Mechanisms of Astrocytic Calcium Wave Propagation and in Vitro Hippocampal Cell Death Are Determined by the Magnitude of Mechanical Injury

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URL: https://repository.upenn.edu/edissertations/172/

Abstract

It has been estimated that traumatic brain injury (TBI) costs the U.S. economy over $60 billion a year, with over 5.3 million people suffering some level of long term effects. Despite these figures, no effective pharmacological treatment has been successfully developed to help patients recover from TBI. A more complete understanding of both the immediate cellular reactions to injury and the longer-term responses may present new strategies for reducing damage and promoting repair. In this dissertation we first examine the ability for astrocytes to modulate the mechanisms of calcium wave propagation in response to increasing degrees of injury. As injury magnitude increases, the complexity of the calcium wave that is transmitted to surrounding uninjured regions also increases. At very mild levels of stretch calcium waves are primarily transmitted through extracellular ATP. As the level of stretch increases, gap junction communication and metabotropic glutamate receptor activation can be detected. The interaction between these three signaling pathways may transmit information about the severity of injury to surrounding astrocytes and may mediate immediate cellular responses. We also observe that the mechanical properties of cultured astrocytes are altered 24 hours after injury. Rapid stretch induces a decrease in the apparent Young’s modulus on non-nuclear regions of astrocytes, indicating that the cells are softer and more compliant. This change is associated with an upregulation of GFAP, which is a common marker for reactive gliosis. Finally, we investigate the interaction between glutamatergic and purinergic signaling in mediating cell death in hippocampal slices 24 hours following mechanical injury. Within the CA3 region of the hippocampus there is a significant increase in cell death after stretch-injury that can be attenuated by inhibiting the activity of the glutamatergic N-methyl D-aspartate (NMDA) receptors. Alternatively, blocking the activity of P2Y1 receptors is effective in limiting cell death. Our studies suggest that there is a high probability that P2Y1 receptor activity on astrocytes is responsible for inducing the over-excitation of extrasynaptic NMDA receptors, which is responsible for a major component of the observed cell death. Further studies into this pathway may lead to new approaches for pharmacological therapies after TBI.

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