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 responses that complicate interpretation of the effects of the primary mechanical trauma. For this reason, several in vitro models of mechanical cell injury have recently been developed that allow more precise control over intra- and extracellular environments than is possible in vivo. Although we recently 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 magnitudes of rapid mechanical cell stretch were used to examine processing of the cytoskeletal protein alpha-spectrin (280 kDa) to a signature 145-kDa fragment by calpain and to the apoptotic-linked 120-kDa fragment by caspase-3 in septo-hippocampal cell cultures. Additionally, effects of stretch injury on cell viability and morphology were assayed. One hour after injury, maximal release of cytosolic lactate dehydrogenase and nuclear propidium iodide uptake were associated with peak accumulations of the calpain-specific 145-kDa fragment to alpha-spectrin at each injury level. The acute period of calpain activation (1-6 h) was associated with subpopulations of nuclear morphological alterations that appeared necrotic (hyperchromatism) or apoptotic (condensed, shrunken nuclei). In contrast, caspase-3 processing of alpha-spectrin to the apoptotic-linked 120-kDa fragment was only detected 24 h after moderate, but not mild or severe injury. The period of caspase-3 activation was predominantly associated with nuclear shrinkage, fragmentation, and apoptotic body formation characteristic of apoptosis. Results of this study indicate that rapid mechanical stretch injury to septo-hippocampal cell cultures replicates several important biochemical and morphological alterations commonly observed in vivo brain injury, although important differences were also noted.