Cigarette Smoke–Related Oxidants and the Development of Sub-RPE Deposits in an Experimental Animal Model of Dry AMD

Abstract

purpose. Oxidative injury to the retinal pigment epithelium (RPE) has been proposed to be an important injury stimulus relevant to the accumulation of subretinal deposits in age-related macular degeneration (AMD). Cigarette smoking is a major risk factor for AMD, and cigarette smoke-related tar contains high concentrations of a potent oxidant, hydroquinone (HQ). This study was an investigation of the effects of cigarette smoke (CS) and HQ in the development of sub-RPE deposits in an experimental mouse model.

methods. Sixteen-month-old C57BL/6 female mice were fed a high-fat diet (HFD) for 4.5 months. Mice were divided into two major experimental groups, one to examine the effects of cigarette smoke and one to study the effects of a defined cigarette smoke component such as HQ. In the first group, mice eyes were exposed to blue-green light (positive controls) or to whole cigarette smoke. A third group with no intervention served as the negative control. In the second experimental group, animals received a purified diet with HQ (0.8%) with low or high fat content for 4.5 months. Mice in both groups were euthanatized at 4.5 months and eyes processed for transmission electron microscopy.

results. As previously demonstrated by our laboratory and others, most mice fed an HFD without other oxidant exposure demonstrated normal morphology or, in a few cases, small nodular basal laminar deposits. Eyes of mice exposed to whole cigarette smoke or to HQ in the food demonstrated a variable degree of basal laminar deposits and diffusely thickened Bruch’s membrane. The choriocapillaris endothelium was variably hypertrophic.

conclusions. Exposure to cigarette smoke or the smoke-related redox molecule, HQ, results in the formation of sub-RPE deposits, thickening of Bruch’s membrane, and accumulation of deposits within Bruch’s membrane. Smoke-related oxidants may be another oxidative injury stimulus to the choriocapillaris and RPE, and may explain the association between cigarette smoking and early AMD.

Age-related macular degeneration (AMD) is the most important cause of vision loss among the elderly in Western nations. ¹ The initial stage of the disease is characterized by the accumulation of lipid-rich deposits under the retinal pigment epithelium (RPE). ^{2 3 4} Several different deposit types have been associated with AMD, including those between the RPE and its basement membrane (basal laminar deposits; BLD) as well as those within Bruch’s membrane (BrM) itself (BLDs and nodular drusen). ² The pathogenesis of the different deposit types remains controversial, and several different pathogenic paradigms have been proposed. ^{5 6 7 8 9 10} One paradigm, called the response to injury hypothesis, proposes that the RPE cell is the target for specific injury stimuli that results in responses causing deposit accumulation. ^{11 12 13} Particularly, we have proposed that a variety of both endogenous and exogenous oxidants can directly interact with the RPE, to result in cell membrane blebbing and dysregulated extracellular matrix production that causes accumulating deposits. ^{11 12 13}  

Our laboratory and others have demonstrated that various types of oxidant challenges to the RPE can result in sub-RPE deposits. ^{10 11 12 13 14 15} We have shown that in aged animals fed diets rich in polyunsaturated fats and exposed to blue light, BLD and thickening of BrM develop. ¹³ Gottsch et al. ¹⁶ have shown that eyes of mice that have light-induced photoactivation of protoporphyrin secondary to hepatic toxicity from the drug griseofulvin, also exhibit deposits. Hahn et al. ¹⁷ have recently shown that accumulation of extracellular iron in mice genetically defective for two iron transport proteins, ceruloplasmin and hephestin, can also cause deposits and RPE degeneration. Thus, these results raise the hypothesis that a final common pathway can produce deposits independent of the initiating oxidant. 

