Date of Award
Doctor of Philosophy (PhD)
Twenty million Americans suffer from peripheral nerve injury (PNI) caused by trauma and medical disorders, with approximately $150 billion spent in annual health-care dollars in the United States. Even with proper surgical reconstruction, less than 50% of the patients achieve satisfactory functional recovery. The gold standard surgical repair for long gaps (>3cm) is the autologous nerve graft, despite its disadvantages such as donor site morbidity, risk of infection, and increased cost. Alternative methods, such as acellular nerve grafts (ANA), are ineffective for large lesion gaps because of the lack of cells and regeneration factors. Recent efforts have been focused on the application of exogenous growth factors, based on their roles during development and post-injury. Of particular interest for this study, following PNI, the distal nerve and denervated muscles increase expression of glial cell line-derived neurotrophic factor (GDNF) to stimulate axonal growth. However, the duration of this response is often insufficient to promote the full reinnervation of end-organ targets, especially for large injury gaps. To extend the period of release of GDNF, our lab has previously engineered transgenic SCs with constitutive overexpression of GDNF (G-SCs). However, we and others have also discovered that such constitutive GDNF overexpression causes axon entrapment in vivo. Specifically, regenerating axons fail to extend beyond the GDNF source and form dense nerve coils at the site of overexpression, a phenomenon termed the “candy-store” effect. The mechanism by which GDNF overexpression causes the “candy-store” effect is unclear. Moreover, the complexity of the in vivo environment presents a major challenge to effectively study the “candy-store” effect. Therefore, in this dissertation, we developed a microfluidic platform to model axon entrapment in vitro and studied the mechanism by which high levels of GDNF can cause the “candy-store” effect.First, we recapitulated neuronal axon entrapment using dissociated chicken embryonic dorsal root ganglion (DRGs) in a 3-chamber microfluidic device. Consistent with in vivo results, G-SCs were able to cause axon entrapment in this platform. Importantly, we found that a high concentration of soluble GDNF (700ng/mL) is sufficient to induce axon entrapment. In addition, axon entrapment caused by high GDNF concentration cannot be overcome by distal sources of high GDNF levels. To further investigate the underlying mechanism, we used DNA microarray to identify differentially expressed genes in neurons treated with high GDNF. Slitrk4 was upregulated by high GDNF treatment, and the knockdowns of Slitrk4 resulted in reduced axon entrapment. Similar to the effects of soluble GDNF, G-SCs resulted in non-reversible axon entrapment that cannot be overcome by distal sources of high GDNF levels. We also examined the effects of SCs preconditioned with high GDNF on neurite extension. While high GDNF preconditioned SCs also induced axon entrapment, such effect was reversible when distal sources of high GDNF levels were present. Together, our results have highlighted the mechanism by which GDNF can cause neuronal axon entrapment and may contribute to developing drug delivery strategies that harness only the beneficial effects of GDNF while avoiding the negative impact.
Dennis Barbour Shelly Sakiyama-Elbert
Paul Bridgman, Daniel Moran, Jonathan Silva, Matthew Wood,