扩增去极化相关神经元 ROS 生成的定量实时成像。

IF 4.2 3区 医学 Q2 NEUROSCIENCES
Frontiers in Cellular Neuroscience Pub Date : 2024-10-11 eCollection Date: 2024-01-01 DOI:10.3389/fncel.2024.1465531
Marc André Ackermann, Susanne Monika Buchholz, Katharina Dietrich, Michael Müller
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引用次数: 0

摘要

扩散性去极化(SD)会导致神经元/神经胶质细胞大量去极化,扰乱离子平衡并破坏神经元网络功能。SD造成的代谢负担还可能产生大量活性氧(ROS)。然而,要研究这方面的时空细节,需要适当的光学工具。因此,我们早些时候产生了转基因氧化还原指示剂小鼠。它们在兴奋性投射神经元中表达细胞膜氧化还原传感器roGFP,这是一种对还原/氧化敏感的绿色荧光蛋白,可通过激发进行比率测量,并对氧化还原改变做出可逆反应。我们利用成年雄性roGFPc小鼠分析了浸没切片CA1金字塔层中与SD相关的ROS产生情况。SD是由K+微注射、缺氧或线粒体解偶联(FCCP)诱导的。细胞外直流电位偏转伴随着roGFP氧化的扩散波阵面,证实了明显的神经元ROS生成。在缺氧诱导 SD 之前,会出现中度氧化,随着直流电位发生偏转,氧化会加剧。K+ 诱导 SD 后,一些切片的 roGFP 氧化在 10-15 分钟内缓慢恢复。在 FCCP 或缺氧诱导的 SD 中,恢复是有限的。撤出细胞外 Ca2+ 能明显抑制 SD 相关的 roGFP 氧化,并改善其可逆性,这证实了神经元 Ca2+ 负荷在 SD 相关 ROS 生成中的关键作用。线粒体解偶联、抑制 NADPH 氧化酶或黄嘌呤氧化酶都不能抑制 SD 相关的 roGFP 氧化。因此,SD 过程中 ROS 的产生既涉及线粒体,也涉及非线粒体来源。这种基于roGFP小鼠定量氧化还原成像的方法首次实现了对SD相关ROS动态的分析,是一种先进的方法,将有助于进一步破译SD在脑病理生理学中的分子认识。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Quantitative, real-time imaging of spreading depolarization-associated neuronal ROS production.

Spreading depolarization (SD) causes a massive neuronal/glial depolarization, disturbs ionic homeostasis and deranges neuronal network function. The metabolic burden imposed by SD may also generate marked amounts of reactive oxygen species (ROS). Yet, proper optical tools are required to study this aspect with spatiotemporal detail. Therefore, we earlier generated transgenic redox indicator mice. They express in excitatory projection neurons the cytosolic redox-sensor roGFP, a reduction/oxidation sensitive green fluorescent protein which is ratiometric by excitation and responds reversibly to redox alterations. Using adult male roGFPc mice, we analyzed SD-related ROS production in CA1 stratum pyramidale of submerged slices. SD was induced by K+ microinjection, O2 withdrawal or mitochondrial uncoupling (FCCP). The extracellular DC potential deflection was accompanied by a spreading wavefront of roGFP oxidation, confirming marked neuronal ROS generation. Hypoxia-induced SD was preceded by a moderate oxidation, which became intensified as the DC potential deflection occurred. Upon K+-induced SD, roGFP oxidation slowly recovered within 10-15 min in some slices. Upon FCCP-or hypoxia-induced SD, recovery was limited. Withdrawing extracellular Ca2+ markedly dampened the SD-related roGFP oxidation and improved its reversibility, confirming a key-role of neuronal Ca2+ load in SD-related ROS generation. Neither mitochondrial uncoupling, nor inhibition of NADPH oxidase or xanthine oxidase abolished the SD-related roGFP oxidation. Therefore, ROS generation during SD involves mitochondria as well as non-mitochondrial sources. This first-time analysis of SD-related ROS dynamics became possible based on quantitative redox imaging in roGFP mice, an advanced approach, which will contribute to further decipher the molecular understanding of SD in brain pathophysiology.

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来源期刊
CiteScore
7.90
自引率
3.80%
发文量
627
审稿时长
6-12 weeks
期刊介绍: Frontiers in Cellular Neuroscience is a leading journal in its field, publishing rigorously peer-reviewed research that advances our understanding of the cellular mechanisms underlying cell function in the nervous system across all species. Specialty Chief Editors Egidio D‘Angelo at the University of Pavia and Christian Hansel at the University of Chicago are supported by an outstanding Editorial Board of international researchers. This multidisciplinary open-access journal is at the forefront of disseminating and communicating scientific knowledge and impactful discoveries to researchers, academics, clinicians and the public worldwide.
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