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Accumulation of Cholesterol with Age in Human

Bruch’s Membrane
Christine A. Curcio,1,2 C. Leigh Millican,1 Tammy Bailey,1 and Howard S. Kruth 3

PURPOSE. To determine the cholesterol composition of normal thought to arise from smaller LDL particles (22 nm) by extra-
human Bruch’s membrane and choroid as a function of age and cellular matrix–mediated trapping of LDL, degradation of LDL
retinal location. protein or phospholipid components, and fusion of the remain-
METHODS. Human eyes with grossly normal maculas were pre- ing lipid components (for review, see Ref. 14).
served ⬍4 hours after donor death. Cryosections of retina and Late age-related maculopathy (ARM), or age-related macular
choroid from the macula and temporal equator were stained degeneration,15 is the leading cause of untreatable new vision
with filipin to reveal esterified (EC) or unesterified (UC) cho- loss in elderly individuals,16 –18 but its causes are poorly under-
lesterol (n ⫽ 20, 17–92 years). Filipin fluorescence in Bruch’s stood. The most prominent clinical and histopathologic lesions
membrane was quantified with digital microscopy. Maculas of early and late ARM involve Bruch’s membrane, a thin con-
were prepared for lipid-preserving electron microscopy (n ⫽ nective tissue between the basal surface of the retinal pigment
18, 16 – 87 years) and for ultrastructural analysis after lipid epithelium (RPE) and the choriocapillaris that is traversed by
extraction (n ⫽ 2, 85 and 89 years). Punches of macular molecules essential for photoreceptor and RPE function. The
Bruch’s membrane, 8 mm in diameter, were assayed for cho- only known risk factor for early ARM (i.e., drusen and RPE
lesterol content by enzymatic fluorometry (n ⫽ 10, ⬎70 years). changes) is advanced age.16 –18 It is therefore important to
understand how age-related changes in Bruch’s membrane
RESULTS. EC and UC in Bruch’s membrane increased with age in predispose some individuals for subsequent disease.19,20 In
the macula. EC was sevenfold higher in macula than in periph- Bruch’s membrane of the macula there is a progressive accu-
ery. Sixty percent of total cholesterol was esterified, and mulation of lipids stainable by oil red O21, and Bruch’s mem-
Bruch’s membrane EC was 16- to 40-fold enriched relative to brane/choroid extracts contain phospholipids, triglycerides,
plasma. Solid, 100-nm-diameter particles occupied ⬎30% of the UC, and EC, in descending order of abundance.22 It is thought
inner collagenous layer in eyes ⬎60 years. Cholesterol accu- that lipid accumulation renders Bruch’s membrane increas-
mulated in choroidal arteries and in small age-related drusen. ingly hydrophobic with age, impeding diffusion between the
CONCLUSIONS. Human Bruch’s membrane ages like arterial in- RPE and choroidal vessels.21,23,24
tima and other connective tissues for which plasma lipopro- On the basis of comparison with other tissues, it is logical to
teins are the known source of extracellular cholesterol. Age- hypothesize that the oil red O-positive material that increases
related maculopathy and atherosclerotic cardiovascular disease with age in human Bruch’s membrane is EC-rich particles. This
may share common pathogenic mechanisms. (Invest Ophthal- hypothesis is supported by the presence of numerous small,
mol Vis Sci. 2001;42:265–274) round electron-lucent droplets in adult Bruch’s mem-
brane21,25–28 and preliminary polarizing microscopy studies

I n the body, cholesterol assumes two chemical forms, either


unesterified (or free, UC) or esterified to fatty acids (EC).1,2
Further, three physical forms (oily droplets, membranes, and
demonstrating birefringence patterns consistent with EC.29
However, only a small proportion of the cholesterol recovered
in Bruch’s membrane/choroid extracts is esterified.22 Resolv-
crystals) can be distinguished by their relative proportions of ing the apparent discrepancy between morphologic and bio-
UC, EC, and phospholipid.1,2 Lipids that bind the histochemi- chemical data is important for understanding whether lipid
cal stain oil red O increase with age in normal human connec- deposition in Bruch’s membrane is an ocular manifestation of
tive tissues, including the sclera,3 cornea,4,5 and intima, or a systemic process or a phenomenon unique to the eye. We
inner wall, of large arteries.6 – 8 In these tissues, the oil red therefore examined Bruch’s membrane cholesterol in normal
O-positive material comprises small (60 –200 nm) extracellular donor eyes, using the fluorescent probe filipin to reveal EC and
droplets that are highly enriched in EC relative to UC (69% EC, UC as a function of age and retinal location in tissue sec-
22% UC, and 9% phospholipid).9 –13 There is good evidence tions,9,30 an enzymatic fluorometric assay to determine the
that the source of EC in sclera, cornea, and arterial intima is relative proportions of EC and UC of isolated Bruch’s mem-
low-density lipoproteins (LDL) that are transported from brane,31 and lipid-preserving electron microscopy to identify
plasma into connective tissues. EC-enriched particles are EC-rich droplets.11 Our data indicate that Bruch’s membrane is
highly enriched in EC.

