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1 From the Karolinska Institutet, St. Erik’s Eye Hospital, Stockholm, Sweden; 2 The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands; and 3 Karolinska Hospital, Stockholm, Sweden.
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Abstract |
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METHODS. Sprague–Dawley rats received UVR (
MAX = 300 nm)
unilaterally during a 15-minute period. The exposure dose
ranged from 0.1 to 20 kJ/m2, and the rats were kept for up
to 32 weeks after exposure. Intact lenses were photographed and lens
wet and dry masses were measured. The protein density was estimated by
quantitative microradiography. Freeze-dried lens sections were used for
contact x-ray photographs. From the transmission of the
microradiographs, protein density and refractive index profiles were
calculated along the lens radius with a resolution of 2.5 µm.
RESULTS. Lens dry mass in exposed eyes was lower than in nonexposed eyes at one week after exposure. Lens water content was decreased after low UVR doses but increased after high doses. The difference between exposed and nonexposed lenses in dry mass and water content increased with time after exposure. No significant difference was found for the mean protein density in exposed and nonexposed lenses. The protein density increased linearly in the lens cortex, from a minimum in the superficial cortex of 0.26 g/cm3 to a maximum in the deep cortex of 0.81 g/cm3. This corresponded to a refractive index of 1.38 and 1.48, respectively.
CONCLUSIONS. Lenses exposed to UVR grew more slowly than their nonexposed contralaterals. This growth inhibition was dose dependent. Near-threshold doses led to decreased water content in the lens whereas high doses led to swelling. Six months after near-threshold UVR exposure, no global change of the refractive index was found. However, local variations of the refractive index caused a subtle cortical light scattering.
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Introduction |
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UVR may damage the lens by several mechanisms, among them protein cross-linking, DNA damage, dysfunction of enzymes, and membrane damage. UVR injury leads to swelling and disruption of lens epithelial cells and cortical lens fibers.4 13 Threshold exposure to UVR induces programmed cell death (apoptosis) in the lens epithelium 24 hours after exposure.14 Swollen mitochondria, subcapsular vacuoles, chromatin condensation, and nuclear fragmentation are found in the epithelium.13 Long-term, repeated, subthreshold UVR leads to epithelial hyperplasia.15
Previous studies from this laboratory have documented that UVR exposure of the rat leads to increased forward light scattering in the lens.5 6 16 17 18 Two of these studies investigated light scattering data in isolated lenses for different time intervals after exposure (long-term experiment)17 and for different UVR doses (dose–response experiment).18 In both studies, data about the lens wet and dry masses were also collected. Here, our previously unpublished data is analyzed in an attempt to describe the effects of UVR exposure on lens growth and global water content. The relation of lens dry mass and water content may have its impact on cataract development because it is known that osmotic imbalance and changes in lens hydration precede or accompany cataract development.19 20
The transparency of the crystalline lens depends on the regular, orderly spacing of its cells and proteins. Disturbance of this order—for instance, due to protein aggregation, membrane degeneration, fluctuations in protein distribution or phase separation—results in local changes of refractive index. This is the basic explanation for light scattering.21 Therefore, the refractive index distribution in the lens after a near-threshold UVR exposure is investigated in the second part of this article.
It has been shown that there is a linear relationship between the refractive index and the concentration of unconjugated proteins in biological specimens.22 Based on this, Lindström23 and Philipson24 adapted contact microradiography for the estimation of protein density in the crystalline lens. In microradiography, the dry mass content of freeze-dried tissue section is estimated. Because the lens consists only of approximately 2% of material other than water or proteins, the method was applied to get quantitative information on the protein density distribution in the lens down to the cellular level.24 25 26 The present experiment applies a new approach to measure the transmission of the microradiograph; a laser scanning microscope is used in place of a densitometer. In addition, thickness of the specimen is measured with a laser scanning microscope according to a method described by Brismar et al.27
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Methods |
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MAX =
300 nm, full width at half maximum = 10 nm) and was projected on
the cornea of one eye.5
The spectrum of the radiation was
given previously.14
28
Ten minutes before exposure, each
animal was anesthetized by intraperitoneal injection of a mixture of 94
mg/kg ketamine and 14 mg/kg xylazine. Five minutes after injection, the
mydriaticum tropicamide was instilled in both eyes. After another 5
minutes, the eye was exposed to UVR with a narrow beam that covered
only the cornea and the eyelids of the exposed eye. All animals were
kept and treated according to the ARVO Statement for the Use of Animals
in Ophthalmic and Vision Research. In a long-term experiment, animals were exposed to 5 or 20 kJ/m2 UVR and kept for 1, 4, 8, 16, or 32 weeks after exposure, with 20 animals in each group. In a dose–response experiment, animals were exposed to 7 different doses of UVR (0.10, 0.37, 1.3, 3, 5, 8, or 14 kJ/m2) and kept for 1 week after exposure, with 10 animals in each group. In both experiments, the exposure time was always 15 minutes and the different doses of UVR where set by varying the irradiance in the exposure plane. (For the lowest dose of 0.10 kJ/m2 the irradiance was set to 0.11 W/m2 and for the highest dose of 20 kJ/m2 to 22.2 W/m2.) All rats were killed by an overdose of pentobarbitone (200 mg/kg, intraperitoneally), followed by cervical dislocation. Thereafter, the eyes were enucleated, the lens was removed by a posterior scleral incision, placed in balanced salt solution (BSS), and cleared of adherent ciliary body. The majority of lenses from all experiments were photographed in BSS with a stereomicroscope (MZ 6; Leica AG, Heerbrugg, Switzerland) against a black background with a white grid (Fig. 1) . Before measuring the wet mass, the lenses were placed three times shortly on a dry glass plate to remove excessive water from the lens surface. The lenses were then kept for at least 1 week in an oven at 65°C, and lens dry mass was measured.
