Ординатура / Офтальмология / Английские материалы / Aging and Age Related Ocular Diseases_Lutjen-Drecoll_2000

standard subretinal transplants, but connecting cilia were common. Clusters of tight junctions of the kind that form the outer limiting membrane were common in regions where connecting cilia were also seen. Cells identifiable as microglia were uncommon.

Amacrine and bipolar cells were identified by means of their general morphology and the presence of the characteristic synapses. Müller cells were identified on the basis of the appearance of their cytoplasm, the density of the nucleus and the distribution of its chromatin. We did not try to classify each cell, but only to obtain an overview of the cell types that were present.

There was a well-developed basement membrane around the transplants at 4 months (fig. 5), but it was less well developed in 1-month transplants. Müller cells formed a continuous sheath around the transplants at 4 months, usually more than one cell layer deep. In one of the 4-month transplants, the Müller cells were the dominating cell type. They did not show the pillar-like shape that is evident in the normal retina and were connected by junctional complexes morphologically resembling tight junctions and desmosomes (fig. 6b). In 1-month transplants, Müller cells were less prominent than in the 4- month transplants and did not form any continuous sheath around the transplant, even though they were easily found.

Capillaries were occasionally seen at the surface of the transplants. They were never fenestrated and most often showed a comparatively thick endothelium (fig. 6a). However, capillaries facing the normal host pigment epi-

thelium remained fenestrated, also when the transplant was only a few tens of micrometers away in the choroid (fig. 6b). In the 2 cases where transplants were found in the subretinal space, the epithelium had degenerated, showing a loss of microvilli as well as lateral membrane infoldings. In these cases, there were no fenestrations in the capillaries facing the pigment epithelium (fig. 7). There was hardly any cellular reaction in the choroid around the transplant and no signs of scar formation. Macrophages and plasma cells have occasionally been observed, but are no regular constituent at the transplants. There was no sign of any immunological cell attack on the transplant. On the other hand, pyknotic cells and cells which looked like apoptotic cells were frequent at 1 month. Dying cells were infrequent at 4 months, if present at all. Mitoses were regularly observed in 1-month transplants (fig. 2), but were not detectable at 4 months.

Discussion

It is now well established that fetal retinal cells can be transplanted to epiretinal or subretinal sites in mammalian eyes [1±4]. Survival up to 3 or 4 months has been seen in a number of instances, and transplants with the longest survival times so far (about 40 weeks) were obtained by Ehinger et al. [17, 18] with human fetal retinal cells transplanted to rats. There is also a previous report by Bergström [9] that rabbit-to-rabbit retinal cell transplants to the choroid survive and differentiate, and the present

study corroborates this observation. It may be noted that the experimental animals were well outbred.

As seen in this study, retinal transplants are able to survive in the choroid of outbred rabbits for extended periods of time, much longer than needed for rejection responses to develop. When comparing with transplants located between the host photoreceptors and the choroid, there is possibly a reduction in the number of neurons in comparison with the glial Müller cells, and the organization is less exact and without many rosettes. However, the important point is that the normal complement of different constituents remained in the transplants. There can be no doubt that fetal rabbit retinal neurons survive, develop and differentiate also when placed in the well-vascular- ized choroid of rabbits. Further, mitotic figures and apoptotic reactions appeared only in the 1-month transplants and not in the older ones. It is apparent that there is no conventional rejection response against the retinal transplants in the rabbit choroid. It may further be noted how little microglial reaction and few other signs of scar formation there were.

It has previously been shown by Bergström [9] that serum proteins enter the transplant for several days after the transplantation, but subsequently, this passage is blocked. There are no vessels within the transplants at this stage, but there are numerous fenestrated capillaries in its immediate vicinity, and they are the likely source of serum proteins. These capillaries remain fenestrated, also when only a few tens of micrometers away from the transplant. This suggest that the transplant does not release factors that influence the fenestration of the capillaries. Instead, such factors seem to be released by the retinal pigment epithelium as suggested by May et al. [19], and in the present study, it was also seen that the fenestrations were absent if the retinal pigment epithelium had been disturbed (fig. 7).

From the present study it can be seen that the glial Müller cells form a cellular barrier around the transplant, and it appears likely that to a large extent they wall it off from its host so that the immune system does not attack it. However, there was no complete wall in the 1-month translants, only in the 3- to 4-month ones. There were no blood vessels within the transplant at either 1 or 4 months. Since the rabbit retina is not normally vascularized, this was as expected. Tight junctions are not seen in the normal retina, only in the retinal pigment epithelium. In previous studies Eichhorn et al. [20] have shown that pigmented ciliary epithelium cells in vitro also form tight junctions if the nonpigmented epithelium that would normally form these junctions is absent. We assume that sim-

ilar to the events in the cultured ciliary epithelium, transplanted Müller cells develop tight junctions when the pigment epithelium cells that would normally do so are absent.

