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Age-Related Changes in Human RPE Cell Density and Apoptosis Proportion In Situ

¹ From the Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, New York; and the ² Department of Ophthalmology, Washington University School of Medicine, St. Louis, Missouri.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
PURPOSE. To determine the effect of age on the proportion of apoptotic cells in the RPE and cellular density in human eyes in situ.

METHODS. RPE flatmounts prepared from 22 adult human cadaver eyes (11 pairs; ages 19–87) were stained for apoptotic cells by a TUNEL technique. The density of RPE cells was also measured. The flatmount was divided into four concentric regions centered on the fovea (zone 1, 0–1.5 mm radius; zone 2, 1.5–3.0 mm; zone 3, 3.0–12.5 mm; and zone 4, >12.5 mm).

RESULTS. There was a positive correlation between the donor’s age and the proportion of apoptotic RPE cells per eye (r = 0.63; P = 0.04), which increased during the sixth decade and was higher in older (age range, 56–87 years) than in younger (age range, 19–48 years) eyes (0.56 ± 0.14 vs. 0.07 ± 0.07 cells per 100,000 cells, respectively; P = 0.03). Apoptotic RPE cells were located mainly in zone 1 in older eyes. Cell density decreased in the RPE as the distance from the fovea increased (r = 0.66, P < 0.05). The decrease was most prominent in zone 4 (r = -0.76, P = 0.007).

CONCLUSIONS. The proportion of apoptotic human RPE increased significantly with age. Apoptotic RPE cells are confined mainly to the macula of older human eyes. The observation that RPE cell death occurs in the macula but the density of RPE cells remains unchanged in the macula and decreases in the periphery suggests that migration of peripheral RPE cells may compensate for the death of macular RPE cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The retinal pigment epithelium (RPE) is a hexagonal monolayer of cells that forms the barrier between the choriocapillaris and neurosensory retina in the normal human eye.¹ The RPE has many physiological functions, including maintenance of the blood–outer retinal barrier, phagocytosis, recycling of the tips of the photoreceptor outer segments, and isomerization of visual pigments.¹ RPE cell loss occurs as a function of age. The number of RPE cells in otherwise normal human eyes decreases by approximately 0.3% per year.² Dysfunction of the RPE may play a role in the pathogenesis of a wide variety of sight-threatening diseases, including age-related macular degeneration (AMD).³

Apoptosis is a genetically controlled mechanism of cell death characterized by a highly specific sequence of events that include cytoplasmic and nuclear condensation and fragmentation of nuclear chromatin.^{4 5 6} Apoptosis plays a crucial role in physiologic homeostasis and embryogenesis and is an active process that occurs in many disease states, such as loss of ganglion cells in glaucoma,^{7 8 9 10 11 12} death of photoreceptors in retinal detachment,^{13 14} death of RPE and endothelial cells in choroidal neovascularization,¹⁵ and loss of glial cells in Alzheimer’s and Parkinson’s disease.^{16 17} The purpose of the present study was to determine whether human RPE cells undergo apoptosis in situ and whether there are spatial and age-related differences in the distribution of apoptotic RPE cells in vivo. We simultaneously measured the spatial and age-related variation in RPE density in human eyes in situ.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
The 22 human cadaveric eyes (11 pairs) used in this study were obtained from the Mid-America Tissue Bank (St. Louis, MO). The study protocol adhered to the tenets of the Declaration of Helsinki for research involving human subjects, including identifiable human tissue. The age of the donors ranged from 19 to 84 years. All donors were white. There were six males and three females; the tissue bank did not record the sex of two donors. Human cadaveric eyes were stored in a moist chamber after enucleation and transported to our laboratory. The time between death and tissue fixation ranged from 14 to 24 hours (average, 20 ± 3.5).

