Hypergravitation selectively augments neuronal in vitro differentiation

Abstract

Disturbed neuronal connectivity is the ultimate cause of disability in individuals with neurological disease including spinal cord injury, head trauma, and stroke. Functional neurological recovery is limited through an unfavorable balance between neuronal regrowth and glia scar formation. Neuronal growth requires dynamic cytoskeletal protein rearrangements. Because hypergravity stabilizes microtubules while de-stabilizing actin filaments, we hypothesized that experimental hypergravity would shift the balance between neuronal and astroglial growth in vitro. We exposed murine primary hippocampal neurons during different developmental stages to 2g using the DLR hypergravity platform. The platform unlike commercial laboratory centrifuges models physiological hypergravity and allows for cell cultivation and live-cell imaging. We assessed neuritogenesis, neuronal polarization and maturation processes including synaptogenesis and synaptic integration in mature neural networks. Moreover, we studied primary astrocytes to shed light on their role during glial scar formation at neural lesion sites. Exposure of hippocampal neurons to 24h of 2g hypergravity increased neurite number by 30% and neurite projection length by 20% compared to 1g. At later developmental stages, mature synaptic contacts were formed under hypergravity conditions. In contrast, astrocytes showed decreases in cell spreading and lamellipodial protrusions with hypergravity.We conclude that experimental hypergravity ameliorates neuronal cell growth and synaptic contacts while halting astrocyte spreading and migration in vivo. Given the importance of this balance for neuronal regeneration in human neurological disease, we will now study the underlying mechanisms in more detail using, both, DLR hypergravity and clinostat-induced hypogravity platforms.