Biomedical subjects

David F Meaney

Publications and source records attributed to David F Meaney.

At least 19 recordsLinked to original sources

PubMed2006

Pharmacologically induced calcium oscillations protect neurons from increases in cytosolic calcium after trauma.

Increases in cytosolic calcium ([Ca(2+)](i)) following mechanical injury are often considered a major contributing factor to the cellular sequelae in traumatic brain injury (TBI). However, very little is known on how developmental changes may affect the calcium signaling in mechanically injured neurons. One key feature in the developing brain that may directly impact its sensitivity to stretch is the reduced inhibition which results in spontaneous [Ca(2+)](i) oscillations. In this study, we examined the mechanism of stretch-induced [Ca(2+)](i) transients in 18-days in vitro (DIV) neurons exhibiting bicuculline-induced [Ca(2+)](i) oscillations. We used an in vitro model of mechanical trauma to apply a defined uniaxial strain to cultured cortical neurons and used increases in [Ca(2+)](i) as a measure of the neuronal response to the stretch insult. We found that stretch-induced increases in [Ca(2+)](i) in 18-DIV neurons were inhibited by pretreatment with either the NMDA receptor antagonist, APV [D(-)-2-Amino-5-phosphonopentanoic acid], or by depolymerizing the actin cytoskeleton prior to stretch. Blocking synaptic NMDA receptors prior to stretch significantly attenuated most of the [Ca(2+)](i) transient. In comparison, cultures with pharmacologically induced [Ca(2+)](i) oscillations showed a substantially reduced [Ca(2+)](i) peak after stretch. We provide evidence showing that a contributing factor to this mechanical desensitization from induced [Ca(2+)](i) oscillations is the PKC-mediated uncoupling of NMDA receptors (NMDARs) from spectrin, an actin-associated protein, thereby rendering neurons insensitive to stretch. These results provide novel insights into how the [Ca(2+)](i) response to stretch is initiated, and how reduced inhibition - a feature of the developing brain - may affect the sensitivity of the immature brain to trauma.

2-Amino-5-phosphonovalerate↗

PubMed2006

Matrices with compliance comparable to that of brain tissue select neuronal over glial growth in mixed cortical cultures.

Cortical neurons and astrocytes respond strongly to changes in matrix rigidity when cultured on flexible substrates. In this study, existing polyacrylamide gel polymerization methods were modified into a novel method for making substrates capable of engaging specific cell-adhesion receptors. Embryonic cortical dissociations were cultured on polyacrylamide or fibrin gel scaffolds of varying compliance. On soft gels, astrocytes do not spread and have disorganized F-actin compared to the cytoskeletons of astrocytes on hard surfaces. Neurons, however, extend long neurites and polymerize actin filaments on both soft and hard gels. Compared to tissue culture plastic or stiff gel substrates coated with laminin, on which astrocytes overgrow neurons in mixed cultures, laminin-coated soft gels encourage attachment and growth of neurons while suppressing astrocyte growth. The number of astrocytes on soft gels is lower than on hard even in the absence of mitotic inhibitors normally used to temper the astrocyte population. Dissociated embryonic rat cortices grown on flexible fibrin gels, a biomaterial with potential use as an implant material, display a similar mechano-dependent difference in cell population. The stiffness of materials required for optimal neuronal growth, characterized by an elastic modulus of several hundred Pa, is in the range measured for intact rat brain. Together, these data emphasize the potential importance of material substrate stiffness as a design feature in the next generation of biomaterials intended to promote neuronal regeneration across a lesion in the central nervous system while simultaneously minimizing the ingrowth of astrocytes into the lesion area.

Acrylic Resins↗

PubMed2006

Mechanisms and consequences of neuronal stretch injury in vitro differ with the model of trauma.

