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

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 stimulate the slices.

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

#### **Figure 7.**

*Astrocyte Ca2+ response to a shear pulse in a hippocampal slice. (a) The slice is co-loaded with Flou-4 (green) and SR101 (red). (b) Time sequence of Ca2+ images showing Ca2+ peaks at different times in selected cells. (c) Typical traces of astrocyte Ca2+ response of individual cells. (d) Statistics of peak amplitudes and frequency.*

conditions (with the caveat that these may be treated as samples of extreme TBI). To discriminate the astrocytes from neurons, the slices were loaded with SR101 that serves as a marker for astrocytes, as shown in **Figure 7(a)**. Shear stimulated slices showed an acute Ca2+ increase in selected cells that peaked in 1 to 4 s and returned to baseline levels within 20 s, consistent with observations in cell cultures. Most of the cells showed one dominant peak, but some (~20% of cells) responded with multiple peaks (trace 4, **Figure 7(c)**). The average peak Ca2+ was much higher than the spontaneous Ca2+ transients that were 10–20% of the shear-induced peak (traces 5 and 6, **Figure 7(c)**). These Ca2+ peaks were eliminated with 10 μM Gd3+, which is a nonspecific MSC blocker. This confirms the observations that shear stress-induced transient Ca2+ peaks are via MSCs.

It is worthwhile pointing out that the fluid shear stress generates well-controlled forces on the apical surface of cell cultures. In the slide experiments, once the deformation reaches a deeper layer of the cells, the poroelastic nature of the tissue will also modify the forces, so the stimulus profile itself may change as it propagates.

#### **9. Conclusion**

Using advanced technologies that can generate fast shear stimuli mimicking forces that cause TBI, we have demonstrated that cell response to mechanical stimuli is nonlinear and the features of the stimuli play a critical role. Using FRET-based

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**Author details**

Mohammad Mehdi Maneshi and Susan Z. Hua\*

\*Address all correspondence to: zhua@buffalo.edu

provided the original work is properly cited.

and Biophysics, University at Buffalo, Buffalo, New York, USA

Department of Mechanical and Aerospace Engineering, Department of Physiology

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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

**Acknowledgements**

force probes, the nonlinearity is shown to be a direct result of nonuniform force distribution within the cytoskeleton. Rapid shear pulses generate a heterogeneous distribution of cytoskeletal forces in cells, in both time and space. The cytoskeletal forces and their modulation on cell membrane tension open MSCs that mediate Ca2+ response. These early response signals can be small and transient. However, the integration of these signals leads to pathology and the progression of TBI.

This work was supported by the National Institutes of Health grant NS085517 and National Science Foundation grants CMMI-1537239 and CMMI-2015964.

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

force probes, the nonlinearity is shown to be a direct result of nonuniform force distribution within the cytoskeleton. Rapid shear pulses generate a heterogeneous distribution of cytoskeletal forces in cells, in both time and space. The cytoskeletal forces and their modulation on cell membrane tension open MSCs that mediate Ca2+ response. These early response signals can be small and transient. However, the integration of these signals leads to pathology and the progression of TBI.