From the Departments of 1Ophthalmology and 2Physiological Op- METHODS


tics, University of Alabama at Birmingham; and 3Section on Experimen-
tal Atherosclerosis, National Heart Lung and Blood Institute, Bethesda, Human Eyes
Maryland.
Supported in part by National Institutes of Health Grants EY06109 Eyes were obtained from human donors ⬍ 4 hours from death. Use of
and P30 EY03039 (Vision Science Research Center, UAB); unrestricted human tissues was approved by institutional review at the University of
funds to the Department of Ophthalmology from Research to Prevent Alabama at Birmingham (protocol number X900525013). Twenty eyes
Blindness, Inc.; and the Alabama Eye Institute. preserved by immersion in 4% paraformaldehyde in 0.1 M phosphate
Submitted for publication July 21, 2000; revised September 22, buffer (PB) after removal of the anterior segment were prepared for
2000; accepted September 29, 2000.
filipin histochemistry (ages 17–22 years, n ⫽ 3; 32–53 years, n ⫽ 6; and
Commercial relationships policy: N.
Corresponding author: Christine A. Curcio, Department of Oph- 61–92 years, n ⫽ 11). Twenty eyes preserved in 2.5% glutaraldehyde
thalmology, University of Alabama at Birmingham, Eye Foundation and 1% paraformaldehyde in 0.1 M PB were used for lipid-preserving
Hospital, Rm H20, 700 South 18th Street, Birmingham, AL 35294-0009. electron microscopy (ages 16 –51 years, n ⫽ 8; 61– 87 years, n ⫽ 10)
curcio@uab.edu or for ultrastructural analysis after lipid extraction (ages 85– 89 years,

Investigative Ophthalmology & Visual Science, January 2001, Vol. 42, No. 1
Copyright © Association for Research in Vision and Ophthalmology 265

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266 Curcio et al. IOVS, January 2001, Vol. 42, No. 1

