ABSTRACT
2-Ethylhexyl diphenyl phosphate (EHDPP) is a frequently utilized organophosphorus flame retardant (OPFR) and has been extensively detected in environmental media. Prolonged daily exposure to EHDPP has been linked to potential retinal damage, yet the adverse impacts on the retina are still generally underexplored. In this research, we explored oxidative stress, inflammation, and the activating mechanisms initiated by EHDPP in mouse retinal photoreceptor (661â¯W) cells following a 24â¯h exposure period. Our research demonstrated that EHDPP led to a decline in cell viability that was directly proportional to its concentration, with the median lethal concentration (LC50) being 88⯵M. Furthermore, EHDPP was found to elevate intracellular and mitochondrial levels of reactive oxygen species (ROS), trigger apoptosis, induce cell cycle arrest at the G1 phase, and modulate the expression of both antioxidant enzymes (Nrf2, HO-1, and CAT) and pro-inflammatory mediators (TNF-α, IL-1ß, and IL-6) within 661â¯W cells. These findings indicate that retinal damage triggered by EHDPP exposure could be mediated via the Nrf2/HO-1 signaling pathway in these cells. Collectively, our investigation revealed that oxidative stress induced by EHDPP is likely a critical factor in the cytotoxic response of 661â¯W cells, potentially leading to damage in retinal photoreceptor cells.
Subject(s)
Apoptosis , Cell Survival , Flame Retardants , Oxidative Stress , Reactive Oxygen Species , Animals , Oxidative Stress/drug effects , Flame Retardants/toxicity , Mice , Apoptosis/drug effects , Reactive Oxygen Species/metabolism , Cell Survival/drug effects , Organophosphorus Compounds/toxicity , Inflammation/chemically induced , Organophosphates/toxicity , NF-E2-Related Factor 2/metabolism , Cell Line , Photoreceptor Cells/drug effects , Retina/drug effectsABSTRACT
We explored the effect of inhibition of thioredoxin interacting protein (Txnip) on neuroprotection in Müller cells under high glucose. Wild-type (WT) and Txnip knockout (Txnip-/-) mice were used to establish a streptozotocin (STZ)-induced diabetes model and a Müller cells high glucose model. We detected BDNF expression and PI3K/AKT/CREB pathway activation levels in the retina and Müller cells of each group in vivo and in vitro experiments. The Txnip-/- STZ group showed higher expression of BDNF and phosphorylation of PI3K/AKT/CREB in retina, and less retinal photoreceptor apoptosis was observed in Txnip-/- diabetic group than in WT. After using an inhibitor of PI3K signaling pathway, BDNF expression was reduced; In vitro co-cultured with Müller cells in different groups, 661 W cells showed different situations, Txnip-/- Müller cells maximum downregulated Cleaved-caspase 3 expression in 661 W, accompanied by an increase in Bcl-2/Bax ratio. These findings indicate that inhibiting endogenous Txnip in mouse Müller cells can promote their expression and secretion of BDNF, thereby reducing HG induced photoreceptor apoptosis and having important neuroprotective effects on DR. The regulation of BDNF expression by Txnip may be achieved by activating the PI3K/AKT/CREB pathway. This study suggests that regulating Txnip may be a potential target for DR treatment.
Subject(s)
Apoptosis , Carrier Proteins , Diabetes Mellitus, Experimental , Ependymoglial Cells , Phosphatidylinositol 3-Kinases , Signal Transduction , Animals , Apoptosis/drug effects , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/genetics , Ependymoglial Cells/metabolism , Ependymoglial Cells/drug effects , Ependymoglial Cells/pathology , Carrier Proteins/metabolism , Carrier Proteins/genetics , Mice , Signal Transduction/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Mice, Knockout , Photoreceptor Cells/metabolism , Photoreceptor Cells/drug effects , Photoreceptor Cells/pathology , Gene Knockdown Techniques , Brain-Derived Neurotrophic Factor/metabolism , Brain-Derived Neurotrophic Factor/genetics , Thioredoxins/metabolism , Thioredoxins/genetics , Male , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/genetics , Diabetic Retinopathy/pathology , Photoreceptor Cells, Vertebrate/metabolism , Photoreceptor Cells, Vertebrate/pathology , Photoreceptor Cells, Vertebrate/drug effects , Disease Models, AnimalABSTRACT
Zebrafish possess the ability to completely regenerate the retina following injury, however little is understood about the damage signals that contribute to inducing Müller glia reprogramming and proliferation to regenerate lost neurons. Multiple studies demonstrated that iron contributes to various retinal injuries, however no link has been shown between iron and zebrafish retinal regeneration. Here we demonstrate that Müller glia exhibit transcriptional changes following injury to regulate iron levels within the retina, allowing for increased iron uptake and decreased export. The response of the zebrafish retina to intravitreal iron injection was then characterized, showing that ferrous, and not ferric, iron induces retinal cell death. Additionally, iron chelation resulted in decreased numbers of TUNEL-positive photoreceptors and fewer proliferating Müller glia. Despite the contribution of iron to retinal cell death, inhibition of ferroptosis did not significantly reduce cell death following light treatment. Finally, we demonstrate that both the anti-ferroptotic protein Glutathione peroxidase 4b and the Transferrin receptor 1b are required for Müller glia proliferation following light damage. Together these findings show that iron contributes to cell death in the light-damaged retina and is essential for inducing the Müller glia regeneration response.
