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

The area of anomalous scattering light of the HC subgroup was significantly wider than that of LC, and the nuclear light scattering had increased (fig. 5, 6). On the correlation between cataract and temperature, Miranda [16] reports that an analysis of the data reported in the literature suggests that the onset and prevalence of senile cataract follow the same trend as that of presbyopia. That is, ‘senile cataract’ (age-related cataract) develops earlier and is more prevalent in warmer regions. Miranda [16] also speculates that lifelong exposure of the lens to regular small increases in temperature may constitute an accelerating cofactor in the aging process by accelerating the metabolic rate of the lenticular epithelium [16]. It is reported that Brown Norway rats often show an increase of nuclear light scatter with aging [17]. For most biological systems, a temperature increase of 10 °F doubles the reaction rate [18, 19]. The increase of the scattered light in the lens nuclear regions also points to the possibility that high-temperature breeding promotes aging alternations in the rat. As for the relationship between cataractogenesis and temperature, aside from special research such as that concerning the glassblower cataract, very little attention has been paid to the significance of the ambient temperature of living environments. The relationship between cataract and environmental temperature should be the focus of more research in the future.

Acknowledgment

This study was supported by a Grant for Collaborative Research from the Kanazawa Medical University (C99-6).

References

1Hockwin O: Multifactorial pathogenesis of ‘senile cataract’. Nova Acta Leopoldina NF 1997; 75/299:25–36.

2Zigman S, Datiles M, Torcynski E: Sunlight and human cataracts. Invest Ophthalmol Vis Sci 1979; 18:462–467.

3Cruickshanks KJ, Klein BE, Klein R: Ultraviolet light exposure and lens opacities: The Beaver Dam Eye Study. Am J Public Health 1992;82:1658–1662.

4Taylor HR, West SK, Posenthal FS, Munoz B, Newland HS, Abbey H, Emmett EA: Effect of ultraviolet radiation on cataract formation. N Engl J Med 1988;319:1429–1433.

5Sasaki K, Sasaki H, Kojima M, Shui YB, Hockwin O, Jonasson F, Cheng HM, Ono M, Katoh N: Epidemiological studies on UV-related cataract in climatically different countries. J Epidemiol 1999;9:S33–S38.

6Hockwin O, Kojima M, Sakamoto Y, Wegener A, Shui YB, Sasaki K: UV damage to the eye lens: Further results from animal model studies: A review. J Epidemiol 1999;9:S39–S47.

7Michael R, Vrensen G, van Marle J, Löfgen S, Söderberg P: Repair in the rat lens after threshold ultraviolet light radiation injury. Invest Ophthalmol Vis Sci 2000;41:204–212.

8Yamada Y, Kojima M, Vrensen GFJM, Takahashi N, Sasaki K: Acute ultraviolet B induced lens epithelial cell photo-damage and its repair process. J Jpn Ophthalmol Soc 2001;105:102–110.

9Kojima M, Yamada Y, Shui YB, Hata I, Sakamoto Y, Sasaki H, Takahashi N, Sasaki K: Ultraviolet exposure as a risk factor in cataract formation. Environ Sci 2000;7:269–280.

10Hockwin O, Wegener A, Bessems G, Bours J, Korte I, Müller-Breitenkamp U, Schmidt J, Schmitt C: Models and methods for testing toxicity: Lens; in Hockwin O, Green K, Rubin LF (eds): Manual of Oculotoxicity Testing of Drugs. Stuttgart, Gustav Fischer, 1992, pp 254–317.

11Kojima M, Sasaki K: Application of a new Scheimpflug camera (EAS-1000) to animal cataract model. Ophthalmic Res 1992;24(suppl 1):3–9.

12Kojima M, Shui YB, Murano H, Sasaki K: Inhibition of steroid-induced cataract in rat eyes by administration of vitamin-E ophthalmic solution. Ophthalmic Res 1996;28(suppl 2):64–71.

13Diabetes Epidemiology Research International Group: Geographic patterns of childhood insulindependent diabetes mellitus. Diabetes 1988;37:1113–1119.

14MacDonald MJ, Liston L, Carlson I: Seasonality in glycosylated hemoglobin in normal subjects. Does seasonal incidence in insulin-dependent diabetes suggest specific etiology? Diabetes 1987; 36:265–268.

