Recently, a novel protein family, named as neural regeneration peptides (NRPs), was predicted across the rat, human and mouse genomes by one of my supervisors, Dr. Sieg. Synthetic forms of these proteins have been previously shown to act as potent neuronal chemoattractants and have a major role in neural regeneration. In light of these properties, these peptides are key candidates for drug development against an array of neurodegenerative disorders. The aim of this PhD project was to provide confirmation of the existence of a member of the NRP coding gene family, annotated in the mouse genome. This gene, called mouse frameshift nrp (mouFSnrp), was hypothesised exist as a -1bp frameshift to another predicted gene AlkB. This project involved the identification of the mouFSnrp gene, and the characterisation of its expression pattern and ontogeny during mouse neural development. Through the work described in this thesis, the mouFSnrp gene was identified in mouse embryonic cortical cultures and its protein coding gene sequence was verified. mouFSnrp expression was shown to be present in neural as well as non-neural tissues, via RT-PCR. Using non-radioactive in situ hybridisation and immunohistochemical colocalisation studies, interesting insights into the lineage and ontogeny of mouFSnrp expression during brain development were revealed. These results indicate that mouFSnrp expression originates in neural stem cells of the developing cortex, and appears to be preferentially continued via the radial glial lineage. mouFSnrp expression is carried forward via the neurogenic radial glia into their daughter neuronal progeny as well as postnatal astrocyte. In the postnatal brain, mouFSnrp gene transcripts were also observed in the olfactory bulb and the hippocampus, both of which are known to have high neurogenic potential. In general, the radial glial related nature of mouFSnrp expression appears to be a hallmark of the mouFSnrp expression pattern through out neural development. This thesis provides the first confirmation of the existence of a completely novel gene, mouFSnrp, and its putative -1 translational frameshifting structure. Further, preliminary data presented in this thesis regarding the mouFSnrp in situ expression pattern during mouse brain development may suggest a key role of the gene in neuronal migration and neurogenesis in mice. / FRST Bright Futures Enterprise Fellowship

Recently, a novel protein family, named as neural regeneration peptides (NRPs), was predicted across the rat, human and mouse genomes by one of my supervisors, Dr. Sieg. Synthetic forms of these proteins have been previously shown to act as potent neuronal chemoattractants and have a major role in neural regeneration. In light of these properties, these peptides are key candidates for drug development against an array of neurodegenerative disorders. The aim of this PhD project was to provide confirmation of the existence of a member of the NRP coding gene family, annotated in the mouse genome. This gene, called mouse frameshift nrp (mouFSnrp), was hypothesised exist as a -1bp frameshift to another predicted gene AlkB. This project involved the identification of the mouFSnrp gene, and the characterisation of its expression pattern and ontogeny during mouse neural development. Through the work described in this thesis, the mouFSnrp gene was identified in mouse embryonic cortical cultures and its protein coding gene sequence was verified. mouFSnrp expression was shown to be present in neural as well as non-neural tissues, via RT-PCR. Using non-radioactive in situ hybridisation and immunohistochemical colocalisation studies, interesting insights into the lineage and ontogeny of mouFSnrp expression during brain development were revealed. These results indicate that mouFSnrp expression originates in neural stem cells of the developing cortex, and appears to be preferentially continued via the radial glial lineage. mouFSnrp expression is carried forward via the neurogenic radial glia into their daughter neuronal progeny as well as postnatal astrocyte. In the postnatal brain, mouFSnrp gene transcripts were also observed in the olfactory bulb and the hippocampus, both of which are known to have high neurogenic potential. In general, the radial glial related nature of mouFSnrp expression appears to be a hallmark of the mouFSnrp expression pattern through out neural development. This thesis provides the first confirmation of the existence of a completely novel gene, mouFSnrp, and its putative -1 translational frameshifting structure. Further, preliminary data presented in this thesis regarding the mouFSnrp in situ expression pattern during mouse brain development may suggest a key role of the gene in neuronal migration and neurogenesis in mice. / FRST Bright Futures Enterprise Fellowship

