Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy
Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy
复制标题
使用共焦激光显微镜对活体大鼠视神经有髓轴突进行体外钙成像
DOI：
10.1016/s0165-0270(00)00304-6
复制
发表时间：
2000
影响因子：
3
通讯作者：
P. Stys
中科院分区：
文献类型：
--
作者：
Yubo Ren;A. Ridsdale;E. Coderre;P. Stys
细胞内Ca 2+在可兴奋细胞的生理反应中起主要作用，并且内部Ca 2+的过度积累是细胞损伤和死亡的关键决定因素。人们对神经元内部Ca 2+动力学进行了许多研究。在神经再生中，几乎没有哺乳动物中央有髓轴突的这种信息，这在很大程度上是由于染料加载和成像这种精细的有髓结构的技术困难。我们开发了一种技术，允许在37°C下使用共聚焦显微镜在体外对活体大鼠视神经轴突中的离子化Ca 2+进行成像，同时进行电生理记录。使用低Ca 2 +/低Na+溶液将Ca 2+敏感指示剂俄勒冈州绿色488 BAPTA-2的K+盐和Ca 2+不敏感参比染料磺酰罗丹明101一起加载到大鼠视神经中。用神经丝-160抗体双重染色的免疫化学方法证实的轴突轮廓，通过S101荧光从切割末端清楚地观察到800 μm。Ca 2+信号在静止时非常低，仅高于背景荧光强度，表明健康组织，并且在旨在释放内部Ca 2+储存的咖啡因（20 mM）暴露后显著增加。成像区域的健康进一步证实了血影蛋白分解的虚拟缺乏，这是由受损CNS组织中的钙蛋白酶激活引起的。在37°C下记录60 min后，红色和绿色荧光衰减至不低于对照的70%，60 min后绿色：红色荧光比率略微增加21%。与共聚焦图像同时记录的电生理反应也基本保持稳定。
Intracellular Ca2+plays a major role in the physiological responses of excitable cells, and excessive accumulation of internal Ca2+is a key determinant of cell injury and death. Many studies have been carried out on the internal Ca2+dynamics in neurons. In constrast, there is virtually no such information for mammalian central myelinated axons, due in large part to technical difficulty with dye loading and imaging such fine myelinated structures. We developed a technique to allow imaging of ionized Ca2+in live rat optic nerve axons with simultaneous electrophysiological recording in vitro at 37°C using confocal microscopy. The K+salt of the Ca2+-sensitive indicator Oregon Green 488 BAPTA-2 and the Ca2+-insensitive reference dye Sulforhodamine 101 were loaded together into rat optic nerves using a low-Ca2+/low-Na+solution. Axonal profiles, confirmed immunohistochemically by double staining with neurofilament-160 antibodies, were clearly visualized by S101 fluorescence up to 800 μm from the cut ends. The Ca2+signal was very low at rest, just above the background fluorescence intensity, indicating healthy tissue, and increased significantly after caffeine (20 mM) exposure designed to release internal Ca2+stores. The health of imaged regions was further confirmed by a virtual absence of spectrin breakdown, which is induced by calpain activation in damaged CNS tissue. Red and green fluorescence decayed to no less than 70% of control after 60 min of recording at 37°C, with the green:red fluorescence ratio increasing slightly by 21% after 60 min. Electrophysiological responses recorded simultaneously with confocal images remained largely stable as well.
缺氧期间中枢神经系统白质的药理学保护：苯妥英、卡马西平和地西泮的作用。
DOI：
--
发表时间：
1993
期刊：
The Journal of pharmacology and experimental therapeutics
影响因子：
--
作者：
Fern,R;Ransom,BR;Stys,PK;Waxman,SG
通讯作者：
Waxman,SG
影响因子：
3.4
作者：
VANEGERAAT, JM;STASASKI, R;WIKSWO, JP
通讯作者：
WIKSWO, JP
影响因子：
4
作者：
ROE, MW;LEMASTERS, JJ;HERMAN, B
通讯作者：
HERMAN, B