Cytochrome c release and caspase activation in traumatic axonal injury

Axonal injury is a feature of traumatic brain injury (TBI) contributing to both morbidity and mortality. The traumatic axon injury (TAI) results from focal perturbations of the axolemma, allowing for calcium influx triggering local intraaxonal cytoskeletal and mitochondrial damage. This mitochondria l damage has been posited to cause local bioenergetic failure, leading to a xonal failure and disconnection; however, this mitochondrial damage may als o lead to the release of cytochrome c (cyto-c), which then activates caspas es with significant adverse intraaxonal consequences. In the current commun ication, we examine this possibility. Rats were subjected to TBI, perfused with aldehydes at 15-360 min after inj ury, and processed for light microscopic (LM) and electron microscopic (EM) single-labeling immunohistochemistry to detect extramitochondrially locali zed cytochrome c (cyto-c) and the signature protein of caspase-3 activation (120 kDa breakdown product of alpha-spectrin) in TAI. Combinations of doub le-labeling fluorescent immunohistochemistry (D-FIHC) were also used to dem onstrate colocalization of calpain activation with cyto-c release and caspa se-3-induction. In foci of TAI qualitative-quantitative LM demonstrated a parallel, signifi cant increase in cyto-c release and caspase-3 activation over time after in jury. EM analysis demonstrated that cyto-c and caspase-3 immunoreactivity w ere associated with mitochondrial swelling-disruption in sites of TAI. Furt hermore, D-IFHC revealed a colocalization of calpain activation, cyto-c rel ease, and caspase-3 induction in these foci, which also revealed progressiv e TAI. The results demonstrate that cyto-c and caspase-3 participate in the termin al processes of TAI. This suggests that those factors that play a role in t he apoptosis in the neuronal soma are also major contributors to the demise of the axonal appendage.