Subject(s)
Cell Proliferation/drug effects , Ependymoglial Cells/drug effects , Ferrous Compounds/toxicity , Photoreceptor Cells/drug effects , Radiation Injuries, Experimental/etiology , Retinal Degeneration/chemically induced , Animals , Animals, Genetically Modified , Apoptosis , Deferiprone/pharmacology , Ependymoglial Cells/metabolism , In Situ Nick-End Labeling , Intravitreal Injections , Light , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Photoreceptor Cells/radiation effects , Radiation Injuries, Experimental/metabolism , Receptors, Transferrin/metabolism , Retinal Degeneration/metabolism , Zebrafish , Zebrafish Proteins/metabolismABSTRACT
A new phenylethanoid, hebitol IV (1), along with fifteen known glycosides (2-16), were isolated from water extract of the flower buds of Buddleja officinalis. Their structures were elucidated on the basis of 1 D-NMR, 2 D-NMR and MS data. Molecular docking showed the potential activities of the natural products against VEGFR-2. Bioassay results revealed that the compounds 10 and 14 exhibited strong inhibitory activity against VEGFR-2 with IC50 values of 0.51 and 0.32 µM, respectively. Moreover, the potential retinal protective effects of 10 and 14 were then investigated in the mouse model featuring bright light-induced retinal degeneration. The results demonstrated remarkable photoreceptor protective activities of 10 and 14 in vivo.
Subject(s)
Buddleja , Glycosides , Photoreceptor Cells , Retina , Animals , Buddleja/chemistry , Glycosides/chemistry , Glycosides/pharmacology , Mice , Molecular Docking Simulation , Photoreceptor Cells/drug effects , Plant Extracts/chemistry , Plant Extracts/pharmacology , Retina/cytology , Retina/drug effects , Retina/radiation effects , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitorsABSTRACT
Purpose: This study aims to evaluate the effect of citicoline administration in suppressing retinal damage due to methanol intoxication. This study hypothesizes that citicoline will minimize the loss of retinal ganglion cells (RGCs), minimize disruption of photoreceptors, suppress ganglion layer edema, increase expression of bcl-2 as the antiapoptotic protein, and decrease expression of caspase-3 as the proapoptotic protein. Methods: Fifteen Sprague-Dawley rats were divided into 5 groups, including the control group (A); methanol groups, observed on day 3 (B1) and day 7 (B2); and methanol+citicoline groups, observed on day 3 (C1) and day 7 (C2). Rats in groups B and C were placed in an inhalation chamber filled with N2O:O2 during the experiment, then methanol was administered orally. Citicoline, 1 g/kg every 24 h, was orally administered for group C. Enucleation was performed and retinas of rats were prepared for histology and immunohistochemistry examination to evaluate photoreceptor morphology and RGC density, as well as bcl-2 and caspase-3 expression. Results: RGC density of citicoline-treated intoxicated rats was higher than no-citicoline methanol-intoxicated rats on both day 3 (P < 0.001) and day 7 (P < 0.001). The ganglion layer thickness of citicoline-treated intoxicated rats was thinner than no-citicoline intoxicated rats, which means citicoline-treated rats had milder ganglion layer edema. Citicoline-treated rats showed higher bcl-2 and lower caspase-3 expression than no-citicoline rats. No differences were found in photoreceptor findings among groups. Conclusions: This study demonstrated citicoline's potential benefits for management of ocular methanol intoxication. However, more preclinical and clinical trials are needed to obtain a preferred dosage and timing of citicoline administration.
