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Changes in the lens related to the reduction of transparency
Elrp. EyeRes.(1973)16,29-39 Changes in the Lens Related to the Reduction of Transparency B.
PHILIPSOX*
( ‘hanges in the lens mill cause fluctuations in refractive index which, in turn, will increase the scattering of light. Such alterations were studied by quantitative microradiography, electron microscopy, and a light scattering technique. Certain types of opacities of the human cortical cataract were shown to start as an enlargement of the intercellular spaces, followed by t,he breakage of lens cell membranes, loss of some of the protein matrix and the creation of large refractive interfaces between zones with different protein concentrations. The human nuclear cataract differed comp1etel.v from the various types of cortical opacities. Here, t,le only change detected was an aggregation of the protein molecules into dense clusters of about 500-1000 A in diameter. Aggregates of some similarity were also found in the cold cataracts within the nucleus of calf lenses. In these two kinds of nuclear cataracts the reduced transparency can be explained by the presence of these aggregates which are visible under electron microscope.
1. Introduction Knowledge of the refractive index in the various parts of the lens is fundamental in understanding transparency of the normal lens or the reduction of transparency in cataractous lenses.Fluctuations in refractive index are the physical basefor increased light scattering which is by far the most important phenomenon in most cataracts and much more important than resonanceabsorption in lens pigments. E’urthermore, light scattering in the lens not only reduces the incident intensity of light, but also causesglare and distortion of the image formation of the retina. Changesin morphology, in quantitative distribution of protein, and in molecular organization, may all affect the refractive index. In this study, somechanges will be reported which seem to be strongly linked to the increasedlight scattering in lens opacities. Only principal findings related to the reduction of transparency in the lens will be reported and discussed.Subsequent publications will contain more detailed information about changes in different types of human cataracts. 2. Material and Methods Material
Transparent human lenses from individuals ranging in age from 50 to 75 years were obtained from autopsies or taken from eye bank donor eyes within 8 ht after death. Lenses with cataracts were received at intracapsular surgery. Bovine lenses from 2-8week-old calves were obtained immediately at, slaughter. All lenses were immediately placed in ice-cold Earle’s buffer (pH 7.2) and then examined with a slit lamp microscope. Methods
I
The quantitative mlcroradiographic procedure was the same as described earlier (Philipson, 196913; Philipson and LindstrGm, 1969). This method involves the following main stepsin the procedure: 1, freeze sectioning; 2, freeze drying; 3, exposureof sections about 10 pm thick to soft X-rays; 4, photometric evaluation. By this method it is possible to determine the dry weight in the lens in volumes on the order of 10 pm3 to an estimated accuracy of 0.5 _ lo-l2 g (Philipson, 1969a). The dry mass of the lens can be taken to represent the protein content which then includes between 4 and 8% of non-protein con* Present Sweden.
address:
Department
of Ophthalmology, 29
Karolinska
Hospital
5-10401
Stockholm
60,
30
B. PHILIPSON
stituents. Furthermore, t,he refra&ive index can be calculated, as it is x linear function of the protein concentration. Consequently, both the protein concentration and the refractive index can be determined with high accuracy within small volumes of the lens. The electron microscopic preparation procedure utilized was the same as described hy Philipson, HLnninen and BaIazs (1973). In this procedure thin slices of lens material were fixed by glutaraldehyde or formaldehyde vapor in a closed weighing flask at 4°C. Small pieces were then postfixed in 0~0, l-2:4 and stained en bloc with O-5 -2O-;, uranyl a.wtate. The t,issue was then dehydrated in graded ethanol and embedded in Arwldite-Epon resill. Sections were cut with a diamond knife on a LKB-microtome and stained wit,h lead citr;\tr and uranyl acetate. Examination was performed under a Philips 30!) electron microw~pe at. ticI kV. Light scattering from sections about, 0.5 nm itr thickness was deternlined acwrtlirr,~ t( i the procedure described by Vinciguerra and Bet.telheitu (1971). A 1ow Tmwer heliun-ite1111 lasw (A = 632.8 nm) was utilized as a light source.