Recently, a novel protein family, named as neural regeneration peptides (NRPs), was predicted across the rat, human and mouse genomes by one of my supervisors, Dr. Sieg. Synthetic forms of these proteins have been previously shown to act as potent neuronal chemoattractants and have a major role in neural regeneration. In light of these properties, these peptides are key candidates for drug development against an array of neurodegenerative disorders. The aim of this PhD project was to provide confirmation of the existence of a member of the NRP coding gene family, annotated in the mouse genome. This gene, called mouse frameshift nrp (mouFSnrp), was hypothesised exist as a -1bp frameshift to another predicted gene AlkB. This project involved the identification of the mouFSnrp gene, and the characterisation of its expression pattern and ontogeny during mouse neural development. Through the work described in this thesis, the mouFSnrp gene was identified in mouse embryonic cortical cultures and its protein coding gene sequence was verified. mouFSnrp expression was shown to be present in neural as well as non-neural tissues, via RT-PCR. Using non-radioactive in situ hybridisation and immunohistochemical colocalisation studies, interesting insights into the lineage and ontogeny of mouFSnrp expression during brain development were revealed. These results indicate that mouFSnrp expression originates in neural stem cells of the developing cortex, and appears to be preferentially continued via the radial glial lineage. mouFSnrp expression is carried forward via the neurogenic radial glia into their daughter neuronal progeny as well as postnatal astrocyte. In the postnatal brain, mouFSnrp gene transcripts were also observed in the olfactory bulb and the hippocampus, both of which are known to have high neurogenic potential. In general, the radial glial related nature of mouFSnrp expression appears to be a hallmark of the mouFSnrp expression pattern through out neural development. This thesis provides the first confirmation of the existence of a completely novel gene, mouFSnrp, and its putative -1 translational frameshifting structure. Further, preliminary data presented in this thesis regarding the mouFSnrp in situ expression pattern during mouse brain development may suggest a key role of the gene in neuronal migration and neurogenesis in mice. / FRST Bright Futures Enterprise Fellowship

Recently, a novel protein family, named as neural regeneration peptides (NRPs), was predicted across the rat, human and mouse genomes by one of my supervisors, Dr. Sieg. Synthetic forms of these proteins have been previously shown to act as potent neuronal chemoattractants and have a major role in neural regeneration. In light of these properties, these peptides are key candidates for drug development against an array of neurodegenerative disorders. The aim of this PhD project was to provide confirmation of the existence of a member of the NRP coding gene family, annotated in the mouse genome. This gene, called mouse frameshift nrp (mouFSnrp), was hypothesised exist as a -1bp frameshift to another predicted gene AlkB. This project involved the identification of the mouFSnrp gene, and the characterisation of its expression pattern and ontogeny during mouse neural development. Through the work described in this thesis, the mouFSnrp gene was identified in mouse embryonic cortical cultures and its protein coding gene sequence was verified. mouFSnrp expression was shown to be present in neural as well as non-neural tissues, via RT-PCR. Using non-radioactive in situ hybridisation and immunohistochemical colocalisation studies, interesting insights into the lineage and ontogeny of mouFSnrp expression during brain development were revealed. These results indicate that mouFSnrp expression originates in neural stem cells of the developing cortex, and appears to be preferentially continued via the radial glial lineage. mouFSnrp expression is carried forward via the neurogenic radial glia into their daughter neuronal progeny as well as postnatal astrocyte. In the postnatal brain, mouFSnrp gene transcripts were also observed in the olfactory bulb and the hippocampus, both of which are known to have high neurogenic potential. In general, the radial glial related nature of mouFSnrp expression appears to be a hallmark of the mouFSnrp expression pattern through out neural development. This thesis provides the first confirmation of the existence of a completely novel gene, mouFSnrp, and its putative -1 translational frameshifting structure. Further, preliminary data presented in this thesis regarding the mouFSnrp in situ expression pattern during mouse brain development may suggest a key role of the gene in neuronal migration and neurogenesis in mice. / FRST Bright Futures Enterprise Fellowship

