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Millions suffer a traumatic brain injury (TBI) each year wherein the outcomes associated with injury can vary greatly between individuals. This study postulates that variations in each biomechanical parameter of a head trauma lead to differences in histological and behavioral outcome measures that should be considered collectively in assessing injury. While trauma severity typically scales with the magnitude of injury, much less is known about the effects of rate and duration of the mechanical insult. In this study, a newly developed voice-coil fluid percussion injury system was used to investigate the effects of injury rate and fluid percussion impulse on a collection of post-injury outcomes in male rats. Collectively the data suggest a potential shift in the specificity and progression of neuronal injury and function rather than a general scaling of injury severity. While a faster, shorter fluid percussion first presents as a mild TBI, neuronal loss and some behavioral tasks were similar among the slower and faster fluid percussion injuries. This study concludes that the sequelae of neuronal degeneration and behavioral outcomes are related to the complete temporal profile of the fluid percussion and do not scale only with peak pressure.

Forces and displacements play essential roles in the development and maintenance of all living organisms. The biomechanics in living systems includes traction in cell motility, motor proteins driving cellular transport, tensegrity of the cytoskeleton, and mechanotransduction of signal pathways. In the past few decades, we have learned a great deal about how biomechanics is important in the development, maintenance, and repair of the nervous system. In this review, we critically assess recent advances in using mechanical stimuli toward exploiting the directed growth and formation of nervous tissues. We discuss current systems that recapitulate the mechanical environment surrounding neural tissues as engineering solutions to explore the relationship forces play in modifying neurons and their processes, as well as exploit these processes to enhance regeneration and repair.

Myelin loss in brain is a common occurrence in traumatic brain injury (TBI) that results from impact-induced acceleration forces to the head. Fast and abrupt head motions, either resulting from violent blows and/or jolts, cause rapid stretching of the brain tissue, and the long axons within the white matter tracts are especially vulnerable to such mechanical strain. Recent studies have shown that mechanotransduction plays an important role in regulating oligodendrocyte progenitors cell differentiation into oligodendrocytes. However, little is known about the impact of mechanical strain on mature oligodendrocytes and the stability of their associated myelin sheaths. We used an in vitro cellular stretch device to address these questions, as well as characterize a mechanotransduction mechanism that mediates oligodendrocytes’ responses. Mechanical stretch caused a transient and reversible myelin protein loss in oligodendrocytes. Cell death was not observed. Myelin protein loss was accompanied by an increase in intracellular Ca(2+) and Erk1/2 activation. Chelating Ca(2+) or inhibiting Erk1/2 activation was sufficient to block the stretch-induced loss of myelin protein. Further biochemical analyses revealed that the stretch-induced myelin protein loss was mediated by the release of Ca(2+) from the endoplasmic reticulum (ER) and subsequent Ca(2+)-dependent activation of Erk1/2. Altogether, our findings characterize an Erk1/2-dependent mechanotransduction mechanism in mature oligodendrocytes that destabilizes the myelination program.

Trauma to the brain is a biomechanical problem where the initiating event is a dynamic loading (blunt, inertial, blast) to the head. To understand the relationship between the mechanical parameters of the injury and the spatial and temporal deformation patterns in the brain, there is a need to develop a reusable and adaptable experimental traumatic brain injury (TBI) model that can measure brain motion under varying parameters. In this effort, we aim to directly measure brain deformation (strain and strain rates) in different brain regions in a human head model using a drop tower. Methods: Physical head models consisting of a half, sagittal plane skull, brain, and neck were constructed and subjected to crown and frontal impacts at two impact speeds. All tests were recorded with a high-speed camera at 1000 frames per second. Motion of visual markers within brain surrogates were used to track deformations and calculate spatial strain histories in 6 brain regions of interest. Principal strains, strain rates and strain impulses were calculated and reported. Results: Higher impact velocities corresponded to higher strain values across all impact scenarios. Crown impacts were characterized by high, long duration strains distributed across the parietal, frontal and hippocampal regions whereas frontal impacts were characterized by sharply rising and falling strains primarily found in the parietal, frontal, hippocampal and occipital regions. High strain rates were associated with short durations and impulses indicating fast but short-lived strains. 2.23 m/s (5 mph) crown impacts resulted in 53% of the brain with shear strains higher than 0.15 verses 32% for frontal impacts. Conclusions: The results reveal large differences in the spatial and temporal strain responses between crown and forehead impacts. Overall, the results suggest that for the same speed, crown impact leads to higher magnitude strain patterns than a frontal impact. The data provided by this model provides unique insight into the spatial and temporal deformation patterns that have not been provided by alternate surrogate models. The model can be used to investigate how anatomical, material and loading features and parameters can affect deformation patterns in specific regions of interest in the brain.