3. Results and Discussion The distribution of protein in t.he normal hurttatt transparent lens n-its st,udiwi 1)~. quantitative microradiography (Fig. 1). The dry matis which consists of ilborlt YV’,, protein was shown t*o he distributed evenly t’hroughont the km. No sharp fluckuntiullx in the protein distribution were found in the normal lens. The concentratjion of’ prc)tjtbin revealed a gradient which increased smoothly from cortex to thr nucleus. Gmilar to that determined in t)he rat lens (Philipson. 1969b). Howwer, the protcGtt conctwt’rat,ion in the center of t-he human lens was n~uch lower than t,hat in the adult rat l(~tt~ The refractive index is a linear function of the protein concentrat’ion. (~‘o~lsecl~~~t~tl~-, the absence of steep and irregular changes in prot’ein distribution is inlportant for limiting the fluctuation in refractive index and for keeping t)he lens transparwt The fine structure of t,he lens was studied bp clect,ron microscop!;. This ih rwt :I,
Frc:. 1. Rlicroradiogram transparent mass (=
lens. protein)
showing the distribution The mass is evenly distributed are detected. ( x 266)
and
of dry mass in the nn sharp fluctuations
cortex from it II~~~~III in the concentration
I~unran of tlr>
LENS
C’HAKGES
AND
TRAKSPARENCY
31
quantitative method, but alterations in fine structure may still give information about such changes that are likely to be accompanied by strong fluctuations in refractive index. In the optical zone of the normal lens we find very few cell nuclei and organelles. It has been shown by many investigators (U’anko and Gavin, 1959, 1961; jakus, 1964; Cohen. 1965; Huwabara, 1968) that both nuclei and cell organelles are confined mainly to the lens epithelium or to the most peripheral cortex. Consequently, light scattering from such structures is reduced to a minimum. The protein matrix seems to be very uniformly dispersed within the cells in all layers of both the human and the bovine lens (Fig. 2). Furt$ermore. there seems to be an absence of large, densely packed, molecular agm&s. These two factors limit the light scattering by destructive interference and by reduction of the initial scattering, respectively.
Ftc:. 2. Electronn+xwgraph from wosx.sectionetl. Sate the frequent stained cell matrix. ( 9450)
the inner portion of a human transparent) lens. The lens fiber cells are interdivitations of the cell membranes and the even distribution rlf
Even in the normal transparent lens. some light is scattered from certain parts OI structures. Naturally, some light is scattered from the surface of the lens due to the large difference in refractive index between the aqueous humor and the lens capsule. However. the smoothness of the outer surface of the capsule and the virtually right, angle of incidence of light to this surfacelimits this scattering to a relatively small magnitude normally. The bovine lens cell membranes have been shown earlier to scatter light (Vinciguerra and Bettelheim. 1971). In this study it could also be shown t,hat the human lens fiber cell walls scatter light (Fig. 3). Helium neon laser light was passed through thin slices of lens substance about 22500 p:n thick. The diffraction patteru registered from the human cortex corresponded to spacings of about 7 pm. The diffraction maxima was most likely caused by the lens fiher membranes or the intercellular spaces. The human lens is not as regular as the calf lens in its cell dimension and consequently, the diffraction maxima was not as strong and well defined as in the bovine lens. It has been assumed that such light scattering causes the halo around bright objects (Simpson, 1953).
3”
1:. PHILIPSOX
Another source for light scattering is the lens sutures. In this region. intricate interdigitations of the cell nmnhranes are found to form \)odies as large as approximately 0.5 pm in dianleter. Similar structures have been shown earlier by Wanko and Gavin (1961). These structures will act as cmters for light, scattering atld aw certainly very iniport~ant in niaking the sutures visiljle.
FIG. 3. Diffractogram showing low angle laser light lens cortex. Pattern was registered without analyzer.
Portid
scattering
from
a section
of human
trmslmrw~
cataracts
Human cortical cataracts arc a very large group of cataracts wit,11opacitiw of wry different appearance. All arc characterized by the fact that the? Aart, as mlall local opacit,ies in a relatively transparent cortex. Two comnion major types of cortical cataracts arc the subcapsular and suprsnuclear cata.rncts (Nordmann, 1952). The opacities of a subcapsula,rcataract appear as sonlewhat~irregular men~branessituattltl it) the peripheral cortex and are relatively parallel to the lens capsule. The SU~I’ILnuclear cuneiform cataract is characterized by radial or concentric opacities deep within the cortex. Theseopacities often appear as spokesor riders. All lensesincludetl in this st,udy showedearly stagesof cataract where t’he opacities still were surroundetl hv transparent cortex. In this investigation studies of mature cataracts have been avoidetl since they will a,dd very little inforrnatiou about t.he primary mechnnisnlsof cataractogenesis. In the earliest stagesof the cortical cataract. the opacities might appear only as a local slightly increased turbidity. When such opacities were examined by microradiography, no definite changescould be detected. When studied bv electron microscopy, the lens fiber cells appeared intact. but the intercellular spaceswere enlarged (Fig. 4). The cell membraneswere generally still intact, but sometimesbroken nlernbranes and the beginning of the dissolution of lens cells were found. Often, the broken
LEXS CHANGES ANDTRANSPARENCY
FIG. 4. Electronmicrograph cataract. The intercellular
33
from the inner cortex of a human lens during the early stage of cuneiform spaoes are enlarged, but the cell membranes are mainly intact. ( x 26,600)
membranes became attached to themselves or adjacent membranes forming vesiclelike structures (Fig. 5) and closing off the extracellular fluid. The subcapsular and cuneiform cataracts were studied both by microradiography and electron microscopy. To illustrate the principal changes of these cataracts, a lens from a 74-year-old patient was used. This lens showed subcapsular membrane-like opacities in both the
FIG. 5. Electronmicrograph from a human lens with a somewhat more advanced stage of cuneiform cataract than that found in Fig. 4. The intercellular spaces are enlarged, and the cell membranes have formed vesicle-like structures enclosing parts of the cell matrix. ( x 21,700)
B. PHILIPSOS
34
0. 3-
m
‘;-
0.: 3-
‘E 0 b.
