Investigative Ophthalmology & Visual Science, Vol. 30, No.

8, August 1989
Copyright Association for Research in Vision and Ophthalmology

Cell Loss in the Aging Retina

Rebrionship to Lipofuscin Accumubrion ond Mocubr Degenerorion
C. Kathleen Dorey,*t Gloria Wu,*t4: David Ebensrein,* Armando Garsd,* and John J.

We examined the impact of aging on the numbers of photoreceptors and retinal pigment epithelium
(RPE) cells, and the number of photoreceptors per RPE cell profile, in selected regions of 30 human
eyes. The mean ratio of photoreceptors to RPE cell was higher in the macula than in the paramacula
(P < 0.01) or the equatorial area {P < 0.001). We found evidence for an age-related loss of RPE in both
whites (P < 0.02) and blacks (P < 0.0006), although the rate of loss in whites was significantly slower
than in blacks. Photoreceptor loss in blacks was inversely correlated with age (P < 0.04). In whites,
however, photoreceptor loss was very significantly and directly correlated with lipofuscin concentra-
tion in the opposing RPE (P < 0.0001) and unrelated to age. The disparity in the rates of photorecep-
tor and RPE cell loss produced, in older eyes, a higher ratio of photoreceptors per RPE cell profile. In
the macula, the ratio for whites over 50 years of age was significantly higher (P < 0.05) than that in
blacks over 50. Our data suggest that the increased phagocytic and metabolic load on the RPE cell in
the macula causes a preferential age-related accumulation of lipofuscin in the RPE, which ultimately
leads to photoreceptor death. This may prove a useful model of age-related macular degeneration and
Stargardt's disease. Invest Ophthalmol Vis Sci 30:1691-1699,1989

The predilection of age-related retinal degeneration
for the macula has never been explained. One possi-
ble explanation is that macular degeneration occurs
when lipofuscin accumulation in the RPE cell
reaches a threshold. We and others have reported that
the RPE in this area exhibits the greatest accumula-
tion of lipofuscin1'2 and that the annular pattern in
macular degeneration is remarkably well correlated
with the area of greatest lipofuscin concentration.3
The preferential accumulation of lipofuscin in the
macula may be partially explained by the topo-
graphic distribution of RPE melanin and the negative
linear relationship between lipofuscin and melanin
concentrations in individual human RPE cells.1
However, since the coefficient of determination, r2,
was only 0.28 (P < 0.001) for melanin, other factors
may also influence lipofuscin concentration (eg, age,
genetics or light damage). Moreover, although both
blacks and whites exhibit age-related accumulation of
lipofuscin,4 the rate of accumulation4 and the quanti-
From the *Macular Disease Research Center, Eye Research In-
stitute, and fDepartment of Ophthalmology, Harvard University,
and ^Retina Associates, Boston, Massachusetts.
Presented in part at the 59th Annual ARVO Spring Meeting,
Sarasota, Florida, May 1987.
Supported by grant EY-06544 from the National Eye Institute
(CKD), and the Louis Morganstern Memorial Scientist Fund.
Submitted for publication: May 18, 1988; February 27, 1989.
Reprint requests: C. Kathleen Dorey, PhD, Eye Research Insti-
tute, 20 Staniford Street, Boston, MA 02114.
ties within the RPE cell1 are greater in whites who are
at greater risk for macular degeneration.5'6 To date,
no direct evidence exists to show that retinal degen-
eration is due to lipofuscin.
Both the site specificity of degeneration and the
increased lipofuscin in the macula have been attrib-
uted to increased exposure to environmental light.78
However, a study by Kooijman,9 recently confirmed
by Pflibsen and colleagues,10 revealed no evidence of
increased retinal illumination in the posterior pole,
but rather a homogeneous retinal light distribution
regardless of pupil size. Thus, if the amount of light
exposure is uniform, the higher levels of lipofuscin
accumulation must depend on factors other than
light.
Most of the lipofuscin within the RPE may be
safely assumed to derive from phagocytosis of oxida-
tively damaged (and nondegradable) lipids in the
photoreceptor outer segments.11"14 Therefore, RPE
cells phagocytosing greater quantities of outer seg-
ment lipoids may accumulate more lipofuscin. Evi-
dence for this concept is found in a study by Katz et
al15 that demonstrated that lipofuscin deposition de-
creased significantly in retinal degenerate (RD) rats
only after photoreceptors were lost. In a study of the
aging human macula, Gartner and Henkind16 re-
ported a reduced thickness in the outer nuclear layer
and an increase in the number of distally displaced
photoreceptor nuclei; they interpreted the latter find-
ing as evidence of a photoreceptor loss. Since the loss

