**Acknowledgements**

*Recent Advances in Biomechanics*

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

*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* 

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.

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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**9. Conclusion**

**Figure 7.**

*frequency.*

transient Ca2+ peaks are via MSCs.

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