15Ohtsuka Y, Yabunaka N, Noro H, Watanabe I, Agishi Y: Effect of ambient temperature on aldose reductase activity. J Jpn Diabetes Soc 1994;37:827–832.

16Miranda MN: Environmental temperature and senile cataract. Trans Am Ophthalmol Soc 1980;78: 255–264.

17Kojima M: Studies of experimental cataract using regional lens analysis. J Jpn Soc Cataract Res 1993;5:1–12.

18Schwartz B, Feller MR: Temperature gradients in the rabbit eye. Invest Ophthalmol 1962;1:513–521.

19Kojima M, Okuno T, Miyakoshi M, Sasaki K: Effect of environmental temperature on cataract progression in diabetic rats. J Eye 2000;17:555–558.

Masami Kojima, PhD, Department of Ophthalmology, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa-ken 920-0293 (Japan)

Tel. 81 76 286 2211, ext. 3414, Fax 81 76 286 1010, E-Mail m-kojima@kanazawa-med.ac.jp

of lens fibers as seen most obviously by scanning electron microscopy (SEM) is that lens fibers have three types of interlocking devices, namely edge protrusions, ball-and-socket junctions and microplicae or tongues and grooves. The numerous ball-and-socket junctions are found on the apical and lateral surfaces of the cortical fibers, but great differences in density and size from superficial to deep cortical layers exist. However they remain restricted to the cortical and the perinuclear regions [2, 3]. It was pointed out that similar ball-and-socket junctions in adult human lenses from donor eyes are present in identical regions as in other mammals. In old postmortem eyes ruptures of lens fiber membranes are found at the same sites as the ball-and-socket junctions and grooves and ridges [4].

Human age-related cataract is one of the major causes of blindness. SEM studies of early opacities in ageing human donor lenses showed that radial shades, running parallel to the course of the fibers, occurred in the deep cortex and that in addition circular shades, which are running perpendicular to the course of the fibers, occurred in the same region [5]. In order to unravel the mechanisms underlying age-related lens changes in humans, numerous animal studies have been carried out mostly using albino rats. Age-related changes in albino Fischer rats showed that early lens changes occurred in the epithelial cells and in the superficial layer of the cortical fibers, subsequently leading to loss of clarity in the lens [6]. On the other hand, there are only few reports about age-related lens changes in pigmented rat strains.

Recently it has been reported that caloric restriction has a more protective effect in delaying the development of cataract in pigmented than in albino mouse and rat strains over their life spans [7]. Moreover, it has been shown that albino rats are more susceptible to ultraviolet B irradiation with respect to an increase in light scatter [8]. In addition, albino rats are more sensitive to lightinduced cataract [9, 10]. However, there are only few reports comparing lens fiber organization between albino and pigmented animals. In order to identify the reason for the differences in vulnerability to cataractous insults, we have investigated in this study the morphological characteristics of lens fibers in SpragueDawley albino and Brown Norway pigmented rats.

Methods

Nine female albino Sprague-Dawley and 3 female pigmented Brown Norway rats at 4 months of age were used in this study. After sedation and sacrifice, the eyes were removed and immediately fixed in 4% paraformaldehyde for several days. After removing the lenses, they were divided in two halves along the visual axis. One half was dissected for SEM. Cortical layers were carefully peeled off to expose lens fibers at different depths below the surface. The dissected pieces were dehydrated in a graded series of ethanols and dried by immersion in hexamethyldisilazane (Sigma Chemical, St. Louis, Mo.) (2 , 1 h), followed by

Fig. 1. SEM ultrastructure of lens fibers in pigmented Brown Norway rats in the anterior pole (a, d, g), the equatorial region (b, e, h), and the posterior pole (c, f, i ) is shown: taken in the superficial cortex (a, b, c), in the intermediate cortex (d, e, f ) and in the deep cortex (g, h, i). At all sites the lens fibers are connected by interdigitating edge protrusions. In the deep cortex (g, h, i) the fiber edges become undulated. Note that many ball-and-socket junctions are present on the lateral membranes of the equatorial lens fibers from superficial to deep cortical layers. Especially, large ball-and-socket junctions are present in the equatorial intermediate cortical layer (e).

