Effect of EPO and NGF co-transfection on peripheral nerve injury in vitro
Secil Demir, M.Sc, National University of Ireland, Galway, Galway, Ireland and Abhay Pandit, PhD, Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland
Following denervating injury of the sciatic nerve, several proteins are modulated in the distal region but not in the proximal injured nerve. Using next-generation proteomics analyses and biomaterial stimulated regenerative response in rat critical gap (15 mm) injury model, we have identified Erythropoietin (EPO) protein to be associated in the activation of PI3K/Akt pathway. Therefore, it is hypothesized that overexpression of EPO in conjunction with Nerve Growth Factor (NGF) will act as upstream regulator of the PI3K/Akt pathway in peripheral nerve injury model inducing tissue regeneration. To reveal the regenerative effect of EPO and NGF proteins on sciatic nerve injury, functional and biological response of neurons and schwann cells were assessed in vitro.
Materials and Methods:
Protein expression of EPO and NGF non-viral vector (Sino Biological) transfected neurons (E14 rat DRGs) and primary schwann cells were analysed using Operetta High Content Analysis System (PerkinElmer). Myelination of schwann cells as functional response to EPO/NGF modulated expression was assessed by immunocytochemistry, western blotting and qPCR. Also, functional response of neurons to EPO/NGF transfection was evaluated using live calcium imaging, microelectrode arrays and qPCR techniques.
Immunocytochemistry results showed that DRG neurons and schwann cells express EPO protein in their cytoplasm. The highest axonal elongation in neurons (Figure 1 A,B) and myelination rate in schwann cells (Figure 1 E) were observed if EPO/NGF ratio is increased. In vitro electrophysiology and calcium imaging results showed that the DRG neurons are more active in the culture if EPO/NGF ratio reduced (Figure 1 C,D). qPCR results showed that different ratio of EPO and NGF transfection has differential effect on expression of Akt and mTOR genes (Figure 1 F,G,H).
Real-time PCR results confirmed that expression of EPO and NGF is related to the PI3K/Akt pathway. Functional and biological responses of neurons and schwann cells showed that EPO and NGF genes are working synergistically in cells. Effect of EPO and NGF gene overexpression in peripheral nerve injury target cells will give us insight into peripheral nerve regeneration process on molecular level.
Science Foundation Ireland (SFI) (Grant no: 13/RC/2073) for providing financial support to this project.
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