Ca(2+)-induced myelin pathology precedes axonal spheroid formation and is mediated in part by store-operated Ca(2+) entry after spinal cord injury

The formation of axonal spheroid is a common feature following spinal cord injury. To further understand the source of Ca(2+) that mediates axonal spheroid formation, we used our previously characterized ex vivo mouse spinal cord model that allows precise perturbation of extracellular Ca(2+). We performed two-photon excitation imaging of spinal cords isolated from Thy1(YFP+) transgenic mice and applied the lipophilic dye, Nile red, to record dynamic changes in dorsal column axons and their myelin sheaths respectively. We selectively released Ca(2+) from internal stores using the Ca(2+) ionophore ionomycin in the presence or absence of external Ca(2+). We reported that ionomycin dose-dependently induces pathological changes in myelin and pronounced axonal spheroid formation in the presence of normal 2 mM Ca(2+) artificial cerebrospinal fluid. In contrast, removal of external Ca(2+) significantly decreased ionomycin-induced myelin and axonal spheroid formation at 2 hours but not at 1 hour after treatment. Using mice that express a neuron-specific Ca(2+) indicator in spinal cord axons, we confirmed that ionomycin induced significant increases in intra-axonal Ca(2+), but not in the absence of external Ca(2+). Periaxonal swelling and the resultant disruption in the axo-myelinic interface often precedes and is negatively correlated with axonal spheroid formation. Pretreatment with YM58483 (500 nM), a well-established blocker of store-operated Ca(2+) entry, significantly decreased myelin injury and axonal spheroid formation. Collectively, these data reveal that ionomycin-induced depletion of internal Ca(2+) stores and subsequent external Ca(2+) entry through store-operated Ca(2+) entry contributes to pathological changes in myelin and axonal spheroid formation, providing new targets to protect central myelinated fibers.