Xu R X, Nakamura T, Nagao S, Miyamoto O, Jin L, Toyoshima T, Itano T
Department of Neurological Surgery, Kagawa Medical University, Japan.
Neurosurgery. 1998 Jul;43(1):107-14; discussion 114-5. doi: 10.1097/00006123-199807000-00070.
Apoptosis of neuronal cells plays a key role in many developmental and pathological processes of the central nervous system. Deoxyribonucleic acid (DNA) of cells undergoing apoptosis is cleaved by an endonuclease into oligonucleosoma-sized fragments. These fragments can be labeled using in situ terminal deoxynucleotidyl transferase so that the apoptotic cells can be visualized by in situ apoptotic staining. The model of cold-induced rat brain edema was used to further examine this hypothesis. The protective effect of hypothermia was also studied in this model of cold-induced brain injury.
Using a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate-biotin nick end labeling technique, the neuronal cells with DNA fragmentation in different regions of the brains of rats subjected to cold-induced brain injury were detected. The internucleosomal fragments of DNA in apoptotic cells were examined using agarose gel electrophoresis. The animals were randomly divided into three groups: 1) sham (n = 8); 2) cold-induced brain injury, killed at 12, 24, 48, 72, and 168 hours after cold lesion (n = 10 for each time point); 3) hypothermia, both mean temporalis and rectal temperatures were reduced by surface cooling to 32 degrees C (standard deviation, 0.1 degrees C) for 3, 6, and 12 hours (n = 10 for each time point) beginning 1 hour after cold-induced brain injury.
The apoptotic cells were detectable for up to 72 hours after the initial brain injury and reached a peak at approximately 24 to 48 hours, with a mean peak value of 24.29 +/- 5.26, 15.37 +/- 4.10, 15.81 +/- 3.56, 13.94 +/- 2.48, 10.46 +/- 2.23, and 7.68 +/- 2.48% in the cortex, subcortex, white matter, CA1, CA3, and dentate gyrus, respectively, and had a significant increase, compared with the control value (mean +/- standard error, P < 0.01). Agarose gel electrophoresis of DNA extracted from cortex and hippocampus containing apoptotic cells revealed a "DNA ladder" at 180- to 200-base pair intervals. In animals subjected to the same brain injury that underwent 32 degrees C hypothermia, the numbers of apoptotic cells were reduced evidently and DNA fragmentation was inhibited.
The data suggest that apoptosis occurs after cold-induced brain injury and that DNA fragmentation may be associated with apoptotic cell death. Moderate hypothermia shows specific effect on inhibition of apoptotic cell death and cellular DNA fragmentation after cold-induced brain injury in rats.
神经元细胞凋亡在中枢神经系统的许多发育和病理过程中起关键作用。经历凋亡的细胞的脱氧核糖核酸（DNA）被一种核酸内切酶切割成寡核小体大小的片段。这些片段可用原位末端脱氧核苷酸转移酶进行标记，从而通过原位凋亡染色使凋亡细胞可视化。采用冷诱导大鼠脑水肿模型来进一步检验这一假说。还在这个冷诱导脑损伤模型中研究了低温的保护作用。
使用末端脱氧核苷酸转移酶介导的脱氧尿苷5'-三磷酸-生物素缺口末端标记技术，检测遭受冷诱导脑损伤的大鼠大脑不同区域中具有DNA片段化的神经元细胞。使用琼脂糖凝胶电泳检测凋亡细胞中DNA的核小体间片段。将动物随机分为三组：1）假手术组（n = 8）；2）冷诱导脑损伤组，在冷损伤后12、24、48、72和168小时处死（每个时间点n = 10）；3）低温组，在冷诱导脑损伤后1小时开始，通过体表降温将颞部平均温度和直肠温度降至32℃（标准差，0.1℃），持续3、6和12小时（每个时间点n = 10）。
在最初脑损伤后长达72小时可检测到凋亡细胞，在约24至48小时达到峰值，皮质、皮质下、白质、CA1、CA3和齿状回中凋亡细胞的平均峰值分别为24.29±5.26%、15.37±4.10%、15.81±3.56%、13.94±2.48%、10.46±2.23%和7.68±2.48%，与对照值相比有显著增加（平均值±标准误，P < 0.01）。对含有凋亡细胞的皮质和海马提取的DNA进行琼脂糖凝胶电泳，显示在180至200碱基对间隔处有“DNA梯带”。在遭受相同脑损伤并进行32℃低温处理的动物中，凋亡细胞数量明显减少，DNA片段化受到抑制。
数据表明冷诱导脑损伤后发生凋亡，且DNA片段化可能与凋亡细胞死亡有关。适度低温对抑制大鼠冷诱导脑损伤后的凋亡细胞死亡和细胞DNA片段化具有特异性作用。
