Mouse Model Of Age-related Macular Degeneration Research Articles

Ginsenoside Rg3 Improved Age-Related Macular Degeneration Through Inhibiting ROS-Mediated Mitochondrion-Dependent Apoptosis In Vivo and In Vitro.

Age-related macular degeneration (AMD) is marked by a progressive loss of central vision and is the third leading cause of irreversible blindness worldwide. The exact mechanisms driving the progression of this macular degenerative condition remain elusive, and as of now, there are no available preventative measures for dry AMD. According to ancient records, ginseng affects the eyes by brightening them and enhancing wisdom. Modern pharmacological research shows that the active ingredients in ginseng, ginsenosides, may be used to prevent or improve eye diseases that threaten vision. Some articles have reported that ginsenoside Rg3 can treat diabetic retinopathy in mice, but no reports exist on its effects and mechanisms in AMD. Therefore, the role and mechanism of ginsenoside Rg3 in AMD warrant further study. This study aims to investigate the effects of Rg3 on AMD and its underlying molecular mechanisms. We established a mouse model of AMD to examine the impact of ginsenoside Rg3 on NaIO3-induced apoptosis in the retina and to explore the related intrinsic mechanisms. The in vivo results indicated that ginsenoside Rg3 prevents NaIO3-induced apoptosis in retinal pigment epithelial cells by inhibiting reactive oxygen species production and preventing the reduction in mitochondrial membrane potential. Additionally, we assessed the levels of protein expression within the apoptosis pathway. Ginsenoside Rg3 decreased the expression of Bax, cleaved caspase-3, and cleaved caspase-9 proteins. Additionally, it increased the expression of Bcl-2 by decreasing P-JNK levels. Moreover, our in vivo results showed that ginsenoside Rg3 enhanced retinal structure, increased the relative thickness of the retina, and decreased the extent of disorganization in both the inner and outer nuclear layers. Ginsenoside Rg3 may safeguard the retina against NaIO3-induced cell apoptosis by attenuating reactive-oxygen-species-mediated mitochondrial dysfunction, in which the JNK signaling pathway is also involved. These findings suggest that ginsenoside Rg3 has the potential to prevent or attenuate the progression of AMD and other retinal pathologies associated with NaIO3-mediated apoptosis.

Retinal pigment epithelium-specific ablation of GPx4 in adult mice recapitulates key features of geographic atrophy in age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in the elderly population, particularly the late-stage of dry AMD known as geographic atrophy (GA), lacks effective treatment options. Genetic mouse models of AMD have revealed the significance of impaired lipid metabolism and anti-oxidative capacity in early/intermediate stage of AMD, but remains unclear in GA that severely damages visual function. Here, to investigate the potential relevance of peroxidized lipids in RPE for late-stage dry AMD, GPx4fl/fl mice underwent subretinal injections of RPE-specific AAV-Cre vector or control AAV vector. RPE-specific GPx4 deficiency led to rapid RPE degeneration resembling key features of late-stage dry AMD, including preceding loss of RPE cell polarity, accumulation of acrolein, malondialdehyde, and 4-hydroxynonenal, photoreceptor loss, lipofuscin-laden subretinal melanophage infiltration, and complement activation. Treatment with α-tocopherol and ferrostatin-1 mitigated RPE degeneration, and shrunk mitochondria were observed in GPx4 deficient mice, suggesting involvement of ferroptosis. Unexpectedly, necrostatin-1s, an inhibitor of necroptosis, also ameliorated RPE degeneration, and activation of RIP3 and MLKL along with inactivation of caspase-8 was observed, indicating crosstalk between ferroptosis and necroptosis pathways. Our findings shed light on the intricate mechanisms underlying RPE degeneration in AMD and highlight GPx4/lipid peroxidation as potential therapeutic targets. RPE-specific ablation of GPx4 in mice provides a valuable tool for further elucidating the interplay between lipid peroxidation, cell death pathways, and AMD pathogenesis, offering new insights for preclinical research and therapeutic development targeting GA.

Novel Therapeutic Effects of Euphorbia heterophylla L. Methanol Extracts in Macular Degeneration Caused by Blue Light in A2E-Laden ARPE-19 Cells and Retina of BALB/c Mice.