Subject(s)
Cytidine Diphosphate Choline/pharmacology , Methanol/poisoning , Nootropic Agents/pharmacology , Retina/drug effects , Toxic Optic Neuropathy/pathology , Animals , Caspase 3/drug effects , Disease Models, Animal , Male , Photoreceptor Cells/drug effects , Proto-Oncogene Proteins c-bcl-2/drug effects , Rats , Rats, Sprague-Dawley , Retinal Ganglion Cells/drug effectsABSTRACT
Background: Y-27632 is a potent ophthalmic drug for the treatment of ocular hypertension, a globally prevalent eye disease. However, the sustained delivery of Y-27632 by a therapeutic carrier to lesion sites located in the inner segments of the eye for effectively treating the ocular disorder still remains challenging. Methods: To realize the goal, a strategy based on solvothermal-assisted deposition/infiltration in combination with surface modification is utilized to synthesize hollow mesoporous ceria nanoparticles (HMCNs) with tailorable shell thicknesses and drug release profiles. The shell thickness of HMCNs is rationally exploited for achieving sustained drug release and advanced therapeutic benefits. Results: The shell thickness can regulate release profiles of Y-27632, displaying that thick and thin (~40 nm and ~10 nm) shelled HMCNs reveal burst release characteristics (within 2 days) or limited drug loading content (~10% for the 40 nm thick). As a compromise, the HMCNs with moderate shell thickness (~20 nm) possess the most sustained drug release over a period of 10 days. In a rabbit model of glaucoma, a single instillation of the optimized Y-27632-loaded HMCNs can effectively treat glaucoma for 10 days via simultaneously repairing the defected cornea (recovery of ~93% ATP1A1 mRNA levels), restoring the reduced thickness of outer nuclear layer to normal (~64 µm), and restoring ~86% of the impaired photoreceptor cells. Conclusion: A comprehensive study on the importance of HMCN shell thickness in developing long-acting nano eye drops for the efficient management of glaucoma is proposed. The findings suggest a central role of nanobiomaterial structural engineering in developing the long-life eye drops for pharmacological treatment of intraocular diseases.
Subject(s)
Amides/pharmacology , Nanoparticles/chemistry , Nitroimidazoles/chemistry , Ocular Hypertension/drug therapy , Pyridines/pharmacology , Animals , Cell Line , Delayed-Action Preparations/chemistry , Disease Models, Animal , Drug Carriers/chemistry , Drug Delivery Systems/methods , Drug Liberation/drug effects , Eye/drug effects , Glaucoma/drug therapy , Humans , Ophthalmic Solutions/pharmacology , Photoreceptor Cells/drug effects , RabbitsABSTRACT
Müller glia can act as endogenous stem cells and regenerate the missing neurons in the injured or degenerating retina in lower vertebrates. However, mammalian Müller glia, although can sometimes express stem cell markers and specific neuronal proteins in response to injury or degeneration, do not differentiate into functional neurons. We asked whether bFGF and insulin would stimulate the Müller glia to migrate, proliferate and differentiate into photoreceptors in rd1 mouse. We administered single or repeated (two or three) intravitreal injections of basic fibroblast growth factor (bFGF;200⯵g) and insulin (2⯵g) in 2-week-old rd1 mice. Müller glia were checked for proliferation, migration and differentiation using immunostaining. A single injection resulted within 5 days in a decrease in the numbers of Müller glia in the inner nuclear layer (INL) and a corresponding increase in the outer nuclear layer (ONL). The total number of Müller glia in the INL and ONL was unaltered, suggesting that they did not proliferate, but migrated from INL to ONL. However, maintaining the Müller cells in the ONL for two weeks or longer required repeated injections of bFGF and insulin. Interestingly, all Müller cells in the ONL expressed chx10, a stem cell marker. We did not find any immunolabeling for rhodopsin, m-opsin or s-opsin in the Müller glia in the ONL.
Subject(s)
Cell Movement/drug effects , Ependymoglial Cells/drug effects , Fibroblast Growth Factor 2/administration & dosage , Insulin/administration & dosage , Neuroglia/drug effects , Photoreceptor Cells/drug effects , Animals , Cell Movement/physiology , Ependymoglial Cells/chemistry , Ependymoglial Cells/metabolism , Intravitreal Injections , Mice , Mice, Inbred CBA , Mice, Transgenic , Nerve Regeneration/drug effects , Nerve Regeneration/physiology , Neuroglia/chemistry , Neuroglia/metabolism , Photoreceptor Cells/chemistry , Photoreceptor Cells/metabolism , Retinal Degeneration/metabolism , Retinal Degeneration/pathologyABSTRACT
Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel). We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina. The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.
