Comet assay as a novel approach for studying DNA damage in focal cerebral ischemia: differential effects of NMDA receptor antagonists and poly(ADP-ribose) polymerase inhibitors.
The single-cell gel electrophoresis (comet assay) was used to evaluate the possibility of detecting single-strand breaks of brain DNA in the early phase of ischemia. Four hours after occlusion of the middle cerebral artery (MCAO) in rats, the percentage of DNA migrating into the comet tail (indicating the presence of breaks) increased from 11.4 +/- 4.70 to 34.7 +/- 9.2 (means +/- SD) in the caudate and from 9.9 +/- 4.3 to 42.8 +/- 14.1 in the cortex. Interestingly, a subpopulation of cells exhibiting higher resistance to the ischemic insult was present in the caudate putamen, but not in the cortex. Administration of MK801, an N-methyl-d-aspartate (NMDA) glutamate receptor antagonist, (1 mg/kg subcutaneously, 10 minutes before MCAO), reduced the ischemia-induced DNA breaks and the infarct volume, suggesting that excessive stimulation of NMDA receptors contributes to the formation of both DNA damage and infarct volume. In contrast, DPQ, an inhibitor of poly(ADP-ribose) polymerase (PARP) (10 mg/kg intraperitoneally, 2 hours before and 1 hour after MCAO), reduced the infarct volume but not DNA damage, suggesting that the neuroprotective actions of PARP inhibitors occur at a later step of the processes leading to postischemic neuronal death.
Giovannelli
Lisa
L
Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy.
Cozzi
Andrea
A
Guarnieri
Ilaria
I
Dolara
Piero
P
Moroni
Flavio
F
D000818:Animals; D002545:Brain Ischemia / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D020552:Comet Assay; D004247:DNA; D004249:DNA Damage; D004277:DNA, Single-Stranded; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D020244:Infarction, Middle Cerebral Artery / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D018696:Neuroprotective Agents / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors*
D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D007546:Isoquinolines; D018696:Neuroprotective Agents; D010880:Piperidines; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D016194:Receptors, N-Methyl-D-Aspartate; C108729:3,4-dihydro-5-(4-(1-piperidinyl)butoxy)-1(2H)-isoquinolinone; D016291:Dizocilpine Maleate; D004247:DNA