|Molecular Vision 2004;
Received 11 December 2003 | Accepted 8 March 2003 | Published 8 March 2004
Laser scanning analysis of cold cataract in young and old bovine lenses
Alice Banh, Jacob G.
School of Optometry, University of Waterloo, Waterloo Ontario, Canada
Correspondence to: Jacob G. Sivak, School of Optometry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1; Phone: (519) 888-4567 x3714; FAX: (519) 725-0784; email: firstname.lastname@example.org
Purpose: This research involves the use of a laser scanning instrument to evaluate the formation of cold cataracts in young and older bovine lenses.
Methods: Bovine lenses from 18 (n=14) week old calf eyes and approximately 10 (n=7) year old animals were extracted in a sterile environment. The lenses were placed in a specialized glass chamber with temperature controlled circulating culture medium. The temperature cycle inside the chamber started at 37 °C, slowly cooled to 4 °C, and then warmed back up to 37 °C. A laser scanning system (ScanToxTM) was used to analyze the optical quality of bovine lenses during a cooling and warming cycle.
Results: The relative light transmittance was measured as a function of pixel excitation caused by refracted beams and compared to pre-treatment measures. Each lens from each age group showed a significant decrease in transmittance at 4 °C, which recovered when the lenses were warmed to 37 °C. Lenses from young eyes showed less loss of refracted beam intensity than lenses from older eyes (32% versus 34%), although the difference was not significant.
Conclusions: The results of the present study indicate cold cataracts can be induced in both old and young bovine lenses, as shown by using a scanning laser instrument.
The supramolecular organization of the proteins of the crystalline lens is essential for its transparency [1-3]. The cold cataract phenomenon, demonstrated in young lenses from rats, rabbits, cows, and humans involves the completely reversible opacification of the lens nucleus [4,5]. Cold cataract is caused by lowering lens temperature to well below body temperature. For example cold cataract formation occurs in bovine lenses when the temperature falls below 17 °C . It is reversed by warming the lens back to body temperatures. The fact that it is completely reversible and that the focal length profile of the lens (spherical aberration) is identical before and after, indicates that cold cataract is a result of supramolecular change .
It has been shown that the precipitation of γ-crystallin in the lens is correlated with the formation of cold cataract [2,8-11]. The precipitation of γ-crystallin at low temperatures is simply due to a sharp decrease in solubility as it approaches its isoelectric point . It is also suggested that cold cataract formation is due to phase-separation associated with the γ-crystallins. A protein-rich phase and a protein-poor phase coexist at and below the critical temperature (Tc) and these cause light scattering [4,9,11]. The opacity developed is due to localized changes in refractive index within the lens [5,12]. The cold cataract phenomenon is not reversible in all species. In most fishes (except the yellow fin tuna), the lenses undergoes an irreversible cold cataract effect at -10 °C to -20 °C . Fish lenses contain an abundance in low molecular weight proteins that are similar to the mammalian γ-crystallin and these cause the irreversible effects of the cold cataract .
The primary goal of this research was the examination of the optical effects of cold cataract formation in young and old bovine lenses using an in vitro lens culture approach in conjunction with a specially designed laser scanning instrument. Previous experiments were unable to demonstrate the formation of cold cataract in whole lenses of older animals [5,10,11].
Bovine lens culture
Fresh bovine eyes were purchased from two local abattoirs, Delft Blue Inc., Cambridge, Ontario (18 week old [n=14] calf eyes) and Better Beef Ltd., Guelph, Ontario (approximately 10 year old [(n=7)] eyes). Eyes were dissected under sterile conditions and lenses were carefully excised on the same day, as described in previous work [14,15]. The lenses were then placed in a two-chambered cell made of borosilicate glass and silicon rubber and immersed in culture medium. The culture medium consisted of M199 (M3769; Sigma, St. Louis, MO) with Earl's salts, and the addition of L-glutamine (0.1 g/L), sodium bicarbonate (2.2 g/L), Hepes (5.92 g/L), penicillin/streptomycin (100 units/ml), and 3% dialyzed fetal bovine serum. The lenses were incubated at 37 °C in an atmosphere of 4.0% CO2 air. Lenses were incubated for 24 h before any treatment to ensure that they were not damaged during the dissection.
Laser scanning instrument
The laser scanning system (ScanToxTM) was used to analyze the optical quality of the lenses. The laser scanner consists of a collimated laser source (on an X-Y table) that projects a beam onto a mirror mounted at 45° on a carriage assembly. The reflected beam then goes up through the scanner table surface and through the lens under examination. There are two digital cameras that capture images of the light beam being transmitted through the lens. The image information collected by the digital cameras is transferred to a computer, and the refracted direction and pixel excitation level for each of the beam positions is recorded. Each scan involved 22 laser positions across an 11 mm diameter with a step size of 0.5 mm, thus there were eleven measurements on each side of the optical center of each bovine lens tested. The results are given in relative scatter (or intensity) of light across the lens, based on pixel excitation. Cold cataracts are located in the nuclear region of the bovine lens and the increased scatter associated with cold cataract was detected by the laser scanning system.