Subject(s)
Drug Discovery , Organoids/physiology , Retina/physiology , Toxicity Tests , Apoptosis/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Embryoid Bodies/drug effects , Embryoid Bodies/physiology , Fenofibrate/toxicity , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Organoids/drug effects , Photoreceptor Cells/drug effects , Photoreceptor Cells/metabolism , Retina/cytology , Retina/drug effects , Sphingosine/analogs & derivatives , Sphingosine/toxicityABSTRACT
PURPOSE: The phosphodiesterase inhibitor sildenafil is a promising treatment for neurodegenerative disease, but it can cause oxidative stress in photoreceptors ex vivo and degrade visual performance in humans. Here, we test the hypotheses that in wildtype mice sildenafil causes i) wide-spread photoreceptor oxidative stress in vivo that is linked with ii) impaired vision. METHODS: In dark or light-adapted C57BL/6 mice ± sildenafil treatment, the presence of oxidative stress was evaluated in retina laminae in vivo by QUEnch-assiSTed (QUEST) magnetic resonance imaging, in the subretinal space in vivo by QUEST optical coherence tomography, and in freshly excised retina by a dichlorofluorescein assay. Visual performance indices were also evaluated by QUEST optokinetic tracking. RESULTS: In light-adapted mice, 1 hr post-sildenafil administration, oxidative stress was most evident in the superior peripheral outer retina on both in vivo and ex vivo examinations; little evidence was noted for central retina oxidative stress in vivo and ex vivo. In dark-adapted mice 1 hr after sildenafil, no evidence for outer retina oxidative stress was found in vivo. Evidence for sildenafil-induced central retina rod cGMP accumulation was suggested as a panretinally thinner, dark-like subretinal space thickness in light-adapted mice at 1 hr but not 5 hr post-sildenafil. Cone-based visual performance was impaired by 5 hr post-sildenafil and not corrected with anti-oxidants; vision was normal at 1 hr and 24 hr post-sildenafil. CONCLUSIONS: The sildenafil-induced spatiotemporal pattern of oxidative stress in photoreceptors dominated by rods was unrelated to impairment of cone-based visual performance in wildtype mice.
Subject(s)
Oxidative Stress , Phosphodiesterase Inhibitors/pharmacology , Photoreceptor Cells/drug effects , Sildenafil Citrate/pharmacology , Vision, Ocular , Animals , Male , Mice , Mice, Inbred C57BL , Photoreceptor Cells/metabolismABSTRACT
Retinal detachment (RD) is a severe sight-threatening complication that can be caused by a multitude of retinal diseases. It has been evidenced that minocycline exerts neuroprotective effects by targeting microglia in the pathogenesis of massive ocular lesions including RD, but mechanisms remain elusive. We carried out this research to elucidate the potential mediators that link RD-induced vision loss with microglia reactivity by discussing effects of minocycline on cytokine levels and A20, a negative regulator of inflammation. Minocycline or vehicle was intraperitoneally administrated immediately after RD and continued daily before animals being euthanized. The oxygen glucose deprivation assay was undertaken on the co-cultured BV-2 and 661W cells to mimic the condition of RD in vitro, where A20 siRNA was adopted to knock down the A20 expression in BV-2 cells. Photoreceptor cells apoptosis, inflammatory response and microglia activity following RD with or without minocycline were evaluated. Photoreceptor cells apoptosis and inflammatory response were induced after RD, which could be largely counteracted by minocycline. Minocycline postponed the migration and proliferation of microglia and facilitated their transition to the M2 subtype following RD. Blocking A20 expression in BV-2 cells with siRNA crippled the effect of minocycline. Collectively, minocycline yields a promoting effect on photoreceptor cells survival post-RD by modulating the transformation of microglia phenotypes, in which process A20 may play a "bridge" role.
Subject(s)
Anti-Bacterial Agents/pharmacology , Inflammation/prevention & control , Microglia/drug effects , Minocycline/pharmacology , Retinal Detachment/complications , Tumor Necrosis Factor alpha-Induced Protein 3/genetics , Animals , Blotting, Western , Coculture Techniques , Disease Models, Animal , Gene Expression Regulation/physiology , In Situ Nick-End Labeling , Inflammation/etiology , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Microglia/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Phenotype , Photoreceptor Cells/drug effects , Photoreceptor Cells/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Transfection , Tumor Necrosis Factor alpha-Induced Protein 3/metabolismABSTRACT
Methylphenidate (MPH) is used as the first-line treatment for attention-deficit hyperactivity disorder. However, there are concerns that this treatment may be associated with increased risk of retinal damage. This study was to investigate cytotoxicity of MPH on photoreceptor cells and explore its underlying mechanisms. MPH-caused cell toxicity was established in 661 W cells. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromid and lactate dehydrogenase assays. Oxidative stress was measured by the markers: glutathione (GSH) reductase, catalase, and superoxide dismutase activities as well as GSH, reactive oxygen species, and malondialdehyde levels. Gene and protein expression was detected by real-time polymerase chain reaction (PCR) and western blot, respectively. Results showed that MPH decreased 661 W cell viability, increased caspase-3/9 activities, and induced oxidative stress. Furthermore, MPH treatment increased messenger RNA (mRNA) expression of Beclin-1 and microtubule-associated protein 1A/1B-light chain 3B (LC3B) protein expression in 661 W cells, suggesting autophagy was induced. MPH treatment also upregulated p-JAK1/p-STAT1 protein expression. These data demonstrated that MPH could increase oxidative stress in photoreceptor cells to cause cell toxicity via autophagy, providing the scientific rationale for the photoreceptor cell damage caused by the MPH administration.
