Galactose cataract: Changes in protein distribution during development. Bo Philipson

Transcription

1 Galactose cataract: Changes in protein distribution during development Bo Philipson Ticenty-day-old male rats toere fed on a diet containing 40 per cent galactose. Freeze-dried sections of cataractous and control lenses were studied by quantitative microradiography. During cataract development the protein concentration decreased in the cortex and, at early stages of cataract, also in the nucleus. In lenses with a distinct nuclear cataract, however, the lens nucleus had about the same high protein concentration as that in the controls. At this stage of cataract there was a great difference in protein concentration between the central and the peripheral lens regions, presumably partly because of changes in the concentrations of dulcitol and electrolytes. The zone between the cortex and the nucleus had a steep protein gradient and contained irregular interfaces separating regions with different refractive indices. The nuclear cataract may be explained by reflection of light at these interfaces. Scattering of light from the vacuoles in the cortex, most prominent at the early stage of cataract, is discussed. Other scattering processes in the cortex are also considered. Key words: galactose cataract, lens cortex, lens nucleus, lens epithelium, lens fibers, proteins, histopathology, subcapsular vacuoles of the lens, lens hydration, electrolytes, galactitol, rats. T.he clinical and histological features of experimental sugar cataract, particularly that induced by galactose, have been extensively investigated. 1 ' 2 The formation of cataract is characterized by a decrease in the total protein content and increased hydration. 3 " 5 The quantitative distribution of protein has been studied in normal lenses and in lenses with different stages of x-ray cataract. 0 ' 7 Concerning normal transparent From the Department of Medical Physics, Karolinska Institutet, Stockholm 60, Sweden. This work was supported by grants from the Swedish Medical Research Council (12X-587), Svenska Siillskapet for medicinsk forskning and from Karolinska Institutet (Reservationsanslaget). Manuscript submitted Nov. 8, 1968; manuscript accepted Dec. 11, lenses, it was concluded that the changes in refractive index were regular and continuous, except for moderate intracellular variations. When lenses with x-ray cataract were concerned, the main loss of transparency was ascribed to mechanisms of reflection in steep protein gradients, corresponding to interfaces between regions with different refractive indices. The aim of the present investigation was to correlate the changes in structure and in quantitative distribution of protein with the impairment of lens transparency during development of galactose cataract. Material and methods Animals and diet. Sprague-Dawley male rats, 20 days old, were fed on a diet consisting of 40 per cent by weight of anhydrous d-galactose and 60 per cent ground chow (Anticimex 210). The diet was mixed and given in unlimited amounts. Control animals were fed ground chow alone.

