Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.

The fluorescent pigment lipofuscin accumulates with age in the cytoplasm of cells of the CNS. Because of its broad excitation and emission spectra, the presence of lipofuscin-like autofluorescence complicates the use of fluorescence microscopy (e.g., fluorescent retrograde tract tracing and fluorescence immunocytochemistry). In this study we examined several chemical treatments of tissue sections for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 1-10 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5) or 1% Sudan Black B (SB) in 70% ethanol reduced or eliminated lipofuscin autofluorescence in sections of monkey, human, or rat neural tissue. These treatments also slightly reduced the intensity of immunofluorescent labeling and fluorescent retrograde tract tracers. However, the reduction of these fluorophores was far less dramatic than that for the lipofuscin-like compound. We conclude that treatment of tissue with CuSO4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.     

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.     

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.     

Inhibition of autophagy with 3-methyladenine results in impaired turnover of lysosomes and accumulation of lipofuscin-like material

Autophagy (which includes macro-, micro-, and chaperone-mediated autophagy) is an important biological mechanism for degradation of damaged/obsolete macromolecules and organelles. Ageing non-dividing cells, however, progressively accumulate oxidised proteins, defective organelles and intralysosomal lipofuscin inclusions, suggesting inherent insufficiency of autophagy. To learn more about the role of macroautophagy in the turnover of organelles and lipofuscin formation, we inhibited autophagic sequestration with 3-methyladenine (3 MA) in growth-arrested human fibroblasts, a classical model of cellular ageing. Such treatment resulted in a dramatic accumulation of altered lysosomes, displaying lipofuscin-like autofluorescence, as well as in a moderate increase of mitochondria with lowered membrane potential. The size of the late endosomal compartment appeared not to be significantly altered following 3 MA exposure. The accumulation of lipofuscin-like material was enhanced when 3 MA administration was combined with hyperoxia. The findings suggest that macroautophagy is essential for normal turnover of lysosomes. This notion is supported by reports in the literature of lysosomal membrane proteins inside lysosomes and/or late endosomes, as well as lysosomes with active hydrolases within autophagosomes following vinblastine-induced block of fusion between lysosomes and autophagosomes. The data also suggest that specific components of lysosomes, such as membranes and proteins, may be direct sources of lipofuscin.     

Quantification of Ceroid and Lipofuscin in Skeletal Muscle

Ceroid and lipofuscin are autofluorescent granules thought to be generated as a consequence of chronic oxidative stress. Because ceroid and lipofuscin are persistent in tissue, their measurement can provide a lifetime history of exposure to chronic oxidative stress. Although ceroid and lipofuscin can be measured by quantification of autofluorescent granules, current methods rely on subjective assessment. Furthermore, there has not been any evaluation of variables affecting quantitative measurements. The article describes a simple statistical approach that can be readily applied to quantitate ceroid and lipofuscin. Furthermore, it is shown that several factors, including magnification tissue thickness and tissue level, can affect precision and sensitivity. After optimizing for these factors, the authors show that ceroid and lipofuscin can be measured reproducibly in the skeletal muscle of dystrophic mice (ceroid) and aged mice (lipofuscin).     

Stimulation of the D5 dopamine receptor acidifies the lysosomal pH of retinal pigmented epithelial cells and decreases accumulation of autofluorescent photoreceptor debris

Optimal neuronal activity requires that supporting cells provide both efficient nutrient delivery and waste disposal. The incomplete processing of engulfed waste by their lysosomes can lead to accumulation of residual material and compromise their support of neurons. As most degradative lysosomal enzymes function best at an acidic pH, lysosomal alkalinization can impede enzyme activity and increase lipofuscin accumulation. We hypothesize that treatment to reacidify compromised lysosomes can enhance degradation. Here, we demonstrate that degradation of ingested photoreceptor outer segments by retinal pigmented epithelial cells is increased by stimulation of D5 dopamine receptors. D1/D5 receptor agonists reacidified lysosomes in cells alkalinized by chloroquine or tamoxifen, with acidification dependent on protein kinase A. Knockdown with siRNA confirmed acidification was mediated by the D5 receptor. Exposure of cells to outer segments increased lipofuscin-like autofluorescence, but SKF 81297 reduced autofluorescence. Likewise, SKF 81297 increased the activity of lysosomal protease cathepsin D in situ. D5DR stimulation also acidified lysosomes of retinal pigmented epithelial cells from elderly ABCA4(-/-) mice, a model of recessive Stargardt's retinal degeneration. In conclusion, D5 receptor stimulation lowers compromised lysosomal pH, enhancing degradation. The reduced accumulation of lipofuscin-like autofluorescence implies the D5 receptor stimulation may enable cells to better support adjacent neurons.     

