We describe a novel valve-based microfluidic axon injury micro-compression (AIM) platform that enables focal and graded compression of micron-scale segments of single central nervous system (CNS) axons. The device utilizes independently controlled "push-down" injury pads that descend upon pressure application and contact underlying axonal processes. Regulated compressed gas is input into the AIM system and pressure levels are modulated to specify the level of injury. Finite element modeling (FEM) is used to quantitatively characterize device performance and parameterize the extent of axonal injury by estimating the forces applied between the injury pad and glass substrate. In doing so, injuries are normalized across experiments to overcome small variations in device geometry. The AIM platform permits, for the first time, observation of axon deformation prior to, during, and immediately after focal mechanical injury. Single axons acutely compressed (~5 s) under varying compressive loads (0-250 kPa) were observed through phase time-lapse microscopy for up to 12 h post injury. Under mild injury conditions (< 55 kPa) ~73% of axons continued to grow, while at moderate (55-95 kPa) levels of injury, the number of growing axons dramatically reduced to 8%. At severe levels of injury (> 95 kPa), virtually all axons were instantaneously transected and nearly half (~46%) of these axons were able to regrow within the imaging period in the absence of exogenous stimulating factors.
Although a number of cytoskeletal derangements have been described in the setting of traumatic axonal injury (TAI), little is known of early structural changes that may serve to initiate a cascade of further axonal degeneration. Recent work by the authors has examined conformational changes in cytoskeletal constituents of neuronal axons undergoing traumatic axonal injury (TAI) following focal compression through confocal imaging data taken in vitro and in situ. The present study uses electron microscopy to understand and quantify in vitro alterations in the ultrastructural composition of microtubules and neurofilaments within neuronal axons of rats following focal compression. Standard transmission electron microscopy processing methods are used to identify microtubules, while neurofilament identification is performed using antibody labeling through gold nanoparticles. The number, density, and spacing of microtubules and neurofilaments are quantified for specimens in sham Control and Crushed groups with fixation at <1min following load. Our results indicate that the axon caliber dependency known to exist for microtubule and neurofilament metrics extends to axons undergoing TAI, with the exception of neurofilament spacing, which appears to remain constant across all Crushed axon diameters. Confidence interval comparisons between Control and Crushed cytoskeletal measures suggests early changes in the neurofilament spatial distributions within axons undergoing TAI may precede microtubule changes in response to applied loads. This may serve as a trigger for further secondary damage to the axon, representing a key insight into the temporal aspects of cytoskeletal degeneration at the component level, and suggests the rapid removal of neurofilament sidearms as one possible mechanism.
This study developed and validated finite element (FE) models of swine and human thoraxes and abdomens that had subject-specific anatomies and could accurately and efficiently predict body responses to blunt impacts. Anatomies of the rib cage, torso walls, thoracic, and abdominal organs were reconstructed from X-ray computed tomography (CT) images and extracted into geometries to build FE meshes. The rib cage was modeled as an inhomogeneous beam structure with geometry and bone material parameters determined directly from CT images. Meshes of soft components were generated by mapping structured mesh templates representative of organ topologies onto the geometries. The swine models were developed from and validated by 30 animal tests in which blunt insults were applied to swine subjects and CT images, chest wall motions, lung pressures, and pathological data were acquired. A comparison of the FE calculations of animal responses and experimental measurements showed a good agreement. The errors in calculated response time traces were within 10% for most tests. Calculated peak responses showed strong correlations with the experimental values. The stress concentration inside the ribs, lungs, and livers produced by FE simulations also compared favorably to the injury locations. A human FE model was developed from CT images from the Visible Human project and was scaled to simulate historical frontal and side post mortem human subject (PMHS) impact tests. The calculated chest deformation also showed a good agreement with the measurements. The models developed in this study can be of great value for studying blunt thoracic and abdominal trauma and for designing injury prevention techniques, equipments, and devices.
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