Topographical localization of lipofuscin pigment in the brain of the aged fat‐tailed dwarf lemur (Cheirogaleus Medius) and grey lesser mouse lemur (Microcebus Murinus): Comparison to iron localization

The present study was undertaken to explore the distribution of lipofuscin in the brain of cheirogaleids by autofluorescence and compare it to other studies of iron distribution. Aged dwarf (Cheirogaleus medius) and mouse (Microcebus murinus) lemurs provide a reliable model for the study of normal and pathological cerebral aging. Accumulation of lipofuscin, an age pigment derived by lipid peroxidation, constitutes the most reliable cytological change correlated with neuronal aging. Brain sections of four aged (8–15 year old) and 3 young (2–3 year old) animals were examined. Lipofuscin accumulation was observed in the aged animals but not in the young ones. Affected regions include the hippocampus (granular and pyramidal cells), where no iron accumulation was observed, the olfactory nucleus and the olfactory bulb (mitral cells), the basal forebrain, the hypothalamus, the cerebellum (Purkinje cells), the neocortex (essentially in the pyramidal cells), and the brainstem. Even though iron is known to catalyse lipid oxidation, our data indicate that iron deposits and lipofuscin accumulation are not coincident. Different biochemical and morphological cellular compartments might be involved in iron and lipofuscin deposition. The nonuniform distribution of lipofuscin indicates that brain structures are not equally sensitive to the factors causing lipofuscin accumulation. The small size, the rapid maturity, and the relatively short life expectancy of the cheirogaleids make them a good model system in which to investigate the mechanisms of lipofuscinogenesis in primates. Am. J. Primatol. 49:183–193, 1999. © 1999 Wiley‐Liss, Inc.     

Effects of Chlorpromazine on the Activities of Antioxidant Enzymes and Lipid Peroxidation in the Various Regions of Aging Rat Brain

In this work, the effect of chronic intraperitoneal administration of chlorpromazine (5 and 10 mg/kg) on the antioxidant enzymes superoxide dismutase (SOD), catalase (CA), glutathione reductase (GR), and glutathione peroxidase (GP); lipid peroxidation; and lipofuscin accumulation in the brains of rats ages 6, 9, and 12 months was studied. Chlorpromazine increased the activities of SOD, GR, and GP in particulate fraction from cerebrum, cerebellum, and brain stem in a dose-dependent manner. While GR and SOD associated with soluble fraction increased, GP associated with soluble fraction was not affected. CA did not change after chlorpromazine administration in any regions of the brain of rats from all age groups. Chlorpromazine, thus, had a somewhat different action on antioxidant enzymes in different subcellular fractions. Chlorpromazine inhibited lipid peroxidation, both in vivo and in vitro, and it also inhibited accumulation of lipid peroxidation fluorescent products (lipofuscin), which was studied histochemically and biochemically as well. The data indicate that chlorpromazine inhibition of lipid peroxidation and of accumulation of lipofuscin can result from elevation of the activity of brain antioxidant enzymes.     

Effect of Maharishi Amrit Kalash an ayurvedic herbal mixture on lipid peroxidation and neuronal lipofuscin accumulation in ageing guinea pig brain

The effects of ayurvedic herbal mixture Maharishi Amrit Kalash(MAK) were studied on brain lipid peroxidation, oxygen consumption, and lipofuscin accumulation in 10 months and 32 months old guinea pigs. Brain regions studied were cerebral cortex, hypothalamus, cerebellum and spinal cord. Parameters assessed were lipid peroxidation, oxygen consumption, and lipofuscin accumulation. The endogenous lipid peroxide was found to be increased significantly (P < 0.05) in the 32-month-old animals. Neuronal lipofuscin accumulation in the neurons of cerebral motor cortex, cerebellum and cervical spinal cord was increased (P < 0.05) in the older animals. Oxygen consumption was found to be decreased significantly(P < 0.05) in the 32-month old guinea pigs. Treatment with MAK at a dose of 500 mg/kg body weight daily for two months reduced the lipid peroxidation and lipofuscin pigment accumulation significantly in brain regions and it also helped in restoring the normal oxygen consumption in the older animals. This indicates antioxidant properties of MAK.     

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.     

Effects of prolonged ACTH-stimulation on adrenocortical accumulation of lipofuscin granules in aged rats

Subcellular deposition of lipofuscin granules is a marker of aging. Human and rodent adrenal cortices accumulate lipofuscin granules with age, but the mechanism that leads to the accumulation is not known. The ultrastructural appearance of lipofuscin granules resembles that of secondary lysosomes. Since adrenocortical subcellular events are predominantly influenced by ACTH action, we therefore studied the effect of prolonged ACTH-stimulation on adrenocortical accumulation of secondary lysosome-like granules, designated herein as lipofuscin granules. Using aged Fischer 344 male rats as a model, we found that a 7 day ACTH stimulation exerts a reducing effect on adrenocortical lipofuscin accumulation. Thus, adrenocortical accumulation of lipofuscin granules with age in vivo may not be an irreversible process.     

