Introduction
Two different forms of cell death have been distinguished morphologically following ischemia: necrotic and apoptotic cell death. Necrosis has been traditionally referred to brain infarction. However, more recent reports have suggested that apoptosis may become of greater importance than previously thought in focal ischemic injury, by participating to the extension of the lesion. The aim of the present study was (i) to carefully depict the temporal and spatial cell death progression during the enlargement of the lesion, with particular attention to apoptosis and (ii) to evaluate the effects of the lipophilic antioxidant iron chelator 2,2′-dipyridyl (DP).
Methods
Cortical ischemia was performed by chemical photothrombosis. Histological measurements (conventional histology, immuno-histofluorescence for the cleaved caspase-3) were followed over a period of 24 h after the onset of ischemia. Biochemical measurements (DNA fragmentation, immunoblot analysis of cleaved caspase-9 and 3, and cleaved PARP-1, a major substrate caspase-3 substrate) were followed in parallel in three punched tissue regions, distincts in term of neuronal survey: (i) the lesion core that definitively underwent infarction, (ii) surrounding tissue that could be rescued by systemic administration of DP, (iii) tissue, outside this latter region.
Results
The lesion volume remained stable for at least 4 h after photothrombosis, thus representing the initial core of the infarct. Two separate waves of neuronal cell death could be distinguished. In the first wave, shrunken dark neurons were massively present as early as 2 h following the onset of ischemia. From this initial neuronal abnormal population, progressive and time-dependent changes of both necrotic and apoptotic cell death were observed leading to ghost neurons and apoptotic bodies after 24 h. The extension of the lesion coincided with a second wave of cell death. After 12 h, the lesion volume had markedly increased. Massive and rapid neuronal loss occurred at the infarct border which appeared as a sharply demarcated pale region with abundant ghost neurons and apoptotic bodies. Procaspase and poly(ADP-ribose) polymerase-1 (PARP-1) cleavages were also detected in the infarct core and surrounding damaged tissue. DP treatment completely blocked the enlargement of the lesion, the infarct border being rescued from the infarction. Furthermore, neuronal density was increased in the infarct core. Finally a large decrease of apoptotic bodies was associated with a significant drop of caspase and PARP-1 cleavages suggesting that the protective effect of DP closely correlated with limitation of apoptotic cell death.
Conclusion
Overall results indicate that (i) neuronal apoptotic and necrotic death initially evolve in concert in the infarct core from an initial population of damaged neurons, probably depending locally on the vulnerability and environment of each cell and that (ii) surrounding tissue leads massively to death in a second wave. These results also favour the view that apoptosis contributes to the expansion of the lesion following photothrombosis and point out the possibility that, at least in some circumstances, neurons in the ischemic core can be salvaged.