RESUMEN
Significance: Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). Aim: To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. Approach: AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( λ ex / λ em ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. Results: A repeatability analysis revealed < 11.8 % change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Conclusions: Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.
Asunto(s)
Oftalmoscopía , Epitelio Pigmentado de la Retina , Humanos , Oftalmoscopía/métodos , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/química , Adulto , Masculino , Femenino , Envejecimiento/fisiología , Persona de Mediana Edad , Anciano , Adulto Joven , Imagen Óptica/métodos , Lipofuscina/metabolismo , Lipofuscina/análisis , Lipofuscina/química , Estudios de FactibilidadRESUMEN
Age-related macular degeneration (AMD) is a debilitating eye disease that tends to affect people over the age of 55. Lipofuscins are autofluorescent, toxic byproducts of the visual cycle thought to contribute toward the progression of the disease. Targeting the accumulation of lipofuscin through catabolism may serve as a method for the early treatment of AMD. Thus, an enzymatic approach capable of degrading lipofuscin, cycloretinal (all-trans retinal dimer), was examined. A peroxidase from the organism Marasmius scorodonius (MsP1) has shown capability of degrading this toxic metabolite into nontoxic byproducts. A catalytic triad within MsP1 (D228, H365, and R388) was identified through multiple-sequence alignment and homology modeling and confirmed by kinetic analysis. MsP1-associated cleavage products were detected by gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and liquid chromatography-tandem mass spectrometry (LC-MSMS). MsP1 degradation byproducts of cycloretinal show reduced cytotoxicity within cell culture (ARPE-19), demonstrating its potential as a gene therapeutic to alleviate the buildup of lipofuscin within AMD.
Asunto(s)
Lipofuscina , Degeneración Macular , Humanos , Cromatografía Líquida de Alta Presión , Cinética , Lipofuscina/química , Degeneración Macular/metabolismo , PeroxidasasRESUMEN
A major limitation of mechanistic studies in aging brains is the lack of routine methods to robustly visualize and discriminate the cellular distribution of tissue antigens using fluorescent immunohistochemical multi-labeling techniques. Although such approaches are routine in non-aging brains, they are not consistently feasible in the aging brain due to the progressive accumulation of autofluorescent pigments, particularly lipofuscin, which strongly excite and emit over a broad spectral range. Consequently, aging research has relied upon colorimetric antibody techniques, where discrimination of tissue antigens is often challenging. We report the application of a simple, reproducible, and affordable protocol using multispectral light-emitting diodes (mLEDs) exposure for the reduction/elimination of lipofuscin autofluorescence (LAF) in aging brain tissue from humans, non-human primates, and mice. The mLEDs lamp has a broad spectral range that spans from the UV to infrared range and includes spectra in the violet/blue and orange/red. After photo quenching, the LAF level was markedly reduced when the tissue background fluorescence before and after mLEDs exposure was compared (p < 0.0001) across the spectral range. LAF elimination was estimated at 95 ± 1%. This approach permitted robust specific fluorescent immunohistochemical co-visualization of commonly studied antigens in aging brains. We also successfully applied this method to specifically visualize CD44 variant expression in aging human cerebral white matter using RNAscope fluorescent in-situ hybridization. Photo quenching provides an attractive means to accelerate progress in aging research by increasing the number of molecules that can be topologically discriminated by fluorescence detection in brain tissue from normative or pathological aging.
Asunto(s)
Envejecimiento , Encéfalo , Receptores de Hialuranos , Primates , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Encéfalo/metabolismo , Variación Genética , Humanos , Receptores de Hialuranos/genética , Inmunohistoquímica , Hibridación in Situ , Lipofuscina/química , Ratones , Primates/genéticaRESUMEN
Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE), where its fluorescence properties are used to assess retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in the early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE and lipofuscin-laden cells to visible light, and to determine whether an abundant component of lipofuscin, docosahexaenoate (DHA), can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible light leads to a decrease in its long-wavelength fluorescence at about 610 nm, with a concomitant increase in the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure of lipofuscin-laden cells to light leads to a loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes in fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra, together with quantitation of the intensity of long-wavelength fluorescence, can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, and hence useful for screening the retina for increased oxidative damage and early AMD-related changes.