The deformation to the brain that occurs during traumatic brain injury (TBI) results in a complex strain distribution throughout the brain tissue. Recently, many in vitro models of neuronal injury have been developed to simplify the mechanics which occur during TBI. We hypothesized that the type of mechanical insult imparted onto neurons would significantly alter both the mechanism and severity of the neuronal response to injury. In this study, primary cortical neurons were cultured on an elastic substrate and subjected to graded levels (0%, 10%, 30%, 50%) of either uniaxial (cells stretched in one direction only) or biaxial (cells simultaneously stretched in two directions) stretch. We found that neurons stretched in either injury paradigm exhibited immediate increases in intracellular free calcium ([Ca2+]i), but the magnitude of the ([Ca2+]i) rise was nearly an order of magnitude higher in biaxially stretched neurons compared to uniaxially stretched neurons. Moreover, while the ([Ca2+]i) transient after uniaxial stretch was blocked with specific channel antagonists (APV, CNQX, nimodipine, TTX), a substantial ([Ca2+]i) transient persisted in biaxially stretched neurons. We theorized that the additional calcium influx after biaxial stretch entered through nonspecific pores/tears formed in the membrane, since biaxially stretched neurons exhibited significant uptake of carboxyfluorescein, a molecule typically impermeant to cell membranes. Despite the large ([Ca2+]i) transients, neither injury profile resulted in death within 24 h of injury. Interestingly, though, uniaxially stretched neurons exhibited enhanced [Ca+2]i influx following NMDA stimulation 24 h after trauma, compared to both control and biaxially stretched neurons. These data point out that the type of mechanical insult will influence the acute mechanisms of injury in vitro, can cause differences in the response to potential secondary excitotoxic injury mechanisms, and emphasizes the need to further study how these mechanical conditions can separately affect cell fate following mechanical injury.

Animals↗

PubMed2005

Traumatic mechanical injury to the hippocampus in vitro causes regional caspase-3 and calpain activation that is influenced by NMDA receptor subunit composition.

Apoptotic or necrotic cell death in the hippocampus is a major factor underlying the cognitive impairments following traumatic brain injury. In this study, we examined if traumatic mechanical injury would produce regional activation of calpain and caspase-3 in the in vitro hippocampus and studied how the mechanically induced activation of NR2A and NR2B containing N-methyl-d-aspartate receptors (NMDARs) affects the activation of these proteases following mechanical injury. Following a 75% stretch, significant levels of activated caspase-3 and calpain-mediated spectrin breakdown products were evident only in cells within the dentate gyrus, and little co-localization of the markers was identified within individual cells. After 100% stretch, only calpain activation was observed, localized to the CA3 subregion 24 h after stretch. At moderate injury levels, both caspase-3 and calpain activation was attenuated by blocking NR2B containing NMDARs prior to stretch or by blocking all NMDARs prior to stretch injury. Treatment with an NR2A selective NMDAR antagonist had little effect on either activated caspase-3 or Ab38 immunoreactivity following moderate injury but resulted in the appearance of activated caspase-3 in the dentate gyrus following severe mechanical stretch. Together, these studies suggest that the injury induced activation of NR2A containing NMDARs functions as a pro-survival signal, while the activation of NR2B containing NMDARs is a competing, anti-survival, signal following mechanical injury to the hippocampus.

Animals↗

PubMed2005

Development of transplantable nervous tissue constructs comprised of stretch-grown axons.

Pursuing a new approach to nervous system repair, fasciculated axon tracts grown in vitro were developed into nervous tissue constructs designed to span peripheral nerve or spinal cord lesions. We optimized the newfound process of extreme axon stretch growth to maximize the number and length of axon tracts, reach an unprecedented axon growth-rate of 1cm/day, and create 5cm long axon tracts in 8 days to serve as the core component of a living nervous tissue construct. Immunocytochemical analysis confirmed that elongating fibers were axons, and that all major cytoskeletal constituents were present across the stretch-growth regions. We formed a transplantable nervous tissue construct by encasing the elongated cells in an 80% collagen hydrogel, removing them from culture, and inserting them into a synthetic conduit. Alternatively, we induced axon stretch growth directly on a surgical membrane that could be removed from the elongation device, and formed into a cylindrical construct suitable for transplant. The ability to rapidly create living nervous tissue constructs that recapitulates the uniaxial orientations of the original nerve offers an unexplored and potentially complimentary direction in nerve repair. Ideally, bridging nerve damage with living axon tracts may serve to establish or promote new functional connections.