Lectin histochemistry of lipofuscin and certain ceroid pigments

Little is known at present about the saccharide components of lipofuscin (age pigment) and ceroid pigments in situ. The purpose of this study was, therefore, to study in detail the lectin reactivities of lipofuscin in neurons and cardiac myocytes of old humans and rats. In addition, those of diverse ceroid pigments found in human aortic atheromas, in the livers of choline-deficient rats, in the uteri of vitamin E-deficient rats and in the crushed epididymal fat pad of rats, are included. Cryostat and deparaffinized sections from all these tissues were either extracted with a solvent mixture of chloroformmethanol-water (10103, v/v) and incubated with 7 different biotinylated lectins or left untreated. Delipidation was done in order to study whether it was possible to discriminate between the saccharide moieties of glycolipids and glycoproteins of lipofuscin and ceroid pigments in situ. Other similarly treated sections were used to study the autofluorescence, sudanophilia, acid-fastness and reactivity to PAS. The frequency and intensity of lectin binding and standard histochemical properties of all the pigments were evaluated semi-quantitatively and blind. The results indicated that mannose was in general the most consistently detected sugar residue in lipofuscin granules of humans and rats, and that this pigment may also contain acetylglucosamine, acetylgalactosamine, sialic acid, galactose and fucose. However, notable differences were found not only in the lipofuscin saccharide components of different cell types of humans and rats, but also in those in the same type of cells in both species. Although mannose was not detected in the hepatic ceroid of choline-deficient rats, this saccharide moiety was almost always present in the other ceroid pigments. Each of the ceroids also contained other types of saccharides although the frequency of the latter varied between different ceroid pigments. While lipofuscin and each of the ceroid pigments showed somewhat different lectin binding patterns, the variability in the frequency of reactivity to lectins suggests that these patterns may not be permanent but transient. In this sense, it appears that lectin histochemistry may not allow these pigments to be differentiated. Furthermore, the extractive procedures used in this study did not enable us to determine whether the saccharides detected in the pigments in situ corresponded to glycolipids or glycoproteins.     

Changes in Pollen Autofluorescence Induced by Ozone

The changes in the autofluorescence spectra of intact Philadelphus grandiflorus, Epiphyllum hybridum, and Plantago major pollen have been observed with microspectrofluorometric method after 100-h exposure to ozone (3 h per day for 5 d per week in total dose 5.0 cm³ m^-3). The fluorescence maximum at 530 - 550 nm disappeared in carotenoid-containing pollen of Philadelphus grandiflorus and Epiphyllum hybridum and new maximum at 475 - 480 nm arose that correlated with lipofuscin-like substances observed in the extracts from the pollen grains. The carotenoid-less pollen of Plantago major showed only the increase of the maximum at 470 nm, and no lipofuscin in the extracts. It is supposed that main indicator of the pollen damage by ozone in carotenoid-containing microspores are the lipofuscin-like compounds. This revised version was published online in July 2006 with corrections to the Cover Date.     

Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts.

We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence-like growth arrest after about 4 wk and shortened postmitotic life span from 1-1/2 years down to 3 months. During the first 8 wk of hyperoxia-induced 'aging', overall protein degradation (breakdown of [(35)S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin-specific activity (using the fluorogenic substrate z-FR-MCA) and cytoplasmic proteasome-specific activity (measured with suc-LLVY-MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid-loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by inhibiting the proteasome.     

