Recent findings have revealed a crucial contribution of the adhesion molecule neuroligin-1 to the precise organization and regulation of intercellular synaptic connections within the central nervous system, and disruption of neuroligin-1 signaling in vivo fosters cognitive abnormalities. Despite considerable recent progress, several uncertainties remain regarding the exact synaptic function of neuroligin-1. Principle among these uncertainties is whether neuroligin-1 primarily promotes initiation of de novo synaptic connections or maturation of functional, pre-existent connections. To begin to address this, experiments must be devised that are capable of dissociating activity-dependent and -independent effects of neuroligin-1 signaling on pre- and postsynaptic compartments. An additional uncertainty is how and when synapses containing neuroligin-1 are specified as either excitatory or inhibitory. Elucidating these synapse specification cascades will prove crucial in defining the contribution of neuroligin-1 to overall network balances of excitation and inhibition that guide proper cognitive development. A final uncertainty is how alternate adhesion complexes may coordinate with neuroligin-1 to initiate or maintain synaptic connections. Differentiating redundant from complementary functions among adhesion systems will help reconcile unresolved discrepancies between in vitro and in vivo experiments and ultimately provide a clearer understanding of synapse formation and function in vivo. Herein I detail significant new findings clarifying each of these uncertainties. Utilizing a specific transfection protocol, I first demonstrate that neuroligin-1 is capable of robustly inducing presynaptic differentiation independent of proper postsynaptic development and synaptic activity. Second, employing both multi-molecular perturbations and a delimited biological model of the synapse, I show that the postsynaptic scaffolding molecule PSD95 specifically acts downstream of neuroligin-1-mediated synapse initiation. Third, the model synapse is again employed to differentiate between separate synaptic functions of neuroligin-1 and alternate adhesion molecule SynCAM1. Building from these distinct synaptic functions, I provide preliminary evidence that SynCAM1 matures inactive neuroligin-1-initiated synapses. Fourth, I present the first direct evidence that neuroligin-1 contributes to dendritic morphogenesis in mammalian neurons, consistent with recent findings within the Xenopus system. Collectively, these results evince a robust capacity of neuroligin-1 in initial stages of synaptogenesis and contribute to a new theory of neuroligin-1 function in both activity-dependent synapse initiation and activity-dependent synapse maturation.

Pressure ulcers are prevalent and costly, particularly for individuals with impaired mobility and sensation. They are primarily caused by high pressure near bony prominences. Multiple other factors include shear force, friction, temperature, and moisture. Recent research at the University of Pittsburgh was conducted on local cooling effects with respect to skin blood flow. A reduction of skin temperature to 25°C provided a significant benefit to local tissue in healthy controls and subjects with spinal cord injuries. This concurs with prior animal studies which demonstrated reductions in breakdown at lower interface temperatures. Pressure ulcers have been historically managed by providing support surfaces, such as wheelchair seat cushions, to redistribute pressure at the body interface. Few practical interventions exist to control temperature at this interface; most employ passive cooling methods, which are limited by their inability to modulate applied cooling in response to changes in microenvironment. This studys goal was to develop tightly controlled, local cooling elements for integration into a pressure-redistributing support surface. A holistic view of temperature control methods in an iterative design process was taken. Features, benchmarks, and design specifications were generated using available information from the literature. Idea generation and subsequent evaluation led to the modification of a multi-cell air cushion capable of controlling temperature in specific high risk areas. Proof of concept experiments were conducted with respect to interface cooling to a target temperature, redistribution of pressure, and heat and water vapor transmission. The design delivered local cooling over hour-long trials on able-bodied test subjects. No significant difference in skin temperature (p>0.16) was found after 15 minutes of cooling from our target temperature (25°C). The modified cushion showed similar (p=0.79) peak pressure index values when compared to the same cushion design without the cooling elements. A thermodynamic rigid cushion loading indenter mimicked the environmental conditions of the body on our prototype for 3-hour duration tests. Significantly lower temperatures were observed after 1 hour of cooling (p<0.003). No effect was noted for relative humidity. These experiments successfully demonstrated plausible, integrated cooling elements in a multi-cell air cushion for the delivery of local cooling for pressure ulcer prevention.