Natural products with high antioxidant activity are considered as innovative prevention strategies to effectively prevent age-related macular degeneration (AMD) in the early stage because the generation of reactive oxygen species (ROS) leading to the development of drusen is reported as an important cause of this disease. To investigate the prevention effects of the methanol extracts of Euphorbia heterophylla L. (MEE) on AMD, its effects on the antioxidant activity, inflammatory response, apoptosis pathway, neovascularization, and retinal tissue degeneration were analyzed in N-retinylidene-N-retinylethanolamine (A2E)-landed spontaneously arising retinal pigment epithelia (ARPE)-19 cells and BALB/c mice after exposure to blue light (BL). The MEE contained 10 active components and showed high free radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and nitric oxide (NO) radicals. The pretreatments of high-dose MEE remarkably suppressed the production of intracellular ROS (88.2%) and NO (25.2%) and enhanced (SOD) activity (84%) and the phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) in A2E + BL-treated ARPE-19 cells compared to Vehicle-treated group. The activation of the inducible nitric oxide synthase (iNOS)-induced cyclooxygenase-2 (COX-2) mediated pathway, inflammasome activation, and expression of inflammatory cytokines was significantly inhibited in A2E + BL-treated ARPE-19 cells after the MEE pretreatment. The activation of the apoptosis pathway and increased expression of neovascular proteins (36% for matrix metalloproteinase (MMP)-9) were inhibited in the MEE pretreated groups compared to the Vehicle-treated group. Furthermore, the thickness of the whole retina (31%), outer nuclear layer (ONL), inner nuclear layer (INL), and photoreceptor layer (PL) were significantly increased by the MEE pretreatment of BALB/c mice with BL-induced retinal degeneration. Therefore, these results suggest that the MEE, with its high antioxidative activity, protects against BL-induced retinal degeneration through the regulation of the antioxidative system, inflammatory response, apoptosis, and neovascularization in the AMD mouse model.

Protective effect of ghrelin in oxidative stress-induced age-related macular degeneration in vitro and in vivo

Oxidative damage to human retinal pigment epithelial (RPE) cells is the main cause of age-related macular degeneration (AMD), in our previous work, we showed that ghrelin has an antioxidative effect on human lens epithelium (HLE) cells, however, the studies of using ghrelin in treating the degenerative diseases of the retina have rarely been reported. In this article, we assessed the effect of ghrelin on preventing oxidative stress induced by hydrogen peroxide (H2O2) in ARPE-19 cells and its mechanism. We observed that pretreatment with ghrelin protected ARPE-19 cells from H2O2-induced cell oxidative injuries and apoptosis responses. Furthermore, an oxidative stress-induced mouse model of AMD was established via injection of sodium iodate (NaIO3) to tail veins, and treatment with ghrelin preserved retinal function, and protected photoreceptors.

Pre-Clinical Studies of a Novel Bispecific Fusion Protein Targeting C3b and VEGF in Neovascular and Nonexudative AMD Models.

KNP-301 is a bi-specific fragment crystallizable region (Fc) fusion protein, which inhibits both C3b and vascular endothelial growth factor (VEGF) simultaneously for patients with late-stage age-related macular degeneration (AMD). The present study evaluated in vitro potency, in vivo efficacy, intravitreal pharmacokinetics (IVT PK), and injectability of KNP-301. C3b and VEGF binding of KNP-301 were assessed by surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA), and cellular bioassays. A laser-induced choroidal neovascularization (CNV) model and a sodium iodate-induced nonexudative AMD model were used to test the in vivo efficacy of mouse surrogate of KNP-301. Utilizing fluorescein angiography (FA) and spectral-domain optical coherence tomography (SD-OCT) scans, the reduction in disease lesions were analyzed in a CNV mouse model. In the nonexudative AMD mouse model, outer nuclear layer (ONL) was assessed by immunofluorescence staining. Lastly, intravitreal pharmacokinetic study was conducted with New Zealand white rabbits via IVT administration of KNP-301 and injectability of KNP-301 was examined by a viscosity test at high concentrations. KNP-301 bound C3b selectively, which resulted in a blockade of the alternative pathway, not the classical pathway. KNP-301 also acted as a VEGF trap, impeding VEGF-mediate signaling. Our dual-blockade strategy was effective in both neovascular and nonexudative AMD models. Moreover, KNP-301 had an advantage of potentially less frequent dosing due to the long half-life in the intravitreal chamber. Our viscosity assessment confirmed that KNP-301 meets the criteria of the IVT injection. Unlike current therapies, KNP-301 is expected to cover patients with late-stage AMD of both neovascular and nonexudative AMD, and its long-term PK profile at the intravitreal chamber would allow convenience in the dosing interval of patients.

