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Near-infrared autofluorescence (NIA) is a non-invasive retinal imaging technique for examination of the retinal pigment epithelium (RPE) based on the autofluorescence of melanin. Melanin has several functions within the RPE cells, in one of them it serves as a protective antioxidative factor within the RPE cells and is involved in the phagocytosis of photoreceptor outer segments. Disorders that affect the photoreceptor-RPE complex result in alterations of RPE cells which are detectable by alterations of NIA. Therefore, NIA allows to detect early alterations in inherited and acquired chorioretinal disorders, frequently prior to ophthalmoscopical visualisation and often prior to alterations in lipofuscin associated fundus autofluorescence (FAF) or optical coherence tomography (OCT). Although NIA and FAF relate to disorders affecting the RPE, findings between both imaging methods differ and the area involved has been demonstrated to be larger in NIA compared to FAF in several disorders (e.g., age-related macular degeneration, retinitis pigmentosa, ABCA4-gene associated Stargardt disease and cone-rod dystrophy, light damage), indicating that NIA detects earlier alterations compared to FAF. In addition, due to the absence of blue-light filtering which limits foveal visualisation in FAF, foveal alterations can be much better detected using NIA. A reduced subfoveal NIA intensity is the earliest sign of autosomal dominant BEST1-associated disease, when FAF and OCT are still normal. In other disorders, a normal subfoveal NIA intensity is associated with good visual acuity. This review summarizes the present knowledge on NIA and demonstrates biomarkers for various chorioretinal disorders.

The present article gives a survey over the current scientific knowledge of the canine neuronal ceroid-lipofuscinosis (NCL). NCL is a heterogenous group of lysosomal storage diseases in humans and animals. In consequence of a gene mutation, there is an accumulation of ceroid-lipofuscin in neurons, cells of the retina and the skin and other cells. The stored ceroid-lipofuscin in neurons leads to an impaired cell function and subsequently to cell death. Recently, the underlying genetic defect was discovered in several dog breeds. Genetic testing permits an ante mortem diagnosis of the disease, which up to now was only possible with a positive biopsy result. Another advantage is the identification of carrier animals to eliminate the deleterious alleles., Die vorliegende Arbeit gibt eine Übersicht und Zusammenfassung über den aktuellen Wissensstand der neuronalen Ceroid-Lipofuszinose (NCL) beim Hund. Die NCL ist eine heterogene Gruppe lysosomaler Speicherkrankheiten bei Mensch und Tier. Aufgrund einer Genmutation kommt es zur Akkumulation von Ceroid-Lipofuszin in Nervenzellen, Zellen der Netzhaut, Haut und anderen Körperzellen. Die Ansammlung von Ceroid-Lipofuszin in den Neuronen führt zu einer fortschreitenden Funktionsstörung und letztlich zum Tod dieser Zellen. In neuerer Zeit wurde bei einigen Hunderassen der zugrunde liegende Gendefekt identifiziert. Gentests erlauben die Krankheit am lebenden Tier zu diagnostizieren, was bisher nur mittels Biopsie möglich war. Zusätzlich können heterozygote Anlageträger identifiziert und von der Zucht ausgeschlossen werden.

The accumulation of autofluorescent bodies in retinal pigment epithelium (RPE) cells has an impact on the pathogenesis of retinal diseases, including age-related macular degeneration. While current in vivo fluorescence microscopy allows a lateral resolution of fluorophores in a micrometer range, with ex vivo microscopy a lateral resolution down to 200 nm is possible. For the first time, we used structured illumination microscopy for ex vivo high-resolution fluorescence microscopy of RPE cells.Histological sections were prepared from a 68-year-old patient. With epifluorescence microscopy, fluorescent RPE cells were detectable. Structured illumination uses inhomogeneous illumination for resolution of previously nonresolvable structures, similar to the Moiré effect. Images were taken from RPE cells at different excitation wavelengths (488, 568, and 647 nm) and were reconstructed with special software. The different excitation patterns of the fluorescent granules in the RPE cells were colour-coded and analysed.With structured illumination microscopy, autofluorescence signals of RPE cells were detectable, and a lateral resolution of 110 nm could be achieved. Using varying wavelengths, different pigments were excitable. Lipofuscin gave the highest signals, at 488 and 568 nm. The improved resolution showed inhomogeneous intragranular fluorophore patterns.Structured illumination microscopy enabled us to generate images of fluorescent structures in RPE cells ex vivo with a lateral resolution of 110 nm. With the use of different excitation wavelengths, intracellular fluorescence patterns in single cell compartments are visible and allow further differentiation.