Kinematic state feedback is important for neuroprostheses to generate stable and adaptive movements of an extremity. State information, represented in the firing rates of populations of primary afferent neurons, can be recorded at the level of the dorsal root ganglia (DRG). Previous work in cats showed the feasibility of using DRG recordings to predict the kinematic state of the hind limb using reverse regression. Although accurate decoding results were attained, these methods did not make efficient use of the information embedded in the firing rates of the neural population. This dissertation proposes new methods for decoding limb kinematics from primary afferent firing rates. We present decoding results based on state-space modeling, and show that it is a more principled and more efficient method for decoding the firing rates in an ensemble of primary afferent neurons. In particular, we show that we can extract confounded information from neurons that respond to multiple kinematic parameters, and that including velocity components in the firing rate models significantly increases the accuracy of the decoded trajectory. This thesis further explores the feasibility of decoding primary afferent firing rates in the presence of stimulation artifact generated during functional electrical stimulation. We show that kinematic information extracted from the firing rates of primary afferent neurons can be used in a 'real-time' application as a feedback for control of FES in a neuroprostheses. It provides methods for decoding primary afferent neurons and sets a foundation for further development of closed loop FES control of paralyzed extremities. Although a complete closed loop neuroprosthesis for natural behavior seems far away, the premise of this work argues that an interface at the dorsal root ganglia should be considered as a viable option.

Angiogenesis, often thought of as the first step of wound healing, is an organized series of events, beginning with vessel destabilization, followed by endothelial cell proliferation and migration, ending with vessel maturation. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been shown to be important in vascular permeability and endothelial cell proliferation, and migration (early stage angiogenesis), while platelet derived growth factor (PDGF) and sphingosine 1-phosphate (S1P) have been shown to stimulate vascular stability (late stage angiogenesis). For this reason, it was hypothesized that inducing angiogenesis by sequentially delivering angiogenic growth factors, controlling their presence and absence, would better mimic the temporal role of each factor during the progression of native angiogenesis in situ. To this end, we utilized a delivery system based on porous cellulose hollow fibers that, for the first time, permits sequential delivery of an early stage factor followed by a late stage growth factor in vivo, where previous attempts have only resulted in different rates of delivery. Our delivery system addresses the idea that factors involved in one stage of angiogenesis may inhibit other stages of angiogenesis, causing absence of one factor to be just as important as the presence of another factor. Using a modified murine Matrigel plug model, it is apparent that delivery strategies where VEGF alone is delivered before S1P alone as well as delivery strategies where bFGF alone is delivered before PDGF alone, not only lead to greater recruitment of endothelial cells, but also higher maturation index of associated vessels. Sequential delivery was also optimized by examining varying delivery schedules. Additionally, the hollow fiber delivery system, was analyzed for its transport properties, where it was discovered that transport from the lumen of the hollow fiber to the surrounding environment was not only based on diffusion of the factor, but osmosis-driven convection as well. Sequential delivery strategies such as this one have potential to improve wound healing strategies involving angiogenesis as well as other types of tissue formation that occur in a series of organized stages.

Conducting polymers are class of polymer that can be synthesized directly on conductive substrates and incorporate various functional molecules into it. Its conductivity and customizability make it an ideal interface material for neuronal network research. In the first phase of this thesis, the incorporation of laminin fragments into conducting polymer films is investigated. The laminin fragments are shown to produce low impedance surfaces for neuronal recording. Furthermore, it is shown that the incorporated laminin fragments promote the adhesion of neurons to the surface. These results could provide a means for promoting a stable interface for chronic recording devices. In the second phase of this thesis, In vitro multielectrode arrays provide a framework for studying polypyrrole-mediated controlled release of neurochemicals from microelectrodes, and neuronal network dynamics in a controlled setting. We have developed a technique to achieve transient and local inhibition of synaptic transmission in cultured networks. Conducting polymer films containing the glutamate receptor antagonist CNQX are synthesized directly on the microelectrodes in the recording array. Release of CNQX is achieved through a brief electrical pulse. Through single cell patch-clamp recording, the effectiveness of CNQX release on inhibiting excitatory post-synaptic currents (EPSC) is characterized as a function of distance and time from the releasing electrode, and evidence is shown supporting a diffusion-mediated process following release. At the network level, simultaneous patch-clamp and extracellular recordings are used to characterize stimulus-evoked responses from the network. Cross correlation and a model-based variable clustering technique identify functional connectivity in a neuronal network response to electrical stimuli. Use of the controlled release of CNQX in conjunction with these techniques will allow us to examine the functional clustering of neurons in response to a given stimulation, and how a functional cluster is affected by transient, local inhibition in the network.

