Neutrophil depletion reduces myocardial apoptosis and attenuates NFκB activation/TNFα release after ischemia and reperfusion

Background. This study tested the hypothesis that depletion of neutrophils (PMNs) reduces myocardial apoptosis via reducing oxidant generation and inhibiting NF kappa B-mediated signaling pathways after ischemia/reperfusion. Methods. Anesthetized rats were randomly divided into one of four groups: Control: 30 min ischemia and 3 h of reperfusion; PMN depletion: anti-PMN serum was injected 6 h before ischemia; N-acetylcysteine (NAC): NAC was given twice before ischemia and at reperfusion. Sham: the ligature was placed without coronary occlusion. Apoptosis was detected by TUNEL staining and DNA fragmentation. PMN accumulation was studied by immunohistochemical staining. Levels of TNF-alpha, IL-6, and caspase-3 were detected by Elisa kits. Expression in NF kappa B, Bcl-2, and Bax was assessed by Western blotting analysis. Results. Relative to Control, depletion of PMNs or NAC treatment reduced levels of plasma TNF alpha (567 +/- 130* and 231 +/- 72* versus 1994 +/- 447 pg/ml) and IL-6 (791 +/- 473* and 666 +/- 300* versus 3724 +/- 1233, pg/ml), accompanying a reduction in PMN accumulation (12 +/- 1* and 13 +/- 0.6* versus 20 +/- 1 mm(2) myocardium) in ischemic myocardium. Both groups showed a reduction in expression of nuclear NF kappa B relative to Control (62 +/- 9* and 67 +/- 8* versus 124 +/- 16 arb.u), consistent with reduced N kappa B binding activity. The number of apoptotic cells (%) in area at risk myocardium was comparably reduced in anti-PMN and NAC groups relative to Control (12 +/- 1* and 14 +/- 0.9* versus 20 +/- 1), consistent with reduced appearance of DNA ladders. Furthermore, activated caspase-3 was significantly reduced and Bcl-2 was increased relative to Control. No difference in all parameters measured was detected during the course of experiment in the Sham group. Conclusion. These data suggest that the oxidants generated from activated PMNs after ischemia/reperfusion trigger myocardial apoptosis, which is further supported by an anti-oxidant therapy with NAC, potentially mediated by enhanced NF kappa B-TNF alpha signaling pathway, activated caspase-3 and down-regulated Bcl-2. *P < 0.05 versus Control. (c) 2006 Elsevier Inc. All rights reserved.