Ординатура / Офтальмология / Английские материалы / Age-Related Changes of the Human Eye_Cavallotti, Cerulli_2008
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C. A. P. Cavallotti and M. Schveoller |
Increase in Granules of Lipofuscin
With advancing years, granules of lipofuscin appear inside the cells of the RPE. These granules represent the lysosomal accumulation of residual bodies, including the nondegradable final products of metabolism and the consumed acromeres of the photoreceptor, as already described.6 Each cell of the RPE is in a continuous process of intracellular renewal. Sometimes this process of molecular degradation is not complete and results in the increase of metabolic debris and interference with other metabolic activity in these cells. The residual materials are useless molecular aggregations—normally called lipofuscin granules—that contain damaged RPE cells and the membranes of rod and cone phagocytes (i.e., incompletely degraded cellular debris).8 The incomplete molecular degradation seems to be due to altered substrates, which are not therefore recognized by the enzymatic systems. These molecular alterations result from the harmful effects that free radicals have on the RPE cells and their photoreceptors—rich in polyunsaturated fatty acids that become peroxides.The damaged molecules are consumed by the RPE cells and accumulate in their cytoplasm, thus compromising their metabolism and inducing cell death. The acromeres of the retinal photoreceptors can be abnormal due to oxidative damage, or the abnormality can be hereditary. The reason why the acromeres of the photoreceptors are sensitive to oxidative stress is their richness in polyunsaturated fats. Exposure to light, and particularly short wavelength radiation, increases the production of free radicals and accumulation of lipofuscin in the macular RPE. In addition, environmental factors such as cigarette smoke reduce the level of antioxidants and promote the formation of free radicals, contributing to the accumulation of cellular debris.9 Lipofuscin exists inside the RPE cells in the form of granules that appear yellow-green under UV light excitation. Topographically, the majority of the granules of lipofuscin store in the posterior pole of the eye bulb while their quantity decreases in the fovea. This distribution of lipofuscin with respect to the location remains constant during the entire life, and correlates with the density of photoreceptors. The lipofuscin granules contain lipids and proteins. The cytoplasmic volume of the RPE occupied by lipofuscin increases with age, from 8 percent at age 40 to 29 percent at age 80. Furthermore, the cytoplasmatic volume occupied by lipofuscin in the macula is greater than that in the periphery— 19 percent in the macula as compared to 13 percent in the periphery. The excessive accumulation of lipofuscin in the RPE cells slows the metabolic activity of the cells and predisposes them to age-related macular degeneration (AMD).10
Lipofuscin is the generic name given to a heterogenic group of lipid-protein aggregates that are found in aging cells in all human tissues. Different from the other tissues, the lipofuscin does not derive from the degradation of intracytoplasmatic organelles in the retina, but from the incomplete degradation of the products derived from the phagocytosis of the outer portions of the photoreceptors following the peroxidation of the unsaturated fatty acids. Once accumulated, the lipofuscin causes the death of the RPE cells because it acts as a true generator of free radicals11—lipofuscin can release lysosomotropic amines. Finally, other
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authors consider lipofuscin to be an inert substance that acts directly through the congestion of the cytoplasm (in some tissues lipofuscin can occupy up to 30% of the cell volume).12
Increase in Melanin Granules
Melanin has a double role in the retina—to reduce the chromatic aberration, increasing the visual acuteness, and to protect against oxidative stress by acting as a cellular antioxidant. Its concentration increases from the equator to the posterior pole, reaching a peak in the macula. The increased concentration of melanosomes in the macula is due to the fact that the RPE cells here are larger and concentrated into a smaller area with respect to the smaller extra-macular RPE cells dispersed in a much larger area.13 The differential distribution of melanosomes is maintained during the first 40 years of life, but a significant reduction in the melanin granules is seen in all regions of the retina afterwards.14 To make a comparison considering three age groups (10-20; 21-60; 61-100), the reduction in the quantity of melanosomes between the first and the third class is 35 percent. Talking in terms of cell volume, therefore, around 8 percent is occupied by melanin in the first two decades of life, which reduces to 6 percent in the second age group and finally further diminishes to 3.5 percent in the third age group.15
