One of the major hurdles when studying neuronal-glial interactions is trying to understand how two different cell types communicate effectively to accomplish a particular task. Myelination is the result of complex neuronal-glial interactions and is necessary for the efficient and rapid transmission of action potentials throughout the vertebrate nervous system. During development of the central nervous system (CNS), neurons and oligodendroglia interact with each other in numerous ways to achieve precise and timely myelination. It is generally accepted that inductive and/or inhibitory signals produced by axons control whether they will become myelinated1
. Neurons also modulate proliferation and migration of oligodendrocyte precursor cells (OPCs) by providing mitogenic cues2
and trophic factors3
. Conversely, oligodendroglia support the function and the wellbeing of neurons4, 5
. Due to the intimate nature of neuronal-oligodendroglial interactions, it is challenging to study certain processes related to myelination using in vitro co-culture methods, as these systems make it hard to uncouple indirect (neuronal) from direct (oligodendroglial) effects when axonal-oligodendroglial interactions are manipulated.
It is well established that large diameter axons tend to be myelinated while small diameter axons remain unmyelinated6
. This correlative observation combined with the finding that increasing axon diameter stimulates myelination of normally unmyelinated axons7
suggests that axonal fiber diameter may play an important role in regulating myelination. However, demonstrating causation or sufficiency in the contribution of axonal fiber diameter on myelination remains elusive, especially as the presence of molecular cues persist continuously along axons. Previous co-culture setups used to study myelination do not allow discriminating between diameter-dependent molecular signaling mechanisms and the biophysical property of fiber diameter itself. Therefore, uncoupling axon diameter from molecular cues expressed on the surface of axons is currently unachievable as the presence and degree of molecular signaling may be directly linked to axon caliber. In the peripheral nervous system (PNS), for example, production of neuregulin 1 type III (NRG) above a certain threshold by the axons dictates initiation of myelination by Schwann cells and NRG levels are thought to be dependent on axon diameter, providing a mechanism by which axon diameter controls the initiation of myelination. On the other hand, CNS oligodendrocytes, do not seem to respond to NRG1 levels to initiate axonal wrapping and the role of axon caliber on triggering myelination in the CNS remains unclear. Evaluating the causal relationship between axon diameter and oligodendrocyte myelination is complicated further, by the fact that axon diameters can change in an organism over time. Myelination promotes radial growth of axons during development8–10
, and the longitudinal expansion of myelin internodes associated with the age-related growth of vertebrates decreases the overall diameter of the axon11
. Effectively uncoupling the role of fiber diameter from an axonal signal necessitates manipulation of one without the other.
Recent efforts to investigate the need for axonal signals for the initiation of oligodendrocyte myelination reveal that initiation of myelination does not depend on axonal signals and may in fact be due to a permissive axonal environment, as oligodendrocytes myelinate paraformaldehyde-fixed axons12
. To further investigate this phenomenon and to overcome previously mentioned obstacles we have developed an alternative culture system to study myelination-processes and use it to examine the biophysical role of fiber diameter on the initiation of myelination in the absence of molecular cues. To this end, we have utilized electron-spinning technology to generate polystyrene nanofibers as an artificial scaffold for oligodendrocyte myelination13, 14
. Previous studies utilizing either glass microfibers15
and/or Vicryl nanofibers16
obtained promising results with oligodendrocytes ensheathing and wrapping the fibers, however, the influence of fiber diameter was not examined. By engineering nanofibers with varying diameters (0.2 to 4.0 um), we demonstrate that fiber diameter is sufficient for initiating concentric wrapping by rat primary oligodendrocyte cultures. Fibers were examined by light and electron microscopy and the minimum fiber diameter threshold was quantified and determined to be 0.4 um. Oligodendrocytes show a preference for wrapping and ensheathing fibers of diameters larger than 0.5 μm, with a 5-fold increase in the frequency of myelin-like segments formed by oligodendrocytes in these fibers. Further studies show that both OPCs and mature oligodendrocytes exhibit similar sensitivities to the biophysical properties of fiber diameter and that OPCs have the capacity to ensheath fibers prior to the expression of differentiation markers such as myelin basic protein (MBP).
The use of nanofibers in this reduced culture system allows unique studies related to myelination and has revealed the causal nature and sufficiency of fiber diameter on oligodendrocyte wrapping. This culture setup provides new opportunities to uncouple the axonal and glial contribution on myelination and should impart valuable insight into the identification of potential therapeutic strategies for remyelination.