Selective modulation of endothelial cell [Ca2+]i response to flow by the onset rate of shear stress.

The response of endothelial cells (ECs) to their hemodynamic environment strongly influences normal vascular physiology and the pathogenesis of atherosclerosis. Unique responses to the complex flow patterns in lesion-prone regions imply that the temporal and spatial features of the mechanical stimuli modulate the cellular response to flow. We report the first systematic study of the effects of temporal gradients of shear stress on ECs. Flow was applied to cultured ECs using a novel cone-and-plate device allowing precise and independent control of the shear stress magnitude and the onset rate. Intracellular free calcium concentration ([Ca2+]i) increased rapidly following the onset of flow, and the characteristics of the transient were modulated by both the shear stress magnitude and onset rate. ECs were most sensitive to shear stress applied at physiological onset rates. Furthermore, the relative contribution of extracellular calcium and IP3-mediated release were dependent upon the specific flow regime.

In vitro cell shearing device to investigate the dynamic response of cells in a controlled hydrodynamic environment.

Mechanical stresses and strains play important roles in the normal growth and development of biological tissues, yet the cellular mechanisms of mechanotransduction have not been identified. A variety of in vitro systems for applying mechanical loads to cell populations have been developed to gain insight into these mechanisms. However, limitations in the ability to control precisely relevant aspects of the mechanical stimuli have obscured the physical relationships between mechanical loading and the biochemical signals that mediate the cellular response. We present a novel in vitro cell shearing device based on the principles of a cone and plate viscometer that utilizes microstepper motor technology to control independently the dynamic and steady components of a hydrodynamic shear-stress environment. Physical measurements of the cone velocity demonstrated faithful reproduction of user-defined input wave forms. Computational modeling of the fluid environment for the unsteady startup confirmed small inertial contributions and negligible secondary flows. Finally, we present experimental results demonstrating the onset rate dependence of functional and structural responses of endothelial cell cultures to dynamically applied shear stress. The controlled cell shearing device is a novel tool for elucidating mechanisms by which mechanical forces give rise to the biological signals that modulate cellular morphology and metabolism.

Dynamic mechanical deformation of neurons triggers an acute calcium response and cell injury involving the N-methyl-D-aspartate glutamate receptor.

A biomechanical in vitro model of traumatic brain injury was used to examine cellular response to physical insults and the underlying mechanisms that lead to cell dysfunction. A cell shearing injury device was used to deform human NTera-2 neurons at high loading rates during the investigation of mechanisms of cytosolic free calcium increases, which may be detrimental to a cell. Cytosolic free calcium rose immediately to almost three times baseline and was associated with lactate dehydrogenase release at 24 hr, indicating significant cell injury. Low loading rates did not elicit these responses. A major portion of the calcium increase and subsequent cell injury was dependent on the presence of extracellular free calcium. Blocking the N-methyl-D-aspartate glutamate receptor complex with dizocilipine maleate attenuated calcium increases by 45% in injured neurons and blocked a significant part (50%) of the lactate dehydrogenase release. In addition, pretreatment with nifedipine or riluzole also significantly reduced cytosolic free calcium but did not affect cell injury, whereas tetrodotoxin had no affect on either outcome parameter. These results suggest that the increased membrane permeability and immediate calcium influx associated with this model of mechanical injury trigger several cellular pathways, including N-methyl-D-aspartate receptor-mediated cell damage.

Cervical cord neurapraxia: classification, pathomechanics, morbidity, and management guidelines.

