Repeat intravital imaging of the murine spinal cord reveals degenerative and reparative responses of spinal axons in real-time following a contusive SCI.

Spinal cord injury (SCI) induces a secondary degenerative response that causes the loss of spared axons and worsens neurological outcome. The complex molecular mechanisms that mediate secondary axonal degeneration remain poorly understood. To further our understanding of secondary axonal degeneration following SCI, we assessed the spatiotemporal dynamics of axonal spheroid and terminal bulb formation following a contusive SCI in real-time in vivo. Adult 6-8 week old Thy1YFP transgenic mice underwent a T12 laminectomy for acute imaging sessions or were implanted with a custom spinal cord imaging chamber for chronic imaging of the spinal cord. Two-photon excitation time-lapse microscopy was performed prior to a mild contusion SCI (30 kilodyne, IH Impactor) and at 1-4 h and 1-14 days post-SCI. We quantified the number of axonal spheroids, their size and distribution, the number of endbulbs, and axonal survival from 1 h to 14 days post-SCI. Our data reveal that the majority of axons underwent swelling and axonal spheroid formation acutely after SCI resulting in the loss of ~70% of axons by 1 day after injury. In agreement, the number of axonal spheroids rapidly increased at 1 h after SCI and remained significantly elevated up to 14 days after SCI. Furthermore, the distribution of axonal spheroids spread mediolaterally over time indicative of delayed secondary degenerative processes. In contrast, axonal endbulbs were relatively sparse and their numbers peaked at 1 day after injury. Intriguingly, axonal survival significantly increased at 7 and 14 days compared to 3 days after SCI revealing a potential endogenous axonal repair process that mirrors the known spontaneous functional recovery after SCI. In support, ~43% of tracked axonal spheroids resolved over the course of observation revealing their dynamic nature. Furthermore, axonal spheroids and endbulbs accumulated mitochondria and excessive tubulin polyglutamylation suggestive of disrupted axonal transport as a shared mechanism. Collectively, this study provides important insight into both degenerative and recoverable responses of axons following contusive SCI in real-time. Understanding how axons spontaneously recover after SCI will be an important avenue for future SCI research and may help guide future clinical trials.