Prolonged helium postconditioning protocols during early reperfusion do not induce cardioprotection in the rat heart in vivo: role of inflammatory cytokines

Abstract

Postconditioning of myocardial tissue employs short cycles of ischemia or pharmacologic agents during early reperfusion. Effects of helium postconditioning protocols on infarct size and the ischemia/reperfusion-induced immune response were investigated by measurement of protein and mRNA levels of proinflammatory cytokines. Rats were anesthetized with S-ketamine (150 mg/kg) and diazepam (1.5 mg/kg). Regional myocardial ischemia/reperfusion was induced; additional groups inhaled 15, 30, or 60 min of 70% helium during reperfusion. Fifteen minutes of helium reduced infarct size from 43% in control to 21%, whereas 30 and 60 minutes of helium inhalation led to an infarct size of 47% and 39%, respectively. Increased protein levels of cytokine-induced neutrophil chemoattractant (CINC-3) and interleukin-1 beta (IL-1β) were found after 30 or 60 min of helium inhalation, in comparison to control. 30 min of helium increased mRNA levels of CINC-3, IL-1β, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in myocardial tissue not directly subjected to ischemia/reperfusion. These results suggest that the effectiveness of the helium postconditioning protocol is very sensitive to duration of noble gas application. Additionally, helium was associated with higher levels of inflammatory cytokines; however, it is not clear whether this is causative of nature or part of an epiphenomenon.

Figure 1

Infarct size and biomarkers after 120 minutes of reperfusion. Data are shown as mean ± S.E.M. ^* P < 0.05 versus CON. Amount of experiments in each group is shown below individual bars. (a) Infarct sizes as percentage of the area at risk. Underneath the graph, pictures of representative slices of myocardium are shown for each group. (b) Troponin T and LDH levels in the circulation after 120 minutes of reperfusion.

Figure 2

Protein levels of CINC-3 (a), IL-6 (b), IL-1β (c), and TNF-α (d). Protein levels are determined in myocardial tissue after completion of the experimental protocol: all animals underwent 15 min of stabilization, 25 min of ischemia, and 120 min of reperfusion, except for Sham animals. Sham animals were not exposed to ischemia and reperfusion. Animals receiving helium postconditioning received 15, 30 or 60 min of helium. All data are shown as mean ± S.E.M. Amount of experiments in each group is shown below individual bars. Groups were tested with one-way ANOVA plus a Dunnet post hoc test comparing the control group against all other groups; ^* P < 0.05, significant in comparison to CON.

Figure 3

CINC-3 (a), IL-6 (b), IL-1β (c), and TNF-α (d) mRNA expression. Messenger RNA was analyzed in myocardial samples taken at the end of the experimental protocol. All animals underwent 15 min of stabilization and 25 min of ischemia (except for Sham) and 5, 15, or 30 min of reperfusion with or without helium. All data are shown as mean ± S.E.M. Amount of experiments in each group is shown below individual bars. To compare control (I/R) with helium intervention (I/R + He) at the various time points of reperfusion (5, 15, and 30 minutes), a two-way repeated measures ANOVA with Bonferroni correction for multiple testing was done. ^* P < 0.05 I/R in comparison to He.