Subject(s)
Methylphenidate/toxicity , Photoreceptor Cells/drug effects , Animals , Autophagy , Glutathione , Malondialdehyde , Oxidative Stress , Reactive Oxygen SpeciesABSTRACT
The eyeless C. elegans exhibits robust phototaxis behavior in response to short-wavelength light, particularly UV light. C. elegans senses light through LITE-1, a unique photoreceptor protein that belongs to the invertebrate taste receptor family. However, it remains unclear how LITE-1 is regulated. Here, we performed a forward genetic screen for genes that when mutated suppress LITE-1 function. One group of lite-1 suppressors are the genes required for producing the two primary antioxidants thioredoxin and glutathione, suggesting that oxidization of LITE-1 inhibits its function. Indeed, the oxidant hydrogen peroxide (H2O2) suppresses phototaxis behavior and inhibits the photoresponse in photoreceptor neurons, whereas other sensory behaviors are relatively less vulnerable to H2O2. Conversely, antioxidants can rescue the phenotype of lite-1 suppressor mutants and promote the photoresponse. As UV light illumination generates H2O2, we propose that upon light activation of LITE-1, light-produced H2O2 then deactivates LITE-1 to terminate the photoresponse, while antioxidants may promote LITE-1's recovery from its inactive state. Our studies provide a potential mechanism by which H2O2 and antioxidants act synergistically to regulate photosensation in C. elegans.
Subject(s)
Antioxidants/pharmacology , Caenorhabditis elegans Proteins/metabolism , Hydrogen Peroxide/pharmacology , Membrane Proteins/metabolism , Photoreceptor Cells/metabolism , Animals , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , Membrane Proteins/genetics , Mutation , Photoreceptor Cells/drug effects , Phototaxis , Suppression, GeneticABSTRACT
BACKGROUND: Inflammation and particularly interleukin-1ß (IL-1ß), a pro-inflammatory cytokine highly secreted by activated immune cells during early AMD pathological events, contribute significantly to retinal neurodegeneration. Here, we identify specific cell types that generate IL-1ß and harbor the IL-1 receptor (IL-1R) and pharmacologically validate IL-1ß's contribution to neuro-retinal degeneration using the IL-1R allosteric modulator composed of the amino acid sequence rytvela (as well as the orthosteric antagonist, Kineret) in a model of blue light-induced retinal degeneration. METHODS: Mice were exposed to blue light for 6 h and sacrificed 3 days later. Mice were intraperitoneally injected with rytvela, Kineret, or vehicle twice daily for 3 days. The inflammatory markers F4/80, NLRP3, caspase-1, and IL-1ß were assessed in the retinas. Single-cell RNA sequencing was used to determine the cell-specific expression patterns of retinal Il1b and Il1r1. Macrophage-induced photoreceptor death was assessed ex vivo using retinal explants co-cultured with LPS-activated bone marrow-derived macrophages. Photoreceptor cell death was evaluated by the TUNEL assay. Retinal function was assessed by flash electroretinography. RESULTS: Blue light markedly increased the mononuclear phagocyte recruitment and levels of inflammatory markers associated with photoreceptor death. Co-localization of NLRP3, caspase-1, and IL-1ß with F4/80+ mononuclear phagocytes was clearly detected in the subretinal space, suggesting that these inflammatory cells are the main source of IL-1ß. Single-cell RNA sequencing confirmed the immune-specific expression of Il1b and notably perivascular macrophages in light-challenged mice, while Il1r1 expression was found primarily in astrocytes, bipolar, and vascular cells. Retinal explants co-cultured with LPS/ATP-activated bone marrow-derived macrophages displayed a high number of TUNEL-positive photoreceptors, which was abrogated by rytvela treatment. IL-1R antagonism significantly mitigated the inflammatory response triggered in vivo by blue light exposure, and rytvela was superior to Kineret in preserving photoreceptor density and retinal function. CONCLUSION: These findings substantiate the importance of IL-1ß in neuro-retinal degeneration and revealed specific sources of Il1b from perivascular MPs, with its receptor Ilr1 being separately expressed on surrounding neuro-vascular and astroglial cells. They also validate the efficacy of rytvela-induced IL-1R modulation in suppressing detrimental inflammatory responses and preserving photoreceptor density and function in these conditions, reinforcing the rationale for clinical translation.
