The use of microfluidic chambers to study axonal transport in PTEN and SOCS3 dependent axonal regeneration
Zhigang He and Thomas L. Schwarz
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Duration: 1 year Project Completion: 2013
Background and Aim
Rodents are extensively used to study nerve injury. The mouse spinal cord injury model, widely used in nerve injury research, is extremely debilitating. In vivo studies have allowed major advancements in the comprehension of the incapacity of adult central nervous system axons to regenerate. Notably, in vivo studies have shown that axonal regeneration after nerve injury was possible in adult mice if PTEN or SOCS3 were deleted in knock-out mice (1,2,3). However, studying in vivo mechanistic adaptation at the cellular level remains challenging. Axonal transport is an important cellular mechanisms evidenced by the numerous neurodegenerative diseases that have been related to an axonal transport impairment. Improving axonal transport in the injured and diseased central nervous system has been proposed as a promising strategy to improve neuronal repair. However, the contribution of each cargo to the repair mechanism is unknown. Because the transport of specific cargos after axonal insult has not been examined systematically in a model of enhanced regenerative capacity, it is unknown whether the transport of all cargos would be modulated equally in injured central nervous system neurons. In order to test the transport adaptation of cellular cargos during axonal regeneration, an in vitro system allowing straightforward manipulation and analysis is required. We used microfluidic chambers (4) to mimic nerve injury and regeneration in vitro. This method allowed us to (i) injure axons without affecting the cell body, (ii) manipulate neuron cell bodies and axons specifically, and (iii) analyze axonal transport during axonal regeneration at a single axon resolution.
Method and Results
Using a microfluidic culture system we compared neurons co-deleted for PTEN and SOCS3, an established model of high axonal regeneration capacity, to control neurons. We measured the axonal transport of three cargos (mitochondria, synaptic vesicles and late endosomes) in regenerating axons and found that the transport of mitochondria, but not the other cargos, was increased in PTEN/SOCS3 co-deleted axons relative to controls. The results reported here suggest a pivotal role for this organelle during axonal regeneration and validate the microfluidic culture system to identify cellular mechanisms occurring in regenerating axons.
Conclusions and Relevance for 3R
A lab testing the regenerative capacity of axons using the spinal cord injury model (transgenic mice, drug treatment) will use roughly 5,000 mice per year. Some of these mice are used to test hypotheses that will not give any satisfactory results. To increase the chance of obtaining positive results in vivo while decreasing the number of mice used, we propose to validate the microfluidic chambers as an in vitro system that would be a primary test to establish promising hypotheses worth testing in vivo, if possible. We estimate that by first testing the hypotheses in a reliable in vitro system would save one third of the mice used per year. We hope that our study will establish microfluiding chambers as a gold standard in vitro system in the field of the study of spinal cord injury/axonal regeneration.
The results of this project is published in:
Cartoni, R., Pekkunaz, G., Wang, C., Schwarz,T.L., He, Z. An Elevated Mitochondrial Transport Rate characterizes high Regeneration Capacity Neurons in CNS neurons (2017). PLoS One 12(9).
(1) Park K, Liu K, Hu Y, Smith P, Wang C, Cai B, et al. Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science. Vol 322, 2008b: 963-6.
(2) Smith P, Sun F, Park K, Cai B, Wang C, Kuwako K, et al. SOCS3 deletion promotes optic nerve regeneration in vivo. Neuron. Vol 64, 2009: 617-23.
(3) Sun F, Park KK, Belin S, Wang D, Lu T, Chen G, et al. Sustained axon regeneration induced by co-deletion of PTEN and SOCS3. Nature; 480: 372-5.
(4) Taylor AM, Blurton-Jones M, Rhee SW, Cribbs DH, Cotman CW, Jeon NL. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods. Vol 2, 2005a: 599-605.