Early Detection of DNA Strand Breaks in the Brain After Transient Focal Ischemia: Implications for the Role of DNA Damage in Apoptosis and Neuronal Cell Death

Abstract

Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSB5) detected by PANT were present in neurons after as little as 1 mm of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TU-NEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16-72 h of reperfusion, many SSB-and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis. Key Words: DNA damage-Single-strand breaks-Apoptosis-Cerebral ischemia.

Mapping the initial DNA breaks in apoptotic Jurkat cells using ligation-mediated PCR

“…The existence of ss breaks in apoptotic DNA fragments has been clearly demonstrated (Figure 1). 7,51,52 The rate of nuclear proteolysis might also be a factor determining the extent of DNA fragmentation, since rapid protein degradation and collapse of nuclear structure might stop DNA cleavage at any given stage. This would explain the fact that DNA degradation never proceeds to completion and that the DNA ladder is not always observed even in cells containing nucleases capable of internucleosomal DNA cleavage.…”

Behavioral and Anatomical Correlates of Chronic Episodic Hypoxia during Sleep in the Rat

“…The slight disparities between TUNEL and SS-DNA labeling at day 14 of EHYP could reflect sensitivity differences between the two methods in the detection of apoptosis. Alternatively, because TUNEL is considered to be a less specific marker of apoptosis (Grasl Kraupp et al, 1995;Frankfurt et al, 1996), other cells in the process of death by nonapoptotic mechanisms may have also been labeled (Chen et al, 1997;Labat Moleur et al, 1998). It needs to be emphasized that the magnitude of tissue hypoxia used in this study was relatively mild compared with the levels of hypoxia required for induction of apoptosis using a single exposure (Banasiak and Haddad, 1998).…”

Src kinase inhibition decreases thrombin-induced injury and cell cycle re-entry in striatal neurons

“…TUNEL labeling was performed according to a published method (Chen et al, 1997), with minor modifications. In brief, fixed cells in slide chambers were incubated in the terminal deoxynucleotidyl-transferase (TDT) buffer (Invitrogen, Carlsbad, CA) for 10 minutes at RT and then incubated for 2h at 37°C in TDT buffer containing 10 mM biotin-aha-dUTP (Invitrogen, Carlsbad, CA) and 300 U/ml TDT (Invitrogen, Carlsbad, CA).…”