Ischemic cell death is a complex process and the initial distinction between apoptosis and necrosis appears to be an oversimplification. We previously reported that in ischemic neurons with disrupted plasmalemma, apoptotic mechanisms were also active. In the present study, we investigated cellular co-localization of another necrotic feature, lysosomal rupture, with apoptotic mechanisms in the mouse brain and assessed the potential interactions between cysteine proteases. The lysosomal enzymes were spilled into the cytoplasm 1-4 h after ischemia/reperfusion, suggesting that lysosomal membrane integrity was rapidly lost, as occurs in necrosis. The same neurons also exhibited caspase-3 and Bid cleavage, and cytochrome-c release. Caspase-3 activity preceded cathepsin-B leakage in most neurons, and declined by 12 h, while lysosomal leakage continued to increase. Concurrent inhibition of cathepsin-B and caspase-3 provided significantly better neuroprotection than obtained with separate use of each inhibitor. These data suggest that necrotic and apoptotic mechanisms may act both in concert as well as independently within the same cell beginning at the onset of ischemia to ensure the demise of damaged neurons. Therefore, combined inhibition of cysteine proteases may abrogate potential shifts between alternative death pathways and improve the success of stroke treatments. (c) 2010 Elsevier Inc. All rights reserved.