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.
Document Type
Article
Publication Date
1-1-2024
Digital Object Identifier (DOI)
https://doi.org/10.3389/fmolb.2024.1378536
Funding Information
The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by VA Merit Award 2I01BX003405 (PS) and Kentucky Spinal Cord and Head Injury Research Trust #20-7A (PS), NIH R01 NS112693-01A1 (PS). This publication was made possible by the University of Kentucky CNS Metabolism (CNS-Met) COBRE, supported by a grant from the National Institute of General Medical Sciences–NIGMS (P20 GM148326) from the National Institutes of Health.
Repository Citation
Vekaria, Hemendra J; Kalimon, Olivia J.; Prajapati, Paresh; Velmurugan, Gopal V.; and Sullivan, Patrick G, "An efficient and high-throughput method for the evaluation of mitochondrial dysfunction in frozen brain samples after traumatic brain injury." (2024). Neuroscience Faculty Publications. 84.
https://uknowledge.uky.edu/neurobio_facpub/84
Notes/Citation Information
© 2024 Vekaria, Kalimon, Prajapati, Velmurugan and Sullivan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.