Blast-induced neurotrauma (BINT) is not only a signature injury to soldiers in combat field and training facilities but may also a growing concern in civilian population due to recent increases in the use of improvised explosives by insurgent groups. Unlike moderate or severe BINT, repeated low-level blast (rLLB) is different in its etiology as well as pathology. Due to the constant use of heavy weaponry as part of combat readiness, rLLB usually occurs in service members undergoing training as part of combat readiness. rLLB does not display overt pathological symptoms; however, earlier studies report chronic neurocognitive changes such as altered mood, irritability, and aggressive behavior, all of which may be caused by subtle neuropathological manifestations. Current animal models of rLLB for investigation of neurobehavioral and neuropathological alterations have not been adequate and do not sufficiently represent rLLB conditions. Here, we developed a rat model of rLLB by applying controlled low-level blast pressures (10 psi) repeated successively five times to mimic the pressures experienced by service members. Using this model, we assessed anxiety-like symptoms, motor coordination, and short-term memory as a function of time. We also examined levels of superoxide-producing enzyme NADPH oxidase, microglial activation, and reactive astrocytosis as factors likely contributing to these neurobehavioral changes. Animals exposed to rLLB displayed acute and chronic anxiety-like symptoms, motor and short-term memory impairments. These changes were paralleled by increased microglial activation and reactive astrocytosis. Conversely, animals exposed to a single low-level blast did not display significant changes. Collectively, this study demonstrates that, unlike a single low-level blast, rLLB exerts a cumulative impact on different brain regions and produces chronic neuropathological changes in so doing, may be responsible for neurobehavioral alterations.

At birth, there are 100 billion neurons in the human brain, with functional neural circuits extending through the spine to the epidermis of the feet and toes. Following birth, limbs and vertebrae continue to grow by several orders of magnitude, forcing established axons to grow by up to 200 cm in length without motile growth cones. The leading regulatory paradigm suggests that biomechanical expansion of mitotic tissue exerts tensile force on integrated nervous tissue, which synchronizes ongoing growth of spanning axons. Here, we identify unique transcriptional changes in embryonic rat DRG and cortical neurons while the corresponding axons undergo physiological levels of controlled mechanical stretchin vitro. Using bioreactors containing cultured neurons, we recapitulated the peak biomechanical increase in embryonic rat crown-rump-length. Biologically paired sham and “stretch-grown” DRG neurons spanned 4.6- and 17.2-mm in length following static or stretch-induced growth conditions, respectively, which was associated with 456 significant changes in gene transcription identified by genome-wide cDNA microarrays. Eight significant genes found in DRG were cross-validated in stretch-grown cortical neurons by qRT-PCR, which included upregulation ofGpat3, Crem, Hmox1, Hpse, Mt1a, Nefm,Sprr1b, and downregulation ofNrep.The results herein establish a link between biomechanics and gene transcription in mammalian neurons, which elucidates the mechanism underlying long-term growth of axons, and provides a basis for new research in therapeutic axon regeneration.

Blast exposure has been identified to be the most common cause for traumatic brain injury (TBI) in soldiers. Over the years, rodent models to mimic blast exposures and the behavioral outcomes observed in veterans have been developed extensively. However, blast tube design and varying experimental parameters lead to inconsistencies in the behavioral outcomes reported across research laboratories. This review aims to curate the behavioral outcomes reported in rodent models of blast TBI using shockwave tubes or open field detonations between the years 2008-2019 and highlight the important experimental parameters that affect behavioral outcome. Further, we discuss the role of various design parameters of the blast tube that can affect the nature of blast exposure experienced by the rodents. Finally, we assess the most common behavioral tests done to measure cognitive, motor, anxiety, auditory, and fear conditioning deficits in blast TBI (bTBI) and discuss the advantages and disadvantages of these tests.

Biomaterial-based scaffolds used in nerve conduits including channels for confining regenerating axons and 3-dimensional (3D) gels as substrates for growth have made improvements in models of nerve repair. Many biomaterial strategies, however, continue to fall short of autologous nerve grafts, which remain the current gold standard in repairing severe nerve lesions (

Soldiers are often exposed to more than one traumatic brain injury (TBI) over the course of their service. In recent years, more attention has been drawn to the increased risk of neurological deficits caused by the 'blast plus' polytrauma, which typically is a blast trauma combined with other forms of TBI. In this study, we investigated the behavioral and neuronal deficits resulting from a blast plus injury involving a mild-moderate blast followed by a mild blunt trauma using the fluid percussion injury model. We identified that the blast injury predisposed the brain to increased cognitive deficits, chronic ventricular enlargement, increased neurodegeneration at acute time points and chronic neuronal loss. Interestingly, a single blast and single blunt injury differed in their onset and manifestation of cognitive and regional neuronal loss. We also identified the presence of cleaved RIP1 from caspase 8 mediated apoptosis in the blunt injury while the blast injury did not activate immediate apoptosis but led to decreased hilar neuronal survival over time.