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Quantitative Microradiography
Lenses extracted for microradiography were quick-frozen in
isopentane precooled in liquid nitrogen to –160°C. Midsagittal
sections were obtained with a microtome cryostat (HM 500 OM; Microm
Laborgeräte GmbH, Walldorf, Germany) set for a default thickness
of 16 µm. After freeze-drying, the lens sections were placed on a
special holder for x-ray exposure together with a reference system. The
reference system was a step wedge of six layers of standard polyester
film (Mylar; Spectro-film, DuPont, Wilmington, DE). The x-ray
plate (High Resolution Plates type 1A; Eastman Kodak, Rochester, NY)
was placed in contact with the specimen and the reference system and
was exposed to 3 kV x-ray filtered through 9 µm
aluminum24
(Fig. 2)
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 | (1) |
The lens sections shrink in all dimensions during the freeze-drying process. By comparison of the diameter of the fresh intact lenses and the diameter of the freeze-dried sections the shrinkage in x–y dimension was estimated. The sections shrunk to a fraction of 0.834 ± 0.011 of their original diameter (mean with confidence interval, n = 12). Because the lens has a symmetrical structure in all dimensions, the same shrinkage was also assumed for the z-dimension (specimen thickness). Hence the overall volume shrinkage was estimated to (0.834)3 = 0.580. All results from microradiography were corrected by this factor in this article.
The dry mass density as derived from microradiography was set equal to protein density. This caused a systematic error of about + 4%, because the dry mass in the lens consists of more than 95% of proteins. However, because the refractive index is related to the total amount of dry material rather than to the pure protein fraction, this reduction was not introduced.24
From the protein density, the refractive index can be calculated using
the Gladstone–Dale formula22
: n =
nm + 
* C. Here, nm is
the refractive index of the medium (nm = 1.333
for water), is the specific refractive increment (in
cm3/g), and C the concentration of proteins (in
g/cm3). The specific refractive increment for the
majority of proteins is 0.1845
cm3/g.22
In the current
experiment, a value of 0.180 cm3/g is used, as
suggested by Philipson24
to correct for lipids and
potassium in the lens.
Thickness Estimation
A series of confocal line scans across the specimen were
acquired with a Confocal Laser Scanning Microscope (Zeiss LSM 410; Carl
Zeiss, Jena, Germany) using a 40x objective with 0.65 numerical
aperture. The signal intensities of the line scans were low above and
below the specimen and high inside the specimen due to autofluorescence
of the specimen material. For each line scan the focus position
(z-position) was changed, resulting in a vertical profile
image (x–z profile) of the specimen. The
confocal plane of the microscope had a thickness of 1.3 µm (full
width at half maximum of the axial point spread function). The
resolution of the stored image was 10 pixels/µm in
z-direction.
Three vertical profile images were acquired from each specimen. Then, from each vertical profile image, three intensity profiles (Fig. 3A ) were computed which were perpendicular to the specimen surface. The full width at half maximum of the bell-shaped intensity profiles (Fig. 3B ) was used as a measure for the specimen thickness.27 The full width at half maximum was calculated from a Gaussian fit of each single intensity profile and then averaged.
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Vignetting (intensity loss at the edge of images) in the transmission images was minimized by careful adjustment of the scanning microscope. Measurements in background images revealed that the center-to-edge vignetting was always less than 3%.
