Abstract
Background—Proper mitochondrial function is essential to maintain normal cellular bioenergetics and ionic homeostasis. In instances of severe tissue damage, such as traumatic brain and spinal cord injury, mitochondria become damaged and unregulated leading to cell death. The relatively unexplored field of mitochondrial transplantation following neurotrauma is based on the theory that replacing damaged mitochondria with exogenous respiratory-competent mitochondria can restore overall tissue bioenergetics.
New Method—We optimized techniques in vitro to prepare suspensions of isolated mitochondria for transplantation in vivo. Mitochondria isolated from cell culture were genetically labeled with turbo-green fluorescent protein (tGFP) for imaging and tracking purposes in vitro and in vivo.
Results—We used time-lapse confocal imaging to reveal the incorporation of exogenous fluorescently-tagged mitochondria into PC-12 cells after brief co-incubation. Further, we show that mitochondria can be injected into the spinal cord with immunohistochemical evidence of host cellular uptake within 24 hours.
Comparison to Existing Methods—Our methods utilize transgenic fluorescent labeling of mitochondria for a nontoxic and photostable alternative to other labeling methods. Substrate addition to isolated mitochondria helped to restore state III respiration at room temperature prior to transplantation. These experiments delineate refined methods to use transgenic cell lines for the purpose of isolating well coupled mitochondria that have a permanent fluorescent label that allows real time tracking of transplanted mitochondria in vitro, as well as imaging in situ.
Conclusions—These techniques lay the foundation for testing the potential therapeutic effects of mitochondrial transplantation following spinal cord injury and other animal models of neurotrauma.
Document Type
Article
Publication Date
8-1-2017
Digital Object Identifier (DOI)
https://doi.org/10.1016/j.jneumeth.2017.05.023
Funding Information
This work was supported by NIH/NINDS T32 Training Grant 5T32 NS077889 (JLG), NIH/NINDS F31NS093904-01A1 (JLG), Conquer Paralysis Now “Out-of-Box” Award, NIH/NINDS R21NS096670 (AGR), University of Kentucky Spinal Cord and Brain Injury Center Chair Endowment (AGR), NIH/NINDS 2P30NS051220.
Related Content
Refer to Web version on PubMed Central for supplementary material.
Repository Citation
Gollihue, Jenna L.; Patel, Samir P.; Mashburn, Charles B.; Eldahan, Khalid C.; Sullivan, Patrick G.; and Rabchevsky, Alexander G., "Optimization of Mitochondrial Isolation Techniques for Intraspinal Transplantation Procedures" (2017). Physiology Faculty Publications. 138.
https://uknowledge.uky.edu/physiology_facpub/138
Supplemental Video
Notes/Citation Information
Published in Journal of Neuroscience Methods, v. 287, p. 1-12.
© 2017 Elsevier B.V. All rights reserved.
This manuscript version is made available under the CC‐BY‐NC‐ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/.
The document available for download is the author's post-peer-review final draft of the article.