Acute lung injury (ALI) is a critical manifestation of sepsis/septic shock. Heat shock protein A12B (HSPA12B), an endothelial cell-expressed heat shock protein, shows a negative regulation of lipopolysaccharide (LPS)-induced inflammation in myocardium and endothelial cells. However, it is unclear whether HSPA12B exerts protective effects against ALI during sepsis/septic shock. In this study, we treated HSPA12B transgenic mice (Tg) and wild type littermates (WT) with LPS for 6 h to induce endotoxemia. LPS treatment significantly caused pulmonary injuries as evidenced by microarchitecture destruction, vascular leakage and neutrophil recruitment in lungs of WT mice. However, the LPS-induced pulmonary injuries were significantly attenuated in Tg mice. Moreover, the LPS-induced activation of extracellular signal-regulated kinases (ERKs) and upregulation of intercellular adhesion molecule-1 (ICAM-1) and Cyclooxygenase-2 (Cox-2) were inhibited in Tg lungs compared with that in WT mice. Additionally, Tg lungs showed a significant lower level of vascular endothelial growth factor (VEGF) compared with WT mice. Our results demonstrate a pulmonary protective effect of HSPA12B against endotoxin challenge, which indicates management of HSPA12B expression could serve as a potential therapeutic target for ALI during sepsis/septic shock.

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Thesis (Ph.D. in Clinical Science) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 96-120). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

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Introdução: A prática regular de exercício tem sido grandemente associada a efeitos benéficos em doenças pulmonares crônicas como asma e doença pulmonar obstrutiva crônica. Poucos estudos têm avaliado os benefícios do exercício aeróbico na lesão pulmonar aguda (LPA). Objetivo: Neste estudo nós investigamos os mecanismos envolvidos no papel do exercício físico em diminuir os danos pulmonares causados pela LPA induzida por lipopolissacarídeo (LPS). Métodos: Camundongos Balb/c foram divididos em quatro grupos: Controle (CTR), Exercício (Exe), LPS e Exercício+LPS (Exe+LPS). Os animais dos grupos Exe e Exe+LPS foram treinados em baixa intensidade por 60 minutos/dia, 3x/semana, durante 5 semanas. A instilação intratraqueal de LPS (200 /animal) foi realizada 48 horas após o último teste físico nos grupos LPS e Exe+LPS. Vinte e quatro horas após a instilação de LPS nós analisamos os níveis de óxido nítrico exalado (NO), a mecânica respiratória e a densidade de neutrófilos no tecido pulmonar. Nós analisamos também os níveis de extravasamento de proteína, contagem de células totais e diferenciais e os níveis de IL-1, IL-6, KC, IL-10 and TNF- no lavado bronco-alveolar (LBA). Os níveis de IL-6 e IL-10 também foram avaliados no plasma e tecido pulmonar. A expressão de receptores de glicocorticóide (Gre) e da enzima superóxido dismutase (SOD) foi analisada no tecido pulmonar. As atividades enzimáticas de glutationa peroxidade (GPX), catalase (CAT), glutationa redutase (GR), e SOD foram determinadas no homogenato de pulmão por espectrofotometria. O nível de malonaldeído (MDA) foi quantificado no homogenato de pulmão. Resultados: A instilação de LPS resultou em aumento nos níveis de NO exalado (p<0,01), aumento do número de células e neutrófilos no LBA (p<0,001), aumento do número de neutrófilos no parênquima pulmonar (p<0,001), aumento dos valores de resistência e elastância pulmonar (p=0,01), aumento dos níveis de extravasamento de proteína (p0,02), aumento