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Department of Biomedical Engineering

Seminar Thursday, March 26, 2009


Gene Gurkoff, PhD


Post-Traumatic Neuronal Activity and Cell Death


GG. Gurkoff and BG. Lyeth

Traumatic brain injury (TBI) is characterized by an array of pathophysiologies.  A comprehensive understanding of TBI, from cell culture dish, to in-vivo models (mouse, rat and pig) all the way to human, is critical to devising new treatment paradigms for head injured patients. One of the hallmarks of TBI in-vivo is the acute accumulation of extracellular potassium ([K+]e).  This phenomenon has been observed in both human tissue and in rodent models.  The effect of this extracellular potassium on surrounding tissue is unknown, though many believe it may be toxic or make cells vulnerable to secondary insult.  In a model of post-traumatic seizures, however, post-natal day 19 rats were protected against secondary seizure induced cell death when seizures occurred in the first hours post-injury.  We hypothesize that an accumulation of [K+]e will lead to a reduction in neuronal activity and therefore protection against injury-induced cell death.  In order to determine the effect of [K+]e on individual neurons it is critical that we, to the best of our ability, mimic what is seen in the extracellular in-vivo environment in the dish.  To that end we utilized mixed neuronal/astrocytic cultures and a range of potassium shifted ringers (PSR) combined with either glutamate excitotoxicity (1mM) or Ellis mechanical injury. Increasing the concentration of [K+]e  from normal ringers (3 mM) to 30 or 65 mM significantly reduced cell death in response to glutamate injury when quantified 24-hours post-injury. At the highest concentration (90 mM) levels of cell death were equivalent to the normal ringers group.  In contrast, increasing [K+]e during mechanical injury increased cell death at all [K+]e concentrations.   To assess whether cell death following mechanical injury was related to increases in neuronal activity we analyzed the effects of PSR on intracellular free calcium ([Ca++]i) using the ratio-metric dye, fura-2.  First a baseline [Ca++]i measurement was acquired for 5 minutes in standard ringers.  Cells were then bathed in PSR for an additional 5 minutes prior to injury to assess the effect of buffer alone.  Finally cells received either 1mM glutamate, moderate or severe mechanical injury.  PSR alone, regardless of concentration, increased [Ca++]i, however following injury, regardless of injury, there was a potassium concentration-related reduction in [Ca++]i.  Taken together these data indicate that manipulating neuronal activity by raising [K+]e protects neurons from pure glutamate excitotoxicity but does not protect them from mechanical injury even though it reduces [Ca++]i. These data highlight the complexities of the post-injury pathophysiology and the differences between mechanical and excitotoxic insults.   Further they demonstrate that successful interruption of one mechanism of cellular dysfunction and death does not guarantee a positive outcome.  Finally, these data underscore the utility of using an in-vitro model to better understand many of the TBI-related phenomena observed in-vivo.  Ongoing studies are taking advantage of this technique to further understand the consequences of [K+]e as well as to explore the effect of other extracellular changes on neuronal viability and function.