Li P A, Rasquinha I, He Q P, Siesjö B K, Csiszár K, Boyd C D, MacManus J P
Center for Biomedical Research, The Queen's Medical Center, John A. Burns School of Medicine, University of Hawaii, Honolulu, 98613, USA.
J Cereb Blood Flow Metab. 2001 May;21(5):568-76. doi: 10.1097/00004647-200105000-00011.
Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3'-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.
以往的组织病理学结果表明，高血糖（HG）导致缺血性损伤加重的一种机制涉及DNA片段化。通过末端脱氧核苷酸转移酶介导的DNA缺口标记（TUNEL）染色、脉冲场凝胶电泳（PFGE）和连接介导的聚合酶链反应（LM-PCR），研究了正常血糖（NG）和HG大鼠在前脑缺血30分钟后的DNA片段化情况。PFGE检测到高分子量DNA片段，而LM-PCR技术检测到低分子量DNA片段。LM-PCR程序是在具有平端（无Klenow聚合酶）和3'-凹陷末端（有Klenow聚合酶）的测试样品DNA上进行的。此外，通过免疫细胞化学研究了细胞色素c释放和半胱天冬酶-3激活情况。结果表明，HG导致细胞色素c释放，激活半胱天冬酶-3，并加剧缺血诱导的DNA片段化。因此，在HG大鼠中，而非对照或NG大鼠中，在扣带回皮质、新皮质、丘脑和纹状体背外侧嵴中发现了TUNEL染色的细胞，在这些部位通过传统组织病理学观察到神经元死亡，并且通过共聚焦显微镜检测到胞质细胞色素c和活性半胱天冬酶-3。在新皮质中，HG大鼠检测到了平端和交错端片段，而NG大鼠未检测到。在扣带回皮质进行了电子显微镜（EM）分析，在该部位观察到大量TUNEL阳性神经元。尽管通过TUNEL染色和电泳技术检测到了DNA片段化，但EM分析未能表明存在凋亡性细胞死亡。结论是，HG触发了细胞死亡途径并加剧了缺血诱导的DNA片段化。
