Impact of Age and Sex in Ultraviolet Radiation Cataract in the Rat

Abstract

purpose. The purpose of this study was to determine the influence of age and sex on the development of ultraviolet radiation (UVR) cataract in rats. Current safety limits for lens damage due to UVR do not consider age or sex.

methods. Four age groups of Sprague-Dawley rats (3, 6, 17, and 52 weeks) were exposed to 300-nm UVR at either 5 or 8 kJ/m², delivered during 15 minutes. The interval between irradiation and cataract assessment was 1 or 8 weeks. Moreover, two groups of 6-week-old male and female rats were exposed to 5 kJ/m² UVR, with cataract assessment after 1 week. The severity of cataract was quantified by measurement of forward light-scattering in isolated lenses.

results. The youngest age group showed development of anterior subcapsular, equatorial, and nuclear cataract, whereas the three older groups exhibited the first two types. The two younger age groups had significantly more cataract than the other groups. The degree of cataract increased from 1 to 8 weeks after irradiation. There was no difference in cataract severity between sexes.

conclusions. Young rats are more sensitive to UVR than old rats. Nuclear UVR cataract develops in young rats but not in adult rats. With the chosen waveband and dose, the time for maximum cataract development to occur is longer than 1 week. There is no difference in UVR sensitivity between the sexes.

The present study investigates the impact of age, sex, and postexposure time on sensitivity in the lens to 300-nm UVR. Cataract is the major cause of low vision and blindness in the world. ¹ Epidemiologic studies have shown a correlation between cataract and solar ultraviolet radiation (UVR). ^{2 3 4} Case reports support the relationship between UVR and cataract in humans. ^{5 6 7} Experimental studies have shown that UVR wavelengths of approximately 300-nm are the most harmful to the lens ^{8 9} and that 300-nm UVR penetrates a short distance into the lens. ^{10 11}  

Because of safety concerns, experiments on UVR cataract are not conducted in humans. The similarity in lens structure and function between humans and other mammals render animal models useful. In vivo studies on development of UVR cataract involve the natural defenses of the whole eye and body, although whole lens or lens cell cultures are appropriate alternatives in some situations. 

A problem when comparing results of animal studies is that different research groups use different species and strains and animals of both sexes and of various ages. Some enzyme activities in the lens differ between sexes. ¹² Other parameters that render comparisons difficult are dose, ^{13 14} exposure time, ¹⁵ fractionation of dose, ^{16 17} and time between UVR exposure and cataract assessment. ^{18 19} After in vivo UVR exposure of young rats, the time for maximum intensity of forward light-scattering depends on the dose. ¹⁸ However, this has not been investigated in old or very young rats. 

The surface ectodermal origin of both lens and cornea may, at least partly, explain the similar responsiveness to UVR of the two tissues. Pitts et al. ⁸ have shown that, in the rabbit, the threshold 300-nm UVR dose for corneal damage is 0.2 kJ/m² and in the lens is 1.5 kJ/m² (in the cornea plane). ²⁰ Given the low transmittance of UVR in the cornea, ^{10 21} the dose incident at each tissue is of the same order. Therefore, the cornea is the dose-limiting tissue for acute radiation damage. However, although corneal damage from UVR is reversible, lens damage is not. Cumulative long-term solar UVR may ultimately result in cataract. Current safety standards for avoidance of lens UVR damage are based on experiments in adult animals. ⁸ The cornea of the young animal is thinner and therefore attenuates less radiation. The proliferation of cells in the young lens is more active than in the older, which renders the young lens potentially more sensitive to UVR. Repair mechanisms may vary with age. Further, structural and biochemical differences in lenses of different age may vary the response to UVR. 

The purpose of the present study was to investigate whether age has an impact on sensitivity to 300-nm UVR and, if so, whether there is a difference in age-dependent sensitivity between observations at 1 week and 8 weeks after exposure. Further, the study was intended to demonstrate whether there is a sex difference in sensitivity to 300-nm UVR. 

Material and Methods

Animals

A total of 203 outbred albino Sprague-Dawley rats (Bkl/SD, M&K Universal, Sollentuna, Sweden) in the age range 2 to 52 weeks were used, including 10 rats for pilot experiments. Ethics approval was obtained from the Northern Stockholm Animal Experiments Ethics Committee. The experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Irradiation

All animals were anesthetized 15 minutes before exposure to UVR. To facilitate intraperitoneal injection of an anesthetic mixture of 6 to 10 mg/kg xylazine (Rompun Veterinary; Bayer AB, Göteborg, Sweden) and 40 to 70 mg/kg ketamine (Ketalar; Parke-Davis, Täby, Sweden), the oldest rats were sedated with isoflurane gas (Forene; Abbot Scandinavia AB, Solna, Sweden) before the injection. The pupils of both eyes were dilated with 0.5% tropicamide (Mydriacyl; Alcon Sverige AB, Stockholm, Sweden) 10 minutes before exposure to minimize possible variation in pupil size during exposure. 

