Omae Tadaki, Yoshioka Hiroshi, Tanaka Taro, Nagai Hideyuki, Saji Makoto, Noda Kazuko, Kobayashi Shizuka, Sugimoto Tohru
Department of Pediatrics, Kyoto Prefectural University of Medicine, 465 Kajii-cho Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-0841, Japan.
Brain Dev. 2008 May;30(5):313-20. doi: 10.1016/j.braindev.2007.08.002. Epub 2008 Mar 20.
Synaptic release of the excitatory amino acid glutamate is considered as an important mechanism in the pathogenesis of ischemic brain damage in neonates. Synaptotagmin I is one of exocytosis-related proteins at nerve terminals and considered to accelerate the exocytosis of synaptic vesicles by promoting fusion between the vesicles and plasma membrane. To test the possibility that antisense in vivo knockdown of synaptotagmin I modulates the exocytotic release of glutamate, thus suppressing the excitotoxic intracellular processes leading to neuronal death following ischemia in the neonatal brain, we injected antisense oligodeoxynucleotides (ODNs) targeting synaptotagmin I (0.3 (AS), 0.15 (0.5 AS), or 0.03 microg (0.1 AS), or vehicle) into the lateral ventricles of 7-day-old rats by using a hemagglutinating virus of Japan (HVJ)-liposome mediated gene transfer technique. At 10 days of age, these rats were subjected to an electrical coagulation of the right external and internal carotid arteries, then the insertion of a solid nylon thread into the right common carotid artery toward the ascending aorta up to 10-12 mm from the upper edge of the sternocleidomastoid muscle. Cerebral ischemia was induced by clamping the left external and internal carotid arteries with a clip, and ended by removing the clip 2h later. Twenty-four hours after the end of ischemia, the extent of ischemic brain damage was neuropathologically and quantitatively evaluated in the neocortex and striatum. While the relative volume of damage in the cerebral cortex and striatum of the vehicle group was extended to 40% and 13.7%, respectively, that in the AS group was significantly reduced to 4.8% and 0.6%. In the 0.5 AS group, the relative volume of ischemic damage in the cerebral cortex and striatum was reduced to 20.5% and 15.4%, respectively, and the difference between the 0.5 AS group and vehicle group was statistically significant in the neocortex, but not in the striatum. These results indicated that antisense in vivo knockdown of synaptotagmin I successfully attenuated ischemic brain damage in neonatal rats and that the effect was dose-dependent. It was also suggested that this treatment was more effective in the neocortex than in the striatum in neonatal rats.
兴奋性氨基酸谷氨酸的突触释放被认为是新生儿缺血性脑损伤发病机制中的一个重要机制。突触结合蛋白I是神经末梢与胞吐作用相关的蛋白之一，被认为可通过促进囊泡与质膜之间的融合来加速突触囊泡的胞吐作用。为了验证体内敲低突触结合蛋白I的反义核酸是否能调节谷氨酸的胞吐释放，从而抑制新生儿脑缺血后导致神经元死亡的兴奋性毒性细胞内过程，我们采用日本血凝病毒（HVJ）-脂质体介导的基因转移技术，将靶向突触结合蛋白I的反义寡脱氧核苷酸（ODN）（0.3μg（AS）、0.15μg（0.5 AS）或0.03μg（0.1 AS），或溶剂）注入7日龄大鼠的侧脑室。在10日龄时，对这些大鼠进行右侧颈外动脉和颈内动脉的电凝，然后将一根实心尼龙线插入右侧颈总动脉并朝向升主动脉，直至距胸锁乳突肌上缘10 - 12毫米处。通过用夹子夹闭左侧颈外动脉和颈内动脉诱导脑缺血，并在2小时后移除夹子结束缺血。缺血结束后24小时，对新皮层和纹状体的缺血性脑损伤程度进行神经病理学和定量评估。溶剂组新皮层和纹状体的相对损伤体积分别扩大到40%和13.7%，而AS组则显著降低至4.8%和0.6%。在0.5 AS组中，新皮层和纹状体的缺血损伤相对体积分别降至20.5%和15.4%，0.5 AS组与溶剂组之间的差异在新皮层具有统计学意义，但在纹状体中无统计学意义。这些结果表明，体内敲低突触结合蛋白I的反义核酸成功减轻了新生大鼠的缺血性脑损伤，且该效应具有剂量依赖性。还表明这种治疗在新生大鼠的新皮层中比在纹状体中更有效。
