Carotenoid, tocopherol, and retinol concentrations in elderly human brain.

BACKGROUND: Antioxidants, such as tocopherols and carotenoids, have been implicated in the prevention of degenerative diseases. Although correlations have been made between diseases and tissue levels of antioxidants, to date there are no reports of individual carotenoid concentrations in human brain. OBJECTIVE: To measure the major carotenoids, tocopherols, and retinol in frontal and occipital regions of human brain. DESIGN: Ten samples of brain tissue from frontal lobe cortex and occipital cortex of five cadavers were examined. Sections were dissected into gray and white matter, extracted with organic solvents, and analyzed by HPLC. RESULTS: At least 16 carotenoids, 3 tocopherols, and retinol were present in human brain. Major carotenoids were identified as lutein, zeaxanthin, anhydrolutein, alpha- cryptoxanthin, beta- cryptoxanthin, alpha-carotene, cis- and trans-betacarotene, and cis- and trans-lycopene. Xanthophylls (oxygenated carotenoids) accounted for 66-77% of total carotenoids in all brain regions examined. Similar to neural retina, the ratio of zeaxanthin to lutein was high and these two xanthophylls were significantly correlated (p <0.0001). The tocopherol isomers occurred in the brain over a wider range of mean concentrations (0.11-17.9 nmol/g) than either retinol (87.8 - 163.3 pmol/g) or the identified carotenoids (1.8-23.0 pmol/g). CONCLUSIONS: The frontal cortex, generally vulnerable in Alzheimer's disease, had higher concentrations of all analytes than the occipital cortex which is generally unaffected. Moreover, frontal lobes, but not occipital lobes, exhibited an age-related decline in retinol, total tocopherols, total xanthophylls and total carotenoids. The importance of these differences and the role(s) of these antioxidants in the brain remain to be determined.

Age-related accumulation and spatial distribution of lipofuscin in RPE of normal subjects.

PURPOSE: To characterize the age-related accumulation of lipofuscin in a population of normal subjects, resolve differences in estimated accumulation rates obtained in previous studies, and characterize the spatial distribution of lipofuscin in the normal fundus. METHODS: Spectrophotometric measurements were made at the fovea and 7 degrees temporal to the fovea in 145 normal subjects (age range, 15-80 years). Spatial distribution along the four cardinal meridians was measured in selected subjects by both spectrophotometry and autofluorescence imaging. To minimize contributions of extraneous fluorophores, macular pigment, and melanin, all measurements used excitation at 550 nm, integrating emission between 650 and 750 nm. RESULTS: Lipofuscin fluorescence increased linearly until age 70, then declined. The rate of accumulation was significantly slower in the fovea than at the temporal site; accumulation rates in vivo were greater than previously observed in microscopic studies. Fluorescence was approximately 40% lower in the fovea than at 7 degrees eccentricity and was asymmetrically distributed around the fovea. The fluorescence was maximal at approximately 11 degrees temporally, approximately 7 degrees nasally, approximately 13 degrees superiorly, and approximately 9 degrees inferiorly. At the same eccentricity, fluorescence was always less along the inferior meridian than along any other. CONCLUSIONS: Light absorption by RPE melanin can explain differences between the in vivo and ex vivo estimates of the rate of lipofuscin accumulation. Declining fluorescence at old age may represent removal of atrophic RPE cells. The spatial distribution of lipofuscin generally matches that of rods and reflects, rather than predicts, the pattern of age-related loss of rod photoreceptors.

The architectural transcription factor high mobility group I(Y) participates in photoreceptor-specific gene expression.

