Experimental Strategies for Promoting Neurite Outgrowth of Human Model Neurons

Injuries to the nervous system and the spinal cord in particular are correlated with an irreversible loss of sensory input and voluntary limb control. Patients suffer from serious restrictions to their life while high costs for the public health systems remain a major challenge. Up to now, therapies that focus on secondary complications, are pre-clinical, or show no functional repair. Injuries to the spinal cord are differentiated into an acute phase, namely the initial trauma and chronic phase with trauma-related damages. The pathophysiology is characterized by contusions due to dislocated vertebra, shearing and in the worst case scenario complete transection of axons. During the chronic phase, happening within seconds after the initial trauma, glial scar sealing the initial trauma zone is generated. As a consequence, a hostile environment for axonal regeneration containing several growth inhibiting factors is formed. To increase neurite outgrowth after injury, this thesis examines three key aspects for removing the block of regeneration. First, a primary culture system of human model neurons to measure neurite outgrowth was developed. The rather long neuronal differentiation protocol could be shortened to 4 weeks. Now, high numbers of human neurons in a developing state could be generated in contrast to low cell numbers of neurons in a mature state with the former protocol. Next, I used these neurons to measure the growth promoting effect of Ibuprofen and related pharmaceuticals known to inhibit a ubiquitous regeneration blocking pathway. Culturing human neurons with elevated Ibuprofen levels increased neurite lengths. A pull-down assay showed that Ibuprofen decreased RhoA levels. ROCK inhibition resulted in longer neurite lengths without decreasing RhoA levels. Moreover, initiation of primary neurites increased after treatment with a ROCK inhibitor, but not for RhoA inhibition. The second approach used a unique ensheathing cell type to provide a growth permissive environment for ingrowing neurons in the injured spinal cord. Since rodents and primates differ in their in vitro reaction, I used canine cells as a large animal model system. I characterized the motility of ensheathing glial cells originating from the olfactory mucosa or olfactory bulb and compared them with Schwann cells in a scratch wound assay. Ensheathing cells from the bulb showed a higher motility and activation of the protein kinase C pathway showed a strong increase in cell migration. Schwann cells and mucosa ensheathing cells showed a lower motility and did not respond to protein kinase activation. Next I co-cultured canine ensheathing glia with human neurons and examined their ability to support neurite outgrowth and formation. Ensheathing cells from the bulb showed better growth promoting capacities than cells from the mucosa or Schwann cells. The last part of this thesis evaluated Nephila spp. spider silk as promising biomaterial for transplantation as guiding scaffold to achieve enhanced neurorestoration. I cultured human neurons on silk woven steel frames and showed adhesion and outgrowth within several days. For better optical evaluation, I developed a new crossed fiber array system. Neurons showed initial contact within a few hours cultured on fiber arrays. Growth cones of surrounding neurons get in contact and pulled their cell somata towards the fibers. Neurons showed extensive aggregation on and near spider silk fibers, whereas the surrounding substrate showed a sparse neuronal distribution providing further evidence for Nephila spp. silk as nerve repair scaffold. This thesis describes maturation related effects in cultured neurons and examines three different in vitro approaches with a view to develop therapies for functional nerve repair after spinal cord injury.