RESUMO Objetivo: Avaliar se o azul de metileno (AM) pode minimizar os efeitos da lesão de isquemia-reperfusão sobre o pulmão não isquêmico em um modelo de transplante pulmonar em roedores. Métodos: Quarenta ratas Sprague-Dawley foram divididas em 20 doadoras e 20 receptoras. As 20 ratas receptoras foram divididas em dois grupos (n = 10) de acordo com o tratamento (solução salina a 0,9% vs. AM a 1%). Todos os animais foram submetidos a transplante pulmonar unilateral. As receptoras receberam 2 ml de solução salina ou de AM por via intraperitoneal antes do transplante. Após 2 h de reperfusão, os animais foram sacrificados, e foram realizadas análises histopatológicas e imuno-histoquímicas no pulmão não isquêmico. Resultados: Houve diminuição significativa da inflamação - a contagem de neutrófilos e a expressão de intercellular adhesion molecule-1 (ICAM-1, molécula de adesão intercelular-1) foram maiores no grupo salina em comparação com o grupo AM - e da apoptose - a expressão de caspase-3 foi maior no grupo salina, e a expressão de Bcl-2 foi maior no grupo AM. Conclusões: O AM é uma droga eficaz para a proteção de pulmões não isquêmicos contra inflamação e apoptose após transplante pulmonar unilateral em ratos.

ABSTRACT Objective: To evaluate whether methylene blue (MB) could minimize the effects of ischemia-reperfusion injury in the nonischemic lung on a lung transplantation rodent model. Methods: Forty female Sprague-Dawley rats were divided into 20 donors and 20 recipients. The 20 recipient rats were divided into two groups (n = 10) according to the treatment (0.9% saline vs. 1% MB solutions). All animals underwent unilateral lung transplantation. Recipients received 2 mL of saline or MB intraperitoneally prior to transplantation. After 2 h of reperfusion, the animals were euthanized and histopathological and immunohistochemical analyses were performed in the nonischemic lung. Results: There was a significant decrease in inflammation-neutrophil count and intercellular adhesion molecule-1 (ICAM-1) expression in lung parenchyma were higher in the saline group in comparison with the MB group-and in apoptosis-caspase-3 expression was higher in the saline group and Bcl-2 expression was higher in MB group. Conclusions: MB is an effective drug for the protection of nonischemic lungs against inflammation and apoptosis following unilateral lung transplantation in rats.

OBJECTIVE: To evaluate the respiratory systems of male and female rats maintained in individually ventilated cages (IVCs) from birth until adulthood. METHODS: Female Wistar rats were housed in individually ventilated cages or conventional cages (CCs) and mated with male Wistar rats. After birth and weaning, the male offspring were separated from the females and kept in cages of the same type until 12 weeks of age. RESULTS: The level of food consumption was lower in male offspring (IVC=171.7±9; CC=193.1±20) than in female offspring (IVC=100.6±7; CC=123.4±0.4), whereas the water intake was higher in female offspring (IVC=149.8±11; CC=99.2±0) than in male offspring (IVC=302.5±25; CC=249.7±22) at 11 weeks of age when housed in IVCs. The cage temperature was higher in individually ventilated cages than in conventional cages for both male (IVCs=25.9±0.5; CCs=22.95±0.3) and female (IVCs=26.2±0.3; CCs=23.1±0.3) offspring. The respiratory resistance (IVC=68.8±2.8; CC=50.6±3.0) and elastance (IVC=42.0±3.9; CC=32.4±2.0) at 300 µm/kg were higher in the female offspring housed in ventilated cages. The ciliary beat values were lower in both the male (IVCs=13.4±0.2; CC=15±0.4) and female (IVC=13.5±0.4; CC=15.9±0.6) offspring housed in individually ventilated cages than in those housed in conventional cages. The total cell (IVC=117.5±9.7; CC=285.0±22.8), neutrophil (IVC=13.1±4.8; CC=75.6±4.1) and macrophage (IVC=95.2±11.8; CC=170.0±18.8) counts in the bronchoalveolar lavage fluid were lower in the female offspring housed in individually ventilated cages than in those housed in conventional cages. CONCLUSIONS: The environmental conditions that exist in individually ventilated cages should be considered when interpreting the results of studies involving laboratory animals. In this study, we observed gender dimorphism in both the water consumption and respiratory mechanics of rats kept in ventilated cages.

