Ординатура / Офтальмология / Английские материалы / Progress in Lens and Cataract Research_Hockwin_2002

additionally irradiated with UV-B. The experiments presented clearly demonstrate that dietary zinc and vitamin E deficiencies do interact with UV radiation damage in the cornea and lens of Brown Norway rats.

Copyright © 2002 S. Karger AG, Basel

Introduction

The eye is naturally exposed to electromagnetic radiation over a wider range of wavelengths, from ultraviolet ( 290 nm) via the visible spectrum (400–780 nm) to near infrared (781–1,400 nm). The daily dosages, however, may vary dramatically, depending on the weather conditions, the geographic location and the reflection characteristics of the environment. Independent of these factors, the anatomical position of the eye essentially determines the exposure of the eye tissues [1]. Due to their absorption characteristics for UV, cornea and lens are primary target tissues for UV damage [2]. Their reactions to UV-A and UV-B damage, however, differ remarkably. The cornea absorbs little UV-A but most of the more damaging UV-B, which causes typical pathological changes like droplet keratopathy and development of pterygia [3]. It has been questioned for many years whether enough UV-B reaches the human eye lens in order to produce permanent damage [4]. Müller-Breitenkamp et al. [5] have clearly demonstrated, however, that chronic UV-B irradiation from a sunlamp causes cortical cataract formation in man. They also were able to provide exact dosages [6].

Epidemiological studies have shown the high frequency of cataracts in those regions of the earth which have a high yearly exposure to sunlight and UV [7–9]. The observed cataract morphologies, however, vary considerably, pointing out that several factors are involved in their development and severity, corresponding to the well-established concept of coand syncataractogenic effects of noxious influences [10].

Apart from a range of general or ocular diseases also affecting the lens, selective nutritional deficiencies are regarded as important factors in cataract development [11]. To study the effects of ultraviolet radiation in combination with nutritional factors under controlled conditions, animal models in pigmented rats combined with modern optical monitoring and measurement techniques are the methods of choice for such investigations [12].

Dietary deficiencies of zinc and vitamin E have been chosen as nutritional factors to study their interaction with chronic UV-A and UV-B irradiation over a period of 2.5 months. These factors are involved in the oxidative defense mechanisms of the lens and in membrane stability, both biochemical properties being affected by UV irradiation damage.

Zinc is essential for many functions in the body [13]. Its highest concentrations in the human eye are found in the retina (464 g/mg) and choroid

(472 g/mg) followed by the ciliary body (227 g/mg). The lowest concentrations are found in the cornea (41 g/mg) and lens (21 g/mg) [14]. As an essential structural constituent of superoxide dismutase (SOD) zinc is important for maintaining the redox potential of the lens and its glucose breakdown [15]. By affecting the glucose degradation, zinc deficiency also reduces the concentration of reduced glutathione (GSH) in the lens, another essential component of the lens redox potential [16].

In the cornea, among other effects zinc is essential for wound healing [17]. In both tissues, as in other tissue of the body, zinc deficiency also affects the normal function of DNA and RNA synthesis [18], an effect which could interact with UV irradiation.

Vitamin E is a potent natural antioxidant, which is stored in the membranes of the lens cells. Its presence in the membranes protects unsaturated fatty acids from oxidation, thus the concentration of vitamin E is important for the fluidity and functioning of the membranes. UV irradiation has an effect on the membranes of the lens and Jacques et al. [19] could demonstrate in in-vitro experiments that the effects of several cataract factors including radiation damage are slowed down by supplementation of vitamin E. However only few research efforts have been made that study the effects of nutritional deficiencies of vitamin E and zinc in combination with chronic UV irradiation on the transparency of the cornea and lens. Cai et al. [20] investigated morphological and biochemical changes in rat lenses from vitamin E-deficient animals. Besides vacuolation in the equatorial region they could demonstrate a decrease of the activities of SOD and GSH reductase and an increase of the concentration of malondialdehyde in the lens, all indications for an increased rate of oxidative damage. The present study involved Scheimpflug photography and densitometric image analysis to investigate the effects of nutritional deficiencies of zinc and vitamin E alone and in combination with UV-A or UV-B irradiation on the transparency of the cornea and lens of rats in-vivo. In addition lens wet weight and selected biochemical parameters are determined after the sacrifice of the animals in defined layers of the lens, using the Bonn Freeze-Sectioning Technique [21].