Lectin histochemical study of lipopigments with special regard to neuronal ceroid-lipofuscinosis

Summary Concanavalin A (ConA) binding to lipopigments (LPs) of the lipofuscin type was proved to be due to the high content of mannose. The nature of the mannose bearing compound was twofold. One part was soluble in modified chloroform-methanol-water mixture (10:10:3) corresponding possibly to the oligosaccharyl diphosphodolichol (oligo-PP-Dol) described to be increased in LPs especially of inherited types. The second part, most probably a glycoprotein (GP), was entirely resistant to various extraction procedures. The ratio of the two components varied. The deposition of the typical lipofuscin (age pigment) was dominated by the GP component. Its amount was greatest in neurolipofuscin (especially in the olivary nucleus) and in the myocardium but very little in hepatocytic lipofuscin. In human neuronal ceroid lipofuscinoses (of early juvenile, and juvenile types) both components were found in large quantities in the storage granules of the affected neurons. The protein type variant of the storage material (Elleder 1978) displayed the highest degree of lipid-bound mannose accumulation, the GP component being extremely low or entirely absent. In the late infantile, infantile and Kufs variants studied in paraffin sections only, the GP component was detetable, too as in the case of the secondary neuronal LP in mucopolysaccharidoses and gangliosidoses. In the dog model of NCL lipid bound mannose clearly predominated, the GP component being concentrated in the cytoplasm and on the periphery od some storage granules. The nature of the GP component, a new finding of LP analysis, is discussed. The metabolic relationship between the two components is uncertain. Neither could be identified as the component resposible for autofluorescence. However, both are most probably responsible for PAS positivity of lipofuscins. The ceroid-type histiocytic LP did not display any detectable increase in any of the mannose bearing components.Abbreviations NCL neuronal ceroid lipofuscinosis - HNCL human NCL - CNCL canine NCL - ONCL ovine NCL - LP lipopigment - GP glycoprotein     

Lectin histochemical study of lipopigments with special regard to neuronal ceroid-lipofuscinosis. Results with concanavalin A

Concanavalin A (ConA) binding to lipopigments (LPs) of the lipofuscin type was proved to be due to the high content of mannose. The nature of the mannose bearing compound was twofold. One part was soluble in modified chloroform-methanol-water mixture (10:10:3) corresponding possibly to the oligosaccharyl diphosphodolichol (oligo-PP-Dol) described to be increased in LPs especially of inherited types. The second part, most probably a glycoprotein (GP), was entirely resistant to various extraction procedures. The ratio of the two components varied. The deposition of the typical lipofuscin (age pigment) was dominated by the GP component. Its amount was greatest in neurolipofuscin (especially in the olivary nucleus) and in the myocardium but very little in hepatocytic lipofuscin. In human neuronal ceroid lipofuscinoses (of early juvenile, and juvenile types) both components were found in large quantities in the storage granules of the affected neurons. The "protein type variant" of the storage material (Elleder 1978) displayed the highest degree of lipid-bound mannose accumulation, the GP component being extremely low or entirely absent. In the late infantile, infantile and Kufs variants studied in paraffin sections only, the GP component was detectable, too as in the case of the secondary neuronal LP in mucopolysaccharidoses and gangliosidoses. In the dog model of NCL lipid bound mannose clearly predominated, the GP component being concentrated in the cytoplasm and on the periphery od some storage granules. The nature of the GP component, a new finding of LP analysis, is discussed. The metabolic relationship between the two components is uncertain. Neither could be identified as the component resposible for autofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)     