Animals↗

PubMed2005

Temporal window of vulnerability to repetitive experimental concussive brain injury.

OBJECTIVE: Repetitive concussive brain injury (CBI) is associated with cognitive alterations and increased risk of neurodegenerative disease. METHODS: To evaluate the temporal window during which the concussed brain remains vulnerable to a second concussion, anesthetized mice were subjected to either sham injury or single or repetitive CBI (either 3, 5, or 7 days apart) using a clinically relevant model of CBI. Cognitive, vestibular, and sensorimotor function (balance and coordination) were evaluated, and postmortem histological analyses were performed to detect neuronal degeneration, cytoskeletal proteolysis, and axonal injury. RESULTS: No cognitive deficits were observed in sham-injured animals or those concussed once. Mice subjected to a second concussion within 3 or 5 days exhibited significantly impaired cognitive function compared with either sham-injured animals (P < 0.05) or mice receiving a single concussion (P < 0.01). No cognitive deficits were observed when the interconcussion interval was extended to 7 days, suggestive of a transient vulnerability of the brain during the first 5 days after an initial concussion. Although all concussed mice showed transient motor deficits, vestibulomotor dysfunction was more pronounced in the group that sustained two concussions 3 days apart (P < 0.01 compared with all other groups). Although scattered degenerating neurons, evidence of cytoskeletal damage, and axonal injury were detected in selective brain regions between 72 hours and 1 week after injury in all animals sustaining a single concussion, the occurrence of a second concussion 3 days later resulted in significantly greater traumatic axonal injury (P < 0.05) than that resulting from a single CBI. CONCLUSION: These data suggest that a single concussion is associated with behavioral dysfunction and subcellular alterations that may contribute to a transiently vulnerable state during which a second concussion within 3 to 5 days can lead to exacerbated and more prolonged axonal damage and greater behavioral dysfunction.

Animals↗

PubMed2004

Extreme stretch growth of integrated axons.

Large animals can undergo enormous growth during development, suggesting that axons in nerves and white matter tracts rapidly expand as well. Because integrated axons have no growth cones to extend from, it has been postulated that mechanical forces may stimulate axon elongation matching the growth of the animal. However, this distinct form of rapid and sustained growth of integrated axons has never been demonstrated. Here, we used a microstepper motor system to evaluate the effects of escalating rates of stretch on integrated axon tracts over days to weeks in culture. We found that axon tracts could be stretch grown at rates of 8 mm/d and reach lengths of 10 cm without disconnection. Despite dynamic and long-term elongation, stretched axons increased in caliber by 35%, while the morphology and density of cytoskeletal constituents and organelles were maintained. These data provide the first evidence that mechanical stimuli can induce extreme "stretch growth" of integrated axon tracts, far exceeding any previously observed limits of axon growth.

Animals↗

PubMed2004

Traumatic axonal injury induces proteolytic cleavage of the voltage-gated sodium channels modulated by tetrodotoxin and protease inhibitors.

We demonstrated previously that dynamic stretch injury of cultured axons induces structural changes and Ca2+ influx modulated by tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (NaChs). In the present study, we evaluated potential damage to the NaCh alpha-subunit, which can cause noninactivation of NaChs. In addition, we explored the effects of pre-injury and post-injury treatment with TTX and protease inhibition on proteolysis of the NaCh alpha-subunit and intra-axonal calcium levels ([Ca2+]i) over 60 min after trauma. After stretch injury, we found that [Ca2+]i continued to increase in untreated axons for at least 60 min. We also observed that the III-IV intra-axonal loop of the NaCh alpha-subunit was proteolyzed between 5 and 20 min after trauma. Pre-injury treatment of the axons with TTX completely abolished the posttraumatic increase in [Ca2+]i and proteolysis of the NaCh alpha-subunit. In addition, both pre-injury and post-injury inhibition of protease activity attenuated long-term increases in [Ca2+]i as well as mitigating degradation of the NaCh alpha-subunit. These results suggest a unique "feed-forward" deleterious process initiated by mechanical trauma of axons. Na+ influx through NaChs resulting from axonal deformation triggers initial increases in [Ca2+]i and subsequent proteolysis of the NaCh-subunit. In turn, degradation of the alpha-subunit promotes persistent elevations in [Ca2+]i, fueling additional pathologic changes. These observations may have important implications for developing therapeutic strategies for axonal trauma.

