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


Address correspondence and reprint requests to Dr. J. Chen at Department of Neurology, S506-Biomedical Science Tower, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, U.S.A.


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 (SSBs) detected by PANT were present in neurons after as little as 1 min 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 TUNEL 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.

Abbreviations used: DSB, double-strand break; GFAP, glial fibrillary acidic protein; MCA, middle cerebral artery; MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; NSE, neuron-specific enolase; PANT, DNA polymerase I-mediated biotin-dATP nick-translation; PBS, phosphate-buffered solution; SSB, single-strand break; TdT, terminal deoxynucleotidyl-transferase; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling.