**2.3 Earliest cellular events in injury models**

Two early events that are caused by external forces are known: a Ca2+ influx and an associated neurotransmitter release [25–27]. An increase in intracellular Ca2+ is universally seen in brain cells that have been subjected to mechanical perturbations regardless of the nature of the forces [28–30]. There are many Ca2+-dependent processes that can be activated during TBI, including cell swelling [31–33], cytoskeletal disruption [34–36], membrane poration [37, 38], failure of ATP-dependent membrane pumps [39, 40], and mitochondrial dysfunction and apoptosis [41–44]. The Ca2+ loaded or injured neurons release neurotransmitters including glutamate [45, 46]. The release of glutamate and other excitatory amino acids, in turn, can lead to cytotoxic injury and cell death [47–49]. These occurrences clearly show that the early responses of brain cells to mechanical stimuli are responsible for the subsequent pathology.

## **3. Shear forces and their assays**

#### **3.1 TBI-induced internal forces**

The external force results in the development of internal forces within the skull, causing brain injury. Several types of internal forces can occur depending on the type of incidents. For instance, a blast-generated shockwave initially changes the pressure inside the skull. The abrupt motion of the head causes the acceleration or deceleration to generate compression, stretching, and shear forces at multiple places inside the skull [2].

*In vitro* studies using cultured cells show that excessive pressure (above 10 atm) is required to stimulate a cellular response. This level of pressure is much higher than the overall pressure measured *in vivo* within the brain [50]. Using a blast chamber to apply rapid pressures and shear forces to cell cultures, Raven et al. showed that cells are more responsive to shear forces than to hydrostatic pressures [51].

Tensile forces are primarily localized at impact points and have been commonly studied using stretching methods. In stretching models, cell cultures or tissues are placed on a flexible substrate that can be stretched with a vacuum pulse [52] or by piezoelectric actuators [53]. In these studies, cells produced a measurable Ca2+ response only when they experienced very large strains (up to 40%) [54–56]. In contrast, an overall cell deformation of ~4% due to shear pulses was sufficient to cause a similar Ca2+ response [57]. These studies show that brain cells are more susceptible to shear forces than other types of internal force.

Most of the studies that have investigated diffusive brain injury due to shear forces have focused on the injury of neurons. A sliding between two tissue layers can break the long thin nerve fibers (called axons) of neurons by their extension across the layers [58, 59]. However, shear stresses have been found to induce drastic responses in cells (neurons and astrocytes) even without any noticeable damage to the axons, leading to cell damage and cell death [60]. In other words, subcellular signaling is likely to be a more causal pathway than obvious physical damage in these cells.

#### **3.2 Microfluidic assays for shear forces**

Earlier studies variously used a rotating disk, air blow, or pulsed media to apply shear stresses to the cells [61–63]. Following the application of force stimuli, it was found that transient shear deformations tended to increase the membrane permeability to Ca2+ and small dye molecules and decrease the cell viability [64]. A blast chamber was also used to apply rapid pressure and shear forces to cell cultures. It was found that Ca2+ response was more sensitive to shear forces than to hydrostatic pressures [51]. These methods provide controlled shear stimuli, followed by live cell measurements. In real scenarios of TBI, the forces increase in milliseconds, and small elastic deformations in cells occur in real time [16]. Thus, the challenge for the shear stress assays is the ability to rapidly ramp the shear forces and an ability to reliably measure cells' response in real time.

**151**

the head.

**Figure 2.**

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

the schematic of the experimental setup.

specific internal forces need to be defined.

**4. The response of cells to transient shear forces**

In this chapter, we introduce a high-speed programmable microfluidic assay tool to apply precise shear forces to cultured cells [30]. The fluid shear is controlled by a fast pressure servo [65] that is capable of generating pressure pulses with a time resolution of ~1 ms in the microfluidic chamber [30, 65]. The millisecond resolution allows the ability to program arbitrary waveforms emulating the waveforms recorded in TBI [66]. The microfluidic chamber allows high-resolution optical microscopy, which enables *in situ* measurements of changes in intracellular Ca2+ and protein forces in single cells during the very application of forces. **Figure 2** displays

*Schematic of the pressure servo-driven microfluidic chamber. High-speed pressure servo is connected to a* 

*microfluidic chamber to generate controlled fluid shear stimuli on cultured cells.*

