Distinct axo-protective and axo-destructive roles for Schwann cells after injury in a novel compartmentalised mouse myelinating coculture system.

Myelinating Schwann cell (SC)- dorsal root ganglion (DRG) neuron cocultures have been an important technique over the last four decades in understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. While methods using rat SCs and rat DRG neurons are commonplace, there are no established protocols in the field describing the use of mouse SCs with mouse DRG neurons in dissociated myelinating cocultures. There is a great need for such a protocol as this would allow the use of cells from many different transgenic mouse lines. Here we describe a protocol to coculture dissociated mouse SCs and DRG neurons and induce robust myelination. Use of microfluidic chambers permits fluidic isolation for drug treatments, allows cultures to be axotomised to study injury responses, and cells can readily be transfected with lentiviruses to permit live imaging. We used this model to quantify the rate of degeneration after traumatic axotomy in the presence and absence of myelinating SCs and axon aligned SCs that were not induced to myelinate. We find that SCs, irrespective of myelination status, are axoprotective and delay axon degeneration early on. At later time points after injury, we use live imaging of cocultures to show that once axonal degeneration has commenced SCs break up, ingest, and clear axonal debris