Asunto(s)
Ácidos Docosahexaenoicos/química , Lipofuscina/química , Epitelio Pigmentado de la Retina/citología , Línea Celular , Supervivencia Celular , Endocitosis , Humanos , Luz , Microscopía Fluorescente , Oxidación-Reducción , Fotólisis , Epitelio Pigmentado de la Retina/químicaRESUMEN
Retinal lipofuscin which accumulates with age in the retinal pigment epithelium (RPE) is subjected to daily exposures to high fluxes of visible light and exhibits potent photosensitising properties; however, the molecules responsible for its photoreactivity remain unknown. Here, we demonstrate that autooxidation of docosahexaenoate (DHE) leads to the formation of products absorbing, in addition to UVB and UVA light, also visible light. The products of DHE oxidation exhibit potent photosensitising properties similar to photosensitising properties of lipofuscin, including generation of an excited triplet state with similar characteristics as the lipofuscin triplet state, and photosensitised formation of singlet oxygen and superoxide. The quantum yields of singlet oxygen and superoxide generation by oxidised DHE photoexcited with visible light are 2.4- and 3.6-fold higher, respectively, than for lipofuscin, which is consistent with the fact that lipofuscin contains some chromophores which do contribute to the absorption of light but not so much to its photosensitising properties. Importantly, the wavelength dependence of photooxidation induced by DHE oxidation products normalised to equal numbers of incident photons is also similar to that of lipofuscin-it steeply increases with decreasing wavelength. Altogether, our results demonstrate that products of DHE oxidation include potent photosensitiser(s) which are likely to contribute to lipofuscin photoreactivity.
Asunto(s)
Ácidos Docosahexaenoicos/química , Luz , Lipofuscina/química , Retina/metabolismo , Humanos , Oxidación-Reducción , Procesos Fotoquímicos , Oxígeno Singlete/química , Superóxidos/químicaRESUMEN
Purpose: To investigate the major organelles of the retinal pigment epithelium (RPE) in wild-type (WT, control) mice and their changes in pigmented Abca4 knockout (Abca4-/-) mice with in situ morphologic, spatial, and spectral characterization of live ex vivo flat-mounted RPE using multicolor confocal fluorescence microscopy (MCFM). Methods: In situ imaging of RPE flat-mounts of agouti Abca4-/- (129S4), agouti WT (129S1/SvlmJ) controls, and B6 albino mice (C57BL/6J-Tyrc-Brd) was performed with a Nikon A1 confocal microscope. High-resolution confocal image z-stacks of the RPE cell mosaic were acquired with four different excitation wavelengths (405 nm, 488 nm, 561 nm, and 640 nm). The autofluorescence images of RPE, including voxel-by-voxel emission spectra, were acquired and processed with Nikon NIS-AR Elements software. Results: The 3-dimensional multicolor confocal images provided a detailed visualization of the RPE cell mosaic, including its melanosomes and lipofuscin granules, and their varying characteristics in the different mice strains. The autofluorescence spectra, spatial distribution, and morphologic features of melanosomes and lipofuscin granules were measured. Increased numbers of lipofuscin and reduced numbers of melanosomes were observed in the RPE of Abca4-/- mice relative to controls. Conclusions: A detailed assessment of the RPE autofluorescent granules and their changes ex vivo was possible with MCFM. For all excitation wavelengths, autofluorescence from the RPE cells was predominantly contributed by lipofuscin granules, while melanosomes were found to be essentially nonfluorescent. The red shift of the emission peak confirmed the presence of multiple chromophores within lipofuscin granules. The elevated autofluorescence levels in Abca4-/- mice correlated well with the increased number of lipofuscin granules.