Traumatic brain injury (TBI) contributes a major cause of death, disability, and mental health disorders. Most TBI patients suffer long-term post-traumatic stress disorder, cognitive dysfunction, and disability. The underlying molecular and cellular mechanisms of such neuropathology progression in TBI remain elusive. In part, it is due to non-standardized classification of mild, moderate, and severe injury in various animal models of TBI. Thus, a better diagnosis and treatment requires a better understanding of the injury mechanisms in a well-defined severity of mild, moderate, and severe injury in different models that may potentially reflect the various types of human brain injuries. The purpose of this review article is to highlight the classification of mild, moderate, and severe injury in various animal models of TBI with special focus on mixed injury that represents a translational concussive head injury. We will classify animal models of TBI broadly into focal injury, diffuse injury, and mixed injury. Focal injury, a localized injury, is represented by animal models of controlled cortical impact, penetrating ballistic-like brain injury, and Feeney or Shohami weight drop injury. A global diffuse injury is best represented by shock tube model of primary blast injury, and Marmarou or Maryland weight drop model. A mixed injury consists of focal and diffuse injury which reproduces the concussive clinical syndrome, and it is best studied in animal model of lateral fluid percussion injury.

Acoustic startle response (ASR) is a defensive reflex that is largely ignored unless greatly exaggerated. ASR is suppressed after moderate and severe traumatic brain injury (TBI), but the effect of mild TBI (mTBI) on ASR has not been investigated. Because the neural circuitry for ASR resides in the pons in all mammals, ASR may be a good measure of brainstem function after mTBI. The present study assessed ASR in Sprague-Dawley rats after mTBI using lateral fluid percussion and compared these effects to those on spatial working memory. mTBI caused a profound, long-lasting suppression of ASR. Both probability of emitting a startle and startle amplitude were diminished. ASR suppression was observed as soon as 1 day after injury and remained suppressed for the duration of the study (21 days after injury). No indication of recovery was observed. mTBI also impaired spatial working memory. In contrast to the suppression of ASR, working memory impairment was transient; memory was impaired 1 and 7 days after injury, but recovered by 21 days. The long-lasting suppression of ASR suggests long-term dysfunction of brainstem neural circuits at a time when forebrain neural circuits responsible for spatial working memory have recovered. These results have important implications for return-to-activity decisions because recovery of cognitive impairments plays an important role in these decisions.

© 2015 Elsevier B.V. Background: Injury to the brain can occur from a variety of physical insults and the degree of disability can greatly vary from person to person. It is likely that injury outcome is related to the biomechanical parameters of the traumatic event such as magnitude, direction and speed of the forces acting on the head. New method: To model variations in the biomechanical injury parameters, a voice coil driven fluid percussion injury (FPI) system was designed and built to generate fluid percussion waveforms with adjustable rise times, peak pressures, and durations. Using this system, pathophysiological outcomes in the rat were investigated and compared to animals injured with the same biomechanical parameters using the pendulum based FPI system. Results in comparison with existing methods: Immediate post-injury behavior shows similar rates of seizures and mortality in adolescent rats and similar righting times, toe pinch responses and mortality rates in adult rats. Interestingly, post injury mortality in adult rats was sensitive to changes in injury rate. Fluoro-Jade labeling of degenerating neurons in the hilus and CA2-3 hippocampus were consistent between injuries produced with the voice coil and pendulum operated systems. Granule cell population spike amplitude to afferent activation, a measure of dentate network excitability, also showed consistent enhancement 1 week after injury using either system. Conclusions: Overall our results suggest that this new FPI device produces injury outcomes consistent with the commonly used pendulum FPI system and has the added capability to investigate pathophysiology associated with varying rates and durations of injury.

Traumatic brain injury (TBI), even at mild levels, can activate matrix metalloproteinases (MMPs) and the induction of neuroinflammation that can result in blood brain barrier breakdown and neurodegeneration. MMP2 has a significant role in neuroinflammation and neurodegeneration by modulating the chemokine CXCL12α (stromal cell derived factor SDF-1α) signaling pathway and the induction of apoptosis. SDF-1α is responsible for cell proliferation and differentiation throughout the nervous system and is also implicated in various neurodegenerative illnesses. We hypothesized that TBI leads to MMP2 activation and cleavage of the N-terminal 4 amino acid residues of CXCL12α with generation of the highly neurotoxic fragment SDF-1(5-67). Using an in vitro stretch-injury model of rat neuronal cultures and the in vivo fluid percussion injury (FPI) model in rats, we found that oxidative stress has a significant role in the activation of MMP2. This is initiated by the induction of free radical generating enzyme NADPH oxidase 1 (NOX1). Induction of NOX1 correlated well with the signatures of oxidative stress marker, 4HNE in the injured neuronal cultures and cerebral cortex of rats. Further, using MMP2 siRNA and pharmacological MMP2 inhibitor, ARP100, we established the neurodegenerative role of MMP2 in cleaving SDF-1α to a neurotoxic fragment SDF-1(5-67). By immunofluorescence, western blotting and TUNEL experiments, we show the cleaved form of SDF leads to apoptotic cell death in neurons. This work identifies a new potential therapeutic target to reduce the complications of brain damage in TBI.

