Minger S L, Geddes J W, Holtz M L, Craddock S D, Whiteheart S W, Siman R G, Pettigrew L C
The Stroke Program of the University of Kentucky Chandler Medical Center, USA.
Brain Res. 1998 Nov 9;810(1-2):181-99. doi: 10.1016/s0006-8993(98)00921-4.
Excitatory amino acids may promote microtubular proteolysis observed in ischemic neuronal degeneration by calcium-mediated activation of calpain, a neutral protease. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. Spontaneously hypertensive rats were treated with 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)quinoxaline (NBQX), a competitive antagonist of the neuronal receptor for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or cis-4-[phosphono-methyl]-2-piperidine carboxylic acid (CGS 19755), a competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor. After treatment, all animals were subjected to permanent occlusion of the middle cerebral artery for 6 or 24 h. Infarct volumes measured in animals pretreated with CGS 19755 after 24 h of ischemia were significantly smaller than those quantified in ischemic controls. Rats pretreated with NBQX showed partial amelioration of cytoskeletal injury with preserved immunolabeling of microtubule-associated protein 2 (MAP 2) at 6 and 24 h and reduced accumulation of calpain-cleaved spectrin byproducts only at 6 h. Prevention of cytoskeletal damage was more effective after pretreatment with CGS 19755, as shown by retention of MAP 2 immunolabeling and significant restriction of calpain activity at both 6 and 24 h. Preserved immunolabeling of tau protein was observed at 6 and 24 h only in animals pretreated with CGS 19755. Western analysis performed on ischemic cortex taken from controls or rats pretreated with either NBQX or CGS 19755 suggested that loss of tau protein immunoreactivity was caused by dephosphorylation, rather than proteolysis. These results demonstrate a crucial link between excitotoxic neurotransmission, microtubular proteolysis, and neuronal degeneration in focal cerebral ischemia.
兴奋性氨基酸可能通过钙介导的中性蛋白酶钙蛋白酶的激活，促进在缺血性神经元变性中观察到的微管蛋白水解。我们在无再灌注的局灶性脑缺血动物模型中检验了这一假设。自发性高血压大鼠用2,3 - 二羟基 - 6 - 硝基 - 7 - 氨磺酰基 - 苯并 - (F)喹喔啉（NBQX，一种α - 氨基 - 3 - 羟基 - 5 - 甲基 - 4 - 异恶唑丙酸（AMPA）神经元受体的竞争性拮抗剂）或顺式 - 4 - [膦酰基甲基] - 2 - 哌啶羧酸（CGS 19755，一种N - 甲基 - D - 天冬氨酸（NMDA）受体的竞争性拮抗剂）进行治疗。治疗后，所有动物均接受大脑中动脉永久性闭塞6或24小时。缺血24小时后用CGS 19755预处理的动物中测量的梗死体积明显小于缺血对照组中量化的梗死体积。用NBQX预处理的大鼠在6小时和24小时时显示细胞骨架损伤部分改善，微管相关蛋白2（MAP 2）的免疫标记得以保留，仅在6小时时钙蛋白酶切割的血影蛋白副产物积累减少。如在6小时和24小时时MAP 2免疫标记的保留以及钙蛋白酶活性的显著限制所示，用CGS 19755预处理后对细胞骨架损伤的预防更有效。仅在CGS 19755预处理的动物中在6小时和24小时时观察到tau蛋白的免疫标记得以保留。对取自对照组或用NBQX或CGS 19755预处理的大鼠的缺血皮层进行的蛋白质印迹分析表明，tau蛋白免疫反应性的丧失是由去磷酸化而非蛋白水解引起的。这些结果证明了局灶性脑缺血中兴奋性毒性神经传递、微管蛋白水解和神经元变性之间的关键联系。