We report the case of a 74-year-old female with an extreme picture of melanosis coli of the whole colon after chronic use of anthraquinone laxatives for the treatment of constipation over many decades. Endoscopic work-up revealed an impressive deep black pigmentation of the whole colon mucosa which could be verified by histopathology as a widespread lipofuscin granulation. In addition, various adenomas but no colorectal carcinoma could be detected. The term melanosis coli describes a brown or black pigmentation of the colonic mucosa. Induction of melanosis coli by anthraquinone laxatives and their derivatives can be regarded as verified. The question if melanosis coli predisposes for colorectal neoplasia is discussed controversially. Based on the current literature, an association of melanosis coli between colorectal adenomas, but not colorectal carcinomas, is under discussion but the mechanisms to effect the development of colorectal neoplasia are not completely understood. Considering our case and the current scientific backround, we conclude that due to pharmaceutical side effects of anthraquinone derivatives such as electrolytic shift and water loss in addition to the risk of developing melanosis coli, anthraquinone laxatives should not be used for long-term therapy of constipation.

An intact retinal pigment epithelium (RPE) represents an essential condition for the visual process. This post-mitotic RPE monolayer combines different functions such as degradation of photoreceptor outer segments, vitamin A cycle, support of retinal metabolism and maintenance of the outer blood-retina barrier. As a consequence of excessive metabolism, high oxygen levels, exposition to light of short wave length and ensuing radical formation, the RPE is highly dependent on protective systems. In spite of differentiated defence mechanisms, aging processes cause cumulative RPE damage, representing a major component of age-related macular degeneration (AMD), the leading cause of irreversible severe vision loss in people over 50 years old. A better understanding of the underlying pathophysiology will help to develop new prophylactic options which is becoming more and more important with increasing life expectancy.

A pronounced fundus autofluorescence (lipofuscin) occurs in eyes with AMD. Parapapillary lipofuscin accumulation in the retinal pigment epithelial cells was observed in eyes with advanced glaucoma histologically. The aim of this study was to evaluate the parapapillary autofluorescence (PAF) in vivo in healthy eyes (controls), and in eyes with primary open angle glaucoma (POAG), pseudoexfoliation glaucoma (PSXG) or normal tension glaucoma (NTG).Controlled cross-sectional analysis was performed on 281 consecutive eyes (98 controls, 95 POAG, 32 PSXG, 56 NTG). Eyes with fundus pathologies were excluded. The confocal scanning laser ophthalmoscope HRA II (Heidelberg Retina Angiograph II) was used after lipofuscin-excitation with an argon blue laser (488 nm) to detect PAF in the spectrum above 500 nm. PAF area and PAF distance to the optic nerve head were analyzed using the HRA standard software. Two experienced ophthalmologists classified independently the stage of glaucomatous optic nerve head atrophy (GONHA) using 15 degrees fundus photographs.Vital optic nerve heads had smaller PAF areas (stage 0: 0.07 +/- 0.09 mm (2)) in contrast to advanced stages of GONHA (stages 1 to 4: 0.27 +/- 0.46 mm (2); p0.001; logistic regression Cox and Snell: r = 0.7; p = 0.015). The PAF distance to the optic nerve head was lower in controls (0.12 +/- 0.08 mm) than in eyes with POAG, PSXG, or NTG (0.25 +/- 0.21 mm, Bonferroni: p0,004). The PAF area correlated significantly with the stage of GONHA (stage 1: 0.23 +/- 0.23 mm (2), stage 2: 0.24 +/- 0.19 mm (2), stages 3 and 4: 0.34 +/- 0.73 mm (2), p0.01). No significant difference of PAF area was found between the glaucoma types. However, the distance between PAF and optic nerve head was higher in POAG (0.28 +/- 0.26 mm) than in NTG (0.24 +/- 0.07 mm) or in PSXG (0.18 +/- 0.07 mm, Bonferroni: p0.03).A pronounced fundus autofluorescence was detected as a sign of increased lipofuscin accumulation in the parapapillary atrophic zone of eyes with POAG, PSXG, and NTG in contrast to controls. The PAF analysis may provide an indicator for glaucomas in the future.

Fundus autofluorescence (FAF) originates from age- and disease-dependent accumulation of lipofuscin in the lysosomal compartment of the retinal pigment epithelium (RPE). FAF imaging is a noninvasive method to detect intrinsic RPE fluorescence in vivo. We describe features of a novel confocal scanning laser ophthalmoscope (cSLO) for FAF imaging and compare images to the previous cSLO system.FAF images were obtained with a cSLO using an optically pumped solid state laser (OPSL) instead of an argon laser for generation of excitation light at 488 nm. For detection of emitted FAF signals500 nm a barrier filter was used.The novel cSLO allows FAF imaging with a resolution of up to 5 microm/pixel to delineate normal and pathological features in various retinal pathologies including early-stage and advanced atrophic or neovascular age-related macular degeneration, macular edema, and retinal dystrophies. Further technical improvements include an internal fixation target and an enlarged optical focus adaption range.Improved image quality using the novel cSLO for FAF imaging is of clinical relevance for diagnosis and precise phenotyping of retinal diseases. This method may also be useful to monitor therapeutic effects targeting RPE lipofuscin accumulation as a common pathogenetic pathway in various degenerative and hereditary retinal diseases.

