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).

BACKGROUND: 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. PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s44164-024-00070-7.