Subject(s)
Interleukin-1beta/immunology , Peptides/pharmacology , Photoreceptor Cells/pathology , Receptors, Interleukin-1/antagonists & inhibitors , Retinal Degeneration/pathology , Animals , Disease Models, Animal , Inflammation/immunology , Inflammation/pathology , Interleukin-1beta/metabolism , Macrophages/immunology , Macrophages/pathology , Male , Mice , Photoreceptor Cells/drug effects , Retinal Degeneration/immunologyABSTRACT
BACKGROUND: Retinitis pigmentosa (RP) is a group of inherited eye disorders with progressive degeneration of photoreceptors in the retina, ultimately leading to partial or complete blindness. The mechanisms underlying photoreceptor degeneration are not yet completely understood. Neuroinflammation is reported to play a pathological role in RP. However, the mechanisms that trigger neuroinflammation remain largely unknown. To address this question, we investigated the role of cyclooxygenase-1 (COX-1), a key enzyme in the conversion of arachidonic acid to proinflammatory prostaglandins, in the rd10 mouse model of RP. METHODS: We backcrossed COX-1 knockout mice (COX-1-/-) onto the rd10 mouse model of RP and investigated the impact of COX-1 deletion on neuroinflammation in the resulting COX-1-/-/rd10 mouse line, using a combination of immunocytochemistry, flow cytometry, qPCR, ELISA, and a series of simple visual tests. RESULTS: We found that genetic ablation or pharmacological inhibition of COX-1 alleviated neuroinflammation and subsequently preserved retinal photoreceptor and function and visual performance in rd10 mice. Moreover, we observed that the pharmacological inhibition of the prostaglandin E2 (PGE2) EP2 receptors largely replicated the beneficial effects of COX-1 deletion, suggesting that EP2 receptor was a critical downstream effector of COX-1-mediated neurotoxicity in rd10 mice. CONCLUSION: Our data suggest that the COX-1/PGE2/EP2 signaling pathway was partly responsible for significantly increased neuroinflammation and disease progression in rd10 mice, and that EP2 receptor could be targeted therapeutically to block the pathological activity of COX-1 without inducing any potential side effects in treating RP patients.
Subject(s)
Cyclooxygenase 1/deficiency , Disease Models, Animal , Inflammation Mediators/metabolism , Retinitis Pigmentosa/enzymology , Animals , Cell Line , Cyclooxygenase 1/genetics , Cyclooxygenase Inhibitors/pharmacology , Cyclooxygenase Inhibitors/therapeutic use , Electroretinography/methods , Inflammation Mediators/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Photoreceptor Cells/drug effects , Photoreceptor Cells/enzymology , Retinitis Pigmentosa/drug therapy , Retinitis Pigmentosa/geneticsABSTRACT
Which pathogenic mechanisms underlie age-related macular degeneration (AMD)? Are they different for dry and wet variants, or do they stem from common metabolic alterations? Where shall we look for altered metabolism? Is it the inner choroid, or is it rather the choroid-retinal border? Again, since cell-clearing pathways are crucial to degrade altered proteins, which metabolic system is likely to be the most implicated, and in which cell type? Here we describe the unique clearing activity of the retinal pigment epithelium (RPE) and the relevant role of its autophagy machinery in removing altered debris, thus centering the RPE in the pathogenesis of AMD. The cell-clearing systems within the RPE may act as a kernel to regulate the redox homeostasis and the traffic of multiple proteins and organelles toward either the choroid border or the outer segments of photoreceptors. This is expected to cope with the polarity of various domains within RPE cells, with each one owning a specific metabolic activity. A defective clearance machinery may trigger unconventional solutions to avoid intracellular substrates' accumulation through unconventional secretions. These components may be deposited between the RPE and Bruch's membrane, thus generating the drusen, which remains the classic hallmark of AMD. These deposits may rather represent a witness of an abnormal RPE metabolism than a real pathogenic component. The empowerment of cell clearance, antioxidant, anti-inflammatory, and anti-angiogenic activity of the RPE by specific phytochemicals is here discussed.