The ACAID system has been well investigated in rodents and primates and reviewed by Streilein et al. [5] and Streilein [21, 22]. It has been demonstrated in the anterior segment and the retina, but Eichhorn et al. [20] have shown that it may not be present in the primate choroid. In pilot experiments with rat eyes, we have also failed to see surviving transplants so that the choroid is perhaps not immunoprivileged in these animals. The ACAID system has not been well examined in rabbits, whose eyes are special in several respects. For instance, there is a rapid and easily elicited liberation of prostaglandins in the anterior segment, resulting in an acute inflammatory reaction and breakdown of the blood-retina and blood-aqueous barriers. Among other things, this makes it difficult to assess whether or not there is an ACAID system in the rabbit, even though Mondino et al. [23] presented indirect evidence from results with intravitreal injections of bacteria that an ACAID type of reaction may be present.

Fenestrated capillaries occur in the choriocapillaris at a distance of only about 50±100 Ìm for at least 3±4 months after the transplantation and probably for much longer. In the loose meshes of the choroid, this is not a very long distance for serum globulins to diffuse or for immunocompetent blood cells to move. Further, the Müller cell encapsulation is only partial for several weeks after the transplantation. We therefore suggest that strictly anatomical structures are not likely to be solely responsible for the protection of the transplants in the rabbit choroid, but also other factors may participate, such as the ACAID immunosuppressing system [for reviews, see 5, 21, 22]. We consequently suspect that the ACAID or a similar system is active in the rabbit choroid, although direct proof for this is currently not available, or that the rabbit retina in a yet unknown way protects itself from rejection.

Acknowledgements

We are grateful to Ms. Elke Kretschmer and Ms. Karin Arnér for expert technical assistance and to Mr. Marco Gösswein for preparing the photographs. This work was supported by grants to B.E. from the EU BioMed2 program, the RP Foundation, the Segerfalk Foundation, the H. and L. Nilssons Stiftelse, the T. and R. Söderberg Foundation, the Swedish Medical Research Council (project 14X-2321), the Riksbankens Jubileumsfond and the Faculty of Medicine at the University of Lund and by grants to E.L.D. from the IZKF.

References

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Key Words

Cataract W Oxidative stress W Lens

Abstract

The authors review the available evidence supporting the possible role of oxidative stress in cataract formation from an epidemiological and a clinical point of view. They discuss in more detail what is presently known about the molecular mechanisms of response of the mammalian lens to an oxidative insult and report unpublished data on gene modulation upon oxidative stress in a bovine lens model. Main research endeavors that seem to be a most promising source of new insights into the problem of age-related cataract formation are briefly discussed.

Copyright © 2000 S. Karger AG, Basel

Introduction and Background

Age-related cataract is the world's most frequent cause of curable blindness and population projections suggest that the number of cataract-blind persons could reach

Supported by Biomed grant BMH4-CT96-1593.

close to 40 million by the year 2025 [1]. The disease entails a progressive reduction of the transparency of the eye lens which deteriorates the quality of the optic image formed on the retina and reduces visual acuity eventually causing blindness. Three main types of lens opacity are described in age-related cataract (nuclear, cortical and posterior subcapsular). Pure forms of cataract (with only one type of opacity present) are found more frequently in early less advanced forms of the disease but, as the cataract becomes severer, different types of opacity frequently coexist in the same lens producing the so called mixed type of cataract. Although cortical cataract is probably the most frequently observed type of opacity, nuclear and posterior subcapsular cataracts (which primarily affect the optical axis of the eye lens) are certainly the types of cataract that more frequenly cause visual deterioration and lead patients to surgery [2].

As for other types of age-related degenerative eye diseases, the oxidative insult suffered by eye tissues during the lifelong exposure to visual/UV light radiation has been the subject of extensive investigation over the past decades as a possible determinant of cataract formation. Reactive oxygen species, like the superoxide anion, are formed by photochemical reactions of O2 in the presence of electron donors such as riboflavin and are converted to hydrogen peroxide via ascorbic-acid-mediated reduction.