We investigated the hypothesis that high Ca(2+) influx during traumatic brain injury induces the activation of the caspase-1 enzyme, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury and an in vivo model of fluid percussion injury (FPI). We first established that stretch injury causes a rapid increase in the intracellular Ca(2+) level, which activates interleukin-converting enzyme caspase-1. The increase in the intracellular Ca(2+) level and subsequent caspase-1 activation culminates into neuroinflammation via the maturation of IL-1β. Further, we analyzed caspase-1-mediated apoptosis by TUNEL staining and PARP western blotting. The voltage-gated sodium channel blocker, tetrodotoxin, mitigated the stretch injury-induced neuroinflammation and subsequent apoptosis by blocking Ca(2+) influx during the injury. The effect of tetrodotoxin was similar to the caspase-1 inhibitor, zYVAD-fmk, in neuronal culture. To validate the in vitro results, we demonstrated an increase in caspase-1 activity, neuroinflammation and neurodegeneration in fluid percussion-injured animals. Our data suggest that neuronal injury/traumatic brain injury (TBI) can induce a high influx of Ca(2+) to the cells that cause neuroinflammation and cell death by activating caspase-1, IL-1β, and intrinsic apoptotic pathways. We conclude that excess IL-1β production and cell death may contribute to neuronal dysfunction and cognitive impairment associated with TBI.

Optical coherence tomography (OCT) is a versatile imaging technique and has great potential in tissue characterization for breast cancer diagnosis and surgical guidance. In addition to structural difference, cancerous breast tissue is usually stiffer compared to normal adipose breast tissue. However, previous studies on compression optical coherence elastography (OCE) are qualitative rather than quantitative. It is challenging to identify the cancerous status of tissue based on qualitative OCE results obtained from different measurement sessions or from different patients. Therefore, it is critical to develop technique that integrates structural imaging and force sensing, for quantitative elasticity characterization of breast tissue. In this work, we demonstrate a quantitative OCE (qOCE) microsurgery device which simultaneously quantifies force exerted to tissue and measures the resultant tissue deformation. The qOCE system is based on a spectral domain OCT engine operated at 1300 nm and a probe with an integrated Febry-Perot (FP) interferometric cavity at its distal end. The FP cavity is formed by the cleaved end of the lead-in fiber and the end surface of a GRIN lens which allows light to incident into tissue for structural imaging. The force exerted to tissue is quantified by the change of FP cavity length which is interrogated by a fiber-optic common-paths phase resolved OCT system with sub-nanometer sensitivity. Simultaneously, image of the tissue structure is acquired from photons returned from tissue through the GRIN lens. Tissue deformation is obtained through Doppler analysis. Tissue elasticity can be quantified by comparing the force exerted and tissue deformation.

Neural tissue engineering offers tremendous promise to combat the effects of disease, aging, or injury in the nervous system. Here we review neural tissue engineering with respect to the design of living tissue to directly replace damaged or diseased neural tissue, or to augment the capacity for nervous system regeneration and restore lost function. This article specifically addresses the development and implementation of tissue engineered three-dimensional (3-D) neural constructs and biohybridized neural-electrical microsystems. Living 3-D neural constructs may be "pre-engineered" in vitro with controlled neuroanatomical and functional characteristics for neuroregeneration, to recapitulate lost neuroanatomy, or to serve as a nervous tissue interface to a device. One application being investigated is developing constructs of axonal tracts that, upon transplantation, may facilitate nervous system repair by directly restoring lost connections or by serving as a targeted scaffold to promote host regeneration by exploiting axon-mediated axonal regeneration. In another application, living nervous tissue engineered constructs are being investigated to biohybridize neural-electrical interface microsystems for functional integration with the nervous system. With this design, in vivo neuritic ingrowth and synaptic integration may occur with the living component, potentially exploiting a more natural integration with the nonorganic interface. Overall, the use of tissue engineered 3-D neural constructs may significantly advance regeneration or device-based deficit mitigation in the nervous system that has not been achieved by non-tissue engineering approaches.

Damage to the peripheral nervous system is surprisingly common and occurs primarily from trauma or a complication of surgery. Although recovery of nerve function occurs in many mild injuries, outcomes are often unsatisfactory following severe trauma. Nerve repair and regeneration presents unique clinical challenges and opportunities, and substantial contributions can be made through the informed application of biomedical engineering strategies. This article reviews the clinical presentations and classification of nerve injuries, in addition to the state of the art for surgical decision-making and repair strategies. This discussion presents specific challenges that must be addressed to realistically improve the treatment of nerve injuries and promote widespread recovery. In particular, nerve defects a few centimeters in length use a sensory nerve autograft as the standard technique; however, this approach is limited by the availability of donor nerve and comorbidity associated with additional surgery. Moreover, we currently have an inadequate ability to noninvasively assess the degree of nerve injury and to track axonal regeneration. As a result, wait-and-see surgical decisions can lead to undesirable and less successful "delayed" repair procedures. In this fight for time, degeneration of the distal nerve support structure and target progresses, ultimately blunting complete functional recovery. Thus, the most pressing challenges in peripheral nerve repair include the development of tissue-engineered nerve grafts that match or exceed the performance of autografts, the ability to noninvasively assess nerve damage and track axonal regeneration, and approaches to maintain the efficacy of the distal pathway and targets during the regenerative process. Biomedical engineering strategies can address these issues to substantially contribute at both the basic and applied levels, improving surgical management and functional recovery following severe peripheral nerve injury.

