Early Cell Response to Mechanical Stimuli during TBI

*Mohammad Mehdi Maneshi and Susan Z. Hua*

## **Abstract**

Traumatic brain injury (TBI) refers to brain damage resulting from external mechanical forces such as a blast or crash. The tissue and cell deformations caused by shear forces are the most common pathological features in TBI and lead to long-term symptoms. Our current understanding of TBI derives mainly from *in vivo* studies of poststimulus pathology and the effects on brain function. Little is known about the early responses of brain cells during mechanical stimuli. In this chapter, we evaluate the early cell response to the rapid shear forces *in vitro*. We introduce advanced technologies capable of generating fast shear stimuli mimicking forces occurring in TBI and reporting internal forces in specific proteins at the time of injury. We define the threshold shear forces for calcium influx using an astrocyte model. We describe the spatiotemporal distribution of cytoskeletal forces and correlate them with variations in cell membrane tension. This chapter makes a strong argument that cells' response to external forces is nonlinear. The heterogeneous distribution of cytoskeletal tension and the gradient of protein forces play a key role in the cells' response to mechanical stimuli.

**Keywords:** traumatic brain injury (TBI), cell mechanics, astrocytes, shear stress, cytoskeletal forces, cell membrane tension

#### **1. Introduction**

Traumatic brain injury (TBI) is a form of acquired brain injury that is caused by abrupt external mechanical forces. TBI occurs in both civilians and military veterans from events such as an explosive blast, a blunt impact, and uncontrolled acceleration, deceleration, or rotation of the head. The injury occurs in two phases: a primary injury caused by mechanical forces that occur at the incipient moment of injury and a secondary injury that involves subsequent biochemical and metabolic dysfunction [1–3]. Depending on the type of forces, the injury is normally classified as either a focal injury that results in cerebral contusions in a specific location or a diffuse brain injury that occurs over a widespread area due to shear forces generated by rapid acceleration or deceleration [4–6]. While the focal injuries are common in moderate to severe brain injuries, diffuse injuries are the main cause of mild TBI [7]. The deformations caused by shear forces are more difficult to define because they occur throughout the brain and are often invisible to commonly available imaging techniques at the early stages of injury [8, 9].

Most TBI patients exhibit mild or minimally observable damage upon the initial shock, but serious pathology can develop within hours and days [8, 10, 11]. Cell death may proceed from multiple locations within the brain. Many of these sites contain cells with no initially observable injury or proximity to the damage site [12]. Thus, brain cells can respond to low mechanical forces that do not cause an immediate structural damage. Understanding early cell response to mechanical stimuli will help provide valuable insight into the origin and evolution of long-term pathological changes.

Our current understanding of TBI is derived from both *in vitro* and *in vivo* studies. *In vivo* studies provide data on the state of cells well after (hours to days) the injury and probably reflect injuries from downstream processing [13–15]. *In vitro* models, using cultured cells, examine cells' response at the expense of normal physiological interactions [2, 16]. The applied forces for *in vitro* models are often not on the same timescale as TBI. Recent research has begun using the so-called next-generation injury assessment tools to study specific cell responses in real time.

This chapter examines the earliest cell responses during TBI as seen in astrocytes via changes in Ca2+ levels *in vitro*. In this chapter, we will introduce advanced technologies that can generate fast shear stimuli mimicking forces that cause TBI and can measure internal forces in specific proteins at the very point of time of injury. We will determine the features of force stimuli that are most susceptible to cells and that lead to long-term alterations. The mechanical properties of cells will be evaluated by real-time measurements of forces in specific cytoskeletal proteins and in cell membranes.

#### **2. Brain structure and traumatic brain injury**

#### **2.1 Head structure and brain cells**

The brain by its very nature is made of soft substances that are submerged in a thin layer of fluid inside the skull. Underneath the skull, there are multiple layers of tissues separated by intracranial spaces containing water-like liquid, called the cerebrospinal fluid (CSF), as shown in **Figure 1**. It is believed that this fluid plays an important role in the shock-absorbing capacity of the brain. Further inwards, it is the brain itself that consists of a network of functional brain cells. These cells are arranged into several specialized areas, each performing distinct physiological functions.

During TBI, the transient external forces cause linear and angular accelerations, resulting in a range of injuries to the brain. Two main types of injuries may occur: the impact injury due to the brain directly hitting the skull at the point of impact that also generates a whipsaw effect at the opposite side of the brain [17]; and the shear injury due to relative movements between the brain and surrounding tissues and between tissues of different densities [18]. For example, with a sudden acceleration, movement of the brain lags behind that of the skull, producing shear stresses at various interfaces between the brain and the cortical tissues. Similarly, in a deceleration injury, the brain continues its inertial path after the skull has been abruptly halted. Shear injuries commonly occur at gray/white matter junctions, but they are also found in the deeper white matter of the corpus callosum, brain stem, and cerebral cortex [19, 20]. The shear strains and stresses are responsible for producing the loss of consciousness during diffuse axonal injury that accounts for ~60% of hospitalized TBI cases [9, 21].

