To study real-time changes in the Ca2+ spiking patterns using P19-derived neuron-like cells (i.e. neuronal cell differentiation was specifically induced) in response to a millimeter wave exposure.
Transient elevations in cytosolic Ca2+ concentration, referred to as spikes, are a nearly universal mode of signaling in both excitable and non-excitable cells. While the role of oscillatory Ca2+ signals is not fully understood, it is evident that spatial and temporal patterns of Ca2+ dynamics (e.g. spiking amplitude and frequency and spatial distribution) are important characteristics of cellular regulatory pathways.
To elucidate the possible Ca2+ influx/efflux pathways the cells were treated with pharmacological inhibitors (amongst others ω-conotoxin GVIA: N-type Ca2+ channel blocker, thapsigargin: Ca2+ ATPase inhibitor, cytochalasin D: actin polymerization inhibitor, U73122: phospholipase C inhibitor).
To study the effect of millimeter wave exposure on actin filament organization, the authors used fibroblasts, which are known to express abundant actins (in contrast to P19-derived neuronal cells).
In a control series of experiments, cells were investigated at the desired temperature in the range 25-42°C.
| Exposure | Parameters |
|---|---|
|
Exposure 1:
94 GHz
Exposure duration:
continuous for up to 60 min
|
|
| Frequency | 94 GHz |
|---|---|
| Type | |
| Exposure duration | continuous for up to 60 min |
| Exposure source | |
|---|---|
| Chamber | exposure chamber consisting of 24 mm x 30 mm cover slips separated by cover-slip-thick spacers |
| Setup | fixed tuned Gunn oscillator connected to WR10 waveguide operating in TE10 mode, directed to the cell exposure chamber placed on a microscope stage; 2.54 mm x 1.27 mm exposure exit aperture touching the glass of the exposure chamber |
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| power density | 3.1 kW/m² | minimum | measured | - | - |
| power density | 18.6 kW/m² | maximum | measured | - | - |
Millimeter wave exposure at 18.6 kW/m² significantly increased the Ca2+ spiking frequency in active cells (i.e. in cells exhibiting Ca2+ oscillation). However, at the lower power densities (3.1-7.8 kW/m²), no statistically significant increase in Ca2+ spiking frequency compared to unexposed cells was found.
The N-type calcium channels, phospholipase C, and actin cytoskeleton appear to be involved in mediating increased Ca2+ spiking (as shown by specific inhibitors which effectively suppressed spiking). Reorganization of the actin microfilaments by a 94 GHz field seems to play a crucial role in modulating not only Ca2+ activity but also cell biomechanics (decrease of cell elasticity). A millimeter wave exposure at 18.6 kW/m² for 30 minutes caused some damage to the actin structure (in contrast, a hyperthermia treatment at 42°C for 30 min did not result in substantial actin reorganization).
Many but not all observed cellular responses to millimeter wave exposure were similar to thermally induced effects. For example, exposure to a 94 GHz field induced nitric oxide production in some morphologically distinct neuronal cells that could not be reproduced by thermal heating of the cells up to 42°C.
The data may be beneficial in developing novel approaches to control cellular behaviors by external physical stimulation for tissue engineering and regenerative medicine applications.
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