Biomedical subjects

Michelle C LaPlaca

Publications and source records attributed to Michelle C LaPlaca.

9 recordsLinked to original sources

PubMed2007

Three dimensional MEMS microfluidic perfusion system for thick brain slice cultures.

In vitro tissue culture models are often benchmarked by their ability to replicate in vivo function. One of the limitations of in vitro systems is the difficulty in preserving an orchestrated cell population, especially for generating three-dimensional tissue equivalents. For example, tissue-engineering applications involve large high-density constructs, requiring a perfusing system that is able to apply adequate oxygen and nutrients to the interior region of the tissue. This is particularly true with respect to thick tissue sections harvested for in vitro culture. We have fabricated a microneedle-based perfusion device for high-cell-density in vitro tissue culture from SU-8 photosensitive epoxy and suitable post-processing. The device was tested for its ability to improve viability in slices of harvested brain tissue. This model was chosen due to its acute sensitivity to disruptions in its nutrient supply. Improved viability was visible in the short term as assessed via live-dead discriminating fluorescent staining and confocal microscopy. This perfusion system opens up many possibilities for both neurobiological as well as other culture systems.

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PubMed2006

Thermoreversible laminin-functionalized hydrogel for neural tissue engineering.

Traumatic injury to the central nervous system (CNS) triggers cell death and deafferentation, which may activate a cascade of cellular and network disturbances. These events often result in the formation of irregularly shaped lesions comprised of necrotic tissue and/or a fluid-filled cavity. Tissue engineering represents a promising treatment strategy for the injured neural tissue. To facilitate minimally invasive delivery of a tissue engineered system, a thermoreversible polymer is an attractive scaffold candidate. We have developed a bioactive scaffold for neural tissue engineering by tethering laminin-1 (LN) to methylcellulose (MC), a thermoresponsive hydrogel. The base MC chain was oxidized via sodium m-periodate to increase MC tethering capacity. Protein immobilization was facilitated by a Schiff base reaction between primary amine groups on LN and the carbonyl groups of the oxidized MC chain. Immunoassays demonstrated tethering of LN at 1.6 +/- 0.5 ng of LN per milligram of MC. Rheological measurements for different MC-LN constructs indicated MC composition- and MC treatment-dependent effects on solution-gelation transition temperature. Cellular assays with primary rat cortical neurons demonstrated enhanced cell adhesion and viability on LN-functionalized MC when compared with base and oxidized MC. This bioadhesive thermoresponsive scaffold may provide a robust delivery vehicle to injured CNS tissue for neural cell transplantation strategies.

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PubMed2006

Neuronal response to high rate shear deformation depends on heterogeneity of the local strain field.

Many cellular models of traumatic brain injury (TBI) deform cells in a planar (2-D) configuration, a contrast from the three-dimensional (3-D) architecture of the brain, resulting in strain fields that may fail to represent the complex deformation patterns seen in vivo. Cells cultured in 3-D may more accurately represent in vivo cellular behavior than planar models due to differences in cytostructure, cell-cell/cell-matrix interactions and access to trophic factors; however, the effects of culture configuration on the response to high rate deformation have not been evaluated. We examined cell viability following a defined mechanical insult to primary cortical neurons distributed throughout a bioactive matrix (3-D) or in a monolayer sandwiched between layers of a bioactive matrix (2-D). After high rate loading (20 or 30 sec(1); 0.50 strain), there was a significant decrease in neuron viability for both configurations versus unloaded control cultures; however, neurons in 3-D presented greater cell death based on matched bulk loading parameters. Computer simulations of bulk loading predicted local cellular strains, revealing that neurons in 3-D were subjected to a heterogeneous strain field simultaneously consisting of tensile, compressive and shear strains; conversely, neurons in 2-D experienced a less complex deformation regime varying mainly based on shear strains. These results show differential susceptibility to mechanical loading between neurons cultured in 2-D and 3-D that may be due to differences in cellular strain manifestation. Models of TBI that accurately represent the related cellular biomechanics and pathophysiology are important for the elucidation of cellular tolerances and the development of mechanistically driven intervention strategies.

