RESUMEN

Emerging evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR), preceding the development of microvascular abnormalities. Here, we assessed the impact of neuroinflammation on the retina of diabetic-induced rats. For this aim we have used a two-photon microscope to image the photoreceptors (PRs) at different eccentricities in unstained retinas obtained from both control (N = 4) and pathological rats (N = 4). This technique provides high-resolution images where individual PRs can be identified. Within each image, every PR was located, and its transversal area was measured and used as an objective parameter of neuroinflammation. In control samples, the size of the PRs hardly changed with retinal eccentricity. On the opposite end, diabetic retinas presented larger PR transversal sections. The ratio of PRs suffering from neuroinflammation was not uniform across the retina. Moreover, the maximum anatomical resolving power (in cycles/deg) was also calculated. This presents a double-slope pattern (from the central retina towards the periphery) in both types of specimens, although the values for diabetic retinas were significantly lower across all retinal locations. The results show that chronic retinal inflammation due to diabetes leads to an increase in PR transversal size. These changes are not uniform and depend on the retinal location. Two-photon microscopy is a useful tool to accurately characterize and quantify PR inflammatory processes and retinal alterations.

Asunto(s)

Diabetes Mellitus Experimental , Retinopatía Diabética , Animales , Retinopatía Diabética/diagnóstico por imagen , Retinopatía Diabética/patología , Ratas , Diabetes Mellitus Experimental/patología , Masculino , Células Fotorreceptoras de Vertebrados/patología , Modelos Animales de Enfermedad , Retina/patología , Retina/diagnóstico por imagen , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Microscopía/métodos

RESUMEN

Photoreceptor oil droplets (ODs) are spherical organelles placed most commonly within the inner segment of the cone photoreceptors. Comprising neutral lipids, ODs can be either non-pigmented or pigmented and have been considered optically functional in various studies. Among living amphibians, ODs were only reported to occur in frogs and toads (Anura), while they are absent in salamanders and caecilians. Nonetheless, the limited understanding of their taxonomic distribution in anurans impedes a comprehensive assessment of their evolution and relationship with visual ecology. We studied the retinae of 134 anuran species, extending the knowledge of the distribution of ODs to 46 of the 58 currently recognized families, and providing a new perspective on this group that complements the available information from other vertebrates. The occurrence of ODs in anurans shows a strong phylogenetic signal, and our findings revealed that ODs evolved at least six times during the evolutionary history of the group, independently from other vertebrates. Although no evident correlation was found between OD occurrence, adult habits and diel activity, it is inferred that each independent origin involves distinct scenarios in the evolution of ODs concerning photic habits. Furthermore, our results revealed significant differences in the size of the ODs between nocturnal and arrhythmic anurans relative to the length of the cones' outer segment.

Asunto(s)

Anuros , Evolución Biológica , Filogenia , Animales , Anuros/fisiología , Gotas Lipídicas , Bufonidae/fisiología , Células Fotorreceptoras de Vertebrados/fisiología

RESUMEN

Seckel syndrome is a type of microcephalic primordial dwarfism (MPD) that is characterized by growth retardation and neurodevelopmental defects, including reports of retinopathy. Mutations in key mediators of the replication stress response, the mutually dependent partners ATR and ATRIP, are among the known causes of Seckel syndrome. However, it remains unclear how their deficiency disrupts the development and function of the central nervous system (CNS). Here, we investigated the cellular and molecular consequences of ATRIP deficiency in different cell populations of the developing murine neural retina. We discovered that conditional inactivation of Atrip in photoreceptor neurons did not affect their survival or function. In contrast, Atrip deficiency in retinal progenitor cells (RPCs) led to severe lamination defects followed by secondary photoreceptor degeneration and loss of vision. Furthermore, we showed that RPCs lacking functional ATRIP exhibited higher levels of replicative stress and accumulated endogenous DNA damage that was accompanied by stabilization of TRP53. Notably, inactivation of Trp53 prevented apoptosis of Atrip-deficient progenitor cells and was sufficient to rescue retinal dysplasia, neurodegeneration and loss of vision. Together, these results reveal an essential role of ATRIP-mediated replication stress response in CNS development and suggest that the TRP53-mediated apoptosis of progenitor cells might contribute to retinal malformations in Seckel syndrome and other MPD disorders.This article has an associated First Person interview with the first author of the paper.

