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OBJECTIVE: To evaluate the safety and tolerability of a mineralocorticoid, in a single-dose intravitreal (IVT) injection of 1 mg/0.1 mL and 2 mg/0.1 mL fludrocortisone acetate (FCA) in subjects with geographical atrophy (GA) secondary to age-related macular degeneration. METHODS AND ANALYSIS: This phase 1b study was a two-part dose-escalation prospective study. Part 1 involved a single participant treated with 1 mg/0.1 mL and monitored up to 28 days before being reviewed by a safety review committee. Two subsequent participants were then dosed with the same dose. Part 2 involved a single participant dosed with 2 mg/0.1 mL and monitored up to 28 days when a further five participants were dosed. All participants were followed up for 6 months after baseline.A full ophthalmic assessment was performed at study visits which included GA area, best-corrected visual acuity (BCVA), low-luminance BCVA (LL-BCVA) and intraocular pressure (IOP). Adverse events (AEs) were reported from the first dose of FCA until the end-of-study visit. RESULTS: There were no serious AEs (ocular or systemic) observed with IVT FCA at either 1 mg/0.1 mL or 2 mg/0.1 mL among nine participants. There was no evidence of increased IOP or cataract development.Neither BCVA or LL-BCVA changed significantly in the study-eye over the follow-up period (p=0.28 and 0.38, respectively). Mean GA area increased in the study (0.5 mm2, p=0.003) and fellow-eyes (0.62 mm2, p=0.02) over 6 months. Differences between eyes were not significant (p=0.64), and at the lower end of population norms. CONCLUSION: IVT FCA is clinically safe and well tolerated and did not increase IOP.
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Atrofia Geográfica , Mineralocorticoides , Atrofia , Fludrocortisona/análogos & derivados , Atrofia Geográfica/tratamiento farmacológico , Humanos , Mineralocorticoides/efectos adversos , Estudios Prospectivos , Agudeza VisualRESUMEN
The pathogenesis of outer retinal degenerations has been linked to the elevation of cytokines that orchestrate pro-inflammatory responses within the retinal milieu, and which are thought to play a role in diseases such as geographic atrophy (GA), an advanced form of AMD. Here we sought investigate the anti-inflammatory and mechanistic properties of fludrocortisone (FA), as well as triamcinolone acetonide (TA), on Müller cell-mediated cytokine expression in response to inflammatory challenge. In addition, we investigated the neuroprotective efficacy of FA and TA in a photo-oxidative damage (PD), a model of outer retinal degeneration. Expression of CCL2, IL-6, and IL-8 with respect to FA and TA were assessed in Müller cells in vitro, following simulation with IL-1ß or TNF-α. The dependency of this effect on mineralocorticoid and glucocorticoid signaling was also interrogated for both TA and TA via co-incubation with steroid receptor antagonists. For the PD model, C57BL/6 mice were intravitreally injected with FA or TA, and changes in retinal pathology were assessed via electroretinogram (ERG) and optical coherence tomography (OCT). FA and TA were found to dramatically reduce the expression of CCL2, IL-6, and IL-8 in Müller glia in vitro after inflammatory challenge with IL-1ß or TNF-α (P < 0.05). Though FA acts as both a mineralocorticoid and glucocorticoid receptor agonist, co-incubation with selective steroid antagonists revealed that the suppressive effect of FA on CCL2, IL-6, and IL-8 expression is mediated by glucocorticoid signaling (P < 0.05). In PD, intravitreal FA was found to ameliorate outer-retinal atrophy as measured by ERG and OCT (P < 0.05), while TA had no significant effect (P > 0.05). Our data indicate potent anti-inflammatory and mechanistic properties of corticosteroids, specifically FA, in suppressing inflammation and neurodegeneration degeneration associated with outer retinal atrophy. Taken together, our findings indicate that corticosteroids such as FA may have value as a potential therapeutic for outer retinal degenerations where such pro-inflammatory factors are implicated, including AMD.
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Fludrocortisona/farmacología , Neuroprotección , Degeneración Retiniana/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patologíaRESUMEN
BACKGROUND: MicroRNA (miRNA) play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the RNA Induced Silencing Complex (RISC). Identifying the retinal miRNA/mRNA interactions in health and disease will provide important insight into the key pathways miRNA regulate in disease pathogenesis and may lead to potential therapeutic targets to mediate retinal degeneration. METHODS: To identify the active miRnome targetome interactions in the healthy and degenerating retina, AGO2 HITS-CLIP was performed using a rodent model of photoreceptor degeneration. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data was performed to identify the cellular location of AGO2 and key members of the microRNA targetome in the retina. AGO2 findings were verified by in situ hybridization (RNA) and immunohistochemistry (protein). RESULTS: Analysis revealed a similar miRnome between healthy and damaged retinas, however, a shift in the active targetome was observed with an enrichment of miRNA involvement in inflammatory pathways. This shift was further demonstrated by a change in the seed binding regions of miR-124-3p, the most abundant retinal AGO2-bound miRNA, and has known roles in regulating retinal inflammation. Additionally, photoreceptor cluster miR-183/96/182 were all among the most highly abundant miRNA bound to AGO2. Following damage, AGO2 expression was localized to the inner retinal layers and more in the OLM than in healthy retinas, indicating a locational miRNA response to retinal damage. CONCLUSIONS: This study provides important insight into the alteration of miRNA regulatory activity that occurs as a response to retinal degeneration and explores the miRNA-mRNA targetome as a consequence of retinal degenerations. Further characterisation of these miRNA/mRNA interactions in the context of the degenerating retina may provide an important insight into the active role these miRNA may play in diseases such as AMD.