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PubMed2005

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.

Action Potentials↗

PubMed2005

High rate shear strain of three-dimensional neural cell cultures: a new in vitro traumatic brain injury model.

The fidelity of cell culture simulations of traumatic brain injury (TBI) that yield tolerance and mechanistic information relies on both the cellular models and mechanical insult parameters. We have designed and characterized an electro-mechanical cell shearing device in order to produce a controlled high strain rate injury (up to 0.50 strain, 30 s(-1) strain rate) that deforms three-dimensional (3-D) neural cultures (neurons or astrocytes in an extracellular matrix scaffold). Theoretical analysis revealed that these parameters generate a heterogeneous 3-D strain field throughout the cultures that is dependent on initial cell orientation within the matrix, resulting in various combinations of normal and shear strain. The ability to create a linear shear strain field over a range of input parameters was verified by tracking fluorescent microbeads in an acellular matrix during maximal displacement for a range of strains and strain rates. In addition, cell death was demonstrated in rat cortical astrocytes and neurons in response to high rate, high magnitude shear strain. Furthermore, cell response within the 3-D neuronal cultures depended on orientation, with higher predicted shear strain correlating with an increased loss of neurites, indicating that culture configuration may be an important factor in the mechanical, and hence cellular, response to traumatic insults. Collectively, these results suggest that differential responses exist within a 3-D culture subjected to mechanical insult, perhaps mimicking the in vivo environment, and that this new model can be used to investigate the complex cellular mechanisms associated with TBI.

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PubMed2004

Specific beta1 integrins mediate adhesion, migration, and differentiation of neural progenitors derived from the embryonic striatum.

Early inductive signals within the embryonic mammalian forebrain establish two major germinal regions along the dorsal-ventral axis. The dorsal germinal zone eventually forms the cerebral cortex while the ventral ganglionic eminence primarily forms the striatum and globus pallidus. The mechanisms leading to patterning of specific forebrain structures from these distinct germinal regions are not fully understood but may involve the adhesive and migratory properties of regionally specified cells and their interactions with the extracellular environments in which they reside. In the present study, we isolated ganglionic eminence neural progenitor cells (geNPC), precursors of the adult striatum, from the ventral forebrain germinal zone and analyzed adhesion, migration, and differentiation of geNPC on various extracellular matrix (ECM) substrates in vitro. Specifically, we evaluated the role of beta1 integrins, a family of cell surface receptors important in neural development, in mediating geNPC behavior on ECM molecules expressed in embryonic brain tissue. Adhesion and migration of geNPC were significantly enhanced on laminin (LN) and fibronectin (FN) relative to other ECM substrates. Antibody perturbation experiments revealed that although geNPC express several beta1 integrins (alpha1beta1, alpha2beta1, alpha3beta1, alpha5beta1, alpha6beta1, alphavbeta1), adhesion and migration on LN and FN were primarily mediated by alpha6beta1 and alpha5beta1, respectively, and these interactions were confirmed by biochemical cross-link/extraction procedures. Finally, neuronal differentiation of geNPC was enhanced on LN, indicating a role for LN in geNPC differentiation. beta1 integrin-ECM interactions may contribute to basic mechanisms of striatal development and may explain the potent migratory capacity of geNPC transplanted into the adult brain.

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PubMed2003

Susceptibility of hippocampal neurons to mechanically induced injury.