n ⫽ 2). Ten 4% paraformaldehyde-preserved eyes (⬎70 years) were 0.8% to 8.6% for filipin-labeled sections. Three sections on the same
assayed for cholesterol content. All eyes were inspected internally and slide differed by ⬍3%.
lacked grossly visible chorioretinal pathology in the macula.32
Cholesterol Assay
Cryosectioning
Total cholesterol and UC content was determined with an enzymatic
Seven millimeter-wide samples containing retina, RPE, and choroid fluorometric assay.31 Paraformaldehyde-preserved eyes were used be-
from the macula and periphery were cryosectioned for histochemistry. cause of availability and because formalin fixation minimally changes
Macular samples included the fovea and the temporal half of the optic cholesterol content.34 Punches of macular and peripheral retina, RPE,
nerve head. Peripheral samples included the temporal equator and ora and choroid were obtained with an 8-mm-diameter trephin. The mac-
serrata. Samples were infiltrated in 4:1 and 2:1 30% sucrose and Histo ular punch was centered on the fovea, and the peripheral punch was
Prep medium (Fisher, Norcross, GA) for 30 minutes each, frozen in centered on the temporal equator. The retina was detached, the RPE
⫺70°C isopentane, sectioned at 10 ␮m, collected on glass slides, and was removed with a camel hair brush, and major choroidal vessels
dried at 40 to 60°C. were removed by a combination of brush and fine forceps. Grossly
visible large drusen in peripheral retina were avoided. Small pieces
Filipin Histochemistry were removed before and after dissection to assess the completeness
We localized EC and UC using the fluorescent polyene antibiotic of choroid removal in 1-␮m histologic sections. Bruch’s membrane,
filipin, which binds specifically to sterols and interacts with the 3␤- retina, and choroidal vessels were rinsed with water, placed in sepa-
hydroxy group of cholesterol.30 For EC detection,9 native UC was rate pre-weighed 0.25-ml plastic tubes, lyophilized, weighed, and
extracted from cryosections by two 5-minute rinses in 70% ethanol, shipped overnight on dry ice. Lipids were extracted with chloroform/
native EC was hydrolyzed with cholesterol esterase (EC 3.1.1.13; methanol (2:1, by volume), distributed to sample tubes, dried by
Boehringer Mannheim, Indianapolis, IN) at a concentration of 1.65 heating, and redissolved with 95% ethanol. An assay solution was
units/ml in 0.1 M potassium PB (pH 7.4) for 3 hours at 37°C, and UC added for 30 minutes at 37°C. The assay for total cholesterol used
newly released by the hydrolysis of EC was stained with filipin (5 mg cholesterol esterase to hydrolyze native EC, cholesterol oxidase to
filipin, dissolved in 1 ml dimethylformamide and diluted in 100 ml PB generate H2O2, and peroxidase to catalyze the reaction of H2O2 with
saline; Sigma, St. Louis, MO). Control sections were incubated in the p-hydroxyphenylacetic acid. The resulting fluorescent product excited
enzyme vehicle. For UC detection,33 sections were hydrated and incu- at 325 nm and emitted at 425 nm. For determination of UC, cholesterol
bated for 30 minutes in the above filipin solution without prior extrac- esterase was omitted. EC was the difference of total cholesterol and
tion and hydrolysis. Control sections were incubated in the filipin UC. Cholesterol content was expressed as nmol/g dry weight.
vehicle. All sections were counterstained with Mayer’s hematoxylin.
Electron Microscopy
Photography Glutaraldehyde-preserved maculas were divided horizontally through
Filipin-processed sections were photographed on ASA100 black and the fovea. One tissue block was processed conventionally.28 The other
white film (TMax100; Eastman Kodak, Rochester, NY) using an Op- tissue block was processed by the osmium-tannic acid-paraphe-
tiphot fluorescence microscope (Nikon, Melville, NY) equipped with nylenediamine (OTAP) method to preserve small extracellular lipid
a 420-nm excitation filter, 520-nm barrier filter, and a 60⫻ plan particles.11 Tissues were postfixed with 1% osmium tetroxide in 0.1 M
apochromat objective. Negative film was scanned to create composite sodium cacodylate (NaCaco) buffer (2.5 hours), 1% tannic acid in 0.05
images using Photoshop (Adobe, San Jose, CA). M NaCaco buffer (30 minutes), and fresh 1% para-phenylenediamine
in 70% ethanol (30 minutes). Silver sections were cut from adjoining
Quantification of Filipin Fluorescence block faces with a diamond knife, collected on mesh grids, and stained
with lead citrate. Electron micrographs spanning the full thickness of
The mean fluorescence intensity of Bruch’s membrane in filipin-stained
Bruch’s membrane were taken at 7500 to 8000⫻ and enlarged three
and control sections was determined by digital microscopy. In each
times. Five micrographs per preparation were examined, with two
section (one per condition per eye), three 240-␮m-long segments of
within and three between intercapillary pillars. Other OTAP-processed
Bruch’s membrane were digitized. Sections were viewed on a Leitz
blocks from 10 eyes were sectioned in a horizontal plane.
Orthoplan microscope with a 50⫻ Fluotar oil objective (Wetzlar,
Germany) and a Chromatechnology 83000 (Brattleboro, VT) fluores-
cence filter set (excitation, 346 nm; barrier, 460 nm). Images were Extraction Studies
captured at a resolution of 0.18 ␮m/pixel using a Photometrics CH250 Cryosections were extracted with ethanol and chloroform/methanol
CCD video camera (Roper Scientific, Tucson, AZ) and IP Lab Spectrum (2:1, with 1% hydrochloric acid) before filipin histochemistry and
3.2 software (Scanalytics, Fairfax, VA). All images were exposed for 2 digital microscopy. Tissue blocks of the macula of two eyes were
seconds to prevent saturation of the camera. To minimize variability subject to the same solvents before osmication and processing for
due to fluorescence quenching, digitized sites were separated by 1.5 to conventional electron microscopy. The number of electron micro-
2 mm and were identified and focused using bright-field illumination. graphs containing lipid-rich components was determined by an ob-
To minimize variability due to oblique sectioning planes and haze from server unaware of the solvent.
RPE autofluorescence, only sites where the RPE was attached to
Bruch’s membrane and the inner edge of Bruch’s membrane and RPE
Stereologic Analysis of OTAP Preparations
pigment granules could be focused simultaneously were used. In
digitized images, an observer placed 4.56-␮m square sampling win- The proportion of the inner collagenous layer occupied by lipid-rich
dows over Bruch’s membrane. The sampling window spanned the particles (area fraction) was measured in 16 eyes of different ages using
thick macular Bruch’s membrane in older eyes and included chorio- point-counting stereology.35 A transparency containing a 4-mm2 grid
capillary endothelium and lumina in addition to thin Bruch’s mem- was taped to each of three prints for each eye, and the inner collage-
brane in other specimens. Five sampling windows were placed across nous layer was delimited. Intersections of the grid overlying lipid
the length of each three digitized images per section, with at least two particles and other inner collagenous layer constituents were scored
within and three between intercapillary pillars (total, 15 windows). by an observer. The area fraction was the ratio of intersections over-
Sampling windows avoided drusen and basal deposits. Fluorescence lying particles to the total number of intersections. The median num-
intensities in each window were summed and expressed in arbitrary ber of points scored per eye was 1112 (range, 621-1682), and the SEM
units ⫻10⫺6. The SEM was ⬍3% for unlabeled control sections and was ⬍10%. Results from younger and older eyes were compared using

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IOVS, January 2001, Vol. 42, No. 1 Cholesterol Accumulation in Bruch’s Membrane 267

FIGURE 1. Histochemical detection of cholesterol in human Bruch’s membrane, using filipin (epifluores-
cence, A through C). Arrows, Bruch’s membrane; arrowheads, RPE. The filipin fluorescence in (A)
through (C) can be easily distinguished from RPE autofluorescence by color (green versus orange). Bar,
20 ␮m. (A) EC in Bruch’s membrane is labeled by filipin after extraction and hydrolysis. The RPE and retina
are unlabeled. (B) Control section, which was extracted but not hydrolyzed, is devoid of filipin fluores-
cence. Bruch’s membrane is autofluorescent. (C) Filipin labels UC in Bruch’s membrane and in cellular
membranes of retina, RPE, and choroid (Ch) in an unextracted section. ONL, outer nuclear layer.

