Blast Cellular Mechanics


Overview
Kolsky Bar
The cellular and molecular mechanism of blast induced TBI and its secondary progression are not fully understood. Based on the assumption that blast-induced overpressure, or high stress impulse, is the main cause of TBI, we investigated the effects of impulsive pressurization on neuronal cell functions. A novel impulsive pressurization apparatus capable of controlling pressure level and duration time has been built using the Kolsky bar set-up.

SH-SY5Y human neuroblastoma cells, which are differentiated to neuronal cells with the exposure to retinoic acid, are placed inside the pressure chamber and exposed to impulsive pressurization of which the level mimics blast-induced pressure. By examining axon shape change, cell apoptosis, and neuronal cell marker (MAP2) gene expression, we expect to reveal the cell/molecular changes after pressurization. This will be the first step towards revealing correlation between pressurization conditions (magnitude, duration) and neuronal cell responses in blast TBI conditions.
Project Outline


Establish Biomimetic Pressurization Conditions
  1. Achieve physiologically relevant impulsive pressure to extend the lower loading limit to 0.2 MPa and closer to suggested mild TBI levels.
  2. 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.
  3. 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
  1. 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.
  2. 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.
  3. 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.

Kolsky Bar

KolskyBarCloseup
Blast-mimicking impulsive pressurization is conducted through use of the Kolsky bar. The direct compression Kolsky bar works by storing strain energy in the bar between the engaged friction clamp and the compressing scissors jack. When the friction clamp is released, by fracturing the locking bolt, the energy in the stored section is released as a near square wave of strain. When the wave reaches an impedance change, such as is at the sample, part of the wave will bounce back while the remainder will pass through the material, in this case an in-vitro cell containment chamber.
Kolsky Bar Diagram

Bubble Size Effect: Test 1



Small Bubble Graph

A small bubble is ejected and its associated transmitted bar history shows little difference from the incident bar history.

Bubble Size Effect: Test 2



Collapsed Bubble Graph

The bubble is collapsed and not ejected, resulting in a shortened transmitted pulse.

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).

Pressurization
Containment Chamber
Cells cultured on glass cover slip were treated with RA to induce axonal outgrowth and exposed to 0.5, 1, and 2 MPa in the pressure chamber for about 0.5 ms. The SH-SY5Y cells were incubated for 0 h and 24 h.
Cell Viability
Cell Viability Graph
Cells incubated for 24h then treated with 0.2 mg/ml MTT for 3h at 37°C. The absorbance was measured at 570 nm.
Fluorescence Staining

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.