Most approaches to tissue engineering and regenerative medicine include a scaffold component. The scaffold material could be synthetic or natural and may be intended simply as a delivery vehicle for cells or growth factors or as an integral component of an engineered tissue or organ. Regardless of the type of material used, the scaffold must be cell friendly and promote host cell attachment, proliferation, migration, differentiation, and eventual three-dimensional tissue organization. Biologic scaffolds composed of extracellular matrix have shown minimal scar tissue formation with constructive remodeling of the damaged or missing tissue structures in a variety of clinical and pre-clinical applications. However, ECM scaffolds are typically characterized by a two-dimensional sheet and are limited to the mechanical and material properties inherent to the tissue from which it was derived. A soluble form of ECM scaffold would expand the clinical utility of ECM by allowing the delivery of the scaffold via minimally invasive methods to the site of interest. The present work describes the enzymatic digestion of an ECM scaffold derived from the porcine urinary bladder (urinary bladder matrix or UBM). UBM was successfully digested with pepsin and papain under different conditions. The enzymatically digested UBM showed chemotactic and mitogenic properties towards progenitor cells while inhibition was found toward endothelial cells. In addition, pepsin digested UBM was able to re-polymerize into a gel form. The present study investigated the in vitro cell growth of different cell types on the surface of the gels and measured the rheological properties of the UBM gel. Synthetic scaffold materials are an alternative to naturally derived ECM scaffolds. However, synthetic materials lack the bioactivity and beneficial host tissue response characteristic of ECM-derived scaffolds and often result in fibrous encapsulation when implanted in vivo. Poly(ester-urethane)urea (PEUU) is a biodegradable polymer that can be manufactured into an elastomeric scaffold by electro-spinning techniques with an ultrastructural morphology similar to the ECM. The present work successfully combined the soluble form of the UBM with the PEUU to create hybrid scaffolds. The in vitro characterization of the scaffolds and the in vivo response are described. Hybrid scaffolds showed a change in the host tissue response and higher degradation rates in vivo in a subcutaneous location when compared to the polymer alone. Finally, the potential use of the PEUU and a PEUU/UBM hybrid scaffold as a left ventricular patch in a canine model is discussed.

A significant number of individuals with disabilities are denied powered mobility because they lack the visual, motor, or cognitive skills required to operate a powered wheelchair safely. The Drive-Safe System (DSS) is an add-on, distributed, shared control navigation assistance system for powered wheelchairs, intended to provide safe and independent mobility to these individuals. The DSS is a human-machine system in which the user and machine share navigation control. The user is responsible for high-level control of the system, such as choosing the destination, path planning, and some navigation actions, while the DSS overrides unsafe maneuvers through autonomous collision avoidance, automatic wall following, and door crossing. This dissertation reports the design and development of the DSS, followed by results from rigorous engineering and clinical evaluations. The engineering tested technical aspects of the DSS such as sensor coverage, maximum safe speed, maximum detection distance, and power consumption. Clinical evaluations included testing the DSS with Orientation & Mobility (O&M) specialists, ambulatory and non-ambulatory visually impaired individuals, and able-bodied controls. We compared the performance of the DSS with conventional navigation aids such as canes that are commonly used in conjunction with wheelchairs based on measures such as time for task completion and number of collisions. Additionally, we collected data with the NASA-TLX to gain insight into users subjective experience with the DSS. Results indicate that the DSS was able to provide a uniform and reliable sensor coverage field around the wheelchair and could successfully detect obstacles as small as 3 inches in height to overhanging obstacles at a height of 55 inches. The DSS significantly reduced the number of collisions compared to using a cane. Users rated the DSS favorably despite the fact they took longer to navigate the same obstacle course than they would using a cane. Visually impaired participants reported experiencing less physical demand, and had to exert less effort in order to achieve better performance when using the DSS, compared to using a cane. These findings suggest that the DSS can be a viable solution for powered mobility in populations with visual impairment.