drying overnight on filter paper. The pieces were mounted with carbon glue on special stubs, coated with platinum and studied in a Philips XL20 scanning electron microscope (Philips Industries, Eindhoven, The Netherlands). Animals were raised and sacrificed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Results

As shown in figures 1 and 2 lens fibers in both pigmented and albino rats are characterized by their very regular organization with mutually interdigitating

Fig. 2. SEM ultrastructure of lens fibers in albino Sprague-Dawley rats at the same locations as indicated in figure 1 for the pigmented rats. Note that only few and small ball- and-socket junctions are seen on the lateral membranes of lens fibers in this rat strain in all cortical layers, and that their sizes are smaller. Moreover, many membrane ruptures on lens fiber are seen.

edge protrusions keeping them strongly together, thereby minimizing the extracellular space. There is a variation in the form and dimension of the edge protrusions from anterior through equatorial to posterior and at different depths below the lens capsule. In the deep equatorial cortex the fibers have an undulating surface, which may be an adaptation to the sphere-like shape of the lens. There is no complex network of microplicae on the surfaces of the fibers.

In pigmented rats many ball-and-socket junctions on the lateral surfaces of the fibers are observed. There is a variation in density, shape and size between the superficial and the deep cortical layers (fig. 1). In particular, there are large ball-and-socket junctions in the intermediate equatorial cortical layer (fig. 1e). In addition, the ball-and-socket junctions are restricted to the cortical layers and are absent in the perinuclear cortex and nucleus.

In contrast, only few and small ball-and-socket junctions (fig. 2a–f ) are found in albino rats. A remarkable observation are the ruptures on the fiber membranes in the superficial anterior and superficial and intermediate equatorial cortex of albino rats (fig. 2a, d, g) and their absence in the posterior cortex.

With the exception of a slightly more irregular arrangement of cell nuclei in albino rats, there are no significant differences in SEM morphology between the bow regions of the lens of albino and pigmented rat strains. This is supported by a separate histological study using Hoechst staining for visualizing nuclear DNA.

Discussion

This SEM study shows that a main difference between the lenses of pigmented Brown Norway and albino Sprague-Dawley rats is the abundance of ball-and-socket junctions on the lateral membranes of the fibers in the pigmented strain (fig. 1). Especially in the intermediate, equatorial cortex these ball-and- sockets are large (fig. 1e). Most likely ball-and-socket junctions are formed during an early step of differentiation in the superficial equatorial layer and are growing upon further maturation towards the deeper cortex. However, they disappear in the perinuclear cortex and are fully absent in the nucleus. This formation may be related to the loss of nuclei, mitochondria, Golgi apparatus and endoplasmic reticulum upon final maturation of the lens fibers which occurs in this region of the lens [11–14]. On the other hand, there are only few small ball- and-socket junctions on the lateral surfaces of the fiber membranes in all cortical layers in the albino strain (fig. 2a–f ). We must assume that for unknown reasons in albino rats the formation of ball-and-socket junctions is disturbed. The fact is that the segregation of balls appears in the anterior intermediate cortex (fig. 2d).

Lightand electron-microscopic studies in rats have shown that under cataractogenic conditions as for instance threshold UV-B irradiation [15–17] and pCMPS treatment [18] first signs of increased light scatter and opacification are found in the equatorial cortex and are related to the presence of extracellular spaces in the intermediate cortical zone. Similar extracellular spaces are also found in clear ageing human donor lenses [19]. In these studies it was also shown that in the same lens region ruptures of fiber membranes are occurring. In two of these studies [18, 19] it was shown that the extracellular spaces are filled with fluid containing high levels of free calcium as detected by a histochemical method specific for free calcium. This means that in rat lenses under the experimental conditions used and in old human lenses there must be a significant leakage of cellular calcium into the extracellular space indicating a disturbance of calcium homeostasis. It has been emphasized by Duncan et al. [20] that calcium homeostasis plays a strategic role in the maintenance of lens

transparency and that calcium imbalance is accompanied by an imbalance of other ions. This calcium disturbance may also be responsible in the longer term for the observed membrane ruptures by for instance activation of calciumactivated calpain II [21] and consequently for the formation of high-molecular- weight aggregates or proteolysis of membrane-associated proteins [22, 23]. Comparable ultrastructural changes have been described for other experimental cataracts such as diabetic, galactosemic and naphthalenic ones which are also accompanied by a rise in calcium and early increased light scatter and eventually cataract [22, 23].