Melanin is a complex heterogenic biopolymer, containing free radicals which can be identified using electron spin resonance spectroscopy. Using this technique a 40 percent reduction in melanin content is observed with aging.16 Three possible mechanisms may explain the loss of melanin from RPE cells—expulsion of the granules, lysosomal degradation, and chemical damage. The expulsion of the granules may be a possibility, notwithstanding the fact that the granules are not found in the Bruch’s membrane nor in the interphotoreceptor space. Lysosomal degradation is highly elevated due to its function of degrading the acromeres of the photoreceptors.17 With aging comes an increase in the number of melano-lysosomes, accompanied by a change in the appearance of the melanin granules. Notwithstanding that the morphology of the melanosomes changes following an interaction with the lysosomes, it is likely at the melanin is not degraded and that the changes derive from the degradation of the proteins of the matrix on which the melanin is deposited. The third mechanism is that of chemical degradation. The irradiation of human eyes with intense blue light induces a nonuniform photobleaching of the melanosomes. The lack of uniformity of the bleaching seems to be due to the fact that lipofuscin is also found in the complex granules of aged RPE cells and that it is more photoreactive than melanin, and may act as a photosensitizer. Blue light, therefore, would induce oxidative photodegradation of melanin by the formation of superoxide anion and hydrogen peroxide. If, on one hand, the photodegradation of melanin (oxidation or irreversible bleaching) does not have any biological significance in tissues with high turnover (such as hair and skin), then, on the other hand, this event gains a high importance when it occurs in those tissues with low turnover, such as in RPE cells that are post-mitotic cells.17
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Increase of Complex Granules
With the increase in age, melanin, lipofuscin, and lysosomes can join together to form complex structures (melanolipofuscin and melanolysosomes) inside the RPE. These granules have a regional distribution similar to that of lipofuscin—i.e., the highest concentration is in the macula and it decreases in the periphery and in the fovea. In regard to the percentage of cellular volume occupied by complex granules, this varies from 3.3 percent in the first decade of life and reaches to 8-10 percent in the sixth decade. The association of lysosomes with pigment granules (for example, melanosomes, lipofuscin granules, and complex pigments) explains their age-dependant variation observed in - lysosomal enzyme levels and the activity of RPE cells. The increase in pigment granules with age causes an increase in lysosomes and in the activity of various enzymes, such as acid phosphatase and Cathepsin D, to maintain the normal degradation cycle that follows the ingestion of the acromeres of the photoreceptors by the RPE cells.17
Personal Results
In our experiments, only samples of the human retina coming from autopsies, including RPE, were used. Because post-mortem phenomena may bring about early modifications in the data obtained from the eye tissues, the samples were harvested in the same ocular side and at the same time after the death. Our studies were approved by the local Ethical Committee and patients or their relatives gave their informed written consent. The investigations were performed according to the guidelines of the Declaration of Helsinki.18 After removal, the eye-bulb was dissected with a razor blade and samples of intact retinal tissue (located precisely in the same site, equatorially in the nasal region) were harvested. The presence of melanin granules can interfere with morphological ad/or histo-chemical observations on RPE. For these reasons, it is necessary to wish for the strong age-related decrease of melanin granules as it happens in older individuals. On the contrary, in young individuals it is necessary to fade the melanin granules by means of light (exposure for 60min at a ultra-violet lamp ) or by immersion in an oxidant solution of 3 percent H2O2 for 60 min.
Light Microscopy
Samples of the human retina were immediately prefixed in 2 percent osmium tetroxide at pH 7.4 in veronal-acetate buffer for 5 minutes at 4 °C. After fixation, the specimens were washed with veronal-acetate buffer (pH 7.4), dehydrated in a graded ethanol series and embedded in paraffin. Thin sections (about 4 m) were made for morphological staining with toluidine blue (0.05% for 1 minute).19
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Staining of Lipids
Lipids were stained by means of special histo-chemical techniques for light microscopic analysis. To determine the composition and distribution of the lipids, three different stains were used: the bromine–Sudan black B stain, which stains all classes of lipids; Bromine–acetone–Sudan black B, which stains only phospholipids; and Oil red O, which stains neutral lipids (especially esters of saturated and unsaturated fatty acids).20
Transmission Electron Microscopy (TEM)
Samples from the autopsy were fixed in buffered 2 percent glutaraldehyde for two hours, washed in buffer and then post-fixed in buffered 2 percent osmium-tetroxyde for two hours, dehydrated, and embedded in araldite. Ultra-thin sections were made using a Reichert Ultra-microtome. These sections were counterstained by uranylacetate and lead citrate21 and observed with a Zeiss EM 109 electron microscope.
