Cholera toxin subunit B (CTB) and Fluorogold(FG) are two widely utilized retrograde tracers to assess the number and function of retinal ganglion cells (RGCs). However, the relative advantages and disadvantages of these tracers remain unclear, which may lead to their inappropriate application. In this study, we compared these tracers by separately injecting the tracer into the superior Colliculi (SC) in rats, one or 2 weeks later, the rats were sacrificed, and their retinas, brains, and optic nerves were collected. From the first to second week, FG displayed a greater number of labeled RGCs and a larger diffusion area in the SC than CTB; The number of CTB labeled RGCs and the diffusion area of CTB in the SC increased significantly, but there was no distinction between FG; Furthermore, CTB exhibited more labeled RGC neurites and longer neurites than FG, but no difference was evident between the same trace; The optic nerves labeled using CTB were much clearer than those labeled using FG. In conclusion, both CTB and FG can be used for the retrograde labeling of RGCs in rats at 1 or 2 weeks. FG achieves retrograde labeling of a greater number of RGCs than CTB, whereas CTB better delineates the morphology of RGCs. Furthermore, CTB seems more suitable for retrograde labeling of some small, non-image forming nuclei in the brain to which certain RGC subtypes project their axons.
Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases characterized by the loss of photoreceptor cells through apoptosis. N-methyl-N-nitrosourea (MNU) is an alkylating toxicant that induces photoreceptor cell death resembling hereditary RP. This study aimed to investigate the role of nuclear factor κB (NF-κB) in MNU-induced photoreceptor degeneration. Adult rats received a single intraperitoneal injection of MNU (60 mg/kg bodyweight). Hematoxylin and eosin staining demonstrated progressive outer nuclear layer (ONL) loss after MNU treatment. Transmission electron microscopy revealed nuclear pyknosis, chromatin margination in the photoreceptors, increased secondary lysosomes, and lobulated retinal-pigmented epithelial cells in MNU-treated rats. Numerous photoreceptor cells in the ONL showed positive TUNEL staining and apoptosis rate peaked at 24 hours. Enhanced depth imaging spectral-domain optical coherence tomography showed ONL thinning and decreased choroid thickness. Electroretinograms showed decreased A wave amplitude that predominated in scotopic conditions. Western blot analysis showed that nuclear IκBα level increased, whereas nuclear NF-κB p65 decreased significantly in the retinas of MNU-treated rats. These findings indicate that MNU leads to selective photoreceptor degradation, and this is associated with the inhibition of NF-κB activation.
Retinal degenerative diseases ultimately result into irreversible photoreceptor death or loss. At present, the most promising treatment for these diseases is cell replacement therapy. Müller glia are the major glia in the retina, displaying cardinal features of retinal progenitor cells, and can be candidate of seed cells for retinal degenerative diseases. Here, mouse retinal Müller glia dissociated and cultured in vitro amplified and were dedifferentiated into Müller glia-derived progenitors (MGDPs), demonstrating expression of stem/progenitor cell markers Nestin, Sox2 and self-renewal capacity. MicroRNAs (miRNAs) play unique roles in the retinogenesis, so we hypothesized miRNAs would contribute to photoreceptor lineage commitment of MGDPs. By TargetScan, Miranda, and Pictar bioinformatics, gain/loss-of-function models, dual luciferase assay, we identified and validated that miR-28 targeted the photoreceptor-specific CRX transcription factor. Anti-miR-28 could induce MGDPs to differentiate into neurons strongly expressing CRX and Rhodopsin, while miR-28 mimic suppressed CRX and Rhodopsin expression. Knockdown of CRX by siRNA blocked the expression of CRX and Rhodospin upregulated by anti-miR-28, indicating that anti-miR-28 potentially induced photoreceptor commitment of MGDPs by targeting CRX, but more experiments are necessary to confirm their role in differentiation.
Retinal Müller glial cells have the potential of neurogenic retinal progenitor cells, and could reprogram into retinal‐specific cell types such as photoreceptor cells. How to promote the differentiation of Müller cells into photoreceptor cells represents a promising therapy strategy for retinal degeneration diseases. This study aimed to enhance the transdifferentiation of rat Müller cells‐derived retinal stem cells (MC‐RSCs) into photoreceptor‐like cells and explore the signalling mechanism. We dedifferentiated rat Müller cells into MC‐RSCs which were infected with Otx2 overexpression lentivirus or control. The positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus was significantly higher compared to control. Furthermore, pre‐treatment with Crx siRNA, Nrl siRNA, or GSK‐3 inhibitor SB‐216763 reduced the positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus. Finally, Otx2 induced photoreceptor precursor cells were injected into subretinal space of N‐methyl‐N‐nitrosourea induced rat model of retinal degeneration and partially recovered retinal degeneration in the rats. In conclusion, Otx2 enhances transdifferentiation of MC‐RSCs into photoreceptor‐like cells and this is associated with the inhibition of Wnt signalling. Otx2 is a potential target for gene therapy of retinal degenerative diseases.
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