Alterations in the intrinsic ocular fundus fluorescence are under discussion particularly in connection with age-related macular degeneration. However, further knowledge of the chemical substrate of fluorescence is necessary.Ocular fundus autofluorescence was observed using a fundus camera. The fluorescence emission was recorded using a 3-chip CCD camera. For comparison, the fluorescence spectra of lipofuscin, A2-E, FAD, NADH(2) and AGE's were recorded by the Jena ophthalmo-spectrometer.In contrast to the homogeneous intrinsic fluorescence of a normal fundus, the fluorescence images of patients suffering from AMD showed remarkable local differences. The detected fluorescence spectra showed remarkable overlaps.We introduced a new technique enabling the detection of spectral differences in images of ocular fundus autofluorescence. Besides the fluorescence of lipofuscin, we found a green one in patients suffering from nonexudative AMD.

Adult vitelliform macular dystrophy (AVMD) was first described in 1974 (Gass) but is still often misdiagnosed. Large studies using modern morphological and functional diagnostic methods do not exist.The records of 67 consecutive AVMD patients (1994-2003) were reviewed regarding color vision, perimetry, RPE autofluorescence, fluorescein angiography, EOG, ERG, and mfERG.The mean age was 54.8 years. Symptoms, visual loss, color vision deficits, and visual field defects were highly variable. Autofluorescence was increased centrally in 77% of the eyes. In the ERG, the 30 Hz flicker response was reduced in 71% of the eyes. MfERGs showed a marked central amplitude reduction in 62% of the eyes and a continual normalization of the P1 amplitude towards the periphery.The enhanced autofluorescence indicates increased lipofuscin in the vitelliform lesions. The electroretinographic recordings reveal a moderate generalized cone dysfunction with increased severity towards the fovea. Ophthalmoscopy, autofluorescence, and recording of mfERG are prerequisites to diagnose AVMD correctly.

The pigmentary type of orthochromatic leukodystrophy (van Bogaert-Nyssen disease) is a hardly known neurological disorder usually with late onset that is very difficult to diagnose in vivo. Neuropathologically, the disorder features noninflammatory demyelination and the presence of pigmented macrophages and astrocytes that may contain iron. Clinically, van Bogaert-Nyssen disease can lead to death within a few years and is characterized by dementia, psychiatric abnormalities, epileptic seizures, spastic pareses, and occasionally extrapyramidal motor symptoms. This report presents a typical case and an overview of the literature. Furthermore, galactocerebroside could be documented in remaining macrophages and astrocytes by immunohistochemistry. This possibly indicates a dysfunction in sphingolipid breakdown and could relate the pigmented form of orthochromatic leukodystrophy to the genetically defined globoid cell leukodystrophy (Krabbe's disease). Thus, the rather heterogeneous pool of orthochromatic leukodystrophies could be further narrowed.

The ageing process is accompanied by enhanced oxidative damage. All cellular components including proteins are affected by oxidation. Within the cell, the proteasome is responsible for the degradation of these oxidised proteins. During the ageing process this function of the proteasome is increasingly diminished, therefore oxidised proteins accumulate. Furthermore lipofuscin, a highly cross-linked and modified protein aggregate, is formed. This aggregate accumulates within cells and is able to inhibit the proteasome. The nucleus of the cells is less affected by these changes due to the lack of intranuclear lipofuscin accumulation.

To assess the level of autofluorescence (lipofuscin) of atrophic parapapillary zones in different stages of glaucomatous optic disc atrophy.Controlled cross-sectional prospective analysis of 79 consecutive eyes (15 normals as controls, 26 with ocular hypertension, 38 with primary open angle glaucoma). Eyes with retinal diseases or retinal pigment epithelial pathologies were excluded. The confocal scanning laser ophthalmoscope (HRA, Heidelberg Retina Angiograph) was used after lipofuscin excitation with argon blue laser (488 nm) to detect parapapillary autofluorescence in a spectrum above 500 nm. Size, extension of the parapapillary autofluorescent area and its mean distance to the optic nerve head were measured using the HRA standard software. Additional optic nerve head photographs taken with the 15 degrees Zeiss telecentric fundus camera (30 degrees camera with 2 x magnifier) were examined by two experienced ophthalmologists to determine the stage of glaucomatous optic disc atrophy (stages 0 to 4).Very small autofluorescent areas were found in vital discs (optic nerve glaucoma stage 0) in the parapapillary atrophic area (0.08 +/- 0.12 mm (2)) in contrast to glaucomatous discs in stage 1 (0.24 +/- 0.26 mm (2)) and stages 2, 3 and 4 (0.59 +/- 1.29 mm (2), logistic regression analysis r = 0.71; P = 0.029). The circular extension of the autofluorescent area correlated borderlined with the stage of the glaucomatous disc atrophy (higher glaucoma stages: r = 0.82; P = 0.09). The autofluorescent area was larger in OHT than in controls (0.11 mm (2) vs. 0.04 mm (2), P0.03). The circular extension of the autofluorescent area was longer in OHT than in controls (0.5 mm vs. 1.15 mm, P0.04).As a sign of pronounced lipofuscin accumulation in the parapapillary atrophic zone higher degrees of fundus autofluorescence can be detected in OHT and manifest primary open angle glaucoma in contrast to normals. The lipofuscin accumulation is correlated with the stage of progression of glaucoma and the stage of optic disc atrophy. The detection of active parapapillary autofluorescent areas especially in OHT may offer the ophthalmologist an important tool for early diagnosis.