Epidemiologic studies have indicated that cigarette smoke is the single greatest environmental risk factor for both dry and wet AMD. ¹⁸ Cigarette smoke contains more than 4000 potentially toxic substances, but especially it contains abundant potential oxidants. ^{19 20} Particularly, tar within cigarette smoke particulate matter contains numerous pro-oxidant compounds that belong to the quinone family. ^{21 22 23 24} Hydroquinone (HQ), a benzene derivative, is the most abundant quinone in cigarette tar. ^{21 22 23 24 25} High levels of HQ can be detected in the plasma and urine of smokers, indicating that it entered the circulation from the lungs and could interact with cellular targets throughout the body. ²⁵ HQ can diffuse intracellularly and compete with the normal substrate of mitochondrial oxidases associated with electron transport. In the mitochondrion, HQ undergoes redox cycling with its corresponding semiquinone radical resulting in the overproduction of superoxide, which in turn damages mitochondrial membranes and leaks into the cytoplasm. ^{26 27} In the cytoplasm, superoxide can be converted into hydroxyl anion and hydroxyl radicals, which mediate a variety of biochemical reactions such as protein oxidation and lipid peroxidation. ^{23 26 27} We have demonstrated that the addition of HQ to cultured RPE cells results in the induction of cell membrane blebbing, dysregulation of MMP-2, and major over- and underexpression of a wide range of genes involved in the regulation of the extracellular matrix. ^{14 15}  

In this study, we sought to test the hypothesis that mice that were exposed to whole cigarette smoke, or specifically to oral HQ, would develop sub-RPE deposits and other evidence of outer retinal damage. 

Methods

Mice

C57BL/6 female mice of 16 months of age were purchased from the National Institute of Aging (Bethesda, MD). This mice strain and age group were chosen because when fed a high-fat diet (HFD), they have plasma hyperlipidemia and sub-RPE deposit of moderate severity when exposed to oxidative stress. The guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research were followed, and the Division of Veterinary Resources approved all experiments. All mice were maintained on 12-hour light–dark cycles, with ambient light maintained by fluorescent lighting, and cages were kept on middle shelves of the cage rack. 

Experimental Protocols

Studies were divided into two protocols. To evaluate the impact of inhaled cigarette smoke, three groups (n = 5/ group) of 16-month-old female mice were selected. One group was exposed to whole cigarette smoke as described later, another group was exposed only to blue light (positive controls), and a third group (negative control) was not exposed to oxidant injury. All mice consumed an HFD, as described elsewhere. ^{11 12 13}  

To evaluate the effect of one important smoke-related pro-oxidant in tar (by which cigarette smoke might be affecting the induction of sub-RPE deposits), HQ was given orally in food to two groups (n = 5/group) of 16-month-old female mice. The control group received a regular purified diet (diet 1, described later), whereas the other group received a purified HFD (diet 2, described later). Right eyes were exposed to blue light, and the left eyes were not. 

At the end of the experimental period, the mice were killed and the eyes immediately removed for transmission electron microscopy (TEM). Semiquantitative grading of deposit severity was performed as previously described and described later herein. ^{11 12 13}  

Cigarette Smoke Machine and Protocol

Mice were exposed to inhaled cigarette smoke (mainstream and sidestream) by use of a custom-built microprocessor-controlled cigarette-smoking machine (Model TE-10z; Teague Enterprises, Davis, CA) in which whole cigarette smoke is pumped into sealed chambers containing the mice. After initial acclimatization for 2 weeks to increasing doses of cigarette smoke, mice were exposed to a high concentration of cigarette smoke daily for 3.5 months excluding Saturdays and Sundays, beginning 1 month after starting the HFD. The cigarette smoke was generated from Kentucky Research Cigarettes 2R4F Reference Cigarettes (Tobacco Health Research Institute, University of Kentucky, Lexington, KY), which have a declared content of 44.6 mg total suspended particulate (TSP) and 2.45 mg nicotine each after smoking. The cigarettes were kept for a minimum of 24 hours before their use in a standardized atmosphere humidified with a mixture of 60% glycerol and 40% H₂O (i.e., hygrometer = 60% humidity). Based on preliminary experiments, we decided to burn 10 2R4F cigarettes at one time 2 h/d. This accounted for a whole-body exposure to smoke generated by 6300 cigarettes. Under these conditions, the TSP and carbon monoxide (CO) in the exposure chambers were 250 mg/m³ and ∼600 to 750 ppm, respectively, for the duration of the experiment. 