Asunto(s)

Anomalías Múltiples/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/metabolismo , Degeneración Nerviosa/patología , Displasia Retiniana/patología , Células Madre/patología , Animales , Apoptosis , Ceguera/patología , Muerte Celular , Proliferación Celular , Daño del ADN , Modelos Animales de Enfermedad , Embrión de Mamíferos/patología , Desarrollo Embrionario , Ratones , Degeneración Nerviosa/complicaciones , Neurogénesis , Células Fotorreceptoras de Vertebrados/patología , Retina/patología , Displasia Retiniana/complicaciones , Síndrome , Proteína p53 Supresora de Tumor/metabolismo , Visión Ocular

RESUMEN

PDLIM's protein family is involved in the rearrangement of the actin cytoskeleton. In the present study, we describe the localization of PDLIM1 in chicken photoreceptors. This study provides evidence that this protein is present at the cone pedicles, as well as in other synapses of the chicken retina. Here, we demonstrate the expression pattern of PDLIM1 through immunofluorescence staining, immunoblots, subcellular fractionation, and immunoprecipitation experiments. Also, we consider the possibility that PDLIM1 may be involved in the synaptic vesicle endocytosis and/or the presynaptic trafficking of synaptic vesicles back to the nonready releasable pool. This endocytotic/exocytotic coupling requires a tight link between exocytic vesicle fusion at defined release sites and endocytic retrieval of synaptic vesicle membranes. In turn, photoreceptor ribbon synaptic structure depends on the cytoskeleton arrangement, both at the active zone-related with exocytosis-as well as at the endocytic zone-periactive zone. To our knowledge, the PDLIM1 protein has not been observed in the pre synapses of the retina. Thus, the present study describes the expression and subcellular localization of PDLIM1 for the first time, as well as its modulation by visual environment in the chicken retina.

Asunto(s)

Proteínas Aviares/metabolismo , Proteínas con Dominio LIM/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Sinapsis/metabolismo , Animales , Pollos

RESUMEN

Injured retinas in mammals do not regenerate and heal with loss of function. The adult retina of zebrafish self-repairs after damage by activating cell-intrinsic mechanisms, which are regulated by extrinsic signal interactions. Among relevant regulatory extrinsic systems, purinergic signaling regulates progenitor proliferation during retinogenesis and regeneration and glia proliferation in proliferative retinopathies. ATP-activated P2X7 (P2RX7) and adenosine (P1R) receptors are involved in the progression of almost all retinopathies leading to blindness. Here, we examined P2RX7 and P1R participation in the retina regenerative response induced by photoreceptor damage caused by a specific dose of CoCl2 . First, we found that treatment of uninjured retinas with a potent agonist of P2RX7 (BzATP) provoked photoreceptor damage and mitotic activation of multipotent progenitors. In CoCl2 -injured retinas, blockade of endogenous extracellular ATP activity on P2RX7 caused further neurodegeneration, Müller cell gliosis, progenitor proliferation, and microglia reactivity. P2RX7 inhibition in injured retinas also increased the expression of lin28a and tnfα genes, which are related to multipotent progenitor proliferation. Levels of hif1α, vegf3r, and vegfaa mRNA were enhanced by blockade of P2RX7 immediately after injury, indicating hypoxic like damage and endothelial cell growth and proliferation. Complete depletion of extracellular nucleotides with an apyrase treatment strongly potentiated cell death and progenitor proliferation induced with CoCl2 . Blockade of adenosine P1 and A2A receptors (A2A R) had deleterious effects and deregulated normal timing for progenitor and precursor cell proliferation following photoreceptor damage. ATP via P2RX7 and adenosine via A2A R are survival extracellular signals key for retina regeneration in zebrafish.

Asunto(s)

Regeneración Nerviosa/fisiología , Neuronas/patología , Células Fotorreceptoras de Vertebrados/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animales , Muerte Celular/fisiología , Cobalto/toxicidad , Degeneración Nerviosa/inducido químicamente , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/patología , Pez Cebra

RESUMEN

Death of retinal photoreceptors is the basis of prevalent blinding diseases. Since steroids might have a therapeutic role in retinal degenerations, we compared the protective effects of dexamethasone and progesterone on photoreceptor death induced by mifepristone and light exposure. Therefore, we studied the effective protection doses for each steroid in the two models. In addition, we analyzed changes in the levels of pro- and antiapoptotic molecules, glucocorticoid receptors α and ß (GRα and GRß), and rhodopsin under conditions of successful protection and photoreceptor survival. Mifepristone and light exposure selectively damaged photoreceptors. In light exposed retinas, photoreceptors mainly disappeared in the dorsotemporal region, while mifepristone produced a uniform damage. Dexamethasone and progesterone, at the same dose of 4 mg/kg/day for 2 days, preserved over 88% photoreceptor nuclei in both models. Assessment of cell death regulators showed that, in control retinas, both steroids activated BCL-XL, a prosurvival molecule, and decreased BID, a proapoptotic regulator. After steroid treatment of damaged retinas, BCL-XL, BCL2 and BAX showed characteristic patterns depending on the use of dexamethasone or progesterone on mifepristone or light exposed retinas. By contrast, BID decreased with any injury-steroid combination. Changes in GRα or GRß levels did not correlate with survival but were consistent with a mechanism of ligand induced downregulation of receptor expression. GRß might be upregulated by progesterone. Both dexamethasone and progesterone increased retinal rhodopsin stores, suggesting a link between photoreceptor protection and transduction pathways. Results show that dexamethasone and progesterone induced comparable but not identical protection responses in each model.

