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Molecular Pathogenesis of Genetic and Inherited Diseases
Decreased Thickness and Integrity of the Macular
Elastic Layer of Bruch’s Membrane Correspond to
the Distribution of Lesions Associated with Age-
Related Macular Degeneration
N.H. Victor Chong,*
†
Jason Keonin,*
Phil J. Luthert,
‡
Christina I. Frennesson,
§
David M. Weingeist,* Rachel L. Wolf,*
Robert F. Mullins,* and Gregory S. Hageman*
From the Department of Ophthalmology and Visual Sciences,*
The University of Iowa Center for Macular Degeneration, The
University of Iowa, Iowa City, Iowa; the Department of
Ophthalmology,
†
King’s College, London, United Kingdom; The
Institute of Ophthalmology,
‡
University College London and
Moorfields Eye Hospital, London, United Kingdom; and the
Department of Neuroscience and Locomotion,
§
Division of
Ophthalmology, Linkoping University, Linkoping, Sweden
Age-related macular degeneration (AMD) is a leading
cause of blindness in the elderly. In its severest form,
choroidal neovessels breach the macular Bruch’s
membrane, an extracellular matrix compartment
comprised of elastin and collagen laminae, and grow
into the retina. We sought to determine whether
structural properties of the elastic lamina (EL) corre-
spond to the region of the macula that is predilected
toward degeneration in AMD. Morphometric assess-
ment of the macular and extramacular regions of 121
human donor eyes, with and without AMD, revealed a
statistically significant difference in both the integrity
(P<0.0001) and thickness (P<0.0001) of the EL
between the macular and extramacular regions in
donors of all ages. The EL was three to six times
thinner and two to five times less abundant in the
macula than in the periphery. The integrity of the
macular EL was significantly lower in donors with
early-stage AMD (Pⴝ0.028), active choroidal neovas-
cularization (Pⴝ0.020), and disciform scars (Pⴝ
0.003), as compared to unaffected, age-matched con-
trols. EL thickness was significantly lower only in
individuals with disciform scars (Pⴝ0.008). The larg-
est gaps in macular EL integrity were significantly
larger in all categories of AMD (each P<0.0001), as
compared to controls. EL integrity, thickness, and
gap length in donors with geographic atrophy did not
differ from those of controls. These structural prop-
erties of the macular EL correspond spatially to the
distribution of macular lesions associated with AMD
and may help to explain why the macula is more
susceptible to degenerative events that occur in this
disease. (Am J Pathol 2005, 166:241–251)
Bruch’s membrane is a stratified extracellular matrix com-
plex that lies between the retinal pigment epithelium
(RPE) and the choroidal capillary bed, or choriocapillaris.
It is comprised of two collagen-rich layers, referred to as
the inner and outer collagenous layers, that flank a cen-
tral domain of elastin and elastin-associated proteins.
1,2
A number of age-related changes have been described
in Bruch’s membrane,
3–23
the most prominent of which
are drusen and basal laminar deposits.
24–30
In addition,
increases in thickness, enhanced basophilia and Su-
danophilia, accumulation of membranous debris, de-
creases in hydraulic conductivity, and fragmentation and
calcification of Bruch’s membrane, have been described.
These age-related alterations in Bruch’s membrane
could lead to a loss of the normal function of Bruch’s
membrane and promote degenerative changes in the
aging eye. Various lines of evidence suggest that Bruch’s
membrane functions as a physical barrier to the egress of
cells and vessels from the choroid into the sub-RPE and
subretinal spaces. Disruption of, or damage to, this bar-
rier is associated with loss of vision in a variety of ocular
Supported in part by the National Institutes of Health (grants EY11515 to
G.S.H. and EY014563 to R.F.M.); The Ruth and Milton Steinbach Fund,
Inc. (to G.S.H.); a Research to Prevent Blindness Lew R. Wasserman Merit
Award (to G.S.H.); and The University of Iowa Center on Aging (grant
AG00214 to R.F.M.).
Accepted for publication September 16, 2004.
Address reprint requests to Gregory S. Hageman, Ph.D., Department of
Ophthalmology and Visual Sciences, Center for Macular Degeneration,
The University of Iowa, 11190E PFP, 200 Hawkins Dr., Iowa City, IA 52240.
E-mail: gregory-hageman@uiowa.edu.
American Journal of Pathology, Vol. 166, No. 1, January 2005
Copyright © American Society for Investigative Pathology
241
diseases, often resulting from the growth of new blood
vessels—termed choroidal neovascular membranes
(CNVM)—from the choroid into the sub-RPE and/or sub-
retinal spaces. CNVM formation occurs in a number of
macular degenerative diseases, including age-related
macular degeneration (AMD), the leading cause of irre-
versible blindness in the developed world.
31
Moreover,
CNVM formation can be induced experimentally by ther-
mal damage to Bruch’s membrane in normal monkey
eyes after laser photocoagulation
32
and potentially, after
laser treatment of human CNVM.
33
Interestingly, extra-
macular treatment is relatively ineffective, suggesting that
the macula is inherently susceptible to choroidal neovas-
cularization (CNV).
