Autologous Mitochondrial Replacement Strategies to Promote Recovery after Spinal Trauma

We have found that pharmacologically maintaining normal mitochondrial bioenergetics after traumatic spinal cord injury (SCI) is an important factor in fostering both neuroprotection and functional recovery. However, no studies have examined the effects of transplanting exogenous mitochondria into spinal cord tissue after traumatic injury in an attempt to normalize overall cellular bioenergetics and spare adjacent tissues. Accordingly, our novel proposal will test whether supplementing exogenous healthy mitochondria, isolated from various exogenous or endogenous sources to be tested, into the injured spinal cord promotes functional neuroprotection. Using a rat model of severe contusion SCI, we have preliminary data showing that allogeneic transgenically-labeled (turboGFP) mitochondria microinjected around the injury site increases overall mitochondrial bioenergetics of the injured cord assessed 24 hrs later. Moreover, using specific antibodies to both inner and outer mitochondrial membranes, confocal imaging also reveals that grafted turboGFP mitochondria integrate within naïve and injured host cells. Based on these seminal findings, we propose to conduct a series of transplantation strategies to deliver mitochondria around the injury sites with the ultimate goal of identifying potential autologous cell sources to prevent secondary tissue injury that is mediated, in large part, by mitochondrial dysfunction. This project has translational potential wherein mitochondria, notably autologous, can be transplanted around the SCI site to spare penumbral tissues. Specific Aim 1- Comparatively assess whether transplanted mitochondria isolated from muscles are equally effective as turboGFP mitochondria on cellular bioenergetics and inflammatory markers following acute contusion SCI A total of 96 rats will be used in this aim to compare turboGFP versus muscle-derived mitochondria labeled with mitotracker green FM. There will be 4 treatment groups - 1) Sham Laminectomy, 2) Sham Laminectomy + Mitochondria injections, 3) Contusion + Vehicle injections and 4) Contusion + Mitochondria injections [2 labeled mitochondrial cell sources x (N=8/group for bioenergetics and N=4/group for histology)]. Rats will undergo an L1/L2 contusion SCI and immediately after injury, mitochondria will be injected into four injection sites surrounding the injury. At 24 hours post injury, spinal cord homogenates will be used to analyze oxidative stress and inflammatory markers, while isolated total mitochondria will be used to determine mitochondrial respiration. Others will be perfused for immunohistochemistry and confocal microscopy to verify grafted mitochondrial integration within host cells. Specific Aim 2- Establish whether transplantation of autologous mitochondria increase tissue sparing and hindlimb locomotor recovery following contusion SCI A total of 20 rats will be separated into two groups 1) Mitochondria Injected and 2) Vehicle injected. Total number of animals proposed = 20 (i.e. n=10/group). An important caveat is that this aim is based on our belief that muscle-derived mitochondria will be beneficial. If the results do not support the use of this cellular source, then we will employ a cell-line mediated transfer approach based on our preliminary data. Rats will be injured as in Aim 1, followed immediately by injection of mitochondria and survive for 4 weeks while undergoing weekly testing to evaluate hindlimb overground locomotion and terminal gait analyses. Afterwards all animals will be transcardially perfused with paraformaldehyde and the tissue will be processed for histology. Taken together, these aims will show whether autologous mitochondrial transplantation is a viable method for increasing functional neuroprotection after SCI. Whether transplanted mitochondria elicit immune responses will be equally important seminal studies to establish potential immunogenicity. Mitochondrial integrity is vital in maintaining healthy tissue after injury and has been targeted using different drug and anti-oxidant approaches following SCI. If successful, this experiment could pave the way for a novel therapeutic approach for treating acute SCI via mitochondrial supplementation strategies.