Several lines of evidence suggest that excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial (RPE) cells with age and in various hereditary retinal diseases, plays a pathogenetic role. The lipofuscin retinoid component A2-E (N-retinylidene-N-retinylethanolamine) inhibits lysosomal degradation. Here we sought to evaluate additional toxic mechanisms of A2-E, whereby possible detergent-like effects on various membranes in human RPE cells were investigated by latency measurements.A postnuclear supernatant prepared from cultured human RPE cells was used to isolate intact lysosomes by fractionation of cellular organelles in two sequential gradients. Destabilization of the lysosomal membrane was tested by incubating the purified lysosomal fraction in the presence of A2-E and subsequent measurement of the latency of the lysosomal luminal marker beta-hexosaminidase. In order to compare the effect of A2-E on other cellular membranes, latencies of the specific markers succinate dehydrogenase and UDP-galactosyltransferase were assessed using partially purified mitochondria and microsomes. Intactness of the plasma membrane was tested by including A2-E in the culture medium before leakage of lactate dehydrogenase into the medium was determined.A more than 100-fold purification of the lysosomal fraction was achieved. Except for a minor activity of the mitochondrial marker, no contamination with other cell fractions was observed. Intactness of the purified lysosomes was well preserved during incubation in isotonic media and provided the basis for investigations on a possible detergent-like action of A2-E on lysosomal integrity. At concentrations above 2 microM A2-E, progressive leakage of the lysosomal marker was observed. In comparison leakage of the mitochondrial marker was induced at significantly lower concentrations (1 microM), whereas ER/Golgi membranes and the plasma membrane were relatively insensitive to a detergent effect of the retinoid.The described practical and fast methodology to obtain highly purified and intact lysosomes from RPE cells, provides a very suitable tool for investigations on compounds affecting the lysosomal structure. The results suggest that A2-E causes disintegration of the lysosomal membrane at relatively low concentrations which may implicate an involvement of such a mechanism in triggering lipofuscin-induced dysfunction of aged RPE in vivo. Secondary to disintegration of the lysosomal membrane, damage to mitochondria might be an additional pathogenic mechanism. Our data provide evidence for surfactant-like properties of A2-E on biomembranes which might be operative in retinal diseases associated with excessive lipofuscin accumulation including age-related macular degeneration.

The detection of metabolic changes opens the possibility for intervention of reversible pathological alterations. Measurements of oxygen saturation are limited to the blood vessel system. Detection of alterations in oxygen concentrations are up to 3 orders of magnitude more sensitive by autofluorescence of coenzymes than by measurement of oxygen saturation. Because of limited transmission of the ocular media no specific excitation of endogenous fluorophores can be realised. For this reason it was investigated if the fluorescence lifetime after pulse excitation can be detected at the human fundus. Applying a laser scanner ophthalmoscope and mode-locked Ar(+) laser as well as time-correlated single photon counting, lifetime images of the living fundus were obtained. In mono-exponential approximation, a mean lifetime of 5 ns was detected from the optic disc and large vessels whereas about 1.5 ns were detected in the parapapillary area. By evaluating the frequency of lifetimes, lipofuscin, free FAD, and collagen are probably detectable. Comparative measurements were performed in fundus specimens and on free FAD.

Melanin granules in the retinal pigment epithelium (RPE) have many important functions which are not yet completely understood. Melanin in the RPE protects the cell from damage caused by oxidative stress. This pigment acts as a free radical sink and diminishes cytotoxic lipid peroxidation. Thus, melanin protects against light toxicity and against cytotoxic effects caused by ocular inflammation. Many enzymes, e.g. superoxide dismutase or carboanhydrase, are only activated in the presence of zinc. Melanin can store zinc and release it when required. The absence of melanin in patients with oculocutaneous albinism is accompanied by photophobia, poor visual acuity and nystagmus. Furthermore melanin is said to protect against damaging lipofuscin accumulation in the RPE. Melanosomes are involved in the lysosomal degradation pathways and possibly take part in the degradation of rod outer segments (ROS) in the RPE.

