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1From the Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida; and the 2Vascular Biology Institute, University of Miami School of Medicine, Miami, Florida.
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Abstract |
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METHODS. APO B100 and wild-type C57BL/6 2-month-old mice were fed a high-fat diet for 4.5 months. After the first month, the right eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light (20 mJ of argon 488 nm) over 2 weeks. Three months later, transmission electron microscopy (TEM) of the retina was performed to evaluate whether sub-RPE deposits correlate with plasma cholesterol and triglyceride levels. Several eyes were stained with filipin to detect cholesterol and osmium-thiocarbohydrazide-osmium (OTO) to detect neutral lipids in Bruch’s membrane (BrM). A third group of APO B100 2-month-old mice were pretreated with vitamin E subcutaneously twice a week throughout the experiment and underwent the same light-exposure protocol.
RESULTS. Mice fed a high-fat diet had a more elevated plasma triglyceride and cholesterol level than those that consumed a regular diet. Young APO B100 mice fed a high-fat diet had blood lipid levels higher than those in young wild-type mice that consumed high-fat diets, and these two groups had higher lipid levels than animals with regular diets, as shown previously in wild-type C57BL/6 (old and young). Eyes of APO B100 mice treated with blue-green light showed a high frequency of "moderate BLD", whereas the nonexposed eyes did not. In contrast, no BLD formed in either eye of the wild-type young mice fed a high-fat diet. In individual affected mice, only a weak correlation was observed between deposit severity and plasma lipid concentration. None of the eyes in mice with sustained hyperlipidemia with or without BLD demonstrated obvious widespread neutral lipid or cholesterol deposition in BLD or BrM. However, vitamin E–treated mice showed minimal formation of BLD.
CONCLUSIONS. Although a high-fat diet is a necessary precondition for this model of BLD, the findings demonstrated a convincing direct correlation between plasma lipidemia and deposit severity. The results suggest that age, as shown in previous studies, and high-fat predispose to formation of BLD by altering hepatic and/or RPE lipid metabolism in ways more complicated than plasma hyperlipidemia alone.
Several groups have demonstrated that in aging and in AMD, BrM contains increasing amounts of various lipids, including unesterified and esterified cholesterol,4 5 6 neutral fats,6 7 8 phospholipids,6 7 8 unsaturated fatty acids,9 and peroxidized lipids.9 Epidemiologic data indicate that dietary fat, especially polyunsaturated fats, is associated with AMD.10 Recently, work from our and other laboratories has identified an association between high-fat diet, ocular blue-light exposure, and the development of sub-RPE deposits, especially BLD.11 12 13 In our previous research, old, but not young, C57BL/6 mice were susceptible to deposit formation. The susceptibility correlated with the development of plasma hyperlipidemia after consumption of a high-fat diet, which only occurred in old C57BL/6 mice.14 We concluded that an age-related abnormality in lipid metabolism might be related to the susceptibility of the RPE to blue-light–induced deposit formation.
The mechanism of abnormal lipid metabolism and sub-RPE deposit formation is unknown, but at least three pathogenic possibilities have been proposed. Marshall has postulated that plasma lipids directly infiltrate BrM.15 Alternatively, Curcio et al. have proposed that lipid infiltration may be the result of abnormal RPE lipid metabolism (Curcio C, et al. IOVS 2002;43:ARVO E-Abstract 862). Finally, dietary lipids significantly accumulate within cell membranes of the retina, the RPE, and probably the choriocapillaris endothelium. Increased content of polyunsaturated fatty acids (PUFAs) can render cells more susceptible to lipid peroxidation.16
We sought to provide additional evidence of a relationship of dietary fat and abnormal lipid metabolism with sub-RPE deposit formation. In humans, apolipoprotein B100 (APO B100) is the lipoprotein associated with LDL cholesterol particles.17 Conceptually, the APO B100 molecule is inserted in the phospholipid membrane of the LDL particle and contains the LDL receptor-binding domain, which is involved in the uptake and internalization of plasma LDL by tissues.18 Mice typically express only a splice variant (APO B48), which lacks the binding site for the LDL receptor and limits internalization of LDL particles.19 Transgenic mice expressing the human APO B100 molecule have been developed and demonstrate a marked increase in LDL cholesterol on challenge with a high-fat diet and increased transport of LDL cholesterol into peripheral tissues.20
We tested the hypothesis that the age-related susceptibility of C57BL/6 mice to high-fat-diet–induced BLD was directly related to abnormal lipid biology, but not necessarily to the aging eye. Therefore, we determined the susceptibility of young APO B100 transgenic mice to high-fat-diet–induced BLD, evaluated BLD for the presence of cholesterol and triglyceride deposition, and determined whether pretreatment with the antioxidant 
-tocopherol (vitamin E) reduces the severity and frequency of BLD formation.