RESUMO OBJETIVO: A investigação de intensidades padrão de exercício físico é importante para melhor compreender e desenvolver programas de reabilitação para enfisema. Nosso objetivo foi avaliar os efeitos de diferentes intensidades (moderada e alta) de exercício físico sobre o desenvolvimento de enfisema induzido por protease (instilação endotraqueal de papaína) em ratos. MÉTODOS: Ratos Wistar machos foram divididos aleatoriamente em cinco grupos que receberam instilação intra-traqueal de solução de papaína ou veículo: (i) papaína, exercício de alta intensidade, (ii) papaína, exercício moderado, (iii) soro fisiológico, exercício de alta intensidade, (iv) soro fisiológico, sedentário e (v) papaína, sedentário. Quarenta dias após a instilação intra-traqueal os grupos de exercício foram submetidos a um protocolo de treinamento físico em esteira durante 10 semanas, 5 dias/ semana, a 0.9 km/h (papaína e solucão salina exercício de alta intensidade), ou em 0.6km/h (papaína exercício moderado). Medimos elastância do sistema respiratório e resistência, as fibras de colágeno no parênquima pulmonar, e a intercepção linear média pulmonar. RESULTADOS: Todos os grupos de animais que receberam instilação de papaína apresentaram maior destruição da parede alveolar em comparação com animais que receberam apenas solução salina. O grupo papaína exercício de alta intensidade apresentou maiores valores de intercepto linear médio em comparação com grupos de enfisema que foram treinados em uma intensidade moderada ou não submetidos ao exercício. CONCLUSÃO: A alta intensidade de treinamento físico produziu mais destruição alveolar em um modelo experimental de enfisema em ratos quando comparada ao exercício de intensidade moderada, ou a nenhum exercício.

OBJECTIVE: Investigation of standard intensities of physical exercise is important to better comprehend and develop rehabilitation programs for emphysema. We aimed to evaluate the effects of different intensities (moderate and high-intensity) of physical exercise on the development of a protease-induced (papain intratracheal instillation) emphysema in rats. METHODS: Male Wistar rats were randomly separated into five groups that received intratracheal instillation of papain solution or vehicle: (i) papain high intensity exercise, (ii) papain moderate exercise, (iii) saline high intensity exercise, (iv) saline sedentary and (v) papain sedentary. Forty days after intratracheal instillation, the exercise groups were submitted to an exercise-training protocol on a treadmill during 10 weeks, 5 days/week, at 0.9 km/h (Papain and saline high exercise), or at 0.6 km/h (papain moderate exercise).We measured respiratory system elastance and resistance, the collagen fiber lung parenchyma, and the pulmonary mean linear intercept. RESULTS: All animal groups that received papain instillation presented higher alveolar wall destruction compared to animals that received only saline solution. The papain high intensity exercise group presented higher values of mean linear intercept compared to emphysema groups that were trained at a moderate intensity or not submitted to exercise. CONCLUSION: High intensity exercise training worsened alveolar destruction in an experimental model of emphysema in rats when compared to moderate intensity exercise, or to no exercise.

OBJECTIVES:Despite the recent success regarding the transplantation of tissue-engineered airways, the mechanical properties of these grafts are not well understood. Mechanical assessment of a tissue-engineered airway graft before implantation may be used in the future as a predictor of function. The aim of this preliminary work was to develop a noninvasive image-processing environment for the assessment of airway mechanics.METHOD:Decellularized, recellularized and normal tracheas (groups DECEL, RECEL, and CONTROL, respectively) immersed in Krebs-Henseleit solution were ventilated by a small-animal ventilator connected to a Fleisch pneumotachograph and two pressure transducers (differential and gauge). A camera connected to a stereomicroscope captured images of the pulsation of the trachea before instillation of saline solution and after instillation of Krebs-Henseleit solution, followed by instillation with Krebs-Henseleit with methacholine 0.1 M (protocols A, K and KMCh, respectively). The data were post-processed with computer software and statistical comparisons between groups and protocols were performed.RESULTS:There were statistically significant variations in the image measurements of the medial region of the trachea between the groups (two-way analysis of variance [ANOVA], p<0.01) and of the proximal region between the groups and protocols (two-way ANOVA, p<0.01).CONCLUSIONS:The technique developed in this study is an innovative method for performing a mechanical assessment of engineered tracheal grafts that will enable evaluation of the viscoelastic properties of neo-tracheas prior to transplantation.