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Hydrogen peroxide is present in the aqueous humor of the mammalian eye [3] and has been reported to be increased in the aqueous humor of eyes with age-related cataract [4]. Many of the protein and membrane alterations observed in cataractous human lenses are of oxidative origin, and incubation of animal lenses in the presence of hydrogen peroxide (or other oxidants) reproduces many of these changes and may lead to lens opacification. The high concentration of reduced glutathione (GSH) protects the lens from the damaging effect of hydrogen peroxide. The resulting oxidized glutathione (GSSG) is then converted to GSH in situ by a redox system involving glutathione reductase and reduced nicotinamide-adenine dinucleotide phosphate generated by the pentose monophosphate shunt. GSH is therefore crucial in the lens to protect protein SH groups preventing soluble protein cross-linking and the formation of insoluble protein aggregates, as well as to preserve normal permeability and transport function of cell membranes [5, 6]. Blocking GSH regeneration in cultured lenses enhances the toxic effect of hydrogen peroxide. GSH levels in the lens progressively decrease with age and are drastically reduced in cataractous lenses.

In keeping with this view, epidemiological evidence from observational studies supports the possibility that persons with a higher dietary intake of antioxidants are at a lower risk of developing age-related cataract than individuals with a lower intake, and preliminary results from some intervention studies suggest that treatment with antioxidant vitamins might delay progression of age-relat- ed lens opacities. In this paper we shall briefly review the available evidence supporting the role of oxidative stress in cataract formation from an epidemiological and clinical point of view and will discuss in more detail what is presently known about the molecular mechanisms of response of the mammalian lens to oxidative insults.

Epidemiological Evidence:

Observational and Intervention Studies

Experimentally the lens has been shown to be damaged by exposure to ultraviolet B radiation [7, 8]. It is therefore not surprising that the association between sunlight, UVB exposure and cataract formation has been investigated in several observational studies. First suggested by Hiller et al. [9], an association between UVB exposure and cortical cataract has been found by several studies which also revealed an association between an increasing average annual UVB light exposure and risk of cataract [10], and a dose-response relationship between sunlight exposure and

cortical opacities [odds ratio, OR = 2.26 (95% confidence interval [CI] 1.14±4.46) for high vs. very low exposure] after adjusting for potential confounders [11]. A positive association between UVB exposure and increasing odds of cortical opacities (OR = 1.10; 95% CI, 1.04±1.30) was reported also by West et al. [12] and between UVB and posterior subcapsular cataract by Bochow et al. [13]. On the same note the greater prevalence of cortical opacities in the lower nasal quadrant of the lens also provides indirect support to a possible role of sunlight in cataract etiology [14]. Other studies however reported negative or inconclusive results [15±18]. These results may be explained, at least in part, by the high percentage of nuclear cataracts in the corresponding study samples, by different ranges of sunlight exposure among participants, by a different population mobility during lifetime or by residual confounding. Certainly the main difficulty of this type of assessment appears to be the limitation of self-reported assessment of lifetime exposure which can only be partially controlled by the use of detailed questionnaires. Overall, however, the available evidence indicates a dose-relat- ed association between sunlight (and the inherent UVB exposure) and cortical and, perhaps, posterior subcapsular cataracts.

Since 1991 several studies have explored the possible role of known antioxidants in preventing the development of age-related lens opacities. This issue has been addressed in observational studies which evaluated the antioxidant status of participants by assessing dietary intake or measuring plasma levels of vitamins C, E and carotenoids or use of multivitamin supplements. Despite some inconsistencies regarding the specific cataract type or nutrient, many of these studies produced data suggesting that persons with higher antioxidant plasma levels or intake are at lower risk of developing age-related lens opacities [19±22]. Other studies found negative or inconclusive results [23, 24]. Besides possible limitations inherent to the methods of nutritional assessments, cataract definition and geographic characteristics, a lower range of variability of nutrition in a given population with respect to another might explain some of the observed inconsistencies between studies which used similar design and assessment methods [21, 23].

Obviously, final proof of this `antioxidant' hypothesis will only come from randomized clinical trials comparing the effectiveness of multivitamin supplementation in preventing the development of cataract or in delaying its progression. Two large ongoing trials sponsored by the National Eye Institute are currently testing this possibility. At the moment the only available data are those derived

from two large and controlled cancer prevention trials that have been conducted in China using vitamin-mineral supplements. One of these studies enrolled 2,141 patients with esophageal dysplasia (dysplasia trial) randomized to placebo or to a mineral-multivitamin supplemention reinforced with ß-carotene. The second study (general population trial) enrolled 3,249 participants from the general population who were randomized to one of 4 possible treatments (retinol/zinc; riboflavin/niacin; ascorbic acid/ molybdenum; selenium/·-tocopherol/ß carotene) or to placebo. At the end of the follow-up, the treatment group of the first study and the group treated with riboflavin/ niacin of the second study showed, for the age range between 65 and 74 years, a significant reduction of the prevalence of N cataract with respect to controls (OR = 0.57, 95% CI 0.36±0.90 for the first study; OR = 0.45, 95% CI 0.31±0.64 for the second study) [25].

