Vicente-Gutierrez Carlos, Bolaños Juan P
Institute of Functional Biology and Genomics (IBFG), University of Salamanca, CSIC, Salamanca, Spain.
Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
Bio Protoc. 2020 Mar 20;10(6):e3550. doi: 10.21769/BioProtoc.3550.
Mitochondrial reactive oxygen species (mROS) are naturally produced signalling molecules extremely relevant for understanding both health- and disease-associated biological processes. The study of mROS in the brain is currently underway to decipher their physiopathological roles and contributions in neurological diseases. Recent advances in this field have highlighted the importance of studying mROS signalling and redox biology at the cellular level. Neurons are especially sensitive to the harmful effects of excess mROS while astrocytic mROS have been shown to play a relevant physiological role in cerebral homeostasis and behaviour. However, given the complexity of the brain, investigating mROS formation in a specific cell-type in adult animals is methodologically challenging. Here we propose an approach to specifically assess mROS abundance in astrocytes that combines i) a targeting strategy based on the use of adeno-associated virus (AAV) vectors expressing the green fluorescent protein (GFP) under an astrocyte (glial fibrillary acidic protein or GFAP) promoter, along with, ii) a robust and widely extended protocol for the measurement of mROS by flow cytometry using commercial probes. The significance of this work is that it allows the selective study of astrocytic mROS abundance by means of easily accessible technology.
线粒体活性氧（mROS）是天然产生的信号分子，对于理解与健康和疾病相关的生物过程极为重要。目前正在进行关于大脑中mROS的研究，以破译它们在神经疾病中的生理病理作用和贡献。该领域的最新进展突出了在细胞水平研究mROS信号传导和氧化还原生物学的重要性。神经元对过量mROS的有害影响特别敏感，而星形胶质细胞的mROS已被证明在脑内稳态和行为中发挥相关的生理作用。然而，鉴于大脑的复杂性，在成年动物的特定细胞类型中研究mROS的形成在方法上具有挑战性。在这里，我们提出了一种专门评估星形胶质细胞中mROS丰度的方法，该方法结合了：i）一种靶向策略，基于使用在星形胶质细胞（胶质纤维酸性蛋白或GFAP）启动子下表达绿色荧光蛋白（GFP）的腺相关病毒（AAV）载体；以及ii）一种使用商业探针通过流式细胞术测量mROS的强大且广泛应用的方案。这项工作的意义在于，它允许通过易于获取的技术对星形胶质细胞的mROS丰度进行选择性研究。
