Mechanism of neuronal damage following mild in-vitro traumatic neuronal injury.
Published: 2004
Language: eng
Number of pages: 274
Description: Thus, sublethal stretch injury results in elevated ROS that are tolerated by the cultures, but imparts neurons with decreased resiliency to secondary excitotoxic insults specifically associated with NMDAR-triggered NO production via the coupling of NMDA receptors to PSD-95. The result of this interaction is further ROS production specifically associated with toxic nitrogen radical species, leading to non-apoptotic cell death.Traumatic brain injury mechanisms are categorized into primary injury and secondary injury. Primary injury is defined as the injury resulting from the initial mechanical event (direct injury), whereas secondary injury is the delayed damage arising from the signaling pathways triggered by the primary insult. Unlike primary injury, secondary injury may be amenable to treatment and pharmacological intervention.Using primary cortical neuronal cultures grown on flexible silastic membranes, we established parameters needed to mechanically stretch cells to a degree and duration that itself produces sublethal injury. Although the cells survived the stretch, it imparted them with an enhanced vulnerability to secondary excitotoxic insults specifically associated with NMDAR activity. The increased vulnerability to NMDA was dependent on Ca2+ influx through NMDARs. Stretch had no detectable effect on NMDA receptor function or synaptic activity, indicating that postsynaptic mechanisms downstream from NMDARs may be responsible.Following sublethal stretch, neurons exhibited a transient reduction in mitochondrial potential. Secondary NMDA insults prevented complete mitochondrial repolarization, and cultures exhibited both TUNEL staining and DNA laddering on gel electrophoresis---hallmarks of apoptosis. However, caspase 3 activation was not observed, and treatment with a pan caspase inhibitor z-vad-FMK did not attenuate vulnerability to NMDA, suggesting against a classical apoptotic mechanism. Moreover, apoptosis-inducing factor (AIF) and endonuclease-g did not translocate into the nucleus, suggesting against caspase independent apoptotic mechanisms.Stretched neurons exhibited a rapid production of reactive oxygen species (ROS) which were well tolerated by the cultures. The secondary NMDA insult caused greater ROS production and increased cell death, TUNEL immunoreactivity, and protein nitration. NOS inhibition reduced the enhanced vulnerability and DNA fragmentation. Dissociating NMDARs from PSD-95 with TAT fusion peptides also reduced the enhanced vulnerability of stretched cultures to NMDA, and reduced ROS production and nitrotyrosine immunoreactivity.
Notes: Adviser: Michael Tymianski.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2169.