Excessive accumulation of lipofuscin in the retinal pigment epithelium with age and in various hereditary and degenerative retinal diseases, is of pathogenetic significance. We have shown that the major lipofuscin fluorophor A2-E (N-retinylidene-N-retinylethanolamine) affects the lysosomal degradation of human RPE cells and damages the cellular metabolism by phototoxic properties. Herein we sought to determine mechanisms for the inhibitory effect on lysosomal function apart from pH elevation.Potter-Elvejem homogenates of RPE cells were used to measure the activity of 24 lysosomal enzymes before and after incubation with A2-E.This is the first time that RPE cells have been screened for a large spectrum of lysosomal hydrolases including proteases, lipidases, glycosidases, nucleases, sulfatases and phosphatases. The activities of these hydrolases were readily detectable in cultured RPE cells. Incubation of RPE cell homogenates even with high A2-E concentrations (up to 10 microM) did not affect the activity of isolated lysosomal enzymes.The results suggest that a direct inhibition of lysosomal enzyme activity would not explain the inhibitory effect on lysosomal degradation. A2-E increases the acidic intralysosomal pH thereby probably hindering pH-dependent lysosomal enzymatic activities. The understanding of the inhibitory effects of A2-E on RPE cell metabolism may contribute to new approaches for treatment of retinal diseases with excessive lipofuscin accumulation such as ARMD or M. Stargardt.

Visualization of the retinal pigment epithelium (RPE) in vivo has proven difficult for various reasons, including the optical properties of the eye and the small size of the cellular elements, forming a single layer between the neurosensory retina and Bruch's membrane. With the advent of scanning laser ophthalmoscopy it is now possible to image topographic distribution and intensity fundus autofluorescence derived from accumulations of lipofuscin granules in RPE cells. Excessive lipofuscin storage in the RPE cytoplasm occurs not only in association with age but also with many hereditary and degenerative retinal diseases including age-related macular degeneration, Stargardt disease, Best disease, and pattern dystrophies. Lipofuscin in the RPE derives mainly from incomplete degradation of phagocytosed distal segments of photoreceptor outer segments, and is composed of various biomolecules including lipids, protein, and retinoids. Some of its constituents have recently been shown to possess toxic properties in vitro and may play a pathophysiological role in diseases such as age-related macular degeneration.Visualizing lipofuscin in vivo may help to better understand the significance of these metabolic alterations in the pathogenesis of retinal disorders. In addition,fundus autofluorescence imaging can be useful in the preclinical diagnosis of hereditary retinal disease. Dynamic alterations of intrinsic RPE fluorescence change may be applicable for monitoring effects at the level of the RPE of novel therapeutic modalities. Furthermore, identification of high-risk characteristics in patients with age-related macular degeneration with this technique may be helpful for indicating and adjusting future therapies.

A fluorescent compound of lipofuscin, A2-E, has been shown to impair lysosomal function and to increase the intralysosomal pH of human retinal pigment epithelial (RPE) cells. This study addressed the phototoxic potential of A2-E on RPE cells.A2-E accumulation was confirmed by fluorescence microscopy and fluorescence-activated cell sorter analysis. Acridine orange staining allowed assessment of lysosomal integrity and intralysosomal pH. Phototoxic properties of A2-E were determined by exposing A2-E-free and A2-E-fed RPE cell cultures to short-wavelength visible light and assessing cell viability and lysosomal integrity.Intralysosomal accumulation of A2-E was confirmed. Acridine orange staining showed that the A2-E was located in the lysosomal compartment and induced an elevation of intralysosomal pH. Exposure of A2-E fed cells to light resulted in a significant loss of cell viability by 72 h which was not observed in either RPE cells maintained in the dark or A2-E-free cultures exposed to light. Toxicity was associated with a loss of lysosomal integrity.A2-E is detrimental to RPE cell function by a variety of mechanisms including inhibition of lysosomal degrading capacity, loss of membrane integrity, and phototoxicity. Such mechanisms could contribute to retinal aging and to retinal diseases associated with excessive lipofuscin accumulation.