Contribution of ferroptosis and SLC7A11 to light-induced photoreceptor degeneration.

Progressive photoreceptor cell death is one of the main pathological features of age-related macular degeneration and eventually leads to vision loss. Ferroptosis has been demonstrated to be associated with retinal degenerative diseases. However, the molecular mechanisms underlying ferroptosis and photoreceptor cell death in age-related macular degeneration remain largely unexplored. Bioinformatics and biochemical analyses in this study revealed xC-, solute carrier family 7 member 11-regulated ferroptosis as the predominant pathological process of photoreceptor cell degeneration in a light-induced dry age-related macular degeneration mouse model. This process involves the nuclear factor-erythroid factor 2-related factor 2-solute carrier family 7 member 11-glutathione peroxidase 4 signaling pathway, through which cystine depletion, iron ion accumulation, and enhanced lipid peroxidation ultimately lead to photoreceptor cell death and subsequent visual function impairment. We demonstrated that solute carrier family 7 member 11 overexpression blocked this process by inhibiting oxidative stress in vitro and in vivo. Conversely, solute carrier family 7 member 11 knockdown or the solute carrier family 7 member 11 inhibitor sulfasalazine and ferroptosis-inducing agent erastin aggravated H2O2-induced ferroptosis of 661W cells. These findings indicate solute carrier family 7 member 11 may be a potential therapeutic target for patients with retinal degenerative diseases including age-related macular degeneration.

Identification of the CDH18 gene associated with age-related macular degeneration using weighted gene co-expression network analysis.

Purpose: Age-related macular degeneration (AMD) is a chronic and progressive macular degenerative disease that culminates in a gradual deterioration of central vision. Despite its prevalence, the key biomarkers for AMD have not yet been fully elucidated. In this study, we aimed to efficiently identify biomarkers crucial for diagnosing AMD. Methods: Three datasets pertaining to retinal pigment epithelium (RPE)/choroid tissues associated with AMD were selected from the GEO database. The GSE50195 dataset was utilized to conduct weighted gene co-expression network analysis (WGCNA) for identifying module genes linked to AMD. KEGG and GO enrichment analyses were subsequently conducted on these module genes. GSE29801 and GSE135092 datasets were subjected to differential expression analysis to pinpoint the DEGs intersecting with the module genes. Subsequently, wet AMD (wAMD) and dry AMD (dAMD) mouse models were developed, from which RPE/choroid tissues were harvested to validate the hub genes via RT-qPCR and Western blot. Results: Using the WGCNA, we selected the "antiquewhite4" module (r = 0.91 and p = 7e-07), which contains a total of 325 genes. Through the intersection of module genes with DEGs, nine hub genes were identified. Pathways involved in complement and coagulation cascades, ECM-receptor interactions, unsaturated fatty acid biosynthesis, and fatty acid elongation play important roles in AMD. Notably, CDH18 demonstrated notable variance across all three datasets. Post validation using RT-qPCR experiments revealed a significant downregulation of CDH18 in both dAMD and wAMD. EGLN3 was expressed at low levels in wAMD. In dAMD, EYA2, LTB, and PODXL were significantly downregulated, whereas APOC1 was notably upregulated. Western blot confirmed that CDH18 was lowly expressed in dAMD and wAMD mouse models. Conclusion: CDH18 was identified as the key gene involved in the pathogenesis of AMD. An imbalance of the complement and coagulation cascades is a potential mechanism of AMD. This study provides a novel idea for diagnosing and treating AMD in the future.

Retinoic acid related orphan receptor α is a genetic modifier that rescues retinal degeneration in a mouse model of Stargardt disease and Dry AMD

Degeneration of the macula is associated with several overlapping diseases including age-related macular degeneration (AMD) and Stargardt Disease (STGD). Mutations in ATP Binding Cassette Subfamily A Member 4 (ABCA4) are associated with late-onset dry AMD and early-onset STGD. Additionally, both forms of macular degeneration exhibit deposition of subretinal material and photoreceptor degeneration. Retinoic acid related orphan receptor α (RORA) regulates the AMD inflammation pathway that includes ABCA4, CD59, C3 and C5. In this translational study, we examined the efficacy of RORA at attenuating retinal degeneration and improving the inflammatory response in Abca4 knockout (Abca4−/−) mice. AAV5-hRORA-treated mice showed reduced deposits, restored CD59 expression and attenuated amyloid precursor protein (APP) expression compared with untreated eyes. This molecular rescue correlated with statistically significant improvement in photoreceptor function. This is the first study evaluating the impact of RORA modifier gene therapy on rescuing retinal degeneration. Our studies demonstrate efficacy of RORA in improving STGD and dry AMD-like disease.