The severity of TBI is conventionally classified as mild, moderate, or severe, depending on the extent of the damage to the brain tissue. In the 1950s, the head injury tolerance to external forces began to be evaluated by dropping embalmed corpse heads on a rigid surface and examining the resulting profile of the skull fracture. Linear skull fracture came to be used as the criterion of injury. These studies led to the first quantitative tolerance curve to be established, now known as the Wayne State Tolerance Curve (WSTC) [67, 68]. This curve describes the influence of magnitude and duration of external loads on the severity of injury to

However, the internal forces are not directly proportional to the external load, which comprise a more complex load profile depending on the type of forces. It is now also known that most cells show a physiological response at much lower internal force levels that are below the injury criteria. Therefore, the thresholds for *Early Cell Response to Mechanical Stimuli during TBI DOI: http://dx.doi.org/10.5772/intechopen.93295*

**Figure 2.**

*Recent Advances in Biomechanics*

**3. Shear forces and their assays**

**3.1 TBI-induced internal forces**

regardless of the nature of the forces [28–30]. There are many Ca2+-dependent processes that can be activated during TBI, including cell swelling [31–33], cytoskeletal disruption [34–36], membrane poration [37, 38], failure of ATP-dependent membrane pumps [39, 40], and mitochondrial dysfunction and apoptosis [41–44]. The Ca2+ loaded or injured neurons release neurotransmitters including glutamate [45, 46]. The release of glutamate and other excitatory amino acids, in turn, can lead to cytotoxic injury and cell death [47–49]. These occurrences clearly show that the early responses of brain cells to mechanical stimuli are responsible for the subsequent pathology.

The external force results in the development of internal forces within the skull, causing brain injury. Several types of internal forces can occur depending on the type of incidents. For instance, a blast-generated shockwave initially changes the pressure inside the skull. The abrupt motion of the head causes the acceleration or deceleration to generate compression, stretching, and shear forces at multiple places inside the skull [2]. *In vitro* studies using cultured cells show that excessive pressure (above 10 atm) is required to stimulate a cellular response. This level of pressure is much higher than the overall pressure measured *in vivo* within the brain [50]. Using a blast chamber to apply rapid pressures and shear forces to cell cultures, Raven et al. showed that cells

Tensile forces are primarily localized at impact points and have been commonly studied using stretching methods. In stretching models, cell cultures or tissues are placed on a flexible substrate that can be stretched with a vacuum pulse [52] or by piezoelectric actuators [53]. In these studies, cells produced a measurable Ca2+ response only when they experienced very large strains (up to 40%) [54–56]. In contrast, an overall cell deformation of ~4% due to shear pulses was sufficient to cause a similar Ca2+ response [57]. These studies show that brain cells are more

Most of the studies that have investigated diffusive brain injury due to shear forces have focused on the injury of neurons. A sliding between two tissue layers can break the long thin nerve fibers (called axons) of neurons by their extension across the layers [58, 59]. However, shear stresses have been found to induce drastic responses in cells (neurons and astrocytes) even without any noticeable damage to the axons, leading to cell damage and cell death [60]. In other words, subcellular signaling is likely to

Earlier studies variously used a rotating disk, air blow, or pulsed media to apply shear stresses to the cells [61–63]. Following the application of force stimuli, it was found that transient shear deformations tended to increase the membrane permeability to Ca2+ and small dye molecules and decrease the cell viability [64]. A blast chamber was also used to apply rapid pressure and shear forces to cell cultures. It was found that Ca2+ response was more sensitive to shear forces than to hydrostatic pressures [51]. These methods provide controlled shear stimuli, followed by live cell measurements. In real scenarios of TBI, the forces increase in milliseconds, and small elastic deformations in cells occur in real time [16]. Thus, the challenge for the shear stress assays is the ability to rapidly ramp the shear forces and an ability to

are more responsive to shear forces than to hydrostatic pressures [51].

susceptible to shear forces than other types of internal force.

be a more causal pathway than obvious physical damage in these cells.

**3.2 Microfluidic assays for shear forces**

reliably measure cells' response in real time.

**150**

*Schematic of the pressure servo-driven microfluidic chamber. High-speed pressure servo is connected to a microfluidic chamber to generate controlled fluid shear stimuli on cultured cells.*

In this chapter, we introduce a high-speed programmable microfluidic assay tool to apply precise shear forces to cultured cells [30]. The fluid shear is controlled by a fast pressure servo [65] that is capable of generating pressure pulses with a time resolution of ~1 ms in the microfluidic chamber [30, 65]. The millisecond resolution allows the ability to program arbitrary waveforms emulating the waveforms recorded in TBI [66]. The microfluidic chamber allows high-resolution optical microscopy, which enables *in situ* measurements of changes in intracellular Ca2+ and protein forces in single cells during the very application of forces. **Figure 2** displays the schematic of the experimental setup.