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Proteínas del Ojo/genética , Degeneración Macular/metabolismo , MicroARNs/genética , Retina/metabolismo , Animales , Proteínas Argonautas/metabolismo , Modelos Animales de Enfermedad , Proteínas del Ojo/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoprecipitación , Inflamación , Luz/efectos adversos , Degeneración Macular/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/aislamiento & purificación , MicroARNs/metabolismo , Estrés Oxidativo , Complejo Silenciador Inducido por ARN/metabolismo , Degeneración Retiniana/etiología , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Análisis de la Célula Individual , TranscriptomaRESUMEN
Purpose: Dysregulation of the complement cascade contributes to a variety of retinal dystrophies, including age-related macular degeneration (AMD). The central component of complement, C3, is expressed in abundance by macrophages in the outer retina, and its ablation suppresses photoreceptor death in experimental photo-oxidative damage. Whether this also influences macrophage reactivity in this model system, however, is unknown. We investigate the effect of C3 ablation on macrophage activity and phagocytosis by outer retinal macrophages during photo-oxidative damage. Methods: Age-matched C3 knockout (KO) mice and wild-type (WT) C57/Bl6 mice were subjected to photo-oxidative damage. Measurements of the outer nuclear layer (ONL) thickness and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess pathology and photoreceptor apoptosis, respectively. Macrophage abundance and phagocytosis were assessed with immunolabeling for pan-macrophage and phagocytic markers, in conjunction with TUNEL staining in cohorts of C3 KO and WT mice. Results: The C3 KO mice exhibited protection against photoreceptor cell death following photo-oxidative damage, which was associated with a reduction in immunoreactivity for the stress-related factor GFAP. In conjunction, there was a reduction in IBA1-positive macrophages in the outer retina compared to the WT mice and a decrease in the number of CD68-positive cells in the outer nuclear layer and the subretinal space. In addition, the engulfment of TUNEL-positive and -negative photoreceptors by macrophages was significantly lower in the C3 KO mice cohort following photo-oxidative damage compared to the WT cohort. Conclusions: The results show that the absence of C3 mitigates the phagocytosis of photoreceptors by macrophages in the outer retina, and the net impact of C3 depletion is neuroprotective in the context of photo-oxidative damage. These data improve our understanding of the impact of C3 inhibition in subretinal inflammation and inform the development of treatments for targeting complement activation in diseases such as AMD.
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Complemento C3/genética , Macrófagos/metabolismo , Estrés Oxidativo/efectos de la radiación , Fagocitosis/genética , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Animales , Apoptosis/genética , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Luz , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Retina/efectos de la radiación , Degeneración Retiniana/patologíaRESUMEN
Purpose: To examine the foveal avascular zone (FAZ) in patients with congenital achromatopsia (ACHM). Methods: Forty-two patients with genetically confirmed ACHM were imaged either with Optovue's AngioVue system or Zeiss's Plex Elite 9000, and the presence or absence of a FAZ was determined. For images where a FAZ was present and could be confidently segmented, FAZ area, circularity index, and roundness were measured and compared with previously published normative values. Structural optical coherence tomography images were acquired to assess the degree of foveal hypoplasia (number and thickness of inner retinal layers present at the fovea). Results: A FAZ was present in 31 of 42 patients imaged (74%), although no determination could be made for 11 patients due to poor image quality (26%). The mean ± SD FAZ area for the ACHM retina was 0.281 ± 0.112 mm2, which was not significantly different from the previously published normative values (P = 0.94). However, their FAZs had decreased circularity (P < 0.0001) and decreased roundness (P < 0.0001) compared to the normative cohort. In the patients with ACHM examined here, the FAZ area decreased as the number and thickness of the retained inner retinal layers increased. Conclusions: Our data demonstrate that despite the presence of foveal hypoplasia, patients with ACHM can have a FAZ. This is distinct from other conditions associated with foveal hypoplasia, which generally show an absence of the FAZ. In ACHM, FAZ formation does not appear to be sufficient for complete pit formation, contrary to some models of foveal development.
