The effects of rapid stretch injury on rat neocortical cultures

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, a multi-well high throughput injury device is engineered 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 six 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. Classically, in vitro research in TBI has shown increases in [Ca2+]i levels following injury. The Ca2+ sensitive fluorescent dye, Fluo-4AM, is used to observe the effects of strain rate on the changes in [Ca2+]i levels following injury. Neuronal cultures are injured at three strain levels: 20%, 40% and 60% strain. At each of these strain levels, two strain rates are applied; 30s-1 (slow) and 70s-1 (rapid). At each strain level, the data show that neurons injured at 70s-1 experience larger maximum F/F0 and longer sustained Ca2+ fluorescence than neurons injured at 30s-1. It is also shown that at high strain rates TTx no longer blocks increases in [Ca2+]i levels after injury. Traumatic injury to the brain is known to cause dysfunction in surviving neurons. The effects of simulated traumatic injury of rat neocortical neurons cultures are investigated. These neurons are subjected to a stretch injury of 60% strain over 20 ms using a custom in vitro injury device. Spontaneous and stimulated electrical properties are measured 20-60 minutes after stretch using current and voltage clamp techniques. The same measurements are performed in non-stretched neurons. All neurons display TTX-inhibitable action potentials when basal membrane potential was set at -60 mV, and many display bursting behavior in response to depolarizing current injection. No differences in resting membrane potential (-40 ± 1 mV [n=20]) or input resistance (1.0 ± 0.1 GΩ [n=20]) are observed in injured and non-injured neurons. Interestingly, stretch injury reduces the frequency of spontaneous action potentials (33 ± 6 min-1 [ n=13] and 11 ± 3 min-1 [n=16] in non-injured and injured neurons, respectively) and decreases spontaneous bursting activity by almost 90%. ADP50 and action potential amplitude are unchanged. However, A D P90 is significantly prolonged in injured neurons and characterized by a less pronounced action potential after-hyperpolarization. These data are consistent with an alteration in kinetics of K+ currents in injured neurons. Since spontaneous action potentials are blocked by 20 µM bicuculline and 3 mM kyneuri nic acid, the frequency of subthreshold depolarizations is measured to estimate overall neuronal network activity. The frequency of spontaneous subthreshold depolarizations is not significantly different in injured and non-injured neurons. These data show that spontaneous electrical signaling is acutely altered and suggest that action potential initiation is reduced by in vitro stretch in neuronal cell cultures

Development of high-speed imaging techniques for C. elegans nervous system studies

We report high-speed imaging techniques for C. elegans nervous systems studies. We introduce C. elegans, the main model organism in this dissertation, and neuroscientific and biomedical studies using C. elegans involving calcium imaging, nerve regeneration, and drug screening. We review technologies including confocal microscopy and microfluidic devices used in the neuroscientific and biomedical studies We discuss development of a high-speed laser scanning confocal microscope capable of flexible control of imaging conditions, fast imaging speed, and large field-of-view. We provides the design principles used in the development of the confocal microscope including the optical, electrical, and software implementation, and the details of the confocal microscope we built based on the design principles. We present the performance characterization of the confocal microscope, then a few sample images obtained with the confocal microscope. We present development of time-lapse volumetric confocal imaging of whole animal C. elegans Ca²⁺ dynamics. We provide the design of the time-lapse volumetric confocal imaging system including a microfluidic device to accommodate the whole animal within the field-of-view of the imaging system. We examine the feasibility of the volumetric confocal imaging of a whole animal, and demonstrate imaging of the whole animal C. elegans neurons’ response to NaCl within a 630 × 150 × 25 μm³ volume at 2 Hz rate. We report a high-throughput automated imaging platform for C. elegans nerve regeneration study. We describe the design of the automated imaging platform and the automation flow, and characterizes the performance of the platform. The imaging platform can obtain high-resolution 3D confocal images of 20 animals in 10 minutes. We show sample images of C. elegans anterior lateral microtubule nerve regeneration examples acquired via the automated imaging platform. We demonstrate a planar laser activated neuronal scanning platform (PLANS), a high-throughput animal examination system for drug screening. We explain the construction of PLANS involving the optics, the microfluidic device, and the electronics. The PLANS system can scan an animal in less than 5 ms with a spatial sampling resolution of 3 μm FWHM. We show sample scanning results of a Huntington’s disease model of C. elegans. We summarize the studies discussed in this dissertation, and suggest relevant future research to follow up on the studies.Electrical and Computer Engineerin