The nonhistone chromosomal proteins high mobility group I(Y) [HMG I(Y)] have been shown to function as architectural transcription factors facilitating enhanceosome formation on a variety of mammalian promoters. Specifically, they have been shown to act as a "molecular glue" mediating protein-protein and protein-DNA contacts within the enhanceosome complex. HMG I(Y) proteins are expressed at high levels in embryonic and transformed cells and have been implicated in transcriptional regulation in these cells. Terminally differentiated cells, however, have been reported to express only minimal, if any, HMG I(Y). In contrast to these observations, we show here that adult mouse retinal photoreceptors, which are terminally differentiated cells, express high levels of these proteins. Using retinoblastoma cells as an approximate model, we further demonstrate in transiently transfected cells that inhibition of HMG I(Y) expression and mutation of HMG I(Y) binding sites significantly reduce rhodopsin promoter activity. DNase I footprint analysis indicates that HMG I protein interacts with a discrete site within the rhodopsin proximal promoter. This site overlaps with the binding site for Crx, a paired-like homeodomain transcription factor that is essential for photoreceptor functioning and that when mutated causes several forms of human photoreceptor degeneration. Both biochemical and functional experiments demonstrate that HMG I(Y) physically associate with Crx and that their interaction with DNA is required for high-level transcription of the rhodopsin gene. These data provide the first demonstration that HMG I(Y) can be important for gene activation in terminally differentiated cells.

Pentoxifylline inhibition of vasculogenesis in the neonatal rat retina.

PURPOSE: The zeta isozyme of protein kinase C (PKC) is essential for activation of the transcription factor nuclear factor (NF)kappaB and transcription of vascular endothelial growth factor (VEGF). This study examined the antiangiogenic potential of an existing drug, pentoxifylline (PTX), which inhibits PKC-dependent activation of NFkappaB and is reported to prevent hypoxia-induced expression of VEGF. METHODS: Neovascularization was induced by maintaining neonatal rats for 10 full days in 80% oxygen, interrupted daily by 30 minutes in room air followed by a progressive return to 80% oxygen. On experimental day 11, they were placed in room air until they were killed on day 17. Daily intraperitoneal injections of PTX in saline (25 or 75 mg/kg per day), or saline alone, were administered from day 6 through day 16. Retinal neovascularization was scored, and avascular areas (AVAs) were measured in ADPase stained retinas. RESULTS: PTX inhibited radial extension of retinal vessels, causing increases in AVA of 65% (P < 0.01) and 33% (P < 0.15) at the lower and upper doses, respectively. A significant increase in mean neovascular score was seen at the lower dose (P < 0.0001), but analysis of variance indicated that neovascularization was strongly and positively influenced by the AVA (P < 0.0001) and only weakly stimulated by PTX (P < 0.05). CONCLUSIONS: Systemic PTX significantly inhibited VEGF-mediated retinal vasculogenesis, but was not effective in reducing neovascularization in the oxygen-exposed neonatal rat.

Autofluorescence distribution associated with drusen in age-related macular degeneration.

PURPOSE: To determine whether drusen in patients with age-related maculopathy and macular degeneration (ARM/AMD) are associated with focal changes in retinal pigment epithelium (RPE) lipofuscin fluorescence. METHOD: A new autofluorescence imaging device was used to study lipofuscin distribution associated with individual drusen in 20 patients with ARM/AMD. Paired monochromatic and autofluorescence fundus images were used for detailed analysis of the topography of autofluorescence at specific sites containing drusen. In four eyes, image analysis was used to compare the spatial distribution of the autofluorescence with the location of drusen and to quantify the autofluorescence distribution over individual drusen (54 drusen). REsuLTs. A specific pattern of autofluorescence was frequently found to be spatially associated with hard drusen and soft drusen between 60 and 175 microm in size. The pattern is characterized by a central area of decreased autofluorescence surrounded, in most cases, by an annulus of increased autofluorescence. The location of this pattern was highly correlated with the position of individual distinct drusen. The central low autofluorescence focus was on average 16% below the surrounding background, and the annulus, when present, was on average 6% more fluorescent than the background. Soft drusen larger than 175 microm and confluent soft drusen show either multifocal areas of low autofluorescence or a more heterogeneous distribution. CONCLUSIoNs. Autofluorescence imaging permits measurement of RPE lipofuscin at specific sites. RPE overlying drusen have altered autofluorescence, suggesting changes in RPE health.

Microglia increase as photoreceptors decrease in the aging avian retina.

