Hortobágyi T, Görlach C, Benyó Z, Lacza Z, Hortobágyi S, Wahl M, Harkany T
Department of Pathology, University of Szeged, Szeged, Hungary.
Neuroscience. 2003;121(4):983-90. doi: 10.1016/s0306-4522(03)00482-2.
Focal traumatic injury to the cerebral cortex is associated with early activation of the neuronal isoform of nitric oxide synthase (nNOS), where high concentrations of nitric oxide-derived free radicals elicit extensive DNA damage. Subsequent activation of the nuclear repair enzyme poly(ADP-ribose) polymerase (PARP) causes a severe energy deficit leading to the ultimate demise of affected neurons. Little is known about the temporal relationship of nNOS and PARP activation and the neuroprotective efficacy of their selective blockade in traumatic brain injury. To determine the relationship of nNOS and PARP activation, brain injury was induced by cryogenic lesion to the somatosensory cortex applying a pre-cooled cylinder after trephination for 6 s to the intact dura mater. Pre-treatment with 3-bromo-7-nitroindazole (BrNI; 25 mg/kg, i.p.), and pre- or combined pre- and post-treatment with 3-aminobenzamide (AB; 10 mg/kg (i.c.v.) or 10 mg/kg/h (i.p.)) were used to inhibit nNOS and PARP, respectively. Cold lesion-induced changes in the somatosensory cortex and neuroprotection by BrNI and AB were determined using immunocytochemistry and immunodot-blot for detection of poly(ADP-ribose; PAR), the end-product of PARP activation, and the triphenyltetrazolium-chloride assay to assess lesion volume. PAR immunoreactivity reached its peak 30 min post-lesion and was followed by gradual reduction of PAR immunolabeling. BrNI pre-treatment significantly decreased the lesion-induced PAR concentration in damaged cerebral cortex. Pre-treatment by i.c.v. infusion of AB markedly diminished cortical PAR immunoreactivity and significantly reduced the lesion volume 24 h post-injury. In contrast, i.p. AB treatment remained largely ineffective. In conclusion, our data indicate early activation of PARP after cold lesion that is, at least in part, related to nNOS induction and supports the relevance of nNOS and/or PARP inhibition to therapeutic approaches of traumatic brain injury.
大脑皮质局灶性创伤性损伤与一氧化氮合酶（nNOS）的神经元亚型早期激活有关，高浓度的一氧化氮衍生自由基会引发广泛的DNA损伤。随后，核修复酶聚（ADP - 核糖）聚合酶（PARP）的激活会导致严重的能量不足，从而导致受影响神经元最终死亡。关于nNOS和PARP激活的时间关系以及它们在创伤性脑损伤中选择性阻断的神经保护效果，人们知之甚少。为了确定nNOS和PARP激活之间的关系，在完整硬脑膜上钻孔后，用预冷的圆柱体对体感皮质进行6秒的冷冻损伤来诱导脑损伤。分别用3 - 溴 - 7 - 硝基吲唑（BrNI；25 mg/kg，腹腔注射）预处理，以及用3 - 氨基苯甲酰胺（AB；10 mg/kg（脑室内注射）或10 mg/kg/h（腹腔注射））进行预处理或联合预处理和后处理来抑制nNOS和PARP。使用免疫细胞化学和免疫斑点印迹法检测聚（ADP - 核糖；PAR）（PARP激活的终产物），并用氯化三苯基四氮唑试验评估损伤体积，以确定冷冻损伤引起的体感皮质变化以及BrNI和AB的神经保护作用。PAR免疫反应性在损伤后30分钟达到峰值，随后PAR免疫标记逐渐减少。BrNI预处理显著降低了损伤诱导的受损大脑皮质中的PAR浓度。通过脑室内注射AB进行预处理显著降低了皮质PAR免疫反应性，并显著减小了损伤后24小时的损伤体积。相比之下，腹腔注射AB治疗基本无效。总之，我们的数据表明冷冻损伤后PARP早期激活，这至少部分与nNOS诱导有关，并支持nNOS和/或PARP抑制在创伤性脑损伤治疗方法中的相关性。