34
AMD-associated CNVM formation occurs in ⬃4% of
the population older than the age of 75
35
and is primarily
restricted to the macula. It accounts for 90% of severe
visual loss associated with AMD, even though only ⬃10
to 15% of individuals with AMD develop CNV. The macula
is a unique 6-mm-diameter region of the posterior pole
that lies directly in the visual axis of human and nonhu-
man primates (Figure 1A). This region of the retina de-
velops postnatally and subserves fine acuity vision. The
reason for the increased susceptibility of the macula to
degeneration and CNVM formation in primates has not
been elucidated.
We have speculated that one possible explanation for
the tendency of new vessels to breach the macular
Bruch’s membrane, as opposed to the extramacular re-
gions, might be that there are regional differences in the
structure and/or composition of Bruch’s membrane. It is
likely that the integrity and nature of its collagen and
elastin components primarily dictate the basic structural
and functional properties of Bruch’s membrane. Hence,
one might predict that the disruption of one or more
layers of Bruch’s membrane might precede CNV in AMD.
Accordingly, we recently observed what we interpreted
to be robust differences in the integrity and thickness of
the elastic lamina (EL) of Bruch’s membrane between the
macular and extramacular regions in a series of human
eyes. To evaluate further the topographical variation in
the thickness and continuity of the EL, we assessed these
parameters in a larger series of normal, unaffected donor
eyes and compared them to those obtained from donors
with early and late stages of AMD.
The data collected herein show that the macular EL is
substantially thinner and more porous than that in the
extramacular region. This information has lead to the
development of a working hypothesis that the structural
attributes of the macular EL of Bruch’s membrane may be
related to, at least in part, the predilection of this region to
degeneration in AMD, other macular dystrophies, and
other conditions characterized by CNVM formation.
Materials and Methods
Human Donor Eyes and Morphological
Procedures
The 121 human eyes used in this study were obtained
from MidAmerica Transplant Services (St. Louis, MO), the
Iowa Lions Eye Bank (Iowa City, IA), the Heartland Eye
Bank (Columbia, MO), the Central Florida Lions Eye and
Tissue Bank (Tampa, FL), and the Virginia Eye Bank
(Norfolk, VA) after informed consent. Institutional Review
Board committee approval for the use of human donor
tissues was obtained from Human Subjects Committees
at St. Louis University and the University of Iowa. All eyes
were processed within 4 hours of death. The gross patho-
logical features, as well as the corresponding fundus
photographs and angiograms when available, of all eyes
Figure 1. Ocular regions used in this study. A: Color funduscopic photograph of the left eye of a middle-aged individual without any clinical signs of AMD. The
macula (dashed circle) is centered on the fovea (arrow) and lies within the major retinal vessels (arrowheads) that emanate from the optic nerve head (ONH).
The superior (S), inferior (I), temporal (T), and nasal (N) axes are marked for orientation. B: Diagrammatic representation of the distribution of the 2-mm-diameter
retinal-choroidal-scleral punches collected from a 14-year-old donor. The solid black circle depicts the location of the optic nerve head, the curved black lines
emanating from the optic disk represent the major retinal vessels, and the blue circle denotes the punch that was centered on the fovea. The remaining circles
represent serial 2-mm-diameter punches taken along the superior, inferior, temporal, and nasal axes. C: Illustration showing the macular and extramacular
(mid-peripheral) sites (circles) from which 4-mm-diameter punches were collected for EL thickness and integrity measurements in the large set of AMD and
unaffected donors. Landmarks are the same as those described in B.
242 Chong et al
AJP January 2005, Vol. 166, No. 1
in this repository were read and classified by retinal spe-
cialists. Fundi were classified according to a modified
version of the International AMD grading system.
36
For
this investigation, donors were placed into one of four
categories: young unaffected (⬍62 years), age-matched
unaffected (ⱖ62 years), early-stage AMD (the youngest
was 62 years old), and late-stage AMD. Late-stage AMD
donors were subdivided into those with 1) geographic
atrophy (GA), 2) active CNVMs, and 3) disciform scars
preceded clinically by CNVM (CNV/DS). Note that some
individuals refer to early-stage AMD as age-related
maculopathy, or ARM; this term will not be used herein.
Donors were classified as unaffected if they had no mac-
roscopic or funduscopic signs of macular pathology or
any ophthalmic history of AMD. Early AMD donors were
defined based on an ophthalmic history of AMD and the
presence of significant numbers of macular drusen, pig-
ment disruption, and/or other clinical signs of early AMD.
Polyclonal antisera directed against tropoelastin
(PCAB 94, gift from Dr. Robert Mecham, Washington
University, St. Louis, MO; PR398, Elastin Products,
Owensville, MO), as well as monoclonal antibodies di-
rected against bovine elastin (clone BA-4, Sigma, St.
Louis, MO; MM436, Elastin Products) and human aortic
␣
-elastin (PR533, Elastin Products) were used to examine
the EL of Bruch’s membrane. For experiments using Elas-
tin Products’ antibodies, antigens were retrieved accord-
ing to the manufacturer’s instructions. Reactivity of anti-
bodies with the EL of Bruch’s membrane was analyzed in
a series of young (⬍10 years), middle-aged (20 to 60
years), and aged (⬎60 years) donors. Posterior poles, or
wedges of posterior poles spanning between the ora
serrata and the macula, were fixed in 4% (para)formal-
dehyde in 100 mmol/L sodium cacodylate, pH 7.4, as
described previously.