Asunto(s)
Lipofuscina/metabolismo , Melanosomas/metabolismo , Orgánulos/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Femenino , Imagenología Tridimensional , Lipofuscina/química , Melanosomas/química , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Fluorescente , Epitelio Pigmentado de la Retina/diagnóstico por imagenRESUMEN
Lipofuscin granules accumulate in the retinal pigment epithelium (RPE) with age, especially in patients with visual diseases, including progressive age-related macular degeneration (AMD). Bisretinoids and their photooxidation and photodegradation products are major sources of lipofuscin granule fluorescence. The present study focused on examining the fluorescence decay characteristics of bisretinoid photooxidation and photodegradation products to evaluate the connection between fluorescence lifetime and spectral characteristics of target fluorophore groups. The primary objective of the study was to apply experimental spectral analysis results of lipofuscin granule fluorescence properties to interpretation of fluorescence lifetime imaging ophthalmoscopy data. Fluorescence analysis of the lipofuscin granule fluorophores in RPE collected from cadaver eyes was performed. The fluorescence lifetimes were measured by picosecond-resolved time correlated single photon counting technique. A global analytical method was applied to analyze data sets. The photooxidation and photodegradation products of bisretinoids exhibited a longer fluorescence lifetime (average value approximately 6 ns) and a shorter wavelength maximum (530-580 nm). Further, these products significantly contributed (more than 30%), to total fluorescence compared to the other fluorophores in lipofuscin granules. Thus, the contribution of oxidized lipofuscin bisretinoids to autofluorescence decay kinetics is an important characteristic for fluorescence lifetime imaging microscopy data analysis. The higher average fluorescence lifetime in AMD eyes was likely due to the higher abundance of oxidized bisretinoids compared with non-oxidized bisretinoids. Because higher level of oxidized bisretinoids is indicative of pathological processes in the retina and RPE, the present findings have the potential to improve fluorescence lifetime imaging approaches for early diagnosis of degenerative processes in the retina and RPE.
Asunto(s)
Fluorescencia , Colorantes Fluorescentes/química , Lipofuscina/química , Epitelio Pigmentado de la Retina/química , Adolescente , Adulto , Anciano , Humanos , Persona de Mediana Edad , Espectrometría de Fluorescencia , Adulto JovenRESUMEN
The deposition of beta-amyloid (Aß) in the brain is detected in Alzheimer's disease and during ageing. Until now, ultrastructural studies of changes caused by Aß in the dentate gyrus are very scarce. The effects of Aß 1-42 injection into the CA1 field of rat hippocampus were studied by electron microscopy. In 2 weeks after injection of aggregated Aß in low concentrations, destructive changes were seen in the structure of dentate gyrus cells, which consisted in a decrease in the number of dentate gyrus neurons and axo-dendritic synapses. These changes were accompanied by enlargement of the endoplasmic reticulum cisterns and widening of the active zones of synapses. Thus, injection of aggregated Aß 1-42 into the hippocampus led to irreversible (a decrease in the number of neurons and axo-dendritic synapses, agglutination of synthetic vesicles) and adaptive changes (an increase in the sizes of endoplasmic reticulum cisterns and active zones of synapses) in dentate gyrus neurons aimed at the maintenance of functional activity of the nervous system.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/administración & dosificación , Región CA1 Hipocampal/ultraestructura , Giro Dentado/ultraestructura , Neuronas/ultraestructura , Fragmentos de Péptidos/administración & dosificación , Sinapsis/ultraestructura , Enfermedad de Alzheimer/inducido químicamente , Péptidos beta-Amiloides/química , Animales , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/patología , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/patología , Gránulos Citoplasmáticos/ultraestructura , Giro Dentado/efectos de los fármacos , Giro Dentado/patología , Modelos Animales de Enfermedad , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/patología , Retículo Endoplásmico/ultraestructura , Inyecciones Intraventriculares , Lipofuscina/química , Masculino , Microscopía Electrónica , Neuronas/efectos de los fármacos , Neuronas/patología , Fragmentos de Péptidos/química , Agregado de Proteínas , Ratas , Ratas Wistar , Sinapsis/efectos de los fármacos , Sinapsis/patologíaRESUMEN
All-trans-retinal (atRAL) is a highly reactive carbonyl specie, known for its reactivity on cellular phosphatidylethanolamine in photoreceptor. It is generated by photoisomerization of 11-cis-retinal chromophore linked to opsin by the Schiff's base reaction. In ABCA4-associated autosomal recessive Stargardt macular dystrophy, atRAL results in carbonyl and oxidative stress, which leads to bisretinoid A2E, accumulation in the retinal pigment epithelium (RPE). This A2E-accumulation presents as lipofuscin fluorescent pigment, and its photooxidation causes subsequent damage. Here we describe protection against a lethal dose of atRAL in both photoreceptors and RPE in primary cultures by a lipidic polyphenol derivative, an isopropyl-phloroglucinol linked to DHA, referred to as IP-DHA. Next, we addressed the cellular and molecular defence mechanisms in commonly used human ARPE-19 cells. We determined that both polyunsaturated fatty acid and isopropyl substituents bond to phloroglucinol are essential to confer the highest protection. IP-DHA responds rapidly against the toxicity of atRAL and its protective effect persists. This healthy effect of IP-DHA applies to the mitochondrial respiration. IP-DHA also rescues RPE cells subjected to the toxic effects of A2E after blue light exposure. Together, our findings suggest that the beneficial role of IP-DHA in retinal cells involves both anti-carbonyl and anti-oxidative capacities.
