Stretch injury causes calpain and caspase-3 activation and necrotic and apoptotic cell death in septo-hippocampal cell cultures

Traumatic brain injury (TBI) results in numerous central and systemic respo nses that complicate interpretation of the effects of the primary mechanica l trauma. For this reason, several in vitro models of mechanical cell injur y have recently been developed that allow more precise control over intra- and extracellular environments than is possible in vivo. Although we recent ly reported that calpain and caspase-3 proteases are activated after TBI in rats, the role of calpain and/or caspase-3 has not been examined in any in vitro model of mechanical cell injury. In this investigation, varying magn itudes of rapid mechanical cell stretch were used to examine processing of the cytoskeletal protein alpha-spectrin (280 kDa) to a signature 145-kDa fr agment by calpain and to the apoptotic-linked 120-kDa fragment by caspase-3 in septo-hippocampal cell cultures. Additionally, effects of stretch injur y on cell viability and morphology were assayed. One hour after injury, max imal release of cytosolic lactate dehydrogenase and nuclear propidium iodid e uptake were associated with peak accumulations of the calpain-specific 14 5-kDa fragment to alpha-spectrin at each injury level. The acute period of calpain activation (1-6 h) was associated with subpopulations of nuclear mo rphological alterations that appeared necrotic (hyperchromatism) or apoptot ic (condensed, shrunken nuclei). In contrast, caspase-3 processing of alpha -spectrin to the apoptotic-linked 120-kDa fragment was only detected 24 h a fter moderate, but not mild or severe injury. The period of caspase-3 activ ation was predominantly associated with nuclear shrinkage, fragmentation, a nd apoptotic body formation characteristic of apoptosis. Results of this st udy indicate that rapid mechanical stretch injury to septo-hippocampal cell cultures replicates several important biochemical and morphological altera tions commonly observed in vivo brain injury, although important difference s were also noted.