The effect of dietary antioxidants on lipofuscin accumulation in the crustacean brain

Lipofuscin accumulation is associated with ageing at the subcellular level. A strong correlation between lipofuscin and age has been found in crustaceans using histological techniques. This association has been proposed as the basis for a methodology to age crustaceans and in some cases lipofuscin levels were found to be better correlated with age than size. The experiment presented here was designed to test the potential effect of diet, in particular dietary antioxidants, on lipofuscin accumulation and age estimation.The shrimp, Penaeus japonicus, was reared in an aquaculture facility and fed commercial pellets with modified vitamins C and E contents. One group was fed with levels of vitamins C and E of 1000 and 150 mg/kg, respectively, and another group with 2500 and 5000 mg/kg, respectively. The experiment started when the shrimp were 19 weeks old. Samples were obtained at this point and at ages 33 and 43 weeks. Lipofuscin was measured in the nerve cords (antennal neuropils and oesophageal connectives) in an area adjacent to the brain.Dietary antioxidants significantly affected lipofuscin levels. High vitamin content in the diet resulted in lower percentage of the observed area covered with lipofuscin, lower lipofuscin granule density and lower average granule size. Gender had no effect on any of these variables and granule size did not significantly change within each treatment. Lipofuscin area and granule density increased with age in both vitamin treatments.These results suggest that age estimation using lipofuscin indices may be biased when: (1) wild populations are dispersed over diverse environments; (2) the age estimation of wild individuals is based on the results obtained using laboratory-reared individuals.     

A comparative study on the effects of ageing and training on the levels of lipofuscin in various tissues of the rat

1. A modified procedure, based on the autofluorescent properties of the age pigment, lipofuscin, has been applied to obtain quantitative data on the accumulation of the proteolipid fraction of the pigment in a variety of tissues of the rat (Rattus norvegicus). The tissues investigated included muscle (fast-twitch white and red fibers), heart, brain and spleen. 2. The effects of a life-long treadmill training program on the concentration of lipofuscin in the different tissues, was determined in a cross-sectional study at various ages between 41/2 and 19 months. 3. All the tissues investigated revealed an increase in the concentration of lipofuscin with training. The increase exceeded the increment observed in ageing control animals. 4. It is postulated that the elevated lipofuscin levels observed with physical training reflect an ultrastructural adaptive change in the cells. A relative deficiency of oxygen in the tissues could be the important metabolic factor triggering this mechanism.     

Estimating age and growth in long‐lived temperate freshwater crayfish using lipofuscin

1. In ecological studies on freshwater crayfish, determination of basic population parameters is often complicated by the lack of a suitable age estimation method.
2. Previously, lipofuscin age pigment in the olfactory lobe cell masses (OLCM) of short‐lived tropical crayfish has been used for accurate age determination. Here we present the first test of this method on a longer‐lived, temperate species, the signal crayfish, Pacifastacus leniusculus .
3. Confocal fluorescence microscopy and image analysis of histological sections were used to quantify OLCM lipofuscin in a reference sample of Swedish P. leniusculus from several known year‐classes, reared under naturally variable temperature conditions. Lipofuscin concentration was linearly associated with age ( r² = 92.4%) and produced much more accurate age estimates than conventional body size‐based procedures.
4. A model derived from the crayfish of known‐age was used to estimate the ages of wild P. leniusculus from an English stream. The relationship between lipofuscin‐estimated age and carapace length suggested relatively slow growth in this wild population, consistent with a high population density and severe competition. The analysis also extended the known longevity of P. leniusculus to approximately 16 years.
5. The lipofuscin method for determining age and growth may be widely applicable to freshwater crayfish, with probable further potential both within and outside the Crustacea.     

Occurrence of the autofluorescent pigment, lipofuscin, in polar crustaceans and its potential as an age marker

In crustaceans, the lack of reliable methods often prevents the determination of individual age. The quantification of the autofluorescent age pigment, lipofuscin, has revealed promising results in boreal and tropical species. We studied the presence of morphological lipofuscin and its possible application as an age marker in five Arctic and five Antarctic species, comprising decapods, amphipods and a euphausiid. Lipofuscin granules were located in the brain, using confocal fluorescence microscopy, and quantified from digital images. The pigment was found in 94 of 100 individuals and in all 10 species, and granules occurred in easily detectable amounts in 5 species. Two scavenging amphipod species, the Antarctic Waldeckia obesa and the Arctic Eurythenes gryllus, revealed the most conspicuous and numerous granules. There was a broad, though weak, correlation of lipofuscin concentration with individual body size within a species, but not with absolute body size of one species compared to another. In larvae of the decapod Chorismus antarcticus, lipofuscin accumulation was quantified over the 1st 4 months after larval release. Morphological lipofuscin is a potential index of age in those investigated species with a sufficient accumulation rate of the pigment.     