Asunto(s)

Dexametasona/farmacología , Glucocorticoides/farmacología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Progesterona/farmacología , Traumatismos Experimentales por Radiación/prevención & control , Degeneración Retiniana/prevención & control , Animales , Apoptosis/efectos de los fármacos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Western Blotting , Caspasa 3 , Supervivencia Celular/fisiología , Antagonistas de Hormonas/toxicidad , Inmunohistoquímica , Luz/efectos adversos , Masculino , Ratones Endogámicos BALB C , Mifepristona/toxicidad , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Traumatismos Experimentales por Radiación/etiología , Traumatismos Experimentales por Radiación/metabolismo , Receptores de Glucocorticoides/metabolismo , Degeneración Retiniana/etiología , Degeneración Retiniana/metabolismo , Rodopsina/metabolismo , Proteína bcl-X/metabolismo

RESUMEN

Introdução: O aumento da potência dos fotopolimerizadores à base de diodos emissores de luz (LED) contribuiu para a redução do tempo de fotopolimerização dos procedimentos restauradores e ortodônticos. Entretanto, é desconhecido o efeito do LED sobre a retina de quem faz o uso crônico desse equipamento sem usar filtros de proteção. Objetivo: Avaliar os possíveis efeitos do uso crônico de um aparelho fotopolimerizador LED de alta potência nas retinas de ratos Wistar. Material e métodos: Neste estudo experimental in vivo, seis ratos machos saudáveis foram utilizados. Os olhos esquerdos dos animais foram expostos à luz do fotopolimerizador à base de LED (Valo Ortho - Ultradent), a uma potência de 3.200mW/cm2 , por 144 segundos, à distância de 30cm, três vezes ao dia, durante 7 dias. Os olhos direitos foram cobertos com um tampão plástico removível preto, opaco, compondo a amostra controle. No oitavo dia, os animais foram anestesiados, submetidos à eutanásia, seus olhos foram dissecados e processados histologicamente. As lâminas foram digitalizadas utilizando-se uma câmera acoplada a um microscópico óptico e as suas imagens foram analisadas por histomorfometria bi e tridimensional. Resultados: Não foram encontradas diferenças intergrupos estatisticamente significativas para o volume total da retina (p=0,655) nem para o volume das camadas retinianas ganglionar (p=0,375), plexiforme interna (p=0,327), nuclear interna (p=0,693), plexiforme externa (p=0,177), nuclear externa (p=0,355) e o prolongamento de cones e bastonetes (p=0,871), quando avaliadas individualmente. As análises histomorfométricas bidimensionais apresentaram redução estatisticamente significativas (p<0,05) para as áreas celulares, com redução de 25,32% para a camada nuclear interna, 17,59% para a camada nuclear externa e 16,76% para a camada ganglionar. Apesar da densidade numérica das camadas nucleares interna e externa terem reduzido, e da camada ganglionar terem aumentado, as mesmas não apresentaram diferença estatisticamente significativas. Conclusão: A exposição crônica do LED de alta potência (3.200mW/cm2 ) induziu atrofia celular nas camadas nuclear interna, nuclear externa e ganglionar (AU).

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Introduction: The increase in the power of light-emitting diode (LED) light cure has contributed to reduce the curing time of restorative and orthodontic procedures. However, the effect of the LED on the retina of those who make the chronic use of light units without using protection filters is unknown. Objective: To evaluate the possible effects of the chronic use of a high-power LED light curing device on the retinas of Wistar rats. Material and methods: In this experimental study in vivo, six healthy male rats were used and their ocular structures being the objects of study. The left eyes of the animals were exposed to high potency LED light, 3.200mW/cm2 (Valo Ortho - Ultradent), for 144 seconds at a distance of 30cm, three times a day, for 7 days. The right eyes were covered with removable plastic opaque tampon, composing the control sample. On the eighth day, the animals were anesthetized, euthanized, the eyes dissected and histologically processed. The slides were scanned using a camera coupled to an optical microscope and their images analyzed by two and three dimensional histomorphometry. Results: No statistically significant intergroup differences were found for total retinal volume (p = 0.655) or for the volume of the retinal layers ganglionic (p = 0.375), internal plexiform (p = 0.327), internal nuclear (p = 0.693), external plexiform (p = 0,177), external nuclear (p = 0.355) and the extension of cones and rods (p = 0.871), when evaluated individually. The histomorphometric analyzes presented statistically significant reduction for the cellular areas, with a reduction of 25,32% for the internal nuclear layer, 17,59% for external nuclear layer and 16,76% for ganglionic layer. Although the numerical density of the internal and external nuclear layers decreased, and the ganglion layer increased, they showed no statistically significant difference. Conclusion: Chronic exposure of the high-power LED (3.200mW/cm2 ) induced cellular atrophy in the internal nuclear layer, external nuclear layer and ganglionic layer (AU).