The radiation system was from Oriel Instruments (Stratford, CT). The radiation from a 350-W high-pressure mercury arc lamp was collimated, passed through a 10-cm water filter, projected through a double 0.12 m monochromator set to 300 nm with 10 nm full width at half maximum (FWHM). The exposure was unilateral, covering the eyelids and eye. The irradiance in the corneal plane was quantified with a thermopile (Oriel Instruments) calibrated by the Swedish National Bureau of Standards. The relative radiation spectrum was obtained with a spectrometer and gray-body radiator (PC2000; Ocean Optics, Inc., Dunedin, FL). Ocular exposure time was 15 minutes. After exposure, saline solution was instilled in both eyes every 10 to 15 minutes until recovery of spontaneous blinking. 

Lens Forward Light-Scattering

At a predetermined time after exposure, the rats were asphyxiated with CO₂, the eyes were enucleated, and the lenses were dissected from remnants of ciliary body, zonular fibers, and vitreous. The isolated lens was immersed in room temperature-controlled Ringer’s acetate and photographed, and the intensity of forward light-scattering in the lens was quantified with a device (Fig. 1) described in detail elsewhere. ²² In this device, the lens is immersed in Ringer’s acetate on top of a dark-field ring illumination. The optics and the detector collect and quantify the forward-scattered light from the lens. Light-scattering was measured three times in each lens. 

The intensity of forward light-scattering is correlated with light-scattering from a commercially available lipid emulsion of diazepam (Stesolid Novum; Alpharma AB, Stockholm, Sweden; Fig. 2 ). This allows conversion of relative current readings of intensity of forward light-scattering to absolute equivalent diazepam concentration (EDC). The absolute readings have the advantage of being comparable over time. The standard curve demonstrates that if the concentration of scatterers becomes too high, the intensity of forward light-scattering decreases. This decrease is secondary to back-scattering and absorption. The absorption is an effect of increased path length of the scattered light due to multiple light-scattering. If the light-scattering in the lens is high enough to decrease the intensity of forward scattered light the complete lens appears white in incident illumination and dark in the center with a peripheral brighter ring in dark-field illumination. In such cases, the lenses were classified as severely damaged and the light-scattering data were omitted from statistical analyses. 

The light-scattering data were normalized by a log(EDC+1) transformation, as suggested by Söderberg et al. ²² The transformed unit was denominated tEDC. 

Experimental Design

Age-Sensitivity Experiments.

In the first age-sensitivity experiment, four groups of 20 rats aged 3, 6, 17, and 52 weeks were included. The rats received 8 kJ/m² unilaterally. The time interval between UVR exposure and cataract measurement was 1 week. 

In the second age-sensitivity experiment, three groups of 22 rats aged 6, 17, and 52 weeks were included. The rats received 8 kJ/m² unilaterally. The time interval between exposure and cataract measurement was 8 weeks. 

Sex-Sensitivity Experiment.

A total of 22 male and 22 female 6-week-old rats were included. The rats received 5 kJ/m² unilaterally. The time interval between UVR exposure and cataract measurement was 1 week. 

Statistics

The significance level and confidence interval (CI) coefficient were set to 0.05 and 0.95, respectively. All tests were two-sided. 

Results

Three rats that died during anesthesia were replaced to maintain an even sample size among groups. 

Light-Scattering in Nonexposed Lenses: Age Sensitivity Experiments

The control lenses were all subjectively clear (Fig. 3) , with forward light-scattering in the range of 0.15 to 0.28 tEDC (Fig. 4) . The variance was unequal among age groups, as indicated by the Levene test performed on pooled, nonexposed lenses, using actual age at the day of light-scattering measurements as the independent variable. Therefore the nonparametric Kruskal-Wallis analysis of variance (ANOVA) was used, revealing a significant difference in light-scattering among the age groups. The two oldest groups, 53 (52 + 1) and 60 (52 + 8) weeks, exhibited significantly more light-scattering than the other groups, as revealed by nonparametric multiple comparison test for unequal sample sizes. 

Age Sensitivity 1 Week after UVR Exposure

The UVR-exposed lenses exhibited anterior subcapsular cataract and equatorial cataract, sometimes with spokes extending toward the posterior suture. 

In the 3-week-old group, 12 of 20 irradiated lenses were severely damaged. Nine of the 12 severely damaged lenses (Figs. 5A 5B) and three of the lenses with quantifiable intensity of forward light-scattering had nuclear cataract in addition to cortical cataract. 

