Timeline of the neuroprotective treatment and MPTP neurotoxin exposure in zebrafish larvae. (A) MPTP treatment: From the time of hatching until 4 days post-fertilization (dpf), the larvae were maintained in E3 Embryo medium. Subsequently, two doses of MPTP were administered: one dose at 4 dpf and another dose at 5 dpf. (B) Neuroprotective treatments (NPTs): In this group, neuroprotective doses (NPTs) were administered daily at 1 dpf, 2 dpf, and 3 dpf. At 4 dpf, the larvae received the first dose of MPTP, followed by the second dose at 5 dpf. At 6 dpf, the larvae were collected for further analysis.

Zebrafish larvae survival following exposure to MPTP with pre-treatment with (A) ascorbic acid (B) vanillic acid, and (C) ferulic acid at various concentrations to determine the dose to be used in further experiments. Neuroprotection treatments were conducted from 1 dpf to 3 dpf. At 4 dpf, embryos were exposed to 0.25 mM of MPTP concentrations and lethality was quantified every 24 hpt until the zebrafish reached 6 dpf.

Effects of neuroprotective compounds on th1 and dat gene expression. Relative gene expression of (A) dat and (B) th1 at analyzed between control, MPTP, ascorbic acid, vanillic acid, and ferulic acid (n = 3 pools of 20 larvae). One-way ANOVA with Tukey’s multiple comparisons test. ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001).

Effects of natural phenolic compounds on mRNA level of genes related to mitochondrial fission. Relative gene expression of (A) pink1, (B) parkin, and (C) fis1 analyzed between control, MPTP, ascorbic acid, vanillic acid, and ferulic acid (n = 3 pools of 20 larvae). One-way ANOVA with Tukey’s multiple comparisons test. ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001).

Effects of natural phenolics compounds on mRNA level of genes related to mitochondrial fusion. Relative gene expression of (A), opa1 and (B) mfn1 analyzed between control, MPTP, ascorbic acid, vanillic acid, and ferulic acid (n = 3 pools of 20 larvae). One-way ANOVA with Tukey’s multiple comparisons test. * (p < 0.05).

Effects of neuroprotective compounds on mRNA level p53 gene (related to apoptosis). Relative gene expression of p53 analyzed between control, MPTP, ascorbic acid, vanillic acid, and ferulic acid (n = 3 pools of 20 larvae). One-way ANOVA with Tukey’s multiple comparisons test. *** (p < 0.001), **** (p < 0.0001).

Effects of MPTP, ascorbic acid, vanillic acid, and ferulic acid on three DAnergic clusters located in the vDC of 6 dpf Tg(dat:eGFP) zebrafish larvae. (A) Schematic representation of eGFP+ neuronal clusters 8 (yellow), 12 (green), and 13 (blue) within the vDC, captured from a dorsal view oriented with the anterior facing the left. (B–G) eGFP+ cells within the vDC (red boxes) through confocal fluorescence imaging following exposure to 0.25 mM MPTP, 50 μM ascorbic acid, 250 μM vanillic acid, and 100 μM ferulic acid (n = 10–15). All images were acquired using a maximum projection of 2 μm z-stacks, scale bar = 100 μm.

Quantification of DAnergic neurons within the vDC of zebrafish larvae. Developing Tg(dat:eGFP) larvae were exposed to (A) 0.25 mM MPTP, (B) 50 μM ascorbic acid, (C) 250 μM vanillic acid, and (D) 100 μM ferulic acid (n = 10–15) and live imaged using a confocal microscope to capture vDC DAnergic clusters 8, 12, and 13. (A): Two-way ANOVA with Sidak’s multiple comparisons test. (B–D): Two-way ANOVA with Tukey’s multiple comparison. **** (p < 0.0001).

Restorative effects of ascorbic acid, vanillic acid, and ferulic acid on behavior. The swimming activity of 6 dpf larvae (n = 20) in NTC, DMSO, MPTP, ascorbic acid, vanillic acid, and ferulic acid solutions was assessed for (A) distance, (B) average velocity, and (C) inactivity duration. Bars represent the mean ± the SEM. One-way ANOVA with Tukey’s multiple comparisons test. *** (p < 0.001), **** (p < 0.0001).