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 Extrac tion Reagent con taining protease Enzastaurin LY317615 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 Inhibitors,Modulators,Libraries 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 con trolled by Ponceau S staining. After blocking the membranes with 5% non fat dry milk for 60 minutes, the following pri mary antibodies were applied, anti phospho ERK1 2, anti total ERK1 2, anti phospho p38 MAPK, anti total p38 MAPK, anti phospho JNK, anti total JNK, anti phospho STAT3, anti total STAT3, pan Cadherin.

Moreover, anti HSP 70, anti HO 1, and anti Inhibitors,Modulators,Libraries B actin were used. As secondary antibodies, HRP Inhibitors,Modulators,Libraries coupled anti rabbit IgG and anti mouse IgG antibodies were used. As chemoluminiscence Inhibitors,Modulators,Libraries reagents Supersignal Pico and Femto were used. Signals were detected on x ray films. Statistical analysis One way Anova for repeated measurement was used to analyse changes at different time points followed by a post hoc Tukey test. Nonparametrical ana lysis by Friedman Test gave similar results. Analysis be tween healthy animals and T1 of the I R group was done by Students t Test. All analyses were performed by Graphpad Prism 5. 0. Results Haemodynamic parameters Table 1 displays the haemodynamic and physiological parameters of the animals in the I R group.

CPB priming with 15 ml 6% hydroxyethyl starch resulted in an ex pected decrease of haemoglobin concentration from 12. 3 g dl before CPB to 4. 5 g dl at the end of the entire experiment and a decrease of the haematocrit from 35. 8 % before CPB to 9. 4 % at Inhibitors,Modulators,Libraries the end of the experiment. Furthermore, a leucocytosis during the rewarming and reperfusion period was observed. Considering the haemo dilution by the CPB priming, the leucocyte numbers were calculated in relation to the haematocrit to obtain com parable values. As the reference range of the leucocytes varies from 3 to 15 103 mm3, for each animal the leuco cyte count was normalised to the individual start value. Regarding the MAP, no significant differences were observed between the different time points throughout the operation.

Heart rate and temperature changes were in accordance with the gradual alternation of the flow rate during the cooling and rewarming period. Blood pH values and partial pressures remained stable such information or were corrected. Clinical biochemistry The plasma samples of the healthy animals and of the time points T1, T2 and T5 were analysed for crucial clinical blood parameters as summarized in Table 2. Plasma AST, creatinine, troponin and potassium levels are exemplarily shown in Figure 2.