Retinal Degeneration In Mice Research Articles

Alleviation effect of lutein Pickering emulsion formed by casein-dextran conjugates through Maillard reaction against blue light retinal degeneration

With the increasing prevalence of electronic devices, awareness of the risks linked to blue light exposure has significantly heightened. Lutein, a powerful antioxidant, safeguards eye tissue by filtering blue light, while supplementation with docosahexaenoic acid (DHA) enhances retinal function. Adequate intake of these nutrients can help reduce the potential damage from prolonged blue light exposure. The protective effects of lutein and algal oil stabilized with Pickering emulsion were investigated using casein-dextran (CD) conjugates via Maillard reaction. Microstructural analysis revealed a three-dimensional network structure surrounding oil droplets formed by CD conjugates. With the increase of the oil phase ratio from 55 % to 80 %, the average size of Pickering emulsion droplets decreased. Pickering emulsion demonstrated higher viscoelasticity, excellent recovery, thixotropy, and good thermal stability as the oil phase ratio increased. The retention of lutein in CD-75 % Pickering emulsions showed significant improvement under various conditions. Simulated gastrointestinal digestion demonstrated that CD-75 % Pickering emulsions effectively enhanced the lutein bioaccessibility from 19.97 % to 48.99 %. In vivo experiments showed that lutein-loaded Pickering emulsion could effectively relieve blue light-induced retinal degeneration in mice. These findings suggested that Pickering emulsion can serve as a delivery system to protect lutein, offering a nutritional intervention to mitigate blue light-induced retinal degeneration.

IL-23 Priming Enhances the Neuroprotective Effects of MSC-Derived Exosomes in Treating Retinal Degeneration.

Neuroinflammation is a characteristic feature of neurodegenerative diseases. Mesenchymal stem cell-derived exosomes (MSC-exo) have shown neuroprotective effects through immunoregulation, but the therapeutic efficacy remains unsatisfactory. This study aims to enhance the neuroprotective capacity of MSC-exo through IL-23 priming for treating retinal degeneration in mice. MSC were primed with IL-23 stimulation in vitro, and subsequently, exosomes (MSC-exo and IL-23-MSC-exo) were isolated and characterized. Two retinal degenerative disease models (NaIO3-induced mice and rd10 mice) received intravitreal injections of these exosomes. The efficacy of exosomes was assessed by examining retinal structural and functional recovery. Furthermore, exosomal microRNA (miRNA) sequencing was conducted, and the effects of exosomes on the M1 and M2 microglial phenotype shift were evaluated. IL-23-primed MSC-derived exosomes (IL-23-MSC-exo) exhibited enhanced capability in protecting photoreceptor cells and retinal pigment epithelium (RPE) cells against degenerative damage and fostering the restoration of retinal neural function in both NaIO3-induced retinal degeneration mice and rd10 mice when compared with MSC-exo. The exosomal miRNA suppression via Drosha knockdown in IL-23-primed MSC would abolish the neuroprotective role of IL-23-MSC-exo, highlighting the miRNA-dependent mechanism. Bioinformatic analysis, along with further in vivo biological studies, revealed that IL-23 priming induced a set of anti-inflammatory miRNAs in MSC-exo, prompting the transition of M1 to M2 microglial polarization. IL-23 priming presents as a potential avenue for amplifying the immunomodulatory and neuroprotective effects of MSC-exo in treating retinal degeneration.

Microalga Chlorella sp. biomass containing high lutein prevents light-induced photooxidation and retinal degeneration in mice

Age-related macular degeneration (AMD) is commonly associated with retinal degeneration. Carotenoids, particularly lutein, can protect against AMD. Chlorella sp. has been widely used as a dietary supplement and contained high levels of lutein. Chlorella sp. biomass ethanol extract containing high lutein concentrations (CEE) was tested for its retinoprotective effects in retinal pigment epithelial (RPE) cells and light exposure-induced retinal degeneration in mice. The inhibitory activity of CEE on photooxidation of Bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E) was assessed in ARPE-19 cells. Retinal degeneration was induced by the exposure to 5000 lx of white LED light for 3 h. For one week, CEE (10, 25 and 50 mg/kg) and lutein (25 mg/kg) were administered orally. A retinal electroretinography (ERG) and a histological examination were conducted. Furthermore, oxidative DNA damage and apoptosis were quantified in the retina. CEE and lutein inhibited the A2E photooxidation and oxidized A2E-induced RPE injury. The results of an in vivo experiment also demonstrate that CEE and lutein have powerful anti-oxidative and anti-apoptotic effects on mice with light-induced retinal degeneration. There were fewer histological changes in the ONL layer in mice that were treated with CEE and lutein. CEE and lutein have both a- and b-wave amplitude increments in ERG analysis. The TUNEL-positive cells and 8-OHdG expression in the retinas were reduced by CEE and lutein. CEE may prevent retinal photoreceptors from light-induced structural and functional damage. The active ingredient, lutein, is responsible for this retinoprotective activity. CEE can provide the additional benefit in patients with retinal degenerative diseases.

