Tuesday  |  Salon 8  |  10:20 AM–10:40 AM

#15826, Determination of Critical Cellular Traumatic Brain Injury Thresholds

Traumatic brain injury (TBI) caused by inertial, blunt or blast forces poses a significant threat to both civilian and military communities. However, the exact force magnitudes and modalities that initiate TBI at the cellular level, i.e., critical cellular thresholds of injury, are still unknown. This is essential to understanding TBI, as clinical effects of TBI stem from cellular signaling cascades arising from mechanical insult to the head. Considering both the structural and cellular diversity of the brain, we developed a unique experimental in vitro approach for resolving impact strain levels at the resolution of single cells to determine the first cell-based injury thresholds for traumatic brain injuries.

We fabricated neural cell networks following our previously established 3D in vitro TBI model. Primary Sprague-Dawley rat cortices are isolated from p0-p1 rat pups. Isolated cortical cells are suspended in rat tail collagen-I hydrogel, cast into 3D molds, and allowed to undergo synaptogenesis over 7 days in vitro (DIV) prior to mechanical deformations. Samples are stained with Calcein AM and a 3D reference image at 7 DIV is acquired using an inverted Nikon-A1 multiphoton microscope. Then, samples are subjected to precisely controlled mechanical loadings, applied by a custom device capable of inducing deformation under tension at rates up to 240 s-1 using voice coil linear actuators. Samples are then returned to the incubator and fixed at 24 hours post injury in 4% paraformaldehyde and 8% sucrose and immunolabeled for enzymatic markers of interest (e.g., cleaved caspase-3, RIPK1). Cell population viability is assessed by registering post-injury images with pre-injury images and quantifying expression of fluorescent cellular injury markers. To fully resolve the cellular injury thresholds, we perform segmentation followed by mapping the local impact strains onto the branched, 3D cell structures with an in-house developed MATLAB code package, generating a statistical model that predicts injury at the resolution of a single cell. Through this detailed analysis, we evaluate the strain magnitude, rate, and modality-dependent neural injury and resolve cellular injury thresholds. This approach has a broad application for in vitro and in situ mechanical impact experimentation and provides the first comprehensive analysis toolbox that resolves cellular TBI thresholds and could lead to the development of future generations of protective equipment.

Annalise Daul   University of Wisconsin-Madison
Jessica Park   University of Wisconsin-Madison
Luke Summey   University of Wisconsin-Madison
Jamie Sergay   University of Wisconsin-Madison
Jing Zhang   University of Wisconsin-Madison
Christian Franck   University of Wisconsin-Madison