29
After 2 to 4 hours of fixation, eyes
were transferred to 100 mmol/L sodium cacodylate and
rinsed (3 ⫻10 minutes), infiltrated, and embedded in
acrylamide. These tissues were subsequently embedded
in OCT, snap-frozen in liquid nitrogen, and stored at
⫺80°C. In addition, unfixed posterior poles, or wedges
thereof, were embedded directly in OCT, without acryl-
amide infiltration or embedment. Both fixed and unfixed
tissues were sectioned to a thickness of 6 to 8
mona
cryostat. Immunolabeling was performed as described
previously,
29,30
using Alexa 488-conjugated secondary
antibodies (Molecular Probes, Eugene, OR). Adjacent
sections were incubated with secondary antibody alone,
to serve as negative controls. Some immunolabeled
specimens were viewed by confocal laser-scanning mi-
croscopy.
37
These cellular nuclei in these sections were
counterstained with TO-PRO-3 (Molecular Probes).
Ocular tissues used for transmission electron micro-
scopical studies were fixed by immersion fixation in one-
half strength Karnovsky’s fixative, within 4 hours of death,
for a minimum of 24 hours. Trephine-punched specimens
(see below) were fixed, transferred to 100 mmol/L sodium
cacodylate buffer, pH 7.4, and subsequently dehydrated,
embedded in epoxy resin, sectioned, and photographed,
as described previously.
38,39
Morphometric Analyses
To establish the baseline structural and topographical
characteristics of the EL, irrespective of aging or AMD,
three separate analyses were performed. In the first anal-
ysis, baseline topographic data were collected from a
series of oriented, 2-mm-diameter, full-thickness punches
of RPE-choroid-sclera that were collected using a tre-
phine punch. These punches were taken at 2-mm inter-
vals from the fovea to the ora serrata in the temporal,
nasal, inferior, and superior quadrants in an eye from a
14-year-old donor and subsequently prepared for elec-
tron microscopy (Figure 1B). The average thickness and
integrity of the EL in each of these punches were mea-
sured. A second series of 2-mm-diameter punches de-
rived from two additional donors aged 7 and 25 were
measured (these data were similar to those of the 14-
year-old and are not depicted in this article).
In the second analysis, measurements of EL thickness
and integrity were made at two defined locations, 1 to 2
mm and 12 to 13 mm, from the foveal center, in the
infero-temporal quadrant, in a series of donors (Figure
1C). Fifty-six eyes from 56 unaffected (non-AMD) donors,
ranging in age from 6 hours after birth to 96 years of age
(mean age, 51.4 years), were used in this analysis. At
least five donor eyes from each decade of life were
included in this analysis. Oriented, 4-mm-diameter, full-
thickness punches of RPE-choroid-sclera were taken us-
ing a trephine punch and prepared for electron micros-
copy, as described above.
In the third analysis, similar measurements of macular
EL thickness and integrity were made from punches
taken from 64 eyes derived from 64 human AMD donors,
ranging in age from 62 to 99 years of age (Figure 1C).
These included 24 donors with macular drusen, pigment
disruption, and other signs of early-stage AMD; 15
donors with GA; 9 donors with active, patent CNVMs;
and 16 donors with disciform scars (DS) that were
preceded by documented CNVMs or in which a CNVM
was present in the second eye. The mean ages of
donors in these four categories were 79.0, 82.8, 83.7,
and 88.2 years, respectively.
Outcome Measures
Four random photographic images were taken from each
punched specimen using a JEOL JEM 1220 microscope
(JEOL USA Inc., Peabody, MA), as described previous-
ly.
38
Images were collected at ⫻5000 actual magnifica-
tion for the first analysis (see above) and ⫻2500 actual
magnification for the second and third analyses (see
above). The thickness of the EL of Bruch’s membrane
was measured at 20 points (five equal-distance points
from each of the four images per specimen) using a
micrometer. The average value of the 20 points was used
in the statistical analyses. The integrity of the EL was
defined as the total length of visually detectable elastin
divided by the overall length of visible Bruch’s membrane
within each of the four images. Thus, 100% integrity
represented a fully intact (nonporous) EL and 0% integrity
represented complete absence of elastin. The average
Topographical Variation in Bruch’s Membrane 243
AJP January 2005, Vol. 166, No. 1
integrity value derived from the four images of each
punch was used for statistical analyses. The largest, or
maximum, gap length (ie, discontinuity in the EL) present
within the elastin lamina from the four micrographs used
for each donor was identified and measured. The largest
gap length from all donors in any given category was
averaged (Figure 2F).
Statistical analyses were performed using Microsoft
Excel (Microsoft Inc., Redmond, WA) and S-Plus Statisti-
cal Packages (Insightful Corp., Seattle, WA). The Mann-
Whitney U-test (Wilcoxon rank-sum test) was used to
compare data series and linear regression analysis was
used to explore age-related changes. To compare nor-
mal, unaffected donors to donors with AMD, statistical
analyses were performed using only the normal, unaf-
fected donors ⬎61 years of age (23 unaffected, 23 early-
stage AMD, and 40 late-stage AMD total). The mean age
of normal donors ⬎61 was 79.8 years; this mean age was
not significantly different from that of each of the three
groups of donors with AMD. Some data are represented
graphically in the form of box and whiskers plots. These
graphs show values for the median (bolded line within
box), the first and third quartiles as a solid box, and the
minimum and maximum observation range as whiskers.