a t-test. Diameters of lipid-rich particles were measured in six eyes years old. Macular EC was much higher than peripheral EC at
using a digitizing tablet. all ages ⬎ 30 years (median macula/peripheral ratio for eyes,
6.9), and the difference increased significantly with age (P ⫽
Statistical Analysis 0.02). Macular and peripheral EC in individual eyes were sig-
The relationship among filipin fluorescence intensities and age in the nificantly associated when the effect of age was removed using
macula and periphery was analyzed with multiple linear regression, multiple linear regression analysis (Table 1). Regarding UC, the
adjusting for age to examine the independence of associations (e.g., macula exhibited a significant age-related increase (Fig. 2C),
macular EC and peripheral EC) with the effect of age removed. We but the peripheral retina did not (Fig. 2D). Unlike EC, UC was
report the slopes of regression lines as crude parameter estimates and present in the youngest specimens (likely because choriocap-
the slopes after adjustment. A P value of 0.05 was considered signifi- illary endothelial membranes37 were included in the measure-
cant. ments) and did not increase as markedly with age in the macula
as EC. Macular and peripheral UC in individual eyes were
RESULTS not significantly related when the effect of age was removed
(Table 1).
Histochemistry
The identity of neutral lipids in Bruch’s membrane was deter-
mined by histochemistry (Fig. 1). In sections that were ex-
tracted with ethanol and hydrolyzed with cholesterol esterase
to reveal EC, Bruch’s membrane exhibited an intense, diffusely
distributed fluorescence (Fig. 1A). The RPE and retina were
negative for EC, with the exception of occasional retinal arter-
ies (not shown), and the choroid was lightly labeled (see
below). The specificity of this reaction was verified by elimi-
nating cholesterol esterase (Fig. 1B). Because Bruch’s mem-
brane fluorescence intensity was similar in all control sections
of each eye (data not shown), we conclude that it represents
background autofluorescence.36 In sections that were exposed
to filipin without prior extraction and hydrolysis, cellular mem-
branes of the neural retina, RPE, and choroid were fluorescent,
as was Bruch’s membrane (Fig. 1C). From these results, we
conclude that Bruch’s membrane contains both EC and UC.
Using sections of macula and periphery that were stained
with filipin for EC and UC, we investigated the effects of age
and retinal location on cholesterol deposition in Bruch’s mem-
brane (Fig. 2 and Table 1). The range of mean fluorescence
intensities (⫻10⫺6 units) for each eye was 1.2 to 18.0 for EC, FIGURE 2. Filipin fluorescence due to EC and UC increases with age in
4.0 to 14.2 for UC, and 1.1 to 2.6 for controls in the macula and Bruch’s membrane. For EC in the macula (A) and periphery (B) and UC
in the macula (C) and periphery (D), fluorescence intensity (⫻10⫺6
1.1 to 5.3, 0.3 to 4.6, and 1.0 to 4.0, respectively, in the arbitrary units) for each eye is expressed as the mean sum of intensities
periphery. EC increased significantly with age in both macula within 15 4.86-␮m square windows placed on Bruch’s membrane in
and periphery (Figs. 2A, 2B). In the macula, EC was absent the experimental section minus the mean sum of intensities in control
from donors ⬍ 20 years old, increased sharply with age to sections of the same eye. Negative differences between experimental
reach high and variable levels (fourfold range) in donors ⬎ 60 and control means were set to zero.

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268 Curcio et al. IOVS, January 2001, Vol. 42, No. 1

TABLE 1. Histochemically Detected Cholesterol: Age and TABLE 2. Esterified Cholesterol in Choroidal Arteries
Retinal Location
Macula Periphery
Slope Parameter,
Multiple Linear ⬍60 years 1/7 2/7
Regression* ⬎60 years 9/11 9/11

Region Fluorescence Versus Crude Adjusted† Values are number of eyes with filipin-labeled arteries per total
number of eyes.
Macula EC Age 0.14‡ —§
Peri EC 1.31 ⫺1.26
Macula UC Age 0.09‡ — accumulates with age in choroidal arteries, as it does in Bruch’s
Peri UC 0.86 ⫺0.04 membrane. In contrast to Bruch’s membrane, however, EC
Periphery EC Age 0.02‡ — deposition in choroidal arteries appears significantly later in
Mac EC 0.06 ⫺0.81‡
life, reaches qualitatively lower levels, and is unrelated to
Periphery UC Age 0.01 —
Mac UC 0.07 ⫺0.01 retinal location.