In the UV-B and pCMPS studies [16, 18] it was observed that the extracellular spaces are most evident in a deeper aspect of the lens bow. In this respect it is worth noting that in normal cells which means for the LECs and differentiating lens fibers most of the cellular calcium is sequestered in the mitochondria and the endoplasmic reticulum including the nuclear envelope. Especially the endoplasmic reticulum and nuclear envelope stores are involved in the physiological regulation of cellular calcium as outlined by Duncan et al. [20]. It is common knowledge that upon final maturation the cellular organelles including the cell nucleus are broken down and have fully disappeared in the deep cortex without affecting the cellular integrity of the lens fibers [11–14]. In the deep bow region it was found that the mitochondria and endoplasmic reticulum in the process of breakdown are characterized by the presence of significant amounts of free calcium [18, 19]. This indicates that the calcium stores are disturbed and are forced to release their sequestered calcium into the cytosol. This is corroborated by the finding of Duncan et al. [24, 25] that the free calcium concentration (pCa) is higher in the intermediate as compared to the superficial cortex and is going down again in the deep cortex. Upon further maturation the cellular free calcium is no longer present in the cytosol but mainly found along the fiber membranes and as shown by electron-microscopic tomography is aggregated to the extracellular face of the fiber membranes [18, 19, 26]. In addition, it was found that in the cytoplasm of the deep cortical fibers vesicular elements are found densely filled with free calcium [18, 19]. On account of their size and location in transmission electron-microscopic (TEM) images they must be identical with the ball-and-socket junctions described in the present and previous papers. TEM and freeze fracture observations further revealed that these structures are surrounded by gap junctional membranes [27]. Because of their location in the region of organelle and nuclear breakdown and release of free calcium this may indicate that the ball and sockets have a specific function related to the sequestering of free calcium and thus preventing the aggregation of the crystallins by free calcium.

On account of the evidence given above it is tempting to speculate that both the extracellular space between the fiber membranes and the ball-and-socket

junctions are crucial for sequestering the calcium liberated during the breakdown of the mitochondria, endoplasmic reticulum and nuclear envelope and may thus prevent the calcium to exert its deleterious effect on crystallins. It has been discussed that the binding of free calcium on the fiber membranes is due to the presence of phospholipids which allow ionic binding of the calcium to the negative charges of the phosphorous atoms [25]. Why and how the calcium is accumulating in the ball-and-socket vesicles is unknown but it cannot be excluded that the shift in the connexins upon fiber maturation has something to do with it. In view of this we can further speculate that the efficiency with which the liberated calcium can be sequestered, which is of strategic importance for the lens to remain transparent, may depend on the abundance and size of the ball- and-socket junctions. Because of this we can finally hypothesize that the low density and small size of the ball-and-socket junctions in the albino rat as compared to that in the pigmented rat may be the reason why albino rats are more vulnerable to cataractogenic insults than pigmented rats. As a consequence of this we have to be very careful with extrapolating cataract risk factors simply on the basis of studies in albino rats.

References

1Spencer WH: Lens; in Spencer WH (ed): Ophthalmic Pathology, ed 3. Philadelphia, Saunders, 1985, vol 1, chap 5, pp 423–479.

2Willekens B, Vrensen GFJM: The three-dimensional organization of lens fibers in the rhesus monkey. Graefes Arch Clin Exp Ophthalmol 1982;219:112–120.

3Vrensen GFJM, Sanderson J, Willekens B, Duncan G: Calcium localization and ultrastructure of clear and pCMPS-treated rat lenses. Invest Ophthalmol Vis Sci 1995;36:2287–2295.

4Vrensen GFJM, Kappelhof J, Willekens B: Morphology of aging human lens. Lens Eye Toxic Res 1990;7:1–30.

5Vrensen GFJM, Willekens B: Biomicroscopy and scanning electron microscopy of early opacities in the aging human lens. Invest Ophthalmol Vis Sci 1990;31:1582–1591.

6Uga S, Obara Y, Takehana M, Nishigori H, Hikida M, Mibu H: Morphological study of age-related changes in Fischer rat lens. Jpn J Ophthalmol 1996;40:33–41.