Unilateral eyestalk ablation reduces neurolipofuscin accumulation rate in the contralateral eyestalk of a crustacean, Pacifastacus leniusculus

Use of the lipofuscin ageing method as a crustacean fisheries research tool requires a calibration of tissue lipofuscin concentration to chronological age that is applicable to the natural population under investigation. Current approaches, involving known-age individuals or analysis of cohorts in neurolipofuscin concentration frequency distributions of the wild population, have advantages and disadvantages. A possible alternative that could be applied to individuals of unknown age involves initial biopsy of lipofuscin-loaded tissue from an eyestalk followed, after a known time period, by sampling of the second eyestalk, providing two successive lipofuscin measurements from the same individual and, thus, the neurolipofuscin accumulation rate in the intervening period. We tested the feasibility of this approach by examining the effect of eyestalk removal itself on subsequent lipofuscin accumulation in the remaining eyestalk using known-age individuals of a convenient decapod model, the signal crayfish, Pacifastacus leniusculus. By comparison with untreated controls, a 61% reduction in average neurolipofuscin accumulation rate in the remaining eyestalk occurred. It is hypothesized that this represents either slowed lipofuscinogenesis due to reduced oxidative metabolism or glycosylation, or increased lipofuscin loss due to enhanced proteolytic or phagocytic activity. It is recommended that the proposed ablation technique not be used for calibration of lipofuscin-based age determinations due to its unpredictable effect on lipofuscin accumulation.     

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.     

Effect of ambient oxygen concentration on lipofuscin accumulation in cultured rat heart myocytes--a novel in vitro model of lipofuscinogenesis

The objective of this study was to elucidate the factors involved in the accumulation of lipofuscin in post-mitotic cells. The hypothesis that oxidative stress accelerates the rate of lipofuscin accumulation was tested by examining the effects of 5%, 20%, and 40% ambient oxygen concentration on lipofuscin content in cultured rat cardiac myocytes. Lipofuscin was quantified by microspectrofluorometry at 7 and 12 days of in vitro age. Lipofuscin-emitted yellow autofluorescence increased in direct relationship to ambient oxygen concentration with age. Transmission electron microscopic examination of the cells after 3, 8, and 12 days in culture indicated a progressive time and oxygen dependent increase in the frequency and size of lipofuscin organelles. The results are interpreted to suggest that oxidative stress is one of the causal factors in the accumulation of lipofuscin.     

The accumulation of age pigments during larval development of the spider crab,Hyas araneus (Decapoda,Majidae)

• 1.1. Lipofuscin, body carbon and respiration rates were measured in Hyas araneus from hatching to metamorphosis. Lipofuscin was measured spectrofluorometrically from the chloroform phase of chloroform/methanol extracts.
• 2.2. Excitation/emission spectra of both the chloroform and the methanol/aqueous phase showed one distinct fluorescence peak in the chloroform (410–415 nm emission/340–350 nm excitation) and the methanol/aqueous phase (405/350 nm) of zoea I (directly after hatching) and megalopa (0 and 24 days old).
• 3.3. Individual lipofuscin concentrations increased continuously during zoea I and halfway through zoea II, but remained constant through the entire megalopa despite high metabolic activity in this stage.
• 4.4. Individual lipofuscin concentrations were positively correlated with body carbon and carbonspecific lipofuscin was negatively correlated.
• 5.5. Moulting caused considerable loss of lipofuscin. During the first two larval ecdyses 17–18% were lost, with the shed moults containing only 3.4–4.5% of the lipofuscin found in late premoult individuals.
• 6.6. The different patterns of lipofuscin accumulation in respective larval stages is discussed in regard to mitotic activity of tissues. While in the zoea, growth is more related to lipid formation and biomass accumulation, in the megalopa morphogenetic processes require substantial epidermal growth, i.e. protein accumulation. However, the question why in the megalopa no increase in lipofuscin is found, remains unanswered.

     

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.     

Lipofuscin-like fluorophores originated from malondialdehyde

The accumulation of fluorescent age pigment or lipofuscin is a frequently observed age-associated cellular alteration in a variety of post-mitotic cells of many species. These pigments are observed within granules composed, in part, of damaged protein and lipid. In the present mini-review, I provide a comprehensive summary of fluorescent adducts originated from malondialdehyde (MDA).     

Hydroxynonenal-generated crosslinking fluorophore accumulation in Alzheimer disease reveals a dichotomy of protein turnover

Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently bind amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated in intimate association with the pathological lesions of Alzheimer disease (AD), suggesting that oxidative stress is a major component of AD pathogenesis. Some HNE-protein products result in protein crosslinking through a fluorescent compound similar to lipofuscin, linking lipid peroxidation and the lipofuscin accumulation that commonly occurs in post-mitotic cells such as neurons. In this study, brain tissue from AD and control patients was examined by immunocytochemistry and immunoelectron microscopy for evidence of HNE-crosslinking modifications of the type that should accumulate in the lipofuscin pathway. Strong labeling of granulovacuolar degeneration (GVD) and Hirano bodies was noted but lipofuscin did not contain this specific HNE-fluorophore. These findings directly implicate lipid crosslinking peroxidation products as accumulating not in the lesions or the lipofuscin pathways, but instead in a distinct pathway, GVD, that accumulates cytosolic proteins.     

