3R-Project 129-11

The use of microfluidic chambers to study axonal transport in PTEN and SOCS3 dependent axonal regeneration

Zhigang He and Thomas L. Schwarz
F.M. Kirby Neurobiology Center and Children's Hospital and Department of Neurology, Harvard Medical School
Boston, MA 02115, USA
zhigang.he@childrens.harvard.edu, thomas.schwarz@childrens.harvard.edu

Keywords: mice; axons; neurons; spinal cord; spinal cord repair / diseases; cell cultures: organ-specific; perfusion chamber; reduction; replacement

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). In order to test the hypothesis that would decipher the mechanism by which PTEN/SOCS3 deletion induces axonal regeneration, an in vitro system allowing straightforward manipulation and analysis is required. We propose to use microfluidic chambers (4) to mimic nerve injury and regeneration in vitro. This method permits (i) to injure axons without affecting the cell body, (ii) an easy manipulation of neuron cell bodies or axons specifically, and (iii) a single axonal analysis. In the present project, the method will be further evaluated in order to prove its suitability as a replacement method for specific in vivo studies.

Method and Results
in progress (present status)
Using freshly isolated cortical neurons from knock-out mice (PTEN, SOCS3 or PTEN/SOCS3) cultured in microfluidic chambers (Fig.1), we will test the regenerative capacity of these neurons after injury.

Figure 1: (A)
First panel: Schematic view of a microfluidic chamber. Adapted from (4). Neurons plated in the somal side will project their axons through the 450 µm long microgrooves and reach the axonal side.
Second and third panel: immunohistochemistry of E18 mouse cortical neurons culture (DIV7) in microfluidic chambers.
Second panel: Red; anti-3-Tubulin (axonal marker), Green: anti-GFAP (glial marker), Blue: DAPI (nuclear marker). Third panel: Red; anti-3-Tubulin, Green: anti-MAP2 (dendrites marker), Blue: DAPI. 450 µm microgrooves allow a pure isolation of neurons.

(B)
3-Tubulin immunohistochemistry of E18 mouse cortical neurons culture (DIV7) in microfluidic chambers.
No Injury (first panel), immediately after injury (second panel) and 3 days after injury (third panel).

(C) Regrowing axons can be observed 3 days after injury.

Neurons isolated from E18 embryos and from post natal pups will be used to assess the cellular response to axonal injury of the different genotypes as they develop. We will also use the unique feature of the microfluidic chambers to perform in vitro axonal injury in order to decipher the role played by axonal transport to support robust axonal regeneration induced by the deletion of PTEN and/or SOCS3. In vitro axonal injury will be inflicted on neurons after 6 days in culture. To study to corelation between fast axonal transport and axon regeneration we will track the transport of different markers of the fast axonal transport (endosomes, mitochondria, synapti vesicle) using live imaging techniques in regenerative axons 20 hour post injury (after 7 days in culture). The microfluidic chambers will allow us to follow these highly dynamic organelles in single axon minutes or hours post injury. Genetic manipulation or drug treatment specifically applied in the somal or axonal compartment of the chamber will be used to assess the mechanism by which axonal transport supports axonal regeneration. Finally, to fully validate the use of microfluidic chambers in the field of spinal cord injury/axonal regeneration, the regenerative capacity of knock-out axons injured in vitro will be compared with the results obtained in the lab using classical in vivo nerve injury models (optic nerve injury and spinal cord injury). Axonal transport rate post injury and during regeneration will be compared with the rate of transport observed in vivo using a live imaging technique of the mouse optic nerve that we are currently developing in the lab.

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 would save one third of the mice used per year. We hope that our study will establish microfluiding chambers as a gold standard system in the field of the study of spinal cord injury/axonal regeneration.

References
(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.