Injuries to the peripheral nervous system (PNS) are major and common source of disability, impairing the ability to move muscles and/or feel normal sensations, or resulting in painful neuropathies. Annually traumatic nerve injuries resulting from collisions, gunshot wounds, fractures, motor vehicle accidents, lacerations, and other forms of penetrating trauma, affected more than 250,000 patients just in the U.S. The clinical gold standard to bridge long non-healing nerve gaps is to use a nerve autograft- typically the patient’s own sural nerve. However, autografts are not ideal because of the need for secondary surgery to ‘source’ the nerve, loss of function at the donor site, lack of appropriate source nerve in diabetic patients, neuroma formation, and the need for multiple graft segments. Despite our best efforts, finding alternative ‘nerve bridges’ for peripheral nerve repair remains challenging – of the four nerve ‘tubes’ FDA approved for use in the clinic, none is typically used to bridge gaps longer than 10 mm due to poor outcomes. Hence, there is a compelling need to design alternatives that match or exceed the performance of autografts across critically sized nerve gaps. Here we demonstrate that early modulation of innate immune response at the site of peripheral nerve injury inside biomaterials-based conduit can favorably bias the endogenous regenerative potential after injury that obviates the need for the downstream modulation of multiple factors and has significant implications for the treatment of long peripheral nerve gaps. Moreover, our study strongly suggests that more than the extent of macrophage presence, their specific phenotype at the site of injury influence the regenerative outcomes. This research will advance our knowledge regarding peripheral nerve regeneration, and help developing technologies that are likely to improve clinical outcomes after peripheral nerve injury. The significant results presented here are complementary to a growing body of evidence showing the direct correlation between macrophage phenotype and the regeneration outcome of injured tissues.

This study was concerned with the ultrastructural changes that occur in axons and glial cells during peripheral nerve regeneration in the cockroach <i>Periplaneta americana</i>. Metathoracic nerve 5 was cut and regeneration of the proximal stump was studied using electron microscopy. Nerve 5 was surrounded by an acellular layer, the neural lamella. Underneath this structure was a layer of glial cells which formed the perineurium. Lanthanum penetration stopped between the perineurial cell processes, revealing them to be the site of the blood-brain barrier (BBB). Underlying the perineurium were the axons, surrounded by the subperineurial glial cells. Extracellular matrix was present between subperineurial glial processes. After cutting nerve 5, the initial changes in the proximal stump were a result of the degeneration of sensory axons. Haemocytes accumulated outside the nerve and morphologically similar granule-containing (g-c) cells appeared inside the nerve. After the first week signs of regeneration were distinguishable. These included axonal sprouting, glial proliferation and extracellular matrix production. Many small axonal sprouts were formed by regrowing axons. These became grouped into bundles, surrounded by glial processes, as the nerve outgrowth elongated. Glial proliferation by cell division began after the first week, and reached a maximum rate between two and three weeks. It is possible that mitosis of glial cells may be triggered by contact with the sprouting axons. Freeze-fracture studies of the tip of the growing nerve showed that formation of gap and septate unctions took place between the glial cells. This junctional assembly was asynchronous. Reinnervation of the coxal muscles occurred 8 weeks after the nerve was cut. At this stage the nerve was composed of several axonal bundles, each containing large and small axons. The nerve did not completely resemble the control even after 16 to 20 weeks of regeneration. Lanthanum incubation showed that the tracer was again excluded by the perineurial cells, indicating that the BBB of the regrown nerve reappeared at 8 weeks. Glial repair was studied following selective glial disruption using localised application of ethidium bromide. This treatment killed the perineurial and subperineurial glial cells. The repair of the glial system involved the transitory appearance of g-c cells in the nerve. 11 days after ethidium bromide treatment, new glial cells were present and lanthanum was excluded by the perineurial cell layer. Preinjection of microspheres into the haemolymph, which were taken up by phagocytic haemocytes, reduced the numbers of g-c cells that appeared in the nerve after ethidium bromide treatment. This lengthened the time required for glial repair. Cell division of neuroglial cells was observed. Cells derived from haemocytes and glial cell division were probably involved in the replacement of the damaged glial cells after ethidium bromide treatment. This study shows that glial cells play an important role in peripheral nerve regeneration in insects, forming the environment through which the regenerating axons grow.