PolySialic Acid Nanoparticles Actuate Complement-Factor-H-Mediated Inhibition of the Alternative Complement Pathway: A Safer Potential Therapy for Age-Related Macular Degeneration.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

Targeting ZIP8 mediated ferroptosis as a novel strategy to protect against the retinal pigment epithelial degeneration

The degeneration of retinal pigment epithelium (RPE) plays an important role in the development of age-related macular degeneration (AMD). However, the underlying mechanism remains elusive. In this study, we identified that ZIP8, a metal-ion transporter, plays a crucial role in the degeneration of RPE cells mediated by ferroptosis. ZIP8 was found to be upregulated in patients with AMD through transcriptome analysis. Upregulated ZIP8 was also observed in both oxidative-stressed RPE cells and AMD mouse model. Importantly, knockdown of ZIP8 significantly inhibited ferroptosis in RPE cells induced by sodium iodate-induced oxidative stress. Blocking ZIP8 with specific antibodies reversed RPE degeneration and restored retinal function, improving visual loss in a mouse model of NaIO3-induced. Interestingly, the modification of the N-glycosylation sites N40, N72 and N88, but not N273, was essential for the intracellular iron accumulation mediated by ZIP8, which further led to increased lipid peroxidation and RPE death. These findings highlight the critical role of ZIP8 in RPE ferroptosis and provide a potential target for the treatment of diseases associated with retinal degeneration, including AMD.

Microglia at sites of atrophy restrict the progression of retinal degeneration via galectin-3 and Trem2.

Outer retinal degenerations, including age-related macular degeneration (AMD), are characterized by photoreceptor and retinal pigment epithelium (RPE) atrophy. In these blinding diseases, macrophages accumulate at atrophic sites, but their ontogeny and niche specialization remain poorly understood, especially in humans. We uncovered a unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and human AMD. In disease models, conditional deletion of galectin-3 in microglia led to phagocytosis defects and consequent augmented photoreceptor death, RPE damage, and vision loss, indicating protective roles. Mechanistically, Trem2 signaling orchestrated microglial migration to atrophic sites and induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection but in a galectin-3-dependent manner. In elderly human subjects, we identified this highly conserved microglial population that expressed galectin-3 and Trem2. This population was significantly enriched in the macular RPE-choroid of AMD subjects. Collectively, our findings reveal a neuroprotective population of microglia and a potential therapeutic target for mitigating retinal degeneration.

Fluorescence Lifetime Imaging Ophthalmoscopy of Mouse Models of Age-related Macular Degeneration.

To investigate fluorescence lifetime of mouse models of age-related macular degeneration (AMD) by fluorescence lifetime imaging ophthalmoscopy (FLIO). Two AMD mouse models, apolipoprotein E knockout (ApoE-/-) mice and NF-E2-related factor-2 knockout (Nrf2-/-) mice, and their wild-type mice underwent monthly ophthalmic examinations including FLIO from 3months of age. After euthanasia at the age of 6 or 11months, blood plasma was collected to determine total antioxidant capacity and eyes were enucleated for Oil red O (ORO) lipid staining of chorioretinal tissue. In FLIO, the mean fluorescence lifetime (τm) of wild type shortened with age in both spectral channels. In short spectral channel, τm shortening was observed in both AMD models as well, but its rate was more pronounced in ApoE-/- mice and significantly different from the other strains as months of age progressed. In contrast, in long spectral channel, both model strains showed completely opposite trends, with τm becoming shorter in ApoE-/- and longer in Nrf2-/- mice than the others. Oil red O staining at Bruch's membrane was significantly stronger in ApoE-/- mice at 11months than the other strains. Plasma total antioxidant capacity was highest in ApoE-/- mice at both 6 and 11months. The two AMD mouse models exhibited largely different fundus fluorescence lifetime, which might be related to the different systemic metabolic state. FLIO might be able to indicate different metabolic states of eyes at risk for AMD. This animal study may provide new insights into the relationship between early AMD-associated metabolic changes and FLIO findings.

Age-Related Changes in the Glycolytic Enzymes of M2-Isoform of Pyruvate Kinase and Fructose-1,6-Bisphosphate Aldolase: Implications to Age-Related Macular Degeneration.

