TY - JOUR
T1 - Detection of single- and double-strand DNA breaks after traumatic brain injury in rats
T2 - Comparison of in situ labeling techniques using DNA polymerase I, the Klenow fragment of DNA polymerase I, and terminal deoxynucleotidyl transferase
AU - Clark, R. S.B.
AU - Chen, M.
AU - Kochanek, P. M.
AU - Watkins, S. C.
AU - Jin, K. L.
AU - Draviam, R.
AU - Nathaniel, P. D.
AU - Pinto, R.
AU - Marion, D. W.
AU - Graham, S. H.
PY - 2001
Y1 - 2001
N2 - 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/×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/×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/×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.
AB - 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/×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/×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/×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.
KW - Apoptosis
KW - Controlled cortical impact
KW - Klenow
KW - PANT
KW - TUNEL
UR - http://www.scopus.com/inward/record.url?scp=0034902082&partnerID=8YFLogxK
U2 - 10.1089/089771501750357627
DO - 10.1089/089771501750357627
M3 - Article
C2 - 11497094
AN - SCOPUS:0034902082
SN - 0897-7151
VL - 18
SP - 675
EP - 689
JO - Journal of Neurotrauma
JF - Journal of Neurotrauma
IS - 7
ER -