D. Kacy Cullen

Strain rate-dependent induction of reactive astrogliosis and cell death in three-dimensional neuronal-astrocytic co-cultures

A mechanical insult to the brain drastically alters the microenvironment as specific cell types become reactive in an effort to sequester severely damaged tissue. Although injury-induced astrogliosis has been investigated, the relationship between well-defined biomechanical inputs and acute astrogliotic alterations is not well understood. We evaluated the effects of strain rate on cell death and astrogliosis using a three-dimensional (3-D) in vitro model of neurons and astrocytes within a bioactive matrix. At 21 days post-plating, co-cultures were deformed to 0.50 shear strain at strain rates of 1, 10, or 30 s(-1). We found that cell death and astrogliotic profiles varied differentially based on strain rate at 2 days post-insult. Significant cell death was observed after moderate (10 s(-1)) and high (30 s(-1)) rate deformation, but not after quasi-static (1 s(-1)) loading. The vast majority of cell death occurred in neurons, suggesting that these cells are more susceptible to high rate shear strains than astrocytes for the insult parameters used here. Injury-induced astrogliosis was compared to co-cultures treated with transforming growth factor beta, which induced robust astrocyte hypertrophy and increased glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs). Quasi-static loading resulted in increased cell density and CSPG secretion. Moderate rate deformation increased cell density, GFAP reactivity, and hypertrophic process density. High rate deformation resulted in increased GFAP reactivity; however, other astrogliotic alterations were not observed at this time-point. These results demonstrate that the mode and degree of astrogliosis depend on rate of deformation, demonstrating astrogliotic augmentation at sub-lethal injury levels as well as levels inducing cell death.

Developing a tissue-engineered neural-electrical relay using encapsulated neuronal constructs on conducting polymer fibers

Neural-electrical interface platforms are being developed to extracellularly monitor neuronal population activity. Polyaniline-based electrically conducting polymer fibers are attractive substrates for sustained functional interfaces with neurons due to their flexibility, tailored geometry and controlled electro-conductive properties. In this study, we addressed the neurobiological considerations of utilizing small diameter (<400 microm) fibers consisting of a blend of electrically conductive polyaniline and polypropylene (PA-PP) as the backbone of encapsulated tissue-engineered neural-electrical relays. We devised new approaches to promote survival, adhesion and neurite outgrowth of primary dorsal root ganglion neurons on PA-PP fibers. We attained a greater than ten-fold increase in the density of viable neurons on fiber surfaces to approximately 700 neurons mm(-2) by manipulating surrounding surface charges to bias settling neuronal suspensions toward fibers coated with cell-adhesive ligands. This stark increase in neuronal density resulted in robust neuritic extension and network formation directly along the fibers. Additionally, we encapsulated these neuronal networks on PA-PP fibers using agarose to form a protective barrier while potentially facilitating network stability. Following encapsulation, the neuronal networks maintained integrity, high viability (>85%) and intimate adhesion to PA-PP fibers. These efforts accomplished key prerequisites for the establishment of functional electrical interfaces with neuronal populations using small diameter PA-PP fibers-specifically, improved neurocompatibility, high-density neuronal adhesion and neuritic network development directly on fiber surfaces.

Three-dimensional neural constructs: a novel platform for neurophysiological investigation

Morphological and electrophysiological properties of neural cells are substantially influenced by their immediate extracellular surroundings, yet the features of this environment are difficult to mimic in vitro. Therefore, there is a tremendous need to develop a new generation of culture systems that more closely model the complexity of nervous tissue. To this end, we engineered novel electrophysiologically active 3D neural constructs composed of neurons and astrocytes within a bioactive extracellular matrix-based scaffold. Neurons within these constructs exhibited extensive 3D neurite outgrowth, expressed mature neuron-specific cytoskeletal proteins, and remained viable for several weeks. Moreover, neurons assumed complex 3D morphologies with rich neurite arborization and clear indications of network connectivity, including synaptic junctures. Furthermore, we modified whole-cell patch clamp techniques to permit electrophysiological probing of neurons deep within the 3D constructs, revealing that these neurons displayed both spontaneous and evoked electrophysiological action potentials and exhibited functional synapse formation and network properties. This is the first report of individual patch clamp recordings of neurons deep within 3D scaffolds. These tissue engineered cellular constructs provide an innovative platform for neurobiological and electrophysiological investigations, serving as an important step towards the development of more physiologically relevant neural tissue models.

