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 deoxynucleotidyl transferase
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 deoxynucleotidyl transferase
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DOI：
10.1089/089771501750357627
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发表时间：
2001-07-01
影响因子：
4.2
通讯作者：
Graham, SH
中科院分区：
文献类型：
--
作者：
Clark, RSB;Chen, MZ;Graham, SH
DNA损伤是创伤性脑损伤（TBI）的常见后遗症。用于原位鉴定DNA损伤的可用技术包括DNA聚合酶I介导的生物素-dATP缺口翻译（PANT）、DNA聚合酶I介导的生物素-dATP缺口末端标记（Klenow）的Klenow片段和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）。虽然TUNEL已被广泛用于主要检测双链DNA断裂，但在TBI后使用PANT主要检测单链DNA断裂和Klenow检测单链和双链DNA断裂尚未报道。因此，使用PANT、Klenow和TUNEL方法处理来自未处理大鼠和在施加二次损伤的受控皮质撞击后0、0.5、1、2、6、24和72 h的大鼠（n = 3-5只/组）的冠状脑切片。损伤后0.5h，PANT检测到损伤侧大脑半球有DNA断裂细胞，6 h达高峰（皮质= 66.3 +/- 15.8，齿状回58.6 +/- 12.8，CAI = 15.8 +/- 5.9，CA 3 = 12.8 +/- 4.2个细胞/ x 400视野，平均值+/- SEM，所有p < 0.05，相对于未处理）。通过Klenow，早在30分钟就检测到具有DNA断裂的细胞，并且在24小时时达到最大值（皮质= 56.3 +/- 14.3，齿状回78.0 +/- 16.7，CA 1 = 25.8 +/- 4.7，CA 3 = 29.3 +/- 15.1个细胞/ x 400视野，所有p < 0.05，相对于未处理的）。具有DNA断裂的细胞直到2小时才通过TUNEL检测到，并且在24小时时达到最大值（皮质= 47.7 +/-21.4，齿状回63.0 +/-11.9，CA 1 = 5.6 +/-5.4，CA 3 = 6.9 +/-3.7个细胞/x400视野，皮质和齿状回p < 0.05，相对于未处理的）。双标记免疫荧光显示PANT阳性细胞主要为神经元。这些数据表明，TBI导致广泛的DNA损伤，包括损伤的皮质和海马中的单链和双链断裂。多种类型的DNA断裂的存在暗示了TBI后DNA损伤演变中的几种途径。
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, 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 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/x400 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.