Autofluorescence method to measure macular pigment optical densities fluorometry and autofluorescence imaging

Non-invasive measurement of the optical density of the human macular pigment by the autofluorescence method takes advantage of the fluorescence of lipofuscin in the human retinal pigment epithelium. Measuring the intensity of fluorescence above 550 nm, where macular pigment has essentially zero absorption, and stimulating the fluorescence with two wavelengths, one well absorbed by macular pigment and the other minimally absorbed by macular pigment, provides a single-pass measurement of the macular pigment optical density. The method is implemented either by fluorometry of lipofuscin to yield the optical density of the macular pigment in a 2 degrees diameter central area, or by autofluorescence imaging to yield a high-resolution map of the macular pigment distribution.     

Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants.

The accumulation of lipofuscin by retinal pigment epithelium may be an important feature in the pathogenesis of age-related macular degeneration, suggesting the possibility that this common cause of blindness might be prevented or delayed by antioxidants. In support of this idea, we now report significantly reduced formation of lipofuscin when the antioxidant substances lutein, zeaxanthin, lycopene (carotenoids), or alpha-tocopherol were added to rabbit and bovine (calf) retinal pigment epithelial (RPE) cells exposed to normobaric hyperoxia (40%) and photoreceptor outer segments. Rabbit and calf RPE cells were grown for 2 weeks with addition of one of the test substances every 48 h. The cellular uptake of carotenoids and alpha-tocopherol was assayed by HPLC after 2 weeks. The lipofuscin-content was measured by static fluorometry (rabbit cells) or by image analysis (calf cells). Both rabbit and calf RPE showed similar results with significantly lower amounts of lipofuscin in antioxidant-treated cells. The effect of carotenoids is especially interesting, since the result is not dependent on their protective effect against photo-oxidative reactions. The chain-breaking abilities of these antioxidants in peroxidative reactions of lipid membranes and quenching of free radicals seem to be of importance for inhibition of lipofuscin formation.     

Lipofuscin Accumulation in Cultured Retinal Pigment Epithelial Cells Causes Enhanced Sensitivity to Blue Light Irradiation

Lipofuscin accumulates with age within secondary lysosomes of retinal pigment epithelial (RPE) cells of humans and many animals. The autofluorescent lipofuscin pigment has an excitation maximum within the range of visible blue light, while it is emitting in the yellow-orange area. This physico-chemical property of the pigment indicates that it may have a photo-oxidative capacity and, consequently, then should destabilize lysosomal membranes of blue-light exposed RPE. To test this hypothesis, being of relevance to the understanding of age-related macular degeneration, cultures of heavily lipofuscin-loaded RPE cells were blue-light–irradiated and compared with respect to lysosomal stability and cell viability to relevant controls. To rapidly convert primary cultures of RPE, obtained from neonatal rabbits, into aged, lipofuscin-loaded cells, they were allowed to phagocytize artificial lipofuscin that was prepared from outer segments of bovine rods and cones. Following blue-light irradiation, lysosomal membrane stability was measured by vital staining with the lysosomotropic weak base, and metachromatic fluorochrome, acridine orange (AO). Quantifying red (high AO concentration within intact lysosomes with preserved proton gradient over their membranes) and green fluorescence (low AO concentration in nuclei, damaged lysosomes with decreased or lost proton gradients, and in the cytosol) allowed an estimation of the lysosomal membrane stability after blue-light irradiation. Cellular viability was estimated with the delayed trypan blue dye exclusion test. Lipofuscin-loaded blue-light–exposed RPE cells showed a considerably enhanced loss of both lysosomal stability and viability when compared to control cells. It is concluded that the accumulation of lipofuscin within secondary lysosomes of RPE sensitizes these cells to blue light by inducing photo-oxidative alterations of their lysosomal membranes resulting in a presumed leakage of lysosomal contents to the cytosol with ensuing cellular degeneration of apoptotic type. The suggested mechanism may have bearings on the development of age-related macular degeneration. © 1997 Elsevier Science Inc.     