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Defectos de la Visión Cromática/congénito , Fóvea Central/patología , Adolescente , Adulto , Anciano , Niño , Defectos de la Visión Cromática/diagnóstico por imagen , Defectos de la Visión Cromática/patología , Femenino , Fóvea Central/irrigación sanguínea , Fóvea Central/diagnóstico por imagen , Humanos , Masculino , Persona de Mediana Edad , Retina/diagnóstico por imagen , Retina/patología , Vasos Retinianos/diagnóstico por imagen , Vasos Retinianos/patología , Tomografía de Coherencia Óptica , Adulto JovenRESUMEN
INTRODUCTION: MicroRNAs (miRNAs) are small, non-coding RNA molecules that have powerful regulatory properties, with the ability to regulate multiple messenger RNAs (mRNAs) and biological pathways. MicroRNA-223-3p (miR-223) is known to be a critical regulator of the innate immune response, and its dysregulation is thought to play a role in inflammatory disease progression. Despite miR-223 upregulation in numerous neurodegenerative conditions, largely in cells of the myeloid lineage, the role of miR-223 in the retina is relatively unexplored. Here, we investigated miR-223 in the healthy retina and in response to retinal degeneration. METHODS: miR-223-null mice were investigated in control and photo-oxidative damage-induced degeneration conditions. Encapsulated miR-223 mimics were intravitreally and intravenously injected into C57BL/6J wild-type mice. Retinal functional responses were measured using electroretinography (ERG), while extracted retinas were investigated by retinal histology (TUNEL and immunohistochemistry) and molecular analysis (qPCR and FACS). RESULTS: Retinal function in miR-223-/- mice was adversely affected, indicating that miR-223 may be critical in regulating the retinal response. In degeneration, miR-223 was elevated in the retina, circulating serum, and retinal extracellular vesicles. Conversely, retinal microglia and macrophages displayed a downregulation of miR-223. Further, isolated CD11b+ inflammatory cells from the retinas and circulation of miR-223-null mice showed an upregulation of pro-inflammatory genes that are critically linked to retinal inflammation and progressive photoreceptor loss. Finally, both local and systemic delivery of miR-223 mimics improved retinal function in mice undergoing retinal degeneration. CONCLUSION: miR-223 is required for maintaining normal retinal function, as well as regulating inflammation in microglia and macrophages. Further investigations are required to determine the targets of miR-223 and their key biological pathways and interactions that are relevant to retinal diseases. Future studies should investigate whether sustained delivery of miR-223 into the retina is sufficient to target these pathways and protect the retina from progressive degeneration.
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Purpose: The use of small non-coding nucleic acids, such as siRNA and miRNA, has allowed for a deeper understanding of gene functions, as well as for development of gene therapies for complex neurodegenerative diseases, including retinal degeneration. For effective delivery into the eye and transfection of the retina, suitable transfection methods are required. We investigated the use of a lipid-based transfection agent, Invivofectamine® 3.0 (Thermo Fisher Scientific), as a potential method for delivery of nucleic acids to the retina. Methods: Rodents were injected intravitreally with formulations of Invivofectamine 3.0 containing scrambled, Gapdh, Il-1ß, and C3 siRNAs, or sterile PBS (control) using a modified protocol for encapsulation of nucleic acids. TdT-mediated dUTP nick-end labeling (TUNEL) and IBA1 immunohistochemistry was used to determine histological cell death and inflammation. qPCR were used to determine the stress and inflammatory profile of the retina. Electroretinography (ERG) and optical coherence tomography (OCT) were employed as clinical indicators of retinal health. Results: We showed that macrophage recruitment, retinal stress, and photoreceptor cell death in animals receiving Invivofectamine 3.0 were comparable to those in negative controls. Following delivery of Invivofectamine 3.0 alone, no statistically significant changes in expression were found in a suite of inflammatory and stress genes, and ERG and OCT analyses revealed no changes in retinal function or morphology. Injections with siRNAs for proinflammatory genes (C3 and Il-1ß) and Gapdh, in combination with Invivofectamine 3.0, resulted in statistically significant targeted gene knockdown in the retina for up to 4 days following injection. Using a fluorescent Block-It siRNA, transfection was visualized throughout the neural retina with evidence of transfection observed in cells of the ganglion cell layer, inner nuclear layer, and outer nuclear layer. Conclusions: This work supports the use of Invivofectamine 3.0 as a transfection agent for effective delivery of nucleic acids to the retina for gene function studies and as potential therapeutics.
