Establish Biomimetic Pressurization Conditions
- Achieve physiologically relevant impulsive pressure to extend the lower loading limit to 0.2 MPa and closer to suggested mild TBI levels.
- Pressurization of neuronal cells while being cultured on biomimicking extracellular matrix (ECM) protein layer which may provide a cell pressurization condition more physiologically relevant to that experienced by the neuronal cells embedded in brain tissues subjected to impulsive pressure loading.
- Investigate long-term neuronal cell response after impulsive pressurization by examining them for up to 4 weeks after pressurization.
Reveal the Molecular Mechanism of Neuronal Functional Loss
- Examine the molecular mechanism of TBI by investigating calcium-mediated calpain activation in SH-SY5Y neuronal cells and its role in the breakdown of microtubule-associated spectrin protein. Calpain-activated spectrin damage has received considerable attention as one of the key mediators in axonal injury.
- Conduct parametric studies including systematic studies using optimized ECM layer thickness and cell physiology effect from cell-to-cell interaction and neuronal cell co-culture with astrocyte.
- Primary cells for pressurization. As part of the animal model study, we plan to apply the same dynamic loading technique and biological diagnostics to investigate the impulsive pressurization effects on the primary neuronal cells cultured from the model animal’s brain sample.
Bubble Size Effect: Test 1
Bubble Size Effect: Test 2
Differentiation of SH-SY5Y cells
SH-SY5Y human neuroblastoma cells were maintained in DMEM with 10% FBS and 1% penicillin/streptomycin at 37°C. The cells were differentiated to neuronal cells with 10 μM retinoic acid (RA).
Cells were observed by immuno- fluorescence, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Actin cytoskeletons were stained with rhodamine phalloidin and nuclei were stained with DAPI.