Peripheral nerve injuries are frequently encountered in trauma, sports accidents, military activities, and degenerative muscle diseases. Like most neurological conditions, patients exhibit pain, sensory and motor deficits, and functional disability. Despite all the advances in biomedical science and technology, achieving full function and organ reinnervation after these injuries remains a major challenge. High costs of healthcare, loss of employment, and social disruption have provided the impetus for active research focusing on improved strategies for repair and regeneration. Stem cell therapy holds tremendous potential for the treatment of pathologic conditions and has consequently emerged as a new area of focus in regenerative medicine. Stem cells isolated from skeletal muscle have been shown to be both pluripotent and of significant therapeutic value; however, their ability to undergo neurogenic differentiation has yet to be investigated. Here, we report that progenitor cells isolated from skeletal muscles of both mouse and human, using our established preplate technique, adopt neuronal and glial phenotypes under controlled culture conditions. Transplantation of these cells into a critical-size sciatic nerve defect allowed full nerve restoration with induction of axonal regeneration through myelin-producing Schwann-like cells. Functional recovery resulted in improved gait of cell-transplanted mice. Multi-lineage progenitor cells have been recently identified in blood vessel walls, notably in skeletal muscle, and venous grafts have been used effectively to bridge nerve defects experimentally and clinically, through unknown cellular mechanisms. In a sex-mismatch model, we identified donor-derived Y chromosomes co-localized with host Schwann cells nuclei, indicating nerve repair through vein grafting are mediated by vascular cells. A sustained decrease in nerve regeneration by decellularized or irradiated venous grafts also highlights the contribution of blood vessel-derived cells to nerve repair. Together, these findings not only identify the cellular basis for the efficacy of therapeutic vein wrapping, but also reinforce the emerging view of muscle cell-mediated therapy for peripheral neuropathies.

Glaucoma is the second leading cause of blindness worldwide, which induces irreversible structural damage (retinal ganglion cell loss and retinal nerve fiber layer (RNFL) thinning) on the retina. Optical coherence tomography (OCT) provides RNFL thickness measurements, which have become an essential clinical measure for objective glaucoma assessment. RNFL thickness is measured on a cross-sectional retinal image sampled along a 3.4mm circle centered around the optic nerve head (ONH). With the conventional time-domain OCT (TD-OCT), its operator dependent scan registration is responsible for the majority of measurement variability. Recently, spectral domain OCT (SD-OCT) technology has been introduced. SD-OCT provides faster scanning (up to 100x) and finer axial resolution (up to 2x) compared to TD-OCT, allowing three-dimensional (3D) volume sampling. 3D SD-OCT data can be visualized as an en face image of the retina. This allows us to create a virtual OCT image along any sampling line (curved or straight), which permits virtually perfect scan registration. The objective of this study is to improve the clinical utility of OCT RNFL measurement by establishing data comparability across the multiple OCT generations and models. First, we developed an algorithm to match the TD-OCT scan location within the corresponding 3D SD-OCT volume. Scan location matching (SLM) enables computation of the calibration equation between TD-OCT and SD-OCT for direct comparison of measurements, bridging the old technology with new ones. Second, the performance of the SLM method was measured using various SD-OCT devices with different spatial sampling methods. By making TD-OCT measurements at one time point comparable to the most recent SD-OCT measurement using SLM, glaucoma progression can be assessed on one to one basis. However, due to the variable TD-OCT scan registration over multiple visits, one can still not analyze the trend of glaucoma progression because RNFL thickness measured at different locations is not directly comparable even after calibration. Therefore, we developed a mathematical model of the retinal nerve fiber bundle distribution pattern to normalize the off-centered TD-OCT RNFL thickness to a virtually centered one. The outcome of this study would facilitate more accurate and reliable glaucoma disease/progression detection in cross-sectional as well as longitudinal clinical settings.

Functional magnetic resonance imaging (fMRI) is currently one of the most widely used non-invasive neuroimaging modalities for mapping brain activation. Techniques such as blood oxygenation level dependent (BOLD) fMRI or cerebral blood volume (CBV)-weighted fMRI are based on the assumption that hemodynamic responses are tightly regulated by neural activity. However, the relationship between fMRI responses and neural activity is still unclear. To investigate this relationship, the unique properties of temporal frequency tuning of primary visual cortex neurons was used as a model since it can be used to separate the neural input and output activities of this area. During moving grating stimuli of 1, 2, 10 and 20 Hz temporal frequencies, two fMRI studies, areal and laminar studies, were conducted with different spatial resolution in a 9.4-T Varian spectrometer. In areal studies, BOLD fMRI was able to detect the difference in tuning properties between area 17 (A17), area 18 (A18) and lateral geniculate nucleus. In A17, the BOLD tuning curve seemed to reflect the local field potential (LFP) low frequency band (<12 Hz) rather than spiking activity and LFP gamma band (25-90 Hz). In laminar studies, a high spatial resolution protocol was adopted to resolve the different cortical layers in A17. In addition to BOLD fMRI, CBV-weighted fMRI was performed to eliminate the contamination from the superficial draining veins. These results showed that BOLD and CBV tuning curves do not reflect the underlying spiking activity or the LFP activity at infragranular layers (the bottom layer of three cortical layers). This implies that the hemodynamic response may not be regulated on a laminar level. Therefore, caution should be taken when interpreting BOLD responses as the sole indicator of different aspects of neural activity in areal and laminar scales.