Blue-Light Exposure

Briefly, in the right eye, a brief repetitive exposure to nonphototoxic levels of argon laser 488 nm blue-green light (Model 910A Argon Laser; Coherent, Palo Alto, CA) was delivered to induce transient RPE oxidant production 4 weeks after starting the HFD. ¹³ 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 uses a probe producing a 200-μm spot, and the energy intensity was calibrated before each application by a photometer (210 Power Meter; Coherent) held at a standardized distance from the probe. Retinal illumination required a specially designed biconcave lens (focal distance, −6.5 mm at 670 nm; 153.8 dioptric power) to neutralize the optical power of the natural lens and to enlarge the retinal spot size. 

HQ Diet

Mice were fed one of two purified diets based on a basal purified synthetic diet that provides all the essential nutrients to support maintenance, growth, gestation, and lactation in laboratory mice (Purina Basal Diet 5755; PMI Nutrition International Test Diet, Richmond, IN) for 4.5 months. A purified diet was chosen to incorporate HQ as part of the mice diet. Diet 1 (product 493357; Purina Test Diets) consisted of 19% protein, 4.50% fat, 4.29% fiber, 66.05% carbohydrates, 1.8% linoleic acid, 0.40% oleic acid, 0.40% palmitic acid, and 1.05% lauric acid. Diet 2 (product 493350; Purina Test Diets), consisting of 19% protein, 18% fat, 4.29% fiber, 52.55% carbohydrates, 7.14% linoleic acid, 1.69% oleic acid, 1.47% palmitic acid, and 4.20% lauric acid. All diets had 0.8% of HQ, 0.5 ppm vitamin K, and 0.02% of taurine. Diet 1 had a normal content of fats and diet 2 had a high content of both saturated and nonsaturated fatty acids, resembling the diets we normally used as rodent chows for the experimental model for sub-RPE deposits. 

Serum Levels of HQ and Cotinine

Blood (800 μL) was removed by cardiac puncture and serum was obtained for determination of HQ concentration measured using gas chromatography (National Medical Services, Willow Grove, PA). Also, cotinine levels were measured at the time of death of smoke-exposed mice with an enzyme immunoassay (Cotinine MicroPlate, catalog no. 1124EA; OraSure Technologies, Inc., Bethlehem, PA). 

Histology and TEM

The 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 in 3% glutaraldehyde and 2% paraformaldehyde in PBS (0.1 M, pH 7.3) overnight. 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 the retina, choroid, and sclera was submitted for electron microscopic sectioning. The tissue was fixed in 1% osmium tetroxide for 1 hour, rinsed in PBS, dehydrated in EtOH and then embedded in Spurr’s resin. Thick (0.7–1.0 μm) and ultrathin sections (0.1 μm) were cut on a microtome (Porter Blum MT-2). 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 CX-100 transmission electron microscope (JEOL, Tokyo, Japan). 

Semiquantitative Grading System

For each specimen, a single cross-section was examined, and low-power transmission electron micrographs (i.e., magnification, ×7200) were made of the entire section from perioptic to ciliary body portion (usually, approximately 10 micrographs). Then, one representative high-power micrograph (i.e., magnification, ×25,000) was made from each low-power section by an individual unaware of the experimental conditions and used for semiquantitative scoring. 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 on each of five different categories of abnormalities (from 0 to 3 points for each): continuity of BLD (score: 0, no BLD16-month-old; 1, occasional BLD16-month-old i.e., focal nodule; 2, BLD16-month-old extending under less than two RPE cells; and 3, BLD16-month-old extending under two or more RPE cells); maximum thickness of BLD (score: 0, no BLD; 1, flat BLD16-month-old; 2, deposit thickness <25% of RPE cell cross-sectional thickness; and 3, deposit thickness ≥25% of RPE cell cross-sectional thickness); nature of deposit content (score: 0, no BLD16-month-old; 1, homogeneous BLD16-month-old; 2, any banded structures within BLD16-month-old; and 3, three or more banded structures within BLD16-month-old); presence of BrM abnormalities (score: 0, no abnormalities; 1, collagenous thickening, no deposit; 2, thickening with circular profiles or nonspecific debris; and 3, presence of basal linear deposits represented as banded structures, granular material or membranous debris); and assessment of other choriocapillaris endothelial damage or invasion (score 0, no alterations; 1, loss of fenestrations; 2, loss of fenestrations and thickening; and 3, choriocapillaris invasion into BrM). BrM thickness was also directly measured in three different standardized locations in each image, and then averaged to provide a mean score for that micrograph. The mean of 10 micrographs was used to assign and “average” BrM thickness for an individual specimen. 