Subject(s)
Autophagy/genetics , Macular Degeneration/metabolism , Phytochemicals/therapeutic use , Retinal Pigment Epithelium/metabolism , Choroid/drug effects , Choroid/metabolism , Choroid/pathology , Humans , Macular Degeneration/drug therapy , Macular Degeneration/etiology , Macular Degeneration/pathology , Oxidation-Reduction , Photoreceptor Cells/drug effects , Photoreceptor Cells/metabolism , Retina/metabolism , Retina/pathology , Retinal Pigment Epithelium/pathologyABSTRACT
Age-related macular degeneration (AMD) is one of the leading causes of visual loss in western countries, it has no cure, and its incidence will grow in the future, for the overall population aging. Albino rats with retinal degeneration induced by exposure to high-intensity light (light-damage, LD) have been extensively used as a model of AMD to test neuroprotective agents. Among them, trophic factors (NGF and BDNF) have been shown to play a significant role in photoreceptors' survival. Interestingly, cord blood serum (CBS) is an extract full of chemokines and trophic factors; we, therefore, hypothesized that CBS could be an excellent candidate for neuroprotection. Here, we investigate whether CBS-based eye drops might mitigate the effects of light-induced retinal degeneration in albino rats. CBS treatment significantly preserved flash-electroretinogram (f-ERG) response after LD and reduced the "hot-spot" extension. Besides, CBS-treated animals better preserved the morphology of the outer nuclear layer, together with a reduction in microglia migration and activation. Interestingly, the treatment did not modulate reactive gliosis and activation of the self-protective mechanism (FGF2). In conclusion, our results suggest that CBS-based eye drops might be successfully used to mitigate retinal neurodegenerative processes such as AMD.
Subject(s)
Fetal Blood/chemistry , Macular Degeneration/drug therapy , Neuroprotective Agents/pharmacology , Ophthalmic Solutions/pharmacology , Photoreceptor Cells/drug effects , Animals , Epidermal Growth Factor/analysis , Epidermal Growth Factor/pharmacology , Female , Humans , Interleukins/analysis , Interleukins/pharmacology , Light/adverse effects , Macular Degeneration/etiology , Microglia/drug effects , Nerve Growth Factors/analysis , Nerve Growth Factors/pharmacology , Neuroprotective Agents/chemistry , Neuroprotective Agents/therapeutic use , Ophthalmic Solutions/chemistry , Ophthalmic Solutions/therapeutic use , Rats , Rats, Sprague-Dawley , Serum/chemistryABSTRACT
Photoreceptor cells are first-order retinal neurons that directly contribute to the formation of vision. Photoreceptor degeneration is the primary cause of vision impairment during the course of retinopathies such as retinitis pigmentosa and age-related macular degeneration, for which photoreceptor-targeted therapies are currently unavailable. Shihu Yeguang Pill (SYP), a classic formula in traditional Chinese medicine, has a long histology of clinical application for the treatment of a wide range of retinopathies in China. However, whether SYP is pharmacological effective at protecting photoreceptor cells is unclear. The current study thus directly addressed the pharmacological implications of SYP in photoreceptor degeneration in a mouse model characterized by bright light-induced retinal degeneration. Non-invasive full-retinal assessment was carried out to evaluate the effect of SYP on the retinal structure and function through optical coherence tomography and electroretinography, respectively. In addition, photoreceptor apoptosis, second-order neuron impairment and reactive changes in retinal microglial and müller cells, hallmark pathologies associated with photoreceptor degeneration, were assessed using immunohistochemistry and real-time PCR analyses. The results showed that SYP treatment attenuated bright light-induced impairment of the retinal structure and function. Moreover, SYP treatment suppressed photoreceptor apoptosis, alleviated the impairment of bipolar and horizontal cells and mitigated the reactive changes of müller and microglial cells in the bright light-exposed retinas. Real-time PCR analyses showed that dysregulated expression of pro-apoptotic c-fos and c-jun and anti-apoptotic bcl-2 as well as proinflammatory TNF-α in the bright light-exposed retinas was partially normalized as a result of SYP treatment. In summary, the work here demonstrates for the first time that SYP treatment protects the retinas from developing bright light-induced photoreceptor degeneration and associated alterations in second-order neurons and glial cells. The findings here thus provide experimental evidence to better support the mechanism-guided clinical application of SYP in the treatment of related retinal degenerative diseases.