Any extreme values, defined as those further than 1.5⫻
the interquartile range from the median, are represented
as solitary, horizontal lines.
Results
Immunohistochemistry
The reactivity of anti-elastin antibodies with the macular
EL of Bruch’s membrane differed markedly from that of
extramacular regions in all donor eyes examined (Figure
2, A and B). Immunoreactive elastin was attenuated and
highly discontinuous in the maculas of most donors, as
compared to more peripheral regions where it was con-
tinuous and thick. This same pattern was observed with
all elastin antibodies tested. The differences between
these same structural attributes in the macular and ex-
tramacular regions are even more striking when viewed
Figure 2. Confocal (A,B) and transmission electron microscopical (C–F) images of the elastic layer of Bruch’s membrane. Images were collected from macular
(A,C,E) and extramacular (B,D,F) regions of Bruch’s membrane, as indicated in Figure 1. Aand B: Labeling with an antibody directed against tropoelastin in
a 78-year-old donor without macular disease. Tissues prepared for confocal microscopy were counterstained with TO-PRO-3 to visualize cellular nuclei (blue);
RPE-associated lipofuscin is autofluorescent (red). Note that the elastin immunoreactivity of the elastic layer (green) is far more robust in the extramacular region
(B) that it is in the macular region (A). The EL (arrows) in the macular region of an 82-year-old donor, depicted in Cand D, is thinner and contains more
numerous discontinuities than in the extramacular region. E: A higher magnification image of Bruch’s membrane directly adjacent to the foveal pit; the elastin
fibers in this region are very sparse and thin (arrow). When the extramacular EL is viewed en face (F), its porosity (asterisk) is evident. These discontinuities
contribute to the integrity and gap length values measured in this study. Original magnifications, ⫻400 (A,B). Scale bar, 2
m(C,D).
244 Chong et al
AJP January 2005, Vol. 166, No. 1
by transmission electron microscopy (Figure 2, C and D).
In the center of the fovea, elastin was particularly sparse
and thin (Figure 2E). These data provided the impetus for
a more robust evaluation of these structural parameters in
a larger set of samples derived from donors with and
without a history of AMD.
EL Integrity and Thickness Maps Derived from a
Young Donor
The mean integrity of the EL measured in the series of
punches derived from a 14-year-old donor (Figure 1B) is
depicted in Figure 3, A and B. The mean integrity was
lowest (⬃40%) in the central macula (fovea) and in-
creased rapidly, approaching ⬃80% at the main retinal
vascular arcades (⫹8mmand⫺8 mm) in the temporal,
superior, and inferior regions. In the nasal quadrant, the
integrity was lowest around the optic disk, especially on
the nasal aspect, and rapidly increased in regions ap-
proaching 8 to 10 mm from the fovea, just outside the
major vascular arcades. The mean thickness values ex-
hibited a similar distribution, except there was not a sig-
nificant fovea-associated dip (Figure 3, C and D). A cal-
culated color-coded schematic map of the mean EL
integrity of this eye is depicted in Figure 4A. In general,
the area of the fundus with the thinnest and most porous
EL corresponds spatially to the same region where the
majority of lesions associated with AMD are manifest
(Figure 4, B and C).
EL Integrity and Thickness in the Macular and
Extramacular Regions of Donors without AMD
Scatter plots for the mean integrity and thickness of the
EL of Bruch’s membrane in a series of normal, unaffected
human donors are depicted in Figure 5. The mean integ-
rity value of the EL in this series of normal human donors
was 37.2% (SD ⫽7.9%) in the macula and 92.3% (SD ⫽
4.7%) in the extramacula (Figure 5A). The mean thick-
ness of the EL from the same series of donors was 134.2
nm (SD ⫽32.0 nm) in the macula and 391.7 nm (SD ⫽
120.1 nm) in the extramacula (Figure 5B). This represents
a statistically significant difference in both the integrity
(P⬍0.00001) and thickness (P⬍0.00001) of the EL
between the macular and extramacular regions. When
the unaffected donors were split into young and age-
matched groups, aging effects in both the macular and
extramacular regions were observed. The thickness, but
not the integrity, of the EL was significantly higher in the
age-matched group, as compared to the young group, in
both the macular and peripheral regions (P⫽0.033 and
P⬍0.001, respectively). Scatter plots comparing the mean
integrity and mean thickness values for each donor (Figure
6) suggest that a strong relationship exists between integrity
and thickness of the EL in all regions in which these param-
eters were measured (exponential fit R
2
⫽0.8735).