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.

Object Although neuron transplantation to repair the nervous system has shown promise in animal models, there are few practical sources of viable neurons for clinical application and insufficient approaches to bridge extensive nerve damage in patients. Therefore, the authors sought a clinically relevant source of neurons that could be engineered into transplantable nervous tissue constructs. The authors chose to evaluate human dorsal root ganglion (DRG) neurons due to their robustness in culture. Methods Cervical DRGs were harvested from 16 live patients following elective ganglionectomies, and thoracic DRGs were harvested from 4 organ donor patients. Following harvest, the DRGs were digested in a dispase–collagenase treatment to dissociate neurons for culture. In addition, dissociated human DRG neurons were placed in a specially designed axon expansion chamber that induces continuous mechanical tension on axon fascicles spanning 2 populations of neurons originally plated ∼ 100 μm apart. Results The adult human DRG neurons, positively identified by neuronal markers, survived at least 3 months in culture while maintaining the ability to generate action potentials. Stretch-growth of axon fascicles in the expansion chamber occurred at the rate of 1 mm/day to a length of 1 cm, creating the first engineered living human nervous tissue constructs. Conclusions These data demonstrate the promise of adult human DRG neurons as an alternative transplant material due to their availability, viability, and capacity to be engineered. Also, these data show the feasibility of harvesting DRGs from living patients as a source of neurons for autologous transplant as well as from organ donors to serve as an allograft source of neurons.

We performed depth resolved elasticity measurement based on a quantitative optical coherence elastography system with integrated Fabry-Perot force sensor. We performed depth resolved stiffness measurement on a muti-layer phantom under different axial force.

We developed a miniature quantitative optical coherence elastography (qOCE) instrument with an integrated Fabry-Perot force sensor, for in situ elasticity measurement of biological tissue. The technique has great potential for biomechanics modeling and clinical diagnosis. We designed the fiber-optic qOCE probe that was used to exert a compressive force to deform tissue at the tip of the probe. Using the space-division multiplexed optical coherence tomography (OCT) signal detected by a spectral domain OCT engine, we were able to quantify the probe deformation that was proportional to the force applied, and to quantify the tissue deformation corresponding to the external stimulus. Simultaneous measurement of force and displacement allowed us to extract Young’s modulus of biological tissue. We experimentally calibrated our qOCE instrument, and validated its effectiveness on tissue mimicking phantoms and biological tissues.

Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Studies revealed that the pathogenesis of TBI involves upregulation of MMPs. MMPs form a large family of closely related zinc-dependent endopeptidases, which are primarily responsible for the dynamic remodulation of the extracellular matrix (ECM). Thus, they are involved in several normal physiological processes like growth, development, and wound healing. During pathophysiological conditions, MMPs proteolytically degrade various components of ECM and tight junction (TJ) proteins of BBB and cause BBB disruption. Impairment of BBB causes leakiness of the blood from circulation to brain parenchyma that leads to microhemorrhage and edema. Further, MMPs dysregulate various normal physiological processes like angiogenesis and neurogenesis, and also they participate in the inflammatory and apoptotic cascades by inducing or regulating the specific mediators and their receptors. In this review, we explore the roles of MMPs in various physiological/pathophysiological processes associated with neurological complications, with special emphasis on TBI.

Elongation of nerve fibers intuitively occurs throughout mammalian development, and is synchronized with expansion of the growing body. While most tissue systems enlarge through mitosis and differentiation, elongation of nerve fibers is remarkably unique. The emerging paradigm suggests that axons undergo stretch as contiguous tissues enlarge between the proximal and distal segments of spanning nerve fibers. While stretch is distinct from growth, tension is a known stimulus which regulates the growth of axons. Here, we hypothesized that the axon stretch-growth process may be a natural form of injury, whereby regenerative processes fortify elongating axons in order to prevent disconnection. Harnessing the live imaging capability of our axon stretch-growth bioreactors, we assessed neurons both during and following stretch for biomarkers associated with injury. Utilizing whole-cell patch clamp recording, we found no evidence of changes in spontaneous action potential activity or degradation of elicited action potentials during real-time axon stretch at strains of up to 18 % applied over 5 minutes. Unlike traumatic axonal injury, functional calcium imaging of the soma revealed no shifts in free intracellular calcium during axon stretch. Finally, the cross-sectional areas of nuclei and cytoplasms were normal, with no evidence of chromatolysis following week-long stretch-growth limited to the lower of 25 % strain or 3 mm total daily stretch. The neuronal growth cascade coupled to stretch was concluded to be independent of the changes in membrane potential, action potential generation, or calcium flux associated with traumatic injury. While axon stretch-growth is likely to share overlap with regenerative processes, we conclude that developmental stretch is a distinct stimulus from traumatic axon injury.