2 282 Philipson Investigative Ophthalmology June 1969 Ophthalmoscopical or slit-lamp examination of the lenses was performed daily. After varying intervals, galactose-fed rats together with control rats from the same litter were killed by an overdose of ether and the lenses removed. Lenses from 3 galactose-fed rats and from 3 controls were analyzed after each period of 6, 14, and 28 days, respectively. Preparation and microradiographical procedures. One of the removed lenses from each animal was immediately frozen in isopentane, cooled to about -140 C. by liquid nitrogen. The other lens was weighed and used for histological, chemical, or macroscopical studies. The frozen lenses were freeze-sectioned to a thickness of about 10 ft and then freeze-dried. Most sections were parallel to the optic axis of the lens, but some were equatorial. Microradiographical observations were made in at least 3 central sections of each lens. For quantitative determinations, a reference system composed of thin mylar foils (490 /*gcm, -2 ) was utilized. The microradiographical and densitometric procedures were identical to those described earlier. 0 The thickness of each section was measured in at least 10 different points. The dry mass of the lens was calculated from densitometric readings and thickness determinations and expressed as mass per unit volume of protein. The elemental composition of the dry material was determined microchemically (Mikroanalyslaboratoriet, Uppsala) in at least one lens in each of the 3 experimental groups. The mean values for carbon, hydrogen, and nitrogen were 50.3, 7.1, and 15.9 per cent by weight, respectively. These values differed only slightly from those in the normal lenses. The same ratio 1.02 between the mass absoiption coefficients for mylar and dry lens substance was obtained. However, changes in the concentration of K, Na, Cl, and dulcitol during the development of galactose cataract have been reported. The Na ions may cause a reduction of the ratio to about 1.00 for lenses with mature cataract in which a high Na concentration has been determined. Changes in the concentrations of other ions and of dulcitol will not have a detectable influence on the mass absorption coefficient for dry lens substance. Consequently, 1.02 is a reasonable average value but it may be 1 or 2 per cent too high at the last stages of cataract. Shrinkage of the freeze-dried sections was measured to be about 10 per cent, which was corrected for in the calculation of protein content per unit volume. The nonprotein constituents were estimated to about 4 per cent by dry weight in the normal lens.' 1 - G The lipids constitute the main nonprotein fraction in the normal lens and were shown to have about the same concentration in the cataractous lens. s During the early stages of galactose cataract there is an accumulation of the sugar alcohol, dulcitol, reaching values about 4 per cent by dry lens substance in the cortex and about 2 per cent in the nucleus. 0 ' 10 The changes in the concentrations of K, Na, and Cl will increase the nonprotein fraction by about 1 per cent in the mature cataracts. The dry mass is here taken to represent the protein content and, consequently, protein values for the cataractous lenses include between 4 and 8 per cent nonprotein constituents. Other errors in micro radiography of this system have been discussed previously in detail. 0 Supplementary procedures. Some of the removed lenses were examined with a binocular stereomicroscope and with the slit lamp. One cataractous and one control lens from each of the 6, 14, and 28 day groups were prepared according to standard histological technique and stained with hematoxylin and eosin. Results Ophthalmoscopical and slit-lamp examination of the lenses from galactose-fed rats showed a cataract development similar to that reported by Cotlier 12 and Sippel. 5 After 2 days the equatorial cortex region appeared as a thin dark band on transillumination. On biomicroscopical examination this region was shown to contain vacuoles. The vacuolization migrated forward and involved the major part of the anterior cortex after 6 days of galactose induction. After 14 days the peripheral cortex still contained many vacuoles, but it appeared relatively clear and transparent, despite some delimited opacities. On slit-lamp examination, the interface between this zone and the inner cortex was distinct, because of the scattering of light. The border zone between the central, spherical part of the lens and the cortex was slightly opalescent and showed a discontinuous change in refractive power. The opacification of the outer region of the nucleus proceeded rapidly, but that of the cortex relatively slowly. After 28 days of galactose induction, the whole lens was cloudy and the nucleus nontransparent. The main structural and quantitative differences between cataractous lenses and controls, observed in microradiographs and in histological sections, will be described. In investigations on normal lenses pre-

3 Volume 8 Number 3 Protein distribution in galactose cataract 283 sented earlier, the nucleus of the lens was defined as the solid central region with a protein concentration exceeding 0.6 Gm.- cmr a After 6 days of galactose diet, vacuoles were recognized in microradiographs and in histological sections. In the equatorial cortex and in the major part of the anterior cortex, a zone about 300 /.i thick close to the capsule consisted of hydropic lens fibers and both isolated and confluent vacuoles (Fig. 1, AtoC). After 14 days of galactose induction, the microradiogiaphs and histological sections revealed a reduced number of vacuoles within the peripheral zone of the cortex, Fig. 1. A to C, Microradiographs from the peripheral part of the lens cortex, showing different types of vacuolization in lenses from rats fed galactose for 6 clays (A and B) and for 11 days (C). (Magnification xll5.) Fig. 2. A and B, Microradiograph (A) and photomicrograph (B) from the anterior peripheral cortex in lenses of a rat fed galactose for 14 days. A few vacuoles are present in A. Irregular and swollen cells can be observed in both A and B but are less distinct in A because of the uniform x-ray absorption. The small circular areas in (A) close to the periphery (capsule is lost) with a low x-ray absorption are cell nuclei of the thickened lens epithelium also observed in B. (Magnification x220.)