,4l
C
,37
0, It b) (b)
I 0.2
I
I 0.4
I
I
0.6
mm
Pm.
6
I
I
,333
LENS
CHANGES
AND
TRAXSPAREKCY
35
anterior and the posterior cortex. Furthermore, this lens contained dense radial opacities. When the lens was studied microradiographically, it, was shown that all the dense and distinct opacities were accompanied by locally decreased protein concentrations (Fig. 6). The border zone between the relat.ively transparent cortex and the opacities was associated with steep irregular gradients in protein concentration. The magnitude of these protein concentration gradients was generally in the order of 0.1-0.2 g/cm3. These changes in protein concentration correspond to fluctuation in the refractive index of about 0~020~04. The refractive interfaces formed are irregular and wavy, which will make them act as strong sourcesof light scattering. The light will hit this refractive interface under different anglesof incidence, thereby increasing the scattering of light. Electronmicrographs of the region corresponding to the subcapsular membranous opacity revealed the fine structure of this type of irregular refractive interface (Fig. 7). The radial opacities of the cuneiform cataract, when studied electron microscopically, revealed enlarged extracellular spaceswhich often created large clefts in between the lens fiber cells (Fig. 8). Sometimes lens fibers seemedto dissolve and leave only
PIO. 7. Electronmicrograph of the region between the relatively dissolved peripheral zone of a subcapsular cat,aract. Notice the wavy which explains the high scattering of light. ( x 14,000)
intact inner cortex and the partly interface between the two portions
FIG. 6. (a) Microradiogram from a human lens with cortical cataract, both subcapsular and supranuclear spokes. To the left is the anterior cortex with a reduced X-ray absorption. The capsule is torn off during the preparation procedure. About 0.6 mm from the periphery is another area with reduced X-ray absorption. This area has the same position as the spoke-like opacities, as revealed under slit lamp examination. Note the sharp and irregular interface between the regions with normal X-ray absorption and the region with reduced absorption. ( x 160); (b) the protein concentration and the refractive index determined along the marked line in the lens section shown in (a). Note the low concentration in the dark areas of the miororadiogram and the sudden steep jumps in protein content and refractive index; (c) schematic drawing of the microradiogram in (a). Only the strong refractive interfaces are marked. Two rays of light are shown, and some of the main centers of light reflection or scattering are indicated.
36
B. PHILIP:;ON
the membranes which formed myeline-like bodies in the clefts. In t’he later stages of these cataracts, there were often found opaque regions displaying more advanced ultrastructural changes. The lens fiber cells were pnrtlv dissolved. a,ntl the membranes formett small vesic1e.s enclosing minor amounts of n&-ix. The 1~lectronlnicrographs. as well, gave a suggestion that the distribution of drv tuass was not homogeneous causing refractive index fluctuations (Fig. 9). 111conclusion. the cortical opecitiestudied seemed to start, wit811 an enlargement of the int,erwllular sl)aces. probabl\~ proceedtd t)y mwnbrane cleficicney. This enlargelt~cnt seeurwl to Iw follow-cd t)v t tub ulatrix ant1 tllch breakage of lens cell memt)ranes. which causetl the lo is of’ prott4ii creation of uifnibram~ \wiclt:s with varying f)r.otc>in wiicentrat ion atrd refract i\-(’ IUp, itlt.c,r. itrclex. In the regions lwt.~vveet~the t.ransparent. cortcks antI t.licl opxitics. faces were fotltltl between zones with different ptcirl ~~OIl~t~tlttilti~~ll ilIlt refrartivl~ imles.
Xucleur
cata,ract
The human nuclear cataract is completely difierent from those cataracts which are included in the grouping labeled cortical cataracts. Its early st,ages. which are very common in aged human lenses, are called nuclear sclerosis. This type is characterizctl by a diffuse and generally yellowish scattering of light throughout the nucleus of the lens. No defined or structured opacities are revealed under slit lamp examination. The more advanced stages, called real nuclear cataract,, and giving reduced visual acuity, are much less common than the corresponding cortical types (van Heyningen.
