Gustavo R. Prado

CNS injury biomechanics and experimental models

Traumatic brain injury (TBI) and traumatic spinal cord injury (SCI) are acquired when an external physical insult causes damage to the central nervous system (CNS). Functional disabilities resulting from CNS trauma are dependent upon the mode, severity, and anatomical location of the mechanical impact as well as the mechanical properties of the tissue. Although the biomechanical insult is the initiating factor in the pathophysiology of CNS trauma, the anatomical loading distribution and the resulting cellular responses are currently not well understood. For example, the primary response phase includes events such as increased membrane permeability to ions and other molecules, which may initiate complex signaling cascades that account for the prolonged damage and dysfunction. Correlation of insult parameters with cellular changes and subsequent deficits may lead to refined tolerance criteria and facilitate the development of improved protective gear. In addition, advancements in the understanding of injury biomechanics are essential for the development and interpretation of experimental studies at both the in vitro and in vivo levels and may lead to the development of new treatment approaches by determining injury mechanisms across the temporal spectrum of the injury response. Here we discuss basic concepts relevant to the biomechanics of CNS trauma, injury models used to experimentally simulate TBI and SCI, and novel multilevel approaches for improving the current understanding of primary damage mechanisms.

Neural mechanobiology and neuronal vulnerability to traumatic loading

In order to understand the physical tolerance of neurons to traumatic insults, engineers and neuroscientists have attempted to reproduce the biomechanical environment during a traumatic event using in vitro injury systems with isolated components of the nervous system. This approach allows one to begin to unravel the underlying molecular and biochemical mechanisms that lead to cell dysfunction and death as a function of mechanical inputs. Excess mechanical force and deformation causes structural and functional breakdown, including several key deleterious cellular processes, such as membrane damage, an upset of calcium homeostasis, glutamate release, cell death, and caspase-mediated proteolysis. Understanding of the mechanotransduction events, however, that lead to cellular failure and dysfunction, are not well understood. Mechanically characterized cellular models of traumatic loading are critical to the improved understanding of mechanotransduction in the context of neural injury, the improvement of protective systems, and to provide a controlled setting for testing therapeutic interventions. In this review of the cellular mechanics of traumatic neural loading, we focus on the backdrop and motivation for studying mechanical thresholds in neurons and glial cells and discuss some of the acute responses that may help elucidate improved tolerance criteria and illuminate future research directions.

Mechanical trauma induces immediate changes in neuronal network activity.

During a traumatic insult to the brain, tissue is subjected to large stresses at high rates which often surpass cellular thresholds leading to cell dysfunction or death. The acute response of neurons to a mechanical trauma, however, is poorly understood. Plasma membrane disruption may be the earliest cellular outcome from a mechanical trauma. The increase in membrane permeability due to such disruptions may therefore play an important role in the initiation of deleterious cascades following brain injury. The immediate consequences of an increase in plasma membrane permeability on the electrophysiological behavior of a neuronal network exposed to the trauma have not been elucidated. We have developed an in vitro model of traumatic brain injury (TBI) that utilizes a novel device capable of applying stress at high rates to neuronal cells cultured on a microelectrode array. The mechanical insult produced by the device caused a transient increase in neuronal plasma membrane permeability, which subsided after 10 min. We were able to monitor acute spontaneous electrophysiological activity of injured cultures for at least 10 min following the insult. Firing frequency, average burst interval and spikes within burst were assessed before and after injury. The electrophysiological responses to the insult were heterogeneous, although an increase in burst intervals and in the variability of the assessed parameters were common. This study provides a multi-faceted approach to elucidate the role of neuronal plasma membrane disruptions in TBI and its functional consequences.

Plasma membrane damage as a marker of neuronal injury

Traumatic injury to neurons, initiated by high strain rates, consists of both primary and secondary damage, yet the cellular tolerances in the acute post-injury period are not well understood. The events that occur at the time of and immediately after an insult depend on the injury severity as well as inherent properties of the cell and tissue. We have analyzed neuronal plasma membrane disruption in several in vitro and in vivo injury models of traumatic injury. We found that insult severity positively correlated with the degree of membrane disruptions and that the time course of membrane breaches and subsequent repair varies. This approach provides an experimental framework to investigate injury tolerance criteria as well as mechanistically driven therapeutic strategies. It is postulated that a traumatic insult to the brain or spinal cord results in cellular membrane strain, inducing acute damage that upsets plasma membrane homeostasis. An increased understanding of the pathophysiological mechanisms involved in membrane damage is required in order to specifically target these pathways for diagnostic and treatment purposes and overcome current clinical limitations in the treatment of traumatic brain injury (TBI) and traumatic spinal cord injury (SCI).