Lipofuscin accumulates with age and in association with various retinal diseases. To investigate cellular effects of lipofuscin components in an in vitro RPE cell model, specific loading of the lysosomal compartment is required. Herein a major lipofuscin fluorophor was complexed to LDL and the subsequent subcellular localization of the retinoid was examined.The lipofuscin component N-retinylidene-N-retinylethanolamine (A2-E) was synthesized and coupled to LDL. Human RPE cell cultures were loaded with the A2-E/LDL complex over 4 weeks. Thereafter, RPE cells were harvested by trypsinization and disrupted by nitrogen cavitation. After ultra-centrifugation, the postnuclear supernatant was fractionated on a self-generating gradient and fractions were analyzed by measuring marker enzyme activities of various cellular compartments.A2-Eaccumulated almost exclusively in the lysosomal compartment, as indicated by the identical peaks of the marker enzyme ss-hexosaminidase and the relative fluorescence of A2-E. Only a small amount of A2-E appeared to associate with the cell membrane, as shown by a minor peak of A2-E corresponding to the distribution of phosphodiesterase activity. The lysosomal marker enzyme was not present in the cytosolic fraction.The feeding of A2-E/LDL complexes to cultured RPE-cells proved to be highly effective in specific loading of the lysosomal compartment, providing a suitable in vitro cell culture model for RPE aging and the investigation of A2-E-effects on lysosomal functions in RPE cells. Such a model may contribute to the understanding of the pathogenesis of degenerative diseases of the outer retina associated with excessive lipofuscin accumulation, including age-related macular degeneration, Stargardt's disease and Best's disease.

The lack of histopathological material has placed limitations on our knowledge on lipofuscin in central serous chorioretinopathy (CSCR). This study was designed to document the pathological changes of the retinal pigment epithelium (RPE) in CSCR using in vivo recording of fundus autofluorescence.Fundus autofluorescence was documented in 62 eyes of 44 subjects with CSCR using a laser scanning ophthalmoscope (Zeiss, Oberkochen; excitation wavelength: 488 nm, barrier filter at 521 nm). Images were compared to the respective fundus appearance and fluorescein angiograms.Neurososensory retinal detachments showed diffuse increased autofluorescence corresponding to the detached area. Long-standing lesions showed very irregular autofluorescence with regions greater and less than the background levels of autofluorescence.Focal accumulation of autofluorescent material occurs at the level of the RPE in patients with CSCR, relating to variation in metabolic activity of the RPE. This technique may be useful in selecting patients for laser photocoagulation.

The fundus autofluorescence imaging technique has been modified allowing improved image resolution (768 x 572 pixel). We present results of fundus autofluorescence studies using this technique.Fundus autofluorescence was studied in 286 eyes of 143 patients with retinitis pigmentosa, macular dystrophies and age-related macular degeneration using a confocal laser scanning ophthalmoscope prototype (Zeiss, Oberkochen; excitation wavelength: 488 nm, cut-off filter at 521 nm).The spatial distribution of autofluorescence was different in all diseased eyes investigated compared to the normal pattern of fundus autofluorescence. Each disorder showed a specific fundus autofluorescence appearance.The advanced technique of imaging fundus autofluorescence allows detailed studies of the lipofuscin distribution. In vivo analysis of the dynamics of accumulation and degradation of lipofuscin in eyes with tapeto-retinal dystrophies and age-related macular disease may contribute to elucidation of the pathogenesis of these disorders.

The lack of histopathological material has placed limitation on our knowledge on the composition of focal deposits in eyes with macular dystrophies, malattia leventinese, dominant drusen and age related macular degeneration. This study was designed to study the composition of focal deposits in these eyes by documenting fundus autofluorescence in vivo.Fundus autofluorescence was documented in 343 eyes of 199 subjects with macular dystrophies, malattia leventinese, dominant drusen and age-related macular degeneration using a laser scanning ophthalmoscope (Zeiss, Oberkochen; excitation wavelength: 488 nm, barrier filter at 521 nm).Autofluorescence of focal deposits was increased in eyes with macular dystrophies. In eyes with malattia leventinese and dominant drusen autofluorescence intensity of focal deposits showed a wide spectrum. In contrast, autofluorescence of age-related drusen was within normal limits. Background autofluorescence intensity was increased in eyes with macular dystrophies and within normal limits in eyes with malattia leventinese, dominant drusen and age-related drusen.The technique of in-vivo recording of fundus autofluorescence allows the differential diagnosis between macular dystrophies/malattia leventinese, dominant drusen/age related drusen when otherwise not possible.

It is thought that lipofuscin plays a central role in the pathogenesis of age-related macular degeneration (AMD). The lack of histopathological material has been a severe limitation in our knowledge on lipofuscin in this disease. A new technique has been developed that allows in vivo imaging of fundus autofluorescence derived from lipofuscin in the retinal pigment epithelium (RPE) using a confocal Laser Scanning Ophthalmoscope (LSO). We studied the dynamics of lipofuscin accumulation and degradation in patients with AMD.Serial examinations of the spatial distribution of fundus autofluorescence were performed in 148 eyes of 74 patients with AMD using a LSO over a period of 1-3.5 years.Fundus autofluorescence changed over time in almost all eyes studied. Areas of increased autofluorescence occurred progressively during follow up in eyes with drusen and hyperpigmentation. The size of pathologic autofluorescence increased over time in almost all eyes with geographic atrophy, subretinal neovascularisations and disciform scars. Irregular autofluorescence was seen over most subretinal neovascularisations. Autofluorescence intensity decreased in old subretinal neovascularisations and disciform scars over time.Changes of the distribution of autofluorescence occur in eyes with AMD over time. Fundus autofluorescence imaging allows in vivo analysis of the dynamics of accumulation and degradation of lipofuscin in the RPE in eyes with AMD and documentation of metabolic activity of the RPE.

