An in vitro model of adult mammalian nerve repair

Abstract

The role of pathway-derived growth factors in the support of peripheral axon regeneration remains elusive. Few appropriate knock-out mice are available, and gene silencing techniques are rarely 100% effective. To overcome these difficulties, we have developed an in vitro organotypic co-culture system that accurately models peripheral nerve repair in the adult mammal. Spinal cord sections from P4 mice that express YFP in their neurons are used to innervate segments of P4 peripheral nerve. This reconstructed ventral root is then transected and joined to a nerve graft. Growth of axons across the nerve repair and into the graft can be imaged repeatedly with fluorescence microscopy to define regeneration speed, and parent neurons can be labeled in retrograde fashion to identify contributing neurons. Nerve graft harvested from adult mice remains viable in culture by both morphologic and functional criteria. Motoneurons are supported with GDNF for the first week in culture, after which they survive axotomy, and are thus functionally adult. This platform can be modified by using motoneurons from any genetically modified mouse that can be bred to express XFP, by harvesting nerve graft from any source, or by treating the culture systemically with antibodies, growth factors, or pathway inhibitors. The regeneration environment is controlled to a degree not possible in vivo, and the use of experimental animals is reduced substantially. The flexibility and control offered by this technique should thus make it a useful tool for the study of regeneration biology.

Axonal regeneration is compromised in NFH-LacZ transgenic mice but not in NFH-GFP mice

“…3A; 1 and 3 lower left panel). This is in sharp contrast with the Thy1-YFP model, another ''crush model'', where fluorescence is still detectable all along the nerve 7 days after crush (Vyas et al, 2010). Other cytoskeletal proteins were also analysed in parallel to assess the axonal damage and regeneration.…”

Biofabrication and testing of a fully cellular nerve graft

“…In order to provide a more statistically significant study, we plan to modify the in vitro method of Vyas et al [52] to further test the repair capabilities of the biofabricated grafts. An in vitro study of nerve repair allows for a much larger sample size than our current study presented.…”

Studying Axonal Degeneration and Regeneration Using In Vitro and In Vivo Models: the Translational Potential

“…The technique of explanting ganglia offers the additional benefit of adapting in vivo models of degeneration and regeneration to in vitro studies via explants of nerves that have been crushed or subjected to 'cut and repair' [7]. A good example of this has recently been shown in a spinal cord explant model where one can do 'nerve repair' in vitro and study regeneration of axons in a 3D structure while manipulating the regenerative environment with precision [10]. [11].…”