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Unlike the peripheral nervous system (PNS), adult neurons in the central nervous system (CNS) have limited regenerative capacity after injury. One interesting phenomenon observed nearly four decades ago was that lesion of a peripheral nerve can significantly enhance the regenerative capacity of the central axons of the corresponding dorsal root ganglion (DRG) neurons, termed a ‘conditioning lesion’, but the underlying mechanism is still not fully understood. Since ATP is released after nerve injury and extracellular ATP has a broad range of biological activities, we postulated that ATP might be the injury signalling molecule that triggers the regenerative machinery in the injured neurons. If that were the case, injection of ATP into a peripheral nerve should be able to mimic the effect of a conditioning lesion. To test this theory, we injected ATP into a peripheral (sciatic) nerve after a dorsal column transection and found that ATP injection did promote the regeneration of injured axons into the lesion cavity. We also found that ATP injection activated transcription factor STAT3 and increased the expression of growth associated protein 43 (GAP43) in the corresponding DRG neurons. ATP injection increased the concentrations of ciliary neurotrophic factor and interleukin-6 in sciatic nerve and DRG. These results indicate that intraneural injection of ATP can mimic conditioning lesion to a certain degree. Most interestingly, we found that a second injection of ATP one week after the first one markedly boosted the effects of the first injection as many more axons grew into or across the lesion compared with double saline injection or ATP plus saline injection. Double ATP injection is also more effective in sustaining the expression of phospho- STAT3 and GAP43. Immunohistochemical analysis showed ATP injection caused little Wallerian degeneration at the injection site. Behavioural tests showed no long-term adverse effects to the injected sciatic nerve. In order to explore the underlying mechanism of ATP induced elevation of the regeneration state of DRG neurons and look for more potent purinoceptor agonists to stimulate axonal regeneration, we first tried to identify the expression of purinoceptor subtypes in sciatic nerves using quantitative PCR and immunohistochemistry. We found that mRNAs for all the four P1 and fourteen P2 purinoceptor subtypes were expressed in the sciatic nerve, DRG or dissociated Schwann cells at various levels. Immunohistochemical analysis showed that purinoceptor subtypes are expressed by different types of cells. Due to the expression of nearly all purinoceptor subtypes in the sciatic nerve, it will be a big challenge to identify the receptor subtype(s) responsible for ATP induced axonal regeneration. We have set up a compartmented co-culture system to test various agonists/antagonists of purinoceptors. Taken together, we have shown that intraneural ATP injection can mimic conditioning lesion in promoting sensory axonal regeneration. Identification of the receptor subtype(s) and other molecules involved in the enhanced regeneration capacity of injured neurons may lead to the development of therapeutic agents to effectively promote the axonal regeneration of both peripheral and central neurons.

The present study has developed poly ε-caprolactone (PCL)/ poly lactic acid (PLA) films with specific internal structure suitable to prepare nerve guide conduit for peripheral nerve repair. The film preparation method has been carried out using an environmental chamber to prepare the solvent cast films with the specific surface structure. Different cellular behaviour of neuronal cell cultures was seen on the pitted films with different pits configurations (size and distribution). The consistent surface morphology provided a reliable surface structure for further in vitro and in vivo studies. The effect of a medical grade sterilisation process using gamma radiation at eight doses (0-45kGy) on PCL/PLA films was explored. It has been shown that material properties, including mechanical strength, were significantly affected, while cellular behaviour and responses (NG108-15) were improved. Grooved films with three groove shapes (Sloped, Square, and V shape) were prepared using patterned silicon substrates, photolithography and wet/dry etching. The groove patterns were successfully transferred and good mechanical strength was observed for grooved PCL/ PLA. Oriented growth of NG108-15 cells was observed on the patterned films with an improved alignment and organisation on SL and V shape grooved films. UV-ozone treatment was used to increase hydrophilicity of PCL/PLA films to improve Schwann cells behaviour. No negative effect was observed on cell growth and proliferation on the treated films however the mechanical properties were reduced. Schwann cells expressed typical long spindle-shape morphology with cell-to-cell interaction in longitudinal direction on the treated grooved films. Consistent to in vitro experiment with NG108-15, Schwann cells alignment was also improved on SL and V shape grooves. A three-week in vivo study was carried out to test grooved and non-grooved conduits in a rat sciatic nerve model. The grooved conduits showed better regeneration, with SL-grooved film showing a significant improvement of nerve regeneration. A separate in vivo study evaluated the effect of wall-thickness on nerve regeneration. However, it was shown that the wall thickness had no positive effect, and the conduit with improved mechanical strength adversely affected the nerve regeneration. In conclusion, a nerve guide conduit was developed with the optimised surface structure to support nerve regeneration. The promising in vitro and in vivo studies together with the suitable biomechanical properties and specific surface structure and morphology indicate that the grooved PCL/PLA conduit is a viable treatment for peripheral nerve repair.