High Rate Shear Insult Delivered to Cortical Neurons Produces Heterogeneous Membrane Permeability Alterations

Traumatic brain injury (TBI) occurs when brain tissue is subjected to stresses and strains at high rates and magnitudes, yet the mechanisms of injury and cellular thresholds are not well understood. The events that occur at the time of and immediately after an insult are hypothesized to initiate cell dysfunction or death following a critical cell strain and strain rate. We analyzed neuronal plasma membrane disruption in two in vitro injury models-fluid shear stress delivered to planar cultures and shear strain induction of 3-D neural cultures. We found that insult severity positively correlated with the degree of membrane disruptions in a heterogeneous fashion in both cell configurations. Furthermore, increased membrane permeability led to increases in electrophysiological disturbance. Specifically, cells that exhibited increased membrane permeability did not fire random action potentials, in contrast to neighboring cells that had intact plasma membranes. This approach provides an experimental framework to investigate injury tolerance criteria as well as mechanistically driven therapeutic strategies

Mechanoporation is a potential indicator of tissue strain and subsequent degeneration following experimental traumatic brain injury

Background: An increases in plasma membrane permeability is part of the acute pathology of traumatic brain injury and may be a function of excessive membrane force. This membrane damage, or mechanoporation, allows non‐specific flux of ions and other molecules across the plasma membrane, and may ultimately lead to cell death. The relationships among tissue stress and strain, membrane permeability, and subsequent cell degeneration, however, are not fully understood. Methods: Fluorescent molecules of different sizes were introduced to the cerebrospinal fluid space prior to injury and animals were sacrificed at either 10min or 24h after injury. We compared the spatial distribution of plasma membrane damage following controlled cortical impact in the rat to the stress and strain tissue patterns in a 3‐D finite element simulation of the injury parameters. Findings: Permeable cells were located primarily in the ipsilateral cortex and hippocampus of injured rats at 10min post‐injury; however by 24h there was also a significant increase in the number of permeable cells. Analysis of colocalization of permeability marker uptake and Fluorojade staining revealed a subset of permeable cells with signs of degeneration at 24h, but plasma membrane damage was evident in the vast majority of degenerating cells. The regional and subregional distribution patterns of the maximum principal strain and shear stress estimated by the finite element model were comparable to the cell membrane damage profiles following a compressive impact. Interpretation: These results indicate that acute membrane permeability is prominent following traumatic brain injury in areas that experience high shear or tensile stress and strain due to differential mechanical properties of the cell and tissue organization, and that this mechanoporation may play a role in the initiation of secondary injury, contributing to cell death.

A New Experimental Framework for the Examination of Acute Dysfunction in Traumatic Neural Injury

The functional consequences of traumatic neural injury are dependent on the mechanical insult and the transfer of force to the cells in the affected tissue, yet these events are not well understood. We have developed a new experimental framework using both in vivo and in vitro models of traumatic brain injury and measurement techniques that span levels of complexity and allow examination of plasma membrane integrity and electrophysiological response in the minutes following the insult. In an in vivo model using a normally cell impermeant dye we found significant uptake in cortical and hippocampal regions ipsilateral to a cortical impact at 10 minute post-insult. In addition, extracellular electrophysiological activity within the same time period was reduced in animals subject to injury. In an in vitro model of injury in which cultured primary neurons were subject to hydrodynamic deformation at comparable rates to the in vivo insult, membrane permeability increased immediately. In addition, custom-made multielectrode arrays were used to measure electrophysiological signals during and immediately following the same insult. Data revealed a reduction in firing frequency, supporting the hypothesis that immediate membrane failure has a disruptive effect on firing and overall network activity. Morphologically, cells that displayed dye uptake in both in vitro and in vivo were elongated and shrunk, suggesting downstream changes in cytoskeletal integrity and/or osmotic balance. A working conceptual model of these acute events includes deformation-induced membrane failure that is rate and magnitude dependent and leads to ionic imbalance. These events may directly alter the ability to fire normally and hence lead to reduced spike amplitude and frequency. Collectively, the acute response leaves the cells vulnerable to energy deficits, abnormal signaling, and, ultimately, cell death