Material and Methods

Female Brown Norway rats (BN, Charles River) with an initial mean body weight of 86–89 g were chosen for this study. They were housed in groups of 13 animals in type IV Makrolon® cages in a non-air-conditioned environment. Ambient temperature ranged from 20 to 25 °C and humidity was not regulated. A day-night rhythm of 12 h was maintained. The animals were never exposed to natural sunlight or its components. Special diets highly deficient in zinc and vitamin E, respectively, were prepared by Altromin (Lage, Germany). Non-diet-treated animals received Altromin® Standard Lab Chow for rats. Diet treatment

Table 1. Blood concentrations of zinc and vitamin E determined at the end of the study

n.d. not determined.

was maintained uninterrupted for 10 weeks. Irradiation treatment of the animals started 2 weeks after the beginning of diet application. Irradiation was performed with a Waldmann Sunlamp (UV 800) designed for dermatological application, equipped with 5 separate fluorescent bulbs each for UV-A (Phillips TL-K 40/09 N) and UV-B (Phillips TL 20 W/12). Their spectral maxima were 350 nm (UV-A) and 305 nm (UV-B), respectively. Irradiation sessions were always performed in a dark room with mydriasis induction by the instillation of atropine 1% 10 min prior to the start of irradiation. UV-A dosage per session was 1 J/cm2 equivalent to an irradiation time of 21 min. UV-B dosage per session was 0.2 J/cm2 equivalent to an irradiation time of 17 min. Three such irradiation sessions were performed per week on Monday, Wednesday and Friday.

General parameters controlled during the whole study period were body weight and food consumption in weekly intervals. Specific parameters for the study were slitlamp monitoring together with retroillumination photography in weekly intervals but photographic documentation was only performed in exemplary cases. Slitlamp monitoring comprised a baseline examination and a final examination prior to the sacrifice of the animals. Changes in light scattering and biometry of the cornea and lens were documented on a Kodak Tmax 400 b/w film with an SL-45 Scheimpflug camera (Topcon). Diet-treated groups had their baseline examination with this technique at the start of the diet. Two weeks later, at start of the irradiation treatment, the eyes of all animals were documented. Two other Scheimpflug photographic documentations followed at 4 and 8 weeks of irradiation treatment, the latter being the final documentation just prior to sacrifice. All recorded images were evaluated densitometrically according to standard procedures published elsewhere [22]. To monitor the course of the deficiencies, blood concentrations of zinc and vitamin E were determined [23, 24] (table 1). After sacrifice of the animals, the fresh weight of all lenses was determined, in order to get an insight into effects of the various treatment combinations on the growth characteristics of the lens. In addition, the concentrations of GSH and GSSG [25], the activity of SOD and the concentrations of water-soluble and water-insoluble crystallines were determined [26].

Fig. 1. Slitlamp micrograph of an UV- B-damaged rat cornea showing edema and stromal neovascularization.

Results

Mean body weight development was normal with an increase from around 80 to 180 g in all animals on normal diet and also in those groups on the vitamin E-deficient diet. In contrast, the mean body weight in those groups on the zinc-deficient diet increased only minimally from around 80 to 100 g over the study period.

Slitlamp microscopical examination did not evidence any abnormality in the eyes of the animals from the control group (1), the UV-A irradiated groups (2 8) and the vitamin E-deficient groups (7 8). The corneas and lenses of the animals in the UV-B-irradiated groups (3, 6, 9) showed the expected effects consisting of the formation of an anterior polar cataract in the sutural center and corneal edema sometimes followed by marked corneal neovascularization. The corneal edemas were intense in most UV-B-irradiated animals and the main vessels of the neovascular lesion had a large diameter (fig. 1). The eyes of those animals on a zinc-deficient diet only appeared normal until close to the end of the experiment, when a faint whitish ring of peripheral vacuolation of the corneal stroma and some faint very peripheral stromal vascularization starting from the limbal meshwork became discernable in most animals (fig. 2). Surprisingly, the combination of zinc deficiency and UV-B irraditation did not demonstrate additive but subtractive effects. Corneal edema was less pronounced and the diameter of blood vessels, which had grown into the stroma, remained much smaller (fig. 3). Even the lenticular changes seemed to be less intense in this combination. Neither zinc nor vitamin E deficiency showed any interactive effect with UV-A irradiation. In combination with UV-B irradiation,

Fig. 2. Slitlamp micrograph of the eye of a Zn-deficient diet-treated rat showing peripheral vacuolation and haziness of the cornea.

Fig. 3. Cornea of an UV-B-irradiated rat on a Zn-deficient diet, which shows a much less pronounced reaction to the irradiation damage.

vitamin E deficiency neither had a promoting nor a retarding effect on the pathological reaction of the cornea and lens.

Scheimpflug photography and subsequent densitometric image analysis extended the observations from slitlamp microscopy by evidencing differences indiscernible by slitlamp microscopy. First of all, both in groups 4 (Zn deficiency) and 7 (vitamin E deficiency) mean corneal density was significantly higher than in the control group at the last examination. In addition, both in groups 5 (UV-A Zn deficiency) and 8 (UV-A vitamin E deficiency) mean corneal density was higher than in group 2 (UV-A). At the third and the fourth evaluation dates, mean corneal density in group 6 (UV-B Zn deficiency) was significantly lower than in group 3 (UV-B). In group 9 (UV-B vitamin E deficiency), however, mean corneal density only indicated a trend to be higher than in group 3 (fig. 4).