EC, esterified cholesterol; UC, unesterified cholesterol; Mac, mac- Cholesterol Assay
ula; Peri, periphery. The relative proportions of EC and UC were directly assayed in
* Slope parameter b, where y ⫽ a ⫹ bx Bruch’s membrane, retina, and choroid of 10 eyes from do-
† Adjusted for age.
‡ Significant (P ⬍ 0.05).
nors ⬎ 70 years of age with high expected cholesterol content.
§ —, not in model. Table 3 shows that 39.1 ⫾ 18.8 nmol EC/g dry weight were
recovered from 8-mm-diameter punches of macular Bruch’s
membrane. There was an almost threefold range in macular EC
In addition to the intense filipin fluorescence due to EC in content (15.5– 68.4 nmol/g). The proportion of total choles-
Bruch’s membrane, there was also a faint but specific filipin terol that was esterified was less variable at 59.6% ⫾ 6.7%. In
fluorescence due to EC in the walls of choroidal arteries of the contrast, both the content and proportion of EC was low in
same sections (Figs. 3A, 3B). Veins were unlabeled (Figs. 3A, choroidal arteries (mean, 7.4 nmol/g; 15.0%) and very low
3B). Table 2 shows that EC was present in choroidal arteries of (mean, 2.0 nmol/g; 3.6%) in macular retina of the same eyes.
the macula and periphery only in eyes ⬎ 60 years old. Thus, EC Peripheral Bruch’s membrane in three eyes contained 2.7- to
11-fold less EC than macular Bruch’s membrane. Together,
these biochemical data validate the results obtained using fili-
pin histochemistry.
Histologic analysis revealed that the RPE and major choroi-
dal arteries were completely removed from Bruch’s mem-
brane, and the choriocapillaries and some veins were still
attached. Because EC bound to plasma lipoproteins in the
remaining vessels would affect estimates of Bruch’s membrane
cholesterol content, we computed the magnitude of this po-
tential contamination in the worst case, that is, if the chorio-
capillaries and veins were intact and completely filled with
blood. Using standard clinical measures and published esti-
mates of choroidal morphology,38 – 41 we estimated that the
choriocapillaries underlying an 8-mm-diameter macular punch
contain 0.39 nmol EC in 0.09 ␮l plasma. The choriocapillaries
and veins together contain 0.94 nmol EC in 0.13 ␮l plasma.
Thus, blood remaining in the choriocapillaries could account
for only 2.5%, and choriocapillaries and veins together only 6%,
of the EC determined in our samples. Accordingly, Bruch’s
membrane EC content is enriched at least 16- to 40-fold relative
to plasma.

Ultrastructure
The intensity and diffuse distribution of filipin fluorescence in
Bruch’s membrane (see Fig. 1) suggests that cholesterol-con-
taining particles are densely packed and smaller than the res-
olution limit of light microscopy. To identify these particles,
we used conventional and lipid-preserving electron micros-
copy. Conventional electron microscopy revealed numerous
small round particles with electron-lucent interiors (“drop-
lets”) in Bruch’s membrane of older eyes (Fig. 4A). Droplets
were scattered throughout the collagenous layers. They were
also grouped within coated membrane-bounded bodies
(CMBBs),27 91% of which occurred in the outer collagenous
layer, and were associated with coiled membranes, the pre-
sumed remnants of CMBBs (Fig. 4A). Droplets have been pre-
FIGURE 3. Filipin fluorescence due to EC in choroid. (A) Artery (a) viously described as vesicles25,42 (i.e., membrane-bounded
and vein (v), bright field. (B) EC is in artery wall only. structures with aqueous contents). However, in preparations

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IOVS, January 2001, Vol. 42, No. 1 Cholesterol Accumulation in Bruch’s Membrane 269

TABLE 3. Cholesterol Content of Bruch’s Membrane, Retina, and Choroid

Macula Periphery

Bruch’s Membrane Retina Choroid Bruch’s Membrane

Age (y) EC EC/TC (%) EC EC/TC (%) EC EC/TC (%) EC EC/TC (%)

83 21.3 62.3 — — — — — —
85 15.5 56.0 — — — — — —
76 35.7 62.5 — — — — — —
85 43.3 58.8 0.0 0.0 4.5 13.5 — —
76 20.2 64.8 7.1 8.4 4.7 12.8 — —
71 39.2 47.6 3.2 4.4 3.9 5.9 9.4 15.4
72 26.8 50.2 0.0 0.0 10.5 15.6 9.8 20.9
80 58.6 68.2 4.0 12.0 10.3 28.7 — —
87 62.3 59.4 0.0 0.0 5.8 11.7 5.7 44.3
80 68.4 66.0 0.1 0.1 12.4 16.7 — —
Mean 79.5 39.1 59.6 2.0 3.6 7.4 15.0 8.3 26.9
SD 5.6 18.8 6.7 2.8 4.9 3.5 7.0 2.3 15.4

EC, esterified cholesterol, in nmol/gm dry weight; TC, total cholesterol.