Asunto(s)

Animales , Ratas , Retina , Altas Potencias , Ratas Wistar , Luces de Curación Dental , Ensayo Clínico , Estadísticas no Paramétricas , Células Fotorreceptoras de Vertebrados , Polimerizacion , Luz Azul

RESUMEN

Ceramide (Cer) has a key role inducing cell death and has been proposed as a messenger in photoreceptor cell death in the retina. Here, we explored the pathways induced by C2-acetylsphingosine (C2-Cer), a cell-permeable Cer, to elicit photoreceptor death. Treating pure retina neuronal cultures with 10 µM C2-Cer for 6 h selectively induced photoreceptor death, decreasing mitochondrial membrane potential and increasing the formation of reactive oxygen species (ROS). In contrast, amacrine neurons preserved their viability. Noteworthy, the amount of TUNEL-labeled cells and photoreceptors expressing cleaved caspase-3 remained constant and pretreatment with a pan-caspase inhibitor did not prevent C2-Cer-induced death. C2-Cer provoked polyADP ribosyl polymerase-1 (PARP-1) overactivation. Inhibiting PARP-1 decreased C2-Cer-induced photoreceptor death; C2-Cer increased polyADP ribose polymer (PAR) levels and induced the translocation of apoptosis inducing factor (AIF) from mitochondria to photoreceptor nuclei, which was prevented by PARP-1 inhibition. Pretreatment with a calpain and cathepsin inhibitor and with a calpain inhibitor reduced photoreceptor death, whereas selective cathepsin inhibitors granted no protection. Combined pretreatment with a PARP-1 and a calpain inhibitor evidenced the same protection as each inhibitor by itself. Neither autophagy nor necroptosis was involved in C2-Cer-elicited death; no increase in LDH release was observed upon C2-Cer treatment and pretreatment with inhibitors of necroptosis and autophagy did not rescue photoreceptors. These results suggest that C2-Cer induced photoreceptor death by a novel, caspase-independent mechanism, involving activation of PARP-1, decline of mitochondrial membrane potential, calpain activation, and AIF translocation, all of which are biochemical features of parthanatos.

Asunto(s)

Ceramidas/farmacología , Parthanatos/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/patología , Animales , Factor Inductor de la Apoptosis/metabolismo , Calpaína/metabolismo , Caspasas/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Cypermethrin (CM), widely used for control of indoor and field pests, is one of the most common contaminants in freshwater aquatic systems. We evaluated CM genotoxicity and the activities of superoxide dismutase (SOD) and catalase (CAT) in retinal cells of adult zebrafish. Histological and immunofluorescence techniques show the presence of apoptotic cells in the zebrafish retina after 9 d of treatment with 0.6 µg/L CM. Histone γ-H2AX, a marker of DNA damage, was detected in both outer and inner nuclear layers; caspase-3, an apoptotic marker, was detected in the outer nuclear layer. In the comet assay, the cells were sensitive to hydrogen peroxide-induced DNA damage, showing a dose-dependent response. We observed a positive comet assay response to CM that was dose- and time-dependent. Following exposure to CM, SOD and CAT enzyme activities, and sod and cat mRNA levels, increased. These results indicate that CM causes DNA damage and oxidative stress and can induce apoptosis in retinal cells.

Asunto(s)

Daño del ADN , Estrés Oxidativo , Células Fotorreceptoras de Vertebrados/citología , Piretrinas/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Caspasa 3/metabolismo , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Histonas/metabolismo , Peróxido de Hidrógeno/efectos adversos , Pruebas de Mutagenicidad , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Pez Cebra

RESUMEN

Non-exudative age-related macular degeneration, a prevalent cause of blindness, is a progressive and degenerative disease characterized by alterations in Bruch's membrane, retinal pigment epithelium, and photoreceptors exclusively localized in the macula. Although experimental murine models exist, the vast majority take a long time to develop retinal alterations and, in general, these alterations are ubiquitous, with many resulting from non-eye-specific genetic manipulations; additionally, most do not always reproduce the hallmarks of human age-related macular degeneration. Choroid vessels receive sympathetic innervation from the superior cervical ganglion, which, together with the parasympathetic system, regulates blood flow into the choroid. Choroid blood flow changes have been involved in age-related macular degeneration development and progression. At present, no experimental models take this factor into account. The aim of this work was to analyze the effect of superior cervical gangliectomy (also known as ganglionectomy) on the choroid, Bruch's membrane, retinal pigment epithelium and retina. Adult male C57BL/6J mice underwent unilateral superior cervical gangliectomy and a contralateral sham procedure. Although superior cervical gangliectomy induced ubiquitous choroid and choriocapillaris changes, it induced Bruch's membrane thickening, loss of retinal pigment epithelium melanin content and retinoid isomerohydrolase, the appearance of drusen-like deposits, and retinal pigment epithelium and photoreceptor atrophy, exclusively localized in the temporal side. Moreover, superior cervical gangliectomy provoked a localized increase in retinal pigment epithelium and photoreceptor apoptosis, and a decline in photoreceptor electroretinographic function. Therefore, superior cervical gangliectomy recapitulated the main features of human non-exudative age-related macular degeneration, and could become a new experimental model of dry age-related macular degeneration, and a useful platform for developing new therapies.