The proportion of severely damaged lenses after 8-kJ/m² 300-nm UVR was significantly higher for rats aged 3 weeks (60%) than for rats aged 6 weeks (0%), as indicated by the Fisher exact test. The same UVR exposure created variation of light-scattering among 6-, 17-, and 52-week-old rats, as indicated by Kruskal-Wallis ANOVA, in which the paired-sample difference between exposed and contralateral nonexposed lens was used as primary data. Further, the same UVR dose induced more light-scattering in 6-week-old rats than in 17- and 52-week-old rats, with no difference between the two latter groups, as indicated by a Tukey-type multiple comparison test. 

Age Sensitivity 8 Weeks after UVR Exposure

For all three age groups, the exposure to UVR increased the light-scattering in exposed lenses relative to control lenses (Fig. 4) . Exposed lenses exhibited more extensive cataract compared to same age groups 1 week after exposure (Fig. 5) . 

There were severely damaged lenses in the two younger groups, including almost all (91%) exposed lenses in the 6-week-old group. The proportion was 30% in the 17-week group and 0% in the 52-week group. This indication of correlation between age and the proportion of severely damaged lenses was confirmed by Pearson χ² test of a 2 × 3 contingency table. All groups differed significantly from each other as indicated by Tukey-type multiple comparisons of the ranked proportions, after arcsine transformation for normalization. 

Sex Sensitivity

For both sexes, 5-kJ/m² 300-nm UVR induced light-scattering in the exposed lenses, as indicated by the estimated confidence interval for the mean paired-sample difference between exposed and contralateral nonexposed lens (males 0.35 ± 0.09 tEDC, females 0.34 ± 0.07 tEDC). There was no difference between sexes, as indicated by the confidence interval for the difference between the mean paired-sample difference for males and females (0.003 ± 0.11 tEDC). 

Discussion

We have shown that age is important in rat UVR cataractogenesis, whereas sex is not. Nuclear cataract, which is not commonly associated with UVR cataractogenesis, developed in the youngest animals, 

Initially the ages of the animals were selected so that the log scale age interval would be equal between groups. The planned age of 2 weeks could not be used, because the eyelids are not fully open at this age. A 3-week-old rat is a weanling, whereas a 6-week-old rat is weaned but still prepubertal. A 17-week-old rat is a fertile young adult and a 1-year-old rat is elderly. The approximate healthy lifespan for this strain is 75 weeks. 

The pilot study did not include younger rats, because 5 kJ/m² is shown by our research group to induce detectable cataract in 6-week-old rats. ¹⁸ Instead, 18- and 52-week-old rats were exposed to 5 kJ/m² UVR and the degree of cataract was measured after 1 week. The 95% CI for the mean paired-sample difference in light-scattering between exposed and contralateral nonexposed lens included zero, probably because of the small sample size. It was then decided that a higher dose was necessary to induce detectable light-scattering. The chosen dose of 8 kJ/m² is well above the threshold dose for permanent UVR cataract in rabbits ⁸ and young rats. ^{9 13} We did not anticipate the strong reaction in the 3-week-old rats in the first age experiment. Because the purpose of the second age experiment was to compare postexposure time, the dose was kept at 8 kJ/m². The 3-week-old group was omitted, because they developed severe cataract already at 1 week after exposure. For the 6-week-old rats in the sex experiment, 5 kJ/m² was known from earlier experiments to be appropriate. 

The two- to threefold difference in cataract severity between the two younger and the two older groups at 1 week follow-up should be addressed. Lenses from young rats are smaller and the anterior chamber shallower than in older rats. This means that with the same corneal dose, more radiation would reach the lens of a younger rat. Measurements on cryosectioned rat eyes revealed a 25% difference in corneal thickness between the youngest and oldest rats (Fig. 6) . Combining the actual corneal thickness and anterior chamber depths with the corneal and aqueous absorption coefficients for 300 nm from Maher ²¹ gives a range of lens anterior surface doses (3.8, 3.6, 3.1, and 2.9 kJ/m², from youngest to oldest rats), with 8 kJ/m² corneal dose. Because the dose-response function for UVR cataract is approximately linear in this dose range, ¹³ the effective dose to the lens due to intraocular dimensions does not fully explain the differences in cataract development among the age groups. 