1691

Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933379/ on 08/17/2017

 1692 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Augusr 1989
RETINAL REGIONS EXAMINED
TE - TEMPORAL EQUATOR
PF - PARAFOVEA
u_
FOVEA
NP - NASAL POSTERIOR POLE
NE - NASAL EQUATOR
Fig. 1. Retinal sites studied.
of photoreceptors in the macula should theoretically
decrease the lipofuscin content of the RPE, these ob-
servations suggested to us that excessive lipofuscin in
the macular RPE might cause photoreceptor degen-
eration or that both age-related changes might share a
common etiology. In fact we previously described a
significant correlation between the accumulation of
lipofuscin and the loss of photoreceptors in whites.4
The current study examined cell loss in the aging
retina and its impact on the photoreceptor/RPE
complex. We report thefirstevidence that: (1) there is
an age-related increase in the phagocytic and meta-
bolic load on the macular RPE; and (2) that lipofus-
cin accumulation is correlated with photoreceptor
loss in the human macula.
Materials and Methods
Thirty donor eyes with no known ocular pathology
were studied: 15 from whites (age range 2 weeks to 88
years, mean age 50 years) and 15 from blacks (age
range 8 to 84 years, mean age 49 years). Through
randomization when necessary, only one eye was in-
cluded for each donor.
To obtain general topographic information on the
photoreceptor and RPE nuclei, counts were made at
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933379/ on 08/17/2017
Vol. 30
regular intervals between the fovea and periphery in
several eyes. Based on these results, six sites per eye
were selected for counting of the photoreceptor and
RPE nuclei: the fovea, the nasal posterior pole, two
parafovea, and the two equatorial sites (Fig. 1). The
parafoveal sites were located at half the distance from
the fovea to the disc at either side of the fovea. The
distance between the fovea and the disc was measured
on the nasal aspect of the posterior pole, that point
being designated the nasal posterior pole.
The lipofuscin fluorescence measurements pre-
viously reported1 were also obtained from the same
sites in these eyes.
Donor eyes, obtained less than 12 hr after death,
were fixed in formalin and embedded in paraffin.
Pupil-optic nerve sections 8 jim thick were cut
through the fovea, mounted on slides and deparaffin-
ized, and coverslipped in ultraviolet-inert immersion
oil. Unstained slides were used for lipofuscin mea-
surement; adjacent sections were stained with hema-
toxylin to facilitate counts of nuclei. A duplicate ad-
jacent section was not always available for staining,
so-final counts of RPE nuclei were made on 19
dtfiiors: ten blacks (ages 8, 20, 27, 39, 40, 46, 53, 65,
69 and 80; mean = 43.7 years) and nine whites (ages
6 weeks, 10, 28, 36, 61, 70, 73, 82 and 88; mean = 51
years). Sites were examined using a Zeiss photomi-
croscope III at a magnification of X320. Photorecep-
tor nuclei were counted within a 90 ^m long X 8 /xm
deep area from each site. Estimates of photoreceptor
density assumed triangular spacing. Photoreceptor
and RPE data from identical regions were used to
obtain a ratio for that region.
RPE cell sizes were estimated from the number of
nuclei within an area 90 /tm long X 8 /xm deep (the
thickness of the section). To calculate the diameter of
the cell, we assumed that each nucleus represented a
whole cell, and that the monolayer was composed of
equal sized hexagons with an area in /xm2 of 3.5 R2,
where R is the perpendicular distance in nm from the
middle of a side to the center of the hexagon.
Statistical Methods
Before further analysis, extreme values in each
group were examined to identify aberrant observa-
tions; none were found. The effect of age on any
given variable was first assessed by simple linear re-
gression; however, to compare the relative effects of
several variables on the formation of lipofuscin, and
the loss of photoreceptors or RPE cells, multiple lin-
ear regressions were performed.
To evaluate a model of photoreceptor loss, the
model submitted for statistical analysis expressed the
number of photoreceptors (PRN)j in the macula of a
 No. 8 ELEVATED PHAGOCYTIC LOAD ON RPE CELLS IN THE AGING MACULA / Dorey er ol
single individual (i) as a possible function of that per-
son's age, race, and the level of RPE lipofuscin (LF)
in relative fluorescence units. Since race was known
to influence ratio and lipofuscin concentration, to
permit statistical recognition of a racial influence the
model was expanded by interaction terms (ie, the
product of the variable and a race indicator (RI) set
equal to 1 for whites and 0 for blacks). Specifically,
our model for statistical analysis was the following.
