Real Time Imaging of Mitochondrial Dynamics in the Dorsal Root Ganglion Following Nerve Injury

Abstract

Mitochondria are dynamic and motile organelles that respond rapidly to oxidative stress and cellular energy demands. Their morphological changes, as determined by fission and fusion, have been linked to the progression of neuropathic pain. However, little is known about specific pathology underlying mitochondrial dysfunction after peripheral nerve injury or how pharmacological interventions affect these changes. Our goal is to evaluate mitochondrial dynamics following peripheral nerve injury by characterizing axonal mitochondrial density, movement in axons and fusion-fission events. We dissociated dorsal root ganglia (DRG) from mice with spare nerve injury (SNI) and selectively labelled mitochondria with a live-cell fluorescent dye. Time lapse videos were then captured using a spinning disk confocal microscope. We observed a significant increase in axonal mitochondrial density in DRG cultures obtained from SNI mice when compared to naïve cultures. Representative kymograph images depict a larger proportion of mitochondria moving anterogradely in axons of DRG neurons from SNI mice. Interestingly, we found that the SNI-mitochondria are trafficked at lower velocities. Previous studies have shown that calcium overload in damaged mitochondria may contribute to decreased mitochondrial motility. Additionally, we observe a decrease in perinuclear clustering within the SNI group which may indicate a disruption in mitochondrial networking or a failure to regulate fission events in DRG neurons after injury. Collectively, our findings highlight the benefit of real time imaging of DRG neurons to gain greater insight into specific aspects of mitochondrial dysfunction after nerve injury. Our findings likely have important implications for therapeutic approaches to neuropathic pain. Supported by NIH grant NS065926.