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The compressive strain rates for this study were in the quasi-static range to allow for confocal microscopy and image analysis since the compressed specimens would remain intact for histological processing. In other studies where a split-Hopkinson bar setup was implemented, significantly higher strain rates were achieved to better understand stress wave propagation through brain tissue as observed in high-rate scenarios such as automotive impacts. However, high rate compressed specimens were destroyed beyond recognition, eliminating the possibility for a histological analysis. One of the main objectives in this study was to analyze the fluctuations in the neuron and glial cell arrangement and their correlation to the observed viscoelastic tissue response. The microstructural data could eventually be utilized for assessing the internal changes of neural tissue during the lower threshold of deformation and for evaluating the potential for injury. The strain rate range in this study was chosen based on a similar unconfined compression study on the porcine brain by Miller et al. for strain rates of 0.64, 0.64 x 10^-2, and 0.64 x 10^-5s. The strain rates applied in this study were 0.00625, 0.025, and 0.10s^-1 while the strain levels in which the microstructure was suspended by formalin fixation were 15, 30, or 40%. Each test specimen was compressed once according to the strain rate and strain level designated. The first series of compression experiments were performed to determine if consistent nonlinearity in the stress–strain behavior could be observed when the strain level varied while the strain rate remained fixed. If similar nonlinear stress–strain behavior could be achieved despite variations in the strain levels selected, then confocal images obtained at an intermediate strain (e.g., from brain tissue fixed at 15 or 30% strain) could be assumed to represent the tissue internal microstructure in the compressive loading procedure. In this first series of compression testing at a fixed strain rate of 0.025s^-1, four specimens were compressed to 15% strain, four were compressed to 30% strain, and seven were compressed to 40% strain. The second series of compression experiments were conducted with variations in both the strain rate and the strain level in order to obtain the stress–strain data required to generate structure–property relationships. With the strain level fixed at 40%, 16 specimens were compressed at 0.10s^-1, seven were compressed at 0.025s^-1, and six were compressed at 0.00625s^-1. The original height of each test specimen was determined through a repeatable procedure in which the circular platen was programmed to descend until it initiated contact with the upper surface of the tissue. Contact was confirmed through a load-controlled condition, in which the platen immediately stopped after detecting a load reading of 0.50g. The micromechanical system consisted of a universal displacement actuator platform with a displacement control of 25nm. The built-in actuator sensitivity was utilized for measuring the initial sample height, which was then used for calculating the corresponding loading velocity and the vertical compression for the Mach-1TM programming. A pure slip interface was maintained between the platen surface and the upper tissue surface. While the circular platen remained in a stationary position but still in contact with the test specimen, the chamber was filled with 0.01 M PBS at 25 C to simulate the fluid environment in which the brain is typically surrounded. When full immersion was confirmed, each test specimen was compressed according to the strain rate and strain level specified. Engineering stress and engineering strain were used for data analysis. The cross-sectional area of the specimens was not monitored throughout the entire experiment to avoid tampering with the structural integrity of the tissue. A pre-conditioning step was not incorporated into the testing protocol, because the inherent mechanical integrity of the neural tissue was considered to be incapable of maintaining normal physiological function under cyclic external loading. As a result, a preconditioning was considered to be a contributing factor in the compressive deformation rather than a procedure for tissue preparation. Furthermore, other researchers have elected not to implement any preconditioning steps in their experiments. Toward the end of each experiment, an automated pipette was utilized for siphoning the 0.01 M PBS and replacing it with 10% NBF. Custom-designed polycarbonate cover pieces were immediately positioned over the stainless steel chamber to limit exposure of 10% NBF to the atmosphere while each compressed specimen remained in the fixative for approximately 20–30min. After the preliminary immersion in fixative was complete, each specimen was transferred to a separate storage container filled with a fresh batch of 10% NBF. Stress relaxation was expected to occur because it is an inherent property of viscoelastic tissues such as the brain. Although the fixative was placed on the tissue immediately following compression to 15, 30, and 40%, total fixation did not occur instantly and thus stress relaxation was inevitable to some degree. Stress relaxation was not directly measured in this study, but the authors assumed that the effects were minimal considering that the tissue was undergoing fixation.

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