For each individual i (i = 1 , . . . , 30):
PRNi = Bo + B,(Age)i + B2(LF)i + B3(RI)i
+ B4(RI)i X (LF); + E; (1)
where all the Bs are unknown parameters to be esti-
mated from the data and where Ej is a random term.
To further identify the variables that might be in-
fluencing lipofuscin accumulation, we modified
equation (1) as follows. We included age in years, the
number of photoreceptors (PRN)i opposing the RPE
in the area of lipofuscin (LF); measurement, and a
race indicator (RI)i equal to either 1 or 0 as before.
For each individual i (i = 1,.. ., 30):
LFj = Bo + B,(Age)i + B2(PRN)i + B3(RI)i
+ B4(RI)i X (Age)i + B5(RI)i X (PRN); + E; (2)
We applied the Median Test17 to determine
whether there were significant differences in the pho-
toreceptor load on the RPE cell (ie, the ratio (R) of
photoreceptors to RPE cell) in eyes of different ages
or races, or in different areas within an eye. Since the
plane of section would only rarely coincide with the
diameter of the cell, we have expressed this ratio as
the number of photoreceptors per RPE cell profile.
The Median Test, a variation of the Chi-square test, is
completely nonparametric and makes no assump-
tions about the distributions of ratios. In those cases
where significance was not found with the Median
Test, we confirmed the result with the more powerful
Mann-Whitney test17 (after first testing for equality of
variances with the Ansari-Bradley procedure).
Results
Photoreceptor Number
Figure 2 presents the mean and standard deviation
of paired photoreceptor and RPE cell counts in six
adjacent 15 nm long fields in the macula of 19 indi-
vidual eyes for which RPE cell counts were also avail-
able. The means in the paramacular and equatorial
regions :weresdetermined from sixfieldsin each of the
nasal and temporal sites. The number of photorecep-
tors per 720 /urn2 area in the macula varied from over
200 in ah 8-year-old eye to a low of 75 in an 80-year-
old eye. Within one individual, counts varied very
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933379/ on 08/17/2017
1693
little, and the standard deviations were usually less
than 10% of the mean. In the parafovea, the photore-
ceptor counts ranged from 115 to a low of 50 in the
same 80-year-old eye; standard deviations were rarely
more than 20% of the mean. There was greater heter-
ogeneity in the density of photoreceptors in the equa-
torial region; 11 out of 18 eyes exhibited greater than
20% variation. Although the regression line fitted to
the data indicated an annual decrease of 1-2% in the
photoreceptor number in the macular and para-
macular regions, none of the regions was found to
exhibit a significant age-related loss of photorecep-
tors.
When multiple stepwise regression was used to
identify the variables that influenced the number of
photoreceptors remaining [Eq. (1) in Methods], the
only significant factor was race. Further analysis on
separate racial groups revealed that the determinant
with the most significant association with the number
of photoreceptors remaining was not the same in
black and white eyes. The number of photoreceptors
remaining in the macula of blacks was inversely re-
lated to age. The final model for blacks was: PRN4
= 22 - 0.06 (Age);; (P < 0.006, r2 = 0.43). However,
in whites a strong inverse relationship was deter-
mined between photoreceptor number and the
amount of lipofuscin in the opposing RPE. The rela-
tionship illustrated in Figure 3 was: PRNj = 23.7
- 0.01 (LF)J; (r2 = 0.59; P < 0.0001).
Age was not a significant regressor of photorecep-
tor number in whites (P < 0.9), even though the ac-
cumulation of lipofuscin was age-related, and the in-
dividuals with highest lipofuscin values ranged in age
from 73 to 88 years. The final models predicted very
similar starting numbers of photoreceptors for both
blacks (22/90 ixm) and whites (23.7/90 ^m). Because
displaced photoreceptor nuclei have been considered
evidence of photoreceptor loss, we also considered
their number. Displacement of photoreceptor nuclei
in the macular region was constant in blacks and
whites of all ages (Fig. 4), and the number of dis-
placed nuclei did not correspond to the rate of pho-
toreceptor loss.
Increase in Lipofuscin
Equation (2) in Methods proposed several possible
factors related to the amount of lipofuscin. Among
these, age, race and, in whites only, the number of
photoreceptors were found to be significant. The final
equation found in Table 1 indicated that the amount
of lipofuscin in the RPE of an individual can be satis-
factorily predicted by adding: (1) a basal value (Bo)
and (2) the amount accumulated during that individ-
ual's life. In whites, additional components adjust for
 1694 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / August 1989 Vol. 30

