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M-E Gosselin, CJ Kapustij, UD Venkateswaran, VR Leverenz, FJ Giblin; Raman Spectroscopy Shows a Loss of -SH- and an Increase in -SS- in Intact Lenses of a Hyperbaric Oxygen In Vivo Model for Nuclear Cataract . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2362.
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Purpose: In order to investigate nuclear cataract, we treat guinea pigs with hyperbaric oxygen (HBO). Our previous analyses of lens supernatants using this model have shown decreases in levels of lens GSH and protein sulfhydryl (PSH) groups and increases in protein disulfide (PSSP) and mixed disulfides. The purpose here was to confirm the oxygen-induced loss of -SH and the increase in -SS- in intact, freshly-excised lenses using Raman spectroscopy. Methods: Guinea pigs, initially 18 months old, were treated 3 times per week with HBO (2.5 atm, 100% oxygen, 2.5 h) for a total of 50 (4 months of treatment) and 85 (7 months of treatment) times to induce an increased level of nuclear light scatter in the lenses. Control lenses were from age-matched untreated animals. Intact lenses were placed in Tyrode's medium and analyzed by Raman spectroscopy performed in a 90° geometry. The Raman spectra were excited with a 514.5nm laser and the changes in the levels of -SS- (503cm-1) and -SH (2577cm-1) were measured. The laser beam was focused either in the center of the lens (nucleus) or in the equatorial region (cortex). Raman spectra were analyzed by fitting Lorentzian profiles to the observed data in the -SS- and -SH regions. Results: -SS- levels in the experimental nucleus increased by a factor of 2.1 (p=0.0001) and 2.5 (p=0.001) after 50 and 85 HBO treatments, respectively, compared to aged-matched controls. In the experimental cortex, there was less of an increase in -SS- (1.3 times the controls; p=0.03 and ≷0.1). Control nuclear -SS- and control cortical -SS- increased by 1.2 (p=0.05) and 1.3 (p=0.02) times, as the animals aged from 22 to 25 months-old. -SH levels in the experimental nucleus decreased by 13% (p=0.007) and 35% (p=0.001) after 50 and 85 HBO treatments, respectively. In the experimental cortex, -SH levels decreased by 8% and 14% after 50 and 85 HBO treatments, respectively; however, both values were not statistically significant (p≷0.1). For the control lenses, both the nuclear and cortical -SH levels did not change significantly as the animals aged from 22 to 25 months-old (p≷0.1). Conclusion: An increase in -SS- and a decrease in -SH, occuring primarily in the nucleus of intact, freshly-isolated lenses, were shown to be associated with a loss of lens nuclear transparency in HBO-treated guinea pigs. The results support the hypothesis that nuclear cataract is caused by an elevation of oxidative stress in the central region of the lens, and demonstrates the susceptibility of the lens nucleus to deleterious oxidative effects.
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