in which lipid-rich particles are preserved by the OTAP method ethanol eliminated Bruch’s membrane UC fluorescence, pro-
(Fig. 4B), droplets were solid, electron-dense particles that duced somewhat distorted droplets, and reduced the visibility
formed a single size distribution (minimum/median/maximum of cellular and CMMB membranes. Treatment with chloro-
diameters; 54/98/156 nm for inner collagenous layer; 56/112/ form/methanol eliminated UC and EC fluorescence, converted
225 nm for outer collagenous layer). These results indicate that droplets into distorted empty spaces embedded within an
electron-lucent droplets are not vesicles but rather solid parti- electron-dense matrix, and completely removed all mem-
cles whose lipid-rich contents were extracted by conventional branes. These results are consistent with our expectations.
tissue processing. The distribution of droplets changed with age in a manner
To determine whether droplet cholesterol composition re- consistent with their identification as EC-rich particles. In
sembles that of EC-rich particles isolated from other tissues eyes ⬍ 60 years, droplets were scarce and scattered through
(i.e., 69% EC and 22% UC),5,13 we examined the effects of the inner collagenous layer or associated with CMMB in the
solvents on filipin histochemistry and ultrastructure in conven- outer collagenous layer (Figs. 5A, 5B). In contrast, droplets in
tional preparations. We expected that droplet ultrastructure eyes ⬎ 60 years old were abundant in the inner collagenous
would be affected by solvents that reduce both EC and UC layer and formed a broken or continuous layer external to the
fluorescence. We further expected that the ultrastructure of RPE basal lamina (Figs. 5C and 6, asterisk). Tangential sections
UC-containing membranes in the RPE, choriocapillary endothe- of Bruch’s membrane revealed rows of droplets aligned along
lium, and CMMBs would be affected only by solvents that fibers in the inner collagenous layer (Fig. 6). The proportion of
reduce UC fluorescence. Table 4 shows that treatment with the inner collagenous layer occupied by droplets in eyes ⱖ 67

FIGURE 4. Lipid-rich particles in inter-


capillary pillar of foveal Bruch’s mem-
brane, conventional EM (A) and OTAP
(B), 85-year-old donor. Basal RPE is at
the top. ic, inner collagenous layer; oc,
outer collagenous layer; c, collagen
fibrils (electron dense in A, negative
image in B); arrowheads, RPE basal
lamina; asterisks, electron lucent drop-
lets in (A) and solid particles in (B);
large double arrows, coated mem-
brane-bounded bodies; large single ar-
row, remnants of coated membrane-
bounded bodies; small double arrows,
row of particles external to the RPE
basal lamina.

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270 Curcio et al. IOVS, January 2001, Vol. 42, No. 1

TABLE 4. Effects of Solvents on Bruch’s Membrane Cholesterol

Untreated Ethanol Chloro/Meth

Filipin fluorescence*
Unesterified
cholesterol Intense At background† At background†
Esterified
cholesterol NA‡ Intense‡ At background†
Ultrastructure§
Droplets/vesicles ⫹⫹ ⫹⫹ Distorted
CMMB ⫹ ⫹ ⫺
Coiled
membranes ⫹⫹⫹ ⫹⫹ ⫹
RPE membranes ⫹⫹⫹ ⫹ ⫺
ChC membranes ⫹⫹⫹ ⫹ ⫺

NA, not applicable; ChC, choriocapillaris endothelium; RPE, reti-


nal pigment epithelium; CMMB, coated membrane bounded bodies.
* Fluorescence was quantified in two eyes per condition (see
Methods).
† Significant reduction in intensity by t-test.
‡ By definition, the EC condition involved ethanol treatment (see
Methods).
§ Frequency of findings in two eyes: ⫺, not present; ⫹, present in
⬍1⁄2 of the micrographs; ⫹⫹, present in ⬎1⁄2 of micrographs; ⫹⫹⫹,
present in all micrographs.

years (36% ⫾ 7.0%, n ⫽ 10) was significantly higher than in


eyes ⱕ 51 year (7% ⫾ 2.7%, n ⫽ 6, t ⫽ ⫺11.6, P ⬍ 0.0001).

Cholesterol and Age-Related Pathology FIGURE 6. Oblique section of Bruch’s membrane (OTAP, 85-year-old
Older eyes exhibit many mild pathologic changes in Bruch’s donor). RPE is at top. Arrowheads, RPE basal lamina; asterisk, the band
membrane, including calcification and the accumulation of of lipid-rich particles. Other particles are disbursed among collagen
basal deposits and drusen.28,43,44 In eyes ⬎ 60 years used for fibrils (negative image) in the inner collagenous layer.
filipin histochemistry, maculas contained only small drusen
and patchy basal deposits, and the periphery contained numer- the thickened band of EC-rich particles external to the RPE
ous small drusen and basal deposits, as previously character- basal lamina (see Figs. 4 and 5) and some small drusen.
ized by light microscopy.45 Below we describe patterns of Figure 7C shows an EC-rich band overlying a small accumu-
filipin fluorescence and lipid particle distribution that are con- lation of a homogeneous material. Figure 7D shows EC-rich
sistent with cholesterol involvement in age-related pathology. deposits on the inner surface of Bruch’s membrane that are
In the macula, Bruch’s membrane from eyes ⬎ 60 years was too small to be considered drusen. Figure 7E shows a small
thick and intensely labeled (Fig. 7A). Most eyes (9/11) from druse with an EC-rich rim. Electron micrographs comparable
older donors exhibited intermittent bands of less intense to the histochemical observations in Figures 7D and 7E have
filipin labeling that corresponded to patches of calcifica- been published previously (Ref. 46, Figs. 2 and 5D; Ref. 28,
tion44 (Fig. 7B). Ultrastructural and histochemical observa- Fig. 4B). Finally, the small drusen (Figs. 7F, 7G) and basal
tions (Figs. 7C through 7E) suggested a progression between deposits (Figs. 7H, 7I) present in the periphery of these eyes

FIGURE 5. Lipid-rich particles in eyes


of different ages, OTAP. Arrowheads,
RPE basal lamina; arrows, electron
dense lipid particles. c, negative image
of collagen fiber in cross-section. (A)
16 years; (B) 51 years; (C) 76 years. X,
basal laminar deposit.