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Material and Methods |
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Diet
We have developed a model of sub-RPE deposit formation based on feeding mice a high-fat diet followed by exposure to blue-green light.14 Experimental mice were switched from the regular diet (Diet 5001; PMI Nutrition International Test Diet, Richmond, IN) to the high-fat chow (Diet 5015; PMI Nutrition International Test Diet) at the beginning of the experiments to allow tissue distribution of dietary lipid. The high-fat diet more than doubled the relative percentage of calories from fat but maintained the same number of calories per gram of food (by reducing the percentage of calories from carbohydrates). Dietary fat was increased from 11% of total calories to 25%, equally divided between increased saturated and unsaturated fat. The diets contained the following constituents: 64% carbohydrates, 30% protein, 6.5% total fat, 3.6% unsaturated fat, 1.2% linoleic acid, and 0.05 IU/g vitamin E in the regular chow and 55% carbohydrates, 18% protein, 26% total fat, 8.2% unsaturated fat, 2.5% linoleic acid, and 0.04 IU/g vitamin E in the high-fat chow.
Experimental Model for Sub-RPE Deposits
Briefly, in the right eye, a repetitive exposure to nonphototoxic levels of argon laser 488 nm blue-green light was added to induce transient RPE oxidant production 4 weeks after starting the high-fat chow. Seven 5-second exposures to 20 mJ of argon laser were given 2 to 3 days apart over a 2-week period. The delivery system used a probe producing a 200-µm spot. Retinal illumination required a specially designed biconcave contact lens to neutralize the optical power of the natural lens and to enlarge the retinal spot size. The high-fat chow diet was continued for three additional months, after the 2-week exposure to blue-green light. Then, mice were killed and the eyes immediately removed for transmission electron microscopy (TEM) with special lipid staining.
Experimental Protocol
To determine the effect of plasma hyperlipidemia in the formation of BLD, the deposit model was performed in wild-type C57BL/6 (n = 10) and transgenic APO B100 mice (n = 10). The effect of aging shown in previous work was eliminated in this experiment by choosing mice of a young age (2 months). Another group of 2-month-old APO B100 mice (n = 10) were given vitamin E (90 IU twice a week, subcutaneously) to investigate the protective effect of this vitamin on lipid peroxidation after a diet high in PUFAs and exposure to blue-green light. Control animals for this last experiment were given soybean (vehicle) using the same route of administration and dose.
Serum Levels of Cholesterol and Triglycerides
Total plasma cholesterol and triglycerides were measured by collecting 0.8 mL of whole blood in heparin by cardiac puncture at the time of euthanasia and submitting the samples for analysis by a chemistry analyzer (Beckman Instruments, Fullerton, CA). Sub-RPE deposit severity was correlated with plasma cholesterol and triglyceride levels. For the studies to correlate plasma lipid levels with BLD severity, we also included BLD severity scores from 3- to 6-month-old APO B100 mice and old wild-type C57BL/6 mice (n = 6 or 7 per age group).
Histology and TEM
Mice were killed by anesthetic overdose and perfused with saline followed by a mixture of 3% glutaraldehyde and 2% paraformaldehyde. The eyes were immediately enucleated and the corneas removed and fixed overnight in 3% glutaraldehyde and 2% paraformaldehyde in PBS (0.1 M, pH 7.3). The lens was removed, and the posterior segment (retina, choroid, and sclera) was quadrisected to contain the perioptic nerve portion at the apex and ciliary body at the base. The superotemporal quadrant of retina, choroid, and sclera was sectioned for electron microscopy. The tissue was fixed in 1% osmium tetroxide for 1 hour, rinsed in PBS, dehydrated in EtOH, and embedded in Spur’s resin. Thick and ultrathin sections (0.7–1.0 µm) were cut on a microtome (MT-2 Porter Blum; Sorvall, Newtown, CT). Thick sections were stained with toluidine blue and examined by light microscopy. Ultrathin sections were stained with 4% uranyl acetate and lead citrate and then examined with a transmission electron microscope (model CX-100; JEOL, Tokyo, Japan).
