Membrane Integrity as a Therapeutic Target in Neural Cell Injury

The importance of cell membrane integrity for normal cell function and indeed survival is well established, yet the role of membrane disruption in cellular pathology is seldom considered except as a prelude to, or indication of, cell death. However, evidence from diverse fields strongly implicates membrane disruption as a key precipitating event in the pathological responses to various stimuli. Dynamic mechanical loading of neural cells produces an acute disruption of the plasma membrane as indicated by a rapid and transient release of LDH from the cytoplasm of injured cells. In this report, we show that this cellular level injury is not immediately fatal, but rather gives rise to a cascade of signaling events that lead to cell death in the long term. In our model, over 50% of the cells were dead at 24 hours post injury, the majority of which were apoptotic as assessed by the TUNEL assay using flow cytometry. Though many of the signaling pathways involved in this response to injury have been studied, the link between the initial membrane damage and the subsequent signaling is poorly understood. We report for the first time that treating injured neurons with an agent that promotes resealing of membrane pores can rescue the cells from both necrotic cell death and apoptosis at 24 hours post injury. Treatment with the nonionic surfactant, poloxamer 188 (P188), at 15 minutes post injury restored cell viability at 24 hours to control values. The role of the pro-apoptosis MAP kinase, p38, in cell death following injury was investigated using Western blot analysis. Activation of p38 was increased over 2-fold at 15 minutes post injury. P188 treatment at 10 minutes inhibited p38 activation. However, treatment with a specific inhibitor of p38 activation produced only a partial reduction in apoptosis and had no effect on necrotic cell death. These data suggest multiple signaling pathways are involved in the long term response of neurons to mechanical injury. Furthermore, the putative mechanism of action of P188 to promote membrane resealing suggests that the acute membrane damage due to trauma is a critical precipitating event lying upstream of the many signaling cascades that contribute to the subsequent pathology.