Characteristics of Retinal Vascular Degeneration and the Expression of Vessel-Related Claudin Proteins in Retinal Degeneration Mouse.

This study aimed to investigate the characteristics of retinal vascular degeneration and the expression of vessel-related claudin (CLD) proteins in retinal degeneration mouse (Pde6βrd1/rd1 rd1 mouse). Retinas from wild-type (WT) mice and rd1 mice at postnatal day 3 (P3), P5, P8, P11, P13, P15, P18, and P21 were collected. Immunofluorescence staining was used to assess the retinal vascular plexus, cell proliferation, CLD expression, and retinal ganglion cells (RGCs). The distribution of retinal superficial and deep vessels was determined by isolectin B4 fluorescence staining of retinal flat mounts and frozen sections. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling were used to investigate retinal histological degeneration and apoptosis in rd1 mice, respectively. Quantitative real-time PCR and Western blot were used to measure the expression of vessel-related CLD-1, -2, -3, and -5, vascular endothelial growth factor A (VEGFA), and vascular endothelial growth factor receptor 2 (VEGFR2) in the retinas. Compared to the WT mice, the rd1 mice displayed delayed but completed progressive development in the retinal superficial vascular plexuses (SVPs) and deep vascular plexuses (DVPs). In the rd1 mice, the thickness of retinal layers gradually decreased and the retinas underwent progressive atrophy and degeneration. The deterioration got worse at the late developmental stage. The declined vessel density of SVP and DVP correlated with the decreased thickness of the full and inner parts of the retina and the reduced number of RGCs. DVP degeneration and the thinning of the outer nuclear layer exhibited an obvious reduction at P15. The expression levels of CLD-1, CLD-2, CLD-3, CLD-5, VEGFA, and VEGFR2 decreased and were consistently lower in the rd1 mice than in WT mice since P15. Rd1 mice exhibited progressive vascular degeneration of retinal SVP and DVP, the thinning and atrophy of retinal ONL and RGC, and the downregulation of vessel-related CLD proteins during the late developmental period. Thus, the rd1 mouse is a useful model of not only retinal neuro-degeneration but also retinal vascular degeneration.

Automated quantification of photoreceptor outer segments in developing and degenerating retinas on microscopy images across scales

The functionality of photoreceptors, rods, and cones is highly dependent on their outer segments (POS), a cellular compartment containing highly organized membranous structures that generate biochemical signals from incident light. While POS formation and degeneration are qualitatively assessed on microscopy images, reliable methodology for quantitative analyses is still limited. Here, we developed methods to quantify POS (QuaPOS) maturation and quality on retinal sections using automated image analyses. POS formation was examined during the development and in adulthood of wild-type mice via light microscopy (LM) and transmission electron microscopy (TEM). To quantify the number, size, shape, and fluorescence intensity of POS, retinal cryosections were immunostained for the cone POS marker S-opsin. Fluorescence images were used to train the robust classifier QuaPOS-LM based on supervised machine learning for automated image segmentation. Characteristic features of segmentation results were extracted to quantify the maturation of cone POS. Subsequently, this quantification method was applied to characterize POS degeneration in “cone photoreceptor function loss 1” mice. TEM images were used to establish the ultrastructural quantification method QuaPOS-TEM for the alignment of POS membranes. Images were analyzed using a custom-written MATLAB code to extract the orientation of membranes from the image gradient and their alignment (coherency). This analysis was used to quantify the POS morphology of wild-type and two inherited retinal degeneration (“retinal degeneration 19” and “rhodopsin knock-out”) mouse lines. Both automated analysis technologies provided robust characterization and quantification of POS based on LM or TEM images. Automated image segmentation by the classifier QuaPOS-LM and analysis of the orientation of membrane stacks by QuaPOS-TEM using fluorescent or TEM images allowed quantitative evaluation of POS formation and quality. The assessments showed an increase in POS number, volume, and membrane coherency during wild-type postnatal development, while a decrease in all three observables was detected in different retinal degeneration mouse models. All the code used for the presented analysis is open source, including example datasets to reproduce the findings. Hence, the QuaPOS quantification methods are useful for in-depth characterization of POS on retinal sections in developmental studies, for disease modeling, or after therapeutic interventions affecting photoreceptors.