Animals↗

PubMed2004

Long-term accumulation of amyloid-beta, beta-secretase, presenilin-1, and caspase-3 in damaged axons following brain trauma.

Plaques composed of amyloid beta (Abeta) have been found within days following brain trauma in humans, similar to the hallmark plaque pathology of Alzheimer's disease (AD). Here, we evaluated the potential source of this Abeta and long-term mechanisms that could lead to its production. Inertial brain injury was induced in pigs via head rotational acceleration of 110 degrees over 20 ms in the coronal plane. Animals were euthanized at 3 hours, 3 days, 7 days, and 6 months post-injury. Immunohistochemistry and Western blot analyses of the brains were performed using antibodies specific for amyloid precursor protein (APP), Abeta peptides, beta-site APP-cleaving enzyme (BACE), presenilin-1 (PS-1), caspase-3, and caspase-mediated cleavage of APP (CCA). Substantial co-accumulation for all of these factors was found in swollen axons at all time points up to 6 months following injury. Western blot analysis of injured brains confirmed a substantial increase in the protein levels of these factors, particularly in the white matter. These data suggest that impaired axonal transport due to trauma induces long-term pathological co-accumulation of APP with BACE, PS-1, and activated caspase. The abnormal concentration of these factors may lead to APP proteolysis and Abeta formation within the axonal membrane compartment.

Amyloid Precursor Protein Secretases↗

PubMed2004

Methodological considerations regarding single-cell gene expression profiling for brain injury.

Genomic microarrays are rapidly becoming ubiquitous throughout a wide variety of biological disciplines. As their use has grown during the past few years, many important discoveries have been made in the fields of central nervous system (CNS) injury and disease using this emerging technology. In addition, single-cell mRNA amplification techniques are now being used along with microarrays to overcome many of the difficulties associated with the cellular heterogeneity of the brain. This development has extended the utility of gene expression profiling and has provided researchers with exciting new insights into the neuropathology of CNS injury and disease at a molecular and cellular level. New methodological, standardization, and statistical techniques are currently being developed to improve the reproducibility of microarrays and facilitate the analysis of large amounts of data. In this review, we will discuss the application of these techniques to experimental, clinically relevant models of traumatic brain injury.

Brain Injuries↗

PubMed2004

Effect of acute calcium influx after mechanical stretch injury in vitro on the viability of hippocampal neurons.

We use a new in vitro model to examine the effect of mechanical deformation on neurons. We examined acute changes in cytosolic calcium concentrations ([Ca(2+)](i)) caused by a rapid stretch of cultured hippocampal neurons, using mechanical loading conditions that mimic brain deformations during trauma. We found that stretch-injury of neurons induces a strain-dependent increase in [Ca(2+)](i). Remarkably, the extent of this calcium response exceeded the levels initiated by chemical toxicity with NMDA (100 microM) or glutamate (5 mM) exposure. Propidium iodide labeling at 24 h following stretch showed neuronal death occurred only at the most severe level of mechanical injury. Although NMDA-induced toxicity could be inhibited in calcium free media or by treatment with MK-801, stretch-induced neuronal death was not similarly reduced with either treatment. Unexpectedly, reduction of the acute stretch-induced calcium transient with calcium-free media or MK-801 resulted in an increase in neuronal death at lower stretch levels. These data suggest that mechanical stretch can initiate calcium influx in hippocampal neurons, but substantially modulating the early calcium flux from the extracellular space or through the NMDA channel does not provide an effective means for improving neuronal survival.

Animals↗

PubMed2004

A device to study the initiation and propagation of calcium transients in cultured neurons after mechanical stretch.

