The Lens’ Circulation in 14-Month-Old GPX-1 KO and WT Mice

Abstract

Purpose:: Knockout (KO) of Glutathione Peroxidase-1 (GPX-1) provides a useful model to study long term effects of H₂O₂ when an important protective mechanism is absent. GPX-1 KO mice get an age related nuclear cataract. We previously reported results on gap junction coupling and [Ca²⁺]_i in lenses from 2 month old GPX-1 KO and WT mice. We have extended this study to include fiber cell membrane water permeability p_f, [Na⁺]_i and to 14-month-old mice.

Methods:: Impedance studies of gap junction coupling were performed in intact lenses. [Ca²⁺]_i was measured by injecting the Ca²⁺-sensitive dye FURA2 into lens fiber cells. [Na⁺]_i was similarly measured using the Na⁺-sensitive dye SBFI. Fiber cell membrane vesicle swelling in hypertonic medium was used to determine p_f.

Results:: At 2 months of age, all lenses were transparent. In WT lenses, p_f (µm/s) was 35±3, the outer differentiating fiber gap junction coupling conductance G_DF (S/cm²) was 1.04±.07, and the inner mature fiber coupling conductance G_MF (S/cm²) was 0.42±.08. At 14 months of age, almost all GPX-1 KO mice developed a cataract, but the same age control lenses were transparent. The p_f was 28±4 for WT vs 8±3 for KO. G_DF was 0.46±0.04 for WT vs 0.23±0.03 for KO; G_MF was 0.13±0.02 for WT vs 0.04±0.004 for KO. Lenses from 14-month-old WT mice showed a large accumulation of [Na⁺]_i relative to young lenses, however the data fell on a smooth diffusion curve suggesting the circulation was still functional. For the GPX-1 KO data, there was clear loss of the circulation in central cells as [Na⁺]_i accumulated beyond that predicted for diffusion. [Ca²⁺]_i in the lens core of both 14-month-old WT and KO mice was significantly higher than in young lenses, and the GPX-1 KO data deviated significantly from the prediction for a normal circulation, suggesting loss of calcium homeostasis.

Conclusions:: All lenses are subjected to cumulative oxidative damage that causes significant reductions in gap junction coupling and some loss of p_f. Knockout of GPX-1 increased the damage and caused age onset nuclear cataracts. Our model is that reductions in coupling disrupt the lens circulation. This disruption leads to Ca²⁺ accumulation, which activates Ca-dependent proteases and the resulting proteolysis causes the central opacity.

Keywords: gap junctions/coupling • oxidation/oxidative or free radical damage • calcium