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IOVS, January 2001, Vol. 42, No. 1 Cholesterol Accumulation in Bruch’s Membrane 271

FIGURE 7. Cholesterol and age-related


changes in Bruch’s membrane. (A
through E) Macula; (F through I) pe-
riphery. (A, B) Arrowhead, an EC-free
gap (A, filipin fluorescence) that corre-
sponds to a calcified patch (B, bright
field, hematoxylin). (C) Band of lipid-
rich particles (arrows) overlying a
druse-like deposit on the inner surface
of Bruch’s membrane, OTAP. Arrow-
heads, RPE basal lamina; X, basal lam-
inar deposit. (D) EC in small, drusen-
like deposits; (E) small druse with EC-
rich shell; (F) EC in druse; (G) UC in
druse; (H) EC in basal deposits; (I) UC
in basal deposits.

were virtually all positive for both EC (Figs. 7F, 7H) and UC coated membrane-bounded bodies,27,46 implicating these enig-
(Figs. 7G, 7I). matic structures in lipid trafficking. In the maculas of older
adults, EC-rich particles occupy one third of the inner collag-
DISCUSSION enous layer and nearly 100% of a narrow sublayer external to
the RPE basal lamina. These results provide a basis for the
Throughout adulthood, normal human connective tissues ac- finding that hydraulic conductivity across Bruch’s membrane
cumulate extracellular EC-rich droplets (perifibrous lipid) that and choroid isolated from older donors improves markedly
appear to originate from infused plasma lipoproteins.8 In this when the inner collagenous layer is removed.24
article we provide evidence that Bruch’s membrane shares Our data contrast sharply with those of Holz et al.,22 who
with sclera, cornea, and arterial intima a linear, age-related indicated that the proportion of EC in macular Bruch’s mem-
increase in EC and UC, a high degree (60%) of enrichment with brane was only 14% and that EC content was unrelated to age.
EC, and an abundance of 100-nm-diameter droplets. Our use of The previous study,22 which used thin-layer chromatography
filipin histochemistry and digital microscopy permitted spe- to assay lipids in unfixed tissues and did not remove the
cific labeling and precise localization of samples to Bruch’s choroid from Bruch’s membrane, recovered one tenth as much
membrane, and histochemical results were verified by direct total cholesterol per unit area as we did. Our higher yield may
chemical assay. be due to using tissues that were preserved in paraformalde-
Using ultrastructural techniques that preserve lipid particle hyde quickly after death, presumably cross-linking EC-rich par-
morphology and extraction experiments, we demonstrated ticles to a surrounding proteinaceous matrix. Our higher pro-
that the electron-lucent droplets recognized in human Bruch’s portion of EC may be due to our removing the choroid.
membrane for more than 30 years21,25–27,42 are the EC-rich Because the choroid contains many cells and is 50- to 100-fold
particles suggested by filipin histochemistry. Droplets resem- thicker than Bruch’s membrane, its presence would inflate
ble the solid, EC-rich particles seen in other tissues with regard Bruch’s membrane UC content relative to EC. In humans, two
to size and alignment along matrix fibers.5,47 A membranous thirds of total plasma cholesterol is transported by LDL,38 and
shell of UC and phospholipid12 could account for the vesicular 64% of the cholesterol in LDL is esterified.48 The low propor-
appearance of Bruch’s membrane droplets in tissue examined tion of Bruch’s membrane EC found by Holz et al.,22 relative to
by conventional electron microscopy. Droplets also occur in total lipids, was the basis of that study’s conclusion that plasma

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272 Curcio et al. IOVS, January 2001, Vol. 42, No. 1