4 284 Philipson Investigative Ophthalmology June 1969 Fig. 3. A to C, Micro radiographs (A and B) and photomicrograph (C) from the border zone between the nucleus (to the right) and the cortex (to the left) in lenses from the 28 day group. A relatively sharp interface between the nucleus and the cortex can be observed most distinctly in B and C. Irregular lens fibers are seen on both sides of this border zone in A and B. Small vacuoles with a low content of dry material can be recognized in the nucleus. (Magnification x220.)

5 Volume Numbe about 500 /.i thick. Here the cortical lens cells were disarranged and irregularly shaped. Many of these cells were hydropic and contained a nucleus, the majority being located close to the thickened lens epithelium within the previously mentioned peripheral zone (Fig. 2). Some formed, however, spherical groups in the inner cortex. Similar, but more pronounced, changes were observed in the 28 day group (Fig. 3). The nucleus then had a conspicuously higher x-ray absorption than the cortex with a border zone containing many small and almost empty vacuoles (Fig. 4). Densitometric evaluation of the microradiographs was performed in order to determine the dry mass taken to represent, approximately, the protein content. In 3 lenses from each of the 6, 14, and 28 day groups, the protein content was determined in small areas, 13 p. in diameter. These areas were selected 100 /J. apart along an Protein distribution in galactose cataract 2S5 almost straight line from the anterior to the posterior pole. Large vacuoles were avoided. The protein concentration was expressed in grams per cubic centimeter and plotted in diagrams as a function of the distance from the center of the lens. Representative diagrams from each of the 3 groups were selected to illustrate the distribution of protein (Fig. 5). The mean protein concentration in certain zones is given in Table I. A marked decrease in protein content was observed in all regions at all stages of cataract, except for the central zone at the stage of mature cataract (28 days on the diet). The low protein concentrations were generally more pronounced in the anterior cortex than in the posterior. The border zone, about 200,u thick, between the nucleus and the cortex had a steeper protein gradient at all stages of cataract than that in the controls. In the 14 day group, this gradient was of the or- Fig. 4. A and B, Microradiographs from 2 lens sections in the 28 day group. The anterior cortex and the nucleus are shown. The irregular structure of the peripheral cortex and the distinct border zone between the nucleus and the cortex are conspicuous. Artifact fractures are present in the freeze-dried sections, easily distinguished from the vacuoles. (Original magnification x70.)

6 286 Philipson Investigative Ophthalmology )une 1969 der of 0.13 Gm.-cmr 3 per 100 fx and reached values of about 0.45 Gm.-cm." 3 in the 28 day group. These values should be compared to the mean values for the controls of about 0.06 Gm.-cm." 3 per 100 /.«. In lenses with nuclear cataract, this zone and the peripheral region of the nucleus were composed of sharp but irregular interfaces, because of the steep protein gradient and the vacuoles containing only minor amounts of dry substance (Fig. 3). Determinations of the dry substance in the small vacuoles observed at all stages ranged from 0.01 to 0.02 Gm.-cm." 3 In vacuoles with a diameter exceeding 50 fx, the concentration of dry material was below the limit of detection. Within the hydropic lens fibers, the intracellular varia- J E u o> ANI tkluk POLE POSTERIOR POLE AGE 26 DAYS CONCEI^JTRAT ION DAYS ON DIET PROTI I i I AGE 34 DAYS / ^- f ^ 14 DAYS ON DIET i i i I i i AGE 48 DAYS, 28 DAYS ON DIET " nn mm DISTANCE TB FROM CENTER Fig. 5. Distribution of protein along the optic axis in central sections from 3 stages of galactose cataract (thick line) and from the corresponding control lenses (thin line).