One hundred ten cases of the transient neurological phenomenon, cervical cord neurapraxia (CCN), are presented. The authors established a classification system for CCN, developed a new computerized measurement technique for magnetic resonance (MR) imaging, investigated the relationship of the cervical cord to the canal, and analyzed clinical, x-ray, and MR data. One hundred nine males and one female were included in the study; the average age of the participants was 21 years (range 13-33 years). All episodes occurred during sports participation; 87% occurred while the patient was playing football. Follow-up review lasting an average of 3.3 years was available for 105 patients (95%). Narrowing of the sagittal diameter of the cervical canal in the adult spine was confirmed to be a causative factor. Cervical cord neurapraxia was not associated with permanent neurological injury and no permanent morbidity occurred in patients who returned to contact activities. Of the patients returning to contact activities, 35 (56%) experienced a recurrent episode. The risk of recurrence was increased with smaller spinal canal/vertebral body ratio (p < 0.05), smaller disc-level canal diameter (p < 0.05), and less space available for the cord (p < 0.05). There was no correlation between either the classification of the CCN episode or the disease noted on MR imaging and x-ray films and the risk of recurrence. The authors conclude that: 1) CCN is a transient neurological phenomenon and individuals with uncomplicated CCN may be permitted to return to their previous activity without an increased risk of permanent neurological injury; 2) congenital or degenerative narrowing of the sagittal diameter of the cervical canal is a causative factor; 3) the overall recurrence rate after return to play is 56%; and 4) the risk of recurrence is strongly and inversely correlated with sagittal canal diameter and it is useful in the prediction of future episodes of CCN (p < 0.001). These data will enable the physician to counsel individuals regarding a predicted risk of recurrence based on canal measurements.

An in vitro traumatic injury model to examine the response of neurons to a hydrodynamically-induced deformation.

A novel in vitro system was developed to examine the effects of traumatic mechanical loading on individual cells. The cell shearing injury device (CSID) is a parallel disk viscometer that applies fluid shear stress with variable onset rate. The CSID was used in conjunction with microscopy and biochemical techniques to obtain a quantitative expression of the deformation and functional response of neurons to injury. Analytical and numerical approximations of the shear stress at the bottom disk were compared to determine the contribution of secondary flows. A significant portion of the shear stress was directed in the r-direction during start-up, and therefore the full Navier-Stokes equation was necessary to accurately describe the transient shear stress. When shear stress was applied at a high rate (800 dyne cm-2 sec-1) to cultured neurons, a range of cell membrane strains (0.01 to 0.53) was obtained, suggesting inhomogeneity in cellular response. Functionally, cytosolic calcium and extracellular lactate dehydrogenase levels increased in response to high strain rate (> 1 sec-1) loading, compared with quasistatic (< 1 sec-1) loading. In addition, a subpopulation of the culture subjected to rapid deformation subsequently died. These strain rates are relevant to those shown to occur in traumatic injury, and, as such, the CSID is an appropriate model for studying the biomechanics and pathophysiology of neuronal injury.

An in vitro model of traumatic neuronal injury: loading rate-dependent changes in acute cytosolic calcium and lactate dehydrogenase release.

We developed a new in vitro model of neuronal injury using NT2-N cells to examine the effects of hydrodynamic loading rate on intraneuronal calcium dynamics and lactate dehydrogenase (LDH) release. Our apparatus consisted of a parallel disk viscometer which induced fluid shear stress with well-defined magnitudes and loading rates to cultured cells. We found that the deformation response of the cells was dependent on the severity of the insult, with increased cellular strains generated for higher shear stresses at a constant loading rate. Peak intracellular free calcium concentration correlated with strain, suggesting that mechanical deformation may regulate calcium response. Slowly applied fluid shear stress elicited no response, whereas high loading rates resulted in peak calcium increases 2.9 to 3.6 times baseline values as injury severity was increased. LDH release measured within 5 min after the insult correlated with loading rate. In addition, LDH release continued to increase out to 24 h following high loading rate conditions, demonstrating that the application of fluid shear stress led to prolonged cell damage. The acute response in NT2-N cells subjected to an insult with the CSID is dependent on the loading rate, and these results suggest that initial membrane deformation may trigger subsequent events.

The biochemical pathway mediating the proliferative response of bone cells to a mechanical stimulus.