Groups were compared by determining the mean and standard deviations. Kruskal-Wallis ANOVA and Mann-Whitney test were 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 equal to or greater than 25% of RPE cell cross-sectional thickness, or the presence of any banded structures within the BLD. Differences in the relative frequency were tested using χ² test. 

Results

First, we confirmed that cigarette smoke–exposed mice had increased blood levels of cotinine, the major metabolite of nicotine present in tar. Serum cotinine levels in smoke-exposed mice were 63.7 ± 2.7 ng/mL, whereas control animals demonstrated unmeasurable levels. These results are consistent with blood levels recorded in habitual cigarette smokers. ^{28 29 30 31 32} Similarly, HQ-fed mice demonstrated serum levels of 11.3 ± 0.4 ng/mL, whereas mice fed normal diets had unmeasurable levels. Increase lipidemia was achieved in mice on an HFD with similar levels of plasma cholesterol and triglycerides, as reported previously by our group, especially in old animals. ^{11 12 13}  

Effect of Blue Light and Cigarette Smoke in Sub-RPE Deposits and Bruch16-Month-Old Membrane Changes

We evaluated the development of sub-RPE deposits in the different groups exposed to blue light, cigarette smoke, or neither, by using TEM. As expected, 16-month-old mice not exposed to blue light or cigarette smoke but fed HFD showed a high frequency of “any BLD, ” typically characterized as very mild, focal nodular deposits of homogeneous material between the plasma and basement membrane of the RPE in at least one photomicrograph (Fig. 1) . None of the eyes in this group demonstrated “moderate BLD. ” In general, old animals that received a normal diet or an HFD showed normal morphology of BrM and choriocapillaris endothelium. 

We have used ocular exposure to blue-green light with a brief seven-exposure protocol repeated over a 2-week period to serve as a presumed oxidant stressor to induce sub-RPE deposits. ^{11 12 13} Similar to past results, eyes from old mice fed HFD and exposed to blue light demonstrated pathologic changes in the RPE and BrM characterized by “moderate BLD.” The deposits were often continuous under several adjacent RPE cells, but occasionally they were greater than 20% of the RPE height, demonstrated by patches of banded material and/or granular material (Fig. 2) . BrM was often diffusely thickened, and these changes were significantly greater than animals only in the HFD (Table 1) . Choriocapillaris abnormalities, such as thickened endothelium with loss of fenestration and protrusion into BrM, were frequent findings in this group. Our grading system demonstrated that 80% of eyes exhibited moderate BLD with a mean severity score of 7.8 ± 3.3, significantly greater than the severity score of 1.8 ± 1.2 in the animals not exposed to light but receiving an HFD (Table 1) . 

We evaluated cigarette smoke as another potential oxidant stressor that induces BLD formation. Whole-body exposure of mice to a daily mixture of mainstream and sidestream cigarette smoke was achieved. Sub-RPE deposits were a frequent finding and similar to the blue light model described earlier. In general, deposits were thick and contained abundant patches of banded structures and granular material (Table 1 , Fig. 3 ). Smoke-exposed mice developed 60% of “moderate” BLD (Table 1) . The severity score of 6.6 ± 3.9 was statistically greater than that in the control mice, but no different in severity from the blue-light–exposed mice. 

Some specimens demonstrated increased cellular infiltration within choriocapillaris lumens or within the choriocapillaris pillars, as shown in Figures 3A and 3B . These cells may represent monocytes, but their absolute identity is unknown. Also, the choriocapillaris endothelium was abnormal with increased thickening, loss of fenestrations, and cellular invasion of processes into BrM (Fig. 3) . 