LENS
CHANGES
F’Ic:. 9. Electrnnmicrograph from a relatively dissolved, and the cell membranes hare formed t,s) txh very inhomogeneous. ( i( 21,700)
ANI)
TRANSPARENCY
37
a~lvanced cortical cataract. Most lens fiber cells arc’ vt:siclt\-lilrr bodies. The distribution of cell matrix XPWIS
PIG. 10. Electronmicrograph from the nucleus of a lens with dense nuclear cataract. Densely stained, large aggregatea of cell matrix are revealed. The diameter of the aggregates is about 500-1000 ip. ( :I’ 90,000)
38
B. PHILIPSON
1972). The nuclear cataract is often accompanied by the formation of a yellow-brown pigment, but on rare occasion the cataract appears to be whitish. The distribution of protein, as revealed by microradiography, does not show any detectable fluctua.tion in the protein content. The electron microscopical appearance of the lens fiber cells of the nuclear cataract did not show any difference from that of a normal transparent lens nucleus. The extracellular spaces were seldom larger than 200 A. The membranes appeared the same as in the normal transparent nucleus. In both normal and cataract lenses, gap-junctions are more frequent than in the lens periphery (Philipson, Hanninen and Balazs, unpublished manuscript). However, in dense, whitish nuclear cataracts the matrix was shown to aggregate in dense clusters with a diameter of about 500-1000 H (Fig. 10). This type of dense aggregates was never seen in the transparent, or the slightly “sclerotic” lens nuclei. It has been shown by Benedek (1971) that dense molecular aggregates of this size might scatter light to such a magnitude that it, would explain the turbidity of these cataracts. This type of cataract has been studied more extensively and will be reported in a subsequent report.
FIG. 11. Electronmicrograph from the nucleus made a.t a temperature of about 4°C. Non-stained 2000 A. ( x 21,700)
of a calf lens with cold cataract.. All preparations areas are seen which have a diameter of about
were IOOO-
An interesting analogy to the human nuclear cataract is the cold cataract which can be seen in young lenses from many animals. The calf lens exhibits a dense white nuclear cataract at temperatures of 10°C or below. This cataract was studied electron microscopically, and all preparation procedures were made at low temperatures (4°C). The electronmicrographs showed non-stained areas with a diameter of lOOO2000 A, which were never detected in the transparent calf lens. These areas are more
LENS
CHANGES
AND
TRANSPARENCY
39
likely to be interpreted as spherical aggregates (Fig. 11) than empty spaces. As it has been shown earlier, the concentration of y-crystallin is crucial in the formation of this cataract (Balazs, Teeta and Armand). It is very tempting to assume that the non-stained areas are dense aggregates composed mainly of y-crystallin. Moreover, it seems that the cc-crystallin is very easily stained by this preparation procedure and appears to be almost absent in these formations. Further studies are detiitely needed in order to determine the nature of these non-stained aggregates. However, it is certain that the distribution and the size of these structures could easily explain the very strong scattering of light in the cold cataract. ACKNOWLEDGMENTS
I thank Dr E. Balazs for valuable advice and for providing excellent facilities at the department of connective tissue research, Boston Biomedical Research Institute, Boston, during my stay as a visiting scientist. The technical assistance by L. HBnninen and C. Lindyuist is greatly appreciated. This work was supported in part by a grant (EY-00223) of the National Eye Institute, VSPHS, by the Swedish Medical Research Council (Project No. 13x-3008 and 6OF-3653) and by a Public Health Service International Post doctoral Research Fellowship (No. If05 TW 1801). REFERENCES Balazs, E., Testa, M. and Armand, G. In manuscript. Benedek, G. B. (1971). Applied Optkx 10,459. Cohen, I. (1965). Invest. Ophthulmol. 4, 433. van Heyningen, R. (1972). Exp. Eye Re.s. 13, 136. Jakus,M. (1964). Selected Electron Micrographs, Vol. 1. Little Brown and CO., Boston. Kuwabara, T. (1968). Arch. Ophthdmol. 79, 189. Nordmann,J. (1972). Ophthalmol. Res. 3, 323. Philipson, B. (1969a). Acta Ophthd. SuppZ. 103. Philipson, B. (1969b). Invest. Ophthdmol. 8, 258. Philipson, B. and Lindstriim, B. (1969). Histochemie 17, 201. Simpson, G. C. (1953). Brit. J. Ophthalmol. 37,450. Vinciguerra, M. J. and Bettelheim, F. A. (1971). Exp. Eye Res. 11,214. Wanko, T. and Gavin, M. A. (1959). J. Biophys. Biochem. Cytol. 6,97. Wanko, T. and Gavin, M. A. (1961). In The S&z&we of the Eye, p. 221. (Ed. Smelser, Academic Press, New York.