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Técnicas de Silenciamiento del Gen/métodos , Lipoproteínas/farmacología , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Transfección/métodos , Animales , Muerte Celular/genética , Convertasas de Complemento C3-C5/genética , Modelos Animales de Enfermedad , Portadores de Fármacos/química , Electrorretinografía , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Etiquetado Corte-Fin in Situ , Interleucina-1beta/genética , Lípidos/química , Lípidos/farmacología , Lipoproteínas/química , Ratones , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Ratas , Retina/diagnóstico por imagen , Tomografía de Coherencia ÓpticaRESUMEN
Purpose: Glutathione-S-transferase omega 1-1 (GSTO1-1) is a cytosolic glutathione transferase enzyme, involved in glutathionylation, toll-like receptor signaling, and calcium channel regulation. GSTO1-1 dysregulation has been implicated in oxidative stress and inflammation, and contributes to the pathogenesis of several diseases and neurological disorders; however, its role in retinal degenerations is unknown. The aim of this study was to investigate the role of GSTO1-1 in modulating oxidative stress and consequent inflammation in the normal and degenerating retina. Methods: The role of GSTO1-1 in retinal degenerations was explored by using Gsto1-/- mice in a model of retinal degeneration. The expression and localization of GSTO1-1 were investigated with immunohistochemistry and Western blot. Changes in the expression of inflammatory (Ccl2, Il-1ß, and C3) and oxidative stress (Nox1, Sod2, Gpx3, Hmox1, Nrf2, and Nqo1) genes were investigated via quantitative real-time polymerase chain reaction. Retinal function in Gsto1-/- mice was investigated by using electroretinography. Results: GSTO1-1 was localized to the inner segment of cone photoreceptors in the retina. Gsto1-/- photo-oxidative damage (PD) mice had decreased photoreceptor cell death as well as decreased expression of inflammatory (Ccl2, Il-1ß, and C3) markers and oxidative stress marker Nqo1. Further, retinal function in the Gsto1-/- PD mice was increased as compared to wild-type PD mice. Conclusions: These results indicate that GSTO1-1 is required for inflammatory-mediated photoreceptor death in retinal degenerations. Targeting GSTO1-1 may be a useful strategy to reduce oxidative stress and inflammation and ameliorate photoreceptor loss, slowing the progression of retinal degenerations.
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Proteínas Portadoras/fisiología , Modelos Animales de Enfermedad , Glutatión Transferasa/fisiología , Células Fotorreceptoras/fisiología , Degeneración Retiniana/metabolismo , Animales , Biomarcadores/metabolismo , Western Blotting , Supervivencia Celular/fisiología , Complemento C3/genética , Citocinas/genética , Electrorretinografía , Femenino , Marcadores Genéticos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/metabolismo , Retina/fisiopatología , Degeneración Retiniana/fisiopatologíaRESUMEN
BACKGROUND: The role of the alternative complement pathway and its mediation by retinal microglia and macrophages, is well-established in the pathogenesis of Age-Related Macular Degeneration (AMD). However, the contribution of the classical complement pathway towards the progression of retinal degenerations is not fully understood, including the role of complement component 1q (C1q) as a critical activator molecule of the classical pathway. Here, we investigated the contribution of C1q to progressive photoreceptor loss and neuroinflammation in retinal degenerations. METHODS: Wild-type (WT), C1qa knockout (C1qa-/-) and mice treated with a C1q inhibitor (ANX-M1; Annexon Biosciences), were exposed to photo-oxidative damage (PD) and were observed for progressive lesion development. Retinal function was assessed by electroretinography, followed by histological analyses to assess photoreceptor degeneration. Retinal inflammation was investigated through complement activation, macrophage recruitment and inflammasome expression using western blotting, qPCR and immunofluorescence. C1q was localised in human AMD donor retinas using immunohistochemistry. RESULTS: PD mice had increased levels of C1qa which correlated with increasing photoreceptor cell death and macrophage recruitment. C1qa-/- mice did not show any differences in photoreceptor loss or inflammation at 7 days compared to WT, however at 14 days after the onset of damage, C1qa-/- retinas displayed less photoreceptor cell death, reduced microglia/macrophage recruitment to the photoreceptor lesion, and higher visual function. C1qa-/- mice displayed reduced inflammasome and IL-1ß expression in microglia and macrophages in the degenerating retina. Retinal neutralisation of C1q, using an intravitreally-delivered anti-C1q antibody, reduced the progression of retinal degeneration following PD, while systemic delivery had no effect. Finally, retinal C1q was found to be expressed by subretinal microglia/macrophages located in the outer retina of early AMD donor eyes, and in mouse PD retinas. CONCLUSIONS: Our data implicate subretinal macrophages, C1q and the classical pathway in progressive retinal degeneration. We demonstrate a role of local C1q produced by microglia/macrophages as an instigator of inflammasome activation and inflammation. Crucially, we have shown that retinal C1q neutralisation during disease progression may slow retinal atrophy, providing a novel strategy for the treatment of complement-mediated retinal degenerations including AMD.
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Complemento C1q/biosíntesis , Macrófagos/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Animales , Progresión de la Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The pathogenesis of many retinal degenerations, such as age-related macular degeneration (AMD), is punctuated by an ill-defined network of sterile inflammatory responses. The delineation of innate and adaptive immune milieu among the broad leukocyte infiltrate, and the gene networks, which construct these responses, are poorly described in the eye. Using photo-oxidative damage in a rodent model of subretinal inflammation, we employed a novel RNA-sequencing framework to map the global gene network signature of retinal leukocytes. This revealed a previously uncharted interplay of adaptive immunity during subretinal inflammation, including prolonged enrichment of myeloid and lymphocyte migration, antigen presentation, and the alternative arm of the complement cascade involving Factor B. We demonstrate Factor B-deficient mice are protected against macrophage infiltration and subretinal inflammation. Suppressing the drivers of retinal leukocyte proliferation, or their capacity to elicit complement responses, may help preserve retinal structure and function during sterile inflammation in diseases such as AMD.
