Herbst Eric A F, Holloway Graham P
Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
J Physiol. 2015 Feb 15;593(4):787-801. doi: 10.1113/jphysiol.2014.285379. Epub 2015 Jan 23.
Mitochondrial function in the brain is traditionally assessed through analysing respiration in isolated mitochondria, a technique that possesses significant tissue and time requirements while also disrupting the cooperative mitochondrial reticulum. We permeabilized brain tissue in situ to permit analysis of mitochondrial respiration with the native mitochondrial morphology intact, removing the need for isolation time and minimizing tissue requirements to ∼2 mg wet weight. The permeabilized brain technique was validated against the traditional method of isolated mitochondria and was then further applied to assess regional variation in the mouse brain with ischaemia-reperfusion injuries. A transgenic mouse model overexpressing catalase within mitochondria was applied to show the contribution of mitochondrial reactive oxygen species to ischaemia-reperfusion injuries in different brain regions. This technique enhances the accessibility of addressing physiological questions in small brain regions and in applying transgenic mouse models to assess mechanisms regulating mitochondrial function in health and disease.
Mitochondria function as the core energy providers in the brain and symptoms of neurodegenerative diseases are often attributed to their dysregulation. Assessing mitochondrial function is classically performed in isolated mitochondria; however, this process requires significant isolation time, demand for abundant tissue and disruption of the cooperative mitochondrial reticulum, all of which reduce reliability when attempting to assess in vivo mitochondrial bioenergetics. Here we introduce a method that advances the assessment of mitochondrial respiration in the brain by permeabilizing existing brain tissue to grant direct access to the mitochondrial reticulum in situ. The permeabilized brain preparation allows for instant analysis of mitochondrial function with unaltered mitochondrial morphology using significantly small sample sizes (∼2 mg), which permits the analysis of mitochondrial function in multiple subregions within a single mouse brain. Here this technique was applied to assess regional variation in brain mitochondrial function with acute ischaemia-reperfusion injuries and to determine the role of reactive oxygen species in exacerbating dysfunction through the application of a transgenic mouse model overexpressing catalase within mitochondria. Through creating accessibility to small regions for the investigation of mitochondrial function, the permeabilized brain preparation enhances the capacity for examining regional differences in mitochondrial regulation within the brain, as the majority of genetic models used for unique approaches exist in the mouse model.
传统上，通过分析分离的线粒体中的呼吸作用来评估大脑中的线粒体功能，该技术对组织和时间要求较高，同时还会破坏协作性的线粒体网状结构。我们对原位脑组织进行通透处理，以在完整保留天然线粒体形态的情况下分析线粒体呼吸作用，从而无需进行分离操作，将组织需求量降至约2毫克湿重。通透脑技术与传统的分离线粒体方法相比得到了验证，随后进一步应用于评估患有缺血再灌注损伤的小鼠大脑中的区域差异。应用一种在线粒体内过表达过氧化氢酶的转基因小鼠模型，以显示线粒体活性氧对不同脑区缺血再灌注损伤的作用。该技术提高了研究小脑区域生理问题以及应用转基因小鼠模型评估健康和疾病状态下调节线粒体功能机制的可行性。
线粒体是大脑中的核心能量供应者，神经退行性疾病的症状通常归因于其功能失调。经典的线粒体功能评估是在分离的线粒体中进行的；然而，这个过程需要大量的分离时间、对大量组织的需求以及对协作性线粒体网状结构的破坏，所有这些在试图评估体内线粒体生物能量学时都会降低可靠性。在这里，我们介绍一种方法，通过对现有的脑组织进行通透处理，使其能够直接原位接触线粒体网状结构，从而推进对大脑中线粒体呼吸作用的评估。通透脑制剂允许使用非常小的样本量（约2毫克）对线粒体功能进行即时分析，同时线粒体形态保持不变，这使得能够在单个小鼠大脑的多个亚区域中分析线粒体功能。在这里，该技术被应用于评估急性缺血再灌注损伤时脑线粒体功能的区域差异，并通过应用一种在线粒体内过表达过氧化氢酶的转基因小鼠模型来确定活性氧在加剧功能障碍中的作用。通过为研究线粒体功能创造对小区域的可及性，通透脑制剂增强了检查大脑中线粒体调节区域差异的能力，因为用于独特方法的大多数遗传模型都存在于小鼠模型中。