dos níveis de IL-6 e IL-10 no plasma (p<0,02) e aumento dos níveis de IL-1, IL-6 e KC no LBA (p0,005), comparado ao grupo CTR. O exercício aeróbico (grupo Exe+LPS) diminuiu significativamente os níveis de NO exalado (p=0,006), a densidade de neutrófilos no parênquima pulmonar (p=0,004), os valores de resistência e elastância pulmonar (p = 0,003), aumentou a expressão de IL-6, IL-10 e Gre no tecido pulmonar (p0,04) e aumentou o nível de IL- 1 no LBA (p=0,04) comparado ao grupo LPS. Conclusão: Nossos resultados mostram que o exercício desenvolve um importante papel em proteger o pulmão dos efeitos inflamatórios da LPA induzida por LPS. Os efeitos do exercício são principalmente mediados pelo aumento da expressão de citocinas antiinflamatórias, sugerindo que o exercício aeróbico pode modular o balanço inflamatório, antiinflamatório na fase inicial na SARA. / Background: The regular practice of exercise has been increasingly associated to benefic effects on chronic pulmonary conditions such as asthma and chronic obstructive pulmonary disease. Few studies have also reported the effects of aerobic exercise on acute lung injury (ALI). Objective: In this study we investigated the mechanisms involved in the role of exercise in attenuating the pulmonary changes in a model of lipopolysaccharide (LPS)-induced ALI. Methods: BALB/c mice were divided into four groups: Control (CTR), Exercise (Exe), LPS, and Exercise + LPS (Exe+LPS). Mice from Exe and Exe+LPS groups were trained at low intensity exercise for 60 minutes/day, 3 days/week, during 5 weeks. Intratracheal instillation of LPS (200/mouse) was performed 48 hours after the last physical test in the LPS and Exe+LPS groups. Twenty-four hours after LPS instillation we measured exhaled nitric oxide (NO), respiratory mechanics, and the density of neutrophils in lung tissue. We further analyzed protein leakage, total and differential cell counts and the levels of IL-1, IL-6, KC, IL-10 and TNF- in bronchoalveolar lavage fluid (BALF). IL-6 and IL-10 levels were also evaluated in serum and lung tissue. The expression of glucocorticoid receptors (Gre) and superoxide dismutase (SOD) was analyzed in lung tissue. Enzymatic activity of glutathione peroxidase (GPX), catalase (CAT), glutathione reductase (GR) and SOD was determined in lung homogenates by spectrophotometry. The level of malondialdehyde (MDA) was quantified in lung homogenates. Results: LPS instillation resulted in increased levels of exhaled NO (p<0.01), higher number of total cells and neutrophils in the BALF (p<0.001), higher number of neutrophils in the lung parenchyma (p<0.001), higher values of pulmonary resistance and elastance (p=0.01), increase of protein leakage (p0.02), increase of IL-6 and IL-10 level in serum (p<0.02) and increase in IL-1, IL-6 and KC levels in BALF (p0.005), compared to the CTR group. Aerobic exercise (Exe+LPS group) resulted in significantly lower exhaled NO levels (p=0.006), lower density of neutrophils in the lung parenchyma (p=0.004), lower pulmonary resistance and elastance values (p = 0.003), increased expression of IL-6, IL-10 and Gre in lung tissue (p0.04) and increased IL-1 level in BALF (p=0.04) compared to the LPS group. Conclusion: Our results show that exercise plays an important role in protecting the lung from the inflammatory effects of LPS-induced ALI. The effects of exercise are mainly mediated by the increased expression of anti-inflammatory cytokines, suggesting that aerobic preconditioning can modulate the inflammatory-anti-inflammatory balance in the early phase of ARDS.