The basis for acute seizures following traumatic brain injury (TBI) remains unclear. Animal models of TBI have revealed acute hyperexcitablility in cortical neurons that could underlie seizure activity, but studying initiating events causing hyperexcitability is difficult in these models. In vitro models of stretch injury with cultured cortical neurons, a surrogate for TBI, allow facile investigation of cellular changes after injury but they have only demonstrated post-injury hypoexcitability. The goal of this study was to determine if neuronal hyperexcitability could be triggered by in vitro stretch injury. Controlled uniaxial stretch injury was delivered to a spatially delimited region of a spontaneously active network of cultured rat cortical neurons, yielding a region of stretch-injured neurons and adjacent regions of non-stretched neurons that did not directly experience stretch injury. Spontaneous electrical activity was measured in non-stretched and stretch-injured neurons, and in control neuronal networks not subjected to stretch injury. Non-stretched neurons in stretch-injured cultures displayed a three-fold increase in action potential firing rate and bursting activity 30-60 min post-injury. Stretch-injured neurons, however, displayed dramatically lower rates of action potential firing and bursting. These results demonstrate that acute hyperexcitability can be observed in non-stretched neurons located in regions adjacent to the site of stretch injury, consistent with reports that seizure activity can arise from regions surrounding the site of localized brain injury. Thus, this in vitro procedure for localized neuronal stretch injury may provide a model to study the earliest cellular changes in neuronal function associated with acute post-traumatic seizures.

Motor neurons express particularly high levels of the AMPA receptor subunit GluR1(Q)flip (GluR1(Q)i) during the period in early postnatal life when their dendritic tree grows and becomes more branched. To investigate how GluR1-containing AMPA receptors contribute to dendrite morphogenesis, we characterized a mutant form of GluR1 (containing a histidine in the Q/R editing site) with unique electrophysiological properties. Most notably, AMPA receptors assembled from GluR1(H)i display less calcium permeability than AMPA receptors assembled from GluR1(Q)i. Expression of GluR1(Q)i in vivo or in vitro led to an increase in dendrite branching with no net change in the overall tree size while GluR1(H)i led to a loss of branches and a net reduction in overall tree size. GluR1(H)i-dependent dendrite atrophy is mediated by protein phosphatase 2B. The results suggest that the electrophysiological properties of cell surface AMPA receptors, specifically their permeability to calcium, can be a central determinant of whether the dendrites undergo activity-dependent branching or atrophy.

The changes of excitability in affected neural networks can be used as a marker to study the temporal course of traumatic brain injury (TBI). The cerebellum is an ideal platform to study brain injury mechanisms at the network level using the electrophysiological methods. Within its crystalline morphology, the cerebellar cortex contains highly organized topographical subunits that are defined by two main inputs, the climbing (CFs) and mossy fibers (MFs). Here we demonstrate the use of cerebellar evoked potentials (EPs) mediated through these afferent systems for monitoring the injury progression in a rat model of fluid percussion injury (FPI). A mechanical tap on the dorsal hand was used as a stimulus, and EPs were recorded from the paramedian lobule (PML) of the posterior cerebellum via multi-electrode arrays (MEAs). Post-injury evoked response amplitudes (EPAs) were analyzed on a daily basis for 1 week and compared with pre-injury values. We found a trend of consistently decreasing EPAs in all nine animals, losing as much as 72 ± 4% of baseline amplitudes measured before the injury. Notably, our results highlighted two particular time windows; the first 24 h of injury in the acute period and day-3 to day-7 in the delayed period where the largest drops (~50% and 24%) were observed in the EPAs. In addition, cross-correlations of spontaneous signals between electrode pairs declined (from 0.47 ± 0.1 to 0.35 ± 0.04, p < 0.001) along with the EPAs throughout the week of injury. In support of the electrophysiological findings, immunohistochemical analysis at day-7 post-injury showed detectable Purkinje cell loss at low FPI pressures and more with the largest pressures used. Our results suggest that sensory evoked potentials (SEPs) recorded from the cerebellar surface can be a useful technique to monitor the course of cerebellar injury and identify the phases of injury progression even at mild levels.

