I have investigated whether or not neurotrophic factors can alter the effects of an applied electric field on neurite growth. A direct current electric field (150mV/mm) applied for 5 hours affected the growth of dissociated cells from the neutral tube of stage 20 <I>Xenopus laevis. </I>Neurites turned and grew to the cathode, showed a differential rate of growth, with cathodal-facing neurites growing faster than those facing the anode, and exhibited a polarised branching pattern, with the majority of branches emanating from the cathodal-facing side of the neurite. Exposure of neurites to the neurotrophins neurotrophin 3 (NT-3; 50ng/ml and 100ng/ml), brain derived neurotrophic factor (BDNF; 50ng/ml and 100ng/ml) and neurotrophin 4 (NT-4; 100ng/ml) lowered the threshold field strength necessary to elicit a turning response to 100mV/mm. Only BDNF (100ng/ml) enhanced cathodal orientation at 150mV/mm, with neurites turning three times as far as in the field alone. Additionally, in the presence of these neurotrophins the threshold field that evoked a differential rate of growth and polarised branching was also lowered to 100mV/mm. Neurite turning did not occur in a field of 100mV/mm in the presence of 50ng/ml nerve growth factor (NGF), 100ng/ml ciliary neurotrophic factor (CNTF) or 50ng/ml glial-cell-line derived neurotrophic factor (GDNF), or when the Trk receptor tyrosine kinase inhibitors K-252a and K-252b were added concomitantly with 50ng/ml NT-3. <I>Xenopus </I>growth cones release acetylcholine (Ach) spontaneously. This is enhanced by NT-3, BDNF, NT-4. Nicotinic Ach receptor antagonists abolish field-induced cathodal turning, thus Ach release may be crucial for this response. The neurotrophins NT-3, BDNF and NT-4 may therefore modulate field-directed nerve growth because they enhance Ach release. Interactions <I>in vivo,</I> where neurotrophins and electric fields co-exist, would increase the efficacy of endogenous electric fields as modulators of nerve growth.

Stroke is a leading cause of disability in the world, for which there currently is no effective treatment. One potential method for treating stroke is to stimulate the endogenous neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) of adult brain to replace the tissue lost during stroke. Two growth factors that have shown promise in eliciting functional repair in rodent models of stroke are epidermal growth factor (EGF) and erythropoietin (EPO). However, there is a significant challenge in delivering protein drugs in a minimally invasive yet effective manner. In this thesis, a minimally invasive polymer-based system is developed to control the sequential release of EGF followed by EPO. This system comprises of a hyaluronan-methylcellulose (HAMC) hydrogel and two types of polymeric particles, and is applied epicortically to deliver EGF and EPO to stroke-injured mouse brains in a minimally invasive manner. In this thesis, the following are demonstrated: 1) The ability of therapeutics delivered locally to reach the target site after delivery is crucial for the success of local delivery strategies. PEG-modification leads to enhanced penetration distance of EGF. 2) When delivered epicortically to the stroke-injured mouse brain using HAMC, PEG-EGF penetrates further into the brain compared to unmodified EGF. Both EGF and PEG-EGF stimulated NSPC proliferation in the SVZ, but the extent of stimulation is greater when PEG-EGF is delivered compared to unmodified EGF. 3) The transport of EPO is similar in the uninjured and the stroke-injured brain following epicortical delivery from HAMC. EPO delivered epicortically from HAMC is able to reach the SVZ and can enhance neurogenesis in the stroke-injured brain. 4) A composite delivery system is engineered where PEG-EGF and EPO are individually encapsulated in different polymeric particles, and the particles are embedded in the HAMC hydrogel matrix. Stroke-injured animals that receive composite-mediated growth factor treatments ultimately achieve repair comparable to that achieved using a conventional catheter/osmotic minipump infusion system, without causing tissue damage associated with insertion of the infusion system into the brain.

No description available.

No description available.

A hallmark of retinal diseases is degeneration of neural cells, leading to subsequent vision loss. For such diseases, replenishment of functional neural cells may be an optimal therapy. Unlike humans, the adult red-spotted newt, Notophthalmus viridescens, possesses the remarkable ability to regenerate a complete retina following its removal or injury. The purpose of this study was to develop a reproducible model of retinal damage and regeneration in the newt to understand the process of retinal regeneration. Intense light, shown in other organisms to be a relevant model of visual cell loss, was tested in the newt and resulted in variable loss of retinal function, correlating with the appearance of apoptotic cells. Due to the variability of damage observed, surgical removal of the retina was used to complement the light-damage model. A novel and non-invasive protocol using full-field electroretinography was developed to assess retinal function in vivo following damage. Measures of retinal function with the electroretinogram protocol successfully showed that photoreceptor function is initially lost and subsequently restored during regeneration. These results enhance our understanding of retinal regeneration in the adult newt and serve as a starting point for further studies aimed at determining the molecular mechanisms involved in the regeneration process.

