22 and the increase of lesion area in the different time point intervals. Conclusions: Our results suggest that Th9 and Th22 pathways may contribute to human and experimental periapical lesion stability

New perspective on the pathophysiology of panic: merging serotonin and opioids in the periaqueductal gray

Panic disorder patients are vulnerable to recurrent panic attacks. Two neurochemical hypotheses have been proposed to explain this susceptibility. The first assumes that panic patients have deficient serotonergic inhibition of neurons localized in the dorsal periaqueductal gray matter of the midbrain that organize defensive reactions to cope with proximal threats and of sympathomotor control areas of the rostral ventrolateral medulla that generate most of the neurovegetative symptoms of the panic attack. The second suggests that endogenous opioids buffer normal subjects from the behavioral and physiological manifestations of the panic attack, and their deficit brings about heightened suffocation sensitivity and separation anxiety in panic patients, making them more vulnerable to panic attacks. Experimental results obtained in rats performing one-way escape in the elevated T-maze, an animal model of panic, indicate that the inhibitory action of serotonin on defense is connected with activation of endogenous opioids in the periaqueductal gray. This allows reconciliation of the serotonergic and opioidergic hypotheses of panic pathophysiology, the periaqueductal gray being the fulcrum of serotonin-opioid interaction.