The globes were cleaned of extraocular tissue under a dissecting microscope. Eyes were included if there was no donor history of posterior segment disease and no visible signs of chorioretinal disease, including subretinal hemorrhage, extensive drusen, or irregular pigmentation of the macular RPE. An incision was made through the sclera 3 mm posterior to the limbus until the choroidal vessels were exposed.^{18 19} Tenotomy scissors were introduced through this incision into the suprachoroidal space, and the incision was extended 360° circumferentially. Four radial relaxing incisions were made in the sclera, and the sclera was peeled posteriorly to expose the choroid. A circumferential choroidal incision was made along the ora serrata and extended into the subretinal space, and the RPE–choroid complex was separated gently from the neural retina. Eight radial incisions were made from the ora serrata toward the posterior pole and the RPE–choroid complex was flattened on an unlaminated, hydrophobic, polytetrafluoroethylene membrane (125–175 µm thickness, 0.5 µm pores; Millipore, Bedford, MA) with the RPE facing up. The flatmount was then fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 12 hours.

The RPE-choroid complex was mounted on a glass slide (Fig. 1 , left) and the flatmount preparation was stained with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique.²⁰ Briefly, samples were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate solution on ice for 4 minutes. Cells were incubated with a mixture of fluorescein labeled nucleotides and terminal deoxynucleotidyl transferase (TdT) from calf thymus for 60 minutes. This enzyme catalyzes the polymerization of labeled nucleotides to free 3'-H terminals of DNA fragments. Fluorescence microscopy was used to visualize the apoptotic cells at the end of this period. Some of the flatmount preparations were also stained with propidium iodide to determine the chromatin condensation pattern within the TUNEL-positive cells.

View larger version (49K): [in this window] [in a new window]   Figure 1. Left: flatmount of RPE and choroid. Optic nerve (N), macula (M) and ora serrata (O) marked. Right: flatmount shows the four zones centered on the fovea. Zone 1, 0–1.5 mm radius; zone 2, 1.5–3.0 mm; zone 3, 3.0–12.5 mm; and zone 4, >12.5 mm.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Proportion of Apoptotic RPE Cells
The presence of TUNEL-positive cells was detected with fluorescence light microscopy. Propidium iodide staining confirmed that TUNEL-positive cells also exhibited peripheral chromatin condensation (Fig. 2) .

View larger version (48K): [in this window] [in a new window]   Figure 2. TUNEL and propidium iodide staining. Images obtained from different fluorescent channels were superimposed digitally to demonstrate apoptotic cells. Left: TUNEL-positive staining (green near box center) demonstrates DNA breaks within the nucleus. Right: Higher-magnification of area within the box. Arrows: cell border that was distinguished by vertical focusing with the microscope; arrowheads: border of nucleus. Lobular chromatin condensation was visualized with red propidium iodide staining. Melanin granules (m) were present within the cell cytoplasm.

View larger version (16K): [in this window] [in a new window]   Figure 3. A statistically significant positive correlation between donor age and the number of apoptotic RPE cells per 100,000 cells (Pearson’s r = 0.63, P = 0.04) was observed. Proportion (per 100,000 cells) = 0.014 (age) - 0.38. The proportion of apoptotic RPE cells increased to within 5% of maximum at 30 years of age (AP5) and reached it half-maximum (AP50) by 57 years.

View larger version (21K): [in this window] [in a new window]   Figure 4. Age-related change in proportion of apoptotic RPE cells in the various zones. There was a significant increase in apoptotic cells in zones 1 (r = 0.76, P = 0.03) and 2 (r = 0.89, P = 0.002) with increasing age. Within zone 1 the apoptosis proportion started to increase in the sixth decade (AP5 = 53 years) and reached 50% of its maximum by age 79 (AP50 = 79 years). Within zone 2, the apoptotic proportion started to increase in the ninth decade (AP5 = 83 years) and reached 50% of its maximum value by age 86 (AP50 = 86 years). There was no significant increase in apoptosis with increasing age in zones 3 or 4.