Asunto(s)
Deshidroepiandrosterona/farmacología , Floroglucinol/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Retinaldehído/toxicidad , Animales , Antioxidantes/farmacología , Catalasa/metabolismo , Línea Celular , Supervivencia Celular , Humanos , Lipofuscina/química , Ratones , Mitocondrias/metabolismo , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Oxígeno/química , Consumo de Oxígeno , Fenol/química , Floroglucinol/química , Pigmentación , Sustancias Protectoras/farmacología , Ratas , Especies Reactivas de Oxígeno , Epitelio Pigmentado de la Retina/metabolismo , Retinoides/metabolismo , Relación Estructura-ActividadRESUMEN
The study of brain pathology by fluorescence microscopy finds in the autofluorescence of the tissue an additional difficulty for the recognition of markers of interest. In particular, in the immunofluorescence study of brains from Alzheimer's disease (AD) patients, several approaches have been attempted to eliminate or mask the presence of autofluorescent aggregates. In the present work, we propose a method to characterize by fluorescent microscopy senile plaques discriminating them from autofluorescent aggregates, such as lipofuscin granules.This work describes four protocols carried out in human brain tissue of patients with AD, covering adequate tissue preparation, immunofluorescence acquisition, and data analysis: 1. Tissues processing of frozen samples for optimal epitope conservation. 2. Analysis of the fluorescence emission spectrum of the tissue by performing a confocal microscopy λ-scan. 3. Analysis of fluorescence emission of both intact and formic acid-treated tissues in four channels corresponding to the emission in blue, green, near red, and far-red regions. 4. Analysis a specific immunostaining of amyloid beta in senile plaques, using fluorescent-labeled antibodies by using specific emission channels to avoid detection of tissue autofluorescence.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Lipofuscina/química , Placa Amiloide/diagnóstico por imagen , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía Confocal , Microscopía Fluorescente , Placa Amiloide/metabolismo , Conservación de TejidoRESUMEN
PURPOSE: To examine the ultrastructure of lipofuscin bodies and melanosomes in retinal epithelium of elderly rhesus monkeys and determines changes in their number and morphology as a function of retinal eccentricity. METHODS: Electron microscopy was used to describe and quantify two major organelles in elderly monkey retinal epithelium, lipofuscin bodies and melanosomes, at different retinal loci extending from the macula to the peri-macula, equator, periphery and ora serrata. Osmium tetroxide was used to distinguish lipofuscin bodies from melanosomes. RESULTS: Lipofuscin bodies and melanosomes diminished in number with advanced age but there was an inverse relationship between these two organelles. Lipofuscin bodies were more numerous in the macula and melanosomes more numerous in the peripheral retina. Three types of lipofuscin bodies were identified: 1) smaller and tending to locate in the middle third of the epithelial cell, 2) larger, less common, and located more basally, and 3) extremely rare, melano-lipofuscin, containing a melanosome. When osmicated, all lipofuscin bodies contained electron dense materials. When osmium tetroxide was not used for fixation, the first two types of lipofuscin bodies lost their electron densities while the third type retained its electron density due to the melanosome it contained. CONCLUSION: As previously reported for human retina, lipofuscin is most abundant in the macular and peri-macular epithelium and least abundant in the periphery, whereas melanosomes show the opposite relationship. This distribution pattern could contribute to the macula's greater vulnerability to photo-toxicity. Three types of lipofuscin bodies are found in aging monkey retinal epithelium. All types contain electron dense material, but the most prominent two types lose their densities in the absence of osmium tetroxide during fixation. Most of the electron densities in lipofuscin bodies must contain a material that binds strongly to osmium tetroxide such as polyunsaturated fatty acids.