was an unlikely source for Bruch’s membrane lipids. Although of rabbits with extremely high levels of plasma lipoproteins65
we did not examine other lipid classes (e.g., triglycerides or but not in mice with moderately elevated levels.66 – 68 The role
phospholipids), our demonstration that EC accounts for a high played by plasma lipoproteins in normal retinal homeostasis is
proportion of total cholesterol suggests that this conclusion poorly understood. The RPE has LDL receptor activity,69 and
should be re-evaluated, at least for cholesterol.7 plasma LDL transports docosahexanoic acid destined for pho-
Although it is thought that the RPE produces many Bruch’s toreceptors.70 However, the choriocapillaris endothelium re-
membrane constituents,49,50 there is currently little reason to portedly excludes molecules as large as LDL,71,72 which is
suspect that the RPE is a source of EC-rich droplets. It is inconsistent with our findings. Studies to clarify these issues
possible that the large-diameter (1–2 ␮m), oil red O-positive are warranted.
droplets occasionally seen in RPE cells (lipoidal degenera- If Bruch’s membrane can be conceptualized as an arterial
tion51–53) could be released into Bruch’s membrane upon cell intima, it is appropriate to examine diseases of the intima for
death. However, we did not detect EC within RPE cells using insight into ARM. Atherosclerotic cardiovascular disease
filipin, and the uniformly small size of Bruch’s membrane (CVD), a major cause of morbidity and mortality, is a complex
droplets is inconsistent with their originating from the break- disease that proceeds from the normal infusion of plasma LDL
down of larger droplets.12 Another potential source of Bruch’s and accumulation of EC in thickened intima through advanced
membrane EC is the photoreceptor outer segment membranes lesions containing increased levels of cholesterol and lipopro-
that are regularly ingested by RPE. However, outer segment teins, culminating in neovascularization, inflammation, and
membranes are poor in UC compared with typical plasma thrombosis.73 Although the cholesterol and lipoprotein con-
membranes,54 and it is not yet known if the RPE can esterify tent of ARM-specific lesions28 remain to be determined, our
cholesterol. Although the RPE cannot be conclusively ex- study confirmed that small age-related drusen contain EC and
cluded as a source without further data, it is more parsimoni- UC, extending our previous observation that these drusen
ous to postulate that cholesterol deposition in Bruch’s mem- contain neutral lipids and apo B45 (but see Ref. 74). A plasma
brane reflects a well-described systemic process than to invoke origin for neutral lipids in small drusen is therefore likely,
a new mechanism involving the RPE. consistent with studies demonstrating other plasma proteins in
We found a highly variable but marked (sevenfold) differ- drusen.74,75 EC-rich droplets occur in other age-related and
ence in the degree of cholesterol accumulation of macular and disease-related sub-RPE deposits.76 –78 In dominant late-onset
peripheral Bruch’s membrane. This difference is far greater retinal degeneration, an inherited disorder, a cholesterol-enriched
than can be explained by differences in the number of photo- basal laminar deposit also displays intense apo B immunoreactiv-
receptors in the macula and at the temporal equator (macula/ ity.77 Thus, diverse retinal degenerations with sub-RPE debris
periphery ratio ⫽ 2.7 for rods, 1.9 for cones; calculated from may involve plasma lipoproteins interacting with a locally
Ref. 55). Regional differences in age-related EC accumulation retentive matrix material.
also occur in the cornea, which has higher EC near the limbus Significantly, our study demonstrates that with regard to the
(arcus lipoides) than in the center.5 In the cornea this effect is deposition of extracellular cholesterol, Bruch’s membrane ages
attributed to greater vascular perfusion near the limbus and a like the intima of large, atherosclerosis-prone arteries. In fact,
tightly packed matrix that retards LDL diffusion toward the the proportion of Bruch’s membrane occupied by EC-rich
center.5 A similar combination of a lipoprotein-retaining milieu droplets in older adults is much higher than in normal arterial
and a high blood flow that saturates retentive components intima at the same age (1%–3%).12 Therefore, it is plausible that
could underlie the predilection of macular Bruch’s membrane the progression from aging to ARM shares pathogenic mecha-
for extracellular cholesterol. Notably, the choroid has the high- nisms with atherosclerotic CVD. Seeking this link through
est blood flow in the body, and blood flow in the macula is epidemiologic studies79 is difficult, because the earliest lesions
eightfold higher than that in the periphery.56 High blood flow in CVD occur decades earlier than those in ARM.16,44,80 Our
alone is insufficient to account for regional differences in data now strengthen the rationale for seeking links between
Bruch’s membrane cholesterol, however, because the EC con- these two diseases at the tissue, cellular, and molecular level.
tent of the choroidal arteries was low and we could not detect We caution that atherosclerotic CVD and ARM are complex
a difference in age-related EC accumulation between macular multifactorial diseases, and the geometry, spatial scale, and
and peripheral arteries using histochemistry. Understanding cellular constituents of arterial intima and Bruch’s membrane
the relative roles of blood flow and matrix in EC accumulation differ in important ways. Nevertheless, this analogy can pro-
will benefit from additional information about regional differ- vide a useful conceptual framework for generating testable
ences in Bruch’s membrane composition.26,57 new hypotheses about the pathogenesis of ARM.
Although Bruch’s membrane is the wall of a capillary bed, it
is intriguingly similar to the inner wall of an artery. The arterial Acknowledgments
intima is located between two diffusion barriers: an endothelial
cell layer and a dense elastic layer.58 Throughout life intima The authors thank the Alabama Eye Bank for timely retrieval of donor
thickens adaptively to the mechanical stresses of blood flow eyes; Shawn Williams of the High Resolution Imaging Facility at UAB
and wall tension.59 Collagen, elastin, and proteoglycans at sites for assistance with digital microscopy; Gerald McGwin, Jr., Ph.D., for
of intimal thickening specifically interact and bind with plasma statistical consultation; David Fisher for graphics assistance; and
LDL.60,61 Similarly, Bruch’s membrane is located between the Steven J. Fliesler, Ph.D., for helpful discussions.
choriocapillaris endothelium and the RPE component of the
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