**7.1 The cell membrane**

The cell membrane consists of a lipid bilayer incorporating the membrane proteins including integral proteins such as transmembrane ion channels and receptors and peripheral proteins that loosely attach to the outer side of the cell membrane. Through the functional proteins, the cell membrane selectively controls the transport of ions, water, and macromolecules between the intracellular and extracellular compartments. Inside the cell, the lipid bilayer intimately adheres to the cortical cytoskeleton that provides the support for membrane topography and integrity [118]. On the outside is a hair-like structure called the glycocalyx. Depending on the cell type and local environments, the cell membrane may have tension at its resting state, called pretension. Several factors contribute to the pretension, including internal forces exerted by the cytoskeleton, osmotic pressure from the cytosol, and the forces resulting from cell-substrate interactions at adhesions that can be passed by cytoskeleton [119].

#### **7.2 Effect of shear stress on cell membrane tension**

The bilayer tension can be measured using lipid-soluble molecular rotor probe FCVJ [120] whose mobility is commonly used to extract the lipid bilayer fluidity or viscosity. An increase in bilayer tension increases the fluidity in cell membrane, causing a decrease fluorescent intensity of probes [121]. We measured the bilayer tension in astrocyte membranes using the molecular rotor probe FCVJ with the above-described microfluidic chip. As demonstrated in **Figure 6(a)**, a square pulse of fluid shear (23 dyn/cm2 , 400 ms) generates a gradient in the membrane tension, with higher tension at the upstream edge of the cell and a lower tension (compression) at the distal edge. Both tension and compression recover back to the initial state within ~30 ms. In comparison, the same shear pulse generated a much longerlasting tension in actinin at the upstream edge of the cell. **Figure 6(b)** and **(c)** illustrates these different characteristics. Interestingly, the membrane tension at the front edge increased much slower than compression at the downstream edge, suggesting that there exists a pretension in the membrane probably via the cortical actin cytoskeleton. The pretension resists the effect of shear force at the upstream edge. In addition, buckling (rapid compression) can occur at the downstream edge. It has been shown that buckling of the lipid membrane can occur at a similar timescale that takes ~150 ms to saturate [122].

#### **Figure 6.**

*Change in membrane tension measured using FCVJ molecular rotor incorporated into the astrocyte membrane. (a) Left panel: Membrane tension in response to a shear pulse (23 dyn/cm2 , 400 ms), where downward inflection indicates tension and upward inflection compression. Labels 1–4 correspond to the regions shown in the image in the right panel. (b) and (c) comparison of membrane tension and actinin force, respectively, in response to a same shear pulse. They show membrane tension at upstream edge increases much slower than actinin force.*

A rapid shear pulse generates a gradient in the membrane tension. The spatiotemporal distribution of tension is dependent on the rise time of the shear force. When subjected to a slow ramp-up shear stress, the tension gradient was reduced significantly.

#### **7.3 Membrane tension gradients are coupled to cytoskeletal forces**

It has been shown that mechanosensitive channels can be activated by bilayer tension in the lipid vesicles without the cytoskeleton [123]. However, membrane tension measurements show that the membrane tension at the upstream edge increases rather slowly compared with the Ca2+ rise. This suggests that the Ca2+ channel could not have been activated by the bilayer tension alone and additional mechanisms are likely involved. Slowly ramping the shear stress was not able to change the membrane tension at the upstream edge until it reaches a shear stress threshold. This threshold exactly matches the threshold observed for the rise in the cytoskeletal tension. Therefore, both cytoskeleton and membrane tensions are involved.

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stimulate the slices.

*Early Cell Response to Mechanical Stimuli during TBI DOI: http://dx.doi.org/10.5772/intechopen.93295*

cytoskeleton, thereby increasing its stress [116, 125].

**7.4 Effect of shear stress on cell membrane**

lated by the intact cytoskeleton forces.

Time-dependent analysis shows that shear pulse generates faster and longerlasting tension in actinin at the front edge of the cell compared to membrane tension, suggesting the cytoskeleton imposes the limiting force at the upstream edge of the cells. In the presence of cytochalasin D that disrupts F-actin, this limit is lifted, and a more predominant membrane tension is seen at the upstream edge compared with controls. While disruption of F-actin diminished tension gradients in the cytoskeleton, it also eliminated the fast influx of Ca2+ at the upstream edge of the cell [114]. Thus, cell membrane tension is modulated by the cytoskeleton stresses since the bilayer alone has a minimal ability to bear a large tension gradient.

Moreover, fluid shear by itself is not likely to affect the cell membrane directly since most of the velocity gradient is absorbed by the glycocalyx, leaving little friction at the bilayer [124]. Fluid drag applied to the cell body will pull on the

The effect of shear stress on the lipid membrane response has been variously modeled. The generally proposed model for lipid bilayers is the fluid mosaic model [126]. This model describes the structure of the plasma membrane as a mosaic of components including phospholipids, cholesterol, proteins, and carbohydrates that give the membrane a 2D viscous fluid characteristic. Since the lipid membrane is expected to be intrinsically incompressible, the membrane tension changes could not be explained by solely considering the pure fluid characteristics. Hence, lipid bilayer and MSC interaction via a fluid, or instability of curved stress states of a non-lamellar lipid bilayer, is not easily justifiable within the scope of the fluid mosaic model [127]. Subsequent studies suggest that the lipid membrane is more mosaic rather than fluid [128]. Other models such as the surface model [129] could predict the interaction of lipids and proteins better by allowing both compression and tension changes in the membrane. Based on such models, interactions of lipid and MSCs could be explained by an increase in distance of lipid hydrophobic head groups in the vicinity of proteins and a decrease in outer layer viscosity of membrane [130]. However, without an input

from underneath cytoskeleton, the tension gradient in bilayer is minimal.