Effect of Chronic HQ Exposure on Sub-RPE Deposits and Bruch16-Month-Old Membrane Changes

We hypothesize that circulating oxidants derived from tar in cigarette smoke were responsible for the deposit formation in smoke-exposed mice. Specifically, we added the pro-oxidant benzene derivative, HQ, into the normal diet or modified HFD, as a surrogate for whole cigarette smoke. The results showed moderate BLD in both regular and HFD groups (Fig. 4) . The BLD showed characteristics similar to those developed in mice exposed to cigarette smoke, including electron-dense deposits with patches of banded structures or granular material and associated with degenerating membranous folds. Other alterations were thickened endothelium, loss of fenestration, and cellular invasion of the choriocapillaris into Bruch16-month-old membrane, similar to the findings of cigarette smoke or blue-light–exposed mice (Tables 1 2 ; Figs. 2 3 5 ). The frequency of moderate BLD was 100% independent of the fat content of the diet. Also, the mean severity score was similar in mice receiving HQ, irrespective of fat content (Table 2) . Additional findings in BrM were observed more frequently in HQ-fed mice than in mice exposed to smoke. The Bruch16-month-old membrane was thickened, with empty profiles, coiled membranous debris, membrane-coated bodies, and banded structures (Fig. 5) , typical of those described in some human AMD specimens. RPE cells also showed an increase in lucent bodies, both in cigarette smoke animals and in HQ-fed mice. 

Discussion

In this study, we observed that mice that were chronically exposed to whole cigarette smoke showed development of sub-RPE deposits and BrM thickening consistent with changes in early AMD. Findings were of a magnitude similar to those previously observed with exposure to blue light. We were able to replicate the pathologic changes by feeding mice the pro-oxidant, HQ in food, which resulted in similar pathologic changes, irrespective of the fat content of the diet. These observations support the hypothesis that oxidant injury can initiate a process resulting in deposit formation. However, we did not observe evidence of thick drusen, RPE degeneration, or spontaneous choroidal neovascularization, to suggest that oxidant injury alone, as evaluated in this study, leads to the progressive manifestations of severe AMD. 

Cigarette smoking is the single greatest environmental risk factor for the incidence and prevalence of AMD. ¹⁸ Cigarette smoke contains numerous potential oxidants including nitric oxide, carbon monoxide, and many other toxic chemical moieties. ¹⁹ However, we believe that HQ is particularly relevant because of its relatively high plasma and urine concentration in smokers. ²⁵ Of note, HQ is also a prominent chemical contaminant in automobile exhaust, industrial pollution, and the production of plastics. ^{25 33 34 35} Many individuals living Western life styles have detectable levels of HQ in their blood. ²⁵ Therefore, we believe that cigarettes are a “drug delivery system” for high concentrations of HQ, and the findings observed in this study may be generalizable to nonsmokers. 

The morphologic changes observed in this study were similar to those that we have observed in previous studies in which blue-green light exposure was used after an HFD was fed to aged mice, young mice transgenic for apolipoprotein B100 (which develop hypercholesterolemia), or middle-aged female mice that had been rendered surgically estrogen depleted. ^{11 12 13} Moderately thick BLD contain typical homogeneous electron-dense material with occasional banded structures consistent with long-space collagen. In addition, BrM generally was moderately thickened, often containing vesicular structures and other inclusions. Not infrequently, evidence of endothelial damage with protrusion of endothelial processes into BrM was observed. Although these changes do not represent authentic progressive AMD, they do represent the morphologic features of the early manifestations of AMD. ²  

We have previously hypothesized that the RPE is the key target cell in deposit formation. Specifically, we proposed that HQ and other oxidants trigger a specific cellular process called nonlethal blebbing. ^{10 14 15 25} In preliminary experiments, we have demonstrated that RPE cells that are repeatedly exposed to HQ demonstrate blebbing of cell membrane material that ultimately accumulates under the basal surface as sub-RPE deposits (Cousins SW et al. IOVS 2003;44:ARVO E-Abstract 1619). In other preliminary experiments, we have also demonstrated that mice that received subconjunctival injections of HQ exhibit a rudimentary form of BLD, often demonstrating small vesicular bleblike structures (Reinoso MA et al. IOVS 2005;46:ARVO E-Abstract 3016). 