G. 9.)
PHILIPSOX*
( ‘hanges in the lens mill cause fluctuations in refractive index which, in turn, will increase the scattering of light. Such alterations were studied by quantitative microradiography, electron microscopy, and a light scattering technique. Certain types of opacities of the human cortical cataract were shown to start as an enlargement of the intercellular spaces, followed by t,he breakage of lens cell membranes, loss of some of the protein matrix and the creation of large refractive interfaces between zones with different protein concentrations. The human nuclear cataract differed comp1etel.v from the various types of cortical opacities. Here, t,le only change detected was an aggregation of the protein molecules into dense clusters of about 500-1000 A in diameter. Aggregates of some similarity were also found in the cold cataracts within the nucleus of calf lenses. In these two kinds of nuclear cataracts the reduced transparency can be explained by the presence of these aggregates which are visible under electron microscope.
1. Introduction Knowledge of the refractive index in the various parts of the lens is fundamental in understanding transparency of the normal lens or the reduction of transparency in cataractous lenses.Fluctuations in refractive index are the physical basefor increased light scattering which is by far the most important phenomenon in most cataracts and much more important than resonanceabsorption in lens pigments. E’urthermore, light scattering in the lens not only reduces the incident intensity of light, but also causesglare and distortion of the image formation of the retina. Changesin morphology, in quantitative distribution of protein, and in molecular organization, may all affect the refractive index. In this study, somechanges will be reported which seem to be strongly linked to the increasedlight scattering in lens opacities. Only principal findings related to the reduction of transparency in the lens will be reported and discussed.Subsequent publications will contain more detailed information about changes in different types of human cataracts. 2. Material and Methods Material
Transparent human lenses from individuals ranging in age from 50 to 75 years were obtained from autopsies or taken from eye bank donor eyes within 8 ht after death. Lenses with cataracts were received at intracapsular surgery. Bovine lenses from 2-8week-old calves were obtained immediately at, slaughter. All lenses were immediately placed in ice-cold Earle’s buffer (pH 7.2) and then examined with a slit lamp microscope. Methods
I
The quantitative mlcroradiographic procedure was the same as described earlier (Philipson, 196913; Philipson and LindstrGm, 1969). This method involves the following main stepsin the procedure: 1, freeze sectioning; 2, freeze drying; 3, exposureof sections about 10 pm thick to soft X-rays; 4, photometric evaluation. By this method it is possible to determine the dry weight in the lens in volumes on the order of 10 pm3 to an estimated accuracy of 0.5 _ lo-l2 g (Philipson, 1969a). The dry mass of the lens can be taken to represent the protein content which then includes between 4 and 8% of non-protein con* Present Sweden.
address:
Department
of Ophthalmology, 29
Karolinska
Hospital
5-10401
Stockholm
60,
30
B. PHILIPSON
stituents. Furthermore, t,he refra&ive index can be calculated, as it is x linear function of the protein concentration. Consequently, both the protein concentration and the refractive index can be determined with high accuracy within small volumes of the lens. The electron microscopic preparation procedure utilized was the same as described hy Philipson, HLnninen and BaIazs (1973). In this procedure thin slices of lens material were fixed by glutaraldehyde or formaldehyde vapor in a closed weighing flask at 4°C. Small pieces were then postfixed in 0~0, l-2:4 and stained en bloc with O-5 -2O-;, uranyl a.wtate. The t,issue was then dehydrated in graded ethanol and embedded in Arwldite-Epon resill. Sections were cut with a diamond knife on a LKB-microtome and stained wit,h lead citr;\tr and uranyl acetate. Examination was performed under a Philips 30!) electron microw~pe at. ticI kV. Light scattering from sections about, 0.5 nm itr thickness was deternlined acwrtlirr,~ t( i the procedure described by Vinciguerra and Bet.telheitu (1971). A 1ow Tmwer heliun-ite1111 lasw (A = 632.8 nm) was utilized as a light source.