Prior studies have emphasized a bioenergetic crisis in the retinal pigment epithelium (RPE) as a critical factor in the development of age-related macular degeneration (AMD). The isoforms Fructose-1,6-bisphosphate aldolase C (ALDOC) and pyruvate kinase M2 (PKM2) have been proposed to play a role in AMD pathogenesis. While PKM2 and ALDOC are crucial for aerobic glycolysis in the neural retina, they are not as essential for the RPE. In this study, we examined the expression and activity of PKM2 and ALDOC in both young and aged RPE cells, as well as in the retina and RPE tissue of mice, including an experimentally induced AMD mouse model. Our findings reveal an upregulation in PKM2 and ALDOC expression, accompanied by increased pyruvate kinase activity, in the aged and AMD mouse RPE. Conversely, there is a decrease in ALDOC expression but an increase in PKM2 expression and pyruvate kinase activity in the aged and AMD retina. Overall, our study indicates that aged and AMD RPE cells tend to favor aerobic glycolysis, while this tendency is diminished in the aged and AMD retina. These results underscore the significance of targeting PKM2 and ALDOC in the RPE as a promising therapeutic approach to address the bioenergetic crisis and prevent vision loss in AMD.

Mesoporous Cerium Oxide Nanoparticles with High Scavenging Properties of Reactive Oxygen Species for Treating Age‐Related Macular Degeneration

Age‐related macular degeneration (AMD), the leading cause of vision loss among older individuals, is characterized by damage to photoreceptors and retinal pigment epithelial cells (RPEs). Oxidative stress and chronic inflammation in the retina play notable roles in AMD pathogenesis, rendering them attractive therapeutic targets. Cerium oxide nanoparticles (CeNPs) have shown promise in scavenging reactive oxygen species (ROS) by mimicking antioxidant enzymes, whereas mesoporous materials have emerged as versatile drug carriers. Herein, mesoporous CeNPs (mCeNPs) that integrate the advantages of CeNPs and mesoporous materials are presented. The mCeNPs can be synthesized using 1,1′‐carbonyldiimidazole and imidazole in acetone without heating and pressurization. The resulting mCeNPs exhibit mesoporous structures comprising assembled small CeNPs, exerting excellent ROS‐scavenging capabilities, biocompatibility, and cytoprotective and anti‐inflammatory effects against H2O2‐induced damage in RPEs. Using a sodium iodate‐induced AMD mouse model, it is demonstrated that intravitreal mCeNP administration can exhibit disease‐preventive effects. These findings indicate the therapeutic potential of mCeNPs against AMD.

Thrombospondin-1 proteomimetic polymers exhibit anti-angiogenic activity in a neovascular age-related macular degeneration mouse model.

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the developed world. Current therapy includes monthly intraocular injections of anti-VEGF antibodies, which are ineffective in up to one third of patients. Thrombospondin-1 (TSP1) inhibits angiogenesis via CD36 binding, and its down-regulated expression is negatively associated with the onset of nAMD. Here, we describe TSP1 mimetic protein-like polymers (TSP1 PLPs). TSP1 PLPs bind CD36 with high affinity, resist degradation, show prolonged intraocular half-lives (13.1 hours), have no toxicity at relevant concentrations in vivo (40 μM), and are more efficacious in ex vivo choroidal sprouting assays compared to the peptide sequence and Eylea (aflibercept), the current standard of care anti-VEGF treatment. Furthermore, PLPs exhibit superior in vivo efficacy in a mouse model for nAMD compared to control PLPs consisting of scrambled peptide sequences, using fluorescein angiography and immunofluorescence. Since TSP-1 inhibits angiogenesis by VEGF-dependent and independent mechanisms, TSP1 PLPs are a potential therapeutic for patients with anti-VEGF treatment-resistant nAMD.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Supramolecular Senolytics via Intracellular Oligomerization of Peptides in Response to Elevated Reactive Oxygen Species Levels in Aging Cells.