EL Integrity and Thickness in the Macular
Regions of Donors with Early and Advanced
Forms of AMD
In the macular area, there was an apparent thinning of EL
thickness and a loss of EL integrity from elderly normal
individuals, through early-stage AMD to late-stage AMD
(Table 1; Figures 7, 8, 9, and 10). For macular integrity,
significant differences were observed between unaf-
fected, age-matched controls and the following: all AMD
(P⫽0.003), early-stage AMD (P⫽0.028), late-stage
Figure 3. Values for mean integrity (percent; A,B) and thickness (nm; C,D)
of the elastic layer derived from the 14-year-old donor described in Figure
1B. Plots Aand Cdepict measurements taken from the inferior ora serrata to
the superior ora serrata, passing through the fovea (0), whereas plots Band
Dshow measurements spanning between the nasal and temporal ora serrata,
passing through the fovea (0) and optic nerve head (brackets; ⫺5mmto⫺7
mm). Note the extremely low integrity of the EL in the fovea (Aand B;
spanning between ⫺2mmand⫹2 mm) and adjacent to the optic nerve head
on the nasal side (⫺8mmto⫺10 mm in B). A step increase in integrity
occurs in the vicinity of the major retinal vessels (⬃⫺6mmand⫹6mminA
and B). The EL is thinnest in the macula area and increases abruptly in the
region of the vascular arcades (⬃⫺6mmto⫹6mminCand D).
Figure 4. A: A derived, color-coded schematic of the integrity of the elastic layer
(relative percent scale shown to right) of Bruch’s membrane based on the data
shown in Figure 3, A and B. The black circle represents the optic nerve head
and the curved black lines emanating from it are the approximate positions of
the major retinal vessels. Note that the integrity is lowest in the foveal region
(arrow), central macula, and on the nasal aspect of the optic nerve head. These
regions of decreased integrity correspond spatially to the distribution of the
majority of macular lesions, such as GA (B;arrows depict the margin of macular
atrophy) and CNVMs (C;arrow) that occur in individuals with AMD.
Topographical Variation in Bruch’s Membrane 245
AJP January 2005, Vol. 166, No. 1
AMD (P⫽0.003), active CNVM (P⫽0.02), and CNV/DS
(P⫽0.003). For macular thickness, significant differ-
ences were observed between age-matched controls
and donors with CNV/DS (P⫽0.008), the latter which
accounted for the apparent significant differences ob-
served in the AMD (P⫽0.035) and late-stage AMD (P⫽
0.024) groups (Table 1). These changes in EL thickness
were in the opposite direction to those seen in normal
aging. In contrast to the macula, no significant differ-
ences were found in the extramacular region between
age-matched donors and those with early- or late-stage
AMD. Importantly, no significant differences in EL param-
eters were observed between unaffected, age-matched
controls and individuals with GA. Further analyses were
made between early-stage AMD and late-stage AMD,
between active CNVM and CNV/DS, between all CNV
and GA, between active CNVM and GA, and between
CNV/DS and GA (Table 1). No significant differences in
macular or extramacular EL thickness or integrity were
revealed in these comparisons.
EL Gap Length in the Macular and Peripheral
Regions of Donors with and without AMD
The largest gap length, or discontinuity, in the EL was
assessed for each donor used in this study (Figures 9
and 10). There were no significant differences in largest
elastin layer gap in the extramacular region between the
various control and affected groups. In the macular area,
the largest gaps in the elastin layer were significantly
larger in the early AMD (P⬍0.0001), active CNV (P⬍
0.0001), and CNV/DS (P⬍0.0001) groups (in which the
average gap length was ⬃9to10
m), as compared to
both the age-matched and young unaffected control
groups (in which the average gap width was 4 to 5
m).
No significant differences were observed between do-
nors in the unaffected, age-matched control group and
those in the classic GA group (P⫽0.11).
Discussion
Bruch’s membrane likely provides a scaffold for RPE
adhesion, regulates the diffusion of molecules between
the choroid and retina, and serves as a physical barrier to
cell movement, restricting the passage of cells between
the choroid and retina.
40
Numerous changes have been
described at this interface that have the potential to dis-
rupt the normal physiology of Bruch’s membrane and its
surrounding tissues
3–13,16–19,21,22,25,28,41,42
, however
few of them help to explain the predilection of the macular
Bruch’s membrane to degradation and dysfunction that
occurs in individuals afflicted with AMD. To pursue pre-
liminary findings suggesting the presence of topograph-
ical variation in the EL between the macular and extra-
macular regions we rigorously examined the morphology
of this extracellular structure in a large series of human
donor eyes.
The Macular EL Is Thinner and More Porous in
All Individuals
In this study, we have found that the EL of Bruch’s mem-
brane is three to six times thinner and two to five times
more porous in the macular region than it is in the peripheral
region at all ages. This profound difference was not antici-
pated based on previous investigations.
4,14,17,27,43–47
It is
intriguing to speculate about the mechanisms that might
give rise to the observed topographical variation in the
Figure 5. Scatter plots showing values of EL integrity (top; percent) and thick-
ness (bottom; nm) derived from 121 individual donors, aged 1 day to 99 years.
Note that the integrity of the EL in the macular region is approximately three
times lower, on average, than that of the extramacular regions in all donors. The
thickness of the EL in the macular regions is approximately four to six times
lower, on average, than that of the extramacular region in all donors.
Figure 6. Scatter plot showing the relationship between EL integrity and
thickness for all donors examined in this investigation. Note that there is a
strong relationship between these two parameters in all regions measured
(exponential fit R
2
⫽0.8735).