The Oxidative Insult Cataract Model: Oxidative Stress Response Mechanism in the Lens

The application of oxidative stress conditions to in vitro cultured lenses, either by incubation in the presence of H2O2 or by a photochemical insult in the presence of a photosensitizer (e.g. riboflavin), forms the basis of one of the most widely used models of artificially induced lens opacification. Under organ culture conditions the opacification starts in the equatorial region, is subepithelial in location and eventually spreads to the entire lens cortex. Loss of lens transparency is associated with the alteration of critical biochemical and transport parameters that can be prevented by an H2O2-decomposing enzyme like catalase. The sequence of events leading to the loss of lens transparency has been investigated in detail by Spector et al. [26], who studied the effect on the cultured rat lens of a short photochemically induced oxidative insult resulting in H2O2 levels in the culture medium not higher than 100 ÌM. These authors found that the oxidative insult is associated with an epithelial cell damage, which precedes lens opacification, develops slowly in the postinsult period and is related to the intensity and duration of the stress. The main changes observed in the epithelial cell layer are a progressive loss of cell viability, as revealed by an increased Trypan blue staining and, upon a longer stress time, DNA fragmentation and decreased 3H-thymi- dine incorporation [26, 27]. Interestingly, by comparing human noncataractous eye bank lens epithelial cells to human cataractous samples, Kleiman and Spector [28] found that the proportion of cells showing DNA single-

strand breaks is significantly increased in cataractous lenses. Similarly, a comparison of lens epithelial cells from a group of cataract patients with those of 8 normal human lenses of comparable age revealed an increased number of apoptotic cells in cataractous samples [29]. These results, however, have not been confirmed by Harocopos et al. [30]. In fact, a comparison of cataractous epithelial specimens obtained at surgery with specimens obtained from transparent and cataractous eye bank eyes led these authors to conclude that apoptotic cells are not present in cataractous epithelia if they are immediately fixed after surgery. Dead epithelial cells, only found when the samples are allowed to remain for some time in culture media, are produced by a necrotic, rather than an apoptotic, process and are probably secondary to the surgical trauma. The same authors did not find any evidence for a significant decrease in lens epithelial cell density during cataract formation and ageing. In the photo-oxida- tive cataract model, epithelial damage leads to a reduced uptake and an increased efflux of rubidium, reduced cellular levels of ATP and increased lens wet weight. Another important landmark of lens oxidative damage is the progressive decrease in the GSH/GSSG ratio, reflecting a drastic change in the redox set point which almost completely recovers upon stress removal. An upregulation, which can reach 38-fold for c-jun, 72-fold for c-fos and 5-fold for c-myc, has also been reported for these protooncogenes to occur in a rabbit lens epithelial cell line and in cultured rat lenses exposed to H2O2 levels ranging from 25 to 200 ÌM [31, 32]. The resulting accumulation of c- jun and c-fos, the two subunits of the transcription factor AP-1, may in turn promote the activation of a number of target genes, thus determining a large reprogramming of lens gene expression. The presence of an AP-1 binding site upstream of the gene has further led to the proposal that this gene may be one of the targets of the transactivator AP-1. Unfortunately, however, no evidence that H2O2 can influence ·-A-crystallin expression or that c-jun and c-fos are upregulated in lens epithelia from human cataractous lenses has been obtained so far. A slight, H2O2- induced upregulation of mRNA levels of the antioxidative stress genes of glutathione peroxidase and catalase [31] and the posttranscriptional upregulation of an ubi- quitin-activating enzyme [33] have also been reported in the mammalian lens.

An alternative approach to identify mammalian lens genes modulated in response to a severe oxidative stress is differential display (DD), a nontargeted gene-searching procedure that does not require any prior knowledge of either gene function or identity. One may utilize mRNA

DD analysis to investigate the mechanism of response of the mammalian lens to a severe oxidative stress.

In a preliminary analysis, Ottonello et al. [34] have utilized DD to investigate the in vitro response of bovine lenses exposed for 2.5 h to a severe H2O2 stress that reduced GSH levels to about 80% of controls. Focusing on mRNA modulation during the acute phase of oxidative stress, total RNA, extracted from epithelium/capsule preparations from treated and control lenses, was sub-

jected to mRNA DD analysis using 7 different combinations of primers. A total of 1,400 cDNA amplicons (corresponding to approx. 10% of the estimated mRNA lens population) was visualized in this way. Thirty putatively differential amplicons, 23 upregulated and 7 downregulated, were identified and eluted from display gels (fig. 1). Following reamplification and intermediate hybridization screening, 14 cDNA fragments representative of putative differentially modulated mRNAs (12 upregu-