Recently, a novel protein family, named as neural regeneration peptides (NRPs), was predicted across the rat, human and mouse genomes by one of my supervisors, Dr. Sieg. Synthetic forms of these proteins have been previously shown to act as potent neuronal chemoattractants and have a major role in neural regeneration. In light of these properties, these peptides are key candidates for drug development against an array of neurodegenerative disorders. The aim of this PhD project was to provide confirmation of the existence of a member of the NRP coding gene family, annotated in the mouse genome. This gene, called mouse frameshift nrp (mouFSnrp), was hypothesised exist as a -1bp frameshift to another predicted gene AlkB. This project involved the identification of the mouFSnrp gene, and the characterisation of its expression pattern and ontogeny during mouse neural development. Through the work described in this thesis, the mouFSnrp gene was identified in mouse embryonic cortical cultures and its protein coding gene sequence was verified. mouFSnrp expression was shown to be present in neural as well as non-neural tissues, via RT-PCR. Using non-radioactive in situ hybridisation and immunohistochemical colocalisation studies, interesting insights into the lineage and ontogeny of mouFSnrp expression during brain development were revealed. These results indicate that mouFSnrp expression originates in neural stem cells of the developing cortex, and appears to be preferentially continued via the radial glial lineage. mouFSnrp expression is carried forward via the neurogenic radial glia into their daughter neuronal progeny as well as postnatal astrocyte. In the postnatal brain, mouFSnrp gene transcripts were also observed in the olfactory bulb and the hippocampus, both of which are known to have high neurogenic potential. In general, the radial glial related nature of mouFSnrp expression appears to be a hallmark of the mouFSnrp expression pattern through out neural development. This thesis provides the first confirmation of the existence of a completely novel gene, mouFSnrp, and its putative -1 translational frameshifting structure. Further, preliminary data presented in this thesis regarding the mouFSnrp in situ expression pattern during mouse brain development may suggest a key role of the gene in neuronal migration and neurogenesis in mice. / FRST Bright Futures Enterprise Fellowship

Soft tissue reconstruction in the nervous system is sensitive to the mechanical and chemical cues of the growth microenvironment. Many technologies have been designed to study these stimuli and their effect on the regional extracellular environment (ECM). Because of the hard-to-achieve and costliness of these technologies, biologists are usually reluctant to employ them to study cellular behaviors. In addition, the complexity of the nervous system, particularly in cases of nerve repair and reconstruction, necessitates the development of facile high- throughput investigational tools. The objective for this dissertation is to examine and manipulate neuronal cell-cell and cell-ECM responses to varying nervous system microenvironment stimuli in a 3-D in vitro model. / acase@tulane.edu

Axons are injured after stroke, spinal cord injury, or neurodegenerative disease such as ALS. Most axons do not regenerate. A recent report suggests that not all neurons are poor regenerators, but rather a small subset can regenerate robustly. What intrinsic property of these regenerating neurons allows them to regenerate, but not their neighbors, remains a mystery. This subtype-specific regeneration has also been observed in Drosophila larvae sensory neurons. We exploited this powerful genetic system to unravel the intrinsic mechanism of subtype-specific neuron regeneration. We found that neuron bursting activity after axotomy correlates with regeneration ability. Furthermore, neuron bursting activity is necessary for regeneration of a regenerative neuron subtype, and sufficient for regeneration of a non-regenerative neuron subtype. This optogenetically-induced regeneration is dependent on a bursting pattern, not simply overall activity increase. We conclude that neuron bursting activity is an intrinsic mechanism of subtype-specific regeneration. We then discovered through a reverse genetic screen that an L-type voltage gated calcium channel (VGCC) promotes neuron bursting and subsequent regeneration. This VGCC has high expression in the regenerative neuron and weak expression in the non-regenerative neuron. This suggests that VGCC expression level is the molecular mechanism of subtype-specific neuron regeneration. Together, our findings identify a cellular and molecular intrinsic mechanism of subtype-specific regeneration, which is why some neurons are able to regenerate while the majority of neurons do not. Perhaps VGCC activation or neuron activity pattern modulation could be used therapeutically for patients with nerve injury.

