NEUROPROTECTION AFTER TRAUMATIC SPINAL CORD INJURY THROUGH MITOCHONDRIAL CALCIUM UNIPORTER INHIBITION

Abstract

A common final pathway of secondary spinal cord injury (sSCI) is glutamate excitotoxicity, manifested by calcium (Ca2+) overloading of the neuron. Mitochondria are damaged by excessive intake of cytoplasmic Ca2+ through the mitochondrial calcium uniporter (MCU). When this occurs, the mitochondria become overwhelmed, collapse, and trigger neuronal cell death. We hypothesized that MCU inhibition by Ru265 will improve recovery after traumatic spinal cord injury (tSCI) by preserving mitochondrial integrity and function essential for axonal survival and repair. We demonstrated that intraperitoneal (IP) delivery at a dose previously shown to be neuroprotective in a stroke model (3 mg/kg) promotes seizure activity in mice, prompting us to investigate more localized methods of drug delivery and a lower total dose. We compared single epidural and IP doses of Ru265 (1 mg/kg; non seizure inducing dose, determined empirically), in mice immediately after a tSCI. Mice were sampled 4, 8 and 24 hours afterwards and Ru265 levels within the spinal parenchyma, forebrain, and whole blood determined using mass spectrometry (MS). MS data supports that Ru265 reached higher concentrations in the spinal parenchyma with epidural application (over the lesion), than via IP injection. This effect was also time-dependent with the drug concentration increasing with time. Epidural application was determined to be a feasible and effective method of Ru265 delivery. Following this, mitochondrial integrity of Ru265 treated and control mice was evaluated with transmission electron microscopy. Evaluation of the structural integrity of mitochondria following tSCI revealed that there were significantly more healthy mitochondria (p<0.0001) and significantly fewer damaged mitochondria (p<0.0001) in the Ru265 treated sections compared to control specimens. To determine if this increase in mitochondrial preservation translated to improved neuronal survival, Fluoro-Jade C, a fluorescent label for degenerating neurons was used. Samples treated with Ru265 had significantly fewer degenerating neurons compared to controls (p=0.009) suggesting that Ru265 may improve neuronal survival following tSCI. These findings support that Ru265 provides mitoprotection and as a likely result, neuroprotection following tSCI. To our knowledge this is the first study demonstrating the benefits of MCU inhibition in the context of SCI.