Prohibitin 2 deficiency in photoreceptors leads to progressive retinal degeneration and facilitated Müller glia engulfing microglia debris

Müller glia and microglia are capable of phagocytosing fragments of retinal cells in response to retinal injury or degeneration. However, the direct evidence for their mutual interactions between Müller glia and microglia in the progression of retinal degeneration (RD) remains largely unclear. This study aims to construct a progressive RD mouse model and investigate the activated pattern of Müller glia and the interplay between Müller glia and microglia in the early stage or progression of RD. A Prohibitin 2 (Phb2) photoreceptor-specific knockout (RKO) mouse model was generated by crossing Phb2flox/flox mice with Rhodopsin-Cre mice. Optical Coherence Tomography (OCT), histological staining, and Electroretinography (ERG) assessed retinal structure and function, and RKO mice exhibited progressive RD from six weeks of age. In detail, six-week-old RKO mice showed no significant retinal impairment, but severe vision dysfunction and retina thinning were shown in ten-week-old RKO mice. Furthermore, RKO mice were sensitive to Light Damage (LD) and showed severe RD at an early age after light exposure. Bulk retina RNA-seq analysis from six-week-old control (Ctrl) and RKO mice showed reactive retinal glia in RKO mice. The activated pattern of Müller glia and the interplay between Müller glia and microglia was visualized by immunohistology and 3D reconstruction. In six-week-old RKO mice or light-exposed Ctrl mice, Müller glia were initially activated at the edge of the retina. Moreover, in ten-week-old RKO mice or light-exposed six-week-old RKO mice with severe photoreceptor degeneration, abundant Müller glia were activated across the whole retinas. With the progression of RD, phagocytosis of microglia debris by activated Müller glia were remarkably increased. Altogether, our study establishes a Phb2 photoreceptor-specific knockout mouse model, which is a novel mouse model of RD and can well demonstrate the phenotype of progressive RD. We also report that Müller glia in the peripheral retina is more sensitive to the early damage of photoreceptors. Our study provides more direct evidence for Müller glia engulfing microglia debris in the progression of RD due to photoreceptor Phb2 deficiency.

Overexpression of miR-96 leads to retinal degeneration in mice

Double knockout of miR-183 and miR-96 results in retinal degeneration in mice; however, single knockout of miR-96 leads to developmental delay but not substantial retinal degeneration. To further explore the role of miR-96, we overexpressed this miRNA in mouse retinas. Interestingly, we found that overexpression of miR-96 at a safe dose results in retinal degeneration in the mouse retina. The retinal photoreceptors dramatically degenerated in the miR-96-overexpressing group, as shown by OCT, ERG and cryosectioning at one month after subretinal injection. Degenerative features such as TUNEL signals and reactive gliosis were observed in the miR-96-overexpressing retina. RNA-seq data revealed that immune responses and microglial activation occurred in the degenerating retina. Further qRT‒PCR and immunostaining experiments verified the microglial activation. Moreover, the number of microglia in the miR-96-overexpressing retinas was significantly increased. Our findings demonstrate that appropriate miR-96 expression is required for mouse retinal homeostasis.