INTRODUCTION: Injury to the facial nerve can lead to functional and aesthetic sequelae in patients. Though surgical interventions are available to restore lost motor and sensory function, outcomes are often suboptimal due to inadequate or disorganized axonal regeneration. While engineering improvements to the standard of care are underway, gaps remain in our molecular understanding of peripheral nerve injury to translate these efforts clinically. Over the last few decades, advancements in intravital imaging such as the development of fluorescent reporter mice and use of multiphoton excitation techniques have allowed for markedly enhanced characterization of biological phenomena at higher resolutions, at greater depths, and for longer timescales. Challenges in reliably and serially imaging in vivo within murine models have been overcome through the development of chronic imaging windows in various settings of the body. However, there are very few techniques available presently for imaging the peripheral nerve microenvironment and no prior work detailing use in the facial nerve setting. OBJECTIVE: Longitudinal studies employing intravital imaging techniques carry potential to improve understanding of peripheral nerve regeneration and function. Using multiphoton microscopy and fluorescent reporter mice, we propose a prototype, surgical protocol of implantation, and initial safety and efficacy testing of a facial nerve window to enable chronic imaging for enhanced characterization of the peripheral nerve microenvironment. METHODS: A stainless-steel implant with an affixed glass coverslip and aluminum external fixation component was developed for implantation in a transgenic reporter mouse model to enable chronic intravital imaging of the facial nerve buccal and marginal mandibular branches. A qualitative observational study and clinical assessment scoring study was performed post-surgical implantation to monitor behavior, physical appearance, weight loss, and reactivity to animal handling over the typical time-course of nerve regeneration. Segments of facial nerve branches were harvested from control and window-implanted mice and imaged using widefield epifluorescence microscopy for axon quantification to determine any adverse effects from window compression onto axonal fibers. Two-photon microscopy (2PM) and Simulated Raman Scattering (SRS) were also performed through the window to visualize axon tracts, myelin sheaths, and surrounding collagen matrix in wild-type and transgenic mice models. RESULTS: Qualitative serial observational studies and assessment scoring indicated no obvious functional deficits over the time-course of typical nerve regeneration and normal scores for weight, behavior, physical appearance, and reactivity. Neural histomorphometric analysis indicated no significant difference in mean myelinated axon count of buccal (mean ± SD; control buccal, 947.6 ± 129.9; window-implanted buccal, 799.3 ± 128.6; p = .136) and marginal mandibular branches (control marginal mandibular, 801.3 ± 145.1; window-implanted marginal mandibular, 738.0 ± 197.2; p = .599) between control and window-implanted mice, suggesting that neuropathy was not induced from the window itself. High-resolution images of nerve morphology in healthy and injured transgenic and wild-type mice were obtained using 2PM and SRS. CONCLUSION: Herein, we describe a novel and replicable platform for longitudinal intravital imaging of murine facial nerve. Future studies will evaluate viability of this model for imaging the facial nerve microenvironment, particularly Schwann cell-axon interactions, in the setting of severe nerve injury over a period of several weeks to months. Improved understanding gained through such studies of the structural peripheral nerve microenvironment may allow for advancements in viral vector therapeutics, nerve graft scaffold design, as well as advanced injury diagnostics and tracking. / 2022-06-02T00:00:00Z