Microfluidic engineered high cell density three-dimensional neural cultures

Three-dimensional (3D) neural cultures with cells distributed throughout a thick, bioactive protein scaffold may better represent neurobiological phenomena than planar correlates lacking matrix support. Neural cells in vivo interact within a complex, multicellular environment with tightly coupled 3D cell-cell/cell-matrix interactions; however, thick 3D neural cultures at cell densities approaching that of brain rapidly decay, presumably due to diffusion limited interstitial mass transport. To address this issue, we have developed a novel perfusion platform that utilizes forced intercellular convection to enhance mass transport. First, we demonstrated that in thick (>500 microm) 3D neural cultures supported by passive diffusion, cell densities <or=5.0 x 10(3) cells mm(-3) were required for survival. In 3D neuronal and neuronal-astrocytic co-cultures with increased cell density (10(4) cells mm(-3)), continuous medium perfusion at 2.0-11.0 microL min(-1) improved viability compared to non-perfused cultures (p < 0.01), which exhibited widespread cell death and matrix degradation. In perfused cultures, survival was dependent on proximity to the perfusion source at 2.00-6.25 microL min(-1) (p < 0.05); however, at perfusion rates of 10.0-11.0 microL min(-1) survival did not depend on the distance from the perfusion source, and resulted in a preservation of cell density with >90% viability in both neuronal cultures and neuronal-astrocytic co-cultures. This work demonstrates the utility of forced interstitial convection in improving the survival of high cell density 3D engineered neural constructs and may aid in the development of novel tissue-engineered systems reconstituting 3D cell-cell/cell-matrix interactions.

Neural substrate expansion for the restoration of brain function

Restoring neurological and cognitive function in individuals who have suffered brain damage is one of the principal objectives of modern translational neuroscience. Electrical stimulation approaches, such as deep-brain stimulation, have achieved the most clinical success, but they ultimately may be limited by the computational capacity of the residual cerebral circuitry. An alternative strategy is brain substrate expansion, in which the computational capacity of the brain is augmented through the addition of new processing units and the reconstitution of network connectivity. This latter approach has been explored to some degree using both biological and electronic means but thus far has not demonstrated the ability to reestablish the function of large-scale neuronal networks. In this review, we contend that fulfilling the potential of brain substrate expansion will require a significant shift from current methods that emphasize direct manipulations of the brain (e.g., injections of cellular suspensions and the implantation of multi-electrode arrays) to the generation of more sophisticated neural tissues and neural-electric hybrids in vitro that are subsequently transplanted into the brain. Drawing from neural tissue engineering, stem cell biology, and neural interface technologies, this strategy makes greater use of the manifold techniques available in the laboratory to create biocompatible constructs that recapitulate brain architecture and thus are more easily recognized and utilized by brain networks.

Animal models of traumatic brain injury.

Traumatic brain injury (TBI) is a major health issue comprising a heterogeneous and complex array of pathologies. Over the last several decades, numerous animal models have been developed to address the diverse nature of human TBI. The clinical relevance of these models has been a major point of reflection given the poor translation of pharmacologic TBI interventions to the clinic. While previously characterized broadly as either focal or diffuse, this classification is falling out of favor with increased awareness of the overlap in pathologic outcomes between models and an emerging consensus that no one model is sufficient. Moreover, an appreciation of injury biomechanics is essential in recapitulating and interpreting the spectrum of TBI neuropathology observed in various established models of dynamic closed-head TBI. While these models have replicated many specific features of human TBI, an enhanced context with clinical relevancy will facilitate the further elucidation of the mechanisms and treatment of injury.

In Vitro Neural Injury Model for Optimization of Tissue-Engineered Constructs

Stem cell transplantation is a promising approach for the treatment of traumatic brain injury, although the therapeutic benefits are limited by a high degree of donor cell death. Tissue engineering is a strategy to improve donor cell survival by providing structural and adhesive support. However, optimization prior to clinical implementation requires expensive and time‐consuming in vivo studies. Accordingly, we have developed a three‐dimensional (3‐D) in vitro model of the injured host–transplant interface that can be used as a test bed for high‐throughput evaluation of tissue‐engineered strategies. The neuronal‐astrocytic cocultures in 3‐D were subjected to mechanical loading (inducing cell death and specific astrogliotic alterations) or to treatment with transforming growth factor‐β1 (TGF‐β1), inducing astrogliosis without affecting viability. Neural stem cells (NSCs) were then delivered to the cocultures. A sharp increase in the number of TUNEL+ donor cells was observed in the injured cocultures compared to that in the TGF‐β1‐treated and control cocultures, suggesting that factors related to mechanical injury, but not strictly astrogliosis, were detrimental to donor cell survival. We then utilized the mechanically injured cocultures to evaluate a methylcellulose‐laminin (MC‐LN) scaffold designed to reduce apoptosis. When NSCs were codelivered with MC alone or MC‐LN to the injured cocultures, the number of caspase+ donor cells significantly decreased compared to that with vehicle delivery (medium). Collectively, these results demonstrate the utility of an in vitro model as a preanimal test bed and support further investigation of a tissue‐engineering approach for chaperoned NSC delivery targeted to improve donor cell survival in neural transplantation.

