Background: Diffuse axonal injury (DAI) is a one of the main causes of traumatic brain injury and caused by the impulsive stretching of neuronal axons resulting from rapid deformation of a brain. DAI is characterized by a gradual swelling of the axons which is formed by the accumulation of cellular organelles and proteins, and the swelling is the morphological hallmark of DAI pathology. In recent years, the details of the stress and strain of the whole damaged brain are becoming clear with the rapid development of the computational mechanics and finite element (FE) head models are able to give better prediction to the brain injury and to evaluate the protective safety methods with more detailed neuronal tolerance criteria. In this study, axonal injury induced by precisely controlled impulsive strain and strain rate was evaluated and the tolerance criteria for the functional disorder for each dysfunction and disruption level of axonal transport was obtained by observation of β-amyloid precursor protein (β-APP) in cultured rat cortex neuron. Methods: The uniaxial stretching device which could give various combinations of strains and strain rates to neurons was developed. The various loading conditions for neurons were verified by comparing the experimental displacement history of the substrate taken by microscopy with the FE strain distribution analysis of culturing substrate. The primary rat cortex neurons were stretched by different combinations of strains and strain rates and β-APP was immunostained at 3h after loading and observed by fluorescence microscopy. Results: The number of swellings and bulbs formed on axons by β-APP-accumulation after stretching were observed and counted by fluorescent images. The dysfunction of the axonal transport was defined as the rate of neurons that have β-APP-accumulating axonal swellings and disruption of the axonal transport was defined as the rate of neurons that have β-APP-accumulating axonal bulbs, respectively. The degree of the functional disorder of the axonal transport advanced with the increase of strain and strain rate. Conclusions: The mechanical threshold of dysfunction and disruption of axonal transport were the strain with 0.22 and the strain rate with 27 /s. The intervals between swellings on an axon are constant and do not depend on the axonal injury level nor the magnitude of the strain of the axons.

Diffuse axonal injury (DAI), a major component of traumatic brain injury, is associated with rapid deformation of brain tissue resulting in the stretching of neuronal axons. Focal axonal beading, which is the morphological hallmark of DAI pathology, leads to the disconnection of neurons from tissues and results in cell death. Our goal is to achieve a better understanding of neuronal tolerance and help predict the pathogenesis of DAI from mechanical loading to the head. In the present study, we developed an experimental model that subjected cultured neurons to uniaxial stretch by controlling the direction of axonal elongation with a microfluidic culture technique and examined the effect of strains along the axon on cell damage. Neurites from PC 12 cells that differentiate into neurons with structurally axon-like cylindrical protrusions by nerve growth factor were extended at 0°, 45°, and 90° relative to the tensile direction by using a fabricated polydimethylsiloxane (PDMS) piece with microgrooves in combination with a PDMS substrate. The morphology of the same neurites was observed before and after stretching with a strain of 0.22 at a strain rate of 27 s -1 . As a result, swellings along neurites oriented at 0° increased immediately following stretching and were sustained for 24 h. In contrast, swellings along neurites oriented at 45° and 90° transiently increased within 1 h following stretching. Although more ruptures were observed in neurites oriented at 0° and 90° than in those oriented at 45°, the number of neurites and cells did not differ among orientation conditions. These results suggest that the difference in strain along the axon induces axonal injuries differing in type and degree. They also suggest that the strain on the axon, rather than that on the cell-cultured substrate, is important for evaluating neuronal damage.mixture and cured at 65 °C for 1 h. To obtain the relation between the displacement of the PDMS chamber and the strain of the culture substrate, the PDMS chamber was pulled statically in increments of 0.5 mm to 4 mm and the Green-Lagrange strains of the culture substrate in the PDMS chamber were calculated from microscope images before and after stretching (Pfister, et al., 2003). As a result, the transverse strain, i.e., contraction in the direction perpendicular to tension, was suppressed to less than 10% of the longitudinal strain, i.e., elongation in the direction parallel to tension (Fig. 3B). In this study, the displacement shown in Fig. 3C was applied to the chamber with a strain of 0.22 at a strain rate of 27 s -1 . The strain rate was obtained by dividing the maximum strain by the time to maximum strain. Fig. 5Top and cross-sectional views of microtunnel structure (A) and microtunnel structure on culture substrate of PDMS chamber (B). The microtunnel structure has a culture well of 6 mm in diameter, spacer area of 100 μm in height, and 20 microtunnels of 50 μm in width and 2 mm in length.

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