Quantitative Analysis of Images
For a detailed evaluation of the effects of aging on retinal morphology, a QAI was performed on slides and microphotographs using a Quantimet Analyzer (Leica®) equipped with specific software. This software made it possible to determine (see Table 10.1): a) the thickness of the retina; b) the thickness and the number of the cells of RPE; c) the number of granules in the RPE cells; and d) the electron density of the intracellular sub-structures. Final values must be submitted to statistical analysis of data. The values reported in this paper represent the values of staining for each age group, and are expressed in conventional units (CU) ± S.E.M. CU are arbitrary units furnished and printed directly by the Quantimet system.22
Statistical Analysis of Data
The statistical methods used throughout this study must be interpreted as an accurate description of the data rather than as a statistical inference of such data. The preliminary studies of each value were performed with the aid of basic sample statistics. Mean values, maximum and minimum limits, variations, standard deviation (SD), standard error of the means (SEM), and correlation coefficients were calculated. Correlation coefficients denote a significant level less than 0.001 (P < 0.001), while it is not significant when P > 0.05 (n.s.).23
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Results
All our morphological results are reported in Figs. 10.1 through 10.5, while the histochemical results are summarized in Table 10.1. Fig. 10.1 shows the normal structure of the human retina in a young subject. The trineuronal intraretinal chain is male by photoreceptors, bipolar and ganglion cells. The RPE is detached, by experimental manipulations, from the other retinal layers. Fig. 10.2 shows, as appears in a digital angiography, the retinal fundus in a young subject. In this image, we can see the nasal superior and inferior branches of the ophthalmic artery. These vessels show a normal caliber, without signs of aging or diseases. Fig. 10.3 shows the same image as in Fig. 10.2, but comes from an old man. The retinal vessels show an increased caliber, a snake-like running and numerous dystrophic zones in comparison with RPE. All these findings can be considered as age-related changes.
The major age-related changes concern the metabolism of the lipids. In fact, as said above, the melanin in young subjects can alter the results because of the high pigmentation due to the melanin granules and it is present in all the cells of RPE.
Fig. 10.1 Light microscopic image of a normal human retina in young and healthy subjects . The RPE is detached from the other retina layers. RPE=retinal pigmented epithelium, P=photoreceptors, B = bipolar cells, and G = ganglion cells (Magnification 1600x; Calibration bar 100 m)
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Fig. 10.2 Retinal fundus in a young (22 years of age) and healthy subject as appears in a digital angiography of the ophthalmic artery. In comparison with the head of the optic nerve, the ophthalmic artery is branched in four divisions—one for each quadrant of the retina. In this image, the nasal superior and inferior branches are evident. (field 40° corresponding to a magnification of about 5x)
Fig. 10.3 Retinal fundus in an older (70 years of age) subject. The retinal vessels show an increased caliber. We can see many dystrophic zones in comparison with the RPE (field 40° corresponding to a magnification of about 5x)
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In old subjects, however, the RPE appears depigmented owing to the decrease of the melanin granules. For this reason the samples coming from young subjects can be previously depigmented. Fig. 10.4 shows that the RPE of a young healthy man after depigmentation (for the decrease of the melanin granules) was stained with Sudan Black B and bromine acetone for the detection of the phospholipids. The phospholipids are increased if compared with old subjects (A1). On the contrary, oil red O stains neutral lipids in a young (B) and/or in an old (B1) man.
There is a strong increase of neutral lipids with age. Fig. 10.5 shows that Sudan black B dyes the total lipids in a young (A) and/or in an old man (B). The total lipids are increased with age. Table 10.1 shows the values of QAI of lipids in RPE for young and /or old subjects. Three classes of lipids are dyed—total lipids, phospholipids, and neutral lipids.
After the specific coloration, a quantitative analysis of images was performed and results were expressed in conventional units (see Methods section). The probability or significance index was calculated, comparing the results obtained in young subjects versus older ones. All the tabled results show a high statistic significance (p< 0.001).
Fig. 10.4 Light microscopy of the RPE in a 19-year old eye donor (A and B) and/or a 75-year old donor (A1 and B1). The two figures A and A1 are stained with bromine-acetone-sudan black B (phospholipids), and those on the bottom (B and B1) are stained with oil red (neutral lipids). It can be seen that the intensity of staining with both systems increases with age. Therefore, both phospholipids and neutral lipids show a progressive age-related increase. (Magnification 400x; Calibration bar 10 m)
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Fig. 10.5 Light microscopy of the RPE in an 19-year old eye donor (A ) and/or a 75-year old donor (A1). Both A and A1 are stained with bromine-sudan black B. This method stains all classes of lipids. It can be seen that the intensity of staining increases with age. Therefore, the total lipids show a progressive age-related increase. (Magnification 400x; Calibration bar 10 m)
Table 10.1 QAI of lipids in the RPE of Young and old objects
Class of lipids and staining |
Young |
Old |
Total Lipids Sudah Blach B |
31.3 ± 2.2 |
44.5 ± 3.1* |
Phospholipids Sudan Black B plus Bromine acetone |
62.6 ± 4.4 |
25.7 ± 2.3* |
Neutral lipids Oil Red O |
30.4 ± 2.9 |
49.6 ± 2.6* |
All the value are expressed as Conventional Units (C.U.) ± SEM (see methods) *P<0,001 young versus old.