Diversas estratégias de ventilação mecânica que estabelecem limites na pressão e volume intratorácicas têm sido propostas para pacientes com síndrome de angustia respiratória aguda (SARA). Estas recomendações são baseadas na observação de que a ventilação mecânica com volume corrente excessivo ou pressão positiva expiratória final (PEEP) insuficiente pode ocasionar lesões pulmonares graves, decorrentes de superdistensão de unidades alveolares. O objetivo do atual estudo foi aplicar manobras de recrutamento alveolar e PEEP em pulmões submetidos à lesão pulmonar aguda (LPA) através da administração de HCl . Foram utilizados 24 suínos Landrace - Largewhite, do sexo feminino, pesando entre 25 e 35 Kg. Após a anestesia os animais foram submetidos à ventilação com volume controlado (6 a 8 ml/Kg) e foram randomizados em 4 grupos: GI (6 animais não submetidos a LPA e tratados com PEEP progressivo de 5, 10, 15 e 20 cmH2O e regressivo de 20 a 5 cmH2O); GII (6 animais não submetidos a LPA e tratados com PEEP progressivo de 5, 10, 15 e 20 cmH2O e regressivo de 20 a 5 cmH2O associado a 3 manobras de recrutamento consecutivas, com pressão de 30 cmH2O antes de cada alteração do PEEP); GIII (6 animais submetidos a 1 hora de LPA por HCl e tratados como GI) e GIV (6 animais submetidos a 1 hora de LPA por HCl e tratados como GII). A mecânica respiratória e oxigenação foram avaliadas a cada 20 minutos, acompanhando cada alteração do PEEP. A LPA foi observada através de severas alterações na oxigenação e mecânica respiratória. A administração de MR associada a PEEP foi capaz de restaurar os valores controle, porém, os elevados valores de PEEP e CPAP foram acompanhados de significantes alterações hemodinâmicas quando comparadas com os animais que não foram submetidos a LPA. O derrecrutamento alveolar ocorreu provavelmente quando os valores de PEEP foram retornados para 5cmH2O. As lesões pulmonares foram uniformes nos animais que foram submetidos ao HCl, evidenciadas pela presença de necrose, hemorragia, congestão e infiltrado de células inflamatórias no interstício e nos alvéolos. O modelo experimental de lesão pulmonar aguda foi adequado para estudar MR seguidas por PEEP, pois apresentou importantes alterações dos valores de oxigenação e complacência, observado 1 hora após a instilação do HCl.Os valores de PEEP de 5cmH2O foram incapazes de manter o recrutamento no momento final do estudo, enquanto que os valores de PEEP de 10cmH2O foram suficientes para restabelecer a oxigenação com mínima alteração hemodinâmica. A complacência não melhorou após as manobras. Futuros estudos são necessários para confirmar os resultados obtidos, especialmente para mostrar que a manutenção do PEEP de 10cmH2O é suficientes para manter o recrutamento após as MR / Different mechanical ventilation strategies which define limits of intrathoracic pressures and volumes are being proposed for patients with acute respiratory distress syndrome (ARDS). These recommendations are based on observations that mechanical ventilation with excessive tidal volumes or insufficient values of positive end expiratory pressure (PEEP) can cause severe lung injury due to overinflation. The aim of the present study was to apply recruitment maneuvers (RM) and PEEP in lungs submitted to acute lung injury (ALI) due to the administration of hydrochloride acid. Twenty four female Landrace – Largewhite pigs, weighing 25 to 35 Kg were used. After anesthesia, animals were submitted to volume controlled mechanical ventilation (6 to 8ml/kg) and were randomly allocated in four groups of 6 animals each: GI animals without ALI and treated with progressive values of PEEP (5, 10, 15 and 20 cmH2O) or regressive (20 to 5 cm H2O); GII animals without ALI and treated with progressive values of PEEP (5, 10, 15 and 20 cmH2O) or regressive (20 to 5 cm H2O) plus 3 consecutive recruitment maneuvers with 30 cmH2O; GIII animals submitted to 1 hour of ALI and treated as GI; GIV animals submitted to 1 hour of ALI and treated as GII. Parameters of respiratory mechanics, ventilation and oxygenation were measured each 20 minutes according to the change of the PEEP values. ALI could be observed by the severe changes of oxygenation and respiratory mechanics noted. The use of RM and PEEP were able to restore control values. Nevertheless, application of high values of PEEP and CPAP were accompanied by significant hemodynamic changes which could be evidenced in animals without ALI. Derecruitment probably occurred when PEEP value reached 5 cmH2O. The