246 Chong et al
AJP January 2005, Vol. 166, No. 1
EL. One explanation is that the majority of the elastin in
Bruch’s membrane is synthesized at a time before that of
the final differentiation of the macula, which occurs dur-
ing the first few years of postnatal life.
48
This concept is
supported by the fact that the majority of elastin is de-
posited in most tissues and organs during the latter
stages of gestation.
49
Elastin gene reactivation and neo-
synthesis does occur postnatally, typically in response to
injury and inflammation, however its deposition rarely
results in the reformation of ordered elastin fibers.
50,51
It is also conceivable that differential thinning of
Bruch’s membrane-associated elastin occurs during
postnatal growth of the eye and this event is more pro-
nounced in the macula because of differential rates of
growth and/or stretching in this region. This process may
be exacerbated in myopia, a condition characterized by
an increased axial length of the eye and oftentimes by the
secondary formation of macular atrophy and CNVMs.
52
Our data also suggest that an interesting regional re-
lationship between the porosity and thickness of the EL
exists (Figure 6). Increasing integrity is associated with
increasing thickness in a nearly exponential manner from
the macula to the extramacular region. This relationship,
which might prove to be predictive of AMD, will be ex-
amined in more detail in future studies.
The Macular EL in Individuals with AMD
This study also shows that the integrity of the macular EL
is significantly lower in individuals with early-stage AMD,
as compared to age-matched controls, whereas both the
thickness and integrity of the EL are significantly lower in
individuals with late-stage AMD, including those with ac-
tive CNVMs and DSs (except for those with classic GA,
as discussed below). It will be valuable to understand
better the mechanisms that lead to even greater porosity
Table 1. Comparative Analyses of Elastic Lamina Parameters Using the Mann-Whitney U-Test
Macular
thickness
Macular
integrity
Peripheral
thickness
Peripheral
integrity
Aging comparisons
⬍62 Young versus ⱖ62 age-matched 0.033 0.34 0.001 0.28
Age-matched to AMD comparisons
ⱖ62 Age-matched versus all AMD 0.035 0.003 0.77 0.94
ⱖ62 Age-matched versus early-stage AMD 0.19 0.028 0.42 0.49
ⱖ62 Age-matched versus late-stage AMD 0.024 0.003 0.92 0.55
ⱖ62 Age-matched versus GA 0.15 0.10 0.99 0.95
ⱖ62 Age-matched versus active CNVM 0.44 0.02 0.42 0.94
ⱖ62 Age-matched versus CNV/DS 0.008 0.003 0.79 0.24
AMD subgroup comparisons
Early-stage AMD versus late-stage AMD 0.25 0.45 0.40 0.12
Active CNVM versus CNV/DS 0.089 0.64 0.35 0.51
CNVM and CNV/DS versus GA 0.61 0.41 0.72 0.35
Active CNVM versus GA 0.59 0.64 0.46 0.92
CNV/DS versus GA 0.28 0.40 0.98 0.23
Significant differences are bolded.
Figure 7. Top: Box and whiskers plots showing data for EL thickness (A,B)
and integrity (C,D) derived from the macular (A,C) and extramacular (B,D)
regions of unaffected donors ⬍62 years, age-matched donors ⱖ62 years,
donors with early-stage AMD, and donors with late-stage AMD (includes
donors with GA, active CNVMs, and DSs). Median values are shown as bold
red lines within each box), first and third quartile values are represented as
blue and magenta, respectively, within the solid box, the minimum and
maximum observation ranges are shown as whiskers and extreme values are
represented as solitary, horizontal lines. One young donor with an extreme
macular elastic layer thickness of 275 nm and one age-matched control donor
with an extreme macular elastic layer thickness of 409 nm were omitted from
graph Afor scaling purposes.
Figure 8. Bottom: Box and whiskers plots showing data for EL thickness (A,
B) and integrity (C,D) derived from the macular (A,C) and extramacular (B,
D) regions of donors with early-stage AMD and subgroups of donors with
late-stage AMD, including active CNVMs [CNV (act)], DSs ( CNV/DS), and GA.
Topographical Variation in Bruch’s Membrane 247
AJP January 2005, Vol. 166, No. 1
and thinning of the macular EL in individuals with AMD.
One explanation is that the overall integrity and thickness
of the macular EL do not decrease with advancing age in
individuals who develop AMD, but rather, that these pa-
rameters are lower in affected individuals from the time of
birth. This could occur, for example, if ocular elastin
synthesis was impaired during gestation, if specific iso-
forms of elastin were synthesized that predispose indi-
viduals to disease, or if one or more components of the
elastin synthetic pathway were dysfunctional because of
mutations in the genes encoding elastin or elastin-asso-
ciated proteins. It has been established experimentally,
for example, that exposure to increased amounts of vita-
min D during gestation results in a reduction of aortic
elastin content.
53
Similarly, it has been proposed that the
synthesis of aortic elastin is deficient in fetuses with im-
paired growth.
54
Moreover, the expression of tissue-spe-
cific isoforms of tropoelastin,
55
or the enhanced expres-
sion of the wrong isoform might predispose one to AMD.
