Jenna L Gollihue1, Samir P Patel1, Charlie Mashburn2, Khalid C Eldahan1, Patrick G Sullivan3, Alexander G Rabchevsky4. 1. University of Kentucky, Department of Physiology, Lexington, KY 40536-0509, United States; University of Kentucky, Spinal Cord & Brain Injury Research Center, Lexington, KY 40536-0509, United States. 2. University of Kentucky, Spinal Cord & Brain Injury Research Center, Lexington, KY 40536-0509, United States. 3. University of Kentucky, Department of Neuroscience, Lexington, KY 40536-0509, United States; University of Kentucky, Spinal Cord & Brain Injury Research Center, Lexington, KY 40536-0509, United States. 4. University of Kentucky, Department of Physiology, Lexington, KY 40536-0509, United States; University of Kentucky, Spinal Cord & Brain Injury Research Center, Lexington, KY 40536-0509, United States. Electronic address: agrab@uky.edu.
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 24h. 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.
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 24h. 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.
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