Ischemia Leads to Apoptosis—and Necrosis‐like Neuron Death in the Ischemic Rat Hippocampus

Abstract

Morphological evidence of apoptosis in transient forebrain ischemia is controversial. We therefore investigated the time sequence of apoptosis-related antigens by immunohistochemistry and correlated it with emerging nuclear patterns of cell death in a model of transient forebrain ischemia in CA1 pyramidal cells of the rat hippocampus. The earliest ischemic changes were found on day 2 and 3, reflected by an upregulation of phospho-c-Jun in a proportion of morphologically intact CA1 neurons, which matched the number of neurons that succumbed to ischemia at later time points. At day 3 and later 3 ischemic cell death morphologies became apparent: pyknosis, apoptosis-like cell death and necrosis-like cell death, which were confirmed by electron microscopy. Activated caspase-3 was present in the vast majority of cells with apoptosis-like morphology as well as in a small subset of cells undergoing necrosis; its expression peaked on days 3 to 4. Silver staining for nucleoli, which are a substrate for caspase-3, revealed a profound loss of nucleoli in cells with apoptosis-like morphology, whereas cells with necrosis-like morphology showed intact nucleoli. Overall, cells with apoptosis-like morphology and/or caspase-3 expression represented a minor fraction (<10%) of ischemic neurons, while the vast majority followed a necrosis-like pathway. Our studies suggest that CA1 pyramidal cell death following transient forebrain ischemia may be initiated through c-Jun N-terminal kinase (JNK) pathway activation, which then either follows an apoptosis-like cell death pathway or leads to secondary necrosis.

Activation of Toll-like receptor 4 signaling contributes to hippocampal neuronal death following global cerebral ischemia/reperfusion

“…One is that the neurons of CA1 died a caspase-dependent death, but the presence of cleaved caspase-3 in the CA1 pyramidal layer had declined at 3 days after the ischemic insult, since previous studies reported that caspase-3 was activated as early as 30 min and declined at 24 h and afterwards following cerebral ischemia (Cho et al, 2003;Namura et al, 1998). Another explanation is that the majority of the ischemic CA1 neurons follow a caspase-independent necrosis-like death (Muller et al, 2004), while the neurons in DG area follow a caspase-dependent apoptosis pathway. In addition, compared with WT mice, there were fewer TUNEL positive cells and less activation of caspase-3 in HF in TLR4 -/-mice subjected to GCI/R (Fig.…”

Cell cycle inhibition attenuates microglial proliferation and production of IL‐1β, MIP‐1α, and NO after focal cerebral ischemia in the rat

“…Increasing evidence has suggested that ischemic brain injury induces neuronal apoptosis (Bi et al, 2006;Muller et al, 2004). In this study, TUNEL and neuron doublestaining was performed to examine whether roscovitine can reduce neuronal apoptosis after MCAO.…”

Visualizing Cell Death in Experimental Focal Cerebral Ischemia: Promises, Problems, and Perspectives

“…In the latter case, specific differentiation between apoptotic and necrotic cell death is frequently requested, which is problematic based only on microscopic morphology. This is best illustrated by the longstanding controversy whether postischemic delayed neuronal death of vulnerable hippocampal neurons is apoptotic or necrotic (Deshpande et al, 1992;Muller et al, 2004;Wei et al, 2006). Another major challenge is reliable detection of early ischemic changes.…”