Calvarial bone cells of rats were subjected to either a cyclic biaxial strain of 0.17 per cent (1700 microstrain) or a hydrostatic pressure of 2.5, five, or ten pounds per square inch (17.2, 34.5, or sixty-nine kilopascals). The frequency was held constant at one hertz for both types of mechanical stimulation. When cultured bone cells that had been subjected to a cyclic biaxial strain for two hours were harvested twenty-two hours later, it was found that the level of prostaglandin E2 had increased significantly (p < 0.01) as had cellular proliferation (p < 0.01), as indicated by the incorporation of [3H]-thymidine. The addition to the medium of indomethacin, an inhibitor of prostaglandin synthesis, at a ten-micromolar concentration significantly inhibited (p < 0.01) the increase in prostaglandin E2 synthesis but had no effect on the strain-induced increase in cellular proliferation, as indicated by the incorporation of [3H]-thymidine. Twenty-four hours after exposure to the same cyclic biaxial strain for thirty seconds, other cultured bone cells showed a significant increase in the level of cytoskeletal calmodulin (p < 0.05) and in the DNA content (p < 0.05). N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), a calmodulin antagonist, was added to the medium at a one-micromolar concentration, which had been shown to have no effect on the increase in the DNA content of control cells; W-7 completely blocked the increase in the level of cytoskeletal calmodulin and in the DNA content in the cells that were subjected to a cyclic biaxial strain. The bone cells subjected to a hydrostatic pressure showed a dose-dependent increase in the concentration of cytosolic Ca2+, as measured with Fura 2-AM, a fluorescent indicator of intracellular calcium. With a pressure of ten pounds per square inch (sixty-nine kilopascals), the increase in the concentration of cytosolic Ca2+ was nearly eight times greater than that at 2.5 pounds per square inch (17.2 kilopascals) (126 +/- 15.2 compared with 16 +/- 8.0 nanomolar, mean and standard deviation). The addition to the medium of neomycin, an inhibitor of the inositol phosphate cascade, at a ten-millimolar concentration completely blocked the increase in the concentration of cytosolic Ca2+ in these cells; this concentration of neomycin had been shown to have no effect on proliferation in control bone cells. There was also a dose-dependent relationship between the duration of the stimulus and the cellular proliferation. Remarkably, one cycle of pressure at ten pounds per square inch (sixty-nine kilopascals) and a frequency of approximately one hertz produced a 57 per cent increase in the incorporation of [3H]-thymidine at twenty-four hours (p < 0.001). From these findings, we hypothesized that the inositol phosphate cascade-cytosolic Ca(2+)-cytoskeletal calmodulin system plays a dominant role in the signal transduction of a mechanical stimulus into increased proliferation of bone cells, at least under the conditions reported here.

Acute alterations in [Ca2+]i in NG108-15 cells subjected to high strain rate deformation and chemical hypoxia: an in vitro model for neural trauma.

The short-term (less than 2 min) alterations in the intracellular free calcium concentration in differentiated NG108-15 (neuroblastoma cross glioma) cells exposed to dynamic mechanical deformation with and without superimposed chemical hypoxia were determined. A previously developed device, modified for these studies, was used to apply deformations at a magnitude and rate representative of those experienced by neural tissue in Traumatic Brain Injury. Chemical hypoxia was imposed using a combination of 2-deoxy-D-glucose and salicylate, anaerobic and aerobic metabolic blockers, respectively. Real time measurement of intracellular free calcium concentration using Fura-2 and a custom epifluorescence microscopy system provided a quantitative index of cell response. At high rates of deformation (approximately 10 sec-1), increases in intracellular free calcium concentration were exponentially related to the magnitude of the applied deformation. Chemical hypoxia had no effect on this acute response. At low rates of deformation, small increases in intracellular free calcium concentration were independent of the magnitude of the deformation. These findings indicate that strategies for reducing severity of TBI should focus on minimizing the rate of deformation of neural cells. Together with data from animal, physical, and finite element models, these data can be employed in the development of physiologic injury tolerance criteria for the whole head.

The mechanical properties of the human cervical spinal cord in vitro.

The response of spinal cord tissue to mechanical loadings is not well understood. In this study, isolated fresh cervical spinal cord samples were obtained from cadavers at autopsy and tested in uniaxial tension at moderate strain rates. Stress relaxation experiments were performed with an applied strain rate and peak strain in the physiological range, similar to those seen in the spinal cord during voluntary motion. The spinal cord samples exhibited a nonlinear stress-strain response with increasing strain increasing the tangent modulus. In addition, significant relaxation was observed over 1 min. A quasilinear viscoelastic model was developed to describe the behavior of the spinal cord tissue and was found to describe the material behavior adequately. The data also were fitted to both hyperelastic and viscoelastic fluid models for comparison with other data in the literature.

