AN EXPERIMENTAL MODEL FOR TRAUMATIC AXONAL INJURY BASED ON CYTOSKELETAL EVOLUTION
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Traumatic brain injury (TBI) and spinal cord injury (SCI) are debilitating causes of traumatic death and disability to millions of people worldwide. These injuries occur from damage to the brain and spinal cord resulting from external mechanical stimuli, including rapid linear/rotational acceleration and/or deceleration, blast waves, crush, impact, or penetration by a projectile. A primary pathology of TBI and SCI is traumatic axonal injury (TAI) where rapidly applied loads trigger a progressive series of changes in the cytoskeletal network that provides neural cells with structure and stability. These changes gradually evolve from cytoskeletal alterations to a delayed axonal disconnection, a process potentially amenable for therapeutic intervention. The goal of this work is to implement experimental models for traumatic axonal injury that provide quantitative measures for assessing changes in neurological tissues and to connect these across multiple length scales. To better understand TAI, we have developed a new experimental platform to apply controlled loads on isolated CNS axons. We apply focal compression to neural axons where the applied load is predicted using a validated finite element model of the system. The experimental and finite element models have led to the development of threshold criteria, governing the cellular response of the axons to the applied load, as continued growth, degeneration (TAI), or regrowth. An approach to assess the temporal evolution of the cytoskeleton during the TAI response of the cell was developed using confocal microscopy and transmission electron microscopy. The ability to visualize the live cell in situ and in-vitro response was accomplished through confocal microscopy where fluorescently tagged microtubules and neurofilaments were continuously imaged prior to, during, and immediately following focal compression. Comparisons between unloaded and loaded live cells demonstrate both spatial and temporal changes for cytoskeletal populations within the imaged volume. Transmission electron microscopy connected the changes observed through confocal imaging with alterations in the ultrastructural composition of microtubules and neurofilaments within neural axons. These metrics provide a pathway for connecting changes in cytoskeletal spatial distributions to previously observed changes in measured intensity using confocal microscopy with the same loading platform in situ and in vitro, and may be critical in understanding mechanical failure and degeneration of the cytoskeletal system for neural axons undergoing TAI. Our experimental framework can be applied to developing new connections with existing analytical and computational models for predicting TBI and SCI at smaller length scales. This could manifest itself in the form of new standards and protocols for protection against TAI, and for improvement of protective materials and restraint systems.