Voluntary exercise preserves visual function and reduces inflammatory response in an adult mouse model of autosomal dominant retinitis pigmentosa

Whole-body physical exercise has been shown to promote retinal structure and function preservation in animal models of retinal degeneration. It is currently unknown how exercise modulates retinal inflammatory responses. In this study, we investigated cytokine alterations associated with retinal neuroprotection induced by voluntary running wheel exercise in a retinal degeneration mouse model of class B1 autosomal dominant retinitis pigmentosa, I307N Rho. I307N Rho mice undergo rod photoreceptor degeneration when exposed to bright light (induced). Our data show, active induced mice exhibited significant preservation of retinal and visual function compared to inactive induced mice after 4 weeks of exercise. Retinal cytokine expression revealed significant reductions of proinflammatory chemokines, keratinocyte-derived chemokine (KC) and interferon gamma inducible protein-10 (IP-10) expression in active groups compared to inactive groups. Through immunofluorescence, we found KC and IP-10 labeling localized to retinal vasculature marker, collagen IV. These data show that whole-body exercise lowers specific retinal cytokine expression associated with retinal vasculature. Future studies should determine whether suppression of inflammatory responses is requisite for exercise-induced retinal protection.

Eye Drop with Fas-Blocking Peptide Attenuates Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), characterized by macular retinal degeneration, poses a significant health concern due to the lack of effective treatments for prevalent dry AMD. The progression of AMD is closely linked to reactive oxygen species and Fas signaling, emphasizing the need for targeted interventions. In this study, we utilized a NaIO3-induced retinal degeneration mouse model to assess the efficacy of Fas-blocking peptide (FBP). Intravitreal administration of FBP successfully suppressed Fas-mediated inflammation and apoptosis, effectively arresting AMD progression in mice. We developed a 6R-conjugated FBP (6R-FBP) for eye drop administration. 6R-FBP, administered as an eye drop, reached the retinal region, attenuating degeneration by modulating the expression of inflammatory cytokines and blocking Fas-mediated apoptosis in rodent and rabbit NaIO3-induced retinal degeneration models to address practical concerns. Intravitreal FBP and 6R-FBP eye drops effectively reduced retinal degeneration and improved retinal thickness in rodent and rabbit models. This study highlights the therapeutic potential of FBP, particularly 6R-FBP as an eye drop, in inhibiting Fas-mediated cell signaling and protecting against retinal cell death and inflammation in dry AMD. Future investigations should explore the translational prospects of this approach in primates with eye structures comparable to those of humans.

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model.

Transplantation of photoreceptor cells and retinal pigmentepithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into 'adult' rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

CX3CL1/CX3CR1 Signaling Mediated Neuroglia Activation Is Implicated in the Retinal Degeneration: A Potential Therapeutic Target to Prevent Photoreceptor Death.

Retinal degeneration (RD) is a large cluster of retinopathies that is characterized by the progressive photoreceptor death and visual impairments. CX3CL1/CX3CR1 signaling has been documented to mediate the microglia activation and gliosis reaction during neurodegeneration. We intend to verify whether the CX3CL1/CX3CR1 signaling is involved in the RD pathology. A pharmacologically induced RD mice model was established. AZD8797, a CX3CR1 antagonist, was injected into the vitreous cavity of an RD model to modulate the neuroglia activation. Then, the experimental animals were subjected to functional, morphological, and behavioral analysis. The CX3CL1/CX3CR1 signaling mediated neuroglia activation was implicated in the photoreceptor demise of an RD model. Intravitreal injection of AZD8797 preserved the retinal structure and enhanced the photoreceptor survival through inhibiting the CX3CL1/CX3CR1 expressions. Fundus photography showed that the distribution of retinal vessel was clear, and the severity of lesions was alleviated by AZD8797. In particular, these morphological benefits could be translated into remarkable functional improvements, as evidenced by the behavioral test and electroretinogram (mf-ERG) examination. A mechanism study showed that AZD8797 mitigated the microglia activation and migration in the degenerative retinas. The Müller cell hyper-reaction and secondary gliosis response were also suppressed by AZD8797. The neuroinflammation is implicated in the photoreceptor loss of RD pathology. Targeting the CX3CL1/CX3CR1 signaling may serve as an effective therapeutic strategy. Future refinements of these findings may cast light into the discovery of new medications for RD.

Microglia depletion/repopulation does not affect light-induced retinal degeneration in mice.