1995 William J. Stickel Gold Award. High strain rate tissue deformation. A theory on the mechanical etiology of diabetic foot ulcerations.

Foot ulcerations are one of the most common and dangerous complications associated with chronic diabetes mellitus. Many studies have focused on neuropathy, in conjunction with elevated ground reactive forces, as the principal cause of these ulcerations. The authors discuss the mechanical cause of diabetic ulcerations at the cellular level. It is hypothesized that increased rate of tissue deformation associated with foot slap secondary to progressive motor neuropathy is the actual culprit, and not the magnitude of local pressure applied. The authors present a cellular model that shows that high rates of tissue deformation may result in elevated intracellular calcium concentrations, which may lead to cellular death, while comparable loads gradually applied do not. Furthermore, there is no significant difference in the response observed at 5 psi and 10 psi. Based on these findings, it is hypothesized that techniques such as ankle foot orthoses, which control the velocity of foot strike, may be useful in treating diabetic foot ulcerations.

The effect of N-acetylaspartate on the intracellular free calcium concentration in NTera2-neurons.

We have studied the effect of relatively high concentrations of extracellular N-acetylaspartate (NAA) on the intracellular free calcium concentration [Ca2+]i in NTera2-neurons. While low concentrations of extracellular NAA (0.1, 1 mM) had no effect on the [Ca2+]i, high concentrations of extracellular NAA (3, 10 mM) elicited sharp and statistically significant elevations of [Ca2+]i. Different classes of antagonists of the N-methyl-D-aspartate (NMDA) receptor abolished the NAA induced elevations of the [Ca2+]i, indicating the involvement of the NMDA receptor in NAA-induced elevations of [Ca2+]i.

Modification of the cortical impact model to produce axonal injury in the rat cerebral cortex.

Diffuse axonal injury (DAI) is a form of brain injury that is characterized by morphologic changes to axons throughout the brain and brainstem. Previous biomechanical studies have shown that primary axonal dysfunction, ranging from minor electrophysiologic disturbances to immediate axotomy, can be related to the rate and level of axonal deformation. Some existing rodent head injury models display varying degrees of axonal injury in the forebrain and brainstem, but the extent of axonal damage in the forebrain has been limited to the contused hemisphere. This study examined whether opening the dura mater over the contralateral hemisphere could direct mechanical deformation across the sagittal midline and produce levels of strain sufficient to cause a more widespread, bilateral forebrain axonal injury following cortical impact. Intracranial deformation patterns produced by this modified cortical impact technique were examined using surrogate skull-brain models. Modeling results revealed that the presence of a contralateral craniotomy significantly reduced surrogate tissue herniation through the foramen magnum, allowed surrogate tissue movement across the sagittal midline, and resulted in an appreciable increase in the shear strain in the contralateral cortex during the impact. To evaluate the injury pattern produced using this novel technique, rat brains were subjected to rigid indentor impact injury of their left somatosensory motor cortex (1.5 mm indentation, 4.5-4.9 m/sec velocity, and 22 msec dwell time) and examined after a 2-7 day survival period. Neurofilament immunohistochemistry revealed numerous axonal retraction balls in the subcortical white matter and overlying deep cortical layers in the right hemisphere beneath the contralateral craniotomy. Retraction balls were not seen at these positions in normals, sham controls, or animals that received cortical impact without contralateral craniotomy and dural opening. The results from these physical modeling and animal experiments indicate that opening of the contralateral dura mater permits translation of sufficient mechanical deformation across the midline to produce a more widespread pattern of axonal injury in the forebrain, a pattern that is distinct from those produced by existing fluid percussion and cortical impact techniques.

Strain measurements in cultured vascular smooth muscle cells subjected to mechanical deformation.