3. Results and Discussion The distribution of protein in t.he normal hurttatt transparent lens n-its st,udiwi 1)~. quantitative microradiography (Fig. 1). The dry matis which consists of ilborlt YV’,, protein was shown t*o he distributed evenly t’hroughont the km. No sharp fluckuntiullx in the protein distribution were found in the normal lens. The concentratjion of’ prc)tjtbin revealed a gradient which increased smoothly from cortex to thr nucleus. Gmilar to that determined in t)he rat lens (Philipson. 1969b). Howwer, the protcGtt conctwt’rat,ion in the center of t-he human lens was n~uch lower than t,hat in the adult rat l(~tt~ The refractive index is a linear function of the protein concentrat’ion. (~‘o~lsecl~~~t~tl~-, the absence of steep and irregular changes in prot’ein distribution is inlportant for limiting the fluctuation in refractive index and for keeping t)he lens transparwt The fine structure of t,he lens was studied bp clect,ron microscop!;. This ih rwt :I,
Frc:. 1. Rlicroradiogram transparent mass (=
lens. protein)
showing the distribution The mass is evenly distributed are detected. ( x 266)
and
of dry mass in the nn sharp fluctuations
cortex from it II~~~~III in the concentration
I~unran of tlr>
LENS
C’HAKGES
AND
TRAKSPARENCY
31
quantitative method, but alterations in fine structure may still give information about such changes that are likely to be accompanied by strong fluctuations in refractive index. In the optical zone of the normal lens we find very few cell nuclei and organelles. It has been shown by many investigators (U’anko and Gavin, 1959, 1961; jakus, 1964; Cohen. 1965; Huwabara, 1968) that both nuclei and cell organelles are confined mainly to the lens epithelium or to the most peripheral cortex. Consequently, light scattering from such structures is reduced to a minimum. The protein matrix seems to be very uniformly dispersed within the cells in all layers of both the human and the bovine lens (Fig. 2). Furt$ermore. there seems to be an absence of large, densely packed, molecular agm&s. These two factors limit the light scattering by destructive interference and by reduction of the initial scattering, respectively.
Ftc:. 2. Electronn+xwgraph from wosx.sectionetl. Sate the frequent stained cell matrix. ( 9450)
the inner portion of a human transparent) lens. The lens fiber cells are interdivitations of the cell membranes and the even distribution rlf
Even in the normal transparent lens. some light is scattered from certain parts OI structures. Naturally, some light is scattered from the surface of the lens due to the large difference in refractive index between the aqueous humor and the lens capsule. However. the smoothness of the outer surface of the capsule and the virtually right, angle of incidence of light to this surfacelimits this scattering to a relatively small magnitude normally. The bovine lens cell membranes have been shown earlier to scatter light (Vinciguerra and Bettelheim. 1971). In this study it could also be shown t,hat the human lens fiber cell walls scatter light (Fig. 3). Helium neon laser light was passed through thin slices of lens substance about 22500 p:n thick. The diffraction patteru registered from the human cortex corresponded to spacings of about 7 pm. The diffraction maxima was most likely caused by the lens fiher membranes or the intercellular spaces. The human lens is not as regular as the calf lens in its cell dimension and consequently, the diffraction maxima was not as strong and well defined as in the bovine lens. It has been assumed that such light scattering causes the halo around bright objects (Simpson, 1953).
3”
1:. PHILIPSOX
Another source for light scattering is the lens sutures. In this region. intricate interdigitations of the cell nmnhranes are found to form \)odies as large as approximately 0.5 pm in dianleter. Similar structures have been shown earlier by Wanko and Gavin (1961). These structures will act as cmters for light, scattering atld aw certainly very iniport~ant in niaking the sutures visiljle.
FIG. 3. Diffractogram showing low angle laser light lens cortex. Pattern was registered without analyzer.
Portid
scattering
from
a section
of human
trmslmrw~
cataracts
Human cortical cataracts arc a very large group of cataracts wit,11opacitiw of wry different appearance. All arc characterized by the fact that the? Aart, as mlall local opacit,ies in a relatively transparent cortex. Two comnion major types of cortical cataracts arc the subcapsular and suprsnuclear cata.rncts (Nordmann, 1952). The opacities of a subcapsula,rcataract appear as sonlewhat~irregular men~branessituattltl it) the peripheral cortex and are relatively parallel to the lens capsule. The SU~I’ILnuclear cuneiform cataract is characterized by radial or concentric opacities deep within the cortex. Theseopacities often appear as spokesor riders. All lensesincludetl in this st,udy showedearly stagesof cataract where t’he opacities still were surroundetl hv transparent cortex. In this investigation studies of mature cataracts have been avoidetl since they will a,dd very little inforrnatiou about t.he primary mechnnisnlsof cataractogenesis. In the earliest stagesof the cortical cataract. the opacities might appear only as a local slightly increased turbidity. When such opacities were examined by microradiography, no definite changescould be detected. When studied bv electron microscopy, the lens fiber cells appeared intact. but the intercellular spaceswere enlarged (Fig. 4). The cell membraneswere generally still intact, but sometimesbroken nlernbranes and the beginning of the dissolution of lens cells were found. Often, the broken
LEXS CHANGES ANDTRANSPARENCY
FIG. 4. Electronmicrograph cataract. The intercellular
33
from the inner cortex of a human lens during the early stage of cuneiform spaoes are enlarged, but the cell membranes are mainly intact. ( x 26,600)
membranes became attached to themselves or adjacent membranes forming vesiclelike structures (Fig. 5) and closing off the extracellular fluid. The subcapsular and cuneiform cataracts were studied both by microradiography and electron microscopy. To illustrate the principal changes of these cataracts, a lens from a 74-year-old patient was used. This lens showed subcapsular membrane-like opacities in both the
FIG. 5. Electronmicrograph from a human lens with a somewhat more advanced stage of cuneiform cataract than that found in Fig. 4. The intercellular spaces are enlarged, and the cell membranes have formed vesicle-like structures enclosing parts of the cell matrix. ( x 21,700)
B. PHILIPSOS
34
0. 3-
m
‘;-
0.: 3-
‘E 0 b.