Asunto(s)
Envejecimiento/metabolismo , Lipofuscina/química , Melanosomas/ultraestructura , Tetróxido de Osmio/química , Epitelio Pigmentado de la Retina/ultraestructura , Animales , Microanálisis por Sonda Electrónica , Femenino , Macaca mulatta , Masculino , Melanosomas/química , Microscopía Electrónica , Modelos Animales , Epitelio Pigmentado de la Retina/químicaRESUMEN
The risk of chronic oxidative stress in the retinal pigment epithelium (RPE) increases with age due to accumulation of the photoreactive age pigment lipofuscin (LFG). Here, we asked whether sublethal and weakly lethal photic stress, induced by irradiation of ARPE-19 cells containing phagocytised LFG, affected the cell specific phagocytic activity, which is critically important for proper functioning and survival of the retina, and if natural antioxidants could modify the observed outcomes. ARPE-19 cells preloaded with LFG isolated from human donors of different age or containing LFG enriched with zeaxanthin and α-tocopherol (LFG-A), were irradiated with blue light. Phagocytosis of fluorescein-5-isothiocyanate (FITC)-labelled photoreceptor outer segments was determined by flow cytometry. Photoreactivity of LFG and LFG-A was analysed by measuring photoconsumption of oxygen and photogeneration of singlet oxygen mediated by the granules. LFG-mediated photic stress in ARPE-19 cells induced significant inhibition of their specific phagocytosis. The inhibitory effect increased with age of LFG donors and was reduced by enrichment of the granules with antioxidants. Oxygen consumption and generation of singlet oxygen induced by the photoexcited LFG increased with donor's age and was partially quenched by antioxidants. Although the phototoxic potential of lipofuscin increased with age, natural antioxidants reduced photoreactivity of LFG and their efficiency to induce oxidative stress. This study has demonstrated, for the first time, that mild oxidative stress, mediated by the age pigment lipofuscin, impairs specific phagocytic activity of RPE, and that natural antioxidants can protect this important cellular function by reducing lipofuscin photoreactivity.
Asunto(s)
Lipofuscina/fisiología , Estrés Oxidativo , Fagocitosis , Epitelio Pigmentado de la Retina/metabolismo , Adolescente , Adulto , Envejecimiento , Antioxidantes/farmacología , Línea Celular , Humanos , Luz , Lipofuscina/química , Persona de Mediana Edad , Procesos Fotoquímicos , Epitelio Pigmentado de la Retina/efectos de la radiación , Albúmina Sérica Bovina/química , Adulto Joven , Zeaxantinas/química , Zeaxantinas/farmacología , alfa-Tocoferol/química , alfa-Tocoferol/farmacologíaRESUMEN
Lipofuscins are toxic autofluorescent byproducts of the visual cycle. The accumulation of lipofuscins such as cycloretinal in the retina is thought to play a role in the progression of age-related macular degeneration (AMD). Intriguingly, the milk protein ß-lactoglobulin (BLG) can promote the cyclodimerization of all-trans-retinal to cycloretinal both in vitro and in vivo. Here, site-directed mutagenesis of BLG and mass spectrometric analysis with substrate analogues demonstrate that lysine residues play a key role in catalysis. It is also shown that catalytic activity necessitates the presence of a physical binding site and cannot be mediated by a peptide chain. These studies provide insight into the mechanism of the cyclodimerization process and provide a model system for biocatalysis and biosynthesis of cycloretinal in vivo. In the long term, these studies may pave the way for drug development and inhibitor design as an early treatment regimen for AMD.