Although we favor the RPE as primary target of HQ, it is likely that other cells in the outer retina may also be affected. For example, the choriocapillaris endothelium was abnormal with increased thickening, loss of fenestrations, and cellular invasion of processes into the BrM. Gottsch et al. ¹⁶ suggested that oxidant injury to the endothelium may be the initial insult, somehow resulting in sub-RPE deposit formation. These endothelial alterations may contribute to AMD by affecting oxygen and nutritional/waste transport across BrM (the barrier hypothesis). Furthermore, photoreceptor inner segments contain a high concentration of mitochondria and high levels of unsaturated fatty acids. Therefore, these cells may also be targets of oxidative injury (i.e., blue light, HQ), which will ultimately contribute to the pathogenesis of AMD-related sub-RPE deposits. ¹⁰ Future experiments will determine the specific mechanism for oxidant-induced deposits. 

The role of the HFD is not entirely clear. In our current and previous studies, including those with mice transgenic for apolipoprotein B100, an HFD alone failed to induce moderate BLD in the absence of an oxidant challenge. ^{13 18} In contrast, Bowes Rickman et al. have recently demonstrated that very aged transgenic mice expressing the human apolipoprotein E4 polymorphism and who were fed a high saturated fat diet, did show more severe degenerative changes, including the spontaneous onset of CNV in some eyes. ⁵⁵ The role of dietary fat in AMD remains unclear, but we have postulated that polyunsaturated fats in the diet may concentrate within the RPE cell membrane, where the polyunsaturated fatty acids become substrates for lipid peroxidation. ^{10 13} This mechanism appears to be important for light-induced deposits, but apparently is less important for HQ. The differences may relate to different biochemical mechanisms for light- and HQ-induced blebbing. For example, blue light is believed to produce singlet oxygen, a very potent mediator of lipid peroxidation. ^{10 36 37} HQ stimulates production of superoxide anion, which is thought to induce blebbing through kinase-dependent disassembly of actin filaments, a process not involving lipid peroxidation. ^{26 27 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54} We did not specifically test for the different biochemical pathways in this study. 

Limitations of our study in relation to physiologically levels of HQ must be considered. We are aware of no recent publications using modern analytical methods to quantify plasma HQ levels in humans. Most modern epidemiologic studies have converted to the measurement of urine levels, not applicable to mice. The older epidemiologic literature measured protein-bound HQ, and therefore missed free HQ (probably 90% is free). Protein-bound HQ concentration is approximately 0.2 to 0.5 μg/dL in smokers. In this study, we chose levels in food designed to achieve approximately 10- to 20-fold greater total plasma levels. Unfortunately, we did not measure plasma HQ in the smoke-exposed mice. It is possible that high HQ levels achieved in this study may have overwhelmed an interrelationship with dietary fat that may have become apparent at lower doses. Future studies will address HQ dose dependence. 

Taken together, the observations presented in this study, along with those previously published by our group, ¹⁷ by Gottsch et al., ¹⁶ and by Hahn et al., ¹⁷ indicate that different kinds of oxidant stressors can induce sub-RPE deposits. However, only in the Hahn et al. 16-month-old model of mice genetically null for two iron transport proteins, ceruloplasmin and hephestin (causing extracellular iron accumulation), was significant RPE degeneration observed. ¹⁷ The implication is that additional mechanisms must be superimposed on mild oxidant injury in vivo, to convert BLD and mild linear deposits into full-blown drusen and RPE degeneration. Whether the “second hit” is merely a dose–response to additional oxidant exposure, or instead requires the addition of another cofactor (e.g., genetic, systemic, or environmental factor) remains to be determined. 

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, April, 2004.

 Supported by National Eye Institute Grant R01 EY 015249, the Flight Attendant Medical Research Institute (FAMRI) Award, and the Miami Veterans Affairs Merit Award Grant.