The mounted transmission images of the microradiographs were processed with the Image Processing Toolkit (Reindeer Games, Inc., Gainesville, FL). This included the spatial calibration as well as the calculation of the intensity profiles. Three intensity profiles were obtained inside both equatorial sectors marked in Figure 2 . The intensity profiles were 10 pixels or 25 µm wide and directed from the lens capsule toward the nucleus. A spreadsheet program was used to calibrate the intensity profile data for dry mass density by employing the reference system described previously.
Confidence coefficients and significance levels were set to 0.95 and 0.05, respectively.
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Results |
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Lens Mass
The wet mass of nonexposed lenses increased logarithmically with
age during the observed period from 7 to 38 weeks of the rat’s age
(Fig. 4)
. This increase was similar to, but slower than the increase of the rat
body weight. Rat lens dry mass and water content also started to
develop logarithmically with increasing age in the nonexposed rats.
However, at approximately 30 weeks, there was a change in the growth
curves (Fig. 4)
. The water content of the lens started to decrease and
the dry mass increased more rapidly. From the age of 7 to 22 weeks, the
total lens dry mass content only increased from 43 to 47%, but then
reached a value of 58% at the age of 38 weeks.
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After 5 kJ/m2, the water content was lower in the exposed lenses for all time points after exposure (Fig. 5D) . It was 4% lower at 1 and 4 weeks and 9% lower between 8 and 32 weeks postexposure compared with the nonexposed lenses. In contrast, after 20 kJ/m2, the water content first increased (approximately 15% at 1 and 4 weeks postexposure) and then decreased rapidly with postexposure time (approximately 75% lower at 32 weeks postexposure).
Protein Density
The freeze-dried lens sections had a coronary radius of at most
2000 µm and a mean thickness of 12.7 µm (± 0.7 µm,
n = 12). In the nonexposed lenses, the lens capsule had
a protein density of approximately 0.58 g/cm3. In
the very superficial cortex (at 50 µm below the capsule) the protein
density dropped to a local minimum of 0.26 g/cm3,
corresponding to a refractive index of 1.38 (Fig. 6)
. Toward the lens center, the protein density increased continuously.
The mean value between 800 to 900 µm below the capsule was 0.81
g/cm3, corresponding to a refractive index of
1.48.
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Protein density = 6.70 g/cm2 * distance below capsule + 0.253 g/cm3
Below 900 µm, there was a large variation in the measured protein density or it could not be estimated at all because of missing parts of the specimen. There was no significant difference between the mean protein density distribution of the exposed and nonexposed lenses at 26 weeks after UVR exposure (Fig. 6) . The only difference that could be discerned was the larger variation (confidence interval for the mean) in the deeper cortex of the exposed lenses (600 to 1000 µm below the capsule).
Taking the mean of a measure can sometimes result in removal of important specific information. A look at the protein density distribution for each of the six exposed animals revealed differences in the local variation of the protein density (Fig. 7A 7B) . Therefore, linear regressions were performed for 100-µm wide intervals. From these regressions the residual sum of squares was used as a measure for the local variation of the protein density (Fig. 7C 7D) . For the nonexposed lenses the variation was low in all intervals between 50 and 850 µm. The mean difference between the residual sum of squares for each interval for nonexposed and exposed lenses was calculated. Only for the 650- to 750-µm interval, was there a significant difference between the exposed and nonexposed lenses, as revealed by Student’s t-test. The area of 650- to 750-µm below the lens capsule corresponded to 33% to 38% of the total coronary radius (2000 µm) of the freeze-dried sections.
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Discussion |
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All animal eyes exposed to 20 kJ/m2 and 50% of those exposed to 5 kJ/m2 developed an opaque cornea and some ocular inflammation between 4 and 7 days after exposure. This occurred only in a few cases after exposure to lower doses. However, experiments with pigmented and unpigmented rats have shown that there seem to be no correlation between UVR damage to the cornea and to the lens.30
In the present experiment, lens mass data were recorded in young adult rats ranging in age from 7 to 38 weeks. This should be compared to a life expectancy of rats under laboratory conditions of approximately 100 to 150 weeks. The wet mass of nonexposed, normal lenses increased logarithmically during the observed time (Fig. 4) which is the expected growth development.31 The observation that the dry mass increased faster than the water content shows that the average protein density in the lens increases more rapidly in older rats (Fig. 4) . These results are supported by earlier findings31 that showed that the percentage of water-insoluble proteins increases continuously throughout life. In other words, lens proteins in older rats bind less water than lens proteins in younger rats.