Asunto(s)
Lactoglobulinas/química , Lipofuscina/química , Mutación Missense , Sustitución de Aminoácidos , Catálisis , Humanos , Lactoglobulinas/genética , Lactoglobulinas/metabolismo , Lipofuscina/genética , Lipofuscina/metabolismo , Degeneración Macular/metabolismo , Mutagénesis Sitio-DirigidaRESUMEN
The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.
Asunto(s)
Melaninas/metabolismo , Melanosomas/metabolismo , Animales , Bovinos , Decapodiformes/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Cinética , Luz , Lipofuscina/química , Lipofuscina/metabolismo , Melaninas/química , Melanosomas/efectos de los fármacos , Melanosomas/efectos de la radiación , Oxidación-Reducción/efectos de los fármacos , Oxidación-Reducción/efectos de la radiación , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Superóxidos/química , Superóxidos/metabolismo , Superóxidos/farmacologíaRESUMEN
The fluorescence lifetimes of lipofuscin fluorophores contained in chloroform extracts from retinal pigment epithelium (RPE) of human cadaver eyes without signs of pathology were evaluated by single photon counting. The comparison of fluorescence lifetimes of N-retinylidene-N-retinylethanolamine (A2E) and its photooxidation and photodegradation products has been carried out. It was shown that the contribution of A2E to the total fluorescence of chloroform extract from lipofuscin granules is not major. The results are important for the improvement of noninvasive diagnostic method of degenerative diseases of the retina and RPE-fundus autofluorescence (FAF).
Asunto(s)
Lipofuscina/química , Epitelio Pigmentado de la Retina/metabolismo , Cadáver , Gránulos Citoplasmáticos/metabolismo , Fluorescencia , Humanos , Lipofuscina/metabolismo , Oxidación-Reducción , Fotones , Retinoides/químicaRESUMEN
This paper shows that the aging and death of nematodes, accompanied by the ignition of a blue glow under fluorescent microscopy, are not directly linked to any lipofuscin (aging pigment), nor with the anthranilic acid (a product of degradation of tryptophan residues of proteins). The main contribution in the blue flash of the dying nematodes belongs to parasitic light, scattered on the cuticle and bodies of the worm. The main contribution in the blue region at spectrofluorometry of homogenates, obtained from nematodes, really gives anthranilic acid. However, the content of anthranilic acid, measured by spectrofluorimetry, in adult nematodes is lower than that in the young ones. Artificial aging of nematodes by moderate heating revealed no accumulation of anthranilate and no loss of tryptophan, from which it must be formed. Thus, it is hardly lipofuscin or anthranilic acid. The cause of aging and death of nematodes is the formation of strong cross-links between proteins. This is supported by data on tryptophan fluorescence and light scattering of homogenates: the old worms show a large number of denaturated proteins and large protein particles with a strong cross-links, which are not destroyed be detergent.
Asunto(s)
Proteínas del Helminto/química , Lipofuscina/química , Nematodos/química , ortoaminobenzoatos/química , Envejecimiento , Animales , Proteínas del Helminto/metabolismo , Lipofuscina/análisis , Microscopía Fluorescente , Nematodos/fisiología , Espectrometría de Fluorescencia , ortoaminobenzoatos/análisis , ortoaminobenzoatos/metabolismoRESUMEN
Autofluorescence of aldehyde-fixed tissues greatly hinders fluorescence microscopy. In particular, lipofuscin, an autofluorescent component of aged brain tissue, complicates fluorescence imaging of tissue in neurodegenerative diseases. Background and lipofuscin fluorescence can be reduced by greater than 90% through photobleaching using white phosphor light emitting diode arrays prior to treatment with fluorescent probes. We compared the effect of photobleaching versus established chemical quenchers on the quality of fluorescent staining in formalin-fixed brain tissue of frontotemporal dementia with tau-positive inclusions. Unlike chemical quenchers, which reduced fluorescent probe signals as well as background, photobleaching treatment had no effect on probe fluorescence intensity while it effectively reduced background and lipofuscin fluorescence. The advantages and versatility of photobleaching over established methods are discussed.