 Submitted for publication June 8, 2005; revised August 10 and October 16, 2005; accepted December 22, 2005.

 Disclosure: D.G. Espinosa-Heidmann, None; I.J. Suner, None; P. Catanuto, None; E.P. Hernandez, None; M.E. Marin-Castano, None; S.W. Cousins, None

 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.

 

Figure 1.

 

TEM of the outer retina and choroid of a 16-month-old female mouse. (A) Outer retina and choroid of a mouse fed a regular diet with no exposure to blue light or cigarette smoke. Specimen showed very mild, subtle sub-RPE deposits (arrowheads). The BrM and choriocapillaris were normal. (B) Outer retina and choroid of a mouse fed an HFD with no exposure to blue light or cigarette smoke. Material of similar electron density has accumulated between the plasma and basement membrane of the RPE (arrowheads). The BLD changes are more pronounced with the HFD but the overall severity score is mild. RPE, retinal pigment epithelium; CC, choriocapillaris; BrM, bruch16-month-old membrane; BLD, basal laminar deposit. Magnification, ×25,000.

Figure 2.

 

TEM of the outer retina and choroid from a 16-month-old female mouse fed HFD and exposed to blue light (seven 5-second flashes of 20 mJ of argon light 2 to 3 days apart over a 2-week period). (A) Specimen shows thick sub-RPE deposits (BLD) of moderate severity. (B) Magnified view of the boxed section in (A) showing patches of banded material (arrowheads) in sub-RPE deposits. There was also a thickening of BrM with the choriocapillaris endothelium sending protrusions into the BrM ( ). Abbreviations are as in Figure 1 . Magnification: (A) ×7,200; (B) ×25,000.

Table 1.

 

The Effect of Blue Light and Cigarette Exposure on BLD Severity in Mice

n	Frequency of “Any BLD”					Frequency of “Moderate BLD”					Mean Severity Score					Mean BrM Thickness					EC Invasion
		G I	G II	G III	G I	G II	G III	G I	G II	G III	G I	G II	G III	G I	G II	G III
1	Yes	Yes	Yes	No	Yes	Yes	2.4	9.8	12.67	4.9	8.5	18.58	No	No	No
2	Yes	Yes	Yes	No	No	No	0.43	5.2	6.83	5.52	12.67	9.8	No	No	No
3	Yes	Yes	Yes	No	Yes	Yes	0.58	11.83	4.43	5.22	7.1	7.46	No	Yes	Yes
4	Yes	Yes	Yes	No	Yes	Yes	3.07	3.83	6.57	4.76	5.92	7.35	No	No	Yes
5	Yes	Yes	Yes	No	Yes	No	2.36	8.17	2.29	3.52	9.67	13.18	No	No	No
	100%	100%	100%	0%	80%	60%	1.77 ± 1.19	7.77 ± 3.27	6.56 ± 3.88	4.78 ± 0.77	8.77 ± 2.60	11.27 ± 4.72	0%	20%	40%

 Experimental groups were the following: G1 received high fat diet without oxidant; G2 received high fat diet plus ocular blue light exposure; G3 received high fat diet plus inhaled cigarette smoke. Any BLD: presence of any homogeneous deposit (focal or continuous) between RPE cell membrane and its basement membrane in at least 1 (of 10) micrograph from an individual specimen. Moderate BLD: presence of continuous deposits underlying several cells, banded structures (fibrous long-spacing collagen) and/or thickness more than one quarter cell height in at least three micrographs from an individual specimen. Frequency of “Moderate BLD”: χ²: group 1 versus 2, P = 0.01; group 1 versus 3, P = 0.039; Mean Severity Score: Kruskal-Wallis ANOVA: P = 0.023; Mann-Whitney test: group 1 versus 2, P = 0.009; group 1 versus 3, P = 0.047; group 2 versus 3, P = 0.601. BrM Mean Thickness Score: Kruskal-Wallis ANOVA: P = 0.0068; Mann-Whitney test: group 1 versus 2, P = 0.009; group 1 versus 3, P = 0.009; group 2 versus 3, P = 0.25.

