PDF(Pubmed)
Abstract：
Mitochondrial function analysis is a well-established method used in preclinical and clinical investigations to assess pathophysiological changes in various disease states, including traumatic brain injury (TBI). Although there are multiple approaches to assess mitochondrial function, one common method involves respirometric assays utilizing either Clark-type oxygen electrodes or fluorescent-based Seahorse analysis (Agilent). However, these functional analysis methods are typically limited to the availability of freshly isolated tissue samples due to the compromise of the electron transport chain (ETC) upon storage, caused by freeze-thaw-mediated breakdown of mitochondrial membranes. In this study, we propose and refine a method for evaluating electron flux through the ETC, encompassing complexes I, II, and IV, in frozen homogenates or mitochondrial samples within a single well of a Seahorse plate. Initially, we demonstrate the impact of TBI on freshly isolated mitochondria using the conventional oxidative phosphorylation protocol (OxPP), followed by a comparison with ETC analysis conducted on frozen tissue samples within the context of a controlled cortical impact (CCI) model of TBI. Additionally, we explore the effects of mitochondrial isolation from fresh versus snap-frozen brain tissues and their storage at -80°C, assessing its impact on electron transport chain protocol (ETCP) activity. Our findings indicate that while both sets of samples were frozen at a single time point, mitochondria from snap-frozen tissues exhibited reduced injury effects compared to preparations from fresh tissues, which were either homogenized or isolated into mitochondria and subsequently frozen for later use. Thus, we demonstrate that the preparation of homogenates or isolated mitochondria can serve as an appropriate method for storing brain samples, allowing for later analysis of mitochondrial function, following TBI using ETCP.
摘要：
线粒体功能分析是一种公认的方法，用于临床前和临床研究，以评估各种疾病状态的病理生理变化。包括创伤性脑损伤（TBI）。虽然有多种方法来评估线粒体功能，一种常见的方法涉及利用Clark型氧电极或基于荧光的海马分析(Agilent)的呼吸测定。然而,由于储存时电子传递链（ETC）的折衷，这些功能分析方法通常仅限于新鲜分离的组织样品的可用性，由冻融介导的线粒体膜分解引起。在这项研究中,我们提出并完善了一种通过ETC评估电子通量的方法，包括配合物I，II,IV,在海马板的单个孔中的冷冻匀浆或线粒体样品中。最初，我们使用常规氧化磷酸化方案（OxPP）证明了TBI对新鲜分离的线粒体的影响，然后与在TBI的受控皮质冲击（CCI）模型的背景下对冷冻组织样品进行的ETC分析进行比较。此外,我们探索了从新鲜的和速冻的脑组织中分离线粒体的效果，以及它们在-80°C下的储存，评估其对电子传递链协议(ETCP)活动的影响。我们的发现表明，虽然两组样品都在一个时间点冷冻，与来自新鲜组织的制剂相比，来自速冻组织的线粒体表现出减少的损伤作用，将其均质化或分离到线粒体中，然后冷冻以备后用。因此，我们证明，匀浆或分离的线粒体的制备可以作为存储脑样本的适当方法，允许以后分析线粒体功能，以下使用ETCP的TBI。