Lipofuscin is the main fluorophore of the human fundus. Because lipofuscin is the result of the accumulation of metabolic debris in pigmentepithelial cells (RPE), the autofluorescence can be interpreted as a clinical sign for the metabolic activity of the RPE. In order to get informations of RPE-function in different types of late AMD, the autofluorescence patterns in patients with late AMD were analyzed.A prospective examination of the fundus-autofluorescence of 64 eyes of 52 patients with different types of late AMD was performed using a confocal scanning-laser-opthalmoscope. The autofluorescence images were categorized in respect to the type of late AMD according to the opthalmoscopic and fluoresceine-angiographic findings.Reduced autofluorescence was found in the centre of occult (78.6%) and classic (100%) choroidal neovascularisations (NV) as well as in the occult NV of RPE detachments. A loss of autofluorescence was related to the RPE free area of RPE-tears (100%) and to RPE-atrophy (88.9%) with sometimes increased autofluorescence at the rim. Increased autofluorescence could be seen at the surface of RPE-detachments (71.4%), in the area of the shrink age of RPE in RPE-tears (100%) as well as at RPE-proliferations in small occult NV (100%). Disciforme scars showed variable patterns of autofluorescence.The autofluorescence of the RPE can be analyzed clinically with the described method. Different patterns of autofluorescence could be revealed in different types of late AMD. Increased autofluorescence was found in lesions with proliferative or phagocytotic metabolic activity of the RPE like RPE-detachments, shrinked RPE in RPE-tears or occult NV with RPE-proliferations. The reduced autofluorescence in occult or classical choroidal NV can be interpreted as a sign of decompensation of the RPE and was also seen in areas with RPE-loss.

We investigated fundus autofluorescence in vivo using a novel scanning laser ophthalmoscope.A total of 550 patients with various retinal diseases were examined and compared with normal eyes. Autofluorescence was detected after excitation with an argon blue laser (488 nm), and emission was recorded with a short wavelength cut off above 500 nm.Reduced autofluorescence was observed in the foveal and parafoveal region due to retinal xanthophyll, along the retinal vessels, at the optic nerve head and in areas with atrophy of the retinal pigment epithelium (RPE). Autofluorescence intensity was increased either focally or diffusely in certain degenerative (AMD) or genetically determined retinal diseases (e.g., Stargardt's disease, Best's disease).These findings are in accordance with the view that in vivo fundus autofluorescence originates at the level of the RPE and suggest that it is derived from lipofuscin.

Lipofuscin pigments are considered indigestible residues of lysosomal activity associated with aging. We present the clinical and histopathological features of ten patients with corneal lipofuscinosis.Ten patients (five female, five male, mean age 62 years, range 53 to 77 years) underwent penetrating keratoplasty for vascularized corneal scars. The clinical diagnoses were herpes simplex keratitis (5), zoster keratitis (1), phlyctenular keratitis (1), immunologic graft rejection (2) and corneal injury (1). The mean duration of disease was 6.5 years (range 3 to 11 years). On slit lamp examination both linear and diffuse deposits of yellow pigment were noted in the corneal stroma. All corneal buttons were processed for histopathologic and electron microscopic studies.The areas of yellowish pigmentation in the corneal stroma corresponded histopathologically to clusters of macrophages, keratocytes and occasional giant cells containing PAS-positive granules. The granules measured 1 to 3 micrometers and were also present extracellularly. Results of Gram stain were negative, but the material showed vivid autofluorescence.Corneal lipofuscin can be detected at the slit lamp as focal or diffuse yellowish deposits in both the superficial and deep corneal stroma of patients with long-standing keratitis. This is in contrast to corneal iron deposits, which appear as yellowish pigment in the corneal epithelium. The identification of corneal lipofuscin is possible using histochemical and autofluorescent studies. Further studies should address whether corneal lipofuscin predisposes to suture loosening.

Amiodarone can occasionally lead to a slate-coloured pigmentation in chronically UV-expose skin. A 63 year old patient presented with a pruritic bluish-purple pigmentation of the face 2 years after taking 400 mg of amiodarone daily for 2 years. During the 24 months after the dose was reduced to 200 mg daily, the pigmentation remained unchanged. Amiodarone pigmentation is caused by the UV-induced accumulation of lipofuscin in dermal macrophages, depending on both the daily dose and the duration of therapy. In the differential diagnosis, pigmentation caused by other drugs is the main consideration. Cessation of therapy eventually leads to complete normalization of skin colour.

