Retinal degeneration (RD) is one of the most common causes of visual impairment and blindness and is characterized by progressive degeneration of photoreceptors. Transplantation of neural stem/progenitor cells (NPCs) is a promising treatment for RD, although the mechanisms underlying the efficacy remain unclear. Accumulated evidence supports the notion that paracrine effects of transplanted stem cells is likely the major approach to rescuing early degeneration, rather than cell replacement. NPC-derived exosomes (NPC-exos), a type of extracellular vesicles (EVs) released from NPCs, are thought to carry functional molecules to recipient cells and play therapeutic roles. In present study, we found that grafted human NPCs (hNPCs) secreted EVs and exosomes in the subretinal space (SRS) of RCS rats, an RD model. And direct administration of mouse neural progenitor cell-derived exosomes (mNPC-exos) delayed photoreceptor degeneration, preserved visual function, prevented thinning of the outer nuclear layer (ONL), and decreased apoptosis of photoreceptors in RCS rats. Mechanistically, mNPC-exos were specifically internalized by retinal microglia and suppressed their activation in vitro and in vivo. RNA sequencing and miRNA profiling revealed a set of 17 miRNAs contained in mNPC-exos that markedly inhibited inflammatory signal pathways by targeting TNF-α, IL-1β, and COX-2 in activated microglia. The exosomes derived from hNPC (hNPC-exos) contained similar miRNAs to mNPC-exos that inhibited microglial activation. We demonstrated that NPC-exos markedly suppressed microglial activation to protect photoreceptors from apoptosis, suggesting that NPC-exos and their contents may be the mechanism of stem cell therapy for treating RD.

Resident microglia are the main immune cells in the retina and play a key role in the pathogenesis of retinitis pigmentosa (RP). Many previous studies on the roles of microglia mainly focused on the neurotoxicity or neuroprotection of photoreceptors, while their contributions to synaptic remodeling of neuronal circuits in the retina of early RP remained unclarified. In the present study, we used Royal College of Surgeons (RCS) rats, a classic RP model characterized by progressive microglia activation and synapse loss, to investigate the constitutive effects of microglia on the synaptic lesions and ectopic neuritogenesis. Rod degeneration resulted in synapse disruption and loss in the outer plexiform layer (OPL) at the early stage of RP. Coincidentally, the resident microglia in the OPL increased phagocytosis and mainly engaged in phagocytic engulfment of postsynaptic mGluR6 of rod bipolar cells (RBCs). Complement pathway might be involved in clearance of postsynaptic elements of RBCs by microglia. We pharmacologically deleted microglia using a CSF1 receptor (CSF1R) inhibitor to confirm this finding, and found that it caused the accumulation of postsynaptic mGluR6 levels and increased the number and length of ectopic dendrites in the RBCs. Interestingly, the numbers of presynaptic sites expressing CtBP2 and colocalized puncta in the OPL of RCS rats were not affected by microglia elimination. However, sustained microglial depletion led to progressive functional deterioration in the retinal responses to light in RCS rats. Based on our results, microglia mediated the remodeling of RBCs by phagocytosing postsynaptic materials and inhibiting ectopic neuritogenesis, contributing to delay the deterioration of vision at the early stage of RP.

