RPE-derived exosomes rescue the photoreceptors during retina degeneration: an intraocular approach to deliver exosomes into the subretinal space

Abstract

Retinal degeneration (RD) refers to a group of blinding retinopathies leading to the progressive photoreceptor demise and vision loss. Treatments against this debilitating disease are urgently needed. Intraocular delivery of exosomes represents an innovative therapeutic strategy against RD. In this study, we aimed to determine whether the subretinal delivery of RPE-derived exosomes (RPE-Exos) can prevent the photoreceptor death in RD. RD was induced in C57BL6 mice by MNU administration. These MNU administered mice received a single subretinal injection of RPE-Exos. Two weeks later, the RPE-Exos induced effects were evaluated via functional, morphological, and behavior examinations. Subretinal delivery of RPE-Exos efficiently ameliorates the visual function impairments, and alleviated the structural damages in the retina of MNU administered mice. Moreover, RPE-Exos exert beneficial effects on the electrical response of the inner retinal circuits. Treatment with RPE-Exos suppressed the expression levels of inflammatory factors, and mitigated the oxidative damage, indicating that subretinal delivery of RPE-Exos constructed a cytoprotective microenvironment in the retina of MNU administered mice. Our data suggest that RPE-Exos have therapeutic effects against the visual impairments and photoreceptor death. These findings will enrich our knowledge of RPE-Exos, and highlight the discovery of a promising medication for RD.

Keywords: Subretinal delivery; neural degeneration; therapeutics.

Figure 1.

(A) Upper micrographs: particles derived from RPE appeared as specific spheroidal shape under electron microscopy. Bottom micrographs: the particles of scattering light were observed using the ZETASIZER Nano series-Nano-ZS instrument. When laser is projected on these samples, these white objects can refract the scattering light, indicating that the size of particles is uniform under dynamic light scattering. (B) Concentration and size distribution of the particles samples derived from RPEs by NTA. The diameters of extracted particles ranged between 40 and 100 nm. (C) Western blot assay was used to identify the biomarkers of these particles. The exosomes biomarkers CD81 and TSG101 were detected in these vesicles. (D) Flow cytometry analysis showed that surface proteins CD63 and CD81 were expressed in these vesicles (#1 and #2 represents two replicate samples derived from RPEs).

Figure 2.

(A) RD was induced by an intraperitoneal injection of MNU. The subretinal injection of RPE-Exos was performed instantly after MNU administration. Morphological analysis was performed 2 weeks following treatment. Retinal architecture of MNU administered mice was effective preserved by RPE-Exos treatment. The mean ONL thickness of RPE-Exos treated group was significantly larger compared with MNU group. (B) OCT examination of the RPE-Exos treated group also revealed significant protective effects, seen as a thicker total retina thickness compared with MNU group (ANOVA analysis followed by Bonferroni’s post hoc analysis was performed, ^#p < .01, for differences between groups; n = 8; GCL: ganglion cell layer; IPL: inner plexiform layer; OPL: outer plexiform layer; ONL: outer nuclear layer; INL: inner nuclear layer; OS: outer segments).

Figure 3.

(A) Representative ERG waveforms demonstrated better visual responsiveness in RPE-Exos treated mice compared with MNU group. (B) The a- and b-wave amplitudes were significantly larger in RPE-Exos treated group than those in the vehicle treated group. (C) Optokinetic behavioral tests also showed that the visual acuity and contrast sensitivity in the RPE-Exos treated group were larger compared with the MNU group. (D) Immunohistochemistry work was performed on these retinal sections from central retina. Extensive punctate PNA staining was detected in the retinal sections of the RPE-Exos treated group. No PNA staining was found in the retinal sections of vehicle treated group. Abundant M- and S-cone opsin staining was also found in the retinal sections of RPE-Exos treated group (ANOVA analysis followed by Bonferroni’s post hoc analysis was performed, ^#p < .01, for differences between groups; n = 8).

Figure 4.

(A) As shown in the MEA recording, the electrical response of retina was affected remarkably by MNU toxicity. However, the LFPs of the MNU + RPE-Exos group were effectively preserved. (B) Comparison analysis showed that the LFPs amplitude was significantly larger in the MNU + RPE-Exos group than that in the MNU group. In particular, these photoreceptors in peripheral region were more efficiently rescued by RPE-Exos treatment. (C) MNU administration also affected the firing activities of retinal neurons, as evidenced by an elevated firing frequency in the MNU group. Conversely, the increase of spontaneous firing frequency was relatively slighter in the MNU + RPE-Exos group (ANOVA analysis followed by Bonferroni’s post hoc analysis was performed, ^#p < .01, for differences between groups; n = 8).

Figure 5.

(A) The mRNA levels of these pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, MCP-1, reduced significantly in the RPE-Exos treated group than those the MNU group. The mRNA levels of Bax, Caspase-3, and Calpain-2 decreased significantly in MNU + RPE-Exos group than those in the MNU group, whereas the mRNA level of bcl-2 increased significantly. Analysis of retinal MDA concentration demonstrated a significant reduction in the RPE-Exo treated group than that in the MNU group, indicating that RPE-Exos can alleviate the oxidative stress in retina (ANOVA analysis followed by Bonferroni’s post hoc analysis was performed, ^#p<.01, for differences between groups; n = 8).