Cerebellum is highly susceptible to traumatic injuries, and yet it has rarely been studied in the context of traumatic brain injury (TBI). Much of the TBI research in the cerebellum has been conducted through histochemical techniques, particularly using selective protein staining. In this study, we investigated if fluid percussion injury (FPI) was detectable using electrophysiological recordings of the cerebellar activity with multi-contact array electrodes. Rats were chronically implanted with micro ECoG electrodes on the paramedian lobule (PML). The FPI was induced via a skull opening near the electrode implant on the cerebellum. Spontaneous and evoked potentials (EPs) featured almost immediate alterations in their waveform patterns after the delivery of pressure pulse. Recorded evoked potential amplitudes declined drastically by the next day. We also studied the linear correlation changes between all the electrode contact pairs and observed a significant decrease (R from > 0.7 to

Mechanisms that lead to traumatic brain injury due to blast exposure (bTBI) are not well understood. Complexities of cellular responses involved have made identification of these mechanisms challenging. In-vitro studies of blast induced traumatic brain injury are possible with employment of shock tubes which closely mimic the loading conditions of blasts in a laboratory setting. A novel pneumatic, two-chamber shock tube has been developed to generate a span of mild to severe pressure waveforms seen in TBI literature [1–2]. Unlike other shock tubes utilized in TBI research, it is small, portable, controllable and most importantly safe to operate. The ultimate goal is to use this device to develop a blast induced traumatic brain injury animal model that can be used to explain the injury mechanism(s) and threshold levels of brain injury after blast exposure.

Axon Stretch Growth (ASG) is the process by which nerve fibers elongate in conjunction with growth of the enlarging body. During early embryonic development, neuronal growth cones traverse short distances to reach local synaptic targets. Subsequently, neuronal somata are separated from their targets by developmental growth of the enlarging organism. It is believed that mechanical forces, such as those due to skeletal growth, transduce a second mode of neuronal growth unique from growth cone extension. Such mechanotransduction results in the elongation of short processes into the long nerves characteristic of the adult nervous system. The majority of nerve regeneration studies focus heavily on the extension of growth cones, while neglecting affiliated body growth. Currently, regeneration of adult nerves by growth cone extension is limited to 3cm in total overall length [1, 2], despite the initial capacity for 30 Fold this limitation. As biomedical engineers, our objective is to define and characterize the role biomechanical forces play in promoting growth of the nervous system. Here, utilizing custom motion-controlled bioreactors, we developed a method to perform high magnification imaging of neuronal somata undergoing ASG. Embryonic rat cervical dorsal root ganglia neurons were dissociated and plated onto poly-d-lysine coated cover glass and stretch grown at 0.25mm/day for 1 week.

The purpose of this project is to program a robot that will interact with disabled children by reading stories and having physical therapy routines. The processes currently used to engage children in reading and exercising are monotonous and visually displeasing. A commercial robot, RS Media, manufactured by WowWee Corp., was selected because of its instructiveness, mobility, aesthetics, and programmability. The goal is ultimately to use Linux-based programming to create programs that include text-to-speech software for storytelling and using robot body movements with text-to-speech to create exciting physical therapy routines. The scope of this project is to enhance literacy and physical therapy (exercise) by using this robot as a motivation factor among children.

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004). Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension. Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10 cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3 cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

Strategies for nervous system repair arise from knowledge of growth mechanisms via a growth cone. The distinctive process of axon stretch growth is a robust, long-term growth that may reveal new pathways to accelerate nerve repair. Here, a live imaging bioreactor was engineered to closely explore cellular events initiated by applied tension. The stretch growth potential between adult and embryonic dorsal root ganglion (DRG) neurons was investigated, an important difference in nerve repair. Embryonic axons were capable of unidirectional stretch growth rates of 4?mm/d and reliably reached 4?cm in length within 2 weeks. Adult axons could only reach 2?mm/d and took over 3 weeks to reach 4?cm. Utilizing time-lapse imaging, we observed growth cone motility in coordination with stretch growth. Upon initiation of stretching, growth cones retracted. However, within 10?h of continuous stretching, growth cones extended at a rate of 0.2?mm/d opposite the direction of applied tension, contributing to overall axon elongation. We analyzed fast mitochondrial transport under increasing levels of strain to determine the effect of stretch on axonal transport. Transport began to diminish at 24% strain, and was almost completely absent at 39% strain. Surprisingly, axons recovered and were capable of subsequent stretch growth. When tension was completely released (?5% strain), stretch grown axons retracted at rates up to 6.1??m/sec and slowed as resting tension was restored. This ability to assess the process of axon stretch growth in real time will allow detailed study of how tension can be used to drive axonal growth and retraction.

Type I collagen was extruded into well-defined fibers using a novel controlled wet spinning device. The device significantly improved the yield of collagen fibers on the micron scale. Collagen scaffold materials fabricated from the device will provide contact-guided growth of neuronal cultures. Fibers composed of different weight percentages, 75, 2, and 3.4 wt% collagen dispersions, were used to validate Schwann cell alignment along the fibers. Interestingly, the different concentrations of extruded collagen dispersions demonstrated unique morphologies and directed growth patterns of Schwann cells.