LIPOFUSCIN (AGING) PIGMENT GRANULES OF THE NEWBORN HUMAN LIVER

We have observed pigmented cytoplasmic granules, with the characteristic staining properties of lipofuscin (ceroid, "wear-and-tear") pigment, in newborn human liver. The pigment is found at the periphery of the lobule in hepatocytes and some bile ductular cells. It is acid-fast, PAS-positive after diastase digestion, slightly argyophilic and sudanophilic, and markedly Schmorl's- and peroxidase positive in paraffin sections. Difficult to see in sections stained with hematoxylin and eosin, the pigment can be detected in unstained sections. The granules also resemble lipofuscin found in adult tissues, in their ultra-structural and enzymatic properties. They are polymorphic, contain granular material of moderate and high electron opacity, and are delimited by a single membrane. Acid phosphatase and β-glucuronidase activities are visualized in the newborn granules, identifying them as lysosomes. The granules also contain copper and, to a much lesser extent, iron. The accumulation of lipofuscin pigment in lysosomes in many tissues correlates well with aging, and this process has been interpreted as a reflection of cellular degeneration or wear-and-tear. However, the presence of lipofuscin granules as a constant component of neonatal liver suggests that they are not a measure of cellular senescence.     

A clinical variant of familial Hermansky-Pudlak syndrome

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive inherited disease consisting of (1) partial oculocutaneous albinism (with nystagmus, strabism, and visual acuity loss), (2) platelet storage pool deficiency (with bleeding diathesis), and (3) disorder of "ceroid" metabolism with a multisystem tissue lysosomal ceroid deposition. HPS is less uncommon in Puerto Rico, where the most important studies have been performed, but is a very rare disease in Europe. HPS basic defect remains unknown, even if an HPS-causing gene was identified in chromosome segment 10q23-q23.3, and several mutations have been reported. The aim of this article is to discuss, on the basis of a review of relevant literature, a new familial HPS clinical variant observed in 2 young sisters (aged 16 and 23 years old, respectively), characterized by the typical symptoms of this syndrome. Our patients also suffered from diffuse interstitial pulmonary disease and an unexpectedly increased platelet aggregation and were prone to bacterial infections. Interestingly, we observed urinary tract abnormality in the younger HPS sister and a porencephalic cyst in the older HPS sister; both of these developmental defects have been reported in the Cross syndrome (or oculocerebral hypopigmentation syndrome). It seems that in our patients, an overlapping of the phenotypic manifestations of different rare syndromes may be present. The presence of ceroid-like autofluorescent material in urinary sediment together with the histologic aspects and the autofluorescence of oral mucosa biopsy are consistent with a ceroid-like lipofuscin storage. HPS should be carefully tested for in suspected cases to prevent the severe visual impairment, rapidly progressive pulmonary fibrosis, and other complications associated with this disorder.     

ECL cells of the rat stomach: development of lipofuscin in response to sustained gastrin stimulation

Ageing cells, especially post-mitotic cells, are known to accumulate pigments, i.e. highly electron-dense material, referred to as ceroid or lipofuscin. This material is formed as a consequence of autophagocytosis and peroxidation of the products undergoing degradation. The present study describes the development of lipofuscin in the ECL cells of the rat stomach. These cells produce and secrete histamine in response to gastrin. They are rich in secretory vesicles, which fuse to form vacuoles in hypergastrinaemic rats. Hypergastrinaemia was induced by continuous infusion of human Leu¹⁵-gastrin-17 for 6 days or by daily treatment with omeprazole for 10 weeks. Either treatment caused both vacuoles and lipofuscin bodies to appear in large numbers; the vacuoles disappeared promptly after interruption of the hypergastrinaemia, whereas the lipofuscin bodies remained. Antrectomy-evoked hypogastrinaemia was associated with a reduced number and volume density of lipofuscin bodies. Treatment with α-fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, resulted in depletion of ECL-cell histamine and was found to prevent the omeprazole-evoked formation of vacuoles and lipofuscin. The numbers of both vacuoles and lipofuscin bodies were well-correlated with the serum gastrin concentration, suggesting that gastrin stimulates the development not only of vacuoles but also of lipofuscin, perhaps through enhanced autophagocytosis and/or oxidative stress. Thus, lipofuscin bodies may develop from vacuoles, and both vacuoles and lipofuscin bodies may reflect the efforts of overstimulated ECL cells to cope with the excessive formation of secretory products.     

