Confocal imaging of changes in glial calcium dynamics and homeostasis after mechanical injury in rat spinal cord white matter

Periaxonal glia play an important role in maintaining axonal function in white matter. However, little is known about the changes that occur in glial cells in situ immediately after traumatic injury. We used fluo-3 and confocal microscopy to examine the effects of localized (<0.5 mm) mechanical trauma on intracellular calcium (Ca_i²⁺) levels in glial cells in a mature rat spinal cord white matter preparation in vitro. At the injury site, the glial Ca_i²⁺ signal increased by 300-400% within 5 min and then irreversibly declined indicating cell lysis and death. In glial cells at sites adjacent to the injury (1.5-2 mm from epicenter), Ca_i ²⁺ levels peaked at 10-15 min, and thereafter declined but remained significantly above rest levels. At distal sites (6-9 mm), Ca_i ²⁺ levels rose and declined even slower, peaking at 80-90 min. Injury in zero calcium dampened Ca_i²⁺ responses, indicating a role for calcium influx in the generation and propagation of the injury-induced Ca_i²⁺ signal. By 50-80 min post-injury, surviving glial cells demonstrated an enhanced ability to withstand supraphysiological Ca_i²⁺ loads induced by the calcium ionophore A-23187. Glial fibrillary acidic protein (GFAP) and CNPase immunolabeling determined that the glial cells imaged with fluo-3 included both astrocytes and oligodendrocytes. These data provide the first direct evidence that the effects of localized mechanical trauma include a glial calcium signal that can spread along white matter tracts for up to 9 mm within less than 3 h. The results further show that trauma can enhance calcium regulation in surviving glial cells in the acute post-injury period.