DEVELOPMENT OF INNOVATIVE MULTICOMPARTMENT MICROFLUIDIC PLATFORMS TO INVESTIGATE TRAUMATIC AXONAL INJURY

Compartmentalization of cell body from the axon of a neuron is an important aspect in studying the influence of microenvironments. Microenvironment is an integral part of neuronal studies involving traumatic axonal injuries (TAI). While TAI is one of the possible outcomes of various forms of traumatic insults to the central nervous system (CNS) and peripheral nervous system (PNS), many of the mechanistic details are still unknown, it can be agreed that the level of injury often dictates the functional deficit. This motivates the question, what is occurring at both the morphological and biomolecular scale in CNS and PNS axons during and throughout the recovery phase after injury? And, are there any treatment strategies that could be applied to improve the recovery and regeneration of the axons subject to TAI? Motivated by this, I propose to develop novel microfluidic platforms as a part of my master’s thesis to allow unprecedented, physiologically relevant focal and graded mechanical injury and observation to both CNS and PNS axons. My research for this thesis can be broadly classified into two fold. 1) I examined the regenerative effects of the members of the Glial cell line-derived neurotrophic factor (GDNF), a family of neurotrophic factors after axotomy. This work resulted in the discovery of the fact that GDNF is the most potent neurotrophic factor among the family of growth factors for axon regeneration in dorsal root ganglion (DRG) neurons after in vitro axotomy. It was also found that GDNF locally applied to cell body better promotes axonal regeneration in comparison to when applied locally to axons. 2) Development and refinement of existing axon injury microplatform (AIM) to closely mimic physiological conditions during traumatic injury in CNS neurons. This work was my attempt in improving already existing microfluidic compression platform. I successfully developed a displacement control injury device and demonstrated displacement control as a proof of principle. Further development of these microfluidic platforms will significantly contribute to the field of basic neuroscience, neurobiology, and biomedical engineering

Crosslinked Hyaluronic Acid Hydrogel Networks Designed as Mechanical Actuators

Bioengineers are in constant pursuit of solutions to problems facing the medical and pharmaceutical field by designing biomaterials that closely mimic the target natural systems. A unique collection of polymers, known as polymeric actuators, have been devised with the ability to convert an external stimulus to a change in shape, size or permeability. The current options within polymeric biomaterials with multi-functionality include matrices that are biocompatible, biodegradable, quick transitioning / shape changing, and mechanically tunable. These properties have been harnessed for application such as stents, valves, semi permeable membranes, and dynamic cell culture substrates. For such applications quick and uniform actuator response that does not need to be sustained for more than a few hours is desired. However there exist other areas of biomedical applications, such as wound closure/healing and nerve regeneration, where polymeric actuators have been underutilized. These applications however call for a polymer system that can actuate at controlled slow speeds and sustain this actuation for several days. At present there is a lack of such slow actuating polymer system. Each year over 50,000 peripheral nerve repair procedures are performed (National Center for Health Statistics, 1995). The total annual costs in U.S alone exceed $ 7 billion (American Paralysis Association, 1997). The treatment of a nerve transection is dependent on the size of the injury gap. Similarly, the extent of regeneration and re-innervation in the PNS is also governed by the size of the gap. For a smaller gap (\u3c10 mm) the surgeon can pull the severed nerve ends closer and suture them to repair the injury. For larger gaps autologous nerve transplant is the gold standard treatment despite the inherent disadvantages. Over the past decades biomaterial researchers have tested several polymeric nerve conduits as an alternative to autologous nerve grafts. However none have been able to match the success rates of autologous grafts. There is a lack of an effective biomaterial solution to the problem of a large gap nerve injury. For many years there has been a hypothesis that nervous tissue can be successfully elongated via application of an external mechanical force alone which could be used to treat peripheral nerve gap injuries. Mechanical actuation studies have been shown to produce successful stretch growth in individual axons and axonal bundles. This phenomenon is at play in nature during embryonic growth and development of the body of organisms to adulthood. Applying tensional forces at appropriate rates (\u3c 100 μm/hr) causes sustained axonal stretch growth. The solution we propose in this work is a biomaterial that can be programmed to perform the function of a mechanical actuator at rates suitable for axonal stretch growth. We designed, fabricated, and characterized a novel hyaluronic acid based hydrogel that shrinks over time along a pre-defined axis thereby providing the source for tension that could be used for sustained axonal stretch growth. The shear thinning property of hyaluronic acid (HA) enabled us to test if we could store a retractive stress in a rapidly crosslinked network under shear flow and then controllably release this stress and achieve shrinkage of the network scaffold along one desired axis. We investigated two strategies to achieve this goal. The retractive stress trapped in the crosslinked network was released either by manipulating the main backbone HA chains or by selectively breaking the crosslinks. The shrinkage rates obtained were within the range of stretching rates that have successfully stretched neuronal cells. We also confirmed that the material\u27s cytocompatibility was unaffected by the chemical modifications that HA was subjected to. This polymer system is a novel addition to the existing polymeric actuators and is a step towards filling the void of a slow, long term actuating polymer