,4l
C
,37
0, It b) (b)
I 0.2
I
I 0.4
I
I
0.6
mm
Pm.
6
I
I
,333
LENS
CHANGES
AND
TRAXSPAREKCY
35
anterior and the posterior cortex. Furthermore, this lens contained dense radial opacities. When the lens was studied microradiographically, it, was shown that all the dense and distinct opacities were accompanied by locally decreased protein concentrations (Fig. 6). The border zone between the relat.ively transparent cortex and the opacities was associated with steep irregular gradients in protein concentration. The magnitude of these protein concentration gradients was generally in the order of 0.1-0.2 g/cm3. These changes in protein concentration correspond to fluctuation in the refractive index of about 0~020~04. The refractive interfaces formed are irregular and wavy, which will make them act as strong sourcesof light scattering. The light will hit this refractive interface under different anglesof incidence, thereby increasing the scattering of light. Electronmicrographs of the region corresponding to the subcapsular membranous opacity revealed the fine structure of this type of irregular refractive interface (Fig. 7). The radial opacities of the cuneiform cataract, when studied electron microscopically, revealed enlarged extracellular spaceswhich often created large clefts in between the lens fiber cells (Fig. 8). Sometimes lens fibers seemedto dissolve and leave only
PIO. 7. Electronmicrograph of the region between the relatively dissolved peripheral zone of a subcapsular cat,aract. Notice the wavy which explains the high scattering of light. ( x 14,000)
intact inner cortex and the partly interface between the two portions
FIG. 6. (a) Microradiogram from a human lens with cortical cataract, both subcapsular and supranuclear spokes. To the left is the anterior cortex with a reduced X-ray absorption. The capsule is torn off during the preparation procedure. About 0.6 mm from the periphery is another area with reduced X-ray absorption. This area has the same position as the spoke-like opacities, as revealed under slit lamp examination. Note the sharp and irregular interface between the regions with normal X-ray absorption and the region with reduced absorption. ( x 160); (b) the protein concentration and the refractive index determined along the marked line in the lens section shown in (a). Note the low concentration in the dark areas of the miororadiogram and the sudden steep jumps in protein content and refractive index; (c) schematic drawing of the microradiogram in (a). Only the strong refractive interfaces are marked. Two rays of light are shown, and some of the main centers of light reflection or scattering are indicated.
36
B. PHILIP:;ON
the membranes which formed myeline-like bodies in the clefts. In t’he later stages of these cataracts, there were often found opaque regions displaying more advanced ultrastructural changes. The lens fiber cells were pnrtlv dissolved. a,ntl the membranes formett small vesic1e.s enclosing minor amounts of n&-ix. The 1~lectronlnicrographs. as well, gave a suggestion that the distribution of drv tuass was not homogeneous causing refractive index fluctuations (Fig. 9). 111conclusion. the cortical opecitiestudied seemed to start, wit811 an enlargement of the int,erwllular sl)aces. probabl\~ proceedtd t)y mwnbrane cleficicney. This enlargelt~cnt seeurwl to Iw follow-cd t)v t tub ulatrix ant1 tllch breakage of lens cell memt)ranes. which causetl the lo is of’ prott4ii creation of uifnibram~ \wiclt:s with varying f)r.otc>in wiicentrat ion atrd refract i\-(’ IUp, itlt.c,r. itrclex. In the regions lwt.~vveet~the t.ransparent. cortcks antI t.licl opxitics. faces were fotltltl between zones with different ptcirl ~~OIl~t~tlttilti~~ll ilIlt refrartivl~ imles.
Xucleur
cata,ract
The human nuclear cataract is completely difierent from those cataracts which are included in the grouping labeled cortical cataracts. Its early st,ages. which are very common in aged human lenses, are called nuclear sclerosis. This type is characterizctl by a diffuse and generally yellowish scattering of light throughout the nucleus of the lens. No defined or structured opacities are revealed under slit lamp examination. The more advanced stages, called real nuclear cataract,, and giving reduced visual acuity, are much less common than the corresponding cortical types (van Heyningen.
