Detection of Single- and Double-Strand DNA Breaks After Traumatic Brain Injury in Rats: Comparison ofIn SituLabeling 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.

A dual role for poly‐ADP‐ribosylation in spatial memory acquisition after traumatic brain injury in mice involving NAD+ depletion and ribosylation of 14‐3‐3γ

“…PANT labeling was primarily nuclear and, based on the size, morphology, and location of the stained cells, appeared to occur in neurons. Previous dual-label studies in rats have detected PANT in mostly neuronal phenotypes (Clark et al 2001). PANT was not detected in the contralateral hemispheres, or in deeper brain regions after CCI.…”

“…PANT assay PANT was performed as previously described (Clark et al 2001). Briefly, paraffin-embedded 5-lm coronal sections were deparaffinized then incubated in 3% Triton X-100 (Sigma, St Louis, MO, USA) at room temperature for 1 h, then placed in 3% H 2 O 2 and 30% methanol in PBS for 20 min.…”

“…The transferred membranes were incubated in the monoclonal primary antibody against nitrotyrosine (1 : 500; clone 1A6, Upstate Biotechnology, Lake Placid, NY, USA) or poly(ADPribose) polymers (1 : 1000; SA-216, Biomol, Plymouth Meeting, PA, USA) at room temperature for 1 h, washed in phosphatebuffered saline (PBS) containing 0.1% Tween-20, then incubated in the appropriate secondary antibody (1 : 3000) for 1 h. The membranes were washed in PBS containing 0.1% Tween-20 three times over 60 min, then incubated in commercial enhanced chemiluminescence reagents (NEN Life Science Products, Boston, MA, USA) and exposed to X-ray film. Autoradiogram signals were semiquantified using a gel densitometric scanning program (MCID, St Catherines, Ontario, Canada).PANT assay PANT was performed as previously described (Clark et al 2001). Briefly, paraffin-embedded 5-lm coronal sections were deparaffinized then incubated in 3% Triton X-100 (Sigma, St Louis, MO, USA) at room temperature for 1 h, then placed in 3% H 2 O 2 and 30% methanol in PBS for 20 min.…”

Dissecting the mechanical behavior of neuronal microcompartments by combining magnetic tweezers and protein micropatterns

“…Few studies reported soma shrinkage with pyknotic nuclei [26] (i.e. condensation of chromatin leading to a shrunken nucleus) and damaged DNA [27,28] . However, these alterations are delayed compared to the axonal damages, suggesting that they may be associated with a secondary injury related to axonal swellings.…”

Immunohistochemistry of Neuronal Apoptosis in Fatal Traumas: The Contribution of Forensic Molecular Pathology in Medical Science

“…terminal deoxynucleotidyl-transferase-mediated dUDP nick end-labeling (TUNEL) or in situ nick translation (ISNT) (Clark et al, 2001;Gavrieli et al, 1992;Rink et al, 1995). However, experimental studies have shown that single-stranded DNA (ssDNA) degradation precedes DNA double-strand breaks (DNAdsb) during a delayed neuronal death process caused by reperfusion after transient brain ischemia or intracerebral hemorrhage, possibly due to oxidative stress (Chen et al, 1997;Gong et al, 2001;Love, 1999;Nakamura et al, 2005).…”