Normoxic HIF1A stabilization during ALI.

<p>(<b>A</b>) Wild-type mice (BL6C57) were exposed to ventilator induced lung injury (VILI; pressure-controlled mechanical ventilation at an inspiratory pressure of 45 mbar with an inspired oxygen concentration of 100%, exposure time 180 minutes). To examine the influence of mechanical ventilation on pulmonary HIF1A expression patterns, lungs were stained with antibodies for Hif1a. IgG controls were used at identical concentrations and staining conditions as the target primary antibodies (magnification ×400; <i>n</i> = 4). (B) Frozen lung tissue was lysed and proteins resolved by SDS-PAGE. Resultant Western blots were probed with anti-Hif1a antibody. A representative blot of three is displayed. (C) Quantitative analysis of Western blot in (B) assessed by densitometry. (D) Imaging HIF1A using previously described HIF reporter mice expressing luciferase upon HIF stabilization (“ODD-Luc mice”) during ALI. Prior to imaging, mice were injected with i.p. luciferin (50 mg/kg) and mice were euthanized. Left column (control): Lungs excised without mechanical ventilation. To induce ALI, mice were ventilated with pressure controlled ventilation (45 mbar) at 21% oxygen concentration over 3 h. Middle column, 3 h at 15 mbar; right column, 3 h at 35 mbar. Color bar indicates photons/(cm2·s·steradian) with minimum and maximum threshold values (<i>n</i> = 4). (E) “ODD-Luc mice” were exposed to VILI (45 mbar, 3 h) using 20% or 100% inspired oxygen. Lungs were harvested, homogenized, and analyzed for luciferase gene expression using the Dual-Luciferase Reporter Assay System from Promega. (F) After 3 h of ventilation at 45 mbar, mitochondrial fractions obtained from lung tissue were analyzed for succinate dehydrogenase (SDH, mitochondrial Complex II) activity using ELISA. Activity is given as OD (optical density) change over time (mean ± s.d., <i>n</i> = 3).