Intracellular calcium dynamics in traumatic brain injury: Understanding the role of N-type voltage-gated calcium channels in stretch-induced calcium rise in cortical neurons

Traumatic brain injury (TBI) is one of the leading causes of death and disability in the world. The mechanisms leading to cell damage and death following injury are not full understood, but likely involve an acute rise in neuronal cytosolic calcium levels after trauma. Studies have suggested that N-type voltage-gated calcium channels (VGCCs) participate in calcium elevation following mechanical trauma or hypoxia, but their exact role in injury-induced calcium elevation in neurons has not been previously studied.; Mixed neuronal-glial cell cultures were generated from rat pups using modified techniques. A well-defined model of in vitro TBI was used to injure cells grown on silastic membranes. A microdialysis apparatus was designed to sample cell culture media composition following mechanical injury. Hypoxia and acidosis were induced by exposure to elevated incubator CO2 (EIC). Calcium measurements were performed using the ratiometric fluorescent indicator fura-2, and cell survival and damage were quantified using the fluorescent markers carboxyfluorescein and propidium iodide, respectively. N-type VGCCs were selectively blocked using the omega conotoxin SNX-185.; Stretch injury resulted in a severity-dependent rise of intracellular calcium that returned to pre-injury baseline values when maintained in culture media. Treatment with SNX-185 before injury significantly reduced glutamate release into the culture media and completely prevented intracellular calcium elevation, resulting in increased cell survival. Delayed treatment (five minutes) significantly expedited calcium return to baseline levels but did not improve neuronal survival. EIC exposure also resulted in elevation of intracellular calcium with subsequent cell damage. Delayed treatment with SNX-185 significantly reduced the effects of secondary EIC exposure and was neuroprotective.; N-type VGCCs are critically involved in mediating and maintaining calcium elevation following stretch injury or exposure to hypoxia and acidosis, due, in part, to their role in injury-induced glutamate release. Neuronal survival following injury is primarily determined by the magnitude, and not duration, of elevated intracellular calcium. Therefore, treatment with SNX-185 prior to acute calcium elevation (before primary injury or in a delayed fashion with secondary injury) is robustly neuroprotective. N-type VGCCs play a vital role in cell damage and death following injury, and may be an effective target for therapy in TBI.