Evaluating the Use of Engineered Nervous Tissue Constructs in the Repair of Peripheral Nerve Lesions and Amputations

Severe trauma to the limbs can often result in the lesioning, or even amputation, of the underlying peripheral nerves. In these cases, endogenous neural repair mechanisms are compromised and a path to the end target may be lost, resulting in the need for surgical intervention. Current repair strategies are incapable of maintaining this regenerative pathway, or providing a bridge to a surrogate end target, often resulting in incomplete repair. This thesis describes the development and evaluation of a novel method of addressing peripheral nerve lesions and amputations that utilizes living tissue-engineered neural grafts. These grafts are created by the controlled mechanical separation of axons spanning integrated neuron populations in vitro, resulting in axon tracts spanning several centimeters in length. Techniques were developed to encapsulate and transplant these tracts, with the goal of providing structural and nutrient support, while minimizing macrophage infiltration. The efficacy of these constructs in the treatment of lesions and amputations was then assessed using a rat sciatic nerve transection model. In the first study, the ability of neural constructs to (a) encourage host regeneration from the proximal stump, while also (b) attenuating distal pathway degeneration, was evaluated. At the 4-week time point, the axonal constructs were observed to promote more robust host axonal and tissue regeneration across the graft when compared to unstretched grafts. A measurement of nerve conduction velocities also revealed a statistically significant improvement in the stretch-grown group, correlating with the observed increased fiber regeneration. At the distal pathway, neural constructs were observed to prevent the atrophy of the support cells, and maintain the alignment of the Schwann cell columns for up to 4 months. These results suggest that the use of neural grafts may expand the time window within which successful nerve regeneration can occur. The axon grafts were then shown to support and maintain regenerating host axon fibers for up to 4 weeks in the absence of a distal end target. Finally, axon grafts pre-attached to an implantable electrode substrate were shown to encourage host ingrowth to the vicinity of the substrate, showing promise for the development of a chronic brain-machine interface

Development of Tethered Aligned Engineered Neural Tissue Containing Elongated Neurons for Peripheral Nerve Regeneration

