Contusion spinal cord injury (SCI) is a mechanical insult to the spinal cord which results in immediate tissue damage followed by excitotoxicity, increased production of reactive oxygen species, and compromised mitochondrial function. Each of these contributes to secondary pathophysiological cascades which are responsible for increasing the spread and severity of the injured tissue. Importantly, the loss of mitochondrial function is implicated in most of the pathways in these cascades. As such, this proposal will determine whether supplementation with exogenous healthy mitochondria after SCI can attenuate secondary injury damage, possibly salvaging at-risk tissue that would have otherwise been compromised. Mitochondrial transplantation has been tested successfully in other tissue injuries, but never following SCI. In doing so, it may be possible to restore some functional recovery of the hind limbs over time after injury. A rat model of contusion SCI will be used to tes the overall hypothesis that supplementation of healthy mitochondria into an injured spinal cord will result in improved functional recovery via preservation of compromised tissues. In the first set of experiments, mitochondria labeled with turbo green fluorescent protein (tGFP) will be isolated from cultured cells and transplanted directly into the injured rat spinal cord at three different concentrations and two time points after injury. To test the hypothesis that mitochondrial supplementation restores cellular bioenergetics and reduces oxidative stress, molecular/biochemical outcome measures will be used, including mitochondrial respiration, calcium buffering capacity, and markers of oxidative damage or inflammation. A second experiment is designed to determine which cell types are incorporating the exogenous tGFP mitochondria, employing the injection concentration found to most closely maintain normal mitochondrial bioenergetics. Confocal microscopy of double and triple immunolabeling will allow visualization of cell-type colocalization with exogenous tGFP mitochondria, as well as establish their rostral-caudal distribution. Most critically, injections of isolated tGFP mitochondria after CI will be used to test the hypothesis that mitochondrial transplantation improves long-term hindlimb functional recovery after injury. The extent of hindlimb functional recovery will then be correlated with tissue sparing afforded by mitochondrial supplementation. Taken together, results from these experiments will show that healthy mitochondria can be injected into an injured spinal cord and integrate into host cells, thus increasing both mitochondrial function and cellular bioenergetics. Such maintenance of cellular homeostasis is expected to be correlated with increased tissue preservation and improved long-term hindlimb functional recovery. Once found to be a viable therapeutic, mitochondrial supplementation therapy may be implemented as a novel bioenergetics medicine in the clinic to treat individuals after SCI.
There are a reported 12,000 new cases of traumatic spinal cord injury (SCI) in the United States of America each year, resulting in lifelong debilitating consequences including paralysis, loss of sensation, compromised immune and respiratory systems, and depression. This project proposes that supplementing the acutely injured spinal cord with healthy mitochondria, the major energy producing organelle in cells, will rescue damaged tissue and prevent the injury from spreading in a rodent model of SCI. Translation of this therapy into the clinic may improve the potential for locomotor recovery and have a high impact on the long-term quality of life for individuals living with SCI.
|Gollihue, Jenna L; Rabchevsky, Alexander G (2017) Prospects for therapeutic mitochondrial transplantation. Mitochondrion 35:70-79|
|Gollihue, Jenna L; Patel, Samir P; Mashburn, Charlie et al. (2017) Optimization of mitochondrial isolation techniques for intraspinal transplantation procedures. J Neurosci Methods 287:1-12|