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Purpose: We sought to determine the role and retinal cellular location of microRNA-124 (miR-124) in a neuroinflammatory model of retinal degeneration. Further, we explored the anti-inflammatory relationship of miR-124 with a predicted messenger RNA (mRNA) binding partner, chemokine (C-C motif) ligand 2 (Ccl2), which is crucially involved in inflammatory cell recruitment in the damaged retina. Methods: Human AMD donor eyes and photo-oxidative damaged (PD) mice were labeled for miR-124 expression using in situ hybridization. PDGFRa-cre RFP mice were used for Müller cell isolation from whole retinas. MIO-M1 immortalized cells and rat primary Müller cells were used for in vitro analysis of miR-124 expression and its relationship with Ccl2. Therapeutic efficacy was tested with intravitreal administration of miR-124 mimic in mice, with electroretinography used to determine retinal function. IBA1 immunohistochemistry and photoreceptor row counts were used for assessment of inflammation and cell death. Results: MiR-124 expression was correlated with progressive retinal damage, inflammation, and cell death in human AMD and PD mice. In addition, miR-124 expression was inversely correlated to Ccl2 expression in mice following PD. MiR-124 was localized to both neuronal-like photoreceptors and glial (Müller) cells in the retina, with a redistribution from neurons to glia occurring as a consequence of PD. Finally, intravitreal administration of miR-124 mimics decreased retinal inflammation and photoreceptor cell death, and improved retinal function. Conclusions: This study has provided an understanding of the mechanism behind miR-124 in the degenerating retina and demonstrates the usefulness of miR-124 mimics for the modulation of retinal degenerations.
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MicroARNs/metabolismo , Retina/metabolismo , Degeneración Retiniana/metabolismo , Análisis de Varianza , Animales , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , RatasRESUMEN
Purpose: Systemic increases in reactive oxygen species, and their association with inflammation, have been proposed as an underlying mechanism linking obesity and age-related macular degeneration (AMD). Studies have found increased levels of oxidative stress biomarkers and inflammatory cytokines in obese individuals; however, the correlation between obesity and retinal inflammation has yet to be assessed. We used the leptin-deficient (ob/ob) mouse to further our understanding of the contribution of obesity to retinal oxidative stress and inflammation. Methods: Retinas from ob/ob mice were compared to age-matched wild-type controls for retinal function (electroretinography) and gene expression analysis of retinal stress (Gfap), oxidative stress (Gpx3 and Hmox1), and complement activation (C3, C2, Cfb, and Cfh). Oxidative stress was further quantified using a reactive oxygen species and reactive nitrogen species (ROS and RNS) assay. Retinal microglia and macrophage migration to the outer retina and complement activation were determined using immunohistochemistry for IBA1 and C3, respectively. Retinas and sera were used for metabolomic analysis using QTRAP mass spectrometry. Results: Retinal function was reduced in ob/ob mice, which correlated to changes in markers of retinal stress, oxidative stress, and inflammation. An increase in C3-expressing microglia and macrophages was detected in the outer retinas of the ob/ob mice, while gene expression studies showed increases in the complement activators (C2 and Cfb) and a decrease in a complement regulator (Cfh). The expression of several metabolites were altered in the ob/ob mice compared to the controls, with changes in polyunsaturated fatty acids (PUFAs) and branched-chain amino acids (BCAAs) detected. Conclusions: The results of this study indicate that oxidative stress, inflammation, complement activation, and lipid metabolites in the retinal environment are linked with obesity in ob/ob animals. Understanding the interplay between these components in the retina in obesity will help inform risk factor analysis for acquired retinal degenerations, including AMD.
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Activación de Complemento , Regulación de la Expresión Génica/inmunología , Obesidad/inmunología , Estrés Oxidativo/inmunología , Retina/inmunología , Degeneración Retiniana/inmunología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/inmunología , Complemento C2/genética , Complemento C2/inmunología , Complemento C3/genética , Complemento C3/inmunología , Factor B del Complemento/genética , Factor B del Complemento/inmunología , Factor H de Complemento/genética , Factor H de Complemento/inmunología , Electrorretinografía , Ácidos Grasos/inmunología , Ácidos Grasos/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/inmunología , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/inmunología , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/inmunología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/inmunología , Obesidad/complicaciones , Obesidad/genética , Obesidad/patología , Retina/patología , Degeneración Retiniana/complicaciones , Degeneración Retiniana/genética , Degeneración Retiniana/patologíaRESUMEN
The complement system is highly implicated in both the prevalence and progression of Age-Related Macular Degeneration (AMD). Complement system inhibitors therefore have potential therapeutic value in managing excessive activation of the complement pathways in retinal degenerations. The vaccinia virus complement control protein (VCP) has been shown to be effective as a complement inhibitor in neuroinflammatory models including traumatic brain injury and spinal cord injury. We aimed to investigate the potential of VCP as a therapeutic molecule for retinal degenerations. In this study, we investigated the effect, localisation and delivery of VCP to the rodent retina. Complement inhibition activity of VCP was tested using a hemolytic assay. Photoreceptor cell death, inflammation and retinal stress were assayed to determine if any retinal toxicity was induced by an intravitreal injection of VCP. The effect of VCP was investigated in a model of photo-oxidative retinal degeneration. Localisation of VCP after injection was determined using a fluorescein-tagged form of VCP, as well as immunohistochemistry. Finally, a copolymer resin (Elvax) was trialled for the slow-release delivery of VCP to the retina. We found that a dose equivalent to 20µg VCP when intravitreally injected into the rat eye did not cause any photoreceptor cell death or immune cell recruitment, but led to an increase in GFAP. In photo-oxidative damaged retinas, there were no differences in photoreceptor loss, retinal stress (Gfap) and inflammation (Ccl2 and C3) between VCP and saline-injected groups; however, Jun expression was reduced in VCP-treated retinas. After VCP was injected into the eye, it was taken up in all layers of the retina but was cleared within 1-3 hours of delivery. This study indicates that a method to sustain the delivery of VCP to the retina is necessary to further investigate the effect of VCP as a complement inhibitor for retinal degenerations.
