Detection of single- and double-strand DNA breaks after traumatic brain injury in rats: Comparison of in situ labeling techniques using DNA polymerase 1, the Klenow fragment of DNA polymerase 1, and terminal deoxynucleotidyltransferase

DNA damage is a common sequela of traumatic brain injury (TBI). Available t echniques for the in situ identification of DNA damage include DNA polymera se I-mediated biotin-dATP nick-translation (PANT), the Klenow fragment of D NA polymerase I-mediated biotin-dATP nick-end labeling (Klenow), and termin al deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Wh ile TUNEL has been widely utilized to detect primarily double-strand DNA br eaks, the use of PANT to detect primarily single-strand DNA breaks and Klen ow to detect both single- and double-strand DNA breaks has not been reporte d after TBI. Accordingly, coronal brain sections from naive rats and rats a t 0, 0.5, 1, 2, 6, 24, and 72 h (n = 3-5/group) after controlled cortical i mpact with imposed secondary insult were processed using the PANT, Klenow, and TUNEL methods. Cells with DNA breaks were detected by PANT in the ipsil ateral hemisphere as early as 0.5 h after injury and were maximal at 6 h (c ortex = 66.3 +/- 15.8, dentate gyrus 58.6 +/- 12.8, CAI = 15.8 +/- 5.9, CA3 = 12.8 +/- 4.2 cells/ x 400 field, mean +/- SEM, all p < 0.05 versus naive ). Cells with DNA breaks were detected by Klenow as early as 30 min and wer e maximal at 24 h (cortex = 56.3 +/- 14.3, dentate gyrus 78.0 +/- 16.7, CA1 = 25.8 +/- 4.7, CA3 = 29.3 +/- 15.1 cells/ x 400 field, all p < 0.05 versu s naive). Cells with DNA breaks were not detected by TUNEL until 2 h and we re maximal at 24 h (cortex = 47.7 +/- 21.4, dentate gyrus 63.0 +/- 11.9, CA 1 = 5.6 +/- 5.4, CA3 = 6.9 +/- 3.7 cells/x400 field, cortex and dentate gyr us p < 0.05 versus naive). Dual-label immunofluorescence revealed that PANT -positive cells were predominately neurons. These data demonstrate that TBI results in extensive DNA damage, which includes both single- and double-st rand breaks in injured cortex and hippocampus. The presence of multiple typ es of DNA breaks implicate several pathways in the evolution of DNA damage after TBI.