Assessment of Neuroprotective Effectsof Neurotropin in Cultured Rat DorsalRoot Ganglion Neurons Using High-Throughput Imaging

Background: Neurotropin, a non-protein extract derived from inflamed rabbit skin following inoculation with vaccinia virus, is an analgesic agent. The mechanism of action of neurotropin has been partially clarified. No experimental study, however, has been undertaken to assess oxaliplatin-induced neurotoxicity using quantitative in vitro high-throughput image analysis. In the present study, to elucidate the action of neurotropin on dorsal root ganglion (DRG) neurons, we explored the antioxidative and anti-inflammatory activity of neurotropin. We assessed the viability and morphological changes in cultured rat DRG neurons treated with oxaliplatin and neurotropin. Methods: We plated dissociated DRG neurons from female Sprague-Dawley rats (100-120 g) into 96-well plates and added oxaliplatin and/or neurotropin. The cells were examined with a fluorescence microscope, ImageXpress. The images were analyzed with high content image processing software, MetaXpress. Cells treated with neurotropin and oxaliplatin were lysed in Pierce RIPA Buffer and subjected to western blot analysis. Results: Significant increases in neurite length, neurite area, cell body area, and neurite branching were observed in the presence of neurotropin in DRG neurons treated with oxaliplatin. When the highest concentration of neurotropin, 40 neurotropin units (NU) mNU/mL, was applied, a marked effect was seen. Neurotropin did not affect the phosphorylation of c-Jun N-terminal kinase, but suppressed the phosphorylation of p38. This finding suggests that neurotropin reversed oxaliplatin-induced apoptosis via suppression of the p38 mitogenactivated protein kinase signaling pathway. Conclusion: The results of this in vitro study suggest a possible role for neurotropin as a useful neuroprotective and anti-apoptotic agent.