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Luz , Retina/efectos de los fármacos , Proteínas Virales/farmacología , Animales , Hemólisis/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Oxidación-Reducción/efectos de la radiación , Transporte de Proteínas/efectos de la radiación , Ratas , Retina/metabolismo , Retina/efectos de la radiación , Proteínas Virales/metabolismoRESUMEN
Müller cells, the supporting cells of the retina, play a key role in responding to retinal stress by releasing chemokines, including CCL2, to recruit microglia and macrophages (MG/MΦ) into the damaged retina. Photobiomodulation (PBM) with 670 nm light has been shown to reduce inflammation in models of retinal degeneration. In this study, we aimed to investigate whether 670 nm light had an effect on Müller cell-initiated inflammation under retinal photo-oxidative damage (PD) in vivo and in vitro. Sprague-Dawley rats were pre-treated with 670 nm light (9J/cm2) once daily over 5 days prior to PD. The expression of inflammatory genes including CCL2 and IL-1ß was analysed in retinas. In vitro, primary Müller cells dissociated from neonatal rat retinas were co-cultured with 661W photoreceptor cells. Co-cultures were exposed to PD, followed by 670 nm light treatment to the Müller cells only, and Müller cell stress and inflammation were assessed. Primary MG/MΦ were incubated with supernatant from the co-cultures, and collected for analysis of inflammatory activation. To further understand the mechanism of 670 nm light, the expression of COX5a and mitochondrial membrane potential (ΔΨm) were measured in Müller cells. Following PD, 670 nm light-treated Müller cells had a reduced inflammatory activation, with lower levels of CCL2, IL-1ß and IL-6. Supernatant from 670 nm light-treated co-cultures reduced activation of primary MG/MΦ, and lowered the expression of pro-inflammatory cytokines, compared to untreated PD controls. Additionally, 670 nm light-treated Müller cells had an increased expression of COX5a and an elevated ΔΨm following PD, suggesting that retrograde signaling plays a role in the effects of 670 nm light on Müller cell gene expression. Our data indicates that 670 nm light reduces Müller cell-mediated retinal inflammation, and offers a potential cellular mechanism for 670 nm light therapy in regulating inflammation associated with retinal degenerations.
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Células Ependimogliales/efectos de la radiación , Macrófagos/efectos de la radiación , Microglía/efectos de la radiación , Degeneración Retiniana/radioterapia , Animales , Quimiocinas/metabolismo , Grupo Citocromo c/metabolismo , Modelos Animales de Enfermedad , Células Ependimogliales/fisiología , Interleucinas/metabolismo , Potencial de la Membrana Mitocondrial/efectos de la radiación , Estrés Oxidativo/efectos de la radiación , Ratas , Ratas Sprague-Dawley , Degeneración Retiniana/metabolismoRESUMEN
Purpose: Complement system dysregulation is strongly linked to the progression of age-related macular degeneration (AMD). Deposition of complement including C3 within the lesions in atrophic AMD is thought to contribute to lesion growth, although the contribution of local cellular sources remains unclear. We investigated the role of retinal microglia and macrophages in complement activation within atrophic lesions, in AMD and in models of focal retinal degeneration. Methods: Human AMD donor retinas were labeled for C3 expression via in situ hybridization. Rats were subject to photo-oxidative damage, and lesion expansion was tracked over a 2-month period using optical coherence tomography (OCT). Three strategies were used to determine the contribution of local and systemic C3 in mice: total C3 genetic ablation, local C3 inhibition using intravitreally injected small interfering RNA (siRNA), and depletion of serum C3 using cobra venom factor. Results: Retinal C3 was expressed by microglia/macrophages located in the outer retina in AMD eyes. In rodent photo-oxidative damage, C3-expressing microglia/macrophages and complement activation were located in regions of lesion expansion in the outer retina over 2 months. Total genetic ablation of C3 ameliorated degeneration and complement activation in retinas following damage, although systemic depletion of serum complement had no effect. In contrast, local suppression of C3 expression using siRNA inhibited complement activation and deposition, and reduced cell death. Conclusions: These findings implicate C3, produced locally by retinal microglia/macrophages, as contributing causally to retinal degeneration. Consequently, this suggests that C3-targeted gene therapy may prove valuable in slowing the progression of AMD.