lung lesions were uniform in the HCL-injured animals and consisted of necrosis, hemorrhage, congestion, and inflammatory cells infiltration that involved both the interstitium and the alveoli. The experimental model of lung injury was adequate to the study of RM followed by PEEP since significant changes of the oxygenation and compliance values could be observed 1 hour after acid instillation. PEEP values of 5cmH2O were incapable to maintain recruitment at the end of the observation period, while 10 cmH2O were sufficient to promote the reestablishment of oxygenation index with minimal hemodynamic changes. Compliance did not improve during the maneuvers. Further studies are necessary to confirm the results obtained, especially to show that the maintenance of a PEEP value of 10 cmH2O are sufficient to maintain recruitment after the RM

Diversas estratégias de ventilação mecânica que estabelecem limites na pressão e volume intratorácicas têm sido propostas para pacientes com síndrome de angustia respiratória aguda (SARA). Estas recomendações são baseadas na observação de que a ventilação mecânica com volume corrente excessivo ou pressão positiva expiratória final (PEEP) insuficiente pode ocasionar lesões pulmonares graves, decorrentes de superdistensão de unidades alveolares. O objetivo do atual estudo foi aplicar manobras de recrutamento alveolar e PEEP em pulmões submetidos à lesão pulmonar aguda (LPA) através da administração de HCl . Foram utilizados 24 suínos Landrace - Largewhite, do sexo feminino, pesando entre 25 e 35 Kg. Após a anestesia os animais foram submetidos à ventilação com volume controlado (6 a 8 ml/Kg) e foram randomizados em 4 grupos: GI (6 animais não submetidos a LPA e tratados com PEEP progressivo de 5, 10, 15 e 20 cmH2O e regressivo de 20 a 5 cmH2O); GII (6 animais não submetidos a LPA e tratados com PEEP progressivo de 5, 10, 15 e 20 cmH2O e regressivo de 20 a 5 cmH2O associado a 3 manobras de recrutamento consecutivas, com pressão de 30 cmH2O antes de cada alteração do PEEP); GIII (6 animais submetidos a 1 hora de LPA por HCl e tratados como GI) e GIV (6 animais submetidos a 1 hora de LPA por HCl e tratados como GII). A mecânica respiratória e oxigenação foram avaliadas a cada 20 minutos, acompanhando cada alteração do PEEP. A LPA foi observada através de severas alterações na oxigenação e mecânica respiratória. A administração de MR associada a PEEP foi capaz de restaurar os valores controle, porém, os elevados valores de PEEP e CPAP foram acompanhados de significantes alterações hemodinâmicas quando comparadas com os animais que não foram submetidos a LPA. O derrecrutamento alveolar ocorreu provavelmente quando os valores de PEEP foram retornados para 5cmH2O. As lesões pulmonares foram uniformes nos animais que foram submetidos ao HCl, evidenciadas pela presença de necrose, hemorragia, congestão e infiltrado de células inflamatórias no interstício e nos alvéolos. O modelo experimental de lesão pulmonar aguda foi adequado para estudar MR seguidas por PEEP, pois apresentou importantes alterações dos valores de oxigenação e complacência, observado 1 hora após a instilação do HCl.Os valores de PEEP de 5cmH2O foram incapazes de manter o recrutamento no momento final do estudo, enquanto que os valores de PEEP de 10cmH2O foram suficientes para restabelecer a oxigenação com mínima alteração hemodinâmica. A complacência não melhorou após as manobras. Futuros estudos são necessários para confirmar os resultados obtidos, especialmente para mostrar que a manutenção do PEEP de 10cmH2O é suficientes para manter o recrutamento após as MR / Different mechanical ventilation strategies which define limits of intrathoracic pressures and volumes are being proposed for patients with acute respiratory distress syndrome (ARDS). These recommendations are based on observations that mechanical ventilation with excessive tidal volumes or insufficient values of positive end expiratory pressure (PEEP) can cause severe lung injury due to overinflation. The aim of the present study was to apply recruitment maneuvers (RM) and PEEP in lungs submitted to acute lung injury (ALI) due to the administration of hydrochloride acid. Twenty four female Landrace – Largewhite pigs, weighing 25 to 35 Kg were used. After anesthesia, animals were submitted to volume controlled mechanical ventilation (6 to 8ml/kg) and were randomly allocated in four groups of 6 animals each: GI animals without ALI and treated with progressive values of PEEP (5, 10, 15 and 20 