The research objective of this proposal is to define and analyze how stretching forces, associated with the growth of an organism, initiate unique neurobiological mechanisms to accommodate stretch growth of axons, driving the natural and rapid formation of long nerves and white matter tracts. In a developing embryo, axons navigate via a growth cone over seeming large distances to reach their targets. However, well after axons integrate with their targets and establish synaptic connections, animals and their nervous systems continue to grow several orders of magnitude. It is conceivable that stretching forces, exerted on axons by the enlarging body, serves as the mechanism that initiates and maintains stretch growth of the axon cylinder.
An in vitro tissue engineering method has been developed to recapitulate this fundamentally different and rapid form of axonal growth that occurs during an organism's development. Far exceeding the rate of growth cone extension, this new-found form of nervous system growth, extreme axon stretch growth, can reach at least 10mm per day. These investigations mapped out the biomechanical boundaries that allow integrated axon bundles to quickly adapt to escalating stretch-growth rates, producing large axon fascicles 10cm in length and potentially much longer. Remarkably, these extreme stretch growth conditions also stimulate expansion of axon caliber, while maintaining a normal cytoskeletal ultrastructure and the ability to convey action potentials. Surprisingly, few studies have examined the effects of mechanical stretch on the rapid growth potential of axons.
Axon stretch growth presents a novel opportunity to greatly expand upon the current understanding of nervous system growth with real potential to discover new targets to accelerate regeneration, offering an unexplored direction in nerve repair. Additional scientific benefits of this model could be the ability to engineer structured nervous tissue to study the pathology of nervous system diseases or the neurophysiological behavior of an organized network of neurons.
Students at all levels will be included in this exciting and challenging opportunity to explore new territory in bioengineering and neuroscience. Opportunities and mentoring will also be provided for students with disabilities as well as encouragement and assistance for high school students with disabilities and their college plans.
The objective of this research was to define and analyze how stretching forces, associated with the growth of an organism, initiate unique neurobiological mechanisms to accommodate stretch induced growth of long nerves also called axons. Defining the mechanotransduction of unique and unknown neurobiological mechanisms and cellular processes that accommodate extreme rates of axon stretch growth will contribute new knowledge to the neurosciences and may offer new insight into approaches for nerve growth applicable to regenerative applications. To investigate the dynamic physical and neurobiological changes, a system was developed for the real-time imaging and quantification of axon stretch growth. The system can be viewed at: www.jove.com/video/2753. Using this system, the biomechanical limitations and long-term effects of axon stretch growth were determined. If tension is kept above a resting threshold (stretch is applied), axons grow. Conversely, it was discovered if tension is released to below the resting tension level, axons retract. Under stretch growth conditions, it was found that instantaneous stretch is limited to 39% strain where the axonal transport of mitochondria diminishes. Despite apparent occlusion, axons remained intact for 6 days after which they were capable of subsequent stretch growth. While single strains of 39% diminished transport, axons can only sustain continuous strains of 25% without disconnection. This mechanical limit proceeds until a rate of 4mm/day growth rate for embryonic and 2.5mm/day for adult axon. Interestingly however, they can tolerate stretch rates well beyond their maximum growth rate temporarily without damage. To verify if long term stretch growth is normal we considered some nerve injury markers. In a nerve transection injury, a retrograde signal from the axon to the soma signals a repair process known as central chromatolysis. Following 14 days of stretch growth under the maximum identified 25% strain above, the cytoplasm and nucleus remain unchanged. Under damaging stretch growth conditions, however, strains greater than 38% lead to a large increase in the cytoplasm and nucleus characteristic of a chomatolytic response. Using electrophysiological techniques, stretch growing axons elicited normal action potentials. Furthermore, calcium dysregulation, a common occurrence in stretch injury, was not found. This suggests axon stretch growth is a normal undamaging process. The cellular mechanisms that drive growth cone extension and guidance during development are applied in essentially every approach to enhance regeneration and repair of the adult nervous system. The extreme rates of axon stretch growth present an exciting and potentially groundbreaking opportunity to implicate existing and uncover novel cellular pathways for accelerating nerve repair. The hypothesis of this research was that axon stretch growth induces unique cellular mechanisms to alter protein synthesis and transport to rapidly assemble new axon. Once identified, these processes can be exploited to accelerate regeneration of axons following injury. To investigate changes in gene regulation, a controlled experiment consisting of stretched and non-stretched controls were run on Affymetrix rat gene 1.0 ST DNA Microarrays. Fold changes less than 1.15 were removed and selecting Q-values of 0.05 resulted in the identification of only 487 genes to be significantly changed, which ranged from -2.0 to +5.0 fold. Developing functional gene maps from this data is an ongoing process and we have isolated a set of regenerative associated genes of interest. Using Ingenuity Pathway Analysis Software to study interaction pathways among genes, a limited set of connections were identified. We found the greatest utility of the software by plotting known Regenerative Associated Genes (RAGs) and building upon those genes using the software's grow function. Utilizing this method we found parallels between axon stretch growth and the regenerative growth cascade that follows injury. Interestingly, the overall picture of gene activation is a stress related response opposed to a regenerative, injury initiated response. Specifically, regenerative associated genes that are growth related appear in axon stretch growth. The regenerative associated genes that are injury related, however, are not affected. Indeed, this system might be a powerful method of identifying genes needed for regeneration without the associated injury response.