Lipofuscin-mediated photic stress inhibits phagocytic activity of ARPE-19 cells; effect of donors’ age and antioxidants

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.     

Occurrence of xanthommatin containing pigment granules in the epidermal cells of the silkworm,Bombyx mori

A new type of pigment granule was found in the epidermal cells of the quail mutant of the silkworm, Bombyx mori. Electron microscopic observation shows this granule to be dense and distinct from the translucent pteridine granule. After the granules were isolated by sucrose density gradient centrifugation, the pigment was extracted and identified as xanthommatin.Xanthommatin localizes in the pigment granules binding with a protein. By SDS-polyacrylamide gel electrophoresis, the molecular weight of the pigment protein was estimated to be 13 kDa. The pigment granules may have a role in the biosynthesis and accumulation of xanthommatin.     

Pattern of lipofuscin pigmentation in nitrergic and non-nitrergic,neurofilament immunoreactive myenteric neuron types of human small intestine

Lipofuscin, an autofluorescent age pigment, occurs in enteric neurons. Due to its broad excitation and emission spectra, it overlaps with commonly used fluorophores in immunohistochemistry. We investigated the pattern of lipofuscin pigmentation in neurofilament (NF)-reactive nitrergic and non-nitrergic human myenteric neuron types. Subsequently, we tested two methods for reduction of lipofuscin-like autofluorescence. Myenteric plexus/longitudinal muscle wholemounts of small intestines of five patients undergoing surgery for carcinoma (aged between 18 and 69 years) were double stained for NF and neuronal nitric oxide synthase (nNOS). Lipofuscin pigmentation patterns were semiquantitatively evaluated by using confocal laser scanning microscopy with three different excitation wave lengths (one for undisturbed lipofuscin autofluorescence and two for specific labellings). Two pigmentation patterns could be detected in the five NF-reactive neuron types investigated. In nitrergic/spiny as well as in non-nitrergic/stubby neurons, coarse, intensely autofluorescent pigment granules were prominent. In non-nitrergic type II, III and V neurons, a fine granular, diffusely distributed and less intensely autofluorescent pigment was obvious. After incubation of wholemounts in either CuSO₄ or Sudan black B solutions, unspecific autofluorescence could be substantially reduced whereas specific NF and nNOS fluorescence remained largely unaffected. We conclude that NF immunohistochemistry is useful for morphological representation of subpopulations of human myenteric neurons. The lipofuscin pigmentation in human myenteric neurons reveals at least two different patterns which can be related to distinct neuron types. Incubations of multiply stained whole mounts in both CuSO₄ or Sudan black B are suitable methods for reducing autofluorescence thus facilitating discrimination between specific (immunohistochemical) and non-specific (lipofuscin) fluorescence.     

Maturation-related changes in mass and elemental contents of secretory granules as measured by electron-microprobe

Summary The relationship between granule density, protein content, and Ca and S contents were studied in two secretory granule fractions, from parotid glands of the rat, previously shown to constitute different stages in granule maturation. The density of the lighter fraction was between 1.133 and 1.142 g/ml, while that of the heavier fraction was greater than 1.142 g/ml. The mean protein content of the denser granules was 12% greater than that of the lighter granules (P<0.03), while the dry-mass elemental concentrations in the two granule fractions were unchanged. These results indicate that protein is added to granules during the maturation process (presumably by vesicular traffic), and that the resulting increase in granule density is not driven simply by decrease in water content and/or increased concentrations of inorganic Ca or S in the granules. The elemental concentration values also indicate that the diffusible elements permeate the granule membrane during the fractionation procedures.     

Retinyl palmitate and the blue-light-induced phototoxicity of human ocular lipofuscin

Lipofuscin accumulation in the retinal pigment epithelium is associated with the onset of age-related macular degeneration. Lipofuscin is phototoxic and affects cellular function through the photochemical generation of reactive oxygen intermediates. Mass spectral analysis of solvent extracts of human retinal lipofuscin granules reveals the presence of retinyl palmitate, the substrate for the enzymatic regeneration of 11-cis-retinal. Retinyl palmitate has an appreciable binding constant for phosphatidylcholine liposomes, and based on the glycophospholipids present in lipofuscin, retinal palmitate likely accumulates within the lipid content of the granule. Photochemical oxidation of retinal palmitate generates anhydroretinol, an intracellular signaling retinoid in the signal transduction cascade from the plasma membrane that causes apoptosis by generating reactive oxygen intermediates. These data are used to propose a model for the phototoxicity of lipofuscin.     

