title: Detection of single- and double-strand DNA breaks after traumatic brain injury in rats: comparison of in situ labeling techniques using DNA polymerase I, the Klenow fragment of DNA polymerase I, and terminal deoxynucleotidyl transferase.
abstract: DNA damage is a common sequela of traumatic brain injury (TBI). Available techniques for the in situ identification of DNA damage include DNA polymerase I-mediated biotin-dATP nick-translation (PANT), the Klenow fragment of DNA polymerase I-mediated biotin-dATP nick-end labeling (Klenow), and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). While TUNEL has been widely utilized to detect primarily double-strand DNA breaks, the use of PANT to detect primarily single-strand DNA breaks and Klenow to detect both single- and double-strand DNA breaks has not been reported after TBI. Accordingly, coronal brain sections from naive rats and rats at 0, 0.5, 1, 2, 6, 24, and 72 h (n = 3-5/group) after controlled cortical impact with imposed secondary insult were processed using the PANT, Klenow, and TUNEL methods. Cells with DNA breaks were detected by PANT in the ipsilateral hemisphere as early as 0.5 h after injury and were maximal at 6 h (cortex = 66.3+/-15.8, dentate gyrus 58.6+/-12.8, CA1 = 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 were 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 versus naive). Cells with DNA breaks were not detected by TUNEL until 2 h and were maximal at 24 h (cortex = 47.7+/-21.4, dentate gyrus 63.0+/-11.9, CA1 = 5.6+/-5.4, CA3 = 6.9+/-3.7 cells/x 400 field, cortex and dentate gyrus 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-strand breaks in injured cortex and hippocampus. The presence of multiple types of DNA breaks implicate several pathways in the evolution of DNA damage after TBI.
authors.affiliation: Department of Anesthesiology, The Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh School of Medicine, Pennsylvania 15260, USA. clarkrs@anes.upmc.edu
authors.lastname: Clark RSB
authors.forename: M
authors.initials: M
authors.lastname: Chen
authors.forename: P M
authors.initials: PM
authors.lastname: Kochanek
authors.forename: S C
authors.initials: SC
authors.lastname: Watkins
authors.forename: K L
authors.initials: KL
authors.lastname: Jin
authors.forename: R
authors.initials: R
authors.lastname: Draviam
authors.forename: P D
authors.initials: PD
authors.lastname: Nathaniel
authors.forename: R
authors.initials: R
authors.lastname: Pinto
authors.forename: D W
authors.initials: DW
authors.lastname: Marion
authors.forename: S H
authors.initials: SH
authors.lastname: Graham
mesh_terms: D000818:Animals; D001930:Brain Injuries / Q000235:genetics* / Q000473:pathology; D004249:DNA Damage / Q000235:genetics*; D004253:DNA Nucleotidylexotransferase / Q000235:genetics*; D004256:DNA Polymerase I / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D004195:Disease Models, Animal; D020287:In Situ Nick-End Labeling; D008297:Male; D008856:Microscopy, Fluorescence; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
chemical_list: D004277:DNA, Single-Stranded; D004253:DNA Nucleotidylexotransferase; D004256:DNA Polymerase I