Much effort has been spent over the last decade in producing so called "Machine Vision" systems for use in robotics, automated inspection, assembly and numerous other fields. Because of the large amount of data involved in an image (typically ¼ MByte) and the complexity of many algorithms used, the processing times required have been far in excess of real time on a VAX-class serial processor. We review a number of image understanding algorithms that compute a globally defined "state", and show that they may be computed using simple local operations that are suited to parallel implementation. In recent years, many massively parallel machines have been designed to apply local operations rapidly across an image. We review several vision machines. We develop an algebraic analysis of the performance of a vision machine and show that, contrary to the commonly-held belief, the time taken to relay images between serial streams can exceed by far the time spent processing. We proceed to investigate the roles that a variety of pipelining techniques might play. We then present three pipelined designs for vision, one of which has been built. This is a parallel pipelined bit slice convolution processor, capable of operating at video rates. This design is examined in detail, and its performance analysed in relation to the theoretical framework of the preceeding chapters. The construction and debugging of the device, which is now operational in its hardware is detailed.

No description available.

Despite advances in bone grafting technology for musculoskeletal injury, re-injury or incomplete healing persists. Efforts to modify bone allografts sing proteins and growth factors show improvement in wound healing outcomes. We use FTY720, an agonist of S1P receptors 1 and 3, to improve bone graft integration through bone regeneration and vascularization. Four methods of delivering FTY720 into a bone defect are described: 1) FTY720 loaded onto a PLAGA-coated bone allograft and implanted in a critical size rat cranial defect; 2) FTY720 loaded onto a PLAGA-coated bone allograft and implanted in a rat tibial defect; 3) FTY720 loaded into a Matrigel plug and injected into a mouse tibial fracture; and 4) FTY720 directly adsorbed to human bone xenografts and implanted in a critical size rat cranial defect. In each of these models, FTY720 release was characterized, and bone regeneration and vascularization was monitored within the defect. Additionally, local tissue composition and immune response was evaluated. The results presented here indicate that FTY720 released locally into the bone defect improved new bone formation and vascularization, promoting improved graft integration.

This study aimed to better understand the clearance of myelin debris during Wallerian degeneration in the goldfish visual paths. Myelin debris was first examined immunohistochemically in the presence or absence of regenerating axons. From these preliminary experiments it was apparent that the clearance of myelin debris was not affected by regenerating axons and that the debris was removed in a differential pattern along the visual pathway. Specifically, in the distal stump of the nerve as well as in the optic tract, myelin debris had been effectively cleared by one-month postoperative, while in the cranial segment of the nerve debris persisted for at least 6 weeks after injury. The differential pattern of myelin debris in the optic nerve and tract was then analyzed qualitatively and quantitatively using thick and thin plastic sections at various time points during regeneration. The results suggested that highly activated peripheral macrophages were responsible for the effective clearance of myelin in the distal nerve stump. In the optic tract a number of cellular properties, including their unique population of astrocytes may have enhanced the rate of debris clearance. By contrast, in the cranial segment of the nerve persistent debris was found both intracellularly in phagosomes and extracellularly, suggesting that the resident phagocytes were deficient in effecting both phagocytosis and emigration. Deficient phagocytosis may be a result of the production of anti-inflammatory cytokines in this region, while the failure to emigrate is most likely due to the rigid network of astrocytes in the nerve.

The neuromuscular junction (NMJ) is a synapse formed between a motoneuron and a muscle fiber which transmits the signals required to initiate muscle contraction. The functional state of the NMJ is intimately tied to the structure and function of the motoneuron, such that reductions in postsynaptic activity retrogradely stimulate sustained reorganization of presynaptic motor terminals in an attempt to maintain normal contractile output. In the adult, these plastic changes occur most notably as regenerative responses following traumatic injury and during the progression of motoneuron diseases (MNDs), and can contribute to a considerable amount of functional repair. However, limitations to the regeneration capacity of motoneurons place an upper limit on the effectiveness of endogenous repair mechanisms and can restrict the extent of functional recovery. Using a combination of immunofluorescence, sharp electrode electrophysiology and live labeling of synaptic vesicle recycling during various forms of synaptic growth and regeneration in vivo and in vitro, I have identified that the neural cell adhesion molecule (NCAM) is a key regulator of the regenerative capacity of motoneurons. In vivo experiments revealed that NCAM influences the maturation and stabilization of regenerated synapses via the recruitment and recycling of synaptic vesicles necessary for effective synaptic transmission. The presence of both pre- and post-synaptic NCAM were necessary to maintain the abundance of recycling synaptic vesicles at regenerated synapses, demonstrating a coordinated influence of these molecules in regenerative synaptic plasticity in vivo. To accurately assess the regenerative potential of motoneurons in vitro, it was necessary to develop a system which could reliably and consistently generate mature NMJs amenable to experimental investigation. Motoneurons differentiated from embryonic stem cells were grown for 3-5 weeks in co-culture with muscle fibers and generated mature NMJs which possessed morphological and functional criteria consistent with NMJs formed in vivo. NMJs formed by NCAM-/- motoneurons did not mature and were found to exhibit deficits consistent with their in vivo counterparts. These studies have revealed that NCAM is a key mediator of regenerative plasticity at the NMJ and may be a target for efforts to enhance endogenous repair following traumatic injury or during the progression of neurodegenerative disease.