PURPOSE: There is evidence that microglial activation occurs with normal aging in some regions of the brain of rodents. We investigated the pattern of microglia in the retinas of young and aged quail and pigeons to determine if age-related retinal changes evoked migration of microglia into the outer retina. In quail we also investigated the correlation between activated microglia and age-related photoreceptor loss. METHODS: Microglia were identified with the monoclonal antibody QH1 in cryosectioned eyes from pigeons, ages 2 to 20 years (n = 14), and in paraffin sections from six-month (n = 15) and one-year-old quail (n = 30). Rounded microglia in the photoreceptor layer were counted in consecutive 400x fields from temporal to nasal. Photoreceptor counts were made from 10 quail retina flat mounts. The photoreceptor number was compared to the number of microglia in corresponding regions of the same retina. RESULTS: Rounded microglia were detected among the photoreceptors of pigeons and quail. Significantly more of these microglia were found in peripheral than in central regions close to the pecten (pigeon p < 0.002 and quail p < 0.01). Furthermore, more microglial cells were present among peripheral photoreceptors of older quail (p < 0.03) and pigeons (p < 0.05) than in the younger birds. In the peripheral retina of the older quail, microglia were significantly and inversely related to the number of photoreceptors (r2 = 0.9; p < 0.001). CONCLUSIONS: Increased microglial were observed in the peripheral retina of both old quail and old pigeons. In the quail, the rounded (activated) microglia were distributed preferentially in regions of greatest photoreceptor loss. Microglial activation does not appear to be a general phenomenon of the aging retina, but in quail activation appears directly related to photoreceptor loss. It is unclear at this time how the change in microglia shape and distribution is related to their neuroprotective / neurotoxic potential.

Lipid hydroperoxide stimulates retinal neovascularization in rabbit retina through expression of tumor necrosis factor-alpha, vascular endothelial growth factor and platelet-derived growth factor.

To test the hypothesis that oxidative damage associated with tissue hypoxia plays a role in neovascularization, a lipid hydroperoxide (LHP) was injected into the vitreous of rabbits. Single injections of LHP (50-600 microg) caused a sustained retinal neovascularization visualized clinically by ophthalmoscopy and confirmed by microscopy. Vasodilators, i.e. histamine and nitric oxide, peaked at 6h and 7 days, respectively. The levels of both tumor necrosis factor-alpha and interleukin-1alpha peaked at 12h and dropped to basal levels by 24h. Expression of vascular endothelial growth factor (VEGF) and transforming growth factor-beta peaked at 24h and were sustained throughout the following 3 weeks, and platelet-derived growth factor was also elevated throughout the same period. Upregulation of these five angiogenic cytokines, but not basic fibroblast growth factor, occurred prior to the appearance of neovascularization. Leakage of fluorescein at the tips of new vessels was demonstrated by fluorescein angiography. Linoleic hydroperoxide induced neovascularization, but saturated or unsaturated native C-18 fatty acids had no effect. The cascade of multiple, angiogenic cytokines induced by LHP may interact to promote sustained neovascularization.

Correlation of vascular permeability factor/vascular endothelial growth factor with extraretinal neovascularization in the rat.

OBJECTIVE: To determine whether neovascularization was spatially correlated with the distribution of messenger RNA for vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). METHODS: Neonatal rats were raised 8 days in 80% oxygen with daily intervals of relative hypoxia in room air, then transferred to room air for 5, 7, or 10 additional days. In situ hybridization for VPF/VEGF expression and avascular area were examined in retinas from oxygen-exposed animals and room-air controls. Severity of neovascularization was scored. RESULTS: The inner nuclear layer of oxygen-exposed retinas exhibited a continuous intense band of VPF/VEGF messenger RNA expression across the peripheral avascular zone that dropped sharply in vascular retina. Neovascularization occurred adjacent to regions of greatest expression. Controls had much lower expression and smaller avascular regions. The VPF/VEGF messenger RNA expression was most intense in Müller cells, scattered astrocytes, and amacrine cells, strong in retinal pigment epithelium, and moderate in the remaining inner nuclear layer and ganglion cell layer. CONCLUSION: The expression of VPF/VEGF message was spatially and quantitatively correlated with the neovascularization.

In vivo measurement of lipofuscin in Stargardt's disease--Fundus flavimaculatus.