cmH2O) or regressive (20 to 5 cm H2O); GII animals without ALI and treated with progressive values of PEEP (5, 10, 15 and 20 cmH2O) or regressive (20 to 5 cm H2O) plus 3 consecutive recruitment maneuvers with 30 cmH2O; GIII animals submitted to 1 hour of ALI and treated as GI; GIV animals submitted to 1 hour of ALI and treated as GII. Parameters of respiratory mechanics, ventilation and oxygenation were measured each 20 minutes according to the change of the PEEP values. ALI could be observed by the severe changes of oxygenation and respiratory mechanics noted. The use of RM and PEEP were able to restore control values. Nevertheless, application of high values of PEEP and CPAP were accompanied by significant hemodynamic changes which could be evidenced in animals without ALI. Derecruitment probably occurred when PEEP value reached 5 cmH2O. The lung lesions were uniform in the HCL-injured animals and consisted of necrosis, hemorrhage, congestion, and inflammatory cells infiltration that involved both the interstitium and the alveoli. The experimental model of lung injury was adequate to the study of RM followed by PEEP since significant changes of the oxygenation and compliance values could be observed 1 hour after acid instillation. PEEP values of 5cmH2O were incapable to maintain recruitment at the end of the observation period, while 10 cmH2O were sufficient to promote the reestablishment of oxygenation index with minimal hemodynamic changes. Compliance did not improve during the maneuvers. Further studies are necessary to confirm the results obtained, especially to show that the maintenance of a PEEP value of 10 cmH2O are sufficient to maintain recruitment after the RM

Introdução: A prática regular de exercício tem sido grandemente associada a efeitos benéficos em doenças pulmonares crônicas como asma e doença pulmonar obstrutiva crônica. Poucos estudos têm avaliado os benefícios do exercício aeróbico na lesão pulmonar aguda (LPA). Objetivo: Neste estudo nós investigamos os mecanismos envolvidos no papel do exercício físico em diminuir os danos pulmonares causados pela LPA induzida por lipopolissacarídeo (LPS). Métodos: Camundongos Balb/c foram divididos em quatro grupos: Controle (CTR), Exercício (Exe), LPS e Exercício+LPS (Exe+LPS). Os animais dos grupos Exe e Exe+LPS foram treinados em baixa intensidade por 60 minutos/dia, 3x/semana, durante 5 semanas. A instilação intratraqueal de LPS (200 /animal) foi realizada 48 horas após o último teste físico nos grupos LPS e Exe+LPS. Vinte e quatro horas após a instilação de LPS nós analisamos os níveis de óxido nítrico exalado (NO), a mecânica respiratória e a densidade de neutrófilos no tecido pulmonar. Nós analisamos também os níveis de extravasamento de proteína, contagem de células totais e diferenciais e os níveis de IL-1, IL-6, KC, IL-10 and TNF- no lavado bronco-alveolar (LBA). Os níveis de IL-6 e IL-10 também foram avaliados no plasma e tecido pulmonar. A expressão de receptores de glicocorticóide (Gre) e da enzima superóxido dismutase (SOD) foi analisada no tecido pulmonar. As atividades enzimáticas de glutationa peroxidade (GPX), catalase (CAT), glutationa redutase (GR), e SOD foram determinadas no homogenato de pulmão por espectrofotometria. O nível de malonaldeído (MDA) foi quantificado no homogenato de pulmão. Resultados: A instilação de LPS resultou em aumento nos níveis de NO exalado (p<0,01), aumento do número de células e neutrófilos no LBA (p<0,001), aumento do número de neutrófilos no parênquima pulmonar (p<0,001), aumento dos valores de resistência e elastância pulmonar (p=0,01), aumento dos níveis de extravasamento de proteína (p0,02), aumento dos níveis de IL-6 e IL-10 no plasma (p<0,02) e aumento dos níveis de IL-1, IL-6 e KC no LBA (p0,005), comparado ao grupo CTR. O exercício aeróbico (grupo Exe+LPS) diminuiu significativamente os níveis de NO exalado (p=0,006), a densidade de neutrófilos no parênquima pulmonar (p=0,004), os valores de resistência e elastância pulmonar (p = 0,003), aumentou a expressão de IL-6, IL-10 e Gre no tecido pulmonar (p0,04) e aumentou o nível de IL- 1 no LBA (p=0,04) comparado ao grupo LPS. Conclusão: Nossos resultados mostram que o exercício desenvolve um importante papel em proteger o pulmão dos efeitos inflamatórios da LPA induzida por LPS. Os efeitos do exercício são principalmente mediados pelo aumento da expressão de citocinas antiinflamatórias, sugerindo que o exercício aeróbico pode modular o balanço inflamatório, antiinflamatório na fase