PR - MACULA
220
\

170 i
120 M
1

I
ii

MX
]
70 -
I
* ! 0

20 -
O 20 100

o> E
CO o

LU
cs>
O
LU LU

O o
LU
5 140
20 40 60 80 100
Q.
CC
PR - EQUATORIAL
120 -

100

80 -

60 -

40

20
FT
20 40 60 80 100 40 60 100
AGE (yrs) AGE (yrs)

Fig. 2. Counts of photoreceptors (left) and the overlying RPE cells (right) observed in three retinal regions of 18 human eyes. Mean and
standard deviation of six adjacent microscopefieldsof 15 ^m each are presented in the macula. In the other retinal regions, two areas of six
adjacentfieldswere counted on opposite sides of the fovea, and combined for calculation of the mean. Superimposed regression lines were not
significant for the photoreceptors, but were for the RPE cells,.

their greater lipofuscin accumulation and for the
greater quantity of lipofuscin observed in sites with
fewer photoreceptors. Race interacts with PRN sig-
nificantly. The overall coefficient of determination
(r2) for the final equation in Table 1 was highly signif-
icant, and an excellent fit of the data (r2 = 0.79; P
< 0.0001).
Loss of RPE
The number of RPE nuclei in the posterior pole
decreased significantly with age in whites (Bi
= -0.05, P < 0.02, r2 = 0.64) and in blacks (B,
= -0.07; P < 0.0006; r2 = 0.46); the difference in the
slopes was highly significant (P < 0.0001). Among the
eyes over 20 years old the mean RPE cell count was
8.1 3 , representing an estimated mean area of 88.9
nm2, and a minimum diameter of 10.1 nm.
Phagocytic Load
The rates of cell loss in photoreceptors and RPE
cells were not equal in this sample. In order to assess
the effects of photoreceptor and RPE cell loss on the
relationship between them, we determined the
phagocytic load on the RPE, that is, the ratio of the

Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933379/ on 08/17/2017

 No. 8 ELEVATED PHAGOCYTIC LOAD ON RPE CELLS IN THE AGING MACULA / Dorey er ol 1695

number of photoreceptors to the number of RPE cells o

in the six areas of each eye (Fig. 5), and compared the z
ratios of various regions via the Median Test. Our 25-
results, presented in Table 2, demonstrate that the PR = -(0.01[LF]) + 23.7
ratio is significantly higher in the macula than in the
r 2=0.I
paramacula (P < 0.01), or in the periphery (P
< 0.00002). Moreover, the RPE cells in the macula of p < 0.0001
whites over 50 years of age oppose more photorecep-
tors than do those in the macula of older blacks (P
< 0.05). In fact, the ratio in older whites was signifi-
cantly higher than that found in all other maculas (P
< 0.015). When age effects were not included we were K
unable to identify significant racial differences in the o
ratios found in the macula. X
0 500 1000 1500 2000
Cursory inspection of the data indicated that the
regions closer to the macula tended to have higher RPE LIPORJCSIN CONCENTRATION
(arbitrary units)
ratios. Statistical comparisons of these regions in eyes
of all ages and both races determined that the tem- Fig. 3. Relationship between lipofuscin concentration and the
poral paramacula had more photoreceptors per RPE number of photoreceptors found in the overlying macula of 15
whites. Each data point represents the mean of six independent
cell profile than did the nasal (P < 0.05). If only those observations in the macular region of a single eye. A similar but less
over 50 years of age were considered, the differences significant (P < 0.02) relationship was found in the paramacula.
in ratio were more significant (P < 0.02). Similarly,
the temporal equator had much higher ratios than did
the nasal equator, whether eyes of all ages were con- no correlation between their number and age. Our
sidered (P < 0.001) or only those of donors over 50 study showed an average of 2.8 + 1 displaced photo-
years of age (P < 0.00002). receptor nuclei (DN) per 90 /*m in the fovea, compa-
rable to the average of 33 DN/1500 jim found by
Discussion Gartner and Henkind in 50-70-year-olds. If each dis-
placed nucleus died within 1 year, the rate of photo-
Aging changes are associated with cell loss and de- receptor nuclear loss would be six times that actually
generation in different parts of biological systems.18 observed in our data. This suggested that either cells
In the human retina an age-related loss of ganglion with displaced nuclei required many years to die or
cells has been identified by examining axonal densi- that nuclear displacement was not related to cell
ties in the optic nerve,19 and age-related loss of pho- death. We also found that photoreceptor loss was
toreceptors has been inferred from morphologic stud- constant over age. Therefore we concluded that dis-
ies,20 and from pyschophysical demonstrations of placement of nuclei is not a valid indicator of pho-
age-related declines in color perception,21 visual toreceptor death.
acuity,22 sensitivity23 and foveal cone pigment den-
sity.24 However, we know of no previous attempts to
identify possible causes of cell loss in the retina. E
m 0 "
Photoreceptor Density LEGBJD
16 n FOVEAL
In 1981 Gartner and Henkind reported an obvi- UJ 5" PARAFOVEAL D
ous reduction in the thickness of the outer nuclear _j
layer (ONL) of the retina in older eyes, but did not O 1 DD a
z> 4- D D

count the nuclei remaining. They also described a z

distal displacement of photoreceptor nuclei seen in- oc 3- a D

creasingly in the macular area after age 40. They in- Q.

terpreted the displacement as a stage of cell loss and a aa a a* c
UJ 2- a
concluded that photoreceptor loss was accelerated in o
those over 40 years of age. Also not counting the 1-
nuclei in the ONL, Lai and Rana25 found signifi- _ 0 20 40 60 80 100
cantly more displaced nuclei in the retina of a 20- a AGE (yrs)
year-old monkey than in a 4- or 10-year-old retina. Fig. 4. Number of photoreceptor nuclei displaced into the outer
We too observed distally displaced nuclei but found segment region observed within high-power fields of 15 /<m.

Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933379/ on 08/17/2017

 1696 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1989 Vol. 30

Table 1. Significant covariates of lipofuscin
in human RPE
LF> =
B0 + B1(Age)i + B2(RDi "r- B5(RI)i(!PRN)i + E;
(r2 = 0 .79, P < 0.0001)
Variable B P value
Baseline 2.46
Age 0.004
Rit 1.37
(RI) X (PRN)J - 1.17
* LFj = the lipofuscin in individual i.
t RI = race indicator: 1 = whites, 0 = blacks.
% (RI)j X (PRN)j is the product of the race indicator and the number of
photoreceptors, a term to recognize race-related interaction in loss of pho-
toreceptors.
In these data the maximum peak foveal cone den-
sity was 1.9 times greater than the minimum found in
four retinas with mean age 36.7 years; Curcio et al
found that the foveal cone density in four adult
human retinas (mean age 35) exhibited a 2.9-fold
range of variation.26 The latter group reported a fo-
veal cone density of 200,000 cells/mm2 or a linear
cone spacing of 1 cone/2.2 fim. This value corre-
sponds well with Osterberg's data27 and with the cone
spacing of 120 cones/degree (120 cones/270 ^m) or 1
cone/2.2 /um determined from physiological optics.
In the macaque monkey similar foveal cone densities
were found in studies by Curcio and colleagues26 and
by Schien.28 In order to compare our values for foveal
cone densities it is necessary to process the data to
adjust for the geometry of our samples. We counted
photoreceptors in a 720 /tm2 area (90 /im long by 8
^m thick tissue section). The maximum cone density
estimated from our data was 125 cones/720 ^m2.
This indicates a cone spacing of 1 cone/5.76 /xm2 or a
0.0001 linear spacing of 1 cone/2.4 /xm in the fovea of the
0.0001
new born.
0.0002
Lipofuscin
These data provide possibly the first evidence for
the concept that increased lipofuscin correlates with
photoreceptor loss.3'29"31 The greatest lipofuscin ac-
cumulation in whites was associated with the greatest
photoreceptor loss, (ie, photoreceptor number was
negatively correlated with the accumulation of lipo-
fuscin but not with age). The RPE of some whites had
more lipofuscin than would be expected for their age;
the same eyes had lower numbers of photoreceptors.
Although we demonstrated a significant age-depen-
dent accumulation of lipofuscin in both blacks and
whites, lipofuscin was not a strong determinant of
photoreceptor loss in blacks. This apparent contra-
diction may indicate that below a critical threshold
lipofuscin does not have negative impact on the sur-
vival of photoreceptors. Studies in other model sys-
tems have also suggested that lipofuscin accumula-
tion may reach a threshold which compromises sur-
vival.3132 We consider that lipofuscin accumulation
caused photoreceptor loss and not vice versa, since
Katz and Eldred demonstrate^ that loss of photore-
ceptors in rats resulted in lower levels of lipofus-
cin.15'33 In both of their models, photoreceptor loss
preceded a decrease in lipofuscin. A dying photore-
ceptor might temporarily increase the load on an
RPE cell, but since a photoreceptor is replaced ap-
proximately every 10 days, the decrease in the
amount of material processed will be significant after
only a few months.

RPE Cells
Tso and Friedman34 in 1968 showed that the num-
ber of RPE cells decreased in the posterior pole with
age. Our data supported this; the number of RPE cells
decreased significantly with age both in blacks (P
< 0.02) and in whites (P < 0.01). Also agreeing with
the work of Tso and Friedman, our study found no
PF F PF NP NE significant changes in the equatorial regions.
RETINAL REGION In this study and in others,2 an age-related increase
Fig. 5. Ratio of mean photoreceptor number per RPE cell profile in the height of the RPE has been observed. Unfortu-
in each of six 720 nm2 regions of 19 eyes. Abbreviations as in Figure nately, these data have sometimes been erroneously
1. cited as evidence that RPE cells become taller and

Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933379/ on 08/17/2017

 No. 8 ELEVATED PHAGOCYTIC LOAD ON RPE CELLS IN THE AGING MACULA / Dorey er ol 1697

Table 2. Comparisons of photoreceptor to RPE ratios in regions of human eyes

Region 1 vs. Region 2

Macula vs. Macula Result Significance

Ages Race Ages Race P value*

All W All B A= B N.S.

>50 W >50 B A>Bf 0.05
<50 W <50 B A= B N.S.
>50 All <50 All A= B N.S.
>50 W All others A> B 0.05

Macula vs. Paramacula

All All All All A>B 0.01
>50 All >50 All A>B 0.01
Macula vs. Equator
All All All All A> B 0.0001
>50 All >50 All A>B 0.05
T-Paramacula vs. N-paramacula
All All All All A> B 0.05
>50 All >50 All A> B 0.05
T-Equator vs. N-Equator
All All All All A> B 0.001
>50 All >50 All A>B 0.001

W = whites; B = blacks; T = Temporal; N = Nasal. * Via Median Test,

N.S.: Not Significant. t A > B: A is significantly greater than B.

