Quantitative Automated Mircroscopy (QuAM) elucidates growth factor specific signaling in pain sensitization

Pain is a health problem with the need of new therapeutics. Nevertheless, knowledge of pain sensitization underlying intracellular signaling mechanism is very limited, as appropriate methods to study them on single nociceptor level are missing. Therefore, we established a quantitative automated microscopy (QuAM) to investigate intracellular signaling in single nociceptive neurons derived from rat dorsal root ganglia (DRG). We showed operability of QuAM by detection of DRG-subgroup specific changes of Erk1/2 activation levels in response to stimulation with the pain sensitizing growth factors NGF and GDNF. Thereby, we classified heterogeneous DRG-neurons by a new pain relevant parameter, the signaling status. We quantified the NGF responsive subgroup and characterized its size properties. We found medium sized neurons stronger responsive to NGF than small sized neurons. Next, we used the QuAM assay to analyze functionality of the growth factor EGF in DRG-neurons. So far EGF is described to be central for regeneration of epithelium, but not in the context of pain. Surprisingly, we could not detect an EGF induced Erk1/2 phosphorylation, although the EGF receptor was expressed in DRG-neurons. The cellular results could be correlated with results obtained in pain behavior experiments. While NGF led to mechanical pain sensitization, EGF did not. Nevertheless, EGF had a function in pain. EGF could block PGE₂ mediated hyperalgesia. Thus, EGF is a new modulator of pain sensitization. We even tested further growth factors (FGF, IGF, PDGF and VEGF) for their potential to induce Erk1/2 phosphorylation. We could show that FGF induced pain sensitization in dependence of Erk1/2 activation. This indicates that Erk1/2 activation might be a key event in the process of growth factor induced pain sensitization.