Mechano-stimulation initiated by extracellular adhesion and cationic conductance pathways influence astrocyte...

Traumatic brain injury (TBI) represents a major cause of long-term disability worldwide. Primary damage to brain tissue leads to complex secondary injury mechanisms involving inflammation, oxidative stress and cellular activation/reactivity. The molecular pathways that exacerbate brain cell dysfunction after injury are not well understood and provide challenges to developing TBI therapeutics. This study aimed to delineate mechanisms of astrocyte activation induced by mechano-stimulation, specifically involving extracellular adhesion and cationic transduction. An in vitro model was employed to investigate 2D and 3D cultures of primary astrocytes, in which cells were exposed to a single high-rate overpressure known to cause upregulation of structural and proliferative markers within 72 h of exposure. An inhibitor of focal adhesion kinase (FAK) phosphorylation, TAE226, was used to demonstrate a relationship between extracellular adhesion perturbations and structural reactivity in the novel 3D model. TAE226 mitigated upregulation of glial fibrillary acidic protein in 3D cultures by 72 h post-exposure. Alternatively, incubation with gadolinium (a cationic channel blocker) during overpressure, demonstrated a role for cationic transduction in reducing the increased levels of proliferating cell nuclear antigen that occur at 24 h post-stimulation. Furthermore, early changes in mitochondrial polarization at 15 min and in endogenous ATP levels at 4-6 h occur post-overpressure and may be linked to later changes in cell phenotype. By 24 h, there was evidence of increased amine metabolism and increased nicotinamide adenine dinucleotide phosphate oxidase (NOX4) production. The overproduction of NOX4 was counteracted by gadolinium during overpressure exposure. Altogether, the results of this study indicated that both extracellular adhesion (via FAK activation) and cationic conductance (via ion channels) contribute to early patterns of astrocyte activation following overpressure stimulation. Mechano-stimulation pathways are linked to bioenergetic and metabolic disruptions in astrocytes that influence downstream oxidative stress, aberrant proliferative capacity and structural reactivity.