Asunto(s)

Degeneración Macular/etiología , Ganglio Cervical Superior/cirugía , Animales , Lámina Basal de la Coroides/patología , Lámina Basal de la Coroides/ultraestructura , Coroides/patología , Degeneración Macular/patología , Masculino , Melaninas/metabolismo , Ratones Endogámicos C57BL , Células Fotorreceptoras de Vertebrados/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Ganglio Cervical Superior/patología , cis-trans-Isomerasas/metabolismo

RESUMEN

PURPOSE: To evaluate the role of serum urea and creatinine as surrogate markers for disruption of retinal photoreceptor external limiting membrane (ELM) and inner segment ellipsoid zone (EZ) in Type 2 diabetic retinopathy (DR) using spectral-domain optical coherence tomography, for the first time. METHODS: One hundred and seventeen consecutive cases of Type 2 diabetes mellitus (diabetes without retinopathy [No DR; n = 39], nonproliferative diabetic retinopathy [NPDR; n = 39], proliferative diabetic retinopathy [PDR; n = 39]) and 40 healthy control subjects were included. Serum levels of urea and creatinine were assessed using standard protocol. Spectral-domain optical coherence tomography was used to grade the disruption of ELM and EZ as follows: Grade 0, no disruption of ELM and EZ; Grade 1, ELM disrupted, EZ intact; Grade 2, ELM and EZ disrupted. Data were analyzed statistically. RESULTS: Increase in serum levels of urea (F = 22.93) and creatinine (F = 15.82) and increased grades of disruption of ELM and EZ (γ = 116.3) were observed with increased severity of DR (P < 0.001). Increase in serum levels of urea (F = 10.45) and creatinine (F = 6.89) was observed with increased grades of disruption of ELM and EZ (P = 0.001). CONCLUSION: Serum levels of urea and creatinine are surrogate markers for disruption of retinal photoreceptor ELM and EZ on spectral-domain optical coherence tomography in DR.

Asunto(s)

Creatinina/sangre , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/patología , Retinopatía Diabética/sangre , Retinopatía Diabética/patología , Membrana Epirretinal/patología , Segmento Interno de las Células Fotorreceptoras Retinianas/patología , Urea/sangre , Adulto , Anciano , Análisis de Varianza , Biomarcadores/sangre , Estudios de Casos y Controles , Retinopatía Diabética/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Fotorreceptoras de Vertebrados/patología , Enfermedades de la Retina , Índice de Severidad de la Enfermedad

RESUMEN

Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments. Retinal degeneration and function were compared in WT versus KO mice following light damage. Gene expression changes, endoplasmic reticulum (ER) stress, and glial cell activation were also compared. At baseline, the ERG responses and rhodopsin levels were lower in CD59aKO compared to wild-type (WT) mice. Following LD, the ERG responses were better preserved in CD59aKO compared to WT mice. Correspondingly, the number of photoreceptors was higher in CD59aKO retinas than WT controls after LD. Under normal light conditions, CD59aKO mice had higher levels than WT for GFAP immunostaining in Müller cells, mRNA and protein levels of two ER-stress markers, and neurotrophic factors. The reduction in photon capture, together with the neurotrophic factor upregulation, may explain the structural and functional protection against LD in the CD59aKO.

Asunto(s)

Antígenos CD59/genética , Luz , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Degeneración Retiniana/patología , Animales , Antígenos CD59/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de la radiación , Células Ependimogliales/metabolismo , Enucleación del Ojo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuroglía/efectos de la radiación , Fagocitosis/efectos de la radiación , Células Fotorreceptoras de Vertebrados/metabolismo , ARN Mensajero/metabolismo , Retina/diagnóstico por imagen , Retina/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/veterinaria , Retinaldehído/análisis , Rodopsina/genética , Rodopsina/metabolismo , Regulación hacia Arriba/efectos de la radiación

RESUMEN

In the vertebrate retina, three types of photoreceptors-visual photoreceptor cones and rods and the intrinsically photosensitive retinal ganglion cells (ipRGCs)-converged through evolution to detect light and regulate image- and nonimage-forming activities such as photic entrainment of circadian rhythms, pupillary light reflexes, etc. ipRGCs express the nonvisual photopigment melanopsin (OPN4), encoded by two genes: the Xenopus (Opn4x) and mammalian (Opn4m) orthologs. In the chicken retina, both OPN4 proteins are found in ipRGCs, and Opn4x is also present in retinal horizontal cells (HCs), which connect with visual photoreceptors. Here we investigate the intrinsic photosensitivity and functioning of HCs from primary cultures of embryonic retinas at day 15 by using calcium fluorescent fluo4 imaging, pharmacological inhibitory treatments, and Opn4x knockdown. Results show that HCs are avian photoreceptors with a retinal-based OPN4X photopigment conferring intrinsic photosensitivity. Light responses in HCs appear to be driven through an ancient type of phototransduction cascade similar to that in rhabdomeric photoreceptors involving a G-protein q, the activation of phospholipase C, calcium mobilization, and the release of the inhibitory neurotransmitter GABA. Based on their intrinsic photosensitivity, HCs may have a key dual function in the retina of vertebrates, potentially regulating nonvisual tasks together with their sister cells, ipRGCs, and with visual photoreceptors, modulating lateral interactions and retinal processing.