PURPOSE: Several histopathologic studies have concluded that Stargardt's disease (Fundus flavimaculatus) is associated with abnormally high levels of lipofuscin-like material in the retinal pigment epithelium. The purpose of this study was to determine whether this material has the same fluorescence characteristics as lipofuscin in vivo and whether noninvasive measurements identify a significant elevation in this material. METHODS: Five patients with autosomal recessive Stargardt's disease were included in this study, as were 45 healthy controls. All patients had the angiographic dark choroid sign. The intensity and emission spectra of lipofuscin fluorescence were measured by noninvasive fundus spectrophotometry at 7 degrees temporal to the fovea. RESULTS: The fluorescence intensities in the five patients with Stargardt's disease were significantly higher (P < 0.0001) than those observed in normal subjects of the same age. The emission spectra in the patients are similar in shape to those measured in normals, but flecks appear to shift the spectra toward shorter wavelengths. CONCLUSIONS: The spectral characteristics of the fluorophore observed in patients with Stargardt's disease are consistent with those of retinal pigment epithelial lipofuscin. These patients have abnormally high levels of lipofuscin, confirming previous histopathologic observations. Noninvasive retinal pigment epithelial lipofuscin measurements may be a useful adjunct in the diagnosis of Stargardt's disease.-F. flavimaculatus.

In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics.

PURPOSE: To characterize the intrinsic fluorescence (autofluorescence) of the human ocular fundus with regard to its excitation and emission spectra, age relationship, retinal location, and topography, and to identify the dominant fluorophore among the fundus layers. METHODS: Using a novel fundus spectrophotometer, fluorescence measurements were made at 7 degrees temporal to the fovea and at the fovea in 30 normal subjects and in 3 selected patients. Topographic measurements were made in 3 subjects. Ex vivo measurements of fluorescence of human retinal pigment epithelium (RPE) were obtained and compared to in vivo data. RESULTS: Fundus fluorescence reveals a broad band of emission between 500 and 750 nm, a maximum of approximately 630 nm, and optimal excitation of approximately 510 nm. Exhibiting a significant increase with age, this fluorescence is highest at 7 degrees to 15 degrees from the fovea, shows a well-defined foveal minimum, and decreases toward the periphery. In vivo fluorescence spectra are consistent with those obtained ex vivo on human RPE. Measurements with short wavelength excitation are strongly influenced by ocular media absorption and reveal an additional minor fluorophore in the fovea. CONCLUSIONS: Spectral characteristics, correlation with age, topographic distribution, and retinal location between the choriocapillaris and the photoreceptors suggest that the dominant fundus fluorophore is RPE lipofuscin. The minor fluorophore is probably in the neurosensory retina but has not been identified.

Extraretinal neovascularization induced by hypoxic episodes in the neonatal rat.

PURPOSE: To test the hypothesis that hypoxia induces retinal neovascularization. METHODS: To produce relative hypoxia in the avascular retina, newborn rats were exposed for 11 days to 80% oxygen interrupted daily by short episodes in room air. Episodes in room air lasted 1/2 hour or 1 hour followed by abrupt reintroduction, or 1/2 hour followed by progressive reintroduction to 80% oxygen lasting 3 hours to prolong the period of hypoxia. At the end of the 11 days of interrupted oxygen exposure, the animals were left in room air for 6 days. RESULTS: The incidence of neovascularization exhibited a dose-response relationship to the period out of 80% oxygen. Periods of approximately 3 hours produced neovascularization (in at least one eye) in 94% of the animals. The total area of the peripheral avascular retina was larger in animals exposed to prolonged periods of hypoxia than in those exposed for shorter periods. The incidence of neovascularization was strongly associated with the total area of the peripheral avascular retina and occurred in the inferior quadrant in 78% of the cases. Additional features of stage 3 retinopathy of prematurity (ROP), including arteriovenous shunts and ridges, were observed in some retinas. CONCLUSIONS: These data demonstrate that hypoxic episodes can induce extraretinal neovascularization in the rat and suggest that brief periods of oxygen deficiency could exacerbate progression of ROP. With the high incidence and predictability of localization of neovascularization, this model could be useful for the study of angiogenesis and for evaluation of antiangiogenic or antioxidant substances.