LENS
CHANGES
F’Ic:. 9. Electrnnmicrograph from a relatively dissolved, and the cell membranes hare formed t,s) txh very inhomogeneous. ( i( 21,700)
ANI)
TRANSPARENCY
37
a~lvanced cortical cataract. Most lens fiber cells arc’ vt:siclt\-lilrr bodies. The distribution of cell matrix XPWIS
PIG. 10. Electronmicrograph from the nucleus of a lens with dense nuclear cataract. Densely stained, large aggregatea of cell matrix are revealed. The diameter of the aggregates is about 500-1000 ip. ( :I’ 90,000)
38
B. PHILIPSON
1972). The nuclear cataract is often accompanied by the formation of a yellow-brown pigment, but on rare occasion the cataract appears to be whitish. The distribution of protein, as revealed by microradiography, does not show any detectable fluctua.tion in the protein content. The electron microscopical appearance of the lens fiber cells of the nuclear cataract did not show any difference from that of a normal transparent lens nucleus. The extracellular spaces were seldom larger than 200 A. The membranes appeared the same as in the normal transparent nucleus. In both normal and cataract lenses, gap-junctions are more frequent than in the lens periphery (Philipson, Hanninen and Balazs, unpublished manuscript). However, in dense, whitish nuclear cataracts the matrix was shown to aggregate in dense clusters with a diameter of about 500-1000 H (Fig. 10). This type of dense aggregates was never seen in the transparent, or the slightly “sclerotic” lens nuclei. It has been shown by Benedek (1971) that dense molecular aggregates of this size might scatter light to such a magnitude that it, would explain the turbidity of these cataracts. This type of cataract has been studied more extensively and will be reported in a subsequent report.
FIG. 11. Electronmicrograph from the nucleus made a.t a temperature of about 4°C. Non-stained 2000 A. ( x 21,700)
of a calf lens with cold cataract.. All preparations areas are seen which have a diameter of about
were IOOO-
An interesting analogy to the human nuclear cataract is the cold cataract which can be seen in young lenses from many animals. The calf lens exhibits a dense white nuclear cataract at temperatures of 10°C or below. This cataract was studied electron microscopically, and all preparation procedures were made at low temperatures (4°C). The electronmicrographs showed non-stained areas with a diameter of lOOO2000 A, which were never detected in the transparent calf lens. These areas are more
LENS
CHANGES
AND
TRANSPARENCY
39
likely to be interpreted as spherical aggregates (Fig. 11) than empty spaces. As it has been shown earlier, the concentration of y-crystallin is crucial in the formation of this cataract (Balazs, Teeta and Armand). It is very tempting to assume that the non-stained areas are dense aggregates composed mainly of y-crystallin. Moreover, it seems that the cc-crystallin is very easily stained by this preparation procedure and appears to be almost absent in these formations. Further studies are detiitely needed in order to determine the nature of these non-stained aggregates. However, it is certain that the distribution and the size of these structures could easily explain the very strong scattering of light in the cold cataract. ACKNOWLEDGMENTS
I thank Dr E. Balazs for valuable advice and for providing excellent facilities at the department of connective tissue research, Boston Biomedical Research Institute, Boston, during my stay as a visiting scientist. The technical assistance by L. HBnninen and C. Lindyuist is greatly appreciated. This work was supported in part by a grant (EY-00223) of the National Eye Institute, VSPHS, by the Swedish Medical Research Council (Project No. 13x-3008 and 6OF-3653) and by a Public Health Service International Post doctoral Research Fellowship (No. If05 TW 1801). REFERENCES Balazs, E., Testa, M. and Armand, G. In manuscript. Benedek, G. B. (1971). Applied Optkx 10,459. Cohen, I. (1965). Invest. Ophthulmol. 4, 433. van Heyningen, R. (1972). Exp. Eye Re.s. 13, 136. Jakus,M. (1964). Selected Electron Micrographs, Vol. 1. Little Brown and CO., Boston. Kuwabara, T. (1968). Arch. Ophthdmol. 79, 189. Nordmann,J. (1972). Ophthalmol. Res. 3, 323. Philipson, B. (1969a). Acta Ophthd. SuppZ. 103. Philipson, B. (1969b). Invest. Ophthdmol. 8, 258. Philipson, B. and Lindstriim, B. (1969). Histochemie 17, 201. Simpson, G. C. (1953). Brit. J. Ophthalmol. 37,450. Vinciguerra, M. J. and Bettelheim, F. A. (1971). Exp. Eye Res. 11,214. Wanko, T. and Gavin, M. A. (1959). J. Biophys. Biochem. Cytol. 6,97. Wanko, T. and Gavin, M. A. (1961). In The S&z&we of the Eye, p. 221. (Ed. Smelser, Academic Press, New York.
G. 9.)