Semiquantitative Grading System
For each specimen, a single cross section was examined, and low-power transmission electron micrographs were made of the entire section from the perioptic to the ciliary body portion (usually approximately 10 micrographs). For semiquantitative scoring, two to four representative high-power micrographs were made of each low-power section by an individual unaware of the experimental conditions. The high-power micrographs were graded by two independent examiners for the presence and severity of BLD. A severity score of 0 to 15 points was determined for each section by summation of the median scores of all the micrographs from a section containing one of the five different categories of abnormality (from 0 to 3 points for each): continuity of BLD, maximum thickness of BLD, nature of deposit content (homogeneous, banded structures, membranous debris, and granular material), presence of BrM abnormalities, and assessment of other choriocapillaris endothelial damage or invasion. BrM thickness was also directly measured in three different standardized locations in each image and averaged to provide a mean score for that micrograph. The mean of 10 high-power micrographs was used to assign an average BrM thickness for an individual specimen. Groups were compared by determining the median values, and the Mann-Whitney test was used for statistical analysis of the differences. In addition, the frequency of BLD was determined using two different criteria. "Any BLD" was defined as the presence of any discrete focal nodule of homogenous material of intermediate electron density between the RPE cell membrane and BrM in at least one micrograph within a section. "Moderate BLD" was defined as the presence in at least three micrographs of the following: continuous BLD extending under two or more cells, deposit thickness equaling 20% or more of RPE cell cross-sectional thickness, and/or the presence of any banded structures within the BLD.
Lipid Staining
Eyes and mesentery (used as control tissue) were removed and placed in fixative overnight. The eye was dissected, and the retina was quadrisected as described previously. One of two techniques was used for lipid staining before eyes were embedded for sectioning and stained for TEM. Filipin staining was used for unesterified cholesterol. Filipin (5 mg) was dissolved in 1 mL of dimethylformamide and then diluted to 100 mL in PBS. Tissue for analysis was incubated in this solution for 30 minutes, washed, fixed in 2.5% glutaraldehyde, and postfixed in 1% OsO4. OTO was used to visualize neutral lipid deposition, mostly triglycerides. Tissue to be analyzed was incubated for 30 minutes in 2% OsO4, washed in PBS buffer, and incubated in 1.5% aqueous thiocarbohydrazide for 5 minutes. Then, the tissue was washed and reincubated in OsO4 for 30 minutes.
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Results |
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-tocopherol twice weekly for 4.5 months prevented the accumulation of deposits, compared with untreated mice (Fig. 6) .
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-tocopherol demonstrated a reduced frequency and severity of deposits (score, 0.5). BrM thickness was measured in the latter study. There was a statistically significant difference between the control and the vitamin E–treated group (1.16 ± 0.21 µm vs. 0.69 ± 0.09 µm, P < 0.05 by t-test). These data suggests that the antioxidant properties of -tocopherol appear to prevent blue-green-light–mediated formation of deposits.
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Discussion |
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The barrier hypothesis proposes that deposit formation is secondary to a defect in transport through BrM caused by the accumulation of lipids.25 26 Research has established the age-related accumulation of various lipids, including phospholipid, triglycerides, and unesterified cholesterol, within BrM to be part of normal aging—perhaps more severe in the presence of AMD.6 8 9 Nevertheless, the source of these lipids remains controversial. Originally, it was thought that BrM lipid accumulation was the result of deposition from circulating LDL cholesterol or other plasma sources. Recently, however, Curcio et al. have postulated that BrM lipids may in fact be derived from the RPE (Curcio C, et al. IOVS 2002;43:ARVO E-Abstract 862). Conceivably, these RPE-derived lipids are the product of specific secretion, exocytosis, or blebbing.
In this study, our results demonstrated that transgenic mice with overexpression of APO B100 and increased LDL cholesterol in the blood, are more susceptible to blue-light–induced formation of deposits. Deposits did not form in the absence of blue-light exposure, and they were prevented by pretreatment with the antioxidant -tocopherol. These observations suggest that high-fat diet and blue-light exposure induces oxidant injury, which is related to the formation of deposits. In addition, the data suggest a poor correlation between plasma lipid concentration and deposit formation, and provide no evidence of significant unesterified cholesterol or triglyceride accumulation. Taking these findings together, we believe that they support the hypothesis that defects in hepatic and possibly RPE lipid metabolism in response to dietary lipid challenge play a role in the formation of deposits, but the pathogenesis is much more complex than mere deposition into BrM.
