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)

Abstract

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.

Keywords: Mechanical injury; NSC-34 cells; Nerve regeneration; NeuroAiD II (MLC901); Neurodegeneration; PI3K-AKT pathway.

Fig. 1

a Morphological changes in NSC-34 differentiation study at day 0 and day 7 with Media P, Media I (1 µM RA), Media II (10 µM RA), and Media III (30 µM PGE2). b Neurite outgrowth length (µm) for Media P, Media I (1 µM RA), Media II (10 µM RA), and Media III (30 µM PGE2) up to day 7 of differentiation; the Media II and Media III at day 5 and 7 showed statistically significantly longer neurite outgrowth (p < 0.05*) and (p < 0.01**) compared to Media P and Media I, n = 6

Fig. 2

a Fluorescence images of differentiated NSC 34 cells using Tubulin beta-III (green fluorescence), for the differentiated cells in media P, I, II & III, and b mean fluorescence intensity with Media P, Media I (1 µM RA), Media II (10 µM of RA), and Media III (30 µM PGE2) at day 7, calculated by ImageJ. Cells were fixed in 4% PFA before being stained with the antibody Tubulin beta-III (green fluorescence) and DAPI (blue fluorescence). Media II and III showed significantly higher green fluorescence (p < 0.05*, and p < 0.01**) indicating longer neurite outgrowth compared to Media P and I. The cells were observed under 10 × and 20 × magnifications. The scale bar represents 100 µm and 500 µm, n = 6

Fig. 3

MTT neurotoxicity study of differentiated NSC-34 cells after 24 h and 48 h treatment with MLC901. The result showed that % viability of differentiated NSC-34 cells decreased after conc. 1000 µg/mL, with estimated IC50 value for 24 h was 1178 µg/mL, and for 48 h was 1251 µg/mL, the bars represent the percentage of viable cells; error bars represent standard deviation and line represent the IC50 value for 24 and 48 h, n = 6

Fig. 4

a Time-lapse imaging with MLC901 treatment showing neurite outgrowth of NSC-34 cells at day 0 with injury and day 3 with MLC901 treatment. b The neurite outgrowth for different concentrations of MLC901 at day 0 and day 3. The 1200 µg/mL concentration showed the highest neurite outgrowth and mean cell bodies (p < 0.05) at the injured area compared to UT cells and other treatment concentrations. Morphological changes were observed up to day 3, at 10 × and 20 × magnification with scale bars 100 µm and 500 µm; the mean of 20 cells/ treatment concentration /day was quantified, n = 6

Fig. 5

Neurite outgrowth after time-lapse imaging at day 0, day 1, day 2, and day 3 for UT cells and MLC901, T cells having 800 µg/mL, 1000 µg/mL, 1200 µg/mL, and 1400 µg/mL concentrations, quantified by ImageJ software. All T group cells showed longer neurite (p > 0.05 *) outgrowth on day 3 compared to day 0; however, 1000 and 1200 µg/mL showed significant (p < 0.01**) longer neurite outgrowth compared to UT cells and 1200 µg/mL showed outgrowth (p < 0.001***) compared to UT cells at day 3, n = 6

Fig. 6

a Fluorescence staining using nuclear stain DAPI (blue fluorescence) and Tubulin beta-III (green fluorescence) antibody expression at the scratch area, after mechanical scratch injury model, and b MFI at day 3 with different MLC901 treatments of 800 µg/mL, 1000 µg/mL, 1200 µg/mL, and 1400 µg/mL. The center area indicated the mechanical scratch injury lesion, and boxes showed neurite outgrowth at the injury area, magnification 10 × , scale bar 100 µm. The 1200 µg/mL concentration of MLC901 showed significantly (p < 0.05) higher neurite outgrowth with higher MFI at the injured area compared to 800 µg/mL and 1400 µg/mL, n = 6

Fig. 7

The PI3K/AKT signalling pathways involved in neuron and axon regeneration, indicating the protein markers regulating the different pathway associated with nerve regeneration

Fig. 8

a The expression of various protein markers (p-AKT, p-GSK3β, GAP43, ATF-3, p53, elF2β) for PI3K/AKT pathway, and housekeeping protein (β-Tubulin) for UT, 1000 and 1200 µg/mL of MLC901-treated cells after mechanical scratch injury in NSC-34 cells; b relative expression of p-AKT, p-GSK3β, GAP43, ATF-3, p53, and elF2β normalized with β-Tubulin using ImageJ, and data were expressed as mean ± SD with significance value (*p < 0.05) and (**p < 0.01), respectively, n = 6