Intravital Oxygen Radical Imaging in Normal and Ischemic Rat Cortex
Intravital Oxygen Radical Imaging in Normal and Ischemic Rat Cortex
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正常和缺血大鼠皮层的活体氧自由基成像
DOI：
10.1227/01.neu.0000370055.99998.6b
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发表时间：
2010-07-01
期刊：
影响因子：
4.8
通讯作者：
Terakawa, Susumu
中科院分区：
文献类型：
--
作者：
Wang, Yong;Yamamoto, Seiji;Terakawa, Susumu
目的：应用活体荧光成像技术检测脑缺血再灌流后氧自由基(ROS)的产生。方法：在麻醉成年大鼠大脑皮质内加压注射超氧阴离子自由基(中心点O-2(-))的荧光染料L(5mU)、超氧阴离子自由基(中心点O-2(-))的丝裂霉素(MitoSOX)和羟基自由基(中心点OH)的羟苯基荧光素(20mU/L)。通过闭合的颅窗，用共聚焦显微镜拍摄前脑缺血10分钟的荧光图像。由于血红蛋白吸收激发光和发射光，缺血可能会影响大脑内部荧光强度(FI)的变化。为了检测缺血对FI变化的影响，使用直径为0.2微米的荧光微球模拟一种染料，并以与使用ROS指示剂相同的方式分析FI。结果：校正MitoSOX和羟苯基荧光素的FI后，缺血期ROS指标的FI无明显变化，而原始数据显示ROS指示剂的FI有所增加。在再灌注早期，FI显著增加(n=5/个，P<0.01)(MitoSOX为183%，羟苯基荧光素为189%)，且在动脉邻近区域显著增加。为了测试我们成像的可行性，我们使用了依达拉奉(3.0 mg/kg)。结论：再灌流早期ROS生成增加，而缺血时ROS生成无明显变化，表现为部位选择性，且在邻近动脉区域表现明显。我们的方法对于研究细胞内原位ROS的产生是有用的。
OBJECTIVE: We examined reactive oxygen species (ROS) generation on cerebral ischemia/reperfusion by intravital fluorescence imaging.METHODS: In anesthetized adult rats, a fluorescent dye (5 mu L), MitoSOX (5 mu mol/L) for superoxide radical (center dot O-2(-)), and hydroxyphenyl fluorescein (20 mu mol/L) for hydroxyl radical (center dot OH), was injected into cortices by a pressurized bolus. Through a closed cranial window, fluorescent images were taken with a confocal microscope on 10-minute forebrain ischemia. Because hemoglobin absorbs excitation and emission lights, ischemia may affect the change in fluorescence intensity (FI) inside the brain. To examine the effects of ischemia on the FI change, fluoromicrospheres (0.2-mu m diameter) were used to mimic a dye and FI was analyzed in the same manner as when using ROS indicators. Their FI increased to 129% during ischemia (n = 3/mimicking each dye), and based on the results, FI of ROS indicators was corrected.RESULTS: After correcting the FI of MitoSOX and hydroxyphenyl fluorescein, they showed no change during ischemia, whereas the raw data showed the increase. In the early period of reperfusion, FI significantly (n = 5/each, P < .01) increased (to 183% in MitoSOX and to 189% in hydroxyphenyl fluorescein), and these increases were significant in the areas adjacent to the arteries. To test the feasibility of our imaging, edaravone (3.0 mg/kg) was used. The treatment completely scavenged center dot OH, but did not do so in center dot O-2(-) generation.CONCLUSION: ROS production increased in the early period of reperfusion but not during ischemia, which was location selective, being significant in the areas adjacent to the arteries. Our method was useful for investigating intracellular in situ ROS production.