Lenses exposed to UVR grow more slowly than their nonexposed contralaterals (Fig. 5A 5B) . The lens growth rate decreased with increasing dose. Reduction in lens growth has been reported earlier.32 Even though UVR-induced light scattering had regressed several weeks after a near-threshold exposure (5 kJ/m2),28 there was a decrease in mass of these lenses (Fig. 5B) . The finding that both water content and dry mass decreased by approximately 10% after near-threshold exposure (Fig. 5A 5C) shows that the average protein density is kept constant. Therefore protein density seems to be more important for maintenance of lens transparency than lens mass. The drastic decrease of lens dry mass and water content at 8, 16, and 32 weeks after 20 kJ/m2 (Fig. 5B 5D) is of minor importance, because these lenses are totally opaque already after 4 weeks postexposure (Fig. 1F) .
After near-threshold exposure, most lenses are capable of repair, whereas lenses exposed to high dose UVR become totally opaque.17 28 The lens epithelium may play a key role in this process. If the epithelium has the chance to recover (after low-dose exposure) it may also be able to continue the support of underlying fibers.28 However, if too many epithelial cells die, underlying fibers cannot be supported nor new fibers can develop properly, hence the lens becomes opaque and has no ability to recover.
Near-threshold doses led to decreased water content in the lens whereas high doses led to swelling (Fig. 5B 5D) . This means that a marked water uptake correlates with severe cataract. On the other hand, decreased water content after near-threshold exposure correlates with lens opacities, which are largely repaired several weeks after the exposure. The changes of water content after UVR exposure supports previous morphologic findings28 that UVR causes disturbance of water balance in the lens.
The development of opacities after a near-threshold UVR exposure is known from an earlier study.28 At 1 week postexposure, extracellular spaces in the epithelium and in the outer lens cortex produce a corrugated opaque lens surface and equatorial opacities. Within several weeks after exposure, the lens epithelium recovers, and new fibers develop normally. The lens fibers regain normal water balance and fill up the extracellular spaces. Repair, however, is incomplete, and disarranged fibers remain in the cortex, producing a subtle shell-shaped opacity.
In the present study, a similar subtle opacity was observed in the deep cortex of the lens at 6 months postexposure (Fig. 1H 1J) . At that time point, the opacity was located between 25 and 30% of the coronary lens radius below the capsule. This compares to 13% to 17% below the capsule at 8 weeks postexposure.28 In other words, at a longer postexposure time, the shell-shaped opacity is found deeper in the lens cortex. This finding supports the idea that the superficial fibers damaged at the time of the UVR exposure remain in their growth shell and move relatively deeper into the lens as new, normal fibers are formed and grow on top of the damaged growth shell.
The most basic physical explanation of light scattering is local fluctuations of the refractive index in a medium.21 In many biological tissues, the refractive index is proportional to the protein density.22 Philipson and Fagerholm33 could explain the light scattering from different types of human cataract by sudden changes in the protein density distribution. For example, protein aggregates in the lens fiber cytoplasm have refractive indices that deviate from those of their surrounding and therefore cause light scattering.
Bettelheim used the mean squared deviation from the average refractive index21 as a quantitative measure for the variation in the refractive index. In the present experiment, a similar approach was applied by using the residual sum of squares for a linear regression of the refractive index (Fig. 7) . Both are measures of variation within a sample; the mean squared deviation is equal to the sum of squares divided by the degrees of freedom.
The evaluation of the residual sum of squares revealed local variations in the refractive index between 33 and 38% of the lens radius below the capsule at 6 months postexposure. This location is not exactly the same as observed in the intact lens (25% to 30%). An explanation could be that the light scattering was observed inside a medium which itself has refractive power and which could change the observed location from the actual location by refraction. Below 850 µm under the lens capsule, exposed as well as nonexposed lenses showed high local variations in the refractive index (Fig. 7B 7D) . This effect is due to limitations with the cryosectioning technique. The protein density in the lens nucleus is so high, that proper sections can hardly be made.
The protein density found for the nonexposed eyes increased continuously from the lens cortex (minimum of 0.26 g/cm3) toward the nucleus (0.81 g/cm3; Fig. 6 ). These values as well as the profile of the increase are similar to those reported in the earlier studies by Philipson.24 He found a protein density of 0.30 g/cm3 in the peripheral cortex and 0.88 g/cm3 in the nucleus of Sprague–Dawley rats of comparable age.
At 6 months after near-threshold UVR exposure, no global change of the refractive index was found in exposed versus nonexposed lenses. The local variations in protein density cancel each other out when averaged between the individuals because they are displaced somewhat along the lens radius and they have positive or negative magnitude. The absence of a significant difference of the protein density in the outer cortex, supports the earlier morphologic finding28 that the new fibers that grow after the UVR exposure develop normally.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 30, 2000; revised October 13, 2000; accepted October 30, 2000.
Commercial relationships policy: N.
Corresponding author: Ralph Michael, The Netherlands Ophthalmic Research Institute, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands. ralph.michael{at}altavista.net
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