In vitro neuronal cell injury model: characterization and treatment strategies

Gulyeter Serbest

doi:10.17918/etd-198

A new in vitro model of Traumatic Brain Injury (TBI) was developed. Mechanical loading parameters were precisely controlled and optimized to create a reproducible injury severity in cultured PC2 neuronal cells. The cellular response to trauma was characterized in terms of membrane damage and long-term viability. In addition, the mechanism of cell death and the signaling pathways involved were analyzed. Finally, a novel therapeutic strategy was developed to protect cells from both acute and delayed injuries caused by mechanical trauma. Injury severity increased monotonically with increasing peak shear stress, and it was strongly dependent on loading rate. The loading rate and magnitude were optimized to produce an injury severity resulting with 46 % loss of viability at 24 hours after the initial loading. The acute (<5 min) release of LDH within demonstrated a loss of membrane integrity as a direct result of applied mechanical loading. Flow cytometry of cells fluorescently labeled to indicate apoptosis (TUNEL assay) and necrosis (low propidium iodide staining) was used to identify the mechanism of cell death at 24 hours following trauma. The primary cell death mechanism was apoptosis, although significant necrosis was also detected. The MAP kinases, ERK1/2, JNK1/2, and p38 kinase, were rapidly and transiently phosphorylated after mechanical trauma peaking at 15 min. The concerted activation of these kinases may be the cause of delayed cell death. Treatment of the cells with Poloxamer (P188), a water soluble and non- ionic surfactant, remarkably protected injured cells from both acute and delayed cell death. P188 treatment was also fo und to inhibit p38 activation. However, treatment with a p38 inhibitor, SB203580, only partially rescued cells from apoptosis but not from necrosis. This shows that P188 may act upstream of p38 activation and may affect other pathways in addition to the p38 pathway. Since the loss of membrane integrity has been shown to be a major pathophysiological result of mechanical trauma, the application of P188 to reseal injured membranes after traumatic injury represents a novel approach to long-term neuronal recovery from both acute and delayed injuries in TBI.

In vitro neuronal cell injury model: characterization and treatment strategies

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In vitro neuronal cell injury model: characterization and treatment strategies

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