Asunto(s)
Activación de Complemento/fisiología , Complemento C3/genética , Regulación de la Expresión Génica , Macrófagos/metabolismo , ARN/genética , Retina/metabolismo , Degeneración Retiniana/genética , Animales , Animales Recién Nacidos , Complemento C3/biosíntesis , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Hibridación in Situ , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Microglía/patología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Tomografía de Coherencia ÓpticaRESUMEN
BACKGROUND: Chemokine signalling is required for the homing of leukocytes during retinal inflammation, and is associated with pathogenesis of diseases such as age-related macular degeneration (AMD). Here, we explore the role of interleukin-1ß (IL-1ß) in modulating AMD-associated chemokines Ccl2, Cxcl1, and Cxcl10 during photo-oxidative retinal damage, and the effect on both the accumulation of outer-retinal macrophages, and death of photoreceptors. METHODS: Inhibition of retinal IL-1ß expression was performed using either siRNA or antibody neutralisation, which was intravitreally injected in SD rats prior to photo-oxidative damage. Changes in the expression and localisation of Il-1ß, Ccl2, Cxcl1 and Cxcl10 genes were assessed using qPCR and in situ hybridisation, while the recruitment of retinal macrophages was detected using immunohistochemistry for IBA1. Levels of photoreceptor cell death were determined using TUNEL. RESULTS: Photo-oxidative damage elevated the expression of Il-1ß and inflammasome-related genes, and IL-1ß protein was detected in microglia infiltrating the outer retina. This was associated with increased expression of Ccl2, Cxcl1, and Cxcl10. Intravitreal IL-1ß inhibitors suppressed chemokine expression following damage and reduced macrophage accumulation and photoreceptor death. Moreover, in Müller and RPE cell cultures, and in vivo, Ccl2, Cxcl1 and Cxcl10 were variously upregulated when stimulated with IL-1ß, with increased macrophage accumulation detected in vivo. CONCLUSIONS: IL-1ß is produced by retinal microglia and macrophages and promotes chemokine expression by Müller cells and RPE in retinal degeneration. Targeting IL-1ß may prove efficacious in broadly suppressing chemokine-mediated inflammation in retinal dystrophies such as AMD.
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Quimiocinas/metabolismo , Células Ependimogliales/citología , Interleucina-1beta/metabolismo , Microglía/metabolismo , Degeneración Retiniana/metabolismo , Animales , Modelos Animales de Enfermedad , Inflamación/metabolismo , Macrófagos/metabolismo , Ratas Sprague-Dawley , Retina/metabolismoRESUMEN
PURPOSE: Light is a requirement for the function of photoreceptors in visual processing. However, prolonged light exposure can be toxic to photoreceptors, leading to increased reactive oxygen species (ROS), lipid peroxidation, and photoreceptor cell death. We used the 661W mouse cone photoreceptor-like cell line to study the effects of pyruvate in protecting these cells from light-induced toxicity. METHODS: 661W cells were exposed to 15,000 lux continuous bright light for 5 hours and incubated in Dulbecco's modified eagle medium (DMEM) with various concentrations of pyruvate. Following light damage, cells were assessed for changes in morphology, cell toxicity, viability, and ROS production. Mitochondrial respiration and anaerobic glycolysis were also assessed using a Seahorse Xfe96 extracellular flux analyzer. RESULTS: We found that cell death caused by light damage in 661W cells was dramatically reduced in the presence of pyruvate. Cells with pyruvate-supplemented media also showed attenuation of oxidative stress and maintained normal levels of ATP. We also found that alterations in the concentrations of pyruvate had no effect on mitochondrial respiration or glycolysis in light-damaged cells. CONCLUSIONS: Taken together, the results show that pyruvate is protective against light damage but does not alter the metabolic output of the cells, indicating an alternative role for pyruvate in reducing oxidative stress. Thus, sodium pyruvate is a possible candidate for the treatment against the oxidative stress component of retinal degenerations.
