An isolation method for assessment of brain mitochondria function in neonatal mice with hypoxic-ischemic brain injury
An isolation method for assessment of brain mitochondria function in neonatal mice with hypoxic-ischemic brain injury
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DOI：
10.1159/000121416
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发表时间：
2008-01-01
影响因子：
2.9
通讯作者：
Ten, Vadim S.
中科院分区：
文献类型：
--
作者：
Caspersen, Casper S.;Sosunov, Alexander;Ten, Vadim S.
这项工作的目的是开发一种从新生小鼠的单个大脑半球中分离线粒体的方法。通过所提出的方法分离的正常小鼠大脑半球的线粒体在谷氨酸-苹果酸诱导的磷酸化呼吸过程中表现出良好的呼吸控制比，为 6.39 +/- 0.53。电子显微镜显示线粒体完整。该方法的适用性在从遭受缺氧缺血损伤的幼鼠及其同窝小鼠中分离的线粒体上进行了测试。与幼崽相比，再灌注前的缺氧缺血性损伤导致磷酸化呼吸的显着抑制（p < 0.01）。这与缺血半球中 ATP 含量的严重消耗有关。通过蛋白质印迹法测定锰超氧化物歧化酶和细胞色素 C（线粒体基质和外膜完整性的标记）的表达，以控制比较样品中线粒体的完整性和数量。因此，我们开发了一种从适用于新生小鼠的单个半球中分离脑线粒体的方法。该方法可能作为研究未成熟脑损伤小鼠模型中线粒体功能的有价值的工具。此外，所提出的方法使我们能够检查具有靶向遗传改变的未成熟小鼠的线粒体功能表型。版权所有 (C) 2008 S. Karger AG，巴塞尔。
This work was undertaken to develop a method for the isolation of mitochondria from a single cerebral hemisphere in neonatal mice. Mitochondria from the normal mouse brain hemisphere isolated by the proposed method exhibited a good respiratory control ratio of 6.39 +/- 0.53 during glutamate-malate-induced phosphorylating respiration. Electron microscopy showed intact mitochondria. The applicability of this method was tested on mitochondria isolated from naive mice and their littermates subjected to hypoxic-ischemic insult. Hypoxic-ischemic insult prior to reperfusion resulted in a significant (p < 0.01) inhibition of phosphorylating respiration compared to naive littermates. This was associated with a profound depletion of the ATP content in the ischemic hemisphere. The expression for Mn superoxide dismutase and cytochrome C (markers for the integrity of the mitochondrial matrix and outer membrane) was determined by Western blot to control for mitochondrial integrity and quantity in the compared samples. Thus, we have developed a method for the isolation of the cerebral mitochondria from a single hemisphere adapted to neonatal mice. This method may serve as a valuable tool to study mitochondrial function in a mouse model of immature brain injury. In addition, the suggested method enables us to examine the mitochondrial functional phenotype in immature mice with a targeted genetic alteration. Copyright (C) 2008 S. Karger AG, Basel.