The purpose of this work is to design novel, modular devices in order to reproduce traumatic brain injury in the rat and mouse models: fluid percussion injury (FPI), controlled cortical impact (CCI) and the Shock tube. Commercially available TBI devices are often unimethodical, expensive and sensitive to experimental variability. To achieve maximum control and sensitivity, in our first device, we utilized a closed loop voice coil control device to more accurately and precisely generate the temporal force function delivered by the CCI and FPI methods. This device has generated pressures comparable to the ones from literature where commercially available devices were utilized. Upon exposure to a moderate parasagittal TBI from this FPI device, experimental rats have shown comparable pathological effects as has been discussed in literature for similar levels of injury. The intent of the second device, the shock tube, which uses a slightly different technique but is nonetheless modular and precise; is also to induce blast related TBI in small animals. Initial studies would include in vitro bench testing with a pressure transducer to compare results obtained from literature using other shock tubes.

Adult Schwann cells cultured within three-dimensional (3D) collagen-based nervous tissue constructs demonstrated downstream migration and potential use in the contact-guidance of regenerating adult dorsal root ganglia (DRG) axons. Type I collagen extracted from rat tail tendons was used to prepare collagen dispersions for wet spinning micron-size collagen fibers. Biodegradable poly-L-lactic acid (PLLA) and wet-spun collagen fibers containing various extracellular matrix (ECM) proteins were used to evaluate Schwann cell guidance and survival of primary adult rat DRGs, which presents a more realistic model for studying regenerative processes.

This work involves the study of the origin and effect of space charges in the electrospinning process. Electrospinning is a process used to make solid, nanofibrous polymer mats. These mats are used in tissue engineering as scaffolds for cells. The origin and effect of space charges in this process has not been well-studied.

The purpose of this study is to develop biodegradable nerve guidance conduits seeded with Schwann cells (SC) using chitosan-based biopolymers for improving nerve regeneration. Chitosan, prepared from alkaline hydrolysis of chitin, is a natural polysaccharide biopolymer, having biocompatibility and biodegradability. In this study, chitosan and chitosan-gelatin membranes were prepared, characterized, and evaluated. The SC isolated from rat sciatic nerve was seeded on chitosan and chitosan-gelatin membranes to investigate their interactions. To enhance the SC attachment and growth on the membranes, we incorporated the poly-L-lysine (PLL) into chitosan and chitosan-gelatin network at various ratios of 1:800, 1:200, 1:50 (chitosan or chitosan-gelatin: PLL). We have also investigated the effect of extracellular matrix (ECM) molecules on the proliferation of SC. We observed that SC proliferation on PLL-coated surface was significantly higher than that of ECM coated and uncoated surfaces. With the increase of PLL ratio into chitosan or chitosan-gelatin network, the attachment and growth of SCs were significantly enhanced. This study indicates that the incorporation of PLL into chitosan and chitosan-gelatin membranes significantly improves the bioactivity of the materials. The Schwann cell-seeded films can be used to construct a three-dimensional nerve conduit for enhancing nerve regeneration.

The transition from embryo to adulthood involves a massive growth in the nervous system where axons in nerves extend and expand to accommodate to this growth. Applying mechanical forces to dorsal root ganglia neuronal cultures has previously shown that there is stimulation in axonal growth, gradually elongating the axon over a set period of time. In this research, the main focus is to study with live imaging how natural biomechanical forces, associated with growth of an organism, initiate unique neurobiological mechanisms that help drive the formation of long nerves. For this purpose, a bioreactor was developed for live imaging of stretch-growth as it occurs on the stage of a microscope. The bioreactor is independent from an incubator with external temperature controller and heating system regulated its physiological conditions. Morphology changes and cytoskeletal transport were captured live at magnifications up to 60x over weeks of culturing.

Several key biological mechanisms of traumatic injury to axons have been elucidated using in vitro stretch injury models. These models, however, are based on the experimentation of single cultures keeping productivity slow. Indeed, low yield has hindered important and well-founded investigations requiring high throughput methods such as proteomic analyses. To meet this need, we engineered a multi-well high throughput injury device to accelerate and accommodate the next generation of traumatic brain injury research. This modular system stretch injures neuronal cultures in either a 24-well culture plate format or 6 individual wells simultaneously. Custom software control allows the user to accurately program the pressure pulse parameters to achieve the desired substrate deformation and injury parameters. Analysis of the pressure waveforms showed that peak pressure was linearly related to input pressure and valve open times and that the 6- and 24-well modules displayed rise times, peak pressures, and decays with extremely small standard deviations. Data also confirmed that the pressure pulse was distributed evenly throughout the pressure chambers and therefore to each injury well. Importantly, the relationship between substrate deformation and applied pressure was consistent among the multiple wells and displayed a predictable linear behavior in each module. These data confirm that this multi-well system performs as well as currently used stretch injury devices and can undertake high throughput studies that are needed across the field of neurotrauma research.