Abstract
Neurotrophic factors play a key role in the development, differentiation, and survival of neurons and nerve regeneration. In the present study, we evaluated the effect of certain neurotrophic factors (NGF, BDNF, and GDNF) on axon growth and migration of Nestin-green fluorescent protein (GFP)-positive cells using a 3D model of dorsal root ganglion (DRG) explant culture in Matrigel. Our method generally represents a convenient model for assessing the effects of soluble factors and therapeutic agents on axon growth and nerve regeneration in R&D studies. By analyzing the DRG explants in ex vivo culture for 21 days, one can evaluate the parameters of neurite outgrowth and the rate of cell migration from the DRG explants into the Matrigel. For the current study, we used Nestin-GFP-expressing mice in which neural precursors express Nestin and the green fluorescent protein (GFP) under the same promoter. We revealed that GDNF significantly (two fold) stimulated axon outgrowth (p < 0.05), but not BDNF or NGF. It is well-known that axon growth can be stimulated by activated glial cells that fulfill a trophic function for regenerating nerves. For this reason, we evaluated the number of Nestin-GFP-positive cells that migrated from the DRG into the Matrigel in our 3D ex vivo explant model. We found that NGF and GDNF, but not BDNF, stimulated the migration of Nestin-GFP cells compared to the control (p < 0.05). On the basis of the aforementioned finding, we concluded that GDNF had the greatest stimulating potential for axon regeneration, as it stimulated not only the axon outgrowth, but also glial cell migration. Although NGF significantly stimulated glial cell migration, its effect on axon growth was insufficient for axon regeneration.
Highlights
Peripheral nerve injuries remain one of the most significant causes of disability and have a marked impact on patients’ everyday lives [1]
Length assessment of the outgrowing axons and measurement of glial cell migration without attached dorsal root ganglion (DRG) can lead to misinterpretation of the results
Our obtained results are in accordance with the previously published data because only glial cells migrated from DRGs in 2D cultures
Summary
Peripheral nerve injuries remain one of the most significant causes of disability and have a marked impact on patients’ everyday lives [1]. Cell survival is usually low, and neural cell cultures from DRG can be maintained for only 5–7 days, provided that soluble factors that increase survival are added [14] This is one of the complications in result interpretation when analyzing the effects of various agents on cell migration and neurite formation. Explants, isolated and cultured ex vivo in 3D conditions, present a convenient model because they retain tissue-specific characteristics and long-term interactions between various cell types (neurons and glial cells), ECM, and biologically active molecules. This method can be used in the neurological field and for cardiology, cancer biology, and other fields [15]. The time required for visualization using confocal microscopy depends on the number of experimental points, and usually takes up to 3 h for high-resolution images
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