Long-Term Survival and Integration of Transplanted Engineered Nervous Tissue Constructs Promotes Peripheral Nerve Regeneration

Although peripheral nerve injury is a common consequence of trauma or surgery, there are insufficient means for repair. In particular, there is a critical need for improved methods to facilitate regeneration of axons across major nerve lesions. Here, we engineered transplantable living nervous tissue constructs to provide a labeled pathway to guide host axonal regeneration. These constructs consisted of stretch-grown, longitudinally aligned living axonal tracts inserted into poly(glycolic acid) tubes. The constructs (allogenic) were transplanted to bridge an excised segment of sciatic nerve in the rat, and histological analyses were performed at 6 and 16 weeks posttransplantation to determine graft survival, integration, and host regeneration. At both time points, the transplanted constructs were found to have maintained their pretransplant geometry, with surviving clusters of graft neuronal somata at the extremities of the constructs spanned by tracts of axons. Throughout the transplanted region, there was an intertwining plexus of host and graft axons, suggesting that the transplanted axons mediated host axonal regeneration across the lesion. By 16 weeks posttransplant, extensive myelination of axons was observed throughout the transplant region. Further, graft neurons had extended axons beyond the margins of the transplanted region, penetrating into the host nerve. Notably, this survival and integration of the allogenic constructs occurred in the absence of immunosuppression therapy. These findings demonstrate the promise of living tissue-engineered axonal constructs to bridge major nerve lesions and promote host regeneration, potentially by providing axon-mediated axonal outgrowth and guidance.

Trauma-Induced Plasmalemma Disruptions in Three-Dimensional Neural Cultures Are Dependent on Strain Modality and Rate

Traumatic brain injury (TBI) results from cell dysfunction or death following supra-threshold physical loading. Neural plasmalemma compromise has been observed following traumatic neural insults; however, the biomechanical thresholds and time-course of such disruptions remain poorly understood. In order to investigate trauma-induced membrane disruptions, we induced dynamic strain fields (0.50 shear or compressive strain at 1, 10, or 30?sec(?1) strain rate) in 3-D neuronal-astrocytic co-cultures (>500??m thick). Impermeant dyes were present during mechanical loading and entered cells in a strain rate-dependent manner for both shear and compression. Real-time imaging revealed increased membrane permeability in a sub-population of cells immediately upon deformation. Alterations in cell membrane permeability, however, were transient and biphasic over the ensuing hour post-insult, suggesting initial membrane damage and rapid repair, followed by a phase of secondary membrane degradation. At 48?h post-insult, cell death increased significantly in the high-strain-rate group, but not after quasi-static loading, suggesting that cell survival relates to the initial extent of transient structural compromise. Cells were more sensitive to bulk shear deformation than compression with respect to acute permeability changes and subsequent cell survival. These results provide insight into the temporally varying alterations in membrane stability following traumatic loading and provide a basis for elucidating physical cellular tolerances.

Synapse-to-neuron ratio is inversely related to neuronal density in mature neuronal cultures.

Synapse formation is a fundamental process in neurons that occurs throughout development, maturity, and aging. Although these stages contain disparate and fluctuating numbers of mature neurons, tactics employed by neuronal networks to modulate synapse number as a function of neuronal density are not well understood. The goal of this study was to utilize an in vitro model to assess the influence of cell density and neuronal maturity on synapse number and distribution. Specifically, cerebral cortical neurons were plated in planar culture at densities ranging from 10 to 5000 neurons/mm², and synapse number and distribution were evaluated via immunocytochemistry over 21 days in vitro (DIV). High-resolution confocal microscopy revealed an elaborate three-dimensional distribution of neurites and synapses across the heights of high-density neuronal networks by 21 DIV, which were up to 18 μm thick, demonstrating the complex degree of spatial interactions even in planar high-density cultures. At 7 DIV, the mean number of synapses per neuron was less than 5, and this did not vary as a function of neuronal density. However, by 21 DIV, the number of synapses per neuron had jumped 30- to 80-fold, and the synapse-to-neuron ratio was greatest at lower neuronal densities (< 500 neurons/mm²; mean approximately 400 synapses/neuron) compared to mid and higher neuronal densities (500-4500 neurons/mm²; mean of approximately 150 synapses/neuron) (p<0.05). These results suggest a relationship between neuronal density and synapse number that may have implications in the neurobiology of developing neuronal networks as well as processes of cell death and regeneration.