OBJECTIVE: This study evaluated the performance of lungs that were preserved with different solutions (Celsior, Perfadex or saline) in an ex vivo rat lung perfusion system. METHODS: Sixty Wistar rats were anesthetized, anticoagulated and randomized into three groups (n = 20). The rats were subjected to antegrade perfusion via the pulmonary artery with Perfadex, Celsior, or saline, followed by 6 or 12 hours of ischemia (4ºC, n = 10 in each group). Respiratory mechanics, gas exchange and hemodynamics were measured at 10-minute intervals during the reperfusion of heart-lung blocks in an ex vivo system (IL2-Isolated Perfused Rat or Guinea Pig Lung System, Harvard Apparatus, Holliston, Massachusetts, USA; Hugo Sachs Elektronik, Germany) for 60 minutes. The lungs were prepared for histopathology and evaluated for edema following reperfusion. Group comparisons were performed using ANOVA and the Kruskal-Wallis test with a 5% level of significance. RESULTS: Gas exchange was not significantly different between lungs perfused with either Perfadex or Celsior at the same ischemic times, but it was very low in lungs that were preserved with saline. Airway resistance was greater in the lungs that were preserved for 12 hours. Celsior lungs that were preserved for 6 and 12 hours exhibited lower airway resistance (p = 0.01) compared to Perfadex lungs. Pulmonary artery pressure was not different between the groups, and no significant differences in histopathology and apoptosis were observed between the groups. CONCLUSIONS: Lungs that were preserved with Celsior or Perfadex exhibited similar gas exchange and histopathological findings. Airway resistance was slightly lower in the Celsior-preserved lungs compared with the Perfadex-preserved lungs.

OBJECTIVE: To study the effects of mycophenolate sodium on mucociliary clearance. INTRODUCTION: Mycophenolate is one of the most commonly used immunosuppressive drugs in lung transplantation. Although its pharmacokinetic properties are well defined, its side effects on mucociliary clearance have not yet been studied. METHODS: Sixty rats were subjected to left bronchial section and anastomosis. The right bronchus was used as a control. After surgery, the rats were assigned to two groups based on whether they received saline solution (n = 30) or mycophenolate sodium (n = 30). After 7, 15, or 30 days of treatment, 10 animals from each group were sacrificed, and in vitro mucus transportability, in situ mucociliary transport velocity and ciliary beat frequency were measured. RESULTS: The analysis of mucus transportability revealed that neither mycophenolate nor bronchial section altered any transportability related property for up to 30 days of treatment after surgery (p>0.05). With regard to ciliary beat frequency, the operated left bronchi from the mycophenolate group showed a significant decrease on post-surgical day 30 (p = 0.003). In addition, we found a significant reduction in the in situ mucociliary transport velocity in the mycophenolate-treated group (p = 0.0001). DISCUSSION: These data add important information regarding mucociliary clearance dysfunction following mycophenolate therapy and suggest that mycophenolate might contribute to the high incidence of respiratory tract infections in lung transplant patients. Further studies are needed to investigate the combined action of mycophenolate with other immunosuppressive drugs and to establish methods to protect and recover mucociliary clearance, an important airway defense mechanism.

OBJETIVO: Investigar a função do sistema mucociliar em ratos transplantados sob a influência de imunossupressores. MÉTODOS: Trinta e seis ratos foram submetidos ao transplante pulmonar unilateral e divididos em dois grupos, onde um grupo foi tratado com ciclosporina e outro foi controle. Administrada ciclosporina por via subcutânea na dose de 10 mg/kg diariamente. Os ratos foram sacrificados 2, 15 e 30 dias após o transplante pulmonar. O transporte mucociliar brônquico (TMC) in situ e a freqüência de batimento ciliar (FBC) foram analisados na porção proximal e distal à anastomose brônquica. Realizada correlação dos achados com parâmetros gasométricos e histologia. RESULTADOS: Foi encontrada melhora progressiva e significante no TMC na região proximal e distal a anastomose no grupo que recebeu ciclosporina em 15 e 30 dias (p<0,01). Não houve diferença na FBC nos dois grupos estudados. A análise histológica mostrou que a rejeição foi significantemente maior no grupo controle (p<0,05). A oxigenação foi melhor nos animais que receberam a ciclosporina. CONCLUSÃO: A ciclosporina exerceu influência positiva no transporte mucociliar brônquico, provavelmente por sua ação imunossupressora.

