Chen J, Uchimura K, Stetler R A, Zhu R L, Nakayama M, Jin K, Graham S H, Simon R P
Department of Neurology, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
J Cereb Blood Flow Metab. 1998 Jun;18(6):646-57. doi: 10.1097/00004647-199806000-00007.
Using in situ hybridization, Northern blot analysis, Western blot analysis, and immunocytochemistry, mRNA and protein expression of the novel DNA damage-inducible gene GADD45 was examined in the rat brain at 0.5, 2, 4, 8, 16, 24, 48, and 72 hours after 15 minutes of transient global ischemia. Transient ischemia produced by the four-vessel occlusion method resulted in DNA double-strand breaks and delayed neuronal cell death in vulnerable neurons of the hippocampal CA1 sector, the hilus, dorsal caudate-putamen, and thalamus, as shown by in situ DNA nick end-labeling and histologic staining. GADD45 mRNA was transiently increased in less-vulnerable regions such as the parietal cortex (up to 8 hours after ischemia) and dentate granule cells (up to 24 hours after ischemia) but was persistently increased in vulnerable neurons such as CA1 pyramidal neurons (up to 48 hours). GADD45 immunoreactivity was increased in both vulnerable and less-vulnerable regions at earlier reperfusion periods (4 to 16 hours), but thereafter immunoreactivity was decreased below control levels in most vulnerable regions before delayed cell death and DNA double-strand breaks. At 72 hours after transient ischemia, a moderate increase in GADD45 immunoreactivity was still detectable in some CA3 neurons and in a few surviving neurons in the CA1 region. Double staining performed at 16 to 72 hours after ischemia revealed that GADD45 immunoreactivity was persistently increased in neurons that did not develop DNA damage. Because GADD45 protein may participate in the DNA excision repair process and because it has been shown that this protein is also overexpressed in neurons that survive focal ischemia and kainate-induced epileptic seizures, the results reported here support the hypothesis that GADD45 could have a protective role in neuronal injury.
采用原位杂交、Northern印迹分析、Western印迹分析和免疫细胞化学方法，检测了短暂全脑缺血15分钟后0.5、2、4、8、16、24、48和72小时大鼠脑内新的DNA损伤诱导基因GADD45的mRNA和蛋白表达。四动脉闭塞法所致的短暂性缺血导致DNA双链断裂，并使海马CA1区、齿状回、背侧尾状核-壳核和丘脑等易损神经元中的神经元细胞死亡延迟，原位DNA缺口末端标记和组织学染色结果显示了这一点。GADD45 mRNA在顶叶皮质（缺血后8小时内）和齿状颗粒细胞（缺血后24小时内）等不易损区域短暂增加，但在CA1锥体细胞等易损神经元中持续增加（缺血后48小时内）。在再灌注早期（4至16小时），GADD45免疫反应性在易损和不易损区域均增加，但此后在延迟性细胞死亡和DNA双链断裂之前，大多数易损区域的免疫反应性降至对照水平以下。短暂性缺血72小时后，在一些CA3神经元和CA1区少数存活神经元中仍可检测到GADD45免疫反应性适度增加。缺血后16至72小时进行的双重染色显示，未发生DNA损伤的神经元中GADD45免疫反应性持续增加。由于GADD45蛋白可能参与DNA切除修复过程，且已表明该蛋白在局灶性缺血和海藻酸诱导的癫痫发作后存活的神经元中也过表达，因此本文报道的结果支持GADD45可能在神经元损伤中起保护作用这一假说。