#### **2.2 Brain cells**

Two major cell types in the brain are the neurons and glia. Astrocytes are the most abundant glial cells. While the neurons play the role of processing and transmitting information, astrocytes provide the critical link between the circulatory system and the structural support and the maintenance of neurons (tripartite synapses, neurotransmitter processing, etc.) [22]. **Figure 1** (zoom-in panel) illustrates the configuration of the brain cells. During TBI, astrocytes transmit

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**Figure 1.**

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

mechanical forces to neurons and (by Newton's third law) accept forces from them. They can also propagate damage signals via Ca2+ waves, signal to neurons through neurotransmitters, and alter ion concentrations in the extracellular space [22, 23]. By applying a blast-like overpressure to brain slices, it has been shown that astrocyte injury *precedes* the neuronal injury. This raises the possibility that astrocyte Ca2+

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

may be one of the earliest responses modulating the brain function [24].

**2.3 Earliest cellular events in injury models**

*Schematic illustration of brain structure and brain cells.*

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

*Recent Advances in Biomechanics*

with no initially observable injury or proximity to the damage site [12]. Thus, brain cells can respond to low mechanical forces that do not cause an immediate structural damage. Understanding early cell response to mechanical stimuli will help provide valuable insight into the origin and evolution of long-term pathological changes. Our current understanding of TBI is derived from both *in vitro* and *in vivo* studies. *In vivo* studies provide data on the state of cells well after (hours to days) the injury and probably reflect injuries from downstream processing [13–15]. *In vitro* models, using cultured cells, examine cells' response at the expense of normal physiological interactions [2, 16]. The applied forces for *in vitro* models are often not on the same timescale as TBI. Recent research has begun using the so-called next-generation injury assessment tools to study specific cell responses in real time. This chapter examines the earliest cell responses during TBI as seen in astrocytes via changes in Ca2+ levels *in vitro*. In this chapter, we will introduce advanced technologies that can generate fast shear stimuli mimicking forces that cause TBI and can measure internal forces in specific proteins at the very point of time of injury. We will determine the features of force stimuli that are most susceptible to cells and that lead to long-term alterations. The mechanical properties of cells will be evaluated by real-time measure-

ments of forces in specific cytoskeletal proteins and in cell membranes.

specialized areas, each performing distinct physiological functions.

fuse axonal injury that accounts for ~60% of hospitalized TBI cases [9, 21].

Two major cell types in the brain are the neurons and glia. Astrocytes are the most abundant glial cells. While the neurons play the role of processing and transmitting information, astrocytes provide the critical link between the circulatory system and the structural support and the maintenance of neurons (tripartite synapses, neurotransmitter processing, etc.) [22]. **Figure 1** (zoom-in panel) illustrates the configuration of the brain cells. During TBI, astrocytes transmit

The brain by its very nature is made of soft substances that are submerged in a thin layer of fluid inside the skull. Underneath the skull, there are multiple layers of tissues separated by intracranial spaces containing water-like liquid, called the cerebrospinal fluid (CSF), as shown in **Figure 1**. It is believed that this fluid plays an important role in the shock-absorbing capacity of the brain. Further inwards, it is the brain itself that consists of a network of functional brain cells. These cells are arranged into several

During TBI, the transient external forces cause linear and angular accelerations, resulting in a range of injuries to the brain. Two main types of injuries may occur: the impact injury due to the brain directly hitting the skull at the point of impact that also generates a whipsaw effect at the opposite side of the brain [17]; and the shear injury due to relative movements between the brain and surrounding tissues and between tissues of different densities [18]. For example, with a sudden acceleration, movement of the brain lags behind that of the skull, producing shear stresses at various interfaces between the brain and the cortical tissues. Similarly, in a deceleration injury, the brain continues its inertial path after the skull has been abruptly halted. Shear injuries commonly occur at gray/white matter junctions, but they are also found in the deeper white matter of the corpus callosum, brain stem, and cerebral cortex [19, 20]. The shear strains and stresses are responsible for producing the loss of consciousness during dif-

**2. Brain structure and traumatic brain injury**

**2.1 Head structure and brain cells**

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**2.2 Brain cells**

**Figure 1.** *Schematic illustration of brain structure and brain cells.*

mechanical forces to neurons and (by Newton's third law) accept forces from them. They can also propagate damage signals via Ca2+ waves, signal to neurons through neurotransmitters, and alter ion concentrations in the extracellular space [22, 23]. By applying a blast-like overpressure to brain slices, it has been shown that astrocyte injury *precedes* the neuronal injury. This raises the possibility that astrocyte Ca2+ may be one of the earliest responses modulating the brain function [24].
