Directing Neuronal Outgrowth and Network Formation of Rat Cortical Neurons by Cyclic Substrate Stretch

Abstract

Neuronal mechanobiology plays a vital function in brain development and homeostasis with an essential role in neuronal maturation, pathfinding, and differentiation but is also crucial for understanding brain pathology. In this study, we constructed an in vitro system to assess neuronal responses to cyclic strain as a mechanical signal. The selected strain amplitudes mimicked physiological as well as pathological conditions. By subjecting embryonic neuronal cells to cyclic uniaxial strain we could steer the direction of neuronal outgrowth perpendicular to strain direction for all applied amplitudes. A long-term analysis proved maintained growth direction. Moreover, stretched neurons showed an enhanced length, growth, and formation of nascent side branches with most elevated growth rates subsequent to physiological straining. Application of cyclic strain to already formed neurites identified retraction bulbs with destabilized microtubule structures as spontaneous responses. Importantly, neurons were able to adapt to the mechanical signals without induction of cell death and showed a triggered growth behavior when compared to unstretched neurons. The data suggest that cyclic strain plays a critical role in neuronal development.

Manipulation of Axonal Outgrowth via Exogenous Low Forces

“…A possible explanation for this discrepancy is that axons respond to extremely low forces acting for days as viscoelastic fluid, while neurites show an elastic behavior when subjected to intense forces acting for a shorter time [79]. Indeed, a similar result was obtained by Abraham et al investigating the response of primary cortical neurons to cyclic strain over hours with physiologically relevant amplitudes and repeated frequencies [80]. Therefore, from recent experimental data, long-acting and low magnitude force significantly influences the outgrowth process [72,[81][82][83][84].…”

“…Abraham et al found a dependency in the neurite orientation relative to cyclic strain. They found significant remodeling of the MT cytoskeleton, adaptation to the cyclic strain, and MT formation in the stretch direction [80].…”

Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes

“…nHEK cells were cultured in DermaLife®K Keratinocyte Culture medium (CellSystems, Troisdorf, Germany) with manufacturer's supplements lacking tumor necrosis factor (TNF). For experiments on cortical neurons, cells were isolated from rat embryos and cultured as described previously [66]. All cells were continuously kept at 37 • C and 5% CO2 in a humidified atmosphere.…”

“…Cortical tissue was isolated from 19-day-old Wistar rat embryos by dissection as described before [66] and subsequently fused with the following complex: 2 µg eGFP-mRNA, 1 µg protamine, 2.5 µL FLs, and 250 µL PBS for 30 min at 37 • C. Complex preparation was performed as described above. After fusion, the solution was replaced by prewarmed neurobasal media (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA), B-27 (Thermo Fisher Scientific, Waltham, MA, USA), and gentamicin (Sigma, Taufkirchen, Germany) and tissue fragments were cultured for 4-6 days at 37 • C and 5% CO 2 containing, humidified atmosphere.…”

Isolation and Immunofluorescent Staining of Fresh Rat Pia–Arachnoid Complex Tissue for Micromechanical Characterization

“…Even though not traditionally part of the realm of neuroscience, biomechanics is making its way as a fundamental building block to our understanding of numerous processes of brain tissue development (Siegenthaler & Pleasure, ), homeostasis (Borrell & Marn, ), and regeneration (Decimo, Fumagalli, Berton, Krampera, & Bifari, ). Long regarded as not being mechanically active tissue, the brain and central nervous system have in fact become the recent object of intense mechanical scrutiny, with active research trying to clarify the mechanisms of developmental mechanotransduction (Franze, ) or of neuronal response to stretch and strain (Abraham et al., ; Smith, Wolf, Lusardi, Lee, & Meaney, ), as well as to elucidate how the mechanical environment provided within a tissue by its extracellular matrix affects its regenerative potential after damage (Moeendarbary et al., ). Especially within the traumatic brain injury community, there is a need for obtaining experimentally derived mechanical parameters able to characterize the material response of the meninges (Scott, Margulies, & Coats, ).…”