In vitro and in situ visualization of cytoskeletal deformation under load: traumatic axonal injury

It is difficult to obtain insight into the mechanisms occurring within live cells during mechanical loading, because this complex environment is dynamic and evolving. This is a particular challenge from a subcellular mechanics perspective, where temporal and spatial information on the evolving cytoskeletal structures is required under loading. Using fluorescently labeled proteins, we visualize 3-dimensional live subcellular cytoskeletal populations under mechanical loading using a high-resolution confocal microscope. The mechanical forces are determined using a computational (finite element) model that is validated by integrating instrumentation into the testing platform. Transfected microtubules and neurofilaments of E17 rat neuronal axons are imaged before, during, and after loading. Comparisons between unloaded and loaded live cells demonstrate both spatial and temporal changes for cytoskeletal populations within the imaged volume. NF signal decreases by 24%, yet the microtubule signal exhibits no significant change 20-35 s after loading. Transmission electron microscopy assesses cytoskeletal structure spatial distribution for undeformed and deformed axons. While cytoskeletal degeneration occurs at prolonged time intervals following loads, our data provides insights into real time cytoskeletal evolution occurring in situ. Our findings suggest that, for axons undergoing traumatic injury in response to applied mechanical loads, changes at the substructural level of neurofilaments may precede microtubule rupture and degeneration.