Lipofuscin and N-retinylidene-N-retinylethanolamine (A2E) accumulate in retinal pigment epithelium in absence of light exposure: their origin is 11-cis-retinal

The age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been associated with the development of retinal diseases, particularly age-related macular degeneration and Stargardt disease. A major component of lipofuscin is the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent release of all-trans-retinal. To understand the role of light exposure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors from mice of different ages and strains, reared either in darkness or cyclic light. Lipofuscin levels were determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorption spectroscopy. The identity of A2E was confirmed by tandem mass spectrometry. Lipofuscin and A2E levels in the RPE increased with age and more so in the Stargardt model Abca4(-/-) than in the wild type strains 129/sv and C57Bl/6. For each strain, the levels of lipofuscin precursor fluorophores in dark-adapted rods and the levels and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic light-reared animals. Both 11-cis- and all-trans-retinal generated lipofuscin-like fluorophores when added to metabolically compromised rod outer segments; however, it was only 11-cis-retinal that generated such fluorophores when added to metabolically intact rods. The results suggest that lipofuscin originates from the free 11-cis-retinal that is continuously supplied to the rod for rhodopsin regeneration and outer segment renewal. The physiological role of Abca4 may include the translocation of 11-cis-retinal complexes across the disk membrane.     

Effect of age on lipid peroxides, lipofuscin and ascorbic acid contents of the lungs of male garden lizard

Oxidative damage was assessed through the estimation of lipid peroxides (LP) in the lungs of an ageing short-lived species of reptile, Calotes versicolor, commonly known as the garden lizard. Attempts were also made to trace its relationship with the age pigment, lipofuscin and the antioxidant ascorbic acid. While LP increased with advancing age the contents of both lipofuscin and ascorbic acid did not show appreciable change during maturation ( < 1-1 year old) but declined during senescence phase (1 to 2-4 year old). While the pattern of age associated changes in LP and ascorbic acid indicate similarity with the pattern observed in most of the mammals, the reduction of lipofuscin in older lizards is a significant departure from the common trend.     

Age determination of Antarctic krill using fluorescence and image analysis of size

Summary The ages of 252 mature female krill, collected from Prydz Bay in January 1985, were determined using length frequency analysis and the fluorescent age pigment (FAP) technique. Results of both methods suggest 6 years classes for adult krill. Correspondence between the ages determined by the two techniques is generally within one year. The animals were also analyzed by a computerized image analysis system, which recorded a large suite of size and shape parameters. The accuracy of discriminant functions constructed to relate the image analysis parameters to age approached 90% for the ages defined by length frequency, and 52% for physiological age.     

Age-dependent anatomical changes in an identified neuron in the cns of Aplysia californica

Neurons of Aplysia californica are naturally pigmented and the pigment accumulates with age. In the present study the pigment was examined in the same neuron from Aplysia of three postmetamorphic ages: young, sexually mature, and old. The large central neuron, R₂, was examined by light and electron microscopy to determine if the pigment possessed properties similar to lipofuscin pigment seen in aging mammalian neurons. We used the same microscopic techniques that demonstrate the presence of lipofuscin in mammalian neurons. Light microscopic studies demonstrated a regional correlation between autofluorescence, staining with Sudan Black, and the naturally occurring pigment in old R₂s. Electron microscopic studies revealed the presence of large vacuolated and lamellated membrane-bound bodies in the peripheral cytoplasm of old R₂s, similar to those found in mammalian neurons. The bodies were located in the same region in which autofluorescence and Sudan Black staining were observed. Although the naturally occurring pigment accumulates with age, it acquires characteristics of lipofuscin pigment in the neurons of older sexually mature animals. The presence of these pigment characteristics can be used as an index of aging in Aplysia neurons as they are in mammalian neurons.     

The photoreactivity of the retinal age pigment lipofuscin.

