Reduced Glial Scarring Through Hypergravity Exposure

Abstract

Introduction: Neural regeneration following injuries to the central nervous system (CNS) in mammals is inhibited by several factors. One important mechanism preventing axon regrowth and thus the healing of a CNS injury is the formation of the glial scar. Key players in glial scar formation are reactive astrocytes that migrate into the region of the injury and produce an inhibitory extracellular environment, rich in chondroitin sulfate proteoglycans (CSPGs) and other signaling molecules. These, in turn, have an inhibiting effect on axon growth and even actively induce axon dystrophy, which has severe consequences for patients, e.g., loss of neuronal signaling and in some cases permanent paralysis. We could show that exposure to altered gravity has a direct effect on primary astrocytes and that hypergravity in particular might be a viable tool to reduce glial scarring. Methods: We cultivated primary murine cortical astrocytes in vitro under hypergravity conditions at constant 2g for several days up to weeks by using the DLR Multi Sample Incubator Centrifuge (MuSIC) and compared key cellular characteristics with 1g controls. To investigate cellular dynamics and migration speed under hypergravity, we employed our Hyperscope platform at DLR, a fully automated fluorescent live-cell imaging microscope installed on the :envihab human short-arm centrifuge (SAHC). Additionally, using our group’s expertise in the ground-based simulation of microgravity by means of fast clinorotation (60 rpm) and also flight opportunities for experiments in real microgravity (DLR sounding rocket MAPHEUS8/ATEK) we were able to expose astrocytes to space-like conditions and investigated their responses on a morphological and protein-level. Results: On the one hand we could show that astrocyte spreading, a well-known effect of 2D cultures, is significantly reduced by about 20% due to hypergravity (2g) exposure, while on the other hand cell proliferation is unchanged. The diminished spreading of astrocytes in combination with morphological alterations indicates an impact of altered gravity conditions on the cytoskeleton. Since cellular migration depends on a fully functioning actin and tubulin cytoskeleton, we expected an impact of hypergravity on the migrational behavior of astrocytes. To test this hypothesis, we performed in vitro wound-healing assays (scratch-assays) on both the DLR incubator-centrifuge as well as the Hyperscope platform, enabling a live assessment of the migratory behavior of astrocytes during exposure to hypergravity. As a result, astrocyte migration was confirmed to be diminished by about 33% during an initial phase followed by cell adaptation with a less substantial but prolonged diminished migratory rate with about 10% reduction of cell velocity. Conclusions: Our results show that hypergravity represents a stimulus that inhibits not only cell spreading but also astrocytic migration, which in case of a CNS injury might reduce glial scarring and therefore increase the progression of neural regeneration. Our further steps are the identification of the underlying mechanisms, e.g., cytoskeletal alterations to generate an advanced model of astrocyte responses to altered gravity. For this, the plan is to not only work with increased gravitational loading, but also mechanically unload the cells to see what cellular mechanisms respond to this kind of stimulus.