Penetration of UVR-B into the lens is limited, ^{10 11} and the main target for UVR is presumed to be the epithelium, with its abundance of mitochondria and nuclei. ²³ Major UVR-B chromophores are the aromatic amino acid tryptophan and its oxidation products. ^{24 25 26} Lenses from young humans transmit more UVR than lenses of old humans. ²⁷ If the same holds for 300-nm UVR and rat lenses it may be of importance because mitochondria are targets for UVR and lenses from young rats have a distribution of mitochondria reaching deeper into the lens than older rats. ²⁸  

The cell division rate and the lens growth rate are higher in young rats. The mitotic region is located in the pre-equatorial zone behind the iris, which, in albino animals, transmits a substantial part of UVR. Considering lower UVR absorption in the anterior segment, possibly lower intralenticular UVR absorption, deeper mitochondrial distribution, and higher mitotic activity in young animals, it is clear that they have a higher potential risk of development of UVR cataract. 

The type of cataract in all but the 3-week-old rats was equatorial, with anterior haze and cortical spokes extending to the posterior suture. We did not expect to find the nuclear cataract that occurred in the 3-week-old rats. 

There was an increase in cataract severity between 1 and 8 weeks after irradiation. This finding is consistent with the cataract development occurring after 20 kJ/m² 300-nm UVR but is in disagreement with that after 5-kJ/m² 300-nm UVR. ¹⁸ Michael et al. ¹⁸ showed that with the higher UVR dose, cataract development progressed after 1 week, whereas for the lower dose, no further increase in light-scattering occurred at time points up to 32 weeks. The reason for this disagreement with their 5-kJ/m² dose may be due to the difference in radiation spectrum. Michael et al. used an interference-filter-based source, which produced a wider UVR waveband, including more radiation in the upper UVR-B wavelengths (Fig. 7) , whereas in the present study we used a monochromator (Fig. 7) . The monochromator source delivered more UVR at 300 nm, which is more damaging than UVR at 315 nm. ^{8 9} The true bandwidth (FWHM) was 6.2 nm, markedly narrower than the theoretical 10 nm. 

The current safety limits for optical radiation (UVR, light and infrared radiation) exposure do not consider age. Regardless of the mechanisms for the age dependency, young rats are more sensitive to UVR than older rats. Thus, age should be considered a factor during future revisions of UVR safety limits. 

The example of UVR cutaneous carcinogenesis may serve as a warning, because we know that sunburn episodes in childhood predispose the child to skin cancer in adulthood. ^{29 30} Perhaps the same pattern is valid for UVR and cataract. 

 Supported by the Sigvard and Marianne Bernadotte Research Foundation for Children Eye Care; St. Erik’s Eye Research Foundation; Karolinska Institutet Travel Fund; the Loo and Hans Osterman Foundation for Medical Research; the Swedish Society for Medical Research; the Swedish Radiation Protection Institute; a Swedish Institute guest research fellowship; the Karolinska Institute Research Foundation; and the European Union Research and Development Quality of Life Program, through Marie Curie Individual Fellowship Contract QLK6-CT-1999-51159.

 Submitted for publication September 14, 2001; revised April 29, 2002; accepted May 23, 2002.

 Commercial relationships policy: N.

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Figure 1.

 

Schematic drawing of device for quantitative measurement of cataract. ¹⁹

Figure 2.

 

Standard curve for forward light-scattering in a lipid emulsion of diazepam.

Figure 3.

 

Samples of nonexposed rat lenses of different ages. Grid square diameter is 0.79 mm.

Figure 4.

 

Age dependency of intensity of forward light-scattering after exposure to 300-nm UVR. Exposed (X) and nonexposed (−) lenses. Bars are 95% CI for the mean. Numbers just above or below the bars are sample sizes for the confidence interval estimations. Displayed above the graphs are number of severely damaged exposed lenses (SDLs), not included in the mean or confidence interval estimations. The 3-week-old group was not investigated at 8 weeks after exposure. The exposed lenses in the 6-week-old group was omitted from the 8-week postexposure graph because of the high percentage of severely damaged lenses.

Figure 5.

 

Representative UVR-exposed lenses in the four age groups at two postexposure times. (A) A severely damaged lens with both nuclear and cortical cataract, viewed with incident illumination. (B) Same lens in dark-field illumination, exhibiting nuclear absorption of the probing light. The 3-week group was omitted in the 8-week postexposure experiment. Grid square diameter is 0.79 mm.

Figure 6.

 

Intraocular dimensions in cryosectioned rat eyes of different ages were measured with a microscope calibrated with an object ruler (0.01 mm).

Figure 7.

 

Radiation spectra from high-pressure mercury arc lamp sources equipped with water filter and either interference filter or double monochromator. Total UVR dose was 8 kJ/m² in both spectra. FWHM is the theoretical full width at half maximum.

The authors thank professor Bo Lindström for invaluable help with the statistics and Manoj Kakar and Vino Mody for help with the language.