The presence of the age pigment lipofuscin is associated with numerous age-related diseases. In the retina lipofuscin is located within the pigment epithelium where it is exposed to high oxygen and visible light, a prime environment for the generation of reactive oxygen species. Although we, and others, have demonstrated that retinal lipofuscin is a photoinducible generator of reactive oxygen species it is unclear how this may translate into cell damage. The position of lipofuscin within the lysosome infers that irradiated lipofuscin is liable to cause oxidative damage to either the lysosomal membrane or the lysosomal enzymes. We have found that illumination of lipofuscin with visible light is capable of extragranular lipid peroxidation, enzyme inactivation, and protein oxidation. These effects, which were pH-dependent, were significantly reduced by the addition of the antioxidants, superoxide dismutase and 1,4-diazabicyclo(2,2,2)-octane, confirming a role for both the superoxide anion and singlet oxygen. We postulate that lipofuscin may compromise retinal cell function by causing loss of lysosomal integrity and that this may be a major contributory factor to the pathology associated with retinal light damage and diseases such as age-related macular degeneration.     

The autofluorescent age pigment lipofuscin: key to age, growth and productivity of the Antarctic amphipod Waldeckia obesa (Chevreux, 1905)

Peracarid crustaceans are among the most important taxa in terms of biodiversity and carbon-flow within the Weddell Sea benthos; however, very few data on their age, growth and productivity are available. This study uses the pigment lipofuscin as an age marker in the scavenging amphipod Waldeckia obesa (Chevreux, 1905) from the eastern Weddell Sea. Resin brain sections of 159 trap-caught specimens (1.2 to 7.7 mm coxal plate length L(cox) equal to 5 to 31 mm total length) were recorded digitally by confocal microscopy, and images were analysed. A modal progression analysis of the lipofuscin concentration-frequency distribution revealed five regularly spaced modes presumed to reflect consecutive annual age classes. Single females outside the range of mode V occurred, indicating maximum age of up to 8 years in females. No regular modes were obvious from the comparable length-frequency distribution of 386 individuals. Average yearly pigment accumulation was linear, and accumulation rates did not differ between sexes. The estimates of the growth parameters L(infinity) and k of the von Bertalanffy growth function were 7.47 mm L(cox) and 0.50 per year in females, respectively, and 6.92 mm L(cox) and 0.60 per year in males, respectively. Mortality, estimated from catch curves, amounted to 0.27 per year in females and 0.43 per year in males. P/B ratio, calculated from the mass specific growth rate method, was 0.38 per year for the pooled population (0.25 per year in females, 0.31 per year in males, 2.26 per year in juveniles). The results are discussed with regard to advantages and drawbacks of the methodology, and are compared with results from warmer water habitats.     

Immunochemical detection of a lipofuscin-like fluorophore derived from malondialdehyde and lysine

The accumulation of fluorescent age pigment or lipofuscin is a frequently observed age-associated cellular alteration in a variety of postmitotic cells of many species. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. In the present study, we raised a monoclonal antibody (MAb 1F83) directed to the malondialdehyde-modified protein and identified a lipofuscin-like dihydropyridine fluorophore as the major epitope. This antibody was used to conclusively demonstrate that the fluorophore forms on oxidatively modified low density lipoproteins. In addition, we demonstrated that the materials immunoreactive to MAb 1F83 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells. The results of this study suggest that the reaction between the lipid peroxidation-derived aldehyde and primary amino groups of protein might represent a process common to the formation of the lipofuscin-like fluorophore during aging and its related diseases.     

Quantification of the age-pigment lipofuscin in known-age octopus (Octopus pallidus): A potential tool for age determination

Stylet increment analysis (SIA) is the key method to age octopus, however, currently it is not reliable for all species. The suitability of the age-pigment lipofuscin as an alternative ageing method for octopus was examined. To determine the relationship between age and lipofuscin known-age octopus (Octopus pallidus) were reared in the laboratory from hatching to eight months old. Twenty-eight individuals at three different ages (3, 6 and 8 months old) were collected for lipofuscin analysis. The first two age groups (n = 5 each) were reared under ambient temperatures, while the oldest group (n = 18) was reared under three different controlled temperature regimes (n = 6 per treatment). For comparison, five wild O. pallidus were also collected for lipofuscin analysis and aged using SIA. Lipofuscin was analysed in the brain tissue and quantified at a commercial ageing centre using standard histological methods. Lipofuscin granules were clearly discernable in the brain tissue, and there was a strong exponential relationship between age and lipofuscin (R² = 0.86). Lipofuscin concentration was not related to sex, temperature or body weight in same-age individuals. Except for one individual, the predicted age of the wild animals, based on the relationship between lipofuscin and age, was close to the age determined using SIA. This study is the first to report lipofuscin in an octopus species and shows that lipofuscin has excellent potential as an alternative ageing method for octopus. This research will have important applications for species which cannot be reliably aged using current ageing methods.