Development and implementation of multi-cued guidance strategies for axonal regeneration

Millions of individuals are affected by nerve damage to either their central nervous system (brain, spinal cord and eyes) or their peripheral nervous system. Endogenous positive and negative biochemical cues are expressed during trauma, working in conjunction with one another, regenerating tissue and protecting the body from further damage. However, because of the inherent nature of the nervous system, these mechanisms are typically skewed toward deleterious results and functional recovery is limited or unlikely to occur. Additionally, topographical features and electrical signals aid in nervous system patterning, and they could therefore be beneficial in axonal regeneration as well. The goal of this dissertation project is to develop initial axon guidance models to serve as a platform for neural regenerative strategies. The major component of this research is the development of an immobilization scheme where guidance proteins, notably nerve growth factor (NGF) and semaphorin3A (Sema3A), are synthesized and immobilized to a chitosan biomaterial film in order to evaluate axon and growth cone responses. Characteristic axon outgrowth responses were seen for single protein cues, attraction toward immobilized NGF sources and inhibition and axon turning away from immobilized Sema3A sources. These responses were not seen when proteins were adsorbed to the chitosan substrate. These axonal results showed the validity of the immobilization platform, which was further examined by tethering NGF and Sema3A in the same region in order to fine-tune axon guidance. Axons were more sensitive to lower concentrations of Sema3A signals compared to NGF, when these proteins were co-immobilized, as noted by axon turning and breakdown of axonal cytoskeletal structure. Another axon guidance model was formed through topographical contact guidance of micropatterned channels on coumarin polyester films. These features were able to align processes of central nervous system neurons and glia parallel to the microchannel and plateau features. The polarity resulting from the topographical features is important for initiating axon guidance. Finally, an optic nerve crush (ONC) model was designed in vivo in order to examine retinal ganglion cell (RGC) regeneration when exposed to soluble and immobilized NGF. Analysis of anterograde neuronal tracing and astrocyte infiltration revealed a crush injury was successfully created. Behavioral analysis showed an increase in visual acuity after ONC surgeries, but a better baseline needs to be established in order for a link to be made between enhanced depth perception and applied treatment. Further quantification of optic nerve tissue is needed to reveal if there were any differences in RGC regeneration between groups by means of cholera toxin B (CTB) labeling and RGC responsiveness in the form of NGF receptors. This information will direct us to understand if RGC were capable of receiving survival and regenerative instructions for NGF treatments after ONC. Overall these axon guidance platforms can be tailored toward future injury models in vitro and in vivo where axons need multiple cues for enhanced but controlled pathfinding.