Standardization of a new method for assessing the development of cataract in cultured bovine lenses

Abstract

PurposeTo standardize a new method for assessing cataractogenesis in isolated cultured bovine lenses using L-cysteine as the standard anti-cataract agent. MethodsIntact bovine lenses were cultured in DMEM with L-cysteine in presence or absence of hydrogen peroxide (H2O2). Lens opacity (transmittance) was determined using a plate reader. Lens homogenate glutathione (GSH) and superoxide dismutase (SOD) contents were measured using enzyme immunoassays kits. ResultsDMEM-cultured lenses exhibited a time-dependent loss in transmittance (230–710 nm) up to 120 h, achieving the highest reduction of 38.6 ± 0.09% at 420 nm (p < .001;n = 12). Compared to untreated lenses (time in hours [t] = 0), L-cysteine (10−6 M and 10−5 M) significantly (p < .001;n = 6) increased time-dependent transmittance (420 nm) by 31.6 ± 0.17% and 28.0 ± 0.07%(t = 120), respectively. When compared to DMEM-cultured lenses (t = 0), H2O2 (10 mM, 50 mM and 100 mM) significantly (p < .001;n = 12) reduced transmittance by 57.8 ± 0.1, 57.4 ± 0.04 and 87.7 ± 0.6%(t = 120), respectively. Moreover, L-cysteine significantly (p < .001;n = 6) attenuated H2O2 (50 mM)-induced decrease in transmittance by 12.5 ± 0.05%(10−6 M), 13.0 ± 0.09%(10−5 M), 14.5 ± 0.08%(10−4 M) and 8.6 ± 0.11%(10−3 M)(t = 120), respectively.When compared to untreated lenses (t = 0), the time-dependent decrease (p < .001;n = 5) in lenticular total GSH content and total SOD activity of 46.1 ± 0.06% and 42.0 ± 1.65% (t = 120) was attenuated (p < .001;n = 5) by L-cysteine (10−6 M) by 76.6 ± 0.06% and 7.4 ± 1.98%, respectively. Similarly, the H2O2(50 mM)-induced decline (p < .001; n = 5) in total GSH content and SOD activity of 82.6 ± 0.08% and 86.6 ± 0.66% (t = 120) was attenuated by L-cysteine (10−4 M) by 74.7 ± 1.05% and 161.1 ± 4.9%, respectively. ConclusionMeasurement of spectral transmission coupled with assessment of the activity of antioxidant enzymes in bovine cultured lens can provide a useful tool in studies of cataracts in an animal model of this disease.