Reactive microglia are a hallmark of age-related retinal degenerative diseases including age-related macular degeneration (AMD). These cells are capable of secreting neurotoxic substances that may aggravate inflammation that leads to loss of photoreceptors and impaired vision. Despite their role in driving detrimental inflammation, microglia also play supporting roles in the retina as they are a crucial cellular component of the regulatory innate immune system. In this study, we used the colony stimulating factor 1 receptor (CSF1R)-antagonist PLX3397 to investigate the effects of microglia depletion and repopulation in a mouse model of acute retinal degeneration that mimics some aspects of dry AMD. Our main goal was to investigate whether microglia depletion and repopulation affects the outcome of light-induced retinal degeneration. We found that microglia depletion effectively decreased the expression of several key pro-inflammatory factors but was unable to influence the extent of retinal degeneration as determined by optical coherence tomography (OCT) and histology. Interestingly, we found prominent cell debris accumulation in the outer retina under conditions of microglia depletion, presumably due to the lack of efficient phagocytosis that could not be compensated by the retinal pigment epithelium. Moreover, our in vivo experiments showed that renewal of retinal microglia by repopulation did also not prevent rapid microglia activation or preserve photoreceptor death under conditions of light damage. We conclude that microglia ablation strongly reduces the expression of pro-inflammatory factors but cannot prevent photoreceptor loss in the light-damage paradigm of retinal degeneration.

Liquid-metal-based three-dimensional microelectrode arrays integrated with implantable ultrathin retinal prosthesis for vision restoration

Electronic retinal prostheses for stimulating retinal neurons are promising for vision restoration. However, the rigid electrodes of conventional retinal implants can inflict damage on the soft retina tissue. They also have limited selectivity due to their poor proximity to target cells in the degenerative retina. Here we present a soft artificial retina (thickness, 10 μm) where flexible ultrathin photosensitive transistors are integrated with three-dimensional stimulation electrodes of eutectic gallium–indium alloy. Platinum nanoclusters locally coated only on the tip of these three-dimensional liquid-metal electrodes show advantages in reducing the impedance of the stimulation electrodes. These microelectrodes can enhance the proximity to the target retinal ganglion cells and provide effective charge injections (72.84 mC cm−2) to elicit neural responses in the retina. Their low Young’s modulus (234 kPa), owing to their liquid form, can minimize damage to the retina. Furthermore, we used an unsupervised machine learning approach to effectively identify the evoked spikes to grade neural activities within the retinal ganglion cells. Results from in vivo experiments on a retinal degeneration mouse model reveal that the spatiotemporal distribution of neural responses on their retina can be mapped under selective localized illumination areas of light, suggesting the restoration of their vision.

Photoreceptor-targeted extracellular vesicles-mediated delivery of Cul7 siRNA for retinal degeneration therapy.

Rationale: Photoreceptor loss is a primary pathological feature of retinal degeneration (RD) with limited treatment strategies. RNA interference (RNAi) has emerged as a promising method of gene therapy in regenerative medicine. However, the transfer of RNAi therapeutics to photoreceptors and the deficiency of effective therapeutic targets are still major challenges in the treatment of RD. Methods: In this study, photoreceptor-derived extracellular vesicles (PEVs) conjugated with photoreceptor-binding peptide MH42 (PEVsMH42) were prepared using the anchoring peptide CP05. Transcriptome sequencing was applied to investigate the potential therapeutic target of RD. We then engineered PEVsMH42 with specific small-interfering RNAs (siRNAs) through electroporation and evaluated their therapeutic efficacy in N-methyl-N-nitrosourea (MNU)-induced RD mice and Pde6βrd1/rd1 mutant mice. Results: PEVsMH42 were selectively accumulated in photoreceptors after intravitreal injection. Cullin-7 (Cul7) was identified as a novel therapeutic target of RD. Taking advantage of the established PEVsMH42, siRNAs targeting Cul7 (siCul7) were efficiently delivered to photoreceptors and consequently blocked the expression of Cul7. Moreover, suppression of Cul7 effectively protected photoreceptors to alleviate RD both in MNU-induced mouse model and Pde6βrd1/rd1 mutant mouse model. Mechanistically, PEVsMH42 loaded with siCul7 (PEVsMH42-siCul7)-induced Cul7 downregulation was responsible for preventing Cul7-mediated glutathione peroxidase 4 (Gpx4) ubiquitination and degradation, resulting in the inhibition of photoreceptor ferroptosis. Conclusions: In summary, PEVsMH42-siCul7 attenuate photoreceptor ferroptosis to treat RD by inhibiting Cul7-induced ubiquitination of Gpx4. Our study develops a PEVs-based platform for photoreceptor-targeted delivery and highlights the potential of PEVsMH42-siCul7 as effective therapeutics for RD.

