Ence and presence of superoxide dismutase (SOD) to estimate the proportion of superoxide-induced CPH oxidation. We thus demonstrated the feasibility of the ESR technique, and obtained interesting new PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/26780312 insights into the oxygen-dependence of baseline versus PMA/NADPH oxidase-dependent superoxide generation in the intact lung vasculature.MethodsChemicals and reagents l-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) was purchased from L-Optik (Berlin, Germany). Krebs-Henseleit buffer contained 125.0 mM NaCl, 4.3 mM KC1, 1.1 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgCl2 and 275 mg glucose per 100 ml. FeCl2, deferoxamine (DFO), Anlotinib site diethyldithiocarbamate (DETC), phorbol-12myristate-13-acetate (PMA), and Cu/Zn-superoxide dismutase (SOD) were obtained from Sigma (Deisenhofen, Germany). Apocynin was from Merck Biosciences (Schwalbach, Germany). In-vitro CPH experiments For studying CPH characteristics and the effect of metal chelating agents on background ESR signals, experiments were performed in glass tubes containing 1 mM CPH solution prepared in Krebs-Henseleit buffer at room temperature. The iron chelator DFO was added in the concentrations 20 or 2 mM to the solution and measurements were run every 30 min for a total period of 5 h. Isolated rabbit lung experiments The technique of isolated lung perfusion and ventilation has been described previously in detail [31,32]. Briefly, pathogen-free New Zealand white rabbits of either sex (body weight 2.5?.2 kg) were deeply anesthetized with ketamine (30?0 mg/kg body weight) and xylazine (6?0 mg/kg body weight), and anticoagulated with heparin (1000 U/kg body weight). The lungs were excised whilePage 2 of(page number not for citation purposes)Respiratory Research 2005, 6:http://respiratory-research.com/content/6/1/perfused with Krebs-Henseleit buffer through cannulas in the pulmonary artery and the left atrium. After the lungs were rinsed with at least 1 1 of buffer fluid for removal of blood, the perfusion circuit was closed for recirculation (total system volume 150 ml) and left venous pressure set at 2 mmHg to secure West zone III conditions for perfusion. In parallel with the onset of artificial perfusion, room air ventilation was changed to a mixture of 5.3 CO2, 21.0 O2 and the balance N2 (tidal volume, 30 ml; frequency, 30 strokes/min) with the use of positive endexpiratory pressure of 1 cm H2O. The pH was adjusted to 7.35 ?7.40 by addition of NaHCO3. The isolated lungs were placed in a temperature-equilibrated housing chamber and the whole system was heated to 38.5 . Krebs-Henseleit buffer was incubated with 5 diethyldithiocarbamate overnight to allow sedimentation. Briefly before the experiments, 20 DFO and NaHCOs were added to the supernatant. Lungs included in the study were those that i) had a homogeneous white appearance with no signs of hemostasis, edema or atelectasis, ii) revealed constant mean pulmonary artery and peak ventilation pressure in the normal range, and iii) were isogravimetric during the initial perfusion period of 55 min. For normoxic and hypoxic ventilation maneuvers, a gasmixing chamber (KM 60-3/6 MESO, Witt, Witten, Germany) was employed for step changes in the ventilator O2 content. 5.3 CO2 were used throughout and the percentage of N2 was balanced accordingly. After the initial steady state period with normoxic ventilation (21 O2), CPH (1 mM) was added to the buffer fluid. Five minutes later, one of three different protocols was initiated: 1) A 2.5 or 3.0 h.
