Vekaria Hemendra J, Kalimon Olivia J, Prajapati Paresh, Velmurugan Gopal V, Sullivan Patrick G
Spinal Cord and Brain Injury Research Center, College of Medicine, University of Kentucky, Lexington, KY, United States.
Lexington VA Medical Center, United States Department of Veterans Affairs, Lexington, KY, United States.
Front Mol Biosci. 2024 Jun 5;11:1378536. doi: 10.3389/fmolb.2024.1378536. eCollection 2024.
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 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.
线粒体功能分析是一种在临床前和临床研究中广泛应用的成熟方法，用于评估包括创伤性脑损伤（TBI）在内的各种疾病状态下的病理生理变化。尽管有多种评估线粒体功能的方法，但一种常见的方法是使用克拉克型氧电极或基于荧光的海马分析（安捷伦）进行呼吸测定。然而，由于冻融介导的线粒体膜破裂导致电子传递链（ETC）在储存时受损，这些功能分析方法通常限于新鲜分离的组织样本的可用性。在本研究中，我们提出并完善了一种在海马板的单个孔内评估冷冻匀浆或线粒体样本中通过ETC（包括复合物I、II和IV）的电子通量的方法。最初，我们使用传统的氧化磷酸化方案（OxPP）证明了TBI对新鲜分离的线粒体的影响，随后在TBI的可控皮质撞击（CCI）模型背景下，将其与对冷冻组织样本进行的ETC分析进行比较。此外，我们探讨了从新鲜速冻脑组织中分离线粒体及其在-80°C储存的影响，评估其对电子传递链方案（ETCP）活性的影响。我们的研究结果表明，虽然两组样本都在单个时间点冷冻，但与新鲜组织制备的样本（无论是匀浆还是分离成线粒体并随后冷冻以供后续使用）相比，速冻组织中的线粒体表现出降低的损伤效应。因此，我们证明了匀浆或分离线粒体的制备可以作为储存脑样本的合适方法，允许在TBI后使用ETCP对线粒体功能进行后续分析。