Early work in the field of biomechanics employed rigorous application of the principles of mechanics to the study of the macroscopic structural response of tissues to applied loads. Interest in the functional response of tissues to mechanical stimulation has lead researchers to study the biochemical responses of cells to mechanical loading. Characterization of the experimental system (i.e., specimen geometry and boundary conditions) is no less important on the microscopic scale of the cell than it is for macroscopic tissue testing. We outline a method for appropriate characterization of cell deformation in a cell culture model; describe a system for applying a uniform, isotropic strain field to cells in culture; and demonstrate a dependence of cell deformation on morphology and distribution of adhesion sites. Cultured vascular smooth-muscle cells were mechanically deformed by applying an isotropic strain to the compliant substrate to which they were adhered. The state of strain in the cells was determined by measurement of the displacements of fluorescent microspheres attached to the cell surface. The magnitude and orientation of principal strains were found to vary spatially and temporally and to depend on cell morphology. These results show that cell strain can be highly variable and emphasize the need to characterize both the loading conditions and the actual cellular deformation in this type of experimental model.

Model for short-term intracranial pressure changes following traumatic injury.

Results from primate studies show a transient increase in intracranial pressure (ICP) after a nonimpact inertial loading condition. The measured ICP increase varies linearly with the peak tangential load of these experiments. These experiments point to possible alterations in cerebral blood flow. This paper investigates the possible etiology of this particular phenomenon, and presents a simple analytical model that could explain the changes in intracranial pressure. The model combines the effects of cerebral venous constriction, arterial dilatation, and raised mean blood pressure to yield the characteristic immediate rise and exponential decay of ICP. The main contributor to the increase in intracranial pressure is believed to be vasodilation of cerebral arteries following venous constriction. Passive release of cerebrospinal fluid (CSF) is believed to mediate the long-term decay of intracranial pressure and possibly contribute to local hyperemia.

An analysis of the time-dependent changes in intracellular calcium concentration in endothelial cells in culture induced by mechanical stimulation.

When bovine pulmonary artery endothelial cells in culture are subjected to mechanical strain, their physiology is altered. Experimentally, this mechanical strain is generated by increased tension in the substrate to which the cells are attached and results in altered levels of fibronectin. Studies of the structural response of the endothelial cell suggest that this stimulus is transmitted to the cell membrane, organelles, and cytoskeleton by natural cell attachments in a quantifiable and predictable manner. This report examines altered intracellular calcium homeostasis as a possible messenger for the observed strain-induced physiologic response. In particular, using the intracellular trapped calcium indicator dyes, Quin2 and Fura2, we observed changes in cytosolic free calcium ion concentration in response to biaxial strain of bovine pulmonary artery endothelial cells in culture. The magnitude and time course of this calcium transient resemble that produced by treatment with the calcium ionophore, Ionomycin, indicating that mechanical stimulation may alter cell membrane permeability to calcium. Additional experiments in the presence of EDTA indicated that calcium was also released from intracellular stores in response to strain. In order to explain the stretch-induced calcium transients, a first-order species conservation model is presented that takes into account both the cell's structural response and the calcium homeostatic mechanisms of the cell. It is hypothesized that the cell's calcium sequestering and pumping capabilities balanced with its mechanically induced changes in calcium ion permeability will determine the level and time course of calcium accumulation in the cytosol.

Mechanical and electrical responses of the squid giant axon to simple elongation.

There is a limited amount of information available on the mechanical and functional response of the nervous system to loading. While deformation of cerebral, spinal, or peripheral nerve tissue can have particularly severe consequences, most research in this area has concentrated on either demonstrating in-vivo functional changes and disclosing the effected anatomical pathways, or describing material deformations of the composite structure. Although such studies have successfully produced repeatable traumas, they have not addressed the mechanisms of these mechanically induced injuries. Therefore, a single cell model is required in order to gain further understanding of this complex phenomena. An isolated squid giant axon was subjected to controlled uniaxial loading and its mechanical and physiological responses were monitored with an instrument specifically designed for these experiments. These results determined that the mechanical properties of the isolated axon are similar to those of other soft tissues, and include features such as a nonlinear load-deflection curve and a hysteresis loop upon unloading. The mechanical response was modeled with the quasi-linear viscoelastic theory (Fung, 1972). The physiological response of the axon to quasi-static loading was a small reversible hyperpolarization; however, as the rate of loading was increased, the axon depolarized and the magnitude and the time needed to recover to the original resting potential increased in a nonlinear fashion. At elongations greater than twenty percent an irreversible injury occurs and the membrane potential does not completely recover to baseline.

A proposed tolerance criterion for diffuse axonal injury in man.

