Our previous studies reported that pharmacological maintenance of mitochondrial bioenergetics after experimental spinal cord injury (SCI) provided functional neuroprotection. Recent evidence indicates that endogenous mitochondrial transfer is neuroprotective as well, and, therefore, we extended these studies with a novel approach to transplanting exogenous mitochondria into the injured rat spinal cord. Using a rat model of L1/L2 contusion SCI, we herein report that transplantation of exogenous mitochondria derived from either cell culture or syngeneic leg muscle maintained acute bioenergetics of the injured spinal cord in a concentration-dependent manner. Moreover, transplanting transgenically labeled turbo green fluorescent (tGFP) PC12-derived mitochondria allowed for visualization of their incorporation in both a time-dependent and cell-specific manner at 24 h, 48 h, and 7 days post-injection. tGFP mitochondria co-localized with multiple resident cell types, although they were absent in neurons. Despite their contribution to the maintenance of normal bioenergetics, mitochondrial transplantation did not yield long-term functional neuroprotection as assessed by overall tissue sparing or recovery of motor and sensory functions. These experiments are the first to investigate mitochondrial transplantation as a therapeutic approach to treating spinal cord injury. Our initial bioenergetic results are encouraging, and although they did not translate into improved long-term outcome measures, caveats and technical hurdles are discussed that can be addressed in future studies to potentially increase long-term efficacy of transplantation strategies.

Mitochondrial dysfunction has been implicated in a multitude of diseases and pathological conditions- the organelles that are essential for life can also be major players in contributing to cell death and disease. Because mitochondria are so well established in our existence, being present in all cell types except for red blood cells and having the responsibility of providing most of our energy needs for survival, then dysfunctional mitochondria can elicit devastating cellular pathologies that can be widespread across the entire organism. As such, the field of “mitochondrial medicine” is emerging in which disease states are being targeted therapeutically at the level of the mitochondrion, including specific antioxidants, bioenergetic substrate additions, and membrane uncoupling agents. New and compelling research investigating novel techniques for mitochondrial transplantation to replace damaged or dysfunctional mitochondria with exogenous healthy mitochondria has shown promising results, including tissue sparing accompanied by increased energy production and decreased oxidative damage. Various experimental techniques have been attempted and each has been challenged to accomplish successful transplantation. The purpose of this review is to present the history of mitochondrial transplantation, the different techniques used for both in vitro and in vivo delivery, along with caveats and pitfalls that have been discovered along the way. Results from such pioneering studies are promising and could be the next big wave of “mitochondrial medicine” once technical hurdles are overcome.

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.

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Pioglitazone is an FDA-approved PPAR-γ agonist drug used to for treat diabetes, and it has demonstrated neuroprotective effects in multiple models of central nervous system (CNS) injury. Acute treatment after spinal cord injury (SCI) in rats is reported to suppress neuroinflammation, rescue injured tissues, and improve locomotor recovery. In the current study, we additionally assessed the protective efficacy of pioglitazone treatment on acute mitochondrial respiration, as well as functional and anatomical recovery after contusion SCI in adult male C57BL/6 mice. Mice received either vehicle or pioglitazone (10 mg/kg) at either 15 min or 3 hr after injury (75 kDyn at T9) followed by a booster at 24 hr post-injury. At 25 hr, mitochondria were isolated from spinal cord segments centered on the injury epicenters and assessed for their respiratory capacity. Results showed significantly compromised mitochondrial respiration 25 hr following SCI, but pioglitazone treatment that was initiated either at 15 min or 3 hr post-injury significantly maintained mitochondrial respiration rates near sham levels. A second cohort of injured mice received pioglitazone at 15 min post injury, then once a day for 5 days post-injury to assess locomotor recovery and tissue sparing over 4 weeks. Compared to vehicle, pioglitazone treatment resulted in significantly greater recovery of hind-limb function over time, as determined by serial locomotor BMS assessments and both terminal BMS subscores and gridwalk performance. Such improvements correlated with significantly increased grey and white matter tissue sparing, although pioglitazone treatment did not abrogate long-term injury-induced inflammatory microglia/macrophage responses. In sum, pioglitazone significantly increased functional neuroprotection that was associated with remarkable maintenance of acute mitochondrial bioenergetics after traumatic SCI. This sets the stage for dose-response and delayed administration studies to maximize pioglitazone’s efficacy for SCI while elucidating the precise role that mitochondria play in governing its neuroprotection; the ultimate goal to develop novel therapeutics that specifically target mitochondrial dysfunction.

Mitochondria are essential cellular organelles critical for generating adenosine triphosphate for cellular homeostasis, as well as various mechanisms that can lead to both necrosis and apoptosis. The field of “mitochondrial medicine” is emerging in which injury/disease states are targeted therapeutically at the level of the mitochondrion, including specific antioxidants, bioenergetic substrate additions, and membrane uncoupling agents. Consequently, novel mitochondrial transplantation strategies represent a potentially multifactorial therapy leading to increased adenosine triphosphate production, decreased oxidative stress, mitochondrial DNA replacement, improved bioenergetics and tissue sparing. Herein, we describe briefly the history of mitochondrial transplantation and the various techniques used for both in vitro and in vivo delivery, the benefits associated with successful transference into both peripheral and central nervous system tissues, along with caveats and pitfalls that hinder the advancements of this novel therapeutic.

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Autonomic dysreflexia (AD) is a potentially life-threatening syndrome in individuals with spinal cord injury (SCI) above the T6 spinal level that is characterized by episodic hypertension in response to noxious stimuli below the lesion. Maladaptive intraspinal plasticity is thought to contribute to the temporal development of AD, and experimental approaches that reduce such plasticity mitigate the severity of AD. The mammalian target of rapamycin (mTOR) has gained interest as a mediator of plasticity, regeneration, and nociceptor hypersensitivity in the injured spinal cord. Based on our preliminary data that prolonged rapamycin (RAP) treatment markedly reduces mTOR activity in the cord weeks after high-thoracic (T4) spinal transection, we sought to determine whether RAP could modulate AD development by impeding intraspinal plasticity. Naïve and injured rats were administered RAP or vehicle every other day, beginning immediately after injury for four weeks, and hemodynamic monitoring was conducted to analyze the frequency of spontaneously occurring AD, as well as the severity of colorectal distention (CRD) induced AD. Results showed that after SCI, RAP significantly exacerbated sustained body weight loss and caused a marked elevation in resting blood pressure, with average daily blood pressure rising above even normal naïve levels within one week after injury. Moreover, RAP significantly increased the frequency of daily spontaneous AD and increased the absolute blood pressure induced by CRD at three weeks post-injury. These dynamic cardiovascular effects were not, however, correlated with changes in the density of nociceptive c-fibers or c-Fos+ neurons throughout the spinal cord, indicating that intraspinal plasticity associated with AD was not altered by treatment. These findings caution against the use of RAP as a therapeutic intervention for SCI because it evokes toxic weight loss and exacerbates cardiovascular dysfunction perhaps mediated by increased peripheral nociceptor sensitivity and/or vascular resistance.