DNA Fragmentation Follows Delayed Neuronal Death in CA1 Neurons Exposed to Transient Global Ischemia in the Rat

Abstract

Summary: Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA pre cedes morphological changes in the nucleus and, later, in the cytoplasm, In ischemia, biochemical studies have detected oli gonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than ap optosis, To resolve this apparent discrepancy, we subjected rats to 10 minutes of transient forebrain ischemia followed by I to 14 days of reperfusion, Parameters evaluated in the CAl region of the hippocampus included morphology, in situ end labeling (ISEL) of fr'l-gmented DNA, and expression of p53, Neurons were indistinguishable from controls at postischemic day I but displayed cytoplasmic basophilia or focal condensations at day 2; some neurons were slightly swollen and a few appearedThe neuronal response to brain ischemia depends on the severity and duration of the ischemic insult as well as on intrinsic neuronal factors that impart selective vulner ability to specific brain regions (Scholz, 1953), Brief periods of global ischemia kill vulnerable neurons in the CAl region of the hippocampus whereas longer intervals of vascular occlusion kill neurons in the cerebral cortex and corpus striatum as well (Ito et aI., 1975; Pulsinelli et aI., 1982a), Although seemingly undamaged, neurons in other brain regions such as the CA3 region of the hip pocampus or the paramedian cerebral cortex undergo transient alterations as evidenced by reversible changes 967normaL In situ end labeling was absent At days 3 and 5, approximately 40 to 60% of CAl neurons had shrunken eosin ophilic cytoplasm and pyknotic nuclei, but only half of these were ISEL By day 14, many of the necrotic neurons had been removed by phagocytes; those remaining retained mild ISEL Neither p53 protein nor mRNA were identified in control or postischemic brain by in situ hybridization with riboprobes or by northern blot analysis, These results show that DNA frag mentation occurs after the development of delayed neuronal death in CA I neurons subjected to 10 minutes of global isch emia, They suggest that mechanisms other than apoptosis may mediate the irreversible changes in the CAl neurons in this model. Key Words: Global ischemia-Apoptosis Necrosis-Delayed neuronal death-In situ end labeling-Rat in their subcellular organelles (Petito and Pulsinelli, 1984a,b), The postischemic interval required for the ap parent development of selective neuronal necrosis is in versely proportional to the severity or duration of the ischemic insult-the so-called maturation phenomena described by Ito et al. (Ito et at, 1975), This interval is especially prolonged in the CAl neurons of the hippo campus (Ito et aI., 1975; Pulsinelli et aI, 1982a). The term delayed neuronal death has been applied to this process (Kirino, 1982).During the past few years, the concept of cell death via apoptosis has become increasingly important. This mechanism of cell death is mediated by regulated path ways involving defined gene product...

Reduction of Apurinic/Apyrimidinic Endonuclease Expression After Transient Global Cerebral Ischemia in Rats

“…9,10 However, it is still unclear whether DNA fragmentation in hippocampal neurons after transient global ischemia is entirely due to the apoptotic pathway. 24 Also, the neuronal nature of the cells that exhibited DNA fragmentation is not clear at present. Further studies using double staining consisting of a neuronal marker (NeuN) and TUNEL are needed to address this issue.…”

Exacerbation of Delayed Cell Injury After Transient Global Ischemia in Mutant Mice With CuZn Superoxide Dismutase Deficiency

“…Recent studies, although still somewhat controversial, have identified some apoptotic features by biochemical and morphological evidence such as terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining 14,31,32 and internucleosomal DNA fragmentation, as indicated by the DNA laddering pattern. 33 Several apoptosis-regulatory genes and enzymes are found to be induced in ischemic cells.…”

Overexpression of SOD1 in Transgenic Rats Protects Vulnerable Neurons Against Ischemic Damage After Global Cerebral Ischemia and Reperfusion

“…Recent studies, although still somewhat controversial, have identified some apoptotic features by biochemical and morphological evidence such as TUNEL (MacManus et al, 1993;Sei et al, 1994;Petito et al, 1997) and internucleosomal DNA fragmentation as indicated by the DNA laddering pattern (Héron et al, 1993). Because TUNEL staining indicates DNA damage, and its specificity for apoptosis is questionable, we have used TUNEL staining only as an indication of DNA damage in cells.…”