Period of normoxic ventilation with 21 O2, in the case of a 3 h ventilation period followed by a bolus application of PMA into the pulmonary artery, resulting in a concentration of 1 in the recirculating buffer fluid. Control experiments received a bolus of saline instead of PMA, 2) Consecutive 30 min periods of ventilation with different O2 concentrations (21, 16, 10, 5, 2.5 or 1 O2 in a randomized fashion) for a total period of 3 h, or 3) One 30 min-period of ventilation at either 21, 16, 10, 5, 2.5 or 1 O2, followed by a bolus application of PMA into the pulmonary artery, resulting in a concentration of 1 in the recirculating buffer fluid. Control experiments received a bolus of NaCl instead of PMA.In a portion of the experiments a fiber oxygenator (Hilite 1000, Stolberg, Germany) was used instead of the lung for oxygenation of the buffer fluid.Isolated mouse lung experiments Mouse lung experiments were performed in a protocol analogous to the isolated rabbit lung experiments but in an in-chest preparation as previously described [33]. Lungs were perfused with 0.5 mM CPH at a flow rate of 2.0 ml/min for 120 min at normoxic ventilation (21 O2), followed by a bolus application of PMA into the buffer fluid, resulting in a concentration of 10 . For these investigations either C57/BL6 mice (= wildtype PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/28859980 control) or mice lacking the NADPH oxidase subunit gp91phox (= p91phox-/-). Mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). ESR measurements Oxidation of the spin probe CPH by superoxide forms the nitroxide CP radical. The triple-line spectrum of CP radical was detected by ESR spectroscopy, using a MS 100 spectrometer (Magnettech, Berlin, Germany). The ESR measurements were performed in field scan with the following settings: microwave frequency 9.78 GHz, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 18 mW. All samples, both from the in vitro experiments or from the venous outflow of the isolated lung, were made in 50 glass capillary tubes and measured immediately at room temperature. The ESR amplitude is in MS-275MedChemExpress Entinostat proportion to the amount of CP? reflecting the interaction of ROS with CPH [29,34,35]. Thus, the quantity of trapped ROS was directly calculated from the ESR spectrum of the probe, while the contribution of superoxide radical to the formation of CP was determined in parallel experiments performed in the presence of SOD in the buffer fluid (150 U/ml). The first sample was taken 5 min after CPH addition to the buffer fluid (time set at zero), followed by further sampling every 5 or 30 minutes, as appropriate. The values after PMA addition were assessed every minute in the respective experiments. In the isolated mouse lung samples were taken every 2 min. For quantification, the second-field component of the ESR spectrum was used. To standardize values, the amplitude of this component was divided through the receiver gain. Statistical analysis Data are given as mean ?standard error (SEM). For comparison of two groups, a two-tailed t-test was employed. For multiple comparisons, analysis of variance was used, followed by the Student-Newman-Keuls post hoc test when differences were indicated. Statistical significance was assumed when p < 0.05.Page 3 of(page number not for citation purposes)Respiratory Research 2005, 6:http://respiratory-research.com/content/6/1/ResultsWhen CPH (1 mM) was dissolved in Krebs-Henseleit buffer ESR spectroscopy resulted in a triple band spectrum (Fig.
