Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada.
University of Ottawa Brain and Mind Research Institute, University of Ottawa, Ottawa, ON, Canada.
Methods Mol Biol. 2022;2515:1-15. doi: 10.1007/978-1-0716-2409-8_1.
Defects in mitochondrial oxidative phosphorylation have been observed in numerous neurodegenerative disorders and are linked to bioenergetic crises leading to neuronal death. The distinct metabolic profile of neurons is predominantly oxidative, which is characterized by the oxidation of glucose or its metabolites in the mitochondria to produce ATP. This process involves the tricarboxylic acid cycle, electron transfer in the respiratory chain, and oxygen consumption. Therefore, measurement of oxygen consumption rates (OCR) can be accurately applied to assess the rate of mitochondrial respiration. In this chapter, we describe our optimized protocol for the assessment of OCR specifically in primary mouse cerebellar granule neurons (CGN). The protocol includes isolation and manipulation of mouse CGNs followed by real-time assessment of mitochondrial OCR using a Seahorse XFe96 extracellular flux analyzer.
在线粒体氧化磷酸化中存在的缺陷在许多神经退行性疾病中都有观察到，并且与导致神经元死亡的生物能量危机有关。神经元的独特代谢特征主要是氧化，其特征在于葡萄糖或其代谢物在线粒体中氧化以产生 ATP。这个过程涉及三羧酸循环、呼吸链中的电子转移和耗氧量。因此，测量耗氧量（OCR）可以准确地用于评估线粒体呼吸的速率。在本章中，我们描述了我们优化的用于评估原代小鼠小脑颗粒神经元（CGN）中 OCR 的方案。该方案包括小鼠 CGN 的分离和操作，然后使用 Seahorse XFe96 细胞外通量分析仪实时评估线粒体 OCR。