7 Volume 8 Number 3 Protein distribution in gcdactose cataract 287 Table I. Protein concentration (Gm.-cmr 3 ) in 5 zones from lenses at different stages of cataract Dat/s on diet No. of lenses Zone I Zone II Zone III Zone IV Zone V (0.255 ±0.063) ( ) ±0.024 ( ) (0.645 ±0.040) (0.684 ±0.018) ( ) ±0.067 ( ) (0.888 ±0.003) ( ) ± ( ) ± (0.720 ±0.024) (0.678 ±0.086) ( ) ±0.030 (0.258 ±0.042) (0.292 ±0.032) Values are given as mean ± standard error of the mean. The corresponding mean concentrations of protein in the control lenses are given in parentheses. Zones I and V comprise the peripheral part, 100 fi thick, inside the capsule at the anterior and the posterior pole, respectively. Zones II and IV are situated halfway between the center of the lens and the anterior and posterior pole, respectively. Zone III comprises the center of the lens. tion in protein content was more pronounced, the central region of these fibers containing about 0.02 Gm.-cni." 3 The volume and the wet weight of the lenses were increased at all cataract stages. After 28 days on the diet, this increase was about 15 per cent for volume and about 5 per cent for weight. The volume increase was most prominent in the anterior cortex, partly as a consequence of the decreased size of the nucleus as compared to that of the controls. Discussion The biomicroscopical appearance of the cataractous lenses in the present investigation conforms to that reported in previous investigations. 1 5> 12 ' A decrease in protein concentration, partly caused by an accumulation of water in the lenses during the development of galactose cataract, has been well established » 13 ' 15 Here, a decrease in protein concentration, compared to the controls, was observed at all stages of cataract and in all regions except the lens nucleus at later stages. The reduction of the protein concentration was most pronounced in the peripheral cortex at the early vacuolar stage. When a distinct nuclear cataract had been developed, the protein concentration in the whole cortex was very low, but the protein concentration in the central region was on the same level as that in the corresponding control lenses. The border zone between the cortex and the nucleus was well defined because of a very steep gradient in protein concentration. The increased hydration of the lens in the early stages of galactose cataract has mainly been attributed to the accumulation of dulcitol (d-galactitol). 9 ' 1G This sugar alcohol was shown to be fairly uniformly distributed in all cortical regions of lenses from galactose-fed rats, but the concentration of dulcitol in the cortex was about twice that in the nucleus. 10 Dulcitol is supposed to be retained within the cells and, consequently, water will be drawn into the lens fibers in the different regions of the lens. 9 When the nuclear cataract appeared, it was accompanied by drastic chemical changes as shown by Kinoshita and Merola. 11 The hydration of the lens increased still more which, for the main part, is explained by a flow of Na and Cl ions into the lens, reaching extremely high levels. The concentrations of dulcitol and K were reduced, but these losses were more than compensated by an increase in Na and Cl. The entry of these ions may be explained by changes in membrane permeability presumedly most prominent in the cortex where an advanced cellular damage was recognized. Therefore, the high levels of Na and Cl are probably limited to the cortex in which these ions will create hypertonicity and an increased extracellular space. This will result in an uptake of

