IP3R-mediated intra-axonal Ca2+ release contributes to secondary axonal degeneration following contusive spinal cord injury

Secondary axonal loss contributes to the persistent functional disability following trauma. Consequently, preserving axons following spinal cord injury (SCI) is a major therapeutic goal to improve neurological outcome; however, the complex molecular mechanisms that mediate secondary axonal degeneration remain unclear. We previously showed that IPR-mediated Ca release contributes to axonal dieback and axonal loss following an ex vivo laser-induced SCI. Nevertheless, targeting IPR in a clinically relevant in vivo model of SCI and determining its contribution to secondary axonal degeneration has yet to be explored. Here we used intravital two-photon excitation microscopy to assess the role of IPR in secondary axonal degeneration in real-time after a contusive-SCI in vivo. To visualize Ca changes specifically in spinal axons over time, adult 6-8 week-old triple transgenic Avil-Cre:Ai9:Ai95 (sensory neuron-specific expression of tdTomato and the genetic calcium indicator GCaMP6f) mice were subjected to a mild (30 kdyn) T12 contusive-SCI and received delayed treatment with the IPR blocker 2-APB (100 μM, intrathecal delivery at 3, and 24 h following injury) or vehicle control. To determine the IPR subtype involved, we knocked-down IPR3 using capped phosphodiester oligonucleotides. Delayed treatment with 2-APB significantly reduced axonal spheroids, increased axonal survival, and reduced intra-axonal Ca accumulation within dorsal column axons at 24 h following SCI in vivo. Additionally, knockdown of IPR3 yielded increased axon survival 24 h post-SCI. These results suggest that IPR-mediated Ca release contributes to secondary axonal degeneration in vivo following SCI.