Following peripheral nerve injury, the axons in the distal nerve between the injury site and the muscle degenerate. When the injured site is very proximal, functional recovery from nerve repair is a clinical challenge since neuronal regeneration rate is limited, resulting in muscle atrophy due to the delay in reinnervation, even where the ‘gold standard’ autograft is used. Much research focuses on developing biomaterial scaffolds that mimic the autograft and promote host neurite regeneration from proximal to distal stump, whereas here, we aim to improve long distance repair by populating constructs with functional neurons and glial cells. With an engineered living scaffold populated with neurons exhibiting long neurite extensions supported by glial cells, the gap between proximal stump and muscle could potentially be reconnected promptly once the challenge of integration is overcome. To test the concept, a method was developed using tethered aligned engineered neural tissue (TaeNT) formed from simultaneous self-alignment of Schwann cells and collagen fibrils in a fully-hydrated tethered gel resulting in an anisotropic tissue-like structure. The in vitro results showed neurite elongation and alignment in the co-culture of neurons and Schwann cells in TaeNT, indicating that TaeNT could be an appropriate substrate for growing long neurites with a view to generating therapeutic constructs containing long functional neurons. The implantation of TaeNT containing neurons and Schwann cells in a 10mm-gap rat sciatic nerve for 3 weeks provided information about host-transplant cell interaction including Schwann cell migration and alignment inside the conduit, and neurite elongation across the conduit interface. Furthermore, in an attempt to induce longer neurite growth, TaeNT was proposed as a substrate that could be combined with mechanical tension application using a 3D-printed mould developed to stretch the cellular gels in a controlled manner. A series of newly designed protocols for mechanical tension application to induce growth response for enhanced neural regeneration was developed and discussed correspondingly. In summary, the findings represent the development and investigation of the regenerative potential for engineered living scaffolds containing neurons and Schwann cells suitable for stretch-growth to provide an elongated functional nerve graft. With a view to translation for clinical use, investigating the source of therapeutic cells in the conduit and the functional integration of host and transplanted cells is an important step towards optimising the regenerative potential of the engineered living scaffold

Microfluidic Organ/Body-on-a-Chip Devices at the Convergence of Biology and Microengineering

Recent advances in biomedical technologies are mostly related to the convergence of biology with microengineering. For instance, microfluidic devices are now commonly found in most research centers, clinics and hospitals, contributing to more accurate studies and therapies as powerful tools for drug delivery, monitoring of specific analytes, and medical diagnostics. Most remarkably, integration of cellularized constructs within microengineered platforms has enabled the recapitulation of the physiological and pathological conditions of complex tissues and organs. The so-called organ-on-a-chip technology, which represents a new avenue in the field of advanced in vitro models, with the potential to revolutionize current approaches to drug screening and toxicology studies. This review aims to highlight recent advances of microfluidic-based devices towards a body-on-a-chip concept, exploring their technology and broad applications in the biomedical field.European Regional Development Fund-Project FNUSA-ICRC [CZ.1.05/1.1.00/02.0123]; Fundacao para a Ciencia e a Tecnologia (FCT), Portugal [UID/BIM/04773/2013]; Internal Research Grant Program, Universita Campus Bio-Medico di Romainfo:eu-repo/semantics/publishedVersio

A Role for Focal Adhesions and Extracellular Matrix in Traumatic Axonal Injury

Traumatic Brain Injury (TBI) is linked to a diverse range of diffuse pathological damage for which there is a severe lack of therapeutic options. A major limitation to drug development is the inability to identify causal mechanisms that link head trauma to the multitude of secondary injury cascades that underlie neuropathology. To elucidate these relationships, it is important to consider how physical forces are transmitted through the brain across multiple spatial scales ranging from the whole head to the sub-cellular level. In doing so, the mechanical behavior of the brain is typically characterized solely by its material properties and biological structure. Alternatively, forces transmitted through distinct cellular and extracellular structures have been shown to influence physiological processes in multiple cell types through the transduction of mechanical forces into cellular chemical responses. As an essential component of various biological processes, these mechanotransduction events are regulated by mechanical cues directed through extracellular matrix (ECM) and cell adhesion molecules (CAM) to mechanosensitive intra-cellular structures such as focal adhesions (FAs). Using a series of in vitro models, we have implicated FAs in the cellular mechanism of traumatic axonal injury by showing that forces directed through these structures potentiate injury levels and, moreover, that inhibition of FA-mediated signaling pathways may be neuroprotective. In addition, we show that localizing trauma forces through specific brain ECM results in differential injury rates, further implicating mechanosensitive cell-ECM linkages in the mechanism of TBI. Therefore, we show that FAs play a major role in axonal injury at low strain magnitudes indicating that cellular mechanotransduction may be an important mechanism underlying the initiation of cell and sub-cellular injuries ultimately responsible for the diffuse pathological damage and clinical symptoms observed in diffuse axonal injury. Furthermore, since these mechanisms may present the earliest events in the complex sequelae associated with TBI, they also represent potential therapeutic opportunities.Engineering and Applied Science