Abstract: Protecting white matter following SCI is a major goal to improve neurological recovery following spinal cord injury (SCI). Whether the axoplasmic reticulum (AR), the major endomembrane system and Ca2+ store within axons, contributes to secondary “bystander” central myelinated fiber degeneration following SCI remains unknown. Furthermore, the role of the AR's major release channels, ryanodine receptors (RyR) and inositol 1,4,5-trisphophate receptors (IP3R) in white matter injury remain poorly understood; however, our preliminary data support an important role for RyR in mediating secondary degeneration of axons following a clinically-relevant contusion SCI in vivo. Our overall objective is to protect central myelinated fibers following SCI by targeting AR Ca2+ release channels. We will test the following hypothesis within this proposal. RyR and IP3R mediate intra-axonal Ca2+ store release of Ca2+ within spinal axons and cause secondary axonal degeneration following contusion SCI. Therefore, inhibiting Ca2+ store mediated Ca2+ release by targeting RyR or upstream signaling pathways that converge on RyR and IP3R will protect white matter following SCI. Our specific aims are to: 1. Determine the role of RyR and IP3R in secondary axonal degeneration following contusion SCI in real-time. 2. Determine the role of upstream signaling pathways in AR Ca2+ release in real-time. 3. Evaluate clinically relevant approaches to inhibit AR-mediated intra-axonal CICR to improve neurological recovery following contusion SCI. To accomplish this goal we will utilize two-photon microscopy combined with an ultrafast resonant scanner (capable of collecting images at up 420 frames per second) to assess axonal swelling, spheroid formation, axonal retraction, degeneration (Advillin-Cre: tdTomato transgenic mice), axonal Ca2+ wave generation and Ca2+ accumulation in axons (Advillin-Cre: tdTomato: CaMP6f and Thy1GCaMP6f transgenic mice), myelin integrity (fluorescent lipophilic dyes), and changes in AR (e.g., ER tracker dyes and 3D electron microscopy) as these dynamic events are unfolding in real-time in vivo following a contusion SCI. The technology and approach proposed may help advance the field as live imaging of axons over time allows unequivocal determination of the fate of injured axons and whether they can be rescued in real-time with treatment. Furthermore, it allows direct visualization of myelin and the fate of this vital element simultaneously with axons as these events are unfolding in the injured spinal cord. This proposal is innovative and uses advanced imaging techniques to explore overlooked areas of SCI research. The approach taken may also unveil potential novel therapeutic targets and clinically relevant treatments (e.g. FDA approved carvedilol) to promote neurological recovery after SCI. The underlying mechanisms of white matter injury may also be relevant to multiple sclerosis and other neurological diseases.