Induction of cold cataract
To induce the formation of cold cataract and the recovery process, a modified borosilicate glass two-chambered cell with two glass extensions, an inlet and an outlet was used. A closed circuit of circulating culture medium was established by attaching 4 m of clear flexible tubing (1/4 inch ID x 5/16 inch OD) on each glass extension and connecting the inlet and outlet tubing to the corresponding ports on a small peristaltic variable-flow pump. The tubing was coiled and immersed inside a digitally controlled water bath. This allowed the culture medium to be circulated at temperatures controlled by the water bath. A digital thermometer (accuracy ±1 °C) was placed inside the glass chamber to monitor the temperature of the fluid surrounding the lens. The thermometer was removed each time an optical scan was made and then replaced.
The temperature cycle inside the chamber started at 37 °C, slowly cooled to 4 °C, and then warmed back up to 37 °C. One cooling and warming cycle took approximately 2 h. Figure 1 presents a movie of an optical scan during cold cataract formation in a young bovine lens, while Figure 2 shows the appearance of the cold cataract in a young and old bovine lens. All lenses underwent the same cooling and warming cycle but the first appearance of opacities were different for each lens. For the purpose of comparison between both age groups we chose a temperature that demonstrated the most prominent cold cataract formation for both groups (4 °C is the endpoint before freezing levels).
The relative light transmittance was measured as a function of pixel excitation caused by refracted beams and compared to pre-treatment measures. A decrease in transmittance was proportional to the increased scatter of refracted light caused by lens opacification. Figure 3 represents the normalized relative transmittance measurements for each group. Three critical temperature points were used to demonstrate the effect of the cold cataract; pre-cold cataract (37 °C), cold cataract (4 °C), and recovery or post-cold cataract (37 °C). Each group of lenses showed a significant decrease in transmittance at 4 °C, which recovered when the lenses were warmed to 37 °C.
Statistical analysis with repeated-measure analysis of variance showed that there was an effect of temperature on relative light transmittance (p<0.05), but no age effect (p>0.05). Lenses from young eyes showed less loss of refracted beam intensity than lenses from older eyes (32% versus 34%), although the difference was not significant (Figure 3).
The ocular lens grows throughout life by continuous layering of new fiber cells on top of older lens fibers . Thus, a minimal turnover rate of proteins occurs in the nuclear regions of the lens [16-18]. As the age of the lens increases, there is a decrease in the amount of γ-crystallin. The exception is γS-crystallin, which increases with age [12,17,19-21]. Studies have also shown that age-related changes of γ-crystallin are due to post-translational modification, which may affect the susceptibility to cataract formation by changing the critical temperature of phase separation [22-24]. Hence, the cold cataract phenomenon is most apparent in young lenses (Figure 1). However, it has been noted that cold cataract formation in older lens extractions can still be induced at much lower temperatures [4,10]. This is also supported by a previous experiment, which showed that temperature sensitivity to phase transition of the intact rat lens decreases with age and correlates with a lower critical temperature (Tc) among older lenses . In general however, and as already noted in the introduction, it was widely believed that older mammalian lenses are not susceptible to the formation of cold cataract. Previous work was mainly performed on calf lenses and on isolated lens protein fractions and the γ-crystallin aggregation was detected in respect to light scattering properties [6,8,11].
The results of the present study indicate that there is a decrease in transmitted light in both old and young bovine lenses, as shown by using a scanning laser instrument (Figure 2 and Figure 3). This device is being tested for other cataract conditions and may prove to be a useful method in cataract research. In this context it is important to note that while the measure of pixel excitation used to measure relative transmittance showed that the transmittance of light is decreased at lower temperatures in both old and young lenses, there is no difference between the two even though the cataract is clearly denser in younger lenses (Figure 2). We assume that this is because of the fact the laser beam exiting the lens will excite a larger number of pixels as it broadens with loss of transmittance. However, with continued loss of transmittance, the light available at the edges of the beam will fall below excitation threshold and pixel excitation values will fall. Thus, in its current state this system can objectively measure the onset of cold cataract, and the reversal of cold cataract, but not gradations in the degree of this cataract.
The authors acknowledge the support of the Natural Sciences and Engineering Research council of Canada.
1. Bettelheim FA, Chen A. Thermodynamic stability of bovine alpha-crystallin in its interactions with other bovine crystallins. Int J Biol Macromol 1998 May-Jun; 22:247-52.