Yet another explanation for the observed decrease in
macular EL integrity in individuals with AMD is that the
elastin layer is degraded in these patients more rapidly
than it is in unaffected individuals. Along these lines,
we
20,37
and others
47,56– 60
have provided evidence that
inflammatory and immune-mediated processes, particu-
larly complement activation,
30,61
are associated with the
development of AMD. These processes are prominent at
the RPE-Bruch’s membrane interface, and are typically
more robust in the macular region than in the extramacu-
lar region. A potential consequence of these chronic
inflammatory processes, if analogous to those that occur
in a variety other age-related disease processes,
62,63
would be an overall disruption, degradation, and/or re-
modeling of Bruch’s membrane-associated elastin and
collagen. In cutus laxa, for example, a robust and dra-
matic loss of cutaneous elastic fibers is caused by local
inflammation.
64
If local, chronic inflammation participates in the de-
struction of the Bruch’s membrane, one would predict
that macular CNVM formation would be associated with
other chronic, systemic inflammatory diseases. Indeed,
the development of neovascular membranes is not lim-
ited to AMD.
65,66
On review, there are numerous immune-
mediated and inflammation-based diseases including
presumed ocular histoplasmosis syndrome, membrano-
proliferative glomerulonephritis, rubella, toxoplasma reti-
nochoroiditis, sarcoidosis, Vogt-Koyanagi-Harada dis-
ease, birdshot choroidopathy, Bechet’s disease, chronic
uveitis, and bacterial endocarditis,
67–69
in which CNVMs
can form. Importantly, the majority of these CNVMs form
within the macula, an issue that is addressed in more
detail below.
The Macular Elastic Layer in Classic GA
These data also show that the integrity and thickness of the
macular EL in individuals with GA does not vary significantly
from those of unaffected, age-matched controls. These
data imply that differences or changes in the EL of individ-
uals with AMD may be restricted to a specific phenotype(s)
or genotype(s) of the disease. Importantly, they may also
help to explain the distinct pattern of macular degeneration
that is observed in individuals afflicted with GA.
Consequences of Decreased Macular EL
Integrity in AMD
Whatever the derivation of the unique macula-associated
features of Bruch’s membrane described herein, one
would predict that any degeneration-inducing processes
and/or modifications of the structural components of
Bruch’s membrane (eg, collagen and elastin), whether
genetic or acquired, might preferentially affect the overall
function of the macular region. One consequence of an
EL that is more porous in the macula might be that there
is less substrate for RPE cell adhesion. If proven to be the
case, this would help to explain the propensity for pig-
ment epithelial cell detachments, basal laminar deposit
accumulation, and soft drusen formation—all strong risk
factors for the development of AMD—to form within the
macula.
7,16
Compromised adhesion of macular RPE cells
might also help to explain the dysfunction of these cells
that occurs in AMD.
Figure 9. Transmission electron micrographs depicting the EL of Bruch’s mem-
brane in eyes from a 82-year-old, age-matched control donor (A), an 84-year-old
donor with early AMD (B), and an 83-year-old AMD donor with active CNV (C).
The elastic layers are depicted with arrows and longest gaps in macular EL
integrity (maximum gap lengths) for each donor are bracketed.
Figure 10. Bar graph depicting the average values for the largest gap length,
or discontinuity, in the EL of donors with early-stage AMD and subgroups of
donors with late-stage AMD (active CNVM, CNVM/DS, and GA). Significant
differences in maximum gap length are noted between the both control
groups (young and age-matched) and the early AMD, active CNVM, and
CNVM/DS groups (all P⬍0.0001), but not between the age-matched control
group and the GA group (P⫽0.11).
248 Chong et al
AJP January 2005, Vol. 166, No. 1
Another consequence of a more porous macular EL
might be the predilection of CNVMs to form within the
macula. CNV—a pathological process in which neoves-
sels emanate from the choroidal vasculature, breach the
barrier imposed by Bruch’s membrane, and grow into the
sub-RPE and subretinal spaces—is associated with a
variety of disease processes,
65,66
including AMD. CNVM
formation can occur secondary to genetic alteration and
degeneration of Bruch’s membrane, such as occur in
pseudoxanthoma elasticum, Ehlers Danlos syndrome,
Sorsby’s fundus dystrophy, and Menke’s disease,
70–74
suggesting that genetic conditions resulting in elastin
breakdown can predispose one to CNVM formation.
CNVM formation may also be acquired in certain situa-
tions. For example, disruption of Bruch’s membrane that
occurs as a consequence of mechanical stress in cho-
roidal rupture, myopia, cryoinjury, ocular massage, and
manipulation of the eye during cataract extraction is
often associated with the subsequent formation of
CNVM.
23,52,75,76
Similarly, CNVM formation can be in-
duced iatrogenically in human eyes
77
and experimentally
in monkey eyes by damage to Bruch’s membrane after
laser photocoagulation.
32
One recent study shows that
there is a predilection of the macula to a higher incidence
of CNVM formation after intense laser photocoagulation
in monkeys.
78
Moreover, in mouse models of CNV
79,80
the majority of data provide evidence that neither choroidal
nor retinal neovessels are capable of breaching Bruch’s
membrane unless it is first disrupted,
79–81
consistent with
the notion that a robust Bruch’s membrane may safeguard
against CNV, although one investigator has suggested that
mechanical disruption is not prerequisite.