Figure 3.

 

TEM of the outer retina and choroid from 16-month-old female mice fed HFD and exposed to cigarette smoke. (A) Specimen showed a moderately thick sub-RPE deposits (BLD). There was also severe thickening of the BrM and the presence of a cell that may represent a macrophage ( ). (B) Inset: magnified view of BLD16-month-old BrM, and an invading cell that may be a macrophage ( ) in BrM. (C) TEM of the outer retina and choroid from another 16-month-old female mouse fed HFD and exposed to cigarette smoke. Specimen shows moderately thick sub-RPE deposits (BLD). (D) Inset: a magnified view of (C) showing abundant patches of banded structures in BLD. There is also severe thickening of BrM with the accumulation of a homogeneous material ( ). There are also deposits present in BrM (arrowheads) within this homogeneous material with thickening and protrusion of choriocapillaris endothelium (arrows). Abbreviations are as in Figure 1 . Magnification: (A, C) ×7200; (B, D) ×25,000.

Figure 4.

 

TEM of the outer retina and choroid from a 16-month-old female mouse fed HQ for 4.5 months. (A) The mouse received HQ in regular purified diet (low fat) and the specimen shows sub-RPE deposits that correspond to basal laminar deposits (BLD) accumulated between the plasma and basement membrane of the RPE. Patches of banded structures were commonly seen. The choriocapillaris endothelium was thickened ( ). (B) TEM of the outer retina and choroid from a 16-month-old female mouse fed HQ in a regular purified diet showed moderately thick BLD with banded structures ( ). A cellular process from an unknown cell type containing organelles appeared to be invading the BrM (arrow). (C) TEM of the outer retina and choroid from a 16-month-old female mouse fed HQ in a high-fat, purified diet for 4.5 months showed BLD16-month-old in the sub-RPE space with banded structures ( ) similar to the animals in regular, purified low-fat diet. Arrowheads: BLD associated with degenerating membranous folds. BrM seems thickened by the accumulation of a homogeneous material that resembles the one present in cigarette smoke animals. (D) TEM of the outer retina and choroid from another 16-month-old female mouse fed HQ in a high-fat purified diet showed moderate BLD, compatible with a high mean severity score. Abbreviations are as in Figure 1 . Magnification, ×25,000.

Table 2.

 

The Effect of an HQ Diet on BLD Severity in Mice

n	Frequency of “Any BLD”			Frequency of “Moderate BLD”			Mean Severity Score			Mean BrM Thickness			EC Invasion
		G I	G II	G I	G II	G I	G II	G I	G II	G I	G II
1	Yes	Yes	Yes	Yes	8.14	8	7.2	6.15	No	No
2	Yes	Yes	Yes	Yes	9.67	9.75	16.58	9.68	Yes	Yes
3	Yes	Yes	Yes	Yes	6.67	9.67	9.33	6.25	No	No
4	Yes	Yes	Yes	Yes	10.67	9.28	7.08	7.25	No	No
5	Yes	Yes	Yes	Yes	9.14	9.8	5.71	10.97	No	Yes
	100%	100%	100%	100%	8.86 ± 1.53	9.30 ± 0.75	9.18 ± 4.33	8.06 ± 2.16	20%	40%

 Experimental groups were the following: G1 received normal diet plus hydroquinone in the food; G2 received high fat diet plus hydroquinone in the food. See Table Ifor description of Any and Moderate BLD.

Figure 5.

 

TEM of the outer retina and choroid from 16 month old female mice fed HQ for 4.5 months. (A) High-power micrograph reveals “moderate BLD” with numerous oval-shaped banded deposits (white asterisks). BrM contains empty profiles (black asterisk) and several membrane coated bodies (white arrowheads). The choriocapillaris endothelium is thickened. (B) Another high-power micrograph revealed moderate BLD. BrM was invaded by several processes from the choriocapillaris endothelium ( ) without true lumens. Also oval-shaped banded structures (arrowheads) seem to be present in the inner collagenous layer of BrM. Abbreviations are as in Figure 1 . Magnification, ×25,000.