Asunto(s)
Encéfalo/patología , Formaldehído/química , Iluminación/instrumentación , Lipofuscina/química , Microscopía Fluorescente/economía , Microscopía Fluorescente/métodos , Fotoblanqueo/efectos de la radiación , Análisis Costo-Beneficio , Fluorescencia , Técnica del Anticuerpo Fluorescente , Humanos , Adhesión en Parafina , SemiconductoresRESUMEN
Lipofuscin granules accumulate in the cells of retinal pigment epithelium with age, particularly in patients with hereditary diseases. These granules are heterogeneous, being composed of mixtures of proteins and lipids, including more than 21 different fluorescent compounds. Bisretinoids and their photo-oxidation and photodegradation products represent the main source of lipofuscin fluorescence and exhibit phototoxic properties. This study used time-of-flight secondary ion mass spectrometry (ToF-SIMS) with in-depth probing to assess the depth distribution of N-retinylidene-N-retinylethanolamine (A2E) and its singly and doubly oxidized forms (A2E-ox and A2E-2ox, respectively) within lipofuscin granules and in their surface layer (lipid membrane). ToF-SIMS showed that A2E and its oxidized forms were uniformly distributed throughout lipofuscin granules but were not present at the membrane surface layer. This finding is important for understanding the process involved in the formation of lipofuscin granules and in their toxicity.
Asunto(s)
Lipofuscina/química , Epitelio Pigmentado de la Retina/química , Retinoides/análisis , Espectrometría de Masa de Ion Secundario/métodos , Anciano , Humanos , Persona de Mediana Edad , Oxidación-Reducción , Epitelio Pigmentado de la Retina/citologíaRESUMEN
The purpose of this study was to correlate features on flood-illuminated adaptive optics (AO) images with color fundus, fundus autofluorescence (FAF) and spectral domain optical coherence tomography (SD-OCT) images in patients with retinitis pigmentosa (RP). We imaged 39 subjects diagnosed with RP using the rtx1™ flood-illuminated AO camera from Imagine Eyes (Orsay, France). We observed a correlation between hyper-autofluoresence changes on FAF, disruption of the interdigitation zone (IZ) on SD-OCT and loss of reflective cone profiles on AO. Four main patterns of cone-reflectivity were seen on AO: presumed healthy cone mosaics, hypo-reflective blurred cone-like structures, higher frequency disorganized hyper-reflective spots, and lower frequency hypo-reflective spots. These regions were correlated to progressive phases of cone photoreceptor degeneration observed using SD-OCT and FAF. These results help provide interpretation of en face images obtained by flood-illuminated AO in subjects with RP. However, significant ambiguity remains as to what truly constitutes a cone, especially in areas of degeneration. With further refinements in technology, flood illuminated AO imaging has the potential to provide rapid, standardized, longitudinal and lower cost imaging in patients with retinal degeneration.
Asunto(s)
Oftalmoscopía/métodos , Retina/patología , Retinitis Pigmentosa/diagnóstico , Tomografía de Coherencia Óptica/métodos , Fluorescencia , Humanos , Lipofuscina/química , Lipofuscina/metabolismo , Microscopía Confocal/métodos , Reproducibilidad de los Resultados , Células Fotorreceptoras Retinianas Conos/patología , Epitelio Pigmentado de la Retina/química , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Sensibilidad y EspecificidadRESUMEN
Retinitis pigmentosa and cone/cone-rod dystrophy are inherited retinal diseases characterized by the progressive loss of rod and/or cone photoreceptors. To evaluate the status of rod/cone photoreceptors and visual function, visual acuity and visual field tests, electroretinogram, and optical coherence tomography are typically used. In addition to these examinations, fundus autofluorescence (FAF) has recently garnered attention. FAF visualizes the intrinsic fluorescent material in the retina, which is mainly lipofuscin contained within the retinal pigment epithelium. While conventional devices offer limited viewing angles in FAF, the recently developed Optos machine enables recording of wide-field FAF. With wide-field analysis, an association between abnormal FAF areas and visual function was demonstrated in retinitis pigmentosa and cone-rod dystrophy. In addition, the presence of "patchy" hypoautofluorescent areas was found to be correlated with symptom duration. Although physicians should be cautious when interpreting wide-field FAF results because the peripheral parts of the image are magnified significantly, this examination method provides previously unavailable information.