Surgery of axons in C. elegans using ultrafast laser pulses, and observing their subsequent regrowth opens a new frontier in neuroscience, since such research holds a great potential for the development of novel therapies and cures to neurodegenerative diseases. In order to make the required large-scale genetic screenings in C. elegans possible and thus obtain statistically significant biological data, an automated laser axotomy system needs to be developed. Microfluidic devices hold the promise of improved throughput by integrating different functional modules into a single chip. The first step to developing a microfluidic device for laser axotomy is to devise an on-chip worm trapping method, which maintains a high degree of immobilization to sever axons without using anesthetics. In this thesis, we present a novel method that uses a thin, deflectable PDMS membrane that individually traps worms in a microfluidic device. Axons can successfully be severed with the same accuracy as those using conventional paralyzing techniques. This device also incorporates recovery chambers for housing worms after surgery and for time-lapse imaging of axonal regrowth without the repeated use of anesthetics. Towards accomplishing an automated, high-throughput laser axotomy system, we developed an improved microfluidic design based on the same mechanical immobilization technique. This second generation device allows for serially processing of a large quantity of worms rapidly using a semi-automated system. Integrated to the opto-mechanical platform, a software program utilizing image processing techniques is developed. This semi-automated program can automatically identify the location of worms, their neuronal cell bodies, focus on the axons of interest, and align the laser beam with the axon via a PID based viso-servo feedback algorithm. Statistic data demonstrate that there is no significant difference in axonal reconnection rates between surgeries performed on-chip and using anesthetics. To improve flow control, a three-dimensional novel microfluidic valve structure is designed and fabricated. This novel valve structure allows for a complete sealing of the flow channel, without degrading optical conditions for imaging and laser ablation in the trapping area. Finally, we developed a prototypical microfluidic assembly that will eventually be able to interface a well-plate to automatically deliver population of worms from individual wells to the automated chip for axotomy. This interface consists of a microfluidic multiplexer to significantly reduce the number of solenoid valves needed to individually address each well. / text

Amputation is a life-changing event that results in a drastic reduction in quality of life including extreme loss of function and severe mental, emotional and physical pain. In order to mitigate these negative outcomes, there is great interest in the design of ‘advanced/robotic’ prosthetics that cosmetically and functionally mimic the lost limb. While the robotics side of advanced prosthetics has seen many advances recently, they still provide only a fraction of the natural limbs’ functionality. At the heart of the issue is the interface between the robotic limb and the individual that needs significant development. Amputees retain significant function in their nerves post-amputation, which offers a unique opportunity to interface with the peripheral nerve. Here we evaluate a relatively new approach to peripheral nerve interfacing by using microchannels, which hold the intrinsic ability to record larger neural signals from nerves than previously developed peripheral nerve interfaces. We first demonstrate that microchannel scaffolds are well suited for chronic integration with amputated nerves and promote highly organized nerve regeneration. We then demonstrate the ability to record neural signals, specifically action potentials, using microchannels permanently integrated with electrodes after chronic implantation in a terminal study. Together these studies suggest that microchannels are well suited for chronic implantation and stable peripheral nerve interfacing. As a next step toward clinical translation, we developed fully-integrated high electrode count microchannel interfacing technology capable of functioning while implanted in awake and freely moving animal models as needed for pre-clinical evaluation. Importantly, fabrication techniques were developed that apply to a broad range of flexible devices/sensors benefiting from flexible interconnects, surface mount device (SMD) integration, and/or operation in aqueous environments. Examples include diabetic glucose sensors, flexible skin based health monitors, and the burgeoning flexible wearable technology industry. Finally, we successfully utilized the fully integrated microchannel interfaces to record action potentials in the challenging awake and freely moving animal model validating the microchannel approach for peripheral nerve interfacing. In the end, the findings of these studies help direct and give significant credence to future technology development enabling eventual clinical application of microchannels for peripheral nerve interfacing.

Chronic denervation and chronic axotomy present independent barriers for axonal regeneration. Chronic denervation occurs when nerves are no longer connected to their neuronal cell bodies; chronic axotomy occurs when neurons are not connected to their targets for prolonged periods of time. The harmful effects of chronic denervation can be addressed by the side-to-side bridge surgical technique. Additionally, the negative effects of chronic axotomy can be reversed by GDNF delivery to the nerve. The experiments in this thesis were designed to evaluate nerve regeneration in a rat model of chronic injury after treatment with local GDNF delivery, side to-side bridge protection, or both. The GDNF delivery system consisted of poly(lactic-co-glycolic acid) microspheres embedded in fibrin for controlled delivery of GDNF. Overall, the side-to-side bridges technique was effective in protecting against the negative effects of chronic denervation regardless of treatment with or without GDNF. Local delivery of GDNF did not increase axonal regeneration or functional recovery.