PURPOSE: To investigate the function of the bronchial mucociliary system in transplanted rat lungs with and without the influence of immunosuppression. METHODS: Thirty-six rats underwent single-lung transplantation and were divided into two groups, one of which received cyclosporine treatment, and the control group which did not. Cyclosporine was administered subcutaneously in doses of 10 mg/kg daily. The rats were sacrificed 2, 15 or 30 days after transplantation. In situ bronchial mucociliary transport (MCT) and ciliary beat frequency (CBF) were determined proximal and distal to the bronchial anastomosis. RESULTS: Significant progressive improvement on MCT, proximal and distal to the anastomotic site, was also found in the cyclosporine-treated group at 15 and 30 days (p<0.01). No significant change in MCT was found in the control group. CBF behavior in the two groups. Histological analysis showed that rejection was significantly higher in the control group (p<0.05). CONCLUSION: Cyclosporine has a positive influence on bronchial mucociliary transport but not on CBF due to the effect of the rejection mechanism.

OBJETIVO: Avaliar os efeitos da ciclosporina A sobre a produção de muco das células caliciformes e sobre o transporte mucociliar in situ de ratos. MÉTODOS: Vinte e um ratos machos Wistar foram distribuídos em três grupos: Controle (n=5), Salina (n=8) e Ciclosporina A (n=8). Após 30 dias de terapia, os ratos foram mortos e os pulmões removidos da cavidade torácica. Amostras de muco foram coletadas e a medida da transportabilidade in vitro foi realizada através de um modelo de palato de rã. A velocidade do transporte mucociliar foi medida através da observação direta do deslocamento de partículas aderidas ao muco do epitélio ciliado brônquico. Por fim, efetuamos a quantificação das mucinas estocadas nas células caliciformes do epitélio respiratório. RESULTADOS: O valor médio da concentração sangüínea da ciclosporina no momento do sacrifício dos ratos foi de 1.246,57 ± 563,88 ng/ml. A transportabilidade do muco in vitro foi estatisticamente menor (p < 0.001) no grupo tratado com ciclosporina. Da mesma forma, houve um decréscimo na velocidade de transporte mucociliar nos animais imunossuprimidos em relação aos que receberam o placebo (p = 0.02). Houve diminuição significativa na quantidade de muco ácido (p = 0,01) e neutro (p = 0,02) produzidos pelas células caliciformes nos animais tratados com ciclosporina. A correlação entre a porcentagem de muco e a transportabilidade in vitro foi positiva e significante (r = 0.706, p < 0.001), assim como entre a porcentagem do muco e o transporte mucociliar in situ (r = 0.688, p = 0.001). CONCLUSÃO: O presente estudo mostra que a ciclosporina A age no sistema mucociliar causando um sério prejuízo através da redução na produção de muco ácido e neutro pelas células caliciformes como também a diminuição da velocidade de transporte mucociliar in situ e a transportabilidade do muco in vitro.

PURPOSE: To assay the effects of cyclosporin A on mucus secretion from goblet cells and on mucociliary transport in situ in rats. METHODS: Twenty-one male Wistar rats were assigned to 3 groups: control (n = 5), saline (n = 8), and cyclosporin A (n = 8). After 30 days of drug therapy, the rats were killed, and the lungs were removed from the thoracic cavity. Mucus samples were collected, and the transport rate was evaluated in vitro using a bullfrog palate model. Mucociliary transport was timed in situ by direct view of particles trapped on the mucus moving across the respiratory tract. Finally, the amount of stored mucins in the goblet cells of the respiratory epithelium was measured. RESULTS: Drug dosage measurements showed that cyclosporine blood concentration at the moment the rats were killed was 1246.57 ± 563.88 ng/mL. The in vitro transport rate was significantly lower (P < .001) in the cyclosporin A-treated group. Also, the in-situ mucociliary transport rate was decreased in all cyclosporin A-treated animals when compared to the saline group (P = .02). Mucus quantity measurements showed a significant decrease on both acid (P = .01) and neutral (P = .02) mucus production from goblet cells in the animals submitted to cyclosporin A therapy. The correlation between the percentage of total mucus and in vitro transport rate was positive and significant (r = 0.706, P < .001), as was the correlation between the percentage of total mucus and the in situ mucociliary transport rate (r = 0.688, P = .001). CONCLUSION: This study shows that cyclosporin A plays an important role in the impairment of the mucociliary clearance in rats by reducing both acid and neutral mucus production from goblet cells and causing a decrease in the mucociliary transport velocity.