Visualization of irreparable ischemic damage in brain by selective labeling of double strand blunt-ended DNA breaks.
BACKGROUND
Double-strand DNA breaks with blunt ends represent the most serious type of DNA damage, and cannot be efficiently repaired by cells. They are generated in apoptosis or necrosis and are absent in normal or transiently damaged cells. Consequently, they can be used as a molecular marker of irreparable cellular damage. We evaluated the effects of focal brain ischemia using selective labeling of blunt-ended DNA breaks as a marker of irreversible tissue damage. A new approach permitting such analysis in situ is introduced.


METHODS
Rat brain sections taken 6, 24, 48 and 72 hr after the onset of focal brain ischemia were used. Double-strand DNA breaks were detected directly in the tissue sections via ligation of blunt-ended hairpin-shaped oligonucleotide probes. The probes were attached to the ends of the breaks by T4 DNA ligase. Conventional cresyl violet co-staining and terminal transferase based labeling (TUNEL) were employed to analyze the distribution of labeled cells.


RESULTS
Double-strand blunt-ended DNA breaks rapidly accumulate in brain cells after focal brain ischemia. At 24 hr, they concentrate in the peripheral areas of stroke, which are prone to ischemia-reoxygenation. By 48-72 hr, this type of DNA damage spreads inward, covering the internal areas of the ischemic zone.


CONCLUSIONS
Selective labeling of blunt-ended DNA breaks delineates the dynamics of stroke-induced irreversible DNA damage and provides highly specific detection of brain cells with irreparable DNA injury. It can be used for comparing the efficiency of various anti-ischemic drugs, particularly those that target DNA damage, as well as for monitoring stroke-induced damage.
Didenko
Vladimir V
VV
Department of Neurosurgery, Baylor College of Medicine, Houston, TX, USA.
Ngo
Hop
H
Minchew
Candace L
CL
Boudreaux
Denise J
DJ
Widmayer
Marsha A
MA
Baskin
David S
DS
D000818:Animals; D002545:Brain Ischemia / Q000473:pathology* / Q000628:therapy; D004247:DNA / Q000378:metabolism*; D005456:Fluorescent Dyes; D015335:Molecular Probes; D051381:Rats; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D015335:Molecular Probes; D004247:DNA