Tesis por compendio / [ES] Actualmente las lesiones del sistema nervioso periférico que conllevan una pérdida de continuidad de los haces axonales suelen implicar secuelas de tipo permanente. Es cierto que en el sistema nervioso periférico existe una cierta regeneración natural de los tractos axonales dañados, pero solamente cuando el espacio entre ambos extremos de la lesión es pequeño, como máximo de 5 mm. Si el espacio es mayor que esta distancia la regeneración no sucede de forma natural y se crea un neuroma traumático. Por tanto, estas lesiones largas requieren de una intervención quirúrgica para puentear la lesión, normalmente con un nervio autógrafo del propio paciente o un nervio alógrafo de un cadáver. No obstante, su uso presenta diversos inconvenientes, como la morbilidad del sitio donante donde puede ocurrir un neuroma, la necesidad de realizar una segunda cirugía, la diferencia de tamaño entre nervio receptor y donante o la necesidad de inmunosupresión en el caso de los nervios alógrafos. Por ello, la ingeniería de tejidos trabaja en el desarrollo de los conductos de guiado nervioso que incorporan estrategias para guiar topográficamente la regeneración, así como células y moléculas bioactivas. La presente tesis doctoral presenta un nuevo conducto de guiado nervioso con una aproximación multimodular para su aplicación en la regeneración de lesiones nerviosas largas (a partir de 15 mm) que hace uso de conductos tubulares huecos modulares de ácido hialurónico (HA) que contienen en su interior una estructura tubular de microfibras de ácido poliláctico (PLA). La estructura fibrilar aporta un guiado topográfico necesario para guiar el crecimiento axonal durante la regeneración a la vez que mantiene unidos los diferentes módulos de HA. Por su parte, los conductos de HA son un hidrogel que evita adherencias con el tejido circundante. A su vez, proporcionan un soporte sobre el que pueden crecer células presembradas. En concreto se ha optado por presembrar células de Schwann, las cuales son unas células gliales de soporte críticas para la regeneración del sistema nervioso periférico. Se ha observado que dichas células son capaces recubrir por completo las paredes internas de los conductos de HA formando una estructura tipo vaina, así como de recubrir las microfibras de PLA creciendo en dirección longitudinal. Los experimentos in vivo en modelo de nervio ciático de conejo han mostrado que la aproximación multimodular mejora significativamente la regeneración nerviosa gracias a proporcionar una mejor neovascularización. A su vez, gracias a las células de Schwann presembradas se ha logrado una mejora adicional de la regeneración nerviosa gracias a su efecto favorecedor del crecimiento axonal. Además, se han estudiado diferentes mejoras aplicables al conducto de guiado nervioso con el objetivo de mejorar los resultados obtenidos in vivo. Gracias a la incorporación de fibroína de seda a los conductos de HA se ha logrado mejorar sus propiedades mecánicas y biológicas. Asimismo, también se ha desarrollado un sustrato electroconductor de microfibras de PLA recubiertas con el polímero electroconductor Polipirrol gracias al cual se ha observado in vitro que es capaz de mejorar el crecimiento axonal al aplicar una estimulación eléctrica. Además, mediante un sistema de modificación génica de las células de Schwann por electrotransfección se ha logrado aumentar su secreción del factor neurotrófico derivado del cerebro (BDNF), gracias a lo cual se ha observado que se incrementa la velocidad de crecimiento axonal in vitro. / [CA] Actualment les lesions del sistema nerviós perifèric que comporten una pèrdua de continuïtat dels feixos axonals solen implicar seqüeles de tipus permanent. És cert que al sistema nerviós perifèric hi ha una certa regeneració natural dels tractes axonals danyats, però només quan l'espai entre ambdós extrems de la lesió és petit, com a màxim de 5 mm. Si l'espai és més gran que aquesta distància la regeneració no succeeix de manera natural i es crea un neuroma traumàtic. Per tant, aquestes lesions llargues requereixen una intervenció quirúrgica per pontejar la lesió, normalment amb un nervi autògraf del pacient o un nervi al·lògraf d'un cadàver. No obstant això, el seu ús presenta diversos inconvenients, com la morbilitat del lloc donant on pot ocórrer un neuroma, la necessitat de fer una segona cirurgia, la diferència de mida