Asunto(s)

Células Fotorreceptoras de Vertebrados/fisiología , Células Horizontales de la Retina/fisiología , Opsinas de Bastones/fisiología , Animales , Calcio/fisiología , Células Cultivadas , Pollos , Embrión no Mamífero , Luz , Retinaldehído/fisiología , Opsinas de Bastones/genética , Ácido gamma-Aminobutírico/fisiología

RESUMEN

Oxidative stress is involved in activating photoreceptor death in several retinal degenerations. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, protects cultured retina photoreceptors from apoptosis induced by oxidative stress and promotes photoreceptor differentiation. Here, we investigated whether eicosapentaenoic acid (EPA), a metabolic precursor to DHA, had similar effects and whether retinal neurons could metabolize EPA to DHA. Adding EPA to rat retina neuronal cultures increased opsin expression and protected photoreceptors from apoptosis induced by the oxidants paraquat and hydrogen peroxide (H2 O2 ). Palmitic, oleic, and arachidonic acids had no protective effect, showing the specificity for DHA. We found that EPA supplementation significantly increased DHA percentage in retinal neurons, but not EPA percentage. Photoreceptors and glial cells expressed Δ6 desaturase (FADS2), which introduces the last double bond in DHA biosynthetic pathway. Pre-treatment of neuronal cultures with CP-24879 hydrochloride, a Δ5/Δ6 desaturase inhibitor, prevented EPA-induced increase in DHA percentage and completely blocked EPA protection and its effect on photoreceptor differentiation. These results suggest that EPA promoted photoreceptor differentiation and rescued photoreceptors from oxidative stress-induced apoptosis through its elongation and desaturation to DHA. Our data show, for the first time, that isolated retinal neurons can synthesize DHA in culture. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in retina photoreceptors, and its precursor, eicosapentaenoic acid (EPA) have multiple beneficial effects. Here, we show that retina neurons in vitro express the desaturase FADS2 and can synthesize DHA from EPA. Moreover, addition of EPA to these cultures protects photoreceptors from oxidative stress and promotes their differentiation through its metabolization to DHA.

Asunto(s)

Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Estrés Oxidativo/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Mitocondrias/metabolismo , Paraquat/farmacología , Sustancias Protectoras/farmacología , Ratas Wistar , Retina/metabolismo

RESUMEN

Retina light stimulation triggers phototransduction events as well as different signaling mechanisms in outer segments (sensorial portion) of photoreceptor cells. We have recently reported a novel light-dependent activation of diacylglycerol kinase (DAGK) and protein kinase C (PKC) at the nuclear level of photoreceptor cells. The aim of the present study was to analyze whether ex-vivo light exposure of bovine retinas also modulates insulin-related signaling pathways in nuclei from photoreceptor cells. To this end, a nuclear fraction enriched in small nuclei from photoreceptor cells (PNF) was obtained using a modified nuclear isolation protocol. In PNF obtained from bovine retinas exposed to light or darkness, the presence of insulin receptor (IR) and phosphorylated insulin receptor (pIR), the activation of Akt, p38 and extracellular signal-regulated kinase (ERK1/2) and the local action of insulin on lipid kinases were studied. Immunofluorescence (IF) and Western blot (WB) studies revealed the presence of IR in photoreceptor nuclei. In PNF a light-dependent increase in IR total content was observed. The presence of activated IR (pIR) was also observed in PNF by WB, being its content higher in PNF from light than in to darkness. Light exposure also produced a significant increase in the content of p-Akt (3 fold) and p-p38 (60%) without changes in total Akt and p38. In addition, an increase in the content of total ERK1/2 (2 fold) was found without changes in p-ERK/total ERK ratio, indicating that light induces translocation of p-ERK to the nucleus. Polyphosphoinositide kinase and diacylglycerol kinase (DAGK) activities were measured in isolated nuclei from light-activated or darkness-adapted retinas through the formation of polyphosphoinositides (PPIs) and phosphatidic acid (PA) using nuclear lipid substrates and [γ-(32)P]ATP as radioactive substrate. A light-dependent increase in PPIs and PA formation was detected when isolated nuclei were exposed to 0.8 µM insulin plus 0.2 mM vanadate. WB studies revealed that retina's exposure to insulin under light condition increased nuclear IR content. In addition, PNF exposure to insulin increased ERK1/2 phosphorylation with no changes in total ERK1/2. Our results demonstrate the presence and the functional state of IR in the nucleus from photoreceptor cells. They also show that molecular signaling components linked to tyrosine kinase receptors and MAPK pathways, such as Akt and ERK1/2, respectively, are present in photoreceptor nuclei and are regulated by insulin and light.