Paraoxonase 2 Deficiency Causes Mitochondrial Dysfunction in Retinal Pigment Epithelial Cells and Retinal Degeneration in Mice.

Although AMD is a complex disease, oxidative stress is a crucial contributor to its development, especially in view of the higher oxygen demand of the retina. Paraoxonase 2 (PON2) is a ubiquitously and constitutively expressed antioxidant protein that is found intracellularly associated with mitochondrial membranes and modulates mitochondrial ROS production and function. The contribution of PON2 to AMD has not been studied to date. In this study, we examined the role of PON2 in AMD utilizing both in vitro and in vivo models of AMD with emphasis on mitochondrial function. Mitochondrial localization and regulation of PON2 following oxidative stress were determined in human primary cultured retinal pigment epithelium (hRPE) cells. PON2 was knocked down in RPE cells using siRNA and mitochondrial bioenergetics were measured. To investigate the function of PON2 in the retina, WT and PON2-deficient mice were administered NaIO3 (20 mg/kg) intravenously; fundus imaging, optical coherence tomography (OCT), electroretinography (ERG) were conducted; and retinal thickness and cell death were measured and quantified. In hRPE, mitochondrial localization of PON2 increased markedly with stress. Moreover, a time-dependent regulation of PON2 was observed following oxidative stress, with an initial significant increase in expression followed by a significant decrease. Mitochondrial bioenergetic parameters (basal respiration, ATP production, spare respiratory capacity, and maximal respiration) showed a significant decrease with oxidative stress, which was further exacerbated in the absence of PON2. NaIO3 treatment caused significant retinal degeneration, retinal thinning, and reduced rod and cone function in PON2-deficient mice when compared to WT mice. The apoptotic cells and active caspase 3 significantly increased in PON2-deficient mice treated with NaIO3, when compared to WT mice. Our investigation demonstrates that deficiency of PON2 results in RPE mitochondrial dysfunction and a decline in retinal function. These findings imply that PON2 may have a beneficial role in retinal pathophysiology and is worthy of further investigation.

A Distinct Microglial Cell Population Expressing Both CD86 and CD206 Constitutes a Dominant Type and Executes Phagocytosis in Two Mouse Models of Retinal Degeneration.

Microglial cells are the key regulators of inflammation during retinal degeneration (RD) and are conventionally classified as M1 or M2. However, whether the M1/M2 classification exactly reflects the functional classification of microglial cells in the retina remains debatable. We examined the spatiotemporal changes of microglial cells in the blue-LED and NaIO3-induced RD mice models using M1/M2 markers and functional genes. TUNEL assay was performed to detect photoreceptor cell death, and microglial cells were labeled with anti-IBA1, P2RY12, CD86, and CD206 antibodies. FACS was used to isolate microglial cells with anti-CD206 and CD86 antibodies, and qRT-PCR was performed to evaluate Il-10, Il-6, Trem-2, Apoe, and Lyz2 expression. TUNEL-positive cells were detected in the outer nuclear layer (ONL) from 24 h to 72 h post-RD induction. At 24 h, P2RY12 was decreased and CD86 was increased, and CD86/CD206 double-labeled cells occupied the dominant population at 72 h. And CD86/CD206 double-labeled cells showed a significant increase in Apoe, Trem2, and Lyz2 levels but not in those of Il-6 and Il-10. Our results demonstrate that microglial cells in active RD cannot be classified as M1 or M2, and the majority of microglia express both CD86 and CD206, which are involved in phagocytosis rather than inflammation.

Lycii Fructus, a medicine food homology material, attenuates retinal damage in sodium iodate-induced retinal degeneration mice and inhibits oxidation-induced apoptosis of Müller cells via regulating Nrf2-HO1 signaling pathway

Lycii Fructus (LF) is a medicinal-edible homology material frequently used for treating age-related macular degeneration (AMD) in Chinese medicine. Abundant of literatures verified the retina protection effect of LF on AMD-like animal models. However, previous studies on LF attenuating retinal damage mainly focused on its protection effect on pigment epithelial cells and optic ganglion cells while few reports on Müller cells. In the current study, we found that LF extracts attenuated oxidation damages and inhibited cell apoptosis of retina on a mouse retinal injury model induced by sodium iodate. LF extracts also increased expressions of Müller cell biomarker glutamine synthetase of eyeballs of model mice, which indicated that LF extract protected Müller cell from oxidation injury induced by sodium iodate. In Müller cell MIO-M1 cultures, LF also significantly inhibited cellular apoptosis induced by NaIO3 by regulating Nrf2/HO-1 signaling pathway and the aqueous extracts exerted better active tendency than ethanol extracts. These data implied that the antioxidant effect of LF on protecting Müller cell might contribute to its retina repair function, thereby providing more scientific evidences for the development of LF as an alternative therapy or preventive measures for AMD.