Retinitis pigmentosa (RP) is a progressive hereditary retinal degenerative disease in which photoreceptor cells undergo degeneration and apoptosis, eventually resulting in irreversible loss of visual function. Currently, no effective treatment exists for this disease. Neuroprotection and inflammation suppression have been reported to delay the development of RP. Metformin is a well-tested drug used to treat type 2 diabetes, and it has been reported to exert beneficial effects in neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease. In the present study, we used immunofluorescence staining, electroretinogram (ERG) recordings and RNA-Seq to explore the effects of metformin on photoreceptor degeneration and its mechanism in rd1 mice. We found that metformin significantly reduced apoptosis in photoreceptors and delayed the degeneration of photoreceptors and rod bipolar cells in rd1 mice, thus markedly improving the visual function of rd1 mice at P14, P18, and P22 when tested with a light/dark transition test and ERG. Microglial activation in the outer nuclear layer (ONL) of the retina of rd1 mice was significantly suppressed by metformin. RNA-Seq showed that metformin markedly downregulated inflammatory genes and upregulated the expression of crystallin proteins, which have been demonstrated to be important neuroprotective molecules in the retina, revealing the therapeutic potential of metformin for RP treatment. αA-crystallin proteins were further confirmed to be involved in the neuroprotective effects of metformin in a Ca 2+ ionophore-damaged 661W photoreceptor-like cell line. These data suggest that metformin exerts a protective effect in rd1 mice via both immunoregulatory and new neuroprotective mechanisms.

Retinitis pigmentosa initially presents as night blindness owing to defects in rods, and the secondary degeneration of cones ultimately leads to blindness. Previous studies have identified active roles of microglia in the pathogenesis of photoreceptor degeneration in RP. However, the contribution of microglia to photoreceptor degeneration remains controversial, partly due to limited knowledge of microglial phenotypes during RP. Rationale: In this study, we investigated the pathways of microglial activation and its contribution to photoreceptor degeneration in RP. Methods: A classic RP model, Royal College of Surgeons rat, was used to explore the process of microglial activation during the development of RP. An inhibitor of colony-stimulating factor 1 receptor (PLX3397) was fed to RCS rats for sustained ablation of microglia. Immunohistochemistry, flow cytometry, RT-qPCR, electroretinography and RNA-Seq were used to investigate the mechanisms by which activated microglia influenced photoreceptor degeneration. Results: Microglia were gradually activated to disease-associated microglia in the photoreceptor layers of RCS rats. Sustained treatment with PLX3397 ablated most of the disease-associated microglia and aggravated photoreceptor degeneration, including the secondary degeneration of cones, by downregulating the expression of genes associated with photoreceptor function and components and exacerbating the impairment of photoreceptor cell function. Disease-associated microglial activation promoted microglia to engulf apoptotic photoreceptor cell debris and suppressed the increase of infiltrated neutrophils by increasing engulfment and inhibiting CXCL1 secretion by Müller cells, which provided a healthier microenvironment for photoreceptor survival. Conclusions: Our data highlight a key role of disease-associated microglia activation in the suppression of rod and cone degeneration, which reduces secondary damage caused by the accumulation of dead cells and infiltrated neutrophils in the degenerating retina.

Retinal oscillatory potentials (OPs) consist of a series of relatively high-frequency rhythmic wavelets, superimposed onto the ascending phase of the b-wave of the electroretinogram (ERG). However, the origin of OPs is uncertain and methods of measurement of OPs are diverse. In this study, we first isolated OPs from the rat ERG and fitted them with Gabor functions and found that the envelope of the OP contained information about maximum amplitude and time-to-peak to enable satisfactory quantification of the later OPs. And the OP/b-wave ratio should be evaluated to exclude an effect of the b-wave on the OPs. Next, we recorded OPs after intravitreal injection of 2-amino-4-phosphonobutyric acid (APB), tetrodotoxin (TTX), γ-aminobutyric acid (GABA), strychnine (STR), SR95531 (SR), isoguvacine (ISO), (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) and GABA+TPMPA. We showed that GABA and APB only removed the later OPs, when compared to control eyes. TTX delayed the peak time, and STR, SR and ISO reduced the amplitude of OPs. TPMPA delayed the peak time but increased the ratio of OPs to b-wave. Furthermore, administration of combined GABA and TPMPA caused the later OPs to increase in amplitude with time, compared with those after delivery of GABA alone. Finally, we observed that GABAc and glycine receptors contributed to a low-frequency component of the OPs, while GABAa contributed to both components. These results suggest that the early components of the OPs are mainly generated by the photoreceptors, whilst the later components are mainly regulated by GABAa, GABAc and glycine receptors.