82
Thus, it seems clear that the structural components of
Bruch’s membrane must be compromised for CNV to
occur and it is reasonable to postulate that a neovascular
breach of Bruch’s membrane is primarily opportunistic,
after a failure of its barrier function. However, a satisfac-
tory explanation for this topographical predilection of
CNVM formation to occur in the macula has not been
made previously.
We propose that the data related to macular EL integ-
rity may help to explain this predilection. In the proposed
model of inflammation-induced destruction of Bruch’s
membrane, as an example, the macular region might
likely be compromised before that of the extramacular
region, based on the morphological characteristics of
Bruch’s membrane described herein. In other words,
pan-choroidal inflammation would be expected to have a
more rapid and dramatic effect in the macula, as com-
pared to other regions, because of the more porous
nature of its elastin layer (Figure 11A). Ironically, the
elastin peptides that might be generated as a conse-
quence of elastin fiber destruction in the macula could
play a key role in subsequent neovascular events be-
cause of their highly angiogenic properties.
83,84
In addition, the observation that the largest gap
lengths (discontinuities) within the macular EL are signif-
icantly longer in individuals with AMD (with the exception
of eyes with GA) than in age-matched controls may also
help to explain the predilection toward CNVM formation in
some eyes. For a neovessel to grow from the choroid into
the sub-RPE space, its cells must be able to pass phys-
ically through Bruch’s membrane. Although the smallest
theoretical pore through which choroidal neovessel-as-
sociated endothelial cells can migrate has not been es-
tablished to our knowledge, it is intuitive that the larger
gaps observed in some AMD donors would more readily
allow penetration by developing neovascular fronds.
Along these lines, it is interesting to note that the majority
of images in the literature that depict early choroidal
neovessels penetrating Bruch’s membrane show gaps
greater than 5
m in diameter. Our data show that many
donors with AMD, except for those with GA, have EL gap
widths greater than 5
m. These data also imply that the
decreased integrity observed in many of our AMD donors
can be accounted for pores that are larger in diameter
than in the unaffected controls.
Summary
In summary, the results of this study have documented
the existence of unique structural features associated
with the macular EL of Bruch’s membrane. These at-
tributes correspond spatially to the distribution of the
majority of macular lesions, including GA, CNV, and DS
formation that are associated with AMD, a leading cause
of blindness throughout the world. Our current working
hypothesis is that insults to the structural integrity of
Bruch’s membrane, whether mechanical, inflammatory,
or compositional in nature, are prerequisite for invasion of
choroidal neovessels into the sub-RPE and/or subretinal
spaces, even in conditions in which blood vessel growth
is abnormally enhanced. By extension, we suggest that
once the macular EL is compromised, CNVM formation is
likely to reoccur after mechanical or pharmaceutical de-
struction of an initial neovascular membrane (Figure
Figure 11. Models depicting possible scenarios that might lead to the en-
hanced susceptibility of the macula to CNVM formation (A) and CNVM
recurrence (B). We have proposed that AMD-associated pathways, such as
inflammation, that degrade or disrupt elastin might affect the barrier function
of Bruch’s membrane in the macular region before that of the extramacular
region because the elastic layer is the thinnest and most porous in this region
(Aand B,lane 1). The resulting breaches of Bruch’s membrane in the
macula (asterisks;Aand B,lane 2) might allow CNVMs to penetrate
Bruch’s membrane and to grow into the subretinal and/or sub-RPE spaces. In
treatments intended to destroy these neovessels, such as laser photocoagu-
lation and photodynamic therapy (wavy lines;B,lane 3), the breached
region of Bruch’s membrane might be replaced by fibrotic scar tissue (hatch-
es;B,lane 4). Subsequently, the process of elastin degradation and neovas-
cularization would continue at thinned, porous regions of the EL lying
adjacent to that of the original vascular penetration (B,lanes 4 and 5).
Ultimately, this process might continue until a scar formed over that portion
of the macula (hatches;B,lane 6) where the EL was initially the thinnest
and most porous.
Topographical Variation in Bruch’s Membrane 249
AJP January 2005, Vol. 166, No. 1
11B). Indeed, this phenomenon of macular neovessel
recurrence is frequently observed after CNVM treatment
modalities.
85,86
We conclude that the quantitatively dis-
tinct features of the EL may help to explain the enhanced
susceptibility of the macula to degeneration in AMD and
other diseases characterized by neovascularization in
the macular region.
Acknowledgments
We thank Dr. Stephen Russell for stimulating discussions
and for evaluation of human donor eyes; Dr. Robert
Mecham for his kind gift of anti-elastin polyclonal anti-
bodies; James Borchardt, Ryan Lee, Amber Bain, Sheri
McCormick, and Aleks Rozek for logistical assistance;
Sepidah Amin and Karen Gehrs for helpful discussions;
the staffs of the Iowa Lions Eye Bank, MidAmerica Trans-
plant Services, the Heartland Eye Bank, the Central Flor-
ida Lions Eye and Tissue Bank, and the Virginia Eye Bank
for their dedication in procuring the eyes used in these
studies; and all of the families who unselfishly donated
eyes of loved ones to this study.
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