samples Before the start of CPB, after 25 minutes of cooling, during rewarming at a rectal temperature of 20 C and 35 C and after 60 minutes of reperfusion arterial blood gas analyses were performed and the vital considering signs were documented. Additionally, blood sam ples were taken at T1, T2 and T5. They were transferred to heparin containing tubes and kept at room temperature for 30 minutes to allow coagulation before centrifuging at 4000g. Plasma aliquots were snap frozen and stored at 80 C. For harvesting of organs, animals were perfused with NaCl and organs were removed in the following order heart, lung, liver, and kidney. All organs were immedi ately snap frozen in liquid nitrogen for subsequent mo lecular analysis.
In order to illustrate the study design and the time points Inhibitors,Modulators,Libraries taken for data collection throughout the e peri ment, the e perimental time and temperature flow is given in a scheme. The e perimental setup was designed to mimic standard procedures in the clin ical scenario of cardiothoracic surgery using CPB and DHCA. Similarly, time points of blood sampling have been set to meet critical transition points during CPB. After an initial period of establishment, si animals of the I R group were evaluated. Healthy animals were anaesthetised by injection of pentobarbital. Blood samples were taken by puncture of the left ventricle after anaesthetisation and the rats were perfused with NaCl for 3 5 minutes until organs could be harvested. Animals in the H group did not undergo Inhibitors,Modulators,Libraries any further surgical treatment.
Analysis of metabolic parameters in plasma samples Using blood plasma samples taken before CPB, after 25 minutes of cooling and after 60 minutes of reperfusion following parameters were deter mined by the Central Institute Inhibitors,Modulators,Libraries of Clinical Chemistry Inhibitors,Modulators,Libraries and Cilengitide Laboratory Medicine of the University Hospital Duesseldorf lactate, urea, aspartate transaminase, alanine transaminase, lactate dehydrogenase, creatinine and potassium. These parameters were mea sured spectrophotometrically using commercially available standard Roche Hitachi methodology. Plasma interleukin 6 and TNF levels were determined using an ELISA according to the manufacturers instructions. High sensitive tropo nin, c reactive protein, creatine kinase and MB isoform of CK were determined in plasma samples by a partner laboratory specialised in clinical diagnostics using ELISAs according to the manufacturers instructions.
Analysis of molecular parameters in tissue samples Immunoblot analysis of proteins in tissue samples was per formed as previously described. Briefly, a portion of each tissue was lysed in M Per Mammalian Protein E trac tion Reagent con taining protease third inhibitors and phosphatase inhibitors. The protein content of the lysates was measured by DC Protein assay with bovine serum albumin as standard. Lysates were boiled in Laemmli loading buffer and loaded either onto 10% or 14% SDS PAGE gels. After electrophoresis the gels were trans ferred to PVDF membranes. Equal loading was