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Nerve injury is a very common trauma affecting 300,000 people in Europe every year. Although autografts are currently the gold standard in surgery, they can cause loss of sensation and scar tissue formation. Artificial nerve conduits are a valid alternative for peripheral nerve repair. They can provide a confined environment during the regeneration process, enabling axons sprouting from the proximal to the distal nerve segments as well as reducing scar tissue formation. Poly-e-caprolactone (PCL) is a biocompatible and biodegradable polymer suitable for the fabrication of nerve guidances. In particular, previous works demonstrated that neural cells are able to adhere and proliferate on micropitted PCL films obtained through solvent casting. Also, short term studies showed that axons were able to bridge 1cm injury gap. In this work a 18 weeks long term in vivo experiment using a rat model was performed to investigate the reinnervation of end organ skin and muscle. PCL conduits were compared to autografts, with no significant differences in terms of regeneration and reinnervation. However, Schwann cells (SCs), the most important glial cells in the peripheral nervous system, showed poor attachment in vitro on PCL scaffolds; hence, surface modification was carried out in order to improve the material biocompatibility. The effect of both hydrophilicity and functional groups on SCs was first investigated. PCL films were then hydrolysed and aminolysed to modify the surface with carboxylic and amino groups respectively. Hydrolysed films increased remarkably the surface hydrophilicity, although topography and mechanical properties were not affected. Conversely, the tensile modulus and strength were significantly reduced by aminolysis, but still suitable for the desired application. The two treatments influenced also the morphology of SCs. It was demonstrated that cell elongation was induced by hydrophilic surfaces, whilst cells preferred cell-cell interaction when cultured on aminolysed films. However, cell proliferation was remarkably increased on the latter surfaces, confirming previous results obtained on substrates characterised by amino groups. These results confirmed that a good balance between hydophilicity and surface chemistry is necessary to guarantee the best cell response. In order to enhance both proliferation and morphology of SCs, arg-gly-asp (RGD) sequences were immobilised on the PCL film surface using two different reaction mechanisms. Carbodiimide chemistry was compared to a new mechanism developed in the present study based on the Thiol chemistry. Biological tests performed on these modified films demonstrated the improvement of SC response after the peptide immobilisation using the novel approach. Cell attachment and proliferation were three times higher compared to untreated PCL films. It was also observed that the presence of peptides on the film surface induced the formation of focal adhesion plaques by SCs, important for the perception of cellular signals when in contact with a particular substrate. Hence, a good balance between focal adhesion and adhesion forces was achieved after peptide immobilisation. Overall the results of this study showed that material functionalisation is very important for SC response and it will be fundamental for the production of artificial nerve conduits.

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The failure of mammalian CNS neurons to regenerate their axons derives from a combination of intrinsic deficits and extrinsic obstacles. Following injury, chondroitin sulfate proteoglycans (CSPGs) accumulate within the glial scar that forms at the lesion site in response to the insult. CSPGs inhibit axonal growth and regeneration, an action mediated by their sulfated glycosaminoglycan (GAG) chains, especially those with 4-sulfated (4S) sugars. Arylsulfatase B (ARSB) selectively cleaves 4S groups from the non-reducing ends of GAG chains without disrupting other, potentially growth-permissive motifs. In this thesis, "Modifying Chondroitin Sulfation Enhances Retinal Ganglion Cell Axon Regeneration," I, Craig Pearson, seek to determine the time course and spatial distribution of CSPG accumulation in the glial scar following acute injury, and then to demonstrate that ARSB is effective in reducing the inhibitory actions of CSPGs. I examine the effects of ARSB in an in vitro model of the glial scar and in vivo, using optic nerve crush (ONC) in adult mice. ARSB is clinically approved for replacement therapy in patients with mucopolysaccharidosis VI and therefore represents an attractive candidate for translation to the human CNS. My findings illustrate the importance of CSPGs as a barrier to axon extension following injury, and show compelling evidence that selective modification of the sulfation pattern on GAG chains results in significant enhancement of RGC axonal regeneration. Finally, I combine ARSB treatment with a host of intrinsic pro-regenerative stimuli and show robust, long-distance regeneration of RGC axons through the optic chiasm and into the optic tract. Taken together, the results of this thesis argue for the therapeutic potential of modifying the extracellular matrix to promote regeneration of axons in the CNS.