Repeated In Vitro Impact Conditioning of Astrocytes Decreases the Expression and Accumulation of Extracellular Matrix.

Pathological changes to the physical and chemical properties of brain extracellular matrix (ECM) occur following injury. It is generally assumed that astrocytes play an important role in these changes. What remain unclear are the triggers that lead to changes in the regulation of ECM by astrocytes following injury. We hypothesize that mechanical stimulation triggers genotypic and phenotypic changes to astrocytes that could ultimately reshape the ECM composition of the central nervous system following injury. To explore astrocyte mechanobiology, an in vitro drop test bioreactor was employed to condition primary rat astrocytes using short duration (10 ms), high deceleration (150G) and strain (20%) impact stimuli. Experiments were designed to explore the effect of single and repeated impact (single vs. double) on mechano-sensitive behavior including cell viability; ECM gene (collagens I and IV, fibronectin, neurocan, versican) and reactivity gene [glial fibrillary acidic protein (GFAP), S100B, vimentin] expression; matrix regulatory cytokine secretion [matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinases 1 (TIMP1), transforming growth factor beta 1 (TGFβ1)]; and matrix accumulation [collagen and glycosaminoglycan (GAG)]. Experiments revealed that both ECM and reactive gliosis gene expression was significantly decreased in response to impact conditioning. The decreases for several genes, including collagen, versican, and GFAP were sensitive to impact number, suggesting mechano-sensitivity to repeated impact conditioning. The measured decreases in ECM gene expression were supported by longer-term in vitro experiments that demonstrated significant decreases in chondroitin sulfate proteoglycan (CSPG) and collagen accumulation within impact conditioned 3-D scaffolds accompanied by a 25% decrease in elastic modulus. Overall, the general trend across all samples was towards altered ECM and reactive gliosis gene expression in response to impact. These results suggest that the regulation of ECM production by astrocytes is sensitive to mechanical stimuli, and that repeated impact conditioning may increase this sensitivity.