narrower with age. In fact we know of only two pre-
vious studies that have examined the diameters of
RPE cells in various regions of the eye, only one of
which considered the effect of age. Young's study of
two young monkeys35 was consistent with our data in
the approximate number of photoreceptors per RPE
cell and in the location of the highest ratio of photo-
receptors to RPE cell in the posterior polein the
human macula and in the monkey "perifovea."
Young has made the only precise measurement of
RPE cell areas; in the young monkey fovea and para-
fovea the cells occupy areas of 186.3 and 336.2 ^m2,
respectively.35 Assuming hexagonal packing, the di-
ameters would be 14.6 in the fovea and 19.6 in the
parafovea. Tso and Friedman34 estimated the average
RPE diameter to be 14 nm in the human posterior
pole.
Streeten36 has extensively studied the development
of the RPE from fetal through adult life. She reported
regional differences in the size of the human RPE cell
and an age-related increase in pleomorphism, but de-
scribed an age-related increase in size only in the
areas adjacent to the ora. A further comment in the
discussion that "cell densities tended to decrease in
all areas but the macula, where they tended to in-
crease" has been misapplied to the aging macula.
Streeten demonstrated that between the ages of 0 and
3 years, RPE cell density increased sharply in the
macula and decreased in all other regions. In the eight
pairs of eyes over age 20 in her study, the RPE cell
density ranged from 29-^5 cells/0.004 mm2 or 29-35
cells/3969 nm2. The area of an RPE cell would there-
fore range from 113-137 nm2 and, assuming hexago-
nal packing, the diameters would be from 11.4-
12.5/xm.
In our sample of 15 eyes over 20 years of age, the
mean count of RPE cells in the macula was 8.1 3
cells/720 nm2. With hexagonal packing the average
adult RPE cell would occupy 88.9 ^m2, and would
have a diameter of 10.1 /im, considerably smaller
than the 14 ^m previously reported for macular RPE
cells.34'35 However, our enumeration of RPE cells
considered each nucleus to be equivalent to a cell. In
fact many of the nuclei counted would not represent
cells wholly within the section, and the number of
RPE cells would be overestimated. Consequently
both the size of the RPE cell and the number of pho-
toreceptors per RPE cell profile are underestimates of
the true situation. Based on the discrepancy in size
estimation our data may overestimate RPE cell den-
sity, and underestimate phagocytic load by as much
as 40%. It is likely that Streeten's counting technique
also overestimated the actual number (and underes-
timated RPE size), since she reported that decreasing
the size of the counting window caused an increase in
the estimate of cell density. Although we could not

Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933379/ on 08/17/2017

 1698 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Augusr 1989
reliably distinguish all multinucleated RPE cells,
their contribution to the overestimate would be
small; only 3% of human RPE are binucleate.37
Phagocytic Load
The relative increase in the number of photorecep-
tors served by the older macular RPE cell is a conse-
quence of a disproportionate loss of RPE cells vis-a-
vis the photoreceptors. The phagocytic load on the
RPE can be estimated by considering the ratio of
photoreceptors to RPE cell. We cannot be sure that
the observed increase is entirely due to age, since our
sample of those over 50 years of age contained more
eyes from whites than from blacks. However, it is
clear that the ratio in the macula of older whites is
higher than in any other group. Our sample indicated
that the discrepancy in the rates of cell loss was
greater in whites than in blacks. The increase in ratio
implies that when one RPE cell dies the adjacent cells
expand to fill the space. As the number of photore-
ceptors per remaining RPE cells increases, the meta-
bolic demands on the individual RPE cell will also
increase. Theoretically, the aging RPE cells would
also bear larger phagocytic loads, and consequently
accumulate lipofuscin at a faster rate. Both the
greater accumulation of lipofuscin in whites1 and the
increase in rate of accumulation in older eyes1'2 could
be the natural result of the increased phagocytic load
on the older RPE cells.
Could the observed increase in ratio be incorrect?
As indicated above, all of our estimates of phagocytic
load are very conservative. However, since loss of
RPE is considered the underlying process in the so-
called dry type of macular degeneration, we cannot
exclude the possibility that the older whites in this
case had a prodermal stage of age-related macular
degeneration, and that our observations simply dem-
onstrate early events in this process. Moreover, al-
though our data were carefully obtained and the
differences observed were highly significant, we
recognize the presence of an uncontrolled variable
swelling in the macula after death.2 Our data were
obtained from donor eyes and we may safely assume
that none was fixed sooner than 2 hr after death. It is
conceivable that disproportionate lateral swelling in
the macular RPE cells could contribute to the ob-
served increase in the ratio. However, if we accept
this argument we must also accept its corollary, that
the macula swells more in older whites. In either case
our data present strong evidence that the RPE cells in
the macula of older whites can not be considered
equivalent to the RPE cells in other regions or to the
macula of younger eyes.
In conclusion, we have demonstrated that the
number of photoreceptors per RPE cell is higher in
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933379/ on 08/17/2017
Vol. 30
situations characterized by elevated levels of lipofus-
cin: in the macula more than the periphery, in whites
more than blacks, in older eyes more than younger
ones. Moreover, we have identified: (1) a strong nega-
tive correlation between the amount of RPE lipofus-
cin and the number of photoreceptors present in the
eye; and (2) that age seems to exacerbate the demands
on the RPE, particularly in the macula. Not only is
there an age-related elevation in RPE lipofuscin, but
also a discrepancy between the rates of loss of pho-
toreceptors and RPE cells, producing higher ratios of
photoreceptors to RPE cell in the macula of older
individuals. Taken together, these two lines of evi-
dence suggest the following sequence of events: (1)
the unequal loss of photoreceptors and RPE cells
causes (2) an increase in the phagocytic load on the
RPE cells, particularly in the macula; (3) the elevated
phagocytic load causes the well documented greater
RPE lipofuscin accumulation in the macula, which
may in turn (4) accelerate photoreceptor death. Alter-
natively, (5) the extensive compromise of RPE cyto-
plasm by massive accumulations of lipofuscin may
compromise essential RPE functions enough to in-
crease the vulnerability of the macular RPE in older
whites or even (6) cause the death of the photorecep-
tors and/or RPE cells. We therefore hypothesize that
age-related macular degeneration may be lipofuscin-
related. This hypothesis predicts that factors in-
creasing RPE lipofuscin, including genetic factors
(Stargardt's disease, fundus flavi,38 cone and rod de-
generation,39 Best's disease,40 decreased ocular pig-
mentation16), environmental factors (light-induced
retinal damage1433), or nutritional deficiencies in vi-
tamins C and E7'41 would increase the risk for degen-
eration of photoreceptors, RPE cells,32 or both.
Key words: lipofuscin, cell loss, photoreceptors, retinal pig-
ment epithelium (RPE), phagocytic load, age-related macu-
lar degeneration, Stargardt's disease
Acknowledgments
The authors are grateful to Susan A. Curran for editorial
assistance and to Drs. Stanley Schein of Massachusetts Eye
and Ear Infirmary and Yoshihura Torige of UC Irvine for
their advice concerning the relationship between nuclear
counts and actual cell number in tissue sections. We thank
Dr. Francois Delori for his critical reading of the manu-
script and for many helpful discussions during the course of
this work. The assistance of Karlotta Fitch made it feasible
to use the specimens on which well documented lipofuscin
measurements had already been made. The support and
encouragement of Mr. David Dresnick is gratefully ac-
knowledged.
References
1. Weiter JJ, Delori FC, Wing GL, and Fitch KA: Retinal pig-
ment epithelial lipofuscin and melanin and choroidal melanin
in human eyes. Invest Ophthalmol Vis Sci 27:145, 1986.
 No. 8 ELEVATED PHAGOCYTIC LOAD ON RPE CELLS IN THE AGING MACULA / Dorey er ol
2. Feeney-Burns L, Hilderbrand ES, and Eldridge S: Aging