Density of RPE Cells
The density of RPE cells decreased with increasing distance from the foveal center (Table 1) . The average density was highest in zone 1 and decreased gradually toward the periphery (ANOVA, P < 0.01; Scheffé comparison between each zone, P < 0.01). The overall density of RPE cells also decreased significantly with increasing age (r = -0.62, P = 0.04, RD₅₀ = 53 years, Fig. 5 ). This decrease in the density of RPE cells corresponds to loss of 2.3% of total RPE cells per decade. No significant change in the density of RPE cells was noted in zone 1 (r = 0.08, P = 0.84, RD₅₀ = 53 years), zone 2 (r = 0.26, P = 0.43, RD₅₀ = 53 years), or zone 3 (r = -0.10, P = 0.78, RD₅₀ = 53 years). In zone 4 the density of RPE cells decreased significantly with increasing age (r = -0.76, P = 0.007, RD₅₀ = 50 years, Fig. 6 ). Regardless of age, the density of RPE cells was always highest in zone 1 and decreased toward peripheral regions of Bruch’s membrane (Fig. 7) .

View larger version (15K): [in this window] [in a new window]   Figure 5. Change in density of RPE cells with age. The overall RPE density decreased with increasing age (r = 0.62. P = 0.04). Total density (cells/mm2) = 4,890 - 11.3 (age). Approximately 2.3% of total RPE cells are lost every decade.

View larger version (23K): [in this window] [in a new window]   Figure 6. Age-related change in the density of RPE cells in different zones. There was an age-related decline in the density of RPE cells in zone 4 only. The density did not decrease with age in zones 1 to 3.

View larger version (17K): [in this window] [in a new window]   Figure 7. Spatial variation in the density of RPE cells in situ. The density of RPE cells was highest in zone 1 and decreased with increasing distance from the center of the fovea, regardless of age (r = 0.66, P < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The proportion of apoptotic cells we observed is similar to the proportion of ganglion cells lost by apoptosis in glaucomatous human eyes.⁸ Although the proportion of apoptotic RPE cells in these flatmount preparations was low, it should be remembered that the total amount of time between the initiation of apoptosis and phagocytosis of an apoptotic cell by its neighbors may be as short as 8 hours.²³ Thus, a significant proportion of RPE cells can die by apoptosis over many years, even though we observed a paucity of apoptotic cells in individual flatmount preparations. For example, the apoptotic proportion of 1.79 cells per 100,000 cells observed in the macula of older donor eyes could lead to loss of 1,960 per 100,000 macular RPE cells per year, if it is assume that apoptotic cells can be detected for only 8 hours. Loss of this percentage (1.96%) of macular RPE cells per year could be quite significant, because this implies that nearly 20% of the macular RPE would be lost per decade in older human eyes. Interestingly, apoptosis has been observed in surgically excised choroidal neovascular membranes from AMD-affected eyes,¹⁵ and retinal cell apoptosis occurs as a function of increasing age in tiger salamanders that exhibit an age-related decrease in the optomotor reflex.²⁴

We were intrigued by the observation that age-related RPE cell death occurred mainly in the macula, but the density of RPE cells decreased mainly in zone 4. In vitro, small defects in the RPE monolayer can be resurfaced by flattening of the RPE adjacent to the defect, but resurfacing of larger defects in the RPE requires migration and proliferation of more distant cells.^{25 26 27} Our current observation (death of macular RPE with loss of peripheral density of cells) can be accounted for by a hypothesis in which migration or proliferation of peripheral RPE cells toward the macular area compensates for continuous loss of foveal RPE cells. In fact, in some species there is continuous regeneration of the RPE arising from a source of stem cells in the marginal zone region of the ciliary body.^{28 29} We observed only three mitotic figures during the course of this study that were all located in zone 4.