Chronic denervation and chronic axotomy present independent barriers for axonal regeneration. Chronic denervation occurs when nerves are no longer connected to their neuronal cell bodies; chronic axotomy occurs when neurons are not connected to their targets for prolonged periods of time. The harmful effects of chronic denervation can be addressed by the side-to-side bridge surgical technique. Additionally, the negative effects of chronic axotomy can be reversed by GDNF delivery to the nerve. The experiments in this thesis were designed to evaluate nerve regeneration in a rat model of chronic injury after treatment with local GDNF delivery, side to-side bridge protection, or both. The GDNF delivery system consisted of poly(lactic-co-glycolic acid) microspheres embedded in fibrin for controlled delivery of GDNF. Overall, the side-to-side bridges technique was effective in protecting against the negative effects of chronic denervation regardless of treatment with or without GDNF. Local delivery of GDNF did not increase axonal regeneration or functional recovery.

Teleost retinal ganglion cells can regenerate severed axons following injury, something their mammalian counterparts cannot do. In the teleost, successful regeneration has been attributed in part to microglial cell activities including the phagocytosis of myelin. Although the regulation of microglial phagocytosis has been studied in mammals, in the teleost it is largely unexamined. The present study was designed to identify mediators of microglial phagocytosis released by injured goldfish optic nerve during the course of regeneration. We found that microglial phagocytosis was significantly enhanced in the presence of a 7 day regenerating nerve or medium conditioned by the nerve (CM). When either nerve or CM was incubated with microglia along with an antibody against tumour necrosis factor alpha (TNFalpha), this effect was neutralized. The L929 cell cytotoxicity assay further demonstrated TNFalpha activity in the CM. However, Western blot analysis did not confirm this result. Therefore, further work is necessary to clearly establish the presence of TNFalpha.

Injury to the central nervous system (CNS) results in the formation of a highly inhibitory glial scar consisting mainly of chondroitin sulfate proteoglycans (CSPGs). CSPGs are comprised of a protein core with covalently attached chondroitin sulfate glycosaminoglycan (CS-GAG) side chains. CSPGs and CS-GAGs have been implicated in the regenerative failure of the CNS, though the mechanism underlying inhibition is unclear. Sulfation affects both the physical and chemical characteristics of CS-GAGs and, therefore, it has been hypothesized that certain sulfation patterns are more inhibitory than others. To investigate this hypothesis, specific chondroitin sulfate sulfotransferases (CSSTs), the enzymes responsible for CS-GAG sulfation, were knocked down in vitro using siRNA. C4ST-1, C4ST-2, and C46ST were chosen as targets for gene knockdown in this study based on their expression in neural tissue and the extent of inhibition caused by their respective CS-GAG. It was hypothesized that transfection of primary rat astrocytes with siRNAs designed to prevent the expression of C4ST-1, C4ST-2, and C46ST would decrease specific sulfation patterns of CSPGs, resulting in improved neurite extension in a neurite guidance assay. Through optimization of siRNA dose, astrocyte viability was maintained while successfully knocking down mRNA levels of C4ST-1, C4ST-2, and C46ST and significantly reducing total levels of secreted CS-GAGs. However, no increase in the incidence of neurite extension was observed using conditioned media collected from siRNA transfected astrocytes compared to non-transfected controls. These data suggest that sulfation does not contribute to CSPG-mediated neurite inhibition, though further investigation is necessary to confirm these findings. Significantly, this work has established a paradigm for investigating the role of CSPG sulfation patterns in CNS regeneration.

Acetylcholine is a neurotransmitter that regulates neurite branching, induces neurite outgrowth, and synapse formation. Because of its various roles in neuronal activities, acetylcholine-based materials may also be useful in nerve repair. We present a series of biodegradable polymers with varying concentrations of acetylcholine-like motifs. We hypothesize that neurite sprouting and extension can be enhanced by using materials to present biochemical and physical cues. Acetylcholine-like motifs were incorporated by the polycondensation of diglycidyl sebacate, aminoethyl acetate, and leucine ethyl ester, which permitted control over acetylcholine motif concentration. Interactions between the polymers and neurons were characterized using rat dorsal root ganglia explants (DRG). We screened the potential application of these materials in nerve tissue engineering using the following criteria: 1) neurite sprouting, 2) neurite length, and 3) distribution of the neurite lengths. The ability of DRG to sprout neurites was influenced by the concentration of acetylcholine motifs of the polymer. Addition of acetylcholine receptor antagonists to DRG cultured on the polymers significantly decreased neurite sprouting, suggesting acetylcholine receptors mediate sprouting on the polymers. Future studies may examine how neurons on acetylcholine-based polymers exhibit changes in downstream signaling events and cell excitability that are associated with receptor activation. In preparation for testing the acetylcholine-based polymers in vivo, porous scaffolds with longitudinally oriented channels were fabricated using fiber templating and salt leaching. Micro computed tomography, scanning electron microscopy, and cryo-sectioning revealed the presence of longitudinally oriented channels. Channel volume and average pore size of the scaffolds were controlled by the number of fibers and salt fusion time. Future studies may involve testing the effect of acetylcholine-motifs by coating polymers onto such scaffolds or assessing the effect of the scaffold's dimensional properties on nerve regeneration.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.