Asunto(s)

Núcleo Celular/metabolismo , Diacilglicerol Quinasa/metabolismo , Insulina/farmacología , Células Fotorreceptoras de Vertebrados/metabolismo , Receptor de Insulina/metabolismo , Animales , Western Blotting , Bovinos , Núcleo Celular/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Luz , Fototransducción/efectos de los fármacos , Modelos Animales , Fosforilación , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos

RESUMEN

Light is the visible part of the electromagnetic radiation within a range of 380-780 nm; (400-700 on primates retina). In vertebrates, the retina is adapted to capturing light photons and transmitting this information to other structures in the central nervous system. In mammals, light acts directly on the retina to fulfill two important roles: (1) the visual function through rod and cone photoreceptor cells and (2) non-image forming tasks, such as the synchronization of circadian rhythms to a 24 h solar cycle, pineal melatonin suppression and pupil light reflexes. However, the excess of illumination may cause retinal degeneration or accelerate genetic retinal diseases. In the last century human society has increased its exposure to artificial illumination, producing changes in the Light/Dark cycle, as well as in light wavelengths and intensities. Although, the consequences of unnatural illumination or light pollution have been underestimated by modern society in its way of life, light pollution may have a strong impact on people's health. The effects of artificial light sources could have direct consequences on retinal health. Constant exposure to different wavelengths and intensities of light promoted by light pollution may produce retinal degeneration as a consequence of photoreceptor or retinal pigment epithelium cells death. In this review we summarize the different mechanisms of retinal damage related to the light exposure, which generates light pollution.

Asunto(s)

Contaminación Ambiental/efectos adversos , Luz/efectos adversos , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Traumatismos por Radiación/etiología , Degeneración Retiniana/etiología , Animales , Humanos , Estimulación Luminosa

RESUMEN

IMPORTANCE: This case report describes a man who developed retinal changes in his right eye associated with brilliant blue G migration into the subretinal space during 2 years of follow-up. OBSERVATION: The patient's best-corrected visual acuity in the right eye was 20/70 before surgery, and it improved to 20/25 at 1 year after surgery. Fluorescein angiography showed staining during the late phase in the central macula at all follow-up visits after surgery. Multifocal electroretinography demonstrated normal amplitude and implicit times before surgery but decreased amplitudes and increased implicit times in at least 5 contiguous hexagons after surgery on all 3 examinations performed during the 2-year follow-up period. These functional changes were not topographically correlated with the area of fluorescein staining or with the internal limiting membrane peeled area, but were matched to the area where brilliant blue G accidentally entered the subretinal space. Microperimetry demonstrated reduced retinal threshold sensitivity, particularly in areas with decreased multifocal electroretinography amplitude. CONCLUSIONS AND RELEVANCE: Despite the visual acuity improvement observed in this case, multifocal electroretinography and microperimetry indicate that subretinal brilliant blue G might cause focal macular damage with a decrease of macular function suggestive of a toxic effect.

Asunto(s)

Membrana Epirretinal/cirugía , Indicadores y Reactivos/efectos adversos , Complicaciones Intraoperatorias , Edema Macular/inducido químicamente , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Colorantes de Rosanilina/efectos adversos , Membrana Basal/patología , Membrana Basal/cirugía , Electrorretinografía , Membrana Epirretinal/diagnóstico , Angiografía con Fluoresceína , Humanos , Implantación de Lentes Intraoculares , Edema Macular/diagnóstico , Masculino , Persona de Mediana Edad , Facoemulsificación , Tomografía de Coherencia Óptica , Agudeza Visual/fisiología , Pruebas del Campo Visual , Vitrectomía

RESUMEN

The retina is a key component of the vertebrate circadian system; it is responsible for detecting and transmitting the environmental illumination conditions (day/night cycles) to the brain that synchronize the circadian clock located in the suprachiasmatic nucleus (SCN). For this, retinal ganglion cells (RGCs) project to the SCN and other nonvisual areas. In the chicken, intrinsically photosensitive RGCs (ipRGCs) expressing the photopigment melanopsin (Opn4) transmit photic information and regulate diverse nonvisual tasks. In nonmammalian vertebrates, two genes encode Opn4: the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs. RGCs express both Opn4 genes but are not the only inner retinal cells expressing Opn4x: horizontal cells (HCs) also do so. Here, we further characterize primary cultures of both populations of inner retinal cells (RGCs and HCs) expressing Opn4x. The expression of this nonvisual photopigment, as well as that for different circadian markers such as the clock genes Bmal1, Clock, Per2, and Cry1, and the key melatonin synthesizing enzyme, arylalkylamine N-acetyltransferase (AA-NAT), appears very early in development in both cell populations. The results clearly suggest that nonvisual Opn4 photoreceptors and endogenous clocks converge all together in these inner retinal cells at early developmental stages.