The head injury criterion (HIC) is currently the government-accepted head injury indicator. The HIC is not injury-specific, does not relate to injury severity, nor does it take into account variations in the brain mass or load direction. This report focuses on one type of inertial brain injury, diffuse axonal injury (DAI), and utilizes animal studies, physical model experiments, and analytical model simulations to determine the kinematics of DAI in the subhuman primate and to scale these results to man. A human injury tolerance for moderate to severe DAI, which includes the influences of rotational loads and brain mass, is proposed.

Biomechanical aspects of a fluid percussion model of brain injury.

The fluid percussion model is in widespread use for the study of brain injury. However, the tissue deformation characteristics of the model have not been determined. Studies have suggested that at high levels of fluid percussion, the fluid percussion model is primarily a model of brainstem injury. It was proposed that this occurs as a direct result of the volume influx to the cranial vault at the moment of impact. This study examines the biomechanical deformation produced by the fluid percussion model. The purpose of this investigation was to describe the regional strain distribution in brain tissue at the moment of impact and to determine the effect of volume efflux produced by the percussion device. A cat skull was sectioned parasagittally and filled with an optically transparent gel. A grid pattern was painted in the midsagittal plane and was used to record the surrogate brain tissue deformation in response to fluid percussion loading. Motion of the grid pattern at low and high levels of fluid percussion loading was recorded using a high-speed camera, and a series of photographs developed from the high-speed film were analyzed to determine the intracranial strain distribution at these loading levels. The results of these studies indicated that the maximum site of strain was located in the region of the lower brainstem and that deformations were negligible in other regions of the brain. These studies provide an explanation for the pathophysiologic results obtained in a parallel series of experiments from which it was concluded that high-level fluid percussion is predominantly a model of lower brainstem injury.

Selective vulnerability of hippocampal neurons in acceleration-induced experimental head injury.

Traumatically induced subtotal hippocampal neuronal loss traditionally has been considered a consequence of intracranial hypertension and impaired cerebral perfusion. We have examined the frequency and distribution of hippocampal lesions in an acceleration model of brain injury in 54 anesthetized nonhuman primates undergoing physiologic monitoring and subjected postinjury to comprehensive neuropathologic examination. Hippocampal lesions occurred in 32/54 animals (59%). These lesions always involved the CA-1 hippocampal subfield and were bilateral in 24 animals. Hippocampal involvement was not associated with marked elevation of intracranial pressure or depression of cerebral perfusion pressure. These lesions occurred in the absence of involvement of other brain regions considered selectively vulnerable to hypoxic insults. Hippocampal damage occurred in 46% of animals with mild injury characterized by brief periods of unconsciousness and no residual neurologic deficit. Ninety-four percent of animals with severe injuries and prolonged posttraumatic coma had hippocampal involvement. Traumatically induced selective neuronal necrosis of the hippocampus is a specific lesion not explained by the conventional mechanistic theories of head injury. An alternative hypothesis, such as excitotoxicity involving glutamate or other neurotransmitters, may account for the lesions demonstrated in this study.

Physical model simulations of brain injury in the primate.

Diffuse brain injuries resulting from non-impact rotational acceleration are investigated with the aid of physical models of the skull-brain structure. These models provide a unique insight into the relationship between the kinematics of head motion and the associated deformation of the surrogate brain material. Human and baboon skulls filled with optically transparent surrogate brain tissue are subjected to lateral rotations like those shown to produce diffuse injury to the deep white matter in the brain of the baboon. High-speed cinematography captures the deformations of the grids embedded within the surrogate brain tissue during the applied load. The overall deformation pattern is compared to the pathological portrait of diffuse brain injury as determined from animal studies and autopsy reports. Shear strain and pathology spatial distributions mirror each other. Load levels and resulting surrogate brain tissue deformations are related from one species to the other. Increased primate brain mass magnified the strain amplified without significantly altering the spatial distribution. An empirically-derived value for a critical shear strain associated with the onset of severe diffuse axonal injury in primates is determined, assuming constitutive similarity between baboon and human brain tissue. The primate skull physical model data and the critical shear strain associated with the threshold for severe diffuse axonal injury were used to scale data obtained from previous studies to man, and thus derive a diffuse axonal injury tolerance for rotational acceleration for humans.