The effect of oxygen on melanin precursors released from retinal pigment epithelial cells in vitro.

The autoxidation of dopa to melanin in culture media causes toxicity to retinal pigment epithelial (RPE) cells and endothelial cells. The damage is specific to cell type and to the ambient oxygen concentration. To determine whether RPE cells influence the oxidation of dopa to media, we compared light absorbing dopa derivatives in the media exposed to cells with those found in the media incubated without cells. Dopa was extensively oxidized in the presence of RPE cells, and more light absorbing substances were generated with higher dopa and oxygen concentrations. However, an increase in ambient oxygen concentration decreased the quantity of several dopa derivatives which had been formed. The data provided evidence that RPE modulated dopa metabolism. Quinolic derivatives produced from a tyrosinase reaction and dopa-melanin formation moved the peak absorbance wavelength of dopa into the visible range. The spectrum between the dopa-derived compounds in the media has an absorbance at 240-275 nm and a maximum around 300 nm with a shoulder near 375 nm. Gaussian analysis (peak separation) resolved these spectra into five components: a sharp band at 248 nm, a band at 295 nm, a large band at 359 nm, and two broad bands at 459 and 585 nm.

Comparison of external and internal approaches for transplantation of autologous retinal pigment epithelium.

The feasibility of autologous transplantation of retinal pigment epithelial (RPE) cells from just posterior to the ora serrata to the posterior pole was demonstrated in the rabbit model. Two techniques for introducing the transplanted cells were compared: an internal (anterior transvitreal) and an external (posterior transscleral) penetration to the subretinal space. In both approaches, RPE cells were obtained by biopsy from the peripheral retina of a rabbit eye, cultured, labeled with a fluorescent dye and 3H-thymidine, and transplanted to the posterior pole of the same or contralateral eye. The external approach consistently resulted in a greater number of transplanted cells on Bruch's membrane. The internal technique was more precise because it permitted direct visualization of the placement of the transplanted RPE. Transplantation of autologous RPE is a possibility that should be further pursued.

Comparison of effects of oxygen and antioxidative enzymes on cell growth between retinal pigment epithelial cells and vascular endothelial cells in vitro.

We assayed the proliferation of porcine retinal pigment epithelial (RPE) cells, bovine melanotic and amelanotic RPE cells, and bovine aortic endothelial cells exposed to 20, 10 and 5% oxygen and compared their responses to oxygen and antioxidative enzymes (superoxide dismutase and catalase). Irrespective of the cell type, the cell growth was optimal in 10% oxygen that is most closely approximating to the oxygen concentration prevailing in the cellular environment of the choroid and the retina in vivo. However, the effects of oxygen concentrations were cell specific because bovine endothelial cells were influenced by lowering of oxygen concentrations more significantly than bovine and porcine RPE cells. Moreover, addition of antioxidative enzymes caused significant improvement in growth of porcine RPE cells, but had no significant effects on bovine RPE cells. On the contrary, the bovine vascular endothelial cells represented the only one cell type significantly inhibited by antioxidative enzymes, i.e., a decrease in reactive intermediates of oxygen was seen in the media. Our results show that responses of vascular endothelial cells to reactive species of oxygen were distinctly different from those of RPE cells and more easily influenced by the environment related to hypoxia than RPE cells.

Growth of cultured RPE and endothelial cells is inhibited by blue light but not green or red light.

Blue light, but not green or red light, inhibited growth of retinal pigment epithelial (RPE) cells, aortic endothelial cells, and fibroblasts in vitro. Significant inhibition was observed in all 3 cell types exposed for 18 hr to blue light (425-500 nm) at 42 J/cm2. Damage was prevented by inclusion of superoxide dismutase (SOD) and catalase, providing evidence for a photooxidative mechanism. Dopa (100 microM) also caused oxidative damage that suppressed growth of all 3 cell types. A synergism of dopa and light effects was observed in endothelial cells and fibroblasts, but the agents caused additive effects on RPE cells. Endothelial cells were the most sensitive to dopa, light, and the two combined. Fibroblasts were the only cell type that exhibited greater sensitivity to light than to dopa. These data suggest that oxygen-mediated damage to the growing blood vessels in the retina of a premature infant may be exacerbated by exposure to blue light. A further implication is that restriction of RPE melanogenesis to the prenatal period of darkness and lower oxygen protects the retina from simultaneous oxidative challenge by light and by reactive species generated during oxidation of dopa released to the extracellular environment.