2. Loewenstein MA, Bettelheim FA. Cold cataract formation in fish lenses. Exp Eye Res 1979; 28:651-63.
3. Huang FY, Chia CM, Ho Y. The formation of oxidatively induced high-molecular-weight aggregate of alpha-/gamma-crystallins. Biochem Biophys Res Commun 1999; 260:60-5.
4. Lo WK. Visualization of crystallin droplets associated with cold cataract formation in young intact rat lens. Proc Natl Acad Sci U S A 1989; 86:9926-30.
5. Lerman S, Ashley DL, Long RC Jr, Goldstein JH, Megaw JM, Gardner K. Nuclear magnetic resonance analyses of the cold cataract: whole lens studies. Invest Ophthalmol Vis Sci 1982; 23:218-26.
6. Clark JI, Benedek GB. The effects of glycols, aldehydes, and acrylamide on phase separation and opacification in the calf lens. Invest Ophthalmol Vis Sci 1980; 19:771-6.
7. Sivak JG, Stuart DD, Weerheim JA. Optical performance of the bovine lens before and after cold cataract. Appl Opt 1992; 31(19):3616-20.
8. Bettelheim FA, Christian S. Thermal analysis of cold cataract formation. Lens Res 1983; 1:147-158.
9. Mitton KP, Hess JL, Bunce GE. Causes of decreased phase transition temperature in selenite cataract model. Invest Ophthalmol Vis Sci 1995; 36:914-24.
10. Lerman S, Megaw JM, Gardner K, Ashley D, Long RC Jr, Goldstein JH. NMR analyses of the cold cataract. II. Studies on protein solutions. Invest Ophthalmol Vis Sci 1983; 24:99-105.
11. Siezen RJ, Fisch MR, Slingsby C, Benedek GB. Opacification of gamma-crystallin solutions from calf lens in relation to cold cataract formation. Proc Natl Acad Sci U S A 1985; 82:1701-5.
12. Liu C, Asherie N, Lomakin A, Pande J, Ogun O, Benedek GB. Phase separation in aqueous solutions of lens gamma-crystallins: special role of gamma s. Proc Natl Acad Sci U S A 1996; 93:377-82.
13. Uga S, Ishikawa S, Hikida M, Iwata S. Ultrastructural study of rainbow trout lenses incubated under various conditions. Ophthalmic Res 1986; 18:156-64.
14. Banh A, Vijayan MM, Sivak JG. Hsp70 in bovine lenses during temperature stress. Mol Vis 2003; 9:323-8 <http://www.molvis.org/molvis/v9/a45/>.
15. Bantseev V, McCanna D, Banh A, Wong WW, Moran KL, Dixon DG, Trevithick JR, Sivak JG. Mechanisms of ocular toxicity using the in vitro bovine lens and sodium dodecyl sulfate as a chemical model. Toxicol Sci 2003; 73:98-107.
16. Harding JJ, Dilley KJ. Structural proteins of the mammalian lens: a review with emphasis on changes in development, aging and cataract. Exp Eye Res 1976; 22:1-73.
17. Ma Z, Hanson SR, Lampi KJ, David LL, Smith DL, Smith JB. Age-related changes in human lens crystallins identified by HPLC and mass spectrometry. Exp Eye Res 1998; 67:21-30.
18. McFall-Ngai MJ, Ding LL, Takemoto LJ, Horwitz J. Spatial and temporal mapping of the age-related changes in human lens crystallins. Exp Eye Res 1985; 41:745-58.
19. Hanson SR, Smith DL, Smith JB. Deamidation and disulfide bonding in human lens gamma-crystallins. Exp Eye Res 1998; 67:301-12.
20. Wang K, Li D, Sun F. Dietary caloric restriction may delay the development of cataract by attenuating the oxidative stress in the lenses of Brown Norway rats. Exp Eye Res 2004; 78:151-8.
21. Srivastava OP, Srivastava K. Degradation of gamma D- and gamma s-crystallins in human lenses. Biochem Biophys Res Commun 1998; 253:288-94.
22. Tumminia SJ, Clark JI, Richiert DM, Mitton KP, Duglas-Tabor Y, Kowalak JA, Garland DL, Russell P. Three distinct stages of lens opacification in transgenic mice expressing the HIV-1 protease. Exp Eye Res 2001; 72:115-21.
23. Stephan DA, Gillanders E, Vanderveen D, Freas-Lutz D, Wistow G, Baxevanis AD, Robbins CM, VanAuken A, Quesenberry MI, Bailey-Wilson J, Juo SH, Trent JM, Smith L, Brownstein MJ. Progressive juvenile-onset punctate cataracts caused by mutation of the gammaD-crystallin gene. Proc Natl Acad Sci U S A 1999; 96:1008-12.
24. Kosinski-Collins MS, King J. In vitro unfolding, refolding, and polymerization of human gammaD crystallin, a protein involved in cataract formation. Protein Sci 2003; 12:480-90.