This study was performed to measure and characterize the intrinsic fluorescence of the ocular fundus in patients with age-related macular degeneration (AMD).Fluorescence spectral measurements from discrete retinal locations were made using the fundus spectrophotometer with excitations at 470 and 510 nm. Two normal subjects and seven patients with different stages of AMD were investigated.The spectral characteristics of fundus fluorescence are consistent with those of lipofuscin in the retinal pigment epithelium (RPE). The fluorescence spectrum is broad, with a maximum at about 620 nm. The shape and intensity of the fluorescence spectra are affected by age, site of measurement, pathology, ocular media absorption, and excitation wavelength. Spectra from areas with drusen reveal an additional fluorophore, with maximum around 560 nm, probably emanating from drusen and Bruch's membrane. Measurements in atrophic reveal a decrease of lipofuscin fluorescence and/or a contribution likely due to choroidal and sclera collagen fluorescence. Fluorescence from lipofuscin is more efficiently excited at 510 nm, whereas that of drusen and subretinal structures is relatively more efficient with 470 nm excitation, allowing for discrimination of various fluorophores.The spectral characteristics of RPE lipofuscin could be identified and quantified in AMD patients. In addition, the spectra are affected by other fluorophores such as drusen and choroid contributions in atrophy. Fluorescence spectra measurements in AMD patients allow for discrimination of lipofuscin fluorescence, drusen fluorescence, and choroidal or scleral fluorescence. The non-invasive measurement of lipofuscin and drusen fluorescence in AMD may be helpful in monitoring the disease, understanding its evolution, and testing therapeutic concepts.

Results of macroscopic, microscopic and electronmicroscopic investigations of two cocker spaniels with generalized ceroid-lipofuscinosis are recorded. In the nerve system, the lipofuscin granules were mainly localized, primarily in nervous cells and secondarily in glial cells of the medulla oblongata and spinal cord. Extraneuronal lipofuscin granules were found in the smooth muscle fibres (bowel, bladder, trabeculae of the spleen, prostata etc.) and also in the cells of the exocrine pancreas. Electronmicroscopically the lipofuscin granules in the neurons and in the smooth muscle fibres showed the 'typical' structure of lipofuscin, whereas in macrophages and glial cells the lipofuscin appeared as fingerprint-like, pleomorphic, curvilinear and multi-membranous bodies.

The staining properties of the cationic dye Victoria Blue are summarized in consideration of the findings in the literature and own findings. Dependent on the staining conditions (with or without preoxidation of tissue sections), different structures and substances are stained by Victoria Blue. Without preoxidation elastic fibres, acid glycosaminoglycanes and DNA predominantly stained, but with preoxidation the dye stained insulin, some peptide hormones and secretion products, HBs-antigen, and lipofuscin. The chemism of the histochemical reaction is not quite understood yet.

Histological and morphometrical investigations of the vestibular nerve were performed on surgical specimens from 19 patients suffering from Ménière's disease and on 5 autopsy preparations from patients without inner ear disease. The number of lipofuscin granules and vacuoles in the ganglion cells of specimens from Ménière patients was not increased compared with control specimens of a similar age, nor did the amount of endoneuronal connective tissue in the vestibular nerve specimens differ between the two groups. The density of capillaries in Scarpa's ganglion and vestibular nerve was significantly increased in the Ménière patients compared with the control specimens. Furthermore, the ratio of nerve fibres with a larger diameter was higher in specimens from Ménière patients than in control preparations. There was no difference in the density of myelinated fibres between the two groups.

Lipogenic pigmentation is described in thyroid follicular, parathyroid, and pineal cells, as well as in adrenocortical macrophages. Piracetam activated pituitary prolactin cells and increased paracrystals in thyroid follicular cells; smooth endoplasmic reticulum was increased and glycogen content decreased in numerous hepatocytes randomly distributed over the lobule.

The weight of the heart increases in men and women very rapidly up to the 20th year and after this time grows slowly up to the 90th year. Only after the 90th year there is a decrease of the heart weight. Thickness and length of the heart muscle cells are changing analogous. The capillaries likewise multiply during the first and second decade very quickly and do not diminish in advanced age. We do not find a "withered condition of the top" of the capillary tree and a thickening of the basement membrane. Lipofuscin already appears in heart muscle cells of 7-year-old children; its amount raises continuously. The interstitial connective tissue increases only during the first decade of life. In advanced age a part of the collagenous fibres degenerates. The fibrous framework of the cardiac valves is developing to a solid and stable organ during the first and second decade. After the 50th year the collagenous and elastic fibres show symptoms of degeneration. The different ageing of the blood vessels is demonstrated by the arteries of the iris.