Subject(s)
Apoptosis/drug effects , Drugs, Chinese Herbal/therapeutic use , Light/adverse effects , Photoreceptor Cells, Vertebrate/drug effects , Photoreceptor Cells/drug effects , Retina/drug effects , Retinal Degeneration/prevention & control , Animals , Drugs, Chinese Herbal/pharmacology , Electroretinography , Female , Medicine, Chinese Traditional , Mice, Inbred BALB C , Photoreceptor Cells/pathology , Photoreceptor Cells/radiation effects , Photoreceptor Cells, Vertebrate/pathology , Retina/pathology , Retina/radiation effects , Retinal Degeneration/etiologyABSTRACT
Combined administration of N-Methyl-D-Aspartate (NMDA) and kainic acid (KA) on the inner retina was studied as a model of excitotoxicity. The right eye of C57BL6J mice was injected with 1 µL of PBS containing NMDA 30 mM and KA 10 mM. Only PBS was injected in the left eye. One week after intraocular injection, electroretinogram recordings and immunohistochemistry were performed on both eyes. Retinal ganglion cell (RGC) projections were studied by fluorescent-cholerotoxin anterograde labeling. A clear decrease of the retinal "b" wave amplitude, both in scotopic and photopic conditions, was observed in the eyes injected with NMDA/KA. No significant effect on the "a" wave amplitude was observed, indicating the preservation of photoreceptors. Immunocytochemical labeling showed no effects on the outer nuclear layer, but a significant thinning on the inner retinal layers, thus indicating that NMDA and KA induce a deleterious effect on bipolar, amacrine and ganglion cells. Anterograde tracing of the visual pathway after NMDA and KA injection showed the absence of RGC projections to the contralateral superior colliculus and lateral geniculate nucleus. We conclude that glutamate receptor agonists, NMDA and KA, induce a deleterious effect of the inner retina when injected together into the vitreous chamber.
Subject(s)
Amacrine Cells/drug effects , Excitatory Amino Acid Agonists/toxicity , Kainic Acid/toxicity , N-Methylaspartate/toxicity , Photoreceptor Cells/drug effects , Retinal Ganglion Cells/drug effects , Amacrine Cells/pathology , Amacrine Cells/physiology , Animals , Cells, Cultured , Membrane Potentials , Mice , Mice, Inbred C57BL , Photoreceptor Cells/pathology , Photoreceptor Cells/physiology , Retinal Ganglion Cells/pathology , Retinal Ganglion Cells/physiology , Visual Pathways/drug effects , Visual Pathways/pathology , Visual Pathways/physiologyABSTRACT
Myeloid differentiation factor 88 (MyD88) is an adaptor protein for the Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) families of innate immunity receptors that mediate inflammatory responses to cellular injury. TLR/IL1R/MyD88 signaling is known to contribute to retinal degeneration, although how MyD88 regulates neuronal survival, and the effect of MyD88 on the inflammatory environment in the retina, is mostly unknown. In this study, we tested the hypothesis that blocking MyD88-mediated signaling early in retinal degeneration promotes transition of microglia towards a neuroprotective anti-inflammatory phenotype, resulting in enhanced photoreceptor survival. We also tested whether systemic delivery of a pharmacologic MyD88 inhibitor has therapeutic potential. The rd10 mouse model of retinal degeneration was injected intraperitoneally with increasing doses of a MyD88 blocking peptide or control peptide early in degeneration, and inflammatory responses and photoreceptor survival were measured at specific time points using flow cytometry, cytokine profiling, and electroretinograms. Our results demonstrated that rd10 mice injected with a low dose of MyD88 inhibitor peptide showed increased rod photoreceptor function and reduced apoptosis compared with control peptide and uninjected mice. MyD88 inhibition also resulted in fewer microglia/macrophage cells in the photoreceptor layer whereas total peripheral and retinal macrophage were not changed. Furthermore, increased number of cells expressing the Arg1 marker of neuroprotective microglia in the photoreceptor layer and higher MCP-1 and anti-inflammatory cytokine IL-27 were associated with photoreceptor survival. Therefore, these data suggest that the MyD88 inhibitor modified the retina environment to become less inflammatory, leading to improved photoreceptor function and survival.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Microglia/drug effects , Myeloid Differentiation Factor 88/antagonists & inhibitors , Oligopeptides/pharmacology , Photoreceptor Cells/drug effects , Retinal Degeneration/drug therapy , Animals , Anti-Inflammatory Agents/therapeutic use , Apoptosis , Arginase/genetics , Arginase/metabolism , Cells, Cultured , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Female , Interleukin-27/genetics , Interleukin-27/metabolism , Male , Mice , Microglia/metabolism , Oligopeptides/therapeutic use , Photoreceptor Cells/metabolismABSTRACT
Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661 W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.