Discussion
Our results demonstrate that the old subjects undergo the following age-related changes in RPE cells: a) a strong decrease of the granules of melanin with cellular depigmentation; b) a strong increase of intra-cytoplasm cell bodies; c) a strong increase of the total lipids while phospolipids and fatty acids are decreased; and d) an increase of the electron density of all cellular substructures. RPE cells take part of the so-called choroids retinal complex (named also chorio-retina) formed by choroids, Bruch’s membrane, basal membrane, and RPE. This complex supplies blood and metabolic support to the other cellular layer of the retina (including photoreceptors bipolar and multi-polar cells).
The RPE play a fundamental role in the transport and storage of the retinoids, essential for maintaining the visual cycle. Another function of the RPE is to eliminate components of the acromeres of the photoreceptors through phagocytic activity mediated by cathepsin D and by the integrins that act as membrane receptors mediating the phagocytosis. The phagosomes are linked to lysosomes to form phago-lysosomes. These cellular substructures are digested by the intracellular digestive enzymes located at the basal surface of the cell. The final metabolic products are expelled into the choroidal circulation by exocytosis through the Bruch’s membrane. If they are not completely digested, they can accumulate inside the cells
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as granules of lipofuscin. These granules increase with age,24 especially in the macular area. The constant exposure of the RPE to light, at elevated concentrations of oxygen, together with the high metabolic activity of these cells, creates an environment favorable (especially in the macular area) to the formation of toxic reactive oxygen radicals. The reactive radicals can induce an oxidative stress. In defense against oxidative stress, the RPE cells contain antioxidants such as superoxide dismutase, catalase, reduced glutathione, melanin, and carotenoid. The quantity of these substances decreases with age and, therefore, the antioxidant defenses also decrease with age.
The RPE cells, together with the other retinal cells, allow us to observe the wonders of the world. Unfortunately, all these cells undergo to a senile involution with a strong decrease of their functions. It is well-known that the level of senescence is determined by the interaction between two types of factors: a) promoting aging, and b) counteracting it (theory of senescence). The cellular regeneration capacity is under genetic control and is able to counteracting the senescence.
On the other hand, the theory of the biological clock asserts that the senescence of each species is genetically determined, and that every change is the result of environmental influences and/or some mutations. To fully understand the senile involution of RPE, further research is needed.
References
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Recent Books on RPE Disorders in Old Age (www.amazon.com)
1.The Aging Eye by Sandra Gordon. Harvard Medical School, 2001.
2.Communication Technologies for the Elderly: Vision, Hearing & Speech by Rosemary Lubinski and D. Jeffery Higginbotham, 1997.
3.The Effects of Aging and Environment on Vision by Donald A. Armstrong, et al. 1991.
4.Treating Vision Problems in the Older Adult (Mosby Optometric Problem-Solving Series) by Gerald G. Melore, 2001.
5.Vision and Aging by Alfred A. Rosenbloom and Meredith W. Morgan, 1993.
6.Age-Related Macular Degeneration by Jennifer I. Lim, 2002.
7.The Impact of Vision Loss in the Elderly (Garland studies on the Elderly in America) by Julia J. Kleinschmidt, 1995.
8.Vision in Alzheimer’s Disease (Interdisciplinary Topics in Gerontology) by Alice CroninColomb, et al., 2004.
9.The Senescence of Human Vision (Oxford Medical Publications) by R.A. Weale, 2001.
10.Issues in Aging and Vision: A Curriculum for University Programs and In-service Training by Alberta L. Orr, 1998.
11.Aging with Developmental Disabilities Changes in Vision by Marshall E. Flax, 1996.
12.Trends in Vision and Hearing among Older Americans, by U.S. Dept of Health and Human Services, 2000.
13.Optometric Gerontology: A Resource Manual by Sherrell J. Aston, 2003.