Experimental models of traumatic cortical brain injury in rodents reveal that specific regions of the hippocampus (e.g., CA3 and hilar subfields) are severely injured despite their distance from the initial insult. Hippocampal neurons may be intrinsically more vulnerable to mechanical insult than cortical neurons due to increased NMDA receptor densities and lower energy capacities, as evidenced by increased susceptibility to ischemic insults. The selective vulnerability of hippocampal neurons was evaluated using an in vitro model of TBI in which either primary rat cortical or hippocampal neurons (E17) seeded onto silicone substrates were subjected to graded levels of mechanical stretch. Although cortical neurons exhibited significantly longer increases in stretch-induced membrane permeability, injury of hippocampal neurons resulted in larger increases in intracellular free calcium concentration [Ca(2+)](i) and cell death. [ATP](i) deficits due to stretch were apparent by 60 min after injury in cortical neurons but recovered by 24 h, whereas significant deficits in [ATP](i) were not observed in hippocampal neurons until 24 h after injury. MK801 pretreatment decreased the stretch-induced [Ca(2+)](i) transients in both hippocampal and cortical cultures, thereby negating the regional specificity. However, MK801 pretreatment did not improve hippocampal viability and paradoxically, significantly increased cell death among cortical neurons. As the hippocampus is the primary brain region responsible for the memory deficits and epileptic seizures associated with TBI, understanding why this region is selectively damaged could lead to the development of more accurate mechanical tolerances as well as effective pharmaceutical agents.

Adenosine Triphosphate↗

PubMed2003

Mechanical stretch to neurons results in a strain rate and magnitude-dependent increase in plasma membrane permeability.

The mechanism by which mechanical impact to brain tissue is transduced to neuronal impairment remains poorly understood. Using an in vitro model of neuronal stretch, we found that mechanical stretch of neurons resulted in a transient plasma membrane permeability increase. Primary cortical neurons, seeded on silicone substrates, were subjected to a defined rate and magnitude strain pulse by stretching the substrates over a fixed cylindrical form. To identify plasma membrane defects, various sized fluorescent molecules were added to the bathing media either immediately before injury or 1, 2, 5, or 10 min after injury and removed one minute later. The percent of cells that took up dye depended on the applied strain rate, strain magnitude and molecular size. Severe stretch (10 sec(-1), 0.30) resulted in significant uptake of all tested molecules (ranging between 0.5 and 8.9 nm radii) with up to 60% of cells positively stained. Furthermore, the neurons remained permeable to the smallest molecule (carboxyfluorescein, 380 Da) up to 5 min after severe stretch but were only permeable to larger molecules (>/=10 kDa) immediately after stretch. These transiently formed membrane defects may be the initiating mechanism that translates mechanical stretch to cellular dysfunction.

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PubMed2002

Fibronectin promotes survival and migration of primary neural stem cells transplanted into the traumatically injured mouse brain.

Multipotential stem cells are an attractive choice for cell therapy after traumatic brain injury (TBI), as replacement of multiple cell types may be required for functional recovery. In the present study, neural stem cells (NSCs) derived from the germinal zone of E14.5 GFP-expressing mouse brains were cultured as neurospheres in FGF2-enhanced medium. When FGF2 was removed in vitro, NSCs expressed phenotypic markers for neurons. astrocytes, and oligodendrocytes and exhibited migratory behavior in the presence of adsorbed fibronectin (FN). NSCs (10(5) cells) were transplanted into mouse brains 1 week after a unilateral, controlled, cortical contusion (depth = 1 mm, velocity = 6 m/s, duration = 150 ms) (n = 19). NSCs were injected either directly into the injury cavity with or without an injectable FN-based scaffold [collagen I (CnI)/FN gel; n = 14] or into the striatum below the injury cavity (n = 5). At all time points examined (1 week to 3 months posttransplant), GFP+ cells were confined to the ipsilateral host brain tissue. At 1 week, cells injected into the injury cavity lined the injury penumbra while cells inserted directly into the striatum remained in or around the needle track. Striatal transplants had a lower number of surviving GFP+ cells relative to cavity injections at the 1 week time point (p < 0.01). At the longer survival times (3 weeks-3 months), 63-76% of transplanted cells migrated into the fimbria hippocampus regardless of injection site, perhaps due to cues from the degenerating hippocampus. Furthermore, cells injected into the cavity within a FN-containing matrix showed increased survival and migration at 3 weeks (p < 0.05 for both) relative to injections of cells alone. These results suggest that FGF2-responsive NSCs present a promising approach for cellular therapy following trauma and that the transplant location and environment may play an important role in graft survival and integration.

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