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Age Dependent Gene Expression Responses to Inhaled Lipopolysaccharide Combined with Mechanical Ventilation

ABSTRACT: Children have a lower incidence and mortality from acute lung injury than adults, and infections are the most common event associated with acute lung injury (ALI). To study the effects of age on susceptibility to ALI, we investigated the responses to microbial products combined with mechanical ventilation in juvenile (21 day) and adult (16 week-old) mice. We hypothesized that the increased incidence and severity of lung injury associated with increasing age is due in large part to acquired changes in the way in which inflammatory responses are activated in the lungs in response to microbial products and mechanical ventilation. Juvenile (21 day) and adult (16 week) C57BL/6 mice were treated with an aerosol of E. coli 0111:B4 lipopolysaccharide (LPS) (20 mL of 0.1 mg/mL) for 30 minutes in a sealed aerosol chamber, immediately followed by mechanical ventilation (LPS+MV) using tidal volume = 15 mL/kg, rate = 80 breaths/min, FiO2 = 30% and positive end expiratory pressure = 2 cm H2O for the duration of the study period time = 2 hours. Comparison groups included mice treated with LPS or mechanical ventilation (MV) alone, and untreated age-matched controls. There were N = 4 animals per group except the juvenile mice treated with MV alone and LPS+MV where there were N = 3. Each sample was an individual animal, therefore there were 30 samples. Mice treated with LPS alone were placed into a sealed aerosol chamber as stated above, and then allowed to breath spontaneously with free access to food and water for the duration of the study period time = 2 hours. Mice treated with MV alone were treated with the mechanical ventilation protocol stated above for the duration of the study period time = 2 hours. At the end of the study period, the mice were euthanized, and the lungs were immediately removed and placed into RNAlater (Ambion, Austin, TX) for at least 24 hr prior to isolation of total lung mRNA.

ORGANISM(S): Mus musculus

SUBMITTER: Lincoln Smith

PROVIDER: E-GEOD-18341 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Effects of age on the synergistic interactions between lipopolysaccharide and mechanical ventilation in mice.

Smith Lincoln S LS Gharib Sina A SA Frevert Charles W CW Martin Thomas R TR

American journal of respiratory cell and molecular biology 20091109 4

Children have a lower incidence and mortality from acute lung injury (ALI) than adults, and infections are the most common event associated with ALI. To study the effects of age on susceptibility to ALI, we investigated the responses to microbial products combined with mechanical ventilation (MV) in juvenile (21-d-old) and adult (16-wk-old) mice. Juvenile and adult C57BL/6 mice were treated with inhaled Escherichia coli 0111:B4 lipopolysaccharide (LPS) and MV using tidal volume = 15 ml/kg. Compa ...[more]

PMID: 19901347

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Project description:RATIONALE: Mechanical ventilation (MV) is an indispensable therapy for critically ill patients with acute lung injury and the adult respiratory distress syndrome. However, the mechanisms by which conventional MV induces lung injury remain unclear. OBJECTIVES: We hypothesized that disruption of the gene encoding Nrf2, a transcription factor which regulates the induction of several antioxidant enzymes, enhances susceptibility to ventilator-induced lung injury (VILI), while antioxidant supplementation attenuates such effect. METHODS: To test our hypothesis and to examine the relevance of oxidative stress in VILI, here we have assessed lung injury and inflammatory responses in Nrf2-deficient (Nrf2(-/-)) mice and wildtype (Nrf2(+/+)) animals following acute (2 h) injurious model of MV with or without administration of antioxidant. MEASUREMENTS AND MAIN RESULTS: Nrf2(-/-) mice displayed greater levels of lung alveolar and vascular permeability and inflammatory responses to MV as compared to Nrf2(+/+) mice. Nrf2-deficieny enhances the levels of several pro-inflammatory cytokines implicated in the pathogenesis of VILI. We found diminished levels of critical antioxidant enzymes and redox imbalance by MV in the lungs of Nrf2(-/-) mice; however antioxidant supplementation to Nrf2(-/-) mice remarkably attenuated VILI. When subjected to clinically relevant prolong period of MV, Nrf2(-/-) mice displayed greater levels of VILI than Nrf2(+/+) mice. Expression profiling revealed lack of induction of several VILI genes, stress response and solute carrier proteins and phosphatases in Nrf2(-/-) mice. CONCLUSIONS: Collectively, our data demonstrate for the first time a critical role for Nrf2 in VILI, which confers protection against cellular responses induced by MV by modulating oxidative stress. Experiment Overall Design: The Nrf2 wildtype (Nrf2+/+) and Nrf2-deficient (Nrf2â/â) CD-1/ICR strains of mice were subjected to mechanical ventilation with high (HVT) amounts of tidal volumes (VT) at 30 ml/kg for 2 hours. The animals subjected to spontaneous ventilation (SpV) for 2 hours were used as controls. Lungs were immediately removed and processed for total RNA isolation using TRIzol reagent (LifeTechnologies, Grand Island, NY). The isolated RNA was applied to Murine Genome 430A GeneChip arrays (Affymetrix, Santa Clara, CA), which contain probes for detecting ~14,500 well-characterized genes and 4371 expressed sequence tags according to standard microarray protocol. Scanned output files were analyzed by using Affymetrix GeneChip Operating Software and were independently normalized to an average intensity of 500.

Project description:Acute respiratory distress syndrome (ARDS) is a catastrophic form of acute lung injury (ALI). The necessity for mechanical ventilation (MV) renders patients at risk for ventilator induced lung injury (VILI). Exposure to repetitive cyclic stretch (CS) and/or over-inflation exacerbates injury. Reducing tidal volume (VT) is the only therapeutic strategy shown to mitigate morbidity and mortality. Cyclic stretch has been shown to differentially regulate gene expression in part through the activation of mammalian mitogen-activated protein kinase (MAPK). Although these studies have shown both molecular and cellular alterations, no unifying hypothesis to explain MV-induced lung injury has emerged. In the current study, we hypothesized that coordinated expression of cyclic stretch (CS)-responsive genes relies on the presence of common CS-sensitive regulatory elements. To identify CS-responsive genes, we undertook a comparative examination of the gene expression profile of human bronchial epithelial airway (Beas-2B) cells in response to various injurious stimuli involved in the pathogenesis of acute lung injury (ALI)/Ventilator induced lung injury (VILI): cyclic stretch, tumor necrosis factor alpha (TNF-a), and lipopolysaccharide (LPS). Experiment Overall Design: Human Bronchial Epithelial Cells (Beas-B2) cells grown on silicon elastic plates coated with Type I collagen (Flexercell International, McKeesport, PA) were exposed to six regiments for 4 h: 1) control (static, [control]); 2) mechanical stretch (25 PKa, 30 cycles per min, [stretch]); 3) LPS (1 mcg/ml [LPS]); 4) TNF-Î± (20 ng/ml; [TNF]); 5) mechanical stretch plus LPS [LPS+S], and 6) mechanical stretch plus TNF-Î± [TNF+S]. Total RNA (duplicate experiments) was extracted using TRIZOL reagent (as per manufactures specifications) and purified using Qiagen mRNA purification Kit (as per manufacturers specifications). mRNA was hybridized to Affymetrix Human U133plus2.0 chips. Probe based analysis, background reduction, and quantile data normalization was performed in MeV 4.0 of TM4 using Robust Multi-array Average (RMA).

Dataset Information

Age Dependent Gene Expression Responses to Inhaled Lipopolysaccharide Combined with Mechanical Ventilation

Publications

Effects of age on the synergistic interactions between lipopolysaccharide and mechanical ventilation in mice.

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