We pretreated the fixed flatmount preparations with proteinase K and Triton X-100 because this increases the sensitivity of the TUNEL technique to more than 90% in recognizing morphologically proven apoptotic cells.³⁰ We cannot completely exclude the possibility that the sensitive TUNEL technique detected false positives, because DNA fragmentation can occur in response to apoptosis-independent DNAase activation and pH-induced DNA fragmentation or in late stages of tissue necrosis. However, several facts make it likely that the TUNEL-positive cells we observed represent apoptotic RPE. First, we performed propidium iodide staining of some flatmounts and observed cells that were TUNEL-positive on fluorescence microscopy and simultaneously exhibited chromatin margination on propidium iodide staining (Fig. 2) . Second, there is an excellent correlation between TUNEL staining and morphologic changes consistent with apoptosis in many other systems.³¹ Third, the flatmount preparations were fixed within 24 hours after death. Postmortem changes occurring within 24 hours of death do not alter the number and distribution of apoptotic cells in human tissues, because apoptosis is an active process requiring active enzyme synthesis and activation.³² Fourth, we did not observe any tissue necrosis in these flatmount preparations. Flow cytometry and gel electrophoresis can be used to confirm apoptosis in large populations of cultured cells, but these techniques are not practical in flatmount preparations where the proportion of RPE undergoing apoptosis is very low. In this study, a flatmount preparation was used so that we could simultaneously determine the proportion and distribution of apoptotic RPE, but flatmount preparations require use of an in situ labeling technique. TUNEL staining has a higher sensitivity and specificity than other in situ end-labeling techniques such as DNA polymerase I-based in situ nick translation.³³

We do not know whether the age-related increase in the proportion of apoptotic RPE cells that we observed was part of normal aging or represents cell loss due to disease. In addition, the use of human tissue introduces many variables that cannot be controlled in the laboratory setting, such as the cause of death, time between death and enucleation, presence of chronic or acute hypoxia, hyperglycemia, and effects of the donor’s genotype or phenotype. The effect of these variables cannot be determined in human studies, although many of these factors would be expected to cause apoptosis over wide regions of the RPE rather than the focal loss that we observed in the present study.

The present study does not allow us to determine the cause of apoptosis in our flatmount preparations. However, it is known that numerous structural alterations occur within the RPE substrate as a function of age, including diffuse thickening, collagen cross-linking, calcification of the elastin layer, and the accumulation of basal laminar deposits, basal linear deposits, and drusen.^{21 34 35 36 37 38} Age-related changes in Bruch’s membrane are the hallmark of AMD and are present in up to 40% of eyes of older persons at autopsy.³⁸ The topographic distribution of age-related changes observed in Bruch’s membrane in human donor eyes is similar to the topographic distribution of apoptotic RPE cells in flatmount preparations of the human fundus. In fact, the proportion of human RPE cells that undergo apoptosis after seeding onto Bruch’s membrane in tissue culture increases with the age of the human Bruch’s membrane explant.³⁹ These observations suggest that age-related changes in Bruch’s membrane may be related to apoptosis in the overlying RPE. In the normal human eye, the attachment of adult human RPE to the basal lamina layer of Bruch’s membrane is mediated by a specific interaction between ß₁-integrin receptors on the cell surface and ligands in the extracellular matrix.⁴⁰ Age-dependent extracellular matrix changes within Bruch’s membrane⁴¹ may induce apoptosis in cells in the overlying RPE, because disruption of epithelium–basement membrane interactions leads to apoptosis in a variety of epithelial cells, including the RPE.^{39 42 43 44}

Apoptosis can be inhibited by cellular expression of bcl-2 and p35.⁶ This suggests that the stimuli for apoptosis ultimately converge on a final common pathway, and bcl-2 and p35 interact with components that are "downstream" from the convergence point of these molecular pathways. Thus, one may be able to prevent apoptosis of the RPE in vivo either by interfering with the triggering signal for apoptosis or by using drugs known to interfere with apoptosis farther downstream. The ultimate effect on ocular health and disease of interfering with loss of RPE cells by apoptosis remains to be determined.

	Footnotes

 
Supported in part by unrestricted funds from Research to Prevent Blindness, a grant from the Foundation Fighting Blindness and Grant EY10311 from the National Eye Institute.

Submitted for publication February 11, 2002; revised April 22, 2002; accepted May 20, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.

Corresponding author: Lucian V. Del Priore, Department of Ophthalmology, Columbia University, 635 West 165th Street, New York, NY 10032; ldelpriore{at}yahoo.com.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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