**8. Evaluation of astrocytic Ca2+ response to shear in brain slices**

This leads to the conclusion that shear stress generates tension gradients in the cell cortex and that membrane tension gradients are coupled to cytoskeletal forces to mediate Ca2+ influx. The time-dependent membrane tension gradient is modu-

In the brain, neurons and astrocytes are intimately connected and function through a three-dimensional circuit that passes information waves. The interplay between them is evident in bidirectional glutamatergic astrocyte-neuron signaling in a Ca2+-dependent fashion. A common consequence of TBI is the alternations of this information flow. While *in vitro* experiments described above permit high-resolution measurements, the environment differs from *in vivo*. To better approximate the *in situ* conditions, and to see how much of the *in vitro* results are applicable *in vivo*, similar sets of experiments can be performed to brain slides, since slides would contain the native cell types and their local environments as *in vivo*. To a closer approximation, a modified shear chamber was used to apply fluid shear stress to mechanically

The Ca2+ response in acute slices from rats is demonstrated in **Figure 7**, which

shows how shear stimuli modulate Ca2+ response in cells under physiological

*Early Cell Response to Mechanical Stimuli during TBI DOI: http://dx.doi.org/10.5772/intechopen.93295*

*Recent Advances in Biomechanics*

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significantly.

**Figure 6.**

*actinin force.*

A rapid shear pulse generates a gradient in the membrane tension. The spatiotemporal distribution of tension is dependent on the rise time of the shear force. When subjected to a slow ramp-up shear stress, the tension gradient was reduced

*Change in membrane tension measured using FCVJ molecular rotor incorporated into the astrocyte membrane.* 

*inflection indicates tension and upward inflection compression. Labels 1–4 correspond to the regions shown in the image in the right panel. (b) and (c) comparison of membrane tension and actinin force, respectively, in response to a same shear pulse. They show membrane tension at upstream edge increases much slower than* 

*, 400 ms), where downward* 

It has been shown that mechanosensitive channels can be activated by bilayer tension in the lipid vesicles without the cytoskeleton [123]. However, membrane tension measurements show that the membrane tension at the upstream edge increases rather slowly compared with the Ca2+ rise. This suggests that the Ca2+ channel could not have been activated by the bilayer tension alone and additional mechanisms are likely involved. Slowly ramping the shear stress was not able to change the membrane tension at the upstream edge until it reaches a shear stress threshold. This threshold exactly matches the threshold observed for the rise in the cytoskeletal tension. Therefore, both cytoskeleton and membrane tensions are involved.

**7.3 Membrane tension gradients are coupled to cytoskeletal forces**

*(a) Left panel: Membrane tension in response to a shear pulse (23 dyn/cm<sup>2</sup>*

Time-dependent analysis shows that shear pulse generates faster and longerlasting tension in actinin at the front edge of the cell compared to membrane tension, suggesting the cytoskeleton imposes the limiting force at the upstream edge of the cells. In the presence of cytochalasin D that disrupts F-actin, this limit is lifted, and a more predominant membrane tension is seen at the upstream edge compared with controls. While disruption of F-actin diminished tension gradients in the cytoskeleton, it also eliminated the fast influx of Ca2+ at the upstream edge of the cell [114]. Thus, cell membrane tension is modulated by the cytoskeleton stresses since the bilayer alone has a minimal ability to bear a large tension gradient.

Moreover, fluid shear by itself is not likely to affect the cell membrane directly since most of the velocity gradient is absorbed by the glycocalyx, leaving little friction at the bilayer [124]. Fluid drag applied to the cell body will pull on the cytoskeleton, thereby increasing its stress [116, 125].

#### **7.4 Effect of shear stress on cell membrane**

The effect of shear stress on the lipid membrane response has been variously modeled. The generally proposed model for lipid bilayers is the fluid mosaic model [126]. This model describes the structure of the plasma membrane as a mosaic of components including phospholipids, cholesterol, proteins, and carbohydrates that give the membrane a 2D viscous fluid characteristic. Since the lipid membrane is expected to be intrinsically incompressible, the membrane tension changes could not be explained by solely considering the pure fluid characteristics. Hence, lipid bilayer and MSC interaction via a fluid, or instability of curved stress states of a non-lamellar lipid bilayer, is not easily justifiable within the scope of the fluid mosaic model [127]. Subsequent studies suggest that the lipid membrane is more mosaic rather than fluid [128]. Other models such as the surface model [129] could predict the interaction of lipids and proteins better by allowing both compression and tension changes in the membrane. Based on such models, interactions of lipid and MSCs could be explained by an increase in distance of lipid hydrophobic head groups in the vicinity of proteins and a decrease in outer layer viscosity of membrane [130]. However, without an input from underneath cytoskeleton, the tension gradient in bilayer is minimal.

This leads to the conclusion that shear stress generates tension gradients in the cell cortex and that membrane tension gradients are coupled to cytoskeletal forces to mediate Ca2+ influx. The time-dependent membrane tension gradient is modulated by the intact cytoskeleton forces.