Senolytics, which eliminate senescent cells from tissues, represent an emerging therapeutic strategy for various age-related diseases. Most senolytics target antiapoptotic proteins, which are overexpressed in senescent cells, limiting specificity and inducing severe side effects. To overcome these limitations, we constructed self-assembling senolytics targeting senescent cells with an intracellular oligomerization system. Intracellular aryl-dithiol-containing peptide oligomerization occurred only inside the mitochondria of senescent cells due to selective localization of the peptides by RGD-mediated cellular uptake into integrin αvβ3-overexpressed senescent cells and elevated levels of reactive oxygen species, which can be used as a chemical fuel for disulfide formation. This oligomerization results in an artificial protein-like nanoassembly with a stable α-helix secondary structure, which can disrupt the mitochondrial membrane via multivalent interactions because the mitochondrial membrane of senescent cells has weaker integrity than that of normal cells. These three specificities (integrin αvβ3, high ROS, and weak mitochondrial membrane integrity) of senescent cells work in combination; therefore, this intramitochondrial oligomerization system can selectively induce apoptosis of senescent cells without side effects on normal cells. Significant reductions in key senescence markers and amelioration of retinal degeneration were observed after elimination of the senescent retinal pigment epithelium by this peptide senolytic in an age-related macular degeneration mouse model and in aged mice, and this effect was accompanied by improved visual function. This system provides a strategy for the treatment of age-related diseases using supramolecular senolytics.

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Repeated Topical Administration of 3 nm Cerium Oxide Nanoparticles Reverts Disease Atrophic Phenotype and Arrests Neovascular Degeneration in AMD Mouse Models.

Increased oxidative stress in the retina and retinal pigment epithelium is implicated in age-related macular degeneration (AMD). Antioxidant cerium oxide nanoparticles (CeO2NPs) have been used to treat degenerative retinal pathologies in animal models, although their delivery route is not ideal for chronic patient treatment. In this work, we prepared a formulation for ocular topical delivery that contains small (3 nm), nonaggregated biocompatible CeO2NPs. In vitro results indicate the biocompatible and protective character of the CeO2NPs, reducing oxidative stress in ARPE19 cells and inhibiting neovascularization related to pathological angiogenesis in both HUVEC and in in vitro models of neovascular growth. In the in vivo experiments, we observed the capacity of CeO2NPs to reach the retina after topical delivery and a subsequent reversion of the altered retinal transcriptome of the retinal degenerative mouse model DKOrd8 toward that of healthy control mice, together with signs of decreased inflammation and arrest of degeneration. Furthermore, CeO2NP eye drops' treatment reduced laser-induced choroidal neovascular lesions in mice by lowering VEGF and increasing PEDF levels. These results indicate that CeO2NP eye drops are a beneficial antioxidant and neuroprotective treatment for both dry and wet forms of AMD disease.

Retinal protection by fungal product theissenolactone B in a sodium iodate-induced AMD model through targeting retinal pigment epithelial matrix metalloproteinase-9 and microglia activity

Age-related macular degeneration (AMD) is the leading cause of low vision and blindness for which there is currently no cure. Increased matrix metalloproteinase-9 (MMP-9) was found in AMD and potently contributes to its pathogenesis. Resident microglia also promote the processes of chronic neuroinflammation, accelerating the progression of AMD. The present study investigates the effects and mechanisms of the natural compound theissenolactone B (LB53), isolated from Theissenia cinerea, on the effects of RPE dysregulation and microglia hyperactivation and its retinal protective ability in a sodium iodate (NaIO3)-induced retinal degeneration model of AMD. The fungal component LB53 significantly reduces MMP-9 gelatinolysis in TNF-α-stimulated human RPE cells (ARPE-19). Similarly, LB53 abolishes MMP-9 protein and mRNA expression in ARPE-19 cells. Moreover, LB53 efficiently suppresses nitric oxide (NO) production, iNOS expression, and intracellular ROS levels in LPS-stimulated TLR 4-activated microglial BV-2 cells. According to signaling studies, LB53 specifically targets canonical NF-κB signaling in both ARPE-19 and BV-2 microglia. In an RPE-BV-2 interaction assay, LB53 ameliorates LPS-activated BV-2 conditioned medium-induced MMP-9 activation and expression in the RPE. In NaIO3-induced AMD mouse model, LB53 restores photoreceptor and bipolar cell dysfunction as assessed by electroretinography (ERG). Additionally, LB53 prevents retinal thinning, primarily the photoreceptor, and reduces retinal blood flow from NaIO3 damage evaluated by optic coherence tomography (OCT) and laser speckle flowgraphy (LSFG), respectively. Our results demonstrate that LB53 exerts neuroprotection in a mouse model of AMD, which can be attributed to its anti-retinal inflammatory effects by impeding RPE-mediated MMP-9 activation and anti-microglia.