Neuronal Plasma Membrane Integrity is Transiently Disturbed by Traumatic Loading.

The acute response of neurons subjected to traumatic loading involves plasma membrane disruption, yet the mechanical tolerance for membrane compromise, time course, and mechanisms for resealing are not well understood. We have used an in vitro traumatic neuronal injury model to investigate plasma membrane integrity immediately following a high-rate shear injury. Cell-impermeant fluorescent molecules were added to cortical neuronal cultures prior to insult to assess membrane integrity. The percentage of cells containing the permeability marker was dependent on the molecular size of the marker, as smaller molecules gained access to a higher percentage of cells than larger ones. Permeability increases were positively correlated with insult loading rate. Membrane disruption was transient, evidenced by a membrane resealing within the first minute after the insult. In addition, chelation of either extracellular Ca2+ or intracellular Ca2+ limited membrane resealing. However, injury following chelation of both extracellular and intracellular Ca2+ caused diminished permeability as well as a greater resealing ability compared to chelation of extracellular or intracellular Ca2+ alone. Treatment of neuronal cultures with jasplakinolide, which stabilizes filamentous actin, reduced permeability increases, while latrunculin-B, an actin depolymerizing agent, both reduced the increase in plasma membrane permeability and promoted resealing. This study gives insight into the dynamics of neuronal membrane disruption and subsequent resealing, which was found to be calcium dependent and involve actin in a role that differs from non-neuronal cells. Taken together, these data will lead to a better understanding of the acute neuronal response to traumatic loading.

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 10 min or 24 h 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 10 min post-injury; however by 24 h 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 24 h, 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.

Shear-induced intracellular loading of cells with molecules by controlled microfluidics.

This study tested the hypothesis that controlled flow through microchannels can cause shear-induced intracellular loading of cells with molecules. The overall goal was to design a simple device to expose cells to fluid shear stress and thereby increase plasma membrane permeability. DU145 prostate cancer cells were exposed to fluid shear stress in the presence of fluorescent cell-impermeant molecules by using a cone-and-plate shearing device or high-velocity flow through microchannels. Using a syringe pump, cell suspensions were flowed through microchannels of 50-300 microm diameter drilled through Mylar sheets using an excimer laser. As quantified by flow cytometry, intracellular uptake and loss of viability correlated with the average shear stress. Optimal results were observed when exposing the cells to high shear stress for short durations in conical channels, which yielded uptake to over one-third of cells while maintaining viability at approximately 80%. This method was capable of loading cells with molecules including calcein (0.62 kDa), large molecule weight dextrans (150-2,000 kDa), and bovine serum albumin (66 kDa). These results supported the hypothesis that shear-induced intracellular uptake could be generated by flow of cell suspensions through microchannels and further led to the design of a simple, inexpensive, and effective device to deliver molecules into cells. Such a device could benefit biological research and the biotechnology industry.

Critical Size Bone Defect Healing Using Collagen-Calcium Phosphate Bone Graft Materials.

The need for bone graft materials to fill bony voids or gaps that are not related to the intrinsic stability of the bone that arise due to trauma, tumors or osteolysis remains a clinically relevant and significant issue. The in vivo response of collagen-tricalcium phosphate bone graft substitutes was evaluated in a critical size cancellous defect model in skeletally mature rabbits. While the materials were chemically virtually identical, new bone formation, implant resorption and local in vivo responses were significantly different. Differences in the in vivo response may be due, in part, collagen source and processing which influences resorption profiles. Continued improvements in processing and manufacturing techniques of collagen-tricalcium phosphate bone graft substitutes can result in osteoconductive materials that support healing of critical size bone defects even in challenging pre-clinical models.

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.

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.

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.