Abstract
It is often critical to improve the limited regenerative capacity of the peripheral nerves and direct neural growth towards specific targets, such as surgically implanted bioengineered constructs. One approach to accomplish this goal is to use extrinsic neurotrophic factors. The candidate factors first need to be identified and characterized in in vitro tests for their ability to direct the neurite growth. Here, we present a simple guidance assay that allows to assess the chemotactic effect of signaling molecules on the growth of neuronal processes from dorsal root ganglia (DRG) using only standard tissue culture materials. We used this technique to quantitatively determine the combined and individual effects of the ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth. We demonstrated that these two neurotrophic factors, when applied in a 1:1 combination, but not individually, induced directed growth of neuronal processes towards the source of the gradient. This chemotactic effect persists without significant changes over a wide (10-fold) concentration range. Moreover, we demonstrated that other, more general growth parameters that do not evaluate growth in a specific direction (such as, neurite length and trajectory) were differentially affected by the concentration of the CNTF/GNDF mixture. Furthermore, GDNF, when applied individually, did not have any chemotactic effect, but caused significant neurite elongation and an increase in the number of neurites per ganglion.
Highlights
There is a clear need to design efficient ways to improve the limited intrinsic regenerative capacities of the peripheral nervous system to repair large injuries and facilitate adequate innervation of surgically implanted bioengineered tissue constructs
We developed a simple guidance assay that allows to assess the effect of neurotrophic factors on neurite outgrowth from dorsal root ganglia (DRGs) using only standard tissue culture materials
We developed a simple gradient plate assay that can be used to assess the capacity of candidate chemoattractants to induce directed growth of neurites from DRG explants
Summary
There is a clear need to design efficient ways to improve the limited intrinsic regenerative capacities of the peripheral nervous system to repair large injuries and facilitate adequate innervation of surgically implanted bioengineered tissue constructs. One of the major focuses in the field is to enhance the regrowth of peripheral nerves through the application of extrinsic neurotrophic factors [1]. Directed neurite grow in response to CNTF and GDNF
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