Abstract 137: Superoxide Production in the Presence of Sivelestat During Reperfusion in Isolated Rat Hearts
Introduction: In previous experiments, we note Sivelestat, a neutrophil-elastase inhibitor, preserves post-ischemic cardiac function and reduces infarct size in the absence of neutrophils. Preliminary work suggests Sivelestat may reduce superoxide formation during reperfusion.
Aim: To test the hypothesis that Sivelestat reduces the superoxide burst at full reperfusion when administered during low flow following global ischemia in the rat heart.
Methods: Hearts isolated from male Sprague-Dawley rats (n=6/group) were perfused at 75 mmHg and 37.4º C with Krebs-Henseleit buffer. Following 25 min of global ischemia, hearts were given: a) immediate full reperfusion at 75 mmHg, b) 3 min of low flow at 20% of baseline coronary flow prior to full reperfusion, or c) 3 min of 20% low flow with Sivelestat (100 µg•mL-1) prior to full reperfusion. Hearts were infused continuously for the first two minutes and then at 3 min and 10 min of full reperfusion with the superoxide radical spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, 40 mM). Coronary effluent samples were collected into liquid nitrogen to test for the presence of DMPO-OH, the stable EPR-active product of the reaction of DMPO with superoxide. Frozen effluent samples were thawed and transferred to a quartz flat-cell inside an EPR-300 X-band (9.7 GHz) spectrometer. Quantification of superoxide was performed by comparing the double integral of the least squares fit of the observed signal with that of a known aqueous solution of 4-hydroxy tempol. Significance was set at p=0.05.
Results: A characteristic increase in [DMPO-OH], indicative of superoxide radical formation, was observed in all groups upon full reperfusion. However, no significant differences in [DMPO-OH] were observed between groups.
Conclusions: Measurement of predominantly vascular generated superoxide in the reperfused heart using EPR spin trapping techniques failed to show a Sivelestat-mediated reduction in reperfusion generated superoxide. Future work will focus on the effects of Sivelestat on superoxide generated during the low flow period prior to full reperfusion.
- © 2011 by American Heart Association, Inc.