7Wolf NS, Li Y, Pendergrass W, Schmeider C, Turturro A: Normal mouse and rat strains as models for age-related cataract and the effect of caloric restriction on its development. Exp Eye Res 2000; 70:683–692.

8Löfgren S, Michael R, Ayala M, Söderberg PG: Iris pigmentation and pupil size is important in ultraviolet radiation cataract. Invest Ophthalmol Vis Sci 1999;40:S528.

9Rao GN: Light intensity-associated eye lesions of Fischer 344 rats in long-term studies. Toxicol Pathol 1991;19:148–155.

10Toyoda K, Imaida K, Mitsumori K, Sato H, Maekawa A, Onodera H, Takahashi M: Correlation between cataract and retinopathy due to lighting in F344 rats used in a long-term carcinogenicity study. J Toxicol Environ Health 1992;37:495–509.

11Bassnett S: The fate of the Golgi apparatus and the endoplasmic reticulum during lens fiber cell differentiation. Invest Ophthalmol Vis Sci 1995;36:1793–1803.

12Bassnett S, Beebe DC: Coincident loss of mitochondria and nuclei during lens fiber cell differentiation. Dev Dyn 1992;194:85–93.

13Bassnett S, Mataic D: Chromatin degradation in differentiating fiber cells of the eye lens. J Cell Biol 1997;137:37– 49.

14Bassnett S: Fiber cell denucleation in the primate lens. Invest Ophthalmol Vis Sci 1997;38: 1678–1687.

15Michael R, Vrensen GFJM, VanMarle J, Gan L, Söderberg P: Apoptosis in the rat lens after in vivo threshold dose ultraviolet irradiation. Invest Ophthalmol Vis Sci 1998;13:2681–2685.

16Michael R, Vrensen GFJM, VanMarle J, Löfgren S, Söderberg P: Repair in the rat lens after threshold ultraviolet radiation injury. Invest Ophthalmol Vis Sci 2000;41:204–212.

17Wegener AR, Landwehr H, Breipohl W: Effects of tryptophan and methionine deficiencies on the transparency of cornea and lens in pigmented and albino rats. Invest Ophthalmol Vis Sci 2001; 42:S289.

18Vrensen GFJM, Sanderson J, Willekens B, Duncan G: Calcium localization and ultrastructure of clear and pCMPS-treated rat lenses. Invest Ophthalmol Vis Sci 1995;36:2287–2295.

19Vrensen GFJM, DeWolf A: Calcium distribution in the human eye lens. Ophthalmic Res 1996; 28(suppl 2):78–85.

20Duncan G, Williams MR, Riach RA: Calcium signalling and cataract. Prog Retin Eye Res 1994; 13:623–652.

21David LL, Shearer T: Role of proteolysis in lenses: A review. Lens Eye Toxic Res 1989;6:725–747.

22Phelps Brown N, Bron AJ: Lens Disorders: A Clinical Manual of Catract Diagnosis. London, Butterworth-Heinemann, 1996.

23Bond J, Green C, Donaldson P, Kistler J: Liquefaction of cortical tissue in diabetic and galactosemic rat lenses defined by confocal laser scanning microscopy. Invest Ophthalmol Vis Sci 1996;37: 1557–1565.

24Duncan G, Jacob TJC: Influence of external calcium and glucose on internal total and ionized calcium in the rat lens. J Physiol 1984;357:725–747.

25Duncan G, Bushell AR: Ion analyses of human cataractous lenses. Exp Eye Res 1975;20:223–230.

26VanMarle J, Jonges R, Vrensen GFJM, DeWolf A: Calcium and its localization in human lens fibres: An electron tomographic study. Exp Eye Res 1997;65:83–88.

27Vrensen GFJM, VanMarle J, VanVeen H, Willekens B: Membrane architecture as a function of lens fibre maturation: A freeze-fracture and scanning electron microscopic study in the human lens. Exp Eye Res 1992;54:433–446.

Yoshihisa Yamada, MD, Department of Ophthalmology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-0293 (Japan)

Tel. 81 76 286 2211, ext. 3414, Fax 81 76 286 1010, E-Mail yoshihisay@yahoo.com