Our failure to detect unesterified cholesterol by filipin and triglyceride deposition by OTO staining does not rule out the presence of both of these lipids within BrM. TEM imaging of deposits is a relatively insensitive way to characterize lipid deposition, and thus more sophisticated techniques using other fluorescent staining methods or direct extraction and HPLC may demonstrate the quantitative increase of deposition of lipids in this model. Also, the processing of tissue may result in the loss of some lipids from the specimens. Nonetheless, our data also failed to show a pronounced accumulation within BLD.
Other genetically manipulated mouse models of hyperlipidemia, especially with APO E abnormalities, have given contradictory results regarding deposit formation. Dithmar et al.27 showed the APO E null mice did not show formation of BLD or degeneration of the RPE, but electron-lucent particles were present in BrM, consistent with a mild form of BLD. Kliffen et al.12 showed that typical BLD developed in mice expressing mutant APO E3 after consuming a fat diet, without light exposure. APO E is important in hepatic processing of VLDL remnants, and its absence results in increased VLDL in the blood.17 18 Also, APO E isoforms may contribute to transport and solubilization of extracellular lipids in tissues and therefore may play a role in BrM lipid transport.12 28
Our study differs significantly from these others. APO B100 is the normal ligand for endothelial uptake of LDL cholesterol particles, and it is absent in normal mice.19 LDL cholesterol particles are the physiologic means by which triglycerides, fatty acids, and lipophilic antioxidants are transported into tissues. Their content is strongly influenced by dietary intake and hepatic processing of chylomicrons. Overexpression APO B100 in the mouse mimics the physiology of moderate hypercholesterolemia in humans and facilitates endothelial uptake of LDL particles.20 21 The susceptibility of young APO B100 transgenic and C57Bl/6 mice to BLD rules out age dependence as an absolute necessity in deposit formation, but confirms the complicated relationship between plasma lipid, antioxidants, and formation of sub-RPE deposits.
The determination of a role for -tocopherol in preventing sub-RPE deposits should be considered preliminary. In particular, we used subcutaneous administration of vitamin E to guarantee adequate sustained doses. After oral intake, vitamin E is transported to the liver by chylomicrons and incorporated into LDL particles by specific vitamin E transport proteins.29 In our experiments, vitamin E assimilation would first necessitate drainage of the lymph and gaining access to lymph nodes and macrophage-rich tissues before entry into the circulation with subsequent uptake by the liver. Conceivably, our administration method may have affected anti-inflammatory and antioxidant mechanisms more than after oral ingestion. Future studies will more specifically address the protective mechanism of vitamin E in this model. Of note, the Age-Related Eye Disease Study (AREDS), in which a cocktail of antioxidants including vitamin E was used to treat patients with dry macular degeneration, failed to demonstrate that antioxidants reverse or prevent the progression of drusen formation.30 Nevertheless, in this animal model, the treatment was started before the onset of BLD, a stage in which deposits are not detectable. The preventive mechanism of vitamin E against preclinical formation of sub-RPE deposits was not directly tested in this clinical study.
Although we favor the hypothesis that PUFAs are incorporated into the endothelial cell and/or RPE cell membrane where they become substrates for light-induced lipid peroxidation, we recognize that other mechanisms may also play a role in this process. Studies are under way in our laboratory, testing in vitro and in vivo the mechanisms involved in the response-to-injury paradigm in which lipid peroxidation and immune mechanisms have an important role in the pathogenesis of drusen formation. Because LDL lipoproteins active in the delivery of fatty acids, phospholipids, antioxidants, and cholesterol to peripheral tissue, changes in LDL content may be more important than increases in LDL particle concentrations.
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Footnotes |
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Submitted for publication August 20, 2003; revised September 3, 2003; accepted September 4, 2003.
Disclosure: D.G. Espinosa-Heidmann, None; J. Sall, None; E.P. Hernandez, None; S.W. Cousins, None
Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2000.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Scott W. Cousins, Department of Ophthalmology, William L. McKnight Vision Research Center, 1638 NW 10th Avenue, Miami, FL 33136; scousins{at}med.miami.edu
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). (B) Mesenteric adipocytes demonstrated significant punctuate uptake of OTO stain (arrowhead), consistent with high levels of triglycerides in the intestines used as the positive control CC, choriocapillaris. Magnification: (A) x25,000; (B) x18,000.