Mechanical scratch injury on differentiated motor neuron of NSC-34 cells as an in vitro model for evaluation of neuroregeneration potential of NeuroAiD II (MLC901)

Spinal cord regeneration is considered an ultimate achievement in the field of neuroscience. In vitro, neural stem cell (NSC-34) motor neuron-like cell cultures are powerful tools to study specific molecular pathways involved in neurogenesis. We aimed to demonstrate the usefulness of the in vitro injury model using the mechanical scratch method and to evaluate the effect of MLC901 in injured neuronal cells. In this study, retinoic acid (RA) (1 µM and 10 µM) and 30 µM prostaglandin E2 (PGE2) induction was used to facilitate NSC-34 differentiation into motor neurons (MN). The MN was scratched and treated with different concentrations of NeuroAiD II (MLC901). The time-lapse assay, the AKT/P13K pathway analysis, and Immunocytochemistry (ICC) were performed. The results showed that NSC-34 cell lines were differentiated into mature MN using RA (7 days) and PGE2 (5 days). The mechanical scratch injury model damaged the MN at the scratch area. The time-lapse assay showed treated cells (T) at conc. In total, 1000 and 1200 µg/mL for MLC 901 showed significantly higher neurite outgrowth as compared to untreated cells (UT). The AKT/PI3K pathway analysis showed higher expression of regenerative markers (p-AKT, p-GSK3β, ATF-3, GAP43, p53, and elF2β) at concentrations of 1200 µg/mL than UT. The study showed that the in vitro injury model using mechanical scratch is a useful tool to induce neurodegeneration and may be used to evaluate regenerative treatment options.