Evidence of melanogenesis in porcine retinal pigment epithelial cells in vitro.

Several weeks after porcine retinal pigment epithelial (RPE) cell cultures attain confluence, macroscopically visible brown foci appear. The cuboidal cells that form the foci contain numerous phase dark granules that do not exhibit the autofluorescence characteristic of lipofuscin. The data described here indicate that the granules are melanosomes. Electron microscopy revealed three types of electron-dense granules in these cells: simple spheres 0.3-0.5 microns in diameter, large spheres 1-2 microns in diameter, and lysosomal aggregations of the smaller spheres. The matrix of both spheres is composed of 40-nm microvesicles that were also found free in the cytoplasm and aggregated within vacuolar structures. Reversed-phase high-performance liquid chromatography of RPE cells and their media detected melanogens, i.e. intermediates of melanin biosynthesis, including several indole derivatives. The porcine RPE cultures therefore may be a useful system for studying melanogenic regulation.

Dopa and oxygen inhibit proliferation of retinal pigment epithelial cells, fibroblasts and endothelial cells in vitro.

Some quinones catalyze superoxide formation in a futile cycle involving electron transfer to molecular oxygen. If the quinolic precursors of melanin participated in the futile cycle, the high ambient oxygen surrounding postnatal RPE would make continued melanogenesis risk for the retina. To probe the possibility that arrest of melanogenesis in postnatal RPE is a protective mechanism, we assayed the growth rates of RPE cells, aortic endothelial cells and skin fibroblasts exposed to 0, 10, 50, 100 and 250 microM dopa. To assess the contribution of the futile cycle, we studied the effects of oxygen concentration and the antioxidants, superoxide dismutase and catalase. We found that all three cell types were significantly and dose-dependently inhibited by dopa and that the effects of dopa were oxygen dependent. The powerful inhibition of RPE cells by dopa was counteracted by inclusion of superoxide dismutase and catalase, but not by an inhibitor of dopa oxidase (phenylthiourea), indicating that the mechanism of growth suppression did not involve melanogenesis but, rather, dopa-dependent formation of superoxide in the media. Endothelial cells were more sensitive to the dopa-mediated oxidative damage than were RPE cells or fibroblasts. Fibroblasts were most affected by oxygen alone, and least affected by dopa. These data suggest that suppression of melanogenesis in the postnatal RPE may be an important mechanism for preventing oxidative damage to the retina and the choriocapillaris We propose that the generation of oxygen radicals by the quinone futile cycle is a viable model of the damage of cells in culture by dopa.

Cell loss in the aging retina. Relationship to lipofuscin accumulation and macular degeneration.

We examined the impact of aging on the numbers of photoreceptors and retinal pigment epithelium (RPE) cells, and the number of photoreceptors per RPE cell profile, in selected regions of 30 human eyes. The mean ratio of photoreceptors to RPE cell was higher in the macula than in the paramacula (P less than 0.01) or the equatorial area (P less than 0.001). We found evidence for an age-related loss of RPE in both whites (P less than 0.02) and blacks (P less than 0.0006), although the rate of loss in whites was significantly slower than in blacks. Photoreceptor loss in blacks was inversely correlated with age (P less than 0.04). In whites, however, photoreceptor loss was very significantly and directly correlated with lipofuscin concentration in the opposing RPE (P less than 0.0001) and unrelated to age. The disparity in the rates of photoreceptor and RPE cell loss produced, in older eyes, a higher ratio of photoreceptors per RPE cell profile. In the macula, the ratio for whites over 50 years of age was significantly higher (P less than 0.05) than that in blacks over 50. Our data suggest that the increased phagocytic and metabolic load on the RPE, which ultimately the macula causes a preferential age-related accumulation of lipofuscin in the RPE, which ultimately leads to photoreceptor death. This may prove a useful model of age-related macular degeneration and Stargardt's disease.