Increasing evidence suggests that excessive activation of the
calcium-activated neutral protease mu-calpain could play a major role in
calcium-mediated neuronal degeneration after
acute brain injuries. To further investigate the changes of the in vivo activity of
mu-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated
isoform of
mu-calpain to its 80-kDa precursor was measured by western blotting. This
mu-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24-48 h after injury. We also investigated the effect of
mu-calpain activation on proteolysis of the neuronal cytoskeletal
protein alpha-spectrin. Immunoreactivity for
alpha-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of
alpha-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of
alpha-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using
hematoxylin and
eosin staining demonstrated dark, shrunken neurons within 15 min after
traumatic brain injury. No evidence of
mu-calpain autolysis,
calpain-mediated
alpha-spectrin degradation, or
hematoxylin and
eosin neuronal pathology was detected in the contralateral cortex. Although
mu-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of
calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent
mu-calpain activation plays an important role in cortical neuronal degeneration after
traumatic brain injury. Our data also suggest that specific inhibitors of
calpain could be potential therapeutic agents for the treatment of
traumatic brain injury in vivo.