## **4. The response of cells to transient shear forces**

The severity of TBI is conventionally classified as mild, moderate, or severe, depending on the extent of the damage to the brain tissue. In the 1950s, the head injury tolerance to external forces began to be evaluated by dropping embalmed corpse heads on a rigid surface and examining the resulting profile of the skull fracture. Linear skull fracture came to be used as the criterion of injury. These studies led to the first quantitative tolerance curve to be established, now known as the Wayne State Tolerance Curve (WSTC) [67, 68]. This curve describes the influence of magnitude and duration of external loads on the severity of injury to the head.

However, the internal forces are not directly proportional to the external load, which comprise a more complex load profile depending on the type of forces. It is now also known that most cells show a physiological response at much lower internal force levels that are below the injury criteria. Therefore, the thresholds for specific internal forces need to be defined.

#### **4.1 Shear stress thresholds**

Using the previously described high-speed servo to generate well-defined shear stresses in a microfluidic chamber, the threshold shear stress to cell response in cultured astrocytes was analyzed. Ca2+ rise being the earliest measurable signal in cells, the Ca2+ rise in the cells was measured in real time. The thresholds were defined with multiple input parameters including magnitude, duration, and load rise time.

The cells responded reliably to a shear stimulus of 23 dyn/cm2 with a transient Ca2+ increase, but they did not respond up to a pulse of 11.5 dyn/cm2 , thereby setting an effective threshold [30]. However, the amplitude of the force stimuli is not the only parameter, and a variety of relaxation times has been observed. Changes in pulse duration and rise time also affect the subthreshold responses. For example, a low amplitude of short duration (11.5 dyn/cm2 , 10 ms) did not trigger a measurable Ca2+ elevation, whereas a pulse width of 1000 ms of the same amplitude did. Together, the two parameters establish a multidimensional threshold matrix for pulses with a square profile, as shown in **Figure 3**. The relative significance of each stimulus

**153**

**Figure 4.**

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

frequency (>80 Hz) or a lower frequency (<20 Hz) [30].

**4.2 Cell response is nonlinear**

parameter was evaluated by fitting the profile of Ca2+ response with a two-state Boltzmann equation, which shows the amplitude is the primary determinant [30].

The cell response to shear stimuli is nonlinear. The nonlinear characteristic of Ca2+ response is demonstrated in **Figure 4**. For example, an abrupt increase in shear stress caused the Ca2+ rise, but a slow increase to the same amplitude failed to activate the cells, as shown in **Figure 4(a)**. The cell sensitivity decays rapidly with an increase in force rise time. Moreover, a brief mechanical shock is more important than a sustained shear force. As shown in **Figure 4(b)**, at short intervals of 10 ms, the pulse train caused a response that was three times higher than a single pulse of the same amplitude and a total pulse duration of 100 ms [30]. Thus, the total energy input of the stimulus is not the only determinant in Ca2+ response; the kinetics of the stimulus (force loading rate) also plays an essential role. This emphasizes the viscoelastic/plastic nature of the force transduction processes in cells [64, 69]. By using sine wave stimuli of different frequencies, it has been shown that cells' response is also frequency dependent; the peaks in response occur at a higher

The strain rate dependence of cell injury has been observed in 3D matrix cultured neurons and astrocytes, showing an increase in membrane permeability to small molecules and an increase in post-insult cell death [70, 71]. However, this effect was not observed in hippocampal tissues under biaxial stretch at strain rates ranging from 0.1 to 50 s−1 [72]; this rate of ramping is likely outside the sensitivity

*Nonlinear Ca2+ response of cells. (a) Ca2+ response to shear pulses of different rise times. (b) Ca2+ response to a train of 10 narrow consecutive pulses (10 ms wide) is three times higher than a single broad pulse (100 ms* 

*, 10 ms) with intervals* 

*wide) of the same amplitude. (c) and (d) Ca2+ response to repeated pulses (11.5 dyn/cm2*

*of 1 s and 10s, respectively, showing that the response is nonlinear.*

parameter was evaluated by fitting the profile of Ca2+ response with a two-state Boltzmann equation, which shows the amplitude is the primary determinant [30].