The area of the anterior capsule and epithelium is the primary target for UV damage in the lens. At the third examination, mean capsulo-epithelial density in group 2 (UV-A) was significantly higher than in group 1, an effect which disappeared at the last examination, however. The combinations of UV-A and zinc deficiency (group 5) and UV-A and vitamin E deficiency (group 8), however, both produced a significantly higher mean density in the capsulo-epithelial layer than in group 2 at the final examination. UV-B damage was clearly expressed in group 3 (UV-B), group 6 (UV-B Zn deficiency) and group 9 (UV-B vitamin E deficiency). An important finding at the third examination

Fig. 4. Mean density data of the corneas from all groups at baseline (a), after 4 weeks

(Zn deficiency); c significant difference to group 7 (vitamin E deficiency); d significant difference to group 3 (UV-B); e significant difference to group 2 (UV-A).

was, however, that the mean densities in groups 6 and 9 were significantly lower than in group 3. This finding disappeared at the final examination (fig. 5).

The mean density data from the nuclear region in general did not evidence special effects of the treatment combinations.

The statistical evaluation of lens wet weight and biochemical parameters is shown in table 2 which summarizes again the data mentioned above.

Discussion

The general parameter body weight already demonstrates that zinc deficiency has a more pronounced effect than vitamin E deficiency. This is underlined by comparing the Zn concentrations in the blood of the animals to Zn concentration

Fig. 5. Mean density data of the lens capsulo-epithelial layer from all groups at baseline (a), after 4 weeks (b) and 8 weeks (c) of irradiation (0, 6 and 10 weeks of diet treatment). For an explanation of symbols see legend to figure 4.

in the drinking water at the end of the study period. The fact that both are in the same order of magnitude demonstrates that all Zn stores in the body have been depleted. In addition, the blood concentrations of vitamin E in Zn-depleted animals were down to 50% of the normal values, which is probably due to the reduced food consumption of Zn-deficient animals. This implies, however, that all Zn-deficient animals additionally suffered from a vitamin E deficiency.

Slitlamp microscopical examination evidenced that Zn deficiency alone already caused alterations of corneal morphology by inducing peripheral vacuolation and faint neovascular changes in the limbal area. This observation corresponds to corneal pathology reported in patients with acrodermatitis enteropathica [27]. Zn deficiency and UV-A irradiation did not evidence any interactive effects in the cornea. UV-B irradiation produced the expected edema and neovascular changes in the cornea, which are inflammatory reactions of the tissue to the insult. In combination with Zn deficiency, however, unexpected effects occurred. Instead of an enhancement of the damage, corneal edema was less

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severe and the expression of neovascular changes was less pronounced, demonstrated by the much narrower diameter of the newly formed blood vessels in the corneal stroma. The same trend could be observed in the lens. UV-B irradiation alone induced the formation of an anterior polar cataract, which was less pronounced in those animals which were additionally treated with a Zn-deficient diet. This could be explained by assuming that the tissue reaction to UV-B damage in Zn-depleted animals is less pronounced due to enzyme activity deterioration.

Scheimpflug density data support and extend the visible observations. They confirm that Zn deficiency reduces the reaction of the cornea and lens to UV-B whereas Zn deficiency in combination with UV-A irradiation causes significant density increases in the cornea and anterior lens capsule/epithelium. Vitamin E which did not show such interactive effects at the slitlamp microscope, evidenced significant density increases for the cornea and lens alone and in combination with UV-A. This was not the case in combination with UV-B. This is in contrast to observations from Kojima et al. [28] who found that vitamin E deficiency alone is an entirely subliminal cataract risk factor over more than 10 months.

Biochemical data recorded after death did not show such obvious effects. Zn deficiency in combination with both UV-A and UV-B did cause a significant retardation of lens growth evidenced by a reduced lens wet weight. Concentrations of reduced and oxidized GSH (GSH/GSSG) did not provide further information on underlying pathophysiological processes. The activity of SOD, however, was significantly lower in the lens epithelium in both diet combinations with UV-B irradiation.

The experiments presented here clearly demonstrate that dietary deficiencies of Zn and vitamin E do interact with ultraviolet radiation damage in the cornea and lens. Zn deficiency, however, has more pronounced cocataractogenic effects in combination with UV-A, whereas it reduces tissue reactions to UV-B. Vitamin E deficiency seems to be a much slower cocataractogenic factor in such irradiation combinations. This could in part be explained by the observations from Stephens et al. [29], who demonstrated that vitamin E concentrations in the cornea and lens of the rat are the lowest of all eye tissues, more or less regardless of the dietary levels of vitamin E.

References

1Sliney DH: Ocular exposure to environmental light and ultraviolet: The impact of lid opening and sky condition. Dev Ophthalmol. Basel, Karger, 1996, vol 27, pp 53–75.

2Pitts DG, Cullen AP, Hacker PD: Ocular effects of ultraviolet radiation from 295–365 nm. Invest Ophthalmol Vis Sci 1977;16:932–939.

3Taylor H, West SK, Rosenthal FS, Munoz B, Newland HS, Emmett EA: Corneal changes associated with chronic ultraviolet irradiation. Arch Ophthalmol 1989;107:1481–1484.