The brain is generally considered protected from mechanical forces. However, during traumatic events, cells in brain tissue are exposed to complex mechanical events including transient acceleration, pressure, and direct stretch. This paper presents a model to expose cultured cells of the central nervous system (CNS) to a defined stretch insult. The system is designed to apply a single transient uniaxial stretch to cultured cells; control of both the magnitude and rate of stretch allows study of a broad range of conditions, from physiologic to traumatic. Distinct from previous cell-stretching systems, we control the fraction of cells deformed and examine the response of stretched cells or the surrounding population of unstretched cells, the "mechanical penumbra," of the same culture. Finally, we use this new model with cultured neurons, measuring the acute calcium response in traumatically stretched cells and the propagation of this calcium influx in neighboring, but unstretched cells. Stretched neurons exhibit a strain rate and magnitude-dependent response, unstretched exhibit an "all-or-none" response. This model will further our understanding of the complex interaction between mechanical stimuli, resulting biochemical cascades, and interaction between mechanical and other challenges, thereby furthering our understanding of the initiation and evolution of cellular damage following traumatic CNS injury.

Animals↗

PubMed2004

A quasi-linear, viscoelastic, structural model of the plantar soft tissue with frequency-sensitive damping properties.

Little is known about the structural properties of plantar soft-tissue areas other than the heel; nor is it known whether the structural properties vary depending on location. Furthermore, although the quasi-linear viscoelastic (QLV) theory has been used to model many soft-tissue types, it has not been employed to model the plantar soft tissue. The structural properties of the plantar soft tissue were quantified via stress relaxation experiments at seven regions (subcalcaneal, five submetatarsal, and subhallucal) across eight cadaveric feet. The cadaveric feet were 36.9 +/- 17.4 (mean +/- S.D.) years of age, all free from vascular diseases and orthopedics disorders. All tests were performed at a constant environmental temperature of 35 degrees C. Stress relaxation experiments were performed; different loads were employed for different areas based on normative gait data. A modification of the relaxation spectrum employed within the QLV theory allowed for the inclusion of frequency-sensitive relaxation properties in addition to nonlinear elastic behavior. The tissue demonstrated frequency-dependent damping properties that made the QLV theory ill suited to model the relaxation. There was a significant difference between the elastic structural properties (A) of the subcalcaneal tissue and all other areas (p = 0.004), and a trend (p = 0.067) for the fifth submetatarsal to have less viscous damping (c1) than the subhallucal, or first, second, or third submetatarsal areas. Thus, the data demonstrate that the structural properties of the foot can vary across regions, but careful consideration must be given to the applied loads and the manner in which the loads were applied.

Adolescent↗

PubMed2003

Traumatic axonal injury results in biphasic calpain activation and retrograde transport impairment in mice.

Traumatic axonal injury (TAI) is one of the most important pathologies associated with closed head injury, and contributes to ensuing morbidity. The authors evaluated the potential role of calpains in TAI using a new model of optic nerve stretch injury in mice. Male C57BL/6 mice were anesthetized, surgically prepared, and subjected to a 2.0-mm optic nerve stretch injury (n = 34) or sham injury (n = 18). At various intervals up to 2 weeks after injury, optic nerves were examined for neurofilament proteins and calpain-mediated spectrin breakdown products using immunohistochemistry. In addition, fluorescent tracer was injected into the superior colliculi of mice 1 day before they were killed, to investigate the integrity of retrograde axonal transport to the retina. Optic nerve stretch injury resulted in persistent disruption of retrograde axonal transport by day 1, progressive accumulation and dephosphorylation of neurofilament protein in swollen and disconnected axons, and subsequent loss of neurofilament protein in degenerating axons at day 14. Calpains were transiently activated in intact axons in the first minutes to hours after stretch injury. A second stage of calpain-mediated proteolysis was observed at 4 days in axonal swellings, bulbs, and fragments. These data suggest that early calpain activation may contribute to progressive intraaxonal structural damage, whereas delayed calpain activation may be associated with axonal degeneration.

Animals↗

PubMed2003

Modeling of microstructural kinematics during simple elongation of central nervous system tissue.