human RPE: Morphometric analysis of macular, equatorial
and peripheral cells. Invest Ophthalmol Vis Sci 25:195, 1984.
3. Weiter JJ, Delori FC, and Dorey CK: Central sparing in annu-
lar macular degeneration. Am J Ophthalmol 106:286, 1988.
4. Wu G, Ebenstein D, Weiter JJ, and Dorey CK: Photoreceptor
nuclei: A relationship to aging and to lipofuscin. Invest
Ophthalmol Vis Sci 28:263, 1987.
5. Gregor Z and Joffe L: Senile macular changes in the black
African. Br J Ophthalmol 62:547, 1978.
6. Weiter JJ, Delori FC, Wing GL, and Fitch KA: Relationship of
senile macular degeneration to ocular pigmentation. Am J
Ophthalmol 99:185, 1985.
7. Katz ML, Parker KR, Handelman GJ, Bramel TL, and Dratz
EA: Effects of antioxidant nutrient deficiency on the retina and
retinal pigment epithelium of albino rats: A light and electron
microscopic study. Exp Eye Res 34:339, 1982.
8. Weiter JJ, Delori FC, Wing GL, and Fitch K: Relationship of
senile macular degeneration to ocular pigmentation. Am J
Ophthalmol 99:185, 1985.
9. Kooijman AC: Light distribution on the retina of a wide-angle
theoretical eye. J Opt Soc Am 73:1544, 1983.
10. Pflibsen KP, Pomerantzeff O, and Ross RN: Retinal illumi-
nance using a wide-angle model of the eye. J Opt Soc Am A
5:146, 1988.
11. Essner E and Novikott'AB: Human hepatocellular pigments
and lysosomes. J Ultrastruct Res 3:374, 1960.
12. Brunk U and Ericsson JL: Electron microscopical studies on
rat brain neurons: Localization of acid phosphatase and mode
of formation of lipofuscin bodies. J Ultrastruct Res 38:1, 1972.
13. Dabholkar AS and Ogawa K: Ultracytochemical changes in
the distribution of acid phosphatase in the liver cells of vitamin
E deficient and vitamin E supplemented rats. Acta Histochem
Cytochem 11:195, 1978.
14. Feeney-Burns L and Eldred GE: The fate of the phagosome:
Conversion to "age pigment" and impact on human retinal
pigment epithelium. Trans Ophthalmol Soc UK 103:416,
1984.
15. Katz ML, Drea CM, Eldred GE, Hess HH, and Robison WG
Jr: Influence of early photoreceptor degeneration on lipofuscin
in the retinal pigment epithelium. Exp Eye Res 43:561, 1986.
16. Gartner S and Henkind P: Aging and degeneration of the
human macula. Br J Ophthalmol 65:23. 1981.
17. Conover WJ: Practical Nonparametric Statistics, 2nd Edition.
New York, John Wiley and Sons, 1980, pp. 171,216.
18. Ordy JM and Brizzee KR: Functional and structural age dif-
ferences in the visual system of man and nonhuman primate
models. In Sensory Systems and Communication in the El-
derly, Aging, Vol. 10, Ordy JM and Brizzee KR, editors. New
York, Raven Press, 1979, pp. 13-50.
19. Balazsi AG, Rootman J, Drance SM, Schulzer M, and Douglas
GR: The effect of age on the nerve fiber population of the
human optic nerve. Am J Ophthalmol 97:760, 1984.
20. Marshall J, Grindle J, Ansell PL, and Borwein B: Convolution
in human rods: An ageing process. Br J Ophthalmol 63:181,
1979.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933379/ on 08/17/2017
1699
21. Ohta Y and Kato H: Colour perception changes with age. Mod
Probl Ophthalmol 17:345, 1975.
22. Weale RA: Senile changes in visual acuity. Trans Ophthalmol
Soc UK 95:36, 1975.
23. McFarland RA, Domey RG, Warren AB, and Ward DC:
Dark-adaptation as a function of age: I. A statistical analysis. J
Gerontol 15:149, 1960.
24. Kilbride PE, Hutman LP, Fishman M, and Read JS: Foveal
cone pigment density difference in the aging eye. Vision Res
26:321, 1986.
25. Lai YL and Rana MW: A study of photoreceptor-retinal pig-
ment epithelium complex: Age-related changes in monkeys
(42267). Proc Soc Exp Biol Med 181:371, 1986.
26. Curcio CA, Sloan KR Jr, Packer O, Hendrickson AE, and
Kalina RE: Distribution of cones in human and monkey ret-
ina: Individual variability and radial asymmetry. Science
236:579, 1987.
27. Osterberg GA: Topography of the layer of rods and cones in
the human retina. Acta Ophthalmol 13(Suppl 6):1, 1935.
28. Schein SJ: Anatomy of macaque fovea and spatial densities of
foveal representation. J Comp Neurol 269:479, 1988.
29. Young RW: Pathophysiology of age-related macular degener-
ation. Surv Ophthalmol 31:291, 1987.
30. Young RW: A theory of retinal disease. In New Directions in
Ophthalmic Research, Sears ML, editor. New Haven, Yale
University Press, 1981, pp. 237-270.
31. Boulton M and Marshall J: Effects of increasing numbers of
phagocytic inclusions on human retinal pigment epithelial
cells in culture: A model for aging. Br J Ophthalmol 70:806,
1986.
32. Sohal RW and Donato H: Effects of experimentally altered
lifespan on the accumulations of fluorescent age pigments in
the housefly Musca domeslica. Exp Gerontol 13:335, 1978.
33. Katz ML and Eldred GE: Retinal light damage reduces auto-
fluorescent pigment deposition in the retinal pigment epithe-
lium. Invest Ophthal Vis Sci 30:37, 1989.
34. Tso MOM and Friedman E: The retinal pigment epithelium:
III. Growth and development. Arch Ophthalmol 80:214, 1968.
35. Young RW: The renewal of rod and cone outer segments in the
rhesus monkey. J Cell Biol 49:303, 1971.
36. Streeten B: Development of the human retinal pigment epithe-
lium and the posterior segment. Arch Ophthalmol 81:383,
1969.
37. Tso MOM and Friedman E: The retinal pigment epithelium: I.
Comparative histology. Arch Ophthalmol 78:641, 1967.
38. Eagle RC Jr, Lucier AC, Bernadino VB, and Janoff M: Retinal
pigment epithelial abnormalities in fundus flavimaculatus: A
light and electron microscopical study. Ophthalmology
87:1189, 1980.
39. Rabb MF, Tso MO, and Fishman GA: Cone-rod dystrophy: A
clinical and histopathologic report. Ophthalmology 93:1443,
1986.
40. Weingeist TA, Kobrin JL, and Watzke RC: Histopathology of
Best's macular dystrophy. Arch Ophthalmol 100:1108, 1982.
41. Robison WG Jr, Kuwubara T, and Bieri JG: Deficiencies of
Vitamins E and A in the rat: Retinal damage and lipofuscin
accumulation. Invest Ophthalmol Vis Sci 19:1030, 1980.