Quantification of the age-pigment lipofuscin in brains of known-age, pond-reared prawns Penaeus japonicus (Crustacea, decapoda)

A quantitative study of the lipofuscin content was carried out by image analysis in brains of known-age, pond-reared Penaeus japonicus (Crustacea, Decapoda) with the aim of assessing the applicability of the lipofuscin technique as an estimator of the physiological age in penaeids. With this purpose, three distinct measurements of lipofuscin levels (% area fraction, granule density and mean granule size) were recorded in ten sections of the olfactory lobe cell mass (OLCM) per animal. The image analysis was based on the autofluorescence emitted by the pigment, which accentuates the contrast between the lipofuscin granules and the background tissue. The concentration of lipofuscin increased significantly with age and was independent of sex. The relationship between age and lipofuscin concentration (area fraction and granule density) was best described by a seasonalized von Bertalanffy function, since the accumulation rate of the pigment dramatically slowed down in fall-winter, probably as a result of reduced seasonal metabolism. The present results confirm the potential of the lipofuscin method in the estimation of physiological age in penaeids and suggest that the application of this methodology can be useful in studies of age structure in wild populations and in the assessment of natural resources. J. Exp. Zool. 286:120-130, 2000.     

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.     

The lipofuscin in neuroglial cells of the optic nerve of Formosan rock-monkey (Macaca cyclopis)

The ultrastructure of lipofuscin granules in neuroglial cells of the optic nerve of the Formosan Rock-Monkey was investigated by electron microscopy. In the cytoplasm of astroglial cells, numerous irregular lipofuscin granules were characterized by the presence of large lipid droplets, small electron-dense pigment granules, and some lamellar structures. The lipofuscin granules of the oligodendroglial cells were composed largely of dense, coarse pigment granules, multilinear structures, and a few small lipid droplets. The lipofuscin granules in microglial cells were characterized by numerous lipid droplets in various sizes, small electron-dense pigment granules, and prominent lamellar structures. It was reported that the lipofuscin granules are wear-and-tear materials and products from the cells in lower functional activity. However, our observations suggest that the presence of lipofuscin granules in the neuroglial cells of the optic nerve is likely a characteristic product of active phagocytosis.     

Heavy metal effects on lipid peroxidation in the tissues of mytilus gallopro vincialis lam.

1. The effects of heavy metals on lipid peroxidation in the gills and digestive gland of mussels exposed for six days to Cu²⁺, Cd²⁺ or Zn²⁺ (40 μg/l/animal) were investigated.2. In the tissues of Cu-exposed mussels a significant increase of the level of malondialdehyde (MDA), which is indicative of the peroxidative process, and a decrease of the concentration of glutathione were observed.3. Moreover, in the digestive gland of mussels, copper exposure results in an increase of other carbonyl compounds and in the lysosomal accumulation of lipofuscin granules.4. The exposure of mussels to Zn or to Cd did not elicit any of the above effects.5. The results are discussed in relation to the possible role that Cu-induced lysosomal lipofuscin accumulation may play in heavy metal detoxification.     

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.     

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 in the corpus luteum of macaque ovaries

The present study examined the histochemistry of pigments in the corpus luteum of the ovaries of cynomolgus monkeys (Macaca fascicularis), Japanese monkeys (Macaca fuscata), and rhesus monkeys (Macaca mulatta). Yellowish brown pigments were found in the regressing corpus luteum cells. Histochemical studies revealed that these pigments consisted of lipofuscin, the so-called age pigment. The findings obtained suggest that accumulation of lipofuscin might be related to cellular aging of the corpus luteum.     

Ultrastructure of testicular macrophages in aging mice

Testicular macrophages of aging mice were studied by TEM. Testicular macrophages retained with Leydig cells the close morphological relationships observed in the adult young animals, but digitations were not found. Lipofuscin granules like those of the Leydig cells from aging mice were observed in the cytoplasm. These organelles were generally absent in the testicular macrophages of young adult mice. Testicular macrophages did not display phagocytosis of the lipofuscin granules. In addition, the latter were not found in the intercellular spaces. These observations indicated that lipofuscin granules were formed, at least in a great part, within testicular macrophages as a consequence of metabolic changes occurring with age. Fine lamellar organization was seen in the lipofuscin granules of both Leydig cells and testicular macrophages. Frequently, lipofuscin granules originated from secondary lysosomes containing lipidic vacuoles only. Together with accumulation of the lipofuscin granules, changes of testicular macrophage fine morphology were observed. Endoplasmic reticulum and Golgi apparatus became poorly developed, and coated vesicles were rarely found. Fewer mitochondria were encountered, but their ultrastructure was not altered. These results suggest that in testicular macrophages lipofuscin accumulation is associated with a functional involution.