entre nervi receptor i donant o la necessitat d'immunosupressió en el cas dels nervis al·lògrafs . Per això, l'enginyeria de teixits treballa en el desenvolupament dels conductes de guiatge nerviós que incorporen estratègies per guiar topogràficament la regeneració, així com cèl·lules i molècules bioactives. Aquesta tesi doctoral presenta un nou conducte de guiatge nerviós amb una aproximació multimodular per a la seva aplicació en la regeneració de lesions nervioses llargues (a partir de 15 mm) que fa ús de conductes tubulars buits modulars d'àcid hialurònic (HA) que contenen al seu interior una estructura tubular de microfibres d'àcid polilàctic (PLA). L'estructura fibril·lar aporta un guiatge topogràfic necessari per guiar el creixement axonal durant la regeneració alhora que manté units els diferents mòduls d'HA. Per part seva, els conductes d'HA són un hidrogel que evita adherències amb el teixit circumdant. Alhora, proporcionen un suport sobre el qual poden créixer cèl·lules presembrades. En concret s'ha optat per presembrar cèl·lules de Schwann, les quals són unes cèl·lules glials de suport crítiques per a la regeneració del sistema nerviós perifèric. S'ha observat que aquestes cèl·lules són capaces de recobrir completament les parets internes dels conductes d'HA formant una estructura tipus beina, així com de recobrir les microfibres de PLA creixent en direcció longitudinal. Els experiments in vivo en model de nervi ciàtic de conill han mostrat que l'aproximació multimodular millora significativament la regeneració nerviosa gràcies a proporcionar una millor neovascularització. Alhora, gràcies a les cèl·lules de Schwann presembrades s'ha aconseguit una millora addicional de la regeneració nerviosa gràcies al seu efecte afavoridor del creixement axonal. A més, s'han estudiat diferents millores aplicables al conducte de guiatge nerviós per tal de millorar els resultats obtinguts in vivo. Gràcies a la incorporació de fibroïna de seda als conductes d'HA s'ha aconseguit millorar les seues propietats mecàniques i biològiques. També s'ha desenvolupat un substrat electroconductor de microfibres de PLA recobertes amb el polímer electroconductor Polipirrol gràcies al qual s'ha observat in vitro que és capaç de millorar el creixement axonal quan s'aplica una estimulació elèctrica. A més, mitjançant un sistema de modificació gènica de les cèl·lules de Schwann per electrotransfecció s'ha aconseguit augmentar la secreció del factor neurotròfic derivat del cervell (BDNF), gràcies a la qual cosa s'ha observat que s'incrementa la velocitat de creixement axonal in vitro. / [EN] Currently, lesions of the peripheral nervous system that lead to a loss of continuity of the axonal bundles usually involve permanent sequelae. It is true that in the peripheral nervous system there is some natural regeneration of damaged axonal tracts, but only when the space between the two ends of the lesion is small, at most 5 mm. If the gap is greater than this distance, regeneration does not occur naturally, and a traumatic neuroma is created. Therefore, these long injuries require surgical intervention to bridge the injury, usually with an autograph nerve from the patient or an allograph nerve from a cadaver. However, its use has various drawbacks, such as the morbidity of the donor site where a neuroma can occur, the need to perform a second surgery, the difference in size between the recipient and donor nerves, or the need for immunosuppression in the case of allograft nerves. For this reason, tissue engineering works on the development of nerve guidance conduits that incorporate strategies to topographically guide the regeneration, as well as cells and bioactive molecules. This doctoral thesis presents a new nerve guidance conduit with a multimodular approach for its application in the regeneration of long nerve lesions (from 15 mm) that makes use of modular hollow tubular conduits of hyaluronic acid (HA) that contain in their inside a tubular structure of microfibers of polylactic acid (PLA). The fibrillar structure provides the necessary topographic guidance to guide axonal growth during regeneration while keeping the different HA modules together. For