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Muerte Celular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ácido Pirúvico/farmacología , Degeneración Retiniana/prevención & control , Animales , Recuento de Células , Línea Celular , Modelos Animales de Enfermedad , Luz/efectos adversos , Ratones , Especies Reactivas de Oxígeno/metabolismo , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patologíaRESUMEN
Light-induced degeneration in rodent retinas is an established model for of retinal degeneration, including the roles of oxidative stress and neuroinflammatory activity. In these models, photoreceptor death is elicited via photo-oxidative stress, and is exacerbated by recruitment of subretinal macrophages and activation of immune pathways including complement propagation. Existing light damage models have relied heavily on albino rodents, and mostly using acute light stimuli. These albino models have proven valuable in uncovering the pathogenic mechanisms of such pathways in the context of retinal disease. However, their inherent albinism hinders comparability to normal retinal physiology, and also makes gene technology analysis time-consuming due to the predominance of the pigmented mouse strains in these applications. In this study, we characterise a new light damage model utilising C57BL/6J mice over a 7 day period of chronic light exposure. We use high-efficiency LED technology to deliver a sustained intensity of 100 k lux with negligible modulation of ambient temperature. We show that in the C57BL/6J mouse, chronic light exposure elicits the cardinal features of light damage including photoreceptor degeneration, atrophy of the choriocapillaris, decreased retinal function and increases in oxidative stress markers 4-HNE and 8-OHG, which emerge progressively over the 7 day period of exposure. These changes are accompanied by robust recruitment of IBA1+ and F4/80 + microglia/macrophages to the ONL and subretinal space, followed the strong up-regulation of monocyte-chemoattractants Ccl2, Ccl3, and Ccl12, as well as increases in expression of complement component C3. These findings are in agreement with prior damage models conducted in albino rodents such as Balb/c mice, and support the use of this new model in further investigating the causative features of oxidative stress and inflammation in retinal disease.
Asunto(s)
Luz/efectos adversos , Estrés Oxidativo/fisiología , Degeneración Retiniana , Análisis de Varianza , Animales , Biomarcadores/metabolismo , Muerte Celular/efectos de la radiación , Modelos Animales de Enfermedad , Electrorretinografía , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Inflamación/fisiopatología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras de Vertebrados/patología , Retina/efectos de la radiación , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatologíaRESUMEN
BACKGROUND: The activity of macrophages is implicated in the progression of retinal pathologies such as atrophic age-related macular degeneration (AMD), where they accumulate among the photoreceptor layer and subretinal space. This process is aided by the local expression of chemokines, which furnish these cells with directional cues that augment their migration to areas of retinal injury. While these qualities make chemokines a potential therapeutic target in curtailing damaging retinal inflammation, their wide variety and signalling redundancy pose challenges in broadly modulating their activity. Here, we examine the efficacy of the broad-spectrum chemokine inhibitor NR58-3.14.3-a suppressor of Ccl- and Cxcl- chemokine pathways-in suppressing macrophage activity and photoreceptor death, using a light-induced model of outer retinal atrophy and inflammation. METHODS: Photo-oxidative damage was induced in SD rats via exposure to 1000 lux of light for 24 h, after which animals were euthanized at 0- or 7-day post-exposure time points. Prior to damage, NR58-3.14.3 was injected intravitreally. Retinas were harvested and evaluated for the effect of NR58-3.14.3 on subretinal macrophage accumulation and cytokine expression profile, as well as photoreceptor degeneration. RESULTS: We report that intravitreal administration of NR58-3.14.3 reduces the accumulation of macrophages in the outer retina following exposure to light damage, at both 0- and 7-day post-exposure time points. Injection of NR58-3.14.3 also reduced the up-regulation of inflammatory markers including of Il6, Ccl3, and Ccl4 in infiltrating macrophages, which are promoters of their pathogenic activity in the retina. Finally, NR58-3.14.3-injected retinas displayed markedly reduced photoreceptor death following light damage, at both 0 and 7 days post-exposure. CONCLUSIONS: Our findings indicate that NR58-3.14.3 is effective in inhibiting subretinal macrophage accumulation in light-induced retinal degeneration and illustrate the potential of broad-spectrum chemokine inhibitors as novel therapeutic agents in thwarting retinal inflammation. Although broad-spectrum chemokine inhibitors may not be appropriate for all retinal inflammatory conditions, our results suggest that they may be beneficial for retinal dystrophies in which chemokine expression and subretinal macrophage accumulation are implicated, such as advanced AMD.
Asunto(s)
Inflamación/etiología , Macrófagos/patología , Péptidos Cíclicos/uso terapéutico , Enfermedades de la Retina/complicaciones , Análisis de Varianza , Animales , Proteínas de Unión al Calcio/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Etiquetado Corte-Fin in Situ , Inflamación/tratamiento farmacológico , Inyecciones Intravítreas , Luz/efectos adversos , Macrófagos/efectos de los fármacos , Proteínas de Microfilamentos/metabolismo , Péptidos Cíclicos/farmacología , Células Fotorreceptoras/patología , Ratas , Ratas Sprague-Dawley , Enfermedades de la Retina/etiología , Enfermedades de la Retina/patología , Factores de TiempoRESUMEN
Age-related macular degeneration (AMD) is a multifactorial disorder that affects millions of individuals worldwide. While the advent of anti-VEGF therapy has allowed for effective treatment of neovascular 'wet' AMD, no treatments are available to mitigate the more prevalent 'dry' forms of the disease. A role for inflammatory processes in the progression of AMD has emerged over a period of many years, particularly the characterisation of leukocyte infiltrates in AMD-affected eyes, as well as in animal models. This review focuses on the burgeoning understanding of chemokines in the retina, and their potential role in shaping the recruitment and activation of macrophages in AMD. Understanding the mechanisms which promote macrophage activity in the degenerating retina may be key to controlling the potentially devastating consequences of inflammation in diseases such as AMD.