Human and rat brain lipofuscin proteome

The accumulation of an autofluorescent pigment called lipofuscin in neurons is an invariable hallmark of brain aging. So far, this material has been considered to be waste material without particular relevance for cellular pathology. However, two lines of evidence argue that lipofuscin may play a yet unidentified role for pathological cellular functions: (i) Genetic forms of premature accumulation of similar autofluorescent material in neuronal ceroid lipofuscinosis indicate a direct disease-associated link to lipofuscin; (ii) Retinal pigment epithelium cell lipofuscin is mechanistically linked to age-associated macular degeneration. Here, we purified autofluorescent material from the temporal and hippocampal cortices of three different human individuals by a two-step ultracentrifugation on sucrose gradients. For human brain lipofuscin, we could identify a common set of 49 (among > 200 total) proteins that are mainly derived from mitochondria, cytoskeleton, and cell membrane. This brain lipofuscin proteome was validated in an interspecies comparison with whole brain rat lipofuscin (total > 300 proteins), purified by the same procedure, yielding an overlap of 32 proteins (64%) between lipofuscins of both species. Our study is the first to characterize human and rat brain lipofuscin and identifies high homology, pointing to common cellular pathomechanisms of age-associated lipofuscin accumulation despite the huge (40-fold) difference in the lifespan of these species. Our identification of these distinct proteins will now allow research in disturbed molecular pathways during age-associated dysfunctional lysosomal degradation.     

Primary extracellular ceroid type lipopigment. A histochemical and ultrastructural study

Summary An extracellular ceroid-type (ECC) lipopigment which appears histologically as wavy hyaline membranes or, less frequently, as an amorphous solid or broadly reticulated mass is described. Its ultrastructure is either amorphous or membranous and consists either of simple linear membrane-like aggregates or elaborated trilaminar membranes. The histochemical profile is, as in histiocytic intracellular ceroid, dominated by autofluorescence, strong hydrophobicity, acid and extraction resistance. Staining for aromatic acid residues and periodic acid-Schiff positivity are strong but variable. Lectin receptors are either absent or sparse. The pigment is found solely within the lipid rich tissue debris, bound to processes marked by necrosis of adipose or steatosed tissues and interpreted as originatingde novo extracellularly from liquid unsaturated lipids under the influence of local enzymatic and nonenzymatic lipid oxidation catalysts. A hitherto unknown form of extracellular ceroid is the so-called membranocystic lesion found in Nasu-Hakola's disease, in several other conditions and in annular ceroid in human atheromas.     

Aggregates of oxidized proteins (lipofuscin) induce apoptosis through proteasome inhibition and dysregulation of proapoptotic proteins

Cellular senescence may be accompanied by accumulation of large aggregates of oxidized proteins, also known as lipofuscin. The hypothesis that cellular accumulation of lipofuscin-like materials (LIP) results in cell death as a result of proteasome inhibition was examined. Rat neonatal cardiomyocytes were incubated with synthetic LIP for up to 48 h. This was accompanied by increases in cellular autofluorescence (207% by 48 h; p < 0.05) and electron microscopic evidence of internalization of LIP particles. LIP incubation resulted in loss of viability (-46% by 48 h; p < 0.05) through apoptotic cell death. Although 20S-proteasome activity was increased by 74% after 6 h, both 20S- and 26S-proteasome activities were decreased after 48 h of incubation (-54% (p < 0.05) and -50%, respectively), accompanied by large increases in ubiquitinated proteins. Several proteasome-regulated proapoptotic proteins, including c-Jun (2.9-fold; p < 0.05), Bax (1.8-fold; p < 0.05), and p27(kip1) (3.2-fold; p < 0.05), were observed to be increased by 48 h. Observation of ubiquitinated homologues of Bax and p27(kip1) suggested that part of the increase was due to decreased proteasomal degradation of these proteins. The results of this study are consistent with the conclusion that accumulation of LIP results in inhibition of the proteasome, which initiates an apoptotic cascade as a result of dysregulation of several proapoptotic proteins.     

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.     

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.