Asunto(s)

Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Ritmo Circadiano/fisiología , Células Fotorreceptoras de Vertebrados/fisiología , Retina/embriología , Retina/fisiología , Opsinas de Bastones/metabolismo , Animales , Células Cultivadas , Pollos , Regulación del Desarrollo de la Expresión Génica , Estimulación Luminosa/métodos , Retina/citología , Percepción Visual/fisiología

RESUMEN

In this work, we describe a selective light-dependent distribution of the lipid kinase 1,2-diacylglycerol kinase (EC 2.7.1.107, DAGK) and the phosphorylated protein kinase C alpha (pPKCα) in a nuclear fraction of photoreceptor cells from bovine retinas. A nuclear fraction enriched in small nuclei from photoreceptor cells (PNF), was obtained when a modified nuclear isolation protocol developed by our laboratory was used. We measured and compared DAGK activity as phosphatidic acid (PA) formation in PNF obtained from retinas exposed to light and in retinas kept in darkness using [γ-(32)P]ATP or [(3)H]DAG. In the absence of exogenous substrates and detergents, no changes in DAGK activity were observed. However, when DAGK activity assays were performed in the presence of exogenous substrates, such as stearoyl arachidonoyl glycerol (SAG) or dioleoyl glycerol (DOG), and different detergents (used to make different DAGK isoforms evident), we observed significant light effects on DAGK activity, suggesting the presence of several DAGK isoforms in PNF. Under conditions favoring DAGKζ activity (DOG, Triton X-100, dioleoyl phosphatidylserine and R59022) we observed an increase in PA formation in PNF from retinas exposed to light with respect to those exposed to darkness. In contrast, under conditions favoring DAGKɛ (SAG, octylglucoside and R59022) we observed a decrease in its activity. These results suggest different physiological roles of the above-mentioned DAGK isoforms. Western blot analysis showed that whereas light stimulation of bovine retinas increases DAGKζ nuclear content, it decreases DAGKɛ and DAGKß content in PNF. The role of PIP2-phospholipase C in light-stimulated DAGK activity was demonstrated using U73122. Light was also observed to induce enhanced pPKCα content in PNF. The selective distribution of DAGKζ and ɛ in PNF could be a light-dependent mechanism that in vertebrate retina promotes selective DAG removal and PKC regulation.

Asunto(s)

Núcleo Celular/enzimología , Diacilglicerol Quinasa/metabolismo , Células Fotorreceptoras de Vertebrados/enzimología , Proteína Quinasa C-alfa/metabolismo , Análisis de Varianza , Animales , Bovinos , Núcleo Celular/efectos de la radiación , Adaptación a la Oscuridad , Inhibidores Enzimáticos/farmacología , Luz , Fosforilación , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Retina/enzimología , Retina/efectos de la radiación , Fosfolipasas de Tipo C/antagonistas & inhibidores

RESUMEN

PURPOSE: To determine the half-life of mycophenolic acid (MPA) in the vitreous of New Zealand albino rabbits after intravitreal injection and the retinal toxicity of different doses of MPA. METHODS: Ten micrograms of MPA (Roche Bioscience, Palo Alto, CA) was injected in the vitreous of 16 rabbits, animals were sacrificed at different time-points, and vitreous samples underwent high-performance liquid chromatography. For functional and morphological studies, 5 doses of MPA (0.05, 0.5, 2, 10, and 100 µg) were injected in the vitreous of 20 rabbits. As control, contralateral eyes were injected with aqueous vehicle. Electroretinograms (ERGs) were recorded before injection and at days 7, 15, and 30. Animals were sacrificed on day 30 and retinas were analyzed under light microscopy. RESULTS: MPA half-life in the vitreous was 5.0±0.3 days. ERG revealed photoreceptor functional impairment in eyes injected with 0.5 µg and higher on day 30, while eyes injected with 100 µg presented the same changes already from day 15. No morphological change was found. CONCLUSIONS: MPA vitreous half-life is 5.0 days. Intravitreal injection of 0.5 µg MPA and higher causes dose- and time-related photoreceptor sensitivity decrease in rabbits. The MPA dose of 0.05 µg may be safe for intravitreal use in rabbits.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Inmunosupresores/administración & dosificación , Ácido Micofenólico/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Fenómenos Electrofisiológicos , Electrorretinografía , Semivida , Inmunosupresores/farmacocinética , Inmunosupresores/toxicidad , Inyecciones Intravítreas , Ácido Micofenólico/farmacocinética , Ácido Micofenólico/toxicidad , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Conejos , Retina/efectos de los fármacos , Retina/patología , Factores de Tiempo , Cuerpo Vítreo/metabolismo