Damage to axons and glial cells in the central nervous system (CNS) white matter is a nearly universal feature of traumatic brain injury, yet it is not clear how the tissue mechanical deformations are transferred to the cellular components of the CNS. Defining how cellular deformations relate to the applied tissue deformation field can both highlight cellular populations at risk for mechanical injury, and define the fraction of cells in a specific population that will exhibit damage. In this investigation, microstructurally based models of CNS white matter were developed and tested against measured transformations of the CNS tissue microstructure under simple elongation. Results show that axons in the unstretched optic nerves were significantly wavy or undulated, where the measured axonal path length was greater than the end-to-end distance of the axon. The average undulation parameter--defined as the true axonal length divided by the end-to-end length--was 1.13. In stretched nerves, mean axonal undulations decreased with increasing applied stretch ratio (lambda)--the mean undulation values decreased to 1.06 at lambda = 1.06, 1.04 at lambda = 1.12, and 1.02 at lambda = 1.25. A model describing the gradual coupling, or tethering, of the axons to the surrounding glial cells best fit the experimental data. These modeling efforts indicate the fraction of the axonal and glial populations experiencing deformation increases with applied elongation, consistent with the observation that both axonal and glial cell injury increases at higher levels of white matter injury. Ultimately, these results can be used in conjunction with computational simulations of traumatic brain injury to aid in establishing the relative risk of cellular structures in the CNS white matter to mechanical injury.

Animals↗

PubMed2003

The separate roles of calcium and mechanical forces in mediating cell death in mechanically injured neurons.

In this study, we examine the response of cultured hippocampal neurons to mechanical stretch. To measure the immediate response, we measured the response of a calcium sensitive fluorescent dye (Fura-2AM) prior to and for five minutes following stretch. In separate experiments, we measured the viability of the cells using propidium iodide labeling at 24 hours following a single stretch. Results show that cytosolic calcium increases immediately following stretch to levels much higher than those associated with maximal chemical agonist (NMDA) stimulation. Moreover, the calcium response data indicate a loss in calcium regulation in the neurons at the most severe level of stretch. Finally, at the highest levels of stretch, the cell survivability decreases at 24 hrs following the stretch injury. These data show the association between loss in calcium regulation and early signs of neuronal cell death.

Animals↗

PubMed2002

Expression profiling following traumatic brain injury: a review.

Traumatic brain injury (TBI) elicits a complex sequence of putative autodestructive and neuroprotective cellular cascades. It is hypothesized that the genomic responses of cells in the injured brain serve as the basis for these cascades. Traditional methods for analyzing differential gene expression following brain trauma demonstrate that immediate early genes, cytokines, transcription factors, and neurotrophic factors can all participate in the brain's active and directed response to injury, and may do so concurrently. It is this complexity and multiplicity of interrelated molecular mechanisms that has demanded new methods for comprehensive and parallel evaluation of putative as well as novel gene targets. Recent advances in DNA microarray technology have enabled the simultaneous evaluation of thousands of genes and the subsequent generation of massive amounts of biological data relevant to CNS injury. This emerging technology can serve to further current knowledge regarding recognized molecular cascades as well as to identify novel molecular mechanisms that occur throughout the post-traumatic period. The elucidation of the complex alterations in gene expression underlying the pathological sequelae following TBI is of central importance in the design of future therapeutic agents.

Animals↗

PubMed2002

Roller coasters, g forces, and brain trauma: on the wrong track?

There has been enormous attention in the general press on the possibility that high G force roller coasters are inducing brain injury in riders. Armed with a handful of anecdotal case reports of brain injuries, the U.S. Congress has recently proposed legislation to regulate the level of G forces of roller coasters. However, high G forces are well tolerated during many activities and, therefore, are a poor measure for the risk of brain injury. Rather, accelerations of the head that can be caused by G forces are the key to producing injury. To determine the extent of head accelerations during roller coaster rides, we acquired G force data from three popular high G roller coasters. We used the highest recorded G forces in a simple mathematical model of head rotational acceleration, with the head rigidly pivoting from the base of the skull at a radius representing typical men and women. With this model, we calculated peak head rotational accelerations in three directions. Even for a conservative worst-case scenario, we found that the highest estimated peak head accelerations induced by roller coasters were far below conventional levels that are predicted for head injuries. Accordingly, our findings do not support the contention that current roller coaster rides produce high enough forces to mechanically deform and injure the brain.

Acceleration↗