8 288 Philipson Inuestigatioe Ophthalmology June 1969 water in the cortex from the surrounding fluids, including water from the nucleus. A dehydration of the nucleus may explain the high protein concentration in the nucleus and the very steep gradient in protein content between the cortex and the nucleus after 28 days on galactose diet. Microradiographically determined protein concentrations in different parts of the lenses can be utilized to calculate the refractive indices in these lens portions. 0 ' 17 In this investigation lenses with almost complete opacification had calculated mean refractive indices of about 1.50 in the nucleus and 1.37 in the cortex. In the zone between the cortex and the nucleus, the gradient of the refractive index was then very steep, about 0.13 over 200 /.<. Furthermore, this zone and the peripheral region of the nucleus contained many small and irregular vacuoles. The surfaces of these vacuoles caused very steep and irregular changes in refractive index. The nuclear cataracts may be explained by scattering or rather reflection from these irregular interfaces. 7 The opacification of the cortex in the early stages of cataract might be caused mainly by vacuolization and formation of irregular hydropic lens fibers. The surfaces of the vacuoles will cause losses of light due to partial and total reflection. The vacuoles will also function as small spherical negative lenses diverging the light. These losses of light will vary with the angle of incidence at the surface of the vacuoles and will be very great for angles from 80 to 90 degrees. The diffuse scattering of light from the cortex in mature cataracts is probably caused by several factors, besides those discussed previously. Small protein molecules are supposed to pass through the disrupted fiber membranes and the lens capsule, resulting in a high relative percentage of large protein molecules, such as a- crystalline and albuminoid. 1 Since scattering increases with the second power of the volume of the molecule, these protein molecules have a greater ability to scatter light than the small molecules. Furthermore, changes in the spatial order of the protein molecules may be involved implying a contribution of independent scattering from each molecule. 1S This may be a major factor since the protein concentration was reduced, and the regular cellular structure was destroyed. The relative importance of these different scattering processes cannot be decided from the present investigation. Experiments are, however, in progress to measure light scattering from certain regions of these lenses. I wish to thank Dr. Bo Lindstrom for advice and constructive criticisms during the course of this work. Mrs. Barbro Lindstrom is acknowledged for technical assistance. REFERENCES 1. Gifford, S., and Bellows, J.: Histologic changes in the lens produced by galactose, Arch. Ophth. 21: 346, Friedenwald, J., and Rytel, D.: Contribution to the histopathology of cataract, Arch. Ophth. 53: 825, Dische, Z., Zelmenis, G., and Youlus, J.: Studies on protein and protein synthesis: During the development of galactose cataract, Am. J. Ophth. 44: 332, Patterson, J. W., and Bunting, K. W.: Changes associated with the appearance of mature sugar cataracts, INVEST. OPHTH. 4: 167, Sippel, T. O.: Changes in the water, protein, and glutathione contents of the lens in the course of the galactose cataract development in rats, INVEST. OPHTH. 5: 568, Philipson, B.: Distribution of protein within the normal rat lens, INVEST. OPHTH. 8: Philipson, B.: Distribution of protein within lenses with x-ray cataract, INVEST. OPHTH. 8: Feldman, G. L., and Feldman, L. S.: New concepts of human lenticular lipids and their possible role in cataracts, INVEST. OPHTH. 4: 162, Kinoshita, J., Merola, L., and Dikmak, E.: Osmotic changes in experimental galactose cataracts, Exper. Eye Res. 1: 405, Cotlier, E., and Becker, B.: Rubidium-86 accumulation and dulcitol distributions in lenses of galactose-fed rats, Exper. Eye Res. 4: 340, Kinoshita, J., and Merola, L.: Hydration of

9 Volume 8 Number 3 Protein distribution in galactose cataract 289 the lens during the development of galactose cataract, INVEST. OPHTH. 3: 577, Cotlier, E.: Hypophysectomy effect on lens epithelium mitosis and galactose cataract development in rats, Arch. Ophth. 67: 476, Mitchell, H. S., and Cook, G. M.: Galactose cataract in rats, Arch. Ophth. 19: 22, Lerman, S., and Zigman, S.: The metabolism of the lens as related to aging and experimental cataractogenesis, INVEST. OPHTH. 4: 643, Kinoshita, J., Merola, L., and Hayman, S.: Osmotic effects of the amino acid-concentrating mechanism in the rabbit lens, J. Biol. Chem. 240: 310, van Heyningen, R.: Formation of polyols by the lens of the rat with "sugar" cataract, Nature 184: 194, Barer, R., and Joseph, S.: Refractometry of living cells. Quart. J. Micr. Sc. 95: 399, Trokel, S.: The physical basis for transparency of the crystalline lens, INVEST. OPHTH. 1: 493, 1962.