Shear Forces during Blast, Not Abrupt Changes in Pressure Alone, Generate Calcium Activity in Human Brain Cells

Blast-Induced Traumatic Brain Injury (bTBI) describes a spectrum of injuries caused by an explosive force that results in changes in brain function. The mechanism responsible for primary bTBI following a blast shockwave remains unknown. We have developed a pneumatic device that delivers shockwaves, similar to those known to induce bTBI, within a chamber optimal for fluorescence microscopy. Abrupt changes in pressure can be created with and without the presence of shear forces at the surface of cells. In primary cultures of human central nervous system cells, the cellular calcium response to shockwaves alone was negligible. Even when the applied pressure reached 15 atm, there was no damage or excitation, unless concomitant shear forces, peaking between 0.3 to 0.7 Pa, were present at the cell surface. The probability of cellular injury in response to a shockwave was low and cell survival was unaffected 20 hours after shockwave exposure

Low-field, high-gradient NMR shows diffusion contrast consistent with localization or motional averaging of water near surfaces

Nuclear magnetic resonance (NMR) measurements of water diffusion have been extensively used to probe microstructure in porous materials, such as biological tissue, however primarily using pulsed gradient spin echo (PGSE) methods. Low-field single-sided NMR systems have built-in static gradients (SG) much stronger than typical PGSE maximum gradient strengths, which allows for the signal attenuation at extremely high b-values to be explored. Here, we perform SG spin echo (SGSE) and SG stimulated echo (SGSTE) diffusion measurements on biological cells, tissues, and gels. Measurements on fixed and live neonatal mouse spinal cord, lobster ventral nerve cord, and starved yeast cells all show multiexponential signal attenuation on a scale of b with significant signal fractions observed at b × D0 ≫ 1 with b as high as 400 ms/μm2. These persistent signal fractions trend with surface-to-volume ratios for these systems, as expected from porous media theory. An exception found for the case of fixed vs. live spinal cords was attributed to faster exchange or permeability in live spinal cords than in fixed spinal cords on the millisecond timescale. Data suggests the existence of multiple exchange processes in neural tissue, which may be relevant to the modeling of time-dependent diffusion in gray matter. The observed multi-exponential attenuation is from protons on water and not macromolecules because it remains proportional to the normalized signal when a specimen is washed with D2O. The signal that persists to b × D0 ≫ 1 is also drastically reduced after delipidation, indicating that it originates from lipid membranes that restrict water diffusion. The multi-exponential or stretched exponential character of the signal attenuation at b × D0 ≫ 1 appears mono-exponential when viewed on a scale of (b×D0)1/3, suggesting it may originate from localization or motional averaging of water near membranes on sub-micron length scales. To try to disambiguate these two contributions, signal attenuation curves were compared at varying temperatures. While the curves align when normalizing them using the localization length scale, they separate on a motional averaging length scale. This supports localization as the source of non-Gaussian displacements, but this interpretation is still provisional due to the possible confounds of heterogeneity, exchange, and relaxation. Measurements on two types of gel phantoms designed to mimic extracellular matrix, one with charged functional groups synthesized from polyacrylic acid (PAC) and another with uncharged functional groups synthesized from polyacrylamide (PAM), both exhibit signal at b × D0 ≫ 1, potentially due to water interacting with macromolecules. These preliminary finding motivate future research into contrast and attenuation mechanisms in tissue with low-field, high-gradient NMR

(A) Schematic diagram of the pneumatic device and modified 96 well plate attached to a microscope stage

<p>(<b>B</b>) Pressure profile measurements of the simulated open field blast shockwave compared to a classical Friedlander curve of the same peak pressure and positive phase duration. Average of 6 measurements is shown with standard error.</p

Shear forces are regulated by well fluid volume.

<p>Three consecutive frames of 400 msec duration captured beads before (a), during (b), and after (c) the application of a ∼11 atm peak pressure blast with (180 µl fluid volume) and without shear (380 µl fluid volume). Significant bead motion due to shear is registered in top frame b. Note, a single bead did not move (arrow); this bead, presumably immobilized due to adhesion to the surface, allows one to check for stability of the stage during the blast. In the absence of shear, the application of the same peak pressure blast does not show any bead displacement (bottom frame b).</p

The effect of shear forces on the response of cells to an

<p>∼<b>11 atm peak pressure simulated blast.</b> (<b>A</b>) Sequential fluo-4 calcium imaging of a dissociated primary human fetal CNS cell culture without shear (380 µl well fluid volume); the blast occurred at time 100 seconds. (<b>B</b>) Sequential fluo-4 calcium imaging of the same cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039421#pone-0039421-g003" target="_blank">Figure 3A</a> with shear (150 µl well fluid volume) using the same blast parameters; the blast occurred at time 100 seconds. (<b>C</b>) ΔF/F in time of the cells shown in Figures A and B for 10 minutes before, during, and after the blast. (<b>D</b>) Normalized response of two cells shown in Figure B, panel 1, indicating that the calcium response does not occur simultaneously in cells but sequentially; time to peak response is offset by ∼30 seconds in this example and is consistent with propagation (see Video S4). (<b>E</b>) Integrated Ca²⁺ Response, integral of ΔF/F over time following the blast, without and with shear forces in the same well, for n = 6 pair-matched experiments (11 atm; 10 atm above ambient pressure). The first blast was without shear forces. The response to a lethal peak pressure of 15 atm (14 atm above ambient pressure) with no shear forces is also shown (n = 10) (<b>F</b>) Correlation (r² = 0.99) between Integrated Ca²⁺ Response following a 11 atm peak pressure blast and well fluid volume for the 3 volume conditions evaluated; 150–200, 250–300, and 380 µl with n = 14, 7 and 25, respectively. Fluid volume and Ca²⁺ Response error bars are the range and SEM, respectively.</p

A Flexible Reporter System for Direct Observation and Isolation of Cancer Stem Cells

Summary: Many tumors are hierarchically organized with a minority cell population that has stem-like properties and enhanced ability to initiate tumorigenesis and drive therapeutic relapse. These cancer stem cells (CSCs) are typically identified by complex combinations of cell-surface markers that differ among tumor types. Here, we developed a flexible lentiviral-based reporter system that allows direct visualization of CSCs based on functional properties. The reporter responds to the core stem cell transcription factors OCT4 and SOX2, with further selectivity and kinetic resolution coming from use of a proteasome-targeting degron. Cancer cells marked by this reporter have the expected properties of self-renewal, generation of heterogeneous offspring, high tumor- and metastasis-initiating activity, and resistance to chemotherapeutics. With this approach, the spatial distribution of CSCs can be assessed in settings that retain microenvironmental and structural cues, and CSC plasticity and response to therapeutics can be monitored in real time. : In this article, Wakefield and colleagues show that a subpopulation of cancer cells with characteristics of cancer stem cells can be identified and visualized in vitro and in vivo using a lentiviral-based fluorescent reporter that responds to the presence of the stemness master transcription factors SOX2 and OCT4