inicial na SARA. / Background: The regular practice of exercise has been increasingly associated to benefic effects on chronic pulmonary conditions such as asthma and chronic obstructive pulmonary disease. Few studies have also reported the effects of aerobic exercise on acute lung injury (ALI). Objective: In this study we investigated the mechanisms involved in the role of exercise in attenuating the pulmonary changes in a model of lipopolysaccharide (LPS)-induced ALI. Methods: BALB/c mice were divided into four groups: Control (CTR), Exercise (Exe), LPS, and Exercise + LPS (Exe+LPS). Mice from Exe and Exe+LPS groups were trained at low intensity exercise for 60 minutes/day, 3 days/week, during 5 weeks. Intratracheal instillation of LPS (200/mouse) was performed 48 hours after the last physical test in the LPS and Exe+LPS groups. Twenty-four hours after LPS instillation we measured exhaled nitric oxide (NO), respiratory mechanics, and the density of neutrophils in lung tissue. We further analyzed protein leakage, total and differential cell counts and the levels of IL-1, IL-6, KC, IL-10 and TNF- in bronchoalveolar lavage fluid (BALF). IL-6 and IL-10 levels were also evaluated in serum and lung tissue. The expression of glucocorticoid receptors (Gre) and superoxide dismutase (SOD) was analyzed in lung tissue. Enzymatic activity of glutathione peroxidase (GPX), catalase (CAT), glutathione reductase (GR) and SOD was determined in lung homogenates by spectrophotometry. The level of malondialdehyde (MDA) was quantified in lung homogenates. Results: LPS instillation resulted in increased levels of exhaled NO (p<0.01), higher number of total cells and neutrophils in the BALF (p<0.001), higher number of neutrophils in the lung parenchyma (p<0.001), higher values of pulmonary resistance and elastance (p=0.01), increase of protein leakage (p0.02), increase of IL-6 and IL-10 level in serum (p<0.02) and increase in IL-1, IL-6 and KC levels in BALF (p0.005), compared to the CTR group. Aerobic exercise (Exe+LPS group) resulted in significantly lower exhaled NO levels (p=0.006), lower density of neutrophils in the lung parenchyma (p=0.004), lower pulmonary resistance and elastance values (p = 0.003), increased expression of IL-6, IL-10 and Gre in lung tissue (p0.04) and increased IL-1 level in BALF (p=0.04) compared to the LPS group. Conclusion: Our results show that exercise plays an important role in protecting the lung from the inflammatory effects of LPS-induced ALI. The effects of exercise are mainly mediated by the increased expression of anti-inflammatory cytokines, suggesting that aerobic preconditioning can modulate the inflammatory-anti-inflammatory balance in the early phase of ARDS.

The goal of lung-protective mechanical ventilation is to provide life-sustaining support of gas exchange while minimizing the risk of ventilator-induced lung injury. Multi-frequency oscillatory ventilation (MFOV) was proposed as an alternative lung-protective modality, in which multiple frequencies of pressure and flow oscillations are delivered simultaneously at the airway opening and allowed to distribute throughout the lung in accordance with regional mechanical properties. The distribution of oscillatory flow is frequency-dependent, such that regions overventilated at one frequency may be underventilated at another. Thus the central thesis of this work was that ventilation heterogeneity is frequency-dependent, and therefore ventilation with multiple simultaneous frequencies can be optimized to reduce the risk of ventilator-induced lung injury. Simulations in computational models of distributed oscillatory flow and gas transport demonstrated the sensitivity of regional ventilation heterogeneity to subject size, ventilation frequency, and injury severity. Although the risk of injury in the model associated with strain or strain rate individually was minimized by single-frequency ventilation, the risk of injury associated with mechanical power in lung parenchymal tissue was minimized by MFOV. In an experimental model of acute lung injury, MFOV was associated with reductions in the magnitude and spatial gradient of regional lung strain estimated by four-dimensional CT image registration, as well as increased rates of regional gas transport estimated by wash-in of xenon tracer gas. In conclusion, computational models demonstrated the potential for optimization of MFOV waveforms, and experimental trials demonstrated evidence of improved regional ventilation during MFOV.