Axon stretch growth: towards functional repair of the spinal cord: a translational exercise

Abstract

Background: Injury to the spinal cord often visually presents as a local injury, damaging neurons that reside in the spinal cord, their projecting axons and supporting infrastructure such as oligodendrocytes. However, damage also occurs to ascending and descending axons that communicate with the brain. Fundamentally, repair of these injuries requires two distinct restorative approaches. The local injury will require stabilisation of the local environment, the rescue of injured neurons and support infrastructure, replacement of lost cells and restoration of intra-spinal communication. The latter ascending and descending axon injury will require proximal and distal axon reunification to restore supra-spinal communication with the brain. This thesis presents the results of an early translational exercise that takes a non-linear approach to facilitate investigation into axon stretch growth (ASG) - an intrinsic mechanism that allows axons to adapt to body height and size throughout life. Pioneering research has shown that in-vitro exploitation of ASG has the potential to bridge significant gaps associated with injuries to long supra-spinal nerve tracts within the spinal cord. Although translational science can be applied across the research spectrum, the traditional practice is to intervene once the research has matured. Here, the intervention occurs early, in an environment of limited funding within a progressive school of basic sciences. At the time of intervention, no infrastructure or experience in ASG research was evident within the faculties. Translational Methods: The absence of a robust in-vitro adult motor neuron culture was identified as a potential barrier in ASG translation. Collaborations in anatomy, neuroscience, and toxicology were formed. Three separate animal ethics applications were required. Publication of a protocol followed. The infrastructure required to conduct necessary in-vitro investigations into ASG was determined. Multidisciplinary collaborations were formed with mechanical, electrical and electronics engineers. Design, engineering, and commissioning of the equipment followed. The lack of a definitive translational animal model has been previously identified as a significant barrier to spinal cord injury research. Specifically, a suitable large animal model has yet to be clearly defined for ASG research. Collaborations with comparative anatomy, a large animal research centre, and a senior spinal surgeon progressed development of a sheep model. Separate multi-institutional animal ethics applications were also required. Results: A robust peer reviewed method was established to hydraulically extrude the spinal cord of adult Sprague-Dawley rats in under 60 seconds, and a serum free culture protocol simplified to maximise the yield of motor neurons and reduce culture costs. Adult motor neurons harvested and cultured using this protocol are capable of in-vitro survival for periods exceeding 21 days. A decommissioned infant humidicrib was successfully converted into a portable temperature (32 – 39°C ± 0.1°C), and carbon dioxide controlled imaging incubator. Additional modifications incorporating umbilical support for multiple tailored bioreactors was also developed. A tailored ASG bioreactor was prototyped, tested, and commissioned. Axon stretch growth of motor neurons has been initiated in the bioreactor. The literature review suggested that non-human primates were the optimal model for final translational confirmation. However, there was sufficient evidence to indicate that ungulates (i.e. sheep or pig) may be an alternative for ASG research. Relevant information on the sheep was collated, and basic investigation on their anatomy progressed.

Conclusion: Early applied translational science (as practised here) is strategic and cost effective, showing that the overall strategy facilitates research, while potentially identifying barriers that could delay progress or cause late translational failure. The introduction of an “off the shelf” early intervention funding model allocated to translational scientists should be considered as a mechanism to progress basic science investigations that are in conceptual stages of development.