Deletion of Emc1 in photoreceptor cells causes retinal degeneration in mice.

The endoplasmic reticulum membrane protein complex (EMC) plays a critical role in the synthesis of multi-pass membrane proteins. Genetic studies indicated that mutations in EMC1 gene were associated with retinal degeneration diseases, however the role of EMC1 in photoreceptor has not been proved. Here, we show that Emc1 ablation in the photoreceptor cells of mice recapitulated the retinitis pigmentosa (RP) phenotypes, including an attenuated scotopic electroretinogram (ERG) response and the progressive degeneration of rod cells and cone cells. Histopathological examination of tissues from rod-specific Emc1 KO (RKO) mice revealed mislocalized rhodopsin and irregularly arranged cone cells at the age of 2 months. Further immunoblotting analysis revealed decreased levels of membrane proteins and ER chaperones in 1-month-old RKO retinae, and this led us to speculate that the loss of membrane proteins is the main cause of the degeneration of photoreceptors. EMC1 most likely regulated the membrane protein levels at an earlier step in the biosynthetic process before the proteins translocated into the ER. Our study demonstrates the essential roles of Emc1 in photoreceptor cells, and reveals the mechanism through which EMC1 mutations are linked to RP.

A small molecule compound that inhibits blue light-induced retinal damage via activation of autophagy

Dry age-related macular degeneration (AMD) is a type of disease that causes visual impairment due to changes in the macula located in the center of the retina. The accumulation of drusen under the retina is also a characteristic of dry AMD. In this study, we identified a compound (JS-017) that can potentially degrade N-retinylidene-N-retinylethanolamine (A2E), one of the components of lipofuscin, using fluorescence-based screening, which measures A2E degradation in human retinal pigment epithelial cells. JS-017 effectively degraded A2E in ARPE-19 cells and consequently suppressed the activation of the NF-κB signaling pathway and expression of inflammatory and apoptosis genes induced by blue light (BL). Mechanistically, JS-017 induced LC3-II formation and improved autophagic flux in ARPE-19 cells. Additionally, the A2E degradation activity of JS-017 was found to be decreased in autophagy-related 5 protein-depleted ARPE-19 cells, suggesting that autophagy was required for A2E degradation mediated by JS-017. Finally, JS-017 exhibited an improvement in BL-induced retinal damage measured through fundus examination in an in vivo retinal degeneration mouse model. The thickness of the outer nuclear layer and inner/external segments, which was decreased upon exposure to BL irradiation, was also restored upon JS-017 treatment. Altogether, we demonstrated that JS-017 protected human retinal pigment epithelium (RPE) cells from A2E and BL-induced damage by degrading A2E via the activation of autophagy. The results suggest the feasibility of a novel A2E-degrading small molecule as a therapeutic agent for retinal degenerative diseases.

Exosome-mediated inhibition of microRNA-449a promotes the amplification of mouse retinal progenitor cells and enhances their transplantation in retinal degeneration mouse models

Inherited and age-related retinal degenerations are the commonest causes of blindness without effective treatments. Retinal progenitor cells (RPCs), which have the multipotency to differentiate into various retinal cell types, are regarded as a promising source of cell transplantation therapy for retinal degenerative diseases. However, the self-limited expansion of RPCs causes difficulty in cell source supply and restrict its clinical treatment. In this work, we found that inhibition of microRNA-449a (miR-449a) in RPCs can promote proliferation and inhibit apoptosis of RPCs, partially through upregulating Notch signaling. Further optimization of transduction miR-449a inhibitor into RPCs by endothelial cell-derived exosomes can promote the survival of RPCs transplanted invivo and reduce cell apoptosis in retinal degeneration mouse models. In summary, these studies have shown that exosome-miR-449a inhibitor can effectively promote the expansion of RPCs invitro and enhance transplanted RPCs survival invivo, which might provide a novel intervention strategy for retinal degenerations in the future.