their part, the HA conduits are a hydrogel that prevents adhesions with the surrounding tissue. In turn, they provide a support on which preseeded cells can grow. Specifically, it has been decided to pre-seed Schwann cells, which are glial support cells that are critical for the regeneration of the peripheral nervous system. It has been observed that these cells are capable of completely covering the inner walls of the HA conduits, forming a sheath-like structure, as well as covering the PLA microfibers by growing in a longitudinal direction. In vivo experiments in a rabbit sciatic nerve model have shown that the multimodular approach significantly improves nerve regeneration by providing better neovascularization. In turn, thanks to the pre-seeded Schwann cells, an additional improvement in nerve regeneration has been achieved thanks to its promoting effect on axonal growth. In addition, different improvements applicable to the nerve guidance conduit have been studied with the aim of improving the results obtained in vivo. Thanks to the incorporation of silk fibroin into HA conduits, their mechanical and biological properties have been improved. Likewise, an electroconductive substrate of PLA microfibers coated with the electroconductive polymer Polypyrrole has also been developed, thanks to which it has been observed in vitro that it is capable of improving axonal growth by applying electrical stimulation. In addition, by means of a gene modification system of Schwann cells by electrotransfection, it has been possible to increase their secretion of brain-derived neurotrophic factor (BDNF), thanks to which it has been observed that the speed of axonal growth is increased in vitro. / Agradezco la ayuda de los diferentes proyectos del Ministerio de Economía y Competitividad del Gobierno de España que han hecho posible la financiación de esta tesis doctoral: MAT2015-66666-C3-1-R, DPI2015-72863-EXP, AEI RTI2018-095872-B-C21-C22/ERDF y FPU16/01833 del Ministerio de Universidades del Gobierno de España, sin la cual no hubiera podido realizar esta tesis doctoral. / Gisbert Roca, F. (2022). Strategies for Guidance and Electrical and Biological Stimulation in a Neural Regeneration Device [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/189937 / Compendio

[Truncated abstract] There is limited intrinsic potential for repair in the adult human central nervous system (CNS). Dysfunction resulting from CNS injury is persistent and requires prolonged medical treatment and rehabilitation. The retina and optic nerve are CNSderived, and adult retinal ganglion cells (RGCs) and their axons are often used as a model in which to study the mechanisms associated with injury, neuroprotection and regeneration. In this study I investigated the effects of a variety of strategies on promoting RGC survival and axonal regeneration after optic nerve injury, including the use of reconstructed chimeric peripheral nerve (PN) grafts, gene therapy, and intraocular application of pharmacological agents and other factors . . . C3 transferase is an enzyme derived from Clostridium botulinum that inactivates Rho GTPase. Because SC myelin contains MAG and PN also contains CSPGs, I tested the effects of intraocular injection of a modified form of C3 (C3-11), provided by Dr Lisa McKerracher (CONFIDENTIAL data, under IP agreement with Bioaxone Therapeutic, Montreal) on RGC axonal regeneration into PN autografts. My results showed that there was significantly more RGC survival and axonal regeneration in PN autografts after repeated intraocular injection of C3. I also tested whether intraocular injections of CPT-cAMP and/or CNTF can act in concert with the C3 to further increase RGC survival and/or regeneration. Results showed that the effect of C3 and CPT-cAMP plus CNTF were synergistic and partially additive. The use of combination therapies therefore offers the best hope for robust and substantial regeneration. The overall results from my PhD project will help determine how best to reconstruct nerve pathways and use pharmacological interventions in the clinical treatment of CNS injury, hopefully leading to improved functional outcomes after neurotrauma.