Figure 4.

miR-124-3p sEVs do not increase the number of SVZ-derived newly born neurons in the lesioned striatum of 6-OHDA-treated mice.

(A) Design and timeline of the animal experimental procedure. Male C57Bl/6 mice were subjected to unilateral injection of 6-OHDA into the right striatum followed by an i.c.v. injection with miR-124-3p sEVs, SCR sEVs, sEVs, or saline. Then, mice received BrdU injections (every 12 h) during the following 3 days after surgery. Behavioral tests were performed on day -1 and on weeks 1, 2, and 4 after stereotaxic injections. After 4 weeks, mice brains were collected for histological processing. (B) Representative confocal photomicrographs of (B.1) SVZ, striatal and (B.2, B.3) midbrain parenchyma 24 h after i.c.v. injection of non-loaded sEVs labeled with PKH67 (sEV-PKH67; shown in green). White arrows show sEV-PKH67 close to the cell nuclei. TH labeling (red) depicts DA neurons in the SN and nuclei are counterstained with Hoechst (blue). Scale bar: 20 μm. (C) Representative confocal digital images of BrdU (green), NeuN (red), and Hoechst (blue) staining observed in the striatum of saline-treated mice, 6-OHDA-treated mice, or miR-124-3p sEV-treated 6-OHDA mice. Scale bar: 20 μm; white arrows highlight NeuN⁺/BrdU⁺ cells. (D) The bar graph depicts the total number of NeuN⁺BrdU⁺ cells found in the striatum of mice 4 weeks after treatments. Data are expressed as mean ± SEM, n = 3 mice. ∗∗p < 0.01 and ∗p < 0.05 versus saline-treated mice (control); ^#p < 0.05 versus miR-124-3p sEV-treated 6-OHDA-lesioned mice group using one-way ANOVA followed by Tukey’s multiple comparison test. 6-OHDA, 6-hydroxydopamine; SCR sEV, scramble-loaded small extracellular vesicles; sEV, non-loaded small extracellular vesicles; miR-124-3p sEV, miR-124-3p-loaded small extracellular vesicles; i.c.v., intracerebroventricular; DA, dopaminergic; BrdU, 5-bromo-2′-deoxyuridine; SN, substantia nigra; TH, tyrosine hydroxylase; SVZ, subventricular zone.