fig5

Figure 5. Internalization and phagocytic enhancement of RPE cells in RCS rats by OEC-sEVs. (A) Representative images showing the internalization of PKH26-labeled OEC-sEVs (red) in the RPE layer (RPE65, green) at 2 and 4 weeks post-injection; (A1, A4) Images of PKH26-labeled OEC-sEVs (red) and fluorescent staining of RPE65 (green) at 2 and 4 weeks post-injection. Scale bar = 50 μm; (A2, A3, A5, A6) 3D surface reconstruction images of OEC-sEVs and RPE cells rotated 0° (A2, A5) and 45° (A3, A6) in the horizontal plane. Scale bar = 5 μm; (B) Co-localization of PKH26-labeled OEC-sEVs (red) with RPE cells (RPE65, green) in the RCS-PBS and RCS-sEV groups at 2 and 4 weeks post-injection. Scale bar = 50 μm (original images); scale bar = 20 μm (enlarged images); (C) Quantitative analysis of RPE65 fluorescence intensity in the RCS-PBS and RCS-sEV groups at 2 and 4 weeks post-injection. The dashed boxes denote the regions selected for magnification in the subsequent panels. The arrows indicate the presence of PKH26-labeled OEC-sEVs within the RPE layer. n = 4 biologically independent rats per group. Data are represented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, no statistical difference. One-way ANOVA, followed by Tukey’s multiple comparisons test, was conducted for multiple comparisons. ANOVA: Analysis of variance; DAPI: 4’,6-diamidino-2-phenylindole; INL: inner nuclear layer; OEC-sEVs: olfactory ensheathing cell-derived small extracellular vesicles; ONL: outer nuclear layer; PBS: phosphate-buffered saline; RCS: Royal College of Surgeons; RCS-PBS: Royal College of Surgeons rats treated with phosphate-buffered saline; RCS-sEV: Royal College of Surgeons rats treated with small extracellular vesicles; RPE: retinal pigment epithelium; RPE65: retinal pigment epithelium-specific 65 kDa protein; SD: standard deviation; SRS: subretinal space.